Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy.     

Transformation of rodent immortalized and embryo cells by oncogenes. I. Mouse (NIH 3T3) and rat (FR 3T3) immortalized fibroblasts

Immortalized mouse NIH 3T3 cells were transformed by gene transfer of DNA isolated from a human bladder tumor cell line and plasmids containing an activated human Ha-ras oncogene insert. For gene transfer the calcium-phosphate co-precipitation method was used. Transformation was evaluated by morphological focus formation, growth in soft agar and tumor development in nude mice. In addition, immortalized rat FR 3T3 cells were transformed by Ha-ras, too. The co-transfer of ras and myc oncogenes did not enhance focus formation in FR 3T3 cells.     

Detection and identification of activated oncogenes in spontaneously occurring benign and malignant hepatocellular tumors of the B6C3F1 mouse

Species- and strain-specific spontaneously occurring tumors have been observed in rodents maintained under normal laboratory conditions. Elucidation of the molecular mechanisms associated with the development of these spontaneous tumors may provide a better understanding of tumor development associated with exposure to chemical carcinogens. In view of the high frequencies of oncogene activation shown in rodent tumors induced by known chemical carcinogens, we have investigated oncogene activation in spontaneous tumors of the B6C3F1 mouse and Fischer 344/N rat by DNA transfection techniques. A marked difference in the presence of activated oncogenes in spontaneous rat tumors versus spontaneous mouse liver tumors was observed in this study. All rat tumors tested failed to yield activated oncogenes (0/29), whereas 30% (3/10) of mouse hepatocellular adenomas and 77% (10/13) of hepatocellular carcinomas scored positive by DNA transfection. These transforming genes were identified as an activated Ha-ras gene in all the adenoma transfectants and in 8 of the 10 carcinoma transfectants. The two remaining hepatocellular carcinomas contained transforming genes that appear not to be members of the known ras gene family. The B6C3F1 mouse liver system might provide a very sensitive assay not only for assessing the potential of a chemical to activate a cellular proto-oncogene, but also for detecting various classes of proto-oncogenes that are susceptible to mutational activation.     

Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.

The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A----T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells.     

v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid.

Experimental carcinogenesis has led to a concept that defines two discrete stages in the development of skin tumors: (i) initiation, which is accomplished by using a mutagen that presumably activates a protooncogene, and (ii) promotion, which is a reversible process brought about most commonly by repeated application of phorbol esters. We have created a transgenic mouse strain that carries the activated v-Ha-ras oncogene fused to the promoter of the mouse embryonic alpha-like, zeta-globin gene. Unexpectedly, these animals developed papillomas at areas of epidermal abrasion and, because abrasion can also serve as a tumor-promoting event in mutagen-treated mouse skin, we tested these mice for their ability to respond to phorbol ester application. Within 6 weeks virtually all treated carrier mice had developed multiple papillomas, some of which went on to develop squamous cell carcinomas and, more frequently, underlying sarcomas. We conclude that the oncogene "preinitiates" carrier mice, replacing the initiation/mutagenesis step and immediately sensitizing them to the action of tumor promoters. In addition, treatment of the mice with retinoic acid dramatically delays, reduces, and often completely inhibits the appearance of promoter-induced papillomas. This strain has use in screening tumor promoters and for assessing antitumor and antiproliferative agents.     

Activation of ras oncogene in aflatoxin-induced rat liver carcinogenesis.

The presence of activated transforming genes was investigated in four primary aflatoxin-induced rat liver tumors in male Fischer rats, in two cell lines generated from such tumors, in an epithelial liver-derived nontransformed cell line, and in the latter cell line after transformation by aflatoxin B1 in vitro. When DNA extracted from these sources was transfected into NIH 3T3 cells, negative results were obtained from focus assays. Cotransfection of these DNA samples with a gene for resistance to G418, followed by selection for resistance to that antibiotic, and tumorigenicity testing in nude mice demonstrated DNA-mediated transfer of the neoplastic phenotype in all cases except for DNA from the nontransformed cell line. DNA extracted from these primary nude mouse tumors used in a secondary round of transfection with NIH 3T3 cells gave positive results in focus assays, which were conserved through succeeding rounds of transfection. By use of appropriate radiolabeled probes, activated ras oncogenes were detected in all samples. N-ras activation was detected in three of the primary rat liver tumors and both hepatoma cell lines. Ki-ras activation was detected in one primary rat liver tumor, and Ha-ras activation was detected in the cell line transformed in vitro with activated aflatoxin B1. The activated Ki-ras oncogene was further characterized by use of synthetic oligonucleotide probes and was shown to contain a G----A transition at the second nucleotide in codon 12.     

Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice.

The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder, the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras--i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors.     

Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene.

Established REF52 cells (rat embryo fibroblasts) completely resist stable transformation by ras oncogenes, and simian virus 40 large tumor (T) antigen collaborates with ras to convert REF52 cells to tumorigenic state. A temperature-sensitive simian virus 40 large T antigen (encoded by tsA58) allowed the T24 Ha-ras oncogene to transform REF52 cells in a temperature-dependent manner. Two thirds of the clones transformed with tsA58 and ras became arrested in G2 or late S phase when shifted to a nonpermissive temperature for T antigen stability. Thus, ras induced growth arrest rather than stable transformation in the absence of a functional collaborating oncogene. These results indicate that collaborating oncogenes can regulate cellular responses to ras and have implications regarding therapeutic strategies to control tumor cells expressing activated ras oncogenes.     

Precancerous lesions upon sporadic activation of beta-catenin in mice

BACKGROUND & AIMS: Inappropriate activation of beta-catenin in adult tissues is associated with a wide variety of cancers, especially in the digestive tract. Classic transgenic and knockout murine models in which beta-catenin is activated in large fields of cells have provided experimental support in favor of a role for this molecule in tumorigenesis. However, these models do not reproduce the sporadic nature of the majority of human cancers, beginning with the activation of an oncogene at random in a single cell. METHODS: We used the "hit and run" strategy to generate a mouse model in which the expression of an activated form of beta-catenin occurs sporadically in vivo. RESULTS: Sporadic, multifocal lesions were observed in the stomach of 3% of mice aged 8 months and older. These lesions were associated with loss of Sonic hedgehog (Shh), and a causal relationship between beta-catenin activation and Shh inhibition was established in gastric cells in vitro. No lesion was detected in the intestine or in the liver. In addition, one third of female mutant mice developed benign perimammary papillomas. Mutant mice were also hypersensitive to chemically induced premalignant skin lesions. CONCLUSIONS: These results challenge the view that activation of beta-catenin induces malignant cancerogenesis, because they show in mice that sporadically activated beta-catenin is sufficient for tumor initiation, yet without further malignant progression, and that it sensitizes cells to environmental hits. This model represents a powerful tool to investigate the interplay between genetic and environmental factors in tumor progression.     

Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA.

NIH 3T3 mouse fibroblasts form nonmetastasizing fibrosarcomas upon transformation by the Ha-ras oncogene isolated from the EJ human bladder carcinoma cell line and subcutaneous inoculation into immunocompetent NFS/NCr mice. DNA from a human metastatic tumor was transfected into these Ha-ras transformants, and one of the resulting colonies yielded a lung metastasis after subcutaneous inoculation. DNA was isolated from this metastasis and subjected to a second round of transfer into Ha-ras-transformed NIH 3T3 cells. Inoculation of these transfected cultures into mice led once again to formation of metastases, this time at a higher frequency. Examination of four of the resulting metastases revealed discrete human DNA fragments that were common to all four. These findings demonstrate that the metastatic phenotype can be transferred via DNA from cell to cell and is associated with the presence of a discrete DNA segment. This segment is not identical to the myc oncogene or to any of the frequently detected ras tumor oncogenes.     

Frequency of molecular alterations affecting ras protooncogenes in human urinary tract tumors.

Members of the ras gene family are activated as oncogenes in many different human cancers. To systematically determine the frequency at which such genes might be involved in the neoplastic process affecting a specific target tissue, urothelial cells, we surveyed a large series of urinary tract tumors for ras oncogenes by DNA transfection and by molecular genetic analysis. Harvey (Ha)-ras oncogenes were detected in 2 of 38 tumors by transfection, molecularly cloned in biologically active form, and shown to contain single base changes at codon 61 leading to substitutions of arginine and leucine, respectively, for glutamine at this position. One additional Ha-ras oncogene was identified in a bladder carcinoma by restriction polymorphisms at codon 12. In one of 21 tumors, we observed a 40-fold amplification of the Kirsten (Ki)-ras gene. No amplification of other ras genes was detected in any of the tumors analyzed. Our findings strengthen the conclusion that codons 12 and 61 are the major "hot spots" of ras oncogene activation and suggest that quantitative alterations in expression due to gene amplification may provide an alternative mechanism for ras gene activation in primary human tumors.     

Persistence of Ha-ras-induced metastatic potential of SP1 mouse mammary tumors despite loss of the Ha-ras shuttle vector.

Previous studies have shown that the SP1 mouse mammary adenocarcinoma cell line, which is tumorigenic but nonmetastatic, acquires metastatic potential when transfected with the activated human Ha-ras gene. In addition, the process of calcium phosphate-mediated DNA transfection, as well as treatment with the calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, can also result in heritable changes in the malignant behavior of SP1 cells. It was of interest, therefore, to determine whether the metastatic consequences of Ha-ras oncogene expression in SP1 cells are a primary effect of the transfected gene or whether heritable secondary changes are induced by Ha-ras oncogene expression. In the latter case, continued expression of the Ha-ras oncogene would not be required to maintain the metastatic phenotype. To test this hypothesis we introduced the Ha-ras oncogene into SP1 cells on a shuttle vector in which maintenance of the vector was dependent on selection for resistance to the antibiotic G418. Subclones which had lost the transfected Ha-ras gene were subsequently isolated following growth in nonselective medium. The Ha-ras-transfected clones and the revertant subclones were found to be equally metastatic, indicating that transfection with the Ha-ras gene does induce stable secondary changes in the metastatic phenotype of SP1 cells.     

Ha-ras oncogene activation in mammary glands of N-methyl-N-nitrosourea-treated rats genetically resistant to mammary adenocarcinogenesis.

A single dose of N-methyl-N-nitrosourea given to sexually immature female Buf/N rats produces a high incidence of mammary adenocarcinomas. A large percentage of these tumors contain the Ha-ras oncogene, activated by a G----A transition at the second nucleotide of codon 12. Copenhagen rats, on the other hand, are completely resistant to mammary tumor induction by a number of carcinogens, including N-methyl-N-nitrosourea. Here we show, using a sensitive method involving PCR, that codon 12 Ha-ras mutations occur in the mammary glands of both Buf/N and Copenhagen rats 30 days after N-methyl-N-nitrosourea treatment. These mutations were evenly distributed among individual mammary glands and were present in purified mammary epithelial cells. In Buf/N rats, the fraction of cells containing a mutated Ha-ras allele increased by a factor of 10-100 between 30 and 60 days, whereas in Copenhagen rats, there was no such increase during this time period. We conclude that the resistance of the Copenhagen rat to mammary carcinogenesis is not due to a defect in initiation but rather appears to be due to the inability of cells containing a mutated ras allele to undergo sustained clonal expansion.     

Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice.

Ras oncogenes owe their transforming properties to single point mutations in the sequence coding for the active site of the p21 protein. These mutations lead to changes in cellular proliferation and induce tumorigenic properties. Point mutations represent a well-defined target for antisense oligonucleotides that can specifically suppress the translation of the targeted mutant mRNA. We show that the stability and cellular disponibility of antisense oligonucleotides can be markedly improved by adsorption to polyalkylcyanoacrylate nanoparticles. Nanoparticle-adsorbed antisense oligonucleotides directed to a point mutation (G-->U) in codon 12 of the Ha-ras mRNA selectively inhibited the proliferation of cells expressing the point-mutated Ha-ras gene at a concentration 100 times lower than free oligonucleotides. In addition they markedly inhibited Ha-ras-dependent tumor growth in nude mice after subcutaneous injection. These experiments show that inhibition of ras oncogenes by antisense oligonucleotides can block tumor development even though ras oncogenic activation might be an early event in tumor progression.     

Regulation of parathyroid hormone-related protein gene expression in murine keratinocytes by E1A isoforms: a role for basal promoter and Ets-1 site

PTHrP gene expression was evaluated in a murine keratinocyte line, Pam 212K, transformed with E1A and ras. We found that the 12S-E1A oncogene, with or without ras transformation, markedly reduced PTHrP mRNA expression. Using transient transfection assays, we found that the 12S isoform repressed activity from a 5'PTHrP-driven reporter gene. E1A-induced repression of PTHrP reporter constructs appears to be mediated by sequences within minimal promoter region. The 13S-E1A isoform did not repress PTHrP reporter gene activity, and a 13S-deletion mutant that lacked the repressor domains activated a subset of reporter constructs. Mutation of an Ets-1 binding site upstream of the basal promoter substantially decreased activation of reporter constructs by this 13S-deletion mutant. These findings suggest that the E1A oncoprotein may serve as a model for both activation and repression of PTHrP gene expression.     

The human Ha-ras oncogene induces genomic instability in murine fibroblasts within one cell cycle.

Many human tumors contain an activating mutation in one of the ras protooncogenes. Additionally, these tumor cells are often heteroploid and characterized by chromosome breaks and rearrangements that are consequences of the genomic instability that is thought to contribute to tumor progression. The concurrence of ras mutations and genomic instability in tumors prompted us to ask whether selective induction of an activated Ha-ras gene could render a genome unstable. The NIH 3T3 cells used in this study contained mutant p53 genes and carried a selectively inducible activated (EJ) Ha-ras transgene under the control of bacterial lactose regulatory elements. When stably transfected cells were induced to express activated Ha-ras by isopropyl beta-D-thiogalactoside administration, there was a marked increase in the number of gross chromosomal aberrations including acentric fragments, multicentric chromosomes, and double minutes, which occurred within the time frame of a single cell cycle from the time of induction. To confirm that these aberrations occurred within the first cell cycle after mutant Ha-ras induction, the cells were arrested in G1 phase by serum depletion and, subsequently, released by administration of isopropyl beta-D-thiogalactoside or serum. The mitoses from cells released with isopropyl beta-D-thiogalactoside contained a 3-fold elevation in the fraction of chromosomes containing aberrations compared to mitoses from parallel cell cultures that were released with serum. Thus, the induction of activated Ha-ras gene expression in these cells results in genomic instability that can be detected as aberrant chromosomes at the next mitosis.     

Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse.

Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo.     

A molecular approach to leukemogenesis: mouse lymphomas contain an activated c-ras oncogene.

By inducing mouse thymomas with carcinogens and gamma-radiation, we have studied the potential of tumor DNA to induce foci in rodent fibroblasts. A high percentage of the tumors used transformed the cultured cells, and the oncogenic phenotype segregated with extra copies of the c-ras gene family. There appears to be selectivity in the activated gene because so far all analyzed tumors induced by carcinogen have activated the N-ras gene, and those induced by radiation have activated the K-ras gene. The K-ras gene is the cellular counterpart of the viral ras oncogene in Kirsten murine sarcoma virus, but the N-ras has not yet been found in a retrovirus. The transformed cells have a marked increase in expression of the oncogene at the RNA and protein level. This model system might be a powerful tool in the study of leukemogenesis.     

Ras activation of genes: Mob-1 as a model.

The ras oncogenes function by indirectly controlling expression of a subset of yet-undefined genes that are crucial for cell growth and differentiation. In a differential display strategy, numerous genes were identified on the basis of their differential expression in rat embryo fibroblasts transformed by the cooperation of mutant Ha-ras and p53 genes. We demonstrate here that one such gene, designated mob-1, is a downstream target of the Ras signaling pathway. The 417-bp mob-1 promoter, which contains dual NF-kappa B and AP-1 binding sites, confers the Ras inducibility. Oncogenic Ras as well as serum growth factors that activate endogenous Ras can induce mob-1 expression, but with a fundamental difference in that the oncogenic induction is constitutive whereas the serum induction is transient. mob-1 encodes a small secretory protein with a high degree of homology to IP-10, a member of a proinflammatory cytokine family. These findings link chronic inflammatory response to constitutive ras activation and tumorigenesis. Mob-1 may serve as a secreted marker for oncogenic Ha-ras mutations.