PCR Array Technology in Biopsy Samples Identifies Up-Regulated mTOR Pathway Genes as Potential Rejection Biomarkers After Kidney Transplantation.

Background: Antibody-mediated rejection (AMR) is the major cause of kidney transplant rejection. The donor-specific human leukocyte antigen (HLA) antibody (DSA) response to a renal allograft is not fully understood yet. mTOR complex has been described in the accommodation or rejection of transplants and integrates responses from a wide variety of signals. The aim of this study was to analyze the expression of the mTOR pathway genes in a large cohort of kidney transplant patients to determine its possible influence on the transplant outcome. Methods: A total of 269 kidney transplant patients monitored for DSA were studied. The patients were divided into two groups, one with recipients that had transplant rejection (+DSA/+AMR) and a second group of recipients without rejection (+DSA/-AMR and -DSA/-AMR, controls). Total RNA was extracted from kidney biopsies and reverse transcribed to cDNA. Human mTOR-PCR array technology was used to determine the expression of 84 mTOR pathway genes. STRING and REVIGO software were used to simulate gene to gene interaction and to assign a molecular function. Results: The studied groups showed a different expression of the mTOR pathway related genes. Recipients that had transplant rejection showed an over-expressed transcript (≥5-fold) of AKT1S1, DDIT4, EIF4E, HRAS, IGF1, INS, IRS1, PIK3CD, PIK3CG, PRKAG3, PRKCB (>12-fold), PRKCG, RPS6KA2, TELO2, ULK1, and VEGFC, compared with patients that did not have rejection. AKT1S1 transcripts were more expressed in +DSA/-AMR biopsies compared with +DSA/+AMR. The main molecular functions of up-regulated gene products were phosphotransferase activity, insulin-like grown factor receptor and ribonucleoside phosphate binding. The group of patients with transplant rejection also showed an under-expressed transcript (≥5-fold) of VEGFA (>15-fold), RPS6, and RHOA compared with the group without rejection. The molecular function of down-regulated gene products such as protein kinase activity and carbohydrate derivative binding proteins was also analyzed. Conclusions: We have found a higher number of over-expressed mTOR pathway genes than under-expressed ones in biopsies from rejected kidney transplants (+DSA/+AMR) with respect to controls. In addition to this, the molecular function of both types of transcripts (over/under expressed) is different. Therefore, further studies are needed to determine if variations in gene expression profiles can act as predictors of graft loss, and a better understanding of the mechanisms of action of the involved proteins would be necessary.

Human and mouse DOCK10 splicing isoforms with alternative first coding exon usage are differentially expressed in T and B lymphocytes.

DOCK10 is a member of the dedicator of cytokinesis (DOCK) family of Rho GTPase activators preferentially expressed in lymphocytes. In this paper, we analyzed DOCK10 mRNA diversity produced because of alternative splicing. Alternative first coding exon usage led to 2 main protein-coding transcripts, DOCK10.1 and DOCK10.2. Full-length cDNA clones of both isoforms were obtained from both normal human peripheral blood mononuclear cells and mouse spleen for the first time for human DOCK10.1, mouse DOCK10.1, and mouse DOCK10.2. Human and mouse DOCK10.1 clones corresponded to the protein coding assemblies provided by the National Center for Biotechnology Information as Reference Sequences for DOCK10. Our analysis especially focused on human cDNA clones, of which 63% were alternatively spliced forms involving diverse exons and introns. DOCK10.1 expression was enriched in normal T cells, and DOCK10.2 expression was enriched in normal B cells and chronic lymphocytic leukemia (CLL) B cells. Both isoforms were upregulated in response to interleukin-4 in B cells, both normal and CLL, but not in T cells. Our data suggest that cell-specific mechanisms regulate expression of the alternative first exon variants of DOCK10 in vertebrates.

Dock10, a novel CZH protein selectively induced by interleukin-4 in human B lymphocytes.

The Dock or CZH proteins are a family of activators for Rho GTPase proteins. The Zizimin subfamily is composed of three members: Dock9, Dock10, and Dock11. We have identified DOCK10 as an interleukin-4 (IL4)-inducible gene in chronic lymphocytic leukemias (CLLs). Subsequently, we have obtained the full-length cDNA sequence, which encodes a 2180 amino acid protein. Dock9 (2069 amino acids) and Dock11 (2073 amino acids) share more identity between them (58%) than with Dock10 (52% and 50%, respectively). Among normal human tissues, DOCK10 and DOCK11 mRNAs were mainly expressed in peripheral blood (PB) leukocytes. Dock10 protein was expressed at similar levels in normal PB-B and PB-T cells. Dock10 protein levels were heterogeneous in CLLs. IL4 consistently increased Dock10 mRNA and protein levels in CLL and normal PB-B cells. In contrast, IL4 did not affect the levels of Dock10 expression in normal PB-T cells. IL4 neither increased DOCK9 or DOCK11 mRNA levels in CLL cells. Dock10 protein distributed in the cytoplasm and nucleus of CLL cells, and IL4 increased its expression in both cellular compartments. The rapid and distinctive induction of Dock10 expression by IL4 in CLL and normal PB-B cells suggests a role for Dock10 in IL4-induced B-cell activation. Dock10 could represent a point of convergence for IL4 signalling and small Rho GTPase function in B cells.

Identification of apoptosis-related PLZF target genes.

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localization is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.