Advances toward somatic cell gene therapy.
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Biomedical subjects
D Samid
Publications and source records attributed to D Samid.
Increased radiation resistance in transformed and nontransformed cells with elevated ras proto-oncogene expression.
The cellular Ha-ras oncogene, activated by missense mutations, has been implicated in intrinsic resistance to ionizing radiation. This study shows that the overexpression of the unmutated gene (proto-oncogene) may also be involved in how the cells respond to radiation. The experimental system consisted of mouse NIH 3T3-derived cell lines which carry multiple copies of a transcriptionally activated human c-Ha-ras proto-oncogene. Both tumorigenic (RS485) and revertant nontumorigenic subclones (PR4 and 4C3) which have high levels of ras expression exhibited a marked increase in radioresistance as measured by D0 compared to control NIH 3T3 cells. Other nontransformed cells with elevated levels of ras (phenotypically revertant line 4C8-A10) also had a significantly increased resistance to radiation, further indicating an association between ras and radioresistance. The increased radioresistance of the RS485 and phenotypic revertants could not be explained by a differential expression of the myc or metallothionein I genes or by variations in cell cycle. The correlation between increased ras proto-oncogene expression and radioresistance suggests that the ras encoded p21, a plasma membrane protein, may participate in the cellular responses to ionizing radiation.
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2',5'-Oligoadenylate synthetase gene expression in revertants of ras-transformed NIH3T3 fibroblasts.
Persistent revertant (PR) cells of Ha-ras-transformed NIH3T3 fibroblasts, isolated after prolonged treatment with interferon (IFN), have been previously described. PR cells remain nontumorigenic even after IFN withdrawal. To investigate the mechanisms responsible for the stable phenotypic reversion, we have now examined the potential involvement of an endogenous IFN and the 2',5'-oligoadenylate (2-5A) synthetase pathway. Northern blot analysis revealed an increased level of 2-5A synthetase transcripts in PR cells compared to parental Ha-ras-transformed cultures. Although inducible on treatment with exogenous IFN alpha/beta, this mRNA was not detectable in untreated NIH3T3 cells. 2-5A synthetase expression following IFN treatment was also significantly higher in PRs than in the normal or ras-transformed NIH3T3. The increased levels of synthetase mRNA correlated with a similarly elevated enzymatic activity in cell extracts from PR cells. This increased expression was biologically functional, since the revertant cells were more resistant to the cytolytic action of mengovirus than normal or ras-transformed NIH3T3 fibroblasts. Another class of IFN-induced genes, H-2 class I antigens, showed enhanced expression in PRs. Antibodies directed against mouse IFN alpha/beta did not reduce the constitutive expression of 2-5A synthetase in PR cells. Furthermore, conditioned medium from PR cultures or cocultivation with PRs failed to induce the enzyme message in NIH3T3 cells. Finally, there was no detectable elevation in the mRNA specific for IFN beta in the PR cultures, as determined using a sensitive polymerase chain reaction amplification protocol. These results show that the Ha-ras revertants constitutively produce a functional 2-5A synthetase, which appears to be independent of the production of an endogenous interferon alpha or beta.
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Transfection of EK-3, a subline of NIH 3T3, with the oncogene Ha-ras does not abolish its anchorage dependence.
EK-3 cells, previously isolated by us from cultures of NIH 3T3, require both ras and myc oncogenes for efficient transformation, while their parent cells are readily transformed by ras alone. We transfected the EK-3 cells with the v-Ha-ras oncogene and obtained several sublines which integrated this gene and transcribed it successfully. The ras-NIH 3T3 formed foci of multilayered cells that were piling up in culture, while the ras-EK-3 cells remained contact inhibited. Furthermore, when the growth of the cells in soft agar was examined, a clear difference was observed. Cells of the ras-NIH 3T3 clonal lines showed high efficiency of growth (10%), while the ras-EK-3 cells exhibited low efficiency (0.2%). The latter being quite similar to that of the non-transfected NIH 3T3 and EK-3 cells (0.05%). The results presented now, showing that ras-EK-3 cells are more anchorage dependent than the ras-NIH 3T3 cells, clearly indicate that differences, previously shown to exist between EK-3 and NIH 3T3 cells, persist in their daughter cell lines derived following transfection with the Ha-ras oncogene.
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Transcriptional inhibition of Ha-ras in interferon-induced revertants of ras-transformed mouse cells.
Elevation in Ha-ras expression, due to transcriptional activation or gene amplification, is associated with oncogenic transformation of NIH 3T3 cells. We have previously shown that murine interferon (IFN)-alpha/beta induced phenotypic reversion of NIH 3T3 cells transformed by long terminal repeat (LTR)-activated c-Ha-ras. The revertants produced decreased amounts of ras-encoded Mr 21,000 protein. We have now determined the molecular level at which LTR-ras regulation occurred. Nuclear run-on experiments revealed a selective inhibition of ras transcription in IFN-treated revertants. There was no apparent additional posttranscriptional control by IFN as judged by the unchanged half-life of ras transcripts. Inhibition of ras RNA synthesis was seen only in conjunction with long-term IFN treatment and was limited to the revertants, a population of cells that maintained sensitivity to IFN during the prolonged exposure. The reduction in ras activity appeared responsible in part for the loss of tumorigenicity in treated cells and was stable for several weeks after treatment had been discontinued.
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Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.
Prolonged alpha/beta interferon (IFN-alpha/beta) 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.
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Development of transformed phenotype induced by a human ras oncogene is inhibited by interferon.
Mouse IFN inhibited the development of transformed foci in NIH 3T3 cultures transfected with the viral Ha-MuSV(ras) and Mo-MuSV(mos) oncogenes, or with the human bladder carcinoma ras EJ/T24 DNA. IFN treatment five or seven days after transfection was still effective in inhibiting the oncogenic transformation, but did not inhibit significantly the biochemical transformation induced by pSV2-neo or Ecogpt DNA, so that inhibition of ras-induced transformation was not a result of a general effect on the transfection process. Treatment with IFN did not alter the expression of ras EJ/T24 DNA after the transformed phenotype had been established.
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Interferon-induced modulation of human ras oncogene expression.
Treatment of NIH 3T3 cells with interferon (IFN) after transfection with human bladder carcinoma EJ/T24 c-Ha-ras1 oncogene DNA caused inhibition of ras-induced cell transformation. Furthermore, the effect of IFN on oncogene expression in an established tumor line was studied. A tumor line of NIH 3T3 (RS485) was transformed by human c-Ha-ras1 activated by a viral long terminal repeat. Treatment of the tumor cells with IFN was associated with a progressive appearance of reverted, flat colonies which exhibited a normal phenotype with respect to morphology and growth. The revertants were well spread, contact inhibited cells; they did not grow in soft agar and were not tumorigenic in nude mice. Revertants retained their normal phenotype, although they contained transfecting human c-Ha-ras1 DNA; but, they produced significantly decreased levels of the onc-encoded protein p21 and c-Ha-ras1 mRNA as compared to RS485 cells.
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Biological and morphological characteristics of phenotypic revertants appearing in interferon-treated mouse cells transformed by a human oncogene.
Phenotypic revertants appearing in interferon-treated mouse cells that had been transformed by an activated human c-Ha-ras1 oncogene (cell line RS485) were tested for several biological properties. The cloned revertants regained the growth characteristics of the untransformed parental NIH 3T3 cell line; unlike RS485 cells the revertants failed to form colonies in soft agar or to form rapidly growing tumors in nude mice. Animals inoculated with RS485 cells developed tumors within one week. In contrast, revertant clonal line 4C3 failed to form tumors four months after transplantation. Revertant 4C8 cells were tumorigenic; however, the developing tumors had increased latency, slower growth rate, and remained smaller than tumors of RS485 cells. Histopathological analysis revealed that revertant-associated fibrosarcomas were less anaplastic, less cellular and had relatively infrequent mitotic figures as compared to fibrosarcomas of RS485 cells. The result suggest the IFN-induced revertants exhibit a significantly less malignant phenotype than their parental transformed cells and that the biological differences are maintained after IFN treatment is discontinued.
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Mechanisms of antioncogenic activity of interferon.
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Biochemical correlates of phenotypic reversion in interferon-treated mouse cells transformed by a human oncogene.
Mouse interferon (IFN) induced a phenotypic reversion in RS 485, a clonal line of NIH 3T3 oncogenically transformed by a human c-Ha-rasl gene activated by Ha-MuSV long terminal repeats (LTRs). Transfected c-Ha-ras DNA, unchanged in quantity and distribution, as compared to the parental RS 485 transformed cells, was still present in these revertants; however, there was a significant reduction in the amount of c-Ha-ras specific mRNA and of c-Ha-ras specified p21 protein.
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A phase II study of 5-aza-2'deoxycytidine (decitabine) in hormone independent metastatic (D2) prostate cancer.
AIMS AND BACKGROUND: Decitabine (5-aza-2'-deoxycytidine) is an S-phase-specific pyrimidine analog with hypomethylation properties. In laboratory models of prostate cancer (PC-3 and DU-145), decitabine induces cellular differentiation and enhanced expression of genes involved in tumor suppression, immunogenicity, and programmed cell death. METHODS: We conducted a phase II study of decitabine in 14 men with progressive, metastatic prostate cancer recurrent after total androgen blockade and flutamide withdrawal. Decitabine was administered at a dose of 75 mg/m2/dose i.v. as a 1 hour infusion every 8 hours for three doses. Cycles of therapy were repeated every 5 to 8 weeks to allow for resolution of toxicity. RESULTS: Two of 12 patients evaluable for response had stable disease with a time to progression of more than 10 weeks. This activity was seen in 2 of 3 African-American patients. Toxicity was similar to previously reported experience. No significant changes in urinary concentrations of the angiogenic factor bFGF, a potential biomarker of tumor activity, were identified over time in 7 unselected patients with progressive disease. CONCLUSIONS: We conclude that decitabine is a well tolerated regimen with modest clinical activity against hormone-independent prostate cancer. Further investigations in patients of African-American origin may be warranted.
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