Series Of Melanoma Cell Lines Research Articles

Synthesis, Characterization, In vitro Antiproliferative and Cytotoxic Effect of Ruthenium(II)-Bipyridine-Benzoylpicolinic Acid Complex on SK-MEL-28 and Normal L6 Cell Lines

The most seriously spread malignant tumor of melanocytes throughout the body is a melanoma type of skin cancer. SK-MEL-28 cell line is one of a series of melanoma cell lines. The objective of the present investigation is to study the anticancer activity and cytotoxicity of novel [Ru(bpy)2(bzpic)2]2+ (bpy = 2,2ʹ-bipyridine and bzpic = 3-Benzoylpicolinic acid) complex on SK-MEL-28 and normal L6 cell lines using MTT assay method. The morphology of the cancerous SK-MEL-28 cell line at various concentrations of the [Ru(bpy)2(bzpic)2]2+ complex is assessed by 2-fold dilution method, and the in vitro antiproliferative effect of the complex on the cancerous SK-MEL-28 cell line is analyzed by direct microscopic observation method. The percentage viability of the complex on cancerous SK-MEL-28 cell line at various concentrations is determined. The IC50 value of the complex against the SK-MEL-28 cell is found to be 39.109 µg/mL which shows good antiproliferative effect. The IC50 value of the complex against normal L6 cell line is found to be 55.315 µg/mL. The results revealed that the percentage of growth inhibition of the cell is based on dose-dependent manner and the formation of formazan crystal indicates this. The fluorescent microscopy observation clearly determines that the synthesized [Ru(bpy)2(bzpic)2]2+ complex shows late apoptosis on SK-MEL-28 cell line and early apoptosis on L6 cell line. Hence, it is evident that the synthesized complex exhibits good in vitro antiproliferative effect with lower cytotoxicity.
 HIGHLIGHTS
 
 Synthesis of [Ru(bpy)2(bzpic)2]2+ complex
 Direct and fluorescent microscopic observation of [Ru(bpy)2(bzpic)2]2+ complex on SK-MEL-28 and normal L6 cell lines
 In vitro antiproliferative and cytotoxic effects of [Ru(bpy)2(bzpic)2]2+ complex on SK-MEL-28 and normal L6 cell lines
 
 GRAPHICAL ABSTRACT

ITOC2 – 009. Role of signal transduction and microRNAs on the immunogenicity of melanoma cells

Melanoma is a heterogeneous malignancy with a distinct clinical outcome of patients due to different genetic features and various strategies of melanoma cells leading to immune evasion. For a better understanding of the aggressive melanoma phenotype the molecular mechanisms of aberrant expression of the classical HLA class I antigen processing and presentation machinery (APM) and of the non-classical HLA-G antigen is analysed. A reduced mRNA and/or protein expression of various APM components was identified in a large series of melanoma cell lines and lesions, which correlated with an increased tumour grading and lack of immune therapy response. In addition, microRNAs (miRs) targeting HLA class I APM components as well as HLA-G thereby affecting the immune response have been identified suggesting an important role for posttranscriptional control in this process. These miRs might be used as novel targets for the treatment of melanoma or for selection of melanoma patients undergoing the most effective (immuno)therapy. Furthermore, a direct link between the interferon (IFN) signalling pathway and the constitutive HLA class I expression was found. Altered expression or loss of single components of the IFN-<i>γ</i> signal cascade was detected, which was accompanied by deficient IFN-<i>γ</i> response due to structural alterations, epigenetic control or transcriptional/post-transcriptional deregulation. These were directly associated with a downregulation of basal HLA class I APM component expression in melanoma lesions and melanoma cell lines and confirmed by silencing of STAT1, JAK1 and JAK2. Thus these data provide novel insights into the complex regulation of HLA class I expression and the rational for selecting patients for specific immunotherapies.

Role of signal transduction and microRNAs on the immunogenicity of melanoma cells

Melanoma tumors are heterogeneous and involve different processes generating cells with variable metastatic capacity, which also results in distinct clinical outcome of patients and variations in therapy responses. This might be due to different strategies leading to evasion of T and or NK cell-mediated surveillance, which is accompanied by disease progression and a poor survival of melanoma patients. Therefore, the characterization of immune escape mechanisms might contribute to a better understanding of the development of the aggressive melanoma phenotype. This study analysed the underlying molecular mechanisms of HLA class I abnormalities and aberrant HLA-G expression in melanoma. A reduced mRNA and/or protein expression of various components of the MHC class I antigen processing machinery (APM) was identified in a large series of melanoma cell lines and lesions, which could be correlated with an increased tumor grading. In addition, adoptive T cell therapy also caused an escape from immune cell recognition by down-regulation components of the antigen processing machinery (APM). Recently, microRNAs (miRs) targeting HLA class I APM components were identified suggesting an important role for posttranscriptional control in this process. These miRs have functional activities on APM components, thereby also affecting HLA class I surface expression. Thus, these miRs might be used as novel targets for the treatment of melanoma or for selection of melanoma patients undergoing the most effective (immuno)therapy. In addition, a direct link of the IFN signalling pathway and the constitutive HLA class I APM component expression was found in melanoma. Down-regulation or loss of single components of the IFN-γ signal cascade was detected, which could be due to structural alterations, epigenetic mechanisms, such as methylation and/or histone deacetylation, or transcriptional/post-transcriptional deregulation. This was accompanied by a heterogeneous response of melanoma cells to IFN-γ treatment. Alterations in the IFN-γ signalling pathway were directly associated with a down-regulation of constitutive HLA class I components as demonstrated in melanoma lesions as well as in melanoma cell lines. Using shRNA-mediated silencing a direct effect of STAT1, JAK1 and JAK2 on MHC class I surface expression was confirmed. These data may provide novel insights into the complex regulation of HLA class I expression in melanoma and gives a rational for excluding patients for specific immunotherapies. Furthermore, strategies will be developed for restoration of the HLA class I positive phenotype.

Synthesis of new naphthoisoxazole amide derivatives and study of their biological evaluations

A series of naphthoisoxazole amide derivatives 4a–h have been synthesized from naphthoisoxazole acetic acid 3. 2-Acetyl-1-naphthol-1 on reaction with pulverized sodium and diethyl carbonate gave 4-hydroxy naphthopyrone 2 which on Posner reaction gave naphthoisoxazole acetic acid 3. The cytotoxic activity study for inhibiting melanoma cell survival was evaluated on a series of melanoma cell lines. The anticonvulsant activity of these compounds has been evaluated in Wistar rats. A compound called 4h has been found to have anticonvulsant activity comparable to that of the standard drug phenytoin.

Abstract 3866: TWEAK receptor (Fn14) Is a novel target in melanoma: Characterization of unique targeted therapeutics

Abstract Previous studies have indicated that Fn14, the cell surface receptor for the cytokine TWEAK, is over-expressed in multiple human solid tumor types, including brain, breast, and lung cancers, and overexpression can be a negative prognostic indicator. We analyzed a series of melanoma cell lines and tumor tissue microarrays and detected Fn14 expression in ∼60% of the melanoma cell lines, including both B-Raf WT and B-Raf V600E lines. Fn14 expression was elevated in 178/190 (93.6%) of primary melanoma specimens and in 87/150 (58%) of melanoma metastases tested. Fn14 expression was not elevated in normal skin tissues. Initial development and characterization of an immunoconjugate designated ITEM4-rGel targeting Fn14 receptor has been published (Zhou et al., Mol. Cancer Ther. 10:1276 (2011)). We have now developed an Fn14-targeted immunotoxin more suitable for long-term clinical use. Specifically, we generated a humanized, dimeric single-chain version of ITEM-4 and fused this scFv to rGel. The resulting anti-Fn14 immunotoxin, designated hSGZ, bound to Fn14 with a Kd of ∼1.4 nM as determined by Biacore analysis. Confocal immunofluoresence studies showed that hSGZ specifically and rapidly (within 2 hrs) internalized into Fn14-expressing MDA-MB-435 melanoma cells. Cytotoxicity studies showed that hSGZ was highly cytotoxic to a panel of different melanoma cell lines (IC50 ranged from 0.1 pM to 1.1 nM) and was 2.2 to 2.8 ×105 fold more potent than free rGel. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. When hSGZ was combined with 5-FU, cisplatin, doxorubicin, etoposide or dacarbazine, we found an additive effect on melanoma cell growth inhibition. Mechanistic studies showed that hSGZ induced melanoma cell death consistent with a necrotic mechanism. Additionally, Fn14-targeted immunotoxins increased Fn14 expression and triggered cell signaling events similar to those induced by the TWEAK ligand. Finally, treatment of mice bearing human melanoma MDA-MB-435 and breast MDA-MB-231 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared to controls (P&amp;lt; 0.05). Fn14 appears to be an excellent new target for melanoma and the Fn14-targeting construct hSGZ appears to warrant further development as a novel therapeutic agent against Fn14-positive tumors. Melanoma lines appear to be the most sensitive tumor type tested but the reasons for this are unclear. Additional studies are in progress to investigate the biodistribution and pharmacokinetics of hSGZ in tumor-bearing mice. This work was conducted, in part, by the Clayton Foundation for Research (MGR); and supported by NIH grant NS055126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3866. doi:1538-7445.AM2012-3866

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

IGFBP7 Is Not Required for B-RAF-Induced Melanocyte Senescence

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma.

Immune surveillance of tumour cells by CD8(+) cytotoxic T cells plays a key role in the establishment and control of an anti-tumour response. This process requires the generation of antigenic peptides, which are largely produced by the proteasome in combination with other proteases located in either the cytoplasm and/or the endoplasmic reticulum (ER). The ER-resident aminopeptidases ERAP1 and ERAP2 trim or even destroy HLA class I-binding peptides thereby shaping the peptide repertoire presented for T cell recognition. So far there exists limited information about the expression pattern of ERAP1 and/or ERAP2 in human tumours of distinct histotypes. Therefore, the expression profiles and modes of regulation of both aminopeptidases were determined in a large series of melanoma cell lines. A heterogeneous expression ranging from high to reduced or even total loss of ERAP1 and/or ERAP2 mRNA and/or protein expression was detected, which often could be induced/upregulated by interferon-gamma treatment. The observed altered ERAP1 and/or ERAP2 expression and activity levels were either mediated by sequence alterations affecting the promoter or enzymatic activities, leading to either transcriptional and/or post-transcriptional downregulation mechanisms or limited or excessive processing activities, which both might have an impact on the antigenic peptide repertoire presented on HLA class I molecules.

Abstract A79: Expression of argininosuccinate synthetase (ASS) and response to arginine deiminase (ADI) treatment in a series of melanoma cell lines

Abstract Background: Arginine deiminase (ADI), an enzyme that catalize the conversion of arginine to citrulline, has been used in clinical trials for the treatment of tumors which have been shown to be sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). We here evaluated the effect of ADI treatment on ASS expression in a series of melanoma cell lines. Methods: Twenty-five melanoma cell lines (6 derived from primary melanomas and 19 from MM metastases) and normal fibroblast cell as control underwent cell proliferation assays in presence or absence of ADI. The percentage of cell proliferation was estimated on day 6 of the ADI treatment by a colorimetric assay; the IC50 value was assessed for each cell line. Western blot analysis on lysates from treated and untreated cells were performed for evaluating the expression levels of both the ASS and the ornithine carbamoyl transferase (OTC) proteins. A subset of tissue sections from primary melanomas (N=10) and hepatocellular carcinomas (N=20) was investigated by immunohistochemistry for ASS expression. Results: Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition with a median IC50 value of 1.3 mU/mL (range, 0.4–2.0); growth of the remaining 4 (16%) MM cell lines was not inhibited and the IC50 was not reached. As expected, proliferation of the control fibroblasts was not inhibited. Expression analysis revealed that none of the 21 ADI-responsive cell lines presented detectable levels of the ASS protein before the ADI treatment; conversely, the 4 melanoma cell lines and the normal fibroblasts, which did not reached an IC50 value, showed a constitutive ASS expression in both treated and untreated cells. After treatment with ADI at a concentration corresponding to the IC50 values, 7/21 (33%) melanoma cell lines presented a markedly increased expression levels of the ASS protein; interestingly, six of them were from paired primary and secondary lesions of three patients. The median value of IC50 in this subset was of 1.8 mU/mL (range, 1.2–2.0). Nearly all samples (13/14; 93%) with absence of the ASS expression in both treated and untreated cells originated from MM metastases; among them, three cell lines were from paired primary and secondary lesions of the same patient. The median value of IC50 in this subset was of 1.2 mU/mL (range, 0.4–1.7). A constitutive expression of the OTC protein was found in all melanoma cell lines and no change in OTC expression was observed after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens (10 melanomas and 20 hepatocellular carcinomas). Conclusions: In our series, the most effective cell growth inhibition was observed in melanoma cells with absence of ASS expression either before or after the ADI treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A79.

Effect of celastrol on temozolomide cytotoxicity in melanoma cells and inhibition of NF-kB signaling

9076 Background: Temozolomide (TMZ) exhibits clinical activity in the treatment of melanoma and glioblastoma, but response rates are low. Identification of agents that improve TMZ's efficacy and overcome resistance is of great interest. In this study, we have identified celastrol as a natural product that significantly enhances TMZ-induced cytotoxicity by testing a library of drugs and natural products for cytotoxic activity against glioma and melanoma cell lines and have examined its mechanism of action in melanoma cells. Methods: A preliminary screening of a library of 2000 drugs and natural products was performed and a short list of drugs was identified as able to enhance TMZ-induced cell killing in TMZ-resistant cancer cell lines. The effects of these compounds were further confirmed in five melanoma cell lines. A cell proliferation assay was used to compare growth inhibitory effects of single agent TMZ versus combination treatments. Synergy in inhibiting cell proliferation was assessed using combination-index methods. The expression of NF-kB, IkB, MAPK, and PARP were examined using Western blot analysis. The effect of treatments on the cell cycle was examined by flow cytometry. The localization of NF-kB in melanoma cells was evaluated through immunofluorescence microscopy. Results: Combining celastrol and TMZ synergistically inhibited cell proliferation, enhanced cell cycle arrest, and increased apoptosis in a series of melanoma cell lines, compared to treatment with TMZ alone. We further found that celastrol inhibited proteasome activity, TNF-α induced IkB phosphorylation and NF-kB translocation to the nucleus. Inhibition of NF-kB with siRNA mimicked the ability of celastrol to sensitize melanoma cells to TMZ-induced cell killing, suggesting inhibition of NF-kB was indeed involved in TMZ/celastrol-induced cytotoxicity. Furthermore, combination treatment induced phosphorylation of JNK. Conclusions: Our data suggest that combined use of TMZ with celastrol, a natural product derived from a vine extract that has been used orally in Chinese medicine for over a thousand years, may enhance the chemotherapeutic efficacy of TMZ in melanoma. No significant financial relationships to disclose.

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the "European Searchable Tumour Cell Line and Data Bank" (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/ ) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.

Id1 expression is transcriptionally regulated in radial growth phase melanomas

Id genes have been demonstrated to be upregulated in a wide variety of human malignancies and their expression has been correlated with disease prognosis; however, little is known about the mechanisms of Id gene activation in tumors. We have previously shown that the helix-loop-helix transcription factor, Id1, is highly expressed in primary human melanomas during the radial growth phase and that Id1 is a transcriptional repressor of the familial melanoma gene CDKN2A. Here we use a series of melanoma cell lines that recapitulate the phenotypic characteristics of melanomas at varying stages of malignant progression to evaluate the expression levels of Id1 in this model system and determine the mechanism of Id1 dysregulation in these tumor cells. We find elevated protein levels of Id1 to be present consistently in radial growth phase tumor cells in accordance with our primary tumor data. Id1 transcript levels were also found to be elevated in these radial growth phase melanoma cells without any appreciable evidence of gene amplification and Id1 promoter activity was found to correlate with Id expression levels. We therefore conclude that Id1 expression is primarily regulated at the transcriptional level in radial growth phase melanomas and expect that therapies that target Id1 gene expression may be useful in the treatment of Id-associated malignancies.

Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression.

BackgroundGene expression profiling has revolutionized our ability to molecularly classify primary human tumors and significantly enhanced the development of novel tumor markers and therapies; however, progress in the diagnosis and treatment of melanoma over the past 3 decades has been limited, and there is currently no approved therapy that significantly extends lifespan in patients with advanced disease. Profiling studies of melanoma to date have been inconsistent due to the heterogeneous nature of this malignancy and the limited availability of informative tissue specimens from early stages of disease.Methodology/Principle FindingsIn order to gain an improved understanding of the molecular basis of melanoma progression, we have compared gene expression profiles from a series of melanoma cell lines representing discrete stages of malignant progression that recapitulate critical characteristics of the primary lesions from which they were derived. Here we describe the unsupervised hierarchical clustering of profiling data from melanoma cell lines and melanocytes. This clustering identifies two distinctive molecular subclasses of melanoma segregating aggressive metastatic tumor cell lines from less-aggressive primary tumor cell lines. Further analysis of expression signatures associated with melanoma progression using functional annotations categorized these transcripts into three classes of genes: 1) Upregulation of activators of cell cycle progression, DNA replication and repair (CDCA2, NCAPH, NCAPG, NCAPG2, PBK, NUSAP1, BIRC5, ESCO2, HELLS, MELK, GINS1, GINS4, RAD54L, TYMS, and DHFR), 2) Loss of genes associated with cellular adhesion and melanocyte differentiation (CDH3, CDH1, c-KIT, PAX3, CITED1/MSG-1, TYR, MELANA, MC1R, and OCA2), 3) Upregulation of genes associated with resistance to apoptosis (BIRC5/survivin). While these broad classes of transcripts have previously been implicated in the progression of melanoma and other malignancies, the specific genes identified within each class of transcripts are novel. In addition, the transcription factor NF-KB was specifically identified as being a potential “master regulator” of melanoma invasion since NF-KB binding sites were identified as consistent consensus sequences within promoters of progression-associated genes.Conclusions/SignificanceWe conclude that tumor cell lines are a valuable resource for the early identification of gene signatures associated with malignant progression in tumors with significant heterogeneity like melanoma. We further conclude that the development of novel data reduction algorithms for analysis of microarray studies is critical to allow for optimized mining of important, clinically-relevant datasets. It is expected that subsequent validation studies in primary human tissues using such an approach will lead to more rapid translation of such studies to the identification of novel tumor biomarkers and therapeutic targets.

MLANA/MART1 and SILV/PMEL17/GP100 Are Transcriptionally Regulated by MITF in Melanocytes and Melanoma

The clinically important melanoma diagnostic antibodies HMB-45, melan-A, and MITF (D5) recognize gene products of the melanocyte-lineage genes SILV/PMEL17/GP100, MLANA/MART1, and MITF, respectively. MITF encodes a transcription factor that is essential for normal melanocyte development and appears to regulate expression of several pigmentation genes. In this report, the possibility was examined that MITF might additionally regulate expression of the SILV and MLANA genes. Both genes contain conserved MITF consensus DNA sequences that were bound by MITF in vitro and in vivo, based on electrophoretic mobility shift assay and chromatin-immunoprecipitation. In addition, MITF regulated their promoter/enhancer regions in reporter assays, and up- or down-regulation of MITF produced corresponding modulation of endogenous SILV and MLANA in melanoma cells. Expression patterns were compared with these factors in a series of melanoma cell lines whose mutational status of the proto-oncogene BRAF was also known. SILV and MLANA expression correlated with MITF, while no clear correlation was seen relative to BRAF mutation. Finally, mRNA expression array analysis of primary human melanomas demonstrated a tight correlation in their expression levels in clinical tumor specimens. Collectively, this study links three important melanoma antigens into a common transcriptional pathway regulated by MITF.

Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro.

Sequence analysis of two clones found repressed in melanoma cell lines in earlier studies showed 9F2 to be identical with the TRP-1 gene and 6F5 with TRP-2 containing a long untranslated 3' end. For further investigation of the expression of the tyrosinase gene family in normal and malignant melanocytic cells, a series of melanoma cell lines and of cultured melanocytes were analyzed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). The Northern blots were probed with cDNA fragments specific for TRP-1, TRP-2, and tyrosinase, for nested tyrosinase-PCR the outer primers specified a 284 bp and the nested primers a 207 bp fragment. Investigations on 14 established melanoma cell lines grown in different media compared with seven normal human melanocyte (NHM) cultures revealed that all three pigment genes were expressed in NHM, whereas pigment gene expression was found repressed in nearly all melanoma cell lines and was completely absent in 4 of 14 specimen. In particular, tyrosinase and TRP-2 genes were found always to be expressed together, and TRP-1 mRNA alone was absent in four melanoma cell lines. Negativity of cultured melanoma cells for tyrosinase mRNA was confirmed by nested RT-PCR, and gene deletion was ruled out by genomic Southern blots. The gene expression seemed independent from the type of medium used for cultivation. These findings indicate repressed or lacking expression of pigment genes in melanoma cell lines, most likely due to regulatory mechanisms, and that differences may exist between tyrosinase and TRP-2 on one hand and TRP-1 on the other. Overall, it seemed that RT-PCR for tyrosinase has limited value for identifying melanoma cells in the peripheral blood of melanoma patients; TRP-1, TRP-2, and other, additional markers may be required.