(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.