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Down-Regulation of 1D-Myo-Inositol 1,4,5-Trisphosphate 3-Kinase a Protein Expression in Oral Squamous Cell Carcinoma

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Down-Regulation of 1D-Myo-Inositol 1,4,5-Trisphosphate 3-Kinase a Protein Expression in Oral Squamous Cell Carcinoma Kato 14-11 23/2/06 12:28 Page 873 INTERNATIONAL JOURNAL OF ONCOLOGY 28: 873-881, 2006 873 Down-regulation of 1D-myo-inositol 1,4,5-trisphosphate 3-kinase A protein expression in oral squamous cell carcinoma HISASHI KATO1, KATSUHIRO UZAWA1,2, TAKESHI ONDA4, YOSHIKUNI KATO1, KENGO SAITO1, DAI NAKASHIMA1, KASTUNORI OGAWARA1,2, HIROKI BUKAWA2, HIDETAKE YOKOE2 and HIDEKI TANZAWA1-3 1Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University; 2Division of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital; 3Center of Excellence (COE) Program in the 21st Century, Graduate School of Medicine, Chiba University, Chiba 260-8670; 4First Department of Oral and Maxillo-Facial Surgery, Tokyo Dental College, Chiba 261-8502, Japan Received November 14, 2005; Accepted December 22, 2005 Abstract. Functional proteomics is a useful method to explore (ITPKA) protein that was down-regulated in OSCC cell lines. changes in protein expression in human diseases, including ITPKA phosphorylates inositol 1,4,5-trisphosphate, which carcinomas. To identify tumor-associated proteins as bio- regulates the calcium (Ca2+) level within the cell by releasing markers or molecular targets of human oral squamous cell Ca2+ from intracellular stores, and is responsible for regulating carcinomas (OSCCs), we performed two-dimensional poly- the levels of a large number of inositol polyphosphates that acrylamide gel electrophoresis and matrix-assisted laser are important in cellular signaling. Western blots revealed desorption/ionization-time of flight mass spectrometry. dramatically down-regulated ITPKA expression in all OSCC Comparison of the protein expression profiles of OSCC cell cell lines examined. Real-time quantitative reverse tran- lines and normal oral keratinocytes identified six proteins scriptase-polymerase chain reaction showed down-regulated with markedly different expression levels. Of the six proteins, ITPKA mRNA expression in nine of 12 (75%) OSCC cell we found a 1D-myo-inositol 1,4,5-trisphosphate 3-kinase A lines. Immunohistochemistry analysis showed that 40 of 100 OSCC clinical samples had a significant decrease in ITPKA. Poorly differentiated tumors showed significantly lower _________________________________________ immunoreactivity of the protein compared to well- and moderately-differentiated tumors. These data suggest that Correspondence to: Dr Katsuhiro Uzawa, Department of Clinical ITPKA may be related to carcinogenesis by the modulation Molecular Biology, Graduate School of Medicine, Chiba University, of inositol polyphosphates and Ca2+ homeostasis and that 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan ITPKA may be a potential novel molecular target, biomarker, E-mail: [email protected] parameter, or all of these of cellular differentiation and of intracellular Ca2+ homeostatic characteristics in clinical Abbreviations: 2D-PAGE, two-dimensional polyacrylamide gel medicine. electrophoresis; MALDI-TOF-mass, matrix-assisted laser desorption/ ionization-time of flight mass spectrometry; OSCCs, oral squamous Introduction cell carcinomas; NOKs, normal oral keratinocytes; ITPKA, 1D- myo-inositol 1,4,5-trisphosphate 3-kinase A; RT-PCR, reverse Biological responses consist of protein components resulting transcriptase-polymerase chain reaction; IHC, immunohisto- from transcriptional control, post-transcriptional regulation, chemistry; Ins(1,4,5)P , inositol 1,4,5-trisphosphate; Ca2+, calcium; 3 and post-translational modifications. Thus, proteome analysis DAG, 1,2-diacylglycerol; GTP, guanosine triphosphate; ER, can provide important information in the study of the bio- endoplasmic reticulum; Ins(1,4) P2, inositol 1,4-bisphosphate; IPK3, inositol 1,4,5-trisphosphate 3-kinase; Ins(1,3,4,5) P , inositol logical sciences. To identify the proteins associated with the 4 development of human oral squamous cell carcinoma (OSCC), 1,3,4,5-tetrakisphosphate; ATP2C1, ATPase, Ca2+ transporting, we compared the protein expression of OSCC cell lines with type 2C, member 1; ATP2A2, sarcoendoplasmic reticulum Ca2+ ATPase type 2; SCCs, squamous cell carcinomas; PMF, peptide- normal oral keratinocytes (NOKs), using two-dimensional mass fingerprinting; PBS, phosphate-buffered saline; OPLs, oral polyacrylamide gel electrophoresis (2D-PAGE) and matrix- premalignant lesions; LOH, loss of heterozygosity; GAPDH, assisted laser desorption/ionization-time of flight mass spectro- glyceraldehyde-3-phosphate dehydrogenase metry (MALDI-TOF-mass). We found that 1D-myo-inositol 1,4,5-trisphosphate 3-kinase A protein (ITPKA) has markedly Key words: ITPKA, oral squamous cell carcinoma, two-dimensional decreased expression in OSCC cell lines. To our knowledge, electrophoresis, MALDI-TOF-mass spectrometry, calcium signal ITPKA has not been reported in oncogenesis. ITPKA phos- transduction, inositol polyphosphates phorylates inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which regulates intracellular calcium (Ca2+) homeostasis (1-3). Kato 14-11 23/2/06 12:28 Page 874 874 KATO et al: ITPKA IN ORAL CANCER Ca2+ is a ubiquitous intracellular signal responsible for changes in ITPKA to carcinogenesis in the oral squamous controlling numerous cellular processes, including fertilization, epithelium, we also analyzed the mRNA and protein levels in cell growth, transformation, secretion, smooth muscle con- OSCC cell lines and NOKs and expression levels in immuno- traction, sensory perception, and neuronal signaling (4,5). histochemistry (IHC) of OSCC clinical samples. Intracellular Ca2+ levels are buffered to a low concentration (100 nM) and rapidly rise to 1 μM or more in response to Materials and methods incoming signals (6). A second messenger, Ins(1,4,5)P3, is part of various internal Ca2+ signals that regulate many different Cell cultures. We used the human OSCC cell lines HSC-2, cellular functions (7). When the extracellular agonists, such HSC-3, HSC-4, Ho-1-N-1, Ca9-22, SKN3, Kosc2, KON, as hormones and neurotransmitters, occupy a cell surface SAT, SAS (Human Science Research Resources Bank, Osaka, receptor, phosphatidylinositol 4,5-bis-phosphate in plasma Japan), OK92 (established from carcinoma of the tongue in our department), and Sa3 and H1 (kindly provided by Dr membranes is hydrolyzed to Ins(1,4,5)P3 and 1,2-diacyl- glycerol (DAG) by phospholipase C activity that is enhanced Fujita at Wakayama Medical University, Wakayama, Japan). by guanosine triphosphate (GTP). The primary function of All cell lines were maintained at 37˚C (humidified atmosphere 2+ 5% CO2/95% air) on 150x20-mm tissue culture dishes (Nunc, Ins(1,4,5)P3 is to mobilize Ca from intracellular stores, probably in the endoplasmic reticulum, by binding to Roskilde, Denmark) and cultured in Dulbecco's modified Eagle's medium F-12 HAM (Sigma Chemical Co., St. Louis, Ins(1,4,5)P3 receptors, whereas DAG is a direct activator of protein kinase C (1-3,8-12). This bifurcating messenger system MO) with 10% fetal bovine serum (Sigma) and 50 units/ml operates throughout the life of a typical cell in a variety of penicillin and streptomycin. specialized cells in animals and plants (7). The levels of Five NOK strains from five patients who had undergone Ins(1,4,5)P are tightly regulated and rapidly metabolized by dental surgery served as the controls in the experimental 3 procedures. All patients provided written informed consent two mechanisms: dephosphorylation via an Ins(1,4,5)P 5- 3 before the start of the study. The normal oral specimens were phosphatase to inositol 1,4-bisphosphate (Ins(1,4)P ) or 2 washed in Dulbecco's phosphate-buffered saline (PBS) (Sigma) phosphorylation via an inositol 1,4,5-trisphosphate 3-kinase and then placed overnight in 0.25% trypsin-EDTA solution (IPK3) to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (Sigma) at 4˚C. After the epithelial tissue separated from the (3). Human IPK3 exists as three isoforms, ITPKA, B, and C, connective tissue, it was disaggregated by incubation in of which ITPKA is the most highly characterized isoform 0.25% trypsin-EDTA solution for 15 min with gentle pipetting and is expressed intensely in rat brain and testes (3,13-15). at 37˚C. Isolated epithelial cells then were seeded into Collagen ITPKA protein encoded by the gene located on chromosome I Cellware 60-mm dish biocoat cell environments (Becton- 15q14-q21 is approximately 51 kDa and consists of 461 Dickinson Labware, Bedford, MA) and cultured in Keratino- amino acids. IPK3 activity is stimulated in the presence of cyte Basal Medium-2 (Cambrex, Walkersville, MD) with Ca2+/calmodulin via phosphorylation by cyclic AMP-dependent 0.4% bovine pituitary extract, 0.1% human epidermal growth protein kinase, protein kinase C, or Ca2+/calmodulin-dependent factor, 0.1% insulin, 0.1% hydrocortisone, 0.1% transferrin, protein kinase II (3,14,16-18). 0.1% epinephrine, and 0.1% GA-1000 (Cambrex). It is well known that impaired intracellular Ca2+ homeo- stasis, which ITPKA partly regulates, causes epithelial Tissue samples for IHC. Tumors or oral leukoplakias with disorders. For instance, mutations in ATP2C1 (SPCA1), en- patient-matched normal oral tissue were obtained at the time coding a Ca2+ pump (Ca2+-transport ATPase), cause Hailey- of surgery at Chiba University Hospital after informed consent Hailey disease, which is an autosomal dominant disorder was obtained from the patients, according to a protocol that characterized by persistent blisters and erosions of the skin was reviewed and approved by the institutional review board with impaired adhesion of keratinocytes
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