Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Lin, Stanley L.; Garber, Ellen A.; E, Wang; Caliguiri, Lawrence A.; Schellekens, Huub; Goldberg, Andrew V.; Tamm, Igor

doi:10.1128/mcb.3.9.1656

Cited by 44 publications

(15 citation statements)

References 47 publications

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“…Therefore, the reduction in 32p radioactivity in the p60sr, fraction could have resulted from inhibition (probably inactivation, see below) of the protein kinase activity of p6Osrc but not from a reduction in the amount of p6Osrc by these antibiotics in the cells. This is in contrast to the recent report that interferon selectively inhibits synthesis of p6Osrc in RSV-transformed rat cells, resulting in reversion to the normal phenotype (23).…”

Section: Discussioncontrasting

confidence: 54%

Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus.

Uehara¹,

Hori²,

T³

et al. 1986

Mol. Cell. Biol.

196

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Three benzenoid ansamycin antibiotics (herbimycin, macbecin, and geldanamycin) were found to reduce the intracellular phosphorylation of p6osrc at a permissive temperature (33°C) in a rat kidney cell line infected with a temperature-sensitive mutant of Rous sarcoma virus. This effect was accompanied by morphological changes from the transformed to the normal phenotype. The filamentous staining pattern of actin fibers was observed in the cells treated with these antibiotics at 33°C. Removal of the antibiotics allowed the cells to revert to the transformed morphology. Ansamitocin, another benzenoid ansamycin, and naphthalenoid ansamycins such as streptovaricin and rifamycins did not show this effect. Pulse-labeling of the antibiotic-treated cultures with 32P, showed a marked reduction of 32P radioactivity incorporated into p6orc. A parallel experiment with[35S]methionine showed that synthesis of p6tWc was slightly inhibited. The immune complex prepared by mixing the herbimycin-treated cell extracts with antibody against p6tYrc was inactive in vitro in phosphorylating the complex itself. On the contrary, the immune complex derived from untreated cells was active in vitro even in the presence of the antibiotics. These results suggest that benzoquinonoid ansamycins have no direct effect on src kinase but destroy its intracellular environment, resulting in an irreversible alteration of p60src and loss of catalytic activity. Viral oncogenes have been well characterized at the molecular level, and the studies in this field have provided insights into the understanding of neoplastic transformation. In Rous-sarcoma-virus (RSV)-transformed cells, the functional expression of p6Osrc, the src gene product, is required for manifestation of the oncogenic event (1,3,29). The available evidence strongly suggests that the protein kinase activity of p6Osrc is responsible for the phenotypic changes of cells cultured in vitro (6,22,31): morphological transformation, high saturation density of cell growth, enhanced glucose uptake, the lowered requirement for serum concentration, and growth in soft agar (1,4,14,18).As a means of finding potential antitumor antibiotics, we have been screening compounds which inhibit the function of such oncogenes and thereby suppress the expression of transformed phenotypes. As a tester strain, we have been using normal rat kidney (NRK) cells transformed with a mutant of RSV whose src gene product, the tyrosine protein kinase, is temperature sensitive. By using this assay, we recently found a culture broth of a Streptomyces species which was active in converting transformed cell morphology to normal morphology at a permissive temperature and identified the active agent as herbimycin (34). In herbimycintreated cells, intracellular phosphorylation of p6Osrc was inhibited and the cytoplasmic organization of cytoskeletal proteins was rebuilt. Herbimycin did not inhibit the p6Osr,_ associated kinase activity in vitro, however. Two other benzenoid ansamycins, macbecin and geldanamycin, both of

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Section: Discussioncontrasting

confidence: 54%

Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus.

Uehara¹,

Hori²,

T³

et al. 1986

Mol. Cell. Biol.

196

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“…In contrast, no such reversion was observed in repeated studies with IFN-treated RS504, an NIH 3T3 cell line transformed by the mutated EJras (33). Revertants of RS485 that were propagated in the continuous presence of 200 IU of IFN-cx/p per ml were nontumorigenic (34) and produced four-to eightfold less ras mRNA and ras-encoded p21 than parental RS485 (32).Our present studies showed that in contrast to the reversibility of cellular changes seen with short-term (several days) exposure to IFN (14,20,25,30), the phenotypic reversion of RS485 established after 1 to 2 months of IFN treatment persisted long after treatment was discontinued. The persistent revertants (PRs) expressed high levels of p21, yet were not tumorigenic.…”

contrasting

confidence: 52%

“…Our present studies showed that in contrast to the reversibility of cellular changes seen with short-term (several days) exposure to IFN (14,20,25,30), the phenotypic reversion of RS485 established after 1 to 2 months of IFN treatment persisted long after treatment was discontinued. The persistent revertants (PRs) expressed high levels of p21, yet were not tumorigenic.…”

contrasting

confidence: 41%

Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.

Samid¹,

Flessate²,

Friedman³

1987

Mol. Cell. Biol.

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Prolonged alpha/beta interferon (IFN-a/,8) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion.Abnormalities in ras expression have been observed in a wide variety of naturally occurring or induced tumors (6,36,41). The effect of interferon (IFN) on ras expression is of particular interest since IFNs have been shown to inhibit the growth of tumors in vivo (1) and to induce phenotypic reversion in some transformed, cultured cells (3. 5, 11. 20. 25). The mechanism of action of IFN in such systems is unknown.The human c-Ha-rasl, a homolog of the transforming gene of Harvey murine sarcoma virus, can acquire transforming potential for NIH 3T3 cells by a point mutation (EJras) (39) or by transcriptional activation after ligation to a retroviral long terminal repeat (LTR) regulatory element (4). We have previously demonstrated that with prolonged treatment, mouse IFN-ao/ induced phenotypic reversion in NIH 3T3 cells transformed by an LTR-activated human c-Ha-rasl (clonal line RS485) (32, 34). The revertants retained the activated ras. In contrast, no such reversion was observed in repeated studies with IFN-treated RS504, an NIH 3T3 cell line transformed by the mutated EJras (33). Revertants of RS485 that were propagated in the continuous presence of 200 IU of IFN-cx/p per ml were nontumorigenic (34) and produced four-to eightfold less ras mRNA and ras-encoded p21 than parental RS485 (32).Our present studies showed that in contrast to the reversibility of cellular changes seen with short-term (several days) exposure to IFN (14,20,25,30), the phenotypic reversion of RS485 established after 1 to 2 months of IFN treatment persisted long after treatment was discontinued. The persistent revertants (PRs) expressed high levels of p21, yet were not tumorigenic. While the PRs resisted retransformation by a variety of viral or cellular oncogenes, they were readily retransformed after exposure to DNAdemethylating drugs. The latter suggested possible mechanisms of the action of IFN. MATERIALS AND METHODS Cells

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“…In our cases, the p21 expression was greater in advanced (stage III) MM and closely related with BMPC infiltration. Alpha-interferon reverses a number of transformed, tumorigenic cell lines (Brouty-Boye et al, 1985) and some reversions are associated with a significant reduction in the expression of the oncogenes implicated in the transformation process (Lin et al, 1983;Samid et al, 1984). If we speculate that the effectiveness of this agent in MM is due to a similar mechanism, its activity may be evaluated with the FCM method we employed to detect p21 expression.…”

Section: Resultsmentioning

confidence: 99%

Ras oncogene expression and DNA content in plasma cell dyscrasias: a flow cytofluorimetric study

Danova

Riccardi²,

Ucci³

et al. 1990

Br J Cancer

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Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Cited by 44 publications

References 47 publications

Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus.

Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus.

Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.

Ras oncogene expression and DNA content in plasma cell dyscrasias: a flow cytofluorimetric study

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