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Loss of Novel mda-7 Splice Variant (mda-7s) Expression is Associated with Metastatic Melanoma11Work carried out in Vienna, Austria.

2004; Elsevier BV; Volume: 123; Issue: 3 Linguagem: Inglês

10.1111/j.0022-202x.2004.23321.x

ISSN

1523-1747

Autores

Matthew J. Allen, Barbara Pratscher, Florian Roka, Clemens Krepler, Volker Wacheck, Christian Schöfer, Hubert Pehamberger, Markus Müller, Trevor Lucas,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

Expression of melanoma differentiation associated gene-7 (mda-7) also known as interleukin 24 (IL-24) decreases during melanoma cell differentiation and induces apoptosis in melanoma cells but not in melanocytes. Here we identify a novel splice variant of the cancer growth suppressor gene mda-7/IL-24 (mda-7s) that is differentially expressed in RNA preparations from normal human melanocytes, transformed melanocytes, nevi, subcutaneous metastasis, lymph node metastasis, and melanoma cell lines. The 450 bp mda-7s mRNA encodes a protein of 63 residues with a molecular weight of 12 kDa. mda-7s lacks exons 3 and 5 of the full-length transcript and contains only 14 amino acids of homology to MDA-7 located within the signal peptide region of the wild-type sequence. Despite minimal homology, MDA-7S coprecipitates full length MDA-7 and reduces secretion of cotransfected MDA-7. mda-7 and mda-7s are coexpressed in all RNA preparations other than subcutaneous and lymph node metastasis where mda-7s expression is lacking. mda-7s expression is therefore linked to a non-metastatic phenotype. Expression of melanoma differentiation associated gene-7 (mda-7) also known as interleukin 24 (IL-24) decreases during melanoma cell differentiation and induces apoptosis in melanoma cells but not in melanocytes. Here we identify a novel splice variant of the cancer growth suppressor gene mda-7/IL-24 (mda-7s) that is differentially expressed in RNA preparations from normal human melanocytes, transformed melanocytes, nevi, subcutaneous metastasis, lymph node metastasis, and melanoma cell lines. The 450 bp mda-7s mRNA encodes a protein of 63 residues with a molecular weight of 12 kDa. mda-7s lacks exons 3 and 5 of the full-length transcript and contains only 14 amino acids of homology to MDA-7 located within the signal peptide region of the wild-type sequence. Despite minimal homology, MDA-7S coprecipitates full length MDA-7 and reduces secretion of cotransfected MDA-7. mda-7 and mda-7s are coexpressed in all RNA preparations other than subcutaneous and lymph node metastasis where mda-7s expression is lacking. mda-7s expression is therefore linked to a non-metastatic phenotype. interleukin melanoma differentiation-associated gene-7 Melanoma currently affects one person in 70 with an increasing lifetime risk and represents, in the advanced stages of disease, a paradigm of oncological treatment resistance (Soengas and Lowe, 2003Soengas M.S. Lowe S.W. Apoptosis and melanoma chemoresistance.Oncogene. 2003; 22: 3138-3151Crossref PubMed Scopus (701) Google Scholar). In the progression of melanocytic transformation within normal nevi to radial and the vertical growth phase melanoma eventually leading to metastatic melanoma, cells of the melanocytic lineage lose contact with keratinocytes and become resistant to apoptosis induction by chemotherapeutic agents with a corresponding reduction of median survival of patients (Houghton and Polsky, 2002Houghton A.N. Polsky D. Focus on melanoma.Cancer Cell. 2002; 2: 275-278Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The melanoma differentiation-associated gene 7 (mda-7), also known as interleukin 24 (IL-24), contains seven exons and is transcribed from a locus at 1q 34.2–41 which contains several members of the IL-10 family of cytokines (Huang et al., 2001Huang E.Y. Madireddi M.T. Gopalkrishnan R.V. et al.Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.Oncogene. 2001; 20: 7051-7063Crossref PubMed Scopus (213) Google Scholar). The full-length human mda-7 1718 nt cDNA encodes a protein of 206 amino acids in the major open-reading frame, with a predicted molecular weight of 23.8 kDa (Jiang et al., 1996Jiang H. Su Z.Z. Lin J.J. Goldstein N.I. Young C.S. Fisher P.B. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.Proc Natl Acad Sci USA. 1996; 93: 9160-9165Crossref PubMed Scopus (279) Google Scholar), which, due to glycosylation, migrates with an apparent molecular weight of 35 kDa (Wang et al., 2002Wang M. Tan Z. Zhang R. Kotenko S.V. Liang P. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2.J Biol Chem. 2002; 277: 7341-7347Crossref PubMed Scopus (236) Google Scholar). Originally cloned as a gene upregulated during terminal differentiation of melanoma cells induced by recombinant human fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (Jiang et al., 1995Jiang H. Lin J.J. Su Z.Z. Goldstein N.I. Fisher P.B. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression.Oncogene. 1995; 11: 2477-2486PubMed Google Scholar), MDA-7 expression levels progressively decrease during the transformation of melanocytic cells to metastatic melanoma (Ekmekcioglu et al., 2001Ekmekcioglu S. Ellerhorst J. Mhashilkar A.M. et al.Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.Int J Cancer. 2001; 94: 54-59Crossref PubMed Scopus (116) Google Scholar). At supraphysiological levels, MDA-7 selectively induces growth suppression and apoptosis in diverse human cancers including melanoma, breast, colon, prostate, small cell lung, and pancreatic carcinoma whereas no quantitatively significant effect is found in normal human cells such as melanocytes and endothelial cells (Sarkar et al., 2002Sarkar D. Su Z.Z. Lebedeva I.V. et al.mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK.Proc Natl Acad Sci USA. 2002; 99: 10054-10059Crossref PubMed Scopus (291) Google Scholar). MDA-7 overexpression in a replication-defective adenovirus (Ad.mda-7) in melanoma cells induces activation of the growth arrest and DNA damage (GADD) family genes (GADD153, GADD34, GADD45α, and GADD45γ) via p38 MAPK. This is associated with a reduction of BCL2 protein levels leading to induction of apoptosis in melanoma cells but not in normal melanocytes (Sarkar et al., 2002Sarkar D. Su Z.Z. Lebedeva I.V. et al.mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK.Proc Natl Acad Sci USA. 2002; 99: 10054-10059Crossref PubMed Scopus (291) Google Scholar). It has also been reported that Ad.mda-7 transfection not only confers growth suppression, but also arrests the cell cycle at G2/M in melanoma cells (Ekmekcioglu et al., 2001Ekmekcioglu S. Ellerhorst J. Mhashilkar A.M. et al.Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.Int J Cancer. 2001; 94: 54-59Crossref PubMed Scopus (116) Google Scholar). Ad.mda-7 (INGN 241) has entered clinical trials for solid tumors (Fisher et al., 2003Fisher P.B. Gopalkrishnan R.V. Chada S. et al.mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: From the laboratory into the clinic.Cancer Biol Ther. 2003; 2: S23-S37Crossref PubMed Scopus (151) Google Scholar). As an IL-10 family cytokine, MDA-7 is secreted by concanavalin A-activated peripheral blood mononuclear cells (Wang et al., 2002Wang M. Tan Z. Zhang R. Kotenko S.V. Liang P. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2.J Biol Chem. 2002; 277: 7341-7347Crossref PubMed Scopus (236) Google Scholar) and demonstrates immunostimulatory activity (Caudell et al., 2002Caudell E.G. Mumm J.B. Poindexter N. et al.The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24.J Immunol. 2002; 168: 6041-6046Crossref PubMed Scopus (267) Google Scholar). Secreted MDA-7 binds two functional heterodimeric receptors IL-20R1/IL-20R2 and IL-22R1/IL-20R2 on target cells such as keratinocytes, which leads to stat activation (Dumoutier et al., 2001Dumoutier L. Leemans C. Lejeune D. Kotenko S.V. Renauld J.C. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types.J Immunol. 2001; 167: 3545-3549Crossref PubMed Scopus (355) Google Scholar;Wang et al., 2002Wang M. Tan Z. Zhang R. Kotenko S.V. Liang P. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2.J Biol Chem. 2002; 277: 7341-7347Crossref PubMed Scopus (236) Google Scholar). Although mda-7 mRNA is found in normal human melanocytes and metastatic melanoma cell lines at lower levels, MDA-7 protein levels decrease in more advanced melanoma and metastatic disease (Su et al., 1998Su Z.Z. Madireddi M.T. Lin J.J. et al.The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice.Proc Natl Acad Sci USA. 1998; 95: 14400-14405Crossref PubMed Scopus (240) Google Scholar;Ekmekcioglu et al., 2001Ekmekcioglu S. Ellerhorst J. Mhashilkar A.M. et al.Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.Int J Cancer. 2001; 94: 54-59Crossref PubMed Scopus (116) Google Scholar). In recent years, the identification of tissue-specific differential splicing of key gene products has greatly increased the complexity of the transcriptome and led to further understanding of the control of functional protein expression (Sorek and Amitai, 2001Sorek R. Amitai M. Piecing together the significance of splicing.Nat Biotechnol. 2001; 19: 196Crossref PubMed Scopus (26) Google Scholar). During a search of GenBank-expressed sequenced tags, we identified examples of mda-7 splicing of exons 1–4 in prostate and colon tissue. To examine the role of differential mda-7 splicing in the melanocytic lineage, we amplified mda-7 sequences by PCR and identified a splice variant of mda-7 (mda-7s) in normal human melanocytes, which lacks both exons 3 and 5. MDA-7S has a molecular weight of 12 kDa, contains homologies to wild-type MDA-7 derived only from exon 2, but heterodimerizes with wild-type MDA-7 and reduces secretion of the wild-type protein. Subsequent screening of melanoma RNA samples identified a complete lack of mda-7s expression in subcutaneous metastatic tumor material, with lymph node metastases demonstrating positive association with a non-metastatic phenotype. The full-length 1718 bp mda-7 mRNA (accession NM_006850.1) transcript (Jiang et al., 1995Jiang H. Lin J.J. Su Z.Z. Goldstein N.I. Fisher P.B. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression.Oncogene. 1995; 11: 2477-2486PubMed Google Scholar) is spliced from a 5532 bp locus at chromosome 1q 34.2–41 (Huang et al., 2001Huang E.Y. Madireddi M.T. Gopalkrishnan R.V. et al.Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.Oncogene. 2001; 20: 7051-7063Crossref PubMed Scopus (213) Google Scholar) on GenBank clone RP11-462N18 (accession AC023534.3). Seven exons are spliced to form the mature transcript in which the reading frame extends from exons 2 to 7. To investigate the possible role of differential splicing in the control of mda-7 expression and function, we conducted BLAST searches of expressed sequence tag homologies to the wild-type mda-7/IL-24 sequence. Two clones (accession numbers AW949784.1 and AW949792.1) isolated from colon tumor material and a sequence isolated from normal prostate gland tissue (AA370518.1) that showed direct splicing of mda-7 exons 1–4 were identified. RT-PCR is a highly sensitive method for detection of even single molecules of cDNA. To investigate the possibility of differential splicing in the melanocytic lineage, cDNA preparations from normal human melanocytes were PCR amplified by primer sets spanning the coding region of mda-7. In addition to the full-length mda-7 transcript, an additional smaller amplificant was also seen in all melanocytic preparations (Figure 3a, lanes 1–4). Isolation, cloning, and sequencing of this transcript demonstrated the presence of a novel splice variant (mda-7s) where exon 2 sequences, which contain the mda-7 start codon, are spliced directly to exon 4, and exon 4 sequences are spliced directly to exon 6 (Figure 1a and b). The coding sequence of mda-7s and positions of inter-exonic splicing are shown in Figure 1a. The predicted open reading frame of mda-7s has 63 amino acid residues derived from sequences located in exons 2, 4, 6, and 7 (Figure 1a). Protein homology to wild-type MDA-7 is limited to 14 amino acids derived from exon 2 (Figure 1a), since the exon 2 to exon 4 transition results in a frame shift, which is maintained after the splicing of exon 4 to exon 6. These 14 amino acids represent the initial residues of the 49 residue wild-type N-terminal amino acid signal peptide sequence but lack the signal peptidase cleavage sites between residues 49 and 50 of wild-type MDA-7 (Sauane et al., 2003Sauane M. Gopalkrishnan R.V. Sarkar D. et al.MDA-7/IL-24: Novel cancer growth suppressing and apoptosis inducing cytokine.Cytokine Growth Factor Rev. 2003; 14: 35-51Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). MDA-7S also contains no predicted cleavage sites suggesting MDA-7S is not secreted. Due to the low sequence homologies, MDA-7S also lacks the IL-10 signature motif, N-glycosylation sites, and the protein kinase c and casein kinase II phosphorylation motifs present in the wild-type sequence (Sauane et al., 2003Sauane M. Gopalkrishnan R.V. Sarkar D. et al.MDA-7/IL-24: Novel cancer growth suppressing and apoptosis inducing cytokine.Cytokine Growth Factor Rev. 2003; 14: 35-51Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar).Figure 1Differential splicing of mda-7 RNA. The mda-7 splice variant (mda-7s) was amplified and cloned from normal human melanocytes. (a) mda-7s is spliced (↓) aberrantly between exons (E) 2 and 4, and exons 4 and 6 shown in the sequence traces (b) generating a reading frame between exons 2 and 7. Homologies between the 206 residue wild-type MDA-7 and the 63 amino acid MDA-7S are restricted to 14 residues (underlined) derived from exon 2.View Large Image Figure ViewerDownload (PPT) The molecular mass of C-terminal HA-tagged MDA-7S as determined by western blotting of transfected extracts is 12 kDa (data not shown) approximating to the predicted MDA-7S molecular mass (7.3 kDa) which increases to 9.2 kDa incorporating the HA peptide. It is unlikely that this slight increase in observed molecular weight is due to glycosylation since no N-linked sequences are found and low probilities of O-linked glycosylation are predicted with bioinformatics (data not shown). The transfected MDA-7CTGFP fusion protein migrates as predicted with a molecular weight of 62 kDa equaling the sum of MDA-7 (35 kDa) and cycle 3 GFP (27 kDa) molecular masses (data not shown). Since a common property of IL-10 family cytokines is homodimerization (Fickenscher et al., 2002Fickenscher H. Hor S. Kupers H. Knappe A. Wittmann S. Sticht H. The interleukin-10 family of cytokines.Trends Immunol. 2002; 23: 89-96Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), we investigated whether MDA-7S interacts in vitro with wild-type MDA-7. HEK293 cells were cotransfected with the pMHmda-7s-HA, pMH-HA, pmda-7CTGFP and pCTGFP plasmids. Following immunoprecipitation with either anti-HA or anti-GFP antibodies, associated proteins were detected by western blotting with antibodies directed against GFP or HA, respectively. The 12 kDa HA tagged MDA-7S associated with the wild-type 62 kDa MDA-7CTGFP fusion protein when precipitated with either the anti-HA (Figure 2a) or anti-GFP antibodies (Figure 2b). Cotransfected pMHmda-7s-HA and pCTGFP (Figure 2a) or pMH-HA and pmda-7CTGFP (Figure 2b) showed no interaction. To examine whether mda-7s interaction affects the mda-7-mediated induction of apoptosis in melanoma cells, MelJUSO cells were first transfected with pmda-7CTGFP and monitored by flow cytometry. Twenty-four hours post transfection, 1%–2% of cells were green fluorescent and all cells were permeable to the vital dye propidium iodide after 48 h. Cotransfection with mda-7s did not influence the kinetics of cell death induction. Since IL-10 family cytokines are known to homodimerize and MDA-7/MDA-7S homologies are confined to the leader peptide sequence, we investigated the role of MDA-7S on the secretion of MDA-7CTGFP. As previously reported (Caudell et al., 2002Caudell E.G. Mumm J.B. Poindexter N. et al.The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24.J Immunol. 2002; 168: 6041-6046Crossref PubMed Scopus (267) Google Scholar), secreted MDA-7 accumulates in the culture media of transfected HEK293 cells. In cells cotransfected with pmda-7CTGFP and pMHmda-7s-HA, reduced secretion of wild-type MDA-7CTGFP is observed (Figure 2c). To evaluate the expression of mda-7s during the transformation of melanocytes to metastatic melanoma, mda-7 expression was analyzed by PCR. Normal human melanocytes coexpress mda-7 and mda-7s (Figure 3a, lanes 1–4). RNA isolated from spontaneously transformed melanocyte cultures (Figure 3a, lanes 5–7) which are cultured in normal medium and correspond to the original melanocyte culture RNA in lanes 2–4 (Figure 3a), respectively, also express both mda-7 and mda-7s. To examine possible changes in mda-7s expression during melanoma progression, samples were compared from normal nevi, subcutaneous metastasis (stage IV) and lymph node metastasis (stage IV). Similar to melanocyte patterns, coexpression of mda-7 and mda-7s was seen in normal nevi (Figure 3b, lanes 1–3) mirroring the results obtained from spontaneously transformed melanocyte cultures showing no association between mda-7s expression and cellular transformation. Analysis of subcutaneous (Figure 3c, lanes 1–3) and lymph node metastatic (Figure 3c, lanes 4–6) tumor material, however, demonstrated no expression of mda7-s in independent patient samples summarized in Table I, indicating an association between loss of mda-7s expression and metastatic development. With regard to mda-7 expression, we cannot exclude contamination of melanoma samples with other cells such as lymphocytes, which may express mda-7. Differential mda-7s expression patterns were seen in melanoma cell lines. In A375 and MelJUSO cells, coexpression of mda-7 and mda-7s is seen (Figure 3d, lanes 1 and 3) whereas in the 607B cell line, no expression of mda-7s was detected (Figure 3d, lane 2). Expression levels of mda-7s were also assessed in a real time PCR assay. Confirming the RT-PCR data, mda-7s is expressed in melanocytes with normalized threshold cycle values of 28.3±2.4 (mean three experiments) but is not detected in metastatic melanoma samples within 40 cycles of amplification (Figure 3e).Table ISummary of mda-7s expression status during metastatic melanoma progressionPatient sampleTissue typeDisease stageCharacterizationmda-7s profile1Nevi–NormalPositive2Nevi–NormalPositive3Nevi–NormalPositive4SubcutaneousIII/IVMetastaticNegative5SubcutaneousIII/IVMetastaticNegative6SubcutaneousIII/IVMetastaticNegative7Lymph nodeIII/IVMetastaticNegative8Lymph nodeIVMetastaticNegative9Lymph nodeIVMetastaticNegativemda-7s expression is seen in nevi and primary stage I/II melanoma tumors. In subcutaneous stage III/IV disease, lymph node metastatic stage III/IV disease and terminal disease stages, no expression of mda-7s is seen. Open table in a new tab mda-7s expression is seen in nevi and primary stage I/II melanoma tumors. In subcutaneous stage III/IV disease, lymph node metastatic stage III/IV disease and terminal disease stages, no expression of mda-7s is seen. Great interest in mda-7 as a clinically relevant, candidate tumor suppressor gene has been generated due to its growth arrest and apoptosis-inducing properties in melanoma cells which are not seen in normal human melanocytes subjected to ectopic overexpression (Ekmekcioglu et al., 2001Ekmekcioglu S. Ellerhorst J. Mhashilkar A.M. et al.Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.Int J Cancer. 2001; 94: 54-59Crossref PubMed Scopus (116) Google Scholar;Ellerhorst et al., 2002Ellerhorst J.A. Prieto V.G. Ekmekcioglu S. Broemeling L. Yekell S. Chada S. Grimm E.A. Loss of MDA-7 expression with progression of melanoma.J Clin Oncol. 2002; 20: 1069-1074Crossref PubMed Scopus (132) Google Scholar). Both mda-7 RNA and MDA-7 expression are reduced during the progression of melanoma, and significant differences in MDA-7 expression between primary tumors and metastases have been reported (Ellerhorst et al., 2002Ellerhorst J.A. Prieto V.G. Ekmekcioglu S. Broemeling L. Yekell S. Chada S. Grimm E.A. Loss of MDA-7 expression with progression of melanoma.J Clin Oncol. 2002; 20: 1069-1074Crossref PubMed Scopus (132) Google Scholar). Expression levels may therefore be regarded as a diagnostic marker for disease progression and adenoviral vectors encoding mda-7 (INGN 241) have entered clinical trials (Fisher et al., 2003Fisher P.B. Gopalkrishnan R.V. Chada S. et al.mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: From the laboratory into the clinic.Cancer Biol Ther. 2003; 2: S23-S37Crossref PubMed Scopus (151) Google Scholar). During a BLAST search of GenBank expressed sequence tag homologies to the wild-type mda-7/IL-24 sequence, we identified two clones from colon tumor material and a sequence isolated from normal prostate gland tissue that showed direct splicing of mda-7 exons 1–4. To investigate the possible role of differential splicing in the control of MDA-7 expression and function in the melanocytic lineage, we subsequently isolated the mda-7s transcript, which is produced by direct splicing of exons 2, 4, and 6. As common to the other sequences found in databanks, our data clearly show aberrant splicing to exon 4 within the mda-7 gene. Mda-7s RNA also contains the polyadenylation signal and the AU-rich mRNA destabilization sequences in exon 7 that are essential for protein-mediated post-transcriptional regulation of wild-type mda-7 transcripts (Madireddi et al., 2000Madireddi M.T. Dent P. Fisher P.B. Regulation of mda-7 gene expression during human melanoma differentiation.Oncogene. 2000; 19: 1362-1368Crossref PubMed Scopus (55) Google Scholar). In general, alternative splicing often results in functional protein expression (Rinfret and Anderson, 1993Rinfret A. Anderson S.K. IL-2 regulates the expression of the NK-TR gene via an alternate RNA splicing mechanism.Mol Immunol. 1993; 30: 1307-1313Crossref PubMed Scopus (13) Google Scholar) and considerably increases the transcriptome. Splice variant protein expression may also lead to a reduction in functional transcript expression leading to altered pathology (Hau et al., 2002Hau P. Wise P. Bosserhoff A.K. et al.Cloning and characterization of the expression pattern of a novel splice product MIA (splice) of malignant melanoma-derived growth-inhibiting activity (MIA/CD-RAP).Invest Dermatol. 2002; 119: 562-569Crossref PubMed Scopus (11) Google Scholar). This finding has been reported by a number of groups and include the tumor suppressor gene men1 (Mutch et al., 1999Mutch M.G. Dilley W.G. Sanjurjo F. et al.Germline mutations in the multiple endocrine neoplasia type 1 gene: Evidence for frequent splicing defects.Hum Mutat. 1999; 13: 175-185Crossref PubMed Scopus (77) Google Scholar), pten (Butler et al., 1999Butler M.P. Wang S.I. Chaganti R.S. Parsons R. Dalla-Favera R. Analysis of PTEN mutations and deletions in B-cell non-Hodgkin's lymphomas.Genes Chromosomes Cancer. 1999; 24: 322-327Crossref PubMed Scopus (42) Google Scholar), nf-2 (Jacoby et al., 1994Jacoby L.B. MacCollin M. Louis D.N. et al.Exon scanning for mutation of the NF2 gene in schwannomas.Hum Mol Genet. 1994; 3: 413-419Crossref PubMed Scopus (184) Google Scholar), and the transcription factor pax-2 (Tavassoli et al., 1997Tavassoli K. Ruger W. Horst J. Alternative splicing in PAX2 generates a new reading frame and an extended conserved coding region at the carboxy terminus.Hum Genet. 1997; 101: 371-375Crossref PubMed Scopus (29) Google Scholar). In recent years, the human-expressed sequence tag (EST) database has been analyzed by a number of different groups to investigate differential splicing (Sorek and Amitai, 2001Sorek R. Amitai M. Piecing together the significance of splicing.Nat Biotechnol. 2001; 19: 196Crossref PubMed Scopus (26) Google Scholar) and it has been estimated that up to 59% of human genes have at least two tissue-specific splice variants. Since evidence of splice variants conferring dominant negative effects has recently been reported (Korkalainen et al., 2003Korkalainen M. Tuomisto J. Pohjanvirta R. Identification of novel splice variants of ARNT and ARNT2 in the rat.Biochem Biophys Res Commun. 2003; 303: 1095-1100Crossref PubMed Scopus (24) Google Scholar), we investigated the effects of mda-7s on the apoptosis-promoting properties of the wild-type protein. In MelJuso melanoma cells, wild-type mda-7 rapidly induced cell death, which was not inhibited by cotransfection with an mda-7s expression plasmid demonstrating that heterodimeric MDA-7/MDA-7S expression has no effect on the apoptosis-inducing properties of the wild-type protein. In addition, MDA-7S expression levels were not significantly decreased during spontaneously transformed melanocyte cultures described previously (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 (115) Google Scholar), indicating that MDA-7S expression is not directly associated with melanocyte transformation, nor by culture in melanocyte growth medium which contains additional growth factors and phorbol esters. Predicted amino acid sequence homologies between MDA-7S and MDA-7 are limited to the N-terminal 14 amino acids derived from exon 2. MDA-7S, however, lacks the signal peptidase cleavage sites present in wild type MDA-7 and no evidence of secretion is detected in transfected HEK293 supernatants (data not shown). In contrast to other IL-10 family cytokines, MDA-7 (IL-24) has an extended signal peptide sequence, the functional significance of which has not been addressed. We show that MDA-7S has homology to MDA-7 only within this signal peptide sequence and although homodimerization of wild-type MDA-7 has not been reported, MDA-7S and MDA-7 proteins functionally interact in vitro and secretion of transfected MDA-7 is inhibited. Future investigations of the MDA-7S residues responsible for interaction with wild-type MDA-7 should also reveal whether MDA-7 homodimerizes similar to other IL-10 family cytokines. Studies to determine the precise subcellular localization of MDA-7S are in progress and should lead to a fuller understanding of the role of this heterodimerization during the MDA-7 secretory process within melanocytic cells. During the rapid development of malignant melanoma, transformed melanocytic lineage cells develop through a vertical growth phase and become highly metastatic. The exact expression patterns conferring a metastatic phenotype have not been elucidated. Although mda-7s expression is seen in melanocytes, transformed melanocytes and normal nevi, a complete absence of expression is associated with both local (subcutaneous) and distant (lymph node) metastasis. Since secreted MDA-7 binds IL-20R1/IL-20R2 and IL-22R1/IL-20R2 on target cells lead to stat activation (Wang et al., 2002Wang M. Tan Z. Zhang R. Kotenko S.V. Liang P. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2.J Biol Chem. 2002; 277: 7341-7347Crossref PubMed Scopus (236) Google Scholar), the decreases in mda-7 expression seen during the development of metastatic disease may represent a previously unknown mechanism for MDA-7 secretion in the activation of accessory cells. Although the precise role of MDA-7S in this process is unclear, this association demonstrates that MDA-7S expression levels could potentially play a role in advances of diagnosis when determining the progression of a localized lesion and assessing the disease stage of melanoma tumors. In summary, this study has identified a novel mda-7 splice variant (mda-7s) in human melanocytes. The splice variant was determined to have molecular weight of 12 kDa and lacked exons 3 and 5 of mda-7 but functionally interacted with and influenced the secretion of the wild-type protein in vitro. Subsequently, a screen of melanoma patient samples was performed and lymph node metastatic and subcutaneous metastatic tumors were identified as being deficient of mda-7s message when assessed via both RT-PCR and quantitative PCR. MelJUSO, A375 (ATCC, Manassas, Virginia) and 607B melanoma cells, transformed melanocytes (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 (115) Google Scholar), and the HEK293 cell line were maintained in Dulbecco's modified Eagle's culture medium containing 4.5 g per liter glucose (DMEM), supplemented with 10% fetal calf serum (Gibco, Paisley, Scotland) and an antibiotic-antimycotic mix containing 100 U per mL of penicillin, 100 μg per mL of streptomycin and 0.25 μg per mL of amphotericin B (Gibco) in a fully humidified 5% CO2–95% ambient air atmosphere at 37°C. Normal human melanocytes (Clonetics, Remagen, Germany) were cultured in serum-free melanocyte growth medium (MGM-2) containing 10 μg per mL phorbol 12-myristate-13-acetate, 0.5 μg per mL hydrocortisone, 5 μg per mL insulin, 5 μg per mL human fibroblast growth factor, 50 μg per mL gentamycin-sulfate, 50 μg per mL amphothericin-B supplemented with bovine pituitary extract as recommended by the suppliers. Tumor material was collected at the Department of Dermatology at the University of Vienna following approval by the University of Vienna ethics committee. Tumor samples were isolated after routine surgical removal and placed immediately in RNAlater (Qiagen, Hilden, Germany). Melanocyte and melanoma cell line pellets were resuspended and tissue samples homogenized in TRI Reagent (Sigma, St Louis, Mo.) and total RNA was reverse transcribed with Oligo (dT)12–18 and SuperScript II (Gibco) according to the manufacturer's instructions. Institutional Review Board approval was granted for this work. Primer positions are based on the mda-7/IL-24 sequence GenBank accession number NM_006850.1. RT-PCR was independently performed with mda-7 forward (272 5′-GAGATGAATTTTCAACAGAGGC-3′ 293) and reverse (895 5′-CGAGCTTGTAGAATTTCTGCATCC-3′ 870) primers, and the tubulin forward (1263 5′-CATCCAGGAGCTCTTCAAGC-3′ 1282) and reverse (1442 5′-CTCCTCACCGAAATCCTCCT-3′ 1461) primers based on the β-tubulin RNA sequence (AF141349.1). cDNA (1 μL) was amplified in a 100 μL reaction containing 200 μM dNTPs, 1.5 mM MgCl2, 20 mM Tris-HCl (pH 8), 50 mM KCl, and 1 U Platinum Taq polymerase (Invitrogen, San Diego, California) in a GenAmp thermocycler 2400 (Applied Biosystems, Perkin-Elmer, Foster City, California) by initial denaturation at 95°C for 2 min, 35 cycles of amplification at 95°C, denaturation for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min and end extension at 72°C for 7 min. PCR products were separated on 1% agarose gels containing 0.5 μg per mL ethidium bromide at 1 V/cm in TBE buffer. mda-7s fragments were gel purified with the QIAquick PCR Purification Kit (Qiagen) and sequenced on both strands with the amplification primers on an ABI-Prism 3100 Genetic Analyzer (Applied Biosystems). The coding sequence of full length mda-7 was amplified from melanocyte cDNA and gel purified as before and cloned into the pcDNA3.1/CT-GFP-TOPO vector (Invitrogen) as a C-terminal GFP fusion (mda-7-GFP) with the forward (272 5′-GAGATGAATTTTCAACAGAGGC-3′ 293) and reverse (5′-CGAGC TTGTAGAATTTCTGCATCC-3′ 868) primers, the reverse primer deleting the stop codon and inserting a 5′ terminal G residue to maintain the reading frame for GFP, according to the manufacturer's instructions. The reading frame of mda-7s was amplified from melanocyte cDNA using the forward (5′-GGGGTACCCCGAGAT GAATTTTCAACAG-3′ 289) and reverse (5′-CGGAATTCCGCGTC CAACTGTTTGAATG-3′ 801) primers incorporating KpnI and EcoRI restriction sites, respectively. The PCR product was gel purified, restriction digested and cloned as a C-terminal hemagglutinin (HA)-tagged protein into the pMH vector (Roche, Basel, Switzerland) by standard procedures (pMHmda-7s-HA) and sequenced on both strands with a ABI-Prism 3100 Genetic Analyzer (Applied Biosystems). HEK293 and MelJUSO cells were transiently transfected with optimally diluted endotoxin-free pmda-7-GFP and pMHmda-7s-HA plasmid preparations in six-well plates in the presence of Fugene (Roche) at a DNA to lipid ratio of 3:1. As assessed by flow cytometry in the presence of 5 μg per mL propidium iodide, transfection efficiencies were 20%–30% and 1%–2% for HEK293 and MelJUSO cells, respectively. Conditioned media were harvested after 48 h. Cells were harvested by trypsinization and washed first in cold complete medium and subsequently in phosphate-buffered saline (PBS). Cells from cultures in logarithmic growth, where cell viabilities were estimated at greater than 99% by trypan blue exclusion, were lysed in hypotonic lysis buffer (1% Triton X-100, 150 mM NaCl, 25 mM Tris, pH 7.4, 5 μg per mL leupeptin, aprotinin, and pepstatin, 1 μg per mL benzamidine HCl, 1 mM sodium orthovanadate and 1 mM PMSF) on ice at a density of approximately 106 cells per mL. Protein concentrations were determined by a modified Bradford method (Bio-Rad, Hercules, California). For immunoprecipitations, HEK293 cells were harvested in PBS and incubated in 1 mL of cell lysis buffer (PBS containing 5 mM EDTA, 0.5% Triton X-100, 0.1 mM PMSF, 5 μg per mL leupeptin, aprotinin, and pepstatin) for 30 min on a rotary shaker at 4°C. After centrifugation at 15,000 ×g for 30 min at 4°C, supernatants were incubated with 100 μL anti-HA affinity matrix (clone 3F10; Roche) overnight or sequentially with protein G-Agarose (Roche) for 1 h before addition of mouse monoclonal anti-GFP (clone ab1218; Abcam, Cambridge, UK). The matrix was then washed three times in cell lysis buffer at 4°C. Western blotting was performed essentially as described (Lucas et al., 2001Lucas T. Pratscher B. Krishnan S. et al.Differential expression levels of Par-4 in melanoma.Mel Res. 2001; 11: 379-383Crossref PubMed Scopus (15) Google Scholar). Protein loading was routinely controlled by staining of gels and membranes (Gelcode blue; Pierce, Rockford, Illinois). Briefly, cell extracts, immunoprecipitates or conditioned medium were mixed with loading buffer (62.5 mM Tris, pH 6.8%, 10% glycerol, 2% sodium dodecyl sulphate (SDS), 5%β-mercaptoethanol and 0.003% bromophenol blue) at a ratio of 1:1, boiled for 7 min at 95°C and centrifuged at 15,000 ×g for 30 s. Proteins (15 μg per lane) were separated by SDS polyacrylamide gel electrophoresis (PAGE) at 100 V on 12% gels and transferred onto PVDF membranes (Tropix, Foster City, California) at 100 V for 1.5 h. Membranes were blocked for 1 h in 0.2% I-block (Tropix) in PBS and then incubated with monoclonal mouse anti-GFP or rat anti-HA. Membranes were then incubated with alkaline phosphatase conjugated anti mouse immunoglobulins (Tropix) or sequentially with biotinylated monoclonal anti-rat IgG1 (clone RG11/39.4; Pharmingen, San Diego, California) and alkaline phosphatase-conjugated streptavidin (Pharmingen) in 0.2% I-block. Blots were then washed twice in 0.2% I-block and developed using CSDP (Tropix). To detect mda-7s, a forward primer in exon 2 (281 5′-TTTCAACAGAGGCTGCAAAGC-3′ 301) was combined with a reverse primer in exon 4 (546 5′-AGCCGGGCACTCGTGAT-3′ 530) and a fluoresent (303′ 5′-TGTGGACTTTAGCCAGCAAGCTCAGGA-3′ 525) probe spanning the exon 2/4 boundary. Primer positions are based on the wild-type mda-7/IL-24 sequence (NM_006850.1). PCR amplifications were performed on 2 μL of melanocytic or melanoma cDNA samples with 5 pmol of FAM-labeled probe (VBC-genomics, Vienna, Austria) and 5 pmol of primers in a 25 μL reaction containing TaqMan Universal PCR master mix (Applied Biosystems). Real-time PCR was initiated at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min and threshold cycle values were analyzed with Sequence Detector v1.7 software (Applied Biosystems) in a PRISM 7700 sequence detector (Applied Biosystems) as described previously (Lucas et al., 2004Lucas T. Losert D. Allen M. et al.Combination allele-specific real-time PCR for differentiation of beta2-Adrenergic receptor coding single-nucleotide polymorphisms.Clin Chem. 2004; 50: 769-772Crossref PubMed Scopus (9) Google Scholar). Molecular mass predictions were calculated with the Compute Mw tool (http:http://www.us.expasy.org/tools/pi_tool.html), signal peptide recognition at SignalP V1.1 (http://www.cbs.dtu.dk/services/SignalP) and homologies were determined at the ncbi.nlm.nih.gov/blast interface. The mda-7s sequence has the GenBank accession number AY237723. We would like to thank Dr Judith Johnson (University of Munich, Germany) for MelJUSO cells, Dr Peter Schrier (University of Leiden, The Netherlands) for the 607B cell line and Professor Edgar Selzer (University of Vienna, Austria) for supplying the melanocyte cultures. This project was supported by the Austrian National Bank, the Austrian Science Fund, the Komission Onkologie, the Hygiene Fonds, the Virologie Fonds, the Niarchos Foundation, the “City of Vienna innovative interdisciplinary cancer research award”, MedEd, and the Kamillo Eisner Stiftung.

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