ID proteins have been implicated in the regulation of cell proliferation and differentiation in various cell types during normal development as well as in the formation of cancer. Our aim was to delineate the expression of ID2 by immunohistochemistry in primary breast cancer in order to detect potential associations with cell cycle regulatory proteins and/or clinicopathologic parameters. We further overexpressed ID2 in a breast cancer cell line to elaborate potential effects on proliferation and invasiveness. We observed large variations in ID2 expression in primary breast cancer, and the protein was localised to both the nucleus and cytoplasm. Interestingly, a high cytoplasmic ID2 protein level correlated with a favourable prognosis. Overexpressing ID2 in the MDA-MB-468 breast cancer cell line generated a marked cytoplasmic localisation of the protein and reduced the invasive capacity of cells. Modest enhancement of cell proliferation was further detected in ID2-overexpressing cells. In conclusion, ID2 protein expression varies substantially within primary breast tumours and high cytoplasmic levels of ID2 might reflect a less aggressive breast tumour phenotype. ' 2005 Wiley-Liss, Inc.Key words: ID2; breast cancer; proliferation; invasion The cell cycle regulatory machinery is a highly complex and delicate system that often is abrogated in cancer, and tumour cells consequently override one or more cell cycle checkpoints. One important checkpoint is the G 1 -S transition, where cells are committed to DNA replication and subsequent division. The cell cycle regulatory machinery is dependent on the activity of different CDKs and their associated cyclins, whose expression is tightly regulated over the cell cycle. Regulators of kinase activity are inhibitory proteins like p21, p27 and p16. One of the major substrates for the CDKs is pRb. Overexpression or inactivation of cell cycle regulatory proteins leads to abnormal proliferation and lack of checkpoint control. For example, inactivation of pRb has been noted in about 60% of all human cancers either by mutation and gene deletions or by deregulated phosphorylation, sequestration by oncoproteins or loss of cooperating factors.ID2 is a multipotent regulatory molecule harbouring an HLH motif. Through the HLH domain, ID proteins (ID1-ID4 in mammals) bind and sequester the DNA binding bHLH proteins known as E proteins, thereby preventing them from binding DNA alone or in complex with tissue-specifically expressed bHLH proteins. Since these latter bHLH proteins often are associated with differentiation and decreased proliferation, elevated ID protein levels often lead to maintained proliferation, whereas decreased ID protein levels are associated with differentiation.In the normal mammary epithelium, ID2 has been linked to critical steps in the development of functional mammary glands.2 The phenotype of homozygous ID2 knockout mice reveals lactation defects due to poor expansion of lobulo-alveolar tissue in the mammary glands, possibly caused by decreased proliferation of...