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Mcl-1 Antisense Therapy Chemosensitizes Human Melanoma in a SCID Mouse Xenotransplantation Model

2003; Elsevier BV; Volume: 120; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2003.12252.x

1523-1747

Christiane Thallinger, Markus Wolschek, Volker Wacheck, Helmut Maierhofer, Patrick Günsberg, P. Polterauer, Hubert Pehamberger, Brett P. Monia, Edgar Selzer, Klaus Wolff, Burkhard Jansen,

DNA and Nucleic Acid Chemistry

It is well established that high expression of the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL can significantly contribute to chemoresistance in a number of human malignancies. Much less is known about the role the more recently described Bcl-2 family member Mcl-1 might play in tumor biology and resistance to chemotherapy. Using an antisense strategy, we here address this issue in melanoma, a paradigm of a treatment-resistant malignancy. After in vitro proof of principle supporting an antisense mechanism of action with specific reduction of Mcl-1 protein as a consequence of nuclear uptake of the Mcl-1 antisense oligonucleotides employed, antisense and universal control oligonucleotides were administered systemically in combination with dacarbazine in a human melanoma SCID mouse xenotransplantation model. Dacarbazine, available now for more than three decades, still remains the most active single agent for treatment of advanced melanoma. Mcl-1 antisense oligonucleotides specifically reduced target protein expression as well as the apoptotic threshold of melanoma xenotransplants. Combined Mcl-1 antisense oligonucleotide plus dacarbazine treatment resulted in enhanced tumor cell apoptosis and led to a significantly reduced mean tumor weight (mean 0.16 g, 95% confidence interval 0.08–0.26) compared to the tumor weight in universal control oligonucleotide plus dacarbazine treated animals (mean 0.35 g, 95% confidence interval 0.2–0.44) or saline plus dacarbazine treated animals (mean 0.39 g, 95% confidence interval 0.25–0.53). We thus show that Mcl-1 is an important factor contributing to the chemoresistance of human melanoma in vivo. Antisense therapy against the Mcl-1 gene product, possibly in combination with antisense strategies targeting other antiapoptotic Bcl-2 family members, appears to be a rational and promising approach to help overcome treatment resistance of malignant melanoma. It is well established that high expression of the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL can significantly contribute to chemoresistance in a number of human malignancies. Much less is known about the role the more recently described Bcl-2 family member Mcl-1 might play in tumor biology and resistance to chemotherapy. Using an antisense strategy, we here address this issue in melanoma, a paradigm of a treatment-resistant malignancy. After in vitro proof of principle supporting an antisense mechanism of action with specific reduction of Mcl-1 protein as a consequence of nuclear uptake of the Mcl-1 antisense oligonucleotides employed, antisense and universal control oligonucleotides were administered systemically in combination with dacarbazine in a human melanoma SCID mouse xenotransplantation model. Dacarbazine, available now for more than three decades, still remains the most active single agent for treatment of advanced melanoma. Mcl-1 antisense oligonucleotides specifically reduced target protein expression as well as the apoptotic threshold of melanoma xenotransplants. Combined Mcl-1 antisense oligonucleotide plus dacarbazine treatment resulted in enhanced tumor cell apoptosis and led to a significantly reduced mean tumor weight (mean 0.16 g, 95% confidence interval 0.08–0.26) compared to the tumor weight in universal control oligonucleotide plus dacarbazine treated animals (mean 0.35 g, 95% confidence interval 0.2–0.44) or saline plus dacarbazine treated animals (mean 0.39 g, 95% confidence interval 0.25–0.53). We thus show that Mcl-1 is an important factor contributing to the chemoresistance of human melanoma in vivo. Antisense therapy against the Mcl-1 gene product, possibly in combination with antisense strategies targeting other antiapoptotic Bcl-2 family members, appears to be a rational and promising approach to help overcome treatment resistance of malignant melanoma. antisense oligonucleotide dacarbazine severe combined immunodeficient terminal deoxynucleotidyl transferase mediated dUTP nick end labeling Apoptosis is important for the physiologic removal of unwanted cells during development, for tissue homeostasis, and in host defense mechanisms (Thompson, 1995Thompson C.B. Apoptosis in the pathogenesis and treatment of disease.Science. 1995; 267: 1456-1462Crossref PubMed Scopus (5978) Google Scholar;Strasser et al., 1997Strasser A. Huang D.C. Vaux D.L. 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Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.Nature. 1990; 348: 334-336Crossref PubMed Scopus (3465) Google Scholar;Korsmeyer, 1992Korsmeyer S.J. Bcl-2 initiates a new category of oncogenes: Regulators of cell death.Blood. 1992; 80: 879-886Crossref PubMed Google Scholar;Cohen, 1993Cohen J.J. Apoptosis.Immunol Today. 1993; 14: 126-130Abstract Full Text PDF PubMed Scopus (1211) Google Scholar). A delicate balance between antiapoptotic and proapoptotic Bcl-2 family members exists in each cell and determines whether the cell survives or undergoes apoptosis. Mcl-1 (myeloid cell leukemia-1) was first discovered as an early induction gene during the differentiation of a human myeloid leukemia cell line (Kozopas et al., 1993Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Mcl1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to Bcl2.Proc Natl Acad Sci USA. 1993; 90: 3516-3520Crossref PubMed Scopus (848) Google Scholar). The Mcl-1 protein contains structural motifs that characterize it as a member of the Bcl-2 protein family. Like other Bcl-2 family members, the Mcl-1 protein possesses Bcl-2 homology domains BH 1–3 (Akgul et al., 2000bAkgul C. Turner P.C. White M.R. Edwards S.W. Functional analysis of the human MCL-1 gene.Cell Mol Life Sci. 2000; 57: 684-691Crossref PubMed Scopus (93) Google Scholar;Bae et al., 2000Bae J. Leo C.P. Hsu S.Y. Hsueh A.J. Mcl-1S, a splicing variant of the antiapoptotic Bcl-2 family member Mcl-1, encodes a proapoptotic protein possessing only the BH3 domain.J Biol Chem. 2000; 275: 25255-25261Crossref PubMed Scopus (225) Google Scholar). In contrast to Bcl-2 and Bcl-xL, Mcl-1 contains PEST (proline, glutamate, serine, and threonine) sequences (Rogers et al., 1986Rogers S. Wells R. Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis.Science. 1986; 234: 364-368Crossref PubMed Scopus (1895) Google Scholar), which are commonly found in proteins with a high turnover rate. Surprisingly, deletion of 104 amino acids (residues 79–183) that contain putative PEST sequences and other stability regulating motifs did not affect protein stability (Akgul et al., 2000aAkgul C. Moulding D.A. White M.R. Edwards S.W. In vivo localisation and stability of human Mcl-1 using green fluorescent protein (GFP) fusion proteins.FEBS Lett. 2000; 478: 72-76Crossref PubMed Scopus (81) Google Scholar). Moreover, the intracellular distribution of Mcl-1 appears to be more widespread than Bcl-2 because, in addition to its predominant localization at the mitochondrial membranes, Mcl-1 is also distributed over a variety of nonmitochondrial compartments (Yang et al., 1995Yang T. Kozopas K.M. Craig R.W. The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2.J Cell Biol. 1995; 128: 1173-1184Crossref PubMed Scopus (264) Google Scholar;Leuenroth et al., 2000Leuenroth S.J. Grutkoski P.S. Ayala A. Simms H.H. The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis.J Leukoc Biol. 2000; 68: 158-166PubMed Google Scholar;Murphy et al., 2000Murphy K.M. Ranganathan V. Farnsworth M.L. Kavallaris M. Lock R.B. Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells.Cell Death Differ. 2000; 7: 102-111Crossref PubMed Scopus (270) Google Scholar). Mcl-1 has a short half-life (1–3 h) (Rogers et al., 1986Rogers S. Wells R. Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis.Science. 1986; 234: 364-368Crossref PubMed Scopus (1895) Google Scholar;Akgul et al., 2000bAkgul C. Turner P.C. White M.R. Edwards S.W. Functional analysis of the human MCL-1 gene.Cell Mol Life Sci. 2000; 57: 684-691Crossref PubMed Scopus (93) Google Scholar;Schubert and Duronio, 2001Schubert K.M. Duronio V. Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases and phosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis.Biochem J. 2001; 356: 473-480Crossref PubMed Scopus (67) Google Scholar) and is rapidly induced in a variety of cell types (Kozopas et al., 1993Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Mcl1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to Bcl2.Proc Natl Acad Sci USA. 1993; 90: 3516-3520Crossref PubMed Scopus (848) Google Scholar;Akgul et al., 2000bAkgul C. Turner P.C. White M.R. Edwards S.W. Functional analysis of the human MCL-1 gene.Cell Mol Life Sci. 2000; 57: 684-691Crossref PubMed Scopus (93) Google Scholar). The role of Mcl-1 expression in supporting cell survival has been studied in various cell systems. Human neutrophils express neither Bcl-2 nor Bcl-xL, but they do express Mcl-1. Upon shutdown of Mcl-1 expression, this pro-survival gene product is rapidly depleted, resulting in induction of apoptosis (Moulding et al., 2001Moulding D.A. Akgul C. Derouet M. White M.R. Edwards S.W. Bcl-2 family expression in human neutrophils during delayed and accelerated apoptosis.J Leukoc Biol. 2001; 70: 783-792PubMed Google Scholar). Similarly, downregulation of Mcl-1 by antisense oligonucleotides causes a rapid entry into apoptosis in differentiating human myeloblastic leukemia cells (U937) (Moulding et al., 2000Moulding D.A. Giles R.V. Spiller D.G. White M.R. Tidd D.M. Edwards S.W. Apoptosis is rapidly triggered by antisense depletion of Mcl-1 in differentiating U937 cells.Blood. 2000; 96: 1756-1763PubMed Google Scholar). Overexpression of Mcl-1 in Chinese hamster ovary cells (Reynolds et al., 1994Reynolds J.E. Yang T. Qian L. Jenkinson J.D. Zhou P. Eastman A. Craig R.W. Mcl-1, a member of the Bcl-2 family, delays apoptosis induced by c-Myc overexpression in Chinese hamster ovary cells.Cancer Res. 1994; 54: 6348-6352PubMed Google Scholar;Reynolds et al., 1996Reynolds J.E. Li J. Craig R.W. Eastman A. Bcl-2 and Mcl-1 expression in Chinese hamster ovary cells inhibits intracellular acidification and apoptosis induced by staurosporine.Exp Cell Res. 1996; 225: 430-436Crossref PubMed Scopus (125) Google Scholar) and murine myeloid progenitor cells (FDC-P1) (Zhou et al., 1997Zhou P. Qian L. Kozopas K.M. Craig R.W. Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions.Blood. 1997; 89: 630-643Crossref PubMed Google Scholar), on the other hand, has been shown to delay apoptosis induced by miscellaneous stimuli. Taken together, Mcl-1 prolongs cell viability under various cytotoxic conditions (etoposide, calcium ionophore, ultraviolet irradiation, growth factor withdrawal) that cause apoptotic cell death, as has been also shown in murine myleoid progenitor cells (Zhou et al., 1997Zhou P. Qian L. Kozopas K.M. Craig R.W. Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions.Blood. 1997; 89: 630-643Crossref PubMed Google Scholar). The mechanisms through which Mcl-1 promotes cell survival, however, are not clearly understood. Although reports on the interaction of Apaf-1 and antiapoptotic Bcl-2 family members are controversial, one possible mechanism is the inhibition of caspase activation by retaining the adapter molecule Apaf-1 in an inactive state (Moriishi et al., 1999Moriishi K. Huang D.C. Cory S. Adams J.M. Bcl-2 family members do not inhibit apoptosis by binding the caspase activator Apaf-1.Proc Natl Acad Sci USA. 1999; 96: 9683-9688Crossref PubMed Scopus (127) Google Scholar;Conus et al., 2000Conus S. Rosse T. Borner C. Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells.Cell Death Differ. 2000; 7: 947-954Crossref PubMed Scopus (42) Google Scholar). In HL-60 cells, an increase of Mcl-1 has been associated with inhibition of apoptosis by downregulating cell-damage-induced mitochondrial cytochrome c release (Wang and Studzinski, 1997Wang X. Studzinski G.P. Antiapoptotic action of 1,25-dihydroxyvitamin D3 is associated with increased mitochondrial Mcl-1 and RAF-1 proteins and reduced release of cytochrome c.Exp Cell Res. 1997; 235: 210-217Crossref PubMed Scopus (90) Google Scholar). Bcl-xL and Mcl-1 have in common the ability to heterodimerize with Bax, which has been reported to translocate from cytosol to mitochondria following exposure to apoptotic stresses and neutralize its cell death-inducing activity (Akgul et al., 2000bAkgul C. Turner P.C. White M.R. Edwards S.W. Functional analysis of the human MCL-1 gene.Cell Mol Life Sci. 2000; 57: 684-691Crossref PubMed Scopus (93) Google Scholar;Pedersen et al., 2002Pedersen I.M. Kitada S. Leoni L.M. et al.Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1.Blood. 2002; 100: 1795-1801Crossref PubMed Scopus (104) Google Scholar). Recently, it was shown that Mcl-1 is also widely expressed in normal and malignant human melanocytic cell lines and in melanoma metastases (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). Increased Mcl-1 and Bcl-xL levels were observed in thin primary melanomas as well as in metastatic malignant melanomas but not in benign nevi, suggesting that upregulation of these proteins represents an early event associated with malignant transformation (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). Several clinical studies have provided evidence for the hypothesis that high level expression of antiapoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL, and Mcl-1, confers a clinically important chemoresistant phenotype to cancer cells (Reed, 1995Reed J.C. Bcl-2: prevention of apoptosis as a mechanism of drug resistance.Hematol Oncol Clin North Am. 1995; 9: 451-473Abstract Full Text PDF PubMed Google Scholar;Reed, 1998Reed J.C. Dysregulation of apoptosis in cancer.Cancer J Sci Am. 1998; 4: S8-S14PubMed Google Scholar;Strasser et al., 1997Strasser A. Huang D.C. Vaux D.L. The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumourigenesis and resistance to chemotherapy.Biochim Biophys Acta. 1997; 1333: F151-F178PubMed Google Scholar;Kaufmann et al., 1998Kaufmann S.H. Karp J.E. Svingen P.A. Krajewski S. Burke P.J. Gore S.D. Reed J.C. Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse.Blood. 1998; 91: 991-1000PubMed Google Scholar). In human melanoma it was recently reported that Bcl-2 (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 (456) Google Scholar) expression is linked to chemoresistance. Apoptosis and its regulation may play an important pathogenic role in the development and chemoresistance of human melanoma (Serrone and Hersey, 1999Serrone L. Hersey P. The chemoresistance of human malignant melanoma: An update.Melanoma Res. 1999; 9: 51-58Crossref PubMed Scopus (132) Google Scholar). Antisense oligonucleotides (ASO), chemically modified stretches of single-stranded DNA, are pharmacologically potent inhibitors of disease-related protein expression (Webb et al., 1997Webb A. Cunningham D. Cotter F. et al.Bcl-2 antisense therapy in patients with non-Hodgkin lymphoma.Lancet. 1997; 349: 1137-1141Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar;Jansen et al., 2000Jansen B. Wacheck V. 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Antisense therapy for cancer – the time of truth.Lancet Oncol. 2002; 3: 672-683Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). They are designed to bind to their complementary mRNA sequence once they get inside the cell, thereby inhibiting expression of the encoded protein (Myers and Dean, 2000Myers K.J. Dean N.M. Sensible use of antisense: How to use oligonucleotides as research tools.Trends Pharmacol Sci. 2000; 21: 19-23Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Based on the available evidence, Mcl-1 seems to be a suitable molecular target to enhance chemosensitivity in human melanoma. In this study we used Mcl-1 ASO to downregulate Mcl-1 protein expression in human melanoma cells in vitro and in vivo. This downregulation chemosensitized human melanoma to subsequent treatment with dacarbazine (DTIC) in a severe combined immunodeficient (SCID) mouse xenotransplantation model. The human melanoma cell line 518A2 was a kind tift of Peter Schrier, University of Leiden (The Netherlands), which has been described recently (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 (456) Google Scholar). The human melanoma cell line Mel Juso (Lehmann et al., 1989Lehmann J.M. Riethmuller G. Johnson J.P. MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily.Proc Natl Acad Sci USA. 1989; 86: 9891-9895Crossref PubMed Scopus (379) Google Scholar) was kindly provided by Dr. Judith Johnson (University of Munich, Germany). All cell lines were maintained in Dulbecco's modified Eagle's culture medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Paisley, U.K.) and an antibiotic mixture containing 100 units per ml penicillin, 100 μg per ml streptomycin, and 0.25 μg per ml amphotericin B (all Gibco) in a fully humidified 5% CO2, 95% ambient air atmosphere at 37°C. 2′-O-Methoxyethyl/2′-deoxy-nucleotide chimeric phosphorothioate antisense oligonucleotides were kindly provided by ISIS Pharmaceuticals (Carlsbad, CA). The sequence of Mcl-1 antisense oligonucleotide (ISIS 20408) is 5′-TTGGCTTTGTGTCCTTGGCG-3′. A universal control oligonucleotide pool (ISIS 29848) was used, which is synthesized as a mixture of A (adenine), G (guanine), T (thymine), and C (cytosine) bases so that the resulting preparation contains an equimolar mixture of all possible oligonucleotides. The oligonucleotide chemistry of ISIS 29848 is identical to that of ISIS 20408. For cellular uptake studies Mcl-1 ASO is fluorescein-labeled with a 5′-oligonucleotide fluorescein labeling kit (Amersham, Buckinghamshire, U.K.) according to the manufacturer's instructions. For transfection, 300,000 cells were seeded in a 75 cm2 plate 24 h prior to the oligonucleotide treatment. Oligonucleotides were complexed with lipofectin (Gibco) in antibiotic-free medium without serum as described by the supplier. Subsequently, cells were incubated for 4 h with 250 nM complexed oligonucleotides (10 μg per ml lipofectin) in antibiotic-free medium without serum. After washing with DMEM, cells were further cultured in complete medium containing serum. Alternatively, cells were incubated with oligonucleotides (1 μM) for 24 h in medium supplemented with 10% fetal bovine serum. Following incubation with fluorescein-labeled oligonucleotides with and without uptake-enhancing lipids cells were fixed with paraformaldehyde (Sigma, St. Louis, MO) (4% in phosphate-buffered saline). Slides were evaluated under an Axioplan 2 (Zeiss, Vienna, Austria) fluorescence microscope equipped with a Zeiss Axiocam using Axiovision 3.0 (Zeiss) software. Cell extracts were prepared in lysis buffer containing 0.14 M NaCl, 0.4 M triethanolamine, 0.2% Na deoxycholate, and 0.5% Nonidet P-40, supplemented with 1 mM phenylmethylsulfonyl fluoride, 4.0 μg per ml aprotinin, and 4.0 μg per ml leupeptin. The amount of soluble proteins was quantified by means of a modified Bradford analysis (Bio-Rad, Richmond, CA). Total lysates (15 μg per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted onto polyvinylidene fluoride membranes (Millipore, Bedford, MA), and probed with anti-Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-β-tubulin (Sigma) antibody. Alkaline-phosphatase-conjugated goat antimouse and goat antirabbit immunoglobulins (Tropix, Bedford, MA) were used for secondary incubations. Reactive bands were visualized by chemiluminescence using CSPD® as substrate (Tropix). Protein expression levels were quantified by densitometry of autoradiograms with an Ultrascan XL densitometer (Pharmacia, Uppsala, Sweden). Pathogen-free female C.B.-17 scid/scid (SCID) mice (4–6 wk, Harlan Winkelmann, Borchen, Germany) were randomly assigned to experimental groups of six animals each. SCID mice were injected subcutaneously with 2×107 518A2 human melanoma cells into the lower left flank. Seven days after injection, animals had developed melanomas of similar size (nodules of about 5 mm in diameter) and treatment was initiated. Animals were anesthetized and miniosmotic pumps (Alzet 2002, Alzet, Palo Alto, CA) prefilled with saline, antisense, or universal control oligonucleotides were inserted subcutaneously into a paraspinal pocket. Implanted pumps released their contents by continuous infusion at a rate of 20 mg per kg per d over a period of 2 wk. Mice were treated with DTIC (80 mg per kg per d) administered intravenously for 5 d (days 12–16). During the experiments, tumor weight was assessed twice a week by caliper measurement as previously described (Boehm et al., 1997Boehm T. Folkman J. Browder T. O'Reilly M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature. 1997; 390: 404-407Crossref PubMed Scopus (1579) Google Scholar). On day 21 all animals were sacrificed, tumors were weighed, and tumor xenotransplants were fixed in formalin for immunohistochimestry and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) analysis. Histology of human melanoma xenotransplants was evaluated by hematoxylin and eosin staining. The histochemical detection of apoptosis in formalin-fixed sections was performed by TUNEL using fluorescein-dUTP (Boehringer Mannheim, Germany). Tissue sections were counterstained using DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride, Roche Diagnostics, Vienna, Austria). The expression pattern of Mcl-1 in vivo was evaluated by immunohistochemistry with an anti-Mcl-1 antibody (1:50, Santa Cruz Biotechnology) and a DAKO LSAB 2 System using diaminobenzidine as chromogen. Statistical significance of differences in tumor weight among treatment groups was calculated by using one-way ANOVA and the Scheffe test for post hoc testing (SPSS 10.0.7, SPSS, Chicago, IL). p-values less than 0.05 were considered to be of statistical significance. To assess the efficiency of oligonucleotide uptake, 518A2 human melanoma cells were incubated with 250 nM or 1 μM fluorescein-labeled Mcl-1 ASO in the presence or absence of uptake-enhancing lipids, respectively. Twenty-four hours later cells were fixed with paraformaldehyde and evaluated by fluorescence microscopy. Fluorescein-labeled oligonucleotides were found to be localized to the nucleus but also to the cytosol as well as to vesicular compartments (Figure 1). These findings were also demonstrated for another human melanoma cell line (Mel JUSO; data not shown). The two melanoma cell lines used in this work were shown to express the antiapoptotic protein Mcl-1 (Figure 2). Using Mcl-1 ASO at nanomolar concentrations in the presence of the uptake enhancer lipofectin, we observed a pronounced reduction in Mcl-1 levels in our cell lines in vitro (Figure 2). Normalization to β-tubulin demonstrated that ASO treatment caused a significant reduction of Mcl-1 protein levels compared to saline-control-treated cell lines. In 518A2 cells, ASO treatment reduced Mcl-1 levels by 84% [95% confidence interval (CI) 79.03–88.97], whereas in Mel Juso cells a reduction of 83% (CI 95% 74.00–93.68) was detected (data are given as the range of three independent experiments and are expressed relative to lipofectin-treated control cells). In contrast, universal control treatment decreased Mcl-1 levels by 5% (95% CI 4.70–15.37) in 518A2 cells and 11% (95% CI 3.35–19.32) in Mel Juso cells. Similarly, effects were also observed at a higher oligonucleotide concentration in the absence of uptake-enhancing lipids (data not shown). Treatment of melanoma cells with Mcl-1 ASO did not change Bcl-2 or Bcl-xL protein levels (data not shown). To test whether Mcl-1 ASO is capable of downregulating Mcl-1 expression in vivo, Mcl-1 ASO, universal control oligonucleotides, or saline as a vehicle control were administered to SCID mice bearing sub-cutaneously implanted 518A2 tumors. After 14 d of treatment the expression pattern of Mcl-1 was evaluated by immuno-histochemistry (Figure 3). Mcl-1-specific staining was found in all specimens evaluated; however, levels were markedly reduced in Mcl-1 ASO treated tumors compared to mice that received either saline or control oligonucleotides. Based on these findings we next tested whether reduction of Mcl-1 expression has the potential to enhance the chemosensitivity of melanoma xenotransplants. Tumor size measurements with a caliper during the course of the experiment already indicated that tumor growth is reduced in mice treated with Mcl-1 ASO/DTIC compared to mice treated with either universal control oligonucleotide-DTIC or saline/DTIC (Figure 4a). This observation was confirmed by assessing the tumor weight at the end of the experiment (Figure 4b). The combination of Mcl-1 ASO and DTIC resulted in a significantly lower mean tumor weight (mean 0.16 g, 95% CI 0.08–0.26) compared to saline plus DTIC (mean 0.39 g, 95% CI 0.25–0.53) or universal control oligonucleotides plus DTIC (mean 0.35 g, 95% CI 0.27–0.44) (Figure 4b). DTIC alone exerted an antineoplastic response without eradicating the tumors and reduced the mean tumor weight compared with that of untreated animals by approximately two-thirds. Assessing the influence of oligonucleotide monotreatment in an independent pilot experiment, no significant differences were found in the mean tumor weight between mice treated with ASO alone (mean 1.85 g, 95% CI 1.53–2.17) or universal control oligonucleotides (mean 1.85 g, CI 1.35–2.35) compared to those treated with saline (mean 2.00 g, 95% CI 1.27–2.90). To investigate whether this chemosensitization effect by Mcl-1 ASO was linked to a higher rate of apoptotic cell death in tumor xenografts, tumors were examined for apoptotic cell death by TUNEL staining (Figure 4b). Mcl-1 ASO plus DTIC treatment increased the number of apoptotic cells approximately 2-fold (7% apoptotic cells) within 518A2 tumors compared to the saline plus DTIC or the universal control plus DTIC control group (2% and 3% apoptotic cel