): The current proposal encompasses 5 years and focuses on identifying new genes, or altered levels of expression of known genes, and possible mechanism(s) of lynphomagenesis in individuals with AIDS. Approximately 5 to 10% of AIDS patients develop non-Hodgkin's lymphoma (NHL), a large number of which are classified by multiple criteria as high grade B-cell immunoblastic lymphoma (BIBL). This investigation utilizes guidance provided by three established researchers, each of whom provides a unique expertise and set of reagents. Early stages utilize Suppression Subtractive Hybridization (SSH) and DNA macro- and microarray gene fragment screening of a BIBL subset of 15 genetically defined AIDS-NHL tissue samples. This BIBL subset lacks EBV and K S HV/HHV-8 infections and also lacks generic lesions or dysregulated expression of any of the proto-oncogenes or tumor suppressor genes frequently found in AIDS-NHL. Dr. C. Denny, whose laboratory routinely employs Representational Difference Analysis (RDA), will guide SSH cDNA-isolation from the BIBL subset and Dr. S. Nelson will provide support for DNA microarray screening technology should it become necessary. Gene products differentially expressed in AIDS-BIBL versus hyperplastic lymph node material will be isolated and used to establish an index set of gene fragments against which other lymphoma and non-lymphomatous samples will be compared. Commonly dysregulated genes will be evaluated by multiple criteria and selected candidates will be checked for cytokine inducibility, cloned, sequenced, and analyzed for relatedness to known sequences. Once obtained, novel and known candidate cDNAs will be moved into CMV-based and inducible expression vector systems containing FLAG epitope tags under the guidance of Dr. R. Wall. These t a g ged cDNA candidate clones will be stably transformed into three immortalized premalignant murine B-cell lines, established previously in the laboratory of Dr. J. Braun. Transformants created with novel cDNAs will be assayed for resistance to NK cytolysis and MHC class I expression, while previously defined cDNAs will be similarly assayed and studied further based o n a v ailable reagents and current knowledge of their function(s). Transformants exhibiting NK cell resistance and elevated MHC class I expression will be evaluated for in vivo tumor formation in Balb/c-scid and syngeneic (BWF1 or AJ9) mice. Tumorigenicity in immune competent and deficient hosts will be assessed by BIBL-FLAG immunohistochemistry, routine h i stology, mortality curves, and organ morphology/weights. Genes with significant involvement in the etiology and/or progression of lymphomagenesis, or those with reproducible in vitro biochemical effects, will be further characterized by molecular methods.
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