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    Functional analyses of alternative isoforms of PAX3 in melancytes

    Wang, Qiuyu, Parker, Craig J., Kumar, Shant and Kumar, Patricia (2003) Functional analyses of alternative isoforms of PAX3 in melancytes. In: British Cancer Research Meeting 2003, 2nd-5th July 2003, Bournemouth, UK.

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    Abstract

    The paired box gene, PAX3 encodes a transcription factor, importantin cell proliferation, migration and survival during early embryonic neural and muscle development. Until recently, five different isoforms had been identified, while their functions remain unknown. We have discovered two new isoforms, g and h, lacking complete coding for exon 8. We have confirmed their sequences and subcloned seven isoforms, PAX3a-h into pcDNA4 expression vector. The constructed plasmids and empty vector pcDNA4 were stably transfected into a non-tumorigenic murine melanocyte cell line, Melan-a, using Transfectam. Transfected colonies wereanalysed for PAX3 isoform expression using specific primers bysemiquantitative RT-PCR. Promega Cell Proliferation Assay: melan a-PAX3h cells proliferated more rapidly than mock transfectants over 72h (P<0.01), while Melan a-PAX3a, -PAX3b cells proliferated more slowly (P<0.01 )and Melan a-PAX3c, PAX3d, PAX3e, PAX3g showed similar rates of proliferation. A cell growth curve analysis over 7 days confirmed this, except that PAX3e grew more slowly than mock transfectants. Cell transformation assay: mock transfectants and cells containing PAX3a, PAX3b or PAX3e failed to form colonies in soft agar, whereas cells expressing PAX3c, PAX3d, PAX3g, and PAX3h formed colonies. PAX3d expressing cells formed larger colonies than the others, while cells having PAX3h produced more colonies. Together, our results suggest that alternative isoforms of PAX3 have different effects on melanocyte growth and transformation. Further studies are needed to determine whether there is a causal relationship between PAX3 isoforms and tumorigenesis in melanoma or whether one spliced isoform can regulate another, and to determine the molecular mechanisms involved.

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