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Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance

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Abstract

Background: Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line.

Methods: A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (ΔΨm) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH) fluorescence.

Results: Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in ΔΨm (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or ΔΨm. Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis.

Conclusion: SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis and induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.

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References

  1. Bonadonna G. Evolving concepts in the systemic adjuvant treatment of breast cancer.Cancer Res 1992;52:2127–37.

    CAS  PubMed  Google Scholar 

  2. Osborne CK, Clark GM, Ravdin PM. Treatment of early-stage breast cancer: adjuvant systemic therapy of invasive breast cancer. In: Harris JR, Lippman ME, Morrow M, Hellman S, eds.Diseases of the Breast. Philadelphia: Lippincott-Raven, 1996:548–78.

    Google Scholar 

  3. Allred DC, Clark GM, Tandon AK, et al. HER-2/Neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma.J Clin Oncol 1992;10:599–605.

    CAS  PubMed  Google Scholar 

  4. Muss HB, Thor AD, Berry DA, et al. c-erb-2 expression and response to adjuvant therapy in women with node-positive breast cancer.N Engl J Med 1994; 330:1260–6.

    Article  CAS  PubMed  Google Scholar 

  5. Kerr JFR, Winterford CM, Harmon BV. Apoptosis: its significance in cancer and cancer therapy.Cancer 1994;73:2013–26.

    CAS  PubMed  Google Scholar 

  6. Milross CG, Mason KA, Hunter NR, Chung WK, Peters LJ, Milas L. Relationship of mitotic arrest and apoptosis to antitumor effect of paclitaxel.J Natl Cancer Inst 1996;88:1308–14.

    CAS  PubMed  Google Scholar 

  7. Geier A, Bar-Shalom I, Beery R, et al. Induction of apoptosis in MDA-231 cells by protein synthesis inhibitors is suppressed by multiple agents.Cancer Invest 1996;14:435–44.

    CAS  PubMed  Google Scholar 

  8. Dive C, Hickman JA. Drug-target interactions: only the first step in the commitment to a programmed cell death.Br J Cancer 1991;64:192–6.

    CAS  PubMed  Google Scholar 

  9. Fisher DE. Apoptosis in cancer therapy: crossing the threshold.Cell 1994;78:539–42.

    CAS  PubMed  Google Scholar 

  10. Meyn RE, Stephens LC, Hunter NR, Milas L. Apoptosis in murine tumors treated with chemotherapy agents.Anti-Cancer Drugs 1995;6:443–50.

    CAS  PubMed  Google Scholar 

  11. Gregoire V, Van NT, Stephens LC, Brock WA, Milas L, Plunkett W, Hittelman WN. The role of fludarabine-induced apoptosis and cell cycle synchronization in enhanced murine tumor radiation response in vivo.Cancer Res 1994;54:6201–9.

    CAS  PubMed  Google Scholar 

  12. Potten CS. The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice.Cancer Metastasis Rev 1992;11:179–95.

    CAS  PubMed  Google Scholar 

  13. Moreira LF, Naomoto Y, Hamada M, Kamikawa Y, Orita K. Assessment of apoptosis in oesophageal carcinoma preoperatively treated by chemotherapy and radiotherapy.Anticancer Research 1995;15:639–44.

    CAS  PubMed  Google Scholar 

  14. Omura S, Iwai Y, Takahashi Y, Sadakane N, Nakagawa A. Herbimycin, a new antibiotic produced by a strain of streptomyces.J Antibiot 1979;32:255–61.

    CAS  PubMed  Google Scholar 

  15. Uehara Y, Hori M, Tackeuchi T, Umezawa H. Screening of agents which convert ‘transformed morphology’ of Rous sarcoma virus-infected rat kidney cells to ‘normal morphology’: identification of an active agent as herbimycin and its inhibition of intracellularsrc kinase.Jpn J Cancer Res 1985;76:672–5.

    CAS  PubMed  Google Scholar 

  16. Uehara Y, Hori M, Takeuchi T, Umezawa H. Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus.Mol Cell Biol 1986;6:2198–2206.

    CAS  PubMed  Google Scholar 

  17. Murukami Y, Mizuno S, Hori M, Uehara Y. Reversal of transformed phenotypes by herbimycin A insrc oncogene expressed rat fibroblasts.Cancer Res 1988;48:1587–90.

    Google Scholar 

  18. Uehara Y, Murakami Y, Sugimoto Y, Mizuno S. Mechanism of reversion of Rous sarcoma virus transformation by Herbimycin A: reduction of total phosphotyrosine levels due to reduced kinase activity and increased turnover of p60vsrc-1.Cancer Res 1989;49:780–5.

    CAS  PubMed  Google Scholar 

  19. Uehara Y, Murukami Y, Mizuno S, Kawai S. Inhibition of transforming activity of tyrosine kinase oncogenes by herbimycin A.Virology 1988;164:294–8.

    Article  CAS  PubMed  Google Scholar 

  20. Honma Y, Okabe-Kado J, Hozumi M, Uehara Y, Mizuno S. Induction of erythroid differentiation of K562 human leukemic cells by herbimycin A, an inhibitor of tyrosine kinase activity.Cancer Res 1989;49:331–4.

    CAS  PubMed  Google Scholar 

  21. Mancini M, Paty PB, Hockenbery D, Lieberman MD, Anderson BO. Apoptosis (programmed cell death) is induced with differentiation and growth arrest in a human colon cancer by a Bcl-2 independent mechanism.Surg Forum 1995;46:507–9.

    Google Scholar 

  22. Mancini M, Anderson BO, Caldwell L, Sedghinasab M, Paty P, Hockenbery D. Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line.J Cell Biol 1997;138:449–69.

    Article  CAS  PubMed  Google Scholar 

  23. Sambrook J, Fritsch EF, Maniatis T. Expression of cloned genes in cultured mammalian cells.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989:16.39–16.40.

    Google Scholar 

  24. Reers M, Smith TW, Chen LB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential.Biochemistry 1991;30:4480–6.

    Article  CAS  PubMed  Google Scholar 

  25. Smiley ST, Reers M, Mottola-Hartson C, et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1.Proc Natl Acad Sci USA 1991;88:3671–5.

    CAS  PubMed  Google Scholar 

  26. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative products formulation by neutrophils: a graded response to membrane stimulation.J Immunol 1983;130:1910–7.

    CAS  PubMed  Google Scholar 

  27. Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with Hydroethidine and 2′,7′-dichlorofluorescein.J Leukocyte Biol 1990;47:440–8.

    CAS  PubMed  Google Scholar 

  28. Hockenbery DM, Oltvai ZN, Yin X, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis.Cell 1993;75:241–51.

    Article  CAS  PubMed  Google Scholar 

  29. Zamzami N, Marchetti P, Castedo M, et al. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.J Exp Med 1995;182:367–77.

    Article  CAS  PubMed  Google Scholar 

  30. Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.Biochem J 1956;62:315–23.

    CAS  PubMed  Google Scholar 

  31. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.Nature 1990;348:334–6.

    Article  CAS  PubMed  Google Scholar 

  32. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol 1992;119:493–501.

    Article  CAS  PubMed  Google Scholar 

  33. Henkart PA, Grinstein S. Apoptosis: mitochondria resurrected?J Exp Med 1996;183:1293–5.

    Article  CAS  PubMed  Google Scholar 

  34. Petit PX, O'Connor JE, Grunwald D, Brown SC. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications.Eur J Biochem 1990;194:389–97.

    Article  CAS  PubMed  Google Scholar 

  35. Vayssiere JL, Petit PX, Risler Y, Mignotte B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40.Proc Natl Acad Sci USA 1994;91:11752–6.

    CAS  PubMed  Google Scholar 

  36. Petit PX, Lecoeur H, Zorn E, Dauguet C, Mignotte B, Gougeon M-L. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis.J Cell Biol 1995;130:157–67.

    Article  CAS  PubMed  Google Scholar 

  37. Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere J-L, Petit PX, Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo.J Exp Med 1995;181:1661–72.

    Article  CAS  PubMed  Google Scholar 

  38. Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M, Kroemer G. Mitochondrial control of nuclear apoptosis.J Exp Med 1996;183:1533–44.

    Article  CAS  PubMed  Google Scholar 

  39. Marchetti P, Susin SA, Decaudin D, et al. Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA.Cancer Res 1996;56:2033–8.

    CAS  PubMed  Google Scholar 

  40. Reipert S, Berry J, Hughes MF, Hickman JA, Allen TD. Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy.Exp Cell Res 1995;221:281–8.

    Article  CAS  PubMed  Google Scholar 

  41. Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis.Immunol Today 1994;15:7–10.

    Article  CAS  PubMed  Google Scholar 

  42. Peled-Kamar M, Lotem J, Okon E, Sachs L, Groner Y. Thymic abnormalities and enhanced apoptosis of thymocytes and bone marrow cells in transgenic mice overexpressing Cu/Zn-superoxide dismutase: implications for Down syndrome.EMBO J 1995;14:4985–93.

    CAS  PubMed  Google Scholar 

  43. Busciglio J, Jankner BA. Apoptosis and increased generation of reactive oxygen species in Down's syndrome neurons in vitro.Nature 1995;378:776–9.

    Article  CAS  PubMed  Google Scholar 

  44. Krajewski S, Tanaka S, Takayama S, Schibler MJ, Fenton W, Reed JC. Investigation of the subcellular distribution of the Bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane.Cancer Res 1993;53:4701–14.

    CAS  PubMed  Google Scholar 

  45. Riparbelli MG, Callaini G, Tripodi SA, Cintorino M, Tosi P, Dallai R. Localization of the Bcl-2 protein to the outer mitochondrial membrane by electron microscopy.Exp Cell Res 1995;221:363–9.

    Article  CAS  PubMed  Google Scholar 

  46. Sentman CL, Shutter JR, Hockenbery D, Kanagawa O, Korsmeyer SJ. Bcl-2 inhibits multiple forms of apoptosis but not negative selection of thymocytes.Cell 1991;67:879–88.

    Article  CAS  PubMed  Google Scholar 

  47. Kane DJ, Serafian TA, Anton TA, et al. Bcl-2 inhibition of neuronal death: decreased generation of reactive oxygen species.Science 1993;262:1274–7.

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. the Divisions of Clinical Research and Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington

    Mariangela Mancini MD, Monireh Sedghinasab MS, Katya Knowlton MD, Amy Tam MS, David Hockenberg MD & Benjamin O. Anderson MD

  2. Dept. of Surgery, University of Washington, Box 356410, 98195, Seattle, WA

    Mariangela Mancini MD, Monireh Sedghinasab MS, Katya Knowlton MD & Benjamin O. Anderson MD

Authors
  1. Mariangela Mancini MD
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  2. Monireh Sedghinasab MS
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  3. Katya Knowlton MD
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  4. Amy Tam MS
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  5. David Hockenberg MD
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  6. Benjamin O. Anderson MD
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Funded in part by American Cancer Society Career Development Award #95-2 (BOA) and the Howie Fund of the Department of Surgery at the University of Washington.

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Mancini, M., Sedghinasab, M., Knowlton, K. et al. Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance. Annals of Surgical Oncology 5, 287–295 (1998). https://doi.org/10.1007/BF02303787

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  • DOI: https://doi.org/10.1007/BF02303787

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