Molecular mechanisms of growth inhibition by the chymotrypsin-like serine protease inhibitor, AAPF CMK

Cervical cancer is the second most prevalent cancer in woman worldwide (Kamangar et al., 2006). Infection with one of the 15 known high-risk human papillomavirus (HPV) types is etiologically linked to 95% of cervical cancer (zur Hausen, 1996). AAPFCMK is a chymotrypsin-like serine protease inhibitor that has been shown to inhibit the growth of organotypic raft cultures containing cells with high-risk HPV types, with no effect observed on uninfected keratinocytes (Drubin et al. 2006).;We hypothesized that AAPFCMK would arrest the growth of cervical cancer cell lines and cell lines immortalized or transformed by the integration of the SV40 and HPV small DNA tumor viruses through inhibition of the cell cycle based on previous studies (Clawson et al., 1995) (Drubin et al., 2006). We also hypothesized that AAPFCMK growth arrests cell lines infected by the small DNA tumor viruses, SV40 and HPV-16, by blocking the ability of the SV40 Large T-antigen (SV40 LTag), HPV-16 E6 and/or HPV-16 E7 oncoproteins from interacting with the tumor suppressor proteins, p53 and pRb.;In this dissertation, we investigated mechanisms of AAPFCMK growth inhibition, using cell culture model systems involving three human cervical carcinoma cell lines and the THLE-2 liver epithelial cell line immortalized with the SV40 LTag to test the hypotheses. C33a cells are cervical cancer cells with no HPV DNA while SiHa and CaSki cervical cancer cells have integrated HPV-16. Using this model system we examined the effects of AAPFCMK on cellular growth, cell cycle kinetics, DNA synthesis, and p53 and pRb protein expression. Growth of the three cervical cancer cell lines was inhibited after AAPFCMK treatment. C33a cells arrested at the G2/M phase of the cell cycle after AAPFCMK treatment. The HPV-16 SiHa and CaSki cells also exhibited an arrest of the cell cycle after AAPFCMK treatment; however, CaSki cells exhibited an arrest at the G1 /S phase of the cell cycle while SiHa cells exhibited a significant decrease in cellular proliferation resulting in a global arrest of the cell cycle.;Western analysis of HPV-16 E7 and the E7 protein downstream target E2F1 showed no significant difference in protein expression levels after AAPF CMK treatment in both the CaSki and SiHa cells. However, mRNA expression levels of the E2F1 target gene, PCNA, showed a significant decrease in both CaSki and SiHa cells after AAPFCMK treatment. AAPFCMK treatment also inhibited the growth of the SV40 LTag immortalized THLE-2 cell line, but the inhibition of growth was not associated with a change in p53 protein levels, or the downstream target p21. The protein levels of pRb, and the downstream target proteins E2F1 and PCNA, were also unchanged in THLE-2 cells after AAPFCMK treatment. However, cell cycle arrest, decreased cellular proliferation and inhibition of PCNA transcription observed experimentally after AAPFCMK treatment in both CaSki and SiHa cells indicate that AAPFCMK may potentially inhibit the HPV-16 E7 protein binding of p21 and/or inhibit E2F1 from transcribing PCNA.;Western analysis of p53 and p21, the downstream targets of the HPV-16 E6 protein, demonstrated a significant increase of both p53 and p21 protein expression in CaSki cells but no change was observed in the SiHa cells after AAPFCMK treatment. The results of the western analysis were verified by p53 and p21 mRNA expression levels after AAPFCMK treatment in both cell lines. Changes in p53 and p21 expression in SiHa cells should not be dismissed entirely as high levels of p53 and p21 expression in SiHa cells (as compared to CaSki cells), may make any changes difficult to detect. Therefore, AAPFCMK may also potentially interfere with either HPV-16 E6 protein binding to E6AP or with the E6/E6AP complex from binding to and/or ubiquitinating p53. Future research into the potential binding targets of AAPFCMK elucidated in this dissertation may be necessary to utilize AAPFCMK as a potential HPV therapeutic. (Abstract shortened by UMI.)...