@article{house_gray_owen_jima_smart_hall_2023, title={C/EBP beta deficiency enhances the keratinocyte innate immune response to direct activators of cytosolic pattern recognition receptors}, volume={29}, ISSN={["1753-4267"]}, DOI={10.1177/17534259231162192}, abstractNote={ The skin is the first line of defense to cutaneous microbes and viruses, and epidermal keratinocytes play a critical role in preventing infection by viruses and pathogens through activation of the type I interferon (IFN) response. Using RNAseq analysis, here we report that the conditional deletion of C/EBPβ transcription factor in mouse epidermis (CKOβ mice) resulted in the upregulation of IFNβ and numerous keratinocyte interferon-stimulated genes (ISGs). The expression of cytosolic pattern recognition receptors (cPRRs), that recognize viral RNA and DNA, were significantly increased, and enriched in the RNAseq data set. cPRRs stimulate a type I IFN response that can trigger cell death to eliminate infected cells. To determine if the observed increases in cPRRs had functional consequences, we transfected CKOβ primary keratinocytes with the pathogen and viral mimics poly(I:C) (dsRNA) or poly(dA:dT) (synthetic B-DNA) that directly activate PRRs. Transfected CKOβ primary keratinocytes displayed an amplified type I IFN response which was accompanied by increased activation of IRF3, enhanced ISG expression, enhanced activation of caspase-8, caspase-3 and increased apoptosis. Our results identify C/EBPβ as a critical repressor of the keratinocyte type I IFN response, and demonstrates that the loss of C/EBPβ primes keratinocytes to the activation of cytosolic PRRs by pathogen RNA and DNA to induce cell death mediated by caspase-8 and caspase-3. }, number={1-2}, journal={INNATE IMMUNITY}, author={House, John S. and Gray, Sophia and Owen, Jennifer R. and Jima, Dereje D. and Smart, Robert C. and Hall, Jonathan R.}, year={2023}, month={Jan}, pages={14–24} }
 @article{kaur_barnes_pan_detwiler_liu_mahn_hall_messenger_you_piehler_et al._2021, title={TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA}, volume={49}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkab1142}, abstractNote={Abstract}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, author={Kaur, Parminder and Barnes, Ryan and Pan, Hai and Detwiler, Ariana C. and Liu, Ming and Mahn, Chelsea and Hall, Jonathan and Messenger, Zach and You, Changjiang and Piehler, Jacob and et al.}, year={2021}, month={Dec}, pages={13000–13018} }
 @article{kotlarz_mccord_collier_lea_strynar_lindstrom_wilkie_islam_matney_tarte_et al._2020, title={Measurement of Novel, Drinking Water-Associated PFAS in Blood from Adults and Children in Wilmington, North Carolina}, volume={128}, ISSN={["1552-9924"]}, DOI={10.1289/EHP6837}, abstractNote={Background: From 1980 to 2017, a fluorochemical manufacturing facility discharged wastewater containing poorly understood per- and polyfluoroalkyl substances (PFAS) to the Cape Fear River, the primary drinking water source for Wilmington, North Carolina, residents. Those PFAS included several fluoroethers including HFPO-DA also known as GenX. Little is known about the bioaccumulation potential of these fluoroethers. Objective: We determined levels of fluoroethers and legacy PFAS in serum samples from Wilmington residents. Methods: In November 2017 and May 2018, we enrolled 344 Wilmington residents ≥6 years of age into the GenX Exposure Study and collected blood samples. Repeated blood samples were collected from 44 participants 6 months after enrollment. We analyzed serum for 10 fluoroethers and 10 legacy PFAS using liquid chromatography–high-resolution mass spectrometry. Results: Participants’ ages ranged from 6 to 86 y, and they lived in the lower Cape Fear Region for 20 y on average (standard deviation: 16 y). Six fluoroethers were detected in serum; Nafion by-product 2 and PFO4DA were detected in >99% of participants. PFO3OA and NVHOS were infrequently detected. Hydro-EVE was present in a subset of samples, but we could not quantify it. GenX was not detected above our analytical method reporting limit (2 ng/mL). In participants with repeated samples, the median decrease in fluoroether levels ranged from 34% for Nafion byproduct 2 to 65% for PFO4DA in 6 months due to wastewater discharge control. Four legacy PFAS (PFHxS, PFOA, PFOS, PFNA) were detected in most (≥97%) participants; these levels were higher than U.S. national levels for the 2015–2016 National Health and Nutrition Examination Survey. The sum concentration of fluoroethers contributed 23% to participants’ summed PFAS (median: 25.0 ng/mL). Conclusion: Poorly understood fluoroethers released into the Cape Fear River by a fluorochemical manufacturing facility were detected in blood samples from Wilmington, North Carolina, residents. Health implications of exposure to these novel PFAS have not been well characterized. https://doi.org/10.1289/EHP6837}, number={7}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Kotlarz, Nadine and McCord, James and Collier, David and Lea, C. Suzanne and Strynar, Mark and Lindstrom, Andrew B. and Wilkie, Adrien A. and Islam, Jessica Y. and Matney, Katelyn and Tarte, Phillip and et al.}, year={2020}, month={Jul} }
 @article{tam_hall_messenger_jima_house_linder_smart_2019, title={C/EBP beta suppresses keratinocyte autonomous type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer}, volume={40}, ISSN={["1460-2180"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85083447649&partnerID=MN8TOARS}, DOI={10.1093/carcin/bgz012}, abstractNote={Abstract}, number={9}, journal={CARCINOGENESIS}, author={Tam, Hann W. and Hall, Jonathan R. and Messenger, Zachary J. and Jima, Dereje D. and House, John S. and Linder, Keith and Smart, Robert C.}, year={2019}, month={Sep}, pages={1099–1109} }
 @article{messenger_hall_jima_house_tam_tokarz_smart_2018, title={C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event}, volume={9}, ISSN={2041-4889}, url={http://dx.doi.org/10.1038/S41419-018-1103-Y}, DOI={10.1038/S41419-018-1103-Y}, abstractNote={Abstract}, number={11}, journal={Cell Death & Disease}, publisher={Springer Science and Business Media LLC}, author={Messenger, Zachary J. and Hall, Jonathan R. and Jima, Dereje D. and House, John S. and Tam, Hann W. and Tokarz, Debra A. and Smart, Robert C.}, year={2018}, month={Oct} }
 @inbook{smart_hall_2018, place={Hoboken, NJ}, edition={5th}, title={Carcinogenesis}, booktitle={Molecular and Biochemical Toxicology}, publisher={J Wiley and Sons}, author={Smart, R.C. and Hall, J.R.}, editor={Smart, R.C. and Hodgson, E.Editors}, year={2018} }
 @inbook{tsuji_smart_2018, place={Hoboken, NJ}, edition={5th}, title={Molecular Techniques in the Study of Gene Function}, booktitle={Molecular and Biochemical Toxicology}, publisher={J Wiley and Sons}, author={Tsuji, Y. and Smart, R.C.}, editor={Smart, R.C. and Hodgson, E.Editors}, year={2018} }
 @book{smart_hodgson_2018, place={Hoboken, NJ}, edition={5th}, title={Molecular and Biochemical Toxicology}, publisher={J. Wiley and Sons}, year={2018} }
 @book{molecular and biochemical toxicology_2018, edition={5th}, publisher={J. Wiley and Sons}, year={2018} }
 @inbook{smart_hodgson_2018, place={Hoboken, NJ}, edition={5th}, title={Molecular and Biochemical Toxicology: Definition and Scope}, booktitle={Molecular and Biochemical Toxicology}, publisher={J Wiley and Sons}, author={Smart, R.C. and Hodgson, E.}, editor={Smart, R.C. and Hodgson, E.Editors}, year={2018} }
 @article{hall_messenger_tam_phillips_recio_smart_2015, title={Long noncoding RNA lincRNA-p21 is the major mediator of UVB-induced and p53-dependent apoptosis in keratinocytes}, volume={6}, ISSN={["2041-4889"]}, DOI={10.1038/cddis.2015.67}, abstractNote={Abstract}, journal={CELL DEATH & DISEASE}, author={Hall, J. R. and Messenger, Z. J. and Tam, H. W. and Phillips, S. L. and Recio, L. and Smart, R. C.}, year={2015}, month={Mar} }
 @article{hall_bereman_nepomuceno_thompson_muddiman_smart_2014, title={C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint}, volume={13}, ISSN={1538-4101 1551-4005}, url={http://dx.doi.org/10.4161/15384101.2014.962957}, DOI={10.4161/15384101.2014.962957}, abstractNote={The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21.}, number={22}, journal={Cell Cycle}, publisher={Informa UK Limited}, author={Hall, Jonathan R and Bereman, Michael S and Nepomuceno, Angelito I and Thompson, Elizabeth A and Muddiman, David C and Smart, Robert C}, year={2014}, month={Oct}, pages={3602–3610} }
 @article{thompson_zhu_hall_house_ranjan_burr_he_owens_smart_2011, title={C/EBP alpha Expression Is Downregulated in Human Nonmelanoma Skin Cancers and Inactivation of C/EBP alpha Confers Susceptibility to UVB-Induced Skin Squamous Cell Carcinomas}, volume={131}, ISSN={["0022-202X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79956039252&partnerID=MN8TOARS}, DOI={10.1038/jid.2011.31}, abstractNote={Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. CCAAT/enhancer-binding protein-α DNA damage response immunohistochemical keratin 5 minimum erythemic dose phosphate-buffered saline squamous cell carcinoma 12-O-tetradecanoylphorbol-13-acetate The epidermis is routinely subject to DNA damage by UVB, which is considered to be the principal carcinogenic component of sunlight. Exposure to UVB results in DNA damage in the form of cyclobutane pyrimidine dimers, 6–4 photoproducts, DNA strand breaks, and DNA crosslinks (Brash, 1997Brash D.E. Sunlight and the onset of skin cancer.Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (259) Google Scholar; de Gruijl et al., 2001de Gruijl F.R. van Kranen H.J. Mullenders L.H. UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer.J Photochem Photobiol B. 2001; 63: 19-27Crossref PubMed Scopus (385) Google Scholar). If not repaired or if misrepaired, this DNA damage can result in mutations in the genome, and can ultimately contribute to the development of skin cancers (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1643) Google Scholar). Solar radiation is responsible for >1,000,000 nonmelanoma skin cancer cases per year in the United States, and these cases account for 40% of all new cancer cases diagnosed each year in the United States (American Cancer Society, 2008American Cancer SocietyCancer Facts & Figures 2008.in: 2008http://www.cancer.org/Research/CancerFactsFigures/cancer-facts-figures-2008Google Scholar). To maintain genome integrity and to prevent heritable mutations that lead to cancer, cells respond to DNA damage produced by intrinsic or environmental factors by engaging the DNA damage response (DDR) network. This network entails signaling pathways involving cell cycle checkpoints, DNA repair, transcription programs, and apoptosis. Cell cycle checkpoints can occur in any phase of the cell cycle and are characterized as a pause in the cell cycle that allows time for the repair of damaged DNA. Defective checkpoints can contribute to genome instability and cancer pathogenesis (Kastan and Bartek, 2004Kastan M.B. Bartek J. Cell-cycle checkpoints and cancer.Nature. 2004; 432: 316-323Crossref PubMed Scopus (2028) Google Scholar). C/EBPα (CCAAT/enhancer-binding protein-α) is one of the six members of the C/EBP family of basic leucine zipper transcription factors (Ramji and Foka, 2002Ramji D.P. Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation.Biochem J. 2002; 365: 561-575Crossref PubMed Google Scholar; Johnson, 2005Johnson P.F. Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors.J Cell Sci. 2005; 118: 2545-2555Crossref PubMed Scopus (221) Google Scholar). C/EBPα has been established as a tumor-suppressor gene in human acute myeloid leukemia (Pabst et al., 2001Pabst T. Mueller B.U. Zhang P. et al.Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.Nat Genet. 2001; 27: 263-270Crossref PubMed Scopus (705) Google Scholar). Additionally, there is circumstantial evidence for its function as a tumor suppressor based on diminished C/EBPα expression in a multitude of human tumor types including liver (Xu et al., 2001Xu L. Hui L. Wang S. et al.Expression profiling suggested a regulatory role of liver-enriched transcription factors in human hepatocellular carcinoma.Cancer Res. 2001; 61: 3176-3181PubMed Google Scholar), lung (Halmos et al., 2002Halmos B. Huettner C.S. Kocher O. et al.Down-regulation and antiproliferative.Cancer Res. 2002; 62: 528-534PubMed Google Scholar), breast (Gery et al., 2005Gery S. Tanosaki S. Bose S. et al.Down-regulation and growth inhibitory role of C/EBPalpha in breast cancer.Clin Cancer Res. 2005; 11: 3184-3190Crossref PubMed Scopus (76) Google Scholar), endometrial (Takai et al., 2005Takai N. Kawamata N. Walsh C.S. et al.Discovery of epigenetically masked tumor suppressor genes in endometrial cancer.Mol Cancer Res. 2005; 3: 261-269Crossref PubMed Scopus (63) Google Scholar), and head and neck squamous cell carcinomas (SCCs; Bennett et al., 2007Bennett K.L. Hackanson B. Smith L.T. et al.Tumor suppressor activity of CCAAT/e.Cancer Res. 2007; 67: 4657-4664Crossref PubMed Scopus (70) Google Scholar). The traditional view of C/EBPα in cell biology involves its role in cellular differentiation and metabolism (Roesler, 2001Roesler W.J. The role of C/EBP in nutrient and hormonal regulation of gene expression.Annu Rev Nutr. 2001; 21: 141-165Crossref PubMed Scopus (81) Google Scholar); in cancer, the traditional view holds that it has a tumor-suppressor role, where the loss of expression/function results in an impaired differentiation commitment accompanied by deregulated proliferation (Schuster and Porse, 2006Schuster M.B. Porse B.T. C/EBPalpha: a tumour suppressor in multiple tissues?.Biochim Biophys Acta. 2006; 1766: 88-103PubMed Google Scholar; Koschmieder et al., 2009Koschmieder S. Halmos B. Levantini E. et al.Dysregulation of the C/EBPalpha differentiation pathway in human cancer.J Clin Oncol. 2009; 27: 619-628Crossref PubMed Scopus (155) Google Scholar). However, recent studies indicate that the role of C/EBPα in cells/cancer is more complex and multifaceted than originally thought (Yoon and Smart, 2004Yoon K. Smart R.C. C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.Mol Cell Biol. 2004; 24: 10650-10660Crossref PubMed Scopus (53) Google Scholar; Loomis et al., 2007Loomis K.D. Zhu S. Yoon K. et al.Genetic ablation of CCAAT/enhancer binding protein {alpha.Cancer Res. 2007; 67: 6768-6776Crossref PubMed Scopus (34) Google Scholar; Ranjan et al., 2009Ranjan R. Thompson E.A. Yoon K. et al.C/EBPalpha expression is partially regulated by C/EBPbeta in response to DNA damage and C/EBPalpha-deficient fibroblasts display an impaired G1 checkpoint.Oncogene. 2009; 28: 3235-3245Crossref PubMed Scopus (8) Google Scholar). Cell culture studies have revealed an unexpected role for C/EBPα in the DDR in keratinocytes where C/EBPα is induced following UVB-induced DNA damage, and it regulates the G1 checkpoint. Diminished or ablated expression of C/EBPα results in G1 checkpoint failure following UVB-induced DNA damage (Yoon and Smart, 2004Yoon K. Smart R.C. C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.Mol Cell Biol. 2004; 24: 10650-10660Crossref PubMed Scopus (53) Google Scholar; Ranjan et al., 2009Ranjan R. Thompson E.A. Yoon K. et al.C/EBPalpha expression is partially regulated by C/EBPbeta in response to DNA damage and C/EBPalpha-deficient fibroblasts display an impaired G1 checkpoint.Oncogene. 2009; 28: 3235-3245Crossref PubMed Scopus (8) Google Scholar). In further support of a nonparadigmatic C/EBPα tumor-suppressor function, mice lacking C/EBPα in their epidermis do not display alterations in differentiation or proliferation and are susceptible to chemical carcinogen-induced skin tumorigenesis (Loomis et al., 2007Loomis K.D. Zhu S. Yoon K. et al.Genetic ablation of CCAAT/enhancer binding protein {alpha.Cancer Res. 2007; 67: 6768-6776Crossref PubMed Scopus (34) Google Scholar). Given the function of C/EBPα as a mediator of the G1 checkpoint in keratinocytes in response to UVB, it is possible that C/EBPα functions as a suppressor of UVB-induced tumorigenesis. Whereas C/EBPα expression is diminished in mouse skin SCCs (Oh and Smart, 1998Oh H.S. Smart R.C. Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in skin neoplasms.J Invest Dermatol. 1998; 110: 939-945Crossref PubMed Scopus (79) Google Scholar; Shim et al., 2005Shim M. Powers K.L. Ewing S.J. et al.Diminished expression of C/EBPalpha in skin carcinomas is linked to oncogenic Ras and reexpression of C/EBPalpha in carcinoma cells inhibits proliferation.Cancer Res. 2005; 65: 861-867PubMed Google Scholar), C/EBPα levels have not been examined in human skin precancerous and cancerous lesions. Therefore, the objectives of this study were: to examine the expression of C/EBPα in human skin precancerous and cancerous lesions, to characterize the response of C/EBPα to UVB in human keratinocytes and human skin; and to develop an in vivo SKH-1 mouse model to determine the in vivo physiological significance of C/EBPα in UVB-induced skin tumorigenesis. We examined the expression of C/EBPα in normal human epidermis, precancerous actinic keratoses, keratoacanthomas, SCCs in situ, and invasive SCCs as well as basal cell carcinomas. Immunohistochemical (IHC) staining for C/EBPα in human skin showed that C/EBPα was extensively expressed in the nuclei of nondividing keratinocytes of the suprabasal layers of the epidermis (Figure 1a). C/EBPα expression was also detected, although less frequently, in keratinocytes in the proliferative basal layer of epidermis. Actinic keratoses, a precancerous benign skin growth of which a small percentage progress to SCC, expressed levels of C/EBPα similar to normal epidermis (Figure 1a and b). Keratoacanthomas, once considered as terminally benign but now regarded and treated by many dermatologists as a malignant growth that can progress to SCC, expressed reduced levels of C/EBPα with 20% of keratoacanthomas no longer expressing detectable levels of C/EBPα (Figure 1a and b). Most striking, however, were the SCCs where the majority of both SCCs in situ (80%) and invasive SCCs (100%) no longer expressed detectable levels of C/EBPα (Figure 1a and b). Similarly, the IHC staining for C/EBPα was absent in 14/16 basal cell carcinoma cases (data not shown). Most human nonmelanoma skin cancers are caused by solar radiation, and UVB radiation is considered the most carcinogenic component of sunlight. To characterize the effects of UVB exposure on the expression of C/EBPα in human keratinocytes, we exposed subconfluent proliferating normal human epidermal keratinocytes to 5, 10, or 15mJcm2 UVB. Immunoblot analysis for C/EBPα revealed that the expression of C/EBPα protein was induced at all doses (Figure 2a). To characterize the effects of UVB exposure on the expression of C/EBPα in human skin in vivo, biopsies from UVB-treated human skin (1 minimum erythemic dose (MED) UVB) (N=5) were compared with biopsies from non-sun-exposed human skin (N=5) for C/EBPα expression. C/EBPα levels were increased throughout the epidermis of UVB-treated human skin as determined by the increased C/EBPα IHC nuclear staining intensity as well as by the overall statistically significant increase in the number of keratinocytes staining positively for C/EBPα (Figure 2b–d and Supplementary Figure S1 online). Although UVB treatment increased the percentage of C/EBPα-expressing keratinocytes in the spinous and granular nondividing suprabasal layers by 2.3-fold, we observed a 4.3-fold increase in the number of basal keratinocytes expressing C/EBPα (Figure 2b–d). Download .pdf (.66 MB) Help with pdf files Supplementary Figures S1 and S2 We treated SKH-1 hairless mice, a well-characterized mouse model frequently utilized to study the effects of UVB in skin in vivo (Benavides et al., 2009Benavides F. Oberyszyn T.M. VanBuskirk A.M. et al.The hairless mouse in skin research.J Dermatol Sci. 2009; 53: 10-18Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), with UVB (50mJcm2) and, similar to human skin, we observed a significant increase in C/EBPα-expressing keratinocytes in the suprabasal and basal proliferative compartments of epidermis (Figure 3a). There was an ∼2-fold increase in the number of C/EBPα positively stained suprabasal cells, and a 3-fold increase in the number of C/EBPα positively stained basal keratinocytes (Figure 3b). To begin to determine the physiological significance of UVB-induced epidermal C/EBPα in vivo, we generated an epidermal-specific C/EBPα knockout SKH-1 mouse, hereafter referred to as conditional knockout (CKOα). CKOα mice were generated from C/EBPαfl/fl SKH-1 mice and keratin 5 (K5) Cre (K5Cre) SKH-1 mice where the K5 promoter directs Cre recombinase expression to the epidermis and other stratified squamous epithelia. As shown in Figure 3c, C/EBPα protein was not detectable in epidermal protein extracts prepared from CKOα mice. CKOα mice did not display any abnormal gross or morphological skin phenotype (data not shown). The most susceptible human population to UVB-induced damage is classified as type 1 according to the Fitzpatrick Classification Scale. These individuals have very fair and often freckled skin, have blond or red hair, always sun burn, and are highly susceptible to solar radiation-induced nonmelanoma skin cancer. The MED of UVB for this susceptible population is ∼20–25mJcm2. Therefore, we exposed CKOα and K5Cre control mice to 20mJcm2 UVB three times weekly to test whether the loss of C/EBPα in skin keratinocytes confers susceptibility to UVB-induced tumorigenesis. As shown in Figure 4a and b, CKOα mice were highly susceptibility to UVB-induced skin tumorigenesis as evidenced by decreased latency (18 weeks), increased tumor incidence (85 vs. 25%), and a 6-fold increase in tumor multiplicity. Histological analysis of all skin tumors at the termination of experiment revealed that the majority of tumors (∼90%) in both genotypes were SCCs (in situ or invasive). Most importantly, we observed that 48% of SCCs in CKOα mice were invasive SCCs compared with 15% in the UVB-treated K5Cre control mice (Figure 4c). Invasive carcinomas were identified by severe dysplasia to anaplastic growth, marked atypia in all cell layers, and most importantly, invasion through the panniculus carnosus and/or basement membrane (Supplementary Figure S2 online). To demonstrate that these tumors were the result of UVB treatment and not advanced age, 8 CKOα mice were held for 52 weeks and no skin tumors of any kind were detected. Collectively, these results indicate that loss of C/EBPα confers susceptibility to UVB-induced skin cancer at a biologically relevant dose and loss of C/EBPα accelerates skin tumor progression. To determine whether the ablation of epidermal C/EBPα alters the ability of keratinocytes to undergo a cell cycle arrest in response to UVB treatment in vivo in mouse skin, we treated K5Cre control mice and CKOα mice with UVB, and then examined the number of actively replicating keratinocytes post-UVB by measuring the incorporation of the nucleotide analog BrdU (1hour BrdU pulse before skin collection). As shown in Figure 5a, UVB-treated control mice (K5Cre) displayed a significant cell cycle arrest; at 4hours post-UVB treatment, there was ∼60% decrease in the number of BrdU-positive S-phase basal keratinocytes in the epidermis and this decrease was sustained at 6 and 10hours post-UVB (Figure 5a). At 12hours post-UVB, the number of BrdU-positive S-phase basal keratinocytes returned to untreated control levels (data not shown). Although UVB-treated CKOα mice displayed a similar cell cycle arrest as UVB-treated control mice at 4hours post-UVB treatment, this inhibition was not sustained and cells resumed their progression in the cell cycle prematurely. At 6hours post-UVB, the number of BrdU-positive S-phase cells was significantly increased and by 10hours post-UVB treatment, there was a 3-fold increase in BrdU-positive S-phase basal keratinocytes in CKOα epidermis compared with UVB-treated control mice. Representative examples of BrdU-positive S-phase staining at 10hours post-UVB in K5Cre and CKOα mouse epidermis are shown in Figure 5a (right panel). These results indicate that the loss of C/EBPα in epidermis results in an impaired cell cycle arrest in response to UVB in vivo. To further investigate the role of C/EBPα in UVB-induced cell cycle checkpoints in epidermis in vivo, we utilized an in vivo model of cell cycle regulation involving the induction of synchronous cell proliferation induced by the potent mitogen, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Foley et al., 1993Foley J. Ton T. Maronpot R. et al.Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats.Environ Health Perspect. 1993; 101: 199-205Crossref PubMed Scopus (96) Google Scholar; Kim et al., 1997Kim T.W. Porter K.L. Foley J.F. et al.Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha-ras genotype.Mol Carcinog. 1997; 20: 115-124Crossref PubMed Scopus (9) Google Scholar; Rodriguez-Puebla et al., 1998Rodriguez-Puebla M.L. Robles A.I. Johnson D.G. et al.Synchronized proliferation induced by 12-O-tetradecanoylphorbol-13-acetate treatment of mouse skin: an in vivo model for cell cycle regulation.Cell Growth Differ. 1998; 9: 31-39PubMed Google Scholar). A single topical treatment of TPA resulted in synchronous entry of G1 keratinocytes into S phase as determined by BrdU-positive basal cells at 12hours post-TPA treatment and S-phase cells further increased at 14 and 16hours in control mice (1hour BrdU pulse before skin collection; Figure 5b). TPA-treated CKOα mice responded similarly and the numbers of S-phase cells were not significantly different from TPA-treated control mice at all time points (P>0.3, data not shown). To determine the effect of UVB on this entry into S phase, K5Cre and CKOα mice were treated with TPA, and 4hours later, a time when keratinocytes are still in G1, were either treated with UVB (50mJcm2) or left untreated. The number of BrdU-positive S-phase cells were then determined at 14hours post-TPA treatment and 10hours post-UVB. As shown in Figure 5c, TPA-treated K5Cre and CKOα mice not exposed to UVB displayed a 3-fold increase in the number of BrdU-positive S-phase keratinocytes. UVB treatment of TPA-treated mice resulted in dramatic decreases in the number of BrdU-positive S-phase cells in the epidermis of both genotypes compared with TPA treatment alone (Figure 5c). However, CKOα mice displayed ∼3 times more BrdU-positive S-phase cells than similarly treated K5Cre mice. Collectively, our findings suggest that the loss of epidermal C/EBPα results in an impaired cell cycle arrest involving the G1- to S-phase transition in response to UVB in vivo in mouse epidermis. Because p53 is an important player in the DDR and a key mediator of checkpoints following DNA damage, we wanted to determine whether the impaired cell cycle arrest at the G1- to S-phase transition observed in the CKOα mice following UVB treatment could be related to deficiencies in p53. We performed immunoblot analysis for p53 on epidermal lysates from both UVB-treated (50mJcm2) and untreated K5Cre and CKOα mice, and observed an induction in p53 protein in both genotypes following UVB treatment (Figure 5d). To further determine whether p53 was induced to a similar extent in both genotypes, IHC staining and quantitation of p53-positive basal cells were performed, and no statistically significant differences were found (Figure 5d). C/EBPα is abundantly expressed in the mouse and human epidermis (Swart et al., 1997Swart G.W. van Groningen J.J. van Ruissen F. et al.Transcription factor C/EBPalpha: novel sites of expression and cloning of the human gene.Biol Chem. 1997; 378: 373-379Crossref PubMed Scopus (29) Google Scholar; Maytin and Habener, 1998Maytin E.V. Habener J.F. Transcription factors C/EBP alpha, C/EBP beta, and CHOP (Gadd153) expressed during the differentiation program of keratinocytes in vitro and in vivo.J Invest Dermatol. 1998; 110: 238-246Crossref PubMed Scopus (119) Google Scholar; Oh and Smart, 1998Oh H.S. Smart R.C. Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in skin neoplasms.J Invest Dermatol. 1998; 110: 939-945Crossref PubMed Scopus (79) Google Scholar) and in this study we report that C/EBPα is induced by UVB in human epidermal keratinocytes and human epidermis. Analysis of C/EBPα expression in human skin and human skin precancerous and cancerous lesions revealed that C/EBPα expression is downregulated in SCCs in situ and in invasive SCCs and that expression of C/EBPα is normal in actinic keratoses. These results indicate that loss of C/EBPα expression is not an early event in SCC development but occurs during progression from actinic keratoses to invasive SCCs. The mechanism through which C/EBPα is downregulated in human and mouse SCCs is unknown; however, promoter hypermethylation is a candidate as C/EBPα promoter hypermethylation has been shown to be important in the decreased expression of C/EBPα in breast cancer as well as head and neck cancers (Gery et al., 2005Gery S. Tanosaki S. Bose S. et al.Down-regulation and growth inhibitory role of C/EBPalpha in breast cancer.Clin Cancer Res. 2005; 11: 3184-3190Crossref PubMed Scopus (76) Google Scholar; Bennett et al., 2007Bennett K.L. Hackanson B. Smith L.T. et al.Tumor suppressor activity of CCAAT/e.Cancer Res. 2007; 67: 4657-4664Crossref PubMed Scopus (70) Google Scholar). p53 is frequently mutated in human skin cancer and this is an early essential event (Brash, 1997Brash D.E. Sunlight and the onset of skin cancer.Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Although actinic keratoses are the precursor lesion to SCCs, only a small percentage of actinic keratoses (1–10%) actually progress to SCCs, even though both actinic keratoses and SCCs share a high frequency (70%) of mutant p53 and similar p53 mutation spectra (Callen et al., 1997Callen J.P. Bickers D.R. Moy R.L. Actinic keratoses.J Am Acad Dermatol. 1997; 36: 650-653Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Ortonne, 2002Ortonne J.P. From actinic keratosis to squamous cell carcinoma.Br J Dermatol. 2002; 146: 20-23Crossref PubMed Scopus (101) Google Scholar; Giglia-Mari and Sarasin, 2003Giglia-Mari G. Sarasin A. TP53 mutations in human skin cancers.Hum Mutat. 2003; 21: 217-228Crossref PubMed Scopus (197) Google Scholar; Stockfleth and Kerl, 2006Stockfleth E. Kerl H. Guidelines for the management of actinic keratoses.Eur J Dermatol. 2006; 16: 599-606PubMed Google Scholar). These results indicate that mutant p53 is not sufficient for the progression of actinic keratoses to SCCs and that additional events are required for actinic keratosis progression. One such event may be the loss of C/EBPα, as C/EBPα expression is retained in actinic keratoses, but C/EBPα expression is silenced in SCCs. These results suggest that loss of C/EBPα expression may be a critical event in the progression of precancerous actinic keratoses to invasive SCCs. Our current results in CKOα mice demonstrate that the ablation of C/EBPα in mouse epidermis confers susceptibility to UVB-induced SCCs at a biologically relevant dose and results in the acceleration of skin cancer progression to invasive SCCs. Additionally, earlier studies showed that mice lacking C/EBPα in their epidermis are also more susceptible to chemical carcinogen-induced skin tumorigenesis, and tumors that develop in these mice exhibited an accelerated rate of malignant progression to invasive SCCs (Loomis et al., 2007Loomis K.D. Zhu S. Yoon K. et al.Genetic ablation of CCAAT/enhancer binding protein {alpha.Cancer Res. 2007; 67: 6768-6776Crossref PubMed Scopus (34) Google Scholar). Collectively, these results indicate an important role for the loss of C/EBPα in skin cancer progression induced by UVB radiation or chemical carcinogens. The recent comprehensive genomic characterization of human cancers has revealed that genomic instability is a common characteristic of cancer, that human cancer cells contain hundreds if not thousands of mutations, and that the mutation rate in cancer cells greatly exceeds that in normal cells (Lengauer et al., 1998Lengauer C. Kinzler K.W. Vogelstein B. Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3268) Google Scholar; Bielas et al., 2006Bielas J.H. Loeb K.R. Rubin B.P. et al.Human cancers express a mutator phenotype.Proc Natl Acad Sci USA. 2006; 103: 18238-18242Crossref PubMed Scopus (276) Google Scholar; The Cancer Genome Atlas Research Network, 2008The Cancer Genome Atlas Research NetworkComprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (5212) Google Scholar; Ding et al., 2008Ding L. Getz G. Wheeler D.A. et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2004) Google Scholar; Nancarrow et al., 2008Nancarrow D.J. Handoko H.Y. Smithers B.M. et al.Genome-wide copy number analysis in esophageal adenocarcinoma using high-density single-nucleotide polymorphism arrays.Cancer Res. 2008; 68: 4163-4172Crossref PubMed Scopus (73) Google Scholar). It is the accumulation of genome alterations in critical genes over time that drives the cancer process. There is substantial evidence for a mutator phenotype in cancer in which the mutation rate in cancer cells is much greater than in normal cells, resulting in increases in random and clonal somatic mutations (Bielas and Loeb, 2005Bielas J.H. Loeb L.A. Quantification of random genomic mutations.Nat Methods. 2005; 2: 285-290Crossref PubMed Scopus (75) Google Scholar; Bielas et al., 2006Bielas J.H. Loeb K.R. Rubin B.P. et al.Human cancers express a mutator phenotype.Proc Natl Acad Sci USA. 2006; 103: 18238-18242Crossref PubMed Scopus (276) Google Scholar; Loeb et al., 2008Loeb L.A. Bielas J.H. Beckman R.A. Cancers exhibit a mutator phenotype: clinical implications.Cancer Res. 2008; 68 (discussion 3557): 3551-3557Crossref PubMed Scopus (159) Google Scholar). Studies in genetically engineered mice and keratinocytes in culture have revealed a critical role for C/EBPα in the DDR network where following DNA damage, C/EBPα is highly induced and has a critical role in the G1-cell cycle checkpoint (Yoon and Smart, 2004Yoon K. Smart R.C. C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.Mol Cell Biol. 2004; 24: 10650-10660Crossref PubMed Scopus (53) Google Scholar; Ranjan et al., 2009Ranjan R. Thompson E.A. Yoon K. et al.C/EBPalpha expression is partially regulated by C/EBPbeta in response to DNA damage and C/EBPalpha-deficient fibroblasts display an impaired G1 checkpoint.Oncogene. 2009; 28: 3235-3245Crossref PubMed Scopus (8) Google Scholar). The genetic ablation or knockdown of C/EBPα in keratinocytes results in G1 checkpoint failure in which cells inappropriately enter S phase following U}, number={6}, journal={JOURNAL OF INVESTIGATIVE DERMATOLOGY}, author={Thompson, Elizabeth A. and Zhu, Songyun and Hall, Jonathan R. and House, John S. and Ranjan, Rakesh and Burr, Jeanne A. and He, Yu-Ying and Owens, David M. and Smart, Robert C.}, year={2011}, month={Jun}, pages={1339–1346} }
 @article{deterding_williams_humble_petrovich_wei_trempus_gates_zhu_smart_tennant_et al._2011, title={CD34 antigen: Determination of specific sites of phosphorylation in vitro and in vivo}, volume={301}, ISSN={["1387-3806"]}, DOI={10.1016/j.ijms.2010.05.027}, abstractNote={CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins’ functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309–382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.}, number={1-3}, journal={INTERNATIONAL JOURNAL OF MASS SPECTROMETRY}, author={Deterding, Leesa J. and Williams, Jason G. and Humble, Margaret M. and Petrovich, Robert M. and Wei, Sung-Jen and Trempus, Carol S. and Gates, Matthew B. and Zhu, Feng and Smart, Robert C. and Tennant, Raymond W. and et al.}, year={2011}, month={Mar}, pages={12–21} }
 @article{gaddameedhi_selby_kaufmann_smart_sancar_2011, title={Control of skin cancer by the circadian rhythm}, volume={108}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1115249108}, DOI={10.1073/pnas.1115249108}, abstractNote={Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.}, number={46}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Gaddameedhi, S. and Selby, C. P. and Kaufmann, W. K. and Smart, R. C. and Sancar, A.}, year={2011}, month={Oct}, pages={18790–18795} }
 @article{ming_feng_shea_soltani_zhao_han_smart_trempus_he_2011, title={PTEN Positively Regulates UVB-Induced DNA Damage Repair}, volume={71}, ISSN={["0008-5472"]}, DOI={10.1158/0008-5472.can-10-4614}, abstractNote={Abstract}, number={15}, journal={CANCER RESEARCH}, author={Ming, Mei and Feng, Li and Shea, Christopher R. and Soltani, Keyoumars and Zhao, Baozhong and Han, Weinong and Smart, Robert C. and Trempus, Carol S. and He, Yu-Ying}, year={2011}, month={Aug}, pages={5287–5295} }
 @article{omori_matsumoto_zhu_smart_ninomiya-tsuji_2010, title={Ablation of TAK1 Upregulates Reactive Oxygen Species and Selectively Kills Tumor Cells}, volume={70}, ISSN={0008-5472 1538-7445}, url={http://dx.doi.org/10.1158/0008-5472.can-10-1227}, DOI={10.1158/0008-5472.can-10-1227}, abstractNote={Abstract}, number={21}, journal={Cancer Research}, publisher={American Association for Cancer Research (AACR)}, author={Omori, Emily and Matsumoto, Kunihiro and Zhu, Songyun and Smart, Robert C. and Ninomiya-Tsuji, Jun}, year={2010}, month={Oct}, pages={8417–8425} }
 @article{house_zhu_ranjan_linder_smart_2010, title={C/EBP alpha and C/EBP beta Are Required for Sebocyte Differentiation and Stratified Squamous Differentiation in Adult Mouse Skin}, volume={5}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79952118221&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0009837}, abstractNote={C/EBPα and C/EBPβ are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPα and C/EBPβ in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/EBPα or C/EBPβ alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPα and C/EBPβ in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPα and C/EBPβ in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R), two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPα and C/EBPβ are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal.}, number={3}, journal={PLOS ONE}, author={House, John S. and Zhu, Songyun and Ranjan, Rakesh and Linder, Keith and Smart, Robert C.}, year={2010}, month={Mar} }
 @inbook{smart_2010, place={New York}, edition={4th}, title={Chemical Carcinogenesis and Mutagenesis}, booktitle={Modern Toxicology}, publisher={J. Wiley and Sons}, author={Smart, R.C.}, editor={Hodgson, E.Editor}, year={2010} }
 @article{kim_chiera_linder_trempus_smart_horowitz_2010, title={Overexpression of Transcription Factor Sp2 Inhibits Epidermal Differentiation and Increases Susceptibility to Wound- and Carcinogen-Induced Tumorigenesis}, volume={70}, ISSN={["1538-7445"]}, DOI={10.1158/0008-5472.can-10-1213}, abstractNote={Abstract}, number={21}, journal={CANCER RESEARCH}, author={Kim, Tae-Hyung and Chiera, Shannon L. and Linder, Keith E. and Trempus, Carol S. and Smart, Robert C. and Horowitz, Jonathan M.}, year={2010}, month={Nov}, pages={8507–8516} }
 @article{lee_shuman_guszczynski_sakchaisri_sebastian_copeland_miller_cohen_taunton_smart_et al._2010, title={RSK-Mediated Phosphorylation in the C/EBP beta Leucine Zipper Regulates DNA Binding, Dimerization, and Growth Arrest Activity}, volume={30}, ISSN={["0270-7306"]}, DOI={10.1128/mcb.00782-09}, abstractNote={ABSTRACT The bZIP transcription factor C/EBPβ is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPβ signaling, we investigated C/EBPβ activation by oncogenic Ras. We show that C/EBPβ DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90RSK cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPβ activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g↔e′ salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPβ homodimer formation, whereas heterodimerization with C/EBPγ was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPβ in RasV12-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPβ-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.}, number={11}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Lee, Sook and Shuman, Jon D. and Guszczynski, Tad and Sakchaisri, Krisada and Sebastian, Thomas and Copeland, Terry D. and Miller, Maria and Cohen, Michael S. and Taunton, Jack and Smart, Robert C. and et al.}, year={2010}, month={Jun}, pages={2621–2635} }
 @article{ranjan_thompson_yoon_smart_2009, title={C/EBP alpha expression is partially regulated by C/EBP beta in response to DNA damage and C/EBP alpha-deficient fibroblasts display an impaired G(1) checkpoint}, volume={28}, ISSN={["1476-5594"]}, DOI={10.1038/onc.2009.176}, abstractNote={We observed that CCAAT/enhancer-binding protein (C/EBP)alpha is highly inducible in primary fibroblasts by DNA-damaging agents that induce strand breaks, alkylate and crosslink DNA as well as those that produce bulky DNA lesions. Fibroblasts deficient in C/EBPalpha (C/EBPalpha(-/-)) display an impaired G1 checkpoint as evidenced by an inappropriate entry into the S-phase in response to DNA damage, and these cells also display an enhanced G1/S transition in response to mitogens. The induction of C/EBPalpha by DNA damage in fibroblasts does not require p53. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extracts prepared from ultraviolet B (UVB)- and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated fibroblasts showed increased binding of C/EBPbeta to a C/EBP consensus sequence and chromatin immunoprecipitation (ChIP) analysis also showed increased C/EBPbeta binding to the C/EBPalpha promoter. To determine whether C/EBPbeta has a function in the regulation of C/EBPalpha, we treated C/EBPbeta(-/-) fibroblasts with UVB or MNNG. We observed that C/EBPalpha induction was impaired in both UVB- and MNNG-treated C/EBPbeta(-/-) fibroblasts. Our study shows a novel function for C/EBPbeta in the regulation of C/EBPalpha in response to DNA damage and provides definitive genetic evidence that C/EBPalpha has a critical role in the DNA damage G1 checkpoint.}, number={36}, journal={ONCOGENE}, author={Ranjan, R. and Thompson, E. A. and Yoon, K. and Smart, R. C.}, year={2009}, month={Sep}, pages={3235–3245} }
 @article{ewing_zhu_zhu_house_smart_2008, title={C/EBP beta represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf)}, volume={15}, ISSN={["1476-5403"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-54049144611&partnerID=MN8TOARS}, DOI={10.1038/cdd.2008.105}, abstractNote={CCAAT/enhancer-binding protein-β (C/EBPβ) is a mediator of cell survival and tumorigenesis. When C/EBPβ−/− mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19Arf and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19Arf is dramatically elevated in C/EBPβ−/− epidermis and that C/EBPβ represses a p19Arf promoter reporter. To determine whether p19Arf is responsible for the proapoptotic phenotype in C/EBPβ−/− mice, C/EBPβ−/−;p19Arf−/− mice were generated. C/EBPβ−/−;p19Arf−/− mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19Arf is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPβ−/− epidermis, we generated K14-ER:Ras;C/EBPβ−/− mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPβ−/− mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPβ represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPβ may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.}, number={11}, journal={CELL DEATH AND DIFFERENTIATION}, author={Ewing, S. J. and Zhu, S. and Zhu, F. and House, J. S. and Smart, R. C.}, year={2008}, month={Nov}, pages={1734–1744} }
 @inbook{smart_ewing_loomis_2008, place={New York}, edition={4th}, title={Carcinogenesis}, url={http://dx.doi.org/10.1002/9780470285251.ch24}, DOI={10.1002/9780470285251.ch24}, abstractNote={This chapter contains sections titled: Introduction and Historical Perspective Human Cancer Categorization of Agents Associated with Carcinogenesis Somatic Mutation Theory Epigenetic Mechanism of Tumorigenesis Multistage Tumorigenesis Tumor Viruses Cellular Oncogenes Tumor Suppressor Genes Mutator Phenotype/DNA Stability Genes Interactions of Oncogenes and Tumor Suppressor Genes Genetically Modified Mouse Models Conclusions Suggested Reading}, booktitle={Molecular and Biochemical Toxicology}, publisher={John Wiley & Sons, Inc.}, author={Smart, Robert C. and Ewing, Sarah J. and Loomis, Kari D.}, year={2008}, month={Jul}, pages={537–586} }
 @book{smart_hodgson_2008, place={New York}, title={Molecular and Biochemical Toxicology}, ISBN={9780470285251 9780470102114}, url={http://dx.doi.org/10.1002/9780470285251}, DOI={10.1002/9780470285251}, abstractNote={Much more than an introductory text, this crucial new edition has been completely revised to provide timely and thorough coverage of the underlying biochemical, molecular, and cellular mechanisms through which toxicants produce their adverse effects. Toxicological issues are covered from the molecule to the cell to the organ level. Complex methods used in toxicology are also described in a straightforward, easy-to-understand style.}, publisher={John Wiley & Sons, Inc.}, year={2008}, month={Aug} }
 @inbook{hodgson_smart_2008, place={New York}, edition={4th}, title={Molecular and Biochemical Toxicology: Definition and Scope}, url={http://dx.doi.org/10.1002/9780470285251.ch1}, DOI={10.1002/9780470285251.ch1}, abstractNote={This chapter contains sections titled: Introduction Toxicology Biochemical Toxicology Cellular Toxicology Molecular Toxicology Proteomics and Metabolomics Conclusions Suggested Reading}, booktitle={Molecular and Biochemical Toxicology}, publisher={John Wiley & Sons, Inc.}, author={Hodgson, Ernest and Smart, Robert C.}, editor={Smart, R.C. and Hodgson, E.Editors}, year={2008}, month={Jul}, pages={1–4} }
 @inbook{smart_2008, place={New York}, edition={4th}, title={Overview of Molecular Techniques in Toxicology: Genes and Transgenes}, url={http://dx.doi.org/10.1002/9780470285251.ch2}, DOI={10.1002/9780470285251.ch2}, abstractNote={This chapter contains sections titled: Applicability of Molecular Techniques to Toxicology Overview of the Genetic Code and Flow of Genetic Information Molecular Cloning Southern and Northern Blot Analyses Polymerase Chain Reaction (PCR) Some Methods to Evaluate Gene Expression and Regulation Methods to Evaluate Gene Function Suggested Reading}, booktitle={Molecular and Biochemical Toxicology}, publisher={John Wiley & Sons, Inc.}, author={Smart, Robert C.}, editor={Smart, R.C. and Hodgson, E.Editors}, year={2008}, month={Jul}, pages={5–24} }
 @article{yoon_zhu_ewing_smart_2007, title={Decreased survival of C/EBP beta-deficient keratinocytes is due to aberrant regulation of p53 levels and function}, volume={26}, ISSN={["1476-5594"]}, DOI={10.1038/sj.onc.1209797}, abstractNote={Recent studies have identified roles for C/EBPbeta in cellular survival and tumorigenesis, however, the mechanisms through which C/EBPbeta regulates these processes are not fully understood. Previously, we demonstrated that C/EBPbeta(-/-) mice are resistant to carcinogen-induced skin tumorigenesis and in response to topical carcinogen treatment display a 17-fold increase in keratinocyte apoptosis compared to wild-type mice. Here, we have investigated the mechanisms through which C/EBPbeta regulates apoptosis in response to carcinogenic stress. Analysis of carcinogen-treated C/EBPbeta(-/-) mouse skin revealed a striking increase in the number of p53 immunopositive keratinocytes in the epidermis of C/EBPbeta(-/-) mice compared to wild-type mice and this increase was temporally associated with a concomitant anomalous increase in apoptosis. The increased levels of p53 were functional as Mdm2, Bcl-2, C/EBPalpha and p21 were differentially regulated in the epidermis of carcinogen-treated C/EBPbeta(-/-) mice. The increase in p53 protein was not associated with an increase in p53 mRNA levels. To determine whether p53 is required for the increased apoptosis in C/EBPbeta(-/-) mice, C/EBPbeta/p53 compound knockout mice were generated. Carcinogen-treated C/EBPbeta/p53 compound knockout mice did not display increased apoptosis demonstrating p53 is required for the proapoptotic phenotype in C/EBPbeta(-/-) mice. Our results demonstrate that altered keratinocyte survival in C/EBPbeta(-/-) mice results from aberrant regulation of p53 protein and function and indicate C/EBPbeta has a role in the negative regulation of p53 protein levels in response to carcinogen-induced stress.}, number={3}, journal={ONCOGENE}, author={Yoon, K. and Zhu, S. and Ewing, S. J. and Smart, R. C.}, year={2007}, month={Jan}, pages={360–367} }
 @article{loomis_zhu_yoon_johnson_smart_2007, title={Genetic ablation of CCAAT/Enhancer binding protein alpha in epidermis reveals its role in suppression of epithelial tumorigenesis}, volume={67}, ISSN={["0008-5472"]}, DOI={10.1158/0008-5472.CAN-07-0139}, abstractNote={Abstract}, number={14}, journal={CANCER RESEARCH}, author={Loomis, Kari D. and Zhu, Songyun and Yoon, Kyungsil and Johnson, Peter F. and Smart, Robert C.}, year={2007}, month={Jul}, pages={6768–6776} }
 @article{sterneck_zhu_ramirez_jorcano_smart_2006, title={Conditional ablation of C/EBP beta demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis}, volume={25}, ISSN={["1476-5594"]}, DOI={10.1038/sj.onc.1209144}, abstractNote={The CCAAT/enhancer binding protein β (C/EBPβ) is implicated in the regulation of many different molecular and physiological processes. Mice with a germline deletion of C/EBPβ (C/EBPβ−/−) display phenotypes in a multitude of cell types and organ systems, including skin where C/EBPβ−/− mice exhibit increased apoptosis in epidermal keratinocytes in response to carcinogen treatment and are completely resistant to carcinogen-induced skin tumorigenesis. To determine the contribution of systemic versus cell autonomous functions of C/EBPβ to specific phenotypes, mice with a conditional ‘floxed’ C/EBPβ null allele were generated. Epidermal-specific deletion of C/EBPβ was achieved by Cre recombinase expression from a keratin 5 (K5) promoter. Similar to C/EBPβ−/− mice, K5-Cre;C/EBPβfl/fl mice were completely refractory to 7,12 dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis and these mice displayed increased DMBA-induced apoptosis in epidermal keratinocytes compared to wild-type mice. In contrast, mice lacking the related gene, C/EBPδ, were not resistant to DMBA-induced skin tumorigenesis, indicating a unique role of C/EBPβ in skin tumor development. Our findings demonstrate that C/EBPβ exerts an essential, keratinocyte-intrinsic role in cell survival in response to carcinogen treatment and the elimination of C/EBPβ in keratinocytes is sufficient to confer complete resistance of the skin to chemical carcinogenesis.}, number={8}, journal={ONCOGENE}, author={Sterneck, E and Zhu, S and Ramirez, A and Jorcano, JL and Smart, RC}, year={2006}, month={Feb}, pages={1272–1276} }
 @article{omori_matsumoto_sanjo_sato_akira_smart_ninomiya-tsuji_2006, title={TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis}, volume={281}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M603384200}, abstractNote={Transforming growth factor β-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF-κB in several cell types. However, the functional role of TAK1 in an in vivo setting has not been determined. Here we have demonstrated that TAK1 is the major regulator of skin inflammation as well as keratinocyte death in vivo. Epidermal-specific deletion of TAK1 causes a severe inflammatory skin condition by postnatal day 6-8. The mutant skin also exhibits massive keratinocyte death. Analysis of keratinocytes isolated from the mutant skin revealed that TAK1 deficiency results in a striking increase in apoptosis in response to tumor necrosis factor (TNF). TAK1-deficient keratinocytes cannot activate NF-κB or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-κB (and possibly JNK)-mediated cell survival signaling. Finally, we have shown that deletion of the TNF receptor can largely rescue keratinocyte death as well as inflammatory skin condition in epidermal-specific TAK1-deficient mice. Our results demonstrate that TAK1 is a master regulator of TNF signaling in skin and regulates skin inflammation and keratinocyte death.}, number={28}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Omori, Emily and Matsumoto, Kunihiro and Sanjo, Hideki and Sato, Shintaro and Akira, Shizuo and Smart, Robert C. and Ninomiya-Tsuji, Jun}, year={2006}, month={Jul}, pages={19610–19617} }
 @article{shim_powers_ewing_zhu_smart_2005, title={Diminished expression of C/EBP alpha in skin carcinomas is linked to oncogenic Ras and reexpression of C/EBP alpha in carcinoma cells inhibits proliferation}, volume={65}, number={3}, journal={Cancer Research}, author={Shim, M. and Powers, K. L. and Ewing, S. J. and Zhu, S. and Smart, R. C.}, year={2005}, pages={861–867} }
 @article{yoon_smart_2004, title={C/EBP alpha is a DNA damage-inducible p53-regulated mediator of the G(1) checkpoint in keratinocytes}, volume={24}, ISSN={["1098-5549"]}, DOI={10.1128/MCB.24.24.10650-10660.2004}, abstractNote={ABSTRACT The basic leucine zipper transcription factor, CCAAT/enhancer binding protein α (C/EBPα), is abundantly expressed in keratinocytes of the skin; however, its function in skin is poorly characterized. UVB radiation is responsible for the majority of human skin cancers. In response to UVB-induced DNA damage, keratinocytes activate cell cycle checkpoints that arrest cell cycle progression and prevent replication of damaged DNA, allowing time for DNA repair. We report here that UVB radiation is a potent inducer of C/EBPα in human and mouse keratinocytes, as well as in mouse skin in vivo. UVB irradiation of keratinocytes resulted in the transcriptional up-regulation of C/EBPα mRNA, producing a >70-fold increase in C/EBPα protein levels. N-Methyl-N′-nitro-N-nitrosoguanidine, etoposide, and bleomycin also induced C/EBPα. UVB-induced C/EBPα was accompanied by an increase in p53 protein and caffeine, an inhibitor of ataxia-telangiectasia-mutated kinase, and ataxia-telangiectasia-mutated and Rad3-related kinase inhibited UVB-induced increases in both C/EBPα and p53. UVB irradiation of p53-null or mutant p53-containing keratinocytes failed to induce C/EBPα. UVB irradiation of C/EBPα knockdown keratinocytes displayed a greatly diminished DNA damage G1 checkpoint, and this was associated with increased sensitivity to UVB-induced apoptosis. Our results uncover a novel role for C/EBPα as a p53-regulated DNA damage-inducible gene that has a critical function in the DNA damage G1 checkpoint response in keratinocytes.}, number={24}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Yoon, K and Smart, RC}, year={2004}, month={Dec}, pages={10650–10660} }
 @article{shuman_sebastian_kaldis_copeland_zhu_smart_johnson_2004, title={Cell cycle-dependent phosphorylation of C/EBP beta mediates oncogenic cooperativity between C/EBP beta and H-Ras(V12)}, volume={24}, ISSN={["1098-5549"]}, DOI={10.1128/MCB.24.17.7380-7391.2004}, abstractNote={ABSTRACT CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPβ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPβ in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPβ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPβ, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPβ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPβ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.}, number={17}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Shuman, JD and Sebastian, T and Kaldis, P and Copeland, TD and Zhu, SY and Smart, RC and Johnson, PF}, year={2004}, month={Sep}, pages={7380–7391} }
 @inbook{smart_2004, title={Chemical Carcinogenesis}, url={http://dx.doi.org/10.1002/0471646776.ch12}, DOI={10.1002/0471646776.ch12}, abstractNote={General aspects of cancer biology as well as human cancer causes, incidence and mortality rates are discussed. The identification and classification of potential human carcinogens is discussed. The mechanisms through which chemical carcinogens induce cancer are described as is the involvement of oncogenes and tumor suppressor genes in this process. Finally the usefulness and limitation of mutagenicity assays for the identification of carcinogens is discussed.}, booktitle={A Textbook of Modern Toxicology}, publisher={John Wiley & Sons, Inc.}, author={Smart, Robert C.}, editor={Hodgson, E.Editor}, year={2004}, month={Mar}, pages={225–250} }
 @inbook{hodgson_leblanc_meyer_smart_2004, place={New York}, title={Introduction to Biochemical and Molecular Methods in Toxicology}, ISBN={9780471646778 9780471265085}, url={http://dx.doi.org/10.1002/0471646776.ch2}, DOI={10.1002/0471646776.ch2}, abstractNote={A brief introduction to recently developed molecular and cellular methods currently in use in toxicology, including cell culture techniques (suspension cell culture, monolayer cell culture, indicators of toxicity in cultured cells), molecular cloning techniques (molecular cloning, cDNA and genomic libraries, Northern and Southern blot analysis, PCR, evaluation of gene expression, regulation and function) as well as immunochemical methods.}, booktitle={A Textbook of Modern Toxicology}, publisher={John Wiley & Sons, Inc.}, author={Hodgson, Ernest and Leblanc, Gerald A. and Meyer, Sharon A. and Smart, Robert C.}, editor={Hodgson, E.Editor}, year={2004}, month={Mar}, pages={13–22} }
 @article{shim_smart_2003, title={Lithium stabilizes the CCAAT/enhancer-binding protein alpha (C/EBP alpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity}, volume={278}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.M301356200}, abstractNote={CCAAT/enhancer-binding protein α (C/EBPα), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBPα protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBPα is a short-lived protein with a half-life of ∼1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBPα protein. Higher molecular weight species of ubiquitinated C/EBPα were detected in BALB/MK2, and in vitro studies confirmed that C/EBPα is degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBPα kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBPα protein levels, (ii) increased electrophoretic mobility of C/EBPα, and (iii) no increase in C/EBPα mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBPα may target it for proteasomal degradation. However, a mutant C/EBPα containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBPα levels indicating the effects of LiCl on C/EBPα are GSK3-independent. LiCl treatment of BALB/MK2 cells inhibited C/EBPα degradation and produced a 6-fold increase in the half-life of C/EBPα protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBPα. These results demonstrate C/EBPα is degraded via a ubiquitin-dependent proteasomal pathway, and LiCl stabilizes C/EBPα through a GSK3-independent pathway involving direct inhibition of proteasome activity.}, number={22}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Shim, M and Smart, RC}, year={2003}, month={May}, pages={19674–19681} }
 @misc{smart_oh_2003, title={Method of treating alopecia}, volume={6,555,532}, number={2003 Apr. 29}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Smart, R. C. and Oh, H.-S.}, year={2003} }
 @article{smart_oh_2003, title={Method of treating alopecia}, volume={6,555,532}, number={2003 Apr. 29}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Smart, R. C. and Oh, H.-S.}, year={2003} }
 @article{porter_chanda_wang_gaido_smart_robinette_2002, title={17 beta-estradiol is a hormonal regulator of mirex tumor promotion sensitivity in mice}, volume={69}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/69.1.42}, abstractNote={Mirex, an organochlorine pesticide, is a potent non-phorbol ester tumor promoter in mouse skin. Previous studies have shown that female mice are 3 times more sensitive to mirex tumor promotion than male mice and that ovariectomized (OVX) female mice are resistant to mirex promotion, suggesting a role for ovarian hormones in mirex promotion. To determine whether the ovarian hormone 17-beta estradiol (E2) is responsible for the sensitivity of female mice to mirex promotion, female mice were initiated with DMBA; 2 weeks later groups of mice were OVX and implants, with or without E2, were surgically implanted subcutaneously. These mice were treated topically twice weekly with mirex for 26 weeks. E2 implanted OVX mice demonstrated high normal physiologic levels of serum E2 throughout the tumor promotion experiment. E2 implants restored by 80% the intact mirex-sensitive phenotype to the OVX mice. Consistent with a role for E2 and ERalpha and ERbeta, treatment of DMBA-initiated female mice with topical ICI 182,780, an estrogen-receptor antagonist, reduced mirex tumor multiplicity by 30%. However, in cells co-transfected with ERalpha or ERbeta and estrogen-responsive promoter reporter, mirex did not stimulate promoter reporter activity, suggesting that the promotion effect of mirex is downstream of ERalpha/beta. Finally, a tumor promotion study was conducted to determine whether E2 implants could increase the sensitivity of male mice to mirex promotion. E2 implants in male mice did increase sensitivity to mirex promotion; however, the implants did not produce the full female sensitivity to mirex tumor promotion. Collectively, these studies indicate that E2 is a major ovarian hormone responsible for mirex tumor promotion sensitivity in female mice.}, number={1}, journal={TOXICOLOGICAL SCIENCES}, author={Porter, KL and Chanda, S and Wang, HQ and Gaido, KW and Smart, RC and Robinette, CL}, year={2002}, month={Sep}, pages={42–48} }
 @article{zhu_yoon_sterneck_johnson_smart_2002, title={CCAAT/enhancer binding protein-beta is a mediator of keratinocyte survival and skin tumorigenesis involving oncogenic Ras signaling}, volume={99}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.012437299}, abstractNote={The basic leucine zipper transcription factor CCAAT/enhancer binding protein-β (C/EBPβ) is expressed in many cell types, including keratinocytes. C/EBPβ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C/EBPβ signaling are not currently understood. We report here thatC/EBPβ-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz[a]anthracene-initiation/12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenicRasmutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed inC/EBPβ-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treatedC/EBPβ-null mice compared with wild-type mice. In v-Ha-rastransgenic mice,C/EBPβ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C/EBPβ. Oncogenic Ras potently stimulated C/EBPβ to activate a C/EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1/2 phosphorylation site (T188) in C/EBPβ abolished this Ras effect. Finally, we observed that C/EBPβ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C/EBPβ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C/EBPβ as a target for tumor inhibition.}, number={1}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Zhu, SY and Yoon, K and Sterneck, E and Johnson, PF and Smart, RC}, year={2002}, month={Jan}, pages={207–212} }
 @article{tiano_loftin_akunda_lee_spalding_sessoms_dunson_rogan_morham_smart_et al._2002, title={Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis}, volume={62}, number={12}, journal={Cancer Research}, author={Tiano, H. F. and Loftin, C. D. and Akunda, J. and Lee, C. A. and Spalding, J. and Sessoms, A. and Dunson, D. B. and Rogan, E. G. and Morham, S. G. and Smart, R. C. and et al.}, year={2002}, pages={3395–3401} }
 @inbook{hodgson_smart_2001, place={New York}, title={Biochemical Toxicology: Definition and Scope}, booktitle={Introduction to Biochemical Toxicology}, publisher={J. Wiley and Sons}, author={Hodgson, E. and Smart, R.C.}, editor={Hodgson, E. and Smart, R.C.Editors}, year={2001}, pages={1–10} }
 @inbook{smart_akunda_2001, place={New York}, edition={3rd}, title={Carcinogenesis}, booktitle={Introduction to Biochemical Toxicology}, publisher={J. Wiley and Sons}, author={Smart, R.C. and Akunda, J.K.}, editor={Hodgson, E. and Smart, R.C.Editors}, year={2001}, pages={343–398} }
 @book{hodgson_smart_2001, place={New York}, edition={3rd}, title={Introduction to Biochemical Toxicology}, publisher={J. Wiley and Sons}, year={2001} }
 @book{introduction to biochemical toxicology_2001, edition={3rd}, publisher={J. Wiley and Sons}, year={2001} }
 @misc{smart_oh_2001, title={Method of treating alopecia}, volume={6,204,258}, number={2001 Mar. 20}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Smart, R. C. and Oh, H.-S.}, year={2001} }
 @article{smart_oh_2001, title={Method of treating alopecia}, volume={6,204,258}, number={2001 Mar. 20}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Smart, R. C. and Oh, H.-S.}, year={2001} }
 @inbook{smart_2001, place={New York}, edition={3rd}, title={Molecular Techniques in Toxicology}, booktitle={Introduction to Biochemical Toxicology}, publisher={J. Wiley and Sons}, author={Smart, R.C.}, editor={Hodgson, E. and Smart, R.C.Editors}, year={2001}, pages={11–32} }
 @article{wang_kim_tiano_langenbach_smart_2001, title={Protein kinase C-alpha coordinately regulates cytosolic phospholipase A(2) activity and the expression of cyclooxygenase-2 through different mechanisms in mouse keratinocytes}, volume={59}, ISSN={["0026-895X"]}, DOI={10.1124/mol.59.4.860}, abstractNote={Transgenic mice (K5-PKC alpha) in which the keratin 5 promoter directs the expression of protein kinase C-alpha (PKC alpha) to epidermal keratinocytes display a 10-fold increase in PKC alpha protein in their epidermis and alterations in phorbol ester-induced cutaneous inflammation [J Cell Science 1999;112:3497-3506]. In the current study, we have used these K5-PKC alpha mice to examine the role of PKC alpha in keratinocyte phospholipid metabolism/eicosanoid production and cutaneous inflammation. Primary keratinocytes from wild-type and transgenic mice were prelabeled in culture with [(3)H]arachidonic acid (AA) and subsequently treated with TPA. Compared with wild-type keratinocytes, K5-PKC alpha keratinocytes displayed a 2-fold increase in AA release. TPA treatment resulted in the phosphorylation of cPLA(2). PKC inhibitors GF-109203X or H7, but not mitogen-activated protein/extracellular signal-regulated protein kinase (MEK) inhibitor PD 98059, could inhibit phosphorylation and AA release. Topical 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of K5-PKC alpha mice resulted in a 5-fold increase in epidermal COX-2 induction and a 2- to 3-fold increase in prostaglandin (PG) E(2) levels above that observed in TPA-treated wild-type mice. PD 98059, GF-109203X, or H7 could block cyclooxygenase-2 (COX-2) induction by TPA. Because C/EBP beta, a basic leucine zipper transcription factor, can be activated via a PKC alpha/mitogen-activated protein kinase pathway and can influence COX-2 expression, we examined whether C/EBP beta is involved in TPA-induced epidermal COX-2 expression. TPA-induced COX-2 expression was similar in C/EBP beta nullizygous and wild-type mice. In summary, our results indicate that epidermal PKC alpha coordinately regulates cPLA(2) activity and COX-2 expression resulting in increased levels of AA and PGE(2). Furthermore, PKC alpha-induced AA release and cPLA(2) phosphorylation are independent of MEK, whereas PKC alpha-induced COX-2 expression and PGE(2) production are MEK-dependent and C/EBP beta-independent events.}, number={4}, journal={MOLECULAR PHARMACOLOGY}, author={Wang, HQ and Kim, MP and Tiano, HF and Langenbach, R and Smart, RC}, year={2001}, month={Apr}, pages={860–866} }
 @article{chanda_robinette_couse_smart_2000, title={17 beta-Estradiol and ICI-182780 regulate the hair follicle cycle in mice through an estrogen receptor-alpha pathway}, volume={278}, ISSN={["1522-1555"]}, DOI={10.1152/ajpendo.2000.278.2.e202}, abstractNote={Estradiol (E2) applied topically twice weekly to mouse skin at doses as low as 1 nmol inhibited hair growth by blocking the transition of the hair follicle from the resting phase (telogen) to the growth phase (anagen). In contrast, application of ≤10 nmol of other steroids produced limited inhibition. Topical treatment with the estrogen receptor (ER) antagonist ICI-182780 reversed the effects of E2, and when applied alone, ICI-182780 caused a telogen-to-anagen transition. Both E2and ICI-182780 were highly effective at their site of application but not at distant sites, indicating the direct rather than secondary systemic nature of their effects. Western analysis detected a 65-kDa ER-α immunoreactive dermal protein, and Northern analysis revealed the presence of a 6.7-kb ER-α mRNA. A ribonuclease protection assay confirmed the presence of ER-α transcripts but failed to detect ER-β transcripts. These findings implicate a skin-specific ER-α pathway in the regulation of the hair follicle cycle.}, number={2}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM}, author={Chanda, S and Robinette, CL and Couse, JF and Smart, RC}, year={2000}, month={Feb}, pages={E202–E210} }
 @article{owens_wei_smart_1999, title={A multihit, multistage model of chemical carcinogenesis}, volume={20}, ISSN={["0143-3334"]}, DOI={10.1093/carcin/20.9.1837}, abstractNote={Carcinogenesis involves the accumulation of genetic changes within a single cell. Tumor promotion functions in the initial clonal expansion of an initiated cell but is generally not considered to influence later stages. To investigate whether tumor promotion can influence later stages of carcinogenesis we developed a two-hit 7, 12-dimethylbenz[a]anthracene (D) protocol designed to enrich for keratinocytes that contain at least two D-induced genetic alterations. FVB/N mice were initiated with D and promoted with 12-O-tetradecanoylphorbol-13-acetate (T) or treated with acetone (A) vehicle for 6 weeks. At 7 weeks after the start of promotion, but before visible papilloma development, groups of mice were treated with a second dose of D or A and 1 week later T promotion was resumed. D/T/A/T mice developed 2.8 papillomas/mouse and D/A/D/T mice demonstrated an additive tumor response and developed 5.8 papillomas/mouse. Importantly, D/T/D/T mice developed 12.4 papillomas/mouse, thereby demonstrating a synergistic tumor response compared with D/A/D/T and D/T/A/T mice. D/T/D/T papillomas exhibited increases in suprabasal S phase cells and keratin 13 expression when compared with D/T/A/T papillomas. D/T/D/T mice developed squamous cell carcinomas (SCCs) 10 weeks earlier than D/T/A/T mice and demonstrated a 96% malignancy incidence and 1.71 SCC/mouse compared with D/T/A/T mice, which demonstrated a 28% malignancy incidence and 0.32 SCC/mouse. Greater than 90% of D/T/A/T and D/T/D/T papillomas and SCCs contained mutant Ha-ras, while a normal Ha-ras allele persisted in all cases, indicating that a gene other than the remaining normal allele of Ha-ras was a target gene for the second D hit. These data demonstrate that: (i) promotion between the first and second hits has a profound outcome on carcinogenesis, presumably by increasing the probability that a second hit will occur in a previously initiated cell; (ii) continued promotion after the second hit is required for full expression of malignancy; (iii) the classic initiation-promotion protocol can be extended to a multihit, multistage model.}, number={9}, journal={CARCINOGENESIS}, author={Owens, DM and Wei, SJC and Smart, RC}, year={1999}, month={Sep}, pages={1837–1844} }
 @article{zhu_oh_shim_sterneck_johnson_smart_1999, title={C/EBP beta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression}, volume={19}, DOI={10.1128/mcb.19.10.7181}, abstractNote={ABSTRACT The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPβ within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPβ in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPβ-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPβ-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPβ-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPβ plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.}, number={10}, journal={Molecular and Cellular Biology}, author={Zhu, S. Y. and Oh, H. S. and Shim, M. and Sterneck, E. and Johnson, P. F. and Smart, Robert}, year={1999}, pages={7181–7190} }
 @article{matsuura_adachi_smart_xu_arata_jetten_1999, title={Correlation between expression of peroxisome proliferator-activated receptor beta and squamous differentiation in epidermal and tracheobronchial epithelial cells}, volume={147}, ISSN={["0303-7207"]}, DOI={10.1016/S0303-7207(98)00214-7}, abstractNote={Previously, several members of the nuclear receptor superfamily have been implicated in the regulation of epidermal differentiation. In this study, we analyze the expression of members of the PPAR nuclear receptor subfamily in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK), human tracheobronchial epithelial (HBE) cells and the epidermis in vivo. Our results demonstrate that induction of differentiation in NHEK by either treatment with the phorbol ester phorbol 12-myristate-13-acetate (PMA), suspension culture or confluence greatly enhances the expression of PPARβ mRNA. Likewise, topical treatment of mouse skin with PMA results in increased PPARβ mRNA expression in the epidermis. In addition, the induction of squamous differentiation in HBE cells was also associated with an upregulation of PPARβ mRNA expression. Finally, in situ hybridization analysis localized PPARβ mRNA to the suprabasal layers of normal human skin. Our results demonstrate that the expression of PPARβ is associated with squamous differentiation suggesting a regulatory role for this receptor in the control of specific genes during this differentiation process.}, number={1-2}, journal={MOLECULAR AND CELLULAR ENDOCRINOLOGY}, author={Matsuura, H and Adachi, H and Smart, RC and Xu, XC and Arata, J and Jetten, AM}, year={1999}, month={Jan}, pages={85–92} }
 @article{smart_oh_chanda_robinette_1999, title={Effects of 17-beta-estradiol and ICI 182 780 on hair growth in various strains of mice}, volume={4}, ISSN={["1529-1774"]}, DOI={10.1038/sj.jidsp.5640231}, abstractNote={17-beta-Estradiol (10 nmol per 200 microl acetone) applied topically twice weekly to the clipped dorsal surface of C57BL/6 or C3H female mouse skin prevented hair growth, as previously described in the CD-1 mouse strain. Twice weekly topical application of the estrogen receptor antagonist, ICI 182 780 (10nmol per 200microl acetone), induced the telogenanagen transition and produced early pigmentation appearance in skin and hair growth in C57BL/6 and C3H female mice. Whereas twice weekly topical application of 10nmol 17-beta-estradiol blocked hair growth, the intraperitoneal administration of this dose twice weekly did not block hair growth, suggesting a direct cutaneous effect of 17-beta-estradiol. We also evaluated the effect of 17-alpha-estradiol, 17-beta-estradiol, and ICI 182 780 on hair growth in male mice. As observed in female mice, 17-beta-estradiol was a potent inhibitor of hair growth and ICI 182 780 stimulated hair growth; however, unlike the results previously observed in female mice, 17-alpha-estradiol was a potent inhibitor of hair growth in male mice. These results demonstrate that (i) the route of administration of 17-beta-estradiol is critical for its ability to block hair growth; (ii) C57BL/6 and C3H mice, two commonly employed mouse strains for hair growth studies, responded to 17-beta-estradiol and ICI 182 780 in a manner similar to that described in CD-1 mice; and (iii) the hair follicles of male and female mice respond similarly to 17-beta-estradiol and ICI 182 780, but display striking sex differences in the response to 17-alpha-estradiol on hair growth.}, number={3}, journal={JOURNAL OF INVESTIGATIVE DERMATOLOGY SYMPOSIUM PROCEEDINGS}, author={Smart, RC and Oh, HS and Chanda, S and Robinette, CL}, year={1999}, month={Dec}, pages={285–289} }
 @misc{smart_oh_1999, title={Methods of treating alopecia}, volume={5,965,551}, number={1999 Oct. 12}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Smart, R. C. and Oh, H.-S.}, year={1999} }
 @article{smart_oh_1999, title={Methods of treating alopecia}, volume={5,965,551}, number={1999 Oct. 12}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Smart, R. C. and Oh, H.-S.}, year={1999} }
 @article{wang_smart_1999, title={Overexpression of protein kinase C-[alpha] in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2, and TNF-[alpha] but not tumor promotion}, volume={112}, number={20}, journal={Journal of Cell Science}, author={Wang, H. Q. and Smart, R. C.}, year={1999}, pages={3497–3506} }
 @article{oh_smart_1998, title={Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in neoplasms}, volume={110}, ISSN={["0022-202X"]}, DOI={10.1046/j.1523-1747.1998.00199.x}, abstractNote={The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that undergo sequential changes in gene expression during differentiation. CCAAT/enhancer binding proteins (C/EBP) are members of the bZIP family of DNA binding proteins/transcription factors. Northern analysis demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta mRNA are expressed in mouse epidermis and their mRNA levels were generally greater than those observed in other tissues known to express high levels of C/EBP. Western analysis of isolated epidermal cell nuclei demonstrated the presence of a 42 and 30 kDa C/EBPalpha protein and 35 kDa C/EBPbeta protein. Immunohistochemical localization of C/EBPalpha and C/EBPbeta in intact interfollicular epidermis revealed that C/EBPbeta expression is exclusive to the nuclei of a three-cell cluster of suprabasal keratinocytes that is morphologically consistent with the central column of the epidermal proliferative unit, and that C/EBPalpha is expressed in the nuclei and cytoplasm of suprabasal keratinocytes and weakly expressed in a perinuclear manner in some basal keratinocytes. In squamous cell carcinomas the expression of C/EBPalpha and C/EBPbeta was greatly diminished as both the intensity of nuclear staining and the number of cells expressing C/EBPalpha and C/EBPbeta were reduced. In isolated primary mouse keratinocytes, calcium-induced differentiation was accompanied by specific temporal changes in the expression of C/EBPalpha, C/EBPbeta, and C/EBPdelta mRNA and C/EBPalpha and C/EBPbeta protein. These results implicate a role for the C/EBP family in the regulation of genes involved in or specifically expressed during the process of squamous differentiation in epidermis.}, number={6}, journal={JOURNAL OF INVESTIGATIVE DERMATOLOGY}, author={Oh, HS and Smart, RC}, year={1998}, month={Jun}, pages={939–945} }
 @misc{smart_oh_1998, title={On the effect of estrogen receptor agonists and antagonists on the mouse hair follicle cycle}, volume={111}, ISSN={["1523-1747"]}, DOI={10.1046/j.1523-1747.1998.00257.x}, abstractNote={The experiments we reported in our letter were described as executed, and the mouse strains investigated were chosen for the reasons stated. The experiments were planned and conducted completely independent of one another but, unfortunately, the same mistake was made in both cases – the concentrations used were not as reported in the original report. Although we take responsibility for this oversight, we also recognize that the dosage listing is not entirely conventional to the field. Before starting the repeat experiments we consulted several independent researchers outside our respective laboratories; in all cases the understood dosage was interpreted exactly as we had. When we repeated the work using the twice weekly protocol and the concentrations used originally by Oh and Smart (Proc Natl Acad Sci 93:12525), β-estradiol did indeed inhibit the normal progression of spontaneous anagen in pigmented mice (C57B16). From additional and subsequent studies that we have since executed, we have learned several important features about the role of estrogen receptor-mediated signaling in murine hair growth control that we did not formerly appreciate. We would hope to share these data in a future report. We are indebted to Drs. Oh and Smart for calling our attention to this interesting phenomenon and regret the confusion our mistake might have caused. Note from the Editor: Due to an editorial office error, Drs. Smart and Oh were not given a chance to reply to the original letter about their paper by Drs. Stenn, Paus, and Filippi (J Invest Dermatol, 110:95 1998). We apologize to all the authors for this mistake.}, number={1}, journal={JOURNAL OF INVESTIGATIVE DERMATOLOGY}, author={Smart, RC and Oh, HS}, year={1998}, month={Jul}, pages={175–175} }
 @article{kim_porter_foley_maronpot_smart_1997, title={Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha ras genotype}, volume={20}, DOI={10.1002/(SICI)1098-2744(199709)20:1<115::AID-MC13>3.0.CO;2-4}, abstractNote={Mirex is a potent tumor promoter in 7,12‐dimethylbenz[a]anthracene (DMBA)–initiated female CD‐1 mouse skin. Like 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), mirex promotes papillomas that have a Ha‐ras mutation; however, unlike TPA promotion, mirex promotion does not involve a general hyperplastic response. We used proliferating cell nuclear antigen (PCNA) and 5‐bromo‐2′‐deoxyuridine (BrdU) immunohistochemical staining to further examine the proliferative capacity of mirex. The numbers of PCNA‐ and BrdU‐positive epidermal S‐phase cells were highly concordant in all treatment groups. Unlike a single application of TPA, a single application of mirex had little or no effect on the number of S‐phase epidermal cells, and chronic application of mirex to mouse skin produced only minimal increases in S‐phase cells. Moreover, mirex did not significantly alter the growth of BALB/MK‐2 keratinocytes in media containing either 0.05 or 1.2 mM Ca++. These results suggest that mirex may have highly specific effects on the proliferation of initiated cells and support the existence of a unique mirex mechanism and/or distinct population of mirex‐promotable mutant Ha‐ras epidermal cells. To begin to address this issue of a distinct population of mirex‐promotable mutant Ha‐ras cells, we conducted a tandem experiment in which DMBA‐initiated mice were treated twice weekly with a maximal promoting dose of mirex. Then, when the number of papillomas reached a plateau, these same mice were treated twice weekly with a maximal promoting dose of TPA. Mice treated with mirex developed a maximum of 6.4 papillomas/mouse. These mice were then promoted with TPA, which produced 8.9 additional papillomas/mouse for a total of 15.3 papillomas/mouse. The maximum tumor yields from other groups of mice treated with only TPA or mirex were 9.8 and 7.3 papillomas/mouse, respectively. Therefore, under these tandem conditions, tumor yields were additive, indicating that there are at least two distinct populations of mutant Ha‐ras cells: one promoted by mirex and the other by TPA. Mol. Carcinog. 20:115–124, 1997. © 1997 Wiley‐Liss, Inc.}, number={1}, journal={Molecular Carcinogenesis}, author={Kim, T. W. and Porter, K. L. and Foley, J. F. and Maronpot, R. R. and Smart, Robert}, year={1997}, pages={115–124} }
 @article{oh_smart_1996, title={An estrogen receptor pathway regulates the telogen-anagen hair follicle transition and influences epidermal cell proliferation.}, volume={93}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.93.22.12525}, DOI={10.1073/pnas.93.22.12525}, abstractNote={The hair follicle is a cyclic, self renewing epidermal structure which is thought to be controlled by signals from the dermal papilla, a specialized cluster of mesenchymal cells within the dermis. Topical treatments with 17-beta-estradiol to the clipped dorsal skin of mice arrested hair follicles in telogen and produced a profound and prolonged inhibition of hair growth while treatment with the biologically inactive stereoisomer, 17-alpha-estradiol, did not inhibit hair growth. Topical treatments with ICI 182,780, a pure estrogen receptor antagonist, caused the hair follicles to exit telogen and enter anagen, thereby initiating hair growth. Immunohistochemical staining for the estrogen receptor in skin revealed intense and specific staining of the nuclei of the cells of the dermal papilla. The expression of the estrogen receptor in the dermal papilla was hair cycle-dependent with the highest levels of expression associated with the telogen follicle. 17-beta-Estradiol-treated epidermis demonstrated a similar number of 5-bromo-2'-deoxyuridine (BrdUrd) S-phase cells as the control epidermis above telogen follicles; however, the number of BrdUrd S-phase basal cells in the control epidermis varied according to the phase of the cycle of the underlying hair follicles and ranged from 2.6% above telogen follicles to 7.0% above early anagen follicles. These findings indicate an estrogen receptor pathway within the dermal papilla regulates the telogen-anagen follicle transition and suggest that diffusible factors associated with the anagen follicle influence cell proliferation in the epidermis.}, number={22}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Oh, H S and Smart, R C}, year={1996}, month={Oct}, pages={12525–12530} }
 @article{goodell_oh_meyer_smart_1996, title={Epidermal Protein Kinase C-β2 Is Highly Sensitive to Downregulation and Is Exclusively Expressed in Langerhans Cells: Downregulation Is Associated with Attenuated Contact Hypersensitivity}, volume={107}, ISSN={0022-202X}, url={http://dx.doi.org/10.1111/1523-1747.ep12363325}, DOI={10.1111/1523-1747.ep12363325}, abstractNote={Treatment of mice with multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol resulted in a preferential decrease in epidermal protein kinase C-beta 2 (PKC-beta 2) compared with PKC-alpha as determined by western analysis. When PKC-alpha was decreased by 40%, PKC-beta 2 could no longer be detected, suggesting that PKC-beta 2 is more sensitive to downregulation, and/or specific epidermal cell types that contain PKC-beta 2 are more sensitive to TPA/diacylglycerol. To address this issue, we isolated Langerhans cells (LCs) from epidermal cell suspensions with immunomagnetic beads and an antibody to the class II major histocompatibility complex. Northern blot analysis revealed a PKC-beta 2 signal in isolated LCs that was 40-fold greater than that observed in unfractionated epidermal cells, and no PKC-beta 2 signal was detected in epidermal cells depleted of LCs, indicating that PKC-beta 2 is expressed exclusively in LCs within the epidermis. Western blot analysis confirmed the presence of PKC-beta 2 in LCs. PKC-beta 2 was highly sensitive to downregulation, because a single application of TPA resulted in a 90% loss of PKC-beta 2 within 6 h without a decrease in the number of LCs. To determine whether the decreased level of PKC-beta 2 within LCs was associated with an alteration in contact hypersensitivity, we treated mice with only a single application of TPA, and 6 hours later mice were sensitized with 2,4-dinitrofluorobenzene on the same dorsal area. Subsequent challenge revealed a 60% decrease in contact hypersensitivity in TPA-treated mice. These data indicate that (i) within the epidermis, PKC-beta 2 is highly sensitive to downregulation and is exclusively expressed in LCs, and (ii) the downregulation of PKC-beta 2 is associated with impaired LC function with respect to contact hypersensitivity.}, number={3}, journal={Journal of Investigative Dermatology}, publisher={Elsevier BV}, author={Goodell, Audrey L. and Oh, Hye-Sun and Meyer, Sharon A. and Smart, Robert C.}, year={1996}, month={Sep}, pages={354–359} }
 @article{owens_jetten_smart_zainal_1996, title={Localization and Expression of Cornifin-α/SPRR1 in Mouse Epidermis, Anagen Follicles, and Skin Neoplasms}, volume={106}, ISSN={0022-202X}, url={http://dx.doi.org/10.1111/1523-1747.ep12345463}, DOI={10.1111/1523-1747.ep12345463}, abstractNote={Recently, cornifin- α /SPRR1 has been identified as a putative precursor protein of the cornified cell envelope. In this study, the expression and localization of cornifin- α /SPRR1 was examined in untreated and tumor promoter-treated mouse skin, hair follicles, and skin neoplasms. Western analysis with antiserum (SQ37A) to a rabbit cornifin- α peptide or antiserum (SQ37C) to a human SPRR1 peptide demonstrated a 31-kDa immunoreactive protein in mouse epidermis and Northern analysis revealed the presence of a 1-kb mRNA. Immunohistochemical staining of mouse skin with SQ37A or SQ37C revealed intense and specific staining of the infundibulum, isthmus, and of Henle's layer of the inner root sheath of the lower anagen hair follicle and weak staining of the telogen follicle and the suprabasal layers of the epidermis. Treatment of mouse skin with 12- O -tetradecanoyl-phorbol-13-acetate (TPA) produced a large increase in cornifin- α /SPRR1 protein and mRNA. Immunohistochemical localization of cornifin- α /SPRR1 in TPA-treated skin indicated that cornifin- α /SPRR1 was increased in the suprabasal epidermis but not in the follicle. sn -1,2-Didecanoylglycerol, a model lipid second messenger, produced an increase in cornifin- α /SPRR1 protein similar to that of TPA, while Mirex, a non-phorbol ester-type promoter had no effect. Topical doses of retinoic acid did not repress TPA-induced cornifin- α /SPRR1 expression. Papillomas demonstrated a 10- and 100-fold increase in cornifin- α /SPRR1 protein and mRNA, and expression was restricted to suprabasal cells. Squamous cell carcinomas exhibited an intermediate level of cornifin- α protein, and expression was restricted to keratinized areas. These data indicate: i) cornifin- α /SPRR1 is expressed in mouse skin; ii) cornifin- α /SPRR1 is localized to specific areas of the anagen hair follicle with weak staining in the telogen follicle and epidermis; iii) epidermal cornifin- α /SPRR1 expression is induced by phorbol ester and sn -1,2-didecanoylglycerol but not mirex; and iv) papillomas and squamous cell carcinomas demonstrate a constitutive increase in cornifin- α /SPRR1 in differentiated areas of the neoplasms.}, number={4}, journal={Journal of Investigative Dermatology}, publisher={Elsevier BV}, author={Owens, David M. and Jetten, Anton M. and Smart, Robert C. and Zainal, Theodor A.}, year={1996}, month={Apr}, pages={647–654} }
 @article{owens_spalding_tennant_smart_1995, title={Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin}, volume={55}, number={14}, journal={Cancer Research}, author={Owens, D.M. and Spalding, J.W. and Tennant, R.W. and Smart, R.C.}, year={1995}, pages={3171–3178} }
 @article{kim_smart_1995, title={Lack of effect of retinoic acid and fluocinolone acetonide on mirex tumor promotion indicates a novel mirex mechanism}, volume={16}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/16.9.2199}, DOI={10.1093/carcin/16.9.2199}, abstractNote={Mirex, a halogenated hydrocarbon, is a potent skin tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. In the present study retinoic acid (RA) and fluocinolone acetonide (FA), classical inhibitors of phorbol ester- and non-phorbol ester-type skin tumor promoters, were examined for their ability to inhibit mirex tumor promotion. Female CD-1 mice were initiated with 200 nmol DMBA and promoted with equipotent promoting doses of either 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) or 200 nmol mirex twice weekly for 25 weeks and RA (2(1 or 5 nmol), FA (0.5 or 2 nmol) or acetone were applied 30 min prior to each TPA or mirex dose. TPA-promoted papilloma formation was strongly inhibited by > 70% with both doses of RA and by > 90% with both doses of FA. In contrast, mirex-promoted papilloma formation was not inhibited by either dose of RA or 0.5 nmol FA and 2 nmol FA weakly inhibited mirex-promoted papillomas by only 32%. TPA- and mirex-promoted papillomas that were refractory to RA and FA demonstrated the same incidence of Ha-ras mutation as TPA- or mirex-promoted papillomas without RA and FA treatment, further indicating that the inhibitory activity of RA and FA is promoter-dependent and not solely dependent on mutant Ha-ras. FA (2 nmol) treatment completely abolished TPA-induced epidermal hyperplasia and proliferating cell nuclear antigen (PCNA) S phase-positive cells, however, FA had no inhibitory effect on the weak proliferative response induced by mirex. Collectively, these results indicate that the promotional activity of mirex, as well as its weak proliferative response, result from a distinct promoter mechanism and/or that mirex promotes a unique population of epidermal cells that are insensitive to FA and RA and cannot be distinguished by their mutant Ha-ras genotype.}, number={9}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Kim, Tae-Won and Smart, Robert C.}, year={1995}, pages={2199–2204} }
 @article{meyer_kim_moser_monteiroriviere_smart_1994, title={SYNERGISTIC INTERACTION BETWEEN THE NONPHORBOL ESTER-TYPE PROMOTER MIREX AND 12-O-TETRADECANOYLPHORBOL-13-ACETATE IN MOUSE SKIN TUMOR PROMOTION}, volume={15}, ISSN={["0143-3334"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:A1994MR10300009&KeyUID=WOS:A1994MR10300009}, DOI={10.1093/carcin/15.1.47}, abstractNote={Mirex, an organochlorine pesticide and non-genotoxic rodent hepatocarcinogen, is also a potent non-phorbol ester-type promoter of mouse skin tumors. Mirex, unlike most other skin tumor promoters, is not a significant epidermal hyperplasiogen even at a maximally promoting dose (200 nmol). Experiments described here examined whether tumor promotion by mirex and 12-O-tetradecanoylphorbol-13-acetate (TPA) are mediated through different mechanisms as indicated by their additivity when co-applied to 7,12-dimethyl-benz[a]anthracene (DMBA, 200 nmol)-initiated female CD-1 mouse skin. Instead of the additive response of 14 plus 5 tumors/mouse predicted from mice promoted for 20 weeks (2x/week) with either mirex (200 nmol) or TPA (2 nmol) respectively, their co-application yielded 35 tumors/mouse. This synergy with TPA was specific to mirex since a structurally related compound, chlordecone (Kepone) was inactive. Mirex plus TPA-promoted papillomas contained a c-Ha-ras A182-->T mutation as frequently (13/14) as those promoted by mirex or TPA alone, suggesting that these DMBA-initiated/co-promoted papillomas were not atypical in this genotypic marker. Promotional synergy with mirex was only observed with a submaximal promoting dose of 2 nmol TPA; 5 or 8 nmol TPA plus mirex gave additive or less tumor multiplicities. This synergistic multiplicity with mirex plus 2 nmol TPA (35 tumors/mouse) approximated the sum of individual responses to 200 nmol mirex (14 tumors/mouse) and the maximally promoting dose of TPA (12 nmol), 24 tumors/mouse, suggesting that mirex potentiated the promotional activity of TPA, as well as promoted through a mirex-specific mechanism. Epidermal DNA synthesis induced by 2 nmol TPA was potentiated by mirex, further supporting a role for mirex in potentiation of epidermal TPA activity. Collectively, these studies suggest that mirex affects two possibly related responses: (i) promotion through a distinct mirex-specific mechanism, and (ii) potentiation of a mechanism mediating the promotional activity of TPA.}, number={1}, journal={CARCINOGENESIS}, author={MEYER, SA and KIM, TW and MOSER, GJ and MONTEIRORIVIERE, NA and SMART, RC}, year={1994}, month={Jan}, pages={47–52} }
 @inbook{smart_1993, place={Norwalk, CT}, edition={2nd}, title={Carcinogenesis}, booktitle={Biochemical Toxicology}, publisher={Appleton and Lange}, author={Smart, R.C.}, editor={Hodgson, E. and Levi, P.E.Editors}, year={1993}, pages={381–414} }
 @article{colapietro_goodell_smart_1993, title={Characterization of benzo[<i>a</i>]pyrene-initiated mouse skin papillomas for Ha-<i>ras</i> mutations and protein kinase C levels}, volume={14}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/14.11.2289}, DOI={10.1093/carcin/14.11.2289}, abstractNote={The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.}, number={11}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Colapietro, Anne-Marie and Goodell, Audrey L. and Smart, Robert C.}, year={1993}, pages={2289–2295} }
 @article{moser_robinette_smart_1993, title={Characterization of skin tumor promotion by mirex: structure-activity relationships, sexual dimorphism and presence of Ha-<i>ras</i> mutation}, volume={14}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/14.6.1155}, DOI={10.1093/carcin/14.6.1155}, abstractNote={In the present study we have compared the tumor-promoting activity of the non-phorbol ester-type skin tumor promoter, mirex, a halogenated cycloalkane pesticide, to the following: (i) chlordane, a halogenated cycloalkane pesticide; (ii) 1,1-bis (4-chlorophenyl)-2,2,2-trichlorethane (DDT), a halogenated bridged aromatic pesticide; and (iii) kepone, a halogenated cycloalkane pesticide, which only differs from mirex by the substitution of two chlorine atoms with an oxygen atom. Topical application of 200 nmol mirex three times weekly for 20 weeks to 7,12-dimethylbenz[a]anthracene (DMBA)-initiated female mouse skin produced approximately 16 tumors/mouse with a 96% incidence of tumor bearing mice. Neither chlordane (2 mumol) or DDT (5 mumol) promoted tumors in DMBA-initiated mouse skin after three times weekly application for 20 weeks. Unexpectedly, DMBA-initiated mice treated with 250 nmol kepone three times weekly for 20 weeks did not develop any tumors, demonstrating that the replacement of two chlorine atoms by an oxygen atom results in loss of the skin tumor-promoting activity of mirex. To further characterize mirex-induced skin tumor promotion, male mice were initiated with a single topical application of 200 nmol DMBA and promoted topically three times weekly for 20 weeks with 200 nmol mirex. As compared to female mice, male mice demonstrated (i) 70% fewer tumors/mouse, (ii) decreased incidence of tumor bearing mice, (iii) increased time to first tumor and (iv) increased latency. To determine the role of ovarian hormones in the increased sensitivity of female mice, mice were initiated with DMBA, ovariectomized (OVX) 2 weeks later and then promoted with mirex. OVX mice exhibited 70% fewer tumors/mouse and a 40% decrease in incidence of tumor-bearing mice as compared to controls. Finally, > 90% of DMBA-initiated/mirex-promoted papillomas from male mice and female mice demonstrated a mutated Ha-ras gene with an A-->T transversion in the middle base of the 61st codon. Collectively, these data indicate that the tumor-promoting ability of mirex is highly structure specific, and ovarian hormones are a factor in the increased sensitivity of female mice to the skin tumor-promoting ability of mirex. Furthermore, mirex appears to clonally expand epidermal cells with a mutated Ha-ras oncogene.}, number={6}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Moser, Glenda J. and Robinette, C.Lee and Smart, Robert C.}, year={1993}, pages={1155–1160} }
 @article{mills_reynolds_smart_1993, title={Diacylglycerol is an effector of the clonal expansion of cells containing activated Ha-<i>ras</i> genes}, volume={14}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/14.12.2645}, DOI={10.1093/carcin/14.12.2645}, abstractNote={Diacylglycerols (DAG) are lipid second messengers which are generated during phospholipase-catalyzed hydrolysis of phospholipids. The model DAG, sn-1,2-didecanoylglycerol (DIC10), is an effective topical tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. We now report that 11/12 of DMBA-initiated/DIC10-promoted papillomas examined contain an A-->T mutation in the 61st codon of the Ha-ras gene, suggesting that DAGs affect the clonal expansion of activated Ha-ras-containing cells. To explore further the DIC10-induced clonal expansion of activated Ha-ras-containing cells, we have examined the tumor-promoting effect of DIC10 in the skin of transgenic TG.AC mice, which harbor a v-Ha-ras transgene. By 9 weeks of promotion, 100% of the TG.AC mice developed squamous papillomas and by 15 weeks these mice developed > 20 papillomas/mouse. Because fatty acids are known to participate in signal transduction pathways, and since cellular lipases could cleave the fatty acid side chains present in DIC10, we have examined the tumor promoting activity of n-decanoic acid to verify the specificity of promotional activity of DIC10. n-Decanoic acid did not function as a tumor promoter. These data implicate DAG as an effector of the clonal expansion of mutated Ha-ras-containing cells, and support a mechanism whereby an increase in endogenous DAG could contribute to the clonal expansion of cells containing a Ha-ras oncogene.}, number={12}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Mills, Kevin J. and Reynolds, Steven H. and Smart, Robert C.}, year={1993}, pages={2645–2648} }
 @article{meyer_moser_monteiroriviere_smart_1993, title={MINIMAL ROLE OF ENHANCED CELL-PROLIFERATION IN SKIN TUMOR PROMOTION BY MIREX - A NONPHORBOL ESTER-TYPE PROMOTER}, volume={101}, ISSN={["0091-6765"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:A1993NB89100044&KeyUID=WOS:A1993NB89100044}, DOI={10.2307/3431879}, abstractNote={Mirex, a chlorinated hydrocarbon previously used as a systemic insecticide and flame retar- dant, is a nongenotoxic hepatocarcinogen in both rats and mice.In liver, mirex induced bio- chemical responses and hyperplasia characteristic of increased cell proliferation, which is con- sistent with its role as a liver tumor promoter.We have recently shown that mirex is a potent nonphorbol ester-type skin tumor promoter in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiat- ed mice.However, unlike its effect in liver, a single topical application of mirex to skin does not induce the acute biochemical responses, such as increased epidermal DNA synthesis and ornithine decarboxylase activity, indicative of increased cell proliferation.Multiple topical applications of mirex over a 1 month period induced only a minimal increase in the number of epidermal nucleated cell layers, which contrasts with definitive hyperplasia induced by a com- parable tumor-promoting dose of 12-0-tetradecanoylphorbol-13-acetate (TPA).Collectively, these data indicated that mirex is promoting through a novel mechanism.Further evidence that mirex promotes tumors through a mechanism distinct from that of the prototypical skin tumor promoter, TPA, was obtained by examining the effect of their simultaneous co-treatment.The co-application of mirex and TPA yielded a tumor multiplicity greater than the sum of the responses of each promoter individually.In summary, our results demonstrate that mirex, a carcinogenic and hyperplastic agent in liver, is also a very effective tumor promoter in mouse skin, but suggest that mirex operates via a novel mechanism in skin that may involve only a minimal role for enhanced cell proliferation.}, number={Suppl. 5}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={MEYER, SA and MOSER, GJ and MONTEIRORIVIERE, NA and SMART, RC}, year={1993}, month={Dec}, pages={265–269} }
 @article{mills_bocckino_burns_loomis_smart_1992, title={Alterations in protein kinase C isozymes α and β<sup>2</sup> in activated Ha-<i>ras</i> containing papillomas in the absence of an increase in diacyiglycerol}, volume={13}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/13.7.1113}, DOI={10.1093/carcin/13.7.1113}, abstractNote={The levels of protein kinase C (PKC) activity, PKC isozymes, as well as the level of endogenous diacylglycerols (DAG) were examined in early emergence mouse skin papillomas and compared to the levels in the epidermis. The papillomas were derived from a two-stage carcinogenesis protocol in which mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly for only 12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, greater than 90% of these early emergence papillomas contained an activated Ha-ras gene with an A----T transversion in the 61st codon. There was a TPA-independent, irreversible decrease in total PKC activity (70%) in the early emergence papillomas compared to that in the epidermis. Immunoblot analysis of epidermis and papillomas taken 4 weeks following the cessation of TPA treatment, a time when PKC catalytic activity has completely recovered to control level in epidermis but not in papillomas, revealed that the levels of PKC-alpha and PKC-beta 2 were dramatically decreased in the cytosol of the papillomas, while the levels of these two isozymes in the particulate fraction were approximately equal to the epidermis. PKC-delta, -epsilon and -zeta immunoreactive proteins were present in both epidermis and papillomas and only minor changes were observed in the papillomas. PKC-delta and PKC-epsilon displayed a particulate fraction localization in both the epidermis and papillomas, while PKC-zeta was found in both subcellular fractions. We were unable to detect PKC-gamma in mouse epidermis or papillomas. Since the level of DAG has been shown to be elevated in some ras-transformed cells, we examined DAG levels in the papillomas, as an increased DAG level could explain the constitutive decreases in the levels of PKC. Measurements of cellular DAG indicated that there was no elevation in the total pool of DAG in the early emergence papillomas. These data demonstrate an irreversible decrease in and alteration of the subcellular distribution of PKC-alpha and beta 2 in DMBA-initiated/TPA-promoted papillomas. These changes are TPA-independent, and occur in the absence of an elevation in the total pool of endogenous DAG. These alterations of PKC isozymes may be important early events in multistage tumorigenesis.}, number={7}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Mills, Kevin J. and Bocckino, Stephen B. and Burns, David J. and Loomis, Carson R. and Smart, Robert C.}, year={1992}, pages={1113–1120} }
 @article{moser_meyer_smart_1992, title={The chlorinated pesticide, mirex is a novel non-phorbol ester tumor promoter in mouse skin}, volume={52}, number={3}, journal={Cancer Research}, author={Moser, G.J. and Meyer, S.A. and Smart, R.C.}, year={1992}, pages={631–636} }
 @article{smart_crawford_1991, title={EFFECT OF ASCORBIC-ACID AND ITS SYNTHETIC LIPOPHILIC DERIVATIVE ASCORBYL PALMITATE ON PHORBOL ESTER INDUCED SKIN-TUMOR PROMOTION IN MICE}, volume={54}, ISSN={["1938-3207"]}, DOI={10.1093/ajcn/54.6.1266s}, abstractNote={The effect of topical application of ascorbic acid (AA) and ascorbyl palmitate (AP) on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced tumor promotion in mouse skin was investigated. A single application of TPA decreased epidermal AA by 45%. Repetitive application of 6 and 28 mumol AA with 2 nmol TPA inhibited tumor multiplicity by 39% and 76%. Repetitive application of 0.16, 0.8, and 4.0 mumol AA with 5 nmol TPA inhibited tumor multiplicity by 16%, 46%, and 91%. Because AA may be poorly absorbed cutaneously, we evaluated the effect of dietary AA. Supplementation of drinking water with AA increased epidermal ascorbic acid levels by 50%. Dietary intake of AA did not inhibit TPA-induced tumor promotion. Preliminary data suggest that the mice not receiving AA developed increased epidermal AA levels in response to the tumor promoting regimen. Recently we have found that dietary AP inhibited TPA-induced biochemical parameters associated with tumor promotion.}, number={6}, journal={AMERICAN JOURNAL OF CLINICAL NUTRITION}, author={SMART, RC and CRAWFORD, CL}, year={1991}, month={Dec}, pages={S1266–S1273} }
 @inbook{smart_moser_1991, title={Pesticides, tumor promotion and risk assessment in Pesticides and the Future: Toxicological Studies of Risks and Benefits}, booktitle={Reviews in Pesticides I}, author={Smart, R.C. and Moser, G.J.}, editor={Hodgson, E. and Roe, R.M. and Motoyama, N.Editors}, year={1991}, pages={349–357} }
 @article{hansen_monteiro-riviere_smart_1990, title={Differential down-regulation of epidermal protein kinase C (PKC) by tetradecanoylphorbol-13-acetate and diacylglycerol: Association with epidermal hyperplasia and tumor promotion}, volume={50}, journal={Cancer Research}, author={Hansen, L. A. and Monteiro-Riviere, N. A. and Smart, R. C.}, year={1990}, pages={5740–5745} }
 @article{mills_smart_1989, title={Comparison of epidermal protein kinase C activity, ornithine decarboxylase induction and DNA synthesis stimulated by TPA or dioctanoylglycerol in mouse strains with differing susceptibility to TPA-induced tumor promotion}, volume={10}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/10.5.833}, DOI={10.1093/carcin/10.5.833}, abstractNote={The purpose of this study was to examine the activity and associated kinetic parameters of epidermal protein kinase C (PKC) following stimulation by sn-1,2-dioctanoylglycerol (DIC8) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and to examine the relationship between levels of epidermal PKC activity and the induction of ornithine decarboxylase by these agents, utilizing various stocks and strains of mice. Importantly, the mouse strains and stock used in this study have known differing susceptibilities to undergo TPA-induced tumor promotion: the CD-1 stock and the DBA/2 strain (both sensitive to TPA-induced tumor promotion) and the C57BL/6 strain (resistant to TPA-induced tumor promotion). TPA-stimulated protein kinase C activity was measured in the 10(5)g supernatant fraction of epidermal homogenates using lysine-rich histone as a phosphate acceptor substrate. The maximal velocities for TPA-stimulated epidermal PKC activity in CD-1, DBA/2 and C57BL/6 were 0.28, 0.29 and 0.27 nmol PO4-histone/mg 10(5)g protein/min, respectively. TPA-stimulated epidermal PKC from CD-1, DBA/2 and C57BL/6 had similar theoretical Vmax values and the apparent concentrations of TPA yielding half-maximal stimulation of PKC were also similar. DiC8-stimulated PKC activity to a greater Vmax; however, the concentration required to yield half-maximal stimulation of PKC was one thousand times greater than that of TPA. There were no strain differences in these parameters when the enzyme was stimulated with DiC8. Thus, the levels of epidermal PKC activity in CD-1, DBA/2 and C57BL/6 mice exhibit no strain differences when stimulated by TPA or DiC8 using lysine-rich histone as a phosphate acceptor substrate. Since sn-1,2-diacylglycerols are known effective inducers of epidermal ornithine decarboxylase (ODC) activity, the induction of epidermal ODC was examined in each mouse strain 5 h after topical application of 2 nmol TPA, 5 nmol TPA or 2.5 mumol DiC8. After topical treatment with TPA, C57BL/6 demonstrated an unexpected 2- and 4-fold increase in ODC activity over CD-1 and DBA/2 mice. After treatment with DiC8, C57BL/6 demonstrated a 6- and 10-fold increase in ODC activity over CD-1 and DBA/2, respectively. Thus, the resistant strain (C57BL/6) demonstrated a 'hyperinducibility' of epidermal ODC activity by TPA or DiC8. The time course for the induction of epidermal ODC was examined in each strain, and at every time point measured (3-15 h), the C57BL/6 strain exhibited this 'hyperinducibility' of ODC relative to the other strains. Epidermal DNA synthesis was stimulated to a similar extent in C57BL/6 and CD-1 mice.(ABSTRACT TRUNCATED AT 400 WORDS)}, number={5}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Mills, K. J. and Smart, R. C.}, year={1989}, month={May}, pages={833–838} }
 @article{moser_smart_1989, title={Hepatic tumor-promoting chlorinated hydrocarbons stimulate protein kinase C activity}, volume={10}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/10.5.851}, DOI={10.1093/carcin/10.5.851}, abstractNote={Various chlorinated hydrocarbons, many of which are known hepatic tumor promoters, have been evaluated for their ability to stimulate protein kinase C (PKC) activity in vitro. Chlordane, kepone, toxaphene, heptachlor, 2,2-bis(4-chlorophenyl)-1,1-dichloroethane, the polychlorinated biphenyl Aroclor 1254, aldrin, 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT) and gamma-hexachlorocyclohexane (lindane) were the most potent stimulators of PKC activity. Of these compounds, chlordane was the most potent organochlorine pesticide. Chlordane (100 microM) stimulated mouse brain PKC activity in the 10(5) g supernatant to a maximum velocity equal to that obtained when the enzyme was maximally stimulated with the skin-tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Chlordane concentrations as low as 1 microM significantly stimulated PKC activity. Chlordane-stimulated PKC activity was calcium-dependent, and in the presence of exogenous calcium, chlordane-stimulated PKC activity was at least 5-fold greater than in the absence of added calcium. In contrast, the addition of calcium only minimally affected (less than 30% increase) the TPA-stimulated PKC activity. Concentrations of TPA and chlordane which maximally stimulate PKC did not produce an additive effect on PKC activity. Chlordane- and TPA- stimulated PKC activity was phospholipid-dependent and could be inhibited by quercetin, a known inhibitor of PKC activity. Chlordane in the presence of calcium also stimulated mouse epidermal and hepatic PKC as well as purified rat brain PKC. These results demonstrate that a wide variety of chlorinated hydrocarbons, which are considered hepatic tumor promoters, stimulate protein kinase C activity in vitro.}, number={5}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Moser, G. J. and Smart, R. C.}, year={1989}, month={May}, pages={851–856} }
 @article{smart_mills_hansen_conney_1989, title={Synthetic lipid second messenger, sn-1,2-didecanoylglycerol: A complete tumor promoter in mouse skin}, volume={49}, number={16}, journal={Cancer Research}, author={Smart, R.C. and Mills, K.J. and Hansen, L.A. and Conney, A.H.}, year={1989}, pages={4455–4458} }
 @article{smart_huang_monteiroriviere_wong_mills_conney_1988, title={COMPARISON OF THE EFFECT OF SN-1,2-DIDECANOYLGLYCEROL AND 12-O-TETRADECANOYLPHORBOL-13-ACETATE ON CUTANEOUS MORPHOLOGY, INFLAMMATION AND TUMOR PROMOTION IN CD-1 MICE}, volume={9}, ISSN={["0143-3334"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:A1988R257800013&KeyUID=WOS:A1988R257800013}, DOI={10.1093/carcin/9.12.2221}, abstractNote={Since sn-1,2-didecanoylglycerol mimics 12-O-tetradecanoylphorbol-13-acetate (TPA) by inducing ornithine decarboxylase activity and stimulating DNA synthesis in mouse epidermis [Smart, R.C., Huang, M.-T. and Conney, A.H. Carcinogenesis, 7, 1865 (1986)], we have investigated morphological changes induced by TPA and sn-1,2-didecanoylglycerol in the epidermis and we have also examined sn-1,2-didecanoylglycerol as a possible complete tumor promoter. It was determined that topical application of 2.5 or 10 mumol of sn-1,2-didecanoylglycerol induced epidermal ornithine decarboxylase activity to about the same extent as the application of 1 or 2 nmol of TPA respectively. Therefore, these doses of TPA and sn-1,2-didecanoylglycerol were used in most of our studies. Single or multiple application (2 X/week for 4 weeks) of 1, 2 or 5 nmol of TPA to the skin of CD-1 mice produced a dose-dependent increase in the number of epidermal non-cornified cell layers, epidermal thickness, leukocyte infiltration and intracellular edema. In contrast, neither single nor multiple application (2 X/week for 4 weeks) of 2.5 or 10 mumol sn-1,2-didecanoylglycerol produced any of these responses. However, when 5 mumol sn-1,2-didecanoylglycerol was applied topically twice a day (10 mumol/day) for 5 days there was a significant increase in the number of epidermal non-cornified cell layers and epidermal thickness. The effects of TPA and sn-1,2-didecanoylglycerol were compared using the mouse ear inflammation model. Application of TPA caused edema, but sn-1,2-didecanoylglycerol had little or no effect. sn-1,2-Didecanoylglycerol was then evaluated as a complete tumor promoter utilizing the mouse skin two-stage model. CD-1 mice were initiated with 200 nmol 7,12-dimethylbenz[a]anthracene and then treated with 1 nmol TPA or 2.5 mumol sn-1,2-didecanoylglycerol twice a week for 28 weeks. A 28 weeks, 28% of the mice treated with TPA had developed tumors, while none of the mice treated with 2.5 mumol sn-1,2-didecanoylglycerol developed tumors. The data indicate that topical application of 2.5 mumol sn-1,2-didecanoylglycerol induced ornithine decarboxylase activity to the same extent as a tumor-promoting dose of 1 nmol TPA, but it did not cause morphological changes in the epidermis when applied once or when applied twice a week for 4 weeks and did not function as a complete tumor promoter when applied twice a week for 28 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)}, number={12}, journal={CARCINOGENESIS}, author={SMART, RC and HUANG, MT and MONTEIRORIVIERE, NA and WONG, CQ and MILLS, KJ and CONNEY, AH}, year={1988}, month={Dec}, pages={2221–2226} }
 @article{huang_smart_wong_conney_1988, title={Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-o-tetradecanoylphorbol-13-acetate}, volume={48}, number={21}, journal={Cancer Research}, author={Huang, M. T. and Smart, R. C. and Wong, C. Q. and Conney, A. H.}, year={1988}, pages={5941–5946} }
 @article{zannoni_brodfuehrer_smart_susick_1987, title={Ascorbic Acid, Alcohol, and Environmental Chemicals}, volume={498}, ISSN={0077-8923 1749-6632}, url={http://dx.doi.org/10.1111/j.1749-6632.1987.tb23775.x}, DOI={10.1111/j.1749-6632.1987.tb23775.x}, abstractNote={It is apparent through the efforts of a number of investigators that ascorbic acid is involved in the metabolism and detoxification of numerous xenobiotics. Interestingly, the vitamin participates a t a variety of levels, including the important hepatic electron transport systems, i.e., cytochrome P-450 mixed function oxygenase (MFO)’-’’ and flavin-containing monooxygenase ( FMO)’2-24; protection against covalent binding of “reactive intermediates” to macromolecular protein^''^^; and more recently involvement in the metabolism and toxicological consequences of a most commonly used and abused drug, alcohol.s’d1.6’.66 Although the precise biochemical mechanism of the vitamin’s participation at these levels warrants further investigation, the role of ascorbic acid in xenobiotic metabolism may have important consequences.}, number={1 Third Confere}, journal={Annals of the New York Academy of Sciences}, publisher={Wiley}, author={Zannoni, V. G. and Brodfuehrer, J. I. and Smart, R. C. and Susick, R. L.}, year={1987}, month={Jul}, pages={364–388} }
 @article{huang_smart_thomas_pickett_lu_1987, title={Characterization of purified DT-diaphorase from liver cytosol of 3-methylcholanthrene-treated rats}, volume={27A:49-54}, journal={Chemica Scripta}, author={Huang, M.-T. and Smart, R.C. and Thomas, P.E. and Pickett, C.B. and Lu, Y.H.}, year={1987} }
 @article{smart_huang_han_kaplan_focella_conney_1987, title={Inhibition of 12-O-Tetradecanoylphorbol-13-acetate Induction of Ornithine Decarboxylase Activity, DNA Synthesis, and Tumor Promotion in Mouse Skin by Ascorbic Acid and Ascorbyl Palmitate}, volume={47}, number={24, Part 1}, journal={Cancer Research}, author={Smart, R.C. and Huang, M.-T. and Han, Z.T. and Kaplan, M.C. and Focella, A. and Conney, A.H.}, year={1987}, pages={6633–6638} }
 @article{smart_huang_chang_sayer_jerina_conney_1986, title={Disposition of the naturally occurring antimutagenic plant phenol, ellagic acid, and its synthetic derivatives, 3-<i>O</i>-decylellagic acid and 3, 3'-di-<i>O</i>-methylellagic acid in mice}, volume={7}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/7.10.1663}, DOI={10.1093/carcin/7.10.1663}, abstractNote={The effect of ellagic acid and some of its more lipophilic derivatives on the mutagenicity of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenz[a]pyrene was examined in Salmonella typhimurium TA100. Ellagic acid, 3,3'-di-O-methylellagic acid, 4,4'-di-O-methylellagic acid and 3-O-decylellagic acid were found to have approximately equal antimutagenic activity. The tissue distribution and elimination of ellagic acid, 3,3'-di-O-methylellagic acid and 3-O-decylellagic acid were examined in CD-1 mice. Little or no ellagic acid (less than 1 nmol/g) was found in blood, lung or liver after the oral administration by gavage of 300 mumol of ellagic acid per kg body weight of after feeding 1% of ellagic acid in the diet for 1 week. Following the i.p. administration of 120 mumol/kg of ellagic acid, the blood and lung levels of ellagic acid were 15-20 nmol/g at 30 min after the dose, and the concentrations of ellagic acid decreased to 1-3 nmol/g at 6-8 h after the dose. A portion of the administered i.p. dose precipitated in the abdominal cavity. After i.v. administration, ellagic acid was eliminated very rapidly from blood, lung and liver, and approximately 70% of the administered dose was recovered in the urine and feces as free ellagic acid and its conjugates. At 2 h after an i.v. injection of 60 mumol/kg of ellagic acid, 46% of the dose was recovered in the urine as ellagic acid and its conjugates. Of this amount, about half was excreted as free ellagic acid and half was excreted as conjugates. An additional 25% of the dose was recovered in the feces (mostly as free ellagic acid) after 7 h. The disposition of 3,3'-di-O-methylellagic acid or 3-O-decylellagic acid after i.v. administration (32 mumol/kg) was examined and compared to the disposition of the same i.v. dose of ellagic acid. The concentrations of ellagic acid, 3,3'-di-O-methylellagic acid and 3-O-decylellagic acid decreased rapidly in the blood, liver and lung, but the concentrations of 3-O-decylellagic acid in the lung throughout the experimental period (2-360 min) was on average 20- to 40-fold higher than the corresponding average concentrations of ellagic acid or 3,3'-di-O-methylellagic acid.}, number={10}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Smart, Robert C. and Huang, Mou-Tuan and Chang, Richard L. and Sayer, Jane M. and Jerina, Donald M. and Conney, Allan H.}, year={1986}, pages={1663–1667} }
 @article{smart_zannoni_1986, title={Effect of dietary ascorbate on covalent binding of benzene to bone marrow and hepatic tissue in vivo}, volume={35}, ISSN={0006-2952}, url={http://dx.doi.org/10.1016/0006-2952(86)90409-0}, DOI={10.1016/0006-2952(86)90409-0}, abstractNote={The bio-elimination and organ retention of orally administered [14C]benzanthrone, an anthraquinone dye intermediate, were determined in control and ascorbic acid-supplemented guinea pigs. Urinary excretion of benzanthrone in control and ascorbic acid-treated animals during 96 hr was 27.9 and 30.5%, respectively, with peak elimination at 48 hr. Faecal elimination in control and supplemented animals during 96 hr was 24.5 and 38.8%, respectively, with a peak at 48 hr. The organ retention of radiolabelled benzanthrone at the end of 96 hr was of the order of 39% in control animals (gastrointestinal tract 16%; liver 22%; testis 1.2%); ascorbic acid supplementation reduced benzanthrone retention to 19.5% (gastro-intestinal tract 12.7%; liver 6.8%). Overall, pretreatment of guinea pigs with ascorbic acid caused a 32% enhancement in the clearance of radiolabelled benzanthrone through the urine and faeces, while organ retention was reduced by about 50%. A prophylactic dose of ascorbic acid may prevent benzanthrone-induced toxic symptoms in exposed workers.}, number={18}, journal={Biochemical Pharmacology}, publisher={Elsevier BV}, author={Smart, Robert C. and Zannoni, Vincent G.}, year={1986}, month={Sep}, pages={3180–3182} }
 @article{smart_huang_chang_sayer_jerina_wood_conney_1986, title={Effect of ellagic acid and 3-<i>O</i>-decylellagic acid on the formation of benzo[<i>a</i>]pyrene-derived DNA adducts <i>in vivo</i> and on the tumorigenicity of 3-methylcholanthrene in mice}, volume={7}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/7.10.1669}, DOI={10.1093/carcin/7.10.1669}, abstractNote={The effect of ellagic acid and its more lipophilic derivative, 3-O-decylellagic acid, on the amount of DNA-bound adducts in the epidermis or lung of CD-1 mice treated with [3H]benzo-[a]pyrene ([3H]B[a]P) was evaluated using several different treatment protocols. The i.v. administration of 50 mumol/kg of ellagic acid or 3-O-decylellagic acid either together with or 5 min before a 0.2 mumol/kg i.v. dose of [3H]B[a]P did not inhibit the formation of pulmonary DNA-bound adducts. Feeding mice a diet that contained 1% ellagic acid for 10 days or the i.p. administration of 120 mumol/kg of ellagic acid 30 min before the i.v. administration of 0.2 mumol/kg of [3H]B[a]P did not inhibit the formation of DNA-bound adducts in the lung. The application of 2,500 nmol of ellagic acid or 3-O-decylellagic acid to mouse skin 5 min before the application of 2, 10 or 50 nmol of [3H]B[a]P had little or no effect on the covalent binding of [3H]B[a]P metabolites to epidermal DNA. Feeding mice a diet containing 1% ellagic acid for 10 days did not inhibit the formation of epidermal DNA-bound adducts after a topical dose of 2 nmol of [3H]B[a]P. Similarly, the topical application of 2,500 nmol of ellagic acid at 2 h, 1 h and 5 min before and at 10 min after the application of 2 nmol of [3H]B[a]P did not inhibit the formation of DNA-bound adducts, but the same dosing regimen of 3-O-decylellagic acid (total dose of 10,000 nmol) resulted in a modest inhibition in the formation of DNA-bound adducts. The topical application of 1,500 nmol of ellagic acid 1 h before the application of 1,500 nmol of 3-methylcholanthrene (3-MC) to CD-1 or BALB/c mice twice weekly did not inhibit the development of skin tumors. Our results indicate that ellagic acid and 3-O-decylellagic acid are not effective in inhibiting [3H]B[a]P DNA adduct formation in mouse skin and lung and that ellagic acid does not inhibit 3-MC-induced skin tumorigenesis in BALB/c or CD-1 mice.}, number={10}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Smart, Robert C. and Huang, Mou-Tuan and Chang, Richard L. and Sayer, Jane M. and Jerina, Donald M. and Wood, Alexander W. and Conney, Allan H.}, year={1986}, pages={1669–1675} }
 @article{smart_huang_conney_1986, title={sn-l, 2-Diacylglycerols mimic the effects of 12-<i>0</i>-tetradecanoylphorbol-13-acetate <i>in vivo</i> by inducing biochemical changes associated with tumor promotion in mouse epidermis}, volume={7}, ISSN={0143-3334 1460-2180}, url={http://dx.doi.org/10.1093/carcin/7.11.1865}, DOI={10.1093/carcin/7.11.1865}, abstractNote={12-O-Tetradecanoylphorbol-13 acetate (TPA) or various acylglycerols were applied topically to CD-1 mice, and biochemical changes associated with tumor promotion in the epidermis were examined. The topical application of 5 mumol of sn-1,2-didecanoylglycerol caused a 40-fold increase in ornithine decarboxylase activity which was similar to that found after the topical application of 2 nmol of TPA. The time course for the induction of ornithine decarboxylase activity by TPA and the time course for its induction by sn-1,2-didecanoylglycerol were similar; both compounds produced rapid increases in ornithine decarboxylase activity with peak induction occurring 4-6 h after application of the inducing chemical. sn-1,2-Dioctanoylglycerol and sn-1-oleoyl-2-acetylglycerol also increased ornithine decarboxylase activity in mouse epidermis, but sn-1,2-dioleoylglycerol, 1,3-didecanoylglycerol and rac-1-monodecanoylglycerol were inactive at the dose tested. trans-Retinoic acid, a potent inhibitor of tumor promotion, markedly inhibited the epidermal induction of ornithine decarboxylase activity that resulted from the topical administration of sn-1,2-didecanoylglycerol or TPA. The effects of TPA and the acylglycerols on epidermal DNA synthesis in vivo were determined by measuring the incorporation of [3H]thymidine into epidermal DNA. The application of sn-1,2-didecanoylglycerol or TPA to mouse skin stimulated epidermal DNA synthesis. The maximum increase occurred 18 h after administration of the inducing chemical, and the increase in DNA synthesis was proportional to the dose of sn-1,2-didecanoylglycerol. Although sn-1,2-didecanoylglycerol, sn-1,2-dioctanoylglycerol and sn-1,2-dioleoylglycerol stimulated epidermal DNA synthesis, sn-1-oleoyl-2-acetylglycerol, 1,3-didecanoylglycerol and rac-1-monodecanoylglycerol had little or no effect. The increase in epidermal DNA synthesis induced by sn-1,2-didecanoylglycerol or TPA was inhibited by the simultaneous application of fluocinolone acetonide, a potent inhibitor of tumor promotion. The results indicate that several sn-1,2-diacylglycerols mimic TPA in vivo with respect to their effects on certain biochemical parameters associated with tumor promotion in mouse skin.}, number={11}, journal={Carcinogenesis}, publisher={Oxford University Press (OUP)}, author={Smart, Robert C. and Huang, Mou-Tuan and Conney, Allan H.}, year={1986}, pages={1865–1870} }
 @article{smart_zannoni_1985, title={Effect of ascorbate on covalent binding of benzene and phenol metabolites to isolated tissue preparations}, volume={77}, ISSN={0041-008X}, url={http://dx.doi.org/10.1016/0041-008x(85)90333-3}, DOI={10.1016/0041-008x(85)90333-3}, abstractNote={[14C]Phenol and [14C]benzene are metabolized in the presence of NADPH and hepatic microsomes isolated from phenobarbital- or benzene-pretreated or untreated guinea pigs to intermediates capable of covalently binding to microsomal protein. When 1 mM ascorbate was included in the incubation mixture containing benzene as the substrate, covalent binding was inhibited by 55%. Increasing the ascorbate concentration to 5 mM inhibited binding by only an additional 17%. In contrast, when phenol was used as the substrate, 1 mM ascorbate inhibited binding by 95%. When DT-diaphorase was included in the incubation mixture containing benzene as the substrate, binding was inhibited by only 18%. This degree of inhibition is in contrast to 70% inhibition with phenol. These results indicate that different metabolites are responsible for a portion of the covalent binding depending upon the substrate employed. GSH inhibited covalent binding greater than 95% with either substrate. The metabolism of phenol to hydroquinone was unaffected by the addition of ascorbate or GSH. The metabolism of benzene to phenol was unaffected by the addition of GSH; however, the addition of ascorbate decreased the formation of phenol by 35%. Tissue ascorbate could be modulated by placing guinea pigs on different dietary intakes of ascorbate. Bone marrow ascorbate concentrations could be modulated 10-fold without any significant change in the GSH concentrations. Bone marrow isolated from guinea pigs on different dietary intakes of ascorbate were incubated with H2O2 and phenol. Bone marrow with low ascorbate concentrations displayed 4-fold more covalent binding of phenol equivalents than those with high ascorbate concentrations. This is an example of how the dietary intake of ascorbate can result in a differential response to a potentially toxic event in vitro.}, number={2}, journal={Toxicology and Applied Pharmacology}, publisher={Elsevier BV}, author={Smart, Robert C. and Zannoni, Vincent G.}, year={1985}, month={Feb}, pages={334–343} }
 @inbook{zannoni_susick_smart_1984, place={New York}, title={Ascorbic acid as it relates to the metabolism of drugs and environmental chemicals}, booktitle={Nutrition in the 20th Century}, publisher={John Wiley and Sons}, author={Zannoni, V.G. and Susick, R.L. and Smart, R.C.}, editor={Wimick, M.Editor}, year={1984}, pages={21–36} }
 @article{smart_zannoni_1984, title={DT-Diaphorase and peroxidase influence the covalent binding of the metabolites of phenol, the major metabolite of benzene}, volume={26}, number={1}, journal={Molecular Pharmacology}, author={Smart, R.C. and Zannoni, V.G.}, year={1984}, month={Jul}, pages={105–111} }