Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors

The liver tumor-prone B6C3F1 mouse (C57BL/6 ♂ × C3H/He♀), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB-induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB-induced tumors but in only one of five spontaneous tumors, whereas the level of Ha-ras mRNA increased in nine of 10 PB-induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors. © 1994 Wiley-Liss, Inc.     

Genetic variation in liver tumor susceptibility, plasma testosterone levels, and androgen receptor binding in six inbred strains of mice

We compared six inbred mouse strains for their relative susceptibilities to liver and lung tumor induction. Male and female mice were treated at 12 days of age with a single i.p. injection of N-ethyl-N-nitrosourea (ENU; 0.25 mumol/g), and tumor multiplicity was analyzed at 32 weeks of age (males) or 44 weeks of age (females). Male mice of the SWR/J and C57BL/6J strains were relatively resistant to hepatocarcinogenesis, averaging 0 and 0.3 tumors per animal, respectively. Male C57BR/cdJ, P/J, and SM/J mice had intermediate susceptibilities, averaging seven to 17 tumors per animal, and male CBA/J mice were the most susceptible, averaging 45 tumors per animal. Female mice were more resistant than male mice: no liver tumors were observed for SWR/J females; C57BL/6J, SM/J, P/J, and CBA/J females averaged less than one tumor per animal and C57BR/cdJ females averaged five tumors per animal. In contrast to the results for liver tumor induction, there was no difference between the sexes in lung tumor susceptibility. Male and female SWR/J mice were the most susceptible, averaging 14 lung tumors per animal; male and female CBA/J mice were moderately susceptible, averaging six tumors per animal and the C57BR/cdJ, C57BL/6J, P/J, and SM/J strains were relatively resistant, averaging less than three tumors per animal. To determine if levels of testosterone, a potent liver tumor promoter in mice, or its receptor contribute to the strain variation in liver tumor susceptibility, we measured levels of plasma testosterone as well as binding properties of the hepatic androgen receptor for the six inbred strains. Plasma testosterone in male mice ranged from 1.8 to 7.4 ng/ml and in females ranged from 0.21 to 0.42 ng/ml, which is consistent with the greater susceptibility of male mice to liver tumor development. However, variation in testosterone levels among the different strains of mice was not correlated with liver tumor susceptibility. We also demonstrated the presence of high affinity androgen receptors in mouse hepatic cytosol. The amounts of this receptor for the six strains tested ranged from 24 to 34 fmol/mg cytosolic protein. The apparent KD of the receptor for [3H]mibolerone (a synthetic androgen) differed between the strains: SWR/J, C57BL/6J, and C57BR/cdJ mice had the highest affinity (KD = 0.22 nM), P/J and CBA/J strains had an intermediate affinity (KD = 0.36 nM), and the SM/J strain had the lowest affinity receptor (KD = 0.45 nM). The strain variation in the affinity or abundance of the androgen receptor was not related to the strain variation in liver tumor induction.     

Significance of strain and sex differences in the development of 252Cf neutron-induced liver tumors in mice.

Mouse liver tumors occurring in C3H/HeN, C57BL/6N and C3B6F1 hybrid (C3H x C57BL) were studied following 252Cf fission neutron irradiation. Three strains of mice of both sexes (about 30 mice/group) were irradiated once with 252Cf at doses of 0, 12.5, 50 and 200 cGy. The groups were observed for 13 months after irradiation. The incidence of liver tumors in the non-irradiated controls was 0% in both sexes of C57BL/6N, 11.7% in males and 0% in females of C3B6F1 and 39.5% in males and 11.4% in females of C3H/HeN mice. In the four strains of mice thus far studied, including B6C3F1 hybrid (C57BL x C3H) which was previously studied, 252Cf irradiation has increased the tumor incidence dose-dependently in males and in females, but less effectively in females. The mean number and size of liver tumors were clearly correlated with tumor incidence. The incidence was always highest in C3H/HeN mice of both sexes, followed by B6C3F1, C3B6F1 and C57BL/6N mice. The influence of sex hormones was studied in B6C3F1 mice of both sexes after 200 cGy of 252Cf irradiation. In males, the incidence of liver tumors was significantly decreased from 55.2% to 23.3% and 25.9% after orchidectomy, and in females it was slightly decreased from 27.6% to 14.8% and 18.8% after ovariectomy. Supplementation of testosterone in orchidectomized mice did not restore the occurrence of liver tumors.     

Genetic control of hepatocarcinogenesis in C57BL/6J and C3H/HeJ inbred mice

Treatment of newborn male C3H/HeJ mice with N, N-dietnyl-nitrosamine(DEN) or N-ethyl-N-nitrosourea (ENU) resulted in the inductionof hepatocellular adenomas and carcinomas with a mean numberof tumors per animal that was 20-to 50-fold higher than thatfor similarly treated C57BL/6J male mice. We used two methodsto study the genetic basis for this difference in susceptibilityto liver tumor induction. Analysis of DEN-induced liver tumormultiplicities as a quantitative genetic trait in segregatingcrosses between C3H/HeJ and C57BL/6J mice indicated that allelicdifferences for at least two loci contributed to the highersensitivity to hepatocarcinogenesis of C3H/HeJ mice relativeto C57BL/6J mice. However, a single locus, which we have denotedHcs (hepato-carcinogen sensitivity), was responsible for 85%of the difference in susceptibility. The C57BL/6J and C3H/HeJalkies at this locus were semi-dominant. This result was confirmedby analysis of hepatocarcinogenesis by ENU in BXH (C57BL/6J? C3H/HeJ) recombinant inbred mice. Four of the nine recombinantinbred strains studied were highly susceptible to the inductionof liver tumors by ENU, three of the strains exhibited the resistantphenotype of the C57BL/6J parent, and two of the strains wereof intermediate sensitivity to hepatocarcinogenesis. Newbornmale C3H/HeJ and C57BL/6J mice did not significantly differin the extent of ethylation of hepatic DNA, or in the relativelevels of N-7-ethylguanine or O⁶-ethylguanine after treatmentwith [1-¹⁴C]DEN.     

p53 mutations are absent from carcinogen-induced mouse liver tumors but occur in cell lines established from these tumors.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined, by single-strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen-induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single-base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C----C:G or C:G----G:C transversions. Mutations at codon 61 of the c-Ha-ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c-Ha-ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro.     

Partial hepatectomy is a promoter of hepatocarcinogenesis in C57BL/6J male mice but not in C3H/HeJ male mice

We have shown previously that the difference between C57BL/6J and C3H/HeJ male mice in their susceptibilities to chemically-induced liver tumors results predominantly from an allelic difference at the Hepatocarcinogen sensitivity (Hcs) locus. This locus modulates the rate of growth of preneoplastic liver lesions and may also play a role in the turnover of normal hepatocytes in the adult liver. To define further the growth regulatory pathway of which the Hcs gene is a component, we asked whether the expression of the Hcs gene would modulate the response of preneoplastic liver lesions to the physiologic growth stimulus generated by a two-thirds hepatectomy. Twelve-day-old male and female C57BL/6J and C3H/HeJ mice were injected with 0.5 mumols N-ethyl-N-nitrosourea/g body weight. At six weeks of age half the animals received a two-thirds hepatectomy. Groups of animals were killed between 14 and 44 weeks of age for analysis of glucose-6-phosphatase (G6Pase)-deficient foci and hepatic tumors. The partial hepatectomy induced a regenerative response that caused both the G6Pase-deficient foci and the surrounding, histochemically normal hepatocytes to undergo several rapid rounds of division. As a result, the G6Pase-deficient foci were larger in the hepatectomized animals than in the sham operated controls. The foci in the non-hepatectomized C57BL/6J male mice grew more slowly than in the C3H/HeJ male mice [volume doubling time (Td) = 2.9 +/- 0.1 weeks and 2.0 +/- 0.6 weeks, respectively]. Following partial hepatectomy, the G6Pase-deficient foci in the C57BL/6J male mice maintained a significantly higher growth rate (Td = 2.2 +/- 0.3 weeks) than the foci in the sham operated C57BL/6J male mice. The partial hepatectomy did not have any long term effect on the growth rate of the G6Pase-deficient foci in the C3H/HeJ male mice nor in female mice of either strain. At 32 weeks of age, the mean liver tumor multiplicity for hepatectomized C57BL/6J male mice was approximately 5.3-fold greater than that for sham operated animals. In contrast, a two-thirds hepatectomy resulted in a 60% reduction in the number of liver tumors in C3H/HeJ male mice relative to sham operated mice. These data demonstrate that partial hepatectomy can act as a promoter of hepatocarcinogenesis in C57BL/6J male mice but not C3H/HeJ male mice. We propose that the Hcs gene and partial hepatectomy may promote hepatocarcinogenesis through the same pathway of growth regulation.     

Proto-oncogene activation in liver tumors of hepatocarcinogenesis-resistant strains of mice.

Activation of the ras family of oncogenes occurs frequently in liver tumors of the B6C3F1 mouse, a strain which is highly sensitive to hepatocarcinogenesis. Many other mouse strains are much more resistant to hepatocarcinogenesis; the aim of this study was to determine the frequency and pattern of oncogene activation in spontaneous and chemically induced liver tumors of three such strains, the C57BL/6J, the C57BL/6 x DBA/2 F1 hybrid (B6D2F1) and the C57BL/6 x Balb/c F1 hybrid (B6BCF1). The C57BL/6, DBA/2 and Balb/c strains are all relatively resistant to spontaneous hepatocarcinogenesis (1.5-3.6% of animals develop liver tumors in 2 years); with regard to chemically induced hepatocarcinogenesis the Balb/c is highly resistant, the C57BL/6 has low susceptibility and the DBA/2 has low to moderate susceptibility. The nude mouse tumorigenicity assay was used to search for activated oncogenes in 15 C57BL/6J liver tumors induced by a single neonatal dose of vinyl carbamate (VC, 0.15 mumol/g body weight). Three tumors contained H-ras genes activated by point mutations at codon 61 and one contained a non-ras oncogene. The polymerase chain reaction and allele-specific oligonucleotide hybridization were used to study H-ras mutations in spontaneous and VC-induced tumors from all three strains of mice. The frequency of H-ras codon 61 mutations in tumors induced by 0.15 mumol/g body weight VC in the C57BL/6J mouse (5/37) was similar to that in spontaneous tumors (2/9); surprisingly, tumors induced by a lower dose of VC (0.03 mumol/g body weight) had a higher frequency of H-ras mutations (12/28). The frequencies of H-ras activation detected in VC (0.03 mumol/g body weight)-induced tumors from the two F1 hybrids studied differed markedly. Only one VC-induced B6BCF1 tumor contained a mutated H-ras gene (1/10), whereas the majority of B6D2F1 tumors contained such mutations (23/33). Several spontaneous B6D2F1 liver tumors contained H-ras codon 61 mutations (6/15). Thus, H-ras activation frequency does not determine susceptibility to hepatocarcinogenesis in inbred mice and their F1 hybrids, since a relatively high frequency of H-ras mutations was observed in two resistant strains and a low frequency was found in the other strain.     

Gain of chromosomes 15 and 19 is frequent in both mouse hepatocellular carcinoma cell lines and primary tumors, but loss of chromosomes 4 and 12 is detected only in the cell lines.

Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6JxC3H/HeJ F(1) and C3H/HeJxC57BL/6J F(1) mice. In the primary tumors, non-random gain of chromosomes 15 and 19 was found in seven and five of 14 hepatocellular carcinomas, respectively. On the other hand, in the cases of both liver epithelial and hepatocellular carcinoma cell lines, frequent changes were loss of chromosomes 4 (4/9 cell lines) and 12 (3/9) as well as gain of chromosomes 15 (5/9) and 19 (4/9). These results indicate that the chromosomal gain is associated with both in vivo carcinogenesis and establishment of cell lines, while the loss is specific for the latter. PCR analysis using polymorphic microsatellite DNA markers revealed that the loss of chromosome 12 as well as chromosome 4 was much more frequent for the C57BL/6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.     

Comparison of pulmonary O6-methylguanine dna adduct levels and Ki-ras activation in lung tumors from resistant and susceptible mouse strains

The role of O⁶-methylguanine (O⁶MG) DNA adduct formation and persistence in the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)—induced lung tumors from resistant C57BL/6 and susceptible A/J mice was investigated. In addition, the frequencies of pulmonary tumor formation and Ki-ras activation were defined in C57BL/6 mice treated with NNK or vinyl carbamate (VC), and the role of the p53 gene in pulmonary carcinogenesis in these resistant mice was examined. One day after treatment with 100 mg/kg NNK, O⁶MG adduct concentrations were twofold to eightfold higher in Clara cells and type II cells than in small cells or whole lungs from both mouse strains. The concentrations of O⁶MG in isolated cells decreased at a similar rate in the two strains of mice. Lung tumors were detected by 27 mo of age in 18% of the C57BL/6 mice after a single 100 mg/kg dose of NNK and in 46% of these mice after a single 60 mg/kg dose of VC. In contrast, the tumor incidence in untreated C57BL/6 mice was 4%. Only one of 22 lung tumors from C57BL/6 mice treated with NNK contained an activated Ki-ras gene that was associated with an O⁶MG DNA adduct, whereas previous studies detected activated Ki-ras oncogenes in most of the NNK-induced lung tumors analyzed from susceptible A/J and resistant C3H mice. The small differences in formation and persistence of the O⁶MG adduct in whole lung or isolated lung cells from A/J and C57BL/6 strains do not account for the differences in either susceptibility for tumor formation or activation of the Ki-ras gene between these strains. In contrast to the low number of NNK-induced tumors with Ki-ras mutations in the resistant mice, 11 of 20 lung tumors from VC-treated mice contained activated Ki-ras genes. Neither p53 tumor suppressor gene mutations nor overexpression of the p53 protein were detected in spontaneous or chemically induced lung tumors in C57BL/6 mice. Thus, although Ki-ras activation was detected in some tumors, pathways independent of ras activation and p53 inactivation also appear to be involved in lung tumorigenesis in this resistant mouse strain.     

Further genetic analyses of skin tumor promoter susceptibility using inbred and recombinant inbred mice

To explore further the genetics of susceptibility of skin tumorpromotion in inbred mice, several aspects of responsivenessto 12-O-etradecanoylphorbol-13-acetate (TPA) were examined inC3H/He mice and segregating crosses between this mouse strainand C57BL/6 mice as well as BXD and BXH recombinant inbred (RI)strains. Dose-response relationships were established for skintumor promotion by TPA following initiation with 7, 12-dimethylbenz(a)anthracenein C3H/He and B6C3F1, as well as several other mouse stocksand strains included for comparison. The relative responsivenessto TPA skin tumor promotion was: SENCAR > > DBA/2 >C3H/He = B6D2F1 > B6C3F1 > > C57BL/6. Analyses of thesusceptibility of B6C3F2 and B6C3F1 x C57BL/6 backcross micesuggested that a minimum of two dominant genetic loci controlresponsiveness to phorbol ester promotion in these mice. Furtheranalysis of BXH and BXD RI strains suggested the presence offour distinct promotion-responsive phenotypes controlled bya minimum of two genetic loci. The existence of a ‘hyper-responsive’phenotype in the sets of RI strains, however, suggests thata third, recessive locus also may play a role in controllingresponsiveness to TPA, promotion. At 48 h after the last offour applications of TPA, marked hyperplasia and an increasein dark basal keratino-cytes were observed in C3H/He mice, whereasin B6C3F1 mice the response in these parameters was intermediatebetween C3H/He and C57BL/6 mice. A marked dermal inflamation,as determined by infiltration of polymorpho-nuclear cells, wasobserved in C3H/He and B6C3F1 mice, whereas little was notedin C57BL/6 mice. Furthermore, histological evaluations of selectedBXD RI strains revealed a significant correlation between themagnitude of the hyperplasia response and the percentage ofmice bearing tumors. The present data, in conjunction with ourprevious studies, confirm that the major gene(s) controllingsusceptibility to tumor promoter induced by TPA in two sensitivestrains (i. e. DBA/2 C3H/He) are similar of closely linked tothose for induction of sustained hyperplasia. In addition, thepresent data provide new evidence for a model where allelicdifferences at a minimum of three loci contribute to geneticdifferences in susceptibility to phorbol ester promotion inDBA/2 and C3H/He versus C57BL/6 mice.     

Lack of Allelic Preference in Amplification and Loss of the c-myc Oncogene in Methylcholanthrene-induced Mouse Sarcomas

Sarcomas were induced in Fl mice between C57BL/6N and C3H/He strains by subcutaneous injection of methylcholanthrene. The c-myc oncogene was found to be amplified in 16 cases among 43 sarcomas of C57BL/6N × C3H/He mice and 1 case among 5 sarcomas of the reciprocal cross. The origin of the amplified allele was determined by the polymerase chain reaction single strand conformation polymorphism analysis. Among the 17 sarcomas, only one had both of the alleles amplified. The rest of the tumors carried the amplified c-myc allele coming either from C57BL/6N (9 cases) or from C3H/He (8 cases). These results indicate that the c-myc allele is amplified randomly in methylcholanthrene-induced mouse sarcomas irrespective of its origin, such as paternal or maternal allele and C57BL/6N or C3H/He allele. In addition to these changes, the unamplified c-myc oncogene was found to be lost in 12 cases out of the 17 sarcomas with the amplification.     

Lack of allelic preference in amplification and loss of the c-myc oncogene in methylcholanthrene-induced mouse sarcomas.

Sarcomas were induced in F1 mice between C57BL/6N and C3H/He strains by subcutaneous injection of methylcholanthrene. The c-myc oncogene was found to be amplified in 16 cases among 43 sarcomas of C57BL/6N x C3H/He mice and 1 case among 5 sarcomas of the reciprocal cross. The origin of the amplified allele was determined by the polymerase chain reaction single strand conformation polymorphism analysis. Among the 17 sarcomas, only one had both of the alleles amplified. The rest of the tumors carried the amplified c-myc allele coming either from C57BL/6N (9 cases) or from C3H/He (8 cases). These results indicate that the c-myc allele is amplified randomly in methylcholanthrene-induced mouse sarcomas irrespective of its origin, such as paternal or maternal allele and C57BL/6N or C3H/He allele. In addition to these changes, the unamplified c-myc oncogene was found to be lost in 12 cases out of the 17 sarcomas with the amplification.     

Analysis of loss of heterozygosity in murine hepatocellular tumors

Because allelotype analysis of tumors has been important in the identification of new tumor suppressor genes, here we studied loss of heterozygosity (LOH) in a well-defined animal experimental system. We analyzed spontaneous liver tumors from C3HHc × C57BL/61 (B6C3F1) mice and urethane-induced hepatocellular tumors from (C3H/He × Mus spretus) × C57BL/6JBy (HSB) interspecific mice. A total of 95 different genetic markers were tested: 13 in 24 B6C3F1 tumors, 76 in 58 HSB tumors, and six in both groups. Minisatellite fingerprinting analysis detected one case of LOH and less than 1% genomic rearrangements in polymorphic and nonpolymorphic bands, respectively. There were no changes at hepatocellular susceptibility loci or at markers homologous to loci frequently lost in human hepatocellular carcinomas. Therefore, our results suggest that LOH and genomic rearrangements are uncommon in mouse hepatocellular tumors. © 1995 Wiley-Liss, Inc.     

Cell proliferation and global methylation status changes in mouse liver after phenobarbital and/or choline-devoid, methionine-deficient diet administration

Our laboratory is testing the hypothesis that hypomethylationof DNA [a decreased content of 5-methylcytosine (5MeC) comparedwith cytosine] facilitates aberrant oncogene expression involvedin tumorigenesis, using a model system of mouse strains withdiffering susceptibilities to liver tumorigenesis. The B6C3F1(C57BL/6 x C3H/He) mouse serves as the relatively susceptiblestrain and C57BL/ 6 serves as the relatively resistant strain.Phenobarbital (PB)and/or administration of a choline-devoid,methionine-deficient diet (CMD) were employed as non-genotoxichepatocarcinogens. We have examined hepatocyte and non-hepatocyteproliferation in conjunction with an assessment of global methylationchanges in liver DNA of B6C3F1 and C57BL/6 mice following thesepromoter treatments. Bromodeoxyuridine incorporation into DNA,used to measure cell proliferation indirectly, was visualizedby immunohistochemistry and quantified by aMacintosh-based imageanalysis system. Increased hepatocyte proliferation was demonstratedfollowing all three treatments.This increase was larger in C57BL/6(the relatively resistant strain) as compared with B6C3F1. Incontrast, global hypomethylation was evident to a larger extentin the B6C3F1 mouse, as compared with C57BL/6. PB led to hypomethylation(>20% decrease as compared with controls) at weeks 1, 2 and4 in B6C3F1, but not in C57BL/6 at the same time points. CMDdiet administration led to hypomethylation in both strains.At week 1, 21 and 9% decreasesin global methylation status wereobserved in B6C3F1 and C57BL/ 6 respectively. Evaluation ofthese data suggests that the heightened sensitivity of the B6C3F1mouse compared with the C57BL/6 is due, in part, to a decreasedcapacity for, or fidelity of, maintaining normal methylationstatus. The relatively resistant strain is better able to maintainthe normal methylation status of DNA in the face of a higherlevel of cell proliferation.     

Comparison of effect of tumor promoter treatments on DNA methylation status and gene expression in B6C3F1 and C57BL/6 mouse liver and in B6C3F1 mouse liver tumors

The effects of different liver tumor-promoting treatments (i.e., a choline-devoid, methionine-deficient (CMD) diet, phenobarbital (PB), or both) on Ha-ras and raf methylation status and expression were determined in mouse strains with different susceptibilities to liver tumor formation: the relatively sensitive B6C3F1 and the relatively resistant C57BL/6. Additionally, B6C3F1 mouse liver tumors, spontaneous or PB induced, were assessed for alterations in global DNA methylation status and expression of Ha-ras and raf. The CMD diet led to hypomethylation of Ha-ras and raf after 12 wk of administration in B6C3F1 and C57BL/6 mice. At this early phase of tumor promotion, the frequency of increased expression of both Ha-ras and raf mRNAs was higher in the B6C3F1 but not the C57BL/6 mice. This is a mechanism that may, in part, underlie the heightened sensitivity of the B6C3F1 mouse to liver tumorigenesis. Subpopulations of B6C3F1 mouse liver tumors displayed altered global methylation status, with both hypomethylation and hypermethylation evident. Carcinomas were significantly more hypomethylated than adenomas. The level of raf mRNA was not changed in spontaneous or PB-induced B6C3F1 mouse liver tumors. Increased expression of Ha-ras was evident in some spontaneous B6C3F1 liver tumors and in most of the PB-induced liver tumors. These experiments support the concept that altered DNA methylation plays a key role in tumorigenesis and indicate that the high propensity of the B6C3F1 mice to liver tumorigenesis may be due, in part, to a decreased ability to maintain normal methylation status. Mol. Carcinog. 18:97–106, 1997. © 1997 Wiley-Liss, Inc.     

Significance of Strain and Sex Differences in the Development of 252Cf Neutron-induced Liver Tumors in Mice

Mouse liver tumors occurring in C3H/HeN, C57BL/6N and C3B6F1 hybrid (C3H × C57BL) were studied following ²⁵²Cf fission neutron irradiation. Three strains of mice of both sexes (about 30 mice/group) were irradiated once with ²⁵²Cf at doses of 0,12.5, 50 and 200 cGy. The groups were observed for 13 months after irradiation. The incidence of liver tumors in the non-irradiated controls was 0% in both sexes of CS7BL/6N, 11.7% in males and 0% in females of C3B6F1 and 39.5% in males and 11.4% in females of C3H/HeN mice. In the four strains of mice thus far studied, including B6C3F1 hybrid (CS7BL × C3H) which was previously studied, ²⁵²Cf irradiation has increased the tumor incidence dose-dependently in males and in females, but less effectively in females. The mean number and size of liver tumors were clearly correlated with tumor incidence. The incidence was always highest in C3H/HeN mice of both sexes, followed by B6C3F1, C3B6F1 and C57BL/6N mice. The influence of sex hormones was studied in B6C3F1 mice of both sexes after 200 cGy of ²⁵²Cf irradiation. In males, the incidence of liver tumors was significantly decreased from 55.2% to 23.3% and 25.9% after orchidectomy, and in females it was slightly decreased from 27.6% to 14.8% and 18.8% after ovariectomy. Supplementation of testosterone in orchidectomized mice did not restore the occurrence of liver tumors.     

Analysis of lung tumorigenesis in chimeric mice indicates the Pulmonary adenoma resistance 2 (Par2) locus to operate in the tumor-initiation stage in a cell-autonomous manner: detection of polymorphisms in the Poli gene as a candidate for Par2

The Pulmonary adenoma resistance 2 (Par2) locus of the BALB/cByJ mouse, located within 0.5 cM of chromosome 18, is responsible for reducing the mean multiplicity of urethane-induced lung tumors relative to those in C57BL/6J, A/J and C3H/HeJ mice. Thus, BALB/B6-Par2 congenic strain genetically identical to BALB/cByJ except carrying C57BL/6J Par2 alleles develops seven times more tumors than BALB/cByJ. To gain clues for identification of Par2 candidate genes, we analysed lung tumorigenesis in BALB/cByJ<-->BALB.B6-Par2 chimeric animals. Of 100 tumors induced by urethane in 16 chimeras, 82 originated from BALB.B6-Par2 cells, indicating the Par2 phenotype to be cell-autonomous. In addition, the BALB.B6-Par2- and BALB/cByJ-derived tumors were similar in mean size, implying that the phenotype is primarily expressed during initiation rather than in the promotion stage of carcinogenesis. Given these results, we surveyed a comprehensive mouse genome database and physically mapped Par2 within a 2.3 Mbp segment containing three known genes, Poli, Mbd2 and Dcc. Among those, the Poli seemed to be the most reasonable Par2 candidate, since it encodes an extremely error-prone DNA polymerase preferentially incorporating G or T opposite template T in vitro, reminiscent of the Kras2 activation because of an A to G or T point mutation within codon 61 with which most urethane-induced lung tumors are initiated. Indeed, our sequencing of Poli cDNAs from BALB/cByJ, C57BL/6J, A/J and C3H/HeJ lungs revealed 21 BALB/cByJ-specific single-nucleotide polymorphisms in the coding region accompanied by seven amino-acid substitutions and an elevated frequency of alternative splicing, while no polymorphisms associated with tumor susceptibility were found for either Mbd2 or Dcc. Notably, we obtained evidence that BALB/cByJ Par2 alleles may selectively decrease the frequency of Kras2-mutated tumors compared with C57BL/6J alleles. Consequently, the Poli is an intriguing Par2 candidate clearly deserving further evaluation.     

Levels of 4-aminobiphenyl-induced somatic H-ras mutation in mouse liver DNA correlate with potential for liver tumor development

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation.     

Foreign-body tumors of mice: strain and sex differences in latency and incidence.

Sarcomas were induced by sc implantation of unplasticized polyvinylchloride acetate films in female and male mice of strains AKR/J, BALB/cJ, BALB/cWat, CBA/H and CBA/H-T6, C3H/HeJ, C57BL/10ScSn, C57BL/6J-bgj, C57BL/cdJ, DBA/-1J l/LnJ, LP/J, SJL/J, X/Gf, 129/J, and hybrids (CBA/H-T6 X AKR/J)F1, (C57BL/10ScSn x CBA/H or CBA/H-T6)F1, (C57BL/6J-bgj x C57BL/6J)F1. The strains and sexes showed marked differences in incidence and mean latency of resulting tumors. Crucial information was provided for the selection of appropriate mouse strains for the study of interrelationships between genotypes, defined somatic properties, and the multifactorial process of foreign-body tumorigenesis.     

The effect of connexin32 null mutation on hepatocarcinogenesis in different mouse strains.

Connexin32 (Cx32) is the major gap junctional protein in mouse liver. We have shown recently that the formation of liver tumours in Cx32-deficient mice is strongly increased in comparison with control wild-type mice, demonstrating that the deficiency in gap junctional communication has an enhancing effect on hepatocarcinogenesis. We have now compared the effect of Cx32 deficiency on liver carcinogenesis in two strains of mice with differing susceptibility to hepatocarcinogenesis. Heterozygous Cx32(+/-) females were crossed with male Cx32 wild-type C57BL/6J (low susceptibility) or C3H/He (high susceptibility) mice. Since the Cx32 gene is located on the X-chromosome, the resulting F1 males segregated to the genotypes Cx32(Y/+) and Cx32(Y/-). Genotyping was performed by PCR-analysis using tail-tip DNA. Weanling male mice were i.p. injected with a single dose of N-nitrosodiethylamine and were killed 16, 21 or 26 weeks later. The number, volume fraction and size distribution of precancerous liver lesions characterized by a deficiency in the marker enzyme glucose-6-phosphatase were quantitated. The results demonstrate that Cx32 deficiency only slightly affects the number of enzyme-altered lesions, but strongly enhances their growth, both in the resistant and the susceptible mouse strain, suggesting that decreased intercellular communication results in tumour promoting activity irrespective of the genetic background of the mouse strain used. Since Cx32-deficient C3H/He hybrids were approximately 5-10 times more sensitive than C3H/He hybrids with an intact Cx32 gene, this mouse strain may prove very useful for toxicological screening purposes.