high- (BALB/cfC3H) mammary-tumor-incidence mouse strains. Both strains contain .... Animals were fed Wayne Lab Bloc and given water ad lib. Carcinogen ...
Proc. Natl. Acad. Sci. USA
Vol. 76, No. 10, pp. 5360-5364, October 1979 Microbiology
Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-tumor-incidence mouse strains (7,12-dimethylbenz[aJanthracene/BALB/c/BALB/cfC3H/molecular hybridization/immunohistochemical staining)
S. DUSING-SWARTZ*, D. MEDINA*, J. S. BUTELf, AND S. H. SOCHER*t *Department of Cell Biology and tDepartment of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77030
Communicated by Roy Hertz, July 6, 1979
Involvement of mouse mammary tumor virus ABSTRACT (MMTV) in 7,12-dimethylbenz[alanthracene (DMBA)induced mammary tumorigenesis was investigated in low- (BALB/c) and high- (BALB/cfC3H) mammary-tumor-incidence mouse strains. Both strains contain endogenous MMTV integrated into the cellular genome. Additionally, BALB/cfC3H mice are infected with exogenous MMTV-S which is responsible for a higher incidence of mammary tumors in breeding females. Administration of DMBA to virgin mice of both strains resulted in a moderate frequency of mammary tumors within 40 wk after treatment. No differences were found in DMBA-induced tumor incidences at 18 wk (6% and 7%) or at 38 wk (29% and 36%) after treatment of BALB/c and BALB/cfC3H mice, respectively. Expression of MMTV in these tumors was examined by assaying for the presence of MMTV RNA by hybridization using MMTV-specific cDNA and by immunohistochemical staining utilizing antibodies against MMTV 52,QOO-dalton glycoprotein, gp52, and 28,000-dalton internal protein, p28. Of 16 BALB/c tumors assayed, 11 did not contain detectable levels of MMTV RNA and the remaining 5 tumors contained only low levels (0.0005-0.0010%) of viral RNA. Importantly, MMTV RNA was not detected in 5 of 27 BALB/cfC3H tumors. The other BALB/cfC3H tumors contained quantities of MMTV RNA ranging from 0.0006 to 0.4170%. Most BALB/cfC3H tumors with detectable levels of MMTV RNA also synthesized viral proteins gp52 and p28. Thus, expression of the complete MMTV genome is not requisite for maintenance of the tumor phenotype in DMBA-induced mammary tumors in either BALB/c or BALB/cfC3H virgin mice under 1 year of age.
BALB/c mice have a low incidence of spontaneous mammary tumors and lack mouse mammary tumor yirus (MMTV) particles demonstrable by electron microscopy (1). BALB/c mice do, however, contain endogenous MMTV in proviral form incorporated into the cellular genome (2-4) at a level of 5-9 copies per diploid cell (4). Low levels of MMTV-specific RNA have been detected in BALB/c mammary tumors and lactating glands (5, 6), and certain transplantable BALB/c mammary tumor lines have been shown to contain appreciable amounts of MMTV RNA (7). Introduction of virulent, milk-transmitted MMTV-S to the BALB/c strain through foster-nursing on high-tumor-incidence C3H mice resulted in the BALB/cfC3H line, which is characterized by a high incidence of early developing mammary tumors in breeding females. The presence of MMTV in preneoplastic lesions and tumors of these high-tumor-incidence mice has been demonstrated by electron microscopic and immunologic means (8), and viral RNA has been detected by molecular hybridization (5, 7). The hormonal milieu that mediates growth and differentiation of the mouse mammary gland also increases the inci-
dence and results in an earlier development of mammary tumors in breeding females (8). Previous studies from this laboratory (6) have shown that those hormonal alterations which occur during pregnancy and lactation enhance expression of the MMTV genome in BALB/cfC3H mice and, to a lesser degree, in BALB/c mice. Such evidence suggests that MMTV and hormones mqay act synergistically in the development of mammary tumors in breeding females. The purpose of this investigation was to assess whether MMTV may also act synergistically with the chemical agent 7,12-dimethylbenz[a]anthracene (DMBA) in murine mammary tumorigenesis. The experimental protocol was designed to determine whether the presence of MMTV-S in BALB/cfC3H virgin mice enhanced the capacity of DMBA to induce mammary tumors and whether tumor induction by DMBA in either BALB/c or BALB/cfC3H virgin mice modified the expression of the MMTV genome. MMTV RNA levels determined by molecular hybridization and the presence of viral proteins detected by immunohistochemistry were used as criteria for expression of the viral genome in the chemical carcinogeninduced tumors. Our results suggest that MMTV genome expression is not obligatory for maintenance of DMBA-induced transformation in mammary tumors of either BALB/c or BALB/cfC3H mice. MATERIALS AND METHODS Mice Strains. Mice of strains BALB/cCrglMe and BALB/ cfC3HCrglMe were bred and maintained in a closed colony of the Department of Cell Biology, Baylor College of Medicine. BALB/cCrglMe mice in this colony have a low tumor incidence (1.9 and 260/230 >2.0. The RNA preparations contained 28 S to 4 S in approximately equal proportions in extracts from DMBA-induced and spontaneous tumors. Purification of MMTV RNA. MMTV virions, isolated and purified by zonal centrifugation of the culture medium from C3H Mm5mt/cl cells, were obtained through the Division of Cancer Cause and Prevention, National Cancer Institute. Viral RNA was extracted with sodium dodecyl sulfate/phenol at pH 8.0 by using a procedure similar to that described above, omitting treatment with NaOAc (7). MMTV RNA was further purified by centrifugation through linear 10-35% sucrose gradients at 297,000 X g for 2 hr at 4°C. The position of 70S viral RNA was determined by comparison with 18S and 28S rRNA standards in parallel gradients. Synthesis of MMTV [3HJcDNA. [3H]dCTP- (Schwarz/ Mann; specific activity, 15-30 Ci/mmol; 1 Ci = 3.7 X 1010
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becquerels) labeled DNA complementary to MMTV RNA was synthesized by using avian myeloblastosis virus DNA-dependen-t RNA polymerase obtained from the Division of Cancer Cause and Prevention, National Cancer Institute. Purified 70S MMTV RNA served as template and calf thymus DNA fragments were utilized as a random primer (6, 16). The MMTV cDNA had a specific activity of 1-2 X 107 cpm/ALg of cDNA. At a ratio of one, the cDNA protected approximately 60% of 125I-labeled MMTV RNA; 100% protection was obtained with a 20-fold excess of cDNA (16), indicating that the MMTV cDNA was representative of the entire MMTV genome. Molecular Hybridization. The level of MMTV RNA in tumor extracts was quantitated by the titration hybridization method (17), with the extent of hybridization ascertained by resistance to SI nuclease digestion (18). The amount of MMTV RNA, expressed as a percentage of total cellular RNA, was determined by comparing the initial rate of hybridization observed with the tumor RNA to that obtained with purified 70S MMTV RNA. The limit of detection of MMTV RNA was established at 0.0005% of total cellular RNA, a level which corresponds to approximately two viral genome equivalents per diploid cell.
RESULTS Effect of DMBA Administration on Mammary Tumor Incidence. The incidence of mammary tumors induced by treatment with DMBA was similar in BALB/c and BALB/ cfC3H virgin mice. At 18 wk after the initial administration of DMBA, 6% of the BALB/c animals and 7% of the BALB/ cfC3H mice had developed mammary tumors (Table 1). By 38 wk, approximately one-third of the DMBA-treated mice of both strains had palpable tumors. No mammary tumors were detected in untreated virgin mice of either strain during this same time period. Histologically, the tumors were either adenocarcinomas or a mixture of adenocarcinoma and keratinized adenoacanthoma in various proportions. Expression of MMTV Genome in DMBA-Induced BALB/c Tumors. DMBA-induced tumors from 16 BALB/c virgin mice were analyzed for expression of the MMTV genome (Table 2). Of these, 11 tumors did not contain detectable MMTV RNA-i.e.,
