Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors compared with normal tissue, its contribution to tumorigenicity is unknown. HB-EGF can be produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (s-HB-EGF), although a significant amount of pro-HB-EGF remains uncleaved on the cell surface. To understand the roles of two forms of HB-EGF in promoting tumor growth, we have studied the effects of HB-EGF expression in the process of tumorigenesis using in vitro and in vivo systems. We demonstrate here that in EJ human bladder cancer cells containing a tetracycline-regulatable s-HB-EGF or pro-HB-EGF expression system, s-HB-EGF expression increased their transformed phenotypes, including growth rate, colony-forming ability, and activation of cyclin D1 promoter, as well as induction of vascular endothelial growth factor in vitro. Moreover, s-HB-EGF or wild-type HB-EGF induced the expression and activities of the metalloproteases, MMP-9 and MMP-3, leading to enhanced cell migration. In vivo studies also demonstrated that tumor cells expressing s-HB-EGF or wild-type HB-EGF significantly enhanced tumorigenic potential in athymic nude mice and exerted an angiogenic effect, increasing the density and size of tumor blood vessels. However, cells expressing solely pro-HB-EGF did not exhibit any significant tumorigenic potential. These findings establish s-HB-EGF as a potent inducer of tumor growth and angiogenesis and suggest that therapeutic intervention aimed at the inhibition of s-HB-EGF functions may be useful in cancer treatment.

We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502-802) interacted with the IFI16 Pyrin domain (aa 1-130). We found that IFI16 was localized in the nucleoplasm and nucleoli. Clear nucleolar IFI16 localization was not observed in HCC1937 BRCA1 mutant cells, but reintroduction of wild-type BRCA1 restored IFI16 nuclear relocalization following IR (ionizing radiation). Coexpression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 collaborated in inducing apoptosis when adenovirus p53 was expressed in DNAdamaged p53-deficient EJ cells. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.

IFI16 is a member of the HIN-200 family (hematopoietic interferon-inducible nuclear antigens with 200 amino acid repeats) that contains a DNA binding domain, a transcriptional regulatory domain, and DAPIN/ PAAD, a protein domain associated with interferon response. It can function as a transcription repressor and directly binds p53. Although the structural and biochemical properties of IFI16 are known, the physiological relevance of these properties in the cellular context is still elusive. Here we report that the inhibition of endogenous IFI16 expression by small interfering RNA (siRNA) induces p21Waf1 mRNA and protein expression through p53 but does not induce pro-apoptotic p53 target genes. This rapid induction of p21 was wild-type p53-dependent and resulted in cell cycle arrest along with a marked reduction of phosphorylated Rb in normally growing cells. We also showed that the repression of IFI16 affects p53 transcriptional activity at the p21 promoter as well as the protein stability of p53 and p21. Our findings identified a new role for IFI16 in modulating p53 function and its target gene regulation in the control of cell cycle regulation.The control of cell cycle regulation plays an important role in maintaining normal cellular growth, proliferation, and differentiation, and as a result, the loss of cell cycle control leads to the development of tumors (1). The p53 tumor suppressor gene plays an important role in the regulation of the cell cycle, and it is known that p53 plays a crucial role in the progression of cancer as evidenced by the inactivation or loss of p53 in a majority of human tumors (2, 3). The activity of p53 in controlling cell growth needs to be tightly restrained in unstressed cells to allow cell proliferation and development (4, 5). p53 protein is positively regulated through a succession of posttranslational modifications including acetylation (5, 6). In its inactive form, p53 is bound to the Mdm2 protein, and binding to Mdm2 inhibits the transcriptional activity of p53 (4 -6) and promotes the degradation of p53 by the 26 S proteosome (7). The main effector of p53-mediated cell cycle arrest is the p53 target gene, p21 (4). p21Waf1/Cip1/Sdi1 is an inhibitor of cyclindependent kinases which are key regulators of the cell cycle (8, 9). p53 activates p21 (10) transcriptionally, and it has been shown that the inhibition of cell proliferation by p53 is largely because of its ability to transcriptionally activate genes involved in controlling cell fate (2, 3).Here we describe IFI16, which we found to be involved in the regulation of p21 through p53. IFI16 is a human member of the HIN-200 1 family of interferon-inducible proteins (11). Members of the HIN-200 family are characteristically induced by interferons and distinctly contain one or more of a conserved 200-amino acid motif. IFI16 was first detected in lymphoid cells (12), but since then, through immunohistochemical staining of different tissues, it was found to be expressed at high levels in highly proliferating cells of various cell t...