5/30/2023 0 Comments Our arrows will blot out the sun( a) Western blot analysis for PAX3 expression in cell lines. The tissue samples were scored as either ‘PAX3 protein expression positive' or ‘PAX3 protein expression negative.' These data are summarized in Figure 1e.įigure 1 PAX3 is expressed in melanoma cell lines and primary tumor samples. In contrast, a significant number (2 1/58) ( p<0.005) of metastatic melanoma samples demonstrated PAX3 immunoreactivity ( Figure 1c). The vast majority of these samples (9/75) were negative for PAX3 expression ( Figure 1d). Out of 133 samples analyzed, 75 were benign pigmented lesions, with 38 characterized as typical and 37 as atypical (dysplastic) nevi. Two major groups of primary pigmented lesions were also tested for in situ expression of PAX3. No PAX7 expression was detected in any of the cell lines examined (representative RT-PCR analysis, Figure 1b). The melanoma cell lines were also screened for the expression of PAX7, a closely related family member of PAX3 that is also expressed in melanocyte precursors. As negative controls, non-melanoma cell lines CFPAC1 (a pancreatic carcinoma cell line, Figure 1a, lane 1) and 3T3 (mouse fibroblasts, Figure 1a, lane 11) that do not express PAX3 are shown. Primary Melanoma and Nevus SamplesĪll the melanoma cell lines (one murine and eight human melanoma cell lines) examined by western blot analysis were found to express varying levels of Pax3 ( Figure 1a). Transfection efficiency was determined by pCMV- β-gal transfection and staining of cells for β-galactosidase activity. The plasmid pcDNA3-Pax7 was transfected into 293T cells using Qiagen Effectene reagent (Qiagen Inc., Valencia, CA, USA). 293T cells transfected with Pax7 plasmid served as a positive control. The following primers were used: G3PDH (human) primers: sense 5′-TGAAGGTCGAGTCAACGGATTTGGT-3′ and antisense 5′-CATGTGGGCATGAGGTCCACCAC-3′, with an expected PCR product of 983 bp and PAX7 primers: forward 5′-GCTCCGGGGCAGAACTACC-3′ and reverse 5′-GCACGCGGCTAATCGAACTC-3′, with expected PCR product of 436 bp. A 2- μl portion of cDNA was used for the PCR experiment. As a positive control, each RNA sample was tested for the expression of the housekeeping gene G3PDH. As a negative control against genomic DNA contamination, each RNA sample was tested by PCR following an RT step in the absence of RT. Reverse transcriptase-PCR (RT-PCR) was performed using SuperScript III First-Strand Synthesis System for RT-PCR following the manufacturer’s protocols (Invitrogen), using random hexamer primers to generate cDNA strands. RNA was isolated from freshly growing cells using Trizol (Invitrogen). Reverse transcriptase-PCR for Pax7 Expression in Melanoma Cell Lines The membranes were stripped using Restore Western Blot Stripping Buffer (Pierce Biotechnology, Rockford, IL, USA), checked for complete removal of antibodies, and reanalyzed for β-tubulin expression (1:400 mouse monoclonal antibody E7 University of Iowa Hybridoma Bank) as a loading control. For western blot analysis detection of PAX6 and β-tubulin, cell lysate (40 μg/well) was loaded on 4–12% gradient gels, transferred to PVDF membranes, and probed with PAX3 mouse monoclonal antibody (1:100 dilution University of Iowa Hybridoma Bank, Iowa City, IA, USA) and western blotted (Western Breeze Chemiluminescent kit Invitrogen, Carlsbad, CA, USA). The mouse fibroblast 3T3 and human pancreatic cancer cell line CFPAC1 are available from ATCC (Manassas, VA, USA). We studied the B16 murine melanoma cell line and eight human melanoma cell lines: 537, 624, 888, A375, HIM2, SK23, SKMEL5, and SKMEL28. ![]() Our data suggest that PAX3-expressing melanomas may be less environmentally dependent and more genetically linked.Īll cell lines were obtained through the University of Chicago Cancer Research Center. Further analysis of our melanoma set revealed that PAX3 expression is strongly correlated with younger patients with low or no evidence of sun damage. We also find that PAX3 is commonly expressed in primary melanoma samples (2 1/58) but significantly less frequently in benign pigmented lesions (9/75). ![]() ![]() Here, we find PAX3 expression in 8/8 melanoma cell lines. This mechanism may also contribute to the uncontrolled cell growth and loss of terminal differentiation in melanomas. Based on this function, PAX3 promotes a melanocytic phenotype but blocks terminal differentiation. The transcription factor PAX3 has a well-established role in the development of melanocytes during embryogenesis, and has recently been characterized as a molecular switch in the mature melanocyte. Although the histogenesis of the tumor is not well understood, it is thought to originate from a rare melanocyte stem cell that resides in the skin. Melanoma is responsible for an estimated 62 000 new American cancer diagnoses and is projected to cause nearly 8000 deaths in 2008 alone.
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