(CANCER RESEARCH 49, 1254-1260. March 1. 1989] Effects of Hyperthermia on Chromatin Condensation and Nucleoli Disintegration as Visualized by Induction of Premature Chromosome Condensation in Interphase Mammalian Cells1
George E. Iliakis and Gabriel E. Pantelias2
Thomas Jefferson University Hospital, Department of Radiation Oncology and Nuclear Medicine, Philadelphia, Pennsylvania 19107 [G. E. I., G. E. P.]; and the National Research Center for Physical Sciences "Demokritos", Aghia Paraskevi Attikis, Athens, Greece [G. E. P.]
ABSTRACT nuclei (13), in chromatin (14-17), and in nuclear matrices (18, 19), and it was proposed that disruption of important nuclear The effects of hyperthermia on chromatin condensation and nucleoli processes by this nuclear protein binding may be the reason for disintegration, as visualized by induction of premature chromosome con cell killing (17). Beyond cell killing the excess nuclear proteins densation in interphase mammalian cells, was studied in exponentially have been implicated in the inhibition of DNA synthesis (5, 6) growing and plateau phase Chinese hamster ovary cells. Exposure to heat reduced the ability of interphase chromatin to condense and the and the inhibition of DNA repair following both ionizing (20, ability of the nucleolar organizing region to disintegrate under the influ 21) and uv (22) irradiation. It is thought that inhibition of ence of factors provided by mitotic cells when fused to interphase cells. these cellular functions may be due to alterations induced in Based on these effects treated cells were classified in three categories. chromatin conformation and in particular to restriction of DNA Category 1 contained cells able to condense their chromatin and disinte supercoiling changes as a result of protein addition to the grate the nucleolar organizing region. Category 2 contained cells able to nuclear matrix (19). These results stress the possible importance only partly condense their chromatin and unable to disintegrate the of chromatin as the principal site of heat-induced damage that nucleolar organizing region. Category 3 contained cells unable to condense ultimately leads to cell death. their chromatin and unable to disintegrate the nucleolar organizing region. Integrity and structural alterations in chromatin can also be The fraction of cells with nondisintegrated nucleoli increased with in studied by utilizing the technique of premature chromosome creasing exposure time at 45.5 < and reached a plateau at almost 100% condensation (23-26). Chromatin of a cell can ordinarily be after about 20 min. Exponentially growing and plateau phase cells showed similar response. Recovery from the effects of heat on chromatin conden visualized as distinct chromosomes only when the cell enters sation and disintegration of the nucleolar organizing region depended mitosis. However, when mitotic cells are fused with cells in upon the duration of the heat treatment. For exposures up to 15 min at interphase by means either of Sendai virus or of PEG,3 disin 45.5°C,agradual reduction in the fraction of cells with nondisintegrated tegration of the nuclear membrane and other nuclear structures nucleoli was observed when cells were allowed for repair at 37°C. takes place. In addition, interphase chromatin undergoes a However, only a very limited amount of repair was observed after a 30- min exposure to 45.5°C.The repair times observed at the chromosome condensation process taking morphology that is characteristic of the cell cycle phase of the interphase cell at the time of level were similar to those reported for the removal of excess protein fusion. This phenomenon is referred to as premature chromo accumulating in chromatin or the nuclear matrix, suggesting a causal some condensation (PCC) and the induced chromosome units relationship between the two phenomena. It is proposed that nuclear protein accumulation on chromatin or in the nuclear matrix reduces the as prematurely condensed chromosomes (also abbreviated as accessibility of chromatin to enzymes responsible for the phosphor) l:itinn PCC, the context clarifies the way in which the abbreviation is reactions necessary for chromatin condensation and disintegration of the used). The process of premature chromosome condensation can nucleolus. be thought of as a prophasing reaction (27). The factors from the mitotic cell responsible for the prophasing reaction are most likely heat and Ca2+ sensitive, Mg2+ dependent, proteins of INTRODUCTION relatively high molecular weight that bind preferentially to chromatin (28-33). The recent consideration of hyperthermia as an adjuvant to In this paper we report on the effects of heat (45.5 and 43°C) radiation therapy (1) and the possibility of probing mechanisms of gene expression using the synthesis of heat shock proteins on chromatin morphology and nuclear organization, as visual as a model system (2) has drawn much attention to the effects ized by PCC, in exponentially growing and plateau phase CHO of heat on living systems. To fully utilize the potential of cells. Selected results obtained with HeLa cells are also pre hyperthermia, or hyperthermia in conjunction with other mo sented and discussed. The results obtained indicate a dramatic dalities in the above-mentioned goals, it is necessary to deter effect of heat on the ability of chromatin to condense and on mine the molecular alterations that it induces as well as the the ability of the nucleolus to disintegrate after induction of lesions that lead to cell death. PCC. This is, to the best of our knowledge, the first report on Hyperthermia has been shown to induce a number of effects this topic. in mammalian cells including inhibition of DNA, RNA, and protein synthesis (3-6), induction of chromosomal aberrations MATERIALS AND METHODS (7, 8), and in ultrastructural studies, disappearance of the intranucleolar chromatin and the granular ribonucleoprotein Cell Culture and Heating. Experiments were performed using Chinese components of the nucleolus (9-12). Furthermore, hyperther hamster ovary cells, strain 10B, grown as monolayers in McCoy's 5A mia was found to increase the protein to DNA ratio in isolated medium supplemented with 10% fetal calf serum and antibiotics (50 units/ml potassium penicillin (i,, 50 Mg/ml streptomycin sulfate). Cells Received 8/22/88; revised 11/28/88; accepted 12/1/88. were grown at 37°Cina humidified incubator, in an atmosphere of 5% The costs of publication of this article were defrayed in part by the payment COj and 95% air. Stock cultures were maintained in vitro by routinely of page charges. This article must therefore be hereby marked advertisement in subculturing (every second day) at an initial density of 10'' cells in 75- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by NCI Grants 1R01 CA45557 and 1R01 CA42026 awarded by the NIH. DHHS. 3The abbreviations used are: PEG, polyethylene glycol; PCC, premature 2Supported by Grant B16-E-206-GR awarded by the Commission of the chromosome condensation or prematurely condensed chromosome; CHO, European Communities, Radiation Protection Program. Chinese hamster ovary; PBS, phosphate buffered saline. 1254 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF HYPERTHERMIA ON INTERPHASE CHROMATIN cm2 tissue culture flasks. After 2 days of growth, cells in these cultures as required by the particular experimental protocol and processed for had reached a density of IO7per flask, and were used to prepare cultures fusion. Slides prepared were allowed to dry overnight at room temper for experiments. For this purpose, 10s cells, suspended in 3-ml growth ature and were subsequently immersed in photographic emulsion (II- medium, were plated in 25-cm2 tissue culture flasks and were allowed ford), after dilution with water (2:3, emulsion:water). Slides were dried to grow for 2 or 4 days in order to obtain exponentially growing or in a slight draft and placed in light tight boxes at 4°Cfor 2-5 days. plateau phase cells, respectively. Exponentially growing cultures had a After this period of time slides were developed and analyzed. density of 1-1.3 x IO6cells/flask and cultures in the plateau phase a density of 6-7 x IO6cells/flask. Flow cytometry measurements showed that exponentially growing cells contained about 45 ±5% cells in Gì RESULTS phase, 45 ±5% cells in S phase and 10 ±5% cells in G2 + M phase. In plateau phase cultures more than 90% (usually between 92 and 95%) Effect of Heat on the Process of Premature Chromosome of the cells were found at a stage in the cell cycle showing a DNA Condensation. Morphological changes induced by heat in chro- content equivalent to that of d cells, with the remaining cells found in matin and the chromosomes, as visualized in interphase cells S (3-5%) and in G2 (4-7%) phases. Cells were checked and found free using the technique of premature chromosome condensation, of mycoplasma contamination. To establish the general validity of the were investigated. Plateau phase cells were used first for these results obtained, experiments were also carried out with I lei .a cells experiments because they mainly contain Gìcells that show (kindly provided by Dr. Roti Roti, Washington University, St. Louis, complete chromatin condensation into discrete chromosomes MO). Cells were grown as monolayers in Joklik-modifled minimum after fusion with mitotic cells, thus simplifying analysis. How essential medium plus 3.5% each of fetal bovine and calf sera (16). ever, similar results were subsequently obtained with cells in Exponentially growing cultures were prepared by incubating for 2 days 3x10* cells in 5 ml of growth medium in 25-cm2 tissue culture flasks. every phase of the cycle (using exponentially growing cultures). Exponentially growing cells were heated in their growth medium Fig. 1 illustrates the chromosome condensation achieved after (pH 7.3-7.6) and plateau phase cells in fresh McCoy's 5A (without fusion and 1-h incubation at 37°C,of an untreated (nonheated) serum) to reduce pH related effects (pH of spent medium from plateau interphase (d) CHO cell with a cell at mitosis. phase cultures 6.8-7.0, pH of fresh medium 7.4-7.6). Heating was Severe morphological changes became apparent after heat. carried out in waterbaths regulated at the desired temperature with an Fig. 2 shows chromatin condensation and morphology as ob accuracy of ±0.05°C.Temperature was monitored with a calibrated served in a fraction of cells from a plateau phase population (against NBS standardized thermometer) mercury thermometer. After heated for 10 min at 45.5°Cand fused for PCC induction with heating, cells were either processed immediately for fusion and induc mitotic cells immediately thereafter. Following exposure to heat tion of premature chromosome condensation, or, they were returned to chromatin partly lost its ability to condense into distinct chro the incubator after reestablishing preirradiation conditions (fresh full mosomes. Despite the fact that chromosome condensation oc growth medium for growing cells and spent medium for plateau phase curred to a certain extent and chromatin fibers were visible, the cells). For trypsinization, cells were washed once with phosphate buff ered saline and were incubated at 37°Cfor 5 min with 0.25% trypsin process did not arrive to the degree of completion observed in nonheated cells (compare with Fig. 1). This observation sug- plus 0.1% EDTA. Cells were counted in a particle counter (Coulter Electronics), and appropriate numbers were inoculated into 15-ml dilution tubes for further processing. l'I (,-mediated Cell Fusion and Preparation of Slides. The procedure developed for polyethylene glycol mediated cell fusion and premature chromosome condensation induction has been described in detail else where (26, 34). Briefly, IO6 mitotic CHO cells obtained by 1-2-h incubation of cell monolayers in the presence of 0.04 Mg/ml nocoda- zole, methyl[5-(2-thienylcarbonyl)-1 H-benzimidazol-2-yl] carbamate (Sigma), were mixed with an equal number of interphase cells in a 15- ml round-bottom culture tube. Alternatively, cells selected in this way but kept frozen as described by Borrelli et al. (35) were used at this stage. There was no difference in the yield of fusion between freshly prepared and frozen mitotic cells. After centrifugation at 200 x g for 5 min the supernatant was discarded, and the pellet resuspended in 0.10 ml of 40% ice-cold PEG solution (M, 1450, Sigma, prepared in PBS, 40% wt/v). The pellet was held in this solution for 1 min and subse quently 2 ml of PBS was slowly added while the tube was gently shaken. The cell suspension was centrifuged (200 x g for 5 min) and the supernatant discarded. The tube was blotted and the pellet resuspended in 0.7 ml of McCoy's 5A growth medium. Finally, 0.07 ml of colcemid was added to the suspension from a stock solution prepared at a concentration of 10 iAI, and the tube was placed in the incubator at 37°Cfor 60 min. In that time, cell fusion and induction of PCC was completed (see Fig. 1). Subsequently 7 ml of hypotonie (0.075 M) KO solution was added to the fused cells for 5 min at room temperature. Cells were fixed twice in 10-ml methanohacetic acid (3:1) solution, dropped on precleaned wet slides, air dried, and stained with 2% Fig. 1. Fusion of a mitotic with a control (not treated) d phase CHO cell. Giemsa. Before analysis, slides were mounted with cover slips, and cells Notice the disintegration of the nuclear membrane and the nucleolus and the where premature chromosome condensation was induced were scored induction of premature chromosome condensation. Chromatin condenses into by means of light microscopy. The scoring criteria are discussed in the distinct chromosomes at this stage of the cell cycle. 21 chromosomes (the modal next section. Routinely, 25-100 cells were analyzed and scored per chromosome number for this cell line) can be counted both in the interphase as well as in the mitotic cell. The morphological differences between metaphase and experimental datum. At no point during analysis and scoring was there prematurely condensed chromosomes allow for unequivocal evaluation of the ambiguity in the distinction between interphase and mitotic chromo effects of heat on chromatin of interphase cells. Cells able to condense their chromatin and to disintegrate their nucleoli after fusion with mitotics are classified somes. in Category 1. In this and the following photographs the exposure time during Autoradiography. In experiments involving autoradiography, cells development was adjusted to show optimally the prematurely condensed chro were preincubated for 0-30 min with 100 ^Ci/ml [3H]uridine, heated mosomes. 1255 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF HYPERTHERMIA ON INTERPHASE CHROMATIN
Fig. 2. Fusion of a mitotic cell with two heated (45.5'C, 10 min) ¡nterphase Gìcells.The process of fusion induced disintegration of the nuclear membrane Fig. 3. Fusion of a mitotic cell with a heated (4S.5'C, 20 min) interphase cell. in the heated interphase cell and initiated some condensation of chromatin. Individual chromatin fibers can be observed. As a result of heat, chromatin Fusion induced disintegration of the nuclear membrane in the interphase cell but, condensation did not come to completion (compare with Fig. I). In addition, due to heat-induced damage, chromatin was unable to condense. Nuclear bodies condensed chromatin was not free but associated with nuclear bodies staining staining light with Giemsa can still be recognized and are indicated by the arrows. light with Giemsa (arrows). These nuclear bodies were not induced during the Cells unable to condense their chromatin and displaying nuclear bodies after process of PCC as indicated by the fact that they could also be observed in fusion with mitotics are classified in Category 3. nonfused nuclei (indicated by the arrows in the nucleus present in the figure). Cells able to partly condense their chromatin and displaying nuclear bodies after fusion with mitotics are classified in Category 2. of heat at the chromosome level and allowed comparisons of the results obtained with those reported in the literature for different endpoints (see "Discussion"). Of course, heated cells gested that exposure to heat reduced the efficacy of chromatin to respond to signals transmitted by unheated mitotic cells. were not solely distributed in states identical to those described Furthermore, as it can be seen in Fig. 2, the chromosomes were in Figs. 1 to 3. In a heated sample a series of intermediate states could be observed that were assigned during analysis in not free and independent of each other as in the case of the closest category. In general the fraction of cells in Categories nonheated cells, but appeared to be organized in the periphery of a nuclear "body" that stained with Giemsa lighter than the 2 and 3 gradually increased with increasing heating time and Category 3 prevailed after longer heating times. This is illus surrounding chromatin. Light staining nuclear bodies could trated by the results shown in Fig. 4, obtained with plateau also be observed in heated nuclei that had not undergone fusion phase cells. Results of one experiment are shown, but the with mitotic cells (indicated by arrows in the figure) suggesting findings were qualitatively reproduced in several experiments. that their induction was not due to the PCC process. Plotted in the figure is the fraction of cells in the different A more severe effect of heat on chromatin condensation is categories as a function of the heating time at either 43 or shown in Fig. 3. As in the previous examples, nonheated mitotic 45.5°C.The fraction of cells in Category 1 rapidly decreased cells were fused with heated (45.5°Cfor 20 min) interphase with increasing heating time. The fraction of cells in Category (Gì)cells.Disintegration of the nuclear membrane did occur in 2 increased first with increasing heating time, reached a maxi this case as well, but contrary to the previous observations, mum at intermediate heating times and decreased later as chromatin was unable to condense. Nuclear bodies were also Category 3 started dominating. Thus, a continuous transition observed at this stage of chromatin condensation (arrows in the to stages of reduced ability to respond to mitotic signals was figure) although, due to the diffused state of chromatin, not as observed with increasing heating time. Cells in G2 phase re directly as in Fig. 2. sponded after exposure to heat in a way essentially identical to Based on the above observations, and in order to establish that observed in d cells. A similar effect was also observed in criteria for an evaluation of the damage induced by heat at the cells heated in S phase, although the morphology of the PCC chromosome level, cells were classified in three categories. A was somewhat different due to the decondensed state of chro cell was classified in Category 1 if it was found able to disinte matin in this phase of the cell cycle. Results similar to those grate nuclear membrane and nucleolus and form distinct chro reported for CHO cells were also obtained with HeLa cells. mosomes, as demonstrated in Fig. 1. A cell was classified in Fig. 5 shows a d cell from this cell line heated at 45°Cand Category 2 if it was able to condense its chromatin, at least fused with a mitotic CHO cell. Partly condensed chromosomes partly, and showed bodies associated with its chromosome associated in this case with darkly staining (probably due to the complement (as demonstrated in Fig. 2). A cell was classified presence of chromatin) nuclear bodies were observed. Based on in Category 3 if it had lost its ability to condense its chromatin the above discussed criteria this cell can be classified in Category following fusion with mitotic cells and showed bodies associated 2. with its chromosome complement (as demonstrated in Fig. 3). The Nature of the Nuclear Bodies Observed in Heated Cells. The above classification of chromatin condensation state One of the most intriguing aspects of the observations discussed after heat, although arbitrary, enabled evaluation of the effects in the previous paragraphs was the presence of nuclear struc- 1256 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF HYPERTHERMIA ON INTERPHASE CHROMAT1N
120
category 1 category 2 category 3
4 6 8 10 12 14 time at 45.5 °C,min
Fig. 5. PCC of a HeLa interphase cell exposed to 45'C for 10 min. The cell 120 can be classified in Category 2. Notice that in this cell line the nuclear bodies that associatethe chromosomes stain darker than in CHO cells indicating the presence •¿ category1 of chromatin in the structure. D category2 E category3
0 10 20 30 40 50 60 70 80 90 100110120 time at 43 C, min Fig. 4. Distribution of plateau phase CHO cells in Categories 1, 2, and 3 as a function of the exposure time to either 43"C (bottom) or 45.5'C (top). Plotted is the percentage of cells in Category 1, 2, or 3 as a function of the treatment time. tures associated with the chromosomes in heated interphase cells that had undergone fusion and premature chromosome Fig. 6. Autoradiography of a heated CHO cells incubated for 20 min before condensation. It was thought that their presence may correlate heat exposure in the presence of 100 ¿iCi/ml [3H|uridine. The figure shows a cell with or even induce several of the modifications observed after that has undergone PCC as well as an interphase nucleus. The accumulation of heat in DNA related enzymatic reactions (see "Introduction"). grains in certain areas of the nucleus indicates the location of the nucleolar organizing region. The grains distributed within the nucleus are attributed to Elucidation of their nature was regarded, therefore, as particu nonribosomal RNA synthesis. In the cell that has undergone fusion and induction larly important. The presence of the nuclear bodies in cells that of PCC, grains appear localized above the area of the nuclear bodies (compare had not undergone fusion, and the association of these bodies with Figs. 2 and 5), suggesting that they may be nondisintegrated remnants of the nucleolar organizing regions. Grains usually distributed within the nucleus with the chromosomes prompted us to hypothesize that they are now spread over the whole area covered by the fused cell complex. Giemsa may be identical to the nucleoli. To test this hypothesis plateau staining in this preparation was adjusted to give optimal discrimination of the phase cells were heated for 8 min after a 20-min preincubation grains with acceptable visibility of the interphase chromosomes. (experiments were also carried out after 0, 10, and 30 min preincubation and gave similar results) in the presence of 100 that they may represent the nucleolar organizing regions, where ¿iCi/ml['HJuridine (label was also present during heating), ribosomal RNA synthesis takes place. Their presence after processed for fusion and prepared for autoradiography as de induction of premature chromosome condensation suggested scribed in "Materials and Methods." One example of the au- changes in these cellular organelles that prevented their disin toradiographs obtained is shown in Fig. 6. A specific accumu tegration under the influence of the factors supplied by the lation of grains in the area of the bodies was observed suggesting mitotic cell. In unheated control cells that had undergone fusion 1257 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF HYPERTHERMIA ON INTERPHASE CHROMATIN grains were evenly distributed throughout the fusion complex. disintegrated nucleoli after fusion in heated interphase cells There was no indication for grain accumulation in any part of were reversible, recovery experiments were performed. For this the interphase cell, suggesting complete disintegration of the purpose, cells either in the exponential or in the plateau phase nucleolar organizing region. As expected, grain accumulation of growth were heated for 8, 15, or 30 min and were subse above the nucleolus was observed in control cells that had not quently returned to preirradiation conditions at 37°C.At var undergone fusion (results not shown). ious times thereafter samples were taken and processed to Dose-Response Relationship for the Effect of Heat on Nu measure the fraction of cells with nondisintegrated nucleoli. cleoli. In the following paragraphs experiments will be described The results obtained are shown in Fig. 8 and 9 and indicate designed to study the effect observed on the disintegration of that the effect of heat on the nucleolar organizing region was nucleolus in heated cells as a function of the exposure time and reversible. The rate of reversion depended strongly upon the the kinetics of its reversion. It was hypothesized that informa heat duration, both in exponentially growing as well as in tion on these functional relationships may help in evaluating plateau phase cells. Thus, 50% of the exponentially growing the importance of this phenomenon in the general response of cells showing nondisintegrated nucleoli immediately after an 8- cells to hyperthermia. Fig. 7 shows the percentage of cells with min exposure at 45.5°Cshowed normal condensation pattern nondisintegrated nucleoli (includes Categories 2 and 3 of heat after 4 h. It required 10 h for the same relative recovery after damage) in exponentially growing and plateau phase CHO cells exposure for 15 min at 45.5°C,and after exposure to 45.5°C exposed to 45.5°Cfor various periods of time. The results for 30 min only a limited amount of recovery was observed shown are the average of two experiments except for the meas within 24 h. It was not practical to follow up recovery after urements with exponentially growing cells at 8, 15, and 30 min longer times at these heat doses, due to the induction of cell that represent the average and the standard error calculated lysis that resulted in the loss of a significant fraction of the from three to five experiments. The dose-effect relationship heated cell population. The results obtained when plateau phase observed in exponentially growing and plateau phase cells was cells were allowed to recover after a heat exposure were quali similar. A fast increase in the fraction of cells displaying non- tatively similar but the recovery rate was somewhat slower disintegrated nucleoli was observed at heating times up to 10 (about 7 h were required for recovery in 50% of the cells after min, reaching a plateau at later times. Practically every cell exposure to 8-min heat). Although the results obtained after displayed nondisintegrated nucleoli when heated longer than 15 min heat exposure appear to be similar in exponentially 20 min. growing and plateau phase cells, cell lysis occurring under these Reversion of the Heat Effect on Nucleolar Organizing Region. conditions might have affected the results. If for example, the In order to establish whether the heat induced biochemical and/ most heavily damaged cells detached or lysed during the repair or structural alterations that caused the appearance of non- time period, an enrichment of the culture with less damaged cells would be expected giving thus the illusion of faster repair.
12 16 20 24 28 Time at 45.5 C, min Fig. 7. Heat dose-response curves (45.5"C) for the induction of nondisinte Time after Heat Treatment grated nucleoli (includes damage Categories 2 and 3) in exponentially growing (•)and plateau phase (H) CHO cells. The results shown are the average of two Fig. 8. Recovery of nondisintegrated nucleoli as a function of time exposure experiments except for the 8, IS. and 30 min points of exponentially growing of exponentially growing CHO cells to heat for 8 (•).IS (A), and 30 min (•). cells that were calculated from three to five experiments. Error bars are plotted, Shown is the average value and the standard error of the percent cells with when greater than the symbols, to show the standard error of three or more nondisintegrated nucleoli calculated from three (only 2- for 30-min exposure) measurements. experiments. 1258 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF HYPERTHERMIA ON INTERPHASE CHROMATIN suggesting that exposure to heat reduces the ability of DNA and chromatin to serve as templates for enzymes normally acting upon them. For example, Warters and Roti-Roti (20, 37) reported that exposure of HeLa cells to heat reduced the removal rate of 5'6'-dihydroxydihydrothymine bases from the DNA, and Warters et al. (38) reported that the accessibility to nuclease attack of DNA decreased after exposure to hyperther- mia. These phenomena have been attributed to the increase in protein mass associated with chromatin (20-21). In a similar way, one can interpret the results obtained at the chromosome level, using the technique of premature chromosome conden sation, as indicating a reduction in the ability of chromatin to serve as template for the enzymes that catalyze histone hi phosphorylation as well as for the series of reactions that lead to the disintegration of the nuclear membrane and the nucleolus and cause condensation of chromatin. At present, the exact type of alterations that cause the re sponse observed at the chromosome level in cells exposed to heat is not known. However, the reported increase in protein content of chromatin (14-17), of nuclei (13) and of the nuclear matrix (18,19) isolated from heated cells may contribute to the effect. It is possible that the accessibilityof chromatin and other nuclear structures to the enzymes operating during premature chromosome condensation be physically obstructed by the ac cumulation of nuclear and cytoplasmic proteins (38). This assumption is supported by the observation that the rate of 0 4 8 1216202428 repair of heat damage at the chromosome level, as measured with the technique of premature chromosome condensation, Time after Heat Treatment and the rate of removal of proteins from chromatin are quali Fig. 9. Same as in Fig. 8 but for plateau phase cells. tatively similar. For example, Tomasovic et al. (14) reported that 12-h incubation of CHO cells heated for 5-15 min at 45.5°Callowedfor full recovery of the heat-induced nonhistone DISCUSSION protein accumulation. Warters et al. (18) reported that the removal of protein mass associated with the nuclear matrix of The results presented in the previous section indicate that CHO cells following exposure to 45°Cfor 15 min was com exposure of cells to heat reduces the ability of chromatin to respond to signals induced in interphase cells following fusion pleted in about 10h. These values are within the range measured with mitotics. These signals are responsible for the induction for the disappearance of the nondisintegrated nucleoli in heated of premature chromosome condensation and effect disintegra CHO cells as shown in Figs. 8 and 9. Furthermore, Roti-Roti tion of the nuclear membrane and the nucleolus, as well as et al. (15) reported that HeLa cells restore their protein to condensation of chromatin (23-26). The effect observed in DNA ratio to control values at rates that depend upon the heat dose administered. After 15 and 30 min heat at 45°Cremoval cluded a reduction, or even complete loss, in the ability of chromatin to condense, as well as a loss in the ability of the of protein was found to be complete after 3 h and 14 h, nucleolus to disintegrate. The micrographs obtained indicated respectively.These values are not very different from those (not that disintegration of the nuclear membrane was not affected shown) obtained with HeLa cells at the chromosome levelusing the technique of premature chromosome condensation. Follow by heat in the dose range studied. This preliminary observation ing 8, 15, and 30 min heat at 45°Cnondisintegrated nucleoli is in agreement with the findings of Newport and Span (36) on the disassembly of the nucleus in mitotic extracts of Xenopus were found to disappear after 4, 12, and 24 h, respectively. eggs. These authors observed that membrane vesicularization, A correlation between heat effect as observed at the chro laniin disassembly, and chromosome condensation were inde mosome level using the technique of premature chromosome pendent processes. Since the factors inducing the process of condensation and cell killing cannot be established at this point. premature chromosome condensation were supplied by non- Comparison of the results obtained using CHO cells (Fig. 7) heated mitotic cells, the effect observed could be attributed to with those obtained using HeLa cells (results not shown) sug structural and/or biochemical alterations induced in the heated gest that there may be no direct correlation between heat- interphase cells. Two alternative hypotheses can be offered to induced cell death and the dose-response relationship for the explain the results observed: first, it is possible that heated induction of nondisintegrated nucleoli. A correlation may exist interphase cells are able to neutralize (for example by dephos- however, between the rate of disappearance of the nondisinte phorylation), in the mitotic cells they fuse with, the factors grated nucleoli in heated cells and heat sensitivity. This sugges inducing premature chromosome condensation. Second, it is tion stems from the observation that HeLa cells, which are possible that heat induces alterations in the cellular structures more heat-resistant than CHO cells, show faster kinetics of and organdies these factors act upon (chromatin, nucleolus, removal of nondisintegrated nucleoli than CHO cells (compare nuclear membrane, etc.) reducing thus their ability to serve as values for HeLa cells discussed above with the results for CHO substrates for the enzymatic reactions involved in the process cells shown in Figs. 8 and 9). It is interesting in this respect of premature chromosome condensation, thus leading to the that a correlation was found between heat sensitivity and the observed effects. rate of excess protein removal from the nuclei in HeLa cells There is a considerable amount of data in the literature exposed to heat under various conditions (13). The authors 1259 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF HYPERTHERMIA ON INTERPHASE CHROMATIN 10. Simard, R., and Bernhard, W. A heat-sensitive cellular function located in observed that compared to control cells, accumulated nuclear the nucleolus. J. Cell Biol., 34:61-76, 1967. proteins were removed faster in cells made thermotolerant by 11. Simard, R., Amalric, F., and Zalta, J. P. Effet de la temperature supra preexposure to heat, and slower in cells heated in the presence optimale sur les ribonucleoproteines et le RNA nucleolaire. 1. Etude Ultra- structurale. Exp. Cell Res., 55: 359-369, 1969. of procaine, a heat sensitizer. These results further indicate that 12. Amalric, F., Simard, R., and Zalta, J. P. Effet de la températuresupra- a correlation may exist between protein accumulation in the optimale sur les ribonucleoproteine et le RNA nucleolair. II. Etude Biochi mique Exp. Cell Res., 55: 370-377, 1969. nucleus of heated cells and the effects observed at the chromo 13. Kampinga, H. H., Luppes, J. G., and Konings, A. W. T. Heat-induced some level using the technique of premature chromosome con nuclear protein binding and its relation to thermal cytotoxicity. Int. J. densation. Hyperthermia, 3: 459-465, 1987. 14. Tomasovic, S. P., Turner. G. N., and Dewey, W. C. Effect of hyperthermia The nucleolus has been reported to be a particularly heat on nonhistone proteins isolated with DNA. Radial. Res., 73:535-552,1978. sensitive cellular organelle (9-12). Exposure of cells to heat has 15. Roti Roti, J. L., and Winward, R. T. The effects of hyperthermia on the been shown to result in a loss of the granular ribonucleoprotein protein-to-DN A ratio of isolated HeLa cell chromatin. Radiât.Res., 74:159- 169, 1978. component and intranucleolar chromatin, as well as in a dis 16. Roti Roti, J. L., Uygar, N., and Higashikubo, R. Nuclear protein following appearance of the nucleolar reticulum in the nucleolus (9-12). heat shock: Protein removal kinetics and cell cycle rearrangements. Radiât. These alterations were found to be reversible and accompanied Res., 107: 250-261, 1986. 17. Roti Roti, J. L., and Winward, R. T. Factors affecting the heat-induced by an inhibition of nucleolar RNA synthesis. The authors increase in protein content of chromatin. Radial. Res., 81: 138-144, 1980. interpreted these findings as indicating that the synthesis of 18. Warters, R. L., Brizgy, L. M., and Lyons, B. W. Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single- nucleolar RNA is heat sensitive and related to the presence of strand-break repair. Int. J. Radiât.Biol., 52: 299-313, 1987. intranucleolar chromatin. The results obtained in this work 19. Warters, R. L., Brizgy, L. M., Sharma, R., and Roti Roti, J. L. Heat shock using the PCC technique suggest that the retraction of the (45'C) results in an increase of nuclear matrix protein mass in HeLa cells. Int. J. Radiât.Biol., 50: 253-268, 1986. intranucleolar chromatin induced after exposure of cells to heat 20. Warters, R. L., and Roti Roti, J. L. Excision of X-ray induced thymine was not complete. Chromatin remained associated in the nu damage in chromatin from heated cells. Radiât.Res., 79: 113-121, 1979. cleolus and caused the observed aggregated appearance of the 21. Mills, M. D., and Meyn, R. E. Effects of hyperthermia on repair of radiation- induced DNA strand breaks. Radiât.Res., 87: 314-328, 1981. chromosomes after fusion and induction of premature chro 22. Bodell, W. J., Cleaver, J. E., and Roti Roti, J. L. Inhibition by hyperthermia mosome condensation. If chromatin completely retracted and of repair synthesis and chromatin reassembly of ultraviolet-induced damage dissociated from the nucleolus, free decondensed chromosomes to DNA. Radial. Res., 100: 87-95, 1984. 23. Johnson, R. T., and Rao, P. N. Mammalian cell fusion: induction of pre must have been observed. Furthermore, the degree of retraction mature chromosome condensation in interphase nuclei. Nature (Lond.), 226: of chromatin from the nucleolus, as judged by the degree of 717-722, 1970. 24. Johnson, R. T., Rao, P. N., and Hughes, S. D. Mammalian cell fusion: II. A staining with Giemsa, may vary from cell line to cell line HeLa cell inducer of premature chromosome condensation active in cells (compare results obtained with CHO and HeLa cells). The from a variety of animal species. J. Cell. Physiol., 77: 151-157, 1970. inability of the nucleolus to disintegrate after heat exposure 25. I uncinimi. W. N., and Rao, P. N. 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1260 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. Effects of Hyperthermia on Chromatin Condensation and Nucleoli Disintegration as Visualized by Induction of Premature Chromosome Condensation in Interphase Mammalian Cells
George E. Iliakis and Gabriel E. Pantelias
Cancer Res 1989;49:1254-1260.
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