Molecular analysis of the INK4A and INK4B gene loci in human breast cancer cell lines and primary carcinomas.
The INK4A and INK4B loci are located at 9p21 and have been implicated in the tumorigenesis of various human malignancies. The INK4A gene encodes two cell cycle regulators, p16(INK4A) and ARF, while INK4B encodes p15(INK4B). Previously, we have shown that the p16(INK4) tumor suppressor was not mutated or deleted in primary breast carcinomas. However, primary and metastatic breast carcinomas exhibited a relative hypomethylation of p16(INK4A), which is associated with expression, compared to normal breast tissue. The present study was conducted to determine if inactivation of p15(INK4B) and INK4A exon 1beta (ARF) are common events in breast carcinoma. Mutational analysis was performed by PCR-SSCP, and mRNA expression was evaluated by RT-PCR. Methylation-specific PCR was used to determine the methylation status of the p15(INK4B) promoter. Our results demonstrate that the p15(INK4B) gene was altered in 3 (21%) of the 14 breast cell lines; one had a silent mutation and two had homozygous deletion of the gene. None of the cell lines showed methylation of p15(INK4B). Two (14%) cell lines had homozygous deletion of INK4A exon 1beta. All normal and malignant breast tissue samples were wild-type and non-methylated for p15(INK4B) and wild-type for exon 1beta. Our results show that these structurally and functionally related genes are not invariably affected together, and the most frequently observed alteration at the INK4A and INK4B loci in breast carcinoma appears to be p16(INK4A) hypomethylation.
Bisogna
M
M
Department of Surgery, The Breast Cancer Research Laboratory, Memorial Sloan-Kettering Cancer Center, 10021, New York, NY, USA.
Calvano
J E
JE
Ho
G H
GH
Orlow
I
I
Cordón-Cardó
C
C
Borgen
P I
PI
Van Zee
K J
KJ
D015139:Blotting, Southern; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D018797:Cell Cycle Proteins; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics*; D018899:CpG Islands; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D019942:Genes, p16; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D025521:Tumor Suppressor Proteins; D014764:Viral Proteins; C068650:arf protein, Enterobacteria phage P22