The S100 protein family is a group of structurally-related calcium-modulated proteins involved in cell cycle progression, cell differentiation and malignant transformation. We recently found the uveal melanoma-associated S100 protein (uv-S100) to be immunologically distinct from S100Beta expressed by skin melanocytes. Because S100s are not glycosylated, antigenic differences among these proteins must reflect differences in amino acid sequences. This premise is corroborated by recent studies of uv-S100 mRNAs by a RNA-polymerase chain reaction (PCR) technique. In this proposal, we will examine the S100 phenotype of uveal melanoma at the molecular level and sequence the coding regions of uv-S100 mRNAs from normal and malignant uveal melanocytes. We postulate that this S100 protein, like many S100s, is tissue-specific and characteristics of uveal melanocytes. Like S100Beta in skin melanocytes, uv-S100 may be involved in cell cycling and/or malignant transformation.
Our aims are: (1) To amplify and quantitate S100Beta RNAs from freshly biopsied primary and metastatic choroidal melanomas. Oligonucleotide primers will be designed from conserved domains of S100Beta. Quantitative PCR will estimate S100 expression normalized to that of actin, which correlates with cell number. (2) To identify and sequence uv-S100 mRNAs. Single-strand conformational polymorphic variants of S100Beta will be sought in PCR products from normal and malignant uveal melanocytes by nondenaturing gel. uv-S100 sequences identified by shifts in electrophoretic mobilities will also be sequenced. (3) To determine if uv-S100 is specific to melanocytes of the uveal tract. Quantitative PCR will be applied to normal and malignant melanocytes of both uveal and skin origin as well as human tumors with different embryological origins. (4) To determine which S100s are expressed in the malignant Callender cell types of uveal melanomas. The levels of expression of both S100Beta and uv-S100 will be examined by quantitative PCR. Localization at the single cell level will be studied by in situ hybridization. (5) To study S100s expression in different phases of the cell cycle by flow cytometry. In summary, uv-S100 is the only known biochemical marker that distinguishes uveal melanocytes from its embryologically-related counterpart in the skin. These studies are the first steps towards understanding its function. Since S100Beta is an important marker in pathology, our studies may also have implications for diagnosis, particularly in defining the Callender uveal melanoma cell types.
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