The Relationship Between Cdc20 Mutation And The Progression Of Colon Cancer In Mice

Objects:Demonstrate the relationship between cdc20 mutation and the promotion of colon cancer, furthermore indicate the molecular mechanism.Methods:Apply homologous recombination technology and blastocyst injection, to generate mice carrying cdc20 a null allele caused by the presence of the loxp cassette. Genotyping the mice by PCR,to yield experimental and control mice. Compare the experimental and control mice by phenotypic analysis and histology analysis, to discover the differences and the molecular mechanism. Derive mouse embryonic fibroblasts (MEFs) of cdc20loxp/+; APCmin/+ cdc20loxp/+, APCmin/+ and WT genotype from embryos for growth curve analysis and karyotype analysis. Compare the difference of growth with growth curve analysis.Detect the separation of sister chromatid and the presence of aneuploid by karyotype analysis. Construct the PMXs-E1A-RAS virus vector and transfect it into four genotype MEFs for foci formation assay to detect the ability of cdc20 in cell transformation.The study aim to certify the relationship of cdc20 mutation in the promotion of colon cancer and the molecular mechanism in vitro.Results:Phenotypic analysis show that the number and the maximum diameter of tumor are no difference between cdc20loxp/+APCmin/+compound mutant mice and control mice.Pathological section display colonic tumors from cdc20loxp/+APCmin/+ compound mutant mice had progressed to much higher grade than those from control mice, pathologic type is adenocarcinma and control mice is adenoma. Cell proliferation assays showed that cdc20loxp/+APCmin/+cells grew at an accelerated rate compared with WT MEFs or MEFs with a single gene mutation. Foci formation assay show that the transformation ability of cdc20loxp/+APCmin/+ MEFs transfect with PMXs-E1A-RAS virus vector is much stronger that other MEFs transfect with PMXs-E1A-RAS virus vector. Cdc20loxp/+APCmin/+ MEFs showed a significant increase in the frequency of aneuploid metaphase compared with WT MEFs, which have a karyotype of 38. Our further analysis revealed that cdc20loxp/+APCmin/+ MEFs contained prematurely separated sister chromatids.Conclusions:(1) Cdc20 carrying a null allele (cdc20loxp/+) can accelerate the promotion of colon cancer in mice. (2) The cdc20loxp/+APCmin/+ compound mutant in MEFs cells is sufficient for transformation, cdc20 is an oncogene. (3) The cdc20loxp/+APCmin/+ MEFs contained prematurely separated sister chromatids and a significant increase in the frequency of aneuploid metaphase. (4)The cdc20 influences the promotion of colon cancer in mice through chromosome instability.