ABSTRACT
Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia, with production also being linked to tumourigenesis. HSPA mRNA transcripts and proteins were examined in three human glioma cell lines, representing astrocytoma, oligodendroglioma and glioblastoma, plus 18 clinical brain tissue samples. GAPDH was used as a control gene throughout these studies and exhibited a consistent level of expression in a normal astrocyte cell line, tumourous cell lines and tissue samples. In contrast, the average HSPA mRNA copy numbers detected in glioblastoma tissue were between 1.8- and 8.8-fold higher than in lower grade glioma and control tissue, respectively, which is suggestive of a grade-related transcription profile. Similar patterns of grade-related expression were also observed in glioma cell lines. This study indicates for the first time that HSPA expression in glioma cells may possibly be grade related, and hence could have potential as a prognostic marker.
Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , HSP70 Heat-Shock Proteins/biosynthesis , Prognosis , Adolescent , Adult , Aged , Biomarkers, Tumor/biosynthesis , Brain Neoplasms/diagnosis , Brain Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Glioma/diagnosis , Glioma/pathology , HSP70 Heat-Shock Proteins/genetics , Humans , Male , Middle Aged , RNA, Messenger/biosynthesis , Survival AnalysisABSTRACT
Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia. This study investigated the level of HSPA gene expression in human cell lines exposed to hypoxic conditions. Three human glioma cell lines were selected for this study, each representing different types of glioma (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in three glioma cell lines were approximately sixfold higher than in a normal astrocyte cell line. The expression of HSPA was induced in normal cell lines immediately after exposure to hypoxia with 33% of cells exhibiting expression. However, the effects of hypoxia on gene expression were marginal in glioma cells, due to the already increased levels of HSPA with both pre- and post-hypoxia samples showing expression in approximately 90% of cells. These results show that whilst the stress caused by both cancer and hypoxia induce HSPA expression the underlying imprint of tumourgenesis leads to sustained expression.
Subject(s)
Brain Neoplasms/enzymology , Glioma/enzymology , HSP70 Heat-Shock Proteins/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Hypoxia , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/pathology , HSP70 Heat-Shock Proteins/genetics , Humans , RNA, Messenger/metabolism , Time Factors , Up-RegulationABSTRACT
Activated ras is known to dysregulate TGF-beta signaling by altering the expression of TGF-beta type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-beta signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 microM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 microM concentrations (P>0.004). The TGF-beta effector gene p21(waf1/cip1) was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-beta responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-beta signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT-PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-beta signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.
