Salinomycin increases chemosensitivity to the effects of doxorubicin in soft tissue sarcomas.

BACKGROUND: Chemotherapy for soft tissue sarcomas remains unsatisfactory due to their low chemosensitivity. Even the first line chemotherapeutic agent doxorubicin only yields a response rate of 18-29%. The antibiotic salinomycin, a potassium ionophore, has recently been shown to be a potent compound to deplete chemoresistant cells like cancer stem like cells (CSC) in adenocarcinomas. Here, we evaluated the effect of salinomycin on sarcoma cell lines, whereby salinomycin mono- and combination treatment with doxorubicin regimens were analyzed. METHODS: To evaluate the effect of salinomycin on fibrosarcoma, rhabdomyosarcoma and liposarcoma cell lines, cells were drug exposed in single and combined treatments, respectively. The effects of the corresponding treatments were monitored by cell viability assays, cell cycle analysis, caspase 3/7 and 9 activity assays. Further we analyzed NF-κB activity; p53, p21 and PUMA transcription levels, together with p53 expression and serine 15 phosphorylation. RESULTS: The combination of salinomycin with doxorubicin enhanced caspase activation and increased the sub-G1 fraction. The combined treatment yielded higher NF-κB activity, and p53, p21 and PUMA transcription, whereas the salinomycin monotreatment did not cause any significant changes. CONCLUSIONS: Salinomycin increases the chemosensitivity of sarcoma cell lines - even at sub-lethal concentrations - to the cytostatic drug doxorubicin. These findings support a strategy to decrease the doxorubicin concentration in combination with salinomycin in order to reduce toxic side effects.

MicroRNA-205, a novel regulator of the anti-apoptotic protein Bcl2, is downregulated in prostate cancer.

Decreased expression of the microRNA miR-205 has been observed in multiple tumour types due to its role in the epithelial to mesenchymal transition, which promotes metastasis. We determined the expression of miR-205 in 111 archival samples of prostate carcinoma and found it to be strongly reduced in most samples, with a median expression level of 16% in comparison to benign tissue from the same patient. Lower miR-205 expression correlated significantly with tumour size and miR-205 levels decreased with increasing Gleason score from 7a=3+4 to 8=4+4. In addition, we describe the anti-apoptotic protein BCL2 as a target of miR-205, relevant for prostate cancer due to its role in prognosis of primary tumours and in the appearance of androgen independence. The repression of BCL2 by miR-205 was confirmed using reporter assays and western blotting. BCL2 mRNA expression in the same collective of prostate cancer tissue samples was associated with higher Gleason score and extracapsular extension of the tumour (pT3). Consistent with its anti-apoptotic target BCL2, miR-205 promoted apoptosis in prostate cancer cells in response to DNA damage by cisplatin and doxorubicin in the prostate cancer cell lines PC3 and LnCap. MiR-205 also inhibited proliferation in these cell lines.

Growth rate of late passage sarcoma cells is independent of epigenetic events but dependent on the amount of chromosomal aberrations.

Soft tissue sarcomas (STS) are characterized by co-participation of several epigenetic and genetic events during tumorigenesis. Having bypassed cellular senescence barriers during oncogenic transformation, the factors further affecting growth rate of STS cells remain poorly understood. Therefore, we investigated the role of gene silencing (DNA promoter methylation of LINE-1, PTEN), genetic aberrations (karyotype, KRAS and BRAF mutations) as well as their contribution to the proliferation rate and migratory potential that underlies "initial" and "final" passage sarcoma cells. Three different cell lines were used, SW982 (synovial sarcoma), U2197 (malignant fibrous histiocytoma (MFH)) and HT1080 (fibrosarcoma). Increased proliferative potential of final passage STS cells was not associated with significant differences in methylation (LINE-1, PTEN) and mutation status (KRAS, BRAF), but it was dependent on the amount of chromosomal aberrations. Collectively, our data demonstrate that these fairly differentiated/advanced cancer cell lines have still the potential to gain an additional spontaneous growth benefit without external influences and that maintenance of increased proliferative potential towards longevity of STS cells (having crossed senescence barriers) may be independent of overt epigenetic alterations.

Salinomycin induces autophagy in colon and breast cancer cells with concomitant generation of reactive oxygen species.

BACKGROUND: Salinomycin is a polyether ionophore antibiotic that has recently been shown to induce cell death in human cancer cells displaying multiple mechanisms of drug resistance. The underlying mechanisms leading to cell death after salinomycin treatment have not been well characterized. We therefore investigated the role of salinomycin in caspase dependent and independent cell death in colon cancer (SW480, SW620, RKO) and breast cancer cell lines (MCF-7, T47D, MDA-MB-453). METHODOLOGY/PRINCIPAL FINDINGS: We detected features of apoptosis in all cell lines tested, but the executor caspases 3 and 7 were only strongly activated in RKO and MDA-MB-453 cells. MCF-7 and SW620 cells instead presented features of autophagy such as cytoplasmic vacuolization and LC3 processing. Caspase proficient cell lines activated autophagy at lower salinomycin concentrations and before the onset of caspase activation. Salinomycin also led to the formation of reactive oxygen species (ROS) eliciting JNK activation and induction of the transcription factor JUN. Salinomycin mediated cell death could be partially inhibited by the free radical scavenger N-acetyl-cysteine, implicating ROS formation in the mechanism of salinomycin toxicity. CONCLUSIONS: Our data indicate that, in addition to its previously reported induction of caspase dependent apoptosis, the initiation of autophagy is an important and early effect of salinomycin in tumor cells.

Site- and grade-specific diversity of LINE1 methylation pattern in gastroenteropancreatic neuroendocrine tumours.

BACKGROUND: Recent data indicate that gastroenteropancreatic neuroendocrine tumours (GEP-NETs) have a hypomethylated long interspersed element (LINE1) promoter. To answer the question, of whether LINE1 may be of value in assessing the malignant potential of GEP-NETs, we analysed LINE1 methylation in different organs. MATERIALS AND METHODS: A total of 58 GEP-NETs of gastric (n=14), pancreatic (n=15), small intestine (n=17), appendix (n=8), colorectal (n=4) and non-neoplastic tissues were analysed using DNA isolation, bisulphite-treatment and pyrosequencing. RESULTS: LINE1 hypomethylation was detected in 50% of gastric, 100% pancreatic, 82% small intestine, 87.5% appendix and 100% colorectal NETs. G1 (p<0.001) and G2 (p<0.05) colorectal, and G1 (p<0.001) and G2 (p<0.001) pancreatic NETs exhibited significant LINE1 hypomethylation compared with non-neoplastic tissues. Higher rates of LINE1 hypomethylation in G2 pancreatic NETs than in G1 NETs (p<0.05) were observed. NETs exhibited a significantly lower frequency of hypomethylation in cases with lymph node metastases (p<0.05). CONCLUSION: LINE1 hypomethylation may serve as a marker of tumour grade and lymph node metastasis.

MicroRNA-148a is down-regulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B.

MicroRNAs (miRNAs: short non-coding RNAs) are emerging as a class of potential novel tumor markers, as their dysregulation is being increasingly reported in various types of cancers. In the present study, we investigated the transcription status of miRNA-148a (miR-148a) in human pancreatic ductal adenocarcinoma (PDAC) and its role in the regulation of the dual specificity protein phosphatase CDC25B. We observed that miR-148a exhibited a significant 4-fold down-regulation in PDAC as opposed to normal pancreatic ductal cells. In addition, we observed that stable lentiviral-mediated overexpression of miR-148a in the pancreatic cancer cell line IMIM-PC2, inhibited tumor cell growth and colony formation. Furthermore, CDC25B was identified as a potential target of miR-148a by in silico analysis using PicTar, Targetscan and miRanda in conjunction with gene ontology analysis. The proposed interaction between miR-148a and the 3' untranslated region (UTR) of CDC25B was verified by in-vitro luciferase assays. We demonstrate that the activity of a luciferase reporter containing the 3'UTR of CDC25B was repressed in the presence of miR-148a mimics, confirming that miR-148a targets the 3'UTR of CDC25B. Finally, CDC25B was down-regulated at the protein level in miR-148a overexpressing IMIM-PC2-cells, and in transiently transfected pancreatic cell lines (as detected by Western blot analysis), as well as in patient tumor samples (as detected by immunohistochemistry). In summary, we identified CDC25B as a novel miR-148a target which may confer a proliferative advantage in PDAC.

Role of erythropoietin receptor expression in malignant melanoma.

Recombinant human erythropoietin (Epo) is used to prevent and treat tumor-related anemia and improve quality of life in cancer patients. Recent evidence suggested that Epo may adversely affect the survival of selected cancer patients by promoting tumor growth, inhibition of apoptosis, and induction of migration. Epo unfolds its effect on the Epo receptor (EpoR). We show--to the best of our knowledge for the first time--significantly increased EpoR expression in clinical melanoma metastases and primary melanomas in comparison with different sets of nevi by quantitative real-time reverse transcriptase-PCR, immunohistochemistry, and western blot analysis. When assessing the functionality of the EpoR-signaling pathway, recombinant human Epo led to the phosphorylation of JAK-2, signal transducers and activators of transcription 3 (STAT3), and ERK1/2 in several of the melanoma cell lines that were analyzed. Besides, Epo counteracted cisplatin-induced cell death in BLM and MV3 cells. Finally, Epo promoted cell migration of MV3 cells, whereas inhibition of the JAK/STAT and ERK1/2 pathways reduced Epo-mediated migration. In summary, we show the overexpression of functional EpoR expression in about half of the analyzed clinical melanoma metastasis specimens and show anti-apoptotic as well as pro-migratory effects of Epo, which is of importance for the treatment of anemia in advanced melanoma.

Transcriptional regulation of ASK/Dbf4 in cutaneous melanoma is dependent on E2F1.

BACKGROUND: Melanoma is a complex genetic disease, the management of which will require an in-depth understanding of the biology underlying its initiation and progression. Recently, we have reported the differential regulation of a novel gene, namely ASK/Dbf4, in melanoma and suggested upregulation of ASK/Dbf4 as a novel molecular determinant with prognostic relevance that confers a proliferative advantage in cutaneous melanoma. As trans-acting factor binding is fundamental to understand the regulation of gene expression, this study focuses on characterization of the specific transcriptional regulation of ASK/Dbf4 in melanoma. OBJECTIVE: We investigated whether ASK/Dbf4 is a transcriptional target of the important cell cycle regulator E2F1 in melanoma. RESULTS: As evidenced by gel supershift assays on nuclear extracts from various melanoma cell lines (SK-MEL-28, MV3, M13, A375 and BLM), E2F1 bound to the ASK/Dbf4 minimal promoter (MP). In addition, cisplatin-mediated abrogation of E2F1 binding to the ASK/Dbf4 MP resulted in a transcriptional decrease in ASK/Dbf4. Further, the current study also demonstrated that ASK/Dbf4 regulation was refractory to UVB, a well-known risk factor for melanoma. CONCLUSIONS: In summary, our study not only elucidated that ASK/Dbf4, a novel cell survival gene in melanoma was transcriptionally regulated by E2F1, but also that the induction of ASK/Dbf4 was refractory to UVB exposure suggesting that its upregulation was not an early event in melanomagenesis.

Cutaneous melanoma: fishing with chips.

DNA microarray technology is a versatile platform that allows rapid genetic analysis to take place on a genome-wide scale and has revolutionized the way cancers are studied. This platform has enabled researchers to characterize mechanisms central to tumorigenesis and understand important molecular events in the multi-step tumor progression model of cutaneous melanoma and other cancers. In melanoma, multiple global gene expression profiling studies using various DNA microarray platforms and various experimental designs have been performed. Each study has been able to capture and characterize either the involvement of a novel pathway or a novel cause-effect-relationship. The use of microarrays to define subclasses, to identify differentially regulated genes within a mutational context to analyze epigenetically regulated genes has resulted in an unprecedented understanding of the biology of cutaneous melanoma that may lead to more accurate diagnosis, more comprehensive prognosis, prediction and more effective therapeutic interventions. Related DNA microarray platforms like array-comparative genomic hybridization (CGH) have also been instrumental to identify many non-random chromosomal alterations; however, studies identifying validated targets as a result of CGH are limited. Thus, there exists significant opportunity to discover novel melanoma genes and translate such discoveries into meaningful clinical endpoints. In this review, we focus on various DNA microarray-based studies performed in cutaneous melanoma and summarize our current understanding of the genetics and biology of melanoma progression derived from accumulating genomic information.

Identification and functional characterization of ASK/Dbf4, a novel cell survival gene in cutaneous melanoma with prognostic relevance.

Malignant melanoma is one of the most aggressive and invasive metastatic tumors derived from melanocytes that have undergone malignant transformation by acquisition of genetic and epigenetic alterations. Oligonucleotide microarray-based screening of distinct stages in the tumor progression model of cutaneous melanoma identified ASK/Dbf4, as a novel determinant for melanoma development. Quantitative real-time polymerase chain reaction-based confirmation of ASK/Dbf4 on a series of benign nevi, dysplastic nevi, primary cutaneous melanomas and cutaneous melanoma metastases; and a number of other controls using normal human melanocytes as calibrator not only revealed a melanoma-specific over-expression but also revealed that higher ASK/Dbf4-expressing melanomas were associated with lower relapse-free survival. Additionally, we also confirmed the observed over-expression of ASK/Dbf4 in melanoma using western blot analysis and immunohistochemistry. As ASK/Dbf4 is known to be a cyclin-like regulatory subunit of mammalian Cdc7 from the studies in yeast, the present study investigated its role in melanoma cells. In keeping with its expected role, our data suggest that up-regulated ASK/Dbf4 is localized in the nucleus and binds to human Cdc7 to form Cdc7-ASK/Dbf4 complexes in several analyzed melanoma cell lines. Further, we demonstrate that small interfering RNA-mediated depletion of ASK/Dbf4 retarded melanoma cell survival and proliferation. In summary, we report the differential regulation of a novel gene, namely ASK/Dbf4, in melanoma and suggest that up-regulation of ASK/Dbf4 is a novel molecular determinant with prognostic relevance that confers a proliferative advantage in cutaneous melanoma.

ERK1/2 is highly phosphorylated in melanoma metastases and protects melanoma cells from cisplatin-mediated apoptosis.

Activation (phosphorylation) of mitogen-activated protein kinase (MAPK) signal transduction through BRAF and RAS causes a variety of functional effects including cell survival and cell death. In this study, we observed high extracellular signal-regulated kinase (ERK)1/2 phosphorylation levels in clinical melanoma metastases and various melanoma cell lines. Treatment of melanoma cell lines with cisplatin, a potent antitumor agent, increased the level of phosphorylated-ERK (P-ERK)1/2 and enhanced chemoresistance through activation of the cell survival protein 90-kDa ribosomal S6 kinase (RSK)1. The mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) was able to block this effect and reduced cell viability and sensitized cells to cisplatin-induced apoptosis, as shown by PARP cleavage, caspase 3 expression, and annexin-V staining. In conclusion, the MAP kinase-ERK pathway is activated in melanoma and reduces the sensitivity of melanoma to cisplatin. Thus, inhibition of ERK1/2 in combination with selected chemotherapeutic agents may hold promise for more effective therapy of melanoma.

Epigenetic silencing of the PTEN gene in melanoma.

Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) seems to be an important tumor suppressor gene in melanoma. Because the PTEN gene is only infrequently deleted or mutated, and because the PTEN protein is low to absent in a significant number of melanomas, we investigated alternative methods of epigenetic silencing. We did quantitative positional methylation analysis (pyrosequencing) on 37 sera from melanoma patients and on 21 pairs of corresponding sera and melanoma specimens in addition to Taqman reverse transcription-PCR. We report significant positional PTEN promoter methylation in 62% of circulating DNA isolated from sera of patients with metastatic melanoma. The percentage of methylation of a selected CpG island in blood showed a correlation with methylation levels in the corresponding melanoma tissue. Moreover, high percentages of PTEN methylation were associated with low PTEN transcription levels. Using the demethylation agent 5-aza-2'-deoxycytidine, reduced methylation and a corresponding increase in PTEN protein were observed in BLM melanoma cells, leading to reduced AKT activity in an in vitro kinase assay. In summary, epigenetic PTEN silencing seems to be a relevant mechanism of inactivating this tumor suppressor gene in melanoma that may promote melanoma development by derepression of the AKT pathway.

STAT5 phosphorylation in malignant melanoma is important for survival and is mediated through SRC and JAK1 kinases.

Altered signaling pathways are key regulators of cellular functions in tumor cells. Constitutive activation of signal transducer and activator of transcription (STAT)3 and -5 may be involved in tumor formation and progression. We have investigated the role of STAT5 in cutaneous melanoma metastases using various RNA and protein techniques. In melanoma specimens, Stat5b transcripts were upregulated approximately 3.8-fold. In 13 of 21 (62%) human melanoma metastases, STAT5 was phosphorylated in comparison to normal human melanocytes and benign nevi. The STAT5 target gene Bcl-2 was frequently upregulated. The investigation of the underlying mechanism revealed specific STAT5 activation by recombinant human epidermal growth factor (rEGF). rEGF-induced activation of STAT5 occurred in vitro through the non-receptor tyrosine kinases transforming gene (src) of Rous Sarcoma virus and Janus kinase 1. Inhibition of Stat5b expression by small interfering RNA strongly reduced the expression of Bcl-2 and led to decreased cell viability and increased apoptosis in the melanoma cell lines A375 and BLM. Transfection with dominant-negative Stat5b caused enhanced cell death and G1 arrest in A375 cells. Our study identifies phosphorylated STAT5 in melanoma and shows regulation through rEGF; STAT5 may thus act as a survival factor for growth of human melanoma and may represent a potential target for molecular therapy.

Epigenetic inactivation of tumor suppressor genes in serum of patients with cutaneous melanoma.

Small amounts of cell-free DNA circulate in both healthy and diseased human blood, while increased concentrations of DNA are present in the serum of cancer patients. Tumor-specific mutations or epigenetic modifications have predominantly been detected in tissue specimens. The purpose of this study was to investigate methylation of five different genes involved in tumor suppression and DNA repair (suppressors of cytokine signaling 1 and 2 (SOCS1, SOCS2)), Ras-association domain family protein 1A (RASSF1a), D-type p16(INK4a) cyclin-dependent kinase inhibitor (CDKN), and O6-methylguanine DNA-methyltransferase (MGMT)) in the serum of 100 patients using methylation-specific PCR. In all, 41 melanoma patients (stage I = 18; stage II = 10; stage III/IV = 13), 13 healthy controls without nevi, and 10 individuals with more than 15 nevi of >5 mm in size were investigated. For comparison, sera from patients with other skin tumors (nine basal cell cancers, five Kaposi's sarcoma), different metastasized cancers (five breast cancers, five colon cancers), and several chronic inflammatory diseases (n = 12) were also analyzed. In addition, we examined if methylation was involved in silencing transcription of these genes in 12 melanoma specimens. SOCS1, SOCS2, RASSF1a, CDKN2a, and MGMT were methylated in 75, 43, 64, 75, and 64% of melanoma samples, respectively. Of the 41 melanoma patients, 83% had one hypermethylated gene, while 66, 51, and 41% had two, three, or four hypermethylated genes, respectively. Also, 20% of these patients showed hypermethylation for all genes, while only 17% showed no methylation. Importantly, the methylation profile of the selected genes from melanoma patients was distinct from the other analyzed tumors. Transcription of SOCS1, SOCS2, CDKN2a, and RASSF1a genes was significantly reduced in fresh melanoma samples, while MGMT showed a 12-fold upregulation at the messenger ribonucleic acid level (P < 0.001). Our findings suggest that epigenetic silencing of the studied tumor suppressor genes is a common and probably important mechanism for melanoma formation. This convenient method using a simple blood sample may contribute to classification of melanoma and awaits clinical validation.

Topical application of plasmid DNA to mouse and human skin.

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.

Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors.

Previously, the RASSF1A, BLU and SEMAPHORIN 3B (SEMA3B) candidate tumor suppressor genes on chromosome 3p21.3 were found to be inactivated and downregulated by genetic and epigenetic changes in lung cancer. We analyzed the methylation status of RASSF1A, BLU and SEMA3B in 35 hepatocellular carcinomas (HCCs) and 15 cholangiocarcinomas (CCs) by methylation-specific PCR and loss of heterozygosity (LOH) at 3p21.3 after microdissection. The presence of mRNA transcripts was confirmed by semiquantitative PCR. SEMA3B hypermethylation was found in 29/35 HCCs (83%) and in all (15/15) patients with CC. BLU promoter hypermethylation was detected in 7/35 (20%) HCCs and 3/15 (20%) CCs. In 2 corresponding specimens of hepatitis B virus-related liver cirrhosis, BLU methylation was also observed, but not in uninvolved normal liver tissue. RASSF1A was methylated in 21/35 HCCs (60%) and in 10/15 CCs (67%). LOH at 3p21.3 occurred in 8/35 (23%) HCCs and 3/15 (20%) CCs. The presence of hypermethylation was statistically associated with LOH of SEMA3B and correlated with downregulation of mRNA transcripts. SEMA3B transcripts increased upon treatment of HCC cell lines with the demethylation compound 5-aza-2-deoxycytidine. In conclusion, our data indicate that 2-hit gene silencing of SEMA3B through epigenetic changes and allele loss is a common and important event in the carcinogenesis of malignant liver tumors.

Signaling networks in cutaneous melanoma metastasis identified by complementary DNA microarrays.

BACKGROUND: Melanoma is a complex multigenic disease, susceptibility to which is determined by several parallel and stepwise progressive pathways affecting growth control, differentiation, cell adhesion, and survival. Melanoma and human cancers in general undergo a continuous development from benign to malignant states, as most thoroughly documented in the multistep mole-to-melanoma transition. OBJECTIVE: To examine how high-throughput microarrays are being used in expression profiling to identify regulated genes, patterns, and pathways that may lead to functional characterization and tumor subclassification. DESIGN: Ten melanoma metastases were analyzed by DNA array technology for important regulated candidate genes, with subsequent confirmation by real-time reverse transcription polymerase chain reaction. RESULTS: Hepatocyte growth factor receptor c-met, growth factor receptor-bound protein 10, B-raf proto-oncogene, and several mitogen-activated protein kinase kinase genes were significantly up-regulated in melanoma metastases and several melanoma cell lines relative to normal human melanocytes (P = .03). Among the up-regulated genes, phosphorylated growth factor receptor-bound protein 10 is known to serve a molecular switch turning on the mitogen-activated protein kinase pathway in response to hepatocyte growth factor receptor binding. CONCLUSIONS: As suggested by the DNA arrays, we found the mitogen-activated protein kinase kinase/extracellular-regulated kinase pathway to be activated in most of the cutaneous melanoma metastasis specimens. These findings are in the context of the current microarray technology in melanoma research. Additional steps are needed to gain insights into the pluralistic signaling milieu of this malignancy as we enter the postgenomic era.

Rapid identification of dysregulated genes in cutaneous malignant melanoma metastases using cDNA technology.

One important application of DNA microarray technology is the simultaneous analysis of gene expression of different mRNAs. Comparison of mRNA patterns of diseased and healthy tissue may help to understand the pathogenesis of a given disorder. In cancer tissue, identified dysregulated genes may serve as new molecular markers for diagnosis or prognosis or may ideally serve as new targets for therapy. Using membrane cDNA array technology, we analyzed gene expression in human melanomas, one of the most aggressive types of cancer with a high metastatic potential and with markedly increased incidence worldwide. To account for the heterogeneity of tumors, we compared total RNA from cutaneous melanoma metastases of 10 different patients with primary human melanocytes. An abundance of genes was dysregulated (up-/downregulated), which involved for example the apoptosis gene growth factor receptor-bound protein 10, Bcl2-associated X membrane protein, Bcl2 antagonist of cell death, glutathione S-transferase theta(1) and glutathione reductase. Ultimately, the identification of melanoma-associated genes may provide a potential therapeutic strategy for identifying and targeting malignant melanoma.

Applications of array technology: melanoma research and diagnosis.

A complex set of genetic alterations occurs within a cell in order to permit neoplastic transformation. Human cancers undergo a continuous development from benign to malignant states, as most thoroughly documented in the mole-to-melanoma transition. Several specific genetic and transcriptional events correlate with the prolonged multistep sequence from early to late clinical stages of the disease. High-throughput microarrays are being used in expression profiling analyses with the aim of discovering genes and their pathways, functional characterization of genes and tumor subclassification. There are, however, many potential pitfalls in the use of microarrays that result in false leads and erroneous conclusions. This review summarizes the current status of the application of microarray technology in melanoma research. It also attempts to outline some of the steps needed to develop the key features to be observed in developing diagnostic and prognostic classification systems based upon gene expression profiling.