Proto-oncogenes encode proteins with three main sites of action: the cell-surface membrane, the cytoplasm and the nucleus. Although the exact biochemical function of most proto-oncogene products is not understood, several of them are known to be involved in signal transduction. A role in gene regulation through DNA binding has been suggested for a recently isolated member of the group of oncogenes acting at the nucleus, v-jun. The C-terminus of the putative v-jun-encoded protein is similar in sequence to the C-terminus of the yeast transcriptional activator GCN4 (refs 8, 9), which forms its minimal DNA-binding domain. GCN4 binds to specific sites whose consensus sequence is highly similar to the recognition sequence of the mammalian transcriptional activator AP-1 (refs 12, 13). Like GCN4, AP-1 binds to promoter elements of specific genes and activates their transcription. Because of the similarity between the recognition sites for GCN4 and AP-1, we examined the possibility that AP-1 could be the product of the c-jun proto-oncogene. The experimental results reported here indicate that the JUN oncoprotein is a sequence-specific transcriptional activator similar to AP-1.

Genistein (4 ¶,5,7-trihydroxyisoflavone) is the most abundant isoflavone found in the soybean. The effects of genistein on various cancer cell lines have been extensively studied but the precise molecular mechanisms are not known. We report here the epigenetic mechanism of the action of genistein on androgen-sensitive (LNCaP) and androgen-insensitive (DuPro) human prostate cancer cell lines. Genistein induced the expression of tumor suppressor genes p21 (WAF1/CIP1/ KIP1) and p16 (INK4a) with a concomitant decrease in cyclins. There was a G 0 -G 1 cell cycle arrest in LNCaP cells and a G 2 -M arrest in DuPro cells after genistein treatment. Genistein also induced apoptosis in DuPro cells. DNA methylation analysis revealed the absence of p21 promoter methylation in both cell lines. The effect of genistein on chromatin remodeling has not been previously reported. We found that genistein increased acetylated histones 3, 4, and H3/K4 at the p21 and p16 transcription start sites. Furthermore, we found that genistein treatment also increased the expression of histone acetyl transferases that function in transcriptional activation. This is the first report on epigenetic regulation of various genes by genistein through chromatin remodeling in prostate cancer. Altogether, our data provide new insights into the epigenetic mechanism of the action of genistein that may contribute to the chemopreventive activity of this dietary isoflavone and have important implications for epigenetic therapy. [Cancer Res 2008;68(8):2736-44]

Monoclonal antibodies toward rabbit liver cytochrome P-450 isozyme 6 (P-450 6) were used to identify several recombinant clones from a pBR322 cDNA library that express 13-lactamase-P-450 6 hybrid proteins. The nucleic acid sequence and predicted amino acid sequence of a rabbit P-450 6 cDNA shows a high degree of homology with rat P-450c and mouse P1-450. When used as a probe to rescreen the library, the P-450 6 cDNA hybridized to several heterologous classes of cDNAs. One such class was shown to encode. P-450 4 by comparison of its predicted amino acid sequence to amino acid sequences of cysteine-containing tryptic peptides derived from P-450 4. DNA sequence analysis of a cDNA clone belonging to a third class demonstrated that it contained a 131-base-pair intervening sequencing when compared to the cDNA coding for P-450 6. This sequence corresponds in location to intron E of the rat P-450c gene. Blot-hybridization analysis demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramatically induced P-450 4 and 6 mRNAs, which differ in size. The sizes of these mRNAs differ from their analogs in the mouse as a result of divergence in the 3' untranslated portions of the mRNAs.The treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) results in the induction of two major cytochrome P-450 isozymes, 4 and 6 (P-450 4 and 6) (1). The induction of P-450 6 occurs in several tissues (2) and at various ages (1). This P-450 is analogous to rat P-450c (3, 4) and mouse P1-450 (5) in both its pattern of expression and catalytic function. In contrast, the induction by TCDD of rabbit P-450 4, which is analogous to rat P-450d (3, 6, 7) and mouse P3-450 (5), is restricted to the liver of mature animals (1, 2). Sequence analysis of cDNAs encoding the rat (8, 9) and mouse (10, 11)-cytochromes P-450 indicates extensive amino acid sequence homology (>90%) between rat P-450c and mouse P1-450 and between rat P-450d and mouse P3-450 (10) as well as regions of substantial nucleic acid sequence homology. In addition, seven exons comprise the gene encoding rat P-450c (12), and similar exogenic sequences are evident for the mouse P1-450 gene (11).We have extended the characterization of this P-450 gene family to P-450 6 and P-450 4 of the rabbit. Using '25I-labeled monoclonal antibodies directed toward P-450 6 to screen a pBR322 expression library, we have identified a cDNA clone encoding P-450 6. This cDNA was used in turn to detect additional clones by colony hybridization permitting us to identify cDNAs encoding P-450 4. We also have isolated and sequenced a cDNA to P-450 6 that appears to have retained an unspliced intron, thus allowing a direct comparison between the structure and location of this region and that of the rat P-450c gene. MATERIALS AND METHODSProduction and Purification of Antibodies. Monoclonal antibodies were generated toward a purified preparation of P-450 6 in a fashion similar to that already described for P-450 1 (13). Each antibody was purified from serum-free cell culture supernatants by ...

Human cytochrome P-450s structurally related to 2,3,7,8-tetrachloro-p-dioxin (TCDD)-inducible rabbit P-450 4 and 6 have been cloned from a human liver cDNA library. The human P-450 4 cDNA clone, hpP-450 4, and the human P-450 6 cDNA clone, hpP-450 6, were identified by hybridization to rabbit P-450 4 and P-450 6 cDNAs, respectively. DNA sequence analysis demonstrates that hpP-450 4 is 83% and 75% homologous to rabbit P-450 4 and P-450 6 mRNAs, respectively, whereas hpP-450 6 is 79% and 72% homologous to rabbit P-450 6 and P-450 4, respectively. A comparison of DNA sequence of the two human cDNA clones shows they are 80% homologous. This is similar to the homology found between the cDNA sequences of rabbit P-450 4 and P-450 6. Northern blot analysis has shown that the human P-450 4 mRNA is approximately 3000 bases, while the human P-450 6 mRNA is 2600 bases in length. Clone hpP-450 4 preferentially hybridizes to TCDD-inducible rabbit P-450 4 and mouse P3-450 mRNAs, whereas hpP-450 6 preferentially hybridizes to TCDD-inducible rabbit P-450 6 and mouse P1-450 mRNAs. Both hpP-450 4 and hpP-450 6 recognize different genomic fragments, indicating that each is encoded by different genes. These results indicate the existence of at least two P-450 genes in humans that are highly homologous to the TCDD-inducible P-450s in rabbits and mice.

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