Expression Of Mammary Tumor Virus Research Articles

HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression

We present evidence that the HIV-1 Rev protein can heterologously regulate expression of the simple beta retrovirus mouse mammary tumour virus (MMTV). Up to 10-fold upregulation was seen in a functional assay system when specific MMTV sequences were substituted for the HIV-1 Rev responsive element (RRE). RNA gel shift analysis showed that purified recombinant Rev could specifically bind to MMTV unique region 3 prime (U3) RNA and that these sequences could compete for wild-type Rev-RRE binding approximately 20-fold more efficiently than a non-specific competitor RNA. Using a combination of in silico and deletion mutation analyses, it was not possible to define any single specific secondary structure responsive to Rev, suggesting that a structure or combination of structures that only form in the context of the complete U3 transcript is/are required to interact with Rev. Taken together, these results suggest that HIV-1 Rev can directly bind to MMTV RNA as well as mediate upregulation of MMTV gene expression.

CDP binding to multiple sites in the mouse mammary tumor virus long terminal repeat suppresses basal and glucocorticoid-induced transcription.

Mouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.

BOTH DIETARY CALORIE AND FAT AFFECT THE GROWTH OF TRANSPLANTED MAMMARY-TUMORS IN RIII/SA MICE - THE EFFECT OF CALORIE IS MORE PROFOUND THAN THE EFFECT OF FAT

The effects of dietary calories and/or fat on the growth of mammary tumor transplants, their expression of mouse mammary tumor virus (MMTV) and the incidence of lung metastasis were examined in RIII/Sa mice. Starting at 3-4 weeks of age, groups of female mice were fed either high isocaloric diets (16 kcal/day/mouse) containing 25% or 5% corn or fish oil or low isocaloric (10 kcal/day/mouse) diets containing 25% or 5% fish or corn oil. After one week, small pieces (2x2x2 mm) of tumor tissue, prepared from a transplantable mammary tumor, were inserted into the fourth pair of mammary glands of mice, and the mice maintained on their respective diets until sacrifice. All mice developed palpable tumors during a period of 3-4 weeks. After 12 weeks, all those mice that were assessed for tumor burden were sacrificed, tumor weight in each mouse determined, and the level of the expression of MMTV in the tumors evaluated by dot blot hybridization. Our results show that low isocalorie diets, regardless of the type or amount of fat, inhibited tumor growth by at least 60% in comparison to high isocalorie diets. However, mice fed low isocaloric diets containing fish oil were also found to produce smaller tumors (20-40%) as opposed to those mice fed similar, but corn oil containing diets. Fatty acid analyses of mammary tumors and liver tissue of mice fed corn oil and fish oil containing diets revealed that while normal and tumor tissues from both groups contained high levels of polyunsaturated fatty acids, the tissues of mice fed corn oil had the n-6, whereas the mice fed fish oil had the n-3 family of fatty acids. The levels of MMTV expression were found to be unaffected by either caloric or fat content in the diet. In a separate set of experiments, the effect of a low calorie diet on lung metastasis was determined, It was found that mice fed a low calorie diet produced significantly less metastatic lung nodules than those mice fed a high calorie diet: the mean survival time for the former group of mice was 106 days, as compared to only 71 days for the latter group of mice. In conclusion, we suggest that the amount of calories in a diet is more important than the amount or the type of fat in suppressing the growth of transplanted tumors and that a low calorie diet may also lower the incidence of lung metastasis.

Modulation of androgen receptor transcriptional activity by the estrogen receptor.

Estrogen/androgen receptor interactions in naturally occurring physiological systems and the effect of their respective steroid hormones on transcriptional activity remain undefined. In an attempt to delineate further the nature of the interaction between these two steroid hormone receptors we have examined the effect of cotransfection of androgen (AR) and estrogen receptor (ER) cDNAs on the expression of the mouse mammary tumor virus long terminal repeat region (MMTV-LTR) linked to the chloramphenicol-acetyltransferase (CAT) reporter gene. In QT6 cells, which contain neither AR nor ER, cotransfection of AR cDNA with the MMTV-LTR-CAT reporter, resulted in transactivation only in the presence of dihydrotestosterone (DHT). Treatment with 10(-8) M each of estradiol-17 beta (E2), dexamethasone, or progesterone did not enhance CAT activity, whereas treatment with the androgens DHT and mibolerone resulted in 87% and 89% CAT activity. Transfection of increasing concentrations of ER cDNA in the presence of 100 ng of AR cDNA and 10(-8) M each of DHT and E2 showed a dose-dependent decrease in CAT activity as compared to the response with DHT alone. Cotransfection of AR and ER cDNA in the presence of 10(-8) M DHT and increasing concentrations of E2 resulted in a dose-dependent decrease in CAT activity. When cells were treated with increasing concentrations of DHT with 10(-8) M E2 no significant increase in CAT activity was observed. In GC cells, which contain endogenous ER but no AR, cotransfection of AR cDNA and treatment with E2 and DHT, also reduced DHT-induced CAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary regulation of mammary tumorigenesis in RIII/Sa mice: investigation of a possible mechanism.

The effects of caloric restriction on the incidence of mammary tumor development, the levels of the expression of mouse mammary tumor virus (MMTV)- and prolactin-RNA, as well as the levels of serum prolactin, were investigated in virgin RIII/Sa mice, a strain known to display a high incidence of spontaneous mammary tumor development. Of the 54 mice fed a low-calorie (LC; 10 kcal/day) diet containing low fat (LF; 5% corn oil) for a period of 72 weeks, only seven mice were found to develop mammary tumors, an incidence of 13%. By contrast, the cumulative tumor incidence in a similar sized group of mice, fed a high-calorie (HC; 16 kcal/day) low fat-containing diet was 73%. Estimation of the relative levels of MMTV-RNA, as determined by Northern and slot blot hybridizations, in the mammary glands of mice fed LCLF and HCLF diets for 8, 10, 16, 28, and 36 weeks revealed that the LCLF diet-fed mice expressed 4-15-fold less RNA than the HCLF diet-fed mice. Interestingly, however, the LCLF diet did not appear to exert any effect on the expression of prolactin RNA even though it reduced the levels of serum prolactin. We suggest that in RII/Sa mice the modulation of MMTV-induced mammary tumors by dietary calorie is linked to the secretion of serum prolactin which, in turn, affects the replication of MMTV required for mammary cell transformation.

Positive and Negative Modification of Dexamethasone-Mediated MMTV gp52 Release by Progesterone Pretreatment of Tumor Cells

Murine mammary tumor cells (C3H Mm5mt/cl) were grown in charcoal-stripped serum to examine the effects of hormonal treatments on the expression and release of the mouse mammary tumor virus (MMTV) envelope glycoprotein (gp52). The ability of progesterone to modify extracellular levels of soluble and virion-associated MMTV gp52 was tested singly and as a pretreatment prior to dexamethasone (DXS) stimulation. Single treatments with DXS or progesterone demonstrated that DXS was substantially more effective at elevating gp52 than progesterone; however, a small but significant increase in gp52 levels was noted after 72 h of progesterone treatment. Progesterone pretreatments at 10- to 100-fold excess of DXS altered subsequent DXS-stimulated gp52 levels. Progesterone effectively antagonized DXS and reduced gp52 levels to those of controls but this reduction was accomplished only at the lowest concentrations of DXS (10(-8) M) and shortest treatment interval (24h). While some pretreatment protocols of varied dose slightly reduced gp52 levels at a point in time after treatment, the majority of progesterone pretreatments at 10(-7), 10(-6), and 10(-5) M resulted in MMTV gp52 levels which were equal to and in many cases significantly greater than those obtained with DXS alone. The ability to augment DXS-stimulated gp52 levels with progesterone pretreatment suggests that, under certain hormonal conditions, gp52 expression and release from mouse cells may be progesterone-dependent as well as glucocorticoid-dependent.

Activation of endogenous MMTV proviruses in murine mammary cancer induced by chemical carcinogen.

A study was undertaken to determine whether activation of expression of silent endogenous mouse mammary tumor virus (MMTV) proviruses may occur during tumor induction by a chemical carcinogen. A series of transplantable mammary tumors induced in BALB/c mice by treatment with dimethylbenz(alpha)anthracene (DMBA), pituitary isograft, or both was examined. The results obtained suggest that chemical carcinogens may induce mammary tumors through more than one pathway. Two of 9 tumor lines produced virus-specific products at levels above those observed during the course of normal mammary gland development. One tumor contained high levels of MMTV-specific envelope [3.8 kilobase (kb)] and genomic length (8.9 kb) RNAs. This tumor expressed core- and envelope-related proteins detectable by immunoblotting (including p28, gp52, and gp36), displayed an acquired provirus with a restriction map different from those of described exogenous MMTV strains, and contained abundant virus particles. The other tumor that expressed high levels of MMTV gene products contained envelope-specific (3.8 kb) and long-terminal-repeat-specific (1.6 kb) messages but no full-length RNA. It exhibited an aberrant 39 kDa, envelope-related protein, but no virus particles. Methylation data implicated the usually silent endogenous Mtv-8 provirus as the source of the abnormal envelope protein. None of the tumors expressed RNA from the putative mammary oncogenes, int-1 or int-2. We propose that chemical carcinogens may activate different cellular genes by mutation and that, in a subset of DMBA-induced mammary tumors, the target genes include endogenous MMTV proviruses that are normally not expressed. The effect on provirus expression varies from tumor to tumor, but is stable over passage of a given tumor. MMTV may be of etiological importance in the genesis of those DMBA-induced tumors which contain high levels of MMTV-specific products, but its action in the BALB/c system is not mediated through enhanced expression of the int-1 or int-2 preferred integration regions.

Reversible Suppression of Mouse Mammary Tumor Virus Replication by Prolonged Glucocorticoid Exposure<xref ref-type="fn" rid="FN2">2</xref>

Expression of mouse mammary tumor virus (MuMTV) in MJY-alpha mammary tumor cells was only transiently stimulated by exposure to 14 microM hydrocortisone (HC). Short-term HC treatment for 24-48 hours resulted in twofold-to-fivefold increases in levels of MuMTV polypeptide synthesis, MuMTV surface antigen expression, and MuMTV production. HC treatment also induced quantitative alterations in glycosylation of MuMTV precursors Pr79env and Pr76env. In contrast, prolonged exposure to 14 microM HC for 14-21 days decreased MuMTV polypeptide synthesis, surface antigen expression, and virion production to levels similar to untreated MJY-alpha cells. The pattern of MuMTV precursor glycosylation following prolonged HC treatment was identical to that detected in unexposed control cultures. Attenuation of glucocorticoid-mediated MuMTV stimulation was reversed either by exposure of treated cells to elevated (28 or 60 microM) concentrations of HC or by intermediate passage of treated cells in HC-free medium, suggesting additional levels of control of MuMTV replication.

Effect of N-(4-hydroxyphenyl)retinamide on murine mammary tumor cells in culture.

The effects of N-(4-hydroxyphenyl)retinamide (HPR), a synthetic analogue of vitamin A, on cell morphology, cell cycle kinetics, cytoplasmic matrix, and expression of murine mammary tumor virus (MuMTV) in MuMTV-infected murine mammary tumor cells (GR-3A) were determined. Cellular uptake of HPR was rapid and linear, with zero-order kinetics, during the first 30 minutes of incubation. Flow cytometric analysis of cells treated with nontoxic levels of HPR (10 microM) for 48 hours revealed a reduction in percent cells in the DNA synthetic (S) phase of the cell cycle with a concomitant increase in percent cells in the G1 phase of the cell cycle. Dexamethasone-stimulated MuMTV expression was not affected after 48 hours of HPR exposure, whereas the virus expression was significantly reduced in cells treated with HPR for seven days. The reduction in MuMTV expression was preceded by changes in cell morphology (decreased cell-cell contact and reduced cell flattening) and altered F-actin aggregation. Continuous exposure to HPR (10 microM) for 14 days resulted in reduced cell proliferation rates and decreased cell plating efficiency of GR-3A cells. Taken together, these results indicate that HPR is rapidly incorporated into GR-3A cells and that the effects of HPR on cell profileration, cytoskeletal organization, and cell morphology appear to precede the effects of this retinoid on the expression of the etiological agent of murine mammary tumorigenesis, MuMTV.

Tissue and organ distribution of mammary tumor virus antigens in low and high mammary cancer strain mice

Expression of mammary tumor virus (MTV) antigen was measured in a wide variety of organs and tissues of a series of high (GR, SHN, SHNf, DD, SLN, SLNf) and low (DDf, DDD, DDDf, KF, KFf, ddY, C57BL, BALB/c) mammary cancer strain mice. Tests were carried out by microimmunodiffusion (micro-ID) and immunoperoxidase tests on formalin-fixed tissues and radioimmunoassays in extracts for 2 viral proteins, MTVp27 from the viral core and MTVgp52 from the viral envelope. Organs with exocrine function, i.e. the mammary gland, salivary gland, coagulating gland and prostate, were mostly positive. The secretory epithelial cells of these organs showed viral antigen expression. Less positivity was encountered in brain, pancreas, stomach, urinary bladder, epididymis, uterus, thymus, spleen, lymph nodes and kidney, plasma and blood cell pools. Unexpectedly the uterus extracts showed MTV antigen expression, occasionally even by immunodiffusion, especially in mice of various DD stocks, but also in the GR strain. Another striking observation was the detection of MTV antigen expression in salivary glands in C57BL strain mice; most other organs of this strain (including the mammary glands) were negative. The implications of results of this extensive survey for MTV antigen expression are discussed for tumorigenesis by MTV of mammary gland and perhaps other tissues in the mouse.

Tumor-Associated Antigens Produced by Mouse Mammary Tumor Cells in Artificial Capillary Culture<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Mouse mammary tumor cells were cultured on a three-dimensional bundle of semipermeable hollow fibers as a prototype system for large-scale in vitro production of mammary tumor-associated antigens. Cells were seeded onto the surface of the hollow fibers that served as an artificial capillary network. The cells grew to tissue-like densities within 3 weeks, achieving the high density and three-dimensional intercellular relationships known to potentiate expression of murine mammary tumor virus (MuMTV) antigens. Significant levels of a 52,000-relative-molecular-weight (Mr) glycoprotein antigen of MuMTV (gp52) were detected on the cell side of the semipermeable membrane of the hollow fibers in a yield at least fiftyfold greater than that produced by monolayer cultures. Antigen could be collected from capillary cultures for 6-12 weeks, rather than days from monolayer cultures. Further, gp52 was collected in an undegraded state. Perfusates contained lesser amounts of cross-reactive antigenic species that were predominantly smaller than 50,000 Mr, which suggests that the fibers may be used to separate antigens from their degradation products. The production of nonviral, mammary tumor-associated antigens by cells on artificial capillaries was also demonstrated. Artificial capillary cell culture systems provide a means to obtain significant quantities of tumor-relevant antigens in a semipurified natural state.

Effects of interferon on the expression of mouse mammary tumour virus in GR cells.

The effects of interferon treatment on the expression of murine mammary tumour virus (MuMTV) by a continuous mammary tumour cell line of GR mouse were investigated. Interferon markedly inhibited the excretion of MuMTV particles in the culture media as measured by hybridization of virus-specific RNA and virus-associated reverse transcriptase activity. There was no inhibition of virus RNA and protein synthesis in those conditions. The steady-state level of intracellular virus RNA and its rate of synthesis were not modified by interferon treatment. The intracellular levels of the major virus envelope and core proteins as measured by immunoprecipitation techniques remained unchanged. Interferon failed to inhibit the synthesis and the processing of the gp52 glycoprotein and p27 core protein precursors. However, the rate of maturation of the glycoprotein precursor was slowed down. Surface labelling of intact cells did not reveal accumulation of virus proteins on the cell membrane upon interferon treatment. The intracellular level of MuMTV-characteristic reverse transcriptase activity was reduced in interferon treated cells, although to a lower extent than extracellular virion-associated enzyme activity. MuMTV particles released from interferon-treated cells revealed no difference in their protein composition. These results are consistent with an inhibition by interferon of a late step in the replication of MuMTV.

Suppression of spontaneous mammary tumorigenesis despite Mtv-1 gene expression in hybrid and backcross C3H-AvyfB X C3H/Sm mice.

AbstractMouse mammary tumor virus (MMTV)‐specific RNA and antigen content of normal and neoplastic mouse mammary tissue were measured in strains C3H/Sm and C3H‐AvyfB, in C3H‐AvyfB♀ × C3H/Sm♂ F1 hybrids and in (C3H‐AvyfB × C3H/Sm) × C3H/Sm♂ backcross segregants. Good correlation between MMTV antigen expression in lactating mammary glands and mammary tumor incidence was observed in the parental mouse strains. The high‐mammary‐cancer strain C3H‐AvyfB (90%) had consistently high levels of MMTV antigen in the mammary glands and tumors, whereas mammary tissues from low‐mammary‐cancer subline C3H/Sm (1.6%), whether normal or neoplastic, had little or no detectable MMTV antigen. In contrast, MMTV RNA content of normal C3H/Sm mammary tissue was consistently as high as or higher than corresponding levels in C3H‐AavfB females. These molecular data, along with the relative mammary tumor incidences, suggest that the endogenous MMTV provirus expression gene Mtv‐l present in C3H mice is fully expressed in the C3H‐AvyfB subline but is silent or more probably absent in the C3H/Sm subline. Our earlier genetic crosses between C3H‐AvyfB and C3H/Sm mice were conducted to determine the effect of the Avy gene on mammary tumorigenesis during its segregation in back‐cross (BC) females produced from matings of the F1 hybrid females with C3H/Sm males. Our mammary tumor incidence data presented in the accompanying paper strongly suggested the possibility that C3H/Sm mice possessed a dominant genetic factor(s) which specifically attenuated the tumorigenic effects of the Avy and Mtv‐l genes on the mammary gland without affecting the Avy gene's enhancement of liver tumorigenesis and obesity. Examination of MMTV RNA and antigen content in (C3H‐AvyfB × C3H/Sm) hybrid and BC females showed that the expression of MMTV antigen was completely suppressed in the agouti (AA) BC females. However, yellow F1 (AvyA) and yellow (AvyA) BC females continued to express MMTV antigen in their mammary glands even though mammary tumor incidence was significantly depressed. MMTV RNA levels were similar in all F1 and BC females regardless of the presence or absence of the Avy gene. Mammary tumor incidence was reduced in all the BC females (whose genome is 3/4 derived from C3H/Sm) relative to the F1 females (whose genome is only 1/2 derived from C3H/Sm) suggesting that more than one gene is involved in the suppression of spontaneous mammary tumor development. Cessation of translational expression of endogenous MMTV occurred only in the agouti (AA) BC females, suggesting an association between this phenomenon and the agouti locus of C3H/Sm. Evaluation of the relative MMTV RNA content of nucleic and cyto‐plasmic fractions from C3H/Sm mammary tissues strongly suggests that transport and/or processing of MMTV RNA is defective in these mice. This defect probably accounts for the absence of detectable MMTV antigens in C3H/Sm and agouti (AA) BC mammary tissues and tumors and may contribute to the observed reduction in mammary tumor incidence.

非近交ddマウスにおける乳癌ウイルス (MuMTV) 感染の実態

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.

Reevaluation of the Effect of Mouse Mammary Tumor Virus Infection on the BALB/c Mouse Hyperplastic Outgrowth<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

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.

Expression of mammary tumour virus in late-appearing mammary carcinomas in presumed virus-free mice

Mammary tumours arising in mice which had been tested for the presence of mammary tumour virus (MuMTV) in the milk were tested for either the presence of infectious MuMTV by bioassay in female BALB/c mice or for the presence of the major MuMTV envelope glycoprotein gp 52. In crosses involving the high cancer strain GRS, 16/17 tumours appearing before 1 yr of age contained a virulent virus. Only 2/19 of the late appearing tumours in virus positive mice, harboured a virus which induced tumours at an early age but 12 contained an infectious late-oncogenic MuMTV. In crosses involving the C 3Hf mouse strain which develops tumours at a late age, only 2/28 tumours in virus positive mice contained a virulent MuMTV but 15 harboured a late-oncogenic strain. Of the 22 tumours which arose in so-called virus-negative mice in all crosses, 7 harboured an infectious MuMTV; it was highly virulent in 3 cases. In the immunoassay, all 41 tumours which developed in mice with virus positive milks were also positive for gp 52. The majority (25/39) of tumours arising in virus negative mice were positive for the viral antigen, although the quantities were relatively low.

Demonstration of components of serum-free culture medium effecting maximum in vitro expression of mouse mammary tumor virus.

By using a chemically defined serum-free (SF) medium for propagation of Mm5mtc1 mouse adenocarcinoma cell cultures and clonal derivatives, medium components including hormones, glucose and individual amino acids were evaluated as to modulation of mouse mammary tumor virus (MMTV) porduction. Insulin, hydrocortisone and dexamethasone each increased MMTV production on a per cell basis over constitutive expression that occurs in SF medium devoid of hormones. Maximum production occurred when all three hormones were present. Hormone-stimulated virus expression also was influenced by glucose concentration. Cell growth and maximum MMTV expression increased when thyroxine, asparagine, proline and serine were omitted from the medium formulation. The resulting modified SF medium provides and ideal system for the propagation of high MMTV-producer clones and for the study of the biochemical regulation of MMTV expression.

Development of a congeneic line of the gr mouse strain without early mammary tumours

Based on the previous observation that Mtv-2 controls the appearance of pregnancy-dependent mammary tumours and the early expression of mammary tumour virus (MTV)-antigens in the milk, an attempt was made to develop a congeneic GR line, without this locus, by introducing genetic material of the C57BL/10 strain. The cross-intercross system was used for this purpose. The mice of the even-numbered generations were selected for the absence of the Mtv-2 locus with the early maammary tumour (EMT) test and during the later cycles with the immunodiffusion (ID) test for MTV-antigens in the milk. After six cycles of cross-intercross, brother-sister mating was started with mice selected for the absence of Mtv-2. After two brother-sister matings the milk-transmitted MTV was eliminated by foster-nursing. Of the foster-nursed subline 233 mice (16 of the first, 118 of the second and 99 of the third generation) were tested for the presence of Mtv-2 with one or two of the following three methods: (1) ID-test, (2) EMT-test and (3) by examination of the development of spontaneous mammary tumours in breeding females. The tests were negative in 226 mice. This indicates that Mtv-2 was absent in nearly all mice of the congeneic subline. The positive reactions in the seven other tested mice were weaker than that observed in the GR strain and in GR mice foster-nursed by BALB/c. The presence of the Mtv-2 gene in the seven positive mice of the congeneic line is doubtful. With the ID test, viral antigens were detectable in 50% of the milk samples from the first lactation period of mice of the segregating backcross I population [GR congeneic X (GR congeneic X GR)], indicating a one-gene difference between the congeneic GR line and the progenitor GR strain.

High frequency variation in mammary tumor virus expression in cell culture

Clonal derivatives of C3HMT murine mammary cell lines in culture demonstrate conversion of mammary tumor virus (MMTV) expression at a rate of approximately 6 per 100 clones. This alteration is largely unidirectional from a relatively high level (MMTV H) to a 10 fold lower level (MMTV L). This high rate of MMTV L variant conversion is in apparent contrast to the presumably mutational rate (∼3 per million cells) that governs development of resistance to 6-thioguanine (TG) in the same mammary cells. In somatic cell hybrids between different MMTV TG r clones and mouse or hamster TK- cells, expression of constitutive levels of MMTV and responsiveness to dexamethasone induction is dominant. Thus MMTV expression is regulated by at least two levels of positive control, constitutive expression and glucocorticoid stimulation, but the former is subject to a high rate of variant formation.

Mouse mammary tumor virus as a model for viral carcinogenesis

Evidence is presented suggesting that genetic control may be the limiting factor in mammary tumor virus (MTV) positive mammary oncogenesis. Studies with hybrid mice involving high and low MTV-expressing murine strains suggest that expression of MTV in milk may not be as important in tumorigenesis as previously thought. The usefulness of the murine MTV model in the study of mammary malignancy is discussed.