High throughput functional genomics: Identification of novel genes with tumor suppressor phenotypes

Abstract

We have used a combination of high throughput functional genomics, computerized database mining and expression analyses to discover novel human tumor suppressor genes (TSGs). A genome-wide high throughput cDNA phenotype screen was established to identify genes that induce apoptosis or reduce cell viability. TSGs are expressed in normal tissue and frequently act by reduction of growth of transformed cells or induce apoptosis. In agreement with that and thus serving as platform validation, our pro-apoptotic hits included genes for which tumor suppressing activities were known, such as kangai1 and CD81 antigen. Additional genes that so far have been claimed as putative TSGs or associated with tumor inhibitory activities (prostate differentiation factor, hRAS-like suppressor 3, DPH2L1-like and the metastasis inhibitor Kiss1) were confirmed in their proposed TSG-like phenotype by functionally defining their growth inhibitory or pro-apoptotic function towards cancer cells. Finally, novel genes were identified for which neither association with cell growth nor with apoptosis were previously described. A subset of these genes show characteristics of TSGs because they (i) reduce the growth or induce apoptosis in tumor cells; (ii) show reduced expression in tumor vs. normal tissue; and (iii) are located on chromosomal (LOH-) loci for which cancer-associated deletions are described. The pro-apoptotic phenotype and differential expression of these genes in normal and malignant tissue make them promising target candidates for the diagnosis and therapy of various tumors.

BASP1 Promotes Apoptosis in Diabetic Nephropathy

“…9 In brief, 596,832 independent clones from a human embryo cDNA expression library and 17,680 defined fulllength cDNA expression plasmids were screened for cell death induction after transient transfection into human embryonic kidney (HEK293) cells. Of these, 194 unique cDNAs induced cell death and DNA fragmentation: 138 contained full-length open reading frames (Figure 1).…”

“…9 In brief, a cDNA expression library generated from human embryo tissue (Scinet, Braunschweig, Germany) and a fulllength cDNA expression plasmid collection have been propagated in E. Coli SURE. In a robot cascade, individual colonies were picked and automatically transferred for cDNA plasmid preparation in a 96-well format followed by transient transfection into HEK293 cells.…”

“…Genome-wide functional assays for the identification of novel cell-death-associated molecules have recently been developed. 9 For this study this strategy was adapted to identify apoptosis-inducing full-length reading frames in human embryonic kidney cells as a primary screening step. The in vitro assay was followed by analysis of the regulation of the corresponding mRNA transcripts in human DN.…”

Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBK1 and the IRF3 pathway

“…The screening platform is based on the gain-offunction principle because of transient expression of large numbers of individual cDNAs in mammalian cells. This technique, performed on a robotics platform, was applied to the identification of cDNAs that directly affect the phenotype of the transfected cells (7,8). For the herein-described identification of factors that are associated with angiogenesis, the technology was adapted to identify activities in cell-culture supernatants.…”

“…To circumvent this bottleneck, we ''inverted'' the usual process of target identification by initiating the search for new targets, not with an in silico screen or profiling but, instead, with a highthroughput functional screen. For this screen, we developed a robotics platform that performs transfection of single cDNAs into mammalian cells, followed by phenotype determination (7,8). The phenotype chosen was vascularization, assayed by determination of human umbilical vein endothelial cells (HUVEC) proliferation.…”