Abstract
Human papillomaviruses (HPV) are known to immortalize oral keratinocytes in vitro, but the underlying mechanisms causing the following resistance to differentiation remain unclear. We investigated the effect of extracellular calcium on the proliferation of HPV16-positive keratinocytes and on the mRNA expression of the viral E6-oncogene. HPV16-positive hypopharyngeal carcinoma cells (UD-SCC-2), spontaneously immortalized- (HMK) and HPV16 E6/E7-immortalized human gingival keratinocytes (IHGK) were grown for 3, 6 and 9 days in Keratinocyte Serum-free Medium with calcium concentrations ranging from 0 mM to 6 mM. Calcium concentrations up to 0.09 mM increased cellular proliferation, which decreased at higher concentrations. A shift of calcium concentration from 0 to 4 mM increased E6 expression in UD-SCC-2 cells 2.4-fold by day 9. Simultaneously, E2 expression increased. The most significant upregulation of E6 and E2 expressions was observed at day 9, grown in high-calcium media and the increase in E6 expression coincided with an increase in involucrin expression, likely indicating cell differentiation. Despite this, HPV-positive cells continued to proliferate even at high-calcium media in contrast to HPV-negative cells. Overexpression of E6 mRNA may be an important feature of HPV16-positive cells to resist the natural calcium gradient in differentiating keratinocytes allowing cell proliferation.
Highlights
E6 are mediated by E6-associated protein (E6-AP) along with E6-binding protein (E6-BP), which is a cellular calcium-binding protein [8,9,10]
Because the Human papillomaviruses (HPV) oncogene E6 is expressed in undifferentiated basal- or parabasal cells where the calcium concentrations are low, we hypothesized that HPV16-positive cell lines would express the HPV16 E6 oncogene when grown in a medium with a low-calcium concentration
Obvious differences between cell lines were found at 1.8 mM calcium, where HMK cells′ proliferation clearly slowed by day 9 when compared with that at lower calcium concentrations
Summary
E6 are mediated by E6-associated protein (E6-AP) along with E6-binding protein (E6-BP), which is a cellular calcium-binding protein [8,9,10]. Differentiation can be induced by elevating the calcium concentration to over 1 mM of extracellular calcium This ‘calcium switch’ causes the cells to exit from cell cycle and commits them to terminal differentiation. We report that HPV16 E6/E7immortalized keratinocytes responded poorly to the calcium switch and continued to proliferate even in high-calcium medium, contradictory to HPV-negative cells. This response might be related to increase in HPV16 E6 and E2 mRNA expression. This information would be vital for understanding the specific mechanisms of resistance to differentiation and later progression towards carcinogenesis in the HPV-positive oral keratinocyte
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