Modification of Alternative Splicing of Bcl-x Pre-mRNA in Prostate and Breast Cancer Cells
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m009256200
ISSN1083-351X
AutoresDanielle R. Mercatante, Carl D. Bortner, John A. Cidlowski, Ryszard Kole,
Tópico(s)RNA Interference and Gene Delivery
ResumoThere is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5′-splice site ofbcl-xL, an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-xL to Bcl-xS, a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-xL, Bcl-xS, and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells. There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5′-splice site ofbcl-xL, an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-xL to Bcl-xS, a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-xL, Bcl-xS, and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells. reverse transcription-polymerase chain reaction base pair(s) poly(ADP-ribose) polymerase fluorescence-activated cell sorting Apoptosis, or programmed cell death, is a highly regulated process controlled by numerous genes that determine a proper response to death signals (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 2Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar, 3Lowe S.W. Lin A.W. Carcinogenesis. 2000; 21: 485-495Crossref PubMed Scopus (1562) Google Scholar, 4Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar); the relative levels of expression of pro- and anti-apoptotic genes appear to be particularly important (5Shinoura N. Yoshida Y. Asai A. Kirino T. Hamada H. Oncogene. 1999; 18: 5703-5713Crossref PubMed Scopus (64) Google Scholar, 6Kawakami K. Tsukuda M. Mizuno H. Nishimura G. Ishii A. Hamajima K. Anticancer Research. 1999; 19: 3927-3932PubMed Google Scholar, 7Taylor S.T. Hickman J.A. Dive C. J. Natl. Cancer Inst. 2000; 92: 18-23Crossref PubMed Scopus (89) Google Scholar, 8Liu Q.Y. Stein C.A. Clin. Cancer Res. 1997; 3: 2039-2046PubMed Google Scholar, 9Han J.S. Núñez G. Wicha M.S. Clarke M.F. Springer Semin. Immunopathol. 1998; 19: 279-288Crossref PubMed Scopus (16) Google Scholar). Deregulation of apoptosis, which contributes to the development, progression, and/or maintenance of cancer (3Lowe S.W. Lin A.W. Carcinogenesis. 2000; 21: 485-495Crossref PubMed Scopus (1562) Google Scholar, 4Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar, 10Wang D.G. Clin. Endocrinol. 1999; 51: 1-9Crossref PubMed Scopus (17) Google Scholar), is frequently caused by mechanisms that alter splicing of regulatory genes (11Mercatante D. Kole R. Pharmacol. Ther. 2000; 85: 237-243Crossref PubMed Scopus (69) Google Scholar, 12Jiang Z.H. Wu J.Y. Proc. Soc. Exp. Biol. Med. 1999; 220: 64-72Crossref PubMed Google Scholar). Therefore, this work focused on the modification of alternative splicing of bcl-x, a member of the bcl-2 family of apoptotic genes that play crucial roles in both inhibiting and activating the apoptotic response to cellular insults (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 2Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). Bcl-x is alternatively spliced to produce two distinct mRNAs and two variant proteins, Bcl-xL and Bcl-xS, that have antagonistic functions; the longer, 241-amino acid protein (Bcl-xL) inhibits apoptosis, whereas the shorter, 178-amino acid protein (Bcl-xS) activates it (13Boise L.H. González-Garcı́a M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2913) Google Scholar).Bcl-xL is highly expressed in many types of cancers, including multiple myeloma (14Tu Y. Renner S. Xu F. Fleishman A. Taylor J. Weisz J. Vescio R. Rettig M. Berenson J. Krajewski S. Reed J.C. Lichtenstein A. Cancer Res. 1998; 58: 256-262PubMed Google Scholar), small cell lung carcinoma (15Reeve J.G. Xiong J. Morgan J. Bleehen N.M. Br. J Cancer. 1996; 73: 1193-1200Crossref PubMed Scopus (91) Google Scholar), and breast cancer (16Olopade O.I. Adeyanju M.O. Safa A.R. Hagos F. Mick R. Thompson C.B. Recant W.M. Cancer J. Sci. Am. 1997; 3: 230-237PubMed Google Scholar). High Bcl-xL expression levels have been associated with an increased risk of metastasis in breast cancer (16Olopade O.I. Adeyanju M.O. Safa A.R. Hagos F. Mick R. Thompson C.B. Recant W.M. Cancer J. Sci. Am. 1997; 3: 230-237PubMed Google Scholar) as well as with an increased resistance to apoptosis induced by methotrexate and 5-fluorouracil (17Liu R. Page C. Beidler D.R. Wicha M.S. Núñez G. Am. J. Pathol. 1999; 155: 1861-1867Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and etoposide and cisplatin (18Schmitt E. Cimoli G. Steyaert A. Bertrand R. Exp. Cell Res. 1998; 240: 107-121Crossref PubMed Scopus (44) Google Scholar). Additionally, an immunohistochemical study (19Krajewska M. Krajewski S. Epstein J.I. Shabaik A. Sauvageot J. Song K. Kitada S. Reed J.C. Am. J. Pathol. 1996; 148: 1567-1576PubMed Google Scholar) showed that Bcl-x, presumably Bcl-xL, was expressed in 100% of prostate tumors and that its immunointensity was stronger in higher grade metastases.Bcl-xS antagonizes the pro-survival properties of Bcl-xL (13Boise L.H. González-Garcı́a M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2913) Google Scholar, 20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and appears to induce apoptosis. For example, high levels of Bcl-xS induced apoptosis in cancer cells from patients with colon and stomach cancers (21Clarke M.F. Apel I.J. Benedict M.A. Eipers P.G. Sumantran V. González-Garcı́a M. Doedens M. Fukunaga N. Davidson B. Dick J.E. Minn A.J. Boise L.H. Thompson C.B. Wicha M. Núñez G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11024-11028Crossref PubMed Scopus (127) Google Scholar) as well as in human mammary tumors in nude mice (22Ealovega M.W. McGinnis P.K. Sumantran V.N. Clarke M.F. Wicha M.S. Cancer Res. 1996; 56: 1965-1969PubMed Google Scholar). However, overexpression of Bcl-xS did not cause apoptosis in an established breast cancer cell line; the latter cells became apoptotic only after additional treatment with the chemotherapeutic agents Taxol and etoposide (23Sumantran V.N. Ealovega M.W. Núñez G. Clarke M.F. Wicha M.S. Cancer Res. 1995; 55: 2507-2510PubMed Google Scholar).Work from this laboratory showed that antisense oligonucleotides could be used to modify the splicing patterns of various genes in cell culture (24Sierakowska H. Sambade M.J. Agrawal S. Kole R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12840-12844Crossref PubMed Scopus (204) Google Scholar, 25Sierakowska H. Sambade M.J. Schumperli D. Kole R. RNA. 1999; 5: 369-377Crossref PubMed Scopus (27) Google Scholar, 26Wilton S.D. Lloyd F. Carville K. Fletcher S. Honeyman K. Agrawal S. Kole R. Neuromuscul. Disorders. 1999; 9: 330-338Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 27Friedman K.J. Kole J. Cohn J.A. Knowles M.R. Silverman L.M. Kole R. J. Biol. Chem. 1999; 274: 36193-36199Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). We therefore hypothesized that blocking the alternative 5′-splice site in intron 2 of Bcl-x with an antisense oligonucleotide should shift splicing from Bcl-xL to Bcl-xS mRNA, thereby increasing the level of pro-apoptotic Bcl-xS protein and decreasing the level of its anti-apoptotic isoform, Bcl-xL. We show here that this shift in splicing could indeed be accomplished and that it induced apoptotic markers in the prostate cancer cell line PC-3. However, in the breast cancer cell line MCF-7, shifting splicing from Bcl-xL to Bcl-xS and induction of apoptosis were much less efficient. Differences in the responses to oligonucleotide treatment were also evident in the inhibition of cell growth of the two cell lines.DISCUSSIONWe have taken advantage of the fact that alternative splicing of Bcl-x pre-mRNA yields two products with antagonistic functions and used an oligonucleotide antisense to the 5′-splice site of Bcl-xL to shift splicing from the anti-apoptotic splice variant, Bcl-xL, to the pro-apoptotic splice variant, Bcl-xS. This approach should be superior to antisense down-regulation of Bcl-xL mRNA (39Taylor J.K. Zhang Q.Q. Monia B.P. Marcusson E.G. Dean N.M. Oncogene. 1999; 18: 4495-4504Crossref PubMed Scopus (83) Google Scholar, 40Leech S.H. Olie R.A. Gautschi O. Simoes-Wust A.P. Tschopp S. Haner R. Hall J. Stahel R.A. Zangemeister-Wittke U. Int. J. Cancer. 2000; 86: 570-576Crossref PubMed Scopus (93) Google Scholar) since, by definition, a decrease in Bcl-xL leads to a concomitant increase in the concentration of the antagonistic Bcl-xS, amplifying the biological effects of the treatment. In fact, an oligonucleotide targeted to the 5′-splice site of Bcl-x pre-mRNA, which led to an increase in the Bcl-xS splice variant and a decrease in the Bcl-xL splice variant, was a better inducer of apoptosis in PC-3 cells than an oligonucleotide targeted to the 3′-splice site, which decreased the expression of the Bcl-xL protein.Treatment of PC-3 and MCF-7 cells with the 5′-Bcl-x AS oligonucleotide led to a dose- and time-dependent shift in splicing of Bcl-x pre-mRNA from the Bcl-xL to Bcl-xSpathway and to a concomitant increase in the level of Bcl-xS protein. However, even though most of the Bcl-xL mRNA disappeared, the Bcl-xL protein remained in the cells in apparent excess over the Bcl-xSvariant. There may be two reasons for this unexpected observation. First, the anti-Bcl-x antibodies may preferentially recognize the longer Bcl-xL polypeptide and under-represent the Bcl-xS splice variant. This possibility seems likely since several anti-Bcl-x antibodies from different manufacturers failed to detect the Bcl-xS polypeptide altogether. Second, the Bcl-xL protein may be very stable, persisting in the cells at high concentrations even though its de novo translation was markedly reduced. The latter interpretation suggests that, especially in PC-3 cells, Bcl-xS hastrans-dominant properties, promoting apoptosis and/or cell death despite the existing excess of anti-apoptotic Bcl-xL. Since Bcl-xS binds to Bcl-xL and inhibits its anti-apoptotic action (20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), one has to conclude either that the binding is not stoichiometric or that this is not the only mechanism responsible for the pro-apoptotic properties of Bcl-xS(20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar).It appears that the minimal apoptotic response of MCF-7 cells to oligonucleotide treatment is mostly due to the low level of Bcl-xS protein generated by the shift in the Bcl-xL/Bcl-xS mRNA ratio. It follows that the ratio of Bcl-xS protein to other apoptotic regulatory proteins that may impact its function may also be lower. However, the identity and nature of the genes that may be responsible for the apoptotic resistance of MCF-7 cells are not clear and require additional studies.For instance, a shift in alternative splicing of Bcl-x pre-mRNA was also observed in another breast cancer cell line, HS578T, 2D. R. Mercatante and R. Kole, unpublished data. and, while this work was in progress, in a lung adenocarcinoma cell line, A549, treated with 2′-O-methoxyethoxy-modified oligoribonucleotides (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar). Among these cell lines, as well as the ones investigated here, thep53 gene is mutated in PC-3 (42Carrol A.G. Voeller H.J. Sugars L. Gelmann E.P. Prostate. 1993; 23: 123-134Crossref PubMed Scopus (268) Google Scholar, 43Isaacs W.B. Carter B.S. Ewing C.M. Cancer Res. 1991; 51: 4716-4720PubMed Google Scholar) and HS578T (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar) cells, but not in A549 (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) or MCF-7 (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar) cells, and Bcl-2 is low in PC-3 (Fig. 10 C) (8Liu Q.Y. Stein C.A. Clin. Cancer Res. 1997; 3: 2039-2046PubMed Google Scholar), HS578T (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar), and A549 (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) cells and high in MCF-7 cells (Fig. 10 C) (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar). Yet treatment with 5′-Bcl-x AS alone led to significant apoptosis only in PC-3 cells, whereas a decrease in Bcl-2 in MCF-7 cells to levels below those in PC-3 cells did not promote oligonucleotide-induced apoptosis. It appears that if the p53 and bcl-2 genes do play a role in modulating the function of the 5′-Bcl-x AS oligonucleotide, their effects may depend on additional cellular background. In this regard, it is notable that Bcl-xL was found in 100% of prostate adenocarcinomas, but Bcl-2 was found in only 25% (19Krajewska M. Krajewski S. Epstein J.I. Shabaik A. Sauvageot J. Song K. Kitada S. Reed J.C. Am. J. Pathol. 1996; 148: 1567-1576PubMed Google Scholar). However, in some breast carcinomas, as well as in some breast cancer cell lines, the levels of Bcl-2 were higher than those of Bcl-xL(45Zapata J.M. Krajewska M. Krajewski S. Huang R.P. Takayama S. Wang H.G. Adamson E. Reed J.C. Breast Cancer Res. Treat. 1998; 47: 129-140Crossref PubMed Scopus (106) Google Scholar).The different responses of PC-3 and MCF-7 cells to the shift in Bcl-x pre-mRNA splicing are not limited to generation of subdiploid DNA, but also to cleavage of PARP, a different apoptotic marker, and to cell death assayed by colony formation. In this regard, 5′-Bcl-x AS promoted cell death in both PC-3 and MCF-7 cell lines, further complicating the interpretation of the results. Approximately 39% of MCF-7 cells and 24% of PC-3 cells survived the treatment with 5′-Bcl-x AS (0.4 μm oligonucleotide for MCF-7 cells and 0.08 μm for PC-3 cells) that resulted in a similar ratio of Bcl-xL to Bcl-xS mRNA. Recalculation of the data in terms of the effective concentration (EC50) of the oligonucleotide yielded EC50 values of 0.32 and 0.05 μm for MCF-7 and PC-3 cells, respectively, a 6.5-fold ratio. This difference is partly explained by reduced nuclear uptake of the oligonucleotide in MCF-7 cells. In contrast to PC-3 cells, in which fluorescent labeled oligonucleotide accumulated predominantly in the nucleus, in MCF-7 cells, the large fraction of the compound remained concentrated in cytoplasmic endosomal vesicles (data not shown), where it was unable to affect splicing, a nuclear process (46Zelphati O. Szoka F.C.J. Pharm. Res. ( N. Y. ). 1996; 13: 1367-1372Crossref PubMed Scopus (220) Google Scholar).The fact that cells differ in their response to a shift in the Bcl-xL/Bcl-xS ratio may impart beneficial specificity to the in vivo applications of the 5′-Bcl-x AS oligonucleotide. That is, although Bcl-xL is expressed in a number of cell types, including several hematopoietic cell lineages (47Krajewski S. Krajewska M. Shabaik A. Wang H.G. Irie S. Fong L. Reed J.C. Cancer Res. 1994; 54: 5501-5507PubMed Google Scholar), the apoptotic stimulus of the oligonucleotide may be effective only in certain susceptible cells with a gene expression profile akin to the prostate cancer PC-3 cell line. In addition, a combination of the oligonucleotide with chemotherapeutic agents exemplified by cisplatin in A549 cells (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) may sensitize only certain types of cells to undergo apoptosis. An additional benefit of cellular sensitization by the oligonucleotide may be a reduction in the dosage of chemotherapeutic agents and hence in the overall toxicity of cancer treatment. Apoptosis, or programmed cell death, is a highly regulated process controlled by numerous genes that determine a proper response to death signals (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 2Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar, 3Lowe S.W. Lin A.W. Carcinogenesis. 2000; 21: 485-495Crossref PubMed Scopus (1562) Google Scholar, 4Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar); the relative levels of expression of pro- and anti-apoptotic genes appear to be particularly important (5Shinoura N. Yoshida Y. Asai A. Kirino T. Hamada H. Oncogene. 1999; 18: 5703-5713Crossref PubMed Scopus (64) Google Scholar, 6Kawakami K. Tsukuda M. Mizuno H. Nishimura G. Ishii A. Hamajima K. Anticancer Research. 1999; 19: 3927-3932PubMed Google Scholar, 7Taylor S.T. Hickman J.A. Dive C. J. Natl. Cancer Inst. 2000; 92: 18-23Crossref PubMed Scopus (89) Google Scholar, 8Liu Q.Y. Stein C.A. Clin. Cancer Res. 1997; 3: 2039-2046PubMed Google Scholar, 9Han J.S. Núñez G. Wicha M.S. Clarke M.F. Springer Semin. Immunopathol. 1998; 19: 279-288Crossref PubMed Scopus (16) Google Scholar). Deregulation of apoptosis, which contributes to the development, progression, and/or maintenance of cancer (3Lowe S.W. Lin A.W. Carcinogenesis. 2000; 21: 485-495Crossref PubMed Scopus (1562) Google Scholar, 4Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar, 10Wang D.G. Clin. Endocrinol. 1999; 51: 1-9Crossref PubMed Scopus (17) Google Scholar), is frequently caused by mechanisms that alter splicing of regulatory genes (11Mercatante D. Kole R. Pharmacol. Ther. 2000; 85: 237-243Crossref PubMed Scopus (69) Google Scholar, 12Jiang Z.H. Wu J.Y. Proc. Soc. Exp. Biol. Med. 1999; 220: 64-72Crossref PubMed Google Scholar). Therefore, this work focused on the modification of alternative splicing of bcl-x, a member of the bcl-2 family of apoptotic genes that play crucial roles in both inhibiting and activating the apoptotic response to cellular insults (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar, 2Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). Bcl-x is alternatively spliced to produce two distinct mRNAs and two variant proteins, Bcl-xL and Bcl-xS, that have antagonistic functions; the longer, 241-amino acid protein (Bcl-xL) inhibits apoptosis, whereas the shorter, 178-amino acid protein (Bcl-xS) activates it (13Boise L.H. González-Garcı́a M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2913) Google Scholar). Bcl-xL is highly expressed in many types of cancers, including multiple myeloma (14Tu Y. Renner S. Xu F. Fleishman A. Taylor J. Weisz J. Vescio R. Rettig M. Berenson J. Krajewski S. Reed J.C. Lichtenstein A. Cancer Res. 1998; 58: 256-262PubMed Google Scholar), small cell lung carcinoma (15Reeve J.G. Xiong J. Morgan J. Bleehen N.M. Br. J Cancer. 1996; 73: 1193-1200Crossref PubMed Scopus (91) Google Scholar), and breast cancer (16Olopade O.I. Adeyanju M.O. Safa A.R. Hagos F. Mick R. Thompson C.B. Recant W.M. Cancer J. Sci. Am. 1997; 3: 230-237PubMed Google Scholar). High Bcl-xL expression levels have been associated with an increased risk of metastasis in breast cancer (16Olopade O.I. Adeyanju M.O. Safa A.R. Hagos F. Mick R. Thompson C.B. Recant W.M. Cancer J. Sci. Am. 1997; 3: 230-237PubMed Google Scholar) as well as with an increased resistance to apoptosis induced by methotrexate and 5-fluorouracil (17Liu R. Page C. Beidler D.R. Wicha M.S. Núñez G. Am. J. Pathol. 1999; 155: 1861-1867Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and etoposide and cisplatin (18Schmitt E. Cimoli G. Steyaert A. Bertrand R. Exp. Cell Res. 1998; 240: 107-121Crossref PubMed Scopus (44) Google Scholar). Additionally, an immunohistochemical study (19Krajewska M. Krajewski S. Epstein J.I. Shabaik A. Sauvageot J. Song K. Kitada S. Reed J.C. Am. J. Pathol. 1996; 148: 1567-1576PubMed Google Scholar) showed that Bcl-x, presumably Bcl-xL, was expressed in 100% of prostate tumors and that its immunointensity was stronger in higher grade metastases. Bcl-xS antagonizes the pro-survival properties of Bcl-xL (13Boise L.H. González-Garcı́a M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2913) Google Scholar, 20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and appears to induce apoptosis. For example, high levels of Bcl-xS induced apoptosis in cancer cells from patients with colon and stomach cancers (21Clarke M.F. Apel I.J. Benedict M.A. Eipers P.G. Sumantran V. González-Garcı́a M. Doedens M. Fukunaga N. Davidson B. Dick J.E. Minn A.J. Boise L.H. Thompson C.B. Wicha M. Núñez G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11024-11028Crossref PubMed Scopus (127) Google Scholar) as well as in human mammary tumors in nude mice (22Ealovega M.W. McGinnis P.K. Sumantran V.N. Clarke M.F. Wicha M.S. Cancer Res. 1996; 56: 1965-1969PubMed Google Scholar). However, overexpression of Bcl-xS did not cause apoptosis in an established breast cancer cell line; the latter cells became apoptotic only after additional treatment with the chemotherapeutic agents Taxol and etoposide (23Sumantran V.N. Ealovega M.W. Núñez G. Clarke M.F. Wicha M.S. Cancer Res. 1995; 55: 2507-2510PubMed Google Scholar). Work from this laboratory showed that antisense oligonucleotides could be used to modify the splicing patterns of various genes in cell culture (24Sierakowska H. Sambade M.J. Agrawal S. Kole R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12840-12844Crossref PubMed Scopus (204) Google Scholar, 25Sierakowska H. Sambade M.J. Schumperli D. Kole R. RNA. 1999; 5: 369-377Crossref PubMed Scopus (27) Google Scholar, 26Wilton S.D. Lloyd F. Carville K. Fletcher S. Honeyman K. Agrawal S. Kole R. Neuromuscul. Disorders. 1999; 9: 330-338Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 27Friedman K.J. Kole J. Cohn J.A. Knowles M.R. Silverman L.M. Kole R. J. Biol. Chem. 1999; 274: 36193-36199Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). We therefore hypothesized that blocking the alternative 5′-splice site in intron 2 of Bcl-x with an antisense oligonucleotide should shift splicing from Bcl-xL to Bcl-xS mRNA, thereby increasing the level of pro-apoptotic Bcl-xS protein and decreasing the level of its anti-apoptotic isoform, Bcl-xL. We show here that this shift in splicing could indeed be accomplished and that it induced apoptotic markers in the prostate cancer cell line PC-3. However, in the breast cancer cell line MCF-7, shifting splicing from Bcl-xL to Bcl-xS and induction of apoptosis were much less efficient. Differences in the responses to oligonucleotide treatment were also evident in the inhibition of cell growth of the two cell lines. DISCUSSIONWe have taken advantage of the fact that alternative splicing of Bcl-x pre-mRNA yields two products with antagonistic functions and used an oligonucleotide antisense to the 5′-splice site of Bcl-xL to shift splicing from the anti-apoptotic splice variant, Bcl-xL, to the pro-apoptotic splice variant, Bcl-xS. This approach should be superior to antisense down-regulation of Bcl-xL mRNA (39Taylor J.K. Zhang Q.Q. Monia B.P. Marcusson E.G. Dean N.M. Oncogene. 1999; 18: 4495-4504Crossref PubMed Scopus (83) Google Scholar, 40Leech S.H. Olie R.A. Gautschi O. Simoes-Wust A.P. Tschopp S. Haner R. Hall J. Stahel R.A. Zangemeister-Wittke U. Int. J. Cancer. 2000; 86: 570-576Crossref PubMed Scopus (93) Google Scholar) since, by definition, a decrease in Bcl-xL leads to a concomitant increase in the concentration of the antagonistic Bcl-xS, amplifying the biological effects of the treatment. In fact, an oligonucleotide targeted to the 5′-splice site of Bcl-x pre-mRNA, which led to an increase in the Bcl-xS splice variant and a decrease in the Bcl-xL splice variant, was a better inducer of apoptosis in PC-3 cells than an oligonucleotide targeted to the 3′-splice site, which decreased the expression of the Bcl-xL protein.Treatment of PC-3 and MCF-7 cells with the 5′-Bcl-x AS oligonucleotide led to a dose- and time-dependent shift in splicing of Bcl-x pre-mRNA from the Bcl-xL to Bcl-xSpathway and to a concomitant increase in the level of Bcl-xS protein. However, even though most of the Bcl-xL mRNA disappeared, the Bcl-xL protein remained in the cells in apparent excess over the Bcl-xSvariant. There may be two reasons for this unexpected observation. First, the anti-Bcl-x antibodies may preferentially recognize the longer Bcl-xL polypeptide and under-represent the Bcl-xS splice variant. This possibility seems likely since several anti-Bcl-x antibodies from different manufacturers failed to detect the Bcl-xS polypeptide altogether. Second, the Bcl-xL protein may be very stable, persisting in the cells at high concentrations even though its de novo translation was markedly reduced. The latter interpretation suggests that, especially in PC-3 cells, Bcl-xS hastrans-dominant properties, promoting apoptosis and/or cell death despite the existing excess of anti-apoptotic Bcl-xL. Since Bcl-xS binds to Bcl-xL and inhibits its anti-apoptotic action (20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), one has to conclude either that the binding is not stoichiometric or that this is not the only mechanism responsible for the pro-apoptotic properties of Bcl-xS(20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar).It appears that the minimal apoptotic response of MCF-7 cells to oligonucleotide treatment is mostly due to the low level of Bcl-xS protein generated by the shift in the Bcl-xL/Bcl-xS mRNA ratio. It follows that the ratio of Bcl-xS protein to other apoptotic regulatory proteins that may impact its function may also be lower. However, the identity and nature of the genes that may be responsible for the apoptotic resistance of MCF-7 cells are not clear and require additional studies.For instance, a shift in alternative splicing of Bcl-x pre-mRNA was also observed in another breast cancer cell line, HS578T, 2D. R. Mercatante and R. Kole, unpublished data. and, while this work was in progress, in a lung adenocarcinoma cell line, A549, treated with 2′-O-methoxyethoxy-modified oligoribonucleotides (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar). Among these cell lines, as well as the ones investigated here, thep53 gene is mutated in PC-3 (42Carrol A.G. Voeller H.J. Sugars L. Gelmann E.P. Prostate. 1993; 23: 123-134Crossref PubMed Scopus (268) Google Scholar, 43Isaacs W.B. Carter B.S. Ewing C.M. Cancer Res. 1991; 51: 4716-4720PubMed Google Scholar) and HS578T (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar) cells, but not in A549 (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) or MCF-7 (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar) cells, and Bcl-2 is low in PC-3 (Fig. 10 C) (8Liu Q.Y. Stein C.A. Clin. Cancer Res. 1997; 3: 2039-2046PubMed Google Scholar), HS578T (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar), and A549 (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) cells and high in MCF-7 cells (Fig. 10 C) (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar). Yet treatment with 5′-Bcl-x AS alone led to significant apoptosis only in PC-3 cells, whereas a decrease in Bcl-2 in MCF-7 cells to levels below those in PC-3 cells did not promote oligonucleotide-induced apoptosis. It appears that if the p53 and bcl-2 genes do play a role in modulating the function of the 5′-Bcl-x AS oligonucleotide, their effects may depend on additional cellular background. In this regard, it is notable that Bcl-xL was found in 100% of prostate adenocarcinomas, but Bcl-2 was found in only 25% (19Krajewska M. Krajewski S. Epstein J.I. Shabaik A. Sauvageot J. Song K. Kitada S. Reed J.C. Am. J. Pathol. 1996; 148: 1567-1576PubMed Google Scholar). However, in some breast carcinomas, as well as in some breast cancer cell lines, the levels of Bcl-2 were higher than those of Bcl-xL(45Zapata J.M. Krajewska M. Krajewski S. Huang R.P. Takayama S. Wang H.G. Adamson E. Reed J.C. Breast Cancer Res. Treat. 1998; 47: 129-140Crossref PubMed Scopus (106) Google Scholar).The different responses of PC-3 and MCF-7 cells to the shift in Bcl-x pre-mRNA splicing are not limited to generation of subdiploid DNA, but also to cleavage of PARP, a different apoptotic marker, and to cell death assayed by colony formation. In this regard, 5′-Bcl-x AS promoted cell death in both PC-3 and MCF-7 cell lines, further complicating the interpretation of the results. Approximately 39% of MCF-7 cells and 24% of PC-3 cells survived the treatment with 5′-Bcl-x AS (0.4 μm oligonucleotide for MCF-7 cells and 0.08 μm for PC-3 cells) that resulted in a similar ratio of Bcl-xL to Bcl-xS mRNA. Recalculation of the data in terms of the effective concentration (EC50) of the oligonucleotide yielded EC50 values of 0.32 and 0.05 μm for MCF-7 and PC-3 cells, respectively, a 6.5-fold ratio. This difference is partly explained by reduced nuclear uptake of the oligonucleotide in MCF-7 cells. In contrast to PC-3 cells, in which fluorescent labeled oligonucleotide accumulated predominantly in the nucleus, in MCF-7 cells, the large fraction of the compound remained concentrated in cytoplasmic endosomal vesicles (data not shown), where it was unable to affect splicing, a nuclear process (46Zelphati O. Szoka F.C.J. Pharm. Res. ( N. Y. ). 1996; 13: 1367-1372Crossref PubMed Scopus (220) Google Scholar).The fact that cells differ in their response to a shift in the Bcl-xL/Bcl-xS ratio may impart beneficial specificity to the in vivo applications of the 5′-Bcl-x AS oligonucleotide. That is, although Bcl-xL is expressed in a number of cell types, including several hematopoietic cell lineages (47Krajewski S. Krajewska M. Shabaik A. Wang H.G. Irie S. Fong L. Reed J.C. Cancer Res. 1994; 54: 5501-5507PubMed Google Scholar), the apoptotic stimulus of the oligonucleotide may be effective only in certain susceptible cells with a gene expression profile akin to the prostate cancer PC-3 cell line. In addition, a combination of the oligonucleotide with chemotherapeutic agents exemplified by cisplatin in A549 cells (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) may sensitize only certain types of cells to undergo apoptosis. An additional benefit of cellular sensitization by the oligonucleotide may be a reduction in the dosage of chemotherapeutic agents and hence in the overall toxicity of cancer treatment. We have taken advantage of the fact that alternative splicing of Bcl-x pre-mRNA yields two products with antagonistic functions and used an oligonucleotide antisense to the 5′-splice site of Bcl-xL to shift splicing from the anti-apoptotic splice variant, Bcl-xL, to the pro-apoptotic splice variant, Bcl-xS. This approach should be superior to antisense down-regulation of Bcl-xL mRNA (39Taylor J.K. Zhang Q.Q. Monia B.P. Marcusson E.G. Dean N.M. Oncogene. 1999; 18: 4495-4504Crossref PubMed Scopus (83) Google Scholar, 40Leech S.H. Olie R.A. Gautschi O. Simoes-Wust A.P. Tschopp S. Haner R. Hall J. Stahel R.A. Zangemeister-Wittke U. Int. J. Cancer. 2000; 86: 570-576Crossref PubMed Scopus (93) Google Scholar) since, by definition, a decrease in Bcl-xL leads to a concomitant increase in the concentration of the antagonistic Bcl-xS, amplifying the biological effects of the treatment. In fact, an oligonucleotide targeted to the 5′-splice site of Bcl-x pre-mRNA, which led to an increase in the Bcl-xS splice variant and a decrease in the Bcl-xL splice variant, was a better inducer of apoptosis in PC-3 cells than an oligonucleotide targeted to the 3′-splice site, which decreased the expression of the Bcl-xL protein. Treatment of PC-3 and MCF-7 cells with the 5′-Bcl-x AS oligonucleotide led to a dose- and time-dependent shift in splicing of Bcl-x pre-mRNA from the Bcl-xL to Bcl-xSpathway and to a concomitant increase in the level of Bcl-xS protein. However, even though most of the Bcl-xL mRNA disappeared, the Bcl-xL protein remained in the cells in apparent excess over the Bcl-xSvariant. There may be two reasons for this unexpected observation. First, the anti-Bcl-x antibodies may preferentially recognize the longer Bcl-xL polypeptide and under-represent the Bcl-xS splice variant. This possibility seems likely since several anti-Bcl-x antibodies from different manufacturers failed to detect the Bcl-xS polypeptide altogether. Second, the Bcl-xL protein may be very stable, persisting in the cells at high concentrations even though its de novo translation was markedly reduced. The latter interpretation suggests that, especially in PC-3 cells, Bcl-xS hastrans-dominant properties, promoting apoptosis and/or cell death despite the existing excess of anti-apoptotic Bcl-xL. Since Bcl-xS binds to Bcl-xL and inhibits its anti-apoptotic action (20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), one has to conclude either that the binding is not stoichiometric or that this is not the only mechanism responsible for the pro-apoptotic properties of Bcl-xS(20Minn A.J. Boise L.H. Thompson C.B. J. Biol. Chem. 1996; 271: 6306-6312Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). It appears that the minimal apoptotic response of MCF-7 cells to oligonucleotide treatment is mostly due to the low level of Bcl-xS protein generated by the shift in the Bcl-xL/Bcl-xS mRNA ratio. It follows that the ratio of Bcl-xS protein to other apoptotic regulatory proteins that may impact its function may also be lower. However, the identity and nature of the genes that may be responsible for the apoptotic resistance of MCF-7 cells are not clear and require additional studies. For instance, a shift in alternative splicing of Bcl-x pre-mRNA was also observed in another breast cancer cell line, HS578T, 2D. R. Mercatante and R. Kole, unpublished data. and, while this work was in progress, in a lung adenocarcinoma cell line, A549, treated with 2′-O-methoxyethoxy-modified oligoribonucleotides (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar). Among these cell lines, as well as the ones investigated here, thep53 gene is mutated in PC-3 (42Carrol A.G. Voeller H.J. Sugars L. Gelmann E.P. Prostate. 1993; 23: 123-134Crossref PubMed Scopus (268) Google Scholar, 43Isaacs W.B. Carter B.S. Ewing C.M. Cancer Res. 1991; 51: 4716-4720PubMed Google Scholar) and HS578T (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar) cells, but not in A549 (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) or MCF-7 (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar) cells, and Bcl-2 is low in PC-3 (Fig. 10 C) (8Liu Q.Y. Stein C.A. Clin. Cancer Res. 1997; 3: 2039-2046PubMed Google Scholar), HS578T (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar), and A549 (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) cells and high in MCF-7 cells (Fig. 10 C) (44Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar). Yet treatment with 5′-Bcl-x AS alone led to significant apoptosis only in PC-3 cells, whereas a decrease in Bcl-2 in MCF-7 cells to levels below those in PC-3 cells did not promote oligonucleotide-induced apoptosis. It appears that if the p53 and bcl-2 genes do play a role in modulating the function of the 5′-Bcl-x AS oligonucleotide, their effects may depend on additional cellular background. In this regard, it is notable that Bcl-xL was found in 100% of prostate adenocarcinomas, but Bcl-2 was found in only 25% (19Krajewska M. Krajewski S. Epstein J.I. Shabaik A. Sauvageot J. Song K. Kitada S. Reed J.C. Am. J. Pathol. 1996; 148: 1567-1576PubMed Google Scholar). However, in some breast carcinomas, as well as in some breast cancer cell lines, the levels of Bcl-2 were higher than those of Bcl-xL(45Zapata J.M. Krajewska M. Krajewski S. Huang R.P. Takayama S. Wang H.G. Adamson E. Reed J.C. Breast Cancer Res. Treat. 1998; 47: 129-140Crossref PubMed Scopus (106) Google Scholar). The different responses of PC-3 and MCF-7 cells to the shift in Bcl-x pre-mRNA splicing are not limited to generation of subdiploid DNA, but also to cleavage of PARP, a different apoptotic marker, and to cell death assayed by colony formation. In this regard, 5′-Bcl-x AS promoted cell death in both PC-3 and MCF-7 cell lines, further complicating the interpretation of the results. Approximately 39% of MCF-7 cells and 24% of PC-3 cells survived the treatment with 5′-Bcl-x AS (0.4 μm oligonucleotide for MCF-7 cells and 0.08 μm for PC-3 cells) that resulted in a similar ratio of Bcl-xL to Bcl-xS mRNA. Recalculation of the data in terms of the effective concentration (EC50) of the oligonucleotide yielded EC50 values of 0.32 and 0.05 μm for MCF-7 and PC-3 cells, respectively, a 6.5-fold ratio. This difference is partly explained by reduced nuclear uptake of the oligonucleotide in MCF-7 cells. In contrast to PC-3 cells, in which fluorescent labeled oligonucleotide accumulated predominantly in the nucleus, in MCF-7 cells, the large fraction of the compound remained concentrated in cytoplasmic endosomal vesicles (data not shown), where it was unable to affect splicing, a nuclear process (46Zelphati O. Szoka F.C.J. Pharm. Res. ( N. Y. ). 1996; 13: 1367-1372Crossref PubMed Scopus (220) Google Scholar). The fact that cells differ in their response to a shift in the Bcl-xL/Bcl-xS ratio may impart beneficial specificity to the in vivo applications of the 5′-Bcl-x AS oligonucleotide. That is, although Bcl-xL is expressed in a number of cell types, including several hematopoietic cell lineages (47Krajewski S. Krajewska M. Shabaik A. Wang H.G. Irie S. Fong L. Reed J.C. Cancer Res. 1994; 54: 5501-5507PubMed Google Scholar), the apoptotic stimulus of the oligonucleotide may be effective only in certain susceptible cells with a gene expression profile akin to the prostate cancer PC-3 cell line. In addition, a combination of the oligonucleotide with chemotherapeutic agents exemplified by cisplatin in A549 cells (41Taylor J.K. Zhang Q.Q. Wyatt J.R. Dean N.M. Nat. Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar) may sensitize only certain types of cells to undergo apoptosis. An additional benefit of cellular sensitization by the oligonucleotide may be a reduction in the dosage of chemotherapeutic agents and hence in the overall toxicity of cancer treatment. We thank Elizabeth Smith for excellent technical assistance.
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