@article{kim_rodriguez_macias_rodriguez-puebla_2022, title={Cyclin-dependent kinase 4 expression alters the number of keratinocyte stem cells in the mouse hair follicle}, ISSN={["1095-8355"]}, DOI={10.1002/cbin.11765}, abstractNote={Hair follicles regenerate periodically by spontaneously undergoing cycles of growth, regression, and relative quiescence. During the hair cycle, follicle stem cells residing in a specialized niche remain quiescent, and they are stimulated to proliferate throughout the growth phase of the hair follicle. Although cell cycle regulators play a prominent role during the activation of hair follicle stem cells, the identity and the role of these regulators have not been confirmed. Herein, we reported that stem cells located in the bulge region of the HF (BuSCs) express high levels of cyclin-dependent kinase 4 (CDK4) through the quiescent phase of the hair cycle. Using gain- and loss-of-function studies, we have determined that the CDK4 protein level affects the number of BuSCs. Transgenic expression of CDK4 in the bulge region of the hair follicles reduces the number of BuSCs, whereas CDK4 ablation resulted in an increasing number of BuSCs. These results suggest that deregulation of CDK4 protein levels contributes to distorting the self-renewal/proliferation balance and, in turn, altering the number of BuSCs.}, journal={CELL BIOLOGY INTERNATIONAL}, author={Kim, Sun Hye and Rodriguez, Liliana R. L. and Macias, Everardo and Rodriguez-Puebla, Marcelo L.}, year={2022}, month={Feb} }
 @article{lee_rodriguez_majumdar_de marval_rodriguez-puebla_2021, title={CDK4 has the ability to regulate Aurora B and Cenpp expression in mouse keratinocytes}, volume={22}, ISSN={["1792-1082"]}, DOI={10.3892/ol.2021.12993}, abstractNote={Cyclin‑dependent kinase 4 (CDK4) is a critical molecule that regulates key aspects of cell proliferation through phosphorylation of the retinoblastoma (Rb) family of proteins. In the last few years, it has been suggested that CDK4 plays alternative roles in cell proliferation and tumorigenesis. The main aim of the present study was to define a novel CDK4 function as a transcriptional regulator of genes involved in chromosome segregation, contributing to the G2/M phase transition. Herein, chromatin‑immunoprecipitation reverse transcription‑quantitative PCR assays were performed to demonstrate that CDK4 could occupy the promoter region of genes associated with chromosomal segregation, such as Aurora‑B (Aurkb) and Centromere Protein P (CENP‑P). Moreover, gain‑ and loss‑of‑function experiments showed that CDK4 participated in the transcriptional regulation of Aurkb and CENP‑P. The finding that Aurkb may have a crucial role in chromosome bi‑orientation and the spindle assembly checkpoint, and that CENP‑P could be required for proper kinetochore function suggests that dysregulation of CDK4 expression induces chromosomal instability and, in some cases, cancer development.}, number={4}, journal={ONCOLOGY LETTERS}, author={Lee, Sung Hyun and Rodriguez, Liliana R. L. and Majumdar, Rima and De Marval, Paula L. Miliani and Rodriguez-Puebla, Marcelo L.}, year={2021}, month={Oct} }
 @article{lee_rodriguez_marval_rodriguez-puebla_2020, title={Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription}, volume={80}, ISBN={1538-7445}, DOI={10.1158/1538-7445.AM2020-5814}, number={16}, journal={CANCER RESEARCH}, author={Lee, Sung Hyun and Rodriguez, Liliana R. and Marval, Paula L. Miliani and Rodriguez-Puebla, Marcelo L.}, year={2020}, month={Aug} }
 @article{lee_wang_kim_kim_rodriguez-puebla_2017, title={Cyclin D3 deficiency inhibits skin tumor development, but does not affect normal keratinocyte proliferation}, volume={14}, ISSN={["1792-1082"]}, DOI={10.3892/ol.2017.6551}, abstractNote={Rearrangement and amplification of the D-type cyclin genes have been reported in human cancer. Previous studies have demonstrated that Ras-mediated skin tumorigenesis depends on pathways that act through cyclin D1 and D2; however, the role of cyclin D3 remains unknown. The present study demonstrates that cyclin D3 ablation does not affect keratinocyte proliferation, but instead increases apoptosis levels in the bulge region of the hair follicle. Consequently, cyclin D3 ablation reduces skin papilloma development in a Ras-dependent carcinogenesis model. Previous results revealed that cyclin D3 preferentially binds to cyclin-dependent kinase 6 (CDK6) in mouse keratinocytes and transgenic expression of CDK6 (K5CDK6 mice) inhibits skin tumor development. Thus, we hypothesized that the inhibitory effect of CDK6 is dependent on cyclin D3 expression. To test this hypothesis, a mouse model that overexpresses CDK6 and does not express cyclin D3 (K5CDK6/cyclin D3-/- compound mouse) was developed. Biochemical analysis of the epidermis of K5CDK6/cyclin D3-/- mice revealed that cyclin D3 ablation resulted in increased expression of cyclin D1 protein, with a consequent elevation in the level of CDK6/cyclin D1 and CDK4/cyclin D1 complexes. These findings were concurrent with the increase skin papilloma malignant progression observed in K5CDK6/cyclin D3-/- mice. In summary the absence of cyclin D3 led to fewer number of papillomas in cyclin D3-ablated mice than in the wild-type owing to increased apoptosis, suggesting that alterations in cell survival are a crucial mechanism for crippling cellular defense against neoplasia. The results of the current study also suggest that although cyclin D3 expression does not alter the tumor suppressive role of CDK6 in skin carcinogenesis, the compensatory increase in cyclin D1 can shift the balance towards malignant progression.}, number={3}, journal={ONCOLOGY LETTERS}, author={Lee, Sung Hyun and Wang, Xian and Kim, Sun Hye and Kim, Yongbaek and Rodriguez-Puebla, Marcelo L.}, year={2017}, month={Sep}, pages={2723–2734} }
 @article{sistrunk_kim_wang_lee_kim_macias_rodriguez-puebla_2013, title={Skp2 Deficiency Inhibits Chemical Skin Tumorigenesis Independent of p27(Kip1) Accumulation}, volume={182}, ISSN={["0002-9440"]}, DOI={10.1016/j.ajpath.2013.01.016}, abstractNote={S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.}, number={5}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Sistrunk, Christopher and Kim, Sun Hye and Wang, Xian and Lee, Sung Hyun and Kim, Yongbaek and Macias, Everardo and Rodriguez-Puebla, Marcelo L.}, year={2013}, month={May}, pages={1854–1864} }
 @article{wang_sistrunk_marval_kim_rodriguez-puebla_2012, title={Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development}, volume={11}, ISSN={["1551-4005"]}, DOI={10.4161/cc.11.2.18774}, abstractNote={We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1-/- compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1-/- compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.}, number={2}, journal={CELL CYCLE}, author={Wang, Xian and Sistrunk, Christopher and Marval, Paula L. Miliani and Kim, Yongbaek and Rodriguez-Puebla, Marcelo L.}, year={2012}, month={Jan}, pages={335–342} }
 @article{sistrunk_macias_nakayama_kim_rodriguez-puebla_2011, title={Skp2 Is Necessary for Myc-Induced Keratinocyte Proliferation but Dispensable for Myc Oncogenic Activity in the Oral Epithelium}, volume={178}, ISSN={["0002-9440"]}, DOI={10.1016/j.ajpath.2011.02.034}, abstractNote={The proto-oncogene c-Myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis. Myc accelerates the rate of cell proliferation, at least in part, through its ability to down-regulate the expression of the cell cycle inhibitor p27(Kip1). Moreover, p27(Kip1) protein levels are regulated by ubiquitin-mediated turnover, leading to destruction by the E3 ubiquitin ligase SCF(Skp2). Therefore, we hypothesize that a lack of Skp2 expression should lead to increased p27(Kip1) levels and further inhibition of Myc-mediated proliferation and tumorigenesis. Myc expression in epithelial tissues of transgenic mice (K5-Myc) led to increased keratinocyte proliferation and the development of spontaneous tumors within the oral cavity. We generated K5-Myc-transgenic mice in an Skp2-null background. Consistent with our hypothesis, we found that Myc-mediated keratinocyte hyperproliferation was abolished by the loss of Skp2. However, Skp2 ablation did not affect Myc-driven tumorigenesis because the incidence, latency, and degree of differentiation of oral tumors were identical between K5-Myc/Skp2(+/+) and K5-Myc/Skp2(-/-) mice. Altogether, these findings suggest that Skp2 and p27(Kip1) are critical for Myc-driven keratinocyte proliferation; however, Myc-mediated tumorigenesis in the oral epithelium is independent of the Skp2-p27(Kip1) axis.}, number={6}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Sistrunk, Christopher and Macias, Everardo and Nakayama, Keiichi and Kim, Yongbaek and Rodriguez-Puebla, Marcelo L.}, year={2011}, month={Jun}, pages={2470–2477} }
 @article{mengoni_vichera_rigano_rodriguez-puebla_galliano_cafferata_pivetta_moreno_vojnov_2011, title={Suppression of COX-2, IL-1 beta and TNF-alpha expression and leukocyte infiltration in inflamed skin by bioactive compounds from Rosmarinus officinalis L.}, volume={82}, ISSN={["1873-6971"]}, DOI={10.1016/j.fitote.2010.11.023}, abstractNote={In the present study, we evaluated the effects of extracts and purified compounds from fresh leaves of Rosmarinus officinalis L. Pretreatment with the major anti-inflammatory compounds, carnosic acid (CA) and carnosol (CS), inhibited phorbol 12-myristate 13-acetate (PMA)-induced ear inflammation in mice with an EC50 of 10.20 μg/cm2 and 10.70 μg/cm2, respectively. To further understand the anti-inflammatory mechanism of these compounds, we analyzed the in vivo expression of several inflammation-associated genes in mouse skin by reverse transcriptase-polymerase chain reaction (RT-PCR). Our data showed that CA and CS reduced the expression of IL-1β and TNF-α but had less effect on fibronectin and ICAM-1 expression. Interestingly, both compounds selectively inhibited COX-2 but not COX-1. Histopathological analysis of hematoxylin and eosin (H&E)-stained tissue revealed a marked reduction in leukocyte infiltration and epidermal ulceration of PMA-treated ears when ears were pretreated with ethanolic extracts or pure CA. In vitro, we showed that ethanolic extract, carnosic acid and carnosol significantly inhibited the overproduction of nitric oxide (NO) in a dose-dependent manner in the RAW 264.7 murine macrophage cell line. For the first time in vivo, we showed that CA and CS differentially regulate the expression of inflammation-associated genes, thus demonstrating the pharmacological basis for the anti-inflammatory properties reported for CA and CS.}, number={3}, journal={FITOTERAPIA}, author={Mengoni, Eleonora S. and Vichera, Gabriel and Rigano, Luciano A. and Rodriguez-Puebla, Marcelo L. and Galliano, Silvia R. and Cafferata, Eduardo E. and Pivetta, Omar H. and Moreno, Sivia and Vojnov, Adrian A.}, year={2011}, month={Apr}, pages={414–421} }
 @article{wang_sistrunk_rodriguez-puebla_2011, title={Unexpected Reduction of Skin Tumorigenesis on Expression of Cyclin-Dependent Kinase 6 in Mouse Epidermis}, volume={178}, ISSN={["0002-9440"]}, DOI={10.1016/j.ajpath.2010.11.032}, abstractNote={Cyclin-dependent kinases (CDKs) 4 and 6 are important regulators of the G(1) phase of the cell cycle, share 71% amino acid identity, and are expressed ubiquitously. As a result, it was assumed that each of these kinases plays a redundant role regulating normal and neoplastic proliferation. In previous reports, we have described the effects of CDK4 expression in transgenic mice, including the development of epidermal hyperplasia and increased malignant progression to squamous cell carcinoma. To study the role of CDK6 in epithelial growth and tumorigenesis, we generated transgenic mice carrying the CDK6 gene under the keratin 5 promoter (K5CDK6). Similar to K5CDK4 mice, epidermal proliferation increased substantially in K5CDK6 mice; however, no hyperplasia was observed. CDK6 overexpression also triggered keratinocyte apoptosis in interfollicular and follicular epidermis as a compensatory mechanism to override aberrant proliferation. Unexpectedly, CDK6 overexpression results in decreased skin tumor development compared with wild-type siblings. The inhibition in skin tumorigenesis was similar to that previously reported in K5-cyclin D3 mice. Furthermore, biochemical analysis of the K5CDK6 epidermis showed preferential complex formation between CDK6 and cyclin D3, suggesting that this particular complex plays an important role in tumor restraint. These studies provide in vivo evidence that CDK4 and CDK6 play a similar role as a mediator of keratinocyte proliferation but differ in apoptosis activation and skin tumor development.}, number={1}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Wang, Xian and Sistrunk, Christopher and Rodriguez-Puebla, Marcelo L.}, year={2011}, month={Jan}, pages={345–354} }
 @article{rojas_benavides_blando_perez_cardenas_richie_knudsen_johnson_senderowicz_rodriguez-puebla_et al._2009, title={Enhanced Skin Carcinogenesis and Lack of Thymus Hyperplasia in Transgenic Mice Expressing Human Cyclin D1b (CCND1b)}, volume={48}, ISSN={["1098-2744"]}, DOI={10.1002/mc.20489}, abstractNote={Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.}, number={6}, journal={MOLECULAR CARCINOGENESIS}, author={Rojas, Paola and Benavides, Fernando and Blando, Jorge and Perez, Carlos and Cardenas, Kim and Richie, Ellen and Knudsen, Erik S. and Johnson, David G. and Senderowicz, Adrian M. and Rodriguez-Puebla, Marcelo L. and et al.}, year={2009}, month={Jun}, pages={508–516} }
 @article{patil_lee_macias_lam_xu_jones_ho_rodriguez-puebla_chen_2009, title={Robe of Cyclin D1 as a Mediator of c-Met- and beta-Catenin-Induced Hepatocarcinogenesis}, volume={69}, ISSN={["1538-7445"]}, DOI={10.1158/0008-5472.CAN-08-2514}, abstractNote={Abstract Activation of c-Met signaling and β-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated β-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of β-catenin. To determine the importance of CCND1 as a mediator of c-Met– and β-catenin–induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, β-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by β-catenin/c-Met. In addition, when activated β-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of β-catenin during HCC pathogenesis, although other molecules may be required to fully propagate β-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and β-catenin. [Cancer Res 2009;69(1):253–61]}, number={1}, journal={CANCER RESEARCH}, author={Patil, Mohini A. and Lee, Susie A. and Macias, Everardo and Lam, Ernest T. and Xu, Chuanrui and Jones, Kirk D. and Ho, Coral and Rodriguez-Puebla, Marcelo and Chen, Xin}, year={2009}, month={Jan}, pages={253–261} }
 @article{macias_marval_de siervi_conti_senderowicz_rodriguez-puebla_2008, title={CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development}, volume={173}, ISSN={["1525-2191"]}, DOI={10.2353/ajpath.2008.071124}, abstractNote={It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21Cip1 and p27Kip1. Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4D158N mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4D158N, but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21Cip1 in K5Cdk2, but not in K5Cdk4D158N, epidermis, suggesting that CDK2 overexpression elicits a p21Cip1 response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis. It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21Cip1 and p27Kip1. Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4D158N mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4D158N, but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21Cip1 in K5Cdk2, but not in K5Cdk4D158N, epidermis, suggesting that CDK2 overexpression elicits a p21Cip1 response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis. Normal cell growth and differentiation requires precise control of the mechanisms that govern the entry into, passage through, and exit from the cell cycle. Progress through the G1 phase of the mammalian cell cycle is mediated by D-type cyclins (D1, D2, and D3), which associate and activate CDK4 and CDK6 kinases, resulting in their catalytic activation and substrate recognition.1Sherr CJ D-type cyclins.Trends Biochem Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (873) Google Scholar, 2Weinberg RA The retinoblastoma protein and cell cycle control.Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar CDK2 is considered a unique kinase that binds to cyclin E regulating S phase entry. The pRb family of proteins, pRb, p107, and p130, are key substrates for cyclin Ds/CDK4,6 and cyclin E/CDK2 complexes, that negatively regulate the passage of cells from G1 to S phase.2Weinberg RA The retinoblastoma protein and cell cycle control.Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar The Cip/Kip family of CDK inhibitors, p21Cip1, p27Kip1, and p57Kip2, form inactive complexes with CDK2-cyclin E and CDK2-cyclin A and also bind CDK4,6/cyclin D complexes but do not interfere with their kinase activities.3Blain S Montalvo E Massague J Differential interaction of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 with cyclin A-cdk2 and cyclin D2-cdk4.J Biol Chem. 1997; 272: 25863-25872Crossref PubMed Scopus (244) Google Scholar, 4Labaer J Garret MD Stevenson LF Slingerland JM Sandhu C Chou HS Fattaey A Harlow E New functional activities for the p21 family of CDK inhibitors.Genes Dev. 1997; 11: 847-862Crossref PubMed Scopus (1209) Google Scholar Thus, cyclin D-CDK4 sequester p27Kip1, controlling the amount available for inhibition of CDK2 activity. We and others have demonstrated that indirect activation of CDK2 occurs by sequestration of p27Kip1 on forced expression of CDK4.5Bouchard C Thieke K Maier A Saffrich R Hanley-Hyde J Ansorge W Reed S Sicinski P Bartek J Eilers M Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.EMBO J. 1999; 18: 5321-5333Crossref PubMed Scopus (405) Google Scholar, 6Perez-Roger I Kim SH Griffiths B Sewing A Land H Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1).EMBO J. 1999; 18: 5310-5320Crossref PubMed Scopus (280) Google Scholar, 7Miliani de Marval P Gimenez-Conti I LaCava M Martinez L Conti C Rodriguez-Puebla M Transgenic expression of CDK4 results in epidermal hyperplasia and severe dermal fibrosis.Am J Pathol. 2001; 159: 369-379Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The role of CDK2 in cell proliferation has been supported by several founder reports in this field. A dominant-negative form of CDK2 prevents growth of cells in culture,8van den Heuvel S Harlow E Distinct roles for cyclin-dependent kinases in cell cycle control.Science. 1993; 262: 2050-2054Crossref PubMed Scopus (970) Google Scholar and microinjection of antibodies against CDK2, cyclin E, or cyclin A block initiation of DNA synthesis in mammalian cells.9Ohtsubo M Theodoras A Schumacher J Roberts J Pagano M Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.Mol Cell Biol. 1995; 15: 1559-1571Google Scholar, 10Pagano M Pepperkok R Verde F Ansorge W Draetta G Cyclin A is required at two points in the human cell cycle.EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1114) Google Scholar, 11Tsai LH Lees E Faha B Harlow E Riabowol K The cdk2 kinase is required for the G1-to-S transition in mammalian cells.Oncogene. 1993; 8: 1593-1602PubMed Google Scholar However, in the last few years the concept that CDK2 is crucial for controlling entry into S phase was challenged when two independent groups reported the generation of Cdk2-null mice.12Berthet C Aleem E Coppola V Tassarollo L Kaldis P Cdk2 knockout mice are viable.Curr Biol. 2003; 13: 1775-1785Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 13Ortega S Prieto I Odajima J Martin A Dubus P Sotillo R Barbero JL Malumbres M Barbacid M Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice.Nat Genet. 2003; 35: 25-31Crossref PubMed Scopus (707) Google Scholar These mice are viable, develop normally, and show defects in meiosis, but not in mitosis. Also, CDK2 appears to be dispensable for cell-cycle inhibition and tumor suppression mediated by p27Kip1 and p21Cip1.14Martín A Odajima J Hunt SL Dubus P Ortega S Malumbres M Barbacid M Cdk2 is dispensable for cell cycle inhibition and tumor suppression mediated by p27(Kip1) and p21(Cip1).Cancer Cell. 2005; 7: 591-598Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar Although CDK2 kinase activity is found elevated in human tumors, CDK2 is not directly affected by mutations and only a small subset of human tumors have Cdk2 gene amplification or elevated protein expression.15Marone M Scambia G Giannitelli C Ferrandina G Masciullo V Bellacosa A Benedetti-Panici P Mancuso S Analysis of cyclin E and CDK2 in ovarian cancer: gene amplification and RNA overexpression.Int J Cancer. 1998; 75: 34-39Crossref PubMed Scopus (113) Google Scholar, 16Kitahara K Yasui W Kuniyasu H Yokozaki H Akama Y Yunotani S Hisatsugu T Tahara E Concurrent amplification of cyclin E and CDK2 genes in colorectal carcinomas.Int J Cancer. 1995; 62: 25-28Crossref PubMed Scopus (114) Google Scholar, 17Shintani S Mihara M Nakahara Y Kiyota A Ueyama Y Matsumura T Wong DT Expression of cell cycle control proteins in normal epithelium, premalignant and malignant lesions of oral cavity.Oral Oncol. 2002; 38: 235-243Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar However, CDK2 regulatory binding partner, cyclin E, is frequently amplified and overexpressed in human tumors.18Akama Y Yasui W Yokozaki H Kuniyasu H Kitahara K Ishikawa T Tahara E Frequent amplification of the cyclin E gene in human gastric carcinomas.Jpn J Cancer Res. 1995; 86: 617-621Crossref PubMed Scopus (145) Google Scholar, 19Keyomarsi K Herliczek T The role of cyclin E in cell proliferation, development and cancer.Prog Cell Cycle Res. 1997; 3: 171-191Crossref PubMed Scopus (110) Google Scholar, 20Eguchi N Fujii K Tsuchida A Yamamoto S Sasaki T Kajiyama G Cyclin E overexpression in human gallbladder carcinomas.Oncol Rep. 1999; 6: 93-96PubMed Google Scholar Likewise, loss of CDK2 inhibitor p27Kip1 is a common event in human and experimental tumors. For these reasons, it has been widely assumed that elevated CDK2 activity plays a causal role in human tumor development. Coincidently, mouse models for CDK2 regulatory proteins, cyclin E and p27Kip1, are susceptible to tumor development.21Kiyokawa H Kineman R Manova-Todorova K Soares V Hoffman E Ono M Khanam D Hayday A Frohman L Koff A Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1).Cell. 1996; 85: 721-732Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar, 22Nakayama K Ishida N Shirane M Inomata A Inoue T Shishido N Horii I Loh DY Mice lacking p27Kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors.Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar, 23Fero ML Rivkin M Tasch M Porter P Carow CE Firpo E Polyak K Tsai LH Broudy V Perlmutter RM Kaushansky K Roberts JM A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice.Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1331) Google Scholar, 24Gray-Bablin J Zalvide J Fox M Knickerbocker C DeCaprio J Keyomarsi K Cyclin E, a redundant cyclin in breast cancer.Proc Natl Acad Sci USA. 1996; 93: 15215-15220Crossref PubMed Scopus (112) Google Scholar, 25Harwell RM Porter DC Danes C Keyomarsi K Processing of cyclin E differs between normal and tumor breast cells.Cancer Res. 2000; 60: 481-489PubMed Google Scholar, 26Keyomarsi K Conte DJ Toyofuku W Fox M Deregulation of cyclin E in breast cancer.Oncogene. 1995; 11: 941-950PubMed Google Scholar Similarly, forced expression of cyclin D1-CDK2 fusion protein under the control of MMTV promoter results in the development of spontaneous mammary tumors.27Corsino P Davis B Law M Chytil A Forrester E Norgaard P Teoh N Law B Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression.Cancer Res. 2007; 67: 3135-3144Crossref PubMed Scopus (20) Google Scholar However these mice exhibit a phenotype similar to MMTV-cyclin D1 mice and the consequences of CDK2 activation alone have not been investigated.28Wang TC Cardiff RD Zukerberg L Lees E Arnold A Schmidt EV Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice.Nature. 1994; 369: 669-671Crossref PubMed Scopus (890) Google Scholar Therefore, it remains unclear whether elevated CDK2 kinase activity per se is a cause or consequence of tumorigenesis.Our previous studies demonstrated that induction of keratinocyte proliferation by forced expression of CDK4 is followed by CDK2 activation.7Miliani de Marval P Gimenez-Conti I LaCava M Martinez L Conti C Rodriguez-Puebla M Transgenic expression of CDK4 results in epidermal hyperplasia and severe dermal fibrosis.Am J Pathol. 2001; 159: 369-379Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Miliani de Marval PL Macias E Conti CJ Rodriguez-Puebla ML Enhanced malignant tumorigenesis in Cdk4 transgenic mice.Oncogene. 2004; 23: 1863-1873Crossref PubMed Scopus (54) Google Scholar, 30Miliani de Marval PL Macias E Rounbehler R Sicinski P Kiyokawa H Johnson DG Conti CJ Rodriguez-Puebla ML Lack of cyclin-dependent kinase 4 inhibits c-myc tumorigenic activities in epithelial tissues.Mol Cell Biol. 2004; 24: 7538-7547Crossref PubMed Scopus (89) Google Scholar We also demonstrated that ablation of Cdk4 results in the reduction of CDK2 activity in mouse epidermis because of redistribution of p21Cip1 and p27Kip1.30Miliani de Marval PL Macias E Rounbehler R Sicinski P Kiyokawa H Johnson DG Conti CJ Rodriguez-Puebla ML Lack of cyclin-dependent kinase 4 inhibits c-myc tumorigenic activities in epithelial tissues.Mol Cell Biol. 2004; 24: 7538-7547Crossref PubMed Scopus (89) Google Scholar More recently, we reported that ablation of Cdk2 in a K5Cdk4 transgenic (Tg) mice reduces tumor development and CDK4-mediated malignant progression to squamous cell carcinoma (SCC).31Macias E Kim Y Miliani de Marval PL Klein-Szanto A Rodriguez-Puebla ML Cdk2 deficiency decreases ras/CDK4-dependent malignant progression, but not myc-induced tumorigenesis.Cancer Res. 2007; 67: 9713-9720Crossref PubMed Scopus (26) Google Scholar These findings suggest that CDK2 kinase activity contributes to CDK4-mediated keratinocyte hyperproliferation, as well as the development and progression of mouse skin tumors. Therefore, we hypothesized that elevated CDK2 kinase activity would enhance normal keratinocyte proliferation and mouse skin carcinogenesis. To investigate this hypothesis, we generated two Tg mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4D158N. The bovine keratin 5 (K5) promoter drives the expression of Cdk2 or mutant Cdk4D158N to the basal cell layer of epidermis and other epithelial tissues. The D158N mutation blocks the kinase activity of CDK4 resulting in a kinase dead protein, although it retains the ability to bind D-type cyclins and p27/p21 allowing it to sequester these inhibitors and indirectly activate CDK2 (noncatalytic function of CDK4). Herein, we demonstrate that indirect activation of CDK2 through overexpression of CDK4D158N results in epidermal hyperplasia associated with increased keratinocyte proliferation. In contrast, the direct overexpression of CDK2 does not affect keratinocyte proliferation. Interestingly, immunoblot analysis revealed elevated levels of p21Cip1 in K5Cdk2 epidermis compared to wild-type (Wt) littermates, but not in K5Cdk4D158N epidermis. The latter suggests that overexpression of CDK2, but not its indirect activation, elicits a p21Cip1 response to maintain keratinocyte homeostasis. More importantly, we report that neither the direct overexpression nor the indirect activation of CDK2 increases the susceptibility to mouse skin tumor development or progression to SCC under a two-stage carcinogenesis protocol. Thus, we conclude that although the CDK4 sequestering activity is enough to induce keratinocyte hyperproliferation, concomitant activation of both CDK4 and CDK2 kinase activities are required to induce malignant phenotype. Furthermore, this work shows that Cdk2 does not behave as an oncogene in ras-mediated tumorigenesis.Materials and MethodsDevelopment of Tg MiceK5Cdk2 and K5Cdk4D158N Tg mice were developed by cloning a cDNA containing the human-Cdk4 carrying the D158N mutation or human-Cdk2 into the vector pBK5, which contains the 5.2-kb bovine keratin 5 regulatory sequences, β-globin intron 2, and the 3′ SV40-polyadenylylation sequences. These constructs were designated as pK5Cdk4D158N and pK5Cdk2. The transgene was excised from the plasmid vector by digestion with BssHII and microinjected into the FVB strain, at Science Park Transgenic Mouse Facility, M.D. Anderson Cancer Center, Smithville, TX. Several founders for each Tg mouse (K5Cdk2 and K5Cdk4D158N) were obtained from the transgenic facility. Positive founders were genotyped by polymerase chain reaction (PCR) using specific primers for the human transgenes Cdk2 and Cdk4, although not annealed to the endogenous genes. Two Tg lines for each transgene were used in these studies.Two-Stage Chemical CarcinogenesisFor the two-stage carcinogenesis, newborn mice were initiated at day 1 after birth with a single topical application of 50 μg of 7,12-dimethylbenz(a)anthracene (DMBA) in 50 μl of acetone on dorsal mouse skin. At day 21, mice received 2.5 μg of tumor-promoting agent in 200 μl of acetone twice a week for 25 weeks. Skin tumors were counted once a week until the end of the experiment at 40 weeks. Malignant progression to SCC was determined by macroscopic observation and further confirmed by histopathological analysis of paraffin-embedded hematoxylin and eosin (H&E)-stained cross sections. Twenty mice were used for each experimental group (K5Cdk4D158N, K5Cdk2, and the respective Wt mice littermates).Western Blots and Kinase AssaysFor immunoblots, protein lysates were collected from epidermal skin scrapes with RIPA lysis buffer, 150 mmol/L NaCl, 1.0% IGEPAL, 0.5% DOC, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris (pH 8.0). For immunoblot analysis of skin tumors, papillomas were snap-frozen in liquid N2 and crushed with a pestle and mortar. Homogenates from epidermal scrapes or papillomas were sonicated and centrifuged at 14,000 rpm at 4°C. Supernatants were boiled in 2× Laemmli sample buffer for Western blot analysis or stored at −80°C. To assess CDK2 kinase activities, proteins were extracted and immunoprecipitated in Nonidet P-40 lysis buffer (Tris, pH 7.5, 150 mmol/L NaCl, 0.5% Nonidet P-40, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethyl sulfonyl fluoride). For CDK4 kinase activity proteins were extracted and immunoprecipitated with Tween 20 buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L EDTA, 2.5 mmol/L EGTA, 10% glycerol, 0.1% Tween 20, 1 mmol/L NaF, 1 mmol/L Na3VO4, and 1 mmol/L dithiothreitol). Briefly, 250 μg of protein lysates were immunoprecipitated with 2.5 μg of antibodies against CDK2 (M-20) or CDK4 (C-22), (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4°C, then incubated with 35 μl of protein A-agarose bead. Beads were washed twice each with IP buffer and once with kinase buffer (50 mmol/L HEPES, pH 7, 10 mmol/L MgCl2, 5 mmol/L MnCl2). Then, 30 μl of kinase buffer, 1 μg of pRb or histone H1 (Upstate Biotechnology Inc., Charlottesville, VA) substrate, 5 μCi of [γ-32P]ATP (6000 Ci/mmol), 1 mmol/L dithiothreitol, and 5 μmol/L ATP were added to the bead pellet and incubated for 30 minutes at 30°C. Sodium dodecyl sulfate sample buffer was added, and each sample was boiled for 3 minutes to stop reaction and electrophoresed through polyacrylamide gels. Western blot and kinase assay bands were quantified using UN-SCAN-IT, gel version 6.1 software for Windows (Silk Scientific, Inc., Orem, UT).ImmunostainingEpithelial cell proliferation was measured by intraperitoneal injection of bromodeoxyuridine (BrdU) 30 minutes before the mice were sacrificed by CO2 asphyxiation. BrdU incorporation was detected by immunohistochemical staining of paraffin-embedded skin sections with mouse anti-BrdU (Ab-2) monoclonal antibody (Calbiochem, San Diego, CA), biotin-conjugated anti-mouse antibody (Vector Laboratories, Inc., Burlingame, CA) and an avidin-biotin-peroxidase kit (Vectastain Elite; Vector Laboratories, Inc.) with diaminobenzidine as chromogen. Apoptotic cells were determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assays as was previously described.32Pierce AM Gimenez-Conti IB Schneider-Broussard R Martinez LA Conti CJ Johnson DG Increased E2F1 activity induces skin tumors in mice heterozygous and nullizygous for p53.Proc Natl Acad Sci USA. 1998; 95: 8858-8863Crossref PubMed Scopus (143) Google Scholar The number of apoptosis-positive cells in the hair follicle was determined in sections of 200 μm2 with a reticule grid. In all cases, 10 to 12 fields were counted per section on a total of eight paraffin-embedded sections representing four mice per genotype.Statistical AnalysisStatistical analysis was performed using GraphPad Prism 4 software (GraphPad Software, San Diego, CA).ResultsGeneration of K5Cdk2 Tg Mice and Gross Analysis of Mouse EpidermisExpression of human Cdk2 was targeted to the epidermis using the 5′ regulatory sequence of the keratin 5 (K5) gene, which drives the expression of transgenes to the basal cell layer of the stratified squamous epithelia.33Ramírez A Bravo A Jorcano J Vidal M Sequences 5′ of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development.Differentiation. 1994; 58: 53-64PubMed Google Scholar Two founder mice were used to establish lines K202 and K203 of K5Cdk2 Tg mice in an FVB/N background (Figure 1A). Immunohistochemical staining using antibodies against CDK2 of paraffin-embedded skin verified that exogenous CDK2 was expressed and restricted to the keratin 5-expressing basal cell layer of the epidermis (Figure 1B). The level of CDK2 overexpression was similar between both K202 and K203 Tg lines (data not shown). Thus, line K202 was used for all experiments described in this article. Western blot analysis verified that K5Cdk2 mice exhibit an 11.3-fold increase of CDK2 protein expression in epidermis (Figure 1C). Consistent with previous reports, K5 promoter also drives expression of transgene in thymus where CDK2 expression shows a fourfold increase compared to Wt littermates (Figure 1C).To determine the in vivo consequences of forced CDK2 expression we examined formalin-fixed paraffin-embedded skin cross sections. Microscopic examination of H&E sections from K5Cdk2 mice shows that epidermal and dermal morphology was indistinguishable from that of Wt littermates (Figure 2A). Further characterization of K5Cdk2 epidermis was performed by quantifying the number of nucleated cells in the epidermis, assaying in vivo keratinocyte proliferation via BrdU incorporation and determining the number of apoptotic cells by tunnel assay. Mild epidermal hyperplasia was observed in K5Cdk2 epidermis as showed by the increased number of nucleated cells in the interfollicular epidermis (Figure 2B, P < 0.05). However, the level of proliferation (BrdU labeling index) was similar between K5Cdk2 and Wt littermates (Figure 2B). To verify the effect of CDK2 overexpression on the survival rate of keratinocytes, we assessed the level of apoptosis in follicular and interfollicular epidermis from K5Cdk2 Tg mice. An increased number of follicular apoptotic cells was found in K5Cdk2 mice in comparison to Wt mice (0.0176 and 0.0043, respectively; P < 0.05) (Figure 2B). On the other hand, no differences were observed in interfollicular apoptosis between K5Cdk2 and Wt littermates (data not shown). These results suggest that overexpression of CDK2 does not affect the rate of normal keratinocyte proliferation, although it may compromise the survival rate of follicular keratinocytes.Figure 2Skin phenotype of K5CDK2 Tg mice. A: Representative paraffin sections of skin from K5CDK2 Tg mice and normal littermates (Wt) stained with H&E. Arrow denotes epidermis (E). D, Dermis. B: Quantification of the number of nucleated cells per 200 μm of interfollicular epidermis in K5CDK2 and Wt mice on H&E-stained cross sections. In vivo proliferation assay using BrdU incorporation (labeling index) in the basal cell layer of mouse epidermis and quantification of the number of apoptotic cells (apoptotic labeling index) in the hair follicle of K5CDK2 and Wt mice using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Four mice for each genotype were used to determine the BrdU labeling index, number of nucleated cells, and apoptosis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biochemical Analysis of K5Cdk2 Mouse EpidermisAs aforementioned, epidermal lysates from K5Cdk2 mice show an 11.3-fold increase of CDK2 protein expression in mouse epidermis in comparison to Wt littermates (Figure 1, Figure 3). To study whether forced expression of CDK2 affects related cell-cycle regulators we assessed the protein levels of CDKs, CKIs (cyclin-dependent kinase inhibitors), and cyclins. In general, no changes in protein expression were observed for CDK4 and CDK6 nor their cyclin binding partner, cyclin D1. Likewise, cyclins A and cyclin E, although they bind to CDK2, were unaltered (Figure 3A). Importantly, p21Cip1 protein expression increased fivefold in K5Cdk2 epidermis in comparison to Wt mice, whereas, p27Kip1 levels appear unchanged (Figure 3A). Several reports have shown that p53/p21 form an inducible barrier that protect cells against cyclin E deregulation34Minella AC Swanger J Bryant E Welcker M Hwang H Clurman BE p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation.Curr Biol. 2002; 12: 1817-1827Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 35He G Siddik ZH Huang Z Wang R Koomen J Kobayashi R Khokhar AR Kuang J Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities.Oncogene. 2005; 24: 2929-2943Crossref PubMed Scopus (195) Google Scholar and co-transfection of cyclin E and CDK2 in culture results in the transactivation of p53 signaling.36Segawa K Hokuto I Minowa A Ohyama K Takano T Cyclin E enhances P53-mediated transactivation.FEBS Lett. 1993; 329: 283-286Abstract Full Text PDF PubMed Scopus (11) Google Scholar Thus, it is possible that elevated CDK2 protein levels in mouse epidermis mimics the action of elevated cyclin E inducing a p53/p21 response and partially blocking CDK2 activity. Nevertheless, we did not observe increased p53 levels or p53 activation (phospho-p53 Ser 15) in K5Cdk2 compared with Wt epidermis (Figure 3A). In addition, we could not detect any accumulation of Mdm2, a p53 regulated gene, in K5Cdk2 epidermis (data not-shown). However, these data cannot rule out the possibility that p53 is involved in p21Cip1 induction. To determine the effect of elevated p21Cip1 levels on CDK2 kinase activity, we performed in vitro kinase assays using histone H1 as substrate. Consistent with the elevated expression of CDK2, CDK2-kinase activity increased 1.7-fold in Tg mice compared to Wt littermates (Figure 3B). To determine whether elevated p21Cip1 levels bind to transgenic CDK2, we immunoprecipitated CDK2 and immunoblotted for p21Cip1(Figure 3C). We found that elevated levels of p21Cip1 bind to CDK2 and likely results in a partial attenuation of CDK2 activity in K5Cdk2 Tg mice. In addition, we immunoprecipitated cyclin E and immunoblotted for CDK2, which shows that transgenic CDK2 binds to endogenous cyclin E (Figure 3C). After prolonged exposure of the membrane we also observed complexes between endogenous CDK2 and cyclin E, p21Cip1 in Wt samples (data not shown). It is widely accepted that p27Kip1 and p21Cip1 act as assembly factors for CDK4/D-type cyclin complexes. Thus, we reasoned that the increase CDK2/p21 binding reduce the amount of p21Cip1 available to bind CDK4 complexes decreasing CDK4 kinase activity. However, CDK4 kinase activity was not altered in keratinocytes from K5Cdk2 Tg mice, showing comparable kinase activity against pRb substrate as in Wt keratinocytes (Figure 3B). Altogether these results suggest that elevated p21Cip1 levels may act as a compensatory mechanism in response to the forced expression of CDK2 to maintain keratinocyte homeostasis. However, p21Cip1 levels do not completely inhibit transgenic CDK2 activity, which remains 1.7-fold higher than Wt mice.Figure 3Cell-cycle regulatory protein expression, CDK kinase activities, and complex formation in K5CDK2 epidermis. A: Epidermal lysates from Wt and K5CDK2 mice were probed with antibodies for CDKs, cyclins, CKIs, total and ser15-phopho-p53, pRb, and actin as loading control. B: CDK in vitro kinase assays for CDK2 and CDK4 using H1 and pRb peptides as substrates. Epidermal lysate from K5CDK2 (Tg) and Wt littermates were immunoprecipitated with antibodies against CDK2 (IP CDK2), CDK4 (IP CDK4), or normal rabbit IgG (Cont.) and incubated with respective substrates, H1 or pRb. C: Fresh epidermal lysates from K5CDK2 Tg mice and Wt littermates were immunoprecipitated with polyclonal antibodies against cyclin E (IP Cyc E) and CDK2 (IP-CDK2) and immunoblotted with polyclonal antibody for CDK2 (IB-CDK2) and p21Cip1 (IB-p21).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Generation of K5Cdk4D158N Tg Mice and Gross Analysis of Mouse EpidermisOur previous studies showed that CDK4 overexpression leads to increased epidermal proliferation and tumorigenesis attributable in part to the increased kinase activity of CDK2.7Miliani de Marval P Gimenez-Conti I LaCava M Martinez L Conti C Rodriguez-Puebla M Transgenic expression of CDK4 results in epidermal hyperplasia and severe dermal fibrosis.Am J Pathol. 2001; 159: 369-379Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Miliani de}, number={2}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Macias, Everardo and Marval, Paula L. Miliani and De Siervi, Adriana and Conti, Claudio J. and Senderowicz, Adrian M. and Rodriguez-Puebla, Marcelo L.}, year={2008}, month={Aug}, pages={526–535} }
 @article{macias_marva_senderowicz_cullen_rodriguez-puebla_2008, title={Expression of CDK4 or CDK2 in mouse oral cavity is retained in adult pituitary with distinct effects on tumorigenesis}, volume={68}, ISSN={["0008-5472"]}, DOI={10.1158/0008-5472.CAN-07-2461}, abstractNote={Abstract The keratin 5 (K5) promoter drives transgenic expression to the basal cell layer of stratified epithelia. Surprisingly, analysis of K5CDK4 and K5CDK2 transgenic mouse embryos showed CDK4 and CDK2 expression not only in the expected tissues, but also in the adenohypophysis. This organ is derived from an upwards growth of the primitive oropharnyx, a K5-expressing tissue. We show that transgenic expression of CDKs in the embryonic oral ectoderm is specifically retained in undifferentiated cells from the pars intermedia of the adenohypophysis. Interestingly, we found that K5CDK4 mice show a decreased number of pituitary stem cells, even though CDK4 is not expressed in the stem cells but in transit-amplifying (TA)–like cells. Interestingly, CDK4-expressing cells, but not CDK2-expressing cells, strongly synergize with lack of p27Kip1 to generate pituitary carcinomas that appear with shortened latency and are drastically more aggressive than those arising in p27−/− mice. Thus, we show that deregulation of CDK expression in the primitive oral epithelium plays a unique function, providing a selective advantage that gives rise to transgene-positive TA-like pituitary cells. Furthermore, retention of CDK4 in these TA-like pituitary cells synergizes with loss of p27Kip1 to induce pituitary adenocarcinomas. This model suggests that forced expression of CDK4 sensitizes cells and synergizes with a second change resulting in tumor development. [Cancer Res 2008;68(1):162–71]}, number={1}, journal={CANCER RESEARCH}, author={Macias, Everardo and Marva, Paula L. Miliani and Senderowicz, Adrian and Cullen, John and Rodriguez-Puebla, Marcelo L.}, year={2008}, month={Jan}, pages={162–171} }
 @article{macias_kim_marval_klein-szanto_rodriguez-puebla_2007, title={Cdk2 deficiency decreases ras/CDK4-dependent malignant progression, but not myc-induced tumorigenesis}, volume={67}, ISSN={["1538-7445"]}, DOI={10.1158/0008-5472.CAN-07-2119}, abstractNote={Abstract We have previously shown that forced expression of CDK4 in mouse skin (K5CDK4 mice) results in increased susceptibility to squamous cell carcinoma (SCC) development in a chemical carcinogenesis protocol. This protocol induces skin papilloma development, causing a selection of cells bearing activating Ha-ras mutations. We have also shown that myc-induced epidermal proliferation and oral tumorigenesis (K5Myc mice) depends on CDK4 expression. Biochemical analysis of K5CDK4 and K5Myc epidermis as well as skin tumors showed that keratinocyte proliferation is mediated by CDK4 sequestration of p27Kip1 and p21Cip1, and activation of CDK2. Here, we studied the role of CDK2 in epithelial tumorigenesis. In normal skin, loss of CDK2 rescues CDK4-induced, but not myc-induced epidermal hyperproliferation. Ablation of CDK2 in K5CDK4 mice results in decreased incidences and multiplicity of skin tumors as well as malignant progression to SCC. Histopathologic analysis showed that K5CDK4 tumors are drastically more aggressive than K5CDK4/CDK2−/− tumors. On the other hand, we show that CDK2 is dispensable for myc-induced tumorigenesis. In contrast to our previous report of K5Myc/CDK4−/−, K5Myc/CDK2−/− mice developed oral tumors with the same frequency as K5Myc mice. Overall, we have established that ras-induced tumors are more susceptible to CDK2 ablation than myc-induced tumors, suggesting that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the oncogenic pathway involved. [Cancer Res 2007;67(20):9713–20]}, number={20}, journal={CANCER RESEARCH}, author={Macias, Everardo and Kim, Yongbaek and Marval, Paula L. Miliani and Klein-Szanto, Andres and Rodriguez-Puebla, Marcelo L.}, year={2007}, month={Oct}, pages={9713–9720} }
 @article{rojas_cadenas_lin_benavides_conti_rodriguez-puebla_2007, title={Cyclin D2 and cyclin D3 play opposite roles in mouse skin carcinogenesis}, volume={26}, DOI={10.1038/sj.onc.1209970}, abstractNote={D-type cyclins are components of the cell-cycle engine that link cell signaling pathways and passage throughout G1 phase. We previously described the effects of overexpression cyclin D1, D2 or D3 in mouse epidermis and tumor development. We now asked whether cyclin D2 and/or cyclin D3 play a relevant role in ras-dependent tumorigenesis. Here, we described the effect of cyclin D3 and cyclin D2 overexpression in mouse skin tumor development. Notably, overexpression of cyclin D3 results in reduced tumor development and malignant progression to squamous cell carcinomas (SCC). Biochemical analysis of keratinocytes shows that overexpression of cyclin D3 results in strong reduction of cyclin D2 and its associated kinase activity. Furthermore, we found that reinstatement of cyclin D2 level in the cyclin D3/cyclin D2 bigenic mice results in a complete reversion of the inhibitory action of cyclin D3. Supporting these results, ablation of cyclin D2 results in reduced tumorigenesis and malignant progression. On the other hand, overexpression of cyclin D2 results in an increased number of papillomas and malignant progression. We conclude that cyclin D3 and cyclin D2 play opposite roles in mouse skin tumor development and that the suppressive activity of cyclin D3 is associated with cyclin D2 downregulation.}, number={12}, journal={Oncogene}, author={Rojas, P. and Cadenas, M. B. and Lin, P. C. and Benavides, F. and Conti, C. J. and Rodriguez-Puebla, M. L.}, year={2007}, pages={1723–1730} }
 @article{chien_rabin_macias_marval_garrison_orthel_rodriguez-puebla_fero_2006, title={Genetic mosaics reveal both cell-autonomous and cell-nonautonomous function of murine p27(Kip1)}, volume={103}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0509514103}, abstractNote={Loss of the cyclin-dependent kinase inhibitor p27 Kip1 leads to an overall increase in animal growth, pituitary tumors, and hyperplasia of hematopoietic organs, yet it is unknown whether all cells function autonomously in response to p27 Kip1 activity or whether certain cells take cues from their neighbors. In addition, there is currently no genetic evidence that tumor suppression by p27 Kip1 is cell-autonomous because biallelic gene inactivation is absent from tumors arising in p27 Kip1 hemizygous mice. We have addressed these questions with tissue-specific targeted mouse mutants and radiation chimeras. Our results indicate that the suppression of pars intermedia pituitary tumors by p27 Kip1 is cell-autonomous and does not contribute to overgrowth or infertility phenotypes. In contrast, suppression of spleen growth and hematopoietic progenitor expansion is a consequence of p27 Kip1 function external to the hematopoietic compartment. Likewise, p27 Kip1 suppresses thymocyte hyperplasia through a cell-nonautonomous mechanism. The interaction of p27 Kip1 loss with epithelial cell-specific cyclin-dependent kinase 4 overexpression identifies the thymic epithelium as a relevant site of p27 Kip1 activity for the regulation of thymus growth.}, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Chien, WM and Rabin, S and Macias, E and Marval, PLM and Garrison, K and Orthel, J and Rodriguez-Puebla, M and Fero, ML}, year={2006}, month={Mar}, pages={4122–4127} }
 @article{marval_macias_conti_rodriguez-puebla_2004, title={Enhanced malignant tumorigenesis in Cdk4 transgenic mice}, volume={23}, ISSN={["1476-5594"]}, DOI={10.1038/sj.onc.1207309}, abstractNote={In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1, suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.}, number={10}, journal={ONCOGENE}, author={Marval, PLM and Macias, E and Conti, CJ and Rodriguez-Puebla, ML}, year={2004}, month={Mar}, pages={1863–1873} }
 @article{rodriguez-puebla_marval_lacava_moons_kiyokawa_conti_2002, title={cdk4 deficiency inhibits skin tumor development but does not affect normal keratinocyte proliferation}, volume={161}, ISSN={["1525-2191"]}, DOI={10.1016/S0002-9440(10)64196-X}, abstractNote={Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue.}, number={2}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Rodriguez-Puebla, ML and Marval, PLM and LaCava, M and Moons, DS and Kiyokawa, H and Conti, CJ}, year={2002}, month={Aug}, pages={405–411} }