Free full text available from Original Article www.cancerjournal.net

The role of the molecule in cancer therapies with radiation and/or hyperthermia

ABSTRACT Takeo Ohnishi In recent years, cancer-related genes have been analyzed as predictive indicators for cancer therapies. Among those genes, the Department of Biology, Nara Medical University gene product of a tumor suppressor gene p53 plays an important role in cancer therapy, because the p53 molecule induces ­ School of Medicine, cycle arrest, and depression of DNA repair after cancer therapies such as radiation, hyperthermia and anti-cancer agents. Kashihara, Nara 634­ An abnormality of the p53 gene might introduce low efficiency in their cancer therapies. Mutations of p53 are observed at a high 8521, Japan. frequency in human tumors, and are recognized in about half of all malignant tumors in human. In the both systems of a human For correspondence: cell culture and their transplanted tumor, the sensitivities to radiation, heat and anti-cancer agents were observed in wild-type p53 Ohnishi T, Department cells, but not in mutated or deleted p53 cells. In this review, we discuss the p53 activation signaling pathways through the of Biology, Nara Medical University modification of p53 molecules such as phosphorylation after radiation and/or hyperthermia treatments. School of Medicine, Shijo-cho 840, Kashihara, Nara 634­ Key words: p53, Predictive indicator, Cancer therapy, X-ray, Hyperthermia, Apoptosis 8521, Japan. E-mail: tohnishi@naramed­ u.ac.jp

INTRODUCTION radio-, heat- and chemo-sensitivities of human cultured tongue squamous cell carcinoma cells are Combinations of radiation and hyperthermia p53-dependent, and are closely correlated with therapies have been widely adopted for interdis­ the induction of apoptosis in in vitro[11-13] and in ciplinary cancer therapy. It is well known that vivo.[14-16] The interactive hyperthermic enhance­ ionizing radiation-induced cell killing is enhanced ment of radiosensitivity was also observed in by hyperthermia in vitro.[1,2] Previous studies have wtp53 cells, but not in mutated p53 (mp53) shown that heat treatment depresses the DNA cells.[17] However, it remains unclear whether the repair of radiation-induced DNA strand breaks hyperthermic enhancement of tumor growth in­ and thymine lesions.[3,4] In addition, it has been hibition by irradiation is p53-dependent. To clarify reported that the activities of DNA polymerases, the problem, we described in this review whether α and β, are sensitive to heat treatment at tem­ the p53 gene products contribute to the hyper­ peratures higher than 40°C.[5,6] Thus, it has been thermic enhancement of tumor growth inhibition understood that the synergistic effects of hyper­ by X-ray irradiation, using transplantable human thermia on radiation-induced cell killing is in­ cultured tongue squamous cell carcinoma cells duced mainly through the inhibition of DNA re­ with an identical genotype except for the p53 pair mechanisms. Moreover, another possible gene status, as an in vivo experimental model mechanism of radiosensitization by hyperthermia from the view point of p53 activation and deacti­ has been suggested to involve the hyperthermic vation through the modification and degradation instability of the Ku subunits of DNA-PK, which of p53 molecules. contribute to the repair of radiation-induced dou­ ble-strand breaks in DNA.[7, 8] p53 and the Signal Pathways

Patients with tumors that have p53 mutations The ancestor of the mammalian p53 tumor sup­ often have a worse prognosis than those with pressor is homologous to drosophila and tumors that don’t have wild-type p53 (wtp53).[9] nematodes.[18] The gene product prevents the For prognosis-predictive assays of cancer therapy, malignant degeneration of a normal cell. In can­ the genetic status of the p53 gene is the most cer cells bearing mp53, genetic stability is lost and important candidate among various cancer-re­ mutations are accumulated, and therefore, ma­ lated genes.[10] We previously reported that the lignant changes in the cancer progress at a high

J Cancer Res Ther - September 2005 - Volume 1 - Issue 3 147 Ohnishi T, et al: The p53 gene status in cancer therapies frequency. p53-mutant and p53-deleted cells account for is enhanced.[40] In addition, there are several reports regard­ about 50% of total advanced cancer cells.[19] The p53 mol­ ing the sumoylation of lysine 386 in p53, which seems to ecules are modified for activation by many kinds of differ­ activate a p53-downstream transcriptional factor.[33, 34, 41-43] ent protein kinases at different portions after cell stress [Fig­ In these modifications, it is thought that the structural ure 1].[20] change of p53 molecules, which bind to specific DNA se­ quences known as p53 CON, is brought about by many kinds p53 regulates the transcription of the phenotypic expres­ of the up-stream genes. On the other hand, p53 molecules sions of target genes by binding to a specific sequence. One are deactivated and degradated by activated MDM2 mol­ of the p53 target genes, WAF1 (wild-type p53 activated frag­ ecules, which are phosphorylated at multi-sites by other ment 1) inactivates PCNA regulating DNA replication,[21] and protein kinases [Figure 1]. induces p53-dependent G1 arrest through the inhibition of cyclin/CDK activity.[22, 23] During arrest, p53-regu­ Moreover, p53 is reported to bind to other , such as lated pathways, such as gadd45 (growth arrest and DNA heat shock proteins (HSPs), which are a famous stress pro­ damage inducible 45) and p53R2 (ribonucleotide reductase tein.[44, 45] Thus, p53 regulates the fate of the cells after small subunit 2), play an important role in the repair of dam­ stresses, such as cancer therapies. aged DNA.[24, 25] Otherwise, DNA damage induces apoptosis by several processes of p53-regulated pathways, such as p53 Dependent Synergism after Hyperthermia and X-ray Bax (Bcl-associated X protein),[26] Fas/APO-1,[27] and PAG608.[28] Treatment In contrast, p53-regulated MDM 2 (murine double minute 2)[29] functions in the negative feedback regulation of p53 We previously reported that radio- and heat-sensitivities of activity.[26] It is reported that these determinations of cell cultured human tongue squamous cell carcinoma cells are cycle arrest or apoptosis are divided by the modification of p53-dependent, and are closely correlated with the induc­ p53 molecules, such as phosphorylation (Figure 1),[30] acetyla­ tion of apoptosis in vitro.[11, 12] To confirm that the hyper­ tion,[31] poly (ADP-ribosyl)ation,[32] and sumoylation[33,34]. thermic enhancement of tumor growth inhibition by X-ray When the cells are stressed, Ser15/Ser20 of p53 is phospho­ irradiation is dependent on the p53 gene status, we used rylated, then MDM 2 is separated from the phosphorylated two kinds of cancer cell lines carrying a different p53 gene p53, and finally p53 is stabilized and activated.[35-37] There­ status, wtp53 and mp53. We compared heat and X-ray in­ after, p53 binds to the promoter of WAF1 or the p53R2 gene duced cell killing and apoptosis frequencies in the wtp53 related to DNA repair, and induces their gene expression. cells and the mp53 cells though Bax and Caspase-3 path­ [11, 12] When there are too many DNA lesions, however, G1 arrest ways. We adopted mild treatments with radiation and and DNA repair do not succeed. In this case, p53 is phos­ hyperthermia for transplantation on nude mice to examine phorylated at Ser46, and then binds to the promoter of the p53AIP1 (p53-regulated Apoptosis- Inducing Protein 1) gene. Therefore, p53AIP1 is accumulated. Apoptosis happens in the damaged cells.[38, 39] Other p53 modifications of acetyla­ tion and sumoylation have also been reported. It is under­ stood that acetylation is performed at the C-terminal re­ gion of p53, and the binding capacity of p53 to specific DNA

Figure 2: A, tumor growth curves of human tongue carcinomas. non-treated group � hyperthermia at 42°C for 20 min alone X-rays at 2 Gy alone combined treatment with X-rays at 2 Gy and hyperthermia at 42°C for 20 min. B, The rate of cells positively stained for apoptosis between SAS/neo tumors (closed column) and SAS/ mp53 tumors (open column) 72 h after treatment. In all cases, three individuals, who were blind to the source of the specimen, counted a Figure 1: p53-centered signal transduction pathway, accelerate, total of 500 cells in three random fields. The error bars indicate SD.*, suppress, phosphate a highly significant difference (P < 0.01) by Student’s t-test

148 J Cancer Res Ther - September 2005 - Volume 1 - Issue 3 Ohnishi T, et al: The p53 gene status in cancer therapies the synergistic effects on tumor growth inhibition. Tumor growth curves are shown in [Figure 2A].

We applied an individual treatment with hyperthermia at 42°C for 20 min or X-irradiation (2 Gy) which showed almost no effect on the growth curves of the both wtp53 and mp53 tumors in the non-treated control groups [Figure 2A]. When the tumors were treated with a combination of X-rays and hyperthermia, the apparent enhancement of tumor growth inhibition was observed in the wtp53 tumors, but not the mp53 tumors.

We examined both the accumulation of apoptosis-related proteins and the incidence of apoptosis by immunohisto­ chemical analysis [Figure 2B]. The apoptosis incidence was apparently higher in the wtp53 tumors 72 h after the com­ Figure 3: Apoptosis pathway induced by radiation, hyperthermia and bined treatment, but not in the mp53 tumors. The fragmen­ anti-cancer agents tation of PARP and Caspase-3 as the activation of Caspase­ 3 showed almost the same pattern as the incidence of REFERENCES [46] apoptosis in the wtp53 tumor. 1. Belli J, Bonte F. Influence of temperature on the radiation response of mammalian cells in tissue culture. Radiat Res 1963;18:272-6. p53 Gene Status as a Predictive Indicator in Cancer 2. Ben-Hur E, Elkind MM, Bronk BV. Thermally enhanced radioresponse Therapy of cultured Chinese hamster cells: inhibition of repair of sublethal damage and enhancement of lethal damage. Radiat Res 1974;58:38­ As previously reported, if the hyperthermic enhancement 51. 3. Clark EP, Dewey WC, Lett JT. Recovery of CHO cells from hyperthermic of radiosensitivity results in heat-induced denaturation of potentiation to X-rays repair of DNA and chromatin. Radiat Res repair enzymes for DNA damage, the synergism of the com­ 1981;85:302-13. bination therapy of radiation and hyperthermia should be 4. Warters RL, Roti Roti JL. Excision of X-ray-induced thymine damage similarly detected in both wtp53 and mp53 tumors. How­ in chromatin from heated cells. Radiat Res 1979;79:113-21. ever, it is very interesting that the synergistic depression 5. Raaphorst GP, Feeley MM, Chu GL, Dewey WC. A comparison of the of tumor growth was found only in the wtp53 tumors46). enhancement of radiation sensitivity and DNA polymerase inactivation by hyperthermia in human glioma cells. Radiat Res These findings suggest that the hyperthermic enhance­ 1993;134:331-6. ment of tumor growth inhibition by irradiation may result 6. Dube DK, Seal G, Loeb LA. Differential heat sensitivity of mammalian in p53-dependent apoptosis due to heat-induced inactiva­ DNA polymerases. Biochem Biophys Res Comm 1976;76:483-7. tion of the cell survival system (s), through either regula­ 7. Matsumoto Y, Suzuki N, Sakai K, Morimatsu A, Hirano K, Murofushi tion of the cell cycle or induction of DNA repair. Thus, mp53 H. A possible mechanism for hyperthermic radiosensitization mediated through hyperthermic lability of Ku subunits in DNA­ tumors may rarely include this function, which promotes dependent protein kinase. Biochem Biophys Res Comm cell killing by the heat-induced interactive inactivation of 1997;234:568-72. the repair enzyme (s) for certain types of sublethal dam­ 8. Burgman P, Ouyang H, Peterson S, Chen D J, Li GC. Heat inactivation age. As participants in the p53-dependent apoptotic proc­ of Ku autoantigen: possible role in hyperthermic radiosensitization. esses, the induction of apoptosis mediated by caspase-3 Cancer Res 1997;57:2847-50. activation was examined in these tumors. In fact, activated 9. Lowe SW. Cancer therapy and p53. Curr. Opin. Oncol 1995;7:547-53. 10. Velculescu VE, El-Deiry WS. Biological and clinical importance of Caspase-3 was confirmed from the proteolysis of several the p53 tumor suppressor gene. Clin Chem 1996;42:858-68. important molecules, such as PARP and Caspase-3 itself so 11. Ota I, Ohnishi K, Takahashi A, Yane K, Kanata H, Miyahara H, et al. called markers of apoptosis.[47,48] The induction of p53-de­ Transfection with mutant p53 gene inhibits heat-induced apoptosis pendent apoptosis showed a pattern similar to the tumor in a head and neck cell line of human squamous cell carcinoma. Int growth inhibition after combined treatments with radia­ J Radiat Oncol Biol Phys 2000;47:495-501. 12. Takahashi A. Different inducibility of radiation- or heat-induced tion and hyperthermia [Figure 3]. Therefore, gene analysis p53-dependent apoptosis after acute or chronic irradiation in human of the p53 gene status of cancer cells can be used as a pre­ cultured squamous cell carcinoma cells. Int J Radiat Biol dictive assay for the effectiveness of combined therapy with 2001;77:215-24. radiation and hyperthermia [Figure 3]. 13. Ohnishi K, Ota I, Takahashi A, Yane K, Ohnishi T. Transfection of mutant p53 gene depresses X-ray- or CDDP-induced apoptosis in a ACKNOWLEDGMENT human squamous cell carcinoma of the head and neck. Apoptosis 2002;7:367-72. 14. Asakawa I, Yoshimura H, Takahashi A, Ohnishi K, Nakagawa H, Ota This work was supported by Grants-in-Aid from the Minis­ I, et al. Growth inhibition in transplanted human tongue tumors try of Education, Science, Sports and Culture of Japan. bearing different p53 gene status with X-ray or C-beam irradiation.

J Cancer Res Ther - September 2005 - Volume 1 - Issue 3 149 Ohnishi T, et al: The p53 gene status in cancer therapies

Anticancer Res 2002;22:2037-43. 32. Valenzuela MT, Guerrero R, Nunez MI, Ruiz De Almodovar JM, Sarker 15. Tamamoto T, Yoshimura H, Takahashi A, Asakawa I, Ota I, Nakagawa M, de Murcia G, et al. PARP-1 modifies the effectiveness of p53­ H, et al. Heat-induced growth inhibition and apoptosis in mediated DNA damage response. Oncogene 2002;21:1108-16. transplanted human head and neck squamous cell carcinomas with 33. Schmidt D, Muller S. Members of the PIAS family act as SUMO different p53 status. Int J Hyperthermia 2003;19:590-7. ligases for c-Jun and p53 and repress p53 activity. Proc Natl Acad 16. Yuki K, Takahashi A, Ota I, Ohnishi K, Yasumoto J, Yane K, et al. Sci USA 2001;99:2872-7. ensitization by glycerol for CDDP-therapy against human cultured 34. Melchior F, Hengst L. SUMO-1 and p53. Cell Cycle 2002;1:245-9. cancer cells and tumors bearing mutated p53 gene. Apoptosis 35. Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, Kastan MB. 2004;9:853-9. DNA damage induces phosphorylation of the amino terminus of 17. Takahashi A, Ohnishi K, Ota I, Asakawa I, Tamamoto T, Furusawa Y, p53. Genes Dev 1997;11:3471-81. et al. p53-dependent thermal enhancement of cellular sensitivity 36. Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human homologs of in human squamous cell carcinomas in relation to LET. Int J Radiat checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at Biol 2001;77:1043-51. multiple DNA damage-inducible sites. Genes Dev 2000;14:289-300. 18. Derry WB, Putzke AP, Rothman JH. Caenorhabditis elegans p53: role 37. Urban G, Golden T, Aragon IV, Cowsert L, Cooper SR, Dean NM, et al. in apoptosis, meiosis, and stress resistance. Science 2001;294:591­ Identification of a functional link for the p53 tumor suppressor 5. protein in dexamethasone-induced growth suppression. J Biol Chem 19. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutation in 2003;278:9747-53. human cancers. Science 1991;253:49-53. 38. Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, et al. 20. Wang X, Ohnishi T. p53-dependent signal transduction induced by p53AIP1, a potential mediator of p53-dependent apoptosis, and its stress. J Radiat Res 1997;38:179-94. regulation by Ser-46-phosphorylated p53. Cell 2000;102:849-62. 21. Bambara RA, Jessee CB. Properties of DNA polymerases delta and 39. Saito S, Goodarzi AA, Higashimoto Y, Noda Y, Lees-Miller SP, Appella epsilon, and their roles in eukaryotic DNA replication. Biochim E, et al. ATM mediates phosphorylation at multiple p53 sites, Biophys Acta 1991;1088:11-24. including Ser (46), in response to ionizing radiation. J Biol Chem 22. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, 2002;277:12491-4. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 40. Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding 1993;75:817-25. by acetylation of the p53 C-terminal domain. Cell 1997;90:595­ 23. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/ 606.

WAF1 undergo normal development, but are defective in G1 41. Zhong S, Muller S, Ronchetti S, Freemont PS, Dejean A, Pandolfi PP. checkpoint control. Cell 1995;82:675-84. Role of SUMO-1-modified PML in nuclear body formation. Blood 24. Kastan MB, Zhan Q, El-Deiry WS, Carrier F, Jacks T, Walsh WV, et al. A 2000;95:2748-52. mammalian cell cycle checkpoint pathway utilizing p53 and 42. Rodriguez MS, Desterro JM, Lain S, Midgley CA, Lane DP, Hay RT. GADD45 is defective in ataxia-telangiectasia. Cell 1992;71:587-97. SUMO-1 modification activates the transcriptional response of p53. 25. Smith ML, Kontny HU, Zhan Q, Sreenath A, O’Connor PM, Fornace A EMBO J 1999;18:6455-61. J Jr. Antisense GADD45 expression results in decreased DNA repair 43. Gostissa M, Hengstermann A, Fogal V, Sandy P, Schwarz SE, Scheffner and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene M, et al. Activation of p53 by conjugation to the ubiquitin-like 1996;13:2255-63. protein SUMO-1. EMBO J 1999;18:6462-71. 26. Miyashita T, Reed JC. Tumor suppressor p53 is a direct 44. Ohnishi T, Matsumoto H, Takahashi A, Shimura M, Majima H. transcriptional activator of the human bax gene. Cell 1995;80:293­ Accumulation of mutant p53 and hsp72 by heat treatment, and 9. their association in a human glioblastoma cell line. Int J 27. Owen-Schaub LB, Zhang W, Cusack JC, Angelo LS, Santee SM, Fujiwara Hyperthermia 1995;11:663-71. T, et al. Wild-type human p53 and a temperature-sensitive mutant 45. Matsumoto H, Wang X, Ohnishi T. Binding between wild-type p53 induce Fas/APO-1 expression. Mol Cell Biol 1995;15:3032-40. and hsp72 accumulated after UV and gamma-ray irradiation. Cancer 28. Israeli D, Tessler E, Haupt Y, Elkeles A, Wilder S, Amson R, et al. A Lett 1995;92:127-33. novel p53-inducible gene, PAG608, encodes a nuclear zinc finger 46. Takahashi A, Ota I, Tamamoto T, Asakawa I, Nagata Y, Nakagawa H, protein whose overexpression promotes apoptosis. EMBO J et al. p53-dependent hyperthermic enhancement of tumour growth 1997;16:4384-92. inhibition by X-ray or carbon-ion beam irradiation. Int J 29. Haupt S, Louria-Hayon I, Haupt Y. P53 licensed to kill? Operating Hyperthermia 2003;19:145-53. the assassin. J Cell Biochem 2003;88:76-82. 47. Streffer C, van Beuningen D. The biological basis for tumour therapy 30. Barak Y, Oren M. Enhanced binding of a 95 kDa protein to p53 in by hyperthermia and radiation. Recent Results Cancer Res 1987;104, cells undergoing p53-mediated growth arrest. EMBO J 24-70. 1992;11:2115-21. 48. Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. 31. Brooks CL, Gu W. Ubiquitination, phosphorylation and acetylation: Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an the molecular basis for p53 regulation. Curr Opin Cell Biol early marker of chemotherapy-induced apoptosis. Cancer Res 1993; 2003;5:164-71. 53:3976-85.

150 J Cancer Res Ther - September 2005 - Volume 1 - Issue 3