Skip to main content
Cell Proliferation logoLink to Cell Proliferation
. 2007 Jul 27;32(6):363–378. doi: 10.1111/j.1365-2184.1999.tb01354.x

Spontaneous apoptosis and expression of cell surface heat‐shock proteins in cultured EL‐4 lymphoma cells

A M Sapozhnikov 1,✉, E D Ponomarev 1, T N Tarasenko 1, W G Telford 2
PMCID: PMC6495567  PMID: 10646688

Abstract

Abstract. The expression of heat‐shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress‐induced injury. However, existing data about the relationship between apoptosis and HSP expression is contradictory. In this paper, a mouse lymphoma cell death model system is used to detect simultaneously both the process of apoptosis and the level of HSP expression. The model was established after discovering that spontaneous apoptosis and spontaneous cell surface HSP expression occurs in EL‐4 mouse lymphoma cells during normal optimal culture conditions. The data show that apoptotic EL‐4 cells had higher levels of hsp25, hsp60, hsp70 and hsp90 exposed on the plasma membrane surface than viable cells. The level of surface HSPs was found to increase through several stages of early and late apoptotic death as measured by flow cytometry, with the highest levels observed during the loss of cell membrane phospholipid asymmetry. Heat shock and actinomycin D significantly increased the proportion of apoptotic cells in culture. However, hyperthermia only stimulated a weak and temporary increase in surface HSP expression, whereas actinomycin D strongly elevated the level of surface and intracellular HSPs, particularly in live cells. These results show an associative relationship between apoptosis and HSP expression. The relationship between the progression of cell death and HSP expression suggests a role for membrane HSP expression in programmed cell death.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

References

  1. Altmeyer A., Maki RG, Feldweg AM et al. (1996) Tumor‐specific cell surface expression of the KDEL containing, endoplasmic reticular heat shock protein gp96. Int . J. Cancer 69, 340. [DOI] [PubMed] [Google Scholar]
  2. Arata S., Hamaguchi S., Nose K. (1995) Effects of the overexpression of the small heat shock protein, HSP27, on the sensitivity of human fibroblast cells exposed to oxidative stress. J. Cell. Physiol. 163, 458. [DOI] [PubMed] [Google Scholar]
  3. Beere HM, Hickman JA, Morimoto RI, Parmar R., Newbould R., Waters CM. (1993) Changes in hsc70 and c‐myc in HL‐60 cells engaging differentiation or apoptosis. Mot. Cell. Diff. 1, 323. [Google Scholar]
  4. Borther CD, Cidlowski JA. (1996) Absence of volume regulatory mechanism contributes to the rapid activation of apoptosis in thymocytes. Am. J. Physiol. 271, C950. [DOI] [PubMed] [Google Scholar]
  5. Buttke TM, Sandstrom PA. (1994) Oxidative stress as a mediator of apoptosis. Immunol. Today 15, 7. [DOI] [PubMed] [Google Scholar]
  6. Camins A., Diez‐Fernandez C., Camarasa J., Escubedo E. (1995) Cell surface expression of heat shock proteins in dog neutrophils induced by mitochondrial benzodiazepine receptor ligands. Immimopharmacology 29, 159. [DOI] [PubMed] [Google Scholar]
  7. Chant ID, Rose PE, Morris AG. (1996) Susceptibility of AML cells to in vitro apoptosis correlates with heat shock protein 70 (hsp70) expression. Br. J. Haematol. 93, 898. [DOI] [PubMed] [Google Scholar]
  8. Clevenger CV, Bauer KD, Epstein AL. (1985) A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry. Cytometrv 6, 208. [DOI] [PubMed] [Google Scholar]
  9. Collins MKL, Marvel J., Malde P., Lopez‐Rivaz A. (1992) Interleukin 3 protects murine bone marrow cells from apoptosis induced by DNA damaging agents. J. Exp. Med. 176, 1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cosulish S., Clarke P. (1996) Apoptosis: does stress kill Curr. Biol. 6, 1586. [DOI] [PubMed] [Google Scholar]
  11. Craig EA, Weissman JS, Horwich AL. (1994) Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell. Cell 78, 365. [DOI] [PubMed] [Google Scholar]
  12. Darzynkiewicz Z., Bruno S., Del Bino G. et al. (1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795. [DOI] [PubMed] [Google Scholar]
  13. De Laurenzi V., Melino G., Savini I., Annicchiarico‐Petruzzelli M., Finazzi‐Agr A., Avigliano L. (1995) Cell death by oxidative stress and ascorbic acid regeneration in human neuroectodermal cell lines. Eur. J.Cancer 31A, 463. [DOI] [PubMed] [Google Scholar]
  14. Der SD, Yang YL, Weissmann C., Williams BR. (1997) A double‐stranded RNA‐activated protein kinase‐ dependent pathway mediating stress‐induced apoptosis. Proc. Natl. Acad. Sci. USA 94, 3279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Di Cesare S., Poccia F., Mastino A., Colizzi V. (1992) Surface expressed heat‐shock proteins by stressed or human immunodeficiency virus (HIV)‐infected lymphoid cells represent the target for antibody‐dependent cellular cytotoxity. Immunology 76, 341. [PMC free article] [PubMed] [Google Scholar]
  16. Erkeller‐Yeksel FM, Isenberg DA, Dhillon VB, Latchman DS, Lydyard PM. (1992) Surface expression of heat shock protein 90 by blood mononuclear cells from patients with systemic lupus erythematosus. J. Autoimmun. 5, 803. [DOI] [PubMed] [Google Scholar]
  17. Ferrarini M., Heltai S., Zocchi MR, Rugarli C. (1992) Unusual expression and localization of heat‐shock proteins in human tumor cells. Int. J. Cancer 51, 613. [DOI] [PubMed] [Google Scholar]
  18. Fratelli M., Gadliardini V., Galli G., Gnocchi P., Ghiara P., Ghezzi P. (1995) Autocrine interleukin‐1β regulates both proliferation and apoptosis in EL46.1 thymoma cells. Blood 85, 3532. [PubMed] [Google Scholar]
  19. Gabai VL, Zamulaeva IV, Mosin AF et al. (1995) Resistance of Ehrlich tumor cells to apoptosis can be due to accumulation of heat shock proteins. FEBS Lett 375, 21. [DOI] [PubMed] [Google Scholar]
  20. Galea‐Lauri J., Richardson AJ, Latchman DS, Katz DR. (1996) Increased heat shock protein 90 (hsp90) expression leads to increased apoptosis in the monoblastoid cell line U937 following induction with TNF‐α and cycloheximide. A possible role in immunopathology. J. Immunol. 157, 4109. [PubMed] [Google Scholar]
  21. Hamilton RF, Li JL, Felder TB, Holian A. (1995) Bleomycin induces apoptosis in human alveolar macrophages. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13), L318. [DOI] [PubMed] [Google Scholar]
  22. Hartl FU. (1996) Molecular chaperones in cellular protein folding. Nature 381, 571. [DOI] [PubMed] [Google Scholar]
  23. Kishimoto H., Surh ChD, Sprent J. (1995) Upregulation of surface markers on dying thymocytes. J. Exp. Med. 181, 649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kluck RM, Chapman DE, Egan M. et al. (1993) Spontaneous apoptosis in NS‐1 myeloma cultures: effects of cell density, conditioned medium and acid pH. Immunobiot. 188, 124. [DOI] [PubMed] [Google Scholar]
  25. Lotem J., Peled‐Kamar M., Groner Y., Sachs L. (1996) Cellular oxidative stress and the control of apoptosis by wild‐type p53, cytotoxic compounds, and cytokines. Proc. Natl. Acad. Sci. USA 93, 9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lynch MP, Nawaz S., Gerschenson LE. (1986) Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen. Proc. Natl. Acad. Sci. USA 83, 4784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mehlen P., Schulze‐Osthoff K., Arrigo AP. (1996) Small stress proteins as novel regulators of apoptosis. J. Biol. Chem. 271, 16510. [DOI] [PubMed] [Google Scholar]
  28. Moss DJ, Bishop CJ, Burrows SR, Ryan JM. (1985) T lymphocytes in infectious mononucleosis. I. T cell death in vitro. Clin. Exp. Immunol. 60, 61. [PMC free article] [PubMed] [Google Scholar]
  29. Multhoff G., Botzler C., Wiesnet M. et al. (1995) A stress‐inducible 72‐kD heat‐shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int. J. Cancer 61, 272. [DOI] [PubMed] [Google Scholar]
  30. Nosseri C., Coppola S., Ghibelli L. (1994) Possible involvement of poly (ADF‐ribosyl) polymerase in triggering stress‐induced apoptosis. Exp. Cell Res. 212, 367. [DOI] [PubMed] [Google Scholar]
  31. Poccia F., Piselli P., Di Cesare S. et al. (1992) Recognition and killing of tumor cells expressing heat shock protein 65 kD with immunotoxins containing saponin. Br. J. Cancer 66, 427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Poccia F., Piselli P., Vendetti S. et al. (1996) Heat‐shock protein expression on the membrane of T cells undergoing apoptosis. Immunology 88, 6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pourzand C., Rossier G., Reelfs O., Borner C., Tyrrell RM. (1997) Overexpression of Bcl‐2 inhibits UVA‐ mediated apoptosis in rat 6 fibroblasts: evidence for the involvement of Bcl‐2 as an antioxidant. Cancer Res. 57, 1405. [PubMed] [Google Scholar]
  34. Raff MC. (1992) Social controls on cell survival and cell death. Nature 356, 397. [DOI] [PubMed] [Google Scholar]
  35. Samali A., Cotter TG. (1996) Heat shock proteins increase resistance to apoptosis. Exp. Cell Res. 223, 163. [DOI] [PubMed] [Google Scholar]
  36. Sambrook J., Fritsch EF, Maniatis T. (1989) Molecular Cloning. A Laboratory Manual Cold Spring Harbor , NY : Cold Spring Harbor Laboratory Press. [Google Scholar]
  37. Schlesinger MJ. (1990) Heat shock proteins. J. Biol. Chem. 265, 12111. [PubMed] [Google Scholar]
  38. Servomaa K., Rytomaa T. (1988) Suicidal death of rat chloroleukaemia cells by activation of the long interspersed repetitive DNA element (LIRn). Cell Tissue Kinet. 21, 33. [DOI] [PubMed] [Google Scholar]
  39. Soloff BL, Nagle WA, Moss AJ, Henle KJ, Crawford JT. (1987) Apoptosis induced by cold shock in vitro is dependent on cell growth phase. Biochem. Biophys. Res. Common. 145, 876. [DOI] [PubMed] [Google Scholar]
  40. Swat W., Ignatowicz L., Kisielow P. (1991) Detection of apoptosis of immature CD4 + 8+ thymocytes by flow cytometry. J. Immunol. Meth. 137, 79. [DOI] [PubMed] [Google Scholar]
  41. Telford WG, King LE, Fraker PJ. (1991) Evaluation of glucocorticoid‐induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Proliferation 24, 447. [DOI] [PubMed] [Google Scholar]
  42. Turek Z., Horvath I., Goloubinoff P. et al. (1997) Evidence for a lipochaperonin: association of active protein‐folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions. Proc. Natl. Acad. Sci. USA 94, 2192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Van DER, Bosch J., Ruller S., Horn D., Schumann R., Schlaak M. (1990) Density‐dependent tumor cell death and reversible cell cycle arrest: mutually exclusive modes of monocyte‐mediated growth control. Exp. Cell. Res. 187, 185. [DOI] [PubMed] [Google Scholar]
  44. Verheij M., Bose R., Lin XH et al. (1996) Requirement for ceramide‐initiated SAPK/JNK signalling in stress‐induced apoptosis. Nature 380, 75. [DOI] [PubMed] [Google Scholar]
  45. Vermes I., Haanen C., Steffens‐Nakken H., Reutelingsperger C. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled annexin V. J. Immunol. Meth. 184, 39. [DOI] [PubMed] [Google Scholar]
  46. Wei Y., Zhao X., Kariya Y., Teshigawara K., Uchida A. (1995) Inhibition of proliferation and induction of apoptosis by abrogation of heat‐shock protein (HSP) 70 expression in tumor cells. Cancer Immunol. Immunother. 40, 73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wyllie AH, Kerr JF, Currie AR. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251. [DOI] [PubMed] [Google Scholar]
  48. Xu Y., Nguyen Q., Lo DC, Czaja MJ. (1997) C‐myc‐dependent hepatoma cell apoptosis results from oxidative stress and not a deficiency of growth factors. J. Cell Physiol. 170, 192. [DOI] [PubMed] [Google Scholar]

Articles from Cell Proliferation are provided here courtesy of Wiley

RESOURCES