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(The FASEB Journal. 1998;12:1571-1579.)
© 1998 FASEB

RESEARCH COMMUNICATION

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

Juliann G. Kiang1, Irene D. Gist, and George C. Tsokos

a Department of Clinical Physiology, Division of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307–5100, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 pretreatment 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)


Key Words: viability • heat • protein kinase C • protein kinase A


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OVEREXPRESSION OF HEAT SHOCK proteins (HSPs),2 specifically the HSP-70 family, displays cytoprotective properties against various insults in many different types of cells (for review, see refs 1, 2). For example, HSPs protect human gastric cells from sepsis-induced injury (3), transplanted pig kidneys from ischemic injury (4), rabbit heart from ischemia/reperfusion-induced injury (5), rat small intestine from ischemia-reperfusion injury (6) and ricin toxicity (7), and rat retina from light injury (8). Similar evidence has also been derived from the study of cultured cells. Protection of human monocytes from hydrogen peroxide toxicity (9) and apoptosis (10), guinea pig gastric mucosal cells from ethanol damage (11), human A-431 cells from NaCN toxicity (12), rat FRTL-5 thyroid cells from hypoxia/reoxygenation injury (13), and human breast cancer T47-D cells from lethal heat injury (14) have been reported. The cytoprotection is absent when HSP-70 production is prevented by treatment with inhibitors (15, 16).

Previous studies have demonstrated more effective tumor cell cytotoxicity when hyperthermia was combined with simultaneous chemotherapy (17–19). When tumor cells were exposed to sublethal heat shock prior to the chemotherapy, the latter was less effective (19–21). Cultured breast cancer MCF-7 and MDA-MB-231 cells that express high levels of HSP-70 and -27 are resistant to treatment with anticancer drugs such as doxorubicin and actinomycin D (22, 23). These studies indicate that expression of HSPs can regulate the effectiveness of cytotoxic drugs in a positive or negative manner. Reports from studies of human epidermoid A-431 cells (24, 25), MDCK cells (26), and rat luteal cells (27) indicate that HSP-70 is regulated by [Ca2+]i and protein kinase C. Nevertheless, the role of Ca2+ and protein kinases in the expression of HSPs in breast cancer cells is not known. Information along this line may prove useful in designing effective anti-breast cancer modalities.

In this study, we used an estrogen receptor-positive cell line, T47-D (28, 29), to assess the ability of heat shock to induce the expression of HSPs and convey cytoprotection. We found that heat shock increased the expression of HSP-72, glucose-regulated protein 78 (GRP-78, members of the HSP-70 family), and GRP-94 (a member of the HSP-90 family). The increased expression of stress proteins protected the cells from subsequent thermal injury caused by lethal temperature exposure. The heat shock-induced expression of HSP-70 and GRP-78 but not GRP-94 was dependent on intracellular free Ca2+ concentration ([Ca2+]i), protein kinase A (PKA), and PKC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Breast cancer T47-D cells (American Type Cell Collection, Rockville, Md.) were grown in RPMI medium containing 10% fetal bovine serum, penicillin (50 µg/ml) and streptomycin (50 U/ml) (Gibco/BRL, Gaithersburg, Md.) and incubated in a 5% CO2 atmosphere at 37°C. Cells were fed every 3–4 days.

Heat shock protein measurement
To investigate the synthesis of HSPs, cells were incubated in medium at 44°C for 20 min and returned to 37°C for different periods of time. At specified times, cells were pulse-labeled with [35S]methionine (2 µCi/ml, 1.7 pmol/ml) for 1 h. Cells were removed from the culture plate with trypsin/EDTA (Gibco/BRL), pelleted by centrifugation at 750 x g for 10 min, and the pellet was lysed in Tris buffer (pH=6.8) containing 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol. Aliquots containing 45 µg protein were resolved on SDS-polyacrylamide slab gels (Novex precasted 10% gel, San Diego, Calif.). Gels were vacuum-dried and exposed to Kodak film (X-Omat film, Kodak, Rochester, N.Y.) overnight. Incorporation of radioactivity into individual protein bands was quantified by scanning the resulting autoradiograph with a laser densitometer (Molecular Dynamics, San Diego, Calif.). Actin was not changed by treatment and was used to normalize the data to eliminate the variation of sample loading (25).

Western blots
SDS-polyacrylamide gels were run as described above. Protein was blotted onto a nitrocellulose membrane (type NC, 0.45 µm, Schleicher & Schuell, Keene, N.H.), using a Novex blotting apparatus and the manufacturer's protocol. After blocking the nitrocellulose by incubation for 90 min at room temperature in phosphate-buffered saline (PBS) containing 2.5% nonfat dried milk, the blot was incubated for 60 min at room temperature with mouse polyclonal antibody against HSP-72, GRP-75, GRP-78, HSP-90, and GRP-94 (StressGen, Calgary, Canada) at a 500x dilution in PBS - 5% BSA containing 0.1% thimerosal. The blot was then washed three times (10 min each) in PBS - 0.1% Tween 20 before incubating the blot for 60 min at room temperature with a 1000x dilution of rabbit anti-mouse IgG peroxidase conjugate (Amersham, Arlington Heights, Ill.) in PBS - 1% gelatin. The blot was washed six times (5 min each) in PBS - 0.1% Tween 20 before detection of the peroxidase activity using the Enhanced Chemiluminescence (Amersham Life Science Products).

Intracellular [Ca2+]i measurements
Suspended cells were loaded with 5 µM fura-2/AM plus 0.2% pluronic F-127 at 37°C for 60 min. Cells were then washed with Na+ Hanks' solution before fluorescence measurements. The method to determine [Ca2+]i has been described previously (25).

Measurements of cell viability
Cell viability was determined by trypan blue exclusion assay. Twenty microliters of cell suspension was mixed with 20 µl of 0.4% trypan blue solution (Sigma Chemical Co., St. Louis, Mo.). A drop of the mixture was placed on the hemocytometer and cells were counted under the microscope. Cells that turned blue represented the unviable cells; others represented the viable cells. The viability was calculated according to the following equation: viability (%) = [number of viable cells/(number of viable cells+number of unviable cells)] x100%.

Statistical analysis
All data are expressed as the mean ±SEM. One-way analysis of variance (ANOVA), two-way ANOVA, studentized-range test, Bonferroni's inequality, and Student's t test were used for comparison of groups with 5% as a significant level.

Chemicals
Chemicals used were albumin, 17ß-estradiol (Sigma Chemical Co.), GF-109203X, H89 (Calbiochem Co., La Jolla, Calif.), BAPTA-AM, fura-2AM, and pluronic acid F-127 (Molecular Probes, Eugene, Oreg.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Heat shock-induced synthesis of HSP-72, GRP-78, and GRP-94 in T47-D cells
Exposure of estrogen receptor-positive human breast cancer T47-D cells (28, 29) to heat shock at 44°C for 20 min resulted in synthesis of new proteins. To identify these newly synthesized proteins, Western immunoblots were performed with specific antibodies directed against HSP-72, GRP-75, GRP-78, HSP-90, and GRP-94. The levels of HSP-72, GRP-78 (members of the HSP-70 family), and GRP-94 (a member of the HSP-90 family) increased in the heat-treated cells ( Fig. 1), whereas the levels of GRP-75 and HSP-90 remained similar to that found in untreated cells (data not shown). Treatment with 1 nM 17ß-estradiol induced only the synthesis of GRP-78 and GRP-94 ( Fig. 1, ref. 14).



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  		Figure 1. Treatment of T47-D cells with heat shock induces an increase in the expression of HSP-72, GRP-78, and GRP-94. Cells were exposed to 44°C for 20 min and then returned to 37°C for 4 h. In a parallel experiment, cells were treated with 1 nM 17ß-estradiol for 24 h as a positive control for comparison. Immunoblotting analysis was performed using antibodies directed against HSP-72, GRP-78, and GRP-94, respectively. C: cell lysate from cells treated with vehicle; HS: cell lysate from cells exposed to 44°C for 20 min, followed by 37°C for 4 h; E: cell lysate from cells treated with 1 nM 17ß-estradiol for 24 h.

The induction of HSP-72 and GRPs was time dependent ( Fig. 2). Using [35S]methionine incorporation ( Fig. 2A), we determined that synthesis of HSP-72 caused by exposure of cells at 44°C for 20 min was elevated within 1 h, reached the maximum between 2 and 4 h, and returned to the baseline by 5 h ( Fig. 2B). The time course of newly synthesized GRP-78 was similar to that observed with HSP-72 ( Fig. 2C). Heat exposure also increased the expression of GRP-94. The time course was different from that for HSP-72 and GRP-78. The increase was observed within 1 h, reached the maximum by 4 h, and returned to baseline within 16 h ( Fig. 2D).



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  		Figure 2. Treatment of T47-D cells with heat shock causes increases in HSP-72, GRP-78, and GRP-94 synthesis. Cells were heated at 44°C for 20 min and then returned to 37°C for various periods of time before being labeled with [35S]methionine for 2 h. A representative gel is shown (A). Densitometrically quantitated data for HSP-72 (B), GRP-78 (C), and GRP-94 (D) are expressed as mean ±SEM (n=3).

Because heat shock produced the maximal synthesis rate of HSP-72 and GRPs 4 h later, the protocol in which cells were exposed to heat shock at 44°C for 20 min and returning to 37°C for 4 h was used in the subsequent experiments.

Cytosolic free Ca2+-regulated synthesis of HSP-72 and GRP-78 but not GRP-94
We previously reported that removal of external Ca2+ attenuated HSP-72 production in human epidermoid A-431 cells (30). We sought to determine whether changes in [Ca2+]i affected the rate of HSPs in human breast cancer T47-D cells. When cells were incubated in Ca2+-free buffer containing 3 mM EGTA (an external Ca2+ chelator) for 30 min prior to exposure to heat shock, the basal level of HSPs was not altered. After exposure to heat shock, the synthesis of GRP-78 ( Fig. 3B) was completely abolished, but HSP-72 ( Fig. 3A) and GRP-94 ( Fig. 3C) were not different from that observed when cells were incubated in medium containing 1.6 mM Ca2+, suggesting that Ca2+ entry into the cells is important for the GRP-78 synthesis.



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  		Figure 3. Effect of [Ca2+]i on the heat shock-induced increases in HSP-72, GRP-78, and GRP-94. T47-D cells were treated in the Ca2+-free buffer containing 3 mM EGTA or in the presence of external Ca2+ at 1.6 mM with 30 µM BAPTA-AM before heat shock at 44°C for 20 min. The [35S]methionine incorporated synthesis of HSP-72 (A), GRP-78 (B), and GRP-94 (C) was quantitated densitometrically. *P < 0.05 vs. respective controls, determined by Student's t test.

To further determine the role of levels of [Ca2+]i in the heat shock-induced synthesis of HSPs, cells were treated with 30 µM BAPTA-AM for 30 min prior to heat shock at 44°C for 20 min. Treatment with this intracellular Ca2+ chelator significantly attenuated the synthesis of HSP-72 ( Fig. 3A) and GRP-78 ( Fig. 3B), and had no effect on the synthesis of GRP-94 ( Fig. 3C). These results suggest that the level of [Ca2+]i is associated with synthesis of HSP-72 and GRP-78 but not GRP-94.

Inhibitors of protein kinases A and C diminished HSP-72 and GRP-78 but not GRP-94
This laboratory previously reported that exposure of cells to heat shock increased the intracellular cAMP level (31). cAMP is also known to reverse the inhibition of protein synthesis that is induced by exposure of murine leukemia lymphoblast L5178Y cells to heat (32). cAMP is known to activate PKA. To determine whether PKA was involved in the process of the heat-induced protein synthesis, cells were treated with H89 (a PKA inhibitor) at 1 µM for 30 min prior to exposure to heat shock. Treatment with H89 before heat shock did not alter the increase in HSP-72 ( Fig. 4A) and GRP-94 ( Fig. 4C) synthesis induced by heat shock but abolished that of GRP-78 ( Fig. 4B). Treatment of cells with H89 alone did not change the basal levels of these proteins. The results suggest that PKA plays a role in synthesis of GRP-78 but not HSP-72 and GRP-94.



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  		Figure 4. Effect of H89 and GF-109203X on the heat shock-induced increases in HSP-72, GRP-78, and GRP-94. Cells were treated with 1 µM H89 or 1 µM GF109–109203X for 30 min prior to heat shock at 44°C for 20 min. The [35S]methionine incorporated synthesis of HSP-72 (A), GRP-78 (B), and GRP-94 (C) was quantitated densitometrically. *P < 0.05 vs. respective controls, determined by Student's t test.

It has been reported that activation of PKC induced the expression of HSP-70 in MDCK cells (26), luteal cells (27), and human epidermoid A-431cells (24). To determine whether PKC was involved in the synthesis of the new protein after heat shock, we treated the cells with GF-109203X at 1 µM for 30 min prior to heat shock. GF-109203X significantly reduced the heat-induced increase in HSP-72 synthesis ( Fig. 4A) and abolished that in GRP-78 synthesis ( Fig. 4B), but failed to alter that in GRP-94 synthesis ( Fig. 4C). Treatment of cells with GF-109203X alone did not change the basal level of these proteins ( Fig. 4). The results suggest that synthesis of HSP-72 and GRP-78 but not GRP-94 is regulated by activation of PKC.

HSP-72 and GRPs protected T47-D cells from thermal injury
It has been documented that HSPs protect cells, tissues, and organs from lethal assaults (for review, see refs 1, 2). To determine whether the heat shock-induced HSPs were involved in cell protection, we exposed HSP overexpressing cells to lethal temperatures. We previously showed that heating of cells at 47°C for 50 min limited significantly the number of viable cells (14). Therefore, in this study we subjected cells to 47°C for 50 min and determined their viability. In cells that did not overexpress HSPs, the cell viability after exposure to lethal heating dropped from 86 ± 9 to18 ± 4% (n=4). In cells overexpressing HSPs (by heating at 44°C for 20 min), the cell viability after exposure to the lethal heating was increased to 60 ± 1% (n=4). Pretreatment of cells with EGTA prior to heat shock, which did not inhibit the synthesis of HSP-72 but completely blocked that of GRP-78 ( Fig. 3), still provided moderate cytoprotection to cells exposed to lethal temperature ( Fig. 5B vs. Fig. 5A). However, pretreatment of cells with BAPTA, which significantly attenuated the expression of HSP-72 and completely blocked the expression of GRP-78, preserved the viability of cells exposed to lethal temperature ( Fig. 5B). Pretreatment of cells with EGTA or BAPTA without subsequent exposure to heat shock did not protect cells after exposure to lethal temperature ( Fig. 5A).



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  		Figure 5. Cytoprotection of T47-D cells from thermal injury. A) Cells were pretreated with 3 mM EGTA, 30 mM BAPTA-AM, 1 µM H89, or 1 µM GF-109203X for 30 min prior to exposure to 47°C for 50 min. B) Cells were pretreated with 3 mM EGTA, 30 mM BAPTA-AM, 1 µM H89, or 1 µM GF-109203X for 30 min prior to exposure to 44°C for 20 min, then returned to 37°C for 4 h. These cells were exposed to 47°C for 50 min to determine the percent of cells viable by trypan blue exclusion assay (n=3) and viable cells are presented as percent cell viability. *P < 0.05 vs. respective controls, determined by two-way ANOVA and studentized-range test.

When cells were pretreated with H89 prior to a 44°C heat shock, in order to inhibit GRP-78 production, the cytoprotection from the lethal heating was completely preserved. Pretreatment of cells with GF-109203X, which reduced the heat-induced increase in HSPs by 50%, also attenuated the cytoprotection from the lethal heating accordingly ( Fig. 5lB). Pretreatment with H89 or GF-109203X did not affect the cell viability at 37°C and did not help non-heat-shocked cells survive better after exposure to 47°C for 50 min ( Fig. 5A).

Heat shock increased [Ca2+]i in T47-D cells
Previously we reported that heat shock induced an increase in [Ca2+]i (33) that regulated the heat shock-induced increase in HSP-72 production (25) and cytoprotection from cyanide toxicity (30) in human epidermoid A-431 cells. [Ca2+]i in resting fura-2-loaded T47-D cells was 87 ± 6 nM (n=18). Removal of external Ca2+ decreased the resting [Ca2+]i to 42 ± 4 nM (n=4), suggesting that Ca2+ entry from the extracellular sources contributes to the resting [Ca2+]i. In the presence of 1.6 mM Ca2+, exposure of cells to heat shock increased the levels of [Ca2+]i. The increase was temperature and time dependent ( Fig. 6A, B). The increase was observed at 39°C for 20 min and reached the maximum at 42°C for 20 min. Heating cells at 44°C for 20 min did not cause a further increase in [Ca2+]i. [Ca2+]i returned to the baseline within 80 min ( Fig. 6C).



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  		Figure 6. Increases in [Ca2+]i by heat shock in T47D cells. A) Cells were exposed to 39, 42, or 44°C for 20 min. B) Cells were exposed to 44°C for 1, 5, 10, or 20 min. C) Cells were exposed to 44°C for 20 min, then returned to 37°C for 20, 40, 60, or 80 min. [Ca2+]i was determined using fura-2 probe. *P < 0.05 vs. controls, determined by Student's t test.

The heat shock-induced increase in [Ca2+]i depends on external Ca2+. Table 1 lists the effect of removal of external Ca2+ and treatment with H89 or GF-109203X on the [Ca2+]i in T47-D cells. When cells were heated in a Ca2+-free buffer containing 100 µM EGTA, heat shock failed to increase [Ca2+]i, suggesting that the increase in [Ca2+]i is a result of Ca2+ entry from extracellular sources. After pretreatment of cells with H89 or GF-109203X (1 µM, 30 min), heat shock still increased [Ca2+]i. However, the increase in [Ca2+]i by heat shock in cells treated with H89 or GF-109203X was significantly less than that in untreated cells, suggesting that PKA and PKC are associated with the [Ca2+]i response to heat shock. Treatment with H89 or GF-109203X alone elevated the basal level of [Ca2+]i.


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  		Table 1. Inhibition of the heat shock-induced [Ca2+]i response by removal of external Ca2+ or treatment with inhibitors of PKA and PKC in T47-D cells1tfa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Exposure of estrogen receptor-positive human breast cancer T47-D cells (11, 34) to heat shock resulted in increases in the levels of HSP-72, GRP-78, and GRP-94 in a time-dependent manner. Although other laboratories reported the presence of low molecular mass HSPs such as HSP-27 in MCF-7 breast and endometrial cancer cells (35–37), we did not detect the induction of HSP-27 in T47-D breast cancer cells after heat shock when using the [35S]methionine labeling method. Use of different cell types and stimulation conditions may account for the discrepancy between our observations and those reported previously.

Previous studies have shown that simultaneous exposure of cancer cells to hyperthermia and chemotherapy resulted in more effective tumor cell cytotoxicity (17, 18). In contrast, when tumor cells were exposed sequentially to nonlethal elevated temperatures and then to chemotherapy, a less cytotoxic effect was observed (19–21). This cross-tolerance between heat shock and chemotherapeutic agents is believed to be related to the induction of HSP-70 and HSP-27 (22). The heat shock-induced stress proteins are capable of conferring thermotolerance similar to that provided by estrogen-induced GRPs (14). Our data indicate that the heat shock-induced increase in HSP-72 in T47-D cells is primarily responsible to the observed thermotolerance, because the cytoprotection was still present even though the heat shock-induced synthesis of GRP-78 was inhibited completely by either chelation of Ca2+ or treatment with PKA or PKC. This observation is different from the estrogen-induced increase in GRP-78 and GRP-94 ( Table 2), which is not regulated by levels of [Ca2+]i, PKA, or PKC ( Table 3), but provides cytoprotection from thermal injury (14).


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  		Table 2. Comparison of effects caused by heat shock and treatment with estrogen2tfa


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  		Table 3. Comparison of signal transduction on heat shock- and estrogen-induced stress proteinsfa

Like HSP-27, which is known to be altered by activation of PKC (37), the heat shock-induced HSP-72 is regulated by increases in [Ca2+]i and PKC activity whereas GRP-78 is regulated by levels of [Ca2+]i, PKA, and PKC. It is known that PKC activation causes the phosphorylation of cytosolic heat shock transcription factor (HSF; ref 38), and the increase in [Ca2+]i promotes the translocation of cytosolic HSF to the nucleus and its binding to heat shock elements (HSE) located on the promoter region of the HSP-70 gene (25, 39, 40). It is highly likely that induction of HSP-72 is mediated by phosphorylation of HSF by PKC, which initiates the transcription and the translation of newly synthesized HSP-72 (for review, see refs 1, 41, 42). Like human A-431 cells (24, 25, 38), the heat shock-induced HSP-72 is Ca2+ and PKC dependent. On the contrary, the promoter region of the GRP-78 gene does not contain HSE (43). Therefore, the mechanism of the heat shock-induced GRP-78 expression is still not clear. Since chelation of Ca2+ and inhibitors of PKA and PKC block the heat shock-induced GRP-78 synthesis, a distinct pathway that requires Ca2+, PKA, and PKC with other transcription factors is possible. Unlike heat shock, the estrogen-induced GRP-78 and GRP-94 in T47-D cells are not regulated by Ca2+, PKA, or PKC (14). An estrogen receptor-mediated pathway of protein induction is involved different from the one mediating heat shock. In thyroid FRTL-5 cells, the thapsigargin-induced increases in GRP-78 and GRP-94 were found to be regulated by PKA and PKC but not [Ca2+]i. These proteins also offer cytoprotection from the thermal injury (R. C. Smallridge, I. D. Gist, G. C. Tsokos, and J. G. Kiang, unpublished data). Apparently the pathway of GRP-78 and GRP-94 induction is stimulus and cell type specific.

Heat shock increased [Ca2+]i in a temperature- and heating duration-dependent manner. The data are in agreement with findings observed in other types of cells (33, 44). The increase is the result of increased Ca2+ influx from extracellular sources, because cells failed to increase [Ca2+]i after heat shock in medium that did not contain Ca2+. This result provides additional support for the relationship between [Ca2+]i and synthesis of HSP-72 and GRP-78. Since the increased [Ca2+]i by heat shock was reduced by inhibitors of PKA and PKC, the effect of PKA and PKC on the synthesis of new HSP-72 and GRP-78 probably is a secondary result of their effect on [Ca2+]i. This relationship between [Ca2+]i, PKA, PKC, and stress proteins is specific to heat shock because estrogen treatment did not increase [Ca2+]i and the estrogen-induced GRP-78 and GRP-94 are not regulated by the level of [Ca2+]i, PKA, and PKC (14; Table 3).

The thermal protection offered by stress proteins induced by heat shock is unwanted in breast cancer cells because it will decrease their removal rate by irradiation or chemotherapeutic agents. Agents or methods that inhibit the induction of stress proteins, particularly the HSP-70 family, are desirable. Both GF-109203X and EGTA may have potential for therapeutic use in this regard ( Fig. 5).

In summary, heat shock induced HSP-72, GRP-78, and GRP-94 in a heating duration-dependent manner in human breast cancer T47-D cells that are associated with thermotolerance. The induction of HSP-72 was down-regulated by decreased [Ca2+]i and PKC activity; GRP-78 but not GRP-94 was regulated by [Ca2+]i, PKA, and PKC. Heat shock also increased [Ca2+]i in a temperature- and time-dependent fashion. The [Ca2+]i response disappeared in Ca2+-free buffer and was attenuated by inhibitors of PKA and PKC.


   ACKNOWLEDGMENTS
 
This work was supported by DOD RADII STO C. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.


   FOOTNOTES
 
1 Correspondence: Department of Clinical Physiology, Division of Medicine, Walter Reed Army Institute of Research, Bldg. #40, Rm. 3117A, Washington, DC 20307–5100, USA. E-mail: Dr._Juliann_Kiang{at}WRSMTP-ccmail.army.mil

2 Abbreviations: ANOVA, analysis of variance; HSPs, heat shock proteins; GRPs, glucose-regulated proteins; [Ca2+]i, intracellular free Ca2+ concentration; PKA, protein kinase A; PKC, protein kinase C; HSF, heat shock transcription factor; HSE, heat shock element; PBS, phosphate-buffered saline.

Received for publication April 7, 1998. Revision received May 27, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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