Effects of heat shock, hypoxia, post-mortem interval and glioma disease state on heat shock gene HSPA expression
Beaman, Glenda Marie
Heat shock protein 70 (HSPA/HSP70) gene expression is induced by a wide range of cellular stress conditions. This study investigated HSPA/HSP70 expression in human cell lines exposed to hypoxic conditions, in cancerous and non-cancerous brain tissue specimens from 18 patients (gliomas and normal conditions), and in post mortem rat brain samples exposed to heat shock.\ud Three human glioma cell lines were chosen for this study, each representing various types of glioma: (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. In addition, 18 clinical brain tissue samples were also examined. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. \ud The average HSPA mRNA copy numbers detected in glioblastoma tissue were 1.8 and 8.8 fold higher respectively than in lower grade glioma and control tissues, which is suggestive of a grade related transcription profile. Similar patterns of grade related expression were also observed in corresponding cell lines. The percentage of cells showing positive for HSPA protein in normal cell lines increased from 0 to 33% immediately after exposure to hypoxia, and gradually declined to 11% 24 h after treatment. However, the effects of hypoxia were marginal in glioma cells, due to the already elevated levels of HSPA. Although hypoxia induced HSPA expression in normal cells, it did not achieve the same level of induction in cancer cells, suggesting that there are other factors which contribute to the induction of HSPA. These results suggest that HSPA is induced in cancer cells, not only by hypoxia, but also by other factors. In addition, this study indicated for the first time that HSPA expression in glioma cells may possibly be grade related, and thus may have value as a prognostic marker. However a greater sample size is needed to validate such findings. \ud This study showed that HSPA is expressed at low levels in normal brain tissue, but was more highly expressed in brain tissue subjected to mild heat shock. The levels of HSPA transcripts in heat shocked post mortem brain tissue showed a marked increase in HSPA expression. \ud GAPDH was used as a control gene for these studies, and exhibited a consistent level of expression in normal and tumourous cell lines and tissue samples under normal and hypoxic conditions, and also in post mortem tissues exposed to heat shock. For Homo sapiens GAPDH, the average transcript numbers for normal and tumourous cell lines and brain tissue samples were approximately 145,000 copies per sample. For Rattus norvegicus GAPDH, levels were higher than for human samples, at an average of 268,300 copies per sample. The consistency of these results confirms that GAPDH was a suitable candidate gene for the purpose of this study.\ud Early in the post-mortem period, HSPA is expressed more highly in tissues subjected to single and multiple heat shocks compared to controls. However, later post-mortem intervals of between 3 - 24 h demonstrated inconsistent and irregular results, with no predictive or reproducible patterns. Therefore, although there is demonstrable de novo expression of HSPA in post mortem brain tissue in response to heat shock, it is difficult to predict the full parameters of this induction, probably as a result of other forms of cellular stress affecting these tissues under our experimental methodology. These initial studies indicate that the use of HSPA with the methodologies employed here are not suitable as an accurate indicator of post-mortem interval.