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The effect of tumor promoters on the in vivo expression of tumor-associated, overexpressed genes was studied. Two of the tumor-associated genes, mal 1 and mal 2 were overexpressed already in the benign papilloma stage of mouse skin carcinogenesis. Overexpression of the other two genes, mal 4 and transin, was specific for the malignant state. Treatment of the normal adult epidermis with the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the incomplete, second-stage promoter 12-O-retinylphorbol-13-acetate (RPA) enhanced transiently the expression of the mal sequences and transin. Fractionation of the adult epidermis on Percoll gradients into basal cells and differentiated, suprabasal cells showed that expression of the mal sequences was enhanced by TPA in both basal and differentiated cells. In contrast, transin expression, which was undetectable in cells of the normal epidermis, was enhanced in only the basal cells of the TPA-treated epidermis. The non-tumor-promoting hyperplastic agent, ethylphenyl propiolate (EPP), applied to the skin at a hyperplastic dose level did not enhance the expression of the mal 4 or transin sequences in the epidermis and had only a slight enhancing effect on the levels of mal 1 and mal 2 transcripts in the epidermis. Our results suggest that the observed stimulated expression of mal 1 and mal 2 is related to proliferative processes, whereas stimulated expression of mal 4 and transin reflects tumor-promoter-specific responses.

FR3T3 rat embryo fibroblast cells express preproenkephalin mRNA after transformation by polyoma virus middle T or Ha-ras oncogenes. This effect was not seen in another rat embryo fibroblast cell line (Rat-1) or in FR3T3 cells transformed by Rous sarcoma virus, bovine papilloma virus type I or SV40. The elevation in preproenkephalin mRNA levels is thus cell specific and oncogene specific. These results contrast with those obtained for transin mRNA, which was observed in both Rat-1 and FR3T3 cells transformed by a number of different oncogenes. The expression of transin RNA correlated with the expression of the transformed phenotype. We suggest that genes induced by oncogenes in a given cell will fall into two classes: those linked to the expression of the transformed phenotype (expression induced by all oncogenes conferring this phenotype) and those induced as a consequence of activation of a specific cellular signaling system (expression induced by a subset of oncogenes linked to the signaling system in question). This system may prove useful in distinguishing oncogene-induced events that are related to cellular transformation from those that are secondary to eliciting the transformed phenotype.

The transin gene is induced by oncogenes and epidermal growth factor (EGF). We report here the isolation of a related gene (transin-2 gene). The structures of these genes are very similar. Indeed, a stretch of 428 nucleotides of the transin gene containing both exon and intron sequences is 98% conserved in the transin-2 gene. However, the putative promoter regions of the genes show little sequence homology, apart from a short element related to a sequence involved in control of transcription by cyclic AMP or a tumour promoter. Expression of the transin-2 gene, unlike that of the transin gene, is not induced by EGF, dibutyryl cyclic AMP or cytochalasin D. Nevertheless, transin-2 RNA is expressed in several transformed rat embryo fibroblast cell lines, and can be induced by a tumour promoter. The proteins transin and transin-2 are approximately 71% homologous in sequence. Both proteins show significant sequence homology with two connective tissue degrading metalloproteases. These homologies raise the possibility that expression of transin and transin-2 in transformed cells might play a role in tumour invasion.

Transin mRNA encodes a secreted metalloprotease which is transcriptionally induced in Rat-1 cells by epidermal growth factor (EGF) and a number of oncogenes. A role for transin in tumor progression is suggested by its overexpression in malignant and metastatic tumors compared to their benign counterparts. In an effort to elucidate mechanisms by which elevated transin expression may be inhibited, it has been determined that both transforming growth factor type beta 1 (TGF beta 1) and increased levels of intracellular cyclic 5'-adenosine monophosphate (cAMP) inhibit EGF and oncogene induction of transin mRNA. The inhibition of transin mRNA occurred at the level of transcription as demonstrated by nuclear run-on assays. EGF binding studies in Rat-1 cells showed no significant effect of cAMP or TGF beta 1 on EGF receptor number or affinity. We have also examined the effects of cAMP and TGF beta 1 on oncogene-induced transin using Rat-1 cells transformed by temperature-sensitive mutants of v-src and K-ras oncogenes. Both inhibitors prevented the induction of transin RNA as well as decreased the levels of transin once elevated at the permissive temperature. Despite the similarities in the actions of TGF beta 1 and cAMP on transin gene expression, TGF beta 1 treatment did not significantly elevate intracellular cAMP levels, thus making it unlikely that cAMP is a second messenger system for TGF beta 1 action. These studies suggest that the inhibitory effects of cAMP and TGF beta 1 occur by distinct pathways at the level of gene regulation.

We have isolated and sequenced a cloned cDNA corresponding to an mRNA present in significantly higher levels in rat cells transformed by polyoma virus, Rous sarcoma virus, and the cellular oncogene H-ras than in the normal parental cell lines. The mRNA transcript is also rapidly induced by the polypeptide growth factor epidermal growth factor, providing a new link between oncogenes and growth factors. Both the growth factor and the oncogenes control expression of the corresponding gene at the transcriptional level. Our results point to the existence of intracellular mechanisms that are common to the action of both growth factors and oncogenes.

Various oncogenes or epidermal growth factor (EGF) induce transcription of a 1.9-kilobase RNA (transin RNA) in rat fibroblasts. The induction by EGF can be blocked by cycloheximide. Thus the response of the transin gene to EGF appears to require de novo protein synthesis. Transin RNA induction is specific to EGF, as neither insulin, platelet-derived growth factor, fibroblast growth factor, nor transforming growth factor beta could elicit the same response. However, transforming growth factor beta could block the EGF induction of transin RNA. Whereas the calcium ionophore A23187 and the tumor promoter TPA, either alone or administered together, did not increase transin RNA levels, TPA could synergise with a serum factor to effect such an increase. Dibutyryl cyclic AMP also induced transin RNA. Treatment of cells with the microfilament-disrupting agent cytochalasin B, but not the microtubule-disrupting agent colcemid, resulted in an increase in transin RNA levels, suggesting a role for the cytoskeleton in control of transin gene expression. The transin RNA does not contain repeated sequences and appears to be encoded by a single-copy gene. The protein sequence encoded by the last four exons of the transin gene shows some homology to two regions of the heme-binding protein hemopexin.