BALB/c alleles for Prkdc and Cdkn2a interact to modify tumor susceptibility in Trp53+/- mice

In mice heterozygous for p53 (Trp53(+/-)), the incidence of mammary tumors varies among strains, with C57BL/6 being resistant and BALB/c being susceptible. Mammary tumor phenotypes were examined in female Trp53(+/-) F1 mice (C57BL/6 x BALB/c;n = 19) and N2 backcross mice [(C57BL/6 x BALB/c) x BALB/c] (n = 224). Susceptibility to mammary tumors segregated as a dominant phenotype in F1 females, but a higher frequency and shorter latency in N2 mice indicated a contribution from recessive-acting modifiers. Segregation of the hypomorphic BALB/c alleles for DNA-dependent protein kinase catalytic subunit (Prkdc) and p16(INK4A) (Cdkn2a) was analyzed in the N2 mice. The time to first tumor (considering all tumor types) was significantly different among the four genotype combinations (P = 0.01). Cdkn2a had a strong effect (P = 0.008) but was restricted to Prkdc(B/B) mice (P = 0.001), indicating a strong interaction between the loci. Differences in mammary tumor occurrence among genotypes for Prkdc and Cdkn2a in N2 mice were not statistically significant. This study indicates that BALB/c Prkdc and Cdkn2a alleles do modify tumor incidence in Trp53(+/-) mice and highlights the complexity of gene interaction effects in determining cancer phenotypes but discounts these alleles as major recessive loci contributing to spontaneous mammary tumor susceptibility.     

Loss of p53 in benzene-induced thymic lymphomas in p53+/- mice: evidence of chromosomal recombination

The purpose of this study was to examine the role of chromosomal recombination in mediating p53 loss in benzene-induced thymic lymphomas in C57BL/6-Trp53 haploinsufficient (N5) mice (p53+/- mice). We characterized loss of heterozygosity (LOH) on chromosome 11 using seven microsatellite markers in 27 benzene-induced and 6 spontaneous thymic lymphomas. Eleven patterns of LOH were found between the induced and spontaneous tumors, with only one pattern being in common between the tumor groups. Nearly 90% (24 of 27) of benzene-induced tumors exhibited loss of the functional p53 allele locus, and 83% (20 of 24) of these tumors retained two copies of the disrupted p53 allele. The results indicate that benzene induces a high frequency of LOH on chromosome 11 in p53+/- mice, likely mediated by aberrant chromosomal recombination.     

Different properties of mammary carcinogenesis induced by two chemical carcinogens,DMBA and PhIP,in heterozygous BALB/c Trp53 knockout mice

Chemical carcinogens, such as 7,12-dimethylbenz[a]anthracene (DMBA) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), are known to induce mammary carcinomas in mice and rats. In the present study, the phenotypic and genotypic characteristics of carcinogen-induced mammary carcinogenesis in heterozygous BALB/c tumor protein p53 (Trp53) knockout mice were examined with reference to published data surrounding human breast cancer. A significantly accelerated induction of mammary carcinomas was observed following a single dose of DMBA (50 mg/kg of body weight at 7 weeks of age), and a modest acceleration was induced by PhIP (50 mg/kg of body weight) administered by gavage 6 times/2 weeks from 7 weeks of age. DMBA-induced mammary carcinomas were histopathologically characterized by distinct biphasic structures with luminal and myoepithelial cells, as well as a frequent estrogen receptor expression, and PhIP-induced carcinomas with solid/microacinar structures consisted of pleomorphic cells. Of note, DMBA-induced mammary carcinomas were characterized by a HRas proto-oncogene (Hras) mutation at codon 61, and gene/protein expression indicating MAPK stimulation. PhIP-induced lesions were suspected to be caused by different molecular mechanisms, including Wnt/β-catenin signaling and/or gene mutation-independent PI3K/AKT signaling activation. In conclusion, the present mouse mammary carcinogenesis models, induced by a combination of genetic and exogenous factors, may be utilized (such as the DMBA-induced model with Trp53 gene function deficiency as a model of adenomyoepithelioma, characterized by distinct biphasic cell constituents and Hras mutations), but PhIP-induced models are required to further analyze the genetic/epigenetic mechanisms promoting mammary carcinomas.     

Loss of JNK2 increases intestinal tumor susceptibility in Apc1638+/- mice with dietary modulation

A recent study has shown that c-Jun NH2-terminal kinases (JNKs) 2 interacts with and inhibits β-catenin signaling in vitro. To determine the role of genetic interaction between JNK2 and β-catenin in vivo and to elucidate JNK2-mediated intestinal carcinogenesis, we crossed the JNK2-/- mice with Apc1638+/- mice that carry inactivated Apc allele and develop intestinal tumor due to β-catenin activation. We found that the introduction of mutant JNK2 into Apc1638+/- mice did not increase intestinal tumorigenesis when the mice were fed a defined AIN-76A control diet. However, loss of JNK2 significantly increased animal body weight in the Apc/JNK2+/- and Apc/JNK2-/- mice. Surprisingly, JNK2 loss was synergistic with a Western-style high-risk diet (high fat and phosphate and low calcium and vitamin D) to accelerate intestinal tumorigenesis. Tumor number increased to 3.56 from 1.89 (on AIN-76A diet) in the Apc/JNK2+/- mice (P<0.01) and increased to 4.14 from 1.92 (on AIN-76A diet) in the Apc/JNK2-/- mice (P<0.01) although there was a slight increase of tumor formation in Apc/JNK2+/+ mice. Intestinal tumorigenesis in Apc/JNK2 double-mutant mice with high-risk diet modulation was associated with β-catenin signaling, peroxisome proliferator-activated receptor-γ and inflammation pathway. Collectively, we concluded that JNK2 may function in controlling fat metabolism and loss of JNK2 increases the risk of obesity, the latter synergizes with high-fat diet to increase intestinal tumor susceptibility. This data strongly suggests the importance of JNK2 in intestinal carcinogenesis and the importance of dietary manipulation for cancer prevention in the population whose JNK2 is inactivated.     

Progesterone induction of mammary carcinomas in BALB/c female mice

Summary We have demonstrated that medroxyprogesterone acetate (MPA), when administered in high doses, induces mammary carcinomas in virgin female BALB/c mice. Since one of the possible explanations for this effect was its progestagenic effects, we decided to investigate whether progesterone (Pg) alone could also induce mammary adenocarcinomas in our model and if MPA at doses lower than those used to establish the model was also carcinogenic. A total of 136 mice were subdivided into 3 groups: Group 1, 44 mice were implanted s.c. with 40 mg Pg silastic pellets at the beginning of the experiment, and 6 months later with a 20 mg Pg pellet; Group 2, 45 mice were similarly treated with MPA pellets; Group 3, 47 mice were inoculated s.c. with 40 mg MPA every three months. At the end of 20 months, 9 animals had developed mammary tumors in Group 1, 18 in Group 2 and 34 in Group 3 (actuarial incidence = 28%, 58%, and 98%, respectively); tumor latency was similar in all groups: 46.2 ± 13.1, 51.3 ± 9.9, and 50.1 ± 2.1 weeks, respectively. Seven (Group 1), 14 (Group 2), and 25 (Group 3) tumors were transplanted into syngeneic mice to determine progestin dependence. All tumors, except one from Group 1, were histologically characterized. In Group 1 (Pg 60 mg), 4 tumors (67%) were infiltrating lobular carcinomas and 2 were ductal carcinomas (33%). In Group 2 (MPA 60 mg), 2 tumors (14%) were lobular and 12 were ductal adenocarcinomas (86%) (Group 1 vs Group 2: p < 0.05), whereas in Group 3 (MPA 160 mg), 8 were lobular carcinomas (32%) and 17 were ductal carcinomas (68%). In syngeneic passages all lobular tumors behaved as progestin independent (PI) and ductal tumors as progestin dependent (PD). All ductal tumors, except one, expressed estrogen receptors (ER) and progesterone receptors (PR), whereas receptor expression was variable in lobular carcinomas. It can be concluded that Pg induces mostly lobular, PI mammary tumors in BALB/c female mice. The fact that most MPA-induced tumors are ductal and PD suggests that the two hormones use different carcinogenic pathways.     

Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/-) mouse tumors is carcinogen-and tissue-dependent

Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.     

Bioactivity of C3H and RIII mammary tumor viruses in virgin female BALB/c mice.

The bioactivities of C3H and RIII mammary tumor virus (MuMTV) in virgin female BALB/c mice differed. The average number of mammary hyperplastic alveolar nodules per mouse after noduligenic tests was 20.1 in BALB/cfC3H and 10.7 in BALB/cfRIII females. Spontaneous mammary tumor incidence after 20 months of observation was 47.5% in BALB/cfC3H and 14.6% in BALB/cfRIII females (P less than 0.01). The frequency of lung metastases in mammary tumor-bearing mice was 63.1% in BALB/cfC3H and 16.6% in BALB/cfRIII females (P less than 0.05), although the clinical duration of mammary tumors was the same. These data demonstrated a lower bioactivity of RIII MuMTV when compared to C3H MuMTV in BALB/c mice and suggested that the causative virus may control all the steps of mouse mammary tumor development, including metastasis.     

Expression of differentiation-specific proteins in preneoplastic mammary tissues in BALB/c mice

The levels of milk-specific mRNAs (alpha-casein, whey acidic protein) and proteins (casein, alpha-lactalbumin) were examined in the DIM series of mammary preneoplastic outgrowth lines and tumors. The constitutive production of casein protein was variable among the preneoplastic DIM outgrowth lines maintained in virgin mice. Outgrowth line DIM-2 consistently expressed a very low level of casein mRNA and protein but no detectable alpha-lactalbumin protein. Outgrowth line DIM-4 expressed a high level of casein protein and no alpha-lactalbumin; however, by transplant generation 9, the levels of casein mRNA and protein were significantly decreased but remained greater than those found in DIM-2. Outgrowth line DIM-3 expressed high levels of casein protein during all transplant generations examined and sporadically exhibited detectable amounts of alpha-lactalbumin. The level of beta-casein mRNA in DIM-3 was 7 times greater than seen in DIM-4 outgrowths, but still only 2% of that measured in the normal mammary gland from lactating mice. The majority of tumors derived from the DIM outgrowth lines expressed very low levels of beta-casein mRNA and total casein protein, although occasionally tumors were observed with very high levels of casein expression. Immunoblot analysis of cellular extracts indicated that alpha, beta-, and gamma-caseins were expressed in the three outgrowth lines to varying degrees. Whey acidic protein mRNA attained barely detectable levels in the best of cases. Only outgrowth line DIM-3 responded to a normal lactogenic stimulus (lactating mouse) with significantly increased levels of milk-specific products. As determined by avidin-biotin peroxidase staining, the percentage of alveoli expressing beta-casein and alpha-lactalbumin proteins in lactating mice increased from 20 and 0%, respectively, in virgin mice to 85 and 40%, respectively, in lactating mice. Similarly, the levels of mRNA for beta-casein and whey acidic protein increased 8- and 5.5-fold, respectively. These results indicate that the cell populations selected by serial transplantation of preneoplastic mammary tissues fall into at least three categories with respect to expression of mammary-specific differentiation products: uninducible, inducible, constitutive. The DIM-3 outgrowth line appears to represent a highly inducible cell population. As concluded in earlier investigations, there was no correlation between secretory activity, morphology of the outgrowths, and tumor-producing capabilities in virgin mice. The effect of a normal lactogenic stimulus on the tumor potential of the DIM-3 line is currently under investigation.     

Kinetics of mammary epithelial cell proliferation in pituitary isografted BALB/c mice

Recently, we have published that treatment of pituitary isograftedBALB/c mice with a single injection of N-methyl-N-nitrosourea(MNU) leads to the rapid development of mammary tumors in over90% of the animals (Guzman et al., Cancer Res., 52, 5732–5737).In the present study, we characterized the changes in proliferativeactivity and lobulo—alveolar differentiation of MECs atdifferent time intervals after isografting animals with pituitaryglands. Virgin BALB/c mice 1, 3, 5 or 8 weeks after pituitaryisografting were either pulse-labeled for 2 h or continuouslyinfused with bromodeoxyuridine (BrdU) and the percentage ofBrdU-labeled MECs was assessed. The S-phase duration (T_s) ofMECs was evaluated by double labeling with [³H]thymidine andBrdU. The population potential doubling time (T_p) was calculatedfrom the values of BrdU-LI and T_s. Three stages of proliferationand differentiation of MECs in pituitary isografted virgin BALB/cmice were observed: (i) A sharp increase in the percentage ofproliferating MECs of the terminal ducts and ductal branchingsin the first 1–2 weeks, (ii) Development of lobulo—alveolarstructures from the terminal ductal and alveolar buds, betweenweeks 3 and 5 with the highest BrdU-LI in week 3 and (iii) Multiplicationof the alveolar structures and decrease in the BrdU-LI betweenweeks 5 and 8. The BrdU-LIs of alveolar cells 5 weeks afterisografting the animals were significantly higher than thoseof the ductal cells. The continuous administration of BrdU for3, 5 or 7 days by using osmotic pumps revealed zones in theducts where almost all MECs were labeled as well as zones lackingproliferate activity. When the BrdU administration was extendedfor 10–14 days, almost all (>95%) ductal and lobularepithelial cells were labeled. A small percentage (<5%),of ductal and lobulo-alveolar MECs cells, remained unlabeledeven after 14 days infusion of BrdU. The T_s and T_p values wereshorter in pituitary isografted animals than in controls, butno significant difference was found for either values betweenthe ductal and alveolar cells in either isografted or controlmice. Changes in proliferation kinetics of mouse MECs in pituitaryisografted animals correlated with the circulating concentrationsof prolactin, progesterone and 17ß-estradiol, butnot with corticosterone, growth hormone or thyroxin. We proposethat these time dependent differences in the structural compositionand proliferative activity of MECs in pituitary isografted animalscan be used as a model system for evaluation of the role ofcell proliferation and differentiation in mammary carcinogenesis.In a parallel study, we used this model system to induce mammarytumors by a single injection of MNU in mice isografted withpituitaries for 1, 3, 5 or 8 weeks. We observed that MNU inducedmammary carcinogenesis in pituitary isografted animals requiredelevated proliferative activity of MECs at the time of carcinogenadministration (Swanson et al., submitted).     

Kinetics of responses to tumor antigens and mouse mammary tumor virus in BALB/cCrgl and BALB/cfC3H mice

Spleen cells from BALB/cCrgl mice responded to murine mammary tumor virus (MuMTV) in cell-mediated immune assays at higher levels than did the spleen cells from the syngeneic BALB/cfC3H mice. Implantation in BALB/cCrgl with a chemically induced mammary tumor and in BALB/cfCrgl mice with spontaneous mammary tumors (SMT) arising in the same strain resulted in sensitization of these animals to the antigens of their tumors. Reactivities peaked 3 weeks after transplantation, whereas no positive reactions could be detected when tumors reached maximum size. A kinetic study with the use of MuMTV antigen(s) showed that the responses of lymphocytes from BALB/cfC3H with SMT followed the same pattern as that obtained with tumor antigens, which indicated that this might be a de novo sensitization. In sharp contrast, a steady type of response to MuMTV was observed with the spleen cells from BALB/cCrgl mice; i.e., the levels of responsiveness to MuMTV did not significantly vary at any stage of tumor development. In vivo studies explored the possible relevance of the in vitro cell-mediated immunity to the host defenses. MuMTV-expressing mammary tumor cells were implanted in BALB/cCrgl and BALB/cfC3H mice. The total incidence of tumors was significantly reduced and a delay occurred in their time of appearance in BALB/cCrgl mice in relation to BALB/cfC3H animals. Thus the in vitro reactivity to MuMTV antigen(s) in BALB/cCrgl mice was found to be coincidental with a degree of protection against the development of MuMTV-expressing mammary tumors.     

Induction of mammary adenocarcinomas by medroxyprogesterone acetate in BALB/c female mice

In a previous paper we reported that medroxyprogesterone acetate (MPA) decreased the incidence of foreign body tumorigenesis in BALB/c mice but that mammary adenocarcinomas appeared in some of the females. The experiment was repeated in 245 virgin females as follows: (1) 40 mice treated with 40 mg of MPA depot s.c. every 2 months during a whole year; (2) 117 mice bearing a foreign body (FB) and treated with MPA; (3) 46 mice bearing a FB; (4) 42 non-treated mice. Mammary adenocarcinomas developed in 16/40 in group 1 and 30/117 in group 2; no mammary tumors appeared in either control groups. The tumors were infiltrating adenocarcinomas often affecting more than one mammary gland; metastases were occasionally observed. Animals killed after 1 year of MPA treatment presented deciduomas. MPA also decreased the incidence of FB-induced sarcomas, confirming previous results.     

Murine mammary tumor virus seroepidemiology in BALB/cfC3H mice: correlation with tumor development

Serum reactivity to murine mammary tumor virus (MuMTV) was inversely related to mammary tumor risk in 8-to 22-week-old BALB/cfC3H breeding females. Mice at low tumor risk exhibited high-titered serum reactivity to MuMTV (50% end point, greater than or equal to 1:40 by radioimmunoassay) approximately 3-6 months earlier than did the mice at high tumor risk. Maternal MuMTV antibody levels were correlated with the serum anti-MuMTV reactivity of their neonatal offspring (2 wk of age). Serologic antiviral reactivity in infected mice did not change during periods of pregnancy and lactation. All infected animals had detectable serum MuMTV reactivity by 33 weeks of age. The virus-neutralizing capabilities of some of these sera were tested, Sera from some of the young, low-tumor-risk animals that had MuMTV-precipitating antibodies also had virus-neutralizing activity. Conversely, none of the sera from the high-tumor-risk animals had detectable MuMTV-neutralizing antibodies.     

Inflammatory effects of the tumor promoter croton-oil in BALB/c mice skin

A single dose of 0.25% croton oil induced an edema when applied to the ears of BALB/c mice. The maximal edematous response resulted from a single application of 4% croton oil. At any dose level, edema maximized 6-7 h after croton oil application, waning thereafter to the control level by 30 h. When topically applied on the shaved back skin, a single dose of 0.25% croton oil induced an inflammatory effect characterized by epidermal hyperplasia and infiltration of polymorphonuclear leucocytes (PMNs) in the dermis. Higher doses of croton oil caused more remarkable inflammatory response. The histological changes which were slightly detected in the skin 4 h after application of croton oil, reached a maximal level by 20-27 h, but substantially subsided by 72 h. Results proved that the tumor-promoter croton oil induces an inflammation in mice skin in a dose- and time-dependent process.     

Influence of dietary fat on the growth of mammary ducts in BALB/c mice

Essential fatty acid (EFA)-deficient diets, when fed to weanling BALB/c mice, retarded the rate of mammary ductal growth. Ductal growth was also markedly retarded in pups fed only EFA-deficient foods from birth (milk from mothers fed EFA-deficient diets for the first 3 wk until weaning, then an EFA-deficient diet for an additional 5 wk). The ability of dissociated mammary epithelial cells to form outgrowths was reduced, and the growth rate of those outgrowths that did form was retarded when such cells were injected into gland-free mammary fat pads in syngeneic hosts fed EFA-deficient diets. Indomethacin, an inhibitor of prostaglandin synthesis, when added at a level of 0.003% to a 15% corn oil-containing diet, resulted in the retardation of ductal growth to about the same extent as did a 15% hydrogenated cottonseed oil-containing diet; however, unlike that associated with EFA-deficient diets, the retardation associated with indomethacin was temporary. EFA appears to be required for normal ductal growth in BALB/c mice, and prostaglandins may be involved in the growth-regulating process.     

Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene–induced mammary tumors in BALB/c and p53 hemizygous mice

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridizations detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occured in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration. © 1994 Wiley-Liss, Inc.