Research at molecular and gene level on enhancement mechanisms combined with irradiation and hyperthermia in culture cell lines

• Project/Area Number: 15591277
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Radiation science
• Research Institution: Okayama University
• Principal Investigator: KAWASAKI Shoji Univ. of Okayama, Medical School, Professor, 医学部, 教授 (20034952)
• Project Period (FY): 2003 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
• Keywords: culture cell / heat shock protein / heating / western bloting / methylation / histone / KNK437 / apoptosis / histone / HSP27 / 温熱 / 細胞分裂遅延 / HSP / ヒストン / メチレーション / アセチレーション / cdc-2遺伝子 / エピゼノイック / 扁平上皮癌 / 核蛋白質

Research Abstract

1 Relationship between radiation/or heating induced cell cycle disturbance and gene expression of 14-3-3a, cdc2 and 25 were studied in human cancer cells lines. When cells were heated for 20 to 40 min. at 45 degree, G2-block(G2 cells accumulation) were induced. At that time, decrease of cdc2 and increase of cdc25-P, was observed. When cells were released from G2-block, it was observed increase of cdc2 and decrease of cdc25-P.
2 We established the method of synchronization of culture human cancer cells. Fractions of S phase cells in population reached about 90 percent 13 hours after stimulation of serum. It was suggested that this method is useful for experiments to determine gene expression after cells were exposed to several conditions.
3 Using a pEYFP-Nuc vector, which contains nucleic acid sequences of a nuclear localization signal, we established a Jurkat-YN cell line that expressed enhanced yellow fluorescent protein (EYFP) in the nucleus. The nuclear forms visualized by EYFP were a lmost equal in quality to those visualized by SYTO59, a nucleic acid stain for living cells. Three-dimensional deformities in the nuclear form were observed during apoptosis before chromatin condensation became apparent, indicating these deformities are characteristic morphological changes of the early stage of apoptosis.
4 We report here an analysis of the mechanisms of the inhibition by KNK437 in 2 human oral squamous cell carcinma cell lines whose heat sensitivities are different (HSC4; heat-resistant and KB; heat-sensitive) with a view to identifying which if any HSPs are targeted for KNK437. Combined administration of KNK437 to the medium after heat shock specifically inhibited the induction of HSPs including HSP27, HSP40, HSP70 and HSP90 in time dependence. The hyperthermic treatment combined with heating and KNK437 produced supra-reductive effects (i.e. HSP27 and HSP90 in HSC4, HSP27, HSP40 and HSP90 in KB). Careful examination of the ability of KNK437 to inhibit the induction of various HSPs may therefore be valuable in future attempts to improve the efficacy of oral cancer hyperthermic therapy.
5 Heat-induced changes in histone H3 methylation especially for H3-Lys4 and H3-Lys9 methylation in combination with KNK-437 have been studied in two human oral cancer cell lines such as HSC4 and KB,. Heating of HSC4 cells at 45C for 20 min and KB cells for 3 min gradually increased H3-Lys4 and H3-Lys9 methylation. Treatment of both cells with KNK-437 before or after heat-treatment inhibited methylation of H3-Lys4. Our results indicate a possibility of alteration in histone structure by heat treatment as a result of change in the methylation of H3 histone. Use of KNK-437, an inhibitor of HSPs gene expression, inhibited methylation of H3 Lys4. From these results, there exist some mechanism to regulate the expressions of genes in DNA by heat treatment.

Research Products

• [Journal Article] Effect of KNK437 on induction of Heat shock proteins with heating in human oral cancer cell lines (2006)
  • Author(s): Shin-ya Nakagawa, Keiji Matsuda, Tomoya Nakano., Ganesh C., Jagetia, Jun-ichi Asaumi, Shoji Kawasaki
  • Journal Title: JPN J Hyperthermic Oncology, 2006 22(2) Pages: 71-81
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effect of KNK437 on induction of Heat shock proteins with heating in human oral cancer cell lines (2006)
  • Author(s): Shin-ya Nakagawa, Keiji Matsuda, Tomoya Nakano, Ganesh C. Jagetia, Jun-ichi Asaumi, Shoji Kawasaki
  • Journal Title: JPN J Hyperthermic Oncology 22-28, 22(2) Pages: 71-81
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Hydrogen peroxide overload increases adriamycin-induced apoptosis of SaOS(2)FM, a manganese superoxide dismutase-overexpressing human osteosarcoma cell line. (2005)
  • Author(s): Wang Y, Kuroda M, Gao XS, Asaumi J, Shibuya K, Kawasaki S, Akaki S, St Clair D, Hiraki Y, Kanazawa S
  • Journal Title: Int J Oncol 26(5) Pages: 1291-1300
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer. (2005)
  • Author(s): Murakami J, Asaumi J, Kawai N, Tsujigiwa H, Yanagi Y, Nagatsuka H, Inoue T, Kokeguchi S, Kawasaki S, Kuroda M, Tanaka N, Matsubara N, Kishi K
  • Journal Title: Cancer Chemother Pharmacol. 56(1) Pages: 22-28
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Manganese superoxide dismutase overexpression changes plating efficiency bidirectionally according to change in redox for SaOS2 human osteosarcoma cell line (2005)
  • Author(s): Komatsu M, Kuroda M, Wang Y, St Clair D, Urano M, Akaki S, Asaumi J, Kawasaki S, Hiraki Y, Kanazawa S
  • Journal Title: Int J Oncol. 26(4) Pages: 853-862
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Hydrogen peroxide overload increases adriamycin-induced apoptosis of SaOS(2)FM, a manganese superoxide dismutase-overexpressing human osteosarcoma cell line. (2005)
  • Author(s): Wang Y, Kuroda M, Gao XS, Asaumi J, Shibuya K, Kawasaki S, Akaki S, St Clair D, Hiraki Y, Kanazawa S.
  • Journal Title: Int J Oncol 26(5)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer. (2005)
  • Author(s): Murakami J, Asaumi J, Kawai N, Tsujigiwa H, Yanagi Y, Nagatsuka H, Inoue T, Kokeguchi S, Kawasaki S, Kuroda M, Tanaka N, Matsubara N, Kishi K.
  • Journal Title: Cancer Chemother Pharmacol. Jul 56(1) Pages: 22-28
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Manganese superoxide dismutase overexpression changes plating efficiency bidirectionally according to change in redox for SaOS2 human osteosarcoma cell line. (2005)
  • Author(s): Komatsu M, Kuroda M, Wang Y, St Clair D, Urano M, Akaki S, Asaumi J, Kawasaki S, Hiraki Y, Kanazawa S.
  • Journal Title: Int J Oncol. Apr 26(4) Pages: 853-862
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Hydrogen peroxide overload increases adriamycin-induced apoptosis of SaOS(2)FM, a managanese superoxide dismutase-overexpressing human osteosarcoma cell line. (2005)
  • Author(s): Wang Y, Kuroda M, Gao XS, Asaumi J, Shibuya K, Kawasaki S, Araki S, St Clair D, Hiraki Y, Kanazawa S.
  • Journal Title: Int J Oncol 26(5) Pages: 1291-1300
• [Journal Article] Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer (2005)
  • Author(s): Murakami J, Asaumi J, Kawai N, Tsujigiwa H, Yanagi Y, Nagatsuka H, Inoue T, Kokeguchi S, Kawasaki S, Kuroda M, Tanaka N, Matsubara N, Kishi K
  • Journal Title: Cancer Chemother Pharmacol. 56(1) Pages: 22-28
• [Journal Article] Manganese superoxide disumtase overexpression changes plating efficiency bidirectionally according to change in redox for SaOS2 human osteosarcoma cell line. (2005)
  • Author(s): Komatsu M, Kuroda M, Wang Y, St Clair D, Urano M, Akaki S, Asaumi J, Kawasaki S, Hiraki Y, Kanazawa S.
  • Journal Title: Int J Oncol. 26(4) Pages: 853-862
• [Journal Article] Manganese superoxide dismutase overexpression changes plating efficiency bidirectionally according to change in redox for SaOS2 human osteosarcoma cell line. (2005)
  • Author(s): Kawasaki S et al.
  • Journal Title: Int J Oncol. 24(4) Pages: 853-862
• [Journal Article] Influence of CpG island methylation status in 06-methylguanine-DNA methyltransferase expression of oral cancer cell lines. (2004)
  • Author(s): Murakami J, Asaumi J, Maki Y, Tsujigiwa H, Nagatsuka H, Kokeguchi S, Inoue T, Kawasaki S, Tanaka N, MacPhee D, Matsubara N, Kishi K
  • Journal Title: Oncol Rep 12(2) Pages: 339-345
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Influence of CpG island methylation status in 06-methylguanine-DN methyltransferase expression of oral cancer cell lines. (2004)
  • Author(s): Murakami J, Asaumi J, Maki Y, Tsujigiwa H, Nagatsuka H, Kokeguchi S, Inoue T, Kawasaki S, Tanaka N, MacPhee D, Matsubara N, Kishi K.
  • Journal Title: Oncol Rep.Aug 12(2) Pages: 339-345
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Influence of CpG island methylation status in O6-methylguanine-DNA methyltransferase expression of oral cancer cell lines. (2004)
  • Author(s): Kawasaki S et al.
  • Journal Title: Oncol Rep. 12(2) Pages: 339-345
• [Journal Article] Effects of demethylating agent 5-aza-2(')-deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines. (2004)
  • Author(s): Kawasaki S et al.
  • Journal Title: Oral Oncol. 40(6) Pages: 597-603
• [Journal Article] pEYFP-Nuc vector is a useful tool for three-dimensional and time-lapse observation of nuclear morphology of Jurkat cells during apoptosis. (2004)
  • Author(s): Kawasaki S et al.
  • Journal Title: Int J Mol Med 13(2) Pages: 235-242
• [Journal Article] Cepharanthine enhances in vitro and in vivo thermosensitivity of a mouse fibrosarcoma, FSa-II, based on increased apoptosis. (2003)
  • Author(s): Kawasaki S et al.
  • Journal Title: Int J Mol Med 13(3) Pages: 405-411
• [Journal Article] A study on heat-induced changes in histone structure in two human oral cancer cell lines
  • Author(s): Keiji Matsuda, Shin-ya Nakagawa, Tomoya Nakano, Ganeth C.Jagetia, Shoji Kawasaki
  • Journal Title: Int J Hyperthermia (in press)
• [Journal Article] Effect of KMK437 on induction of Heat shock proteins with heating in human oral cancer cell lines
  • Author(s): Shin-ya Nakagawa, Keiji Matsuda, Tomoya Nakano, Ganeth C.Jagetia, Jun-ichi Asaumi, Shoji Kawasaki
  • Journal Title: JPN J Hyperthermic Oncology (in press)
• [Publications] Kawasaki S et al.: "Cepharanthine enhances in vitro and in vivo thermosensitivity of a mouse fibrosarcoma, FSa-II, based on increased apoptosis"Int J Mol Med.. 13(3). 405-411 (2004)
• [Publications] Kawasaki et al.: "pEYFP-Nuc vector is a useful tool for three-dimensional and time-lapse observation of nuclear morphology of Jurkat cells during apoptosis"Int J Mol Med.. 13(2). 235-242 (2004)
• [Publications] Kawasaki S. et al.: "65 H89, a protein kinase inhibitor, promotes the development of thermorolerance in"Jap.J.of Hyperthermia Oncol. 19(4). 187-194 (2003)
• [Publications] Kawasaki S. et al.: "Effects of p53 gene therapy in radiotherapy or thermotherapy of human head and neck squamous cell carcinoma cell lines"Oncol Rep.. 10(3). 671-677 (2003)