Activation of the Ki-ras gene in spontaneous and chemically induced lung tumors in CD-1 mice.

As part of an evaluation of the effectiveness of using ras mutation analysis for distinguishing carcinogen-induced from spontaneous tumors, we examined the profile of ras gene point mutations in spontaneous, 7,12-dimethylbenz[a]anthracene (DMBA)-induced, and N-nitrosodiethylamine (DEN)-induced lung tumors from Crl:CD-1(ICR)BR (CD-1) mice. Although all of the lung tumors were assayed for mutations in the Ha-ras, Ki-ras, and N-ras genes (codons 12, 13, and 61), only Ki-ras mutations were found, which is consistent with other studies that have noted a strong preference for Ki-ras gene activation in mouse, rat, and human lung tumors. We found that spontaneous CD-1 mouse lung tumors had a very high frequency of Ki-ras gene activation (17 of 20 tumors; 85%), distributed among codons 12 (5 of 20), 13 (1 of 20), and 61 (11 of 20). DMBA-induced lung tumors had a slightly higher frequency of Ki-ras gene mutations (16 of 16; 100%), again distributed among codons 12 (5 of 16), 13 (2 of 16), and 61 (9 of 16). However, seven of the DMBA tumors had mutations qualitatively different from those found in spontaneous tumors. In contrast to DMBA-induced tumors, DEN-induced tumors had a lower frequency of Ki-ras mutations (36%) when compared with spontaneous lung tumors, suggesting that DEN primarily induces lung carcinogenesis by a mechanism other than ras gene activation. Thus, although spontaneous and induced CD-1 mouse lung tumors have a strong tissue-specific preference for carrying an activated Ki-ras gene, the nature of the initiating carcinogen can influence the frequency or profile of Ki-ras mutations.     

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors

We investigated the role od dosing regimen on ras mutationsin chemically induced CD-1 mouse liver tumors. The spectra ofras gene mutations in liver tumors that were induced by 15 dailyi.p. injections of 7,12-dimethyl-benz[a]anthracene (DMBA), 4-aminoazobenzene(AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine(DEN) were compared to those previously obtained for tumorsinduced by a single but higher dose of each carcinogen. Theprincipal assay used was a direct tumor analysis involving sequencingof polumerase chain reaction (PCR)-amplified tumor DNA; additionalmutations that were present in only a small fraction of tumorcells were detected using a transfection assay or a PCR-engineeredrestriction fragment length polymorphism method. Spontaneousliver tumors had a relatively low frequency of ras mutations,all found in Ha-ras codon 61, and most of these mutations werepresent in only a small fraction of tumor cells. With the exceptionof multiple-dose DEN, each group of single- and multiple-dosecarcinogen-induced tumors exhibited a higher frequency of rasmutations compared with spontaneous tumors. For AAB, N-OH-AAFand DEN, the dosing regimen was found to affect significantlythe profile of rasmutations. For each of these carcinogens,the multiple-dose tumor group (versus single-dose group) hadfewer Ki-ras codon 61 (c     

Activation of C-type virus during chemically induced leukemogenesis in mice.

Repeated percutaneous applications of 7,12-dimethylbenz(a)anthracene on weaning DBA/2 and ST/a mice induced 100% leukemias with short latency periods. Endogenous C-type viruses were activated during the treatment as evidenced by (a) increased expression of the murine leukemia virus major core protein, p30, in blood and spleens and (b) increased frequency of detection of ecotropic virus by cocultivation of the splenocytes with SC-1 cells. The treatment did not affect p30 expression in several nonlymphoid organs, and detection of xenotropic viruses in the splenocytes was decreased. Virus expression did not correlate with the progression of disease in that (a) high p30 levels were generally found in mice with relatively low spleen weights and (b) p30 levels had no obvious connection to survival of the individual. 7,12-Dimethylbenz(a)anthracene treatment had little influence on p30 expression in spleens and blood from C3H and BALB/c mice, which are less sensitive to 7,12-dimethylbenz(a)anthracene-induced leukemogenesis. The results indicate an association of C-type virus activation with chemical induction of leukemia but do not necessarily imply an etiological role of the virus in the disease.     

Tissue-specific activating mutations of Ha- and Ki-ras oncogenes in skin, lung, and liver tumors induced in mice following transplacental exposure to DMBA

Transplacental carcinogenesis represents a good model in which to study the involvement of tissue-specific oncogene activation in carcinogenesis because a single exposure to a carcinogen induces tumors at various sites. We tested tumors of the skin, liver, and lung produced in mice after transplacental 7,12-dimethylbenz[a]-anthracene (DMBA) exposure for possible activation of ras genes. XbaI restriction fragment-length polymorphism analysis has shown that exposure to DMBA in utero may result in appearance of A----T transversion at the second position of codon 61 of Ha-ras oncogene in skin and liver tumors but not in lung tumors. Moreover, DNA samples isolated from spontaneous and DMBA-induced lung and liver tumors were analyzed for mutations at the same position of Ki-ras oncogene using differential hybridization with specific oligonucleotides. Among five spontaneous lung tumors, three cases of A----G transition, and one case of A----T transversion were found, whereas four of ten lung tumors of DMBA-treated animals were positive for A----T mutation. No Ki-ras mutation was detected in one spontaneous and four DMBA-induced hepatomas. In two cases, we revealed Ki-ras A----T mutation in the lung tumor and Ha-ras mutation in the liver tumor taken from the same animal. These results indicate first that DMBA treatment may induce A----T mutation at the second position of codon 61 both in Ha-ras and in Ki-ras and, second, that the role of different activated oncogenes in carcinogenesis may differ, depending on the tissue in which the tumor develops.     

B-raf and Ha-ras mutations in chemically induced mouse liver tumors

The mitogen-activated protein kinase signalling pathway is a central regulator of tumor growth, which is constitutively activated in chemically induced mouse liver tumors. In about 30-50% of cases this effect can be related to activation of the Ha-ras gene by point mutations, whereas in the remaining cases mutations may occur in other members within this pathway, such as Raf kinases. Recently, B-raf has been shown to be frequently mutated in human melanomas and certain other cancers, with a V599E amino-acid change representing the most predominant mutation type. We now screened 82 N-nitrosodiethylamine-induced liver tumors from C3H/He mice for mutations within the hotspot positions in the Ha-ras and B-raf genes. About 50% (39/82) of tumors showed Ha-ras codon 61 mutations and 16 tumors ( approximately 20%) harbored mutations at codon 624 of the B-raf gene, which corresponds to codon 599 in human B-raf. None of the tumors was mutated in both Ha-ras and B-raf. The high prevalence of Ha-ras and B-raf mutations in mouse liver tumors is in striking contrast to human hepatocellular cancers which very infrequently harbor mutations in the two genes. These fundamental differences between the biology of liver tumors in mice and man may be of toxicological relevance.     

Elevated concentrations of serum alpha-fetoprotein in rats with chemically induced liver tumors.

The study was undertaken to determine whether aflatoxin B1 (AFB1)-induced liver tumors in rats produced alpha1-fetoprotein (AFP) and whether the age of the animals would influence such as appearance, a finding suggested by data seen in man. Other liver carcinogens (N-hydroxy-N-2-fluorenylacetamide, N-2-fluorenylacetamide, and diethylnitrosamine) were tested for their ability to induce liver tumors producing AFP. The presence of AFP. The presence of AFP in the serum was determined by double diffusion in agarose and by comparison also by quantitative radioimmunoassay. Using double diffusion, AFP was detected in the majority of tumor-bearing rats that had received either N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide. Sera of diethylinitrosamine-treated rats with liver tumors were all positive, whereas sera of rats bearing AFB1-induced tumors were positive in only a few cases. However, all sera of tumor-bearing rats examined had elevated AFP levels by radioimmunoassay. Nonetheless, the average level of AFP in the sera of rats bearing AFB1-induced tumors was considerably lower, compared to the sera of rats with tumors caused by diethylnitrosamine, N-2-fluorenylacetamide, or N-hydroxy-N2-fluorenylacetamide. Rats started on AFB1 when 6 weeks old had more mixed liver tumors with neoplastic hepatocytes and bile ducts and higher AFP levels than did rats started at 26 weeks of age. However, the histological grade of differentiation of inducted tumors did not seem to influence the AFP level.     

Heme enzyme patterns in genetically and chemically induced mouse liver tumors

Chemically induced rat hepatocyte nodules and hepatomas have repeatedly been shown to be deficient in Phase I drug-metabolizing enzymes. Some of these reduced activities are attributable to a diminution of the heme-containing terminal electron acceptor, cytochrome P-450. We recently demonstrated that spontaneous mouse liver tumors exhibit the same deficiency. Therefore, chemically induced and spontaneous liver tumors share common metabolic alterations which are likely to represent intrinsic characteristics of the tumorigenic process and are independent of its etiology. To determine whether the cytochrome P-450 deficit was the result of an altered heme metabolism, we quantitated four heme-containing proteins in normal mouse liver, spontaneous mouse liver tumors, and those induced by a single injection of diethylnitrosamine: cytochrome P-450; cytochrome b5; tryptophan 2,3-dioxygenase (EC 1.13.11.11); and catalase (EC 1.11.1.6). The amounts of these components in spontaneous tumors relative to normal liver were 0.35, 0.68, 0.76, and 0.51, respectively. Similar values were obtained with chemically induced tumors. The enzymes delta-aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in spontaneous tumor relative to liver were 0.49 and 1.51, respectively. Again, similar values were observed for the chemically induced tumors. Alteration of the latter two enzyme activities may be sufficient for the altered hemoprotein patterns seen in mouse liver tumors. Further, this pattern of metabolic alteration is common to both chemically induced and spontaneous tumors. Thus, tumor resistance to cytotoxic agents activated by the monooxygenase system is not necessarily induced by exposure to these agents, nor as a result of selection.     

Radiation, hyperthermia, and their combination in treatment of chemically induced autochthonous tumors in mice

All autochthonous tumors induced chemically in the thighs of C3H/He mice showed recurrence after single X-irradiation with 20-60 Gy when they were 10 mm in diameter, although this treatment caused temporary, dose-dependent regression. Hyperthermia for 30 min at 43.0 degrees alone had little effect. However, in 2 of 16 mice, hyperthermia after irradiation at 60 Gy resulted in complete cure, i.e., survival of mice without recurrence for more than 120 days after treatment. These results indicate that the combined treatment of radiation and hyperthermia is necessary to obtain the cure of mouse autochthonous tumors.     

Cytogenetic analysis of malignant skin tumors induced in chemically treated TG.AC transgenic mice

TG.AC mice (which carry a v-Ha-ras transgene) rapidly develp papillomas in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Approximately 30% of the papilomas are associated with subsequent development of maliganancies. Early-passage spindle-shaped tumor cells arising from explant cultures of TPA-induced tumors in TG-AC mice were tumorigenic when transplanted to syngeneic recipients. The v-Ha-ras transgene in the transplanted tumors was expressed at a high level. To identify possible genetic changes associated with the development of malignant tumors, explanted cells were cultured in vitro and assessed for karyotypic changes between the second and third passages by analyzing G-banded metaphase chromosomes. For comparison, skin malignancies were induced in nontransgenic FVB/N mice (parent strain) by 7, 12-dimethylbenz[a] anthracene (DMBA) Initiation and TPA promotion, and their G-banded metaphase chromosomes were analyzed. Trisomy (in at least 50% of about 30 metaphases) of chromosome 15 (in five of 15 tumors) and chromosome 6 (four of 15 tumors) was observed in TG.AC mice, independent of chemical treatment or tumor type. Of six tumors from DMBA/TPA-treated FVB/N mice, three had trisomy 10 or 15 (or both), and two appeared normal. The absence of trisomy 7 is notable because c-Ha-ras maps to that chromosome. The absence of trisomy 7 in the six FVB/N DMBA/TPA-induced skin malignancies contrasts with DMBA/TPA- induced karyotypic effects in SENCAR mice. Expression of the v-Ha-ras transgene may have precluded the requirement for endogenous mutant ras and allelic imbalance involving chromosome 7 in TG.AC mice, but it could not have in FVB/N mice. These results suggest the possibility that the observed trissmies are consequential, rather than causal, events in the development of TG.AC or FVB/N skin malignancies. Molecular genetic analysis will be required to understand the changes associated with tumorigenesis in this transgenic line as well as in the parent mouse line. ©1994 Wiley-Liss, Inc.     

Evidence concerning the clonal nature of chemically induced tumors: phosphoglycerate kinase-1 isozyme patterns in chemically induced fibrosarcomas

Subcutaneous fibrosarcomas were induced chemically in mosaic mice heterozygous at the Pgk-1 locus on the X-chromosome. The normal tissues of these animals displayed two electrophoretically distinct isozymes of phosphoglycerate kinase-1-[(PGK-1) EC 2.7.2.3]. The tumors were carefully dissected and analyzed with respect to PGK-1 isozymes. A combination method of multiple sampling of the primary tumor and establishment of tissue culture cell lines from the tumor was utilized. A total of 82 samples was studied from 10 tumors developed in 9 mice. Twenty cell lines were established from 9 tumors; 6 of the tumors were shown to contain neoplastic cells of exclusively PGK-1B activity, 3 of the tumors were shown to contain neoplastic cells of exclusively PGK-1A activity, and 1 tumor contained neoplastic cells with both PGK-1A and PGK-1B activities. The tumor with both A and B activities was hourglass shaped, and the pattern of activity from multiple samples of this tumor suggests that it was formed by the coalescence of a PGK-1A and a PGK-1B tumor. The data presented are concordant with results from chimeric animals and spontaneous tumors in humans and indicate that these tumors display the behavior of clonal growths.     

Overexpression of the N-ras proto-oncogene, not somatic mutational activation, associated with malignant tumors in transgenic mice.

We have produced transgenic mice that carry a foreign gene construct consisting of the N-ras proto-oncogene driven by the mouse mammary tumor virus (MMTV) long terminal repeat. Overexpression of the normal N-ras gene is associated with development of hyperplasias and tumors in a variety of tissues. The tumors are clearly malignant, as evidenced by the presence of metastatic lesions. Extensive analysis of the foreign ras gene in these tumors by use of polymerase chain reaction and sequencing demonstrates in all cases the absence of somatically acquired mutations at those codons normally associated with activation of the ras genes. Thus, these tumors develop from overexpression of the proto-oncogene rather than the presence of the mutated oncogene. These data demonstrate that overexpression of a protooncogene of the ras family can predispose cells in vivo to fully malignant behavior.     

Transplantation immunity to individually unique antigens of chemically induced bladder tumors in mice

We have previously shown that lymphocyte-mediated immunity to common bladder tumor antigens can be demonstrated in vitro. In this study we used two carcinomas and two sarcomas of the urinary bladder of BALB/c mice to immunize syngeneic animals against tumor-specific transplantation antigens (TSTA) in order to determine whether common and/or individually unique antigens could be detected by resistance of the mice to challenge with tumor cells. Strong transplantation resistance was induced against each individual tumor line by immunizing with the respective line. No evidence could be obtained for the presence of TSTA common to the two carcinomas or to the two sarcomas (or to all four tumors).     

Analysis of h-ras, k-ras and N-ras genes for expression, mutation and amplification in laryngeal tumors

Previous work from our laboratory has demonstrated that a clone of Moloney murine sarcoma virus (MoMuSV-349) inoculated intraperitoneally into newborn BALB/c mice induces multiorgan disseminated angiosarcomatous tumors. These tumors develop in two stages: sarcomatous and angiosarcomatous. The present report investigates the relationship between tumor stage development and viral replication within the target cells using the technique of in situ hybridization. A nonradioactive RNA probe (riboprobe) labeled with digoxigenin-linked uridine triphosphate (DIG-UTP) was used to detect the presence of MoMuSV-349 in frozen tissue sections. Infected and control mice were euthanized at various times post-inoculation and tissues were collected for in situ hybridization. The riboprobe hybridized to macrophages, splenic reticular cells, mesothelial cells, megakaryocytes, Kupffer cells and neoplastic (spindled and endothelial) cells in mice infected with MoMuSV-349. The riboprobe failed to hybridize to any tissues in the non-infected mice. Cells hybridizing with the riboprobe were detected as early as 3 days post-inoculation. As the neoplasms progressed from the sarcomatous to angiosarcomatous type there was a shift in cellular target hybridized by the riboprobe from spindled cells to endothelial cells. Positive labeling of endothelial cells during the angiosarcomatous stage of tumor development suggests-that viral replication correlates with cellular proliferation. This demonstrates that in situ hybridization utilizing a non-radioactive riboprobe was useful for a pathogenesis study involving MoMuSV-349.     

Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice

Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation.     

Ki-ras mutations in spontaneous and chemically induced renal tumors of the rat.

A high frequency of point mutations at codon 12 of the Ki-ras gene has previously been reported for rat kidney mesenchymal tumors induced by methylating N-nitroso compounds. In this study, we analyzed renal tumors with divergent histogenesis, i.e., mesenchymal tumors (sarcomas), cortical epithelial tumors (carcinomas), and embryonal tumors (nephroblastomas). Renal mesenchymal tumors and carcinomas were induced in juvenile or young adult Wistar rats by a single dose of N-nitrosodimethylamine (NDMA) while nephroblastomas were induced in Nb hooded rats by a single transplacental dose of N-nitrosoethylurea (NEU). Nephroblastomas developing spontaneously in WAB/Not rats were also examined. Amplification of Ki-ras sequences from formalin-fixed, paraffin-embedded tissue by the polymerase chain reaction was followed by direct DNA sequencing. GGT----GAT point mutations at codon 12 of the Ki-ras gene were found in 9 of 12 (75%) renal mesenchymal tumors and in 9 of 12 (75%) cortical epithelial tumors induced by NDMA. Even higher incidences were observed in nephroblastomas (8/8; 100%) induced by NEU and in spontaneous nephroblastomas (10/11; 91%). These results indicate that Ki-ras mutations are frequent events during the development of kidney tumors irrespective of their histogenesis and suggest that they may play an important role in renal carcinogenesis in rats. These data further indicate that mutational activation of Ki-ras proto-oncogenes in carcinogen-induced rat kidney tumors occurs in a tissue-specific, rather than cell-specific, manner.     

Induction of lung and liver tumors by flouranthene in a preweanling CD-1 mouse bioassay

Fluoranthene (FA), a major enviromental pollutant, induced lungand liver tumors 6–9 months after intraperitoneal injectionof 0.7, 1.75 and 3.5 mg FA into preweanling CD-1 mice. Therewas a dose-dependent increase in lung tumors with a maximumtumor incidence of nearly 45% and a maximum tumor multiplicityof 0.6–0.7 lung tumors/mouse. No significant differencewas noted in lung tumors in the 6 and 9 month bioassays, althoughfewer tumors were consistently noted in mice treated with thetwo lowest doses of FA. Indices of lung tumor incidence (ED₅₀)and multiplicity (TM_1.0) were similar for the two bioassaysand ranged from 18.9–19.5 and 26.2–27.2 µmolrespectively. Male mice had up to two times more lung tumorsthan females but these results were not statistically significant.Liver tumors (nodular hyperplasia) appeared only in FA-treatedmales but no dose-response relationship was evident. However,liver tumors were observed in only 0–10% of the male micein the 6 month treatment groups, but in 20–60% of themales in the 9 month groups. Because the CD-1 preweanling mouseresponded to the weak lung tumorigen FA, it is a viable, limited-termbioassay for measuring tumorigenicity of PAH and combustionemissions.