Abstract
The heat-shock proteins (HSPs) or chaperones can protect and stabilize the folding and functions of normal proteins in cells. Chaperones are generally composed of large, evolutionarily conserved families within which the members are highly related, yet might differ in subcellular localization or gene regulation. We used semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression profile of HSP70 isoforms in human non-small cell lung cancer H460 cells under CdCl2 stress. The expressions of hsp70-1, hsp70-2, hsp70-hom, hsp70-3, grp78, hsp70B, and hsp70B’ were monitored by amplifying from a conserved, common primer set and then differentiated by isoform-specific restriction enzymes. It is known that CdCl2 could elicit different cellular responses and gene expressions in a concentration-dependent and treatment time-dependent manner. Consistent with which, in our system, the expression level or pattern of chaperones varied low concentration (5 uM) and high concentration (40 uM to 60 uM) of CdCl2. We compared the changes of the newly synthesized chaperones by metabolic l ling with [35S]methionine and of the total chaperones by Western blotting analysis in CdCl2 treatment. We further analyzed changes of gene expression of individual isoforms by RT-PCR. From our study, this novel method can be performed to differentiate the members of HSP70 family under stress. Here, it has been found that the main members of HSP70 family in CdCl2-treated cells include hsp70-1, hsp70-2, and grp78. Furthermore, other four members of HSP70 family (hsp70-hom, hsp70-3, hsp70B, and hsp70B’) can be analyzed by this novel method. These four members don’t take part in stress conditions with CdCl2-treated.二、英文附錄 1. ABSTRACT....................................2 2. INTRODUCTION................................3 3. EXPERIMENTAL PROCEDURES.....................7 4. RESULTS....................................11 5. DISCUSSION.................................18 6. REFERENCES.................................23 7. FIGURE LEGENDS.............................30 8. FIGURES....................................34 9. TABLES.....................................45