THE EFFECTS OF RETINOIC ACID ON CELLULAR TRANSFORMATION AND TUMORIGENESIS INVOLVING CELLS WITH KNOWN ONCOGENES (VITAMIN A, RETINOIDS, RETROVIRUS)

Abstract

Vitamin A is known to have an important role in cellular differentiation and proliferation. In addition to regulating normal cellular processes vitamin A has also been shown to possess potent antineoplastic activity. The work in this dissertation characterizes the role of retinoic acid (RA) in cellular transformation and tumorigenesis with known oncogene involvement. These studies were initiated by examining the effects of RA on human carcinoma cell lines which express an activated c-ras gene. The bladder carcinoma, EJ/T24 (c-rasᴴ) and the two lung carcinoma cell lines, LXl (c-rasᴷ) and A2182 (c-rasᴷ), were not sensitive to RA. No inhibition of anchorage- or density-dependent growth was observed. Therefore, since these in vitro markers of transformation indicated a lack of effectiveness of RA on carcinomas containing a c-ras gene, retrovirally transformed cells were tested for RA sensitivity. Kirsten murine sarcoma, Balb/c murine sarcoma virus, and Simian sarcoma virus transformed NIH/3T3 and NRK cells were used in these studies. In contrast to the human carcinoma cell lines, anchorage-independent growth of some of the virally transformed cells was very sensitive to inhibition by RA. Anchorage-independent growth of KNRK and SSVNRK cells was sensitive to high concentrations (5 μM) of RA; whereas, Balb/cMSV3T3 and SSV3T3 were sensitive to 1-20 nM RA. BALB/cMSVNRK anchorage-independent growth was stimulated 3.5 fold by 1 μM RA. KNRK displayed a 60% reduction in anchorage-dependent growth at 10 μM RA while little inhibition was observed with the other retrovirally transformed cells. A high level of sensitivity to RA inhibition of anchorage-independent growth was correlated with the presence of cytoplasmic retinoic acid binding protein (CRABP). This indicated that CRABP may have some role in the inhibition of retrovirally induced cellular transformation. RA was shown to significantly reduce the incidence and size of Balb/cMSV3T3 cell tumors in nude mice. The inhibition of tumorigenesis in vivo therefore confirmed the results observed in vitro. To investigate the mechanism by which RA was acting to inhibit retroviral transformation, v-onc mRNA levels were examined. RA had no effect on v-onc mRNA levels in cell lines sensitive to the inhibition of transformation. The effect of RA on the relative rate of synthesis of p21, the transforming protein of KMSV and Balb/cMSV, was investigated. No effect of RA was observed in any of the cell lines. Also, GDP binding by p21 in KNRK cell was unchanged by RA treatment indicating that the functional activity of this transforming protein was not modified. RA does appear to be effective in inhibiting retrovirally induced cellular transformation and tumorigenesis. Evidence presented here indicates that this inhibition is not due to a direct effect of RA on the expression of the v-onc gene and/or gene product. Therefore, some other essential cooperating event(s) occurring within the cell are being acted upon by RA.