ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIa (TopoIIa) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIa than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIa expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line.

The retinoblastoma protein (pRB) is encoded by the RB1 gene whose promoter contains several putative binding sites for ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa), a transcriptional regulator of the topoisomerase IIalpha gene. ICBP90 has two consensus binding sites for pRB in its primary sequence. Here, we show that pRB and ICBP90 co-immunoprecipitate in cell extracts of proliferating human lung fibroblasts and of proliferating or confluent Jurkat cells. GST pull-down assays and immunocytochemistry, after cell synchronization in late G1 phase, confirmed this interaction. Overexpression of ICBP90 induces downregulation of pRB expression in lung fibroblasts as a result of mRNA decrease. DNA chromatin immunoprecipitation experiment shows that ICBP90 binds to the RB1 gene promoter under its methylated status. Overexpression of ICBP90 increases the S and G2/M phase cell fractions of serum-starved lung fibroblasts as assessed by flow cytometry analysis and increases topoisomerase IIalpha expression. Together, these results show that ICBP90 regulates pRB at the protein and gene transcription levels, thus favoring the entry into the S phase of the cells. We propose that ICBP90 overexpression, found in cancer cells, is involved in the altered checkpoint controls occurring in cancerogenesis.

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Inhibition of apoptosis, resulting from an increase in anti-apoptotic protein, plays a fundamental role in carcinogenesis. Because ICBP90 gene expression is deregulated in cancer cells, we studied its expression in Jurkat cells under apoptotic conditions to see whether ICBP90 is involved in the regulation of apoptosis. We found that ICBP90 expression and the percentage of living cells were dose-dependently decreased in PHA and ionophore A23187-stimulated Jurkat cells, but not in THP-1 cells. These results suggest that apoptosis is dependent upon ICBP90 expression downregulation and that ICBP90 exhibits anti-apoptotic properties.