Identification Of Leukemia Cell Apoptosis-related Gene And Study Of Its Effects

Apoptosis (apoptosis), also known as programmed cell death (PCD), is the active dying process of cells, has an important role in regulating organism development, controling cell growth, maintaining a stable internal environment. Apoptosis is preferred by the medical workers for causing no inflammation and has become a cancer etiology, pathology and oncology therapeutics research focus.At present, most cancer therapy drugs play roles by inducing tumor cells apoptosis, therefore, scholars in cancer gene therapy research hope to find more effective tumor cell apoptosis gene and provide new target for cancer therapy.Activin A belongs to transforming growth factor-β(TGF-β) superfamily member, is a multifunctional growth and differentiation factor, plays an important role in the early embryo formation, neural, hematopoietic cell proliferation and differentiation and other aspects.Previous research also showed that activin A can induce hybridoma B-cell apoptosis, but the mechanism is still unclear. In order to find a new apoptotic genes, we have adopted that activin A stimulated mouse B-cell hybridoma, genes high expression was cloned into pcDNA3 eukaryotic expression vector and was transfected into cultured mouse B-cell hybridization tumors. We found it induced apoptosis in B-cell hybridoma, further research was done to validate its effect of inducing tumor cell apoptosis using a variety of tumor cells. Our study revealed the gene induce human and mouse leukemia cell apoptosis but can not induce cells apoptosis which derive from other organizations,such as L929, Neuro-2a et al. So the gene was named as Leukemia Cell Apoptosis-related Gene, LCARG. Specific research is as follows. Part one:Activin induced LCARG expressionBy RT-PCR, LCARG expression was detected in 2B12 cells, and it was confirmed that activin A induced LCARG expression, which peaked at 12h.Part two:The relationship of LCARG and Activin induced 2B12 apoptosisFurther detection showed that LCARG could enhance activin A to inhibit the proliferation of hybridoma cells, antibody secretion, promote Activin induce apoptosis, on the other hand.Part three:LCARG induced 2B12 cell apoptosis Over expression and SiRNA were used to investigate LCARG induce hybridoma 2B12 apoptosis, including prolifration assay, ELISA assay detected cells antibody-secreting function, morphology observation via Gimsa or Hochest-33258 staining, electron microscope, DNA Ladder,flow cytometry detect apoptosis and cell cycle of 2B12 cell after PI and Annexine V staining, Fluorospectrophotometry detected the activation of Caspase3 in 2B12 cells.The results indicated that LCARG could inhibit the proliferation of hybridoma 2B12 cells, decrease antibody-secreting, show the typical phenomenon of apoptosis such as cells diopter disappear, smaller in size, nuclear membrane shrinkage, chromatin condensation and margination, nucleosome formation; cause early Annexine V membrane eversion, cell cycle arrest and sub-diploid peak obtained, induce Caspase3 activation, cause late DNA breakage and nuclear fragmentation. LCARG-SiRNA inhibited ActivinA induce 2B12 apoptosis.Part four:Further investigation about the pathway of LCARG induce 2B12 apoptosisBax and Bcl-2 are known as the main regulation factors in mitochondrial apoptosis pathway,it is well documented that Bax translocates from cytosol to the outer membrane of mitochondria and regulates mitochondrial permeability, allowing the passage of cytochrome C through the membrane,induced cell apoptosis. Caspase-12 is known as a critical signal factor which works in ERS and Caspase8 is known as a critical signal factor which works in death receptor pathway.Weasten-blotting tests found that LCARG induce an up-regulation of Bax and caused a disruption of the mitochondrial membrane potential as well as an increase of Cytochrome-c content in the cytosol, but have no distinguished influence on Bcl-2 expression. RT-PCR revealed Caspase-12 level in 2B12cells increased after transfed with LCARG 24 houres but Caspase8 mRNA expression level had no significant changes. Above studies have shown that LARG-induced apoptosis in tumor cells mainly related to mitochondrial pathway as well as the means of the endoplasmic reticulum, but may not be related with the death receptor pathway.Part five:The effect of LCARG on other tumor cells such as Jurkat,Yac-1 et alIn order to further verify that LCARG can induce apoptosis in tumor cells, we selected human cell lines Jurkat and Raji cell lines as well as mouse cell lines Yac-1, EL-4, RAW264.7, Hep1-6, Neuro-2a, CHO, and L929 cell lines. The proliferation results shew that LARG inhibited growth of Jurkat, Raji, Yac-1, EL-4, as well as RAW264.7 cell in different degrees; morphological detection of Jurkat and Yac-1 shew that cells diopter disappeared, smaller in size, nuclear membrane shrinkage, chromatin condensation and margination, wrapped into a nucleosome. Flow cytometry detected significant apoptotic sub-diploid peak in Jurkat, Raji, Yac-1, EL-4 and RAW264.7 cell lines, increase Bax protein expression in Yac-1 cells. Further tests on the Yac-1 and RAW 264.7 shew LCARG induce Caspase3 activation, induce DNA break and a typical DNA-Ladder map formated; but can not change the expression of Caspase8 levels. Proliferation test and Flow cytometry shew that LCARG has no effect on Neuro-2a, L929, CHO. These results suggest that the gene can specificly induce leukemia-like tumor cell apoptosis.Part six:The effect of other signaling protein in Activin signal transduction pathway on 2B12 Smad 3 is an enhanced signaling protein and Smad 6 is an inhibited signaling protein in activin signal transduction pathway. We tested whether they could induce 2B12 apoptosis through a series of experiments, cell proliferation asaay and flow cytometry shew Smad3, and Smad6 can not induce apoptosis in 2B12 cells.In summary,This study found a new tumor cell apoptosis gene LCARG, and determined the gene can induce leukemia tumor cell apoptosis, induced apoptosis mainly through mitochondrial pathway and endoplasmic reticulum pathway but may not be related with the death receptor pathway, revealed LCARG is the key signaling molecule that mediated activin A induced apoptosis of tumor cells, described LCARG anti tumor effect in vivo, laid a research base for the development of new tumor apoptosis induced medicine in anti-cancer gene therapy.