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The distribution of mouse mammary tumor virus (MuMTV) antigens was studied in normal, preneoplastic, and neoplastic mammary epithelia from female BALB/cfC3H mice with the use of a polyvalent anti-MuMTV serum and indirect immunoperoxidase techniques. The MuMTV antigens were on the apical surface or in focal cytoplasmic aggregates or diffused throughout the infected cells. As much as 70% of all cells in adenocarcinomas and as much as 100% of all cells in preneoplastic hyperplastic alveolar nodules contained MMTV antigens. Comparable percentages of cells from mammary glands of multiparous mice were MuMTV-positive. Some mammary tissues of nullparous and primiparous mice did not contain detectable MuMTV antigen. The MuMTV antigen-containing cells in lactating mammary glands tended to be in discrete lobuloalveolar clusters surrounded by antigen-negative alveoli. The percentage of MuMTV-positive cells in a given gland was proportional to the amount of virus found in the animal's milk.

Long-term primary cultures of mouse mammary tumor cells proved an excellent source of mouse mammary tumor virus (MMTV). Virus purified from these primary cultures had the same morphologic biochemical, immunologic, and biologic characteristics as MMTV. Quantitation of MMTV-protein equivalents released into the medium was measured by the radioimmunoassay for MMTV. Peak production levels were 20-40 mug MMTV protien equivalents/75-cm-2 flask/24 hours. These cultures produced MMTV for as long as 90 days. MMTV cultivation depended on the initial cell-plating density and hormones. Maximal MMTV release was obtained at a plating density of 1 times 10-6 cells/cm-2 in the presence of insulin and hydrocortisone. Insulin alone gave basal levels of MMTV, and hydrocortisone alone increased MMTV release only three-fold, but insulin and hydrocortisone together effected an eightfold increase in MMTV release. This suggested that hydrocortisone had a primary effect on MMTV release and insulin acted synergistically with hydrocortisone to maximize MMTV release.

Mammary tumorigenesis in some mouse strains is characterized by the appearance of a preneoplastic lesion, the hyperplastic alveolar nodule (HAN). The biology of the HAN has been characterized primarily through the study of stable outgrowth lines of serially transplanted HAN. One outgrowth line, Dl, which was developed and carried in female BALB/c mice, has been described as a low-tumor-incidence line that does not express murine mammary tumor virus (MuMTV) and is susceptible to hormonal, chemical, and viral carcinogens. In this report, a high-tumor-incidence subline of Dl, Dl/UCD, is described. Although Dl/UCD, like Dl, is susceptible to the chemical carcinogen 7,12-dimethylbenz[a]anthracene, an increase in tumor incidence was not observed when Dl/UCD outgrowth was exposed to hormones by means of pituitary isografts. Unlike Dl, Dl/UCD is refractory to the carcinogenic action of MuMTV. Both Dl and the Dl/UCD subline contained the endogenous MuMTV provirus but did not contain exogenous MuMTV provirus sequences. MuMTV antigen was not detected in Dl/UCD outgrowths or tumors. RNA hybridizable to MuMTV complementary DNA was detected in some Dl/UCD outgrowths and tumors but did not appear to correlate with tumorigenesis.

Concentrations of murine mammary tumor virus (MuMTV) antigen in the milk of individual naturally infected BALB/cfC3H and BALB/c mice given injections of MuMTV were related to their risk of developing a mammary tumor. Two distinct groups of MuMTV-infected mice were identified. One group exhibited high viral antigen levels in their milk (greater than or equal to 100 micrograms/ml), whereas the other group exhibited low viral antigen levels in their milk (less than or equal to 3 micrograms/ml). Mice that exhibited high levels developed tumors by 17 months of age, whereas those with low levels did not. Viral expression levels in mice having high risks of tumor development were also related to the length of the latency period preceding overt tumor development. The tumor risk potential of naturally infected mice was frequently that of their mothers. Various doses of MuMTV were injected into BALB/c mice, and the resulting infections differed in latency period, level of viral expression, and potential for neoplastic transformation.

The patterns of the milk-transmitted (exogenous) mouse mammary tumor virus (MuMTV) DNA restriction endonuclease fragments in the nodule and tumor stages of BALB/cfC3H mouse mammary neoplasia were compared with the use of the Southern blot analysis. Acquired MuMTV restriction fragments were detected in DNA from hyperplastic alveolar nodules (HAN), from primary hyperplastic outgrowths (HPO), from families of transplanted HPO, from tumors from HPO, and from serially transplanted tumors. The restriction fragment patterns suggested that the HAN were composed of clonal dominant populations. Transplantation of subdivisions of individual HAN resulted in HPO with DNA restriction patterns suggesting that HAN also contained two or more subpopulations. In all cases, HAN subpopulations shared MuMTV restriction fragments suggesting a common origin. Forty-seven tumors arising from HPO shared MuMTV restriction fragments with the HPO. Most but not all tumors had additional acquired MuMTV restriction fragments not detected in the progenitor HPO, indicating that they were composed of a distinct subpopulation that originated from the HPO. The restriction fragment pattern in some tumor lines was remarkably stable through many transplant generations. Some tumors had no major additional restriction fragments, suggesting that major rearrangements of MuMTV DNA are not required for tumorigenesis.

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.

The BALB/c (C-) mouse hyperplastic outgrowth line (D1) was used to study murine mammary tumor virus (MuMTV) expression in both D1 and tumors derived from D1. D1 was transplanted into virus-infected BALB/cfC3H (C+) and virus-uninfected C- animals. In duplicate studies, tumor incidence was the same in both groups. However, the tumor latency period was longer for D1 transplanted into C+ mice (D1/C+) than for D1 transplanted into C- mice (D1/C-). MuMTV was detected in 85% of D1/C+ outgrowths and in 29% of D1/C+ tumors but was never detected in D1/C- outgrowths or tumors. D1/C- outgrowth and tumors and most of the D1/C+ tissues expressed little or no MuMTV RNA. Some D1/C+ tumors expressed substantial levels of MuMTV RNA. These same tumors also had MuMTV antigen and contained the exogenously acquired C3H-MuMTV provirus in the cellular DNA as shown by DNA fragment patterns following cleavage with Pst I and Eco RI endonucleases. D1/C+ tumors containing no viral RNA were also antigen-negative and lacked the acquired C3H provirus. These studies indicate that D1 has substantially changed in its incidence and in its response to MuMTV. MuMTV infection was not tumorigenic in the traditional sense, a finding that has led to a reevaluation of our current models of virus-host relationships and the biology of precancerous conditions.

Laboratory colonies of feral mice (Mus musculus domesticus) have been established with specific mouse mammary tumor virus (MuMTV) genotype, including colonies lacking any proviral DNA (ev-) or carrying only a single copy of MuMTV DNA (ev+). No evidence of a decline in reproductive capacity has been observed in the first 8 generations. Both the ev- and ev+ mice showed normal mammary gland development and the development of hyperplastic lesions in the older females. The mice were very resistant to spontaneous or chemically induced mammary tumors. However, the occurrence of 1 mammary tumor in an ev- mouse indicates that mammary neoplasias can occur in the absence of MuMTV DNA. The few tumors that do occur in the ev- mice provide a unique opportunity to study the neoplastic process in the absence of proviral DNA.

Murine mammary tumor virus (MuMTV) provirus sequences in the DNA from early-occurring (average age 10 mo) and late occurring (age greater than 20 mo) tumors in BALB/cfC3H mice were analyzed by Eco RI restriction endonuclease mapping procedures. All early tumors were MuMTV antigen-positive mammary adenocarcinomas that contained the 0.92- and 4.0-kilo base (kb) exogenous C3H MuMTV-specific Pst I restriction endonuclease fragments. All but 1 of the late mammary adenocarcinomas had MuMTV antigens detected by peroxidase antiperoxidase staining, and all contained the 0.92- and 4.0-kb exogenous virus Pst I fragments. Three late nonmammary tumors lacked both MuMTV antigens and acquired provirus sequences. Greater numbers of MuMTV sequences were detected in both early and late-arising mammary tumors by Eco RI restriction endonuclease mapping than were detected in tissues from uninfected BALB/c mice. However, neither the number nor the location of MuMTV proviruses correlated with tumor latent period.

Rearrangement of the int-1 and int-2 regions of mouse chromosomes was compared in the C3H and BALB/cfC3H hyperplastic alveolar nodule and its hyperplastic outgrowth (HPO) model systems by examining the DNA of the different stages of the neoplastic progression, with use of the Southern blot technique. Rearrangement of int region DNAs associated with proviral amplification occurred more frequently in spontaneous tumors (19 of 27) than in tumors from HPOs (7 of 37) and rarely occurred in HPOs (1 of 29). However, the int-1 rearrangement maintained in 1 BALB/cfC3H HPO line through 11 transplant generations suggests that the int-1 rearrangement is neither sufficient nor necessary for progression to mouse mammary carcinoma.

Mouse mammary tissues, including ducts, prelactating lobules, hyperplastic outgrowth lines, and tumors, were exposed to varying doses of X-rays and then transplanted to fat pads of nonirradiated BALB/c mice for study. Estimates of the dose of radiation that would allow survival of 50% of the transplants (SD50) were made with the use of probit analysis. Nearly all duct and lobule transplants survived doses of X-rays from 0 to 800 rad. The survival rate declined rapidly following doses above 800 rad, and the calculated SD50 was 1,020 and 1,260 rad for mammary ducts and lobules, respectively. The three hyperplastic outgrowth lines tested gave very different results. Hyperplastic line Z5C1 transplants had better than 90% survival at doses up to 1,200 rad and an SD50 between 1,200 and 1,600 rad. Hyperplastic line Z5D transplants had an SD50 of between 800 and 1,200 rad. Hyperplastic line D1 transplants had a better than 90% survival following doses of 0-600 rad and an SD50 between 600 and 800 rad. The survival of tumor transplants was 100% following doses of X-rays up to 1,200 rad; the SD50 was in excess of 1,600 rad. The mouse mammary transplantation system can be used to study the direct effectmore » of X-rays on normal, premalignant, and malignant mammary tissues and provides a basis for the study of the radiobiology of mammary tissues.« less

The pathogenesis of hepatocellular tumors induced in F344 rats by the antihistaminic methapyrilene was investigated by light and electron microscopy in a serial sacrifice study. Eosinophilic foci of altered hepatocytes were found in portal areas after 1 week of treatment, the eosinophilia being caused by proliferation of mitochondria. Eosinophilic neoplastic nodules developed from such lesions after 16 weeks of treatment. Hepatocellular carcinomas developed after 26 weeks of treatment. Mitochondrial proliferation, which had been found as a marker for hepatocytes altered by this compound at 1 week of treatment, was still present in the hepatocellular carcinomas, which therefore met the morphologic criteria of oncocytomas.

Mouse mammary tumor virus (MuMTV) antigens were detected by immunoperoxidase cytochemistry in spontaneous breast tumors of wild mice from two widely separated areas of southern California. Eleven of 25 (44%) tumors from Lake Casitas (LC) mice and 4 of 5 tumors from Bouquet Canyon mice were positive. Included among the tumors lacking detectable MuMTV antigen were well-differentiated type A and type B carcinomas as well as tumors with an atypical pattern. In the antigen-positive tumors the distribution of staining was patchy and extremely variable in extent (less than 1-70% stained cells). The intensity and extent of staining were generally greater in breast tumors from hybrids of LC wild mice and C5LBL/10Sn or AKR inbred mice. A good correlation was found in the same tumors between immunoperoxidase staining, detection of MuMTV gp52 antigen by radioimmunoassay, and detection of type B particles by electron microscopy. All of the breast tumors in LC mice were positive for type C virus particles.

Mammary epithelial cells from normal virgin BALB/c mice were cultivated in vitro by the feeder cell technique developed and reported previously. These cells were cultured up to the 10th passage, equivalent to 60 cell divisions in culture. They were then tested for normality by several criteria, namely, the ability to regrow into normal mammary glands after implantation into cleared mammary fat pads of both syngeneic and nude mice, chromosome numbers, and response to mammogenic hormones. The cultured cells did form normal mammary ducts after implantation. The fraction of fat pads with ductal outgrowths as well as the size of the outgrowths was proportional to the number of cells implanted. When 10(6) cells were implanted into BALB/c mice, 83% of the fat pads contained outgrowths, filling, on the average, approximately 87% of the fat pad. More ductal outgrowths occurred from implanted cells taken from lower tissue culture passages than from high ones, and the number of outgrowths was greater in BALB/c mice than in nude mice. A small fraction of the cells in culture reacted with antibodies to casein, but there was no evidence of alpha-lactalbumin in the cells. However, ductal outgrowths from implanted cells responded to hormone stimulation of an estrogen deoxycorticosteroid pellet by forming well-developed lobulo-alveolar structures characteristic of pregnancy. Of the cells that were studied in passages 3 and 7, 85% were diploid. An abnormally growing culture in passage 10 was composed of cells in the tetraploid range. These tetraploid cells formed normal mammary ducts when implanted into animals.