(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.]
nuclei (13), in chromatin (14-17), and in nuclear matrices (18,
19), and it was proposed that disruption of important nuclear
processes by this nuclear protein binding may be the reason for
cell killing (17). Beyond cell killing the excess nuclear proteins
have been implicated in the inhibition of DNA synthesis (5, 6)
and the inhibition of DNA repair following both ionizing (20,
21) and uv (22) irradiation. It is thought that inhibition of
these cellular functions may be due to alterations induced in
chromatin conformation and in particular to restriction of DNA
supercoiling changes as a result of protein addition to the
nuclear matrix (19). These results stress the possible importance
of chromatin as the principal site of heat-induced damage that
ultimately leads to cell death.
Integrity and structural alterations in chromatin can also be
studied by utilizing the technique of premature chromosome
condensation (23-26). Chromatin of a cell can ordinarily be
visualized as distinct chromosomes only when the cell enters
mitosis. However, when mitotic cells are fused with cells in
interphase by means either of Sendai virus or of PEG,3 disin
ABSTRACT
The effects of hyperthermia on chromatin condensation and nucleoli
disintegration, as visualized by induction of premature chromosome con
densation in interphase mammalian cells, was studied in exponentially
growing and plateau phase Chinese hamster ovary cells. Exposure to
heat reduced the ability of interphase chromatin to condense and the
ability of the nucleolar organizing region to disintegrate under the influ
ence of factors provided by mitotic cells when fused to interphase cells.
Based on these effects treated cells were classified in three categories.
Category 1 contained cells able to condense their chromatin and disinte
grate the nucleolar organizing region. Category 2 contained cells able to
only partly condense their chromatin and unable to disintegrate the
nucleolar organizing region. Category 3 contained cells unable to condense
their chromatin and unable to disintegrate the nucleolar organizing region.
The fraction of cells with nondisintegrated nucleoli increased with in
creasing exposure time at 45.5 < and reached a plateau at almost 100%
after about 20 min. Exponentially growing and plateau phase cells showed
similar response. Recovery from the effects of heat on chromatin conden
sation and disintegration of the nucleolar organizing region depended
upon the duration of the heat treatment. For exposures up to 15 min at
45.5°C,a gradual 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- condensation process taking morphology that is characteristic
min exposure to 45.5°C.The repair times observed at the chromosome
level were similar to those reported for the removal of excess protein
accumulating in chromatin or the nuclear matrix, suggesting a causal
relationship between the two phenomena. It is proposed that nuclear
protein accumulation on chromatin or in the nuclear matrix reduces the
accessibility of chromatin to enzymes responsible for the phosphor) l:itinn
reactions necessary for chromatin condensation and disintegration of the
nucleolus.
INTRODUCTION
The recent consideration of hyperthermia as an adjuvant to
radiation therapy (1) and the possibility of probing mechanisms
of gene expression using the synthesis of heat shock proteins
as a model system (2) has drawn much attention to the effects
of heat on living systems. To fully utilize the potential of
hyperthermia, or hyperthermia in conjunction with other mo
dalities in the above-mentioned goals, it is necessary to deter
mine the molecular alterations that it induces as well as the
lesions that lead to cell death.
Hyperthermia has been shown to induce a number of effects
in mammalian cells including inhibition of DNA, RNA, and
protein synthesis (3-6), induction of chromosomal aberrations
(7, 8), and in ultrastructural studies, disappearance of the
intranucleolar chromatin and the granular ribonucleoprotein
components of the nucleolus (9-12). Furthermore, hyperther
mia was found to increase the protein to DNA ratio in isolated
Received 8/22/88; revised 11/28/88; accepted 12/1/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
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.
2 Supported by Grant B16-E-206-GR awarded by the Commission of the
European Communities, Radiation Protection Program.
of the cell cycle phase of the interphase cell at the time of
fusion. This phenomenon is referred to as premature chromo
some condensation (PCC) and the induced chromosome units
as prematurely condensed chromosomes (also abbreviated as
PCC, the context clarifies the way in which the abbreviation is
used). The process of premature chromosome condensation can
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
relatively high molecular weight that bind preferentially to
chromatin (28-33).
In this paper we report on the effects of heat (45.5 and 43°C)
on chromatin morphology and nuclear organization, as visual
ized by PCC, in exponentially growing and plateau phase CHO
cells. Selected results obtained with HeLa cells are also pre
sented and discussed. The results obtained indicate a dramatic
effect of heat on the ability of chromatin to condense and on
the ability of the nucleolus to disintegrate after induction of
PCC. This is, to the best of our knowledge, the first report on
this topic.
MATERIALS
AND METHODS
Cell Culture and Heating. Experiments were performed using Chinese
hamster ovary cells, strain 10B, grown as monolayers in McCoy's 5A
medium supplemented with 10% fetal calf serum and antibiotics (50
units/ml potassium penicillin (i,, 50 Mg/ml streptomycin sulfate). Cells
were grown at 37°Cin a humidified incubator, in an atmosphere of 5%
COj and 95% air. Stock cultures were maintained in vitro by routinely
subculturing (every second day) at an initial density of 10'' cells in 753 The abbreviations used are: PEG, polyethylene glycol; PCC, premature
chromosome condensation or prematurely condensed chromosome; CHO,
Chinese hamster ovary; PBS, phosphate buffered saline.
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EFFECTS OF HYPERTHERMIA
ON INTERPHASE CHROMATIN
cm2 tissue culture flasks. After 2 days of growth, cells in these cultures
had reached a density of IO7per flask, and were used to prepare cultures
for experiments. For this purpose, 10s cells, suspended in 3-ml growth
medium, were plated in 25-cm2 tissue culture flasks and were allowed
to grow for 2 or 4 days in order to obtain exponentially growing or
plateau phase cells, respectively. Exponentially growing cultures had a
density of 1-1.3 x IO6 cells/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
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%)
of the cells were found at a stage in the cell cycle showing a DNA
content equivalent to that of d cells, with the remaining cells found in
S (3-5%) and in G2 (4-7%) phases. Cells were checked and found free
of mycoplasma contamination. To establish the general validity of the
results obtained, experiments were also carried out with I lei .a cells
(kindly provided by Dr. Roti Roti, Washington University, St. Louis,
MO). Cells were grown as monolayers in Joklik-modifled minimum
essential medium plus 3.5% each of fetal bovine and calf sera (16).
Exponentially growing cultures were prepared by incubating for 2 days
3x10* cells in 5 ml of growth medium in 25-cm2 tissue culture flasks.
Exponentially growing cells were heated in their growth medium
(pH 7.3-7.6) and plateau phase cells in fresh McCoy's 5A (without
serum) to reduce pH related effects (pH of spent medium from plateau
phase cultures 6.8-7.0, pH of fresh medium 7.4-7.6). Heating was
carried out in waterbaths regulated at the desired temperature with an
accuracy of ±0.05°C.
Temperature was monitored with a calibrated
(against NBS standardized thermometer) mercury thermometer. After
heating, cells were either processed immediately for fusion and induc
tion of premature chromosome condensation, or, they were returned to
the incubator after reestablishing preirradiation conditions (fresh full
growth medium for growing cells and spent medium for plateau phase
cells). For trypsinization, cells were washed once with phosphate buff
ered saline and were incubated at 37°Cfor 5 min with 0.25% trypsin
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 nocodazole, methyl[5-(2-thienylcarbonyl)-1 H-benzimidazol-2-yl] carbamate
(Sigma), were mixed with an equal number of interphase cells in a 15ml 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
as required by the particular experimental protocol and processed for
fusion. Slides prepared were allowed to dry overnight at room temper
ature and were subsequently immersed in photographic emulsion (IIford), after dilution with water (2:3, emulsion:water). Slides were dried
in a slight draft and placed in light tight boxes at 4°Cfor 2-5 days.
After this period of time slides were developed and analyzed.
RESULTS
Effect of Heat on the Process of Premature Chromosome
Condensation. Morphological changes induced by heat in chromatin and the chromosomes, as visualized in interphase cells
using the technique of premature chromosome condensation,
were investigated. Plateau phase cells were used first for these
experiments because they mainly contain Gìcells that show
complete chromatin condensation into discrete chromosomes
after fusion with mitotic cells, thus simplifying analysis. How
ever, similar results were subsequently obtained with cells in
every phase of the cycle (using exponentially growing cultures).
Fig. 1 illustrates the chromosome condensation achieved after
fusion and 1-h incubation at 37°C,of an untreated (nonheated)
interphase (d) CHO cell with a cell at mitosis.
Severe morphological changes became apparent after heat.
Fig. 2 shows chromatin condensation and morphology as ob
served in a fraction of cells from a plateau phase population
heated for 10 min at 45.5°Cand fused for PCC induction with
mitotic cells immediately thereafter. Following exposure to heat
chromatin partly lost its ability to condense into distinct chro
mosomes. Despite the fact that chromosome condensation oc
curred to a certain extent and chromatin fibers were visible, the
process did not arrive to the degree of completion observed in
nonheated cells (compare with Fig. 1). This observation sug-
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%
Giemsa. Before analysis, slides were mounted with cover slips, and cells
where premature chromosome condensation was induced were scored
by means of light microscopy. The scoring criteria are discussed in the
next section. Routinely, 25-100 cells were analyzed and scored per
experimental datum. At no point during analysis and scoring was there
ambiguity in the distinction between interphase and mitotic chromo
somes.
Autoradiography. In experiments involving autoradiography, cells
were preincubated for 0-30 min with 100 ^Ci/ml [3H]uridine, heated
Fig. 1. Fusion of a mitotic with a control (not treated) d phase CHO cell.
Notice the disintegration of the nuclear membrane and the nucleolus and the
induction of premature chromosome condensation. Chromatin condenses into
distinct chromosomes at this stage of the cell cycle. 21 chromosomes (the modal
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
prematurely condensed chromosomes allow for unequivocal evaluation of the
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
in Category 1. In this and the following photographs the exposure time during
development was adjusted to show optimally the prematurely condensed chro
mosomes.
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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
in the heated interphase cell and initiated some condensation of chromatin.
Individual chromatin fibers can be observed. As a result of heat, chromatin
condensation did not come to completion (compare with Fig. I). In addition,
condensed chromatin was not free but associated with nuclear bodies staining
light with Giemsa (arrows). These nuclear bodies were not induced during the
process of PCC as indicated by the fact that they could also be observed in
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.
gested that exposure to heat reduced the efficacy of chromatin
to respond to signals transmitted by unheated mitotic cells.
Furthermore, as it can be seen in Fig. 2, the chromosomes were
not free and independent of each other as in the case of
nonheated cells, but appeared to be organized in the periphery
of a nuclear "body" that stained with Giemsa lighter than the
surrounding chromatin. Light staining nuclear bodies could
also be observed in heated nuclei that had not undergone fusion
with mitotic cells (indicated by arrows in the figure) suggesting
that their induction was not due to the PCC process.
A more severe effect of heat on chromatin condensation is
shown in Fig. 3. As in the previous examples, nonheated mitotic
cells were fused with heated (45.5°Cfor 20 min) interphase
Fig. 3. Fusion of a mitotic cell with a heated (4S.5'C, 20 min) interphase cell.
Fusion induced disintegration of the nuclear membrane in the interphase cell but,
due to heat-induced damage, chromatin was unable to condense. Nuclear bodies
staining light with Giemsa can still be recognized and are indicated by the arrows.
Cells unable to condense their chromatin and displaying nuclear bodies after
fusion with mitotics are classified in Category 3.
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
were not solely distributed in states identical to those described
in Figs. 1 to 3. In a heated sample a series of intermediate
states could be observed that were assigned during analysis in
the closest category. In general the fraction of cells in Categories
2 and 3 gradually increased with increasing heating time and
Category 3 prevailed after longer heating times. This is illus
trated by the results shown in Fig. 4, obtained with plateau
phase cells. Results of one experiment are shown, but the
findings were qualitatively reproduced in several experiments.
Plotted in the figure is the fraction of cells in the different
categories as a function of the heating time at either 43 or
45.5°C.The fraction of cells in Category 1 rapidly decreased
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 struc1256
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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
of chromatin in the structure.
•¿
category
1
D category
2
E category
3
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
condensation. It was thought that their presence may correlate
with or even induce several of the modifications observed after
heat in DNA related enzymatic reactions (see "Introduction").
Fig. 6. Autoradiography of a heated CHO cells incubated for 20 min before
heat exposure in the presence of 100 ¿iCi/ml [3H|uridine. The figure shows a cell
Elucidation of their nature was regarded, therefore, as particu
larly important. The presence of the nuclear bodies in cells that
had not undergone fusion, and the association of these bodies
with the chromosomes prompted us to hypothesize that they
may be identical to the nucleoli. To test this hypothesis plateau
phase cells were heated for 8 min after a 20-min preincubation
(experiments were also carried out after 0, 10, and 30 min
preincubation and gave similar results) in the presence of 100
¿iCi/ml['HJuridine (label was also present during heating),
processed for fusion and prepared for autoradiography as de
scribed in "Materials and Methods." One example of the autoradiographs obtained is shown in Fig. 6. A specific accumu
lation of grains in the area of the bodies was observed suggesting
that has undergone PCC as well as an interphase nucleus. The accumulation of
grains in certain areas of the nucleus indicates the location of the nucleolar
organizing region. The grains distributed within the nucleus are attributed to
nonribosomal RNA synthesis. In the cell that has undergone fusion and induction
of PCC, grains appear localized above the area of the nuclear bodies (compare
with Figs. 2 and 5), suggesting that they may be nondisintegrated remnants of
the nucleolar organizing regions. Grains usually distributed within the nucleus
are now spread over the whole area covered by the fused cell complex. Giemsa
staining in this preparation was adjusted to give optimal discrimination of the
grains with acceptable visibility of the interphase chromosomes.
that they may represent the nucleolar organizing regions, where
ribosomal RNA synthesis takes place. Their presence after
induction of premature chromosome condensation suggested
changes in these cellular organelles that prevented their disin
tegration under the influence of the factors supplied by the
mitotic cell. In unheated control cells that had undergone fusion
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EFFECTS OF HYPERTHERMIA
grains were evenly distributed throughout the fusion complex.
There was no indication for grain accumulation in any part of
the interphase cell, suggesting complete disintegration of the
nucleolar organizing region. As expected, grain accumulation
above the nucleolus was observed in control cells that had not
undergone fusion (results not shown).
Dose-Response Relationship for the Effect of Heat on Nu
cleoli. In the following paragraphs experiments will be described
designed to study the effect observed on the disintegration of
nucleolus in heated cells as a function of the exposure time and
the kinetics of its reversion. It was hypothesized that informa
tion on these functional relationships may help in evaluating
the importance of this phenomenon in the general response of
cells to hyperthermia. Fig. 7 shows the percentage of cells with
nondisintegrated nucleoli (includes Categories 2 and 3 of heat
damage) in exponentially growing and plateau phase CHO cells
exposed to 45.5°Cfor various periods of time. The results
shown are the average of two experiments except for the meas
urements with exponentially growing cells at 8, 15, and 30 min
that represent the average and the standard error calculated
from three to five experiments. The dose-effect relationship
observed in exponentially growing and plateau phase cells was
similar. A fast increase in the fraction of cells displaying nondisintegrated nucleoli was observed at heating times up to 10
min, reaching a plateau at later times. Practically every cell
displayed nondisintegrated nucleoli when heated longer than
20 min.
Reversion of the Heat Effect on Nucleolar Organizing Region.
In order to establish whether the heat induced biochemical and/
or structural alterations that caused the appearance of non-
ON INTERPHASE CHROMATIN
disintegrated nucleoli after fusion in heated interphase cells
were reversible, recovery experiments were performed. For this
purpose, cells either in the exponential or in the plateau phase
of growth were heated for 8, 15, or 30 min and were subse
quently returned to preirradiation conditions at 37°C.At var
ious times thereafter samples were taken and processed to
measure the fraction of cells with nondisintegrated nucleoli.
The results obtained are shown in Fig. 8 and 9 and indicate
that the effect of heat on the nucleolar organizing region was
reversible. The rate of reversion depended strongly upon the
heat duration, both in exponentially growing as well as in
plateau phase cells. Thus, 50% of the exponentially growing
cells showing nondisintegrated nucleoli immediately after an 8min exposure at 45.5°Cshowed normal condensation pattern
after 4 h. It required 10 h for the same relative recovery after
exposure for 15 min at 45.5°C,and after exposure to 45.5°C
for 30 min only a limited amount of recovery was observed
within 24 h. It was not practical to follow up recovery after
longer times at these heat doses, due to the induction of cell
lysis that resulted in the loss of a significant fraction of the
heated cell population. The results obtained when plateau phase
cells were allowed to recover after a heat exposure were quali
tatively similar but the recovery rate was somewhat slower
(about 7 h were required for recovery in 50% of the cells after
exposure to 8-min heat). Although the results obtained after
15 min heat exposure appear to be similar in exponentially
growing and plateau phase cells, cell lysis occurring under these
conditions might have affected the results. If for example, the
most heavily damaged cells detached or lysed during the repair
time period, an enrichment of the culture with less damaged
cells would be expected giving thus the illusion of faster repair.
12
Time at 45.5 C, min
Fig. 7. Heat dose-response curves (45.5"C) for the induction of nondisinte
16
20
24
28
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
experiments except for the 8, IS. and 30 min points of exponentially growing
cells that were calculated from three to five experiments. Error bars are plotted,
when greater than the symbols, to show the standard error of three or more
measurements.
Fig. 8. Recovery of nondisintegrated nucleoli as a function of time exposure
of exponentially growing CHO cells to heat for 8 (•).IS (A), and 30 min (•).
Shown is the average value and the standard error of the percent cells with
nondisintegrated nucleoli calculated from three (only 2- for 30-min exposure)
experiments.
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EFFECTS OF HYPERTHERMIA
0
4
8
1216202428
Time after Heat Treatment
Fig. 9. Same as in Fig. 8 but for plateau phase cells.
DISCUSSION
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 hyperthermia. 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
repair of heat damage at the chromosome level, as measured
with the technique of premature chromosome condensation,
and the rate of removal of proteins from chromatin are quali
tatively similar. For example, Tomasovic et al. (14) reported
that 12-h incubation of CHO cells heated for 5-15 min at
45.5°Callowed for full recovery of the heat-induced nonhistone
protein accumulation. Warters et al. (18) reported that the
removal of protein mass associated with the nuclear matrix of
CHO cells following exposure to 45°Cfor 15 min was com
The results presented in the previous section indicate that
exposure of cells to heat reduces the ability of chromatin to
respond to signals induced in interphase cells following fusion pleted in about 10 h. 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 level using
technique of premature chromosome condensation. Follow
by heat in the dose range studied. This preliminary observation the
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.
A correlation between heat effect as observed at the chro
eggs. These authors observed that membrane vesicularization,
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 heatinterphase 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
rate of excess protein removal from the nuclei in HeLa cells
observed effects.
There is a considerable amount of data in the literature exposed to heat under various conditions (13). The authors
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EFFECTS OF HYPERTHERMIA
observed that compared to control cells, accumulated nuclear
proteins were removed faster in cells made thermotolerant by
preexposure to heat, and slower in cells heated in the presence
of procaine, a heat sensitizer. These results further indicate that
a correlation may exist between protein accumulation in the
nucleus of heated cells and the effects observed at the chromo
some level using the technique of premature chromosome con
densation.
The nucleolus has been reported to be a particularly heat
sensitive cellular organelle (9-12). Exposure of cells to heat has
been shown to result in a loss of the granular ribonucleoprotein
component and intranucleolar chromatin, as well as in a dis
appearance of the nucleolar reticulum in the nucleolus (9-12).
These alterations were found to be reversible and accompanied
by an inhibition of nucleolar RNA synthesis. The authors
interpreted these findings as indicating that the synthesis of
nucleolar RNA is heat sensitive and related to the presence of
intranucleolar chromatin. The results obtained in this work
using the PCC technique suggest that the retraction of the
intranucleolar chromatin induced after exposure of cells to heat
was not complete. Chromatin remained associated in the nu
cleolus and caused the observed aggregated appearance of the
chromosomes after fusion and induction of premature chro
mosome condensation. If chromatin completely retracted and
dissociated from the nucleolus, free decondensed chromosomes
must have been observed. Furthermore, the degree of retraction
of chromatin from the nucleolus, as judged by the degree of
staining with Giemsa, may vary from cell line to cell line
(compare results obtained with CHO and HeLa cells). The
inability of the nucleolus to disintegrate after heat exposure
might be attributed, either to the above described morphological
and biochemical alterations or an accumulation of nuclear
proteins on it.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Ronald Coss for making available
equipment necessary for the experiments in this project, as well as for
fruitful discussions. Special thanks go to Robert Seaner for technical
assistance and to Nancy Mott for secretarial help.
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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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