Initial characterization of heat-induced excess nuclear proteins in HeLa cells.

Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two-dimensional gel electrophoresis. The abundance of cytoskeletal elements which co-purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non-heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat-induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus.     

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with the non-ionic detergent Nonidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaCl-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaCl from the DNA-cellulose column, P5 and P5′ are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaCl eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaCl, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.     

Mechanism(s) of heat killing: accumulation of nascent polypeptides in the nucleus?

To investigate the possibility that nascent polypeptides released from polysomes by heat shock accumulate in the nucleus, cells were pulse labeled with [35S]methionine for two minutes and heated immediately thereafter at 45.5 degrees C for 10 minutes. When isolated nuclei were subjected to gel electrophoresis and subsequently autoradiographed, heated nuclei exhibited an approximately 10-fold increase in radioactive polypeptides in comparison to nonheated controls. These nascent polypeptides were nonspecific molecules covering a wide range of molecular weights. It is plausible that the accumulation of polypeptides in the nucleus results in hyperthermic cytotoxicity. Therefore, we propose that a potential target for heat killing is within the nucleus, at sites where nascent polypeptides accumulate after heat shock.     

Protein cross-migration during isolation of nuclei from mixtures of heated and unheated HeLa cells

To investigate the cross-migration of proteins during nuclear isolation heated and control cells were mixed prior to nuclear isolation. These nuclei were stained with fluorescein isothiocyanate (FITC, a protein-specific stain) and with propidium iodide (PI, a DNA-specific stain). Flow cytometric (FCM) analysis showed two populations distinguishable on the basis of protein content. The protein content of the nuclei in the upper population was identical to that for nuclei isolated from heated cells while that for the lower population had a protein content identical to the protein content of nuclei from control cells. This result shows that the heat-induced increase in nuclear protein content occurred throughout the entire population of nuclei (i.e., in G1, S, and G2 nuclei) and that the measured protein content of nuclei was not affected by the presence of the other population during isolation. The capability of the FCM to sort subpopulations from different regions of a histogram was used to separate the subpopulations after analysis. When control cells were prelabeled with [3H]leucine and mixed with unlabeled heated cells, 11% of the radioactivity was found to be associated with the nuclei from heated cells. Autoradiographs showed grains over approximately 99% of the nuclei from heated cells. When [3H]TdR was used as a label in a similar experiment, only 0-3% of the label was observed to become associated with the population of nuclei from heated cells and autoradiography showed that 97% of these nuclei were not labeled. Comparable results were obtained when the labeled cells were heated and the control cells were left unlabeled. These results show that a small amount of protein (approximately 10% of the nuclear protein) will cross-migrate during nuclear isolation without affecting the net amount of protein in either population.     

Content of nonhistone protein in nuclei after hyperthermic treatment

When nuclei were isolated from Chinese hamster ovary cells after being heated, there was a large increase in the amount of ³H-tryptophan labeled nonhistone protein in the nucleus relative to the whole cell. After 15 min or 30 min of heating at 45.5°C, the nuclear nonhistone protein content increased by 1.6 or 1.8, respectively. In contrast, when the nuclear nonhistone protein content was determined in the intact cell by using autoradiography to quantify ³H-tryptophan labeled protein in the nucleus and cytoplasm in sections of fixed cells, the nuclear nonhistone protein content increased by only 1.14 or 1.28 for 15 or 30 min at 45.5°C, respectively. Therefore, heat does not induce a massive movement of cytoplasmic protein into the nucleus. © 1993 Wiley-Liss, Inc.     

Proteins in nucleocytoplasmic interactions. 3. Redistributions of nuclear proteins during and following mitosis in Amoeba proteus

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.     

Proteins in nucleocytoplasmic interactions. I. The fundamental characteristics of the rapidly migrating proteins and the slow turnover proteins of the Amoeba proteus nucleus

By the transplantation of amino acid-³H-labeled nuclei between cells and the subsequent isolation of nuclei for quantitative assay, we have confirmed that all the nuclear proteins of Amoeba proteus are divisible into two classes that are sharply defined by their physiological behavior. About 40% of the proteins in the nucleus rapidly migrates back and forth between the nucleus and the cytoplasm. These rapidly migrating proteins (RMP) are 25–50 times more concentrated in the nucleus than in the cytoplasm, and migration into the nucleus therefore occurs against a high concentration differential. The remaining 60% of nuclear proteins has been classified as slow turnover proteins (STP) since (as reported in a following paper) virtually all of them ultimately undergo turnover. Turnover in this context means loss of label from the nucleus, by either protein breakdown or protein migration to the cytoplasm. Isolation of nuclei in the detergent Triton X-100 results in a 20% loss of nuclear proteins but conclusions about RMP and STP were not found to be significantly affected by this loss.     

Quercetin suppresses heat shock-induced nuclear translocation of Hsp72

The effect of quercetin and heat shock on the Hsp72 level and distribution in HeLa cells was studied by Western blotting, indirect immunofluorescence and immunogold electron microscopy. In control cells and after quercetin treatment, Hsp72 was located both in the cytoplasm and in the nucleus in comparable amounts. After hyperthermia, the level of nuclear Hsp72 raised dramatically. Expression of Hsp72 in cytoplasm was also higher but not to such extent as that observed in the nucleus. Preincubation of heated cells with quercetin inhibited strong Hsp72 expression observed after hyperthermia and changed the intracellular Hsp72 distribution. The cytoplasmic level of protein exceeded the nuclear one, especially around the nucleus, where the coat of Hsp72 was noticed. Observations indicating that quercetin was present around and in the nuclear envelope suggested an involvement of this drug in the inhibition of nuclear translocation. Our results indicate that pro-apoptotic activity of quercetin may be correlated not only with the inhibition of Hsp72 expression but also with suppression of its migration to the nucleus.     

Nuclear exchange of the U1 and U2 snRNP-specific proteins

The snRNP particles include a set of common core snRNP proteins and snRNP specific proteins. In rodent cells the common core proteins are the B, D, D', E, F and G proteins in a suggested stoichiometry of B2D'2D2EFG. The additional U1- and U2-specific proteins are the 70-kD, A and C proteins and the A' and B" proteins, respectively. Previous cell fractionation and kinetic analysis demonstrated the snRNP core proteins are stored in the cytoplasm in large partially assembled snRNA-free intermediates that assemble with newly synthesized snRNAs during their transient appearance in the cytoplasm (Sauterer, R. A., R. J. Feeney, and G. W. Zieve. 1988. Exp. Cell Res. 176:344-359). This report investigates the assembly and intracellular distribution of the U1 and U2 snRNP-specific proteins. Cell enucleation and aqueous cell fractionation are used to prepare nuclear and cytoplasmic fractions and the U1- and U2-specific proteins are identified by isotopic labeling and immunoprecipitation or by immunoblotting with specific autoimmune antisera. The A, C, and A' proteins are found both assembled into mature nuclear snRNP particles and in unassembled pools in the nucleus that exchange with the assembled snRNP particles. The unassembled proteins leak from isolated nuclei prepared by detergent extraction. The unassembled A' protein sediments at 4S-6S in structures that may be multimers. The 70-kD and B" proteins are fully assembled with snRNP particles which do not leak from isolated nuclei. The kinetic studies suggest that the B" protein assembles with the U2 particle in the cytoplasm before it enters the nucleus.     

Vesicular stomatitis virus M protein in the nuclei of infected cells.

The M protein of vesicular stomatitis virus (VSV) was localized in the nuclei and cytoplasm of VSV-infected cells by subcellular fractionation and immunofluorescence microscopy. Nuclei isolated from VSV-infected Friend erythroleukemia cells were fractionated into a nuclear membrane and a nucleoplasm fraction by DNase digestion and differential centrifugation. G protein was present in the membrane fraction, and M protein was present in the nucleoplasm fraction. Immunofluorescence detection of M protein in the nucleus required that fixed cells be permeabilized with higher concentrations of detergent than were required for detection of M protein in the cytoplasm of VSV-infected BHK cells.     

Isolation of nuclei from mature peritoneal granulocytes

Nuclei from mature neutrophil granulocytes were prepared from peritonal exudates of goats. Fluorescein mercuric acetate was required to stabilize the nuclei and fix nucleoproteins. Following differential centrifugation and detergent treatment, electron microscopy showed the interlobar region to be free of cytoplasmic tabs. All of the DNA of the cell was recovered in the nucleus and 71% of the RNA. The DNA : RNA was 6 : 1 in the intact cells, and 9 : 1 in the isolated nuclei. Protein:DNA was 11 : 1 and 4 : 1 for cells and nuclei, respectively. Representative fractions of histones and tryptophan-rich acidic proteins were prepared with (asp + glu) : (lys + arg + hist) values averaging 0.7 and 1.4 respectively. Histones accounted for 30% of the nuclear proteins while the residual proteins contained the bulk of the cystinyl residues. Granulocytes were characterized by high glycine titers, from 8 to 18% of the nuclear proteins, and 70% of the total free amino acids of the cell.     

Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock

Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.     

The nuclear matrix is a thermolabile cellular structure

Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48 degrees C and 55 degrees C with an onset temperature of approximately 40 degrees C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48 degrees C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48 degrees C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46 degrees C) and thermal radiosensitization (Tm = 47 degrees C), suggesting that denaturation of nuclear matrix proteins with Tm = 48 degrees C contribute to these forms of nuclear damage. Following heating at 43 degrees C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei Isolated from heat-shocked cells and is a form of indirect thermal damage.     

Distribution of proteins between nucleus and cytoplasm of amoeba proteus

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but—because the cytoplasm is 50 times larger than the nucleus—about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained. Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm. New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic “contamination” of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments. We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm’s large volume ensures that CP will be abundant there. Extending Bonner’s idea of “quasi-functional nuclear binding sites” for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.     

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.     

THE CYTONUCLEOPROTEINS OF AMEBAE : I. Some Chemical Properties and Intracellular Distribution

Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.     

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.     

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.     

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.     

Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: Multiple pathways to induce tolerance?

Recent data indicate that cells may acquire thermotolerance via more than one route. In this study, we observed differences in thermotolerance development in HeLa S3 cells induced by prior heating (15 minutes at 44 degrees C) or pretreatment with sodium-arsenite (1 hour at 37 degrees C, 100 microM). Inhibition of overall protein and heat shock protein (HSP) synthesis (greater than 95%) by cycloheximide (25 micrograms/ml) during tolerance development nearly completely abolished thermotolerance induced by arsenite, while significant levels of heat-induced thermotolerance were still apparent. The same dependence of protein synthesis was found for resistance against sodium-arsenite toxicity. Toxic heat, but not toxic arsenite treatments caused heat damage in the cell nucleus, measured as an increase in the protein mass of nuclei isolated from treated cells (intranuclear protein aggregation). Recovery from this intranuclear protein aggregation was observed during post-heat incubations of the cells at 37 degrees C. The rate of recovery was faster in heat-induced tolerant cells than in nontolerant cells. Arsenite-induced tolerant cells did not show an enhanced rate of recovery from the heat-induced intranuclear protein aggregation. In parallel, hyperthermic inhibition of RNA synthesis was the same in tolerant and nontolerant cells, whereas post-heat recovery was enhanced in heat-induced, but not arsenite-induced thermotolerant cells. The more rapid recovery from heat damage in the nucleus (protein aggregation and RNA synthesis) in cells made tolerant by a prior heat treatment seemed related to the ability of heat (but not arsenite) to induce HSP translocations to the nucleus.