Mammary Tumor Virus RNA Research Articles

Steroid regulation of transfected genes in mouse mammary tumour cells

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.

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.

Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

Identification of the Mtv -2 gene responsible for the early appearance of mammary tumors in the GR mouse by nucleic acid hybridization

In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this Mtv-2 gene by nucleic acid hybridization between MTV [(3)H]cDNA and DNA from GR, B10, and GR-Mtv-2(-) mice. B10 and GR-Mtv-2(-) mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-Mtv-2(-) mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2(-) DNA might represent another Mtv gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-Mtv-2(-) mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.

Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells

Glucocorticoid hormone treatment of GR cells, a cultured line derived from mouse mammary tumor tissue, selectively stimulates the rate of transcription of integrated proviral genes specifying mammary tumor virus (MTV). We have incubated isolated nuclei from these cells under conditions in which all three endogenous RNA polymerases appear to be active. RNA synthesized in vitro is distinguished from preexisting nuclear RNA by labeling the in vitro products with [3H]CTP, and the level of MTV RNA synthesis is measured by molecular hybridization with unlabeled viral DNA. Synthesis requires the addition of nucleoside triphosphates, and is inhibited by actinomycin D. Pretreatment of GR cells with dexamethasone, a synthetic glucocorticoid, has no significant effect on the amount of total RNA synthesis in isolated nuclei. In contrast, synthesis of MTR RNA is stimulated 10-20-fold in nuclei from dexamethasone-treated cells relative to untreated control nuclei; the sensitivity of in vitro viral RNA synthesis to inhibition by alpha-amanitin suggests that it is carried out exclusively by RNA polymerase II. The fraction of total RNA synthesis which is viral specific (about 0.2-0.4% in nuclei from dexamethasone-treated cells and 0.01-0.03% in controls) is similar to that detected in pulse labeled RNA from whole GR cells in culture. Our procedures for labeling and hybridization of RNA appear to avoid artifacts recently noted in other in vitro transcription systems.