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PTEN Augments Staurosporine-Induced Apoptosis in PTEN-Null Ishikawa Cells by Downregulating PI3K/Akt Signaling Pathway

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PTEN Augments Staurosporine-Induced Apoptosis in PTEN-Null Ishikawa Cells by Downregulating PI3K/Akt Signaling Pathway Cell Death and Differentiation (2002) 9, 414 ± 420 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00 www.nature.com/cdd PTEN augments staurosporine-induced apoptosis in PTEN-null Ishikawa cells by downregulating PI3K/Akt signaling pathway 1 ,1 1 1 X Wan , Y Yokoyama* , A Shinohara , Y Takahashi and Introduction T Tamaya1 PTEN (or MMAC1) is a tumor suppressor located at 1,2 1 Department of Obstetrics and Gynecology, Gifu University School of Medicine, chromosome 10q23.3. The gene is frequently mutated or 40 Tsukasa-machi, Gifu, 500-8705, Japan heterozygously deleted in certain carcinomas. The PTEN * Corresponding author: Yasuhiro Yokoyama, Department of Obstetrics and protein can function as a protein phosphatase as well as a Gynecology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu, Gifu, lipid phosphatase, but its major function as a tumor 500-8705, Japan. Tel. (81) 58 2672631; Fax (81) 58 2659006; suppressor is considered to regulate the intracellular con- E-mail: [email protected] centration of phosphatidylinositol-3,4,5-triphosphate (PIP3) 3 Received 12.3.01; revised 12.10.01; accepted 15.10.01 by dephosphorylating its D3 position as a lipid phosphatase. Edited by D Vaux A deficiency of the PTEN protein increases the PIP3 level in cells and causes recruitment of cytosolic proteins such as Akt/ PKB protein on the plasma membrane.4 The translocated Akt/ Abstract PKB protein undergoes a conformational change, resulting in the exposure of an activation loop that is then phosphorylated Staurosporine is a potent apoptosis inducer, but its by PI3K-dependent serine/threonine kinase (PDK1).5 Acti- mechanism remains to be clarified. We investigated the vated Akt/PKB phosphorylates proteins on serine/threonine involvement of PTEN in staurosporine-induced apoptosis. residues to potentiate cell survival proteins such as Bad, Ishikawa cells, from an endometrial carcinoma cell line, caspase9,GSK3,p70S6K, 4E-BP1/PHAS-1, IKKa and expressed a high amount of PTEN mRNA but did not express members of the Forkhead transcription family. the PTEN protein because of protein truncations. We isolated PDK1 can mediate activation of atypical PKC (PKC zeta, clones expressing the steady-state level of the PTEN protein PKC lambda).6,7 Thus, deficiency of the PTEN protein can from PTEN-null Ishikawa cells by transfection. The obtained activate both Akt/PKB and some PKC signaling pathways. clones showed reduced proliferative activity and reduced Furthermore, in mitochondria, a cooperative protection between Akt/PKB and PKC signaling pathways from anchorage-independent cell growth with the augmented 8 p27Kip1. These cell lines were sensitized to apoptosis by apoptosis onset has been suggested. Staurosporine is a non-selective protein kinase inhibitor staurosporine. A low concentration of UCN-01 did not affect as well as a potent apoptosis inducer in a broad spectrum apoptosis, but a high concentration augmented apoptosis in of cells. A variety of staurosporine analogs with less toxicity the PTEN-expressing clone. Alpha-sphingosine and H-7 did (e.g. UCN-01, 7-hydroxy-staurosporine) have been devel- not affect apoptosis in these cell lines. PI3K inhibition oped. The property of this kind of agent to induce apoptosis augmented staurosporine-induced apoptosis in the parental is thought to be deeply associated with its anticancer cell line, but not in the PTEN-expressing clone. In the clone, activity. It has been reported that the PKC signaling phosho-Akt/PKB and phospho-Bad (Ser-136) were down- pathway is likely associated with staurosporine-induced regulated. Staurosporine reduced the levels of phospho-Akt/ apoptosis in HT-29 colon carcinoma cells9 andinhuman 10 PKB and phospho-Bad (Ser-136) in all the cell lines, but the embryonic lung fibroblast (HEL) cells, whereas in rat 11 reduction was most significant in the PTEN-expressing clone. thymocytes it is probably indirectly involved. Thus, the These results suggest that inhibition of the PI3K/Akt/PKB involvement of PKC in staurosporine-induced apoptosis is still controversial. signaling pathway might be associated with staurosporine- Because the PTEN protein is a key enzyme in the Akt/ induced apoptosis in Ishikawa cells. PKB-associated signaling and in the PKC signaling path- Cell Death and Differentiation (2002) 9, 414 ± 420. DOI: 10.1038/ ways, we analyzed a possible involvement of the PTEN sj/cdd/4400982 protein in staurosporine-induced apoptosis. Keywords: PTEN gene; endometrial carcinoma cell lines; transfection; growth suppression; staurosporine; apoptosis Results Status of PTEN in the cells Abbreviations: Akt/PKB, protein kinase B; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol-3,4,5- We analyzed the status of PTEN in Ishikawa cells (from an triphosphate endometrial carcinoma cell line). In Western blot analysis, we could not detect the PTEN protein, but PTEN mRNA was abundantly expressed in Northern blot analysis (Figure 1). To PTEN augments staurosporine-induced apoptosis XWanet al 415 search for the reason, we studied the copy number of the and significantly reduced anchorage-independent cell PTEN transcript and possible mutations of each copy. To growth activity as shown in Figure 4. screen for possible mutations, PCR ± SSCP analysis from genomic DNA was performed. In the SSCP analysis, shifted bands from the control were found only in exon 8. Next, the Apoptosis induction by staurosporine region spanning exon 8 in the PTEN transcript was amplified The involvement of PTEN in staurosporine-induced apop- by RT ± PCR, and the amplified DNA fragment was subcloned tosis was analyzed. After 48 h exposure to 1 mMof into pCR2.1, a TA cloning vector, to analyze copy number and staurosporine, the percentage of apoptotic cells was mutations of the transcript. We found that there were two calculated. It was 23.4% (SD, 4.9) in parental Ishikawa copies of PTEN transcript. One copy lacked 4-nucleotides cells, while it was 56.7% (SD, 8.7) in PTEN-IK5, 54.9% (TTAC) from codon 318 to codon 319, and the other lacked A in codon 289 (Figure 2). These sequence alterations were compatible with the results of the sequence analyses on the genomic DNA. Either mutation would produce a stop codon adjacent to the mutation site. Stable transfection of PTEN and characterization After plasmid transfection and G418 selection, a number of clones resistant to G418 had appeared. We screened 27 clones and obtained four clones expressing the steady-state level of the PTEN protein (PTEN-IK5, PTEN-IK14, PTEN- IK18, and PTEN-IK23). In the clones, the expression level of p27Kip1 was increased as compared to that of the parental Ishikawa cells (Figure 3). Among the clones expressing the PTEN protein, the malignant phenotype including proliferation activity and anchorage-independent cell growth was evaluated. All the clones showed significantly reduced cell growth potential Figure 2 (A) SSCP analysis of PTEN exon 8 of Ishikawa cells. As a control, normal endometrium was used. Exon 8 of the PTEN gene (225 nucleotides long) was divided into 2 parts (8-1, 8-2) for the analyses. N, normal endometrium; IK, Ishikawa cells. (B) Sequence analysis of the PTEN transcript. Five hundred and sixty nucleotides spanning exon 8 were amplified by RT ± PCR. PCR products were subcloned into pCR2.1, a TA cloning vector. The sequence of the insert was analyzed in 10 clones. One copy deletes A in Figure 1 Expressions of PTEN protein and PTEN mRNA in AN3CA, JAR and codon 289; subsequently the expected amino acid sequence from the mutation Ishikawa cells. AN3CA cells and JAR cells express PTEN protein and mRNA, site is changed into `K and a stop signal'. The other copy deletes TTAC from whereas Ishikawa cells do not express PTEN protein in spite of abundant codon 318 to codon 319; therefore the expected amino acid sequence is expression of PTEN mRNA changed into `L and a stop signal' Cell Death and Differentiation PTEN augments staurosporine-induced apoptosis XWanet al 416 Figure 3 Western blot analyses of PTEN protein and p27kip1 protein expression. VC, vector control Figure 5 Staurosporine-induced apoptosis in the clones. The cells were exposed to 1 mM of staurosporine for 48 h and then stained with Hoechst 33258. Upper: this multinucleated cell is considered to be an apoptotic cell (arrow). Lower: the percentage of apoptotic cells is shown. Experiments were performed in triplicate. STS, staurosporine; Control, without exposure to staurosporine; VC, vector control Next, we analyzed a possible mechanism by which staurosporine augmented apoptosis in the PTEN-expres- sing clones. In preliminary experiments, we determined the IC50 of UCN-01 against PKC activity. It was 47 nM in Ishikawa cells and 45 nM in PTEN-IK5. A concentra- tion of 50 nM of UCN-01, at which PKC was preferen- tially inhibited, caused apoptosis in the parental cells and in PTEN-IK5, and the percentage of apoptotic cells was equivalent between them. A 20-fold higher con- centration of UCN-01 (1 mM),atwhichanumberof protein kinases would be inhibited, augmented apoptosis in the PTEN-expressing clone. At this concentration, the Figure 4 (A) Cell growth of the PTEN-expressing clones. 56104 cells were percentage of apoptotic cells in PTEN-IK5 increased seeded in culture dishes and cell number was monitored for 7 days. The cell significantly compared with the percentage in the growth of the clones is significantly reduced on the 7th day. (B) Colony parental Ishikawa cells and in the vector control cells formation of the PTEN-expressing clones in soft agar. One thousand cells (P50.01) (Figure 6). Alpha-sphingosine (1 ± 50 mM) and were seeded in soft agar and the formed colonies were counted on the 20th day. Experiments were performed in triplicate. VC, vector control H-7 (1 ± 50 mM), PKC inhibitors, scarcely caused apop- tosis in either cells (data not shown).
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