Nhok Cells

To determine the effect of STE on Bcl-2 and p53 genes in NHOK cells, NHOK cells were treated with 0 to 200|g/ml of STE.51 Keratinocytes were also preincubated with GSPE (50|g/ml and 100|g/ml) before treatment with STE. A reverse-transcription polymerase chain reaction (RT-PCR) technique was used to determine the expression of p53 and Bcl-2 genes in NHOK treated with STE and GSPE.51 The differences in the levels of expression of p53 and Bcl-2 in STE treated with and without GSPE was determined using densitometric analysis of the PCR product bands. The data were digitized using BioRad's Gel Analysis System. The density of each band was then determined using Image Pro Plus (Media Cybernetics, Inc., Silver Spring, MD) software and compared with the control values.51

FIGURE 7.3 Flow cytometric analysis of cell cycle distribution and apoptosis in human oral keratinocytes in response to STE (300|g/ml) and selected antioxidants. Primary keratinocytes were grown to approximately 70% confluence and then treated with STE alone or with the antioxidants for 24h. Following incubation, the cells were removed and treated and described as stated in Figure 7.2. The profiles are representative histograms of triplicate assays. A: control; B: STE (300|g/ml); C: STE (300|g/ml) plus vitamins C plus E (75|M each); and D: STE (300|g/ml) plus GSPE (100|g/ml).

The RT-PCR analysis of STE-treated NHOK cells showed a remarkable decrease in Bcl-2 antiapoptotic gene expression following treatment with STE (200^g/ml) for 24h (Figure 7.4). NHOK cells preincubated with GSPE (50^g/ml and 100^g/ml) for 4h prior to STE treatment showed an increase in Bcl-2 mRNA expression, indicating that GSPE ameliorates the toxic effects of STE.51 A concentration-dependent increase in Bcl-2 mRNA expression was observed following preincubation with GSPE. At 100^g/ml concentration of GSPE, preincubation completely reversed the cytotoxic effect of STE, as demonstrated by near-complete restoration of Bcl-2 mRNA expression in these cells. Thus, NHOK cells preincubated with GSPE for 4h followed by STE treatment showed an increase in Bcl-2 mRNA expression,51 which corresponds to our previous apoptosis study.28

The RT-PCR analysis of the STE-treated NHOK cells for the expression of proapoptotic gene p53 revealed that there was an increase/alteration of two-fold in p53 mRNA expression following

FIGURE 7.4 Modulation of Bcl-2, p53 mRNA expression in normal human oral keratinocytes (NHOK) following treatment with STE and/or GSPE (GSPE-1:50|g/ml; GSPE-2:100|g/ml). A: Bcl-2 mRNA expression; B: p53 mRNA expression.

STE (200|g/ml) treatment. The two-fold increase of the band was quantified using Image Pro Plus software, as described previously. The expression of p53 reduces dramatically beyond 200|g/ml STE, confirming increased apoptotic cell death with higher concentration of STE. Thus, expression of p53 and Bcl-2 genes in NHOK cells is affected following incubation with STE28'51'52 (Figure 7.4).

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