Induction of mammary tumors in C57BL/HAAG female mice by a low oncogenic mammary tumor virus.

Mammary tumor virus (MuMTV)-negative mouse substrain C57BL/Haag foster-nursed by strain DD becomes a line with a moderately high incidence of mammary tumors occurring in the females at a late age. Electron microscopy revealed B particles in the tumors, providing additional evidence for the propagation and transmission of DD-MuMTV in C57BL/HaagfDD. Since both albino strains DD and RIII originated in Europe, develop pregnancy-dependent mammary tumors, carry black and agouti genes (cc, BB, AA), and therefore may be related, evidence is presented which suggests that the two naturally occurring MuMTVs from DD and RIII may also be related, and that they may share a common origin.     

Friend virus-induced transplantable tumors of C57Bl/6 origin containing unusually minute amounts of infectious virus

A transplantable solid tumor and its ascites variant were established from a graft of the enlarged spleen of a C57B1/6 mouse which had been infected with Friend virus shortly after birth. Repeated virus assays on these tumors were mostly negative. The virus content of the tumors was not more than one thousandth of that of the tumors which had been induced by the same virus in A and DDD mice. Under an electron microscope, the chance of observing viral particles in these tumor cells was extremely small. The inoculation of viable ascites tumor cells into allogeneic DDD mice induced neither tumor growth nor generalized leukemia, leaving specific resistance against the subsequent challenge of a histocompatible Friend virus-induced tumor. A single intraperitoneal injection of 2.0 × 10⁷ ascites cells sufficed to retard or suppress the growth of tumor cells used for the test challenge. The use of C57B1/6 mice for the study of Friend leukemia is discussed.     

Quantification of mouse mammary tumor virus structural proteins in hormone-induced mammary tumors of low mammary tumor mouse strains

The expression of the mouse mammary tumor virus (MMTV) in hormone-induced mammary tumors was investigated by means of a radioimmunoassay for two major MMTV proteins, gp52 and p27. MMTV proteins were isolated on lectin affinity- and ion-exchange chromatography columns. The purified viral proteins were electrophoretically homogeneous and retained immunoreactivity after labelling with 125iodine. Standard competition assays showed that group-specific antigenic determinants were reacting. Mammary tumors were induced in three strains of mice with a low natural incidence of mammary tumors, C57BL, O20 and C3Hf, by a combined hormone treatment, consisting of hypophyseal isografts and administration of progesterone and estrone. Mammary tumors and mammary glands of hormone-treated animals were extracted and used for competition radioimmunoassays. In general, the tumorigenic hormone treatment resulted in enhanced amounts of MMTV proteins in the mammary glands, compared to the amounts found in lactating mammary glands of untreated animals. The levels of MMTV proteins in the mammary tumors were lower than in the mammary glands.     

Heterogeneity of expression and induction of mouse mammary tumor virus antigens in mouse mammary tumors

Seven spontaneous BALB/cfC3H mouse mammary tumors were heterogeneous in expression of murine mammary tumor virus-associated cell surface antigens. To determine the basis of this heterogeneity, cells from spontaneous tumors and from five subpopulations isolated from a single spontaneous tumor were examined for expression of viral antigens under both conventional conditions and conditions known to induce synthesis of murine mammary tumor virus antigens (5-iodo-2'-deoxyuridine and dexamethasone). Induction with 5-iodo-2'-deoxyuridine resulted in further manifestation of the antigenic heterogeneity of spontaneous tumors. The five subpopulations from a single tumor differed in the amount of viral antigens present in untreated and in induced cultures. Coculturing showed that viral antigen expression was independent in each subpopulation within a heterogeneous mixture and was not influenced by the presence of other subpopulations with different potentials for viral antigen synthesis. The expression of murine mammary tumor virus structural antigens, a protein with a molecular weight of 28,000 and a glycoprotein with a molecular weight of 52,000, differed within the heterogeneous subpopulations, and was noncoordinate. The data suggest that the antigenic heterogeneity in spontaneous tumors reflects the existence of cells within them that differ in both expression of viral antigens and in their response to inducers of viral antigen synthesis.     

High murine mammary tumor virus expression in milk in a low mammary tumor mouse strain and high incidence of mammary tumor in hybrids produced by crossing with another low mammary tumor mouse strain

Large amounts of murine mammary tumor virus (MMTV) were expressed in milk from a low mammary tumor mouse strain called II-TES, established by crossbreeding DBA/2 mouse strain with two strains of Japanese pet mouse origin. The reciprocal hybrids of the II-TES strain and OZ-F strain, another low mammary tumor strain of Japanese pet mouse origin, developed early-appearing mammary tumors at a high rate, and large quantities of MMTV were expressed in the milk. These findings suggest that different regulatory genes control MMTV expression and mammary tumorigenesis.     

Origin and progression of pregnancy-dependent mammary tumors induced by new mouse mammary tumor virus variants

In order to study mechanisms of progression of mouse mammary tumor virus (MMTV)-induced pregnancy-dependent mammary lesions, we removed and serially transplanted 17 small tumors detected in MMTV-infected pregnant females. This gave rise to the same number of 'in vivo' tumor lines. Hormone-dependency of the passages was determined by comparing tumor development in multiparous versus virgin hosts. We found that the first passages of most of these lesions (11/17) required pregnancy to grow. However, all these tumor lines lost their hormone-dependence through successive passages. The original pregnancy-dependent lesions were mostly multiclonal and showed high levels of estrogen and progesterone receptors. Alternatively, pregnancy-independent tumors arose as clonal dominant populations exhibiting a lower hormone receptor content. Our data show that the progression of hormone-dependent MMTV-induced mammary tumors is an irreversible process associated with the appearance of additional MMTV insertional events as well as alterations in the composition of the tumor cell population.     

Peroxidase activity and iodide uptake in hormone-responsive and hormone-independent GR mouse mammary tumors.

Transplanted mammary tumors growing in the inbred GR/AFib mouse were assayed for peroxidase activity and ability to concentrate injected 125I. Both tumor peroxidase activity and iodide uptake were about ten times greater in the hormone-resonsive (HR) tumors than in the hormone-independent tumors. However, although peroxidases are known for their ability to participate in the iodination of proteins, over 90% of the radioactive iodine found in the tumors was shown to be free iodide. This finding suggests that these two parameters may be independent of each other, but both are higher in HR tumors.     

Elevated frequency of loss of heterozygosity in mammary tumors arising in mouse mammary tumor virus/neu transgenic mice.

Loss of heterozygosity (LOH) analysis was performed on 62 mammary tumors that were induced in (BALB/c x C57BL/6)F1 mouse mammary tumor virus/neu transgenic mice. Eighty-six simple sequence length polymorphism markers were used to cover all of the somatic chromosomes. Frequency of LOH was observed to be significant for chromosomes 4 (50%), 19 (32%), and 8 (21%). On chromosome 4, at least three distinct regions of allelic deletions could be identified: one proximal to 22 cM; the second close to the p16INK4a/p15INK4b locus, which is commonly deleted in various tumors; and the third one in the proximity of Mom1. The frequency of LOH on chromosome 19 was the same for the four markers used. Our data suggested the presence of two distinct LOH loci, one proximal to 47 cM and the other at the distal region. On chromosome 8, possibly two distinct LOH loci could be recognized, one around 52 cM and the other one at 67 cM or distal to it. These regions map close to E-cadherin (Cdh1) and M-cadherin (Cdh15) loci, respectively. Because LOH sites are thought to harbor tumor suppressor genes, this allelotype screening has allowed the mapping of putative tumor suppressor genes that may be implicated, in collaboration with the erbB-2/neu oncogene, in the development of mammary tumors in these transgenic mice.     

Point mutation analysis of ras genes in spontaneous and chemically induced C57Bl/10J mouse liver tumours.

The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.     

Immunology of the mouse mammary tumor virus: comparison of mammary tumor virus with the agent found in C3Hf/Crgl mice

C3Hf/Crgl mice, containing no biologically active mammary tumor virus, nonetheless possess both type-B virus particles and a component which cross-reacts serologically with the mammary tumor virus. Neutralization studies and the immunodiffusion assay for measuring the antigenicity of the mammary tumor virus indicate that transfer of the virus particles from C3Hf to BALB/cCrgl mice is accompanied by transfer of the cross-reacting antigenic component.     

Influence of age on induction of mammary tumors by diethylstilbestrol in C3H/HeN mice with low murine mammary tumor virus titer

C3H/HeN female mice with low murine mammary tumor virus titer (MTV-) were fed diets containing a targeted concentration of 640 ppb diethylstilbestrol [(DES) CAS: 56-53-1; 4,4'-(1,2-diethyl-1,2-ethenediyl)bis-phenol]. Mice were started on DES at 3, 5, 7, or 9 weeks of age. Some continued on the diet throughout the rest of their life-spans, whereas others were killed as soon as they had been fed DES for 2, 4, 6, 8, 10, or 12 weeks. Controls were also examined throughout the study. Among mice killed early, the only observation significantly influenced by age at the start of DES treatment was the presence or absence of corpora lutea (CL). DES did not prevent CL from appearing in mice started on DES at 7 or 9 weeks of age, but it did prevent their appearance in about 25% of the mice started at 5 weeks and in up to 75% of the mice started at 3 weeks of age. In the life-span-exposure groups, CL either disappeared or were never formed in 88% or more of the mice, regardless of age at the start of treatment. Neoplastic or presumptive preneoplastic lesions apparently influenced by DES in the life-span-treatment groups included ovarian tubular adenomas; granulosa cell tumors and luteomas; pituitary cystoid degeneration, hyperplasia, and adenomas; uterine adenocarcinomas and cervical adenosis; mesotheliomas; and mammary hyperplastic alveolar nodules (HANs) and adenocarcinomas. Luteoma and granulosa cell tumor incidences were reduced by DES, regardless of age at the start of treatment. Influence of age at the start of treatment was minimal or not apparent for mesotheliomas, uterine adenocarcinomas, or pituitary adenomas; however, pituitary cystoid degeneration and hyperplasia and cervical adenosis occurred in higher frequency and/or with shorter duration of DES exposure the earlier that treatment was started. A delay in the start of DES treatment was associated with a remarkable delay in HAN and mammary adenocarcinoma development. This was especially apparent in young mice (3-7 wk old) in which a 2-week delay in treatment resulted in a 20-week delay in HAN or tumor onset. Age at the start of treatment was a major factor in susceptibility of C3H/HeN-MTV- female mice to DES-induced mammary tumorigenesis.     

Conversion of C57Bl/6 mice from a tumor promotion-resistant to a -sensitive phenotype by enhanced ornithine decarboxylase expression.

A transgenic mouse model was developed in which ornithine decarboxylase (ODC) can be overexpressed in a tissue-specific and regulated manner. Hair follicle keratinocytes were targeted by use of a bovine keratin 6 (K6) promoter/regulatory region, and regulation was accomplished by using the tetracycline-regulated transactivator/tetracycline-response element system. Double-transgenic mice carrying both transgenes (K6/tetracycline-regulatable transactivator protein (tTA) and tetracycline-response element/Odc) on a C57Bl/6 background had no obvious phenotypic abnormalities in the absence (Odc transgene-expressed) of doxycycline (a tetracycline analog) in the drinking water. However, induction of K6-driven tTA expression by the tumor promoter (12-O-tetradecanoylphorbol-13-acetate) (TPA) led to very high levels of epidermal ODC activity and robust hyperplasia, especially involving hair follicles. Both effects were abolished by inclusion of doxycycline in the drinking water to repress transgene expression. Finally, the number of papillomas that developed in a standard (7,12-dimethybenz[a]anthracene) (DMBA)/TPA protocol was greatly reduced in mice in which transgenic Odc expression was repressed by doxycycline. Our results demonstrated that the higher levels of ODC expression produced in the transgenic model in the induced versus the repressed condition make the normally promotion-resistant C57Bl/6 strain much more sensitive to the short-term and long-term (i.e., tumor-promoting) effects of TPA.     

Autogenous immunity to mouse mammary tumor virus in mouse strains of high and low mammary tumor incidence.

Specific radioimmune precipitation assays were utilized to demonstrate the presence of precipitating antibodies to mouse mammary tumor virus (MMTV) in the high-spontaneous mammary tumor strains of mice: C3H/HeN+, GR/N, BALB/cfC3H, and C57BL/6 X C3H F1 (hereafter called B6C3F1). Antibody titers in C3H/HeN+ mice increased with age, with highest titers observed in tumor-bearing animals. MMTV-precipitating antibodies were not detectable by radioimmune precipitation assay in low-mammary tumor strains (AKR, BALB/c C57BL/6, and C3H/HeN-) but were detectable in MMTV-inoculated BALB/c mice. Appearance of antibodies preceded palpable tumor formation, and antibody titers were directly correlated to virus dose. Natural antibody to MMTV in C3H/HeN+ and B6C3F1 mice coexists with the murine leukemia virus natural antibody as determined by competition radioimmunoassays.     

Dominance of a tumor subpopulation line in mixed heterogeneous mouse mammary tumors

When mixtures of cell lines 168 and 4T07, both derived from the same mouse mammary tumor, were injected into syngeneic mice, the resulting tumors, analyzed over a large size range by colony-forming assays in selective media, consisted primarily of line 4T07, even when the ratio injected was 100:1 or greater in favor of line 168. This result indicated a suppression of growth of line 168, since the volume-doubling time of line 168 tumors in the absence of line 4T07 was one-half that of line 4T07 tumors. That growth suppression was not due to inhibition of line 168 by immunity induced to line 4T07 was shown in two ways: (a) line 168 tumors grew almost as well in mice preimmunized with line 4T07 as in controls, whereas line 4T07 tumor growth was strongly inhibited in preimmunized mice; and (b) the final composition (favoring line 4T07) in mixed tumors was similar in tumors grown in mice immunosuppressed by irradiation to that in nonirradiated controls. The strong suppression of line 168 did not occur when the two cell lines were injected simultaneously at different s.c. sites, nor did it occur when line 168 cells were injected in mixtures with lethally irradiated line 4T07 cells. Line 4T07 cells also suppressed the growth of line 168 cells in monolayer cultures. It was not likely that suppression was due to competition for growth factors, since the effect required cell contact. Suppression probably was not mediated through junctional communication, since these cells do not engage in metabolic cooperation. We suggest that a growth-inhibitory factor produced by line 4T07 mediates the suppression of 168 cells.