No abstract

No abstract

This is the first description of an extensive immunohistochemical analysis of interferon (IFN)-inducible gene IFI16 expression in normal tissues. Immunohistochemical detection of IFI16 in paraffin-embedded tissues is achieved by using a polyclonal antibody raised against its C-terminal fragment that recognizes its three closely migrating isoforms in Western blotting. The results clearly indicate that IFI16 expression is not restricted to the hematopoietic compartment. In normal adult human tissues, it is prominent in stratified squamous epithelia and particularly intense in parabasal cells in the proliferating compartments, but it gradually decreases in the more differentiated suprabasal layers. Understanding of IFI16 expression in vivo is essential for interpretation of the results obtained from in vitro studies and elucidation of its physiologic role. The constitutive expression and wider distribution of IFI16 in normal human tissues, not restricted to the hematopoietic compartment, strongly support the possibility of an important role in cell differentiation that can be further modulated by other stimuli, such as IFN. 815

We have previously demonstrated that overexpression of p204, a member of the I® 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo ®bro-blasts (MEF) inhibits cell proliferation, but does not aect cell growth in MEF derived from Rb7/7 mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo ®broblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a signi®cant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this eect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-a antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb7/7 cells were unsensitive to the IFN-a induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-a antiproliferative activity and (ii) the primary target of p204 leading to ecient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system. Oncogene (2000) 19, 3598 ± 3608.