Irene D. Gist

Regulation of heat shock protein 72 kDa and 90 kDa in human breast cancer MDA-MB-231 cells.

It has been shown that expression of HSPs can negatively regulate the effectiveness of cytotoxic drugs. In this study, we conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in human breast cancer MDA-MB-231 cells. Using [35S]methionine incorporation and Western immunoblots, we established that heat shock increased production of HSP-72 and -90. Cells exposed to 44°C for 20 min displayed increased expression of HSP-72 and -90, that reached a maximum 3-7 h later and returned to baseline levels within 24 h. The synthesis of both HSP-72 and -90 was attenuated when cells were exposed to heat shock in medium devoid of Ca2+ or pretreated with the calcium chelator BAPTA for 30 min prior to heat shock. Similarly, synthesis of HSP-72 and -90 was inhibited when cells were treated with the protein kinase A inhibitor, H89. These data indicate that Ca2+ and PKA are involved in regulation of HSP-72 and -90 protein synthesis. Levels of HSP-72 mRNA in cells exposed to heat shock increased, suggesting that the heat-induced increase in HSP-72 occurs at the transcriptional level. Also, heat shock caused phosphorylation and translocation from the cytosol to the nucleus of heat shock factor 1 (HSF1), a transcription factor for heat shock protein synthesis. Removal of external Ca2+ or treatment with a PKA inhibitor prevented the posphorylation and the translocation of HSF1. Cells overexpressing HSP-72 and -90 induced by exposure to a sublethal temperature displayed cytoprotection from thermal injury. Removal of external Ca2+ and treament with BAPTA, or H89 prior to exposure to sublethal heat shock that reduced the amount of HSP-72 and -90 production still protected cells fromsubsequent thermal injury. The intracellular free calcium concentration ([Ca2+]i) in resting fura-2-loaded MDA-MB-231 cells was 175±8 nM. Heat shock increased [Ca2+]i in a time-and temperature-dependent manner. Exposure of cells to 44°C for 20 min increased [Ca2+]i by 234±13%, which subsequently returned to baseline levels within 30 min. Removal of external Ca2+ eliminated the increase, indicating that the increase in [Ca2+]i was due to Ca2+ influx. Pretreatment of the cells with H89 but not GF-109203X for 30 min led to an attenuation of the increase in [Ca2+]i by a subsequent heat shock. The results suggest that HSP-72 and -90 are regulated by [Ca2+]i and PKA activity in MDA-MB-231 cells. Kiang JG, Gist ID, Tsokos GC: Regulation of Heat Shock Protein 72 kDa and 90 kDa in Human Breast Cancer MDA-MB-231 Cells.

Cytoprotection and regulation of heat shock proteins induced by heat shock in human breast cancer T47-D cells: role of [Ca2+]i and protein kinases

Overexpression of heat shock protein 70 kDa alters the susceptibility of tumor cells to chemotherapeutic agents. We conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in heat shock‐treated T47‐D cells, a human breast cancer cell line that expresses estrogen receptors. Cells exposed to heat shock at 44°C displayed increased expression of heat shock protein 72 kDa (HSP‐72), glucose‐regulated protein 78 kDa (GRP‐78), and GRP‐94 in a time‐dependent manner, as shown by [35S]methionine incorporation and Western blotting experiments. The maximal rate of synthesis occurred between 2 and 4 h after heat shock. Removal of external Ca2+ inhibited the synthesis of the heat shock‐induced GRP‐78 but not of HSP‐72 and GRP‐94, whereas treatment of cells with BAPTA (a Ca2+ chelator) inhibited HSP‐72 and GRP‐78. Treatment with H89 (a protein kinase A inhibitor) blocked the heat shock‐induced GRP‐78 synthesis, whereas GF‐109203X (a protein kinase C inhibitor) attenuated the heat shock‐induced HSP‐72 synthesis and completely blocked synthesis of GRP‐78 but not of GRP‐94. These results indicate that protein kinase C is involved in regulation of the heat shock‐induced synthesis of HSP‐72, whereas PKA and PKC are involved in the regulation of GRP‐78 synthesis. Cells overexpressing HSP‐72 and GRPs after heat shock displayed resistance against lethal temperature (47°C for 50 min) ‐induced death, which was diminished after removal of external Ca2+ and treatment with GF‐109203X. Heat shock increased intracellular free Ca2+ concentration ([Ca2+]i)in a temperature‐ and heating duration‐dependent fashion, and the increase was inhibited in the absence of external [Ca2+]i and significantly reduced by pre‐treatment with H89 and GF‐109203X. The results suggest that different pathways are involved in the induction of synthesis of HSP‐72, GRP‐78, and GRP‐94 by heat shock. It is highly likely that only HSP‐72 and GRP‐78 are involved in the process of cytoprotection from the thermal injury.—Kiang, J. G., Gist, I. D., Tsokos, G. C. Cytoprotection and regulation of heat shock proteins induced by heat shock in human breast cancer T47‐D cells: role of [Ca2+]i and protein kinases FASEB J. 12, 1571–1579 (1998)

Biochemical requirements for the expression of heat shock protein 72 kda in human breast cancer MCF-7 cells.

Heat shock alters the susceptibility of tumor cells to chemotherapeutic agents. Cultured breast cancer MCF-7 and MDA-MB-231 cells that express high levels of heat shock protein 70 and 27 kDa are resistant to treatment with certain anticancer drugs. These findings indicate that expression of HSPs can negatively regulate the effectiveness of cytotoxic drugs. In this study, we conducted experiments to study the regulation of expression of heat shock proteins (HSPs) in human breast cancer MCF-7 cells exposed to heat shock by intracellular free Ca2+ and protein kinase C. Cells exposed to 44°C for 20 min displayed increased expression of HSP-72 and GRP-94, that reached a maximum 4-5 h later and returned to baseline levels within 24 h. Levels of HSP-72 mRNA in cells exposed to heat shock increased, suggesting that the heat-induced increase in HSP-72 occurs at the transcriptional level. The synthesis of HSP-72 but not GRP-94 was inhibited when cells were exposed to heat shock in medium devoid of Ca2+ and attenuated by more than 50% when cells were pretreated with the calcium chelator BAPTA for 30 min prior to heat shock. HSP-72 synthesis was enhanced when cells were treated with the protein kinase C inhibitor, GF-109203X. These data indicate that Ca2+ and PKC are involved in regulation of HSP-72 synthesis. However, removal of external Ca^2+ and treament with BAPTA, GF-109203X, or exposure to sublethal heat shock protected cells from subsequent thermal injury. The intracellular free calcium concentration ([Ca2+]i) in resting fura-2-loaded MCF-7 cells was 156 ± 16 nM (n = 29). Heat shock increased [Ca2+]i in a time- and temperature-dependent manner. Exposure of cells to 44°C for 20 min increased [Ca2+]i by 234 ± 13%, which subsequently returned to baseline levels within 120 min. Removal of external Ca2+ eliminated the increase, indicating that the increase in [Ca2+]i was due to Ca2+ influx. Pretreatment of the cells with BAPTA or GF-109203X for 30 min or a sublethal heat shock to allow HSP-72 overexpression led to an attenuation of the increase in [Ca2+]i by a subsequent heat shock. The results suggest that HSP-72 but not GRP-94 is regulated by [Ca2+]i and PKC activity. The cytoprotection produced by chelation of Ca2+, GF-109203X, or HSP-72 overexpression is probably due to their ability to attenuate the [Ca2+]i, response to heating.

Corticotropin-releasing factor induces phosphorylation of phospholipase C-γ at tyrosine residues via its receptor 2β in human epidermoid A-431 cells

Abstract This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-γ via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2β. The levels of the CRF receptor type 2β were not altered in cells treated with activators of protein kinase C, Ca 2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-β3, -γ1, or -γ2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-γ1 and -γ2 were phosphorylated. The increase in phospholipase C-γ phosphorylation was concentration-dependent with an EC 50 of 4.2±0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2β that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2-iminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid attenuated the phospholipase C-γ phosphorylation. In summary, CRF induces phospholipase C-γ phosphorylation at tyrosine residues, which depends on Ca 2+ and is mediated by activation of protein tyrosine kinases via the CRF receptor type 2β.

Defective methionine metabolism in the brain after repeated blast exposures might contribute to increased oxidative stress

&NA; Blast‐induced traumatic brain injury (bTBI) is one of the major disabilities in Service Members returning from recent military operations. The neurobiological underpinnings of bTBI, which are associated with acute and chronic neuropathological and neurobehavioral deficits, are uncertain. Increased oxidative stress in the brain is reported to play a significant role promoting neuronal damage associated with both brain injury and neurodegenerative disorders. In this study, brains of rats exposed to repeated blasts in a shock tube underwent untargeted profiling of primary metabolism by automatic linear exchange/cold injection GC‐TOF mass spectrometry and revealed acute and sub‐acute disruptions in the metabolism of the essential amino acid methionine and associated antioxidants. Methionine sulfoxide, the oxidized metabolite of methionine, showed a sustained increase in the brain after blast exposure which was associated with a significant decrease in cysteine, the amino acid derived from methionine. Glutathione, the antioxidant synthesized from cysteine, also concomitantly decreased as did the antioxidant ascorbic acid. Reductions in ascorbic acid were accompanied by increased levels of its oxidized metabolite, dehydroascorbic acid and other metabolites such as threonic acid, isothreonic acid, glycolic acid and oxalic acid. Fluorometric analysis of the brains showed acute and sub‐acute increase in total reactive oxygen species. In view of the fundamental importance of glutathione in the brain as an antioxidant, including its role in the reduction of dehydroascorbic acid to ascorbic acid, the disruptions in methionine metabolism elicited by blast exposure might prominently contribute to neuronal injury by promoting increased and sustained oxidative stress. HighlightsRepeated blast exposures disrupted the normal metabolism of methionine in the brain.Brain levels of methionine‐derived cysteine and glutathione decreased after blasts.Repeated blasts decreased brain levels of ascorbic acid and its catabolites.Repeated blasts increased oxidative stress in the brain.Disrupted methionine metabolism might contribute to increased oxidative stress.