Control of cell cycle progression in human mesothelioma ... - Nature

Oncogene (2001) 20, 1085 ± 1093 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Control of cell cycle progression in human mesothelioma cells treated with gamma interferon C Vivo1, F LeÂvy1, Y Pilatte1, J Fleury-Feith1,2, P ChreÂtien1,3, I Monnet1,4, L Kheuang1 and MC Jaurand*,1 1 INSERM E 99.09, UniversiteÂ Paris Val de Marne Paris XII (EA 2345), FaculteÂ de MeÂdecine, 8 rue du GeÂneÂral Sarrail, 94010, CreÂteil Cedex, France; 2Service d'Histologie et Biologie Tumorale, HoÃpital Tenon, rue de la Chine, 75020, Paris, France; 3Service d'Immuno-heÂmatologie, Centre Hospitalier Intercommunal 40, rue de Verdun, 94010, CreÂteil Cedex, France; 4Service de Pneumologie, Centre Hospitalier Intercommunal 40, avenue de Verdun, 94010, CreÂteil Cedex, France

Recombinant human interferon gamma (r-hu-IFNg) exerts both antitumoral activity in the early stages of human malignant mesothelioma and a cytostatic eect in human mesothelioma (HM) cell lines in vitro. The antiproliferative eect of interferons (IFNs) reported in a variety of cells has been attributed to several mechanisms. In order to progress in the understanding of HM cell growth modulation by r-hu-IFNg, modi®cations of cell cycle progression and expression of key cell cycle regulator proteins in response to r-hu-IFNg were examined. Nine HM cell lines were studied, including one resistant to the antiproliferative eect of r-hu-IFNg. Except in the resistant cell line r-hu-IFNg produced an arrest in the G1 and G2-M phases of the cell cycle, associated with a reduction in both cyclin A and cyclin dependent kinase inhibitors (CDKIs) expression. Moreover cyclin B1/cdc2 activity was decreased. The present study provides the ®rst evidence of a G2-arrest in r-huIFNg-treated HM cell lines and indicates that HM cell lines, despite their tumorigenic origin still support cell cycle control. The cell cycle arrest induced by r-hu-IFNg seems to depend on cyclin regulation through p21WAF1/ CIP1 - and p27Kip1-independent mechanisms and is not directly related to the induced DNA damage. Oncogene (2001) 20, 1085 ± 1093. Keywords: Human mesothelioma cell lines; cell cycle control; cyclin dependent kinase inhibitors; cyclin; r-hu-IFNg Introduction Human diuse malignant mesothelioma is a severe neoplasm with a poor prognosis. This tumor is mainly due to asbestos exposure and its incidence will still increase in the coming years, despite the reduction of asbestos use, because of the delay between exposure and the detection of the disease (Peto et al., 1999). The

*Correspondence: MC Jaurand Received 4 April 2000; revised 14 November 2000; accepted 19 December 2000

resistance of mesothelioma to classical therapies led several groups to develop immunotherapy based on the intrapleural inoculation of therapeutical agents such as recombinant human interferon gamma (r-hu-IFNg) or other cytokines (Astoul et al., 1998; Boutin et al., 1994; Bielefeldt-Ohmann et al., 1995a,b; Bowman et al., 1991; Christmas et al., 1993; Fitzpatrick et al., 1995). Although observed in a limited number of cases, complete responses and good tolerance (Astoul et al., 1998; Boutin et al., 1994), encouraged further research on the underlying mechanisms in order to improve the therapeutical eects of cytokines. Interferon gamma (IFNg) is a cytokine produced by activated T lymphocytes and natural killer cells which mediates pleiotropic functions including antiviral and antiproliferative eects on a wide variety of cells, and immunomodulatory activities. When inoculated in the pleural cavity, r-hu-IFNg may aect mesothelioma cells by both a direct induction of tumor cell responses and/ or by indirect immunomodulatory pathways (Monti et al., 1994). We recently demonstrated that human mesothelioma (HM) cell growth may be reduced or abrogated in vitro in the presence of non-toxic doses of r-hu-IFNg, through a mechanism involving IFNg signaling pathway activation and especially the induction of the signal transducer and activator of transcription 1a (STAT 1a) (Buard et al., 1998). However, the precise mechanism whereby IFNg modulates cell proliferation remains unknown and the regulators linked to the IFNg signaling pathway have yet to be identi®ed. The antiproliferative eect of interferons (IFNs) has been reported in a variety of cells. Cell cycle analyses have most commonly reported a G1 arrest following IFN treatment. The upregulation of cyclin-dependent kinase inhibitors (CDKIs) p15, p21WAF1/CIP1 and p27Kip1 is one of the proposed mechanisms for IFN-induced cell cycle arrest. The increase in p15 seems to be cell-line restricted whereas p21WAF1/CIP1 induction appears to occur in cell lines from tissues of various origins (GrandeÂr et al., 1997). Mandal et al. (1998) reported a p21WAF1/CIP1 and both p21WAF1/CIP1 and p27Kip1 induction of expression in cells treated with IFNa and IFNg respectively. In studies comparing the response of

Effect of IFNd on cell cycle in mesothelioma cell lines C Vivo et al

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normal versus transformed or variant cells, p27Kip1 enhancement was restricted to cells showing a growth arrest in response to IFNs (Harvat et al., 1997; Moro et al., 1998) and p21WAF1/CIP1 levels were enhanced in glioblastoma cells exposed to IFNg (Kominsky et al., 1998). It has also been suggested that the antiproliferative activity of IFNs could be due to a STAT 1mediated P21WAF1/CIP1 transcription (Chin et al., 1996; Bromberg et al., 1996). The protein p21WAF1 being a CDKI known to arrest cell cycle progression in response to dierent stimuli including DNA damaging agents (El-Deiry et al., 1994), such mechanism might be relevant with IFNs, since chromosome damage has been reported in IFN-treated cells (Lazutka, 1996). Along these lines, it is known that certain cell types treated with IFNg produce nitric oxide (NO), and it was recently shown that NO produced in response to IFN was capable to induce DNA damage (Boehm et al., 1997; Jaiswal et al., 2000); then the possibility of a cell cycle control in response to a DNA alteration deserves further investigation. However, whereas the involvement of p21WAF1/CIP1 in cell growth arrest has been described in several types of mammalian cells following treatment with IFNg or a, in other cell systems p21WAF1/ CIP1 appears to be down-regulated by interferon (Harvat and Jetten, 1996) demonstrating that speci®c responses are observed in dierent cell types. In order to progress in the understanding of HM cell growth modulation by IFNg, we investigated the cell cycle modi®cations in cells exposed to r-hu-IFNg. The identi®cation of cell cycle checkpoints that can be activated by this cytokine may provide information on the molecular events involved in cell growth arrest and may provide clues on the events generating these eects. We observed that, in response to IFNg, most of the HM cell lines showed an arrest in the G1 and G2M phases of the cell cycle. Analysis of some cell cycle regulators namely, CDKI, p21WAF1/CIP1 and p27Kip1, and cyclins E, A and B1, showed that both cyclin A and CDKI protein levels were lowered by r-hu-IFNg treatment. Moreover cyclin B1/cdc2 activity was decreased in r-hu-IFNg-treated cells in comparison with untreated cells. Our results demonstrate that r-huIFNg may activate cell cycle checkpoints located both at the G1-S transition and at G2-M, through a p21WAF1/CIP1 independent mechanism. They also show that the cell cycle arrest is not directly related to DNA damage induced by r-hu-IFNg. Further research is in progress to determine the molecular mechanisms triggering these eects.

Results Effect of r-hu-IFNg on proliferation of HM cells r-hu-IFNg induced an arrest of S phase progression as assessed by dot plot analyses (Figure 1). However, while some cell lines were clearly devoid of BrdUrd-labeled cells (BL, HB), as shown by the lack of cells in the M compartment, other cell lines (QR, MR) were arrested in Oncogene

S phase since a cell population was found in the Q compartment. These cells corresponded to unlabeled cells with a DNA quantity intermediate between 2C and 4C. Moreover, a cell population exhibiting a low level of BrdUrd incorporation (QR, BT) was present in the M compartment also suggesting an arrest in S phase. The reduction of cell proliferation was con®rmed by the measurement of [3Hd]Thd incorporation. In contrast to the other HM cell lines, the proliferation of FR was not altered by r-hu-IFNg (Figures 1 and 2). In addition, dot plot revealed the presence of cells in G2-M phases even after 72 h of treatment with r-huIFNg suggesting an arrest in G2-M. Accordingly the percentage of cells in G2-M phases was higher in treated cells in comparison with untreated control, except in FR cell line (Figure 3). Statistical analyses carried out after 72 h of treatment con®rmed the G2M enhancement in four HM cell lines. Kinetic studies performed in three HM cell lines (BT, BL and HB) showed decrease in the percentage of cells in S phase associated with an increase in the percentage of cells in G2-M phase after 48 h of treatment (data not shown). Cell cycle regulators protein expression in HM cell lines exposed to r-hu-IFNg We investigated the time course of p21WAF1/CIP1, p27Kip1, p53, cyclin A, cyclin E and cyclin B expression in seven HM cell lines showing dierent levels of cell growth reduction in response to r-hu-IFNg. In the cell lines showing a cell growth reduction after treatment with rhu-IFNg, there was an overall decrease in the expression of p27Kip1, p53 and cyclin A, in comparison with untreated cells (Figures 4 and 5A, lower panels). However, the cell line MR exhibited a slightly dierent behavior since the p53 decrease was greater than in the other cell lines, and cyclin A was moderately underexpressed. Densitometric data were used to assess statistical dierences in protein expression between treated and untreated cells (Wilcoxon test). There was a statistically signi®cant decrease in the expression of p27Kip1 at all times, and after 48 and 72 h, for p21WAF1/Cip1 and p53 (P=0.031). Cyclin A was signi®cantly underexpressed after 24 and 72 h of treatment with IFNg (P=0.031) (Figure 5A). The expression of p21WAF1/Cip1 was also determined soon after treatment with r-hu-IFNg in the cell line HB. The protein levels were similar in both treated and untreated cells 15 min, 1, 3, 6 and 16 h after the beginning of the treatment (data not shown). In contrast to the IFNg- sensitive HM cell lines, FR cell line did not show changes in the level of either proteins investigated, except a moderate decrease of expression of p21WAF1/Cip1, p27Kip1 and p53, after 72 h of treatment with IFNg (Figure 4). Cyclin E expression was not modi®ed after treatment with r-hu-IFNg in all the cell lines (data not shown). Similarly, cyclin B1 expression was not signi®cantly reduced in the treated cell lines, except in QR where the level diminished to 40% after 72 h of treatment (Figure 5B).


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Figure 1 Flow cytometric analysis of r-hu-IFNg eect on DNA content and BrdUrd incorporation. BrdUrd (18 mg/ml) was added for 1 h at the end of the incubation with IFNg. Cells were ®xed in ethanol and stained with PI and FITC-labeled anti-BrdUrd. Dot plot analyses of six HM cell lines: BL, HB, QR, MR, BT, and FR (left: untreated; right: treated for 72 h with r-hu-IFNg) (500 IU/ ml). Double labeling: PI (x axis) and BrdUrd (y axis). The dierent areas N, M and P represent cells in G0-G1, cycling S cells and cells in G2-M respectively. An abnormal pattern is seen on dot plots from r-hu-IFNg-treated HB, QR and MR cells. Cells arrested in S phase are detected in Q compartment

The evaluation of the kinase activity of the cyclin/ CDK complexes is of importance to interpret the eect of r-hu-IFNg on cell cycle arrest. We determined the cyclin B1/cdc2 activity on two cell lines (BT and HB) representative of the eect of r-hu-IFNg on sensitive HM cell lines and on the resistant cell line FR. As expected, a reduction of the cyclin B1/cdc2 activity was observed in both BT and HB cell lines but not in FR (Figure 6). Therefore the enhancement in the percentage of cells in the G2-M phase observed in these cell lines suggests a G2 accumulation and agrees with an arrest of the G2-M transition. Cytogenetic analyses of HM cell lines exposed to r-hu-IFNg In view of the previously reported induction of genetic damages by IFNg in some mammalian cells (Lazutka 1996; Jaiswal et al., 2000), ®ve HM cell lines were examined in this respect (Table 1). The background level of chromosome abnormalities was low (510%) in

QR, BT and FR but BL and MR exhibited a higher rate of spontaneous chromosome abnormalities. Treatment of HM cell lines with r-hu-IFNg resulted in an enhancement of the percentage of metaphases presenting structural chromosome aberrations in all but one cell line, FR. Structural chromosome abnormalities consisted of fragments, breaks and pulverization. This enhancement was observed as early as 6 h after treatment with the cytokine. Therefore we may assume that r-hu-IFNg exerts some clastogenic eect on HM cell lines, that seems associated with the cell ability to transduce r-hu-IFNg dependent-signaling pathways involved in cell proliferation. Discussion The aim of the present work was to improve our knowledge of the eects of IFNg on mesothelioma cell proliferation. Elucidating the mechanism in speci®c cancer cell types is of great interest both to understand Oncogene


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Figure 2 [3Hd]Thd incorporation after a 1 h pulse of [3Hd]Thd in nine HM cell cultures exposed for 72 h to 500 IU/ml of r-huIFNg. The results are expressed as a percentage of [3Hd]Thd incorporation in treated cultures in comparison with untreated cultures. Mean+standard deviation of two or three experiments

Figure 3 Histograms of the percentage of HM cells in G2-M phase determined from dual parameter ¯ow cytometric analyses. HM cell cultures were treated for 72 h as described in legend to Figure 1 and Materials and methods. The results are expressed as a percentage of cells in G2-M phase in treated versus untreated cells

the speci®c growth regulation in these cells, and to precise the dierent mechanisms whereby IFNs exert their eects. A variety of mechanisms has been proposed to account for the antiproliferative action of INFs, suggesting that speci®c responses are given by dierent cell types: activation of nitric oxide (NO) synthase (Boehm et al., 1997) and indoleamine (IDO) biochemical pathways (Ozaki et al., 1988; Takikawa et al., 1988), modulation of growth factor production (Kosaka et al., 1992; Martin et al., 1993) or interaction with growth factor signaling pathways (Xu et al., 1995), down-regulation of perlecan, a member of the proteoglycan gene family (Sharma and Iozzo, 1998) or action on regulators of the cell cycle (Harvat and Oncogene

Jetten, 1996; Mandal et al., 1998; Sibinga et al., 1999). In previous studies we reported that IDO activation or NO synthase induction did not account for the antiproliferative action of IFNg in mesothelioma cell lines (Phan-Bich et al., 1997). In the present study we show that r-hu-IFNg produces a cell cycle arrest that appears to be independent of the inhibition of CDKs by p21WAF1/CIP1 or p27Kip1. Cell cycle analyses suggest that IFNg arrests HM cells in S phase since the number of BrdUrd-labeled cells was strongly reduced by IFNg, and BrdUrdunlabeled cells are found between the 2C and 4C DNA contents shown on dot plots. This was particularly obvious in the cell lines QR, MR and to a lesser extent in HB. The presence of cells in the G0-G1 compartment (N on dot plots), after as long as 72 h of treatment suggests that HM cells were also arrested in G0 ± G1. Harvat et al. (1997) and Moro et al. (1998) reported a p27Kip1-dependent growth arrest in mammary cells, and in a small cell lung cancer cell line exposed to IFNg and IFNa respectively. Similarly a p21WAF1/CIP1 induction was observed in multiple myeloma cells treated with IFNg (Urashima et al., 1997), in IFNatreated human prostate cancer cells (Hobeika et al., 1997) and Daudi cells (Subramaniam and Johnson, 1997; Mandal et al., 1998). In contrast, Gooch et al. (2000) observed a p21WAF1/CIP1-independent inhibition of cell growth in MDA-MB-231 breast cancer cell line exposed to IFNg. Moreover, Harvat and Jetten (1996) demonstrated a down-regulation of p21WAF1/CIP1 in mammary epithelial cells. Similarly, Wang et al. (1999) observed a down-regulation of p21 in human cancer cells exposed to UV-C despite a cell growth arrest. Interestingly, these authors found one cell line exhibiting an arrest in S phase (Wang et al., 1999). Our results agree with these latter ®ndings, since p21WAF1/CIP1 protein expression was reduced in IFNgsensitive HM cell lines. However, while p27Kip1 was induced in mammary epithelial cells, accounting for a CDKI-dependent G1 phase growth arrest (Harvat et al., 1997), our results suggest that CDKIs are not involved in the G1 phase arrest observed in HM cell lines since p27Kip1 protein expression was also reduced. In order to rule out the hypothesis of a biphasic response of HM cells, p21WAF1/CIP1 protein expression was studied within minutes of exposure to r-hu-IFNg. No up-regulation was observed, con®rming that CDKIs were not involved in the HM cell cycle arrest. Therefore, we may assume that IFNg does not activate the rather `typical' G1 to S control checkpoint, a hypothesis also supported by the absence of p53 stabilization. As previously shown by several authors in dierent cell types (Yamada et al., 1994; Tiefenbrun et al., 1996; Harvat and Jetten, 1996) a reduction of cyclin A expression was observed in HM cell lines in response to IFNg. Cyclin A down-regulation appeared to be concomitant with or to precede the cell cycle arrest indicating that it is a cause rather than a consequence of this phenomenon. Several mechanisms can account


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Figure 4 p21WAF1/CIP1 (a), p27KIP1 (b) and p53 (c) protein expression in response to r-hu-IFNg in HM cell lines. Cells were cultured in the absence (7) or presence (+) of r-hu-IFNg (500 IU/ml) for 24, 48 and 72 h. Forty mg of protein extracts were subjected to SDS ± PAGE (12%) electrophoresis, followed by immunoblot analysis with antibodies against the corresponding proteins. ECL detection. Top: typical Western blots of a sensitive (HB) and resistant (FR) cell line. Bottom: Densitometric analysis of p21WAF1/ CIP1 , p27KIP1 and p53 expression following IFNg treatment in six sensitive cell lines (BR, BL, HB, BT, QR, MR) and in the resistant cell line FR

for the IFNg-induced cyclin A down-regulation. IFNg could interfere with the recently described negative competitors of E2F transcription factor that interacts with cyclin A promoter (Liu et al., 1997, 1998). However evidence was recently presented by Sibinga et al. (1999) that cyclin A down-regulation observed following treatment with IFNg was more likely related to an interaction with co-activators than with regulatory sequences. Alternatively, reduction in cyclin A expression may be due to a decreased stability of the protein. When studying the control of cyclin A expression associated with cell growth arrest in response to DNA damaging agents, Guo et al. (2000) found a decrease in the protein level of cyclin A in cells exposed to benzo[a]pyrene, despite a normal activity of the cyclin A promoter. Thus altered translation or stability might account for cyclin A down-regulation. In other systems, Spitkovsky et al. (1997) also found a cyclin A down-regulation in mammalian cells exposed to cisplatin, but the regulation occurred at the transcriptional level. Thus, dierent hypotheses can be drawn and further studies would be necessary to precise the mechanism(s) involved in the modulation of cyclin A expression by IFNg in HM cell lines. In addition to the G1-phase arrest, ¯ow cytometric analyses revealed a G2 and/or M arrest in r-hu-IFNgtreated HM cells (Figure 3). This feature was not

commonly found in other IFN-treated cell types, but recent data show that keratinocytes were arrested in G2/M phase in response to IFNg (Grousson et al., 2000). The increase in the percentage of cells in G2-M phase was in agreement with the reduction in activity of cyclin B1/cdc2 in sensitive HM cell lines following exposure to the cytokine. The cyclin B1/cdc2 protein complex is an important regulator of entry into mitosis. Cyclin B1 usually shows an abrupt decrease at the onset of mitosis and is inactivated after triggering of anaphase (Cordon-Cardo, 1995; Darzynkiewicz et al., 1996). In HM cell lines, cyclin B1associated kinase activity was reduced as soon as 24 h after treatment independently of cyclin B1 expression. Two levels of regulation could account for these results: ®rst, a decrease in cyclin B1 ubiquitination (Townsley and Ruderman, 1998); second, a negative control of cdc25C activity, a phosphatase that activates cdc2 (Tiefenbrun et al., 1996). The ®rst hypothesis seems unlikely since ubiquitination appears to be a mitotic process (Townsley and Ruderman, 1998), thus the arrest in G2-M phase in HM cells is more likely mediated by cdc25C. Therefore r-hu-IFNg would produce a cell accumulation in G2 phase rather than in M phase. Given the cell cycle arrest produced by DNA damaging agents, and the occurrence of chromosomal Oncogene


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Figure 5 Cyclin A (a) and cyclin B1 (b) protein expression in response to r-hu-IFNg in HM cell lines. Cells were cultured in the absence (7) or presence (+) of r-hu-IFNg(500 IU/ml) for 24, 48 and 72 h. Forty mg of total cell lysates were subjected to SDS ± PAGE (10%) electrophoresis followed by immunoblot analysis with the corresponding antibodies ECL detection. Top: typical Western blots of a sensitive (HB) and resistant (FR) cell line. Bottom: densitometric analysis of Cyclin A and Cyclin B1 expression following IFNg treatment. See legend to Figure 4

abnormalities in several cytokine-treated cell lines (Lazutka, 1996), we studied metaphases in r-hu-IFNgtreated cell lines. Our results con®rm that r-hu-IFNg produces an enhancement of structural chromosome abnormalities including breaks, fragments and pulverization. No chromosome damage was observed in the FR cell line lacking JAK2 and therefore unable to activate the JAK/STAT signaling pathway (Buard et al., 1998). However, cell cycle arrest in HM cell lines does not appear as a consequence of DNA damage, as no induction of p53 and p21WAF1/CIP1 was observed after treatment of sensitive HM cell lines with r-hu-IFNg, despite the cells' ability to respond to clastogenic agents such as g-rays (Ungar et al., 1996 and unpublished data from our laboratory). This should be tempered by the observation that a p53-independent G2 phase arrest, associated with chromosome damage, has ben reported in cells exposed to radiation (Herzinger et al., 1995; de Toledo et al., 1998). Thus, an hypothesis may be proposed that r-hu-IFNg induces both S phase and G2 phase arrests in HM cell lines, but some cells may cross the G2 to M transition, resulting in the occurrence of abnormal cells. These cycling cells would be further arrested in G1 phase. The mechanism whereby r-hu-IFNg produces chromosome alterations is unknown. One can wonder whether the presence of IFNg in the nucleus, as shown in r-huIFNg-treated cells (Bader and Wietzerbin, 1994; Subramaniam et al., 1999) would play a role, but the mechanisms remain to be elucidated. Oncogene

In conclusion, our results demonstrate that the proliferation of HM cell lines is reduced by r-hu-IFNg through a p21WAF1/CIP1- and p27KIP1-independent cell cycle arrest. The HM cell lines so far investigated exhibit a similar trend in the type of response, a result of considerable importance regarding the potential use of r-hu-IFNg in the treatment of malignant mesothelioma. The present study provides the ®rst evidence of a G2 arrest in r-hu-IFNg-treated HM cell lines, and shows that most of the cell lines studied here responded to r-hu-IFNg. Our results also indicate that HM cell lines, despite their tumorigenic origin, still support cell cycle control mechanisms, encouraging further studies to improve our knowledge of cell cycle control in HM cells.

Materials and methods Human mesothelioma cell lines HM cell lines were obtained from pleuropneumonectomy or surgical biopsy specimens and from pleural or peritoneal eusions in patients with diuse malignant mesothelioma after patient consent. Cell lines were routinely cultured in RPMI 1640 medium with L-glutamine (Life Technologies, Inc., Cergy Pontoise, France) supplemented with 8% fetal calf serum and 10 mmol/l HEPES buer both from Life Technologies, Inc. (Cergy Pontoise, France); 50 U/ml penicillin and 50 mg/ml streptomycin (ATGC Biotechnologie, Noisy le Grand, France).


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Treatment with r-hu-IFNg r-hu-IFNg (Batch 51091; 36107 IU/mg) was a generous gift from Boehringer (Ingelheim, France). The eect of r-hu-IFNg on HM cell proliferation has already been described using an MTT assay (Zeng et al., 1993). However, the HM cell response was again determined in the present study in view of the fact that r-hu-IFNg was obtained from a dierent source and that the HM cells have been re-frozen and thawed between the two series of experiments. Cell proliferation was determined by tritiated thymidine (3HdThd) incorporation. Cell cycle analysis by flow cytometry

Figure 6 Eect of r-hu-IFNg treatment on cyclin B1/cdc2 kinase activity. Cells were cultured in the absence (7) or presence (+) of r-hu-IFNg (500 IU/ml) for 24, 48 and 72 h. HM cells were harvested and lysed as described in Materials and methods. Total cell lysates were precleared, and 300 mg of total protein were immunoprecipitated with anti-cyclin B1 antibodies. Cyclin B1/ cdc2 kinase activity was measured after washings of the immunoprecipitate using histone H1 as substrate. Proteins were boiled, transferred onto Immobilon-P membranes and the kinase activity was measured by autoradiography (Top) and immunoblotting of the same gel with anti-cyclin B1 antibodies with ECL detection (Bottom)

Table 1 Percentage of metaphases with structural chromosome abnormalities after 72 h of treatment with several concentrations of r-hu IFNg HM cell line

0

r-hu IFNg (IU/ml) 100

500

BL QR BT MR FR

16 4 4 34 8

30 8 12 60 4

22 12 2 68 0

Culture medium was replaced every 3 days. When cells were con¯uent, a mixture of Trypsin-EDTA (0.25% Trypsin, 0.02% EDTA in Ca2+, Mg2+ free phosphate buered saline, PBS) was used to detach the cells. Cells were routinely seeded at the concentrations of 56105 cells/25 cm2 tissue culture ¯ask (Costar, Dutscher, Brumath, France) and maintained at 378C. Nine mesothelioma cell lines diering by their proliferative response following treatment with r-hu-IFNg (Zeng et al., 1993; Buard et al., 1998) were used in the present study. One cell line FR, did not respond to IFNg due to a lack of JAK2 (Buard et al., 1998).

HM cells were plated at 26104 cells/cm2 in 25 cm2 tissue culture ¯asks and allowed to grow until about 50% con¯uent; thereafter medium was replaced with complete medium with or without 500 IU/ml r-hu-IFNg. To determine the number of cells in the dierent phases of the cell cycle, HM cells were double-labeled with bromodeoxyuridine (BrdUrd) and propidium iodide (PI) (both from Sigma, St Quentin-Fallavier, France) as follows: after 72 h of incubation with r-hu-IFNg, BrdUrd (®nal concentration: 18 mg/ml) was added to the culture medium for 1 h. In some experiments, the duration of incubation with BrdUrd was 15, 30 min, 1, 2, 3, 4 or 5 h. After incubation, the cells were collected by trypsinization, washed twice in PBS and ®xed in ethanol (70% in PBS) for 1 h at 48C. To determine the amount of BrdUrd incorporated, 36106 cells were resuspended in 2N HCl at room temperature for 30 min, then washed with 5 ml of 0.5% Tween 20 in PBS (PBST), centrifuged and rinsed until pH reached 7.2 ± 7.4. Thereafter 100 ml of 1 : 10 BrdUrd antibodies (Dako, Trappes, France) were incubated for 30 min at room temperature, followed by three washes with PBST. The pelleted cells were then suspended in 100 ml of mouse IgG antibodies (Dako, Trappes, France) conjugated with ¯uorescein isothiocyanate (FITC), diluted at 1 : 20 in PBST, and incubated 30 min at room temperature. Then cells were washed three times, and stained with 100 ml of PI (0.5 mg/ml) for 1 h, at room temperature, in the dark. To verify the speci®city of the FITC-conjugated antibody, the ®rst antibody was omitted from the incubation procedure. Both nuclear DNA content and the amount of BrdUrd incorporated in DNA were analysed with a ¯ow cytometer Coulter Epics XL WO5039 (Coultronics, Margency, France). The excitation wavelength was 488 nm. Red ¯uorescence emitted by PI was measured at 625 nm. Green ¯uorescence emitted by FITC was measured through a 525 nm band pass ®lter. A dual parameter histogram of the cell cycle phase distribution and calculations of the percentage of cells in G0 ± G1, S and G2-M phases were obtained by analysis of each experimental sample using XL software. Thirty thousand cells were analysed per sample. Each experiment was made in triplicate. Thymidine incorporation assay Cells were plated at 26104 cells/cm2 in 12-well plates and grown for 24 h in complete medium before treatment with 500 IU/ml r-hu-IFNg. After 72 h of treatment, cells were pulsed with 3HdThd (5 mCi/ml, speci®c activity 40 ± 60 mCi/ ml, ICN Biomedicals, Orsay, France) for 60 min. Cells were washed three times with PBS and incubated with 1 ml icecold 10% TCA for 10 min. The TCA-precipitable fraction was then solubilized in 200 ml of 0.2 M NaOH-1M SDS. 3 HdThd incorporation was determined with an LS6000SC Oncogene


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scintillation counter (Beckman, Gagny, France); the incorporation in IFNg-treated cultures was expressed as a percentage of incorporation in untreated control cultures. Cell cycle protein expression Cell extracts HM cells were grown in 75 cm2 ¯asks and treated with 500 IU/ml r-hu-IFNg for 24, 48 and 72 h. A control without r-hu-IFNg was carried out for each time. To prepare extracts, the cell layer was washed three times with 10 ml PBS and scraped with a lysis buer (1 mM EDTA, 5 mM NaF, 1% NP40, 1 mM sodium orthovanadate, 1 mg/ml leupeptin, 25 mM NPGB, 5 mM sodium pyrophosphate, in PBS). Cells were further lysed by repeated passages through a 21-gauge needle, and lysates were cleared by centrifugation at 16 0006 g for 15 min at 48C. Western blots The following antibodies were used: Primary mouse monoclonal antibody against human p21WAF1/CIP1, p27Kip1 and cyclin B1 (Transduction Lab, Med Gene Science, Pantin, France), and p53 D01 (Santa Cruz, Tebu, Le Perray en Yvelines, France). Rabbit polyclonal antibody against human cyclin E was purchased from Santa Cruz. Forty micrograms of total protein extract were boiled for 5 min in SDS loading buer with 1% b mercapto ethanol, and loaded onto 7.5 ± 12% SDS-polyacrylamide gel (SDS ± PAGE), depending on the protein type. After electrophoresis, proteins were transferred electrophoretically to an ImmobilonP membrane (Millipore, St Quentin en Yvelines, France). The protein transfer was controlled with a red ponceau coloration, and the membrane was blocked with 5% non fat milk in PBST. Incubation was carried out for 1 h at room temperature. Peroxidase conjugated anti-mouse IgG secondary antibody (Santa Cruz) was used at 1 : 2000 dilution in PBS containing 5% non fat milk and 0.5% Tween 20. The incubation was performed for 1 h at room temperature and followed by a washing step for 30 min in PBS. Detection was done by the ECL method (Amersham, Life Science, Les Ulis, France). Protein kinase assay Cell extracts were prepared as described above and precleared by adding 0.1 vol of protein A-Sepharose beads, (Pharmacia Biotech, Orsay, France) and rocking for 30 min at 48C. Protein concentrations were determined using the DC protein assay (Bio-Rad, Ivry sur Seine, France) and normalized by the addition of lysis buer. 500 mg of protein extracts were incubated for 2 h at 48C with 3 mg of cyclin B1

antibodies (Santa Cruz, Tebu, Le Perray en Yvelines, France). Immunocomplexes were collected on protein-ASepharose and washed three times with an immunoprecipitation buer (40 mM HEPES pH 7.4; 8 mM MgCl2, 100 mM NaCl, 0.5% Nonidet P-40, 1 mg/ml aprotinin, 1 mg/ml leupeptine) and twice with kinase buer (40 mM HEPES and 8 mM MgCl2). Twenty microliters of reaction buer (40 mM HEPES, 8 mM MgCl2, 166 mM ATP, 5 mCi [g32P]ATP, 10 mg histone H1) were added to the pellet and the mixture was incubated at 308C for 10 min. Reactions were stopped by adding an equal volume of 26sample buer and boiling for 5 min. Subsequently, proteins were run on a 10% SDS ± PAGE gel followed by transfer onto ImmobilonP membranes (Millipore). The membrane was autoradiographed on a hyper®lm-MP (Amersham; Orsay, France) at 7808C, and blotted with cyclin B1 antibodies. Cyclin B1 was detected by the ECL method as described above. Cytogenetic analyses For chromosome studies, cells were treated with 500 UI/ml rhu-IFNg for 6, 24, 48 and 72 h, and exposed to 10 mg/ml colchicine for 30 min. Then cells were treated with KCl for 20 min, and ®xed in methanol/acetic acid (3 : 1) according to a procedure already described (Jaurand et al., 1986). The cells were then air-dried onto glass slides and stained with Giemsa (RAL, Paris, France). Scoring of 50 chromosome spreads permitted the determination of the number of chromosomes and types of aberrations. Statistical analyses Statistical analyses were carried out using appropriate w2 tests and Wilcoxon analyses with a GraphPad PRISM1 software, V2.0 for MacIntosh. Scanning densitometry was performed using Image Assistant (Omnipage Professional, Version 5.0, Caere Inc., MuÈnchen, Germany), and quanti®cations were done with Scan analysis (Version 2.0, Biosoft, Cambridge, UK).

Acknowledgments We gratefully thank Boehringer Ingelheim, France, for providing r-hu-IFNg. We also thank Margaret Lefevre for her help checking the English language of this manuscript. This work was supported by INSERM funds and a grant from the Ligue Nationale contre le Cancer, ComiteÂ du Val d'Oise. C Vivo was a fellow of the Association pour la Recherche contre le Cancer.

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