In a differential display experiment comparing normal breast epithelial cells with breast tumors and breast cancer cell lines we isolated a gene, cyclin D2, showing a low expression in tumor cells compared to normal cells. This result was confirmed using Northern blotting and RT-PCR. In total, low expression was seen in 48/109 (44%) tumors. Low cyclin D2 expression was seen in 10/39 (25%) sporadic breast cancer, and in 38/70 (54%) of familial breast cancer. In order to find the explanation for the down-regulation of cyclin D2 we screened for inactivating mutations in tumors with low expression, and studied methylation of the promoter region in breast cancer cell lines. We also used growth factors in an attempt to stimulate cell lines to express cyclin D2, and transfected the cell lines with a vector expressing cyclin D2. Treatment with 5-azacytidin for four days resulted in transcription of cyclin D2. Our result suggests that methylation of the cyclin D2 promoter or another regulating gene is associated with loss of cyclin D2 expression in breast cancer.

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