Publications by Year: 1997

TSG101 has been identified as a candidate tumor suppressor gene and abnormal transcripts have been identified in a substantial fraction of breast cancers. To determine whether TSG101 expression is commonly altered in other tumors, a series of 15 primary and metastatic prostate cancers were analysed by reverse transcriptase-PCR amplification. Abnormal transcripts with extensive deletions in the coding region were found in nine of these tumors, while only the normal transcript was found in control and benign prostatic hypertrophy tissues. More than one abnormal transcript was found in four of these nine cases and distinct abnormal TSG101 transcripts were found in separate biopsies taken from one tumor. Importantly, the normal TSG101 transcript was undetectable in two metastatic prostate cancers, indicating the absence of TSG101 protein. Sequence analysis demonstrated that there were at least six distinct deletions, with four of these deletions found in more than one tumor sample. The most commonly identified deletion, from bp 153 to 1055, was identical to a deletion reported previously in breast cancer. These results demonstrate that TSG101 transcripts are frequently abnormal in prostate cancer and suggest that loss of TSG101 protein contributes to disease development or progression.

An increasing number of proteins which bind to hormone-dependent nuclear receptors and mediate their effects on gene expression are being identified. The human prostate-specific antigen (PSA) and kallikrein 2 (KLK2) genes are regulated by the androgen receptor (AR). Using electrophoresis mobility shift assays (EMSA), a common nuclear protein(s) which binds upstream of the androgen-responsive elements (AREs) in the PSA and KLK2 promoters was identified. Binding occurred between bp -539 and -399 and bp -349 and -224 in the PSA and KLK2 promoters respectively, which were shown previously to be necessary for AR-mediated transactivation. Glutathione S-transferase (GST)-AR fusion proteins were constructed to determine whether the AR interacted directly with this protein or protein complex. Specific interactions were observed with AR fusion proteins containing the DNA binding domain. EMSA supershift experiments and GST-AR pull-down experiments followed by Western blotting identified a Fos-related protein(s) of approximately 40 kDa as part of this complex. Competition experiments with a double-stranded oligonucleotide containing an AP-1 binding site demonstrated that DNA binding was not mediated by AP-1. These results indicate that a Fos-containing protein complex distinct from AP-1 binds upstream of the AREs in the PSA and KLK2 promoters, interacts with the AR and may participate in regulation of these two androgen-responsive genes.

TCR expression by human fetal intestinal intraepithelial lymphocytes (ilELs) and intestinal lamina propria lymphocytes was analyzed to address whether T cell development occurs in human fetal intestine, the diversity of human fetal iIELs, and whether human fetal iIELs may contribute to the adult iIEL repertoire. ilELs and intestinal lamina propria lymphocytes from second trimester human fetal intestine were analyzed for TCR-alphabeta transcripts. Rearranged TCR-alpha transcripts were undetectable at 14 wk in the intraepithelial lymphocytes (IELs), whereas multiple TCR-beta transcripts were found at this stage. The TCR-alpha repertoire remained restricted relative to TCR-beta at later stages, and the IEL repertoire was restricted relative to the lamina propria lymphocytes at all stages. A previously reported T early alpha message was the major transcript from the TCR-alpha locus early in gestation. A previously undescribed TCR-beta transcript initiating upstream of the Dbeta1 locus and spliced to Cbeta1 or Cbeta2 was also identified and may represent a T early beta message. These results provide evidence for ongoing TCR gene rearrangement in human fetal intestine and suggest that transcription from the TCR-beta locus initiates with a T early beta transcript. The TCR-alpha repertoire (and hence the repertoire of potentially functional IELs) was limited through the second trimester.

Mutations in the androgen receptor (AR), that alter steroid hormone specificity have been identified in a series of androgen-independent prostate cancers. To address the functional properties of these mutant ARs that may have contributed to their selection in vivo, responses to a series of steroid hormones and antiandrogens were assessed. CV-1 cells were cotransfected with wild-type or mutant ARs and a luciferase reporter plasmid regulated by an androgen-responsive element. Dose-response curves were analyzed for 5alpha-dihydrotestosterone, the most active androgen in normal prostate, and androstenedione, a major androgen derived from the adrenals. Although the mutant ARs responded to both of these steroids, the responses were equivalent to or less than the wild-type AR. In contrast, responses to flutamide, a competitive antagonist of the wild-type AR, were markedly increased by three of the mutations. Similar responses were observed with a second antiandrogen, nilutamide. Bicalutamide, another antiandrogen related to flutamide, remained an antagonist for these mutant ARs. Finally, flutamide was observed to be a weak partial agonist of the wild-type AR in this system. These results indicate that flutamide used in conjunction with androgen ablation therapy for prostate cancer may select for tumor cells with flutamide-inducible ARs.

Human small intestine contains a very large population of intraepithelial T lymphocytes (IELs) that are oligoclonal, appear functionally to be cytolytic T cells, and may contribute to the normal and pathological turnover of intestinal epithelial cells. This report addresses the cytolytic function of IELs in normal small intestine by examining their expression of molecules that carry out cell-mediated cytolysis. Immunohistochemical analyses of granzyme B, perforin, Fas ligand, and tumor necrosis factor-alpha demonstrated these proteins were not expressed by small intestinal IELs in situ. These proteins also were not expressed by colonic IELs or by lamina propria lymphocytes in the small or large intestine. Granzyme A, however, was expressed by a large fraction of IELs. In contrast to these in situ results, isolated and in vitro activated IELs were shown to express effector proteins consistent with cytolytic T cells, including granzyme B, Fas ligand, tumor necrosis factor-alpha, and interferon-gamma. These results are most consistent with the vast majority of IELs in normal human small intestine being resting cytolytic T cells and suggest that these cells do not contribute to the apoptotic cell death of epithelial cells in normal intestine.