Bisindolylmaleimide IX Facilitates Tumor Necrosis Factor Receptor Family-mediated Cell Death and Acts as an Inhibitor of Transcription

Abstract

Bisindolylmaleimides (Bis) were originally described as protein kinase C inhibitors. Several studies have shown that Bis potentiate tumor necrosis factor (TNF) receptor family-mediated apoptosis in lymphoid and dendritic cells, but the inhibition of protein kinase C cannot account for these effects (Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., and Jope, R. S. (1999) Nat. Med. 5, 42–48). We investigated the effect of four Bis derivatives (I, II, VIII, and IX) in human prostatic carcinoma cell lines and found that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-α, agonistic anti-Fas monoclonal antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Bis IX synergistically induced caspase activity in combination with apoptosis-inducing ligands and converted the phenotype of cell lines from apoptosis-resistant to -sensitive. Bis IX induced p53 accumulation in LNCaP (lymph node carcinoma of prostate), which expresses wild-type p53 that was not accompanied by the induction of p53-responsive genes, p21/WAF1, and Mdm2. Moreover, the induction of p21/WAF1 and Mdm2 by doxorubicin was abrogated by simultaneous treatment with Bis IX. These effects apparently result from general inhibition of transcription by Bis IX. We have shown by Northern blot analysis that the transcription activity of the hygromycin gene after transient transfection of pcDNA3.1-Hygro plasmid in 293 and HeLa cells was inhibited by Bis IX in a dose-dependent manner. Moreover, DNA binding activity of Bis IX was prevented by actinomycin D, suggesting that actinomycin D and Bis IX have similar mechanisms of interaction with DNA. In addition, we found that actinomycin D and Bis IX induced caspase activity to the same extent during TRAIL-mediated apoptosis. In summary, these results suggest that Bis IX potentiates TNF receptor family-mediated cell death in part as an inhibitor of transcription. Bisindolylmaleimides (Bis) were originally described as protein kinase C inhibitors. Several studies have shown that Bis potentiate tumor necrosis factor (TNF) receptor family-mediated apoptosis in lymphoid and dendritic cells, but the inhibition of protein kinase C cannot account for these effects (Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., and Jope, R. S. (1999) Nat. Med. 5, 42–48). We investigated the effect of four Bis derivatives (I, II, VIII, and IX) in human prostatic carcinoma cell lines and found that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-α, agonistic anti-Fas monoclonal antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Bis IX synergistically induced caspase activity in combination with apoptosis-inducing ligands and converted the phenotype of cell lines from apoptosis-resistant to -sensitive. Bis IX induced p53 accumulation in LNCaP (lymph node carcinoma of prostate), which expresses wild-type p53 that was not accompanied by the induction of p53-responsive genes, p21/WAF1, and Mdm2. Moreover, the induction of p21/WAF1 and Mdm2 by doxorubicin was abrogated by simultaneous treatment with Bis IX. These effects apparently result from general inhibition of transcription by Bis IX. We have shown by Northern blot analysis that the transcription activity of the hygromycin gene after transient transfection of pcDNA3.1-Hygro plasmid in 293 and HeLa cells was inhibited by Bis IX in a dose-dependent manner. Moreover, DNA binding activity of Bis IX was prevented by actinomycin D, suggesting that actinomycin D and Bis IX have similar mechanisms of interaction with DNA. In addition, we found that actinomycin D and Bis IX induced caspase activity to the same extent during TRAIL-mediated apoptosis. In summary, these results suggest that Bis IX potentiates TNF receptor family-mediated cell death in part as an inhibitor of transcription. bisindolylmaleimide protein kinase C tumor necrosis factor TNF-related apoptosis-inducing ligand lymph node carcinoma of prostate monoclonal antibody phosphate-buffered saline cardiolipin androgen-free conditions fetal calf serum dihydrotestosterone doxorubicin actinomycin D cycloheximide death-inducing signaling complex Fas-associated polypeptide with death domain acetyl flice-like inhibitory protein Several bisindolylmaleimide (Bis)1 derivatives were originally described as protein kinase C (PKC) inhibitors (1Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhame L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 2Bit R.A. Davis P.D. Elliot L.H. Harris W. Hill C.H. Keech E. Kumar H. Lawton G. Maw A. Nixon J.S. J. Med. Chem. 1993; 36: 21-29Crossref PubMed Scopus (161) Google Scholar, 3Jacobson P.B. Kuchera S.L. Metz A. Schachtele C. Imre K. Schrier D.J. J. Pharmacol. Exp. Ther. 1995; 275: 995-1002PubMed Google Scholar). Zhou et al. (4Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar) estimated the potentiating effect of nine Bis derivatives on Fas-mediated apoptosis in astrocytoma 1321N1 cells and found that some Bis derivatives were capable of substantially facilitating Fas-mediated apoptosis in these cells. The greatest potentiation was found with Bis VIII and IX; Bis III, X, and XI produced intermediate potentiation, whereas Bis I, II, and IV did not potentiate Fas-mediated apoptosis. The Bis derivatives tested were described as potent inhibitors of PKC, but three other PKC inhibitors structurally unrelated to Bis were tested and were ineffective in potentiating Fas-mediated apoptosis. Based on these results, the authors concluded that the inhibition of PKC could not account for the potentiation of Fas-mediated apoptosis by Bis. The authors also showed that Bis VIII potentiated TNF-mediated apoptosis in 1321N1 cells and greatly facilitated Fas-mediated apoptosis in several human T cell lines and in activated T cells. In contrast, Bis VIII did not facilitate apoptosis in 1321N1 cells treated with dexamethasone or irradiation. They concluded that Bis VIII selectively facilitates apoptosis induced by activation of the TNF receptor family. In addition, several Bis derivatives (I, II, III, IV, and VIII) have been found to facilitate Fas- and TRAIL-mediated apoptosis in human dendritic cells (5Willerns F. Amraoui Z. Vanderheyde N. Verhasselt V. Askoy E. Scaffidi C. Peter M.E. Krammer P.H. Goldman M. Blood. 2000; 95: 3478-3482Crossref PubMed Google Scholar). Bis VIII has also been described to potentiate TNF-α-, Fas-, and TRAIL-mediated apoptosis in Jurkat cells (6Meng X.W. Helderbrant M.P. Kaufmann S.H. J. Biol. Chem. 2002; 277: 3776-3783Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, the critical factors involved in the effects of Bis are still not understood. The objective of the current study was to determine whether Bis can potentiate TNF receptor family-mediated apoptosis in human prostatic carcinoma cell lines. We examined the effect of Bis I, II, VIII, and IX in three human prostatic carcinoma cell lines, DU145, LNCaP, and PC3. These experiments showed that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-α, agonistic anti-Fas mAb (IPO-4), and TRAIL. Further, Bis IX binds to the mitochondria and inhibits transcription by binding with DNA to the actinomycin D (act D)-related sites. These results provide new insight into the mechanisms by which Bis IX potentiates TNF receptor family-mediated apoptosis. The human prostatic carcinoma cell lines LNCaP, PC3, and DU145 were cultured in RPMI 1640 as has been described previously (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar). Human TRAIL was purchased from PeproTech (Rocky Hill, NJ), TNF-α was purchased from R&D Systems (Minneapolis, MN), and agonistic anti-Fas (IgM) mAb was described previously (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar). Bis I, II, VIII, and IX were purchased from Alexis Biochemicals (San Diego, CA). Cycloheximide and actinomycin D were purchased from Sigma. To measure cell viability, we used calcein AM assay (Molecular Probes, Eugene, OR), as described previously (8Rokhlin O.W. Guseva N. Tagiyev A. Knudson M.C. Cohen M.B. Oncogene. 2001; 20: 2836-2843Crossref PubMed Scopus (67) Google Scholar). Apoptosis was estimated by caspase activation using both fluorogenic substrates and Western blot analysis and proteolysis of poly(ADP-ribose)polymerase as described previously (9Rokhlin O.W. Gudkov A.V. Kwek S. Glover R.A. Gewies A.S. Cohen M.B. Oncogene. 2000; 19: 1959-1968Crossref PubMed Scopus (61) Google Scholar). In order to measure caspase activity with fluorogenic substrates, cytosolic extracts were prepared in 1% Triton X-100 buffer, pH 7.2 containing 0.1 mm phenylmethylsulfonyl fluoride and 2 μg/ml pepstatin, leupeptin, and aprotinin. 40 μg of protein lysate was incubated for 60 min in assay buffer (20 mm PIPES, pH 7.2, 100 mm NaCl, 10 mm dithiothreitol, mm and with 40 substrates as a that by and to some extent as a for and as a for Western blot of was as described previously (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar). were with in containing and incubated with the monoclonal or Diego, and The were with or with The were by of the with and were prepared as described A. O.W. Cohen M.B. Cancer Res. 2000; Google Scholar). cells were in (20 mm pH 10 mm mm mm mm mm dithiothreitol, mm 0.1 mm phenylmethylsulfonyl and 2 μg/ml pepstatin, leupeptin, and by in a with The were to and cell and were for min to the The were used as and the of mitochondria in the was examined with by Western blot mitochondria were with 10 Bis IX was incubated for min with of and cytosolic in of buffer, and was with an using an and In addition, of cardiolipin were incubated with Bis IX for and was LNCaP cells were on with and treated with Bis IX for were with and in 1% in and Bis IX was in cells using a or 2 with a and 2 293 and HeLa cells were with μg of pcDNA3.1-Hygro using transfection after was to containing of with after was from cells using to the Northern blot analysis of was by on a 1% and as by the containing hygromycin gene was using as and as The of the hygromycin gene in were with using the DNA with The were using We have shown previously that LNCaP and DU145 are to Fas-mediated apoptosis (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar). We investigated whether Bis could LNCaP and DU145 were treated with four Bis I, II, VIII, and IX. were treated with Bis or in combination with agonistic anti-Fas shown in the most effect was with Bis IX. in combination with induced cell death in of the cells. Bis IX induced cell death in of the cells, whereas anti-Fas induced cell death in of the cells. These results that of the four Bis derivatives Bis IX was the most in LNCaP and DU145 cells from to We investigated whether Bis IX could potentiate and cell We have previously that to Fas- and TRAIL-mediated apoptosis (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google LNCaP to TNF-α treatment but to Fas- and TRAIL-mediated cell and DU145 to treatment with three ligands (9Rokhlin O.W. Gudkov A.V. Kwek S. Glover R.A. Gewies A.S. Cohen M.B. Oncogene. 2000; 19: 1959-1968Crossref PubMed Scopus (61) Google O.W. Guseva Tagiyev Glover R.A. Cohen M.B. 2002; PubMed Scopus Google Scholar). We also investigated whether Bis IX could potentiate cell death in cell lines under treatment with of death-inducing ligands that by cannot cell shown in Bis IX can of cells after of Bis IX in combination with TNF-α, anti-Fas and TRAIL was found to capable of cell death in to of cell lines tested after of in DU145 did we not the potentiating effect of Bis IX under simultaneous treatment with The potentiating effect of Bis IX was after treatment of cells in the of TRAIL. However, the effect of Bis IX was found to after of the potentiating effect of Bis IX was still after of treatment in combination with death-inducing effect was not as after as was after of These suggest that Bis IX some that for to three death-inducing We investigated the activation of under Bis IX treatment in combination with TNF-α, anti-Fas and TRAIL. activity was estimated using three fluorogenic PC3, LNCaP, and DU145 were treated for 2 and with Bis IX and ligands or of caspase activity were after 2 of treatment not but caspase activity was after of treatment in Bis IX induced activity in However, Bis IX activity under simultaneous treatment with TNF-α, and TRAIL in cell lines. Bis IX also activity in and LNCaP, but activity in DU145 was found to activity was in and LNCaP with and Bis IX activity under simultaneous treatment with apoptosis-inducing These results that Bis IX potentiates activation of and suggest that the of cell death cell death of death E. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). To determine whether Bis cell death by we to the of death receptor under Bis IX treatment by these experiments we found of in the cells treated with Bis IX but not with of was on the but not on the of LNCaP treated with Bis IX with cells. were incubated with 10 Bis for with and in and the of Bis I, II, VIII, and IX was using both analysis and a assay The was and and We found that after with cells the of Bis IX and whereas the of Bis I, II, and VIII These that Bis tested in the cells, and the of was for Bis of Bis I, II, VIII, and was by using and and analysis was using and The are from three in a new was by using and and analysis was using and The are from three Bis IX was cells, we the of Bis IX the cells by using that Bis IX was found in a the cells, both and the of the which of a To the of Bis IX in the cells, we treated cells with Bis IX for 2 and cytosolic and examined the in both We found in the but not in the not However, these experiments were to not in the cell after To in mitochondria and we these incubated with Bis and the In addition, Bis IX was in the of which to a shown in μg of the same of as μg of However, the of by in mitochondria and of of the mitochondria μg of mitochondria μg of and these results cannot the of Bis IX in to that some other in to the of Bis IX in shown Bis IX in the of DNA. These Bis IX was in the mitochondria in the of of and cardiolipin in the of Bis IX are from was using and in a new The are from was using and We have shown previously that LNCaP to TNF-α treatment and that apoptosis in LNCaP (9Rokhlin O.W. Gudkov A.V. Kwek S. Glover R.A. Gewies A.S. Cohen M.B. Oncogene. 2000; 19: 1959-1968Crossref PubMed Scopus (61) Google Scholar). In we found that LNCaP to TNF-α treatment after in androgen-free conditions can from 5, of LNCaP cells were by TNF-α were cultured in with but cell death was after in for To whether Bis IX was capable of potentiating cell death in we treated LNCaP with Bis IX or in combination with TNF-α after LNCaP in shown in 5, Bis IX the to TNF-α treatment in LNCaP cultured in In the of Bis IX the of LNCaP was similar to LNCaP cultured in cells were treated with We investigated the of p53 in LNCaP after Bis IX treatment during of these cells in both and in we found that Bis IX treatment p53 in cells cultured in with but not in LNCaP cultured in To whether Bis p53 we investigated p53 in LNCaP cultured in in the of of dihydrotestosterone shown in the induction of p53 by Bis IX treatment was found to 10 the of Bis IX to the of We investigated whether p53 accumulation in cells treated with Bis IX or p53 induction by other of doxorubicin and as shown in induced p53 of the of The same results were with not These suggest that accumulation of p53 not a general in to of p53 but that Bis The we was whether p53 accumulation after Bis IX treatment was capable of the p21/WAF1 and Mdm2. shown in induced of p21/WAF1 and Mdm2. In contrast, the treatment of LNCaP with Bis IX did not result in induction of and Mdm2 the accumulation of the of p21/WAF1 and Mdm2 by was by simultaneous treatment with Bis IX and These the that Bis IX effect as an inhibitor of transcription. inhibitors of II, D can accumulation of p53 with inhibition of p21/WAF1 and Mdm2 protein of an inhibition of M. F. F. Oncogene. 1999; PubMed Scopus Google Scholar, T. Exp. Res. PubMed Scopus Google Scholar). To the that Bis IX as an inhibitor of we treated LNCaP with Bis IX D and cycloheximide 10 and examined the of p21/WAF1, and Mdm2 by Western blot shown in both D and Bis IX induced accumulation of p53 to the same and the of p21/WAF1 and Mdm2. with D and Bis the of these These cannot by proteolysis of p21/WAF1 and Mdm2 in the of p53 did not accumulation of but the of p21/WAF1 and Mdm2 were after Further, both D and Bis IX induced accumulation of but the of p21/WAF1 and Mdm2 were not to the same extent as with To whether Bis IX a general inhibitor of we HeLa cells with pcDNA3.1-Hygro We treated the cells with D and Bis IX and investigated the of hygromycin gene by Northern blot We found that both D and Bis IX inhibited the of gene The same results were with 293 cells not IX and D of hygromycin cells were with μg of pcDNA3.1-Hygro after cells were treated with D or Bis IX. treatment with Bis IX Bis IX 5, Bis IX (20 D of treatment with were on a a and with The the of as a of We also examined the binding of Bis IX to DNA by the of Bis IX in the of of DNA. We have also used Bis as a Bis did not potentiate cell shown in Bis IX in the of and was found to We investigated whether D could with Bis IX binding to DNA. shown in D prevented the binding of Bis IX to DNA. These suggest that D and Bis IX with or on DNA. the that Bis IX binds DNA and in the of DNA in we did not the Bis IX in Bis as an inhibitor of to with DNA that are not by The of Bis IX binding on DNA to interaction with In contrast, Bis IX can in mitochondria Bis IX not in the of that in mitochondria but also in the of To the effects of Bis IX and D on caspase we treated the cell DU145 for with Bis IX and D or with TRAIL. activity was in cell both with caspase substrates and by Western blot shown in both Bis IX and D induced caspase activity to the same extent in combination with TRAIL The same results were after treatment of LNCaP with Bis IX and D in combination with anti-Fas mAb not These results suggest that Bis IX potentiates TNF receptor family-mediated cell death as an inhibitor of transcription and the of apoptosis in cells. for apoptosis-resistant tumor cells to to cells to death-inducing ligands 2001; PubMed Scopus Google Scholar, A. Nat. 2002; PubMed Scopus Google Scholar). of the study by Zhou et al. (4Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar) Bis VIII potentiated Fas-mediated apoptosis in human astrocytoma 1321N1 cells, tumor T cell lines, and activated T cells Moreover, Bis VIII has activity in by the of of T Bis derivatives were originally as PKC inhibitors (1Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhame L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 2Bit R.A. Davis P.D. Elliot L.H. Harris W. Hill C.H. Keech E. Kumar H. Lawton G. Maw A. Nixon J.S. J. Med. Chem. 1993; 36: 21-29Crossref PubMed Scopus (161) Google Scholar, 3Jacobson P.B. Kuchera S.L. Metz A. Schachtele C. Imre K. Schrier D.J. J. Pharmacol. Exp. Ther. 1995; 275: 995-1002PubMed Google Zhou et T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar) found that the inhibition of PKC cannot account for the potentiation of Fas-mediated apoptosis by Bis. Based on in cell death (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar, O.W. Guseva N. Tagiyev A. Knudson M.C. Cohen M.B. Oncogene. 2001; 20: 2836-2843Crossref PubMed Scopus (67) Google Scholar, O.W. Gudkov A.V. Kwek S. Glover R.A. Gewies A.S. Cohen M.B. Oncogene. 2000; 19: 1959-1968Crossref PubMed Scopus (61) Google Scholar, A. O.W. Cohen M.B. Cancer Res. 2000; Google Scholar, O.W. Guseva Tagiyev Glover R.A. Cohen M.B. 2002; PubMed Scopus Google was of to whether Bis could the of human prostatic carcinoma cells to TNF receptor apoptosis. can cell death of death E. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, we found that the of receptor and was by under Bis IX treatment for not These results in to the effect of that the of death E. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Bis IX effect by suggest that Bis IX inhibits the of some that for the to three death-inducing TNF-α, and TRAIL. The of Bis derivatives to the of tumor cells to TNF death ligands not cell Bis VIII apoptosis after treatment of Jurkat cells with agonistic to TNF-α, and TRAIL (6Meng X.W. Helderbrant M.P. Kaufmann S.H. J. Biol. Chem. 2002; 277: 3776-3783Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Bis was found to accumulation in dendritic cells and Fas- and TRAIL-mediated apoptosis (5Willerns F. Amraoui Z. Vanderheyde N. Verhasselt V. Askoy E. Scaffidi C. Peter M.E. Krammer P.H. Goldman M. Blood. 2000; 95: 3478-3482Crossref PubMed Google and Bis VIII potentiated both Fas- and apoptosis in human astrocytoma 1321N1 cells (4Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar). the same the mechanisms by which Bis in cell Bis I, II, and IV were on dendritic cells (5Willerns F. Amraoui Z. Vanderheyde N. Verhasselt V. Askoy E. Scaffidi C. Peter M.E. Krammer P.H. Goldman M. Blood. 2000; 95: 3478-3482Crossref PubMed Google but T cells were not by these Bis derivatives (4Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar). Bis VIII and IX showed the same activity in potentiating Fas-mediated apoptosis in 1321N1 cells (4Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google whereas we found that in DU145 Bis IX was Bis Bis VIII was shown to potentiate Fas-mediated apoptosis in Jurkat cells (6Meng X.W. Helderbrant M.P. Kaufmann S.H. J. Biol. Chem. 2002; 277: 3776-3783Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Bis VIII can the of Fas-associated polypeptide with death domain the of in the was after treatment (6Meng X.W. Helderbrant M.P. Kaufmann S.H. J. Biol. Chem. 2002; 277: 3776-3783Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Bis VIII apparently effect of the We the of Bis IX to the of Bis IX in cells. Bis IX was found to to The of Bis to to mitochondria has also been shown in other study M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). of derivatives of bisindolylmaleimide by and M. J. Biol. Chem. 1993; Full Text PDF PubMed Google were investigated and with with to but was with to the of Bis to with mitochondria not to to the to with To the of Bis cell we examined the effect of Bis IX using the cell We have previously shown that apoptosis in LNCaP (9Rokhlin O.W. Gudkov A.V. Kwek S. Glover R.A. Gewies A.S. Cohen M.B. Oncogene. 2000; 19: 1959-1968Crossref PubMed Scopus (61) Google Scholar). In we that LNCaP to treatment with TNF-α after in We the effect of Bis IX on LNCaP cultured in and in We found that Bis IX the to TNF-α treatment in LNCaP cultured in effect was not on an accumulation of and Bis IX was found to effect as an inhibitor of transcription. We have shown previously that cycloheximide converted the phenotype of prostate cell lines from Fas- and to (7Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kieffer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar). We have also found that to Fas- and apoptosis in cell and these suggest that the by an apoptosis O.W. Hostager B.S. Bishop G.A. Sidorenko S.P. Glover A. Gudkov A.V. Cohen M.B. Cancer Res. 1997; 57: Google Scholar). we found that Bis IX potentiates cell death in prostate tumor cell lines that are to Fas-, and TRAIL-mediated apoptosis. Bis IX cannot caspase However, of treatment with Bis IX in the of death-inducing ligands was found to to caspase activity in cells. These results suggest that on the of a that the to TNF receptor family-mediated We have examined the of and which are to apoptosis in some cell lines K. Cancer Res. 2000; Google Scholar, J. 2000; Full Text Full Text PDF PubMed Google Scholar). However, the of these were after treatment with Bis IX not to which under Bis IX that an for prostate are a of that are to prostate tumor cells. However, these death in cells. Bis IX other Bis for treatment of prostate tumor cells for the Bis IX capable of potentiating TNF receptor apoptosis in LNCaP after cells the effect of Bis IX Bis IX potentiated TNF receptor apoptosis not in LNCaP, which expresses wild-type but also in PC3, which and in DU145, which expresses p53 inhibition of transcription by Bis IX in cells not have of by Zhou et al. (4Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google in experiments were with μg of Bis VIII other for In addition, showed that Bis did not have a effect on activated T cells from Moreover, Bis VIII selectively potentiated apoptosis in activated T cells effect on T cells. a 2001; PubMed Scopus Google Scholar, A. Nat. 2002; PubMed Scopus Google and simultaneous treatment with and TRAIL for treatment of prostate