Faculty Bibliography

This study examined the effect of exogenous TGF -beta1 on platelet derived growth factor alpha and beta (PDGF-alpha, beta) receptor expression in human dermal fibroblasts derived from both normal cutaneous tissues (normal skin [NSk]) and (normal scar [NSc]) and abnormal scar (keloid). TGF-beta and PDGF are present in the early phases of wound healing and are implicated in tissue fibrosis. In this study, replicate samples of NSk, NSc and keloid fibroblasts were grown to subconfluency in DMEM/10% FBS followed by replacement of media with DMEM/0.1%FBS for 24 hrs. One group of cells (NSk, NSc and keloid) were exposed to 10 ng/mL of exogenous TGF-beta1 for 24 hours, while the other group was used as control with no exposure to exogenous TGF-beta1. RadioImmunoBinding assays, Western and Northern blot analysis were performed to examine both PDGF-alpha and PDGF-beta receptor expression at the transcriptional and post-transcriptional levels. cDNA receptor probes were synthesized using polymerase chain reaction (PCR) with selected primer sets derived from published sequences. Beta-actin probe was used as a control to confirm that the same quantity of RNA was used for each experimental condition. TGF-beta1 was found to upregulate the expression of PDGF-alpha receptor for keloid fibroblasts but not for NSk or NSc fibroblasts. No effect was observed for TGF-beta 1 on PDGF-beta receptor expression for any of the cell lines examined

Exposure of HPV-immortalized, but not normal human oral keratinocytes, to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) renders the cells tumorigenic. The underlying mechanism of this differential response of normal and immortalized cells was investigated. Normal primary human oral keratinocytes and three HPV-immortalized human oral keratinocyte cell lines exposed to MNNG were evaluated for survival rate, single and double-strand DNA breaks, and the expression of p53 and bcl-2 proteins. MNNG exposure for 2 h induced both greater cytotoxicity and a more rapid kinetic of cell death in normal keratinocytes than in the immortalized cells. Further, normal keratinocytes were more sensitive to lower concentrations of MNNG that were subtoxic for the immortalized cells. Likewise, with lower concentration of MNNG (50 mu M), significant single-strand DNA breaks in normal keratinocytes were induced whereas no such effect was seen in the immortalized cells. Double-strand DNA fragmentation (apoptosis) was observed in normal keratinocytes exposed to 50 mu M MNNG but not in the immortalized cells. Higher concentrations of MNNG (100 mu M) were toxic to both normal and immortalized cells although the normal cells were still more sensitive and with faster kinetics of cell death. MNNG-induced apoptosis was not attributable to down regulation of the anti-apoptotic product bcl-2 in normal cells; however, exposure of normal keratinocytes to MNNG did result in induction of the apoptotic gene p53. No change in p53 level was seen in the immortalized cells. These findings suggest that the selective sensitivity of normal keratinocytes to MNNG-induced apoptosis is in part due to the induction of p53. The HPV-immortalized cells are resistant to MNNG-induced apoptosis and therefore are capable of undergoing mutations affecting cell proliferation and resulting in tumori-genicity.