Characterization of an antibody that can detect the Kai1/CD82 murine metastasis suppressor.

BACKGROUND: Kai1, also known as CD82, is a member of the tetraspanin family (TM4SF). The human homolog, KAI1, is an activation antigen of T-cells and is a metastasis suppressor for prostate and other cancers. Little is known about the mouse protein because of the lack of antibody reagents. METHODS: Peptide immunized rabbits were used to generate polyclonal antibody to Kai1. The antibody was analyzed using immunoblotting, flow cytometry, and immunohistochemistry. RESULTS: This antibody specifically recognizes murine Kai1 protein, crossreacts with rat Kai1 but not with human KAI1. The normal tissue distribution of this protein in mice is shown to be similar to that of the human homolog. Interestingly, mouse prostatic epithelium showed differential expression within the lobes. CONCLUSION: This antibody, the first described that can specifically detect murine Kai1/CD82, should be very useful in addressing the mechanism of action of Kai1 in metastatic suppression.

Differential cis-regulation of human versus mouse TERT gene expression in vivo: identification of a human-specific repressive element.

In vivo expression of human telomerase is significantly different from that of mouse telomerase. To assess the basis for this difference, a bacterial artificial chromosome clone containing the entire hTERT (human telomerase reverse transcriptase) gene was introduced in mice. In these transgenic mice, expression of the hTERT transgene was similar to that of endogenous hTERT in humans, rather than endogenous mTERT (mouse telomerase reverse transcriptase). In tissues and cells showing a striking difference in expression levels between hTERT in humans and mTERT in mice (i.e., liver, kidney, lung, uterus, and fibroblasts), expression of the hTERT transgene in transgenic mice was repressed, mimicking hTERT in humans. The transcriptional activity of the hTERT promoter was much lower than that of the mTERT promoter in mouse embryonic fibroblasts or human fibroblasts. Mutational analysis of the hTERT and mTERT promoters revealed that a nonconserved GC-box within the hTERT promoter was responsible for the human-specific repression. These results reveal that a difference in cis-regulation of transcription, rather than transacting transcription factors, is critical to species differences in tissue-specific TERT expression. Our data also suggest that the GC-box-mediated, human-specific mechanism for TERT repression is impaired in human cancers. This study represents a detailed characterization of the functional difference in a gene promoter of mice versus humans and provides not only important insight into species-specific regulation of telomerase and telomeres but also an experimental basis for generating mice humanized for telomerase enzyme and its pattern of expression.

Transgenic Ly-49A inhibits antigen-driven T cell activation and delays diabetes.

Activation of islet-specific T cells plays a significant role in the development of type 1 diabetes. In an effort to control T cell activation, we expressed the inhibitory receptor, Ly-49A, on islet-specific mouse CD4 cells. Ag-mediated activation of Ly-49A T cells was inhibited in vitro when the Ly-49A ligand, H-2D(d), was present on APCs. Ag-driven T cell proliferation, cytokine production, and changes in surface receptor expression were significantly reduced. Inhibition was also evident during secondary antigenic challenge. Addition of exogenous IL-2 did not rescue cells from inhibition, suggesting that Ly-49A engagement does not lead to T cell anergy. Importantly, in an adoptive transfer model, Ly-49A significantly delays the onset of diabetes. Together these results demonstrate that the inhibitory receptor Ly-49A effectively limits Ag-specific CD4 cell responses even in the presence of sustained autoantigen expression in vivo.

T-cell receptor transgenic response to an endogenous polymorphic autoantigen determines susceptibility to diabetes.

We have produced a T-cell receptor (TCR) transgenic NOD mouse, 6.9TCR/NOD, in which the expression of both diabetogenic T-cells and naturally occurring autoantigen were simultaneously controlled. The parent T-cell clone, BDC-6.9, and T-cells from 6.9TCR/NOD mice recognize a currently unidentified antigen present in NOD but not in BALB/c islet cells. A gene that codes for the antigen, or a protein that regulates the antigen, was previously mapped to a locus on chromosome 6. We have developed transgenic mice bearing the TCR alpha- and beta-chains from the BDC-6.9 T-cell clone on a NOD congenic background in which the antigen locus on chromosome 6 of the NOD mouse is replaced by a segment from BALB/c. These NOD.C6 congenic mice lack the NOD islet cell antigen to which the BDC-6.9 T-cell clone responds. Diabetes in both male and female 6.9TCR/NOD mice is dramatically accelerated, but in 6.9TCR/NOD.C6 mice lacking the NOD islet cell autoantigen, we have not observed diabetes for up to 1 year of age. Thus, the generation of 6.9TCR transgenic mice provides a model of autoimmune diabetes whereby controlled expression of an endogenous polymorphic autoantigen effectively determines disease development.

Microarray analysis reveals distinct gene expression profiles among different histologic types of endometrial cancer.

Previous studies of oncogene and tumor suppressor gene alterations have suggested that differences exist in the molecular pathogenesis of the various histological types of endometrial cancer. To elucidate further the molecular events involved in endometrial carcinogenesis, we examined global expression patterns of 16 nonendometrioid cancers (13 serous papillary and 3 clear cell), 19 endometrioid cancers, and 7 age-matched normal endometria using cDNA microarrays. Unsupervised analysis of gene expression identified 191 genes that exhibited >2-fold differences (P < 0.001) between the histological groups. Many genes were similarly dysregulated in both nonendometrioid and endometrioid cancers relative to normal endometria. Gene expression differences in only 24 transcripts could distinguish serous from endometrioid cancers, the two most common subgroups. These data provide the basis for investigation of previously unrecognized novel pathways involved in the development of endometrial cancers.

JNK1 and AP-1 regulate PMA-inducible squamous differentiation marker expression in Clara-like H441 cells.

Exposure of distal bronchiolar region to various toxicants and pollutants suppresses Clara cell differentiation marker expression and greatly enhances the induction of squamous cell differentiation (SCD). Here, we demonstrate for the first time phorbol 13-myristate 12-acetate (PMA)-inducible expression of SCD markers, SPRRs, in Clara-like H441 cells. The transcriptional stimulation of human SPRR1B expression is mainly mediated by a -150- to -84-bp region that harbors two critical activator protein (AP)-1 sites. In unstimulated cells, the -150- to -84-bp region is weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA prominently induced the binding of JunB and Fra1. Consistent with this, overexpression of wild-type Jun proteins upregulated the SPRR1B promoter activity. Conversely, a c-jun mutant suppressed both basal and PMA-inducible reporter gene expression. Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly enhanced basal level activity, indicating an opposing role for these proteins in SPRR1B expression in a manner similar to that observed in proximal tracheobronchial epithelial cells (BEAS-2B clone S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun NH(2)-terminal kinase (JNK) 1 mutant significantly reduced the PMA-inducible SPRR1B promoter activity in H441 cells. Thus either temporal expression and/or spatial activation of AP-1 proteins by JNK1 might contribute to the induction of SCD in Clara cells.

Multiple comparisons model-based clustering and ternary pattern tree numerical display of gene response to treatment: procedure and application to the preclinical evaluation of chemopreventive agents.

Microarray technology has greatly aided the identification of genes that are expressed differentially. Statistical analysis of such data by multiple comparisons procedures has been slow to develop, in part, because methods to cluster the results of such comparisons in biologically meaningful ways have not been available. We isolated and analyzed, by Northern blot and GeneChip, replicate liver RNA samples (n = 4/group) from rats fed with control diet or diet containing one of three chemopreventive compounds, selected because their pharmacological activities, including RNA expression response, are relatively well understood. We report on a classification tree, based on the results of nonparametric multiple comparisons, which results in the bipolar hierarchical clustering of genes in relation to their response to treatment. In addition to identifying treatment-responsive genes, application of this procedure to our test study identified the known pharmacological relationships among the treatment groups without supervision. Also, small treatment-specific subsets of genes were identified that may be indicative of additional pharmacophores present in the test compounds.