Regulation of alkaline phosphatase by 1,25-dihydroxyvitamin D3 and ascorbic acid in bone-derived cells.

The bone, liver, and kidney isozyme of alkaline phosphatase (ALP) has been measured in MG-63 human osteosarcoma cells after treatment with ascorbic acid (AA) and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both compounds were required to achieve maximum ALP activity. When grown in the absence of 1,25-(OH)2D3 cells had low basal ALP activity regardless of whether media contained AA. In AA-free medium, 1,25-(OH)2D3 (10 nM) increased ALP activity fourfold. Addition of AA further increased levels of ALP activity induced by 1,25-(OH)2D3 to 10-15 times those found in -AA controls. The earliest effects of 1,25-(OH)2D3 were seen after 24-48 h, and ALP activity continued to increase for 6-8 days. AA and 1,25-(OH)2D3 had similar effects on ALP activity in ROS 17/2.8 rat osteosarcoma cells. In MG-63 cells the effects of AA and 1,25-(OH)2D3 could not be simply explained by the ability of these compounds to inhibit cell growth because another mitotic inhibitor, hydroxyurea, had a minimal effect on ALP activity. 1,25-(OH)2D3-specific induction of ALP +/- AA was totally blocked by inhibitors of protein and RNA synthesis. Maximal ALP induction was obtained when cells were plated at low density. Consistent with our previous report (Franceschi et al. 1988 J Biol Chem 263:18938-18945), 1,25-(OH)2D3 rapidly stimulated type I collagen synthesis and acid-precipitable hydroxyproline production in MG-63 cells and this stimulation was further increased by AA. These results suggest that induction of the osteoblast marker, ALP, is directly or indirectly coupled to collagen matrix synthesis and/or accumulation.     

Steroid Receptor Co-activator-1 Mediates 1,25-Dihydroxyvitamin D3-Stimulated Alkaline Phosphatase in Human Osteosarcoma Cells

For steroid hormone function to occur, nuclear receptors interact with a series of coactivators including steroid receptor coactivator-1 (SRC-1). The SRC-1 binds the vitamin D receptor (VDR) in the presence of ligand in an activation function 2 (AF-2)-dependent manner. In order to understand the role of this interaction in 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-mediated gene expression, the level of SRC-1 expression was altered in MG-63 cells. Previous studies had demonstrated that MG-63 cells express the VDR and that 1,25(OH)₂D₃ regulates expression of alkaline phosphatase (ALP). Analysis of MG-63 cells demonstrated that SRC-1 is expressed. A full-length cDNA coding for SRC-1 was inserted in antisense orientation into an expression vector (anti-SRC-1). The MG-63 cells were transfected with anti-SRC-1 or mock vector and stable transformants were selected. Western blot analysis showed a 95% reduction in SRC-1 protein as compared with mock cells. We determined the effect of normal and reduced SRC-1 expression in MG-63 cells on 1,25(OH)₂D₃-mediated stimulation of ALP. Whereas 10⁻⁸ M 1,25(OH)₂D₃ produced a 3.6-fold stimulation in ALP in mock cells expressing normal levels of SRC-1, it did not alter ALP in cells expressing reduced levels of SRC-1. Thus, SRC-1 is required for 1,25(OH)₂D₃-mediated gene expression of ALP by human MG-63 cells. Received: 7 June 1999 / Accepted: 2 November 1999     

Estramustine phosphate enhances the effects of hyperthermia and induces the small heat shock protein HSP27 in the human prostate carcinoma cell line PC-3

The antimicrotubule drug estramustine phosphate (EMP) has been shown to sensitize prostate carcinoma cells to radiation via synchronization at the G2/M phase of the cell cycle. This synchronization may also render cells more sensitive to hyperthermia, providing a rationale for multimodal treatment approaches. We have investigated the effects of EMP and hyperthermia, as well as the regulation of heat shock proteins (HSP) in the PC-3 prostatic carcinoma cell line. Cells were incubated with four doses of EMP for 48 h followed by a 1-h hyperthermia treatment ranging from 41 degrees C to 44 degrees C. Cell cycle distribution at the end of the EMP incubation was investigated by flow cytometry. Cytotoxicity was assessed by colony formation assays. HSP accumulation was investigated by Western immunoblotting. Doses of 1, 5, 10 and 15 microM EMP synchronized 27, 28, 46, and 68% of PC-3 cells at G2/M. With 5, 10 and 15 microM, a sensitizing effect of EMP was assessed at hyperthermic temperatures of 42, 43 and 44 degrees C. EMP did not alter the expression of HSP72, but substantially induced the synthesis of HSP27 in PC-3 cells. Our data show that EMP sensitizes PC-3 cells to hyperthermia induced cytotoxicity. This observation supports the rationale for multimodal treatment approaches in locally advanced prostate cancer.     

Age effect on HSP70: decreased resistance to ischemic and oxidative stress in HDF

BACKGROUND: Heat pre-conditioning results in induction of heat shock proteins including HSP70 that gives a cytoprotective effect against further stress. However, HSP70 induction is attenuated in aged cells. The lower HSP70-levels may contribute to the impaired stress response seen in the aged, and to the higher rates of chronic wounds in aged, which arise from repeated ischemia-reperfusion injury. The aim of this study was to investigate a possible connection by comparing the viability of heat pre-conditioned aged versus young human dermal fibroblasts (HDF) after exposure to stress. MATERIALS AND METHODS: Young (15-28) and aged (61-77) HDF were heat pre-conditioned (42 degrees C, 1 h) and after recovery (1, 2, or 20 h) treated with carbonyl-cyanide-m-chlorophenylhydrazone (hypoxic stress) or with hydrogen peroxide (oxidative stress) for 1 h. HSP70 levels were determined by Western blot. Cell damage was assessed by quantifying lactic dehydrogenase (LDH) in conditioned media. Aged HDF were transfected with HSP70-plasmid, consecutively heat pre-conditioned and exposed to oxidative stress. RESULTS: HSP70 increased in heat pre-conditioned young HDF by 96, 189, and 237% after 1, 2, and 20-h recovery, respectively, and in aged HDF by 27, 61, and 26%. LDH-release was only decreased in young HDF 20-h after heat-treatment compared with non-heat treated cells (P < 0.001). HSP70-transfection of aged HDF with plasmid reduced LDH-release by 29%. CONCLUSIONS: Heat pre-conditioning fails to protect aged HDF to oxidative or hypoxic stress due in part to impaired HSP70 induction compared to young.     

Heat shock inhibits NF-kB activation in a dose- and time-dependent manner

BACKGROUND: The heat shock response (HSR) attenuates NF-kappaB mediated activation of the acute inflammatory response by inhibiting IkB degradation. The HSR also confers a protective phenotype upon cells through production of heat shock proteins (HSP). However, the exact conditions that induce the HSR and stimulate the production of protective HSP are poorly defined. Consequently, we hypothesized that the inhibition of NF-kappaB activation through the HSR is dependent both on the degree of cellular injury and the length of the recovery period from the heat shock. METHODS: RAW 264.7 murine macrophages were heated to 43 degrees C for 15 (mild heat shock), 45 (moderate heat shock), or 90 min (severe heat shock), allowed to recover at 37 degrees C for 0 to 24 h, and then exposed to 100 ng/ml of Escherichia coli (055:B5) lipopolysaccharide (LPS). Cellular viability, HSP expression, and the activation of NF-kappaB after LPS exposure were determined by alamarBlue assay, immunoblot, and electrophoretic mobility shift assay, respectively. RESULTS: Transient attenuation of NF-kappaB activation and IkappaB preservation was observed only with moderate heat shock and 1 h of recovery. Mild heat shock had no effect on LPS-induced NF-kappaB activation or IkappaB degradation. Severe heat shock completely inhibited NF-kappaB activation and preserved IkappaB protein levels. Heat shock proteins were detectable 30 min after moderate heat shock, with maximal and sustained levels 2 to 24 h after heat shock. CONCLUSION: The attenuation of NF-kappaB activation after heat shock is both dose- and time-dependent.     

Relative potencies of 1,25-(OH)(2)D(3) and 19-Nor-1,25-(OH)(2)D(2) on inducing differentiation and markers of bone formation in MG-63 cells.

19-Nor-1,25-(OH)(2)D(2), an analog of 1,25-(OH)(2)D(3), is used to treat secondary hyperparathyroidism because it suppresses parathyroid hormone synthesis and secretion with lower calcemic and phosphatemic activities. 19-Nor-1,25-(OH)(2)D(2) is approximately 10 times less active than 1,25-(OH)(2)D(3) in promoting bone resorption, which accounts in part for the low potency of this analog in increasing serum calcium and phosphorus. Concern that 19-nor-1,25-(OH)(2)D(2) also could be less potent than 1,25-(OH)(2)D(3) on bone formation led to a comparison of the potency of both compounds on osteoblasts. In the human osteoblast-like cell line MG-63, 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) had a similar potency in upregulating vitamin D receptor content and suppressing proliferation. Both sterols caused a similar reduction in DNA content and proliferating cell nuclear antigen protein expression. Time-course and dose-response studies on 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) induction of the marker of bone formation, osteocalcin, showed overlapping curves. The effects on alkaline phosphatase (ALP) activity also were studied in MG-63 cells that had been co-treated with either sterol and transforming growth factor-beta, an enhancer of 1,25-(OH)(2)D(3)-induced ALP activity in this cell line. Transforming growth factor-beta alone had no effect, whereas 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) increased ALP activity similarly. These studies demonstrate that 19-nor-1,25-(OH)(2)D(2) has the same potency as 1,25-(OH)(2)D(3) not only in inducing vitamin D receptor content, osteocalcin levels, and ALP activity but also in controlling osteoblastic growth. Therefore, it is unlikely that 19-nor-1,25-(OH)(2)D(2) would have deleterious effects on bone remodeling.     

Heat shock attenuates oxidation and accelerates apoptosis in human neutrophils.

BACKGROUND: The heat shock response entails the increased expression of heat shock proteins (hsp) which are capable of protecting cells from subsequent metabolic insults. Here we are interested in determining whether activation of the heat shock response might affect polymorphonuclear leukocyte (PMN) function and/or longevity. METHODS: Freshly isolated human PMN were either left at 37 degrees C or subjected to a 43 degrees C heat shock treatment (60 min) and subsequently returned to 37 degrees C. During the course of the recovery period a number of parameters were examined for the control and heat shock-treated neutrophils: the relative expression of the highly stress-inducible hsp72; respiratory burst activity as measured by intracellular peroxidation in response to phorbol ester addition; cell-surface expression of CD16; and finally, the extent of apoptosis as determined by both annexin V staining and nuclear propidium iodide staining. RESULTS: Heat shock treatment resulted in a progressive increase in hsp72 production, peaking at 8 h following return of the cells to 37 degrees C. Net intracellular oxidant production was diminished by 46% immediately following the heat shock treatment and deteriorated even further over the next 4 h. Finally, a significant early increase in the rate of apoptosis was observed in the cells subjected to the hyperthermic treatment. This increase in the heat-induced rate of apoptosis was associated with a marked reduction in cell-surface CD16 levels. CONCLUSIONS: By decreasing PMN oxidative functions and by accelerating their apoptotic demise, it would appear that heat shock is anti-inflammatory and not cytoprotective for PMN.     

Pretransplant induction of HSP-70 in isolated adult pig islets decreases early islet xenograft survival

The heat-induced HSP-70 expression protects rat islet single cells against lysis mediated by nitric oxide (NO), reactive oxygen, and streptozotocin. The present study was performed to investigate the potential antiinflammatory effect of pretransplant heat shock in adult pig islets for subsequent early islet xenograft survival. Maximum HSP-70 expression in freshly isolated pig islets was induced by hyperthermia at 43 degrees C for 90 min prior to islet regeneration at 37 degrees C for 4-6 h. Heat-stressed and sham-treated islets were incubated in 0.6 mM H2O2 or 1.5 mM Na-nitroprusside at 37 degrees C for 20 h. Early graft survival was evaluated in normoglycemic Lewis rats after simultaneous, contralateral transplantation of heat-shocked islets and sham-treated islets into the renal subcapsular space of the same recipient. Prior hyperthermia significantly reduced specific lysis of islets exposed to NO or H2O2, although protection was only marginal. No differences were observed between viability of heat-shocked and sham-treated islets after NO exposure. In contrast, prior heat shock increased islet viability after H2O2 treatment. The finding that hyperthermia reduced recovery of initially grafted pig insulin 48 h after transplantation by 30% compared to controls contrasted significantly with an increased insulin recovery in heat-exposed islets at the end of simultaneous 37 degrees C culture. The observation, that the heat-induced HSP-70 expression decreases early islet xenograft survival as reflected by recovery of grafted insulin, implies an enhancement of islet immunogenicity and the induction of apoptosis. Future experiments aiming at augmentation of intrinsic defense mechanisms should consider detrimental effects associated with induction of heat shock proteins.     

Regulated overexpression of heat shock protein 72 protects Madin-Darby canine kidney cells from the detrimental effects of high urea concentrations.

Exposure of renal medullary cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved resistance to subsequent exposure to a high urea concentration (600 mM). To establish a causal relationship between HSP72 expression and protection against high urea concentrations, HSP72 was inducibly overexpressed in Madin-Darby canine kidney (MDCK) cells, in the absence of hypertonic stress before urea exposure. For this purpose, the human stress-inducible HSP72 gene was cloned downstream from a dexamethasone (DEX)-inducible promoter in the eukaryotic expression vector pLKneo. This construct allowed robust induction of HSP72 by exposure of stably transfected MDCK cells (MDCK-LK72) to 0.1 microM DEX. Increased HSP72 abundance significantly improved survival rates after 24-h exposure of the cells to medium containing 600 mM urea (14 versus 43%). In mock-transfected or wild-type cells, DEX had no significant effect on HSP72 abundance or urea resistance. In accordance with those findings, lactate dehydrogenase activity in the supernatant was significantly reduced, compared with appropriate control samples, only in MDCK-LK72 cells overexpressing HSP72. Labeling with annexin V-FITC and propidium iodide, followed by flow cytometry, revealed that overexpression of HSP72 was associated with a reduction in the number of apoptotic-lysed cells, a concomitant retardation of apoptosis, and an increase in the number of viable cells. These data support the view that HSP72, which is very abundant in the renal inner medulla, is an important component of the defense mechanism of medullary cells against extreme concentrations of urea.     

热休克蛋白72抑制ATP耗竭时细胞色素C释放所诱导的肾小管上皮细胞凋亡

目的 研究热休克蛋白(HSP)72对ATP耗竭时细胞色素C释放所导致的肾小管上皮细胞凋亡的保护作用及其分子机制。方法 应用代谢抑制剂暂时性阻断细胞内ATP的生成,引起细胞凋亡。应用热处理细胞或编码HSP72 RNA的腺病毒感染细胞,诱导HSP72的表达。以Western印迹检测释放于胞浆内的细胞色素C。荧光肽法测定半胱氨酸天冬氨酸蛋白酶(caspase)3活性。Hoechst33342染色观察细胞凋亡的发生情况。结果 肾小管上皮细胞内ATP耗竭时,释放至胞浆内的细胞色素C的含量增多,caspase 3活性增强;细胞内ATP再恢复时,细胞色素C的释放和caspase 3活性进一步增加,细胞体积缩小,核浓染、固缩,形成凋亡小体。预先热处理后,各组细胞色素C的释放明显减少,caspase 3活性显著抑制(P<0.05,n=3)。高表达HSP72时,各时间点caspase 3活性的抑制程度与热处理组相似,细胞体积缩小,核浓染、固缩,凋亡小体的形成明显减少。结论 HSP72可抑制ATP耗竭时细胞色素C所导致的肾小管上皮细胞凋亡,其机制是抑制凋亡通路中细胞色素C的释放和caspase 3的激活。     

The effects of low-level diode laser irradiation on differentiation,antigenic profile,and phagocytic capacity of osteoblast-like cells (MG-63)

Previous in vivo and in vitro studies have reported that low-level diode laser therapy induces a biostimulatory effect, such as cell proliferation. The aim of the present study was to evaluate whether the laser irradiation of osteoblast-like cells (MG-63) can modify alkaline phosphatase activity (ALP), antigenic profile, and phagocytic capacity. The MG-63 cell line was exposed to diode laser (ezLase) of 940 nm at 1–1.5 W/cm² and 3–4 J. ALP was evaluated by a spectrophotometric technique and antigenic expression analysis (CD 54, CD80, CD86, HLA-DR), and phagocytic activity was analyzed by flow cytometry. At 24 h, the treated groups showed an increased ALP, and the highest increase versus controls (P?=?0.002) was at the dose of 1 W/cm² and 3 J; this modulation of the antigenic profile translated into a reduced expression of CD54, CD86, and HLA-DR and a slightly decreased phagocytic capacity with respect to the nonirradiated control group at the different intensities and fluencies assayed. These results demonstrate that laser therapy can exert a biostimulatory effect on osteoblastic cells at different levels, which may be clinically useful in the regeneration of bone tissue.     

Oncogenic ras results in increased cell kill due to defective thermoprotection in lung cancer cells

Background. The survival response of normal cells to heat stress is an upregulation of heat shock proteins and ras protein activation. We hypothesized that in lung cancer cells the presence of oncogenic ras interferes with thermoprotective mechanisms resulting in cell death.

Methods. An equal number of lung tissue culture cells (normal and cancerous) were subjected to either heat stress and then recovery (43°C for 180 minutes, 37°C for 180 minutes) or recovery alone (37°C for 360 minutes). End points were surviving number of cells, cell-death time course, heat shock protein (HSP70, HSC70, HSP27) expression before and after heat stress, and time course for HSP70 expression during heat stress and recovery. Heated cells were compared with unheated control cells, then this difference was compared between cell types.

Results. Heat stress in normal cells caused an 8% decrease in cell number versus a 78% ± 5% decrease in cancer cells (p < 0.05). In normal cells, heat stress caused a 4.4-fold increase in HSP70, no change in HSC70, and a 1.7-fold increase in HSP27. In contrast, cancer cells initially contained significantly less HSP70 (p < 0.05), and there was a 27-fold increase in HSP70 and a 2-fold increase in HSC70 with no HSP27 detected (comparison significant, p < 0.05). HSP70 time course in normal cells showed that HSP70 increased 100-fold, reaching a vertex at 2 hours and remaining elevated for 24 hours; in cancer cells, HSP70 maximum expression (100-fold) peaked at 5 hours, then decreased to slightly elevated at 24 hours.

Conclusions. Cancer cells with oncogenic ras have defective thermoprotective mechanism(s) causing increased in vitro cell death, which provides an opportunity for thermal treatment of lung cancer.     

Effects of quercetin on the heat-induced cytotoxicity of prostate cancer cells

BACKGROUND: It has been demonstrated that prostate cancer cells are relatively sensitive to heat stress. We have reported that heat treatment at 43 degrees C increases the expression of heat shock protein 70 (hsp70) in prostate cancer cells, leading to apoptosis. Hsp70 is a protein that protects cells against heat damage. Cells with lower levels of hsp70 have been shown to have a higher sensitivity to heat stress. Therefore, downregulation of hsp70 is expected to enhance heat-induced inhibitory effects on cell growth. Quercetin has been reported to be an agent that inhibits hsp70 expression. The present study was undertaken to investigate the effects of quercetin and/or heat on the growth of prostate cancer cells in vitro. METHODS: Three human prostate cancer cell lines were used: Lncap; PC-3; and JCA-1. The cells were treated with quercetin and/or heat. Alterations in the cell cycle and hsp70 expression were examined by means of flow cytometry (FCM). The apoptotic cells were detected by FCM using fluorescein isothiocyanate (FITC) labeled annexin V. RESULTS: Treatment with quercetin alone resulted in an apparent decrease of hsp70-positive cells and an increase of subG1 cells in JCA-1 and LNcap cells. Quercetin inhibited an increase of hsp70 expression after heat treatment and increased the number of subG1 cells with lower levels of hsp70 in JCA-1 and LNcap cells. Quercetin was found to enhance heat-induced inhibitory effects on cell growth and heat-induced apoptosis in both JCA-1 and LNcap cells. CONCLUSION: These results suggest that quercetin may enhance heat-induced cytotoxicity in prostate cancer cell lines through the inhibition of hsp70 production.     

Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog,EB1089

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect.     

Laser-induced thermal stress and the heat shock response in neural cells

BACKGROUND: The Ho: YAG laser is used extensively in orthopedic surgery. It offers a minimally invasive method of ablating tissue with precision. Previous studies have explored the effects of laser use on temperature during experimental foraminoplasty. To date, there has been limited work on the effects of thermal stress on cells in this context.Material and methods Cells were exposed either to heated medium or the Ho: YAG laser in the high-power mode. Heated medium was used as a stressor by (I) exposing groups of cells to a constant temperature of 45 degrees C for varying lengths of time: 5, 10, 15 and 20 min, and (II) exposing cells for a fixed length of time (5 min) to varying temperatures: 45 degrees C, 55 degrees C, 65 degrees C with a control treated at 37 degrees C. A third group was subjected to direct laser treatment. The effects of the treatments were assessed using trypan blue staining as a measure of viability and immunocytochemistry was used to measure changes in heat shock protein (HSP) expression. RESULTS: There was a negative correlation between cell viability and HSP expression, and between cell viability and the severity of the treatment. INTERPRETATION: Our findings suggest a possible role for the Ho: YAG laser in spinal foraminoplasty based on the high level of cell viability in the treatment regimen that most closely mirrored the clinical application of the laser.     

A mechanism of glycine and alanine cytoprotective action: stimulation of stress-induced HSP70 mRNA.

Studies done both in vitro and in vivo have shown that glycine and alanine protect kidney cells from stress injury. However, the mechanism(s) of this cytoprotection is unknown. Our aim was to test the hypothesis that the cytoprotective action is in part due to stimulation of gene(s) expression encoding stress protein synthesis. Experiments were carried out using heat shock as a model for stress in the opossum kidney cell line (OK cells). The induction of HSP70 mRNA was evaluated in cell monolayers exposed to 45 degrees C for 15 minutes followed by a recovery period at 37 degrees C for either 0.5, 1, 2, 3, 4, 6 or 24 hours. The results demonstrate that the maximum level of HSP70 mRNA occurred at approximately three hours after heat treatment. Although the mRNA levels declined thereafter, appreciable amounts were still seen even 24 hours after heat-shock. To examine the effect of glycine or alanine on HSP70 mRNA levels and on the synthesis of stress protein, cultures were preincubated for 30 minutes with Krebs-Henseleit buffer, pH 7.4, supplemented with either 1, 2, 5 or 10 mM glycine or alanine, or with no added amino acids. Comparative studies were performed with 10 mM glutamate, aspartate, arginine or leucine. Following preincubation, cultures were heat-shocked (45 degrees C for 15 min) and then reincubated at 37 degrees C for three hours. Both glycine and alanine enhanced the level of HSP70 mRNA and the synthesis of 72, 73 kDa stress proteins, but neither amino acid induced HSP70 mRNA without concomitant heat treatment.(ABSTRACT TRUNCATED AT 250 WORDS)