Urethan-induced mammary tumorigenesis in a murine mammary tumor virus (MuMTV)-positive mouse strain: evidence for a keratinized nodule as an MuMTV-negative precursor lesion for squamous cell tumors.

Administration of urethan (CAS: 51-79-6; carbamic acid, ethyl ester) in the drinking water of breeding female mice of the murine mammary tumor virus (MuMTV)-positive DD/Tbr strain and the MuMTV-negative DDf strain induced so-called keratinized nodules, which were demonstrable in wholemount preparations of mammary glands. It also induced squamous cell tumors (also called adenoacanthoma) at an extremely early age. The keratinized lesions appearing in the lobular areas of the mammary glands showed heavy infiltration with lymphocytes and as such were very different from hyperplastic alveolar nodules, the preneoplastic lesions for adenocarcinomas. Immunoperoxidase tests with antibodies against the MuMTV revealed that positivity of normal mammary gland epithelium in the DD/Tbr strain was not found in the keratinized nodules, which was further evidence that in squamous cell tumorigenesis of the mammary gland the MuMTV is not expressed overtly even at an early stage in tumorigenesis, in contrast to the case with adenocarcinoma tumorigenesis. These findings substantiate previous conclusions that the MuMTV is not involved in chemical carcinogenesis of the mouse mammary gland.

A new locus (Mtv-4) for endogenous mammary tumor virus expression and early mammary tumor development in the SHN mouse strain.

The SHN mouse strain, which has a high incidence of mammary cancer, developed by inbreeding and selection from Swiss stock mice by Dr. H. Nagasawa and co-workers (Meiji University, Tokyo, Japan), harbored an endogenous mammary tumor virus (MTV) responsible for a high frequency of mammary tumors early in life. The locus was called "Mtv-4" and was only comparable with Mtv-2 of the GR mouse strain in its inducing capacity of mammary cancer. Molecular hybridization with 32P-labeled MTV complementary DNA showed that the characteristic Mtv-2 bands of the GR strain were absent in the SHN strain.

Endogenous type-C viral expression during lymphoma development in irradiated NFS mice.

The expression of the type-C retrovirus and the virus-related components in NFS mice were examined during preleukemic and leukemic phases after fractionated whole-body X irradiation. The NFS mice were highly susceptible to induction of thymoma by fractionated X irradiation. The leukemic tissues were negative for infectious type-C virus, as detected by both the XC-plaque test and mink S+ L- focus-inducing assays, but contained a substantially higher level of viral-specific RNA-dependent DNA polymerase activity and a major core protein p30 than the corresponding tissues from unirradiated age-control mice. In the preleukemic phase, the amount of p30-related antigen increased transiently in spleen. The leukemic cell lines established from radiation-induced lymphomas produced particulate entities with a buoyant density of about 1.15 g/ml. These virus-like particles lacked in vitro infectivity to mouse cells and mink lung cells and leukemogenicity in syngeneic mice. The p30-related antigens of these particles were immunologically similar to that of xenotropic virus derived from NZB mouse.

Comparison of mammary tumorigenesis between litters in relation to mammary tumor virus antigenic expression in the milk of C3H/He mice.

As a possible step to estimate the relationship between mammary tumor virus (MTV) and mammary tumorigenesis in mice, MTV antigenic expression in mother's milk and spontaneous mammary tumorigenesis in their daughters were compared between the 1st, the 2nd and the 3rd litters of the highly inbred strains of C3H/H3 mice with low mammary tumor incidence. While MTV antigenic expression was detected in all undiluted samples at each litter by immunodiffusion test, the amount of antigen as measured by the single radial immunodiffusion method was increased with increasing litter numbers. On the other hand, the development of preneoplastic mammary hyperplastic alveolar nodules was different little between litters and mammary tumor incidence by 13 months of age was very low with no difference in all litters. The pattern of estrous cycles and plasma prolactin level were also similar in each litter. The results suggest that spontaneous mammary tumorigenesis in mice is not always affected quantitatively by the amount of MTV when endocrine and genetical conditions are similar.

The antispermatogenic effect and toxicity of 2, 4-dinitro-6-tert-butylphenyl methanesulfonate on Sprague-Dawley rat.

The antispermatogenic effects of 2, 4-dinitro-6-tert-. butylphenyl methanesulfonate (HE-166) were studied in Sprague-Dawley rats. They were fed 25, 100 and 400 ppm of HE-166 in the basal died for one year. Laboratory data showed no significant changes except for increases in gamma-globulin, alkaline phosphatase, GOT and GPT values in the 100 ppm group. Macroscopically, significant changes were found in the testes in the experimental group, which showed marked atrophy. Histologically, the testes were filled with fibrin exudate in the stroma and there was reduced spermatogonia, cellular debris and giant cells, and even calcification, depending on the dose of HE-166. The anti-fertility effects of HE-166 were also observed by mating rats and checking the pregnancy rate during three generations. These effects might be due to the direct cytotoxic effect of HE-166 on post-meiotic cells as epididymal spermatozoa and testicular sperm and spermatids. As far as tumor incidence was concerned, one case of fibroadenoma of the mammary gland, one case of leiomyosarcoma in the uterus in the 100 ppm group and one case of leiomyoma in the uterus in the 25 ppm group developed at around 8 months, but no other tumors developed.

[Carcinogenicity study of cholestyramine in rats].

Carcinogenicity study on cholestyramine, an anti-hypercholesterolemic agent, was carried out using Fischer rats by feeding with a normal pellet diet (control) and with three sorts of the pellet diets in which cholestyramine was admixed in the common diet at the rates of 1.25, 2.5 and 5%, respectively. The animals were fed with these diets for 26 months and a normal diet for subsequent 2 months. Some of the male rats fed with the 5% admixed diet revealed a slight growth retardation and occasionally hemorrhagic tendency, without resulting in severe macroscopic and microscopic lesions of the internal organs and in shortening of the mean survival time as compared with that of the control group. The rates of tumor-bearing animals were high in all experimental groups including the control, but the rates were not considered to be drug-related. Incidences of individual tumors were not significantly different each other between cholestyramine-fed groups and control group. Thus, it was not recognized that cholestyramine induced any drug-related specific tumors and an accelerated growth of any tumors.

[Carcinogenicity study of cholestyramine in mice].

Carcinogenicity study of cholestyramine, an anti-hypercholesterolemic agent, was carried out by feeding B6C3F1 mice of both sexes with the pellet diet in which cholestyramine was admixed at the rates of 1.25, 2.5 and 5%. The animals were fed on the drug-admixed diet for 18 months and on a normal diet for subsequent 3 months. After 32 weeks the mortality of male mice began to increase in the 5% cholestyramine group and the number of dead or moribund mice increased markedly after 60 weeks. Hemorrhage recognized in pleural cavity, heart and other organs, was suggested to be the main cause of death. Some kinds of tumors occurred in each group, but the tumor-incidences seen in mice of 1.25 and 2.5% cholestyramine groups were similar to those in mice of the control group and the occurrence of the specific tumor or an acceleration of tumor-development by feeding cholestyramine were not observed. Furthermore, the tumor-incidence in mice fed on the 5% cholestyramine diet was less than that in mice of the other three groups.

MuMTV antigen expression and mammary tumor incidence in non-inbred dd mice.

The expression of mammary tumor virus (MTV) antigen in the milk and various organs of three non-inbred dd mouse stocks (ddO, ddN and ddY) was examined by the immunodiffusion (ID) and micro-immunodiffusion (micro-ID) tests. The rate of MTV antigen expression in the milk was 100% at the first lactation in ddO (6/6) and ddN mice (10/10), and 23% in ddY mice (3/13). Mammary tumor incidence was 13% (mean tumor age: 12.0 months), 32% (9.6 months) and 10% (11.5 months) in ddO, ddN and ddY mice, respectively, In F1 hybrids between MTV-free BALB/c females and dd males, a high level of MTV antigen was detected by the ID test in the milk of (BALB/c X ddO) F1, however, the levels in (BALB/c X ddN) F1 and (BALB/c X ddY) F1 mice were low at the first lactation and elevated with the advance of lactation number. Mammary tumor incidence had a trend to be higher and earlier in these F1 hybrids than in non-inbred dd stocks. The development of mammary tumors and detection of MTV antigen in F1 hybrids indicate the extrachromosomal transmission of MTV by male dd mice. The micro-ID test has shown that the mammary tumors, mammary glands, male genital organs except for the testis and the salivary gland expressed MTV antigen, with a high frequency of suggesting that secondary male genital organs may play an important role in MTV infection in mice.

Pulmonary lesions in rats caused by intravenous injection.

Sprague-Dawley rats in a chronic toxicity test revealed specific pulmonary lesions after 180 days of intravenous injection of samples via the tail vein. Microscopically, thickening of arterial walls, periarteritis, edema of perivascular sheath, arteriosclerosis, vasodilatation, arteriofibrosis, abscess and necrosis of arterial walls, embolism and thrombus of small muscular artery or alveolar capillary, and granulomas sometimes accompanying giant cells were found in the lungs. The emboli consisted of hair of the rats, and granulomas with multinucleated giant cell formation and cellular infiltration were prominent around the emboli. The thrombi was characterized by perivascular cuffing, fibrinous and cellular components. These pulmonary lesions are thought to be caused by hair brought from the tail when intravenous injection was carried out.

Mammary tumor virus antigen expression in inbred mouse strains of European origin established in Japan.

Expression of the mammary tumor virus (MTV) antigen in the milk from inbred mouse strains maintained in Japanese laboratories was investigated by means of the immunodiffusion test (ID) and the results were compared with the incidence of mammary tumors in breeding females of identical strains. Japanese strains tested were classified into two groups. The DD/Tbr, DDD and KF strains were derived from "dd" stock mice, and they showed equally high incidences of MTV antigen expression in the milk, though each strain developed mammary tumors with different incidences; 71%, 14% and 22%, respectively. Other strains originated from Swiss albino mice. The incidence of MTV antigen expression in the milk of SHN and SLN strains was the same as that of mammary tumors at an early age.

Mammary tumor induction in inbred mouse strains with urethane is not accompanied by changes in expression of B- and C-type retroviral structural proteins.

Urethane administrated in the drinking water of breeding female mice of five inbred strains (DDf, KF, C3Hf, ddY and C57BL) leads to a higher incidence of mammary tumors at an extremely early age in four of these strains (DDf, KF, C3Hf and ddY). In the case of the DDf strain the tumors are adenoacanthomas, but in the case of the other strains adenocarcinomas are also observed. The effect of urethane in the drinking water on expression of endogenous retroviral structural proteins of the B and C type (MTV and MuLV) was studied in the milk by an immunodiffusion test and in organ extracts by radioimmunoassays (for MTVp27, MTVgp52 and MuLVp30). Organs tested include salivary glands, thymus and spleen from female and male animals. In addition, prostate and seminal vesicles were tested in male animals and uterus and mammary glands in females. Mammary tumors induced by urethane were also treated. Urethane does not induce or enhance endogenous retroviral antigen expression, either in these organs or in the mammary gland, one of the target organs for tumorigenesis by this agent.

Strain difference in the expression of mammary tumor virus antigen in the male genital organs of mice during aging.

Mammary tumor virus (MTV) antigen expression was tested in the male genital organs (testis, epididymis, prostate, seminal vesicle and coagulating gland) of DD/Tbr, KF and DDD strains (dd (German) mouse group), GR strain (Swiss origin), SHN and SLN strains (different Swiss origin), C3H and A strains (American origin), BALB/c and C57BL strains (low mammary tumor strains), and DDf and SHNf strains (MTV eliminated strains). Microimmunodiffusion analysis showed that expression of MTV antigen is tissue extracts was found most frequently in the prostate, sometimes in the seminal vesicle and coagulating gland but rarely in the epididymis and not at all in the testis of mammary cancer strains. No expression was found in these organs from BALB/c and C57BL strains. The earliest expression was found in GR, SHN and SLN strains of Swiss origin. Expression was not lost in SHNf strain, indicating that it is due to genetically transmitted MTV, rather than a milk-factor. Male genital organs from two mammary cancer strains of American origin became positive occasionally at rather higher age than in the strains of Swiss origin. DD/Tbr, KF and DDD strains expressed MTV antigen in the male genital organs at intermediate age between the strains of Swiss origin and American origin. Complete disappearance of MTV antigen expression in these organs of DDf strain indicates that the expression of MTV in DD/Tbr is due to a milk-factor.

Horizontal transmission of the mouse mammary tumor virus in cage mates of the same and opposite sex of low and high mammary cancer strain mice.

Horizontal transmission of mouse mammary tumor virus (MTV) was investigated in cage mates of the same and opposite sex of low (BALB/c) and high mammary cancer strains (DD/Tbr, SHN and GR). By MTVp27 and MTVgp52 radioimmunoassay, MTV antigen expression was found in the salivary glands, mammary glands and secondary male genital organs of the MTV-free BALB/c strain. Infectivity and oncogenicity were also found in DDf or BALB/c mice by inoculating extracts of salivary gland and/or seminal vesicle in high mammary cancer strains. It is suggested that the primary source of the infectious agent in cases of caged animals of the same sex is saliva, while the primary source in cases of caged animals of the opposite sex is the seminal fluid, although additional infection through saliva cannot be ruled out in the latter case.

Histological distribution of MTV antigen in mice detected by immuno-peroxidase staining.

Histological localization of mammary tumor virus (MTV) antigen was investigated using a variety of organs high (DD/Tbr, SHN, SLN, GR) and low (BALB/c) mammary cancer mice strains and immuno-peroxidase staining with MTV antigen. Except for BALB/c strain mice, the mammary gland and mammary tumors were generally positive. Accessory male genital organs including the prostate, seminal vesicle, and coagulating gland also demonstrated a positive reaction, but the testis and female genital organs including uterus and ovaries did not. MTV antigen was also revealed in the serous acini of the salivary gland in both sexes. The site of positive reaction in the accessory male sex organs and salivary gland was located in the apical portion of the secretory epithelial cells and their secretory substance. Localization and intensity of antigenic expression of MTV detected histologically were comparable to results obtained by immunodiffusion test and radioimmunoassay. These evidences support the idea that MTV is transmitted horizontally via seminal fluid or saliva.

Establishment and characterization of a new murine mammary tumor cell line, BALB/c-MC.

A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms "domes" or "hemicysts" as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10(6) cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87%) recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).

Establishment of monoclonal antibodies reactive with epithelial and myoepithelial cells in the breast.

Monoclonal antibodies which are considered to be able to differentiate epithelial and myoepithelial cells in the breast have been developed. Human mammary carcinoma cell line (HBC-4W) was used for immunization. Monoclonal antibodies-B4B2F10 (epi-1), E9E8B7 (myo-1)-with IgM was examined using tissues from diseased breast by avidin-biotin-peroxidase assay. Epi-1 antibody reacted with epithelial cells while myo-1 antibody reacted with myoepithelial cells in the mammary glands, respectively. The reaction was markedly visible, in particular, in fibroadenoma, mastopathy, and papilloma, which showed clear two-cell-type structures. In the infiltrating ductal carcinoma, epi-1 antibody reacted with carcinoma cells, while myo-1 antibody reacted with stromal cells rather than carcinoma cells suggesting that infiltrating ductal carcinoma was mainly of epithelial origin. In the infiltrating lobular carcinoma, however, myo-1 as well as epi-1 antibodies reacted with carcinoma cells. It is suggested that infiltrating lobular carcinoma was of a mixture of epithelial and myoepithelial cell origin.