ABSTRACT
PP5715 is a putative tumor suppressor gene that encodes a protein of 199 amino acids. Expression of PP5715 in E. coli was mainly as inclusion bodies. The recombinant protein was purified by a NTA-Ni2+ affinity column, refolded by dialysis, and shown to suppress growth of two human hepatocellular carcinoma cell lines using the MTT assay or the clonogenic assays. Computer analysis and fusion expression of PP5175 and GFP showed that PP5715 may be a secretory protein. It may bind to receptors on the membrane surface and thus induce regulation of cell growth. These preliminary results suggest that protein PP5715 may be a new tumor suppressor with growth inhibition effects on hepatocellular carcinoma cells.
Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Division/genetics , Escherichia coli/genetics , Genes, Tumor Suppressor , Liver Neoplasms/pathology , Base Sequence , Blotting, Western , Cell Line, Tumor , DNA Primers , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Subcellular Fractions/metabolismABSTRACT
<p><b>OBJECTIVE</b>CT120B gene is a splicing variant of CT120A, which deletes 96 nucleotides and leads to an in-frame loss of 32 amino acids between the codon 136 and 167 as compared with CT120A. This study was undertaken to assess the effects of CT120B expression on lung cancer cell growth and to explore the gene expression profiles.</p><p><b>METHODS</b>CT120B cDNA was transfected into the human lung adenocarcinoma SPC-A-1 cells, and stable cell lines overexpressing CT120B were established. CCK-8 assay and tumorigenecity in a xenograft model were performed to analyze cell proliferation in vitro and in vivo. The differential gene expression induced by overexpressed CT120B was investigated using Atlas cDNA expression array. Flow cytometry was performed to analyze cell cycle and cell apoptosis.</p><p><b>RESULTS</b>Overexpression of CT120B in SPC-A-1 cells resulted in a reduced cell growth rate in vitro, and decrease of the tumorigenicity in nude mice. A total of 38 genes were identified as differential expressions with more than a 2.0-fold change by Atlas cDNA expression array analysis, including downregulated cyclin E1, cdk 2, c-kit, CXCR4 and upregulated caspase 8 gene. Overexpression of CT120B also induced G1 phase arrest, but had no effect on cell apoptosis.</p><p><b>CONCLUSION</b>The G1 cell cycle arrest, but not apoptosis, underlay the growth inhibitory activities of CT120B. The down-regulation of c-kit and CXCR4 expression might also contribute to the suppressive effects on cell growth of CT120B.</p>
Subject(s)
Animals , Humans , Male , Mice , Adenocarcinoma , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , DNA, Complementary , Genetics , G1 Phase , Gene Expression Profiling , Lung Neoplasms , Metabolism , Pathology , Membrane Proteins , Genetics , Metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Proteins , Genetics , Metabolism , Neoplasm Transplantation , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-kit , Metabolism , Receptors, CXCR4 , Metabolism , TransfectionABSTRACT
HCAP1 is a novel hepatic cancer related gene located on human chromosome 17p13.3. The loss of heterozygosity occurred at 17p13.3 in various human cancers. In order to investigate the effects of exogenous HCAP1 gene products on cell proliferation of T lymphoma Jurkat cell line, HCAP1 gene! was transfected into Jurkat cells mediated by liposome, and the cells stably expressing exogenous HCAP1 were screened with G418. The effects of HCAP1 products on cell proliferation were assessed by viable cell count, cell growth curve and colony formation assay in soft agar. The results showed that the HCAP1 transgenic Jurkat cells displayed slow growth rate, extended doubling time and reduced colony formation capability, as compared with the cells transfected with pBK/CMV empty vector (P < 0.01). It is concluded that exogenous HCAP1 gene products could inhibit the proliferation of Jurkat cells.
Subject(s)
Humans , Carcinoma, Hepatocellular , Genetics , Cell Division , Jurkat Cells , Liver Neoplasms , Genetics , Neoplasm Proteins , Genetics , Peptides , TransfectionABSTRACT
In order to investigate expression of novel genes HC56, HC70 and HC90 mapped on chromosome 17p13.3 in human leukemic cells, HC56, HC71 and HC90 genes expression was examined in cells from 35 patients with acute leukemias and 4 leukemic cell lines by using semi-quantitative RT-PCR with incorporation of alpha(32)P dATP, betaM2 gene as endogenous control. The results showed that HC90 gene expression level was lower in patients with acute lymphocytic leukemia and T lymphocytic leukemic cell line Jurkat than that in normal control. Low-level HC71 gene expression was detected in acute myeloid leukemia. There was no expression difference in HC56 between leukemic cells and normal blood cells. It was concluded that both HC70 and HC90 genes were aberrantly expressed in human leukemic cells.
