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[Cancer Research 62, 2098-2103, April 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

The Melanocyte-specific Isoform of the Microphthalmia Transcription Factor Affects the Phenotype of Human Melanoma1

Edgar Selzer2, Volker Wacheck, Trevor Lucas, Elisabeth Heere-Ress, Min Wu, Katherine N. Weilbaecher, Werner Schlegel, Peter Valent, Fritz Wrba, Hubert Pehamberger, David Fisher and Burkhard Jansen

Department of Radiotherapy and Radiobiology [E. S., W. S.], Department of Clinical Pharmacology, Section of Experimental Oncology and Molecular Pharmacology [V. W., T. L., B. J.], Department of Dermatology, Division of General Dermatology [T. L., E. H-R., H. P., B. J.], Department of Internal Medicine I, Division of Hematology and Hemostaseology [P. V.], Department of Clinical Pathology [F. W.], Ludwig Boltzmann Institute for Clinical and Experimental Oncology [H. P.], and Center of Excellence for Clinical and Experimental Oncology, [E. S., H. P.], University Hospital Vienna, 1090 Vienna, Austria; Department of Pediatric Hematology/Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [M. W., K. N. W., D. F.]; and Department of Medical Oncology, Washington University, St. Louis, Missouri 63110 [K. N. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The microphthalmia transcription factor MITF plays a pivotal role in the development and differentiation of melanocytes. The purpose of this work was to investigate the expression and function of the melanocyte-specific isoform MITF-M in human melanoma. We found that MITF-M is repressed in 8 of 14 established melanoma cell lines tested. Transfection of MITF-M into a melanoma cell line (518A2) lacking the M-isoform and into a permanent cell line established from normal melanocytes (NMel-II) resulted in slower tumor growth in a severe combined immunodeficient-mouse xenotransplantation model. The growth difference between vector control-transfected tumors derived from the NMel-II cell line (mean tumor weight ± SD, 3.2 g ± 1.13) and MITF-M (+) transfectants (mean tumor weight ± SD, 1.1 g ± 0.49) was significant (P = 0.018). The mean tumor weight of control-transfected 518A2 tumors was 0.99 g ± 0.22 and of MITF-M (+) transfectants, 0.69 g ± 0.32. The difference in growth between 518A2 controls and the MITF-M (+) transfectants was clear, however it did not reach statistical significance (P = 0.08). In addition to the growth-inhibitory effects, MITF-M expression led to a change in the histopathological appearance of tumors from epitheloid toward a spindle-cell type in vivo. These results indicate a role for the MITF-M isoform in the in vivo growth control and the phenotype of human melanoma. In conclusion, MITF-M may qualify as a marker capable of identifying subgroups of melanoma patients with different tumor biology and prognosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MITF3 is a member of the bHLH-Zip family of transcription factors that can both activate and repress transcription. In vivo, some of these proteins are known to control proliferation and apoptosis. MITF itself plays a central role in the differentiation, growth, and survival of cells of the melanocytic lineage (1) . Until now, most of the knowledge about molecular and phenotypic defects associated with the different alleles and the biochemical properties of the mutant proteins has been obtained in the mouse system (2, 3, 4) . Analysis of these mutations has revealed that genes controlling pigmentation may also regulate other developmental processes such as bone formation, hematopoiesis, fertility, and neural development. In humans, syndromes with mutations in MITF have been described and termed Waardenburg Syndrome IIA and Tietz syndrome (5, 6, 7, 8) . The human homologue of the mouse mi gene has been cloned from normal adult human epidermal melanocytes and encodes a protein with 94.3% overall homology to the mouse gene and identical bHLH-Zip domains (9) . Of potential importance for functional differences within the gene family is differential mRNA processing of the 5'- end between melanocytes and other tissues. At present, four MITF isoforms with distinct NH2 termini have been identified: The melanocyte-specific MITF-M, the heart-type MITF-H, and the recently identified MITF-A and MITF-C isoforms (9, 10, 11, 12, 13) . With the exception of the NH2-terminal sequence encoded by exon 1 and an additional alternate splicing site, the rest of the coding sequences of all currently known isoforms are identical. The 18-bp differentially spliced exon is generated by using an alternative splicing acceptor site of exon 6 located upstream from the basic region (3 , 4) . The splicing variant containing the 6-amino-acid insertion is also referred to as the MITF (+) form. Some of the functional and genetic aspects of these splicing variants have been discussed (1 , 3 , 4) . It was recently shown that MITF is more widely expressed in melanocytes than established human melanoma markers such as HMB-45 or S-100 (14) . These observations were made by immunohistochemical analysis with an antibody that does not discriminate among different MITF isoforms. We, therefore, investigated the expression pattern of MITF isoenzymes with MITF-M gene-specific PCR primers in human melanocytes and melanoma cells. In this work, we demonstrate repression of the melanocyte-specific isoform MITF-M in melanoma cell lines and in spontaneously transformed cell lines established from normal melanocytes. We further demonstrate through transfection experiments that expression of MITF-M is linked to morphology and tumor growth in vivo in two independently established cell lines.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Normal human epidermal melanocytes (adult and neonatal, fourth passage) were obtained from Clonetics Corporation (Remagen, Germany) and cultured in melanocyte growth medium (serum-free) containing phorbol 12-myristate-13-acetate (10 µg/ml), hydrocortisone (0.5 µg/ml), insulin (5 µg/ml), human fibroblast growth factor (1 µg/ml), and gentamicin sulfate and amphothericin-B (both at 50 µg/ml) supplemented with bovine pituitary extract (complete melanocyte growth medium 2). The melanoma cell line A375 was obtained from American Type Culture Collection, and 518A2 melanoma cells were kindly provided by Dr. P. Schrier (University of Leiden, Leiden, the Netherlands). The human melanoma cell lines designated MES were generated from melanoma metastases by E. S. and have been characterized in our laboratory. The human melanoma cell lines 501 Mel and Yupac7 were generously provided by Dr. Ruth Halaban (Yale University School of Medicine, New Haven, CT). The human mast cell line HMC-1 established from a mast cell leukemia patient (15) was kindly provided by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) and was cultured in IMDM containing 10% heat-inactivated FCS (Life Technologies, Inc., NY), L-glutamine, and antibiotics. All melanoma cell lines were maintained in DMEM (4.5 g/liter glucose) supplemented with 10% heat-inactivated FCS. From a panel of different primary melanocyte cultures, three cultures underwent spontaneous morphological transition developing a transformed phenotype accompanied by the development of multiple colonies. Three cell lines were subsequently generated by continuous passaging (16) and were designated NMel-II, -III and -IV. The NMel-II and 518A2 cell lines were transfected with vector constructs in the presence of Superfect Transfection Reagents (Qiagen GmbH, Hilden, Germany). The protocol used for stable transfection of adherent cells was as suggested by the manufacturer, and pooled, stable transfectants were selected in the presence of appropriate concentrations of geniticin. All of the tested colonies (n = 5) that were generated after an initial transfection, expressed MITF-M RNA as assessed by RT-PCR.

Expression Analysis.
Total RNA was isolated with Trizol LS (Life Technologies, Inc.) and reverse-transcribed simultaneously with oligo(dT) and random primers with the Advantage RT-for-PCR kit from Clontech (Palo Alto, CA). RT-PCR was performed on an aliquot representing 5% of the cDNA generated from reverse transcribing 1 µg of total RNA. MITF-M primer design was based on the published sequence of the human MITF mRNA (9) , which was derived from normal adult human epidermal melanocytes (coding sequence: nt. 121-1380). The following upstream primers were used: (a) primer I, specific for the UTR of MITF-M (from nt. 79 to 105): 5'-TCTACCGTCTCTCACTGGATTGGTGCC-3'; (b) MITF-M exon-1 primer: the exon-1 specific primer sequence starts with the initiation codon at position 121 and encompasses the complete NH2-terminal coding sequence of exon-1 (nt. 121–153): 5'-ATGCTGGAAATGCTAGAATATAATCACTATCAG-3'; (c) MITF exon-2 specific primer (nt. 174–200): 5'-CCCCACCAAGTACCACATACAGCAAGC-3'; (d) MITF-IC primer (nt. 445–474): 5'-TCACGAGCGTCCTGTATGCAGATGGATGAT-3'; (e) MITF-A exon-1 specific primer (nt. 243- 269): 5'-GCCTCCAAGCCTCCGATAAGCTCCTCC A- 3'. The antisense (downstream) primers that were used in conjunction with the gene-specific upstream MITF primers were the following sequences: (a) primer IIB (nt. 861–832): 5'-TAAGATGGTTCCCTTGTTCCAGCGCATGTC-3'; (b) primer IIA (nt. 1382–1356): 5'-CGCTAACAAGTGTGCTCCGTCTCTTCC-3'.

The gp100 melanocyte lineage-specific antigen/Pmel17 homologue (human, complete mRNA = 2130 nt.) was amplified with upstream (nt. 4–33): 5'-GGAATCCGGAAGAACACAATGGATCTGGTG-3' and downstream (nt.744–715): 5'-GGCAAAGGTCAGAGGCTGATTTCTCAGGAA-3' primers; tyrosinase primers were: upstream (nt. 476–505): 5'-GCAGACCTTGTGAGGACTAGAGGAAGAATG-3' and downstream (nt. 1348–1319): 5'-GTACTCCTCCAATCGGCTACAGACAATCTG-3'. The human proto-oncogene c-Kit cDNA was amplified with upstream (nt. 2261–2287): 5'-ACATAGAAAGAGATGTGACTCCCGCCA-3' and downstream (nt. 2768–2742): 5'-GTTGGTCTTTTTAGGGGATCTGCATCC-3' primers. Human GPDH control primers yielding a 983-bp product were obtained from Clontech.

Cloning of MITF-M cDNA for Transfection.
The full-length coding sequence of MITF-M, which was also used for the transfection experiments contains a part of the 5'-UTR sequence and was amplified from normal early passage neonatal melanocytes with primer I (see above) and primer IIA (nt.1382–1356): 5'-CGCTAACAAGTGTGCTCCGTCTCTTCC-3' (STOP codon is underlined) and cloned into the pCR3.1 expression vector (Invitrogen Corp., Carlsbad, CA) under the control of the cytomegalovirus promoter. Both MITF-M (+6 amino acids and -6 amino acids) splicing variants were cloned and verified by sequencing.

SCID-Human Mouse Model.
Animal experiments were performed in principle as described recently (17) . In short, pathogen-free SCID mice, 4–6 weeks old, were obtained from B & M (Ry, Denmark). The animals were housed in laminar flow racks and microisolator cages under specific pathogen-free conditions and received autoclaved food and water but no antibiotic prophylaxis. The melanoma cell line 518A2 and the transformed cell line NMel-II were injected s.c. (2 x 10 7 cells/mouse) into the left lower flank. All of the animals developed tumors and were killed after 3–4 weeks.

Statistics.
Student’s t test was used for calculating significance levels. Ps of < 0.05 were considered to be of statistical significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of MITF Expression Profile in Normal Melanocytes, Transformed Cell Lines Developed from Normal Melanocytes, Established Melanoma Cell Lines, and Human Mast Cells.
In contrast to the wide tissue distribution of other MITF isoforms, MITF-M is exclusively expressed in the melanocytic lineage. Little is presently known about how the MITF isoenzyme expression patterns change during transformation of melanocytes to melanoma cells because most of the expression data published to date have been obtained with antibodies that do not discriminate between different isoforms of MITF. To investigate differences in the expression pattern of the melanocyte-specific MITF-M form, we compared the expression pattern of MITF-M between normal human adult and neonatal melanocytes as well as in a panel of established melanoma cell lines. In addition, we compared the expression of MITF between normal melanocytes and spontaneously transformed cell lines that arose from normal melanocytes. RT-PCR analyses with exon-1-specific primers demonstrated repression of MITF-M expression in transformed melanocytes (Fig. 1A)Citation . MITF-M expression was not detected in a human mast cell line (Fig. 1BCitation , Lane 1), although MITF-transcript expression in these cells was confirmed with an exon-2-specific primer that recognizes all known MITF isoforms (Fig. 1C)Citation . MITF-M expression was also absent in 6 of 11 melanoma cell lines established in our laboratory (Fig. 2A)Citation as well as in 2 of 3 widely used melanoma cell lines (Fig. 2B)Citation , which suggests that the loss of MITF-M expression may represent a common event in human melanoma development. In Fig. 2ACitation , RT-PCR data of the above mentioned MITF-M-negative melanoma cell lines (Lanes 1–6) are shown, all of which are positive with an exon-2-specific primer in combination with a downstream primer (Fig. 2ACitation , bottom panel). In contrast to MITF-M expression, exon-2 PCR was positive in all of the melanoma cell lines investigated. RNA expression data of MITF-M-positive melanoma cell lines are summarized in Fig. 2DCitation . Two additional human melanoma cell lines were examined by Western blotting as shown in Fig. 1ECitation . The identification of the MITF-M isoform has been previously made by expression of the recombinant isoform (18) . Whereas one of these cell lines expressed both the MITF-M and non-melanocyte-specific isoforms, the other clearly lacked expression of MITF-M although retaining expression of the nonmelanocytic isoform. Taken together, all of the cell lines examined (transformed and untransformed) expressed at least one isoform of MITF, although not necessarily the M-isoform.



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  		Fig. 1. MITF RT-PCR analysis in normal melanocytes and in transformed melanocytic cell lines. A, multiplex PCR with a MITF-M specific primer combination (I/IIA) and GPDH control primers: Lanes 1 and 3, normal melanocytes. Lanes 2 (NMel-II) and 4 (NMel-IV), transformed cell lines established from normal melanocytes analyzed in Lanes 1 and 3, respectively. B, multiplex PCR of MITF- M (primer I/IIA) and c-Kit expression in the HMC-1 cell line (Lane 1), in normal neonatal melanocytes (Lane 2) and in normal adult melanocytes (Lane 3). C, exon-2 PCR: expression of MITF analyzed with primers amplifying the region between nucleotide position 147 (within exon 2) and primer IIA. Lanes 1–3, to the cDNA samples analyzed in B. As a molecular weight marker a 100-bp DNA ladder was used. Arrows, the positions corresponding to 600 and 1500 bp. D, PCR analysis of MITF 18 bp splicing in normal human melanocytes. Primers used are: MITF-IC primer (nt. 445–474) and primer IIB (nt. 861–832). The resulting PCR products are resolved on a 3% agarose gel. (E) Western blot analysis of MITF isoforms in two human melanoma cell lines. MITF-M mobility has been described previously (18) .

 


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  		Fig. 2. MITF-M expression in various cell lines of melanocytic origin. A, a panel of nine selected cell lines is shown. Expression of the melanocyte-specific isoform was analyzed with MITF-M exon 1-specific (I/IIA) primers. MW, a 100-bp molecular weight marker. Lanes 1–6, MITF-M negative melanoma cell lines established in this laboratory (MES20, -21, -24, -26, -27, and -33); Lane 7, the MITF-M isoform-transfected cell line NMel-II; Lane 8, the NMel-III cell line; Lane 9, the cell line NMel-IV. Bottom panel, the same cDNA samples analyzed with exon-2-specific MITF (exon-2/IIA) primers. B, MITF-M expression analysis in three widely used melanoma cell lines. Lane 1, 518A2; Lane 2, A375; Lane 3: G361. The molecular weight marker is a 100-bp DNA ladder. Arrow in the 1200–1300-bp range, MITF-M PCR product. C, MITF-A expression in normal primary adult and neonatal melanocytes (Lanes 1–4) and in the spontaneously transformed melanocytes, NMel-II, -III, and -IV (Lanes 5–7). D, summary of MITF expression data in normal neonatal melanocytes, in cell lines NMel II, -III, and IV, and in melanoma cell lines. Group A cell lines, MITF-M-negative melanoma cell lines 518A2, A375, MES20, -21, -24, -26, -27, and MES33. Group B cell lines, MITF-M positive melanoma cell lines G361, MD3A, MES29, -31, -32a, and -32b. As representative examples, Exon-1 MITF-A PCR analysis was performed with melanoma cell lines 518A2 and G361. MITF-M expression data of the MITF-M-positive melanoma cell lines, except for G361, are not shown.

 
These observations confirm the recently published finding that the transcription factor MITF is more widely expressed in human melanoma than some of the established markers for this disease (14) . It was of interest to determine whether changes in MITF-M expression might be associated with changes in MITF-A because both isoforms are expressed from the same region of chromosome 3 and share 8 exons (9) . The expression of the MITF-A isoform was, therefore, analyzed in a subset of normal and spontaneously transformed cell lines by MITF-A-specific PCR (see Fig. 2, C and DCitation ) as well as by DNA sequencing of 5'-RACE-PCR products and was found to be commonly expressed (5'- RACE products and sequencing data not shown). For example, in the neonatal transformed cell line NMel-II, in which the M-isoform is down-regulated, MITF-A transcripts were still detected, as was the case with the melanoma cell line 518A2, which was used for the transfection experiments (see Fig. 2, C and DCitation ). In parallel, the expression pattern of the melanocyte lineage-specific antigen gp100, which confers immunoreactivity to the monoclonal antibodies HMB-45 and -50 and NKI-beteb (19) and is widely used in the diagnosis of melanoma, was not expressed in all of the melanoma cell lines investigated (16) . Regulation of the human tyrosinase gene by MITF has been investigated by several authors (20 and references therein). This was of interest to us, because the primary neonatal melanocytes from which the permanent line NMel-II was established, initially showed visible signs of pigmentation, but this characteristic was gradually lost during continuous passage, and the loss was accompanied at the molecular level by down-regulation of tyrosinase expression as assessed by PCR analysis. Expression analysis of tyrosinase mRNA in conjunction with MITF mRNA in NMel-II cells is shown in Fig. 3Citation . Morphological changes in this cell line were also observed with cells displaying a more rounded or epitheloid appearance resembling established melanoma cells in culture (16) . Transfection of the MITF-M isoform into these amelanotic cells did not restore the expression of tyrosinase in vitro (Fig. 3A)Citation . Identical changes were observed with two other cell lines established from normal melanocytes regarding loss of MITF-M expression, pigmentation, and morphological characteristics (cell lines NMel-III and NMel-IV, data not shown).



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  		Fig. 3. RT-PCR and Western blot analysis. A, tyrosinase, gp-100, and MITF-M (primers I/IIA) expression in normal neonatal cells that gave rise to the spontaneously transformed cell line NMel-II. Expression analysis was performed with neonatal cells during early passage (Lane 1) when pigmentation was still visible, during late passage when pigmentation was lost (Lane 2), and with the NMel-II cell line transfected with MITF-M cDNA (Lane 3). B, RT-PCR analysis of the vector control-transfected (Ctrl) and MITF-M (+)-transfected melanoma cell line 518A2. Primer 1 (5'-UTR) used to clone the MITF-M sequence was used for analysis, as well as the MITF-M exon-1- and exon-2-specific primers in conjunction with the same downstream primer (IIB). C, a Western blot analysis of vector and vector plus MITF-M (+) cDNA-transfected cell line 518A2 is shown. D, the relative positions of the primers designated 5‘-UTR (primer I), Exon-1, Exon-2, IIB, and IIA on the cDNA are indicated in the simplified graphical representation of the MITF-M cDNA.

 
Growth Effects of Transfection of the MITF-M cDNA into a Neonatal Transformed Cell Line NMel-II and into a Human Melanoma Cell Line 518A2.
The neonatal transformed cell line NMel-II and the melanoma cell line 518A2, neither of which expresses the MITF-M isoform (see Figs. 1ACitation , and 2BCitation ), were transfected with a wild-type melanocyte-specific MITF (+) cDNA construct or with the cloning vector pCR3.1 alone. MITF RT-PCR analysis of the transfected cell line NMel-II is shown in Figs. 2ACitation and 3ACitation . RT-PCR as well as Western blot analysis of the MITF-M-transfected melanoma cell line 518A2 is shown in Fig. 3, B and CCitation . For the mouse experiments, transfected cell lines, derived from a pool of several thousand clones, were used without further subcloning to avoid the possibility of selecting clones not representative of the entire transfected population. Interestingly, no significant differences in the growth of vector-control- or MITF-M-transfected cell lines were measured in vitro (data not shown). In contrast, experiments in a SCID-human xenotransplantation model showed differences between the growth of MITF-M transfected tumors and the control transfectants in two independent cell lines (Fig. 4)Citation . MITF-M-transfected NMel-II cells grew significantly (P = 0.018) slower than vector controls in SCID mice (Fig. 4A)Citation . As shown in Fig. 4BCitation , transfection of 518A2 cells with MITF-M (+) also resulted in a reduction of tumor growth; however, it did not reach statistical significance compared with the vector controls (P = 0.08). We reestablished cell lines from the 518A2 melanoma xenotransplants and analyzed the MITF-M expression pattern as well as the ex vivo growth rates. The RT-PCR expression analysis showed that MITF-M was still expressed ex vivo in cell cultures generated from the respective xenotransplants. Western blot analysis of MITF-transfected cell line 518A2 is shown in Fig. 3Citation . However, ex vivo 3-day growth data of reestablished cell lines showed, in contrast to the in vivo situation, no statistically significant differences between vector and MITF-M-transfected cell lines (growth and RT-PCR expression data not shown).



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  		Fig. 4. Analysis of tumor growth in SCID mice. A, tumor growth of the cell line NMel-II transfected with MITF-M (+) or empty vector (Ctrl) in SCID mice. Bars, the means of the tumor weights. Mean tumor weight for the pCR 3.1 vector control-transfected tumors was 3.2 ± 1.13 g (n = 3); animal 4 in this group died before termination of the experiment because of tumor load. Mean tumor weight for MITF-M (+) transfectants was 1.1 ± 0.49 g (n = 4). Right panel, mean mouse body weight at the end of the experiment. B, tumor growth of the transfected melanoma cell line (518A2). The 518A2 melanoma cell line was transfected with either the vector alone (Ctrl) or with a MITF-M (+) cDNA. The mean weight of pCR 3.1 vector control transfected tumors was 0.99 ± 0.22 g (n = 6) and of MITF-M (+) transfectants, 0.69 ± 0.32 g (n = 6). Right panel, mean mouse body weight at the end of this experiment. Bars, SD.

 
Effects of Transfection of the MITF-M cDNA on Cellular Morphology.
Transfection of the MITF-M isoform led to changes in morphological characteristics in vitro and in vivo. MITF-M transfectants showed a predominant spindle-shaped morphology whereas most of the cells transfected with the vector were epitheloid in nature (not shown). We analyzed tumors grown in SCID mice established from the vector control-transfected and MITF-M-transfected cell line NMel-II (Fig. 5)Citation . Tumor xenotransplants from the vector control-transfected cell line contained, for the most part, epitheloid cells (Fig. 5A)Citation , and melanin pigmentation was not detectable. In contrast, MITF-M-transfected tumors consisted mainly of spindle-shaped cells, and the tumor cells were surrounded by a thin fibrotic stroma (Fig. 5B)Citation . In select cells of the MITF-M-transfected tumors, melanin pigmentation was seen; this phenomenon was never observed in the vector control-transfected tumors (data not shown).



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  		Fig. 5. H&E stains of mouse melanoma tumors. A, vector control-transfected tumor (NMel-II); mouse tumor composed of epitheloid cells with rounded cytoplasm, large vesicular, and prominent nucleoli. Melanin pigmentation was not visible. Scale bar, 100 µm. B, MITF-M-transfected tumor (NMel-II) consisting predominantly of spindle-shaped cells with elongated (cigar-shaped) vesicular nuclei. The tumor cells lie in branching formations surrounded by thin fibrotic stroma. Scale bar, 100 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Goding (1) recently provided an excellent review of the role of MITF in human melanocytes in which he discussed the possibility that deregulation of developmental pathways in melanoma would also affect MITF expression. His arguments were not only based on the fact that MITF is crucial for the development of cells of the melanocytic lineage (21) but were also based on the notion that most of the signaling molecules or transcription factors implicated genetically in melanocyte development are known to affect either MITF expression or its function. Providing first experimental and clinical support for this hypothesis, there are now new reports not only investigating the distribution of MITF in human melanoma (14) but also showing an influence of MITF expression on survival for this disease (22) . We here demonstrate that the MITF-M isoform is not expressed in 8 of 14 human melanoma cell lines tested. However, a positive signal was always detected with primers amplifying a cDNA region of exon 2 in normal and malignant cells of the melanocytic lineage, which indicates expression of at least one MITF isoform in all of these cell types. Restoration of MITF-M expression by transfection in two cell lines lacking the MITF-M form affected the in vivo growth of these cell lines in a SCID-mouse xenotransplantation model and profoundly affected the histopathological appearance of the tumors generated. These observations raise the question of how changes in the NH2-terminal sequence of MITF might be growth inhibitory. From what is known through analysis of the promoter-driven expression of the MITF-M isoform in melanocytes, expression can be regulated by a variety of cooperating transcription factors, such as Pax3, CREB, Sox10, Lef1, and Brn-2 (1 , 23) . The expression and function of MITF is also subject to regulation by the mitogen-activated-protein-kinase and cAMP pathways (18 , 24 , 25) . In addition, MITF itself is able to regulate the expression of genes such as human tyrosinase (20 , 26) . Transfection of MITF-M into the cell line did not restore visible pigmentation in vitro, but in vivo, some cells that were transfected with MITF-M contained melanin. This finding indicates a regulatory activity of the transfected MITF construct on the pigmentation pathway. The most interesting and unexpected finding was the pronounced effect of MITF on the cellular morphology and on the stroma within the tumors. MITF-M expression was associated with a transition from an epitheloid toward a more spindle-shaped cellular morphology that was more prominent in vivo than in vitro. The photomicrographs of the tumors grown in mice are of an appearance similar to the epitheloid and spindle-cell type melanomas that occur in humans. It is of interest to note that the epitheloid-type of ocular melanoma is known to be associated with a significantly better prognosis than the spindle-cell type (27, 28, 29, 30) . To our knowledge, no such relationship has been published for cutaneous melanoma. Under cell culture conditions, differences in the proliferation rates were statistically not significant between MITF-M transfectants and vector-control transfected cells before and after growth under xenotransplantation conditions. One possible explanation is that the tumor growth assay in the mouse system is more sensitive because these experiments last several weeks.

A recently published report by Salti et al. (22) demonstrated that in a multivariate analysis, the expression of MITF in intermediate-thickness cutaneous melanoma is inversely correlated with overall survival. The author speculated that MITF might be a new prognostic marker in intermediate-thickness malignant melanoma. This finding is of potential interest and relevance for our study because we found that overexpression of MITF-M in a SCID-human mouse model led to significantly reduced growth. However, there are still important methodological differences between these studies because the antibody used for immunohistochemical analysis does not discriminate between different MITF isoforms. Unfortunately, to our knowledge, no antibody is available to date that could discriminate between different MITF isoforms. King et al. (14) found that all melanomas investigated by immunohistochemistry stained positive for MITF, and Salti et al. (22) found a positive staining in ~80% of all tissue specimens. We found evidence for MITF expression in all of the melanoma cell lines when analyzed with primers specific for exon-2 sequences, indicating expression of at least one MITF isoform in all of the melanoma cell lines. In conclusion, our results strongly indicate that proteins of the MITF transcription factor family are widely expressed in malignant cells of the melanocytic lineage and that differential expression of MITF isoforms appears to influence phenotype, tumor biology and growth characteristics. MITF isoform expression patterns may prove useful in identifying patient subgroups with different courses of the disease and different prognoses.


   ACKNOWLEDGMENTS
 
We thank R. Haslinger for technical assistance and Dr. Ruth Halaban for contributing melanoma cell lines.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Austrian Science Fund (FWF), NIH (D. E. F.), the Austrian National Bank, the Kommission Onkologie, the Kamillo Eisner Stiftung, the Anton Dreher Stiftung, the Niarchos Foundation, the Hygienefonds, and the Virologiefonds. Back

2 To whom requests for reprints should be addressed, at University Hospital Vienna, Department of Radiotherapy and Radiobiology, Währinger Gürtel 18-20, 1090 Vienna, Austria. Phone: 43-1-40400-7672; Fax: 43-1-40400-2666; E-mail: Edgar.Selzer{at}AKH-Wien.ac.at Back

3 The abbreviations used are: MITF, microphthalmia transcription factor; bHLH-Zip, basic helix-loop-helix leucine zipper; UTR, untranslated region; RT-PCR, reverse transcription-PCR; SCID, severe combined immunodeficiency/immunodeficient; RACE, rapid amplification of cDNA ends; IMDM, Isocove’s modified Dulbecco’s medium; GPDH, glycerol-3-phosphate dehydropenase. Back

Received 1/22/01. Accepted 1/23/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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