The C-terminal domain of the human vitamin D receptor (hVDR) is essential for dimerization with retinoid X receptors and for transcriptional activation. To define the dimerization domain of the hVDR, a series of internal deletion mutants of the receptor were prepared beginning within the E domain and extending through the F domain to the C terminus. These mutant receptors were tested for dimerization and transcriptional activities by means of gel shift assay and beta-galactosidase assay, respectively, in a yeast system. The dimerization domain of the hVDR was localized to two separate but adjacent regions of the receptor molecule. In these experiments, the activation domain colocalized with dimerization. To more precisely delineate a relationship between these domains, region-specific random mutagenesis was carried out to detect mutants using error-prone PCR and a functional screen strategy employed using transformed yeast. Two classes of inactive receptors were identified: one in which both transcriptional activation and dimerization were compromised and a second in which only transcriptional activation was abolished. Most of the mutations responsible for these phenotypes were single. The studies suggest a separation between dimerization and transactivation domains. We reconstituted each of these hVDR mutants in a mammalian expression vector and evaluated them individually in COS-1 cells. All VDR mutants were transcriptionally active in this cellular background in response to 1,25-dihydroxyvitamin D3 although the potency of the hormone was reduced. The latter observation coincided with the observation that each mutant was compromised to some extent in binding affinity. These data clearly demonstrate the existence of an activation domain in hVDR that is separable from the domain involved in dimerization. Factors that couple hVDR to the general transcription apparatus in yeast through the activation domain in the hVDR, however, appear to be unrelated or dissimilar to those used in COS-1 cells.

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IntroductionPeritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip) paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis.MethodsWe developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS) and a cationic liposome (DFP-10825) and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors.ResultsDFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA) levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect.ConclusionCollectively, the ip delivery of DFP-10825, TS shRNA conjugated with cationic liposome, shows a favorable antitumor activity without systemic adverse events via the stable localization of TS shRNA for a sufficient time and concentration in the peritoneal cavity of the peritoneally disseminated human ovarian cancer-bearing mice.