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Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Male F344 rats were fed N[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for up to 4 wk, then given the basal diet with or without 5% sodium saccharin for up to 100 wk. In a previous study, we demonstrated point mutations in codons 12 and 61 of Ha-ras gene among eleven transitional cell carcinomas (TCC), one undifferentiated carcinoma, and two sarcomas of the urinary bladder (Mol Carcinogen 3:210-215, 1990). In this study, Ha-ras, Ki-ras, and N-ras sequences were examined by polymerase chain reaction (PCR) and direct DNA sequencing. The results confirm the point mutation in codon 61 (CAA to CGA in 5 TCCs and to CTA in one TCC) of the Ha-ras gene. Mutation at codon 12 was not confirmed. No mutation was found in the Ki-ras gene. Sequences of the N-ras gene exons 1 and 2 were determined, and no mutations was detected. These results suggest the involvement of activated Ha-ras gene, but not Ki-N or N-ras gene, in rat urinary bladder carcinogenesis induced by FANFT. Subsequent sodium saccharin administration did not affect the changes in Ha-ras gene.

The Ha-ras oncogene has been shown to be point-mutated and overexpressed in papillomas induced by the two-stage skin tumorigenesis regimen of 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Glucocorticoids inhibit mouse skin tumorigenesis when applied with the initiation agent or with the promoting agent. We have extended these studies to evaluate whether dexamethasone (Dex) treatment could inhibit development of already established tumors. An additional objective of this study was to investigate whether glucocorticoids directly inhibit Ha-ras gene expression at the level of transcription during skin tumorigenesis. In this report we demonstrate that topical Dex treatments significantly suppressed the formation of additional tumors relative to the acetone control group. However, Northern blot analysis of total RNA isolated from representative tumors during a series of sequential weeks of promotion indicated that Dex did not have a direct effect on Ha-ras steady-state mRNA levels despite the decrease in additional tumor numbers in the Dex-treated groups. We also investigated short-term effects of Dex on endogenous Ha-ras expression in normal mouse epidermis. Topical Dex administration had no effect on endogenous Ha-ras steady-state mRNA levels in normal skin after 2 or 24 h. To ensure that endogenous corticosterone levels in the SENCAR mouse were not influencing our results, Ha-ras mRNA levels in epidermis from SENCAR mice adrenalectomized 48 h prior to being killed were compared to Ha-ras levels in normal epidermis by Northern blot analysis. The data from this analysis revealed that bilateral adrenalectomy had no effect on Ha-ras steady-state mRNA levels in epidermis compared to ras levels in normal mouse epidermis. In summary, our results demonstrate that although Dex can inhibit further tumor development in DMBA/TPA-treated mouse epidermis, it does not do so by directly effecting Ha-ras gene expression in mouse epidermis.

During two-stage mouse skin tumorigenesis, the mouse c-Ha-ras oncogene undergoes activation by point mutation after initiation with polycyclic aromatic hydrocarbons. Furthermore, initiated epidermal cells containing an activated Ha-ras oncogene have been shown to be resistant to calcium-induced terminal differentiation. However, the relationship between Ha-ras expression and the differentiation process is not well understood in either normal or initiated cells. Before attempting to explore the role of Ha-ras expression in epidermal differentiation during tumorigenesis, we felt that investigation of Ha-ras gene expression in normal primary epidermal cells undergoing differentiation was warranted, since primary cultures of normal newborn and adult keratinocytes presumably contain the stem cells from which skin tumors arise. In the present studies, northern blot analysis was used to compare Ha-ras expression in normal newborn and adult epidermal cells undergoing differentiation. Steady-state levels of Ha-ras mRNA remained unchanged in primary cultures of normal adult epidermal cells during calcium-induced differentiation, whereas steady-state levels of Ha-ras transcripts decreased during calcium-induced differentiation in primary newborn epidermal cells. Differentiation was induced by switching the adult and newborn keratinocytes from medium containing 0.05 mM Ca2+ to medium containing one of three different calcium concentrations (0.15, 0.5, or 1.2 mM Ca2+). The decrease in Ha-ras mRNA levels observed during differentiation in newborn keratinocytes occurred as an intermediate event in the differentiation process, was specific for the Ha-ras gene, and was not due to a general decrease in transcriptional activity during differentiation. Characteristic patterns of keratin 14 gene expression and cornified envelope formation were observed, verifying that the differentiation process had been induced in both the primary adult and newborn epidermal cells. That adult keratinocytes are resistant to the differentiation-induced reduction in Ha-ras mRNA expression observed in newborn keratinocytes may explain the difference in in vivo tumorigenic potentials of newborn and adult skin.

Male F344 rats were fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazoly]formamide for 6 weeks and then fed 3% or 5% sodium saccharin, 5% sodium ascorbate, 3.12% calcium saccharin, 1.34% sodium chloride, 5.2% calcium saccharin plus 1.34% sodium chloride, or basal diet alone for 72 weeks. Protein and DNA were extracted from 89 bladder tumors [87 transitional cell carcinomas (TCC), 1 papilloma, and 1 sarcoma] from 86 rats p21 expression was examined by Western blotting using a monoclonal antibody against p21 (NCC-RAS-004). H-ras mutations in exons 1 and 2 were examined by direct sequencing of DNA amplified by polymerase chain reaction. Sequencing results demonstrated mutations at codon 61 (CAA to CGA in 15 TCCs; CAA to CTA in 2 TCCs), at codon 12 (GGA to TGG in 1 TCC), and at codon 13 (GGC to GTC in 3 TCCs). Mutations at codon 61 were confirmed by faster mobility of the p21 band in Western blots. The level of p21 expression varied among samples, but many TCCs appeared to express more p21 than controls. The overall incidence of H-ras mutations was 24.4% (21 of 86 rats). The type of chemical used for the promoting phase had essentially no effect on H-ras mutation, suggesting that the effects observed were related to FANFT administration. The frequency of H-ras mutation in each group was negatively related to the incidence of carcinoma (r = -0.85; P less than 0.01). Two groups of tumors (with or without the mutated ras gene) were compared for tumor size (reflected by the bladder weight), histological grading, and the presence of invasion. The size of tumors with mutated ras was significantly smaller than those without mutated ras. There was no difference in the histological grading between the two groups. Although not statistically significant, histological invasion was more frequently observed in tumors with mutated ras (14.3%) than in tumors without mutation (3.1%).

The mouse Ha-ras oncogene is activated by point mutation and overexpressed in developing papillomas during two-stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha-ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5' upstream region of the mouse Ha-ras gene so as to characterize various cis-regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF-1 factor. In addition, we located a 13-base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha-ras 5' upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two-stage skin tumorigenesis. Transient gene expression assays carried out after 48-72 h indicated that a 2.3-kb Ha-ras 5' fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha-ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha-ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes.

Previous results in a number of laboratories have demonstrated that epidermal papillomas and carcinomas induced by the two-stage protocol of initiation and promotion contain a point mutation in the 61st codon of the c-Ha-ras oncogene when the initiating agent used is 7,12-dimethylbenz[a]anthracene (DMBA). In the present report, we have analyzed DNA purified from 'spontaneously initiated' papillomas and carcinomas induced in SENCAR mouse epidermis after repetitive treatments with a tumor-promoting agent. Southern blot hybridization studies of tumor DNA digested with diagnostic restriction endonucleases demonstrated that seven of nine papillomas and carcinomas contained a point mutation in the 61st codon of one allele of the c-Ha-ras oncogene. The implications of our findings with respect to the role which a point-mutated Ha-ras proto-oncogene plays in initiation of skin tumorigenesis are discussed.

Glucocorticoids regulate transcription of specific genes through the interaction of glucocorticoid hormone receptor complexes with DNA binding sites called glucocorticoid response elements (GREs). The GRE consensus sequence has been defined to be the imperfect palindromic sequence 5'-GGTACANNNTGTTCT-3', the most highly conserved portion being the 5'-TGTTCT-3' hexamer. We have identified 5 potential GREs in the 5' upstream noncoding region of the mouse c-Ha-ras oncogene, two with the same hexanucleotide sequence and three with a similar sequence. When subcloned fragments of the mouse c-Ha-ras 5' upstream region (containing the 2 hexamer GREs of exact homology) were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HeLa cells, CAT expression driven from the ras promoter was induced up to 3-fold in the presence of dexamethasone. To determine whether the 5' upstream region of the mouse Ha-ras gene was capable of specifically interacting with the glucocorticoid receptor complex, we performed Southwestern blot analysis showing that cloned DNA fragments from the 5' upstream region of the mouse c-Ha-ras gene were able to bind a 97 kDa protein in whole cell extracts from both primary SENCAR mouse epidermal cells and HeLa G cells. Immunodepletion of the epidermal cell extract with a monoclonal antibody to the glucocorticoid receptor verified that the 97 kDa protein bound by the Ha-ras 5' region was indeed the glucocorticoid receptor protein. Our results demonstrate that the upstream noncoding region of the mouse c-Ha-ras gene binds the glucocorticoid receptor. Furthermore, the presence of glucocorticoids enhances the transcription of the mouse Ha-ras promoter region in a transient gene expression assay.