receptor type1 (IP3R1) - Nature

Oncogene (2005) 24, 1396–1402

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Inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1) modulates the acquisition of cisplatin resistance in bladder cancer cell lines Toshiyuki Tsunoda1, Hirofumi Koga1, Akira Yokomizo1, Katsunori Tatsugami1, Masatoshi Eto1, Junichi Inokuchi1, Akira Hirata1, Katsuaki Masuda1, Koji Okumura1 and Seiji Naito*,1 1

Department of Urology, Graduate School of Medical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

To investigate the molecules that regulate the acquisition of cis-diamminedichloroplatinum (II) (cisplatin) resistance, we performed cDNA microarrays using two pairs of parental and cisplatin-resistant bladder cancer cell lines. We found a markedly reduced expression of inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1), endoplasmic reticulum membrane protein, in cisplatin-resistant cells. The suppression of IP3R1 expression using small interfering RNA in parental cells prevented apoptosis and resulted in decreased sensitivity to cisplatin. Contrarily, overexpression of IP3R1 in resistant cells induced apoptosis and increased sensitivity to cisplatin. These results suggest that cisplatin-induced downregulation of IP3R1 expression was closely associated with the acquisition of cisplatin resistance in bladder cancer cells. Oncogene (2005) 24, 1396–1402. doi:10.1038/sj.onc.1208313 Published online 20 December 2004 Keywords: cisplatin resistance; IP3R1; apoptosis; bladder cancer

Introduction Cisplatin is generally accepted as a DNA-damaging agent and broadly used for anticancer drugs in many kinds of cancers, including those occurring in the testes, head, neck, esophagus, lung, ovary and bladder. However, the correlation between DNA adducts and cisplatin cytotoxicity has not yet been clarified (Burger et al., 1997; Vaisman et al., 1997). The fact that approximately only 1% of intracellular cisplatin reacts with DNA (Eastman, 1983, 1986) suggests that cisplatin may have other intracellular targets as an anticancer drug. Furthermore, the clinical uses of these drugs are often limited because of the appearance of cisplatinresistant tumor cells (Hisano et al., 1996). The elucidation of the mechanisms that control the sensitivity to cisplatin is important to improve the therapeutic outcome. Various mechanisms have been shown to contribute to the cellular resistance to cisplatin. These *Correspondence: S Naito; E-mail: [email protected] Received 23 March 2004; revised 10 September 2004; accepted 20 October 2004; published online 20 December 2004

mechanisms include the overproduction of detoxification factors such as thioredoxin (Yokomizo et al., 1995), drug transporters such as multi-drug resistance-related proteins (MRPs) (Suzuki et al., 2001), DNA repair enzymes (Masuda et al., 1988; Eastman and Schulte, 1998), and apoptosis-related genes such as Bcl-2 or p53 (Eliopoulos et al., 1995; Niender et al., 2001). However, the precise molecular mechanisms are still unclear. Inositol 1,4,5-trisphosphate receptor type1 (IP3R1) is known as an IP3-gated Ca2 þ channel that is located on the endoplasmic reticulum (ER). The ER stores intracellular Ca2 þ and it has been reported to play a critical role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, neural signals and apoptosis by pumping out Ca2 þ from ER to cytosol (Berridge, 1993; Patel et al., 1999; Szalai et al., 1999; Demaurex and Distelhorst, 2003). IP3R1-mediated Ca2 þ spike is suggested to be a privileged signal for the induction of mitochondrial apoptosis (Szalai et al., 1999). Indeed, the decreasing amount of IP3R1 by antisense RNA in Jurkat cells lead to significant inhibition of apoptosis (Jayaraman and Marks, 1997). Contrarily, the overexpression of IP3R1 induces the increasing level of cytosolic Ca2 þ and apoptosis (Szalai et al., 1999). The concentration of intracellular-free calcium of the cisplatin-resistant lung cancer cell line has also been reported to be one-third that of the sensitive parental cell line (Liang and Huang, 2000). These reports suggest the possibility that the IP3R1-mediated Ca2 þ signaling is important in cisplatininduced apoptosis. In this report, we performed cDNA microarrays in cisplatin-sensitive and -resistance bladder cancer cells, and we focused on the IP3R1 gene as one of the genes downregulated in cisplatin-resistant cells. We suggested that this is one reason why cisplatin initiates cells to develop acquired resistance to apoptosis.

Results Gene expression profiling of the molecule that correlates with acquired cisplatin resistance We performed cDNA microarray analyses between two kinds of bladder cancer cell lines, T24 and KK47

IP3R1 associate with cisplatin resistance T Tsunoda et al

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cells (Taya et al., 1977), and their cisplatin-resistant cell lines, T24/DDP10 (Kotoh et al., 1994) and KK47/ DDP20 cells (Kotoh et al., 1997), with or without cisplatin stimulation. The resistance to cisplatin of T24/DDP10 and KK47/DDP20 cells was, respectively, 8.4- and 18.7-fold that of the parental cells (Kotoh et al., 1994, 1997). Using these cell lines, we screened 9182 genes. We found 51 genes downregulated in T24/DDP10 cells (Table 1) and nine genes downregulated in KK47/DDP20 cells (Table 2), and five downregulated genes were common in two resistant cells. There were no common upregulated genes (Tables 1 and 2). IP3R1 is one of the genes downregulated in resistant cells, and we preceded further analyses of IP3R1 in this report. The lower expression of IP3R1 in resistant cells and the decreased expression of IP3R1 in parental cells after cisplatin exposure Northern blot analysis shows that a spontaneous lower mRNA expression of IP3R1 was observed in resistant cell lines (Figure 1a). A lower mRNA expression was also observed in resistant sublines, T24/DDP5 (Kotoh et al., 1994) and KK47/DDP10 cells (Kotoh et al., 1997) (data not shown). Similar results were obtained with regard to the protein expression level (Figure 1b). Cisplatin exposure for 24 h decreased the mRNA expression of IP3R1 in parental cell lines. Same results were also obtained regarding the decreased protein expression level (Figure 1b), and this was also observed in a cisplatin dose-dependent manner (Figure 1c). The other heavy metal, Manganese (II), also induced decreased IP3R1 expression level in parental cells (Figure 1d). Disruption of poly-(ADP-ribose) polymerase (PARP) cleavage in cisplatin-resistant cells The nuclear enzyme PARP cleavage was examined under cisplatin exposure. PARP cleavage was observed after cisplatin exposure in T24 and KK47 cells. On the other hand, PARP cleavage was suppressed in two resistant cell lines, T24/DDP10 and KK47/DDP20 cells (Figure 2). Downregulation of IP3R1 expression and exposure of Ca2 þ chelator decrease PARP cleavage in parental cells To confirm these changes by downregulation of IP3R1 expression, we transiently introduced IP3R1small interfering RNA (siRNA) in parental cell lines (Figure 3a). PARP cleavage was suppressed by IP3R1siRNA under cisplatin exposure in T24 cells (Figure 3b). Clamping the cytosolic Ca2 þ concentration by the exposure of bis-(o-aminophenoxy)-ethane-N,N,N0 ,N-tetra-acetic acid (BAPTA-AM) also decreased PARP cleavage in T24 cells (Figure 3c). MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt) assay showed

Table 1

Differential expression between T24 and T24/DDP10

Accession number

Gene

Differential expression

Downregulated gene (>3-fold) U20391 FOLR1 AF154830 CPS1 U36798 PDE3A NM_003937 KYNU V00522 HLA-DRB3 D26070 IP3R1 AB040057 MST4 BE536069 S100P D32076 MAGEA6 NM_020130 C8orf4 U76702 FSTL3 BE393272 ASS BE616306 ANXA8 AW385690 FN1 X63556 FBN1 BE741354 CD74 X69516 FOLR2 AF096290 SLC27A2 L15702 BF U60521 CASP9 AW377189 EPAS1 X62009 FBN2 M80244 SLC7A5 NM_004481 GALNT2 M62403 IGFBP4 J02939 SLC3A2 S78825 ID1 M18767 C1S X13223 B4galt1 BE676430 CRIP1 X03100 HLA-DPA1 NM_000064 C3 AI421214 PTGES NM_000804 FOLR3 U10694 MAGEA9 NM_003392 WNT5A AV652811 C4BPB AF153609 SGK AI336522 FLJ22182 D28235 COX2 AI418605 HLA-DMA AV704811 ARHB AA421326 FLJ21918 AA307373 KRT7 U88629 ELL2 AJ222700 TSC22 AU127127 SORD AI986271 PRMT3 BF337264 CLU NM_005833 RAB9P40 NM_014793 KIAA0547

18.7 12.5 10.3 8.8 8.3 6.9 6.5 6.4 6.1 6.1 5.3 5.2 5.2 5.1 5.1 4.7 4.7 4.7 4.6 4.6 4.5 4.4 4.2 4.1 4 4 4 4 4 4 4 3.9 3.8 3.8 3.8 3.7 3.4 3.4 3.3 3.3 3.3 3.2 3.2 3.1 3.1 3.1 3.1 3 3 3 3

Upregulated gene (o3-fold) BE877317 G0S2 AA451928 VIM AW072424 CD73 AV705672 IFI27 X78947 CTGF NM_006768 BRAP AW663903 INSIG1 AA410508 FLJ14241 L20688 ARHGDIB AF003594 CTR61 M15476 PLAU

7.7 6.7 6.1 4.6 4.3 3.9 3.6 3.6 3.3 3.1 3.1

decreased sensitivity to cisplatin by downregulation of IP3R1 or exposure of BAPTA-AM in T24 cells (Figure 3d). Oncogene


1398 Table 2 The differential expression between KK47 and KK47/ DDP20 Accession number

Gene

Differential expression

Down-regulated gene U20391 X69516 BE536069 D26070 NM_000804 BF224187 AF153609 BF312905 BF337264

FOLR1 FOLR2 S100P IP3R1 FOLR3 NPR3 SGK CTSB CLU

11.1 5.1 4.9 4.9 4.8 3.8 3.5 3.1 3

Up-regulated gene AF007144 U03877 NM_006113 M18767

DIO2 EFEMP1 VAV3 C1S

4.2 4 3.5 3.1

Upregulation of IP3R1 expression increases PARP cleavage in cisplatin-resistant cells To confirm that IP3R1 modulates the sensitivity of cisplatin, we transiently transfected IP3R1 expression vector in cisplatin-resistant cell lines. We found that an overexpression of IP3R1 restored the cisplatin-induced PARP cleavage in T24/DDP10 cells (Figure 4a). Calcium ionophore A23187 also induced PARP cleavage in resistant cell lines (Figure 4b). MTS assay showed that the overexpression of IP3R1 in resistant cells resulted in increased sensitivity of cisplatin. The introduction of calcium ionophore also showed increased sensitivity in resistant cell lines (Figure 4c).

Discussion To clarify the precise mechanism of cisplatin-induced resistance, it is necessary to avoid the clinical failure by cisplatin treatment. Several molecules that are associated with the acquisition of cisplatin resistance have been identified, including the glutathione metabolismrelated enzymes, metallothionein, thioredoxin, MRPs, DNA repair enzymes and apoptosis-related genes such as Bcl-2 or p53 (Masuda et al., 1988; Eliopoulos et al., 1995; Yokomizo et al., 1995; Eastman and Schulte, 1998; Niender et al., 2001; Suzuki et al., 2001). In fact, intrinsically or artificially, overexpression or downregulation of these proteins contributes to change the sensitivity of cisplatin, and each protein bears each its own role to a greater or lesser degree. Therefore, to detect the molecules that show the change toward acquired cisplatin resistance in bladder cancer cells, we used a cDNA microarray analysis as a useful measure. Surprisingly, in our analyses, no significant differences were observed in thiols, drug transporters, DNA repair enzymes, and apoptosis-related genes (data not shown). Instead of these genes, IP3R1 showed markedly suppressed expression in cisplatin-resistant cells (Table 1 and 2 and Figure 1a). Oncogene

Figure 1 The differential expression of IP3R1 and the state of PARP cleavage after cisplatin exposure in parental and cisplatinresistant bladder cell lines are shown. (a) Northern blot analysis of IP3R1 mRNA expression. The total RNA from parental and cisplatin-resistant cell lines at the indicated times after cisplatin exposure (20 mg/ml) was run in an agarose–formaldehyde gel, then Northern blots were carried out using the probe at the position of 42–412 bp of the coding region of IP3R1. 28S was used as a control. (b) The protein expression of IP3R1 with cisplatin exposure. Each cell line was incubated in the presence of cisplatin at the concentration of 20 mg/ml at 371C for the indicated times. Wholecell lysates were fractionated on a 4–20% polyacrylamide gel. The expression of IP3R1 and b-actin in the parental and cisplatinresistant cell lines was detected by Western blot analysis using antiIP3R1 and anti-b-actin antibodies. (c) The cisplatin-induced downregulation of IP3R1 expression was performed in a dosedependent manner. Each cell line was incubated in the presence of cisplatin at the indicated dose at 371C for 24 h. Western blot analysis was performed as described above. (d) The protein expression of IP3R1 with Manganese (II) exposure. Each cell line was incubated in the presence of Manganese (II) at the indicated dose at 371C for 24 h. Western blot analysis was performed as described above

Figure 2 Cisplatin induced the dominant PARP cleavage in parental cell lines. Each cell line was incubated in the presence of cisplatin at a concentration of 20 mg/ml at 371C for the indicated times. Whole-cell lysates were fractionated on a 4–20% polyacrylamide gel. The expression of PARP p85 fragment in the parental and cisplatin-resistant cell lines was detected by a Western blot analysis using an anti-PARP p85 fragment antibody


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IP3R1 has been reported to play a critical role in apoptosis (Szalai et al., 1999). Three members of the IP3R family had reported (Newton et al., 1994) and targeted disruption of all three IP3R isoforms in the chick DT40 B-cell line-blocked Ca2 þ mobilization and apoptosis by B-cell receptor crosslinking. Moreover, the degree of resistance increased with the number of IP3R genes deleted (Sugawara et al., 1997). Jurkat cells deficient of IP3R1 do not induce apoptosis in response to dexamethasone, ionizing radiation, T-cell receptor, and Fas stimulation, and resist subsequent apoptosis

(Jayaraman and Marks, 1997). Our cDNA microarray analyses showed dominated expression of IP3R1 among IP3R families in both parental cells. The separate cDNA microarray analyses with cisplatin exposure showed the downregulation of IP3R1 and IP3R2 under cisplatin exposure in parental cells. IP3R3 expression was dominated in resistant cells but its expression level remained the same after cisplatin exposure (Figure 5). These studies and our results, which showed that overexpression and suppression of IP3R1 modulate cisplatin sensitivity (Figures 3 and 4), indicate that IP3R1 dominated the IP3R family in cisplatin-induced apoptotic pathway. The regulation of Ca2 þ is also achieved by the ryanodine receptor (RyR) and sarcoplasmic/ER Ca2 þ ATPase (SERCA) (Demaurex and Distelhorst, 2003). RyR is important in the release of Ca2 þ from ER to cytosol (Hajnoćzky et al., 2000), and SERCA pumped Ca2 þ into the ER from cytosol (Pinton et al., 2001). However, our results showed no difference in the mRNA expressions of RyR and SERCA between parental and resistant cell lines (data not shown). Therefore, to determine the association between cisplatin-induced downregulation of IP3R1 expression and apoptosis, we modulated the expression of IP3R1, or the cytosolic concentration of Ca2 þ by chemicals. Our results indicated that the downregulation of IP3R1 expression by IP3R1-si RNA or exposure of Ca2 þ chelator, BAPTA-AM, results in decreased cisplatininduced apoptosis in parental cells (Figure 3b, c and d). Contrarily, overexpression of IP3R1, or the introduction of cytosolic Ca2 þ by A23187, influenced cisplatininduced apoptosis in resistant cells (Figure 4). The modulation of IP3R1 expression seems to be more effective than the control of cytosolic Ca2 þ concentration by chemicals. Indeed, the IP3-linked mitochondrial apoptotic signal is suggested to be privileged as compared with other Ca2 þ signals induced by Ca2 þ buffering (Szalai et al., 1999).

Figure 3 Effects of downregulation of IP3R1 or exposure of Ca2 þ chelator under cisplatin exposure in parental cell lines are shown. (a) The transient transfection of IP3R1 si-RNA induces downregulation of IP3R1 expression. T24 cells were seeded at the density of 6.5 105 in a 35-mm-diameter dish. Following 24 h incubation, T24 cells were transiently transfected with IP3R1 si-RNA (50 pmol/ ml), and then the cells were harvested at the indicated times. IP3R1 expression was detected by a Western blot analysis. (b) The downregulation of IP3R1 expression prevents PARP cleavage. T24 cells were transiently transfected with nontargeting siRNA and IP3R1-siRNA (50 pmol/ml) as described above. Cisplatin was added at 48 h after transfection, and then the cells were harvested at the indicated times. Equal amounts of whole-cell lysates were fractionated on a 4–20% polyacrylamide gel, and Western blot analyses were performed as described in Materials and methods. (c) The effect of Ca2 þ chelator, BAPTA-AM (10 mM), under cisplatin exposure in T24 cells. (d) The downregulation of IP3R1-siRNA reduces the cisplatin sensitivity. Sensitivity to cisplatin of T24 (J), T24 þ IP3R1-siRNA (50 pmol/ml) (’), T24/DDP10 (&) and T24 þ BAPTA (10 mM) (m) cells is shown. Values represent means (7s.d.) from three independent experiments. Asterisks showed significant differences, respectively, established as Po0.05. The numbers represent relative levels of protein assessed by laser densitometry Oncogene


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Figure 4 Effects of introduction of IP3R1 or exposure of Ca2 þ ionophore under cisplatin exposure in resistant cell lines. (a) The introduction of IP3R1 emphasizes PARP cleavage in resistant cells. T24/DDP10 cells were seeded at a density of 1.0 106 in a 6-cmdiameter dish. Following 24 h incubation, T24/DDP10 cells were transiently transfected with pCDNA3 or pCDNA3- IP3R1 (6 mg). Cisplatin was added at 24 h after transfection, and then the cells were harvested at the indicated time. Whole-cell lysates were fractionated on a 4–20% polyacrylamide gel. The expression of IP3R1 and PARP p85 fragments in the parental and cisplatinresistant cell lines was detected by Western blot analysis. (b) The effect of Ca2 þ overload by A23187 (10 mM) under cisplatin exposure in T24/DDP10 cells. (c) The transient transfection of IP3R1 (0.25 mg) restores the cisplatin sensitivity of T24/DDP10 cells. Sensitivity to cisplatin of T24 þ pCDNA3 (0.25 mg) (J), T24/ DDP10 þ pCDNA3-IP3R1 (0.25 mg) (’), T24/DDP10 þ pCDNA3 (&) and T24DDP/10 þ pCDNA3 þ A23187 (10 mM) (m) cells is shown. Values represent means (7s.d.) from three independent experiments. Asterisk show significant differences established as Po0.05. The numbers represent relative levels of protein assessed by laser densitometry

Conclusive molecular mechanisms of IP3R1 downregulation by cisplatin have not yet been identified. Recent study shows that IP3R1 is regulated by transcription factor NF-AT (nuclear factor of activated T cells) (Furutama et al., 1996). We also examined transcriptional activity under cisplatin exposure using the luciferase reporter assay; we found that cisplatin decreased the transcriptional activity of NF-AT in parental cell lines (data not shown). These results will support the IP3R1 mRNA downregulation by cisplatin. Oncogene

Figure 5 cDNA microarray analyses of IP3R gene expression with or without cisplatin exposure are performed. (a) Cy3/Cy5 is T24 vs T24/DDP10 and (b) Cy3/Cy5 is KK47 vs KK47/DDP20 with ( þ ) or without () cisplatin exposure

The protein degradation by caspase-3 is one of the mechanisms that induce IP3R1 protein degradation (Hirota et al., 1999). Actually, we observed a small amount of cleavage product of IP3R1 (data not shown); this may also be the reason that promotes the cisplatin resistance. Recently, Mandic et al. (2003) show that cisplatin induces ER stress by nucleus-independent apoptotic signaling. The other heavy metal, Manganese (II), which induces strong ER stress (Oubrahim et al., 2002), decreased IP3R1 expression in parental cells (Figure 1d). ER stress results in the accumulation of malfolded proteins; the cell triggers eIF-2a phosphorylation, which transiently halts general protein synthesis, giving the cell a chance to correct the environment within ER (Travers et al., 2000; Patil and Walter, 2001). The study that ER stress induced transcriptional suppression also may be the reason that the decreased IP3R1 expression is now in the process of investigation. As a result, the mechanism regarding the acquisition of cisplatin resistance in T24 and KK47 cells is triggered due to a cisplatin-induced IP3R downregulation. Irreversible rapid downregulation of IP3R expression and the subsequent changes of Ca2 þ homeostasis lead cisplatin-sensitive cells to resistant cells.


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In conclusion, our findings will be useful for the development of new treatments through restoring apoptosis sensitivity by modulating the IP3R expression in cisplatin-resistant bladder cancer. Currently, cisplatin is regarded as one of the DNA-damaging agents and is still sometimes used for second-line treatment with other drugs even in cisplatin-resistant patients. Cisplatinresistant cells do not show crossresistance against the drugs, for example, DNA-damaging agents, etoposide (Kotoh et al., 1994; Mandic et al., 2003). These properties suggest that the mechanisms of cisplatin resistance are not involved in the DNA adduct and that the more individualized selection of anticancer drugs is necessary for treatment of bladder cancer. Further analyses of IP3R1 expression level in patients before or after cisplatin treatment are presently in progress to confirm these findings and to use IP3R1 as the molecular marker to predict cisplatin sensitivity or as the therapeutical molecular target in the future.

Materials and methods Drugs and antibodies Cisplatin was obtained from the Nihon Kayaku Co. (Tokyo, Japan). A23187 and BAPTA-AM was from Sigma (Saint Louis, MI, USA). pCDNA3 and pCDNA3-IP3R1 were donated by Dr T Sudhof. Custom SMARTpoolt, IP3R1-siRNA that selectively recognize IP3R1 among its family, and negative control, D-001206-13-05 Non-Targeting siRNA, were from Dharmacon (Lafayette, CO, USA). Anti-PARP p85 fragment antibody was from Promega (Madison, WI, USA). Anti b-actin antibody (sc-7210) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-IP3R1 (1157) antibody, which recognizes the C-terminal region, was from Sigma (Saint Louis, MI, USA). Cell lines and cell culture T24 and KK47 cell lines, established from human transitional cell carcinoma of the urinary bladder (Bubenik et al., 1973; Taya et al., 1977), were used as parent lines. T24/DDP10 cells originated from T24 cells (Kotoh et al., 1994), and KK47/ DDP20 from KK47 cells (Kotoh et al., 1997) were cultured as described previously (Kotoh et al., 1994, 1997). RNA preparations, cDNA microarray analyses, and data analyses Cells were harvested under exponential condition with or without 24 h cisplatin exposure, and the total RNA was isolated using an Isogen (Nippongene, Tokyo, Japan) according to the manufacturer’s protocol. cDNA generation, hybridization, and data collection for cDNA microarray analyses were performed by the Incyte Corporation (Palo Alto, CA, USA). In brief, alterations in gene expression were evaluated by the reverse transcription of poly-(A) þ RNAs in the presence of Cy3 or Cy5 fluorescent-labeling dyes followed by hybridization to a UniGEM V 2.0 microarray chip. Each chip displays a total of 9182 elements, of which 8556 are unique genes/EST clusters. These unique gene/EST clusters can be further defined as 8412 annotated and 144 unannotated sequences. A 16-color log scale was used for visual representation and the differential expression

(log 2) was determined for Cy3 signal: the signal of parental cell lines T24 and KK47 cells vs Cy5 signal: the signal of resistant cell lines T24/DDP10 and KK47/DDP20 cells as viewed in Incyte’s GEMTM Tools software (Incyte Corporation). The subsets of genes were selected for further study based on differential Cy3/Cy5 expression ratios that were equal to or greater than 3. Transfections and immunoblottings T24 cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The T24/DDP10 cells were transiently transfected using PolyFect transfection reagent (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. Proteins were extracted using 1 SDS sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 2% bmercaptoethanol, and 1 mM orthovanadate, at the indicated time points after cisplatin exposure. Equal amounts of total lysate were electrophoresed in 4–20% SDS–polyacrylamide gels, followed by transfer to a polyvinylidene fluoride membrane (Micron Separations Inc., Westborough, MA, USA). Western blotting was performed using the ECL system (Amersham, Buckinghamshire, England) with the antibodies described above. Each blot was performed at least five times using cell lysates extracted at different times with identical results. The RC DC Protein Assay (Bio-Rad, Hercules, CA, USA) was used for quantification of protein concentration according to the manufacturer’s protocol. Drug sensitivity test T24 cells (4.0 104/well) or T24/DDP10 cells (1.0 105/well) were in 96-well flat bottom plates in 100 ml growth medium. Following 24 h incubation, each cell line was transfected as described above. At 24 h after transfection, cells were incubated in the presence of cisplatin at the indicated dose at 371C for 24 h. At 24 h after cisplatin exposure, the colorimetric reaction was initiated by adding 20 ml of CellTiter 96s AQueous One Solution Reagent (Promega, Madison, WI, USA), which contains a novel tetrazolium compound (MTS) and electron coupling reagent (phenazine ethosulfate). Prior to the measurement, the plates were incubated for 3 h at 371C, and then the quantity of formazan products formed from MTS were measured by the amounts of 490 nm absorbance using an ELISA plate reader. All assays were performed in quadruplicate, and the mean values (7s.d.) were determined from three separate experiments. The results were shown as the percentage of control, while the absorbance of the untreated cells by cisplatin was considered to be 100%. For statistical analysis, we used Student’s t-test. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C), No. 14571506, 2002 from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. We are grateful to Dr TC Sudhof, Director of the Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, and Dr M Iino, Director of the Department of Pharmacology, Graduate School of Medicine, University of Tokyo, Japan, who donated the pCDNA3-IP3R1 mammalian expression vector. We also thank M Matsuda for expert advice; N Hakoda, M Yamada, T Kuramachi, and H Matoba for technical assistance; and B Quinn and KM Rudiger for help in preparing the manuscript. Oncogene


1402 References Berridge MJ. (1993). Nature, 361, 315–325. Bubenik J, Baresova M, Viklicky V, Jakoubkova J, Sainerova H and Donner J. (1973). Int. J. Cancer, 11, 765–773. Burger H, Nooter K, Boersma AW, Kortland CJ and Stoter G. (1997). Int. J. Cancer, 73, 592–599. Demaurex N and Distelhorst C. (2003). Science, 300, 65–67. Eastman A. (1983). Biochemistry, 22, 3927–3933. Eastman A. (1986). Biochemistry, 25, 3912–3915. Eastman A and Schulte N. (1998). Biochemistry, 27, 4730–4734. Eliopoulos AG, Kert DJ, Herod J, Hodgkins L, Krajewski S, Reed JC and Young LS. (1995). Oncogene, 11, 1217–1228. Furutama D, Shimoda K, Yoshikawa S, Miyawaki A, Furuichi T and Mikoshiba K. (1996). J. Neurochem., 66, 1793–1801. Hajnoćzky G, Csorda´s G, Madesh M and Pacher P. (2000). Cell Calcium, 28, 349–363. Hirota J, Furuichi T and Mikoshiba K. (1999). J. Biol. Chem., 274, 34433–34437. Hisano T, Ono M, Nakayama M, Naito S, Kuwano M and Wada M. (1996). FEBS Lett., 397, 101–107. Jayaraman T and Marks AR. (1997). Mol. Cell. Biol., 17, 3005–3012. Kotoh S, Naito S, Yokomizo A, Kohno K, Kuwano M and Kumazawa J. (1997). J. Urol., 157, 1054–1058. Kotoh S, Naito S, Yokomizo A, Kumazawa J, Asakuno K, Kohno K and Kuwano M. (1994). Cancer Res., 54, 3248–3252. Liang X and Huang Y. (2000). Biosci. Rep., 20, 129–138. Mandic A, Hansson J, Jinder S and Shoshan MC. (2003). J. Biol. Chem., 278, 9100–9106.

Oncogene

Masuda H, Ozols RF, Lai GM, Fojo A, Rothenberg M and Hamilton TC. (1988). Cancer Res., 48, 5713–5716. Newton CL, Mignery GA and Su¨dohof TC. (1994). J. Biol. Chem., 269, 28613–28619. Niender H, Christen R, Lin X, Kondo A and Howell SB. (2001). Mol. Phamacol., 60, 1153–1160. Oubrahim H, Chock PB and Stadtman ER. (2002). J. Biol. Chem., 277, 20135–20138. Patel S, Joseph SK and Thomas AP. (1999). Cell Calcium, 25, 247–262. Patil C and Walter P. (2001). Curr. Opin. Cell Biol., 13, 349–355. Pinton P, Ferrari D, Rapizzi E, Virgilio FD, Pozzan T and Rizzuto R. (2001). EMBO J., 20, 2690–2701. Sugawara H, Kurosaki M, Takata M and Kurosaki T. (1997). EMBO J., 16, 3078–3088. Suzuki T, Nishito K and Tanabe S. (2001). Curr. Drug Metab., 2, 367–377. Szalai G, Krishnamurthy R and Hajnoćzky G. (1999). EMBO J., 18, 6349–6361. Taya T, Kobayashi T, Tsukahara K, Uchibayashi T, Naito K, Hisazumi H and Kuroda K. (1977). Jpn. J. Urol., 68, 1003–1010. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS and Walter P. (2000). Cell, 101, 249–258. Vaisman A, Varchenko M, Said I and Chaney SG. (1997). Cytometry, 27, 54–64. Yokomizo A, Ono M, Nanri H, Makino Y, Ohga T, Wada M, Okamoto T, Yodoi J, Kuwano M and Kohno K. (1995). Cancer Res., 55, 4293–4296.