The role of claudin-1 and claudin-7 in cervical tumorigenesis.

BACKGROUND/AIM: The claudin family of proteins are key constituents of tight junctions and the aberrant expression of these proteins can contribute to de-stabilisation of tight junctions and thus to loss of cell polarity and cohesion. Increased expression of claudin-1 and claudin-7 has been observed in pre-invasive cervical lesions and cervical carcinomas. The present study attempted to assess the effect of claudin-1 and claudin-7 overexpression on the HeLa cervical carcinoma cell line, in terms of cell proliferation/viability, permeability, invasion and migration. MATERIALS AND METHODS: HeLa cells were stably transfected with expression vectors containing the claudin-1 and claudin-7 genes to produce two separate stable cell lines expressing claudin-1 and claudin-7, respectively. The stable cell lines were examined with regard to their invasion and migration abilities, cell permeability and cell proliferation/viability and compared to non-claudin-1 or -7 transfected HeLa. RESULTS: The present study found that claudin-1 and claudin-7 affected the migratory ability of HeLa cells, reducing their ability to migrate in a gap closure assay compared to non-claudin-transfected HeLa cells. Monolayers of claudin-1 and claudin-7 transfected cells also displayed an increased transepithelial electrical resistance indicating decreased permeability compared to non-claudin-transfected HeLa. The study found that claudin-1 or claudin-7 expression had no effect on the proliferation or viability of HeLa cells. Claudin-1 or -7 expression also did not affect the invasive ability of HeLa cells with both stable cells lines and non-claudin-transfected HeLa cells all showing low invasive ability. CONCLUSION: The results of the present study indicate that claudin-1 and claudin-7 overexpression alone does not contribute to increased tumorigenesis in cervical carcinoma, instead claudin-1 and - 7 expression in HeLa cells contribute to reducing the migratory ability of cells and decrease their permeability.

Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression.

To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression.