Mitotic spindle checkpoint & chromosomal instability
Abstracts
Initial Award Abstract |
|
Cancer is a diseased state which develops from abnormalities in the proteins that control cell birth and cell death. However, the exact mechanisms through which these abnormalities are generated are still debatable. One of the mechanisms that is thought to drive the development of cancer is genetic instability. In a majority of colon cancers, and most other tumor types, genetic instability is manifest at the chromosomal level and is characterized by an abnormal number of chromosomes in the cells. Studies have shown that the majority of cancers have gained or lost chromosomes. The average cancer of the colon, breast, pancreas or prostate may lose as many as 25% of its chromosomes. The sources of these errors probably drive more and more abnormal cell growth. Normally cells try to ensure that each daughter cell receives one copy of each chromosome. The way this happens is through a quality control mechanism called the mitotic spindle checkpoint. This checkpoint allows every chromosome to send a stop signal, arresting cell growth till all the chromosomes are appropriately distributed. Defects in different components of the mitotic spindle checkpoint have consistently been observed in cancer cells. These cancers are characterized by chromosomal instability. For example, frequent impairment of mitotic spindle checkpoint has been observed in human lung cancer cell lines, and in a small fraction of colorectal cancer cells, abnormal chromosomal numbers have been proposed to be associated with defects in two of the human mitotic spindle checkpoint genes, namely BUB1 and BUBR1. However, the exact function of BUB1 and BUBR1 are not understood, although it is thought that both BUB1 and BUBR1 are part of a signalling cascade that relays a signal to inhibit further progression of cell cycle if defects in chromosome distribution are detected. The objective of the proposed study is to investigate this aspect in detail and thus determine whether BUB1 and BUBR1 may function as tumor suppressors. To acheive that objective, I aim to selectively delete these genes in mice in order to examine whether these genes are essential for normal development and whether their disruption leads to genetic instability, thereby promoting the progression to tumorigenesis. Further, the proposed study will also serve as a starting point for the experiments designed to test the prediction that mutations (genetic defects) in mitotic checkpoint genes will frequently be found in human tumors arising from mutations in other cancer related genes. These studies will have implications for the pathogenesis of cancer and may in the long run yield entirely new approaches to treating common forms of cancer. |
Final Report |
|
Cancer is a diseased state which develops from abnormalities in the proteins that control cell birth and cell death. One of the mechanisms that is thought to drive the development of cancer is genetic instability. In a majority of colon cancers, and most other tumor types, genetic instability is manifest at the chromosomal level and is characterized by an abnormal number of chromosomes in the cells (aneuploidy). Although the physiological or molecular basis of this abnormality is unknown, it has been shown that chromosomal instability is associated with the loss of function of the mitotic checkpoint. The mitotic spindle checkpoint helps prevent aneuploidy by ensuring that each daughter cell receives one of the two sister chromatids from each chromosome. Frequent impairment of mitotic spindle checkpoint has been observed in human lung cancer cell lines, and in a small fraction of colorectal cancer cell lines, aneuploidy has been proposed to be associated with human mitotic checkpoint genes BUB1 and BUBR1. However, the exact function of BUB1 and BUBR1 and the mechanistic details of their interaction with other mitotic checkpoint components are still not understood. The objectives of the proposed study were two-fold. Firstly, using systemic (classical knock-out) and selectively deletable (conditional knockout) disruption of these genes in mice my aim was to examine whether these genes are essential for normal mouse development. The initial purpose here was to determine if systemic deletion of these genes causes embryonic lethality in mice and if so to pinpoint the stage of development at which this defect is caused.Two possibilities arise: (i) If their deletion does not lead to embryonic lethality, the strategy would be to analyze these mice for an extended period of time to determine if their disruption leads to genetic instability in such mice and if it promotes the progression to tumorigenesis. (ii) If the systemic disruption of these genes leads to embryonic lethality, fibroblasts derived from the embryos of mice with selectively deletable Bub1 or BubR1 would be cultured and used to further analyze chromosomal segregation in the absence of BUB1 and BUBR1 genes.As a first step towards fulfilling the above objectives, mouse genomic library was screened to determine the genomic locus of BUB1 and BUBR1 genes. After ascertaining the intron-exon boundaries, an appropriate targeting vector was constructed for transfection into embryonic stem (ES) cells. Transfection into ES cells with BUB1 targeting vector generated corresponding ES cell clones, which subsequently were injected into blastocysts for producing mice with systemic deletion of BUB1. The mouse chimeras thus produced were back-crossed to B6 mice to acertain germ-line transmission of the targeted ES cells. The F1 agouti progeny thus obtained is being currently analyzed by tail-DNA PCRs to isolate the mice with targeted Bub1 allele. Once a sufficient number of such mice with targeted Bub1 allele is obtained, they will be inter-crossed to get the homozygotes.In order to further understand the function of human Bub1 and BubR1 proteins, these proteins were also expressed in insect cells and their interaction with other components of the mitotic spindle checkpoint namely hCdc20, hMad1, hMad2 and hCENP-E (also expressed in insect cells) was analyzed. It was seen that BubR1 can interact in vitro with hCENP-E and the latter can induce the kinase activity of BubR1. These studies will be helpful in understanding the functioning of the mitotic spindle checkpoint. |
