Heat-induced modulation of lamin B content in two different cell lines

Previous work in our laboratory indicates that the nuclear matrix protein lamin B is a "prompt" heat shock protein, which increases significantly when human U-1 melanoma and HeLa cells are exposed to 45.5 degrees C for 5-40 min. Using Western blotting, we found that the lamin B content in U-1 and HeLa cells increased to a greater extent during post-heat incubation at 37 degrees C than during the heat dose itself. When HeLa cells were heated at 45.5 degrees C for 30 min, and then incubated at 37 degrees C for up to 7 h, lamin B content was increased significantly (1.69-fold maximum increase at 3 h) compared to unincubated heated cells. Also, thermotolerant HeLa cells showed a greater increase (up to 1.72-fold) in lamin B content during subsequent heating compared to nontolerant cells. The increase in lamin B content in thermotolerant cells, or when heated cells were incubated at 37 degrees C, was also observed in U-1 cells. HeLa cells heated in the presence of glycerol (a heat protector) showed a 1.21-1.72-fold increase in lamin B content compared to cells heated for 10-30 min without glycerol. In contrast, lamin B content decreased 1.23-1.85-fold when cells were heated for 10-30 min in the presence of procaine (a heat sensitizer) compared to cells heated without procaine. These data suggest that lamin B may play an important role in the heat shock response, and that modulation of lamin B content by heat sensitizers or protectors may play a role in regulation of heat sensitivity.     

DNA lesions in hyperthermic cell killing: effects of thermotolerance, procaine, and erythritol

When HeLa S3 cells were subjected to 45 degrees C hyperthermia, DNA lesions were detected by the use of the alkaline unwinding/hydroxylapatite method. The number of lesions formed was not affected when the cells were made thermotolerant by either an acute (15 min 44 degrees C + 5 h 37 degrees C) or a chronic (5 h 42 degrees C) pretreatment before 45 degrees C hyperthermia. The presence of 10 mM procaine (heat sensitizer) or 0.5 M erythritol (heat protector) during hyperthermia also had no effect on the rate of formation of heat-induced alkali labile DNA lesions. These observations do not support a concept where DNA lesions are considered to be the ultimate cause of hyperthermic cell killing. Both drugs, however, influenced the rate of repair of radiation-induced strand breaks when present during preirradiation heat treatment. We conclude that the initial number of heat-induced alkali labile DNA lesions is not directly related to cell survival. It cannot be excluded, however, that differences in posthyperthermic repair of these lesions may lead to a positive correlation between residual DNA damage and survival after the different experimental conditions.     

Translocation of MRE11 from the nucleus to the cytoplasm as a mechanism of radiosensitization by heat.

Zhu, W-G., Seno, J. D., Beck, B. D. and Dynlacht, J. R. Translocation of MRE11 from the Nucleus to the Cytoplasm as a Mechanism of Radiosensitization by Heat. Radiat. Res. 156, 95-102 (2001).Hyperthermia sensitizes mammalian cells to ionizing radiation, presumably by inhibiting the repair of radiation-induced double-strand breaks (DSBs). However, the mechanism by which heat inhibits DSB repair is unclear. The nuclear protein MRE11 is a component of a multi-protein complex involved in nonhomologous end joining (NHEJ) of radiation-induced DSBs. Using one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting, we found that MRE11 is translocated from the nucleus to the cytoplasm when human U-1 melanoma or HeLa cells are heated for 15 min at 45.5 degrees C or when cells are heated after irradiation with 12 Gy of X rays. No such translocation is observed in unheated irradiated cells. The kinetics of migration of MRE11 to the cytoplasm was dependent upon whether the heated cells were irradiated, while the magnitude of redistribution of MRE11 was dependent upon post-treatment incubation time at 37 degrees C. Cytoplasmic MRE11 content reached a maximum 2-4 h after heating; the increase was not due to new protein synthesis. Partial recovery of nuclear MRE11 content was observed when heated cells or heated irradiated cells were incubated for up to 7 h at 37 degrees C after treatment. Western blotting results showing translocation of MRE11 from the nucleus to the cytoplasm after heating and irradiation were confirmed using confocal microscopy and immunofluorescence staining of fixed cells. Our data suggest that radiosensitization by heat may be caused, at least in part, by translocation of the DNA repair protein MRE11 from the nucleus to the cytoplasm.     

Variable interaction of heat and procaine in potentiation of radiation lethality in mammalian cells of neoplastic origin

Mild hyperthermia in conjunction with procaine HCl acts as a potentiator of radiation lethality in HeLa cells, with little toxicity for unirradiated cells. The majority of irradiated cells responds extensively within a four hour period of treatment with the two agents. Potentiation of radiation lethality by the combined treatment was also found in a line of human melanoma cells, and to a lesser extent in a line of human ovarian carcinoma cells. The interaction of heat and procaine in the process of potentiation of radiation lethality was assessed from a series of radiation survival curves, with temperatures ranging from 37 to 42 degrees C and procaine concentrations from 1 to 3 mM. The interactive factor was obtained from the ratio of the Dose Reduction Factor (DRF) due to procaine in heated cells, to the DRF due to procaine in unheated cells; a ratio larger than unity denotes interaction of heat and procaine. The largest interaction was observed when individual agents exerted only a minimal radiopotentiating effect, as if increased effectiveness of one agent pre-empted the effectiveness of the other agent.     

Effect of hyperthermia on DNA loop-size in HeLa S3 cells

Nuclear matrices of heated and non-heated HeLa S3 cells were isolated and average DNA loop-sizes were compared. Heat treatment (30 min at 45 degrees C) resulted in an ultimate survival level of the cells of about 10 per cent. The loop-size determinations were done on nuclear material isolated from the cells directly after heat treatment. In the nuclear matrices isolated from the heated cells about 1.8 times more protein was bound as compared to the matrices from control cells. Enzymatic analysis using DNase I digestion, followed by centrifugation on neutral sucrose gradients, was performed. Also, halo visualization was combined with autoradiography. Both methods revealed no gross alterations in DNA loop-sizes. The possible function of DNA loop organization in the effect of hyperthermic interference with DNA-related processes is discussed.     

Heat shock (45 degrees C) results in an increase of nuclear matrix protein mass in HeLa cells

The nuclear matrix from HeLa cells heated at 45 degrees C was isolated to determine the effect of thermal shock on its composition and structure. The matrix from unheated cells contained about 10 per cent of total cell protein and was observed to be spherical particle with a diameter ranging from 3 to 5 microns with the major constituent polypeptides having molecular weights of 45, 47, 55, 57, 59 and 65 kilodaltons. The nuclear-matrix protein mass increased linearly with increasing exposure time at 45 degrees C with no observable change in its size or shape. The additional proteins were observed in general to have molecular weights greater than 45 kilodaltons, with marked increases in polypeptides of 28.5, 38.5, 60, 66, 75, 81, 88, 100 and 115 kilodaltons. An exponential relationship was observed between heat-induced cytotoxicity and the nuclear matrix protein mass increase. A 15 per cent increase in matrix protein mass was sustained prior to the onset of cytotoxicity, while a 35 per cent increase in matrix protein content was associated with a 63 per cent probability of cell killing. The results indicate that redistribution of cell protein or alterations in the mass or structure of the nuclear matrix may be involved in heat-induced cytotoxicity.     

Heat-induced alterations in DNA polymerase activity of HeLa cells and of isolated nuclei. Relation to cell survival

The activity of DNA polymerase alpha and beta was assayed in heated HeLa S3 cells as well as in nuclei isolated from these cells. The enzyme activity as measured in cells and in nuclei has been compared with the extent of cell survival after the different hyperthermic doses. It was found that although the activity of the cellular DNA polymerases was related to cell survival after single heat doses, no correlation was found when thermotolerant cells were heated. When the activity of the DNA polymerases was determined in nuclei isolated from non-heated and heated cells, more polymerase activity was found in the nuclei of the heated cells. However, the heat sensitivity of DNA polymerase activity was the same for nuclei isolated from control, pre-heated and thermotolerant cells. Heat protection of polymerase activity by erythritol and sensitization by procaine was found when cells, but not when nuclei, were heated in the presence of these modifiers. It is concluded that (the nuclear bound) DNA polymerases are not to be considered as key enzymes in cellular heat sensitivity of HeLa S3 cells.     

Effects of procaine on the intracellular pH of Chinese hamster ovary cells heated at 42.0 or 45.0 degrees C

The local anesthetic procaine greatly sensitizes cells to hyperthermia. Though it is generally accepted that procaine is a membrane-active agent that increases membrane fluidity in cells, the mechanism by which it potentiates heat killing is unknown. In this paper we report changes in intracellular pH (pHi) of Chinese hamster ovary (CHO) cells heated at 42.0 or 45.0 degrees C in the presence of procaine. The pHi was measured with flow cytometry using the dye 1,4-diacetoxy-2,3-dicyanobenzene (ADB). Studies were carried out using cells grown at normal pH (7.3) or cells placed in low-pH (6.6) medium 4 h prior to and during heating (acute low-pH treatment). Low-pH-adapted cells (PHV2), which were obtained previously by continuous culture in pH 6.6 medium, were also used. Normal cells heated in the presence of procaine at pH 7.3 underwent a large decrease in pHi compared to cells heated without procaine. Procaine had little additional effect on the intracellular pH of cells in medium with a pH of 6.6 for 4 h before and during 30 min of heating. PHV2 cells exposed to chronic low-pH conditions were resistant to acidification when heated with or without procaine. The surviving fraction of cells heated with procaine was significantly lower under all pH conditions than that of cells heated without procaine. Cells heated at 42.0 degrees C with procaine also became greatly acidified and their survival was reduced. These data suggest that the reduction in pHi caused by procaine may be part of the mechanism of heat sensitization, but cannot account for it entirely. Furthermore, the degree of procaine sensitization and intracellular acidification is dependent on the extracellular pH, with a larger effect occurring at pH 7.3 than at pH 6.6.     

Characterization of a 42 degrees C-specific heat shock protein of mammalian cells

HeLa cells synthesize a particular heat shock protein that is induced only by heat shock at 42 degrees C, and not at 45 degrees C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957-963). We further characterized the 42 degrees C-specific protein. This protein was induced in mouse FM 3A cells as well as in human HeLa cells. In both cell lines, the protein was resolved into two spots, a basic polypeptide and an acidc one. The mRNA of the protein was induced during the incubation of these cells at 42 degrees C, and the in vitro translation product of mRNA corresponded to the basic, not to the acidic, polypeptide. During the chase period for cells that were labeled with [35S]-methionine, the basic polypeptide of the protein decreased, and the acidic one increased, indicating that the protein was synthesized as the basic polypeptide and then somehow modified to become the acidic one. The 42 degrees C-specific protein was found only in the cytosol fraction, and not in the nuclear or other particulate fractions, in both HeLa and FM 3A cells. The results suggested that the 42 degrees C-specific protein may have some function in the cytoplasm of mammalian cells during mild heat shock.     

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 degrees C and 45 degrees C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 degrees C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells (45 degrees C for 15 min) were incubated at 37 degrees C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 degrees C (step-down heating; SDH) a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks.     

13C-NMR and spectrophotometric studies of alcohol-lipid interactions

The interactions of butanol and mixtures of butanol and ethanol with dipalmitoylphosphatidyl choline (DPPC) liposomes have been investigated by both spectrophotometric measurements and Fourier transform 13C nuclear magnetic resonance spectroscopy. The spectrophotometric experiments indicate that butanol exhibits the same effects on the thermotropic properties of DPPC as the other short chain alcohols, methanol, ethanol and propanol, which have been shown to be characteristic of the alcohol induced transition of the lipid to the interdigitated state. An additive effect of butanol and ethanol on the induction of the interdigitated phase in DPPC was also observed. A decrease in line width and increase in T1 of the choline methyl signal were observed in the 13C-NMR experiments conducted at 32 degrees C when butanol was added to DPPC in increasing amounts suggesting an increase of disorder in the head group region of the lipid. Addition of ethanol to the NMR sample containing butanol produced hysteresis in the heating and cooling curves characteristic of the interdigitated state. In the interdigitated state, the choline methyl signal exhibited a T1 value equal to that when the lipid is in the fluid state. The increase of mobility in the head group region in the interdigitated gel state relative to the bilayer gel can be rationalized by the increase in surface area in that site when the lipid interdigitates.     

Nuclear protein redistribution in heat-shocked cells

An increase was observed in the total protein mass of nuclei isolated from Chinese hamster ovary cells heated at 45°C or 45.5°C. An increase in the fractional recovery of DNA polymerase α and β, and of DNA topoisomerase activity coincided with this increase in the protein mass of nuclei from heated cells. Nuclear protein mass which was soluble in 2.0 M NaCl decreased 0.5 fold, while DNA-associated and nuclear matrix-associated protein mass increased 2.2 and 3.4 fold, respectively. The results indicate that the increase in nuclear protein mass observed in nuclei from heated cells is due in part to an increased binding, or precipitation, of nuclear proteins onto the cell's DNA and nuclear matrix. © 1993 Wiley-Liss, Inc.     

The cell cycle dependence of thermotolerance. III. HeLa cells heated at 45.0 degrees C

We have extended our studies on the cell cycle dependence of thermotolerance to include HeLa cells heated at 45.0 degrees C to compare the results to Chinese hamster ovary (CHO) cells. We found that asynchronous HeLa cells were more resistant to heat than CHO cells but showed a similar development and decay of thermotolerance. Flow cytometry (FCM) was used to study redistributions in the cell cycle after an initial heat dose. Cells heated for 35 min at 45.0 degrees C were delayed in G1 by about 7 h compared to controls, with delays in late S and G2/M phase also. The heat sensitivity varied through the cell cycle; G1 cells were the most resistant to heat, while S-phase cells were uniformly sensitive throughout S phase, and G2 cells were resistant. Thermotolerance could be induced and expressed in early or late S-phase cells, but to a lesser extent than for G1 cells. The results were similar in many respects to CHO cells, but there were significant differences.     

Hyperthermia blocks DNA processing at the nuclear matrix

The capacity of control and heated HeLa cells to process newly polymerized DNA at the nuclear matrix was measured. DNA which had been pulse-labeled with [3H]thymidine was enriched by a factor of up to 6 at the cell's nuclear matrix. During continuous exposure to [3H]thymidine at 37 degrees C this enrichment for pulse-labeled DNA was reversed with a half-time of 7 min. We interpret this processing of newly replicated DNA to be a distribution of newly polymerized DNA throughout replicon-sized nuclear DNA domains. Both processing of newly polymerized DNA at the nuclear matrix and ligation of replicon clusters into the interphase cell chromosome were halted by incubation of cells at temperatures at or above 43 degrees C. When HeLa cells were pulse-labeled during a 30-min incubation at 45 degrees C and replaced at 37 degrees C, the enrichment for 3H-labeled DNA at the nuclear matrix was reversed with an initial half-time of 4 h. The results indicate that exposure of cells to hyperthermic temperatures blocks ongoing nascent DNA processing at the nuclear matrix and results in a retardation of DNA processing in preheated cells replaced at 37 degrees C.     

Temperature-dependent induction of thermotolerance by ethanol

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.     

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.     

Localization and quantitation of hsp84 in mammalian cells

In order to investigate the function of heat shock protein 84 (hsp84) we have isolated the protein from mouse neuroblastoma cells and raised a polyclonal antiserum which was affinity-purified. The specificity of the antibody was established by immunoprecipitation and immunoblotting. Immunofluorescence studies revealed both a cytoplasmic and a nuclear localization of hsp84 in five different mammalian cell lines (mouse neuroblastoma cells and fibroblasts, rat hepatoma cells, and HeLa cells). In none of the five cell lines were found significant differences in the total cellular levels of hsp84 before and immediately after a heat shock (4 h, 42 degrees C) by immunoblot quantification. Furthermore after heat shock the fluorescence of anti-hsp84-labeled nuclei was increased relative to that of the surrounding cytoplasm. The increased fluorescence disappeared upon reincubation at 37 degrees C. The heat-induced increase in contrast between cytoplasmic and nuclear fluorescence could be explained by a combination of three factors: (a) decrease in nuclear projection area, (b) increase in cytoplasmic projection area, and (c) translocation of hsp84. The contribution of these factors to the increase after heat treatment was different for the cell lines.     

Effects of sodium butyrate and dibutyryl cyclic AMP on thermosensitivity of HeLa cells and their production of heat shock proteins

When HeLa cells were incubated at 42 degrees C for 6 h with 1 mM sodium butyrate or when cells treated with 1 mM dibutyryl cyclic AMP for 24 h were incubated at 42 degrees C for 6 h, they were more thermoresistant than heated control cells without such drugs. The production of heat shock proteins was not enhanced by the drug treatment. These results suggest that there is a factor (or factors) other than heat shock proteins that accounts for the thermoresistance of HeLa cells.     

Thermodynamic behaviour and conformational changes of alcohol oxidase of yeasts Hansenula polymorpha

We studied influence of heating, ethanol and sodium azide on the structural and conformational changes of the alcohol oxidase from yeast Hansenula polymorpha. The increase of fluorescence of alcohol oxidase -cofactor, flavin adenine dinucleotide, was revealed when heated to 60 degrees C while the enzymatic activity of alcohol oxidase remained unchanged. Differential scanning microcalorimetry revealed that ethanol stabilized the protein structure and increased the temperature of melting, Based on the data of circular dichroism, we concluded that the percentage of helicities in the secondary structure of alcohol oxidase was not influenced by both ethanol and sodium azide, however ethanol significantly modified the circular dichroism spectrum associated with the tertiary structure of alcohol oxidase.     

Inactivation of splicing factors in HeLa cells subjected to heat shock

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.