Hsp90-Inhibitor Geldanamycin Abrogates G2 Arrest in P53-Negative Leukemia Cell Lines Through the Depletion of Chk1

Hsp90-inhibitor geldanamycin abrogates G2 arrest in p53-negative leukemia cell lines through the depletion of Chk1

K Sugimoto, M Sasaki, Y Isobe, M Tsutsui, H Suto, J Ando, K Tamayose, M Ando and K Oshimi

Division of Hematology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan

Checkpoint protein Chk1 has been identified as an Hsp90 (Pearl and Prodromou, 2001; Neckers, 2002). Geldana- client. Treatment with 100 nM geldanamycin (GM) for mycin (GM), an ansamycin antibiotic, binds tightly to 24 h markedly reduced the Chk1 amount in Jurkat and the ATP-binding pocket of Hsp90 and specifically ML-1 leukemia cell lines. Because Chk1 plays a central disrupts its function, and therefore leads to the role in G2 checkpoint, we added GM to G2-arrested proteasome-dependent degradation of various client Jurkat and HL-60 cells pretreated with 50 nM doxorubi- proteins (Prodromou et al., 1997). They include steroid cin for 24 h. GM slowly released both cell lines from hormone receptors, Raf-1, Bcr-Abl, Akt and so on, doxorubicin-induced G2 arrest into G1 phase. GM also which are important components of the signaling abrogated ICRF-193-induced decatenation G2 checkpoint pathways of cell proliferation and survival (Nimmana- in Jurkat and HL-60 cells. Western blot analysis showed palli et al., 2001; Neckers, 2002; Workman, 2004). that addition of GM attenuates doxorubicin- and ICRF- Because these pathways are constitutively activated in 193-induced Chk1 phosphorylation at Ser345. GM, many tumor cells, GM is expected to have a potent however, failed to abrogate G2 arrest in p53-positive antitumor activity (Gorre et al., 2002; Minami et al., ML-1 cells maybe due to the p21 induction. GM released 2002; Bisht et al., 2003). GM and its less toxic derivative HeLa cells from doxorubicin-induced G2 arrest but 17-allylamino-17-demethoxygeldanamycin (17-AAG) trapped them at M phase. Flow cytometric analysis have been reported to inhibit tumor cell growth by showed that addition of GM converted doxorubicin- inducing apoptosis and, in certain cell types, differentia- induced necrosis into apoptosis in Jurkat cells. Colony tion (Mu¨ nster et al., 2001; Jones et al., 2004; Mitsiades assay indicated that although GM has a weak cytotoxic et al., 2006). GM is reported to enhance tumor cell effect as a single agent, it dramatically intensifies the sensitivity to various cytotoxic agents and ionizing cytotoxicity of doxorubicin and ICRF-193 in Jurkat and radiation through the inhibition of nuclear factor HL-60 cells. These results suggest that abrogation of G2 (NF)-kB activation and its downstream survival signals checkpoint by GM may play a central role in sensitizing (Lewis et al., 2000; Broemer et al., 2004). p53-negative tumor cells to DNA-damaging and decate- The most fundamental mechanism of G2 checkpoint nation-inhibiting agents. is Cdc2 inactivation by negative phosphorylation. Chk1 Oncogene (2008) 27, 3091–3101; doi:10.1038/sj.onc.1210978; plays the central role in DNA damage-induced G2 arrest published online 10 December 2007 by maintaining Cdc25C in the phosphorylated inactive form, which suppresses Cdc2 activation (Elledge, 1996; Keywords: Hsp90; geldanamycin; Chk1; G2 arrest Peng et al., 1997; Sanchez et al., 1997; Weinert, 1997; Bulavin et al., 2003). Caffeine has been shown to abrogate DNA damage-induced G2 arrest by inhibiting Ataxia telangiectasia mutated (ATM) and ATM-related (ATR) kinases, which situated upstream of Chk1 in the Introduction DNA damage checkpoint pathway (Fan et al., 1995; Powell et al., 1995; Russell et al., 1995; Yao et al., 1996; Hsp90 plays an important role in the conformational Sarkaria et al., 1999). UCN-01 is known to sensitize maturation and stabilization of several important tumor cells without functional p53to DNA damaging proteins (known as clients) and is required for the agents by abrogating G2 arrest through the direct protein refolding after exposure to various cell stresses inhibition of Chk1 (Graves et al., 2000; Zhao et al., 2002; Tse and Schwartz, 2004). Furthermore, several agents inactivating Chk1 are reported to abrogate G2 Correspondence: Dr K Sugimoto, Division of Hematology, Depart- arrest and simultaneously sensitize tumor cells to DNA- ment of Internal Medicine, Juntendo University School of Medicine, damaging agents (Kohn et al., 2003; Kawabe, 2004). Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: [email protected] Recently, Chk1 has been identified as an Hsp90 client Received 8 April 2007; revised 24 October 2007; accepted 9 November and 17-AAG-mediated depletion of Chk1 has been 2007; published online 10 December 2007 shown to sensitize tumor cells to replication stress Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3092 induced by nucleoside analog gemcitabine (Arlander was apparent only in ML-1 cells after doxorubicin et al., 2003; Bull et al., 2004). treatment (Figure 1). We therefore examined in this study whether the prototypical Hsp90 inhibitor GM functions as a G 2 GM abrogated DNA damage-induced G arrest in Jurkat abrogator in leukemia cell lines pretreated with anti- 2 cells tumor agents. GM released low-dose doxorubicin- Because GM induced considerable loss of Chk1, we treated Jurkat cells from G arrest into G phase. This 2 1 examined whether or not it acts as a G abrogator. We release from G arrest coincided with the tendency to 2 2 first treated Jurkat cells with 50 nM doxorubicin for 24 h, undergo apoptosis instead of doxorubicin-induced which trapped most of the cells in late-S to G position necrosis. 2 with almost complete loss of G1 population. Then, we added 100 nM GM and monitored their cell cycle distribution. As shown in Figure 2a, G2-arrested Jurkat Results cells began to move to the G1 position between 12 and 18 h after the addition of GM. Without GM, most of the Treatment with geldanamycin depleted Chk1 protein doxorubicin-treated Jurkat cells stayed at G2 phase for We performed western blot analysis for cell cycle-related further 24 h. Subsequent addition of 200 nM nocodazole and Hsp90 client proteins after 24 h of treatment with 12 h later, which trapped cycling cells at M phase, clearly 100 nM doxorubicin, ara-C or GM in ML-1 and Jurkat suppressed the appearance of G1 peak even after 24 h of cells (Figure 1). GM dramatically reduced the amount incubation with GM. GM released HL-60 cells from of Chk1 in addition to Raf and Akt in both cell lines. doxorubicin-induced G2 arrest essentially in the same Cdc2 amount was also reduced in these cell lines after way as Jurkat cells (data not shown). GM treatment. Furthermore, especially in ML-1 cells, Appearance of phosphorylated upper bands of considerable portion of the Rb protein became hypo- Cdc25C, Myt 1 and Bcl-2 are hallmarks of the presence phosphorylated, which corresponds well with the pre- of mitotic cells (Sanchez et al., 1997; Ling et al., 1998). vious report that GM induces G1 arrest through the Although nocodazole treatment for 12 h gave rise to the depletion of cyclin D and the resultant Rb hypopho- retarded upper bands of Cdc25C, Myt 1 and Bcl-2 in sphorylation (Srethapakdi et al., 2000). Induction of p21 rapidly growing Jurkat cells, pre- and simultaneous treatment with 50 nM doxorubicin completely eliminated their upper retardation. Addition of 100 nM GM restored upper shift of Cdc25C, Myt 1 and Bcl-2 in doxorubicin-pretreated Jurkat cells, which confirmed the release from G2 arrest and entrapment at mitosis by nocodazole (Figure 2c). Simultaneous count of mitotic ControlDoxorubicinGeldanamycinAra-C ControlDoxorubicinGeldanamycinAra-C cell percentages confirmed the release from doxorubicin- Rb induced G2 arrest by GM in Jurkat cells. We then examined the states of Chk1 in Jurkat cells treated with mock, 50 nM doxorubicin for 48 h, 100 nM GM for 24 h Raf or with the combination of them, that is, in the last condition GM was added after 24 h of treatment with Akt1 doxorubicin. GM depleted Chk1 more efficiently in Jurkat cells pretreated with doxorubicin than nontreated Chk1 ones, which correlated well with the override of DNA damage-induced G2 arrest (Figure 2d). Time course Chk2 analysis confirmed that GM reduced Chk1 level only gradually in doxorubicin-treated Jurkat cells, which is in Cdc2 good accordance with the relatively long interval between the addition of GM and release from the G2 Cyclin B1 arrest (Figure 2e). These results, which were confirmed essentially in the same condition at least three times, PCNA clearly showed that GM gradually releases Jurkat cells from the DNA-damage G checkpoint. Bcl-2 2

p21 GM abrogated decatenation-induced G2 arrest in Jurkat cells ML-1 Jurkat Because GM depleted Chk1 and thus abrogated DNA Figure 1 GM selectively depleted Raf, Akt1, Chk1 and Cdc2 in damage-induced G2 checkpoint, we next examined ML-1 and Jurkat cells. ML-1 and Jurkat cells were mock-treated whether the agent also releases Jurkat cells from the (control) or treated with 100 nM doxorubicin, 100 nM GM or 100 nM ara-C for 24 h. Cell lysates were prepared, and equal decatenation-induced G2 checkpoint. Treatment with amounts of these were separated and immunoblotted for Rb, Raf, 1.2 mM of ICRF-193for 24 h almost completely trapped Akt1, Chk1, Chk2, Cdc2, cyclin B1, PCNA, Bcl-2 and p21. Jurkat cells at G2 phase essentially without G1 population.

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3093 Addition of GM started to release Jurkat cells from cells (1.1%, data not shown). Addition of GM to 150 nM decatenation-induced G2 arrest into G1 phase in 14 h doxorubicin-treated HeLa cells induced almost the same (Figure 3a). Addition of 200 nM nocodazole 12 h later extent of upper shifts in Cdc25C, Myt 1 and Bcl-2 clearly suppressed the appearance of G1 peak even after irrespective of the nocodazole treatment (Figure 5b). 21 h of incubation with GM. GM contrasted with caffeine Confocal microscopy confirmed nuclear import of cyclin in abrogating ICRF-193-induced G2 arrest more efficiently B1 after the addition of GM in doxorubicin-treated than doxorubicin-induced one (Figure 3b). Furthermore, HeLa cells (Figure 5c). After 24 h, considerable portion western blot analysis confirmed restoration of upper- of them entered mitosis with much larger amount of retarded M phase-specific bands of Cdc25C, Myt 1 and cyclin B1 than normal mitotic HeLa cells. Therefore, Bcl-2 by GM in ICRF-193-treated Jurkat cells (Figure 3c). GM has two independent effects on the behavior of These results clearly showed that GM gradually releases doxorubicin-pretreated HeLa cells, those are, release Jurkat cells from the decatenation G2 checkpoint. Western from the G2 arrest and the following mitotic entrapment. blot analysis against phosphoSer345 Chk1 clearly demon- To inquire why HeLa but not Jurkat cells entrapped at strated that not only doxorubicin but also ICRF-193 the mitotic phase after going over the G2 arrest, we activates Chk1. Indeed, ICRF-193induced more intense examined the activation state of Plk1, an important phosphorylation of Chk1 at Ser345 than doxorubicin, and regulator of M-phase progression (Figure 5d). Although the Chk1 phosphorylation was apparently decreased by addition of GM reduced not only total but also Thr210- the treatment with GM (Figure 3d). phosphorylated Plk1 levels in HeLa cells pretreated with doxorubicin, GM rather increased the phosphorylated Plk1 level in Jurkat cells. Therefore, preserved Plk1 ML-1 cells with functional p53 were resistant to activity in spite of the decreased protein level in Jurkat GM-induced G abrogation 2 cells may enable the completion of mitosis. In contrast to Jurkat and HL-60 cells lacking functional p53, ML-1 cells were arrested not only at G2 but also at G1 phases after 50 nM doxorubicin treatment (Figure 4a). Addition of geldanamycin converted doxorubicin-induced Therefore, cell cycle analysis by flow cytometry was not necrosis to apoptosis in Jurkat cells suitable for the estimation of G2 abrogation by GM. We previously showed that treatment with 50 nM Direct count of mitotic cell percentage showed that about doxorubicin for 48 h induced necrosis in most Jurkat 2.3% of rapidly growing ML-1 cells were mitotic. Total cells without apparent apoptosis (Sugimoto et al., 2002). 48 h of pre- and simultaneous treatment with 50 nM In this study, addition of 100 nM GM at 24 h made more doxorubicin reduced nocodazole-trapped cell percentage than 40% of doxorubicin-treated Jurkat cells TUNEL from 8.7 to 1.3%. Addition of 100 nM GM failed to positive (Figure 6a). Treatment with 100 nM GM alone increase mitotic cell percentage of doxorubicin-treated for 24 h induced few, if any, Jurkat cells TUNEL- ML-1 cells (0.3% of mitotic cells, Figure 4b). Therefore, positive. Therefore, not GM per se but addition of GM ML-1 cells expressing wild-type p53, were resistant to G2 to 50 nM doxorubicin-treated Jurkat cells induced checkpoint abrogation by GM in spite of Chk1 depletion. TUNEL-positive apoptosis. Treatment with 50 nM The preservation of G2 arrest might be explained by p21 doxorubicin for 48 h enhanced intracellular reactive induction. Furthermore, addition of GM had little effects oxygen species (ROS) formation in Jurkat cells, but if any on the apoptotic cell percentage of doxorubicin- reduction of mitochondrial membrane potential (DCm) treated ML-1 cells. was absent. In contrast, addition of 100 nM GM at 24 h generated ROS-increased, DCm-decreased apoptotic cell population in about 40% of doxorubicin-pretreated GM released HeLa cells from G arrest but 2 Jurkat cells (Figure 6b). Although reduction of DC is simultaneously trapped them at M phase m not sufficient for commitment to apoptosis (Chen et al., Because Hsp90 has been recently shown necessary for 1998), combination of increase ROS level and decreased the exit from mitosis in HeLa cells (de Ca´ rcer, 2004), we DC is a hallmark of apoptosis. Therefore, flow examined the effect of GM on HeLa cells pretreated with m cytometric ROS/DC analysis also showed that GM 150 nM doxorubicin. Compared to Jurkat, HL-60 and m converts low-dose doxorubicin-induced necrosis into ML-1 cell lines, HeLa cells are relatively insensitive to apoptosis in Jurkat cells. Although Hsp90 inhibition has doxorubicin. Therefore, 150 nM doxorubicin was neces- been reported to facilitate apoptosis by blocking NF-kB sary to induce G arrest in most of the treated HeLa cells 2 activation (Lewis et al., 2000; Broemer et al., 2004), in 24 h. Although addition of 100 nM GM at 24 h during simultaneous treatment with GM rather enhanced the total 48 h of treatment with 150 nM doxorubicin NF-kB nuclear translocation in 50 nM doxorubicin- failed to change the flow cytometric cell cycle distribu- treated Jurkat cells (Figure 6c). Therefore, at least in tion of HeLa cells, GM apparently increased mitotic cell Jurkat cells, facilitation of apoptosis by delayed addition percentage from 1.3to 48.4% (Figures 5a and b). The of GM cannot be explained by NF-kB inhibition. result indicated that HeLa cells overrode doxorubicin- induced G2 arrest but were then trapped at M phase in the presence of GM. Indeed, even treatment with 100 nM Addition of geldanamycin suppressed low-dose GM alone for 24 h increased mitotic cell percentage of doxorubicin-treated cell survival HeLa cells from 4.5 to 21.1%. Treatment solely with To underline the biological significance and the clinical GM for 24 h induced essentially no apoptosis in HeLa relevance of these results, we carried out the colony

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3094 assay using Jurkat and HL-60 cells treated with 50 nM reduced surviving colony number at most to 40% of doxorubicin for 48 h in the presence or absence of rapidly growing Jurkat cells (Table 1). In case of HL-60 100 nM GM during the last 24 h. As controls, rapidly cells, although the reduction of a surviving colony growing and GM-treated Jurkat and HL-60 cells were number by 100 nM GM alone was at most 67%, also plated. Surviving colony numbers of doxorubicin- subsequent addition of GM dramatically reduced the pretreated Jurkat cells decreased to almost 0% in the surviving colony number of doxorubicin-treated HL-60 presence of GM, although treatment with GM alone cells to 2–3% of that treated by doxorubicin alone.

DXR 50 nM 36 h DXR 50 nM 48 h 500 500 400 400 300 300 200 200 100 100 0 0 0 50 100150200250 0 50 100150200250

DXR 50 nM 36 h + DXR 50 nM 42 h + DXR 50 nM 48 h + GM 100 nM 12 h GM 100 nM 18 h GM 100 nM 24 h

Cell Number 500 400 300 200 100 0 0 50 100150200250 0 50 100150200250 0 50 100150200250 DNA Contents

DXR 50 nM 48 h + GM 100 nM 24 h + 36 42 48 (h) Nocodazole 12 h 500 Cell Harvest Points DXR 50 nM 400 GM 100 nM 300 Nocodazole 200 100 GM 24 h - - - + + 0 DXR 48 h - - + + + 0 50 100150200250 Noc 12 h - + ++ - DNA Contents

GM 24 h - - + + Cdc25C DXR 48 h -++ -

Chk1 Myt1

Chk2 Bcl-2 Mitotic Cell 20.8 25.2 Cdc2 Percentage 2.8 0.4 3.4 Cyclin B1 GM - - 6 12 18 (h) DXR- 24 30 36 42 (h)

Chk1

Cdc2

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3095 Almost no colonies were detected after ICRF-193plus the integrity of various client proteins. For example, subsequent GM treatment in both cell lines. These interaction with Hsp90 is shown to be necessary for the results were confirmed in three independent series of activity of Wee1, an inhibitory regulator of mitotic experiments. entry, in fission yeast (Aligue et al., 1994), although we confirmed that Wee1 level remained constant even after the addition of GM in Jurkat cells (data, not shown). Therefore, G2 abrogation by GM may not be solely Discussion attributed to Chk1 depletion. Involvement of Chk1 in the decatenation checkpoint In this study, we have shown that GM, a representative mechanism had been ruled out because of the lack of its Hsp90 inhibitor, releases Jurkat and HL-60 cells from activation after 2 h of incubation with ICRF-193in a the doxorubicin-induced G2 arrest in accordance with previous report (Deming et al., 2001). However, the Chk1 depletion. Although caffeine and UCN-01 treatment with 1.2 mM of ICRF-193for 24 h clearly usually abrogate doxorubicin-induced G2 arrest in a few induced Chk1 phosphorylation at Ser345 in Jurkat cells, hours (Powell et al., 1995; Yao et al., 1996; Sugimoto whose extent was much stronger than that induced by et al., 2000), it took more than 12 h to override the G2 50 nM doxorubicin. Furthermore, addition of GM arrest by GM, which corresponds to a rather lengthy attenuated the phosphorylation of Chk1 and abrogated process of Chk1 depletion by Hsp90 inhibition. These the decatenation-induced G2 arrest. These results observations are compatible with the hypothesis that indicate that both DNA-damage and decatenation reduction of Chk1 level may play a central role in G2 checkpoints should induce G2 arrest through a common abrogation by GM. Hsp90 is, however, responsible for pathway involving Chk1.

Table 1 Reduction of surviving colony number by geldanamycin in doxorubicin- or ICRF-193-pretreated Jurkat and HL-60 cells No. of surviving colonies (% of colonies without geldanamycin treatment)

Treatment Experiment 1 Experiment 2 Experiment 3

Jurkat (/102) Nontreated 36.0 36.3 38.0 100 nM geldanamycin 17.7 (49%) 14.7 (40%) 23.0 (61%)

Jurkat (/104) 50 nM doxorubicin 13.7 16.0 15.3 50 nM doxorubicin+100 nM geldanamycin 0.0 (0%) 0.0 (0%) 0.3(2%) 1.2 mM ICRF-19310.0 9.39.7 1.2 mM ICRF-193+100 nM geldanamycin 0.0 (0%) 0.0 (0%) 0.0 (0%)

HL-60 (/102) Nontreated 49.7 50.7 41.7 100 nM geldanamycin 44.0 (89%) 34.0 (67%) 29.7 (71%)

HL-60 (/104) 50 nM doxorubicin 100.7 98.7 108.0 50 nM doxorubicin+100 nM geldanamycin 2.7 (3%) 2.5 (3%) 2.3 (2%) 1.2 mM ICRF-1930.0 0.30.3 1.2 mM ICRF-193+100 nM geldanamycin 0.0 (undetermined) 0.0 (0%) 0.0 (0%)

Jurkat and HL-60 cells were treated with 50 nM doxorubicin or 2 mM ICRF-193for 24 h, then split into three, and incubated for further 24 h in the presence or absence of 100 nM geldanamycin, then cell suspension containing 1 Â 104 cells was plated. As controls, nontreated Jurkat and HL-60 cells were incubated without or with 100 nM geldanamycin for 24 h and plated at 100 cells per plate. Each number was the mean value of 3actual colony numbers of identical treatment condition after 20 days of incubation.

Figure 2 GM abrogated doxorubicin-induced G2 arrest through the depletion of Chk1. (a) Treatment with 50 nM doxorubicin exclusively induced G2 arrest in Jurkat cells, and the G2 arrest continued until 48 h of treatment (upper row). Addition of 100 nM GM after 24 h slowly but steadily released G2-arrested cells into M and G1 phases (middle row). Incubation with 200 nM nocodazole during the last 8 h clearly suppressed the appearance of G1 peak, which confirmed GM-induced release into M and G1 phases (lower row). (b) The treatment schedule and harvest points were indicated in the diagram; the open triangles indicate the harvest points for flow cytometric cell cycle analysis/TUNEL assay in (a), and the filled triangle shows the cell harvest point for western blot analysis in (c and d). (c) Jurkat cells were mock-treated, or treated with nocodazole alone for 12 h, plus 50 nM doxorubicin for 48 h or further plus 100 nM GM for 24 h and 50 nM doxorubicin plus 100 nM GM. Cell lysates of these populations were prepared and immunoblotted for Cdc25C, Myt1 and Bcl-2, which are electrophoretically retarded specifically in M phase. Mitotic cell percentages were simultaneously counted and added in the bottom column. Although 50 nM doxorubicin blocked Jurkat cells at G2 phase, further addition of 100 nM GM restored cell cycle transition from G2 into M phase. (d) Jurkat cells were mock-treated, or treated with 50 nM doxorubicin for 48 h, with 100 nM GM for 24 h or combination of both treatments. Although the decrease of Cdc2 was rather mild in the combination treatment, Chk1 was depleted more drastically in doxorubicin-pretreated Jurkat cells than single treatment with 100 nM GM for 24 h. (e) Chk1 and Cdc2 levels gradually decreased during the incubation with 100 nM GM in Jurkat cells pretreated with 50 nM doxorubicin for 24 h.

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3096 GM apparently failed to abrogate G2 arrest in ML-1 even after the depletion of Chk1 in ML-1 cells. The G2 cells expressing wild-type p53. As previously reported in checkpoint of ML-1 cells should thus consist of p53- p53-positive cells after g-radiation (Bunz et al., 1998), dependent and p53-independent pathways (Passalaris p21 should directly block the cyclin B1-Cdc2 activity et al., 1999), and GM abrogates only the latter

ICRF 1.2 µM 39 h + ICRF 1.2 µM 42 h + ICRF 1.2 µM 45 h + GM 100 nM 15 h GM 100 nM 18 h GM 100 nM 21 h 500 400 300 200 100 0 0 50 100150200250 0 50 100150200250 0 50 100150200250

ICRF 1.2 µM 45 h + GM 100 nM 21 h + ICRF 1.2 µM 24 h ICRF 1.2 µM 45 h Nocodazole 9 h Cell Number 500 400 300 200 100 0 0 50 100150200250 0 50 100150200250 0 50 100150200250 DNA Contents

Incubation 48 h + Incubation 30 h + Incubation 24 h Incubation 48 h GM 100 nM 24 h Caffeine 6 h 500 400 M

µ 300

1.2 200 ICRF-193 100 0 0 50 100150200250 0 50 100150200250 0 50 100150200250 0 50 100150200250 500

Cell Number 400 300

50 nM 200

Doxorubicin 100 0 0 50 100150200250 0 50 100150200250 0 50 100150200250 0 50 100150200250 DNA Contents

GM 24 h - - - + GM 24 h - -++ - + + ICRF 48 h - - + + ICRF 48 h - + + ---- Noc 12 h - + + + DOX 48 h - - - - + + -

pChk1 Cdc25C

Chk1 Myt1

Bcl-2

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3097 pathway(s). In contrast, intra-S phase arrest elicited by cells although apparently reduced the phosphorylated cytarabine or gemcitabine seems to be dependent almost Plk1 in HeLa cells. This difference may explain the solely on Chk1 even in ML-1 cells, and therefore readily GM-induced mitotic arrest solely in HeLa cells. abrogated by GM (Arlander et al., 2003; Mesa et al., In this study, we showed that addition of GM 2005). In case of HeLa cells, although GM abrogated converted doxorubicin-induced necrosis into apoptosis doxorubicin-induced G2 arrest, they were trapped at M in Jurkat and HL-60 cells. Colony assay conﬁrmed that phase after the G2 override. Confocal microscopy GM intensiﬁes the cytotoxic effects of doxorubicin and showed that GM-arrested mitotic HeLa cells contain ICRF-193, although GM has a weak cytotoxic effect as much larger amount of cyclin B1 than the normal mitotic a single agent. Addition of GM, however, failed to counterpart. Degradation of cyclin B1 is necessary for enhance the cytotoxicity of doxorubicin against ML-1 the exit from metaphase, and this process is mainly cells, although GM or its derivatives has been shown to regulated by Plk1 (King et al., 1995; Descombbes and sensitize ML-1 cells to cytarabine and gemcitabine Nigg, 1998). Recently, Plk1 has been shown to be an (Arlander et al., 2003; Mesa et al., 2005). The different Hsp-90 client (de Ca´ rcer, 2004), and treatment with GM cytotoxic effects of additional GM in doxorubicin- indeed reduced the total Plk1 level both in Jurkat and treated Jurkat and ML-1 cells seem to correlate with the HeLa cells. However, GM rather increased phosphory- abrogation and persistence of G2 arrest, respectively. lated Plk1 at Thr210 in doxorubicin-pretreated Jurkat Even in ML-1 cells, override of intra-S phase checkpoint

Control DXR 50 nM 24 h 500 400 300 200

Cell Number 100 0 0 50 100 150 200 250 0 50 100 150 200 250 DNA Contents

Mitotic Cell Percentages of ML-1 Cells

Geldanamycin 100 nM 24 h - - + + Nocodazole 200 nM 12 h - + - +

Mitotic Cell Percentages

Mock2.3 8.7 3.7 9.3 Doxorubicin 50 nM 48 h 0 1.3 0 0.3

Apoptotic Cell Percentages

Mock0.8 4.5 1.1 6.0 Doxorubicin 50 nM 48 h 1.4 2.6 3.4 3.9

Figure 4 GM failed to abrogate doxorubicin-induced G2 arrest in ML-1 cells. (a) Treatment with 50 nM doxorubicin for 24 h induced both G1 and G2 arrests in ML-1 cells. (b) Treatment with 100 nM GM for 24 h was incompetent to induce cell cycle transition from 50 nM doxorubicin-induced G2 arrest into M phase. Addition of GM had little effects on doxorubicin-induced apoptosis in ML-1 cells.

Figure 3 GM also abrogated ICRF193-induced G2 arrest through the depletion of Chk1. (a) Treatment with 1.2 mM ICRF-193for 24 h induced G2 arrest in Jurkat cells, and the G2 arrest continued until 45 h of treatment (lower row, left and middle). Addition of 100 nM GM after 24 h slowly but steadily released G2-arrested cells into M and G1 phases (upper row). Incubation with 200 nM nocodazole during the last 9 h clearly suppressed the appearance of G1 peak, which conﬁrmed GM-induced release into M and G1 phases (lower right). (b) Treatment with 100 nM GM for 24 h abrogated G2 arrest in 1.2 mM ICRF-193-treated Jurkat cells as efﬁciently as in 50 nM doxorubicin-treated ones. Compared with GM, treatment with 10 mM caffeine for 6 h was ineffective to abrogate 1.2 mM ICRF-193-induced G2 arrest, although the same treatment completely released Jurkat cells from 50 nM doxorubicin-induced G2 arrest. (c) Jurkat cells were mock-treated, or treated with nocodazole alone for 12 h, plus 1.2 mM ICRF-193for 48 h, or further plus 100 n M GM for 24 h. ICRF-193-induced G2 arrest and abrogation of the arrest by GM were demonstrated by electrophoretical retardation of Cdc25C, Myt1 and Bcl-2. (d) Treatment with 1.2 mM ICRF-193or 50 n M doxorubicin for 48 h phosphorylated Chk1 at Ser345 in Jurkat cells. Addition of 100 nM GM during the last 24 h of both treatments apparently attenuated the Chk1 phosphorylation.

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3098 DXR150 nM 48 h + Mock DXR 150 nM 48 h GM 100 nM 24 h 500 400 300 200 Cell Number 100 0 0 50 100150200250 0 50 100150200250 0 50 100150200250 DNA Contents

GM 24 h - - + ++-- GM 24 h - - + + DXR 48 h -++ ++ - - DXR 48 h - + + - Noc12 h - - - +++ - pPlk1 Cdc25C Plk1 Jurkat Myt 1 pPlk1 Plk1 Bcl-2

Mitotic Cell 1.3 44.0 38.7 HeLa Percentage 4.5 48.4 0.8 21.1

DXR 150 nM 36 h + DXR 150 nM 48 h + DXR 150 nM 24 h Normal mitosis GM 100 nM 12 h GM 100 nM 24 h Cyclin B1 DNA

Figure 5 GM released HeLa cells from G2 arrest but simultaneously trapped them at M phase. (a) Treatment with 150 nM doxorubicin exclusively induced G2 arrest in HeLa cells, which continued at least for 48 h (middle). HeLa cells stayed at G2/M position even 24 h after the addition of 100 nM GM (right). (b) HeLa cells were mock-treated, or treated with 150 nM doxorubicin for 48 h, plus 100 nM GM for 24 h or further plus 200 nM nocodazole for 12 h. Cell lysates were immunoblotted for Cdc25C, Myt1 and Bcl-2, which are electrophoretically retarded speciﬁcally in M phase. Mitotic cell percentages were simultaneously counted and added in the bottom column. Addition of 100 nM GM to doxorubicin-treated HeLa cells apparently increased M phase-arrested population even in the absence of nocodazole treatment. Indeed, treatment with 100 nM GM alone for 24 h induced mitotic arrest in 21.1% of HeLa cells. (c) Confocal laser scanning microscopy monitored intracellular distribution of cyclin B1 after the addition of 100 nM GM in HeLa cells pretreated with 150 nM doxorubicin for 24 h. Most part of cyclin B1 was conﬁned in the cytoplasm in doxorubicin-treated HeLa cells. Addition of GM gradually induced nuclear entry of cyclin B1, and after 24 h considerable portion of HeLa cells were arrested at M phase. These GM-induced M phase HeLa cells contained much more amount of cyclin B1 than normal M phase cells. (d)In doxorubicin-pretreated Jurkat cells, phosphorylated Plk1 at Thr210 rather increased although Plk1 level itself decreased by the addition of GM. In contrast, both total and phosphorylated Plk1 levels decreased by the addition of GM in doxorubicin-pretreated HeLa cells.

by GM leads to the enhancement of the cytotoxic major checkpoint response should be essential for the activity of cytarabine and gemcitabine. These observa- sensitization of various chemotherapeutic agents by tions once more conﬁrm the idea that abrogation of a GM. Although depletion of various Hsp90-client

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3099 Doxorubicicin Doxorubicicin Control 50 nM 48 h Control 50 nM 48 h

103 103 1.4% 4.3% 51.8 102 102 1.9

101 101 )

m 3.30.8 7.7 5.9 0 0 10 ∆Ψ 10 ( 6

103 103 DiOC

TUNEL assay 3.4% 41.3% 34.6 102 102 2.3

101 101 23.9 3.4 6.5 37.7 Geldanamycin 100 nM 24 h Control Geldanamycin 100 nM 24 h Control 100 100 0 50 100150200250 0 50 100150200250 0 50 100150200250 0 50 100150200250 DNA Content Dihydroethidium (ROS Generation)

DXR 50 nM 24 h + Control GM 100 nM 24 h DXR 50 nM 24 h GM 100 nM 24 h B (p65) NF- DNA

Figure 6 Addition of 100 nM GM gradually induced apoptosis in Jurkat cell pretreated with 50 nM doxorubicin for 24 h. (a) Flow cytometric cell cycle analysis/TUNEL assay confirmed apoptosis induction in Jurkat cells by combinational treatment with 50 nM doxorubicin for 48 h plus 100 nM GM for 24 h. Apoptotic cell percentages are indicated in each panel. (b) Jurkat cells treated with the same treatment conditions as in (a) were dual-stained with DiOC6 and dihydroethidium to evaluate the change of DCm and reactive oxygen species (ROS) generation. GM reduced DCm, and 50 nM doxorubicin increased ROS generation. Combination of them generated a Jurkat cell population with reduced DCm and increased ROS, which corresponds to apoptotic cell population. (c) Confocal laser scanning microscopy determined cellular localization of NF-kB in Jurkat cells mock-treated (control), or treated with 50 nM doxorubicin for 24 h, with 100 nM GM for 24 h, or with both. Photographs of the same fields were obtained with optical filters specific for green and far-red lights (NF-kB and DNA signals, respectively). proteins including Akt1 may enhance cytotoxic effects supplemented with 10% fetal calf serum (FCS), 100 U mlÀ1 À1 of doxorubicin and ICRF-193, abrogation of G2 penicillin, 100 mgml streptomycin. The cells were split to checkpoint by GM should play a central role in keep cell density at 3 Â 105 to 1 Â 106 cells per ml. HeLa human cervical cancer cell line was maintained in Dulbecco’s sensitizing p53-negative leukemia cells to DNA-dama- À1 ging and decatenation-blocking agents. modified Eagle’s medium (Sigma) with 10% FCS, 100 U ml penicillin and 100 mgmlÀ1 streptomycin. Logarithmically growing cells were treated with GM and antitumor drugs at indicated concentrations for a described period. GM, Materials and methods doxorubicin, ara-C (cytosine b-D-arabinofuranoside), etopo- side and caffeine were purchased from Sigma, and ICRF-193 Cell culture and drug treatment from Zenyaku kougyo (Tokyo, Japan). To trap cells at Jurkat, HL-60 and ML-1 human leukemia cell lines were M phase, we used 200 nM nocodazole (Sigma) for indicated maintained in RPMI 1640 (Sigma, St Louis, MO, USA) period.

Oncogene Geldanamycin-induced Chk1 depletion abrogates G2 arrest K Sugimoto et al 3100 Western blot analysis 37 1C and subsequently washed twice with cold PBS before Harvested cells were washed once with phosphate-buffered analysis using a CYTRON ABSOLUTE flow cytometer. saline (PBS) and suspended in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 250 mM NaCl, 5 mM EDTA, 1 mM Direct count of mitotic cell percentage dithiothreitol (DTT), 1% NP-40, 2% Complete Mini, In brief, 1 Â 105 cells were centrifuged directly onto a slide glass EDTA-free (Roche Applied Science, Basel, Switzerland)). (400 g, 8 min at room temperature) using SC-2 centrifugal cell The suspension was kept on ice for 20 min, and centrifuged collector (Tomy, Tokyo, Japan), air dried briefly and then at 13000 g for 10 min at 4 1C. The supernatant was recovered stained according to the Wright–Giemsa method. A total of as total cell lysate and 10 mg of the aliquot was separated on a 1000 cells were microscopically examined for each treatment 7.5% polyacrylamide gel. Immunoblotting was performed condition. using 1:2000 dilution of the indicated antibodies described below. We used mouse monoclonal antibodies against Rb, Confocal laser microscopy Chk1, Cdc2, cyclin B1, PCNA, p21 (all from Santa Cruz Jurkat cells were directly attached to a poly-L-lysine-coated Biotechnology, Santa Cruz, CA, USA) and Bcl-2 (DAKO, cover glass by centrifugation at 400 g, fixed in 1% parafor- Glastrup, Denmark), rabbit polyclonal antibodies against maldehyde in PBS (pH 7.4) for 15 min at room temperature Chk2, Cdc25C (Santa Cruz Biotechnology) and Plk1 phos- and permeabilized with 0.1% Triton X-100 in PBS. The cover phorylated (Thr210; BioLegend, San Diego, CA, USA), and a glass was incubated with a 1:50 dilution of anti-NF-kB (p65) rabbit monoclonal antibody against phospho-Chk1 (Ser345; mouse monoclonal antibody (Santa Cruz Biotechnology) in Cell Signaling Technology, Danvers, MA, USA). As a second PBS with 3% FCS for 1.5 h, washed with0.1% Triton X-100 in antibody, alkaline phosphatase-conjugated anti-mouse or anti- PBS and stained with a 1:100 dilution of FITC-conjugated rabbit IgG antibody (ProMega, Madison, WI, USA) was used goat-anti-mouse IgG. DNA was counterstained with a 1:1500 at the dilution of 1:5000. dilution of TOTO-3iodide (642/660) in PBS for 10 min. The stained side of the cover glass was stuck to a slide glass with Flow cytometric cell cycle analysis/TUNEL assay FluoroGuard Antifade Reagent (Bio-Rad, Hercules, CA, In brief, 1 Â 106 cells were harvested by centrifugation for 8 min USA) and sealed with nail polish. The slides were viewed at room temperature at 400 g, washed once with phosphate- and photographed using a Zeiss LSM510 confocal laser buffered saline (PBS) and then fixed in 1% formaldehyde in scanning microscopy (Carl Zeiss, Jena, Germany). HeLa cells PBS (pH 7.4) for 15 min on ice. After washing in PBS, cells grown in 35 mm Glass Bottom Culture Dishes (MatTek were resuspended in 70% cold (À20 1C) ethanol and stored at Corporation, Ashland, MA, USA) were fixed with methanol/ À20 1C for 8 h. After rehydration in PBS, cells were resus- acetone (7:3) for 15 min at À20 1C, dried and re-hydrated in pended in 50 ml of a cacodylate buffer containing 0.2 M PBS. Cells were immunostained essentially the same procedure potassium cacodylate, 25 mM Tris–HCl (pH 6.6), 2.5 mM for cover glass-fixed Jurkat cells except for using anticyclin B1 À1 CoCl2, 0.25 mg ml bovine serum albumin, 5 units of terminal mouse monoclonal antibody (Santa Cruz Biotechnology) as deoxynucleotidyl transferase and 0.5 nmol biotin-dUTP primary antibody. (all reagents were purchased from Boehringer Mannheim, Indianapolis, IN, USA). The cells were incubated in this Clonal cell culture solution at 37 1C for 30 min; rinsed in PBS; resuspended in Methylcellulose cell culture was carried out in a 35-mm 100 ml of a solution containing 4 Â concentrated saline-sodium suspension culture dish (Nalge Nunc, Rochester, NY, USA) citrate buffer, 2.5 mgmlÀ1 fluoresceinated avidin (Boehringer using Methocult H4230 (Stem Cell Technologies, Vancouver, Mannheim), 0.1% Triton X-100 and 5% (w/v) nonfat dry milk; BC, Canada) essentially as described previously (Sugimoto and incubated in this solution for 30 min at room temperature et al., 2002). Rapidly growing Jurkat and HL-60 cells were in the dark. The cells were then rinsed in PBS containing 0.1% treated with 50 nM doxorubicin or with 1.2 mM of ICRF-193 Triton X-100, resuspended in 1 ml of PBS containing 5 mgmlÀ1 for 24 h, and further incubated in the presence or absence of À1 propidium iodide (PI) and 200 mgml RNase A (both from 100 nM GM for additional 24 h. Then living cell numbers were Sigma) and analysed by a CYTRON ABSOLUTE flow counted by trypan blue exclusion test, and 1 Â 104 cells were cytometer (Ortho, Raritan, NJ, USA), as previously described plated for each condition as triplicate. As controls, nontreated (Sugimoto et al., 1998). Jurkat and HL-60 cells were incubated without or with 100 nM GM for 24 h and plated at 100 cells per plate. Colony numbers Flow cytometric reactive oxygen species production/ were assessed after 20 days of incubation at 37 1Cina mitochondrial transmembrane potential analysis humidified atmosphere with 5% CO2. Colonies were defined as aggregates of more than 50 cells. Changes in mitochondrial transmembrane potential (DCm) were evaluated by staining with 1 nM 3,30-dihexyloxacarbo- cyanine iodide (DiOC6[3]) (Molecular Probes, Eugene, OR, Acknowledgements USA). ROS production was determined by staining with 2 mM dihydroethidium (DHE; Molecular Probes). DHE is a cell This work was supported by grants from the Ministry of permeable dye, which becomes fluorescent when oxidized by Education, Science, Sports and Culture in Japan. We thank either H2O2 or superoxide in the cell. Cells were washed once Ms Keiko Hayashi and Ms Hiroko Hiraike for their excellent with PBS and incubated with the dyes in PBS for 15 min at technical assistance.

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Oncogene