Development of new therapy for uterine cervical carcinoma

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1. Alterations in cell morphology induced by HPV16E7 We have shown that SM-alpha actin transcription and translation are completely inhibited in the presence of HPV16E7 protein expressed in Rat embryonal fibroblasts. To explore the mechanism involved in the transcriptional silencing of SM-alpha actin, we have made the C24G (Sub-stitution of codon 24 glycine for cystein) and the C91G mutant E7 protein as Rb-and C-jun binding domains are important for E7-mediated cell transformation. A Wild-type E7 and C24G mutant had a potential to inhibit SM-alpha actin transcription. In turn, aC91G mutant abrogated its function to silence the SM- alpha transcription, suggesting the importnatce of c-ter region of E7. In addition, suppression of MyoD expression by wild-type and 024G mutants were in contrast to the absent of MyoD suppression by 091 G.These suggested that modulation of MyoD expression by E7 and E7-c-jun binding are important for the SM-alpha actin silencing in the HPV16E7 expressing cells … More .2. Molecular diagnosis of cervical cell carcinoma progression We have amplified HPV E6/E7 DNAs obtained from lymph nodes of 64 patients by the use of consensus primers targeted to the E6/E7 and amplified DNAs were subjected to southern blot analyses. Histo-pathological examinations indicated the positive lymph node metastasis in 10 out of 64 patients. However, the PCR-southern blot examination showed the presence of HPV16 E6/E7 DNAs in the lymph nodes from 45 patients out of the remaining lymph node metastasis negative 54 patients. These suggested the sensitivity of PCR-Southern methods to detect lymph node metastasis.3. Development of antisense oligo nucleotides (AS) with enhanced functions. We have developed the psolaren-conjugated AS that is targeted to HPV16 or 18 E6 (PS-P1, PS-K3 and PS-K4). A scramble PS-1scr was used as a control. In the presence of UV irradiation, PS-Pi suppressed the growth of cervical cancer cells, that was dependent on the E6 gene sequence. C4 II cervical cancer cells maybe encoded the endogenous mRNA targeted by both PS-K3 and PS-K4. PS-AS had aAS potential to inhibit the cancer cell growth, that was more than 80 folds, compared to phospborothioate AS.PS-AS did not exhibit the toxic effects to normal keratinocyte growth. Less