ABSTRACT
Inositol hexakisphosphate kinase 2 (IP6K2), a member of the inositol hexakisphosphate kinase family, functions as a growth suppressive and apoptosis-enhancing kinase during cell stress. We created mice with a targeted deletion of IP6K2; these mice display normal embryogenesis, development, growth and fertility. Chronic exposure to the carcinogen 4-nitroquinoline 1-oxide (4-NQO, a UV-mimetic compound) in drinking water resulted in fourfold increased incidence of invasive squamous cell carcinoma (SCC) formation in the oral cavity and esophagus of the knockout (KO) mice compared to the wild-type (WT) littermates. Paradoxically, KO mice displayed relative resistance to ionizing radiation and exhibit enhanced survival following 8-10 Gy total body irradiation. Primary KO fibroblasts displayed resistance to antiproliferative effects of interferon-beta and increased colony forming units following ionizing radiation. Radioresistance of KO fibroblasts was associated with accelerated DNA repair measured by comet assay. Direct microinjection of 5-PP-Ins(1,2,3,4,6)P(5) (the enzymatic product of IP6K2), but not InsP(6) (the substrate of IP6K2) induced cell death in SCC22A squamous carcinoma cells.
Subject(s)
Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Gene Deletion , Genetic Predisposition to Disease/genetics , Mouth Neoplasms/genetics , Phosphotransferases (Phosphate Group Acceptor)/genetics , 4-Nitroquinoline-1-oxide/toxicity , Animals , Apoptosis , Carcinogens/toxicity , Carcinoma, Squamous Cell/radiotherapy , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gamma Rays , Gene Expression Profiling , Immunoblotting , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microinjections , Radiation Tolerance , Radiation, Ionizing , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Interferons (IFNs) regulate the expression of genes that mediate their antiviral, antitumor, and immunomodulatory actions. We have previously shown that IFN-beta suppresses growth of human ovarian carcinoma xenografts in vivo and induces apoptosis of ovarian carcinoma cells in vitro. To investigate mechanisms of IFN-beta-induced apoptosis we employed an antisense technical knockout approach to identify gene products that mediate cell death and have isolated several regulators of interferon-induced death (RIDs). In this investigation, we have characterized one of the RIDs, RID-2. Sequence analysis revealed that RID-2 was identical to human inositol hexakisphosphate kinase 2 (IP6K2). IP6K2 is post-transcriptionally induced by IFN-beta in ovarian carcinoma cells. A mutant IP6K2 with substitutions in the putative inositol phosphate binding domain abrogates IFN-beta-induced apoptosis. These studies identify a novel function for IP6K2 in cell growth regulation and apoptosis.
Subject(s)
Apoptosis/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Interferon-beta/pharmacology , Isoenzymes/metabolism , Ovarian Neoplasms/pathology , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Cell Cycle/drug effects , Cell Division/drug effects , Consensus Sequence , Female , Humans , Oligonucleotides, Antisense/pharmacology , Phosphotransferases (Phosphate Group Acceptor)/genetics , RNA, Messenger/metabolism , Tumor Cells, CulturedABSTRACT
OBJECTIVES: We have previously described that bioactive lysophospholipids-lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC)-are present in ascitic fluids from patients with ovarian cancer. To understand the role of these lipids in ovarian cancer, we investigated the effects of these lipids on interleukin-8 (IL-8) production in ovarian cancer cells. IL-8 is a proinflammatory and proangiogenic factor, which is potentially involved in ovarian cancer development. METHODS: The Clontech PCR-Select cDNA subtraction method (Clontech Laboratories, Inc., Palo Alto, CA) was used to identify genes potentially regulated by LPA in HEY and OCC1 ovarian cancer cell lines. Northern blot analysis was used to confirm and examine IL-8 mRNA regulation by lysolipids. Enzyme-linked immunosorbent assay (ELISA) was used for detecting secreted IL-8. RESULTS: We describe here that LPA, S1P, and SPC increased mRNA levels (2- to 7-fold) and protein secretion (2- to 12-fold) of IL-8 from ovarian cancer cells (HEY, OCC1, and SKOV3) in vitro. These regulations were both dose- and time-dependent. All three lipids increased the stability IL-8 mRNA in HEY cells. In contrast to malignant ovarian cancer cells, immortalized human ovarian epithelial cells did not respond to any of these lipids to increase the secretion of IL-8, although these cells secreted similar basal levels of IL-8 (310 pg/ml/10,000 cells). Two breast cancer cell lines (MCF7 and T47D) secreted lower basal levels of IL-8 (48-80 pg/ml/10,000 cells), compared with ovarian cancer cells (200-500 pg/ml/10,000 cells). MCF7 cells responded to LPA, but not S1P and SPC, by increasing the secretion of IL-8. T47D and MCF10A, an immortalized breast cell line, did not respond to LPA, S1P, or SPC to increase IL-8 secretion. CONCLUSIONS: LPA, S1P, and SPC regulate the mRNA and protein levels of the proinflammatory and proangiogenic factor IL-8 in ovarian cancer cells. The pathological significance of these regulations in ovarian cancer remains to be further investigated.
Subject(s)
Interleukin-8/biosynthesis , Lysophospholipids/pharmacology , Ovarian Neoplasms/metabolism , Phosphorylcholine/analogs & derivatives , Sphingosine/analogs & derivatives , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interleukin-8/genetics , Interleukin-8/metabolism , Ovarian Neoplasms/genetics , Phosphorylcholine/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Sphingosine/pharmacology , Tumor Cells, Cultured , Up-Regulation/drug effectsABSTRACT
In this paper, we discuss the value of scientific conferences to both the discipline area and the individual participants and trace the growth in grant support of biomedical meetings by the National Institutes of Health and, in particular, the National Cancer Institute.
