Search

Acute heat stress leads to the glycosylation of a "prompt" stress glycoprotein, P-SG67/64, identified as calreticulin. In the present study, we used immunoprecipitation to investigate the interactions of P-SG/calreticulin with other proteins during cellular recovery from heat stress. In heat-stressed CHO and M21 cells, both glycosylated and unglycosylated P-SGs interact with HSP90, GRP94, GRP78, and the other prompt stress glycoprotein, P-SG50, in an ATP-independent manner. Specificity of HSP-P-SG interactions was determined by chemical cross-linking with the homo-bifunctional agent DSP (3,3'-dithiobis[succinimidyl propionate]). Characterization of the cross-linked complexes involving calreticulin and heat shock proteins (HSPs) showed an average mass of 400-600 kDa by gel filtration chromatography. Overall, the consistent association of glycosylated and unglycosylated calreticulin with P-SG50 and unglycosylated HSPs suggests that P-SG/calreticulin is an active member of the cast of glycone/aglycone chaperones that cooperate to achieve cellular recovery from acute heat stress.

Cellular heat stress results in elevated heat-shock protein (HSP) synthesis and in thermotolerance development. Recently, we demonstrated that protein glycosylation is also an integral part of the stress response with the identification of two major stress glycoproteins, GP50, associated with thermotolerance, and P-SG67, the "prompt" stress glycoprotein induced immediately during acute heat stress. In the present study, we characterized the subcellular location and redistribution of these proteins during the cellular injury and recovery phase. In unheated and heated CHO cells, both stress glycoproteins were present in each subcellular fraction isolated by differential centrifugation. However, the subcellular redistribution in the course of cellular recovery after heat stress was specific for each stress glycoprotein. GP50 was present in all subcellular fractions before heat stress, but showed relatively little redistribution after heat stress. By 24 h of recovery following stress, GP50 showed partial depletion from lysosomes and microsomes, and was mainly present in the mitochondria. Glycosylated P-SG67 was redistributed in a more complex fashion. It was seen predominantly in the lysosomes and microsomes immediately following heat-stress, but after 6 h of recovery following heat stress, it largely disappeared from the microsomes and was present mainly in the cytosol. By 24 h of recovery following heat stress, it was found predominantly in the nucleus-rich fraction and mitochondria. The localization of GP50 and P-SG67 by subcellular fractionation is consistent with immunolocalization studies and contrasts with the translocation of HSP70 after heat stress from cytosol to nuclei and nucleoli. These results reflect a characteristic distribution for each stress glycoprotein; their presence in virtually all subcellular fractions suggests multifunctional roles for the various stress glycoproteins in the cellular heat stress response.

Thermotolerance and heat resistance are often associated with elevated levels of heat shock proteins (HSPs) and a selective increase in protein glycosylation. In the present study, we have characterized heat stress-induced protein glycosylation in M21 cells, derived from the rat fibroblast line, Rat-1. M21 cells are characterized by constitutive overexpression of human HSP70 gene and show increased heat resistance without loss of its normal capacity for thermotolerance development after heat conditioning (Li et al., 1991, Proc. Natl. Acad. Sci. USA 88, 1681-1685). The data presented here show that the elevated heat resistance in these cells is associated not only with the constitutive overexpression of human HSP70, but also with increased glycosylation of a major stress glycoprotein, GP62 (Mr of 62,000). We further purified GP62 by sequential preparative isoelectric focusing and two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis. The purified protein was digested and partially characterized by microsequencing of two peptide fragments, comprising of 14-15 amino acids each. These fragments had a 100% sequence homology with HSP70 and a 71-100% sequence homology with HSC70 from various species. Western blotting using both HSP70 and HSC70 antibodies showed positive reactivity of GP62 with HSP70. Affinity characterizations showed strong binding of GP62 to wheat germ agglutinin and concanavalin A, consistent with the presence of both alpha-D-mannosyl/glucosyl and N-acetyl-beta-D-glucosylaminyl/glucosamine oligomer residues in GP62. These data confirm the glycosylated status of GP62 and indicate that GP62 is a heat stress-induced glycoprotein with partial homology to HSP70.

ストレスに対する細胞の応答は遺伝子発現制御のモデルであり、変化する環境に細胞が応答するために遺伝的に保存された機構のよい例である。細胞のストレスに対する応答には、糖鎖生物学的な因子がかなり関係している。こうした因子め性質はよく調べられてはいないが、“古典的な”熱ショックタンパク質 (HSPs) が機能するために必要であり、HSPsの機能を補うはたらきがある。この総説は、哺乳動物のストレス応答における糖鎖生物学的成分に焦点をあて、現在までに同定されているいくつかの主要な熱ストレス誘導糖タンパク質についてのべる。たとえば、GP50とGP62では遺伝子発現及びグリコシル化が熱許容性の誘導に伴って起こるが、P-SG67とP-SG64の場合には“敏速な”(すなわち、熱ストレス開始の数分内) グリコシル化が起こる。CHO細胞のGP50は、サーピンファミリーに属するレチノール酸で誘導されるマウスのJ6遺伝子産物とホモロジーがあるが、P-SG67とP-SG64は多くの機能を合わせ持つERタンパク質であるカルレチクリンのグリコシル化された変型である。この総説では、ストレス糖タンパク質のあり得る機能とそのHSPsとの関係について議論する。ストレス糖タンパク質に対する分子プローブを用いることにより、ストレス糖タンパク質の機能を理解する研究と、それらの糖タンパク質を生物工学に利用することが容易になるはずである。

Acute heat shock initiates the phenomenon of "prompt" glycosylation, which is characterized by selective glycosylation of specific cellular proteins called the prompt stress glycoproteins (P-SG). Prompt glycosylation rapidly occurs even during short heating periods, e.g. 10 min at 45 degrees C, and is not affected by the presence of cycloheximide (Henle, K. J., Kaushal, G. P., Nagle, W. A., and Nolen, G. T. (1993) Exp. Cell Res. 207, 245-251). The major P-SG in Chinese hamster ovary cells, P-SG67, was characterized by an M(r) of 67,000 and a pI = 5.1. In the present study, we purified P-SG67 by sequential gel filtration, anion exchange, affinity chromatography with concanavalin A-Sepharose, and two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis. The purified protein was digested and partially characterized by microsequencing of three major peptide fragments. The fragments, comprising a total of 46 amino acid residues, had an almost 100% sequence homology with calreticulin and partial homology with calnexin. Calcium binding studies with 45Ca2+ overlay confirmed that P-SG67 is a Ca(2+)-binding protein. These observations support the notion that P-SG67 is identical to calreticulin and that the glycosylation status of calreticulin can respond to environmental stress conditions.

Cancer is the second largest cause of death after cardiovascular disease in the United States. Systemic chemotherapy is the major treatment modality for a number of common cancers, such as lymphomas, leukemias, and for the majority of disseminated tumors. The emergence of drug-resistant tumor cells is the major cause of subsequent cancer treatment failures. Overcoming drug resistance is a difficult problem that remains unresolved; results to date suggest that tumor drug-resistance will continue to be a major limitation to success with anticancer chemotherapy. Short term, a multi-disciplinary treatment of cancer (eg, via cancer centers) should seek to eradicate cancer effectively at the time of diagnosis, with encouragement of patients to participate in clinical trials (eg, adjuvant chemotherapy). Long-term goals for cancer management should include cancer prevention and early detection through intensive public education, incentives for participation in early detection programs, and continued research, with a focus on mechanisms of tumor drug-resistance.

High intracellular levels of heat shock proteins and enhanced protein glycosylation are two phenomena closely associated with the cellular stress response. GP50 is the major heat-induced glycoprotein in Chinese hamster ovary (CHO) cells; however, GP50 is not well characterized, and its function is unknown. J6 is a gene originally identified in F9 murine teratocarcinoma cells after exposure to retinoic acid. In this study we show that J6 is heat-inducible and codes for a protein that shares characteristics with GP50. Western blotting of CHO cell homogenates, using a J6 polyclonal antibody, showed a single band with a molecular weight identical to that of GP50. Thermotolerant cells showed increased levels of the J6/GP50 protein. Heat-shocked CHO cells also accumulated transiently high levels of J6 mRNA between 2 and 7 h following 10 min at 45 degrees C. These induction kinetics are similar to those for GP50 labeling with D-[3H]mannose and to the activation of major heat shock genes, e.g., hsp70. Hybrid selection of J6 mRNA from CHO cells, followed by in vitro translation, produced a single band on SDS-PAGE with a molecular weight identical to that of deglycosylated GP50. Neither cellular proliferation (exponential growth versus plateau phase) nor the specific heat shock temperature (41.5 degrees C versus 45 degrees C) had significant effects on J6 induction by heat stress. Stress conditions other than hyperthermia, including ethanol, arsenite, and hypoxia, increased J6 mRNA levels. Conversely, J6 mRNA was reduced by quercetin, brefeldin A, okadaic acid, uv, and hydrogen peroxide. Our data support the hypothesis that J6 is a heat shock gene with a gene product identical to the polypeptide moiety of GP50.

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.

RIF-1 mouse tumors express high levels of beta-glucuronidase activity relative to most normal tissues. The high activity can be exploited for targeting specific drugs preferentially to tumor tissues. In this study we examined the kinetics of 8-hydroxyquinoline (8-OHQ) accumulation in tumor and in several normal tissues resulting from the in vivo deconjugation of 8-hydroxyquinolyl-glucuronide (8-OHQ-GlcA). Tumors were acidified with D-glucose and NaHCO3 prior to the administration of 8-OHQ-GlcA; subsequently the deconjugated aglycone, 8-OHQ, accumulated preferentially in tumors and reached peak levels between 30 and 60 min after the 8-OHQ-GlcA injection. Mild hyperthermia of 30 min at 43 degrees C to the tumors further increased their peak 8-OHQ levels by a factor of 2-3. Some normal tissues, mostly kidney, liver, and colon, also accumulated 8-OHQ, but the aglycone appeared early in the normal tissues (near 30 min post-injection) and was significantly reduced by 60 min when 8-OHQ remained high in the tumor. Administration of 8-OHQ-GlcA alone, without prior tumor acidification, failed to produce measurable accumulations of 8-OHQ in tumors and in normal tissues. Tissue clearance of 8-OHQ is mediated primarily by the enzymatic reconjugation of 8-OHQ via UDP-glucuronosyltransferase (UDPGT). UDPGT activity was high in liver, kidney, and bowel, but low in the RIF tumor, spleen, muscle, and brain. Hyperthermia had only a modest effects on UDPGT activity: a heat dose of 30 min at 45 degrees C reduced activity less than 60%. Thus, preferential accumulation and prolonged retention of 8-OHQ in RIF tumors may be caused by a combination of factors: a) high tumor beta-glucuronidase activity, b) selective tumor acidification during hyperglycemia, c) low tumor UDPGT activity, and d) other factors, such as tumor blood flow.

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N-acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau-phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of thermotolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O-glycans, alpha 1,2-fucosyltransferase and alpha 2,6-sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat-induced activation of O-glycan-specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.

Poly(A)RNA was isolated from thermotolerant cells and microinjected into recipient non-tolerant Chinese hamster ovary (CHO) cells. The injected cells expressed thermotolerance to a subsequent test heat treatment both in terms of the end-points of colony formation (cell survival) and resumption of protein synthesis after test heating (translational labelling). The magnitude of thermotolerance expression was dependent on the experimental end-point (increase up to 3.8-fold for translational labelling and approximately 2-fold for survival) and on the time between microinjection and the test heat treatment. Control experiments showed that poly(A)RNA from non-tolerant cells did not alter the heat response of microinjected cells. Proteins corresponding to the poly(A)RNA from thermotolerant cells were analysed by in vitro translation and by labelling of microinjected cells, followed by SDS-PAGE. In vitro translations showed high levels of transcripts for classical heat-shock proteins (HSP 70/72, 89, 110) in poly(A)RNA from thermotolerant versus control cells. However, proteins synthesized in intact cells showed no detectable differences when cells were microinjected with poly(A)RNA from thermotolerant versus control cells, or not injected at all. In principle the data show that microinjection of specific poly(A)RNA fractions can be used for defining the contribution of individual gene products to the cellular heat response.

It is well established that renal proximal tubule (RPT) cells grown under standard in vitro conditions attenuate many of their in vivo properties and functions. Thus, the study of renal stress response mechanisms requires an appropriate cell culture model. In the present study, we compared the heat stress (10 min, 45°C) response of freshly isolated RPT cells with that of RPT cells grown in vitro for 6 days under two different culture conditions: (1) SHAKE conditions, where oxygen levels and physiological functions are maintained via continuous media motion [Nowak G, Schnellmann RG: Am J Physiol 1996;271:C2072–2080] and (2) STILL conditions, involving standard cell culture which leads to partial hypoxia and a marked reduction in physiological functions. The freshly isolated RPT cells progressively synthesized heat shock proteins (HSPs) and stress glycoproteins (SGs) during a 3-hour culture period in vitro. Under these conditions, heat stress did not further increase HSP and SG synthesis. In RPT cells grown under SHAKE conditions, HSP70 synthesis was detected 1 h after heat stress and decreased below detection by 3 h. In contrast, the uptake of radiolabeled mannose into (glycoprotein) GP62 (Mr 62,000), GP50, and GP38 was observed in control SHAKE cultures and was not further increased after heat stress. These results are consistent with immunohistochemistry studies, where similar changes in HSP70 and GP50 expression were noted. RPT cells grown under STILL conditions showed both increased synthesis of HSP70 and increased glycosylation of GP62, GP50, and GP38 as early as 1 h after heat stress, but in contrast to SHAKE conditions, this heat-induced stress response further intensified at 3 h after heat stress. By 7 h after heating, HSP synthesis returned to control levels, while glycosylation of GP62 and GP50 remained elevated. Based on our results, we conclude that freshly isolated RPT cells exhibit a stress response that may be caused by acute cell isolation/culture stress. While this stress response unfolds, freshly isolated RPT cells appear unable to respond to additional heat stress. RPT cells grown under SHAKE and STILL conditions exhibit high rates of SG glycosylation, especially that of GP62, possibly reflecting a ‘stress’ condition associated with growth on plastic substrate. Concurrently, RPT cells from STILL cultures show a higher capacity for responding to acute heat stress than SHAKE cultures, evidenced by the transiently increased HSP synthetic rates. The interpretation of the renal stress response capacity, therefore, must be linked to a specific culture condition.

Elevated heat resistance is often associated with high cellular levels of hsp70. In the rat cell line, M21, increased heat resistance is associated with both the expression of an intact exogenous human hsp70 gene, and of an exogenous stress glycoprotein, GP62. In this study, we examined heat-stress induced alterations in protein glycosylation in two variant lines of M21 that contain specific deletions in the exogenous human hsp70 gene. The deletion mutant, MVH delta Sm, lacks the nucleolar localization sequence for human hsp70 gene, whereas the other mutant, MVH delta Bg, is characterized by deletion of the ATP binding domain in the human hsp70 gene. After heat induction, the MVH delta Sm mutants exhibited constitutive and heat-induced endogenous hsp70 mRNA levels, protein glycosylation, as well as hsp70 and GP62 protein levels comparable to those in the parent cell line, Rat-1. Cellular heat sensitivity of MVH delta Sm mutants was also similar to that of Rat-1 cells, although the mutant cells had a reduced capacity for thermotolerance development. On the other hand, MVH delta Bg mutants before thermotolerance induction, exhibited constitutive endogenous and human exogenous hsp70 mRNA levels similar to those in MVH delta Sm mutants. However, after thermotolerance induction, MVH delta Bg mutants showed lower levels of heat-induced endogenous hsp70 mRNA than MVH delta Sm mutants, an overall reduction in protein glycosylation, low hsp70 and GP62 levels, and increased heat sensitivity when compared to the parent Rat-1 cell line. Incorporation of D-[2-3H]mannose into oligosaccharide precursor pools and glycoproteins was consistent with protein glycosylation pattern for each cell line. Acute heat stress resulted in the selective glycosylation of the 'prompt' glycoprotein, P-SG64, in the two deletion mutants, similar to that in M21 cells expressing the intact human hsp70 gene. The data indicate that both protein glycosylation and hsp70 expression generally correlate with cellular heat resistance and thermotolerance expression. The presence of full or partially deleted copies of human hsp70 modulates thermotolerance and protein glycosylation in a complex manner that is not yet fully understood.

Proteins represent both structural and functional elements of biological organisms, however, their structural and catalytic function is directly linked to the acquisition and maintenance of a complex three-dimensional conformation. A molecular machinery to accomplish protein folding and maintenance in vivo is provided by a variety of molecular chaperones that include both heat shock proteins (Hsps), glucose-regulated proteins (Grps), and a separate class of stress glycoproteins (S-Gps). Different chaperones associate to form functional complexes (chaperone) and work coordinately to accomplish specific functions during the folding of particular proteins. In this review, we will summarize recently acquired new insights into the complexities of chaperones, the current state of S-Gps and their interactions with Hsps, and of specific chaperones that appear to be designed for the folding of cellular glycoproteins. Finally, we discuss the physiological role of chaperones by examining their function in specific cellular processes, namely tumor/host interactions and diseases associated with aberrant prion protein folding.

Heat stress results in the cellular accumulation of heat-shock proteins (HSP) and increased protein glycosylation. Among known stress glycoproteins, GP50 and GP62 are associated with the expression of thermotolerance. In the present study, we characterized subcellular localization and redistribution of GP62 and GP50 in a rodent cell line, M21, before and after cellular heat-stress. M21 cells are heat-resistant cells that overexpress human HSP70 and also have concomitantly high GP62 levels. Cellular fractionation by differential centrifugation showed that GP62 and GP50 was present in each subcellular fraction. However, each stress glycoprotein exhibited a characteristic kinetic pattern of redistribution during cellular recovery after heat stress. For example, glycosylated GP62 was seen predominantly in the mitochondria before heat-stress. Immediately after heat-stress, its presence in the mitochondrial fraction was dramatically reduced, while it increased in lysosomes, microsomes and cytosol. By 1 h after heat stress, it had largely disappeared from microsomal and cytosolic fractions. After 24 h, all subcellular fractions showed only trace amounts of residual GP62. By comparison, GP50 was also highest in the mitochondrial fraction before heat-stress, redistributed like GP62 immediately after heat stress, but remained relatively unchanged thereafter. In contrast to GP62, GP50 showed little redistribution during 24 h after heat-stress and remained at high concentrations in all cell fractions, including microsomes. Distribution of GP50 and GP62 before and after heat stress, based on differential centrifugation, was consistent with immunolocalization data. Following heat stress, both GP50 and GP62 showed a partial overlap in distribution with HSP70. The above results indicate that each stress glycoprotein has a specific subcellular location, both before and after heat stress. The presence of GP62 in virtually all cell fractions is consistent with a multifunctional role for GP62 in the cellular stress response.

Thermotolerance and heat resistance are frequently associated with elevated levels of heat shock proteins (HSPs). Elevated heat resistance is also found to be associated with the overexpression of high levels of HSP70, as seen in M21 cells, derived from the Rat-1 line. In the present study, we report that M21 cells also feature an increase in general protein glycosylation and specific expression of the stress glycoprotein, GP62, both of which correlate with cellular heat resistance. The expression of GP50, a major stress glycoprotein in cell lines such as CHO, however, did not correlate with cellular heat resistance in M21 cells. Protein glycosylation that occurs during acute heat stress ("prompt" glycosylation) was associated with the glycosylation of a major "prompt" stress glycoprotein, P-SG64 (M(r) of 64,000), that was identified by immunoblotting as a glycosylated form of calreticulin. The higher level of protein glycosylation in M21 cells correlated well with increased D-[2-3H]mannose incorporation into precursor pools of dolichyl phosphomannose and dolichyl pyrophosphoryl oligosaccharides and into glycoproteins. Thus, heat resistance in M21 cells is associated not only with expression of high levels of HSP70, but also with a concomitant increase in protein glycosylation. These data support the hypothesis that stress-induced protein glycosylation is a component of cellular stress response, either in association with HSPs or as an independent mechanism.

Constitutive patterns of protein synthesis and protein glycosylation are severely disrupted by acute heat stress. Stressed cells respond by preferential synthesis of specific proteins, e.g., the well-known family of heat shock proteins. We observed another response that rapidly occurs during heating periods as short as 10 min at 45°C. During that period, CHO cells began to glycosylate specific proteins, designated as "prompt" stress glycoproteins (P-SG), while constitutive protein glycosylation ceased. Labeling of P-SGs showed a dose response with time and with temperature and appeared regardless of the label used (D-[ 3 H]mannose or D-[ 3 H]-glucose). On SDS-PAGE, the major P-SG was characterized by M r ≈ 67 kDa (P-SG67) and p I = 5.1. Other less prominent P-SGs appeared at M r 160, 100, 64, 60, and 47 kDa; incorporated label showed little turnover during 24 h at 37°C. Prompt glycosylation was inhibited by tunicamycin, and label incorporated into P-SGs was sensitive to N -glycosidase F, but not to O -glycosidase. Analysis of enzymatically digested P-SG67 indicated that label had been incorporated into both high-mannose (Man 9 G1cNAc) and complex-type oligosaccharides. Brefeldin A did not eliminate P-SG67 labeling, but caused the further appearance of novel, Brefeldin-associated P-SGs. Labeling of P-SG67 oligosaccharides occurred without significant concomitant protein synthesis, suggesting that addition of labeled oligosaccharides largely occurred on mature, rather than nascent proteins. The functional significance of prompt glycosylation remains to be defined, but we propose that this novel phenomenon is an integral part of the cellular heat stress response.

We examined the effect of 45 degrees C hyperthermia on the following glycosidases in CHO cells: beta-galactosidase, beta-hexosaminidase, beta-glucuronidase and alpha-mannosidase. Among these, lysosomal alpha-mannosidase exhibited the most dramatic response to hyperthermia with an increase in activity immediately after 45 degrees C hyperthermia. The increase was linearly dose-dependent with a doubling of activity for every 20 min at 45 degrees C. In contrast to alpha-mannosidase, beta-glucuronidase, beta-galactosidase, and beta-hexosaminidase showed only minor alterations in activity, or none, after hyperthermia of 10 to 60 min at 45 degrees C. Induction of thermotolerance enhanced the heat resistance of beta-galactosidase, but caused increased heat sensitivity for alpha-mannosidase. Intracellular beta-galactosidase, measured by histochemical staining, showed a dramatic redistribution in response to mild hyperthermia (10 min, 45 degrees C); the same effect was not observed for beta-glucuronidase. The data argue against non-specific activation of lysosomes by hyperthermia, and suggest that cells contain lysosomal subpopulations that are characterized by different heat sensitivities and variable glycosidase contents.

Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.

We have examined the relative ability of 16 sugars and sugar analogues to reduce cell killing by hyperthermia of 40 min, 45 degrees C. In general, sugars were added to the culture medium 6 h prior to heating at a concentration of 100 mM (400 mosmol). The results show that D-hexoses, L-hexoses, methylated or thiolated sugars and disaccharides significantly protected cells against thermal damage, increasing survival by factors of 10 to 100. The degree of protection varied for specific sugars and could not be predicted on the basis of sugar conformation or the number of hydroxyl groups. Relative heat protection was partially dependent on the survival assay technique (pre- and post-plating); consistently lower cell survival was measured when cells were subcultured after hyperthermia, both in medium-control and sugar-protected cells. However, the time dependence of heat protection appeared independent of pre- and post-plating. Cell survival after heating was not increased by two sugars: (a) D-idose, and (b) 2-deoxy-D-galactose. The latter sugar, curiously, was also a heat protector but only when cells were trypsinized after hyperthermia. Both of these sugars were relatively more toxic at 37 degrees C under identical treatment conditions. The lack of protection by these two sugars is not understood. Another reported non-sugar heat protector, sodium butyrate, was included as an additional control. Heat protection by butyrate was not observed in CHO cells. The accumulation of intracellular free sugar was measured by gas chromatography after incubating cells for 6 h, 37 degrees C with talose, idose, L-galactose or 1-O-methyl-D-glucose. All of these sugars were found in high concentrations inside of cells. The data are consistent with the hypothesis that polyhydroxy compounds must accumulate intracellularly for cellular heat protection.

Step-down heating (SDH) of Chinese hamster ovary cells consisted of an acute heat treatment at an elevated temperature followed immediately by chronic exposure to a lower hyperthermic temperature [step-down temperature (SDT)]. The survival curve at the SDT showed a reduced D0 (dose required to reduce the cell population to 37% on exponential part of the curve) relative to that of control cells only for SDT of 43 degrees C or less. The ratio r of D0's, D0 (control)/D0 (SDH), varied inversely with the SDT (e.g., r = 13 for SDT = 40 degrees C, whereas r = 1.3 for SDT = 43 degrees C). The degree of sensitization, as measured by r, was independent of the conditioning temperature (CT) and the conditioning time, provided that the CT was above 43 degrees C and the conditioning treatment reduced cell survival to 60% or less. The ratio r could be defined mathematically as a function of the activation energies for heat-induced cell killing above and below 43 degrees C, the SDT, and the CT. Biphasic heat survival curves, which appeared with heating at constant temperature below 43 degrees C, also appeared under SDH conditions, which suggested that thermotolerance, as represented by the resistant phase of the biphasic survival curve, was not affected by SDH. Fractionation experiments to measure the loss of interaction of heat damage remaining from the conditioning treatment with the low-temperature heat damage showed that 63% of the 45 degrees C sublethal damage was removed by 67 minutes at a constant exponential rate.

During the development of thermotolerance, Chinese hamster ovary cells not only synthesized classical heat shock proteins, but also incorporated [3H]D-glucose or mannose into a glycoprotein with a Mr of approximately 50 kD. The glycosylation of the 50 kD protein correlated with the expression of thermotolerance under conditions when tolerance was induced either by acute or chronic heat conditioning. A phosphoprotein with the same molecular weight as the 50 kD glycoprotein was dephosphorylated immediately after heat conditioning. Both phosphate and glucose label in the ion front were enhanced immediately after heating, and may represent elevated levels of sugar phosphates. However, the composition of the ion front material remains to be determined. The data are consistent with a hypothesis that attributes increased heat resistance of thermotolerant cells to the glycosylation of specific heat-sensitive cellular sites.

The antibiotic pentalenolactone, a specific inhibitor of glyceraldehydephosphate dehydrogenase, was used to investigate the effect of glycolytic adenosine triphosphate (ATP) synthesis on the survival response of aerobic and hypoxic Chinese hamster cells treated with 42 degrees C hyperthermia. Data obtained with aerobic cells, incubated in balanced salt solutions supplemented with different substrates for ATP production, showed that 50 microM pentalenolactone blocked ATP synthesis via glycolysis but not by oxidative phosphorylation. The glycolytic inhibition was reversed upon transfer of the cells to antibiotic-free medium, and minimal cytotoxicity (less than 20 per cent) was observed. Hypoxic cultures were obtained by incubating dense cell suspensions (2 X 10(6)/ml) to produce metabolic oxygen depletion. Concomitant with the development of hypoxia, pentalenolactone-treated cells became ATP-depleted; cellular ATP levels were reduced by about 70-fold as compared to hypoxic cells in the antibiotic-free medium. The ATP-depleted cells were more sensitive to killing by hyperthermia. Comparison of the 42 degrees C survival curves for control and the antibiotic-treated hypoxic cells yielded a dose-modifying factor of 4 (5 per cent survival level). The results indicate that inhibition of glycolytic ATP synthesis, for example by pentalenolactone, can selectively sensitize hypoxic cells to the lethal effects of mild hyperthermia.

Incubation of Chinese hamster cells at an elevated but sublethal temperature between fractions of radiation and/or pulses of hyperthermia at 45 degrees C strongly modified the effectiveness of cell killing. Increasing the incubation temperature from 37 to 40 degrees C between fractions of hyperthermia at 45 degrees C followed by radiation substantially enhanced cell killing; while the opposite sequence (radiation leads to incubation at 40 degrees C leads to hyperthermia at 45 degrees C) resulted in less effective cell killing than would have been expected from the independent interaction of hyperthermia and radiation alone. This suggests the use of short pulses of localized hyperthermia in the presence of physiologically tolerable fever followed by irradiation as one approach to the utilization of hyperthermia in cancer therapy.

Chinese hamster V79 fibroblast cells were exposed to brief periods of cold but non-freezing temperatures at different points on the population growth curve. Upon rewarming, cells at the transition from loarithmic to stationary growth exhibited apoptosis (programmed cell death). Cells in other stages of growth, or after reentry into logarithmic growth by refeeding, did not exhibit apoptosis. Apoptosis was expressed by marked cytoplasmic blebbing, by a characteristic non-random fragmentation of DNA into nucleosomal-sized pieces, and by loss of colony-forming ability. The data suggest that cold shock served as a stimulus for susceptible cells to undergo apoptosis. Thus, the experiments decribe a new in vitro system for studying the mechanisms of apoptosis.

The interaction of hyperthermia damage and radiation damage has most often been studied under conditions which favoured repair of sublethal damage. In the present study the interaction of 45°C -hyperthermia damage and radiation damage on the survival of Chinese hamster ovary (CHO) cells was determined at temperatures of 0–4°, 20° and 40°C . Subphysiological temperatures inhibited to various degrees both the repair of sublethal radiation and sublethal 45°C -hyperthermia damage while incubation at 40°C apparently led to the conversion of sublethal 45°C -hyperthermia lesions to lethal lesions. Potentially lethal hyperthermia damage (H-PLD) was repaired at 0–4° and 20°C ; whereas the repair of potentially lethal radiation damage (X-PLD) occurred at 20°C , but not at 0–4° or 40°C . The interaction of 45°C -hyperthermia and radiation damage at temperatures of 0–4°, 20° and 40°C represented a super-position of the separate effects of the two treatment modalities separately combined with incubation at 0–4°, 20° or 40°C . This suggests that ionizing radiation and hyperthermia are affecting distinct targets and that H-PLD and X-PLD do not interact.

Two uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), were tested for their ability to modify the survival of cultured Chinese hamster ovary (CHO) and Chinese hamster V79 cells treated with hyperthermia. The uncouplers were used under conditions that inhibit oxidative ATP synthesis, as judged from measurements of cellular ATP levels. Incubation of CHO cells in glucose-free Hanks' balanced salt solution (HBSS) containing 1 mM DNP for 1 h at 37 degrees C followed by reincubation at 37 degrees C in complete growth medium for 3 or 16 h, showed no substantial changes in the 45 degrees C heat survival curve as compared to heated cells not exposed to DNP. Thus, DNP treatment of CHO cells did not induce thermotolerance. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), tested under similar experimental conditions, did alter cellular heat resistance. The major change in the 45 degrees C survival curve of CHO cells pretreated with CCCP was an increase in the width of the shoulder: the Dq value increased from 14 min to 24 min, for the control and CCCP-treated cells respectively. The D0 value did not change appreciably. In contrast, heat-induced thermotolerance (10 min, 45 degrees C + 16 h, 37 degrees C) was characterized primarily by an increase in the D0 parameter from 4 min (unheated cells) to 17 min. Similar results were observed with CCCP-treated V79 cells. The data demonstrate that heat resistance induced by 1.2 microM CCCP was manifest as an increased cellular capacity to accumulate and/or repair hyperthermia damage, rather than an induction of thermotolerance, and that this effect probably was not related to the action of CCCP as an uncoupler of oxidative phosphorylation.

We have tested the reported ability of procaine to inhibit the induction and the development of thermotolerance in Chinese hamster ovary cells. Thermotolerance was induced either by hyperthermia alone (10 min, 45 degrees C) or by combining hyperthermia and procaine (5 min, 45 degrees C + 10 mM procaine) with heating times adjusted to yield similar cell survival after the conditioning treatments. Both the kinetics of thermotolerance development in fresh medium without procaine and the magnitude of thermotolerance 6 h after heat conditioning were similar for the two treatment groups. Development of thermotolerance in the presence of procaine was tested by adding the drug at 5 or 10 mM to culture medium between, but not during two fractionated heat treatments. Thermotolerance development was observed even in the presence of 10 mM procaine, but only if cell survival was corrected for the 37 degrees C-procaine toxicity. Complete survival curves of cells incubated for 6 h at 37 degrees C in 7.5 mM procaine between heat conditioning and test heating showed a D0 that was only 35 per cent lower than that of thermotolerant controls. The data are consistent with the reported sensitization to heat killing by procaine, but show that thermotolerance induction and development were only minimally perturbed by procaine.

The interactions of BCNU (1,3-bis[2-chloroethyl]-1-nitrosourea) with low pH, glucose and hyperthermia were studied in cultured RIF tumor cells. The effect of a mild heat treatment of 43 degrees C, 1 h at pH 7.4 on cell killing [surviving fraction (S) = 0.27 +/- 0.05, standard error of the mean (S.E.)] was significantly enhanced by pH 6.5 (S = 0.11 +/- 0.02, S.E.) and 50 mM D-glucose (S = 0.14 +/- 0.01, S.E.). When heat (43 degrees C, 1 h) was added to BCNU, cytotoxicity was increased approximately 14-fold over BCNU alone. Moreover, pH 6.5 increased killing with BCNU and heat by an additional factor of 28. The presence of glucose at 37 degrees C at either pH 6.5 or 7.4 reduced BCNU toxicity in a dose dependent fashion. However, the presence of glucose did not reduce cell killing by BCNU at 43 degrees C. As a result BCNU cytotoxicity was enhanced by approximately 2 orders of magnitude when tumor cell acidification (glucose and low pH) was combined with BCNU and heat.

SummaryAsynchronous CHO cells heated in the presence of hypertonic medium (600 mosM) showed an increased sensitivity (lowered D0) to 45°C hyperthermia and the extent varied as a function of the time of heating with respect to the hypertonic shock. Initiation of heating 1 min after hypertonic shock reduced the D0 from 4·4 + 0·4 to 2·1 ± 0·2 min and the extrapolation number, n, from 5·9 to 5·2 while hyperthermia initiated 30 min after the hypertonic shock did not reduce the D0 significantly but did reduce n to 1·7. Hypotonic medium (150 mosM) also reduced n without significantly reducing the D0, and in contrast to hypertonic medium, this effect was independent of the temporal relationship between the osmotic shock and heating. Sensitization of non-thermotolerant cells by hypotonic shock was possible but only when cells were heated immediately upon return to isotonic medium following a one-hour equilibration in hypotonic medium.Thermotolerant CHO cells were sensitized slightly more than non-thermotolerant ce...

When L-alanine was added either to full growth medium or to Hanks' balanced salt solution (HBSS) prior to hyperthermia, survival of heated cells was significantly increased in a concentration-dependent manner. Maximal heat protection was not immediate, but required at least 1 h at 37 degrees C incubation prior to heating. Heat protection was principally reflected in an increased Dq on the 45 degrees C survival curve; for example, with 100 mM L-alanine, the Dq increased from approximately equal to 20 (control) to 30 min at 45 degrees C. Hyperthermia of 1 h at temperatures between 42 degrees C and 45 degrees C indicated that 100 mM alanine had shifted the isotoxic temperature by 0.5 degrees C. Comparable heat protection was also observed with D-alanine and amino acid dimers, such as alanyl-alanine or alanyl-leucine. Leucine at similar concentrations by itself, without alanine, did not protect cells against heat killing, but increased cellular heat sensitivity. The data suggest that heat protection by alanine does not require incorporation of alanine into cellular protein, but is mediated by the free amino acid.

Many tumors show elevated levels of hydrolytic enzymes that may be associated with invasive processes. The RIF- 1 murine tumor has levels of 0-glucuronidase that are more than four times higher than those in liver. Elevated tumor glucuronidase levels can be used as a basis for tumortargeted therapy when systemically administered glucuronides of cytotoxic drugs are deconjugated preferentially at the tumor site. In this study we have used 8-hydroxyquinoline (8-OHQ) as a model compound for such a tumor-targeting concept. We showed that RIF tumors and spleen had the highest P-glucuronidase activity in C3H mice; for example, RIF tumors released approximately seven times more phenolphthalein per gram of tissue from its glucuronide than liver, when compared under identical conditions. In vitro, low concentrations of 8-OHQ that might be achievable in vivo, ranging from 1 to 10 AM reduced cell survival by four orders of magnitude, while I mM 8-hydroxyquinolyl-glucuronide (1 h, 37*C) resulted in only modest (S = 54%) cytotoxicity. Combination treatments of 8-OHQ (2.5 or 5 AM) with either hyperthermia or X radiation did not significantly change the slope of survival curves for RIF tumors in vitro, but suggest that targeted 8-OHQ toxicity combined with local hyperthermia and/or irradiation may be useful for significantly increasing therapeutic gains in vivo. ? 1988 Academic Press, Inc.

Asynchronous Chinese hamster ovary cells were microinjected with glutathione disulfide (GSSG). Successfully injected cells were scored by coinjecting FITC-dextran with GSSG, followed by fluorescent microscopy. After microinjection, cells were incubated for 2.5 h at 37°C to permit thermotolerance development and then heated at 45°C for 40 min. Cellular heat sensitivity was quantitated by counting the number of grains per cell after labeling heated cells with tritiated amino acids and processing for autoradiography. The data show that microinjection of GSSG induced thermotolerance which increased the number of grains per cell up to 500% of controls. Cells that were exposed to similar concentrations of GSSG in culture medium without microinjection or microinjected without GSSG did not develop thermotolerance.

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).

The temperature compatible with biphasic hyperthermia-survival curves in Chinese hamster ovary cells was increased from 42.5 to 44°C by acute heat conditioning (10 min, 45°C). An Arrhenius analysis...

The protein content of chromatin isolated from mammalian cells that are heated at temperatures between 40 and 48°C, is increased relative to that from control cells. The kinetics of this increase in chromatin protein content was defined by heating HeLa cells at temperatures between 40 and 48°C for periods of up to 60 min. At temperatures above 43°C, these kinetics show a rapid initial rate of increase which decays to a slower limiting rate. The initial rate of increase in chromatin protein content obeys the Arrhenius law with an activation enthalpy of 86.8 ± 1.4 kcal/mole. Hyperthermia-cell-survival curves, determined between 44 to 48°C, show an activation enthalpy of 153.8 ± 2.5 kcal/mole for cell killing. At 48°C, the activation entropies are 208.2 cal/mole K for the protein increase and 419.6 cal/mole K for cell killing. Although the activation enthalpy and entropy for the increased protein content of chromatin are about half those for cell killing, the Gibbs free energy of activation is similar for bo...

The heating of cells or tissues generally involves a temperature transient during which the temperature of the heated object rises from an initial to a final level with biological effects occurring during this interval. The method proposed in this paper can estimate these effects by using the data on isobiological effects obtained both in vitro and in vivo. The method has some limitations in that it cannot be applied under conditions where thermotolerance develops or step-down heating occurs. Several examples from both in vivo and in vitro applications serve to illustrate this method as a practical tool in hyperthermia research.

The time-temperature relationship for heat killing of asynchronous, exponentially growing L1A2 cells was defined in the range 40.5-45?C. All survival curves were exponential after an initial shoulder, and an Arrhenius plot for heat killing was linear in the entire temperature range with an activation energy of 724 kJ/mole (173 kcal/mole). Thermotolerance did not develop during continuous heating at these temperatures. However, a heat treatment at 40.5 or 42?C could induce thermotolerance if the cells were subsequently incubated at 37?C. Preheating the cells for 90 min at 42?C followed by a 10-hr incubation at 37?C resulted in maximal thermotolerance. A reheating of these cells in the temperature range 41-45?C showed a reduced heat sensitivity as compared with control cells (single heating). An Arrhenius plot for thermotolerant cells showed an activation energy (703 kJ/mole) similar to that of single heated cells but with a reduced activation entropy, suggesting the same mechanism(s) for heat killing in control and thermotolerant cells. Step-down heating was also documented in this cell line. An initial 10-min heat shock at 45?C followed by heating at 41-42?C reduced the activation energy for heat killing from 778 to 238 kJ/mole (186 to 57 kcal/mole) with a transition point at about 42.5?C. These data suggest that although single heated and thermotolerant cells may be killed by the same process, the greater cellular sensitivity under stepdown heating conditions may reflect a different mechanism of cell killing.

ROTI ROTI, J. L., AND HENLE, K. J. Comparison of Two Mathematical Models for Describing Heat-Induced Cell Killing. Radiat. Res. 81, 374-383 (1980). A computer-based minimization algorithm is utilized to obtain the optimum "fits" of two models to hyperthermic cell killing data. The models chosen are the multitarget, single-hit equation, which is in general use, and the linear-quadratic equation, which has been applied to cell killing by ionizing irradiation but not to heat-induced cell killing. The linear-quadratic equation fits hyperthermic cell killing data as well as the multitarget, single-hit equation. Both parameters of the linear-quadratic equation obey the Arrhenius law, whereas only one of the two parameters of the multitarget, single-hit equation obeys the Arrhenius law. Thus the linear-quadratic function can completely define cell killing as a function of both time and temperature. In addition, the linear-quadratic model will provide a simplified approach to the study of the synergism between heat and X irradiation.

Ethanol (1 M) cytotoxicity in asynchronous Chinese hamster ovary cells was strongly temperature dependent, yielding families of cell survival curves between 34 and 39 degrees C that were similar to those obtained at hyperthermic temperatures in medium without ethanol. Below 36 degrees C, survival curves were biphasic, indicating the development of thermotolerance during ethanol exposures. At room temperature (22 degrees C) ethanol was completely nontoxic with incubation periods up to 6 h. A comparison of survival curves with and without ethanol showed that the major effect of ethanol was an effective temperature shift of circa 6.5 degrees C, i.e., the cell survival curve at 37 degrees C in 1 M ethanol was equivalent to that at 43.6 degrees C in medium without ethanol. In addition to the effective temperature shift, ethanol also resulted in sensitization to "heat" with a temperature dependence that was similar to the stepdown heating effect. When thermotolerance was induced with acute ethanol exposures (25 min, 37 degrees C or 60 min, 35.5 degrees C), the kinetics and the magnitude of tolerance were similar to those after isotoxic conditioning treatments with heat alone (10 min, 45 degrees C). In contrast, equimolar ethanol at 22 degrees C did not induce thermotolerance. These data provide a rationale for conflicting results in the literature regarding thermotolerance induction by ethanol. Both heat sensitization and the induction of thermotolerance are interpreted as the effect of ethanol on the solution properties of intracellular water. These solvent alterations reduce the temperature necessary to elicit cytotoxicity and the development of thermotolerance.

Hyperthermia at either 41.5 or 45°C with variable heating times to reduce cell survival up to three orders of magnitude did not decrease significantly cellular ATP content when measured either immediately or up to 7 hr after a heat treatment. Similarly, cellular ATP content was not significantly reduced with step-down heating, precooling prior to hyperthermia, or thermotolerance induction. The data suggest that heat-induced depletion of intracellular ATP content is not a critical factor in the thermal death of cells heated under normal culture conditions.

The monohydroxy alcohol, ethanol, and the dihydroxy alcohols, propylene glycol and ethylene glycol, enhance the killing of CHO cells by hyperthermia (450C) in vitro. At an equimolar concentration (I M), the sensitization to hyperthermia was greatest for ethanol, intermediary for propylene glycol, and least for ethylene glycol. In contrast, the presence of the trihydroxy alcohol, glycerol, in the culture medium during heating protected cells against heat-induced killing. The hexahydroxy alcohol, sorbitol, neither increased nor decreased significantly the cell killing by 450C hyperthermia. A 1-hr exposure at 370C to any of the alcohols, including glycerol, followed by a 6-hr incubation at 370C in fresh medium did not significantly alter the cellular sensitivity to 450C hyperthermia. Thus alcohols can sensitize CHO cells to or protect them from hyperthermia when present during heating, but do not induce thermotolerance. The results are interpreted in terms of the literature on alcohol denaturation of proteins and membranes and the ability of the alcohols to penetrate the plasma membrane.