Growth Retardation, Polyploidy, and Multinucleation Induced by Clast3, a Novel Cell Cycle-regulated Protein

Rumana Bahar,Jiyang O-Wang,Kiyoko Kawamura,Mika Seimiya,Yanqing Wang,Masahiko Hatano,Seiji Okada,Takeshi Tokuhisa,Takeshi Watanabe,Masatoshi Tagawa

doi:10.1074/jbc.m205345200

Abstract

We have identified a novel gene, Clast3, by subtraction of cDNAs derived from activated and naive B lymphocytes. Clast3 expression is elevated in cycling cells and down-regulated in cells undergoing growth arrest, indicating that its expression is controlled in a cell cycle-dependent manner. The deduced amino acid sequence of Clast3 cDNA exhibits no significant homology to the known proteins in mammalian and other species. Immunofluorescence staining revealed that Clast3 localizes into discrete nuclear foci. Forced expression of Clast3 results in growth retardation, polyploidy, and generation of multinucleated cells. Treatment of Clast3 transfectants with nocodazole, a spindle-damaging agent, greatly enhances the incidence of the multinucleated cells, suggesting that Clast3 overexpression impairs the same checkpoint activated by nocodazole. Down-regulation of Clast3 expression by antisense oligonucleotides results in a decrease of cells at G(2)-M phase and a concomitant increase of apoptotic cells. These findings indicate that Clast3 is a novel cell cycle-regulated protein and that its constitutive overexpression induces polyploidy and multinucleation by interfering with the mitotic spindle checkpoint.

Highlights

We have identified a novel gene, Clast3, by subtraction of cDNAs derived from activated and naive B lymphocytes
We show that Clast3 expression is controlled in a cell cycle-dependent manner and that its overexpression interferes with mitotic spindle checkpoint, leading to polyploidy and multinucleation
Down-regulation of Clast3 Expression Results in a Decrease of Cells at G2-M Phase and an Increase of Sub-G1 Cells—The elevated Clast3 expression in cycling cells implicated a potential role for Clast3 in cell division, we examined whether Clast3 is normally essential for cell cycle progression

Summary

Introduction

We have identified a novel gene, Clast, by subtraction of cDNAs derived from activated and naive B lymphocytes. Down-regulation of Clast expression by antisense oligonucleotides results in a decrease of cells at G2-M phase and a concomitant increase of apoptotic cells These findings indicate that Clast is a novel cell cycle-regulated protein and that its constitutive overexpression induces polyploidy and multinucleation by interfering with the mitotic spindle checkpoint. A number of proteins have been identified that sense the kinetochore microtube attachment and regulate the separation of sister chromatids. These include the kinetochore-associated CENPE, MAD, and BUB proteins, the anaphase-promoting complex/cyclosome and its associated cofac-. Accumulating evidence indicates that dysregulated expression as well as functional inactivation of these checkpoint proteins can cause abnormal mitosis, leading to chromosomal mis-segregation, apoptosis, polyploidy, or multinucleated cells. Overexpression of the Chk tyrosine kinase can induce polyploidy and multinucleation [13], whereas overexpression of putative serine/threonine kinase Sak-a or nuclear matrix protein HET/SAF-B or Rad can lead to multinucleation (14 –16)

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