MAD2ΔC promotes aneuploidy and anchorage independent growth in human prostate epithelial cells

Abstract

Stability and fidelity of cell cycle is cruicial for the maintenance of genome. There are checkpoints in the cell cycle to ensure the precise of each cell division. At the metaphase, mitotic checkpoint detects the improper attachment of chromosomes to the spindle microtubules at the metaphase and generates a “wait” signal to delay the entry of anaphase. Mitotic arrest deficient 2 (MAD2) is one of the key mitotic checkpoint proteins which binds to MAD1 and Cdc20 and inhibits the ubiquitin ligase activity of APC/C. In this study, the role of MAD2 on chromosomal instability and tumourigenesis was investigated. A C-terminal deleted truncated form of MAD2 (MAD2ΔC), which was unable to bind both MAD1 and Cdc20, was stably transfected into an immortalized non-malignant prostate epithelial cell line, Hpr-1 to dominantly inactivate MAD2. The mitotic checkpoint of MAD2ΔC cells was defective and centrosome was amplified in response to a microtubule disrupting drug, nocodazole. However, proliferation rate was not affected by the inactivation of mitotic checkpoint through MAD2ΔC. Spectral karyotyping (SKY) illustrated that MAD2ΔC transfected cells were hyper-tetraploid with additional structural aberrations of chromosome compared to the control. This suggested the importance of MAD2 in mitotic checkpoint and chromosomal instability. Moreover, MAD2ΔC transfected cells induce anchorage-independent growth in soft agar forming assay after challenging with carcinogens DMBA and TPA. Completely loss of p53 response towards DNA-damaging agent, cisplatin and activation of MAPK pathway were found in the MAD2ΔC cells grown in agar. This suggested that inactivation of MAD2 had tumor promoting effect on prostate epithelial cells while additional cellular events were required in the process of MAD2ΔC-induced malignant transformation.