Frequent impairment of the spindle assembly checkpoint in hepatocellular carcinoma.
BACKGROUND
Chromosomal instability (CI) leading to aneuploidy is one form of genetic instability, a characteristic feature of various types of cancers. Recent work has suggested that CI can be induced by a spindle assembly checkpoint defect. The aim of the current study was to determine the frequency of a defect of the checkpoint in hepatocellular carcinoma (HCC) and to establish whether alterations of genes encoding the checkpoint were associated with CI in HCC.


METHODS
Aneuploidy and the function of the spindle assembly checkpoint were examined using DNA flow cytometry and morphologic analysis with microtubule disrupting drugs. To explore the molecular basis, the authors examined the expression and alterations of the mitotic checkpoint gene, BUB1, using Northern hybridization and direct sequencing in 8 HCC cell lines and 50 HCC specimens. Furthermore, the authors examined the alterations of other mitotic checkpoint genes, BUBR1, BUB3, MAD2B, and CDC20, using direct sequencing in HCC cell lines with aneuploidy.


RESULTS
An impaired spindle assembly checkpoint was found in five (62.5%) of the eight aneuploid cell lines. Transcriptional expressions of the BUB1 gene appeared in all cell lines. While some polymorphic base changes were noted in BUB1, BUBR1, and CDC20, no mutations responsible for impairment of the mitotic checkpoint were found in either the HCC cell lines or HCC specimens, which suggests that these genes did not seem to be involved in tumor development in HCC.


CONCLUSIONS
The loss of spindle assembly checkpoint occurred with a high frequency in HCC with CI. However, other mechanisms might also contribute to CI in HCC.
Saeki
Ayuko
A
Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 B-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
Tamura
Shinji
S
Ito
Nobuyuki
N
Kiso
Shinichi
S
Matsuda
Yasuo
Y
Yabuuchi
Iwao
I
Kawata
Sumio
S
Matsuzawa
Yuji
Y
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000782:Aneuploidy; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D064246:Mad2 Proteins; D008297:Male; D008875:Middle Aged; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D008941:Spindle Apparatus / Q000235:genetics*; D014407:Tumor Cells, Cultured
C489444:BUB3 protein, human; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; C120552:MAD2L2 protein, human; D064246:Mad2 Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; C577488:CDC20 protein, human; D012313:RNA; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases