Bcl-2 and bcl-xL Antisense Oligonucleotides Induce Apoptosis in Melanoma Cells of Different Clinical Stages
2002; Elsevier BV; Volume: 118; Issue: 3 Linguagem: Inglês
10.1046/j.0022-202x.2001.01677.x
ISSN1523-1747
AutoresRobert A. Olie, Christoph A. Hafner, Renzo Küttel, Brigitte Sigrist, Rolf A. Stahel, Uwe Zangemeister‐Wittke, Jörg Willers, Reinhard Dummer, Jonathan Hall,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoRecent clinical studies have shown the promise of bcl-2 antisense therapy in patients with melanoma. To further demonstrate the importance of bcl-2 and validate the related antiapoptotic protein bcl-xL as targets for antisense therapy in melanoma, their implication as survival factors in melanoma cells of different clinical stages as well as in normal melanocytes was investigated. Primary cell cultures derived from 17 melanomas, the cell line A375, and normal melanocytes from healthy donors were treated with antisense oligonucleotides targeting either the bcl-xL mRNA or the bcl-2 and the bcl-xL mRNAs simultaneously. Bcl-2 and bcl-xL expression in cells was analyzed by real-time polymerase chain reaction and Western blotting. Cell viability was assessed in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and apoptosis assays. Bcl-2 expression was low in melanoma cells of stages I, II, and III, hardly detectable in A375 cells, but high in normal melanocytes. Bcl-xL expression was high in all cell types tested. As shown in A375 cells and the stage III melanoma cells 0513, both the bcl-xL monospecific oligonucleotide 4259 and the bcl-2/bcl-xL bispecific oligonucleotide 4625 effectively reduced tumor cell viability by induction of apoptosis with IC50 values ranging from 200 to 350 nM. Oligonucleotide 4625 proved to be superior to 4259, as it significantly reduced the viability of cells from all melanoma stages. Both oligonucleotides reduced also the viability of normal melanocytes. Our data suggest that bcl-2 and bcl-xL are promising targets for antisense therapy of melanoma, and that the simultaneous downregulation of their expression may provide additional clinical benefit. Recent clinical studies have shown the promise of bcl-2 antisense therapy in patients with melanoma. To further demonstrate the importance of bcl-2 and validate the related antiapoptotic protein bcl-xL as targets for antisense therapy in melanoma, their implication as survival factors in melanoma cells of different clinical stages as well as in normal melanocytes was investigated. Primary cell cultures derived from 17 melanomas, the cell line A375, and normal melanocytes from healthy donors were treated with antisense oligonucleotides targeting either the bcl-xL mRNA or the bcl-2 and the bcl-xL mRNAs simultaneously. Bcl-2 and bcl-xL expression in cells was analyzed by real-time polymerase chain reaction and Western blotting. Cell viability was assessed in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and apoptosis assays. Bcl-2 expression was low in melanoma cells of stages I, II, and III, hardly detectable in A375 cells, but high in normal melanocytes. Bcl-xL expression was high in all cell types tested. As shown in A375 cells and the stage III melanoma cells 0513, both the bcl-xL monospecific oligonucleotide 4259 and the bcl-2/bcl-xL bispecific oligonucleotide 4625 effectively reduced tumor cell viability by induction of apoptosis with IC50 values ranging from 200 to 350 nM. Oligonucleotide 4625 proved to be superior to 4259, as it significantly reduced the viability of cells from all melanoma stages. Both oligonucleotides reduced also the viability of normal melanocytes. Our data suggest that bcl-2 and bcl-xL are promising targets for antisense therapy of melanoma, and that the simultaneous downregulation of their expression may provide additional clinical benefit. inhibitor of apoptosis protein concentration with 50% inhibitory effect 2′-O-2-methoxyethyl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide Advanced melanoma is an aggressive disease and its incidence is rapidly increasing (Urist and Karnell, 1994Urist M.M. Karnell L.H. The National Cancer Database: report on melanoma.Cancer. 1994; 74: 782-788Crossref PubMed Scopus (32) Google Scholar;Dummer et al, in press). In contrast to localized melanoma, which has a good prognosis after adequate surgery, metastatic melanoma has a poor prognosis due to the development of drug resistance (Jansen et al., 2000Jansen B. Wacheck V. Heere-Ress E. et al.Chemosensitisation of malignant melanoma by BCL2 antisense therapy.Lancet. 2000; 356: 1728-1733Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). As most chemotherapeutic agents act by induction of apoptosis (Fisher, 1994Fisher D.E. Apoptosis in cancer therapy: crossing the threshold.Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1342) Google Scholar), treatment failure has been linked to the inability of melanoma cells to undergo apoptosis in response to cytotoxic damage. Several lines of evidence suggest that apoptosis resistance in melanoma relies on ineffective signaling via death receptors and caspase-8 (Irmler et al., 1997Irmler M. Thome M. Hahne M. et al.Inhibition of death receptor signals by cellular FLIP.Nature. 1997; 388: 190-195Crossref PubMed Scopus (2152) Google Scholar;Griffith et al., 1998Griffith T.S. Chin W.A. Jackson G.C. Lynch D.H. Kubin M.Z. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells.J Immunol. 1998; 161: 2833-2840PubMed Google Scholar;Thomas and Hersey, 1998Thomas W.D. Hersey P. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells.J Immunol. 1998; 161: 2195-2200PubMed Google Scholar), inactivation of the apoptosis effector protein apaf-1 (Soengas et al., 2001Soengas M.S. Capodieci P. Polsky D. et al.Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.Nature. 2001; 409: 143-144Crossref Scopus (840) Google Scholar), and deregulated expression of apoptosis inhibitors such as bcl-2, bcl-xL, and the IAP survivin (Jansen et al., 1998Jansen B. Schlagbauer-Wadl H. Brown B.D. et al.Bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice.Nat Med. 1998; 4: 232-234Crossref PubMed Scopus (457) Google Scholar;Grossman et al., 1999Grossman D. McNiff J.M. Li F. Altieri D.C. Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma.J Invest Dermatol. 1999; 113: 1076-1081https://doi.org/10.1046/j.1523-1747.1999.00776.xCrossref PubMed Scopus (339) Google Scholar). Antisense oligonucleotides are useful for target validation and functionalization, and as therapeutic agents for the inhibition of disease-related gene expression. Antisense oligonucleotides inhibiting bcl-2 or bcl-xL expression have proven to be potent inducers of apoptosis and valuable agents for the treatment of proliferative disorders such as cancer (Cotter et al., 1994Cotter F.E. Johnson P. Hall P. et al.Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model.Oncogene. 1994; 9: 3049-3055PubMed Google Scholar;Jansen et al., 1998Jansen B. Schlagbauer-Wadl H. Brown B.D. et al.Bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice.Nat Med. 1998; 4: 232-234Crossref PubMed Scopus (457) Google Scholar,Jansen et al., 2000Jansen B. Wacheck V. Heere-Ress E. et al.Chemosensitisation of malignant melanoma by BCL2 antisense therapy.Lancet. 2000; 356: 1728-1733Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar;Zangemeister-Wittke et al., 1998Zangemeister-Wittke U. Schenker T. Luedke G.H. Stahel R.A. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines.Br J Cancer. 1998; 78: 1035-1042Crossref PubMed Scopus (131) Google Scholar;Koty et al., 1999Koty P.P. Zhang H. Levitt M.L. Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.Lung Cancer. 1999; 23: 115-127Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar;Leech et al., 2000Leech S.H. Olie R.A. Gautschi O. et al.Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.Int J Cancer. 2000; 86: 570-576Crossref PubMed Scopus (89) Google Scholar;Simões-Wüst et al., 2000Simões-Wüst A.P. Olie R.A. Gautschi O. et al.Bcl-xl antisense treatment induces apoptosis in breast carcinoma cells.Int J Cancer. 2000; 87: 582-590Crossref PubMed Scopus (66) Google Scholar). We previously demonstrated the ability of the 2′-O-2-methoxyethyl (2′-MOE) modified gapmer antisense oligonucleotide 4259 targeting the bcl-xL mRNA to induce apoptosis in lung and breast cancer cells (Leech et al., 2000Leech S.H. Olie R.A. Gautschi O. et al.Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.Int J Cancer. 2000; 86: 570-576Crossref PubMed Scopus (89) Google Scholar;Simões-Wüst et al., 2000Simões-Wüst A.P. Olie R.A. Gautschi O. et al.Bcl-xl antisense treatment induces apoptosis in breast carcinoma cells.Int J Cancer. 2000; 87: 582-590Crossref PubMed Scopus (66) Google Scholar). Although from a mechanistic point of view bcl-2 and bcl-xL are functionally indistinguishable, there is evidence in support of distinct biologic roles of these proteins in protecting cells from apoptosis induced by different stimuli (Simonian et al., 1997Simonian P.L. Grillot D.A. Nunez G. Bcl-2 and Bcl-xL can differentially block chemotherapy-induced cell death.Blood. 1997; 90: 1208-1216Crossref PubMed Google Scholar;Lee et al., 1999Lee J.U. Hosotani R. Wada M. et al.Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-x) on cellular susceptibility to radiation in pancreatic cancer cells.Eur J Cancer. 1999; 35: 1374-1380Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This issue is further complicated by the cellular heterogeneity of tumor tissues and the finding that tumor cells may switch expression from one death antagonist to the other (Han et al., 1996Han Z. Chatterjee D. Early J. Pantazis P. Hendrickson E.A. Wyche J.H. Isolation and characterization of an apoptosis-resistant variant of human leukemia HL-60 cells that has switched expression from Bcl-2 to Bcl-xL.Cancer Res. 1996; 56: 1621-1628PubMed Google Scholar). Based on the uncertainty which of the two antiapoptotic proteins is the more important survival factor in a tumor, the use of antisense oligonucleotides that simultaneously inhibit bcl-2 and bcl-xL expression might be advantageous. The 2′-MOE gapmer antisense oligonucleotide 4625 targets a region of high homology shared by the bcl-2 and the bcl-xL mRNAs. It is fully complementary to bcl-2 and owing to its enhanced binding affinity can overcome the three base mismatches to bcl-xL. Recent studies have shown that oligonucleotide 4625 induces apoptosis in tumor cells of various histologic origins in vitro and has antitumor activity in vivo (Zangemeister-Wittke et al., 2000Zangemeister-Wittke U. Leech S.H. Olie R.A. et al.A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.Clin Cancer Res. 2000; 6: 2547-2555PubMed Google Scholar;Gautschi et al., 2001Gautschi O. Tschopp S. Olie R.A. et al.Activity of a novel bcl-2/bcl-xL-bispecific antisense oligonucleotide against tumors of diverse histologic origins.J Natl Cancer Inst. 2001; 93: 463-471Crossref PubMed Scopus (137) Google Scholar). The implication of bcl-2 and bcl-xL in the development and progression of melanoma is still disputed. Although their upregulated expression was reported in some studies (Tang et al., 1998Tang L. Tron V.A. Reed J.C. et al.Expression of apoptosis regulators in cutaneous malignant melanoma.Clin Cancer Res. 1998; 4: 1865-1871PubMed Google Scholar;Leiter et al., 2000Leiter U. Schmid R.M. Kaskel P. Peter R.U. Krahn G. Antiapoptotic bcl-2 and bcl-xL in advanced malignant melanoma.Arch Dermatol Res. 2000; 292: 225-232Crossref PubMed Scopus (116) Google Scholar), other investigators showed that Bcl-2 and bcl-xL expression levels do not change during tumor progression and do not differ from expression levels found in normal melanocytes (Cerroni et al., 1995Cerroni L. Soyer H.P. Kerl H. Bcl-2 protein expression in cutaneous malignant melanoma and benign melanocytic nevi.Am J Dermatopathol. 1995; 17: 7-11Crossref PubMed Scopus (97) Google Scholar;Plettenberg et al., 1995Plettenberg A. Ballaun B. Pammer J. Mildner M. Strunk D. Weninger W. Tschachler E. Human melanocytes and melanoma cells constitutively express the Bcl-2 proto-oncogene in situ and in cell culture.Am J Pathol. 1995; 146: 651-659PubMed Google Scholar;Selzer et al., 1998Selzer E. Schlagbauer-Wadl H. Okamoto I. Pehamberger H. Potter R. Jansen B. Expression of Bcl-2 family members in human melanocytes, in melanoma metastases and in melanoma cell lines.Melanoma Res. 1998; 8: 197-203Crossref PubMed Scopus (112) Google Scholar). In addition, various studies have demonstrated an inverse correlation between increasing tumor stage and bcl-2 expression (Saenz-Santamaria et al., 1994Saenz-Santamaria M.C. Reed J.A. McNutt N.S. Shea C.R. Immunohistochemical expression of Bcl-2 in melanomas and intradermal nevi.J Cutan Pathol. 1994; 21: 393-397Crossref PubMed Scopus (59) Google Scholar;Van den Oord et al., 1994Van den Oord J.J. Vandeghinste N. De Ley M. De Wolf-Peeters C. Bcl-2 expression in human melanocytes and melanocytic tumors.Am J Pathol. 1994; 145: 294-300PubMed Google Scholar;Ramsay et al., 1995Ramsay J.A. From L. Kahn H.J. Bcl-2 protein expression in melanocytic neoplasms of the skin.Mod Pathol. 1995; 8: 150-154PubMed Google Scholar;Tron et al., 1995Tron V.A. Krajewski S. Klein-Parker H. Li G. Ho V.C. Reed J.C. Immunohistochemical analysis of Bcl-2 protein regulation in cutaneous melanoma.Am J Pathol. 1995; 146: 643-650PubMed Google Scholar;Radhi, 1999Radhi J.M. Malignant melanoma arising from nevi, p53, p16 and Bcl-2 expression in benign versus malignant components.J Cutan Med Surg. 1999; 3: 293-297PubMed Google Scholar). Despite the contradictory findings about the expression and survival function of bcl-2 in melanoma, antisense oligonucleotide G3139 (GenasenseTM) was reported to sensitize melanoma cells to the chemotherapeutic agent dacarbazine and has shown encouraging results in patients with refractory melanoma in a recent phase II clinical study (Jansen et al., 1998Jansen B. Schlagbauer-Wadl H. Brown B.D. et al.Bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice.Nat Med. 1998; 4: 232-234Crossref PubMed Scopus (457) Google Scholar,Jansen et al., 2000Jansen B. Wacheck V. Heere-Ress E. et al.Chemosensitisation of malignant melanoma by BCL2 antisense therapy.Lancet. 2000; 356: 1728-1733Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). In view of the promise of bcl-2 antisense therapy and to gain further knowledge about the role of bcl-2 and bcl-xL as survival factors for melanoma cells, we examined their expression in a melanoma cell line, a series of primary melanoma cell cultures derived from tumors of different clinical stages, and in normal melanocytes. In addition, we used antisense oligonucleotides to selectively downregulate the expression of bcl-xL, or of bcl-2 and bcl-xL simultaneously, and investigated their ability to induce apoptosis in the various target cells. Primary melanoma cell cultures, derived from melanoma samples as previously described (Yue et al., 1997Yue F.Y. Dummer R. Geertsen R. Hofbauer G. Laine E. Manolio S. Burg G. Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I, HLA class-II and ICAM-1 molecules.Int J Cancer. 1997; 71: 630-637Crossref PubMed Scopus (218) Google Scholar), were cultured in RPMI-1640 (Hyclone Europe, Cramlington, U.K.) containing 10% fetal bovine serum (Hyclone Europe). Normal melanocytes were isolated as previously described (Geertsen et al., 1998Geertsen R.C. Hofbauer G.F. Yue F.Y. Manolio S. Burg G. Dummer R. Higher frequency of selective losses of HLA-A and -B allospecificities in metastasis than in primary melanoma lesions.J Invest Dermatol. 1998; 111: 497-502Crossref PubMed Scopus (53) Google Scholar) from foreskin obtained from the Children's Hospital Zürich. They were cultured in medium 154 containing human melanocyte growth supplement (Cascade Biologics, Portland, OR). Both cell culture media were supplemented with 2 mM L-glutamine, 50 IU per ml penicillin, and 50 mg per ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Real-time PCR was performed as described previously (Zangemeister-Wittke et al., 2000Zangemeister-Wittke U. Leech S.H. Olie R.A. et al.A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.Clin Cancer Res. 2000; 6: 2547-2555PubMed Google Scholar). Briefly, total RNA was isolated from cells by use of the RNeasy Mini Kit (Qiagen, Basel, Switzerland). For cDNA synthesis Taqman Reverse Transcription Reagents were used, and for real-time PCR an ABI Prism 7700 Sequence Detection system was used (Perkin Elmer Applied Biosystems, Foster City, CA). Relative quantification of gene expression was performed using the comparative threshold cycle method and rRNA as an internal standard. For bcl-xL amplification, primers with the sequences 5′-TCCTTGTCTACGCTTTCCACG-3′ and 5′-GGTCGC ATTGTGGCCTTT-3′ were used together with a 5′-ACAGTGCCC CGCCGAAGGAGA-3′ Taqman probe. For bcl-2 amplification, primers with the sequences 5′-CATGTGTGTGGAGAGCGTCAA-3′ and 5′-GCCGGTTCAGGTACTCAGTCA-3′ were used together with a 5′-CCTGGTGGACAACATCGCCCTGT-3′ Taqman probe. The bcl-xL and bcl-2 probes were labeled at the 5′ end with the reporter dye molecule 6-carboxy-fluorescein and at the 3′ end with the quencher dye molecule 6-carboxy-tetramethyl-rhodamine. A BLAST search of the NCBI database revealed no homology of the primer and probe sequences to other known human genes. Relative data are presented in comparison with a calibrator, which was either an untreated control sample in the case of treatment with oligonucleotides, or tumor 0513 in the case of basal expression levels in the various tumors. Cell lysates were subjected to Western blot analysis as described previously (Leech et al., 2000Leech S.H. Olie R.A. Gautschi O. et al.Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.Int J Cancer. 2000; 86: 570-576Crossref PubMed Scopus (89) Google Scholar). Briefly, 25 μg of soluble protein per sample were separated on 12% polyacrylamide sodium dodecyl sulfate gels. Transfer to polyvinylidene fluoride membranes was performed in semidry blotting chambers for 1 h. The blots were blocked in Tris-buffered saline containing 5% bovine serum albumin and 5% nonfat milk and then incubated overnight at 4°C with mouse antihuman bcl-2 monoclonal antibody (Dako Diagnostics, Glostrup, Denmark), or rabbit antihuman bcl-xL antibody (Transduction Laboratories, Lexington, KY). Actin staining with a mouse antiactin monoclonal antibody (ICN Biomedicals, Aurora, OH) was used as a loading control. To detect the primary antibodies, blots were incubated with rabbit antimouse or goat antirabbit immunoglobulin peroxidase conjugates, respectively (Sigma, St. Louis, MO) for 1 h at room temperature. Visualization of the immunocomplexes was performed by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech, Dübendorf, Switzerland), followed by exposure to X-ray films. Relative basal protein levels were quantified using Scion software (Scion, Frederick, MD) on scanned films, with the β-actin level as internal standard and the Bcl-2 and Bcl-xL protein levels in tumor 0513 as calibrator. Antisense oligonucleotides with the following sequences were synthesized and purified as described elsewhere (Leech et al., 2000Leech S.H. Olie R.A. Gautschi O. et al.Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.Int J Cancer. 2000; 86: 570-576Crossref PubMed Scopus (89) Google Scholar;Zangemeister-Wittke et al., 2000Zangemeister-Wittke U. Leech S.H. Olie R.A. et al.A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.Clin Cancer Res. 2000; 6: 2547-2555PubMed Google Scholar;Gautschi et al., 2001Gautschi O. Tschopp S. Olie R.A. et al.Activity of a novel bcl-2/bcl-xL-bispecific antisense oligonucleotide against tumors of diverse histologic origins.J Natl Cancer Inst. 2001; 93: 463-471Crossref PubMed Scopus (137) Google Scholar): 4259, 5′-AsAsAsGsTsAsTsCsCsCsAsGsCsCsGsCsCsGsTsT-3′; 4258, 5′-CsAsTsAsTsCsAsCsGsCsGsCsGsCsAsCsTsAsTsG-3′; 4625, 5′-AsAsGsGsCsAsTsCsCsCsAsGsCsCsTsCsCsGsTsT-3′; 4626, 5′-CsAsCs GsTsCsAsCsGsCsGsCsGsCsAsCsTsAsTsT-3′. The underlined nucleo tides are 2′-MOE-modified. Oligonucleotide 4259 is fully comple mentary to the bcl-xL mRNA and has three unmodified mismatching nucleotides to bcl-2. Oligonucleotide 4625 is fully complementary to the bcl-2 mRNA and has three mismatching nucleotides to the bcl-xL mRNA of which two are modified by 2′-MOE. Oligonucleotides 4258 and 4626 are scrambled sequence controls to oligonucleotides 4259 and 4625, respectively. Oligonucleotides were delivered to cells in the form of complexes with the transfection reagent lipofectin (Gibco BRL, Life Technologies, Glasgow, U.K.). Briefly, lipofectin 100 mg per ml was allowed to complex with oligonucleotides (6.5 mM) in serum- and antibiotic-free RPMI, prior to dilution and addition to cells, which had been plated the previous day in RPMI with 10% fetal bovine serum without antibiotics. Following various times of incubation, cells were either harvested or the transfection mixture was replaced by fresh medium (containing fetal bovine serum and antibiotics), and cells were further incubated at 37°C in a humidified atmosphere containing 5% CO2 until harvesting. Cell growth was measured by use of a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously (Leech et al., 2000Leech S.H. Olie R.A. Gautschi O. et al.Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.Int J Cancer. 2000; 86: 570-576Crossref PubMed Scopus (89) Google Scholar). Briefly, for each experiment 3000 cells per well were plated in 96-well plates and incubated overnight before a 20 h transfection with oligonucleotides. Following transfection cells were incubated for another 44 h at 37°C in normal tissue culture medium as described above. Subsequently, 10 µl MTT reagent (Sigma) [(10 mg per ml in phosphate-buffered saline (PBS)] were added and allowed to react for 1.5 h at 37°C prior to the addition of solubilization reagent (100 µl). Substrate cleavage was monitored at 570 nm by use of a SPECTRAmax 340 microplate reader and analyzed using the SOFTmax PRO software (Molecular Devices, Sunnyvale, CA). Untreated cells were taken as control. Tumor cell death was determined based on trypan blue dye uptake. Cells were incubated with 0.04% trypan blue in PBS for 5 min at room temperature, and the percentage of dead cells was calculated by counting the numbers of dead and viable cells using phase contrast microscopy. Caspase-3-like protease activity in cells was measured by use of a colorimetric test system. Detached and attached cells were harvested as described above, pooled, and lyzed in buffer by freeze/thawing essentially as described previously (Olie et al., 2000Olie R.A. Simo¨es-Wu¨st A.P. Baumann B. Leech S.H. Fabbro D. Stahel R.A. Zangemeister-Wittke U. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy.Cancer Res. 2000; 60: 2805-2809PubMed Google Scholar). Lysates were centrifuged at 17,500g at 4°C for 15 min. Cytosolic protein (40 μg) was mixed with 80 mM of the caspase-3-specific substrate DEVD-pNa (Bachem, Dübendorf, Switzerland) and incubated at 37°C. Subsequently, substrate cleavage was monitored at 405 nm using a SPECTRAmax 340 microplate reader and analyzed using SOFTmax PRO software (Molecular Devices). To confirm that substrate cleavage was due to caspase activity, extracts were incubated in the presence of 10 mM of the caspase-3-specific inhibitor DEVD-CHO (Bachem) for 30 min at 37°C, prior to the addition of substrate. The value (in arbitrary absorbance units) of the absorbance signal of the inhibited sample was subtracted from that of the noninhibited sample. Cells were washed with PBS and fixed for 15 min in 5 µg per ml Hoechst 33342 (Sigma) containing 4% paraformaldehyde. Subsequently, cells were washed three times with PBS, centrifuged onto glass slides by cytospin centrifugation, and mounted with Mowiol (Calbiochem, La Jolla, CA). Stained nuclei were photographed by use of a Nikon fluorescence microscope (Nikon, Küsnacht, Switzerland). The Student's t test was used to determine the significance of the differences between scrambled control and antisense-treated cells. The expression of bcl-2 and bcl-xL and their role as cell survival factors during melanoma progression is disputed. We determined the expression levels of bcl-2 and bcl-xL in 17 primary melanoma cell cultures derived from eight primary tumors (clinical stage I), four lymph node metastases (stage II), and five distant metastases (stage III) obtained from 13 patients and in the established melanoma cell line A375, using real-time PCR and Western blot analysis. In addition, normal melanocytes from seven healthy donors were analyzed for comparison. Prior to these analyses, eight of the primary melanoma cell cultures were examined for contamination by nonmelanoma cells and purity was found to be more than 95%. As shown in Figure 1 and Table I, bcl-2 expression was found to be increased in some of the stage III tumor-derived melanoma cells. In contrast to bcl-2, bcl-xL expression was high in all tumor stages. Compared to cells from the stage III tumor 0513, which expressed both bcl-2 and bcl-xL, the A375 cell line showed hardly detectable levels of bcl-2 and high levels of bcl-xL (Figure 1b). Freshly isolated melanocytes expressed levels of bcl-2 and bcl-xL similar to those found in 0513 melanoma cells (data not shown).Table ICharacteristics of tumor samples from patients with melanomaStageaStage I, primary tumor; stage II, lymph node metastasis; stage III, distant metastasis.mRNAbBcl-2 and bcl-xL mRNA and protein levels are given relative to the level in 0513 cells and normalized to rRNA or actin, respectively.ProteinbBcl-2 and bcl-xL mRNA and protein levels are given relative to the level in 0513 cells and normalized to rRNA or actin, respectively.PatientTNMIIIIIIbcl-2bcl-xLbcl-2bcl-xLNo markercNo marker: Percentage of cells in the primary tumor cell cultures lacking melanoma marker as analyzed by staining for Melan A, Tyrosinase, gp100, Mage 1 and Mage 3, and flow cytometry.0514T4N0M0xNDND19103ND0724T1N0M0xNDND181120%1122T2N0M0xNDND16116ND0704T1N0M0x139218112ND1008T3N0M0x105516141ND0803T3N0M0x11441894ND0728T4N2M1x2859882ND1121dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.T3N0M0x20108161090%0917dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.T3N1M0x5691271730%0325T2N1M0x1217121124ND0822No datax78613977<5%0322dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.TxN1M0x2281262ND1209dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.TxN1M1x431301032ND0310dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.TxN1M1x2111711740%0513dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.TxN1M1x100100100100<5%0409dTumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively.TxN1M1x384857990%0928T4N2M1x3431111411015%ND, not determined.a Stage I, primary tumor; stage II, lymph node metastasis; stage III, distant metastasis.b Bcl-2 and bcl-xL mRNA and protein levels are given relative to the level in 0513 cells and normalized to rRNA or actin, respectively.c No marker: Percentage of cells in the primary tumor cell cultures lacking melanoma marker as analyzed by staining for Melan A, Tyrosinase, gp100, Mage 1 and Mage 3, and flow cytometry.d Tumors 1121 and 0917 were obtained from the same patient as were tumors 0322, 1209, and 0310, and tumors 0513 and 0409, respectively. Open table in a new tab ND, not determined. Bcl-2 and bcl-xL are survival factors and their downregulation in tumor cells may have therapeutic implication. To downregulate bcl-xL expression in melanoma cells we used the bcl-xL antisense oligonucleotide 4259, a 2′-MOE-modified gapmer oligonucleotide that preferentially targets the bcl-xL mRNA. In addition, the bcl-2/bcl-xL bispecific 2′-MOE-modified gapmer oligonucleotide 4625 was used, which in a previous study with lung cancer cells was shown to induce tumor cell apoptosis more effectively than a bcl-2 or bcl-xL monospecific antisense oligonucleotide (Zangemeister-Wittke et al., 2000Zangemeister-Wittke U. Leech S.H. Olie R.A. et al.A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.Clin Cancer Res. 2000; 6: 2547-2555PubMed Google Scholar). The ability of the two oligonucleotides to downregulate the expression of their target genes was investigated using the melanoma cell line A375 and primary tumor cells from the stage III melanoma 0513. Upon a 10 h transfection with 600 nM oligonucleotide 4259, bcl-xL and bcl-2 levels were downregulated by 92% and 30%, respectively. Transfection with 600 nM oligonucleotide 4625 reduced bcl-2 by 49% and bcl-xL by 81% (Figure 2a). In 0513 cells oligonucleotide 4259 downregulated bcl-xL and bcl-2 by 69% and 47%,
Referência(s)