The v-mos and H-ras oncogene expression represses glucocorticoid hormone-dependent transcription from the mouse mammary tumor virus LTR.

Abstract

We have subjected the viral mos oncogene (v‐mos), the activated human H‐ras oncogene [H‐ras (A)] and the normal human H‐ras protooncogene [H‐ras (N)] to the transcriptional regulation of glucocorticoid hormones by in vitro recombination with the promoter region of the mouse mammary tumor virus long terminal repeat (MMTV LTR) and transfection into NIH 3T3 cells. Cell clones were selected which exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium. The expression of the chimeric genes as a function of time after hormone stimulation was studied at the level of transcriptional rate, mRNA and protein accumulation. Oncogene expression was stimulated rapidly to high levels, after hormone addition, but declined in the continuous presence of hormone. Measurements of the transcriptional rates in nuclei from LTR v‐mos and LTR H‐ras (A) transfected cells showed a repression of LTR v‐mos and LTR H‐ras (A) transcription after the initial stimulation by hormone. LTR H‐ras (N) transcription was not affected. An independently transfected LTR H‐2Ld construct in LTR v‐mos or LTR H‐ras (A) containing cells is also transcriptionally repressed. These experiments demonstrated a transcriptional repression effect of the oncogene products on the glucocorticoid hormone‐dependent MMTV LTR transcription.

Inhibitor of apoptosis protein (IAP) survivin is upregulated by oncogenic c-H-Ras

“…Approximately 200 G418-resistant colonies of each experimental group were pooled and cell lysates subjected to Western blot analysis, using an antibody against human survivin, as described above after 36 h (Figure 2a, right panel). Since the amounts of endogenous Ras proteins were below detection limit, the half-lifetime of oncogenic c-H-Ras was calculated from densitometric evaluation of the Western blot (data not shown) and was found to be approximately 8 h, which is shorter than that calculated for other Ras-transformed cell lines (Jaggi et al, 1986).…”

“…The nontransformed, G418-resistant embryonic rat cell line, 423, which was initially immortalized by Human Papilloma Virus type 11 DNA, was supertransfected with either the plasmid pEJ6.6, encoding a constitutively activated c-H-ras oncogene or the MMTV-rasA construct, expressing a Dexinducible oncogenic version of the c-H-ras gene (DiR) (Jaggi et al, 1986) together with a hygromycin-resistance gene. In the MMTV-rasA plasmid, the oncogenic version of c-H-ras is induced upon addition of Dex (1 mm) to the culture medium.…”

“…In the MMTV-rasA plasmid, the oncogenic version of c-H-ras is induced upon addition of Dex (1 mm) to the culture medium. The MMTV-LTR contains a sequence element located between nucleotide position À59 and À202 which confers glucocorticoid hormone inducibility (Jaggi et al, 1986). Transfected 423-cells were replated 20 h later and hygromycin (50 mg/ml) was added after 2 days.…”

Oncogenic Ras Leads to Rho Activation by Activating the Mitogen-activated Protein Kinase Pathway and Decreasing Rho-GTPase-activating Protein Activity

“…LTR-H-Ras(A) cells express H-Ras(V12) under control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat and exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium (50). The addition of dexamethasone leads to a significant increase in Ras expression at 8 h with peak expression at 24 h; this is associated with a dramatic change in cell morphology and acquisition of anchorage-independent growth (50).…”

GTP‐blot Analysis of Small GTP‐binding Proteins

“…NIH3T3 fibroblasts transfected with the human c-Ha-ras protooncogene or the Ha-ras (Va112) oncogene under control of a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat were cultured, as described by Jaggi et al [20]. If indicated, dexamethasone was added for 24 h at a concentration of 1 pM, either alone or in combination with lovastatin at a concentration of 20 pM.…”