Mutation analysis of the checkpoint kinase 2 gene in colorectal cancer cell lines

Background Checkpoint kinase 2 （CHK2） is a DNA damage-activated protein kinase which is involved in cell cycle checkpoint control, CHK2 gene could be a candidate gene for colorectal cancer susceptibility, But there are few systematic reports on mutation of CHK2 in colorectal cancer. Methods The mutations of all 14 exons of CHK2 in 56 colorectal cancer cell lines were screened systematically, using denaturing high-performance liquid chromatography （DHPLC） to screen the mismatches of the CHK2 exons amplified products, and then the suspected mutant cell lines were scanned by nucleotide sequence analysis. Results VACO400 in CHK2 exon la was suspected to have mutation by DHPLC and confirmed by sequence, but this was nonsense mutation. C106, CX-1, HT-29, SK01, SW480, SW620 and VACO400 in CHK2 exon lb were confirmed to have the same nonsense mutation in 11609 A〉G. DLD-1 and HCT-15 in CHK2 exon 2 were confirmed to have missense mutation R145W, which was heterozygous C〉T missense mutation at nucleotide 433, leading to an Arg〉Trp substitution within the FHA domain. Conclusions The CHK2 mutation in colorectal cancer is a low frequency event, There are just 10 cell lines to have sequence variations in all the 14 exons in 56 colorectal cancer cell lines and only DLD-1/HCT-15 had heterozygous missense mutation. These findings may give useful information of susceptibility of colorectal cancer as single nucleotide polvmorphvsim.

Human umbilical cord mesenchymal stem cells attenuate diabetic nephropathy through the IGF1R-CHK2-p53 signalling axis in male rats with type 2 diabetes mellitus

diabetes mellitus(DM)is a disease syndrome characterized by chronic hyperglycaemia.A long-term high-glucose environment leads to reactive oxygen species(ROS)production and nuclear DNA damage.human umbilical cord mesenchymal stem cell(HUcMSC)infusion induces significant antidiabetic effects in type 2 diabetes mellitus(T2DM)rats.Insulin-like growth factor 1(IGF1)receptor(IGF1R)is important in promoting glucose metabolism in diabetes;however,the mechanism by which HUcMSC can treat diabetes through IGF1R and DNA damage repair remains unclear.In this study,a DM rat model was induced with high-fat diet feeding and streptozotocin(STZ)administration and rats were infused four times with HUcMSC.Blood glucose,interleukin-6(IL-6),IL-10,glomerular basement membrane,and renal function were examined.Proteins that interacted with IGF1R were determined through coimmunoprecipitation assays.The expression of IGF1R,phosphorylated checkpoint kinase 2(p-CHK2),and phosphorylated protein 53(p-p53)was examined using immunohistochemistry(IHC)and western blot analysis.Enzyme-linked immunosorbent assay(ELISA)was used to determine the serum levels of 8-hydroxydeoxyguanosine(8-OHdG).Flow cytometry experiments were used to detect the surface markers of HUcMSC.The identification of the morphology and phenotype of HUcMSC was performed by way of oil red“O”staining and Alizarin red staining.DM rats exhibited abnormal blood glucose and IL-6/10 levels and renal function changes in the glomerular basement membrane,increased the expression of IGF1 and IGF1R.IGF1R interacted with CHK2,and the expression of p-CHK2 was significantly decreased in IGF1R-knockdown cells.When cisplatin was used to induce DNA damage,the expression of p-CHK2 was higher than that in the IGF1R-knockdown group without cisplatin treatment.HUcMSC infusion ameliorated abnormalities and preserved kidney structure and function in DM rats.The expression of IGF1,IGF1R,p-CHK2,and p-p53,and the level of 8-OHdG in the DM group increased significantly compared with those in the control group,and decreased after HUcMSC treatment.Our results suggested that IGF1R could interact with CHK2 and mediate DNA damage.HUcMSC infusion protected against kidney injury in DM rats.The underlying mechanisms may include HUcMSC-mediated enhancement of diabetes treatment via the IGF1R-CHK2-p53 signalling pathway.