Disruption of gap junctional intercellular communication and/or Connexins (the channel proteins of gap junctions) is frequently reported in malignant cell lines and tumours. Many tumor cells exhibit aberrant gap junctional intercellular communication (GJIC), which can be restored by transfection with Connexin genes. Of the Connexin family, Connexin 43 has attracted the most attention as it is widely expressed in many tissues and Connexin 43 gap junctions correlate with various physiological functions. Connexin 43 is a short-lived protein (half-life of 1-3 h in cultured cells), both lysosomes and proteasomes having been reported to be involved in its degradation. Certain human papillomaviruses (HPV) associated with the development of cancers, especially of the cervix, have been reported to downregulate GJIC in vitro. There is also evidence for reduced gap junctions in cervical dysplasia. The association between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in cervical tumour progression, remains unclear. In HPV-16 associated cervical cancer, the viral oncogene E6 inactivates the tumour suppressor p53, but also has p53- independent functions in tumour progression. One of these may involve interaction with hDlg (the human homologue of Drosophila Discs Large), a tumour suppressor present in epithelial cells at sites of cell-cell contact and which regulates cell polarity and attachment. hDlg contains three PDZ protein-protein interaction domains, the second PDZ domain of which binds E6. Connexin 43 also has a PDZ binding domain in its C-terminus. Previously, it was demonstrated that Connexin 43 relocates from the membrane to the cytoplasm in a novel HPV-16 E6-containing cervical epithelial cell line (named WI2GPXY) that is fully transformed and invasive and deficient in gap-junctional intercellular communication (Aasen T et aI., 2003 Oncogene 22, 7969- 7980). The overall aim of this project was to investigate the relationship of loss of gap junctions to malignant progression by comparing the properties of W12GPXY with those of the non-malignant parental cell line, WI2G, from which W12GPXY was derived. First, microarray was used to identify global differences in RNA expression between the two cell lines, large differences were seen in expression of angiogenesis-related genes and they were confirmed by Real-Time RT-PCR for three genes, IL-8, VEGF and FGF-2. No significant differences were noted for connexin genes but there were differences in MAGUK family members including hDlg. However, protein expression studies by western blot and immunofluorescence staining showed a significant increase (2.9 fold) of HPV-16 E6 in W12GPXY cells, which co-localises with hDlg in the cytoplasm. Connexin 43 also binds hDlg. Treatment ofW12GPXY cells with Leptomycin B to trap E6 in the nucleus or siRNA knockdown of E6 abrogate the relocation and co-location of hDlg and endogenous wild type Connexin 43 and restore Connexin 43 gap junction at points of cell-cell contact. Further, when C33a cells (HPV -negative cervical tumour cells which normally retain large Connexin 43 gap junctions) are transfected with HPV -18 wild type E6, changes in localisation of Connexin 43 and hDlg are consistent with those in W12GPXY cells. However, C33a cells transfected with a mutant E6 lacking the hDlg binding site retain Connexin 43 gap junction plaques. Finally, Connexin 43 associates with hDlg through its PDZ-binding domain and this is required for its relocalisation to the cytoplasm in W12GPXY cells. The results suggest that increased cytoplasmic E6 associated with malignant progression of W12GPXY cells redistributes Connexin 43 from membrane junctions into the cytoplasm by a mechanism involving binding of hDlg to the Connexin 43 C-tenninal tail. The findings have uncovered a new role for hDlg in trafficking of Connexin 43. It also provides a novel mechanism for the loss of gap junctional intercellular communication during malignant progression of squamous epithelial cells. The specific roles played by lysosomes and proteasomes in the degradation of Connexin 43 in W12GPXY cells were also studied. The results suggest the involvement of both proteolytic pathways, although the lysosome seems to be the major compartment for Connexin 43 degradation. Association with HPV-16 E6 and hDlg together with proteasome activity seems to be required for Connexin 43 redirection from the cell membrane and transport into the lysosomal degradation pathway. Taken together, these results suggest that Connexin 43 gap junction intercellular communication was lost from the cell membrane requiring maintenance of E6 and hDlg complexes for proteasomal internalization and consequently transport into lysosomal compartment for degradation in W12GPXY cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:444036 |
| Date | January 2005 |
| Creators | Sun, Peng |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5003/ |
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