Mechanism ofcyclin D1 (CCND1, PRAD1) overexpression in human cancer cells: Analysis of allele-specific expression

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Abstract
The cyclin D1/CCND1 oncogene (PRAD1) is amplified in 15% of primary human breast cancers and overexpressed in 30–50% of breast cancers, suggesting that mechanisms in addition to DNA amplification may lead to deregulated expression of this gene in breast cancer. Cyclin D1 overexpression at a higher frequency than gene amplification is also seen in a variety of other tumors. Cyclin D1 overexpression without amplification could result from a trans-acting regulatory disturbance or could be a consequence of a clonal regulatory mutation in one allele of the gene. We have, therefore, examined whether the overexpression of cyclin D1 mRNA is derived from one parental allele or both alleles in tumor cell lines with or without amplification of the cyclin D1 gene. Eight tumor cell lines, MCF-7, SK-BR-3, ZR-75-1, U-2-OS, SK-LMS-1, DLD1, HCT15, and HT29, out of 20 tumor cells initially examined were found to be heterozygous at the polymorphic NciI site within exon 4 of the cyclin D1 gene. Polymerase chain reaction and NciI digestion (PCR-RFLP) analysis of genomic DNA demonstrated DNA amplification of one allele in the ZR-75-1 cells and HT29 cells and no such imbalance in cyclin D1 gene copy number in the other cells, consistent with Southern blot analyses. Reverse-transcription polymerase chain reaction analysis and NciI digestion (RT-PCR-RFLP) of total cDNA revealed that the overexpressed cyclin D1 mRNA is preferentially derived from the amplified allele in the ZR-75-1 and HT29 cells. In contrast, the other tumor cells overexpressed cyclin D1 mRNA equally from both alleles. This finding strongly suggests that, in breast, sarcoma, and in colon cancer cells with cyclin D1 overexpression and normal gene copy number, elevated levels of cyclin D1 mRNA result from a trans-acting influence on both alleles rather than a clonal somatic mutation or rearrangement in or near a single cyclin D1 gene. Genes Chromosomes Cancer 22:66–71, 1998.