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cDNA clones representing genes whose expression is modulated by treatment with mitogens and tumor promoters were isolated and characterized. TPA-S1 corresponds to an mRNA species whose abundance was increased markedly within 1 h of exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), and TPA-R1 represents an mRNA that was decreased in TPA-treated cells. The induction of TPA-S1 was blocked by actinomycin D but was not affected by cycloheximide, and it was specific for phorbol esters with tumor-promoting activity. The role of protein kinase C in the induction of TPA-S1 is supported by the following lines of evidence. (i) Agents that activated protein kinase C (TPA, platelet-derived growth factor, and diacylglycerol) also increased TPA-S1 mRNA levels. (ii) A potent PKC inhibitor blocked the induction of TPA-S1. (iii) Down-regulation of PKC activity, by treatment of cells with TPA for 24 h, resulted in a loss of responsiveness to TPA-S1 induction by subsequent TPA treatment. DNA sequence analysis of TPA-S1 predicts a cysteine-rich, secreted protein with a molecular weight of 22.6 X 10(3) that exhibits homology with sequences representing a protein with human erythroid-potentiating activity and protease inhibitory activity.

Summary We have generated a series of rat fibroblast cell linesthat stably overexpress a full-length cDNA encoding the β1 form of protein kinase C (PKC). These cell lines contain a 20-to 53-fold increase in PKC activity and exhibit dramatically enhanced morphologic changes following exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). They grow to a high saturation density in monolayer cultures and, when maintained at postconfluence, develop small, dense foci. In contrast to control cells, which display complete anchorage dependence, PKC-overproducing cells form small colonies in soft agar in the absence of TPA and large colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer multiple growth abnormalities in rat fibroblasts. These results provide direct evidence that PKC plays a critical role in growth control and that it mediates several of the cellular effects of the phorbol ester tumor promoters. They also suggest that the activation of PKC may be of central importance in the process of multistage carcinogenesis.

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

RNA sequences homologous to the Long Terminal Repeat (LTR) sequence of Moloney Murine Leukemia Virus proviral DNA are expressed in murine squamous cell carcinomas of the skin induced by chemical carcinogens. These transcripts range in size from 8.2 to less than 2.4 kb but their size profile varies between individual tumors. These RNAs are not detected in the poly A+ RNA fraction obtained from the epidermis of control mice or carcinogen induced skin papillomas. The poly A+ RNAs from the livers and spleens of some of the mice with skin carcinomas also revealed LTR related sequences, whereas these RNAs were not detected in the livers and spleens of control mice or of carcinogen-treated mice that did not develop carcinomas. Thus, chemical carcinogenesis in mouse skin is associated with constitutive expression of endogenous retrovirus related sequences in the carcinomas as well as in certain apparently normal host tissues.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has highly pleiotropic effects on cells in culture and on tissues in vivo, including effects on protein kinase C (PKC) activation and gene expression. In order to determine the mechanism of activation of gene transcription by TPA, DNA sequences whose transcription is modulated in cells undergoing a mitogenic response to TPA were isolated by differential screening of a cDNA library from TPA-treated cells. TPA-S1 corresponds to an mRNA species whose abundance is increased within 1 hr of exposure of quiescent C3H 10T1/2 mouse embryo fibroblasts. TPA-R1 corresponds to an mRNA species whose abundance is decreased in TPA-treated cells. The induction of TPA-S1 is blocked by actinomycin D and is specific for phorbol esters with tumor-promoting activity. The transcription of this sequence is not induced by cycloheximide, nor is there an enhancement of the TPA response. Several lines of evidence demonstrate that PKC activation plays a critical role in the regulation of TPA-S1 expression. The nucleotide and predicted amino acid sequence of TPA-S1 exhibits homology with sequences representing a peptide with erythroid-potentiating activity, a metalloproteinase inhibitor protein, and a murine protein with beta-interferon-like activity. The role of TPA-S1 in tumor promotion is suggested by the expression of this sequence in mouse skin carcinomas induced by dimethyl-benzanthracene-TPA treatment, but not in papillomas or in control tissue. The consideration of signal transduction pathways may be useful in the design of short-term risk assessment assays for agents that act as tumor promoters. Images FIGURE 2. FIGURE 3. FIGURE 4.

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.

Previous reports on retroviral vectors have shown them to be useful for transferring genes into animal cells. Genes placed under the retroviral long terminal repeat (LTR) act as dominant loci in recipient cells and can permanently alter their genotype and phenotype. Previous reports have shown that recombinant retroviruses containing genomic sequences with both introns and exons display a high frequency of deletion and abnormal kinetics of splicing of intron sequences. We report here our findings when a 2.9-kb fragment containing the entire human insulin gene was inserted into a Moloney-derived retroviral vector in the same transcriptional orientation as the LTRs. RNA transcripts synthesized in cells containing such constructs remain unspliced, as assessed by both RNA blot analysis and S1 mapping. Ten subclones derived following viral passage showed no splicing, and failure to splice was observed regardless of cell type or species of origin, or number of viral passages. Thus, genomic sequences containing introns when situated within the context of a retroviral transcript do not in all instances exhibit expected kinetics of splicing.

Research on growth factors, growth factor receptors and signal transduction pathways have provided an exciting conceptual framework for understanding multistage carcinogenesis1. Fig. 1 displays in schematic form how certain extracellular growth factors are perceived by cellular receptors, which are often located at the cell surface, and how the occupancy of these receptors leads to a cascade of signal transduction, through the cytoplasm and eventually into the nucleus, thus altering patterns of gene expression. This figure also emphasizes the central role that a series of protein kinase enzymes plays in several pathways of signal transduction. A general theme that has emerged is that the proto-oncogenes represent a subset of genes that normally code for components in these pathways of signal transduction. Alterations in the structure and function of these proto-oncogenes can convert them to “activated” oncogenes, which cause aberrations in signal transduction and thus disrupt normal growth, differentiation and inter-cellular coordination.

We describe the construction and properties of a retroviral expression vector, designated pMV-7, designed to transfer unselected cDNAs and produce their encoded proteins in recipient cells. The vector is flanked by the long terminal repeats (LTRs) of the Moloney murine sarcoma virus (MSV) and contains the selectable drug resistance gene neo under the regulation of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Unique Eco RI and Hind III sites facilitate the introduction of sequences whose transcription is regulated by the 5' LTR. We have inserted cDNAs encoding: (i) the human lymphocyte antigen T4, (ii) the human lymphocyte antigen T8, and (iii) the murine hypoxanthine-guanine phosphoribosyl transferase (HPRT), into the pMV-7 vector. These constructions were used to transduce recipient cells to the neo+ phenotype. In each case, functional assays demonstrated that 65-92% of the neo+ clones produced the appropriate protein encoded by its corresponding cDNA. These clones were characterized further by analyzing the expression of vector-regulated transcripts. The neo+T4+ clones expressed a single full-length LTR-to-LTR transcript as detected by a T4 probe. The neo+T8+ clones, however, expressed both a full-length LTR-to-LTR transcript and an additional smaller transcript as detected by a T8 probe. This smaller transcript probably resulted from the utilization of cryptic signals which control 3' RNA processing. Furthermore, all of the neo+ clones expressed a transcript that initiated from the tk promoter, contained the neo gene, and used polyadenylation signals provided by the 3' LTR. Thus, the pMV-7 vector is capable of high-efficiency transfer and high-frequency expression of the cDNA-encoded protein.