Stress proteins and glycoproteins (Review).

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.

Tumor drug-resistance: a challenge to therapists and biologists.

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.

Heat shock glycoprotein GP50: product of the retinoic acid-inducible J6 gene.

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.

Prompt protein glycosylation during acute heat stress.

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 degrees 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-[3H]mannose or D-[3H]glucose). On SDS-PAGE, the major P-SG was characterized by M(r) approximately 67 kDa (P-SG67) and pI = 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 degrees 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 (Man9GlcNAc) 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.

Multidrug resistance: prospects for clinical management.

Clinical success in the treatment of tumors with chemotherapy has significantly improved over the past several years. However, treatment failures due to drug resistance of cancer cells has remained a major problem. The classical form of multiple drug resistance is perhaps also the most common type of drug resistance, and represents the overexpression of a transmembrane glycoprotein pump (P-170) that mediates the efflux of a spectrum of structurally and functionally unrelated drugs. Here, we discuss recent evidence that support the concept that the total phenomenon of multiple drug resistance (MDR) involves several other mechanisms in addition to that underlying "classical" MDR. These include the action of other energy-dependent membrane efflux pumps, elevated levels of GSH for drug conjugation and detoxification to facilitate export, enhanced DNA repair facility, gene amplification and oncogene activation. The combination of mechanisms used by any particular cell line is variable and suggests that many of these mechanisms are independent. Successful reversal of drug resistance appears to require the identification of relevant operative resistance mechanisms. An example is the competitive inhibition of P-170 with verapamil, quinine and tamoxifen. A broadly successful strategy for killing drug-resistant cancer cells, however, could be based on either selective energy depletion of cancer cells or the permeabilization of tumor cells with an effective bypass of efflux pumps, since many mechanisms of drug resistance entail the energy-dependent export of toxins. The latter approach may be achieved via membrane lipid modifications or the introduction of membrane pores by biological or physical (electroporation) means.

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating.

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.

Tumor-targeted delivery of 8-hydroxyquinoline.

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.

New approaches to the study of tumor drug resistance.

The development of tumor drug resistance is the major obstacle to successful systemic chemotherapy. Therefore, devising methods for reversing drug resistance is a high priority and could lead to significant improvements in cancer treatment. The mechanisms of tumor drug resistance are manifold and are not well understood. The phenomenon of multidrug resistance (MDR) represents the development of resistance to most drugs, regardless of their chemical structure. Several types of MDR are known, for example, the overexpression of a cell membrane glycoprotein (P-170), increased activity of glutathione S-transferase, elevated levels of glutathione (GSH), and alterations in topoisomerase action. A partial reversal of tumor drug resistance has been achieved by the use of competitive inhibitors for the function of glycoprotein P-170, or by the inhibition of GSH synthesis; however, this strategy has not been substantially successful for improving the response of human tumors to clinical therapy. We have recently used electroporation, in conjunction with the cytotoxic drug, cisplatin (cDDP), in an attempt to circumvent drug resistance in cDDP-resistant mouse tumor cells (RIF/Ptr1). Electroporation is the application of a high-voltage electric shock which is known to create transient pores in plasma membranes of cultured cells. Electroporation plus cDDP treatment increased intracellular cDDP concentration and reversed cellular resistance to cDDP-induced cell killing.

Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures.

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.

Protein glycosylation in heat-sensitive and thermotolerance-deficient mutants of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are capable of developing a high degree of thermotolerance in response to appropriate heat conditioning. In this study we examined the relationship between thermotolerance development and protein glycosylation using four sublines of CHO cells. Two of these CHO sublines are characterized by an increased heat sensitivity and impaired cellular capacity for thermotolerance development. The data show that thermotolerance development after heat conditioning in the heat-sensitive, thermotolerance-deficient mutants was accompanied by reduced labeling of a Mr 50,000 glycoprotein (GP50), in both soluble and insoluble cell fractions. Similarly, activation of UDP-N-acetylgalactosaminyltransferase (Gal-NAcT) after hyperthermia was almost completely abolished in these cell lines. Both of these endpoints have been correlated previously with thermotolerance expression. The data are consistent with the glycosylation hypothesis that attributes increased heat resistance of thermotolerant cells, at least in part, to enhanced glycosylation and accumulation of endogenous glycoproteins, such as GP50.

Tumor cell drug resistance and its reversal.

Tumors that formerly were uniformly fatal can now be cured by cancer chemotherapy. However, successful anticancer therapy is faced by many obstacles, such as excessive normal tissue toxicity and drug resistance. Tumor drug resistance may be either intrinsic or acquired. The multidrug resistance (MDR) is a unique phenomenon and is characterized by tumor resistance to various structurally unrelated drugs. Known mechanisms for MDR include overexpression of a membrane P-glycoprotein 170 and elevated cellular levels of reducing agents, such as glutathione (GSH). Currently available strategies for overcoming drug resistance include competitive inhibitors of the P-glycoprotein 170, inhibitors of GSH synthesis, and adjuvant therapy with hyperthermia. Development of drug resistance is analogous to a physiological detoxification mechanism and may continue to limit the effectiveness of cancer chemotherapy in the near future.

In vitro antiproliferative activity of 4-substituted 2-(2-hydroxyphenyl)thiazolines on murine leukemia cells.

Two previously synthesized and two structurally novel thiazoline iron chelators are described. N4-Benzyl-N1,N8-bis[[2-(2-hydroxyphenyl)thiazolin-4-yl]carbonyl] homospermidine (5) proved to be the most potent antiproliferative and cytocidal compound in the series with in vitro IC50 values of 3 and 1 microM on L1210 and P388 murine cell lines. The N4-acetyl analogue 7 was considerably less active than 5 with IC50 and cell viability values that were similar to those of the structurally simple thiazolines 2 and 3. The antiproliferative activity of 3 and 7 could be substantially reduced or ablated by delivery to cell suspensions as a 1:1 molar mixture with FeCl3, while the activity of 5 was unaffected by Fe(III) chelation. As expected, 3 induced a G1/S cell cycle block at the 100 microM block consistent with interference with DNA synthesis while 10 microM 5 did not affect L1210 cell cycle distribution. Tritiated thymidine incorporation studies confirmed that 5 was incapable of interfering with DNA synthesis at concentrations below 40 microM. Alkaline elution studies indicate that 5 does not cause DNA strand breaks in vitro at concentrations of 10 microM. The N4-benzyl group of 5 appears to impart in vitro potency as the N4-acetyl analogue 7 lacks comparable in vitro antiproliferative and cytocidal activity.

Characterization of a cisplatin-resistant subline of murine RIF-1 cells and reversal of drug resistance by hyperthermia.

The development of tumor cell drug resistance is a major obstacle which often leads to failure of cancer chemotherapy. Therefore, reversing the cell drug resistance would have important implications in cancer treatment. We have developed a cisplatin-resistant mouse tumor cell line from the radiation induced fibrosarcoma (RIF-1) parental line; this line is named RIF/ptr1 versus the parental line RIF/pts1. It is shown that the formation of cisplatin-DNA interstrand cross-links is the same for both cell lines although the intracellular cisplatin concentrations of resistant line is significantly lower. The cytosolic activities of glutathione reductase, glutathione peroxidase, and DT-diaphorase were the same in two cell lines. However, the concentration of glutathione was significantly higher in the resistant line. The resistant line was shown to be more sensitive to the cytotoxicity of heat (43 degrees C) but the combination of heat and drug had the same tumoricidal effect for both cell lines. The addition of verapamil also had a similar effect on both cell lines. We conclude that the major difference between these two lines was the glutathione-related detoxification of platinum. Regardless of drug resistance, the combination of drug and heat can effectively kill both cell lines. Elevated glutathione in RIF/ptr1 cells may be associated both with enhanced heat sensitivity and drug resistance such that combined treatments with drug and heat were equally effective in killing cells of either line.

Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl.

To assess target-tissue exposure to the human urinary bladder carcinogen 4-aminobiphenyl (ABP), we have developed a sensitive immunochemical method for measuring the major arylamine-DNA adduct formed, N-(guan-8-yl)-ABP (Gua-C8-ABP). High-affinity polyclonal antisera from rabbits immunized with N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin were characterized and shown to have high specificity for antigenic determinants on the purine and biphenyl rings of Gua-C8-ABP and minimal cross-reactivity with ABP, deoxyguanosine, or hydrolyzed DNA. Assay standards containing ABP-modified DNA were prepared by reacting [3H]N-hydroxy-ABP with calf thymus DNA. DNA samples were hydrolyzed with trifluoroacetic acid and dried under vacuum, and the residues were dissolved in dimethyl sulfoxide under argon. Using a streptavidin-biotin amplified enzyme-linked immunosorbent assay, DNA hydrolysates competed at 25 micrograms DNA/microtiter well for a limiting amount of anti-keyhole limpet hemocyanin-(Gua-C8-ABP) in the presence of excess solid-phase bovine serum albumin-(Gua-C8-ABP) coating antigen. The limit of sensitivity for this assay using 25 micrograms DNA was 2 adducts/10(8) nucleotides. Gua-C8-ABP adducts in liver and bladder epithelial DNAs were readily quantified after p.o. administration of 5 mg/kg ABP to dogs. This methodology is capable of detecting adducts at levels of biological significance and should be applicable to human target-tissue dosimetry.

Tumor-targeted cell killing with 8-hydroxyquinolyl-glucuronide.

Many tumors show elevated levels of hydrolytic enzymes that may be associated with invasive processes. The RIF-1 murine tumor has levels of beta-glucuronidase that are more than four times higher than those in liver. Elevated tumor glucuronidase levels can be used as a basis for tumor-targeted 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 beta-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 microM reduced cell survival by four orders of magnitude, while 1 mM 8-hydroxyquinolyl-glucuronide (1 h, 37 degrees C) resulted in only modest (S = 54%) cytotoxicity. Combination treatments of 8-OHQ (2.5 or 5 microM) 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.

Enhanced glycosylation of a 50 kD protein during development of thermotolerance in CHO cells.

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.

Uncoupling of oxidative phosphorylation does not induce thermotolerance in cultured Chinese hamster cells.

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.

Protection against heat-induced cell killing by alanine.

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.

Interactions of BCNU, low pH, glucose and hyperthermia in cultured RIF cells.

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.

Apoptosis induced by cold shock in vitro is dependent on cell growth phase.

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 logarithmic 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 describe a new in vitro system for studying the mechanisms of apoptosis.