NP95: methods of assaying for cell cycle modulators

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#The present invention relates to regulation of cellular proliferation. More particularly, the present invention is directed to nucleic acids encoding NP95, which is involved in modulation of cell cycle arrest. The invention further relates to methods for identifying and using agents, including small molecule chemical compositions, antibodies, peptides, cyclic peptides, nucleic acids, RNAi, antisense nucleic acids, and ribozymes, that modulate cell cycle arrest via modulation of NP95; as well as to the use of expression profiles and compositions in diagnosis and therapy related to cell cycle regulation and modulation of cellular proliferation, e.g., for treatment of cancer and other diseases of cellular proliferation.

#FIG. 1 provides a nucleotide (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of human NP95.

#FIG. 2 provides an illustration of the relevant domains of NP95, including the ubiquitin like domain, the zinc finger domain, the nuclear protein domain, and the ubiquitin ligase domain.

#FIG. 3 illustrates cell tracker assay data demonstrating that GFP-fused NP95 is antiproliferative in A549. FIG. 3A illustrates fluorescence analysis of green fluorescent protein (GFP) infected A549.tTA control cells. FIG. 3B illustrates cell tracker assay data from GFP infected A549.tTA control cells. FIG. 3C illustrates fluorescence analysis of NP95 infected A549.tTA cells. FIG. 3D illustrates cell tracker assay date from NP95 infected A549.tTA cells.

#Introduction

#The NP95 gene encodes a nuclear zinc finger protein which is associated with cellular proliferation (see, e.g., Fujimori et al. Mammalian Genome 9:1032-1035 (1998). The NP95 open reading frame contains a potential ATP/GTP binding site, a zinc finger motif, a putative cyclin A/E cdk2 phosphorylation site, and a retinoblastoma binding motif (see, e.g., Miura et al. Exp. Cell Res. 263:202-208 (2001). However, NP95 involvement in cellular transformation, tumorigenesis, and anti-proliferative effects in tumor cells has never been demonstrated. Furthermore, the role of NP95 in cell cycle regulation has not yet been elucidated.

#As described below, the present inventors identified human NP95 in a cDNA library screening assay. As shown in FIG. 3, studies with NP95 show NP95 has a antiproliferative phenotype (using, e.g., GFP positivity and cell tracker assays). These functional studies, presented herein, demonstrate for the first time that inhibition of NP95 will inhibit tumor cell growth. With cellular staining of NP95 infected A549.tTA cells, fluorescence analysis shows that NP95 is localized to the nucleus of NP95 infected cells.