Neurological Surgery 脳神経外科35巻2号

2007年02月発行

総説

DNAメチル化剤temozolomideの分子薬理学

著者： 廣瀬雄一¹ 佐野公俊¹

所属機関： ¹藤田保健衛生大学脳神経外科

ページ範囲：P.117 - P.129

Ⅰ．はじめに

　悪性グリオーマは外科的手術による根治は困難であることが多く，その治療のうえでは化学療法と放射線療法を中心とした補助療法の併用が多くの場合行われている．しかし，現状では同腫瘍の根治に向けた有効な補助療法が確立しているとは言えず，治療開始後約1年以内に再発あるいは進行が認められるため，その有効性を改善することが必要である¹⁹⁾．従来，グリオーマに対する化学療法の中心的薬剤はニトロソウレア製剤であったが，その効果は決して満足できるものではなかった．これに対して，1990年代後半に新世代DNAメチル化剤temozolomide（TMZ）^8,63)が再発グリオーマに対する治療に導入されて好成績を挙げたことが欧米から報告されたことは，近年におけるグリオーマ治療のうえで大きな進歩の1つと考えられる．同剤は，放射線療法とニトロソウレア製剤による化学療法を施行した後の再発膠芽腫症例の半数以上において進行抑制を含む何らかの治療効果を示し^7,68,96)，従来の化学療法剤と比較すると骨髄抑制が弱く，しかも経口投与可能な薬剤であるため外来診療により治療を続けやすいという点からも，臨床的な有用性が注目された⁶⁶⁾．本邦においても2006年9月に認可され，今後，悪性グリオーマ治療における中心的薬剤となる可能性が高い．

　本稿では，TMZを用いた悪性グリオーマ治療の可能性と限界についての理解を通じてさらに治療法の進歩することを期待して，同剤に関する主に薬理学的，細胞生物学的な知見を紹介する．

参考文献

1）Alvarez B, Martinez AC, Burgering BM, Carrera AC：Forkhead transcription factors contribute to execution of the mitotic programme in mammals. Nature 413：744-747, 2001

2）Aquilina G, Crescenzi M, Bignami, M：Mismatch repair, G2/M cell cycle arrest and lethality after DNA damage. Carcinogenesis 20：2317-2325, 1999

3）Atherton-Fessler S, Liu F, Gabrielli B, Lee MS, Peng CY, Piwnica-Worms H：Cell cycle regulation of the p34cdc2 inhibitory kinases. Mol Biol Cell 5：989-1001, 1994

4）Backlund LM, Nilsson BR, Liu L, Ichimura K, Collins VP：Mutations in Rb1 pathway-related genes are associated with poor prognosis in anaplastic astrocytomas. Br J Cancer 93：124-130, 2005

5）Baer JC, Freeman AA, Newlands ES, Watson AJ, Rafferty JA, Margison GP：Depletion of O6-methylguanine-DNA alkyltransferase correlates with potentiation of temozolomide and CCNU toxicity in human tumor cells. Br J Cancer 67：1299-1302, 1993

6）Bobola MS, Tseng SH, Blank A, Bertger MS, Siber JR：Role of O6-methylguanine-DNA methyltransferase in resistance of human brain tumor cell lines to the clinically relevant methylating agents temozolomide and streptozotocin. Clin Cancer Res 2：735-741, 1996

7）Bower M, Newlands ES, Bleehen NM, Brada M, Begnet RJH, Calvert H, Colquhoun I, Lewis P, Brampton MH：Multicentre CRC phase II trial of temozolomide in recurrent or progressive high-grade glioma. Cancer Chemother Pharmacol 40：484-488, 1997

8）Brada M, Judson I, Beale P, Moore S, Reidenberg P, Statkevich P, Dugan M, Barta V, Cutler D：Phase I dose-escalation and pharmacokinetic study of temozolomide (SCH 52365) for refractory or relapsing malignancies. Br J Cancer 81：1022-1030, 1999

9）Branch P, Hampson R, Karran P：DNA mismatch repair binding defects, DNA damage tolerance and mutator phenotypes on human colorectal carcinoma cell lines. Cancer Res 55：2304-2309, 1995

10）Branch P, Aquilina G, Bignami M, Karran P：Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362：652-654, 1993

11）Brazil DP, Yang ZZ, Hemmings BA. Advances in protein kinase B signalling：AKTion on multiple fronts. Trends Biochem Sci 26：657-664, 2001

12）Brell M, Tortosa A, Verger E, Gil JM, Vinolas N, Villa S, Acebes JJ, Caral L, Pujol T, Ferrer I, Ribalta T, Graus F：Prognostic significance of O6-methylguanine-DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas. Clin Cancer Res 11：5167-5174, 2005

13）Bunch RT, Eastman A：Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporin (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 2：791-797, 1996

14）Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B：Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282：1497-1501, 1998

15）Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN：The radiosensitizing agent 7-hydroxystaurosporin (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 60：2108-2112, 2000

16）Chakravarti A, Zhai G, Suzuki Y, Sarkesh S, Black PM, Muzikansky A, Loeffler JS：The prognostic significance of phosphatidylinositol 3-kinase pathway activation in human gliomas. J Clin Oncol 22：1926-1933, 2004

17）Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B：14-3-3σis required to prevent mitotic catastrophe after DNA damage. Nature 401：616-620, 1999

18）Chang BD, Xuan Y, Broude EV, Zhu H, Schott B, Fang J, Roninson IB：Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs. Oncogene 18：4808-4818, 1999

19）Conrad CA, Milisavljevic VP, Yung WKA：Advances in chemotherapy for adult primary brain tumors. Neurol Clin 13：795-812, 1995

20）D’Atri S, Piccioni D, Castellano A, Tuorto V, Franchi A, Lu K, Christiansen N, Frankel S, Rustum YM, Papa G, Mandelli F, Bonmassar E：Chemosensitivity to triazene compounds and O6-alkylguanine-DNA alkyltransferase levels；studies with blasts of leukemic patients. Ann Oncol 6：389-393, 1995

21）D’Atri S, Tentori L, Lacal PM, Graziani G, Pagani E, Benincasa E, Zambruno G, Bonmassar E, Jiricny J：Involvement of the mismatch repair system in temozolomide-induced apoptosis. Mol Pharmacol 54：334-341, 1998

22）Denny BJ, Wheelhouse RT, Stevens MFG, Tsang LLH, Slack JA：NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry 33：9045-9051, 1994

23）Dimri GP, Lee X, Basile G：A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92：9363-9367, 1995

24）Eisenbrand G, Muller N, Denkel E, Sterzel W：DNA adducts and DNA damage by antineoplastic and carcinogenic N-nitrosocompounds. J Cancer Res Clin Oncol 112：196-204, 1986

25）Ermoian RP, Furniss CS, Lamborn KR, Basila D, Berger MS, Gottschalk AR, Nicholas MK, Stokoe D, Haas-Kogan DA：Dysregulation of PTEN and protein kinase B is associated with glioma histology and patient survival. Clin Cancer Res 8：1100-1106, 2002

26）Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, Baylin SB, Herman JG：Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 343：1350-1354, 2000

27）Friedman HS, Johnson SP, Dong Q, Schold SC, Rasheed BK, Bigner SH, Ali-Osman F, Dolan E, Colvin OM, Houghton P, Germain G, Drummond JT, Keir S, Marcelli S, Bigner DD, Modrich P：Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft. Cancer Res 57：2933-2936, 1997

28）Furnari B, Rhind N, Russell P：Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase. Science 277：1495-1497, 1997

29）Gerson SL：MGMT：its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4：296-307, 2004

30）Graves PR, Yu L, Schwarz JK, Gales J, Sausville EA, O’Conner PM, Piwnica-Worms H：The Chk1 protein kinase and the cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 275：5600-5605, 2000

31）Gu Y, Rosenblatt J, Morgan DO：Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J 11：3995-4005, 1992

32）Hawn MT, Umar A, Carethers JM, Kunkel TA, Boland CR, Koi M：Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint. Cancer Res 55：3721-3725, 1995

33）Hegi ME, Diserens A-C, Gorlia T, Hamou M-F, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JEC, Hau P, Mirimanoff RO, Cairncross G, Janzer RC, Stupp R：MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352：997-1003, 2005

34）Hickman MJ, Samson LD：Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc Natl Acad Sci U S A 96：10764-10769, 1999

35）Hirose Y, Berger MS, Pieper RO：p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 61：47-63, 2001

36）Hirose Y, Berger MS, Pieper RO：Abrogation of the Chk1-mediated G2 checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res 61：5843-5849, 2001

37）Hirose Y, Katayama M, Stokoe D, Haas-Kogan DA, Berger MS, Pieper RO：The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents. Mol Cell Biol 23：8306-8315, 2003

38）Hirose Y, Kreklau EL, Erickson LC, Berger MS, Pieper RO： Delayed repletion of O6-methylguanione-DNA methyltransferase resulting in failure to protect the human glioblastoma cells line SF767 from temozolomide-induced cytotoxicity. J Neurosurg 98：591-598, 2003

39）Hirose Y, Katayama M, Berger MS, Pieper RO：Cooperative function of Chk1 and p38 pathways in activating G2 arrest following exposure to temozolomide. J Neurosurg 100：1060-1065, 2004

40）Hirose Y, Katayama M, Mirzoeva OK, Berger MS, Pieper RO：Akt activation suppresses Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence. Cancer Res 65：4861-4869, 2005

41）Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN：Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet 25：55-57, 2000

42）Hunter C, Smith R, Cahill DP, Stephens P, Stevens C, Teague J, Greenman C, Edkins S, Bignell G, Davies H, O’Meara S, Parker A, Avis T, Barthorpe S, Brackenbury L, Buck G, Butler A, Clements J, Cole J, Dicks E, Forbes S, Gorton M, Gray K, Halliday K, Harrison R, Hills K, Hinton J, Jenkinson A, Jones D, Kosmidou V, Laman R, Lugg R, Menzies A, Perry J, Petty R, Raine K, Richardson D, Shepherd R, Small A, Solomon H, Tofts C, Varian J, West S, Widaa S, Yates A, Easton DF, Riggins G, Roy JE, Levine KK, Mueller W, Batchelor TT, Louis DN, Stratton MR, Futreal PA, Wooster R：A hypermutation phenotype and somatic MSH6 mutations in recurrent human malignant gliomas after alkylator chemotherapy. Cancer Res 66：3987-3991, 2006

43）Ianzini F, Mackey MA：Spontaneous premature chromosome condensation and mitotic catastrophe following irradiation of HeLa S3 cells. Int J Radiat Biol 72：409-421, 1997

44）Ichimura K, Bolin MB, Goike HM, Schmidt EE, Moshref A, Collins VP：Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite for human astrocytic gliomas with G1-S transition control gene abnormalities. Cancer Res 60：417-424, 2000

45）Innocente SA, Abrahamson JLA, Cogswell JP, Lee JM：p53 regulates a G2 checkpoint through cyclin B1. Proc Natl Acad Sci U S A 96：2147-2152, 1999

46）Kanamori M, Kon H, Nobukuni T, Nomura S, Sugano K, Mashiyama S, Kumabe T, Yoshimoto T, Meuth M, Sekiya T, Murakami Y：Microsatellite instability and PTEN1 gene mutation in a subset of early onset gliomas carrying germline mutation or promoter methylation of the hMLH1 gene. Oncogene 19：1564-1571, 2000

47）Kandel ES, Skeen J, Majewski N, DiCristofano A, Pandolfi PP, Felliciano CS, Gartel A, Hay N：Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol Cell Biol 22：7831-7841, 2002

48）Kanzawa T, Bedwell J, Kondo Y, Kondo S, Germano IM：Inhibition of DNA repair for sensitizing resistant glioma cells to temozolomide. J Neurosurg 99：1047-1052, 2003

49）Karran P, Marinus MG：Mismatch correction at O6-methylguanine residues in E. coli DNA. Nature 296：868-869, 1982

50）Karran P, Bignami M：DNA damage tolerance, mismatch repair and genomic instability. BioEssays 16：833-839, 1994

51）Karran P, Bignami M：Self-destruction and tolerance in resistance of mammalian cells to alkylation damage. Nuc Acids Res 20：2933-2940, 1992

52）Kat A, Thilly WG, Fang WH, Longley MJ, Li G.M, Modrich P： An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc Natl Acad Sci U S A 90：6424-6428, 1993

53）Katayama M, Kawaguchi T, Berger MS, Pieper RO：DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ. in press

54）King RW, Jackson PK, Kirschner MW：Mitosis in transition. Cell 79：563-571, 1994

55）Levati L, Marra G, Lettieri T, D’Atri S, Vernole P, Tentori L, Lacal PM, Pagani E, Bonmassar E, Jiriciny J, Graziani G：Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents. Genes Chromosom Cancer 23：159-166, 1998

56）Lieung, SY, Chan TL, Chung LP, Chan ASY, Fan YW, Hung KN, Kwong WK, Ho JWC, Yuen ST：Microsatellite instability and mutation of DNA mismatch repair genes in gliomas. Am J Pathol 153：1181-1188, 1998

57）Liu L, Gerson SL：Targeted modulation of MGMT：clinical implications. Clin Cancer Res 12：328-331, 2006

58）Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ：Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14：1448-1459, 2000

59）Louis DN, von Deimling A, Chung RY, Rubio MP, Whaley JM, Eibl RH, Ohgaki H, Wiestler OD, Thor AD, Seizinger BR：Comparative study of p53 gene and protein alterations in human astrocytic tumors. J Neuropathol Exp Neurol 53：31-38, 1993

60）Malaise EP, Fertil B, Chavaudra N, Guichard M：Distribution of radiation sensitivities for human tumor cells of specific histological types：comparison of in vitro to in vivo data. Int J Radiation Oncology Bio Phys 12：617-624, 1986

61）Mason WP, Cairncross JG：Drug Insight：temozolomide as a treatment for malignant glioma--impact of a recent trial. Nat Clin Pract Neurol 1：88-95, 2005

62）Matsuoka S, Huang M, Elledge SJ：Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282：1893-1897, 1998

63）Newlands ES, Blackledge GRP, Slack JA, Rustin GJS, Smith DB, Stuart NSA, Quarterman CP, Hoffman R, Stevens MFG, Brampton MH, Gibson AC：Phase I trial of temozolomide (CCR 81045：M＆B 39831：NSC 362856). Br J Cancer 65：287-291, 1992

64）Nurse P：Checkpoint pathways come of age. Cell 91：865-867, 1997

65）O’Connell MJ, Walworth NC, Carr AM：The G2-phase DNA-damage checkpoint. Trends Cell Biol 10：296-303, 2000

66）Osoba D, Braba M, YungWKA, Prados MD：Health-related quality of life in patients treated with temozolomide versus procarbazine for recurrent glioblastoma multiforme. J Clin Oncol 18：1481-1491, 2000

67）Passalaris TM, Benanti JA, Gewin L, Kiyono T, Galloway DA：The G2 checkpoint is maintained by redundant pathways. Mol Cell Biol 19：5872-5881, 1999

68）Paulsen F, Hoffmann W, Becker G, Belka C, Weinmann M, Classes J, Kortmann RD, Bamberg M：Chemotherapy in the treatment of recurrent glioblastoma multiforme：isofamide versus temozolomide. J Cancer Res Clin Oncol 125：411-418, 1999

69）Pegg AE, Wiest L, Foote RS, Mitra S, Perry W：Purification and properties of O-6-methylguanine-DNA transmethylase from rat liver. J Biol Chem 258：2327-2333. 1983

70）Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms H：Mitotic and G2 checkpoint control：regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277：1501-1505, 1997

71）Pieper RO：Understanding and manipulating O6-methylguanine-DNA methyltransferase expression. Pharmacol Ther 74：285-297, 1997

72）Quinn JA, Desjardins A, Weingart J, Brem H, Dolan ME, Delaney SM, Vredenburgh J, Rich J, Friedman AH, Reardon DA, Sampson JH, Pegg AE, Moschel RC, Birch R, McLendon RE, Provenzale JM, Gururangan S, Dancey JE, Maxwell J, Tourt-Uhlig S, Herndon JE 2nd, Bigner DD, Friedman HS：Phase I trial of temozolomide plus O6-benzylguanine for patients with recurrent or progressive malignant glioma. J Clin Oncol 23：7178-7187, 2005

73）Rabik CA, Njoku MC, Dolan ME：Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy. Cancer Treat Rev 32：261-276, 2006

74）Saito R, Bringas JR, McKnight TR, Wendland MF, Mamot C, Drummond DC, Kirpotin DB, Park JW, Berger MS, Bankiewicz KS：Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging. Cancer Res 64：2572-2579, 2004.

75）Saito R, Bringas JR, Panner A, Tamas M, Pieper RO, Berger MS, Bankiewicz KS：Convection-enhanced delivery of tumor necrosis factor-related apoptosis-inducing ligand with systemic administration of temozolomide prolongs survival in an intracranial glioblastoma xenograft model. Cancer Res 64：6858-6862, 2004

76）Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ：Conservation of the Chk1 checkpoint pathway in mammals：linkage of DNA damage to Cdk regulation through Cdc25. Science 277：1497-1501, 1997

77）Sausville EA, Lush RD, Headlee D, Smith AC, Figg WD, Arbuck SG, Senderowicz AM, Fuse E, Tanii H, Kuwabara T, Kobayashi S：Clinical pharmacology of UCN-01：initial observations and comparison to preclinical models. Cancer Chemother Pharmacol 42 Suppl：S54-59, 1998

78）Sausville EA, Johnson J, Alley M, Zaharevitz D, Senderowicz AM：Inhibition of CDKs as a therapeutic modality. Ann N Y Acad Sci 910：207-221, 2000

79）Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM：The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63：1129-36, 1990

80）Schold SC Jr, Kokkinakis DM, Chang SM, Berger MS, Hess KR, Schiff D, Robins HI, Mehta MP, Fink KL, Davis RL, Prados MD：Phase I trial of temozolomide plus O6-benzylguanine for patients with recurrent or progressive malignant glioma. Neuro-oncology 6：28-32, 2004

81）Shao RG, Cao CX, Shimizu T, O’Connor PM, Kohn KW, Pommier Y：Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function. Cancer Res 57：4029-4035, 1997

82）Shtivelman E, Sussman J, Stokoe D：A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle. Curr Biol 12：919-924, 2002

83）Sidransky D, Mikkelsen T, Schwechheimer K, Rosenblum ML, Cavenee WK, Vogelstein B：Clonal expansion of p53 mutant cells is associated with brain tumor progression. Nature 355：846-847, 1992

84）Sonoda Y, Ozawa T, Aldape KD, Deen DF, Berger MS, Pieper RO：Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma. Cancer Res 61：6674-6678, 2001

85）Steck PA, Pershouse M, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DH, Tavtigian SV：Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15：356-362, 1997

86）Sugiyama K, Shimizu M, Akiyama T, Tamaoki T, Yamaguchi K, Takahashi R, Eastman A, Akinaga S：UCN-01 selectively enhances mitomycin C cytotoxicity in p53 defective cells which is mediated through S and/or G2 checkpoint abrogation. Int J Cancer 85：703-709, 2000

87）Tentori L, Graziani G, Gilberti S, Lacal PM, Bonmassar E, D’Atri S：Triazene compounds induce apoptosis in O6-alkylguanine-DNA alkyltransferase deficient leukemia cell lines. Leukemia 9：1888-1895, 1995

88）Tentori L, Turriziani M, Franco D, Levati L, Roy R, Bonmassar E, Graziani G：Treatment with temozolomide and poly (ADP-ribose) polymerase inhibitors induces early apoptosis and increase base excision repair gene transcripts in leukemic cells. Leukemia 13：901-909, 1999

89）Trepel M, Scheding S, Groscurth P, Horny HP, Malipiero U, Brugger W, Dichgans J, Weller M：A new look at the role of p53 in leukemia cell sensitivity to chemotherapy. Leukemia 11：1842-9, 1997

90）Tsang LLH, Farmer PB, Gescher A, Slack JA：Characterization of urinary metabolites of temozolomide in humans and mice and evaluation of their cytotoxicity. Cancer Chemother Pharmacol 26：429-436, 1990

91）Watanabe K, Sato K, Biernat W, Tachibana O, von Ammon K, Ogata N, Yonekawa, Y, Kleihues P, Ohgaki H：Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies. Clin Cancer Res 3：523-30, 1997

92）Van Meir EG, Kikuchi T, Tada M, Li H, Diserens A-C, Wojcik BE, Huang H-JS, Friedman T, de Tribolet N, Cavenee WK：Analysis of the p53 gene and its expression in human glioblastoma cells. Cancer Res 54：649-652, 1994

93）Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O’Conner PM：UCN-01：a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst 88：956-965, 1996

94）Wiencke JK, Wiemels J：Genotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Mutat Res 339：91-111, 1995

95）Yin MB, Guo B, Vanhoefer U, Azrak RG, Minderman H, Frank C, Wrzosek C, Slocum HK, Rustum YM：Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a novel topoisomerase I inhibitor BNP1350. Mol Pharmacol 57：453-459, 2000

96）Yung WKA, Prados MD, Yaya-Tur R, Rosenfeld SS, Braba M, Friedman HS, Albright J, Olson J, Chang SM, O’Neill AM, Friedman AH, Bruner J, Yue N, Dugan M, Zaknoen S, Levin VA：Multicenter phase II trial of temozolomide in patients with anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. J Clin Oncol 12：2762-2771, 1999

97）Zeng Y, Forbes KC, Wu Z, Moreno S, Piwnica-Worms H, Enoch T：Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature 395：507-510, 1998

98）Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, Bajraszewski N, Vazquez F, Carpenter CL, Kwiatkowski DJ：Loss of Tsc1/ Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 12：1223-1233, 2003