[CANCER RESEARCH 47, 5162-5170, October 1, 1987] Characterization of Chromosome Aberrations Induced by Incubation at a Restrictive Temperature in the Mouse Temperature-sensitive Mutant tsFT20 Strain Containing Heat-labile DNA Polymerase a1 Toshihiko Eld,2 Takemi Enomoto, Yasufumi Murakami,3 Fumio Hanaoka, and Masa-atsu Yamada4 Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT tsFT20 cells derived from a mouse mammary carcinoma cell line, FM3A, which has temperature-sensitive DNA polymerase a activity (Y. Murakami, (I. Yasuda, H. Miyazawa, F. Hanaoka, and M. Yamada, Proc. Nati. Acad. Sci. USA, 82:1761-1765,1985) were rapidly commit ted to death after temperature upshift to 39°C.tsFT20 cells synchronized in S phase were more sensitive to the restrictive temperature than exponentially growing cells. In order to gain insight into the processes from the interruption of DNA synthesis to cell death, we analyzed chromosome aberrations induced in tsFT20 cells which had been incu bated for 2 or 4 h at the restrictive temperature and then cultured at the permissive temperature. The majority of metaphase cells showed exten sive chromosome aberrations such as chromatid gaps, breaks, and ex changes; chromosome pulverizations; their mixed types; and ring chro mosomes. Analyses with the use of cell synchronization and autoradiography revealed that chromosome aberrations were induced only in the cells which synthesized DNA during incubation at 39°C.We classified the chromosome aberrations into five types: gap or break type; exchange type; pulverization type; complex type; and ring type. The temporal order of the appearance of these types of chromosome aberrations was found to be the above described order. It was further found that cycloheximide dramatically repressed the induction of chromosome aberrations, and metaphases with many chromosome aberrations exhibited a large number of sister chromatid exchanges. These results indicate that abnormal cessation of DNA replication in tsFT20 cells at the restrictive tempera ture due to the inactivation of DNA polymerase a results in cell death via induction of double-strand breaks which lead to chromosome aber rations as well as sister chromatid exchanges. directly participating in DNA replication are inhibited because of the lack of specific inhibitors for DNA replication enzymes except for aphidicolin and the unavailability of DNA ts mutants having ts enzymes that participate in DNA replication. In recent years, we have tried to isolate ts mutants related to DNA replication in order to get a tool for the analysis of the molecular mechanism of DNA replication in mammalian cells, and we have isolated several such mutants (12-14). One of these ts mutants, tsFT20, was found to have heat-labile DNA polymerase «activity (12). Recently, we have succeeded in proving that the DNA polymerase a molecule of tsFT20 cells itself is heat-labile by using an ¡mmunoaffinity-purified en zyme.6 tsFT20 cells are typical DNA ts mutants, which show rapid decrease in DNA-synthesizing ability after temperature upshift and are arrested in the S phase. It was found that the rapid decrease in DNA-synthesizing ability correlated well with the decrease in the intracellular level of DNA polymerase a activity (15) and was due to the decrease in the frequency of replicón initiation (16). In addition, it has been observed that ts cells are rapidly committed to death after exposure to the restrictive temperature. In this study we have examined chromosome aberrations induced in tsFT20 cells in order to gain insight into the proc esses from the interruption of DNA replication, due to the inactivation of the DNA polymerase a molecule, to cell death. MATERIALS INTRODUCTION During recent years, it has become well known that besides direct DNA lesions induced by X-rays or chemicals, the indirect DNA lesions induced by imbalance of DNA precursor metab olism also cause extensive chromosome instability in eukaryotic cells. In addition, it is known that when de novo synthesis of thymidylate is blocked, growing cells die. This phenomenon is called thymineless death and has been initially found in prokaryotic cells (1). Thymineless death has been studied in eukar yotic cells treated with drugs that block thymidylate metabolism (2-6) and auxotrophic thy"5 mutants (7-9). Recent studies using thy mutants have shown that thymidine starvation in mammalian cells induces extensive chromosome aberrations and results in thymineless death (10, 11). On the other hand, little is known of what happens in cells in which enzymes Received 3/3/87; revised 6/19/87; accepted 6/24/87. 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. 1This work was supported in part by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan, and by the Naito Foundation. 2To whom requests for reprints should be addressed. 3 Present address: Riken Gene Bank, The Institute of Physical and Chemical Research. 4 Present address: Hatano Research Institute, Food and Drug Safety Center. 5 The abbreviations used are: thy", thymidylate synthase-negative; CMF-PBS, calcium- and magnesium-free phosphate-buffered saline; FdUrd, 5-fluoro-2'deoxyuridine; SCE, sister chromatid exchange; ts. temperature-sensitive. AND METHODS Cell Culture. tsFT20 cells (12) and FM3A clone 28 cells (wild-type) which were originally established from a spontaneous mammary carci noma in a C3H/He mouse (17) were incubated at 33°C(permissive temperature) or at 39°C(restrictive temperature) in suspension culture in RPMI 1640 (Flow Laboratories, England) supplemented with 10% calf serum (Flow Laboratories, North Ryde, Australia). Cells used in these experiments were free of Mycoplasma contamination. Assay for Colony-forming Ability. Exponentially growing tsFT20 cells and wild-type cells or tsFT20 cells synchronized at Gi-S boundary (approximately 1.5 x IO6) were inoculated in glass tubes (1.5 x 10.5 cm) containing 5 ml of the growth medium. The cells were incubated at 39°Cfor indicated periods in a water bath and then collected by centrifugation at 1400 x g for 5 min at 4°C.After a washing with icecold CMF-PBS, the cells were suspended in and diluted with the growth medium to a cell concentration of 100 and 300/ml. One volume of the diluted cell suspension was mixed with 2 volumes of 0.5% agar solution (agar Noble; Difco Laboratories, Detroit, MI) dissolved in RPMI 1640 containing 10% calf serum and antibiotics (100 //g/nil streptomycin sulfate and 100 units/ml penicillin G potassium), and 3 ml of the mixture were poured on 4 ml of 0.5% underlayer agar in a glass dish (60 mm in diameter). After incubation at 33°Cfor 2 weeks in CO2 incubator, the number of colonies in the soft agar was counted (4 dishes/point). Cell Synchronization. Exponentially growing cells were inoculated into the growth medium containing 10% dialyzed calf serum and the antibiotics at a concentration of 1.5 x IO5 cells/ml. The cells were incubated at 33°Cfor 1 day and FdUrd was added at a final concentra" R. Takayama, S. Tada, F. Hanaoka, and M. Ui, unpublished data. 5162 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY tion of 3 x 10~8 M. The cells were incubated at 33°Cfor 14 h to be arrested at d-S boundary. The cells were released from the FdUrd block by two washings with ice-cold CMF-PBS and suspended in warm growth medium containing the antibiotics. Over 80% of the population thus synchronized was arrested at the d-S boundary and most of the remainder was arrested in S phase according to cytofluorometric anal ysis (15). Assays for Chromosome Aberrations and Mitotic Index. The cells (3 x IO5) from exponentially growing cultures or synchronized cultures were inoculated into a glass dish (diameter 30 mm) with 2 ml of the growth medium. After incubation for indicated periods, Colcemid was added to the culture at a final concentration of 0.1 ¿ig/ml,and the cells were incubated at 33 °Cfor indicated periods in a CC>2incubator. The cells were chilled in an ice-water bath and transferred into a glass tube. The cells were collected by centrifugation and washed with ice-cold CMF-PBS. The pellet was resuspended in 1 ml of 0.5% sodium citrate and incubated at 37°Cfor 5 min with shaking, and then 5 ml of the fixative (methanol:acetic acid, 3:1) were added. After standing at room temperature for 5 min, the suspension was centrifuged, and the pellet was washed once with the fixative. The resultant pellet was suspended in a small volume of the fixative. The suspension was dropped on a glass slide and air dried. Cells fixed on the glass were stained with 3% Giemsa solution for 15 min. Mitotic index was determined by exam ining about 500 cells. As for chromosome aberrations, about 200 metaphases were examined at x 400. Chromosome aberrations are classified as follows. "Gap or break type" is defined as the aberration exhibiting a chromosome or chromatid gap or break observed in a metaphase which contains less than six aberrations. "Exchange type" is defined as the aberration exhibiting a chromatid interchange or a chromosome/chromatid interchange observed in a metaphase which contains less than six aberrations. "Complex type" is an aberration consisting of more than six aberrations within a metaphase including chromatid gaps, breaks, exchange types, and the fragmented chromo somes as shown in Fig. 2, b-d. "Pulverization type" is represented by pulverized chromosomes as shown in Fig. le. "Ring type" is the aberration exhibiting a centric ring or a ring derived from a chromosome-chromatid interchange. The frequencies of gap or break type, exchange type, and ring type are expressed as the number of sites exhibiting aberrations per 100 metaphases by examining about 200 metaphases. The frequencies of complex type and pulverization type are expressed as the number of metaphases exhibiting the aberration per 100 metaphases by examining about 200 metaphases. Autoradiographic Analysis. Exponentially growing cells (5 x 10s) were inoculated into a culture flask (25 cm2) containing 5 ml of the growth medium supplemented with 10% dialyzed calf serum and the antibiotics. The cells were incubated at 33"C for 2 days and then incubated at 39°Cfor 4 h. The cells were pulse-labeled with 4 ^Ci/ml [3H]thymidine (20 Ci/mmol; Amersham, United Kingdom) for 10 min. The labeled cells were collected by centrifugation and washed twice with ice-cold CMF-PBS. The cells were suspended at a concentration of 1.5 x 10' cells/ml in the growth medium containing 1 x IO"5 M thymidine and 0. l j/g/ml Colcemid. After incubation at 33°Cfor 24 h, IN tsFT20 CELLS 15 min, washed with water, and soaked in Mcllvaine's buffer (196 mM disodium hydrogen phosphate-2 HIMcitric acid, pH 8.25). The glass slide was covered with a cover glass and sealed with colorless nail polish to hold the Mcllvaine's buffer between the glass slide and the coverglass. The glass slide was exposed to UV at a 5-cm distance from a germicida! lamp (Toshiba GL15) for 10 min on a hot plate set at 60"( , washed with water, and stained with 2% Giemsa solution. The stained meta phases were examined at x 1000. RESULTS Decrease in Colony-forming Ability during Incubation at the Restrictive Temperature. As reported previously (12, 15, 16), tsFT20 cells have heat-labile DNA polymerase a activity. As shown in Fig. la, DNA polymerase a activity in tsFT20 cells decreased immediately after temperature upshift in contrast to the activity in wild-type cells. Fig. \b shows the colony-forming ability of tsFT20 cells after the temperature upshift. Exponen tially growing tsFT20 cells lost colony-forming ability rapidly after incubation at 39°C.When tsFT20 cells were synchronized with FdUrd and shifted to the restrictive temperature after the release from the FdUrd block, the decrease in colony-forming ability of the synchronized cells became much more prominent than that of exponentially growing cells. Only 8% of colonyforming ability was obtained with the synchronized cells after incubation at 39°Cfor 8 h. Little decrease in colony-forming ability was observed with synchronized wild-type cells. Chromosome Aberrations Induced in tsFT20 Cells at the Re strictive Temperature. We next studied the effect of the exposure of tsFT20 cells to the restrictive temperature from the chro mosome-morphological point of view, especially chromosome aberrations. Fig. 2 shows typical metaphase figures containing chromosome aberrations obtained from the cells which have been incubated at 39°Cfor 4 h and then incubated at 33°Cfor 24 h in the presence of Colcemid. Various types of chromosome aberrations were observed: simple chromatid-type chromosome aberrations such as chromatid gaps and breaks (Fig. la; Arrows E-H and J) and chromatid interchanges (Arrows A-D and /); "complex-type" chromosome aberrations characterized by many chromatid gaps, breaks, and exchanges (Fig. 2, b-d); and ~ loo so the cells were fixed on a glass slide and processed to autoradiography as described previously (16). The existence of silver grains and chro mosome aberrations was examined with about 300 metaphases at x 400. Assay for SCEs. Exponentially growing cells (2.5 x IO5)were inoc ulated into a culture flask (25 cm2) containing 5 ml of the growth medium supplemented with 10% dialyzed calf serum and the antibiot ics. 5-Bromo-2'-deoxyuridine (Sigma Chemical Co., St. Louis, MO) 012« 8 0124 e Incubation period al 39 *C (h) Incubation period at 39'C (h) was added to the culture at a concentration of 5 Mg/ml, and then the cells were incubated at 33°Cfor 18 h (approximately 1.2 cell cycles Fig. 1. (a) Changes in the level of intracellular ONA polymerase a activity. under these conditions) in the dark. The cells were incubated at 39°C Exponentially growing tsFT20 cells (•)and wild-type cells (O) were shifted up to 39'C and cultured for the indicated periods. The preparation of crude cell extracts for 4 h and further incubated at 33°Cfor 20 h. Colcemid was added at a concentration of 0.08 Mg/ml to the culture, and the cells were incubated at 33°Cfor 6 h. The cells were collected and fixed on a glass slide as described under "Assays for Chromosome Aberrations and Mitotic Index." Sister chromatids were differentially stained by a modified method of Perry and Wolff (18) as follows. The glass was dipped in 30 /¿g/mlHoechst 33258 solution at room temperature for and assay for DNA polymerase a were performed as described previously (12). (b) Changes in colony-forming ability of exponentially growing tsFT20 cells, synchronized tsFT20 cells, and synchronized wild-type cells. Cells were synchro nized at the d-S boundary with FdUrd as described under "Materials and Methods." After the release from the FdUrd block, the cells were incubated at 39"C for the indicated periods and assayed colony-forming ability as described under "Materials and Methods." Bars, SE. Synchronized tsFT20 cells (•),ex ponentially growing tsFT20 cells (•),wild-type cells (O). 5163 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY IN tsFT20 CELLS F a Fig. 2. Chromosome aberrations induced in tsFT20 cells by incubation at the restrictive temperature. Exponentially growing tsFT20 cells were incubated at 39'C for 4 h and then incubated at 33'C for 24 h in the presence of 0.1 <¿g/mlColcemid. (a) Slightly damaged metaphase figure containing chromatid gaps or breaks (Arrows ill, and J) and chromatid interchanges (Arrows A-D, and /); (¿>) metaphase figure exhibiting slight complex-type chromosome aberrations, in which several chromatid interchanges and breaks are observed; (c) and (</) metaphase figures exhibiting typical complex-type chromosome aberrations; (e) metaphase figure exhibiting pulverization-type chromosome aberrations. Bars, 10 t/m. 5164 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY IN tsFT20 CELLS fsfÃ-ÕL.* ^«^ AM--^ '%^>^ ?.'£)& "*¿W^ t * ¿e.A - :^ - • ^» \ ** ^i. 'W » x. V Fig. 3. Relationship between the induction of chromosome aberrations and DNA replication. Exponentially growing tsFT20 cells were incubated at 39'C for 4 h and pulse-labeled with ['Hjthymidine for 10 min. After being washed, the cells were cultured at 33*C for 24 h in the presence of Colcemid and fixed. Metaphases containing normal chromosomes and pulverization-type chromosomes and that exhibiting complex-type chromosome aberrations, which were stained with Giemsa, are shown in (<•) and (¡I).The samples were destained with isopropyl alcohol and processed for autoradiography. (a) and (h) are autoradiograms of the same metaphases of (c) and (</), respectively. Hun, 10 »mi. chromosome pulverization forming pulverized chromosome fragments (Fig. 2e). The complex-type chromosome aberration and chromosome pulverization characterized the chromosome aberration induced in tsFT20 cells by exposing the cells to the restrictive temperature, and such aberrations were induced at very low frequency in wild-type cells and the revenants derived from tsFT20 cells (data not shown). Analysis of Relationship between Induction of Chromosome Aberrations and Position of Cells in the Cell Cycle. In order to determine the relationship between the induction of chromo some aberrations and DNA replication, tsFT20 cells were pulselabeled for 10 min with [3H]thymidine after incubation at the restrictive temperature for 4 h and then incubated at the per missive temperature for 32 h in the presence of Colcemid. Fig. 3 shows metaphase figures exhibiting chromosome ab errations (c, pulverization type; d, exchange type) and autora diograms of the same metaphases containing silver grains (a, lì). From the results summarized in Table 1, it is revealed that all metaphases exhibiting chromosome aberrations contain sil ver grains, suggesting that chromosome aberrations are induced only in the cells which were in the S phase when they were incubated at the restrictive temperature. On the other hand, the Table 1 Relationship between DNA synthesis and the induction of chromosome aberrations in lsFT20 cells by incubation at the restrictive temperature Exponentially growing tsFT20 cells were pulse-labeled with [3H]thymidine for 10 min after incubation at 39'C for 4 h. After washing, mitotic cells were accumulated at 33*C for 32 h in the presence of Colcemid. They were processed as described under "Materials and Methods." no.Sample Chromosome aberration Grain (+) Chromosome aberration Grain (— ) Chromosome aberration Grain (+) Chromosome aberration Grain (-)Sample 1103 2118 3103 (+) (+) 0 0 0 (— ) 92 116 91 (— ) 55Cell 95Sample 67 metaphases containing silver grains do not always show chro mosome aberrations. To analyze in detail susceptible points to induce chromosome aberrations in the cell cycle, we have performed experiments the scheme of which is shown in Fig. 4. At the indicated times after release from synchronization, cells were shifted to 39°C for 2 h and then incubated at 33°Cfor 24 or 32 h in the presence 5165 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY IN tsFT20 CELLS of Colcemid. As shown in Fig. 5, the cells which were shifted to 39°Cduring S phase exhibited various types of chromosome 20 aberrations at high frequencies (Fig. 5; a, gap or break type; b, exchange type; c, pulverization type; d, complex type). The classification of chromosome aberrations is described under "Materials and Methods." The exchange type consisted of 10 0 20 2h 39 «C 10 0 20 10 33 «C Xh K h 0 colcemid 40 -»32 h release FdUrd sample fixation 20 Fig. 4. Scheme for analysis of the relationship between the induction of chromosome aberrations and the position of cells in the cell cycle. ¿n^FinriH nrppn-,Pn„ r-, n 20 n n I 3 4 S 6 32 Time after n_ n 1 1 • Time otter release from Z 3 i 5 S E B 10 cells. 2 h or 4 h sample fixation it inn it 33 *C Kh t I l t t t t colcemid ( 2h or Ah interval FäUrd synchronization 40 ( h ) ( h ) Fig. S. Histograms indicating the frequencies of various types of chromosome aberrations induced at various phases in the cell cycle. Experiments were per formed as described in Fig. 4. The histograms indicate the frequencies of gap- or break-type (a), exchange-type (b), pulverization-type (c), and complex-type (d) chromosome aberrations observed in mitotic cells which had been incubated at 33°Cfor 24 h (solid column) or for 32 h (broken column) after temperature upshift for 2 h at the indicated times. The phases of cell cycle in which the majority of the synchronized cell population exist are shown under these columns by arrows. The classification of chromosome aberration types is described under "Materials and Methods." Lower columns, tsFT20 cells; upper columns, wild-type 39'C 32 type. The classification of the chromosome aberration types is described under "Materials and Methods." 12 ..-02-M-».—G1-» synchronization from 24 Fig. 7. Histograms indicating the temporal order of the appearance of various types of chromosome aberrations in tsFT20 cells. Experiments were performed as described in Fig. 6. Shadowed regions, period of temperature upshift (2 and 4 10-(a)(</) andand (g),( ;'), Mitotic index; (b)(c) andand (A),(A), gappulverization or break type;type; (c) and type; complex type; (/) (i), andexchange (/), ring H O release 16 40 î ) release Fig. 6. Scheme for analysis of the temporal order of the appearance of various types of chromosome aberrations. mainly interchange-type chromosome aberrations, especially chromatid interchanges and a small number of chromosome/ chromatid interchanges. The cells shifted to the restrictive temperature during non-S phase and wild-type cells showed few chromosome aberrations. It was observed that the metaphases exhibiting heavy chromo some aberrations, complex type and pulverization type, in creased as the cells were incubated for longer period at 33°C after temperature upshift. Temporal Order of the Appearance of Various Types of Chro mosome Aberrations. In order to analyze the temporal order of the appearance of various types of chromosome aberrations, the experiment designed as shown in Fig. 6 was performed. The cells synchronized by the treatment of FdUrd were shifted to 39°Cfor 2 or 4 h after the release from the FdUrd block and then harvested at 2- or 4-h intervals to measure mitotic index and the frequency of each type of chromosome aberration. The results obtained with tsFT20 cells are shown in Fig. 7. Chromosome aberrations appeared in the cells exposed to the restrictive temperature for 2 h as the following order: gap or break type (mode time, 8-10 h); exchange type (14-16 h); pulverization type (18-20 h); complex type (20-22 h); and ring type (38-40 h). Double minute-like chromosomes were often 5166 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY IN tsFT20 CELLS X 30 a/ TJ c 20 u o ¡I IO (b) O 20 IO u C 3 CT O 20 .c o 16 O C O» D a> ov> o ok_ Time after release IO er 32 from synchronization ( h ) cu Fig. 9. Schematic illustration of the fluctuation pattern of mitotic index and various types of chromosome aberrations. The curves were drawn according to the results shown in Fig. 7 (a-f). (a) Mitotic index; (b) chromosome aberrations; Curve A, gap or break type; Curve B, exchange type; Curve C, complex type; Curve D, pulverization type; Curve E, ring type. 40 20 40 Time after release from synchronization ( h ) Fig. 8. Histograms indicating the temporal order of the appearance of various types of chromosome aberrations in wild-type cells. Experiments were performed as described in Fig. 6. The shadowed regions indicate the period of temperature upshift (2 h). (a) Mitotic index; (A) gap or break type; (c) exchange type; (d) complex type. The classification of the chromosome aberration types is described under "Materials and Methods." and exchange type (Fig. 8c), were detected at a considerable frequency but few complex-type aberrations (Fig. Sd) and no pulverization-type aberrations were observed in these cells. The aberrations observed in the wild-type cells may be mainly due to thymidylate stress by the treatment of FdUrd as described previously (6). For easy understanding of the temporal order of the appear ance of various types of chromosome aberrations, Fig. 9 shows the fluctuation pattern of mitotic index and various types of chromosome aberrations, which was made on the basis of the results shown in Fig. 7. Effect of Cycloheximide on the Induction of Chromosome Aberrations in tsFT20 Cells. It has been observed that addition of cycloheximide prevents cells from thymineless death and decreases the frequency of chromosome aberrations induced by thymidylate stress (10, 19). Therefore, we examined the effect of cycloheximide on the induction of chromosome aberrations in tsFT20 cells exposed to the restrictive temperature. tsFT20 cells were incubated at 39°Cfor 4 h in the presence or absence of cycloheximide, and mitotic cells were accumulated by an incubation at 33°Cfor 32 h in the presence of Colcemid. observed in the cells containing ring-type chromosome aberra tions (data not shown). In the cells incubated for 4 h at the restrictive temperature, the temporal order of the appearance of various types of chro mosome aberrations was the same as that of 2-h-exposed cells but it was observed that appearance of heavy aberrations such as complex type and pulverization type was retarded as com pared to that of 2-h-exposed cells. The time courses of the appearance of chromosome aberrations in wild-type cells are shown in Fig. 8. Slight aberrations, gap or break type (Fig. 8A) As shown in Table 2, the induction of heavy chromosome aberrations (complex-type) was repressed dramatically by the addition of cycloheximide. It was found that the addition of cycloheximide alone had little effect on the induction of chro mosome aberrations as reported previously (10) (data not shown). Induction of Sister Chromaticl Exchanges in tsFT20 Cells Exposed to the Restrictive Temperature. From many studies on SCE, it has been suggested that SCE is a specific phenomenon related to DNA replication (20-23). Therefore, the induction 5167 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY IN tsFT20 CELLS Fig. 10. Concurrent occurrence of SCEs and chromosome aberrations. Samples were prepared as described under "Materials and Methods." Bars, 10ptn.(a) Metaphases exhib iting slight complex-type aberration; (/>) metaphase exhibiting heavy complex-type aber ration; IM metaphase containing two sites of exchange-type aberration; (¡I)control meta phase (cultured at 33*C). Table 2 Effect of cycloheximide on the induction of chromosome aberrations in tsFT20 cells Exponentially growing cells were incubated at 39'C for 4 h in the presence or Fig. 5 clearly indicate that chromosome aberrations were in duced only in the cells synthesizing DNA at the time when they were exposed to the restrictive temperature. In addition, it is absence of 3 jig/ml cycloheximide. After the cycloheximide was washed, mitotic cells were accumulated by incubation at 33'C for 32 h in the presence of Colcemid. apparent from the results shown in Fig. 5 that the cells in early and middle S phase are more sensitive to the chromosomeFrequency of chromosome aberrations damaging effect than the cells in the late S phase. It seems of possible that the cells in the late S phase had already proceeded exchange2.53.03.11.83.7Gap, break1.51.55.20.82.3 ConditiontsFT20 into G2 before the influence of temperature upshift became cellsNon+ effective. The relatively high frequency of the induction of chromosome aberrations in the next d phase may be due to Cycloheximide+ Cycloheximide"Wild-type the S-phase cells contaminated in the population because of the decay of synchronization. cellsNon+ The analysis of the temporal order of the appearance of CycloheximideComplex60.50.90.400Types various types of chromosome aberrations revealed that slightly * Cycloheximide was added l h before the temperature upshift. damaged aberrations such as gap or break type appear earlier than heavily damaged aberrations such as complex and pulver of SCEs in tsFT20 cells was examined in relation to chromo ization type (Figs. 7 and 9). The observation that the peaks of complex-type and pulverization-type chromosome aberrations some aberrations. Fig. 10 shows metaphase figures exhibiting typical complexappeared later than the peak of mitosis, which was derived from the cells synchronized at d-S boundary, suggests that the cells type aberrations derived from the cells which have been exposed at 39°Cfor 4 h and then incubated at 33°Cfor 26 h. Apparently exhibiting heavy chromosome aberrations are late in traversing S phase. The gap- or break-type chromosome aberrations that a large number of SCEs are observed in the aberrant chromo somes. As for metaphases exhibiting complex-type and pulver appeared soon after the release from the FdUrd block may be ization-type chromosome aberrations, hyperinduction of SCE derived from cells that were in late S phase during treatment of was observed without exception. FdUrd. From the studies on chromosome aberrations induced by Xray, which is known to cause double-stranded DNA lesions, it DISCUSSION has been suggested that DNA lesions on chromosomes before In this study, we have shown that tsFT20 cells, which have replication produce isochromatid-type aberrations, and DNA lesions after replication produce chromatid-type aberrations ts defects in the DNA replication enzyme, are rapidly commit (24, 25). The majority of aberrations induced in tsFT20 cells ted to cell death and exhibit extensive chromosome aberrations were chromatid-type; therefore, it seems likely that the DNA when the cells are exposed to the restrictive temperature. Re markable aberrations induced in these cells were complex type, lesions that cause the aberrations were induced on newly repli which contains a large number of chromai id gaps, breaks, and cated enromadas. However, the other possibility that singleexchanges, and pulverization type, containing a large number stranded lesions were induced on nonreplicated DNA may also of fragmented chromosomes. The results shown in Table 1 and be probable. 5168 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. ANALYSIS OF CHROMOSOME INSTABILITY Ring-type chromosome aberrations were often observed in the metaphases containing double minute chromosomes which belong to isochromatid-type aberrations (not shown). The mechanism of formation of the pulverized chromosomes is not clear at present; however, it must be noted that the morphology of the pulverized chromosomes is very similar to that of pre mature condensed chromosomes of late S-phase cells (26). The precise mechanism by which DNA lesions are induced in tsFT20 cells is unknown. The observations that a large number of SCEs were induced in the cells with chromosome aberrations (Fig. 10) and that cycloheximide inhibited the in duction of chromosome aberrations (Table 2) may provide a clue to consider the mechanism. It is known that SCEs are formed via DNA double-strand breaks. One can speculate that the mechanism which induces DNA strand breaks in tsFT20 cells is as follows. Upon temperature upshift, although DNA replication once started can proceed normally within a replicón, the initiation of DNA replication at replicónorigins is inhibited due to the inactivation of DNA polymerase a as reported previously (16). Under these conditions, replication forks may be arrested near or at the termination points of active replicons or replicónclusters adjacent to inactive nonreplicated replicons, and subsequent persistence of single-stranded gaps is suscepti ble to endonuclease attack. Furthermore, replicónorigins which are activated to initiate replication but cannot initiate due to limiting supply of functional DNA polymerase a may become the target of endonuclease attack. The inhibiting effect of cycloheximide on the induction of chromosome aberrations can be explained by the decrease in activated replicons because the initiation of DNA replication at replicón origins is suggested to be dependent on protein(s) which turn over very rapidly (27, 28). However, it also may be possible that the abnormal cessa tion of DNA replication induces an enzyme or a protein in volved in DNA strand breaks and that cycloheximide blocks the synthesis of such an inducible protein. Several studies on chromosomal instability have been per formed with mammalian cell mutants. Besides tsF'l 20 cells, only a small number of mutants with defects in DNA poly merase a activity have been reported (13, 29-31), and one of the mutants, aph'-4 strain isolated from Chinese hamster V79 cells, which contains aphidicolin-resistant DNA polymerase a, has been shown to exhibit chromosome instability as well as hypermutability and UV sensitivity. Hori et al. (11) have re ported that extensive chromosome aberrations were induced by thymidylate stress in thy" mutants isolated from FM3A cells. Under slight thymidylate stress, the induction of somatic mu tations at two genetic loci (the 6-thioguanine-resistant locus and the ouabain-resistant locus) was observed in the thy" mu tant (32). In addition, it is well known that the interruption of DNA synthesis by the treatment of inhibitors for DNA precur sor synthesis induces chromosome instability and sometimes results in gene amplification to produce drug-resistant mutants (33). 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Cell, 37: 705713, 1984. 5170 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research. Characterization of Chromosome Aberrations Induced by Incubation at a Restrictive Temperature in the Mouse Temperature-sensitive Mutant tsFT20 Strain Containing Heat-labile DNA Polymerase α Toshihiko Eki, Takemi Enomoto, Yasufumi Murakami, et al. Cancer Res 1987;47:5162-5170. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/47/19/5162 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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