Study On Mechanism Of Aneuploid Cells

Aneuploidy is a hallmark of many tumours and human genetic disorders.In cells chromosome separation has been regulated by kinetochore and spindle assemble checkpoint(SAC).In this paper we will describe the underlying mechanism of aneuploid cells by study on mutations of DNA and protein-complex on kinetochore.Kinetochores are large protein assemblies built on chromosomal loci named centromeres,it appears as trilaminar plates[1] and contains CENPs andαsatellite DNA. The primary function of kinetochore is to create load-bearing attachments between chromosomes and microtubules in a dividing mother cell. The correct partitioning of sister chromatids to the daughter cells depends on such attachment.The lose of kinethchore or its components are the leading cause of chromosome separation error.The function of CENPs is highly concerned by scientist.The expression level or structure abnormality can result in kinetochore's assemble,activation,self-replication and occurs as aconsequence of the nondisjunction of homologous or sister chromosomes in mitosis.CENP-B, a highly conserved protein in humans,is specifically localized at the centromere. This protein binds to the 17bp motif named CENP-B box sequence in alphoid DNA,both the CENP-B box and theαsatellite DNA sequence are required for de novo mammalian artificial chromosome(MAC) formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E.Therefore,CENP-B and CENP-B box is required for kinetochore and cell division.The kinetochore-CENPs can be seen by using silver staining technology or indirect immunofluorescence technique[2],the loss of CENPs on kinetochore is the underlying cause for chromosome mis-separation.In this study we will search possible mutations of CENP-B box on Chr21,in order to explore another molecular mechanism of Down syndrome.Otherwise,we will study on kinetochore-CENPs variation of HEP-2 and SW626 human cell lines,searching for underlying mechanism of aneuploidy.These research will lay a foundation for further studying the mechanism of aneuploidy.PART I STUDY ON CENP-B BOX AND ALPHA-I SATELLITE DNA IN DOWN SNYDROMEObjective:Analyze alpha-I satellite DNA and four CENP-B boxes on Chr21,searching for mutations between Down snydrome and normal children.Methods: 150 Down syndrome patients and 100 normal children were enrolled in this study,extract DNA from peripheral blood and amplify two parts of alpha-I satellite DNA(including four CENP-B boxes).Driect sequence analyze PCR production and using DNAssist 2.0 software to align with standard sequence on NCBI database.Results:1. Successfully extract DNA from 150 Down syndrome patients and 100 normal children.2. Successfully amplified two parts of alpha-I satellite DNA with no false priming.3. Complete all samples direct sequencing.Four CENP-B boxes can be seen clearly.4. By contrasting to NCBI database,we find:○1 CENP-B box is highly conserved in Down syndrome patients and normal children;○2These some variations compare with standard alpha-I satellite DNA:delete C in position 661, T→G in position 670,insert A between position 1035 and 1036, insert C between position 1076 and 1077, A→T in position 1239 and T→C in position 1343.○3 We find many non-reported SNPs in alpha-I satellite DNA.Conclusion:1. alpha-I satellite DNA on chromosomal kinetochore is noncoding higher order repeats.The conservation of this structure may because of this repeats.Even if mutate CENP-B box can not bind CENP-B and affect kinetochore-CENPs formation in vitro. We find mutate CENP-B box neither in Down syndrome patients nor in normal children.Therefore we demonstrate that the molecular mechanism of Chr21 mis-separation in Down syndrome patients may not involved to CENP-B box mutation.2. The mutations on alpha-I satellite DNA may because of the difference between races of people.Besides,the contribution of newly find SNPs is yet to be known.PART II STUDY ON KINETOCHORE VARIATION OF HEP-2 AND SW626 CELL LINESObjective:We study on kinetochroe-CENPs variation of HEP-2 and SW626 solid tumour cell lines.To find out the underlying mechanism of chromosome mis-separation in tumour.Methods:In order to probe into the mechanism of aneuploidy aberration of tumour cells,kinetochore-CENPs complex variation on chromosomes of HEP-2 ,SW626 and normal people lympholeukocyte cells were studied by a simultaneous silver staining of both NOR and kinetochore.Results:1. Analyze 308 division cells of HEP-2 cell line including 16962 chromosomes.we find 146 kinetochore loss chromosomes,105 kinetochore-NOR fussion chromosomes,78 kinetochore duplication laggard chromosomes,38 dissymmetry kinetochore chromosomes and 18 multi-kinetochore chromosomes.2. Analyze 334 division cells of SW626 cell line including 12092 chromosomes.we find 325 kinetochore loss chromosomes,243 kinetochore-NOR fussion chromosomes,28 kinetochore duplication laggard chromosomes,15 dissymmetry kinetochore chromosomes and 8 multi-kinetochore chromosomes.3. Analyze 367 division cells of normal people lympholeukocyte cells including 16882 chromosomes.we find 42 kinetochore loss chromosomes,85 kinetochore-NOR fussion chromosomes,29 kinetochore duplication laggard chromosomes,4 dissymmetry kinetochore chromosomes.4. Compared to normal people lympholeukocyte cells,○1 frequencies of kinetochore loss,kinetochore replication laggard, dissymmetry kinetochore chromosomes and multi-kinetochore chromosomes of HEP-2 cells significantly increased (P<0.01);○2 Frequencies of kinetochore loss,kinetochore-NOR fusion, dissymmetry kinetochore chromosomes and multi-kinetochore chromosomes of SW626 cells increased significantly(P<0.01).Conclusion:1. These some kinetochore-CENPs complex variations between HEP-2 and SW626 cells.2. We propose that kinetochore loss,kinetochore replication laggard, and dissymmetry kinetochore chromosomes in HEP-2 cells and kinetochore loss,kinetochore-NOR fusion, and dissymmetry kinetochore chromosomes in SW626 cells may be a underlying cause of CIN.3. Multi-kinetochore is a newly possible reason for induce chromosomal instability.