Abstract
In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of hepatocytes. Results presented here show that absent nutrients including glucose and amino acids play a substantial part in the induction of apoptosis. The use of chemical apoptosis inhibitors was utilised to investigate key components of hepatic apoptosis where caspases, predominantly caspase 8, were implicated in staurosporine (STS)-induced HepZ apoptosis. Caspase 9 and 3 activation although recorded was of less significance. Interestingly, these results were not consistent with those of mitochondrial membrane depolarisation where inhibition of caspase activation appeared to drive depolarisation. Inhibition of mitochondrial permeability transition and use of anti-oxidants was unsuccessful in reducing apoptosis, caspase activation and mitochondrial membrane depolarisation. In further studies, the anti-apoptotic gene bcl-2 was over-expressed in HepZ, resulting in a cell line that was more robust and resistant to death induced by glucose and cystine deprivation and treatment with STS. Bcl-2 did not however show significant cytoprotectivity where apoptosis was stimulated by deprivation of glutamine and serum. Overall, results indicated that although apoptosis can be curbed by use of chemical inhibitors and genetic manipulation, their success is dependent on apoptotic stimuli.
Article PDF
Similar content being viewed by others
References
C.D. Albright C. Borgman C.N. Craciunescu (2003) ArticleTitleActivation of a caspase-dependent oxidative damage response mediates TGFbeta-1 apoptosis in rat hepatocytes Exp. Mol. Pathol. 74 256–261 Occurrence Handle12782012 Occurrence Handle1:CAS:528:DC%2BD3sXktFGitLY%3D Occurrence Handle10.1016/S0014-4800(03)00002-9
M. Al-Rubeai S.K.W. Oh R. Musaheb A.N. Emery (1990) ArticleTitleModified cellular metabolism in hybridomas subjected to hydrodynamic stresses Biotechnol. Lett. 12 323 Occurrence Handle10.1007/BF01024425
M. Al-Rubeai R.P. Singh M.H. Goldman A.N. Emery (1995) ArticleTitleDeath mechanisms of animal cells in conditions of intensive agitation Biotechnol. Bioeng. 45 463–472 Occurrence Handle1:CAS:528:DyaK2MXjvFensr0%3D Occurrence Handle10.1002/bit.260450602 Occurrence Handle18623245
B. Antonsson F. Conti A. Ciavatta S. Montessuit S. Lewis I. Martinou L. Bernasconi A. Bernard J.J. Mermod G. Mazzei K. Maundrell F. Gambale R. Sadoul J.C. Martinou (1997) ArticleTitleInhibition of bax channel-forming activity by Bcl-2 Science 277 370–372 Occurrence Handle9219694 Occurrence Handle1:CAS:528:DyaK2sXkvVels7s%3D Occurrence Handle10.1126/science.277.5324.370
K. Aoshiba S. Yasui K. Nishimura A. Nagai (1999) ArticleTitleThiol depletion induces apoptosis in cultured lung fibroblasts Am. J. Resp. Cell Mol. Biol. 21 54–64 Occurrence Handle1:CAS:528:DyaK1MXks12rsbs%3D
N.B. Blatt G.D. Glick (2001) ArticleTitleSignaling Pathways and effector mechanisms pre-programmed cell death Bioorg. Med. Chem. 9 1371–1384 Occurrence Handle11408158 Occurrence Handle1:CAS:528:DC%2BD3MXkt1Kjs7o%3D
W.M. Blom H.J. Bont Particlede I. Meirjerman G.J. Mulder J.F. Nagelkerke (1999) ArticleTitlePrevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitor Biochem. Pharmacol. 58 1891–1898 Occurrence Handle10591143 Occurrence Handle1:CAS:528:DyaK1MXns1ant78%3D Occurrence Handle10.1016/S0006-2952(99)00268-3
C. Borner (1996) ArticleTitleDiminished cell proliferation associated with the death protective activity of Bcl-2 J. Biol. Chem. 271 12695–12698 Occurrence Handle8663032 Occurrence Handle1:CAS:528:DyaK28XjtlGjt78%3D
K. Bratch M. Al-Rubeai (2001) ArticleTitleCulture of primary hepatocytes within a flat hollow fibre cassette for potential use as a component of a bioartificial liver support system Biotechnol. Lett. 23 137–141 Occurrence Handle1:CAS:528:DC%2BD3MXhtFCis7o%3D Occurrence Handle10.1023/A:1010387612109
R.C. Budd (2001) ArticleTitleActivation–induced cell death Curr. Opin. Immunol. 13 356–362 Occurrence Handle11406369 Occurrence Handle1:CAS:528:DC%2BD3MXjslWiur8%3D Occurrence Handle10.1016/S0952-7915(00)00227-2
K. Cain S.H. Inayat-Hussain C. Couet G.M. Cohen (1996) ArticleTitleA cleavage-site-directed inhibitor of interleukin-1β-convering enzyme-like proteases inhibits apoptosis in primary cultures of rat hepatocytes Biochem. J. 314 27–32 Occurrence Handle8660294 Occurrence Handle1:CAS:528:DyaK28XhtlKmsbk%3D
S.K. Chiou E. White (1998) ArticleTitleInhibition of ICE-like proteases inhibits apoptosis and increases virus production during adenovirus infection Virology 244 108–118 Occurrence Handle9581784 Occurrence Handle1:CAS:528:DyaK1cXivF2ju7g%3D Occurrence Handle10.1006/viro.1998.9077
J.D. Chung A.J. Sinskey G. Stephanopoulos (1998) ArticleTitleGrowth factor and Bcl-2 mediated survival during abortive proliferation of hybridoma cell line Biotechnol. Bioeng. 57 164–171 Occurrence Handle10099191 Occurrence Handle1:CAS:528:DyaK1cXntFGh
L. Colombaioni L. Colombini M. Garcia-Gil (2002) ArticleTitleRole of mitochondria in serum withdrawal-induced apoptosis of immortalized neuronal precursors Dev. Brain Res. 134 93–102 Occurrence Handle1:CAS:528:DC%2BD38Xis1Oqtb4%3D Occurrence Handle10.1016/S0165-3806(01)00326-1
M. Dalili G. Sayles D.F. Ollis (1989) ArticleTitleGlutamine-limited batch hybridoma growth and antibody production: experiment and model Biotechnol. Bioeng. 36 74–82
M. Fussenegger J.E. Bailey (1998) ArticleTitleMolecular regulation of cell cycle progression and apoptosis in mammalian cells: implications for biotechnology Biotechnol. Prog. 14 807–833 Occurrence Handle9841643 Occurrence Handle1:CAS:528:DyaK1cXntFegt7Y%3D Occurrence Handle10.1021/bp9800891
M. Fussenegger D. Fassnacht R. Schwartz J.A. Zanghi M. Graf J.E. Bailey R. Portner (2000) ArticleTitleRegulated over-expression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivations Cytotechnology 32 45–61 Occurrence Handle1:CAS:528:DC%2BD3cXhtV2lt7c%3D Occurrence Handle10.1023/A:1008168522385 Occurrence Handle19002966
P.R. Galle W.J. Hofman H. Walczak (1995) ArticleTitleInvolvement of the CD95/APO-1/fas) receptor and ligand in liver damage J. Exp. Med. 182 1223–1230 Occurrence Handle7595193 Occurrence Handle1:CAS:528:DyaK2MXovFWrurc%3D Occurrence Handle10.1084/jem.182.5.1223
E.J. Griffiths (2000) ArticleTitleMitochondria – potential role in cell life and death Cardivasc. Res. 46 24–27 Occurrence Handle1:CAS:528:DC%2BD3cXhvVOqsL0%3D Occurrence Handle10.1016/S0008-6363(00)00020-1
S. Gupta (2001) ArticleTitleMolecular steps of death receptor and mitochondrial pathways of apoptosis Life Sci. 69 2957–2964 Occurrence Handle11758823 Occurrence Handle1:CAS:528:DC%2BD3MXos12msb4%3D
W. Hamabe N. Fukushima A. Yoshida H. Ueda (2000) ArticleTitleSerum-free induced neuronal apoptosis-like cell death is independent of caspase activity Mol. Brain Res. 78 186–191 Occurrence Handle10891599 Occurrence Handle1:CAS:528:DC%2BD3cXks12qsL4%3D Occurrence Handle10.1016/S0169-328X(00)00074-7
E. Hatano C.A. Bradham A. Stark Y. Iimuro J.J. Lemarsters D. Brenner (2000) ArticleTitleThe mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes J. Biol. Chem. 276 11814–11823
D. Hockenberry G.L. Nunez C. Milliman R.D. Schreiber S.J. Korsmeyeer (1990) ArticleTitleBcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death Nature 348 334–336
D.C.S. Huang L. O’Reilly A. Strasser S. Cory (1997) ArticleTitleThe anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry The EMBO J. 16 4628–4638 Occurrence Handle1:CAS:528:DyaK2sXlsVClsLg%3D
A. Ishaque M. Al-Rubeai (1999) ArticleTitleRole of Ca2+Mg2+ and K+ ion in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture Apoptosis 4 335–355 Occurrence Handle14634337 Occurrence Handle1:CAS:528:DC%2BD3cXhsVynsw%3D%3D Occurrence Handle10.1023/A:1009643204200
A. Ishaque M. Al-Rubeai (2002) ArticleTitleRole of vitamins in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture Apoptosis 7 231–239 Occurrence Handle11997667 Occurrence Handle1:CAS:528:DC%2BD38XmsVaitLg%3D Occurrence Handle10.1023/A:1015343616059
R.A. Jones V.L. Johnson N.R. Buck M. Dobrota R.H. Hinton S.C. Chow G.E. Kass (1998) ArticleTitleFas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases Hepatology 27 1632–1642 Occurrence Handle9620337 Occurrence Handle1:CAS:528:DyaK1cXjvVCrsrs%3D Occurrence Handle10.1002/hep.510270624
G. Kroemer B. Dallaporta M. RescheRigon (1998) ArticleTitleThe mitochondrial death/life regulators in apoptosis and necrosis Annu. Rev. Physiol. 60 619–642 Occurrence Handle9558479 Occurrence Handle1:CAS:528:DyaK1cXitVOhsbs%3D Occurrence Handle10.1146/annurev.physiol.60.1.619
G. Kroemer N. Zamzami S.A. Susin (1997) ArticleTitleMitochondrial control of apoptosis Immunol. Today 18 44–51 Occurrence Handle9018974 Occurrence Handle1:CAS:528:DyaK2sXotFygtA%3D%3D Occurrence Handle10.1016/S0167-5699(97)80014-X
V. Lacronique A. Mignon M. Fabre B. Viollet N. Rouquet T. Molina A. Porteu A. Henrion D. Bouscary P. Varlet V. Joulin A. Khan (1996) ArticleTitleBcl-2 protects from lethal hepathic apoptosis induced by an anti-Fas antibody in mice Nat. Med. 2 IssueID1 80–86 Occurrence Handle8564847 Occurrence Handle1:CAS:528:DyaK28XivVSmsQ%3D%3D Occurrence Handle10.1038/nm0196-80
A.J. Mastrangelo (1999) Inhibition of apoptosis in mammalian cell culture: the biotechnological relevance of limiting cell death M. Al-Rubeai (Eds) Cell Engineering I Kluwer Academic Publishers Netherlands 162–185
M. Mayer M. Noble (1994) ArticleTitleN-acetyl-l-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro Proc. Natl. Acad. Sci. USA 91 7496–500 Occurrence Handle7914368 Occurrence Handle1:CAS:528:DyaK2cXlt1Gitbg%3D
A.J. McGowan R. Fernandes A. Samali T.G. Cotter (1996) ArticleTitleAnti-oxidants and apoptosis Biochem. Soc. Trans. 24 229–233 Occurrence Handle8674671 Occurrence Handle1:CAS:528:DyaK28Xht1Smur0%3D
S. Mecille B. Massie (1994) ArticleTitleInduction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells Biotechnol. Bioeng. 44 1140–1154
T. Minamikawa D.A. Williams D.N. Bowser P. Nagley (1999) ArticleTitleMitochondrial permeability transition and swelling can occur reversibly without inducing cell death in intact human cells Exp. Cell Res. 246 26–37 Occurrence Handle9882512 Occurrence Handle1:CAS:528:DyaK1MXmtlKisQ%3D%3D Occurrence Handle10.1006/excr.1998.4290
K. Murray C.E. Ang K. Gull J.A. Hickman A.J. Dickson (1996) ArticleTitleNSO myeloma cell death: influence of Bcl-2 over-expression Biotechnol. Bioeng. 51 298–304 Occurrence Handle1:CAS:528:DyaK28XksVCms78%3D Occurrence Handle10.1002/(SICI)1097-0290(19960805)51:3<298::AID-BIT5>3.0.CO;2-8 Occurrence Handle18624362
L.A. O’Reilly D.C.S. Huang A. Strasser (1996) ArticleTitleThe cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry EMBO J. 15 6979–6990 Occurrence Handle9003774 Occurrence Handle1:CAS:528:DyaK2sXosFChtg%3D%3D
P.A. Parone D. James J.C. Martinou (2002) ArticleTitleMitochondria: regulating the inevitable Biochemie 84 105–111 Occurrence Handle1:CAS:528:DC%2BD38XktVCru7k%3D Occurrence Handle10.1016/S0300-9084(02)01380-9
M. Rabinovitz (1992) ArticleTitleThe pleiotypic response to amino-acid deprivation is the result of interactions between components of the glycolysis and protein-synthesis pathways FEBS Lett. 302 113–116 Occurrence Handle1321723 Occurrence Handle1:CAS:528:DyaK38XisFaks7Y%3D Occurrence Handle10.1016/0014-5793(92)80418-G
L. Rao E. White (1997) ArticleTitleBcl-2 and the ICE family of apoptotic regulators: making a connection Curr. Opin. Genet. Dev. 7 52–58 Occurrence Handle9024635 Occurrence Handle1:CAS:528:DyaK2sXhsVeis70%3D Occurrence Handle10.1016/S0959-437X(97)80109-8
L.D. Robb-Gaspers G.A. Rutter P. Burnett G. Hajnóczky R.M Denton A.P. Thomas (1998) ArticleTitleCoupling between cytosolic and mitochondrial calcium oscillations: role in the regulation of hepatic metabolism Biochim. Biophys. Acta 1366 17–32 Occurrence Handle9714714 Occurrence Handle1:CAS:528:DyaK1cXltFWks7c%3D
S. Salvioli C. Barbi J. Dobrucki L. Moretti M. Pinti J. Pedrazzi T.L. Pazienza V. Bobyleva C. Franceshi A. Cossarizza (2000) ArticleTitleOpposite role of changes in mitochondrial membrane potential in diffrent apoptotic processes FEBS Lett. 469 186–190 Occurrence Handle10713268 Occurrence Handle1:CAS:528:DC%2BD3cXhsFGrtLo%3D Occurrence Handle10.1016/S0014-5793(00)01266-7
A. Sanfelui G. Stephanopoulos (1999) ArticleTitleEffect of glutamine limitation on the death of attached Chinese hamster ovary cells Biotechnol. Bioeng. 64 46–53
N.H. Simpson A.E. Milner Al-Rubeai (1997) ArticleTitlePrevention of hybridoma cell death by Bcl-2 during suboptimal culture conditions Biotechnol. Bioeng. 54 1–16 Occurrence Handle1:CAS:528:DyaK2sXhvFyns78%3D Occurrence Handle10.1002/(SICI)1097-0290(19970405)54:1<1::AID-BIT1>3.0.CO;2-K Occurrence Handle18634067
N.H. Simpson R.P. Singh A.N. Emery M. Al-Rubeai (1999) ArticleTitleBcl-2 over-expression reduces growth rate and prolongs G1 phase in continuous chemostat culture of hybridoma cells Biotechnol. Bioeng. 64 174–186 Occurrence Handle10397853 Occurrence Handle1:STN:280:DyaK1Mzis1amsw%3D%3D Occurrence Handle10.1002/(SICI)1097-0290(19990720)64:2<174::AID-BIT6>3.0.CO;2-D
N.H. Simpson R.P. Singh A. Perani C. Goldenzon M. Al-Rubeai (1998) ArticleTitleIn hybridoma cultures, deprivation of any single amino acid leads to apoptotic deathwhich is suppressed by the expression of the bcl-2 gene Biotechnol. Bioeng. 59 90–98 Occurrence Handle10099318 Occurrence Handle1:CAS:528:DyaK1cXjtFKns7Y%3D Occurrence Handle10.1002/(SICI)1097-0290(19980705)59:1<90::AID-BIT12>3.0.CO;2-6
R.P. Singh M. Al-Rubeai C.D. Gregory A.N. Emery (1994) ArticleTitleCell death in bioreactors: a role for apoptosis Biotechnol. Bioeng. 44 720–726 Occurrence Handle1:CAS:528:DyaK2cXlslOrsL8%3D Occurrence Handle10.1002/bit.260440608 Occurrence Handle18618832
R.P. Singh A.N. Emery M. Al-Rubeai (1996) ArticleTitleEnhancement of survivability of mammalian cells by over-expression of the apoptotic–suppressor gene bcl-2 Biotechnol. Bioeng. 52 166–175 Occurrence Handle1:CAS:528:DyaK28XlvFaitLc%3D Occurrence Handle18629863 Occurrence Handle10.1002/(SICI)1097-0290(19961005)52:1<166::AID-BIT17>3.0.CO;2-M
V.P. Skulachev (1996) ArticleTitleWhy are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide-producing mitochondria and cell FEBS Lett. 97 7–10
S.A. Susin N. Zamzami G. Kroemer (1998) ArticleTitleMitochondria as regulators of apoptosis: doubt no more Biochim. Biophys. Acta 1366 151–165 Occurrence Handle9714783 Occurrence Handle1:CAS:528:DyaK1cXltFWksLY%3D
E. Suzuki S. Terada H. Ueda (1997) ArticleTitleEstablishing apoptosis resistant cell lines for improving protein production of cell culture Cytotechnology 23 55–59 Occurrence Handle1:CAS:528:DyaK1cXisl2lsbs%3D Occurrence Handle10.1023/A:1007942929800
A.K. Talley S. Dewhurst S.W. Perry S.C. Dollard (1995) ArticleTitleTumour necrosis factor alpha-induced apoptosis inhuman neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and CrmA Mol. Cell Biol. 15 2359–2366 Occurrence Handle7739519 Occurrence Handle1:STN:280:DyaK2M3lsl2itw%3D%3D
B.T. Tey R.P. Singh L. Piredda M. Piancentini Al-Rubeai (2000a) ArticleTitleBcl-2 mediated suppression of apoptosis in myeloma NS0 cultures J. Biotechnol. 79 147–159 Occurrence Handle1:CAS:528:DC%2BD3cXjt1artLc%3D Occurrence Handle10.1016/S0168-1656(00)00223-6
B.T. Tey R.P. Singh L. Piredda M. Piancentini Al-Rubeai (2000b) ArticleTitleInfluence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody Biotechnol. Bioeng. 68 31–43 Occurrence Handle1:CAS:528:DC%2BD3cXitVOru7Y%3D Occurrence Handle10.1002/(SICI)1097-0290(20000405)68:1<31::AID-BIT4>3.0.CO;2-L
Y. Tsujimoto L.R. Finger J. Yunis P.C. Nowell C.M. Croce (1984) ArticleTitleCloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation Science 226 1097–1099 Occurrence Handle6093263 Occurrence Handle1:CAS:528:DyaL2MXhvVyjug%3D%3D
D.L. Vaux S. Cory J.M. Adams (1998) ArticleTitleBcl-2gene promotes haemopoietic cell survival and cooperates with c-mycto immortalise pre-B cells Nature 335 440–442
M. Woo A. Hakem A.J. Elia R. Hakem G.S. Duncan B.J. Patterson T.W. Mak (1999) ArticleTitleIn vivo evidence that caspase-3 is required for fas-mediated apoptosis of hepatocytes J. Immunol. 163 4911–4916
L.Z. Xie D.I.C. Wang (1996) ArticleTitleEnergy metabolism and TAP balance in animal cell cultivating using a stoichiometically based reaction network Biotechnol. Bioeng. 52 591–601 Occurrence Handle1:CAS:528:DyaK28Xms1WqtL0%3D Occurrence Handle18629932
N. Zamzami P. Marchetti M. Castedo T. Hirsch S.A. Susin B. Masse G. Kroemer (1996) ArticleTitleInhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis FEBS Lett. 384 53–57 Occurrence Handle8797802 Occurrence Handle1:CAS:528:DyaK28XisFWgtbg%3D Occurrence Handle10.1016/0014-5793(96)00280-3
N. Zamzami P. Marchetti M. Castedo C. Zanin J.L. Vayssiére P.X. Petit G. Kroemer (1995) ArticleTitleReduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo J. Exp. Med. 181 1661–1672 Occurrence Handle7722446 Occurrence Handle1:CAS:528:DyaK2MXlsVGhtb8%3D Occurrence Handle10.1084/jem.181.5.1661
X.D. Zhang S.K. Gillespie P. Hersey (2004) ArticleTitleStaurosporine induces apoptosis of melanoma by both caspase-dependent and – independent apoptotic pathways Mol. Cancer Therapeutics 3 IssueID2 187–197 Occurrence Handle1:CAS:528:DC%2BD2cXhsFCitrw%3D
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mukwena, N.T., Al-Rubeai, M. Apoptosis and Its Suppression in Hepatocytes Culture. Cytotechnology 46, 79–95 (2004). https://doi.org/10.1007/s10616-005-8306-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-005-8306-7