Frequent mutations of human Mad2, but not Bub1, in gastric cancers cause defective mitotic spindle checkpoint.
Since the underlying mechanism for the high incidence of aneuploidy in gastric cancer has not clarified, we screened 49 gastric cancers and five gastric cancer cell lines for mutations in the mitotic spindle checkpoint genes, Bub1 and Mad2, and we analyzed the functional consequences of these mutations. The presence of mutations in Bub1 and Mad2 coding sequences was primarily detected by RT-PCR-SSCP and subsequently confirmed by automatic sequencing of either the RT-PCR products and/or the PCR products from genomic DNA. Mad2 was mutated in 44.9% of gastric cancer tissues and one gastric cancer cell line, N87, but not Bub1. Of these, three mutational hotspots at codons 156, 165 and 182 were identified. Mutations at codons 165 and 182 led to amino acid substitutions, whereas the mutation at codon 156 was a silent one. Overexpression of mutant Mad2 in HeLa cells led to the appearance of aneuploid cells in the presence of nocodazole, and this indicated that these mutations caused a defect in MAD2 protein. Wild type and mutant MAD2 protein displayed distinct mobility on two-dimensional gel electrophoresis. Novel mutational hotspots in human Mad2 genes were discovered for the gastric cancers and these mutations caused the functional defects in the spindle checkpoint suggesting that these mutations might be involved in the development and progression of gastric cancer.
Kim
Han-Soo
HS
Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Republic of Korea.
Park
Kyung Hwa
KH
Kim
Sun A
SA
Wen
Jing
J
Park
Seung Woo
SW
Park
Byungkyu
B
Gham
Chang-Woo
CW
Hyung
Woo Jin
WJ
Noh
Sung Hoon
SH
Kim
Ho Kun
HK
Song
Si Young
SY
D000328:Adult; D000368:Aged; D002135:Calcium-Binding Proteins / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D064246:Mad2 Proteins; D008297:Male; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009367:Neoplasm Staging; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D008941:Spindle Apparatus / Q000235:genetics*; D013274:Stomach Neoplasms / Q000235:genetics*
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases