Expression of cytokeratin 10 was induced at concentrations at or above Cmax. However, the effect on this cytokeratin under these conditions was minimal and again most apparent
in tissue harvested on later days (Fig. 5, panels J–L; V–X). The cytokeratin 10 expression may be an attempt by the tissue to protect itself from damage. Cytokeratin 6 expression is related to the wound-healing process and is found in the suprabasal layer. Epidermal injury results in induced cytokeratin 6 expression in keratinocytes undergoing activation at the wound edge [32, 33]. The raft culture is a wound-healing environment and so cytokeratin 6 is typically expressed in raft tissues. In our study, cytokeratin 6 expression was dramatically reduced at all Gefitinib concentration concentrations of ZDV when the drug was added at day 0 (Fig. 6, panels A–L). There was a similar decrease in expression, at all ZDV concentrations, when the drug
was added at day 8 (Fig. 6, panels M–X). A marked decrease in cytokeratin 6 was seen after just 2 days when the drug was added to tissue after 8 GSK1120212 manufacturer days of growth (data not shown). Such an immediate and strong decrease in the expression of cytokeratin 6 at 2 and 4 days post treatment suggests an impaired wound-healing response of the tissue. As ZDV treatment changed the expression patterns of the proliferation markers cytokeratins 5 and 6, we then decided to evaluate the effect of ZDV on the expression of well-characterized cell proliferation markers. PCNA is a nuclear protein associated with DNA polymerase delta which is present throughout the cell cycle in the nuclei of proliferating cells [34]. Cyclin A, however, plays a role in proliferation by regulating entry into the DNA synthesis phase (S phase) of the cell
cycle [35, 36]. Immunohistochemical Liothyronine Sodium analysis of PCNA and cyclin A allows a spatial view of cell proliferation to be obtained. Typically, cells only proliferate in the basal layer of tissue. In this study, PCNA and cyclin A expression in untreated rafts was limited to the basal layer. This expression, particularly of PCNA, was strong and varied little throughout the experiment in untreated tissues. In the ZDV-treated rafts, however, both PCNA and cyclin A were strongly expressed in both the basal and differentiating layers of the tissue (Fig. 7). When applied from day 0, ZDV caused an increase in the expression of PCNA and changed its expression pattern. ZDV treatment also changed the location of PCNA expression in these tissues. While PCNA expression in untreated tissues was confined to the basal layers, treated tissues showed expression of PCNA in differentiating layers of the tissue (Fig. 7a). When tissue was treated with ZDV beginning at day 8, an effect on PCNA expression was seen as early as 2 to 4 days post treatment (Fig. 7b, panels A–F and data not shown). There was an increase in the expression of PCNA in differentiating layers of treated tissues.
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