Identification and characterization of zinc binding sites in protein kinase C.

Metal ion coordination in the regulatory domain of protein kinase C (PKC) is suggested by the conservation of six cysteines and two histidines in two homologous regions found therein. By monitoring x-ray fluorescence from a purified sample of rat PKC beta I overexpressed in insect cells, direct evidence has been obtained that PKC beta I tightly binds four zinc ions (Zn2+) per molecule. Extended x-ray absorption fine structure (EXAFS) data are best fit by an average Zn2+ coordination of one nitrogen and three sulfur atoms. Of the plausible Zn2+ coordination models, only those featuring nonbridged Zn2+ sites accommodate the EXAFS data and all of the conserved potential ligands.

An assessment of the carcinogenic potential of ezetimibe using nonclinical data in a weight-of-evidence approach.

Ezetimibe blocks intestinal absorption of sterols via interaction with the Neimann-Pick C1-Like 1 (NPC1L1) transporter and is approved for use in the treatment of primary hyperlipidemia (heterozygous familial and non-familial), homozygous familial hypercholesterolemia, and homozygous sitosterolemia. A recently completed randomized clinical trial [simvastatin and ezetimibe in aortic stenosis (SEAS)] testing the effectiveness of Vytorin (a combination of simvastatin and ezetimibe) in patients with aortic stenosis reported an unexpected safety finding: an increase in overall cancer incidence and cancer-associated mortality (all types) in the treated groups relative to the placebo control. A subsequent meta-analysis utilizing a much larger database from two ongoing clinical trials indicated that the observed findings in the SEAS trial were likely due to chance and not a true drug-induced effect. Nonetheless, it has been suggested by various commentators on the SEAS trial that ezetimibe may be carcinogenic. The extensive nonclinical database for ezetimibe was used to test the hypothesis that ezetimibe may be a direct or indirect carcinogen. Using two different in silico approaches, ezetimibe showed no structural alerts for genetic toxicity or carcinogenicity. Ezetimibe was not genotoxic in two reverse mutation assays, one in vitro clastogenicity assay, and two mouse micronucleus assays. No evidence of proliferative lesions was observed in three species in studies of 1-12 months in duration. Ezetimibe was not carcinogenic in standard 2-year bioassays in mice and rats. Additionally, in these 2-year bioassays, no drug-related non-neoplastic lesions were noted. The absence of drug-induced non-neoplastic or proliferative lesions in these studies indicates that ezetimibe treatment was not associated with findings characteristic of carcinogens (i.e., DNA reactivity or cell proliferation) Administration of pharmacologic doses of ezetimibe to mice did not alter hepatic expression patterns of genes associated with apoptosis, cell proliferation, or epithelial-mesenchymal transition. No evidence of drug-induced tumors was observed in mice in which the molecular target of ezetimibe (NPC 1L1) was knocked out over the life span of the animal. In conclusion, the nonclinical data do not support the proposed hypothesis based on the single observation from the SEAS trial and, rather, support the conclusion that ezetimibe does not represent a carcinogenic hazard to humans using this drug in a therapeutic setting.

Molecular cloning of gene sequences regulated by tumor promoters and mitogens through protein kinase C.

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.

Effects of 5-azacytidine on methylation and expression of specific DNA sequences in C3H 10T1/2 cells.

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.

Design of a retrovirus-derived vector for expression and transduction of exogenous genes in mammalian cells.

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 PstI 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.

Cellular Moloney murine sarcoma (c-mos) sequences are hypermethylated and transcriptionally silent in normal and transformed rodent cells.

Moloney murine sarcoma virus carries an oncogenic sequence (v-mos) which is homologous to a single copy gene (c-mos) present in the normal cells of several vertebrate species. Because of the possible significance of c-mos sequences in normal development and malignant transformation induced by physical or chemical agents, we have examined the state of integration, methylation, and transcriptional activity of c-mos sequences in a variety of normal rodent tissues, normal cell lines, or cell lines transformed by radiation or chemical carcinogens. DNA-DNA hybridization, utilizing the Southern blotting technique and a plasmid-derived DNA probe representing the v-mos sequence, gave no evidence for rearrangements of the c-mos sequence in the DNAs obtained from these diverse cell types. Parallel studies employing the restriction enzyme isoschizomers HpaII and MspI indicated that in all of these cell types the c-mos sequences were heavily methylated. In addition, analysis of cellular RNAs by blot hybridization with the v-mos probe failed to detect evidence of transcription of the c-mos sequences in any of these cell types. This was in contrast to a Moloney sarcoma virus-transformed cell line in which we found that the integrated v-mos sequence was both undermethylated and extensively transcribed. Thus, it would appear that c-mos sequences do not play a role in the transformation of rodent cells by chemical or physical agents, although the possible role of other endogenous onc sequences remains to be determined.

A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity.

Farnesyl protein transferase (FT), an enzyme that catalyzes the first step in the posttranslational modification of ras and a number of other polypeptides, has emerged as an important target for the development of anticancer agents. SCH66336 is one of the first FT inhibitors to undergo clinical testing. We report a Phase I trial to assess the maximum tolerated dose, toxicities, and biological effectiveness of SCH66336 in inhibiting FT in vivo. Twenty patients with solid tumors received 92 courses of escalating SCH66336 doses given orally twice a day (b.i.d.) for 7 days out of every 3 weeks. Gastrointestinal toxicity (nausea, vomiting, and diarrhea) and fatigue were dose-limiting at 400 mg of SCH66336 b.i.d. Moderate reversible renal insufficiency, secondary to dehydration from gastrointestinal toxicity, was also seen. Inhibition of prelamin A farnesylation in buccal mucosa cells of patients treated with SCH66336 was demonstrated, confirming that SCH66336 inhibits protein farnesylation in vivo. One partial response was observed in a patient with previously treated metastatic non-small cell lung cancer, who remained on study for 14 months. This study not only establishes the dose for future testing on this schedule (350 mg b.i.d.) but also provides the first evidence of successful inhibition of FT in the clinical setting and the first hint of clinical activity for this class of agents.

Orally bioavailable farnesyltransferase inhibitors as anticancer agents in transgenic and xenograft models.

The in vivo evaluation process described here was instrumental in the identification of SCH 66336 as a clinical candidate. Our lead FTI, SCH 66336, and several other FTIs are being evaluated in early-phase clinical trials to establish proof-of-principle for farnesyl transferase inhibition in human patients. The preclinical studies described here suggest that FTIs may have utility against a wide array of human cancers as a single agent and may, at least in some cases, lead to tumor regression. In addition, the results to date in combination with cytotoxic chemotherapeutic agents in animal models indicate that these combinations may enhance the clinical efficacy of FPT inhibitors. Further preclinical studies should help to guide the clinical development of this class of novel antitumor agents.

Identification of pharmacokinetically stable 3, 10-dibromo-8-chlorobenzocycloheptapyridine farnesyl protein transferase inhibitors with potent enzyme and cellular activities.

Farnesyl protein transferase (FPT) is a promising target for the development of cancer chemotherapeutics because it is responsible for the farnesylation of oncogenic p21 Ras proteins which are found in nearly 30% of all human cancers and necessary for cellular development and growth. The recent discovery and progression to phase II clinical trials of trihalobenzocycloheptapyridine Sch-66336 as a potent inhibitor of FPT with oral, in vivo efficacy in mice have spawned extensive structure-activity relationship studies (SAR) of this class of compounds. Of the many trihalobenzocycloheptapyridine analogues prepared, we have identified several which inhibit FPT and cellular proliferation at single-digit nanomolar concentrations and which have good pharmacokinetic properties in mice.

(+)-4-[2-[4-(8-Chloro-3,10-dibromo-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1,2-b]- pyridin-11(R)-yl)-1-piperidinyl]-2-oxo-ethyl]-1-piperidinecarboxamid e (SCH-66336): a very potent farnesyl protein transferase inhibitor as a novel antitumor agent.

We have previously shown that appropriate modification of the benzocycloheptapyridine tricyclic ring system can provide potent farnesyl protein transferase (FPT) inhibitors with good cellular activity. Our laboratories have also established that incorporation of either pyridinylacetyl N-oxide or 4-N-carboxamidopiperidinylacetyl moieties results in pharmacokinetically stable inhibitors that are orally efficacious in nude mice. We now demonstrate that further elaboration of the tricyclic ring system by introducing a bromine atom at the 7- or the 10-position of the 3-bromo-8-chlorotricyclic ring system provides compounds that have superior potency and selectivity in FPT inhibition. These compounds have good serum levels and half-lives when given orally to rodents and primates. In vitro and in vivo evaluation of a panel of these inhibitors has led to identification of 15 (SCH 66336) as a highly potent (IC50 = 1.9 nM) antitumor agent that is currently undergoing human clinical trials.

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

Role of protein kinase C in regulation of gene expression and relevance to tumor promotion.

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.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

On the use of lonafarnib in myelodysplastic syndrome and chronic myelomonocytic leukemia.

Lonafarnib is an orally bio-available farnesyltransferase inhibitor that prevents farnesylation of specific target proteins including Ras. In a multicenter study, 67 patients with advanced myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) were treated with a continuous oral dose of 200-300 mg of lonafarnib and were evaluated for hematologic, pathologic and pharmacodynamic response. The median age of patients was 70 years (range 44-86). There were 32 patients with MDS (RAEB-20 and RAEB-t-12) and 35 with CMML. Overall 16 (24%) of the patients responded with two patients achieving a complete remission and one a partial response. Responses were seen in 6/32 and 10/35 patients with MDS and CMML, respectively. Of the 19 patients who were platelet transfusion-dependent prior to treatment, 5 (26%) became transfusion-free for a median duration of 185 days. A decrease in the farnesylation of the HDJ-2 protein measured in patient-derived cells was observed in the majority of patients during treatment with lonafarnib, but no clear correlation between changes in farnesylation and clinical effect could be made. Gastrointestinal toxicity was significant with 19% of patients discontinuing therapy due to diarrhea, nausea and/or anorexia. Lonafarnib has demonstrable activity in patients with advanced MDS and CMML.

Transduction of the human insulin gene via retroviral vectors fails to yield spliced transcripts.

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.

Carcinogen- and radiation-transformed C3H 10T1/2 cells contain RNAs homologous to the long terminal repeat sequence of a murine leukemia virus.

Carcinogen- or radiation-transformed C3H 10T1/2 murine fibroblasts transcribe a set of poly(A)+RNAs that contain sequences homologous to the long terminal repeat (LTR) sequence of Moloney murine sarcoma virus. These LTR-containing RNAs consist of a series of discrete bands ranging in size from about 38 to 18 S. The higher molecular weight molecules (30-38 S) in this set of RNAs also contain sequences homologous to the gag, pol, and env genes of a murine leukemia virus. A 24S RNA contains sequences homologous to the env gene of murine leukemia virus. A 20S and an 18S RNA also share homology with the LTR probe but fail to hybridize to the gag, pol, or env probes or to a probe for the U3 region of the LTR sequence. Thus, the latter transcripts do not appear to arise from a known endogenous murine leukemia virus genome. Although this entire set of RNAs is absent from normal C3H 10T1/2 cells (or is present at an extremely low level), these RNAs are induced by BrdUrd or 5-azacytidine. The presence of these RNAs may provide highly sensitive molecular markers of transformation of murine cells.

Cellular targets and host genes in multistage carcinogenesis.

Recent studies indicate that although cellular DNA is the critical target in the action of initiating carcinogens, specific membrane-associated receptors mediate the actions of certain tumor promoters. A stereochemical model is presented to explain how three different types of tumor promoters (phorbol esters, indole alkaloids, and polyacetates) can interact with the same class of cellular receptors. Multistage chemical carcinogenesis might involve progressive alterations in the expression of cellular DNA sequences homologous to oncogenes and regulatory sequences in certain retroviruses. We found that the oncogene c-mos is not rearranged or expressed in a series of carcinogen-transformed murine C3H 10T112 cells. These cells do express, however, a unique set of poly(A)+ RNAs that contain sequences homologous to the Moloney leukemia virus long terminal repeat sequence. Studies are in progress to determine the significance of this finding with respect to the carcinogenic process.

Benzo[a]pyrene induction of extrachromosomal viral DNA synthesis in rat cells transformed by polyoma virus.

The effect of the carcinogen benzo[a]pyrene (BP) on the production of specific extrachromosomal DNAs has been studied in transformed rat cells containing integrated polyoma virus DNA. Exposure of cells previously transformed by the polyoma virus mutant ts-a, which codes for a temperature sensitive T antigen, to a non-toxic dose of BP (0.25 micrograms/ml) enhanced the production of free polyoma DNA at the population level. This occurred at 33 degrees C (the permissive temperature) but not at 39 degrees C, indicating that the BP effect was dependent on the function of the T antigen of polyoma virus. In the same cell system, BP did not induce the production of extrachromosomal copies of two endogenous rat retrovirus genomes, the 30S RNA virus and rat leukemia virus. Thus, the effects on integrated polyoma DNA are quite specific and do not reflect a generalized increase in asynchronous DNA replication and excision. Further studies utilizing a carcinogenic metabolite of BP, benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide (anti) (BPDE), on a rat cell line transformed by wild type (WT) polyoma virus demonstrate that the enhancement of polyoma DNA synthesis persists for at least 5 days after a single exposure to BPDE, despite the rapid decay of this compound. In addition, enhanced synthesis of polyoma DNA can be induced by fusion of normal rat fibroblasts previously exposed to BPDE with polyoma transformed rat fibroblasts not exposed to BPDE. It appears, therefore, that the effects we have observed are not due solely to direct carcinogen damage at the level of the integrated polyoma virus DNA, but may instead involve the induction of cellular factor(s) that act indirectly to enhance asynchronous replication of polyoma DNA.

The possible role of cellular genes related to retroviruses in the process of chemical and radiation carcinogenesis.

Chemical carcinogens, radiation, and tumor promoters can not introduce new genetic information into cells. They must, therefore, call upon genes already present in the target cell. We have recently explored the possibility that these host genes share homology with specific nucleic acid sequences present in some of the known retroviruses. In one set of experiments we used a cloned DNA probe prepared from the oncogene (v-mos) of Moloney murine sarcoma virus (Mo-MSV) to examine the state of integration, methylation, and transcription of the homologous cellular oncogene (c-mos) in normal murine cells and in murine cells transformed by radiation or chemical carcinogens. We found that in the transformed cells the c-mos sequence has not undergone rearrangement within the host genome and, as in normal cells, this sequence is hypermethylated and transcriptionally silent. These results are in sharp contrast to the situation in cells transformed by Mo-MSV in which the exogenous v-mos sequence is integrated at new sites within the host genome, is undermethylated and is extensively transcribed. In related studies, we have found that another one gene, c-ras (H), also fails to show any evidence for rearrangement in carcinogen or radiation transformed C3H 10T1/2 cells. Since there is evidence that eukaryotic cells contain numerous onc genes our results do not rule out the possibility that onc genes other than c-mos and c-ras (H) may play a role in the transformation of murine cells by chemicals or radiation. Additional studies utilizing probes to other onc genes are required to evaluate this possibility.