ABC  Vol.6 No.3 , June 2016
Moderate Hyperthermia Induces Apoptosis in Metaphase-Arrested Cells But Not in Interphase Hela Cells
Abstract: Metaphase-arrest agents and hyperthermia are both known to be capable of inducing apoptosis, and they have been used, separately, in cancer treatments. Here, we have examined whether the two treatments together may have a synergistic effect. We find that when H-HeLa cells are arrested in metaphase with spindle poisons (nocodazole or paclitaxel) and then subjected to mild heat treatment (41.5℃), they exhibit morphological changes typical of apoptosis within three hours. Moreover, those changes are blocked by the pan-caspase inhibitor zVAD-fmk, indicating apoptosis, and activated Procaspase 3 is detected by immunoblotting and by staining with the fluoresce-in-labelled caspase inhibitor FAM-VAD-fmk. Interphase cells treated in the same way do not under-go apoptosis, even with spindle poisons present. Induction of apoptosis is more rapid when the cells have been arrested longer in metaphase, suggesting that accumulation or depletion of some cellular component(s) during metaphase-arrest may make them more susceptible to hyperthermia. Further work is in progress to test whether other cell lines exhibit the same behavior and to learn more about the mechanism. The phenomenon is of interest because it may provide clues to how hyperthermia induces cell death and may yield novel therapeutic approaches to block or stimulate apoptosis.
Cite this paper: Paulson, J. , Kresch, A. and Mesner, P. (2016) Moderate Hyperthermia Induces Apoptosis in Metaphase-Arrested Cells But Not in Interphase Hela Cells. Advances in Biological Chemistry, 6, 126-139. doi: 10.4236/abc.2016.63011.

[1]   Green, D.R. (1998) Apoptotic Pathways: The Roads to Ruin. Cell, 94, 695-698.

[2]   Song, Z. and Steller, H. (1999) Death by Design: Mechanism and Con-trol of Apoptosis. Trends in Cell Biology, 9, M49-M52.

[3]   Nicholson, D.W. and Thornberry, N.A. (1997) Caspases: Killer Proteases. Trends in Biochemical Sciences, 22, 299-306.

[4]   Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: Enemies within. Science, 281, 1312-1316.

[5]   Earnshaw, W.C., Martins, L.M. and Kaufmann, S.H. (1999) Mammalian Caspases: Structure, Activation, Substrates and Functions during Apoptosis. Annual Review of Biochemistry, 68, 383-424.

[6]   Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (1998) A Caspase-Activated DNase That Degrades DNA during Apoptosis, and Its Inhibitor ICAD. Nature, 391, 43-50.

[7]   Hickman, J.A. (1992) Apoptosis Induced by Anticancer Drugs. Cancer and Metastasis Reviews, 11, 121-139.

[8]   Cotter, T.G., Glynn, J.M., Echeverri, F. and Green, D.R. (1992) Induction of Apoptosis by Chemotherapeutic Agents Occurs during All Phases of the Cell Cycle. Anticancer Research, 12, 773-780.

[9]   Kaufmann, S.H. and Earnshaw, W.C. (2000) Induction of Apoptosis by Cancer Chemotherapy. Experimental Cell Research, 256, 42-49.

[10]   Reed, J.C. (2002) Apoptosis-Based Therapies. Nature Reviews Drug Discovery, 1, 111-121.

[11]   Pollard, T. and Earnshaw, W.C. (2003) Cell Biology. Saunders, Philadelphia.

[12]   Harmon, B.V., Takano, Y.S., Winterford, C.M. and Gobe, G.C. (1991) The Role of Apoptosis in the Response of Cells and Tumours to Mild Hyperthermia. International Journal of Radiation Biology, 59, 489-501.

[13]   Ahmed, K., Tabuchi, Y. and Kondo, T. (2015) Hyperthermia: An Effective Strategy to Induce Apoptosis in Cancer Cells. Apoptosis, 20, 1411-1419.

[14]   Ahmed, K. and Zaidi, S.F. (2013) Treating Cancer with Heat: Hyperthermia as Promising Strategy to Enhance Apoptosis. Journal of Pakistan Medical Association, 63, 504-508.

[15]   De Nardo, G.L. and De Nardo, S.J. (2008) Update: Turning the Heat on Cancer. Cancer Biotherapy and Radiopharmaceuticals, 23, 671-680.

[16]   Hildebrandt, B. and Wust, P. (2007) The Biologic Rationale of Hyperthermia. Cancer Treatment and Research, 134, 171-184.

[17]   Verdoodt, B., Decordier, I., Geleyns, K., Cunha, M., Cundari, E. and Kirsch-Volders, M. (1999) Induction of Polyploidy and Apoptosis after Exposure to High Concentrations of the Spindle Poison Nocodazole. Mutagenesis, 14, 513- 520.

[18]   Yamada, H.Y. and Gorbsky, G.J. (2006) Inhibition of TRIP1/S8/hSug1, a Component of the Human 19S Proteasome, Enhances Mitotic Apoptosis Induced by Spindle Poisons. Molecular Cancer Therapeutics, 5, 29-38.

[19]   Paulson, J.R. (2007) Inactivation of Cdk1/Cyclin B in Meta-phase-Arrested Mouse FT210 Cells Induces Exit from Mitosis without Chromosome Segregation or Cytokinesis and Allows Passage through Another Cell Cycle. Chromosoma, 116, 215-225.

[20]   Medappa, K.C., McLean, C. and Rueckert, R.R. (1971) On the Structure of Rhinovirus 1A. Virology, 44, 259-270.

[21]   Heinz, B.A., Rueckert, R.R., Shepard, D.A., Dutko, F.J., McKinlay, M.A., Fancher, M., Rossmann, M.G., Badger, J. and Smith, T.J. (1989) Genetic and Molecular Analyses of Spontaneous Mutants of Human Rhinovirus 14 That Are Resistant to an Antiviral Compound. Journal of Virology, 63, 2476-2485.

[22]   Paulson, J.R., Ciesielski, W.A., Schram, B.R. and Mesner, P.W. (1994) Okadaic Acid Induces Dephosphorylation of Histone H1 in Metaphase-Arrested HeLa Cells. Journal of Cell Science, 107, 267-273.

[23]   Paulson, J.R. (1982) Isolation of Mitotic Chromosome Clusters from Meta-phase-Arrested HeLa Cells. Chromosoma, 85, 571-581.

[24]   Paulson, J.R., Patzlaff, J.S. and Vallis, A.J. (1996) Evidence that the Endogenous Histone H1 Phosphatase in HeLa Mitotic Chromosomes Is Protein Phosphatase 1, Not Protein Phosphatase 2A. Journal of Cell Science, 109, 1437-1447.

[25]   Patterson, M.K. (1979) Measurement of Growth and Viability of Cells in Culture. Methods in Enzymology, 58, 141-152.

[26]   Mesner, P.W., Bible, K.C., Martins, L.M., Kottke, T.J., Srinivasula, S.M., Svingen, P.A., Chilcote, T.J., Basi, G.S., Tung, J.S., Krajewski, S., Reed, J.C., Alnemri, E.S., Earnshaw, W.C. and Kaufmann, S.H. (1999) Characterization of Caspase Processing and Activation in HL-60 Cell Cytosol under Cell-Free Conditions: Nucleotide Requirement and Inhibitor Profile. The Journal of Biological Chemistry, 274, 22635-22645.

[27]   Zhu, H., Fearnhead, H.O. and Cohen, G.M. (1995) An ICE-Like Protease Is a Common Mediator of Apoptosis Induced by Diverse Stimuli in Human Monocytic THP.1 Cells. FEBS Letters, 374, 303-308.

[28]   Slee, E.A., Zhu, H., Chow, S.C., MacFarlane, M., Nicholson, D.W. and Cohen, G.M. (1996) Benzyloxycarbonyl-Val-Ala-Asp (OMe) Fluoromethylketone (Z-VAD.FMK) Inhibits Apoptosis by Blocking the Processing of CPP32. Biochemical Journal, 315, 21-24.

[29]   Han, C.R., Jun, D.Y., Lee, J.Y. and Kim, Y.H. (2014) Prometaphase Arrest-Dependent Phosphorylation of Bcl-2 and Bim Reduces the Association of Bcl-2 with Bac or Bim, Provoking Bak Activation and Mitochondrial Apoptosis in Nocodazole-Arrested Jurkat T Cells. Apoptosis, 19, 224-240.

[30]   Th’ng, J.P., Wright, P.S., Hamaguchi, J., Lee, M.G., Norbury, C.J., Nurse, P. and Bradbury, E.M. (1990) The FT210 Cell Line Is a Mouse G2 Phase Mutant with a Temperature-Sensitive CDC2 Gene Product. Cell, 63, 313-324.

[31]   Murray, A.W., Solomon, M.J. and Kirschner, M.W. (1989) The Role of Cyclin Synthesis and Degradation in the Control of Maturation Promoting Factor Activity. Nature, 339, 280-286.

[32]   Rimmington, G., Dalby, B. and Glover, D.M. (1994) Expression of N-Terminally Truncated Cyclin B in the Drosophila Larval Brain Leads to Mitotic Delay at Late Anaphase. Journal of Cell Science, 107, 2729-2738.

[33]   Luo, Q., Michaelis, C. and Weeks, G. (1994) Overexpression of a Truncated Cyclin B Gene Arrests Dictyostelium Cell Division during Mitosis. Journal of Cell Science, 107, 3105-3114.

[34]   Giovinazzi, S., Bellapu, D., Morozov, V.M. and Ishov, A.M. (2013) Targeting Mitotic Exit with Hyperthermia or APC/C Inhibition to Increase Paclitaxel Efficacy. Cell Cycle, 12, 2598-2607.

[35]   Baserga, R. (1962) A Study of Nucleic Acid Synthesis in Ascites Tumor Cells by Two-Emulsion Autoradiography. The Journal of Cell Biology, 12, 633-637.

[36]   Prescott, D.M. and Bender, M.A. (1962) Synthesis of RNA and Protein during Mitosis in Mammalian Tissue Culture Cells. Experimental Cell Research, 26, 260-268.

[37]   Konrad, C.G. (1963) Protein Synthesis and RNA Synthesis during Mitosis in Animal Cells. The Journal of Cell Biology, 19, 267-277.

[38]   Leresche, A., Wolf, V.J. and Gottesfeld, J.M. (1996) Repression of RNA Polymerase II and III Transcription during M Phase of the Cell Cycle. Experimental Cell Research, 229, 282-288.

[39]   Lu, M., Lawrence, D.A., Marsters, S., Acosta-Alvear, D., Kimmig, P., Mendez, A.S., Paton, A.W., Paton, J.C., Walter, P. and Ashkenazi, A. (2014) Opposing Unfold-ed-Protein-Response Signals Converge on Death Receptor 5 to Control Apoptosis. Science, 345, 98-101.

[40]   Berger, A.B., Sexton, K.B. and Bogyo, M. (2006) Commonly Used Caspase Inhibitors Designed Based on Substrate Specificity Profiles Lack Selectivity. Cell Research, 16, 961-963.

[41]   Blanc-Brude, O.P., Mesri, M., Wall, N.R., Plescia, J., Dohi, T. and Altieri, D.C. (2003) Therapeutic Targeting of the Survivin Pathway in Cancer: Initiation of Mitochondrial Apoptosis and Suppression of Tu-mor-Associated Angiogenesis. Clinical Cancer Research, 9, 2683-2692.

[42]   Jeyaprakash, A.A., Klein, U.R., Lindner, D., Ebert, J., Nigg, E.A. and Conti, E. (2007) Structure of a Survivin-Borealin-INCENP Core Complex Reveals how Chromosomal Passengers Travel Together. Cell, 131, 271-285.

[43]   Matthess, Y., Raab, M., Knecht, R., Becker, S. and Strebhardt, K. (2014) Sequential Cdk1 and Plk1 Phosphorylation of Caspase-8 Triggers Apoptotic Cell Death during Mitosis. Molecular Oncology 8, 596-608.

[44]   Pettus, B.J., Chalfant, C.E. and Hannun, Y.A. (2002) Ceramide in Apoptosis: An Overview and Current Perspectives. Biochimica et Biophysica Acta (BBA), 1585, 114-125.

[45]   Bieberich, E., MacKinnon, S., Silva, J., Noggle, S. and Condie, B.G. (2003) Regulation of Cell Death in Mitotic Neural Progenitor Cells by Asymmetric Distribution of Prostate Apoptosis Response 4 (PAR-4) and Simultaneous Elevation of Endogenous Ceramide. The Journal of Cell Biology, 162, 469-479.

[46]   Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P.G., Coso, O.A., Gutkind, S. and Spiegel, S. (1996) Suppression of Ceramide-Mediated Programmed Cell Death by Sphingosine-1-Phosphate. Nature, 381, 800-803.

[47]   Yokoyama, K., Suzuki, M., Kawashima, I., Karasawa, K., Nojima, S., Enomoto, T., Tai, T., Suzuki, A. and Setaka, M. (1997) Changes in Composition of Newly Synthesized Sphingolipids of HeLa Cells during the Cell Cycle: Suppression of Sphingomyelin and Higher-Glycosphingolipid Synthesis and Accumulation of Ceramide and Glucosylceramide in Mitotic Cells. European Journal of Biochemistry, 249, 450-455.

[48]   Lee, J.Y., Leonhardt, L.G. and Obeid, L.M. (1998) Cell-Cycle-Dependent Changes in Ceramide Levels Preceding Retinoblastoma Protein Dephosphorylation in G2/M. Biochemical Journal, 334, 457-461.

[49]   Kondo, T., Matsuda, T., Kitano, T., Takahashi, A., Tashima, M., Ishikura, H., Umehara, H., Domae, N., Uchiyama, T. and Okazaki, T. (2000) Role of c-jun Expression Increased by Heat Shock- and Ceramide-Activated Caspase-3 in HL- 60 Cell Apoptosis. The Journal of Biological Chem-istry, 275, 7668-7676.

[50]   Mehta, S., Blackinton, D., Omar, I., Kouttab, N., Myrick, D., Klostergaard, J. and Wanebo, H. (2000) Combined Cytotoxic Action of Paclitaxel and Ceramide against the Human Tu138 Head and Neck Squamous Carcinoma Cell Line. Cancer Chemotherapy and Pharmacology, 46, 85-92.

[51]   Miyazaki, N., Kurihara, K., Nakano, H. and Shinohara, K. (2002) Role of ATP in the Sensitivity to Heat and the Induction of Apoptosis in Mammalian Cells. International Journal of Hyperthermia, 18, 316-331.

[52]   Falk, M.H. and Issels, R.D. (2001) Hyperthermia in Oncology. International Journal of Hyperthermia, 17, 1-18.

[53]   Alexander, H.R. (2003) Hyperthermia and Its Modern Use in Cancer Treatment. Cancer, 98, 219-221.

[54]   Eikesdal, H.P., Bjerkvig, R., Raleigh, J.A., Mella, O. and Dahl, O. (2001) Tumor Vasculature Is Targeted by the Combination of Combretastatin A-4 and Hyperthermia. Radiotherapy and Oncology, 61, 313-320.

[55]   Murata, R., Overgaard, J. and Horsman, M.R. (2001) Combretastatin A-4 Disodium Phosphate: A Vascular Targeting Agent That Improves the Anti-Tumor Effects of Hyperthermia, Radiation and Mild Thermoradiotherapy. International Journal of Radiation Oncology Biology Physics, 51, 1018-1024.