Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays - PubMed (original) (raw)
Analysis of differential gene expression in human melanocytic tumour lesions by custom made oligonucleotide arrays
N J W de Wit et al. Br J Cancer. 2005.
Abstract
Melanoma is one of the most aggressive types of cancer and resection of the tumour prior to dissemination of tumour cells is still the most effective treatment. Therefore, early diagnosis of melanocytic lesions is important and identification of novel (molecular) markers would be helpful to improve diagnosis. Moreover, better understanding of molecular targets involved in melanocytic tumorigenesis could possibly lead to development of novel interventions. In this study, we used a custom made oligonucleotide array containing 298 genes that were previously found to be differentially expressed in human melanoma cell lines 1F6 (rarely metastasising) and Mel57 (frequently metastasising). We determined differential gene expression in human common nevocellular nevus and melanoma metastasis lesions. By performing nine dye-swap array experiments, using individual as well as pooled melanocytic lesions, a constant differential expression could be detected for 25 genes in eight out of nine or nine out of nine array analyses. For at least nine of these genes, namely THBD, FABP7, H2AFJ, RRAGD, MYADM, HR, CKS2, NCK2 and GDF15, the differential expression found by array analyses could be verified by semiquantitative and/or real-time quantitative RT-PCR. The genes that we identified to be differentially expressed during melanoma progression could be potent targets for diagnostic, prognostic and/or therapeutic interventions.
Figures
Figure 1
Custom made oligonucleotide array design. 70-mer oligo's, corresponding to 298 genes that were previously found to be differentially expressed in cell lines 1F6 and Mel57 and four additional housekeeping genes, were spotted in octaplicate on UltraGAPS slides. The housekeeping genes were randomly localised throughout the array. Additionally, various other controls for labelling and hybridisation efficiency were present on the array, such as oligo's of non-human species, Cot-1 repetitive sequences, polyA sequences and spots containing only spotting buffer.
Figure 2
Verification of differential gene expression in human melanoma cell lines 1F6 and Mel57 by semiquantitative RT–PCR. Differential gene expression was determined by normalisation for _β_-actin and GAPDH. For nearly all of the 25 genes that were tested, differential expression that was found by custom array analyses could be validated by semiquantitative RT–PCR, except for MYL6 and FLJ10349.
Figure 3
Analysis of protein expression of THBD, GDF15 and FABP7 in human melanoma cell lines 1F6 and Mel57. Magnification (× 400) of AgarCyto's (A–H) and cultured 1F6 and Mel57 cells (I, J). (A, B) Negative control of 1F6 and Mel57, respectively, leaving out specific antisera. (C, D, I, J) IHC using THBD specific antibodies; in AgarCyto's as well as cultured cells most 1F6 cells showed a (membranous) THBD specific staining (C, I), whereas Mel57 cells were completely negative (D, J). (E, F) IHC using GDF15 specific antibodies; a more intense cytoplasmic GDF15 staining could be detected in cell line Mel57 (F), compared to 1F6 cells (E). (G, H) IHC using FABP7 specific antibodies; 1F6 cells showed a higher immunoreactivity for FABP7 (cytoplasmic) (G) than Mel57 cells (H). (K) Western blot analysis of GDF15; a distinct preferential protein expression could be detected in Mel57 compared to 1F6. The 35 kDa band represents the pro-form of GDF15 (pro-peptide+mature protein), whereas the 25 kDa band shows the cleaved GDF15 pro-peptide.
Figure 4
Verification of differential gene expression in human melanoctyic tumour progression lesions by semiquantitative RT–PCR. (A) For 12 differentially expressed genes, relative expression to GAPDH was determined by semiquantitative RT–PCR as shown for FABP7. (B) For MYL6 and C7orf20 differential expression could hardly be detected. THBD, FABP7, H2AFJ, RRAGD, MYADM and HR showed a downregulated expression during melanoma progression, with a preferable decrease in MM lesions. The latter was especially true for H2AFJ. Expression of CKS2, NCK2 and GDF15 showed an upregulation associated with increased malignancy of melanocytic tumour lesions. For THBD, FABP7, H2AFJ, RRAGD, MYADM, HR, CKS2, NCK2 and GDF15 differential expression found by semiquantitative RT–PCR was in accordance with our custom array data, whereas CTSL showed an inverse differential expression.
Figure 5
Verification of differential gene expression of FABP7 and GDF15 in human melanoctyic tumour progression lesions by real-time qPCR. For FABP7 as well as GDF15, GAPDH was used for normalisation. FABP7 expression in melanocytic tumour lesions is demonstrated relative to expression in cell line 1F6. A downregulated FABP7 expression was detected during melanocytic tumour progression, which was most prominent in MM lesions. For GDF15, expression in melanocytic tumour lesions is demonstrated relative to expression in cell line Mel57. GDF15 showed an upregulated expression in malignant stages of melanocytic tumour progression. Inclusion of normal skin (NS) tissue and laser dissected melanocytes derived from NN lesions (NN#) in the real-time qPCR analyses revealed that epidermal cells do not significantly influence the differential gene expression of FABP7 and GDF15 that is seen in NN and MM lesions.
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