Glucocorticoid-stimulated accumulation of mouse mammary tumor virus RNA: increased rate of synthesis of viral RNA.

Abstract

Glucocorticoid hormones specifically increase the intracellular concentration of mouse mammary tumor virus (MMTV) RNA in a cultured cell line from a GR mouse mammary carcinoma (GR) and in an MMTV-infected rat hepatoma cell line (M1.19). In contrast, these steroids have no effect on the concentration of MMTV RNA in a lymphoma line, S49, from a Balb/c mouse. Using a molecular hybridization procedure to detect newly synthesized RNA, we Steroid hormones are thought to act as allosteric effectors to increase the affinity of their cytoplasmic receptors for DNAcontaining binding sites in the nucleus (1, 2). The biochemical events that take place at the nuclear sites are not understood, but it is presumed that they are responsible for the physiological alterations induced by steroid hormones. In some steroid responsive systems, there is clear documentation that specific mRNA species accumulate in the hormone-treated cells (3-7). While it is usually assumed that the steroid-receptor complex acts to stimulate the transcription of these specific genes, there has not been a clear demonstration that the steroid-induced accumulation of a specific mRNA is actually due to an increase in its rate of synthesis.Production of mouse mammary tumor virus (MMTV) by cultured cells derived from mammary carcinomas of mice can be augmented by treating the cells with glucocorticoids (8)(9)(10)(11)(12)(13)(14). The effect is paralleled by a concomitant increase in the intracellular concentration of MMTV RNA (9,12,13,15,16); similar effects upon the concentration of viral RNA have also been observed in heterologous cells infected by MMTV (17,18). After an apparent lag of about 15 min, the viral RNA concentration in GR mammary tumor cells increases with a half-time of about 2.5 hr, reaching a new steady-state level, 10-to 20-fold over uninduced levels, by 5-6 hr after hormone addition (13). The induction of viral RNA is sensitive to inhibitors of RNA synthesis but insensitive to inhibitors of protein or DNA synthesis (13, 16). Although these observations are consistent with a model in which the steroid-receptor complex stimulates viral RNA synthesis, they do not exclude the possibility that the hormone acts by altering the processing of viral RNA and/or its degradation.Abbreviations: MMTV, mouse mammary tumor virus; cDNA, radioactive DNA complementary to MMTV RNA; NaDodSO4, sodium dodecyl sulfate. 2879In the present communication, we have used an assay devised by E. Stavnezer and J. M. Bishop (unpublished), which has allowed us to investigate the effects of glucocorticoid hormones on the rate of MMTV RNA synthesis. Our results suggest that glucocorticoid hormones stimulate the rate of transcription of MMTV genes in both mouse mammary tumor cells and in MMTV-infected rat hepatoma cells. MATERIALS AND METHODSCells and Viruses. GR and S49 cell lines have been described (12). HTC cells were infected with MMTV concentrated from the medium of GR cultures (17) and cloned as described (19) Preparation of "Tailed Duplex" DNA. MMTV (approxima...

Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells.

“…Glucocorticoids stimulate production of MMTV RNA in mouse, rat, mink, and cat cells (8,9), apparently as a direct effect of the hormone-receptor complex (9,11 Note Added in Proof. Results similar to those described here concerning the sites of integration of retrovirus DNA have recently been described (31)(32)(33).…”

“…This stimulation of MMTV gene expression appears to occur via a rapid increase in the rate of synthesis of viral RNA (10,11). Results of earlier experiments suggest that the template for viral RNA synthesis is MMTV DNA that is covalently linked to the cellular DNA (i.e., proviral DNA) (12).…”

“…Using restriction endonucleases and the DNA transfer procedure of Southern [Southern, E. M. (1975) Glucocorticoid hormones (e.g., dexamethasone) selectively stimulate the production of mouse mammary tumor virus (MMTV) in both mouse mammary carcinoma cells (1-7) and heterologous cells infected with MMTV (8,9). This stimulation of MMTV gene expression appears to occur via a rapid increase in the rate of synthesis of viral RNA (10,11). Results of earlier experiments suggest that the template for viral RNA synthesis is MMTV DNA that is covalently linked to the cellular DNA (i.e., proviral DNA) (12).…”

Effects of Interferon on the Expression of Mouse Mammary Tumour Virus in GR Cells

“…Confluent GR cells in 75 cmz Falcon flasks were incubated with 3 ml culture medium in the presence of 100 /~Ci/ml [3H]uridine for 1 h. Labelled RNA samples were hybridized for increasing periods of time with 0-2 ~g MuMTV eDNA in solution. The extent of hybrid synthesized was measured by resistance to pancreatic RNase digestion as described previously (Ringold et al, 1977). In both cases trichloroacetic acid-precipitable radioactivity in the hybrids was collected on membrane filters (Whatman GF/C) and quantified in a liquid scintillation counter.…”

“…The extent of hybridization was estimated by the fraction of eDNA resistant to S 1 nuclease. Virus RNA synthesis was measured by annealing rapidly labelled cellular RNA with unlabelled MuMTV-specific eDNA in excess according to published procedures (Ringold et al, 1977). Confluent GR cells in 75 cmz Falcon flasks were incubated with 3 ml culture medium in the presence of 100 /~Ci/ml [3H]uridine for 1 h. Labelled RNA samples were hybridized for increasing periods of time with 0-2 ~g MuMTV eDNA in solution.…”