Analysis of gene expression in apoptosis of human lymphoma U937 cells induced by heat shock and the effects of α-phenyl N-tert-butylnitrone (PBN) and its derivatives
Hyperthermia, a modality of cancer therapy, has been known as a stress to induce apoptosis. However, the molecular mechanism of heat shock-induced apoptosis, especially on roles of intracellular oxidative stress, is not fully understood. First, when human lymphoma U937 cells were treated with heat shock (44∘C, 30 min), the fraction of apoptosis, revealed by phosphatidylserine externalization, increased gradually and peaked at 6 hr after the treatment. In contrast, intracellular superoxide formation increased early during the heat shock treatment and peaked at 30 min after the treatment. When the cells were treated with heat shock in the presence of α -phenyl-N-tert-butylnitrone (PBN) and its derivatives, which are potent antioxidants, the DNA fragmentation was inhibited in an order according to the agents’ hydrophobicity. PBN showing the highest inhibitory effects suppressed not only intracellular superoxide formation but also various apoptosis indicators. cDNA microarray was employed to analyze gene expression associated with heat shock-induced apoptosis, and the time-course microarray analysis revealed 5 groups showing changes in their pattern of gene expression. Among these genes, c- jun mRNA expression showed more than 40 fold increase 2 hr after heat treatment. The expression level of c-jun mRNA verified by quantitative real-time PCR was about 20 fold increase, and c- jun expression was similarly suppressed by PBN and its derivatives. These results suggest that the change of c- jun expression is an excellent molecular marker for apoptosis mediated by intracellular oxidative stress induced by heat shock.
Keywordsapoptosis c- jun gene expression heat shock hyperthermia microarray PBN real-time PCR superoxide
Overgaard J. The current and potential role of hyperthermia in radiotherapy. Int J Radiat Oncol Biol Phys
: 535–549.PubMedGoogle Scholar
van der Zee J, Gonzalez Gonzalez D, van Rhoon GC, van Dijk JD, van Putten WL, Hart AA. Comparison of radiotherapy alone with radiotherapy plus hyperthermia in locally advanced pelvic tumours: A prospective, randomised, multicentre trial. Dutch Deep Hyperthermia Group. Lancet
: 1119–1125.PubMedGoogle Scholar
van der Zee J, Gonzalez Gonzalez D. The Dutch deep hyperthermia trial: Results in cervical cancer. Int J Hyperthermia
: 1–12.PubMedGoogle Scholar
Harima Y, Nagata K, Harima K, Ostapenko VV, Tanaka Y, Sawada S. A randomized clinical trial of radiation therapy versus thermoradiotherapy in stage IIIB cervical carcinoma. Int J Hyperthermia
: 97–105.PubMedGoogle Scholar
Kameda K, Kondo T, Tanabe K, Zhao QL, Seto H. The role of intracellular Ca (2+) in apoptosis induced by hyperthermia and its enhancement by verapamil in U937 cells. Int J Radiat Oncol Biol Phys
: 1369–1379.PubMedGoogle Scholar
Li FJ, Kondo T, Zhao QL, et al.
Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells. Free Radic Res
: 281–299.PubMedGoogle Scholar
Janzen EG. Spin trapping. Account Chem Res
: 31–37.Google Scholar
Kotake Y. Pharmacologic properties of phenyl N-tert-
buthylnitrone. Antioxidant Redox Signal 1999; 1
: 481–499.CrossRefGoogle Scholar
Gorman A, McGowan A, Cotter TG. Role of peroxide and superoxide anion during tumour cell apoptosis. FEBS Lett
: 27–33.PubMedGoogle Scholar
Sellins KS, Cohen JJ. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol
: 3199–3206.PubMedGoogle Scholar
Hopcia KL, McCarey YL, Sylvester FC, Held KD. Radiation-induced apoptosis in HL60 cells: Oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose. Radiat Res
: 315–323.PubMedGoogle Scholar
Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol
: 302–310.PubMedCrossRefGoogle Scholar
Reddy MV, Gangadharam PR. Heat shock treatment of macrophages causes increased release of superoxide anion. Infect Immun
: 2386–2390.PubMedGoogle Scholar
Galan A, Garcia-Bermejo L, Troyano A, et al.
The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays). Eur J Cell Biol
: 312–320.PubMedGoogle Scholar
Bauer G. Reactive oxygen and nitrogen species: Efficient, selective, and interactive signals during intercellular induction of apoptosis. Anticancer Res
: 4115–4139.PubMedGoogle Scholar
Moncada S, Erusalimsky JD. Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat Rev Mol Cell Biol
: 214–220.PubMedGoogle Scholar
Yamashita T, Ohshima H, Asanuma T, et al.
The effects of α-phenyl-tert
-butyl nitrone (PBN) on copper-induced rat fulminant hepatitis with jaundice. Free Radic Biol Med
: 755–761.PubMedGoogle Scholar
Kotake Y, Sang H, Tabatabaie T, Wallis GL, Moore DR, Stewart CA. Interleukin-10 overexpression mediates phenyl-N
-butyl nitrone protection from endotoxemia. Shock
: 210–216.PubMedGoogle Scholar
Slater AF, Nobel CS, Maellaro E, Bustamante J, Kimland M, Orrenius S. Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis. Biochem J
: 771–778.PubMedGoogle Scholar
Narita N, Noda I, Ohtsubo T, et al.
Analysis of heat-shock related gene expression in head-and-neck cancer using cDNA arrays. Int J Radiat Oncol Biol Phys
: 190–196.PubMedGoogle Scholar
Kato N, Kobayashi T, Honda H. Screening of stress enhancer based on analysis of gene expression profiles: Enhancement of hyperthermia-induced tumor necrosis by an MMP-3 inhibitor. Cancer Sci
: 644–649.PubMedGoogle Scholar
Yasumoto J, Kirita T, Takahashi A, et al.
Apoptosis-related gene expression after hyperthermia in human tongue squamous cell carcinoma cells harboring wild-type or mutated-type p53. Cancer Lett
: 41–51.PubMedGoogle Scholar
Lee W, Haslinger A, Karin M, Tjian R. Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature
: 368–372.PubMedGoogle Scholar
Lee W, Mitchell P, Tjian R. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell
: 741–752.PubMedGoogle Scholar
Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol
: E131–136.PubMedGoogle Scholar
Shaulian E, Schreiber M, Piu F, Beeche M, Wagner EF, Karin M. The mammalian UV response: c-Jun induction is required for exit from p53-imposed growth arrest. Cell
: 897–907.PubMedGoogle Scholar
Ham J, Babij C, Whitfield J, et al.
A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron
: 927–939.PubMedGoogle Scholar
Eferl R, Sibilia M, Hilberg F, et al.
Functions of c-Jun in liver and heart development. J Cell Biol
: 1049–1061.PubMedGoogle Scholar
Whitfield J, Neame SJ, Paquet L, Bernard O, Ham J. Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release. Neuron
: 629–643.PubMedGoogle Scholar
Kolbus A, Herr I, Schreiber M, Debatin KM, Wagner EF, Angel P. c-Jun-dependent CD95-L expression is a rate-limiting step in the induction of apoptosis by alkylating agents. Mol Cell Biol
: 575–582.PubMedGoogle Scholar
Kondo T, Matsuda T, Kitano T, et al.
Role of c-jun expression increased by heat shock- and ceramide-activated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis. J Biol Chem
: 7668–7676.PubMedGoogle Scholar
Verheij M, Bose R, Lin XH, et al.
Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature
: 75–79.PubMedGoogle Scholar
Enomoto A, Suzuki N, Liu C, et al.
Involvement of c-Jun NH2-terminal kinase-1 in heat-induced apoptotic cell death of human monoblastic leukaemia U937 cells. Int J Radiat Biol
: 867–874.PubMedGoogle Scholar
Saito K, Yoshioka H, Kazama S, Cutler RG. Release of nitric oxide from a spin trap, N
-butyl-alpha-phenylnitrone, under various oxidative conditions. Biol Pharm Bull
: 401–404.PubMedGoogle Scholar
Saito K, Ariga T, Yoshioka H. Generation of nitric oxide from a spin trapping agents under oxidative conditions. Biosci Biotechnol Biochem
: 275–279.Google Scholar
Wertz IE, O’Rourke KM, Zhang Z, et al.
Human De-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science
: 1371–1374.CrossRefPubMedGoogle Scholar
Nateri AS, Riera-Sans L, Da Costa C, Behrens A. The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling. Science
: 1374–1378.PubMedGoogle Scholar
© Springer Science + Business Media, Inc. 2005