EFFECT OF IONIZING RADIATION ON CELL-CYCLE PROGRESSION AND CYCLIN B1 EXPRESSION IN HUMAN MELANOMA CELLS

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1 Int. J. Caner: 66, (1996) Wiley-Liss, In. i Publiation of the International Union Against Caner Publiation de I'Union lnternationale Contre 18 Canel EFFECT OF IONIZING RADIATION ON CELL-CYCLE PROGRESSION AND CYCLIN B1 EXPRESSION IN HUMAN MELANOMA CELLS Raffaella VILLA*, Nadia ZAFFARONI~, Alessandra BEARZATTO', Aurora COSTA', Adele SICHIROLLO~ and Rosella SILVESTRINI~J 'Divisione di Onologia Sperimentale C, zdivisione di Fisia Sanitaria, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy. In the present study we investigated the effet of y-irradiation (2.5 and 10 Gy) on ell-yle progression of a human melanoma ell line, M 14, haraterized by a moderate radiosensitivity (SF2 = 0.5). Flow ytometri analysis showed a dose-dependent S-phase aumulation, whih was detetable 8 hr after treatment with 2 and 5 Gy and was still persistent at I2 hr after I0 Gy exposure. Suh a delay in S-phase was paralleled or followed by an aumulation of ells in G2M, whih was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Suh an aumulation was, at least in part, due to a blok in G2-M transition, as demonstrated by mitoti index analysis. Bivariate flow ytometri analysis of DNA ontent and ylin B, expression showed that, following 2 and 5 Gy, the fration of ylin B,-expressing ells was superimposable upon that of G2M ells. Conversely, in ells treated with 10 Gy, the fration of ylin BI-expressing ells was half the G2M ell fration. Northern-blot analysis indiated that the radiation-indued derease in ylin B, protein expression was aompanied by a redued ylin 6 mrna level. On the whole, our results indiate a diret inhibitory effet of 10 Gy irradiation on ylin B, expression as a possible ause for the persistent G2 blok in irradiated MI4 ells. o 1996 Wiley-Liss, In. Several experimental studies on mammalian ell lines hav demonstrated that ionizing radiation indues a division delay as a onsequene of several events (Maity et al., 1994). Speifially, a G1-phase arrest has been observed after moderate radiation doses in slowly growing human ells as well as after stimulation of non-dividing ells (Nagasawa et al., 1991), whereas an S-phase delay has been seen after relatively high radiation doses (Nagasawa et al., 1980; Wang and Iliakis, 1992). A G2-phase arrest has been onsistently observed in asynhronously and synhronously growing mammalian ells, regardless of the radiation dose (Su and Little, 1993). There is some evidene to link suh a G2 delay with the response of ells to the killing effet of ionizing radiation. In partiular, it has been observed that irradiation auses a more marked G2 delay in radio-resistant than in radio-sensitive lines (Nagasawa et al., 1994). Suh a finding has led to the hypothesis that a GZ delay would allow repair of lethal damage before entry into mitosis, thereby inreasing the hanes of ell survival (Bates and Lavin, 1989). Reent studies on synhronized HeLa ells (Mushel et al., 1993) and rat embryo fibroblasts (Markiewiz et af., 1994) have shown that the Gz-phase delay indued by radiation is assoiated with some alterations of biohemial events responsible for triggering mitosis, suh as formation of the ylin B-p34 d2 kinase omplex (Hartwell and Weinert, 1989; Mushel et al., 1991; MKenna et al., 1991; Bernhard et al., 1994). Speifially, the results indiated that, in synhronized ells, ionizing radiation an indue a delay in ylin B expression at the mrna or the protein level, depending on the phase of the ell yle at whih ells are exposed (Mushel et al., 1993). In the light of these findings, the present study investigates the effet of different radiation doses on ell-yle progression and ylin B, expression in an asynhronously growing human mefanoma ell line. MATERIAL AND METHODS Cell line The study was performed on the M14 human malignant melanoma ell line (Badarao et al., 1981). Cells were maintained in logarithmi growth phase at 37 C in a 5% COz humidified atmosphre in air, using RPMI medium supplemented with 10% FCS, 2 KM L-glutamine and 0.04% gentamyin. During exponential growth, M14 ells have a doubling time of about 24 hr. All experiments were performed within 10 passages after thawing. irradiation Exponentially growing ells were irradiated at room temperature using a 137Cs y-irradiator (IBL-437; Oris, Frane), at a dose rate of 10 Gylmin. Immediately after irradiation and at various times thereafter, ells were harvested with trypsin: EDTA (0.005:0.02%) and proessed for olony formation experiments or flow ytometri analysis as outlined below. Colony formation assay After treatment with 2-8 Gy ionizing radiation, M14 ells were plated at appropriate onentrations in plasti dishes and inubated at 37 C in an atmosphere of 5% C02,95% air for 10 days. Colonies, onsisting of at least 50 ells, were stained with rystal violet in 70% v/v ethanollwater and ounted under the mirosope. Plating effiieny was alulated from the number of olonies ounted and the number of morphologially intat single ells seeded. The surviving fration of ells was determined, and the data were fitted to the multitarget single-hit model of ell inativation. Flow ytometri analysis: bivariate DNA ontentlylin BI expression Cellular DNA ontent and ylin B1 expression were determined by flow ytometry at different intervals (from 0 to 72 hr) after exposure of ells to 2-10 Gy ionizing radiation. Samples of 1 X lo6 ells were fixed in a 1:l (vlv) solution of aetone in absolute ethanol for 30 min at -2o"C, then entrifuged and washed with 70% ethanol and PBS. The ells were inubated overnight at 4 C in the presene of the monolonal antibody to human ylin B1 (PharMingen, San Diego, CA), whih was diluted 1:400 in PBS ontaining 1% BSA. Cells were then washed and inubated with fluoresein isothioyanate (F1TC)- onjugated goat anti-mouse IgG antibody (Sigma, St. Louis, MO) diluted 1:40 in PBS ontaining 1% BSA. Cells were washed again, resuspended in 10 pglml of propidium iodide (PI) and 0.1% RNase A in PBS, and inubated at room temperature for 30 min in the dark prior to measurement (Gorzya et al., 1992). The negative ontrol was proessed in a similar way, exept that isotype-speifi antibody (mouse IgGl; Sigma) was used instead of the ylin B1 antibody. Cylin B,-expressing ells were defined using a gate window on?to whom orrespondene and reprint requests should be sent, at the Divisione di Onologia Sperimentale C, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1,20133 Milan, Italy. Fax: (39) Reeived: August 4,1995 and in revised form November 23,1995.

2 CYCLIN B, EXPRESSION AFTER IRRADIATION 105 ontrol samples, and the same window was also used for treated samples. The fluoresene of stained ells was measured using a FACSan flow ytometer (Beton Dikinson, San Jod, CA) equipped with an argon laser at 488 nm 80 r G1 1 I I I 0.1 S C 0 U 2 Ol. > L I3 m : r G21M 8o Lo t 6o 20 I I I I I dose(gy1 FIGURE 1 - Clonogeni ell survival of M14 ells. Samples were run in tripliate, and data points represent mean values from 3 independent experiments. SD were always within 5% I I FIGURE 2 - Cell-yle distribution of M14 ells at different intervals after treatment. Control ells (0); ells irradiated with 2 Gy (A); 5 Gy (m) and 10 Gy (0). Data represent mean values from 3 independent experiments. I U - E 50 s g o FIGURE 3 - Variations with time in the mitoti index of M14 ells exposed to 2 Gy (hathed olumns), 5 Gy (open olumns) and 10 Gy (dotted olumns). Values are expressed as the perentage of the mitoti index of ontrol ells at the same time. Samples were run in tripliate, and data represent mean values from 2 independent experiments. S.D. were always within 1%.

3 I I. 106 VILLA E TA. wavelength exitation. The red (PI) and green (FITC) emissions from eah ell were deteted separately using the standard optial filter pak of the FACSan. PI and FITC fluoresene signals were olleted in a linear mode. Data were aquired and proessed with LYSIS I1 software (Beton Dikinson). Cell doublets and higher aggregates were distinguished from single ells on the basis of the differential width and height of the eletroni pulses (Sharpless et al., 1975). A minimum of lo4 ells was measured for eah sample. The perentage of ells in the different yle phases was evaluated on DNA plots by means of Cell Fit software aording to the SOBR modl (Bton Dikinson). Mitoti index Cells were harvested and proessed with the standard proedure for mitosis analysis at different intervals (from 8 to 72 hr) after the exposure of M14 ells to 2-10 Gy ionizing radiation. Briefly, ells were inubated in 75 mm potassium hloride for 20 min at 3TC, entrifuged and fixed in methanolaeti aid (3:l) for 20 min at -20 C. Slides were air-dried overnight and stained with Giemsa. The mitoti index was determined by soring at least 1,500 ells on eah sample. RNA extration and Northern-blot hybridization Total ellular RNA was extrated aording to the method of Chomzynski and Sahi (1987). Twenty kg of RNA wre denatured with formaldehyde and formamide and eletrophoresed through a 1% agarose gel ontaining formaldehyde. The RNA was transferred to a Biotrans nylon filter (ICN Pharmaeutials, Costa Mesa, CA) by apillary blotting in 20X standard saline-itrate (SSC). After baking for 30 min at 80 C and UV ross-linking for 30 se, the filter was prehybridized in 5 x standard saline-phosphate (SSPE), 5 x Denhardt s solution (0.1% BSA, 0.1% Fioll and 0.1% polyvinyl pyrrolidone), 0.5% wiv sodium dodeyl sulfate (SDS), 50% formamid and 100 pg/ml denatured salmon sperm DNA and then hybridized in the same solution at 42 C overnight using a ylin B probe made by [32P]-dCTP random primer labeling of the 1.4- kilobase ylin B DNA (generously supplied by Dr. J. Pines and Dr. T. Hunter, The Salk Institute, San Diego, CA). The quantity of RNA loaded in eah lane was equalized by reprobing the nylon blot with human /3-atin DNA. After hybridization, the filter was washed with 2x SSC followed by IX SSC and 0.1% SDS at 55 C and then with 0.1~ SSC and 0.1% SDS at 65 C. Filters were exposed at -80 C with an intensifying sreen using Hyperfilm MP (Amersham, Bukinghamshire, UK) and developed at 2 days. The level of ylin B gene transript was alulated by optial densitometry of autoradiograms using an LKB ultrasan XL laser densitometer equipped with a Gelsan software pakage and was normalized to human j3-atin expression. RESULTS The M14 melanoma ell line showed a moderate sensitivity to y-irradiation (Fig. 1) with a surviving fration of 0.5 at 2 Gy and a DO value of 0.99 Gy. The perturbations indued by different radiation doses on the distribution of M14 ells in the different yle phases are reported in Figure 2. After exposure to 2 Gy or 5 Gy, the ells in S-phase transiently inreased at 8 hr, then progressed and aumulated in G2M at 12 hr. G2M aumulation was still appreiable after 24 hr and 48 hr in ells treated with 2 Gy and 5 Gy, respetively. Exposure to 10 Gy indued a marked aumulation of ells in S-phase at 8 hr and 12 hr, then ells moved to G*M, where they remained bloked until 72 hr. The early inrease in S-phase was paralleled by a depletion of G,-phase. The extent of suh a depletion was independent of radiation dose, whereas its reovery was inversely proportional to treatment intensity. m - C u u A DNA ontent,.. I-.>+.,I 6 DNA ontent FIGURE 4 - Representative histogram of bivariate flow-ytometri analysis of DNA and ylin B, in M14 ells either untreated (a) or at 24 hr after 10 Gy exposure (b). Signifiant ylin-b1-speifi fluoresene was present only in ells with G,M-DNA ontent. To determine whether the G2M aumulation of ells after y-irradiation was primarily due to an aumulation of ells in G2 or in M-phase, we evaluated the mitoti index at different intervals after radiation exposure (Fig. 3). A dose-dependent redution in the mitoti index of treated ells with respet to ontrol ells was appreiable, starting at 8 hr after treatment. Suh a derease was marked at 8 hr and 12 hr, with a omplete abrogation of mitoses after 10 Gy, and orresponded to the maximum aumulation in G2M-phase of ells treated with 2 Gy and 5 Gy and in S-phase of ells exposed to 10 Gy (Fig. 2). Twenty-four hours after treatment with the lowest radiation doses (2 Gy and 5 Gy), the mitoti index of treated ells attained the values observd in ontrol ells. The ability of ells to enter mitosis was also refleted by the partial repopulation of the GI ompartment (Fig. 2). Conversely, after 10 Gy

4 CYCLIN B, EXPRESSION AFTER IRRADIATION CONTROL GY 107 2oo I 1 I 1 I 1 I L GY C d - 8 a,._ > sot m I 1 1 I I logy I I I I I T L FIGURE 5 - Cell-yle distribution and ylin B1 protein expression in M14 ells at different intervals after 2,5 and 10 Gy irradiation. Symbols: G, (O), S (O), G2M (A), ylin BI (A). Data represent mean values from 3 independent experiments. Cell-yle distribution data are the same as those reported in Figure 2. exposure, a 50% redution in the mitoti index was still evident at 48 hr, in agreement with the persistene of G2M aumulation, as deteted by flow ytometry. To investigate the ellular events responsible for the aumulation of MI4 ells in Gz-phase after ionizing radiation, flow ytometri analysis of ylin B1 expression was performed. Bivariate distribution of ells stained for DNA and ylin B1 showed that th aumulation of ylin BI is restrited to ells with G2M-DNA ontent (Fig. 4). Flow ytometri ylin B1 protein expression was evaluated after different intervals from treatment (Fig. 5). In ontrols as well as in samples exposed to 2 Gy or 5 Gy, the fration of ylin B1-expressing ells was superimposabl upon the fration of G2M ells at all the tim intervals onsidered. Conversely, in ells treatd with 10 Gy, the fration of ylin B,-expressing ells showed an earlier inrease than did the G2M-phase ell fration. Starting at 24 hr after treatment, the 2 frations showed a parallel behavior but the values for the G2M-phas were twie as high as those of ylin B,-expressing ells. To understand the moleular basis of suh a disrepany, we analyzed the effet of 10 Gy irradiation on the expression of ylin B mrna, in omparison to the perentage of G2Mphase ells (Fig. 6), in untreated ells and at different intervals after 10 Gy. In ontrol ells, the 2 events showed a superimposable behavior (Fig. 6a). Conversely, in irradiated ells, a ylin B mrna level muh lower that expeted on the basis of the high perentage of G2M-phase ells was onsistently observed (Fig. 6b). DISCUSSION In the present study we investigated the effet of different y-irradiation doses, from 2 to 10 Gy, on ell-yle progression of a human melanoma ell line (M14) in asynhronous growth. Suh a ell line was haratrized by an intermediate level of radioresistane, ompared to that of other ell lines of the same histotype (Bihay et al., 1992). Ionizing radiation aused a dose-dependent S-phase aumulation of MI4 ells, whih was detetable only 8 hr after treatment with the lowest doses and was still persistent at 12 hr after 10 Gy exposure. Suh a delay in S-phase was paralleled or followed by an aumulation of ells in G2M, whih was transient after the lowest doses, and still persisted at 72 hr after 10 Gy. The radiation-indued ell-yle perturbations we observed in M14 melanoma ells were qualitatively similar to those reported in the litrature for other ell lines. In fat, a small but quantifiable radiation-indued delay in S-phase before arrest in the G2-phase has been demonstrated in different normal and tumor mammalian ell populations (Wang and Iliakis, 1992). The G2M aumulation of irradiated M14 ells was, at least in part, due to a blok in G2M transition, as demonstrated by mitoti index analysis. In mammalian ells a hekpoint for DNA damage by irradiation, resulting in a G2 blok, has been identified (Bernhard et al., 1994). Several findings suggest that suh a blok might be related to radiosensitivity (MKenna et al., 1990). In fat, affeine has been shown to overome the radiation-indued G2 blok in human squamous-ell arinoma ell lines and, as a onsequne, to redue lonogeni ell survival without affeting DNA double-strand breaks (Smeets et al., 1994). A possible mehanism by whih ionizing radiation indues dose-dependent G2 delays ould be a negative interferene with the proesses that ativate the onset of mitosis. Some proesses are ontrolld in ukaryoti ells by a omplex of ylin B and p34d2 protein kinase, alled maturation promot-

5 108 VILLA ETAL. FIGURE 6 - Cylin B mrna level (expressed in arbitrary units based on densitometri sanning of RNA blots) (0) and perentage of G2M-phase ells (0) with time in M14 ells. Cylin B mrna levels were normalized to aount for differenes in loading as revealed by probing with P-atin. (a) Control ells; (b) ells irradiated with 10 Gy. ( and d) Autoradiograms from whih the data plotted in a and b, respetively, were derived. ing fator (MPF) (Draetta and Beah, 1989). Cd2 kinase ativity is modulated by the ell-yle-dependent aumulation of ylin B, whose levels inrease in S and G2-phases. In the phosphorylated form, the MPF omplex is inative, thus preventing premature entry into mitosis of ells that have not ompletely repliated DNA. Cd2 ativation, whih ours upon tyrosine dephosphorylation by the produt of the d25 gene, permits several ritial morphologial hanges typial of G2-M transition (Lewin, 1990; MGowan et al., 1990; Nurse, 1 990). In this study we investigated whether exposure of the M14 ell line to different doses of ionizing radiation alters the expression of ylin B, protein. Results from DNAiylin B1 bivariate analysis indiated that, after 2 Gy and 5 Gy treatment, the fration of ylin B,-expressing lls ould be superimposed upon that of ells in G2-phase. Suh evidene suggests that the inability of lls to enter mitosis was not due to a derease in the expression of ylin B1 or a onsequent redution of the MPF omplex. However, it annot be exluded that the inhibition exerted by low-dose radiation (2 Gy and 5 Gy) on ylin B1 protein expression was too small to be deteted. In ontrast, after 10 Gy exposure, a seletive derease in ylin B1-expressing ells aompanied a G2-phase delay, whih suggests that an inhibition of ylin B1 aumulation ould be responsible for the impairment of ell progression into M-phase. To asertain whether or not the redution of ylin B1 protein expression after 10 Gy exposure was onsequent upon a derased transription level of the ylin B gene, we analyzed ylzn B mrna level at different intervals after irradiation. In ells exposed to 10 Gy, the level was onsistently lower than that expeted on the basis of the high perentage of G2M-phase ells. Our findings suggest a down-regulation of ylin B at the post-transriptional level. However, it should be mentioned that an early inrease in ylin B1-expressing ells was observed notwithstanding a onomitant redution of ylin B mrna level, thus suggesting a possible temporary enhanement of ylin B, protein stability after irradiation. Our data are in agreement with the findings of Datta et al. (1992) who observed a rapid and transient down-regulation of ylin B, as well as of ylin A and d2 and d25 genes, assoiated with a delay in ell-yle progression of human myeloid leukemia ells exposed to 20 Gy. A strit orrespondene between the delay in the timing of ylin B1 expression and the lngth of the G2 phase delay was observed by Mushel et al. (1993) in HeLa ells, as wll as by Markiewiz et al. (1994) in a H-ras/V-my-transformed rat embryo fibroblast ell line. Conversely, Smeets et al. (1994) failed to demonstrate any redution in ylin B expression during aumulation of SCC61 and SQ20B human squamous-ell arinoma ell lines in the GzM-phase after irradiation. However, in their report, a radiation-indued alteration in the intraellular distribution of ylin B, with a redution in the nulear aumulation of the protein, was desribed. Moreover, in these ell lines, a derease in the level of ative, dephosphorylated p34d2 kinase was found after irradiation. Taken together, the experimental data from our studis and those of others strongly suggest that down-regulation of the

6 CYCLIN B, EXPRESSION AFTER IRRADIATION 109 proesses whih ativate the MPF omplex are a possible mehanism of the arrest of irradiated ells in GZ, and that radiation imdairment of suh Droesses an varv in the different ell populations. In fat, even when a diret hhibitory effet on vfin B expression an be exluded, radiation has been seen to interfire negatively with other steps of the omplex pathway leading to an ative MPF (k, ylin B loalization, d2 kinase nulear dephosphorylation). Studies are needed to identify the speifi patterns of interation between radiation and ell-yle ontrol mehanisms as a funtion of ell type, proliferative status and radiation dose. ACKNOWLEDGEMENTS The study was supported in part by a grant from the Assoiazione Italiana per la Riera sul Canro (AIRC). The authors thank Miss B. Johnston for editing and Miss B. Canova for typing the manusript. 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