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Effect of suppression of HPV 18 E7 oncoprotein on expression of human

tumor-associated protein MN/CA IX in HeLa cells

Article  in  Chemical Papers- Slovak Academy of Sciences · January 1998

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Effect of Suppression of H P V 18 E7 Oncoprotein on Expression of Human Tumor-Associated Protein MN/CA IX in HeLa Cells

J. LIESKOVSKÁ, A. GIBADULINOVÁ, M. KALUZOVÁ, J. PASTOREK, and S. PASTOREKOVÁ Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic

High-risk genital human papillomaviruses (HPV typel6 and 18) play an important role in the devel­opment of cervical cancers (1). There is considerable evidence that their oncogenic potential is related to the expression of two viral proteins, E6 and E7. The E7 oncoprotein shows immortalizing and transform­ing activities which are mediated, in part, through the interaction with the members of pRb protein family. This interaction results in transcriptional activation of genes regulating cell proliferation, including cyclins E and A as well as oncoprotein B-myb (1).

The MN/CA IX protein is a newly described hu­man tumor-associated antigen. It was originally de­tected in human cervical carcinoma cell line HeLa (2). Expression of MN/CA IX correlates both with the density of HeLa cells and with tumorigenic phenotype of hybrids between HeLa and normal fibroblasts (3). MN/CA IX protein is expressed also in other human carcinoma cell lines and in certain human carcinomas, but not in the corresponding normal tissues. Liao and Stanbridge have proposed the utility of MN/CA IX as a biomarker of cervical neoplasia (4). Both cDNA and gene encoding MN/CA IX were cloned and character­ized (5, 6).

Several human carcinoma cell lines that contain HPV DNA and express E7 oncoprotein, express also MN/CA IX. This finding evokes question about pos­sible relationship between these two proteins. It was shown that E7 oncoprotein has a capacity to trans-activate the AP-1 family of transcription factors (7). There is one AP-1 site in MN/CA9 promoter at posi­tion —68/ — 60 (6). It is protected at both strands in DNase I footprinting, and binds AP-1 in EMS A (un­published results). These facts raised the possibility that antisense expression of E7 could lead to a de­crease of AP-1 trans-activation and consequently, to diminished transcription of MN/CA9 gene finally re­sulting in lower level of MN/CA IX protein.

We have approached this problem by establishment of inducible antisense expression of E7 in HeLa cells by employing a tightly controlled expression system regulated by tetracycline (tet) (8). HPV 18 E7 cDNA was prepared by PCR and cloned into pUHD10-3 vec­tor. The antisense cDNA construct was co-transfected with pTK-Hygro vector to HeLa/tet-off cells, that contain multiple copies of HPV 18 genome and ex­press tet transactivator. In HeLa/tet-off cells, gene expression is turned on when tet is removed from the culture medium. The transfected cells were subjected to hygromycine selection, cloned and several dozens of individual clones were analyzed for expression of

E7 oncoprotein. They were cultured in the presence and absence, respectively, of tet for 3 days. Then the cellular extracts were subjected to Western blotting using E7-specific M Ab 67G7 (Triton). The clone that showed decreased level of E7 protein after tet with­drawal was analyzed for expression of MN/CA IX by Western blotting using M Ab M75. The analysis did not show any difference at protein level in tet-treated versus untreated cells. It is possible that the decrease of AP-1 trans-activating potential was not sufficient to diminish transcription of MN/CA9 gene. Alterna­tively, AP-1 transcription factor might not be the crit­ical trans-activator of MN/CA9 expression. Neverthe­less, our results suggest that there is no relationship between expression of HPV 18 E7 oncoprotein and MN/CA IX protein.

Data presented in this work are in agreement with the results of tumor tissue studies performed by Brewer et al. (9) and Resnick et al. (10). They found that there is no correlation between the frequency of MN/CA IX staining and the proportion of HPV-positive cells (10) and that low expression or loss of expression of MN in cervical cancers correlates with HPV negativity (9).

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Chem. Papers 52 (Focus Issue) 289 (1998) 289

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