TY - CHAP TI - Introduction to Biochemical and Molecular Methods in Toxicology AU - Hodgson, Ernest AU - Leblanc, Gerald A. AU - Meyer, Sharon A. AU - Smart, Robert C. T2 - A Textbook of Modern Toxicology A2 - Hodgson, E. AB - 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. PY - 2004/3/8/ DO - 10.1002/0471646776.ch2 SP - 13-22 PB - John Wiley & Sons, Inc. UR - http://dx.doi.org/10.1002/0471646776.ch2 ER - TY - CHAP TI - Chemical Carcinogenesis AU - Smart, Robert C. T2 - A Textbook of Modern Toxicology A2 - Hodgson, E. AB - 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. PY - 2004/3/8/ DO - 10.1002/0471646776.ch12 SP - 225-250 PB - John Wiley & Sons, Inc. UR - http://dx.doi.org/10.1002/0471646776.ch12 ER - TY - JOUR TI - C/EBP alpha is a DNA damage-inducible p53-regulated mediator of the G(1) checkpoint in keratinocytes AU - Yoon, K AU - Smart, RC T2 - MOLECULAR AND CELLULAR BIOLOGY AB - The basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha), 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/EBPalpha 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/EBPalpha mRNA, producing a >70-fold increase in C/EBPalpha protein levels. N-Methyl-N'-nitro-N-nitrosoguanidine, etoposide, and bleomycin also induced C/EBPalpha. UVB-induced C/EBPalpha 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/EBPalpha and p53. UVB irradiation of p53-null or mutant p53-containing keratinocytes failed to induce C/EBPalpha. UVB irradiation of C/EBPalpha knockdown keratinocytes displayed a greatly diminished DNA damage G(1) checkpoint, and this was associated with increased sensitivity to UVB-induced apoptosis. Our results uncover a novel role for C/EBPalpha as a p53-regulated DNA damage-inducible gene that has a critical function in the DNA damage G(1) checkpoint response in keratinocytes. DA - 2004/12// PY - 2004/12// DO - 10.1128/MCB.24.24.10650-10660.2004 VL - 24 IS - 24 SP - 10650-10660 SN - 1098-5549 ER - TY - JOUR TI - Cell cycle-dependent phosphorylation of C/EBP beta mediates oncogenic cooperativity between C/EBP beta and H-Ras(V12) AU - Shuman, JD AU - Sebastian, T AU - Kaldis, P AU - Copeland, TD AU - Zhu, SY AU - Smart, RC AU - Johnson, PF T2 - MOLECULAR AND CELLULAR BIOLOGY AB - CCAAT/enhancer binding protein beta (C/EBPbeta) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPbeta 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/EBPbeta 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/EBPbeta 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/EBPbeta, 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/EBPbeta as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPbeta on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells. DA - 2004/9// PY - 2004/9// DO - 10.1128/MCB.24.17.7380-7391.2004 VL - 24 IS - 17 SP - 7380-7391 SN - 1098-5549 ER -