Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture

Ishaque, Adiba; Al‐Rubeai, Mohamed

doi:10.1016/s0022-1759(98)00166-5

Cited by 116 publications

(69 citation statements)

References 24 publications

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“…For instance the AV assay using FITC-AV/PI staining is a sensitive FC method for detecting PS exposure, which is an early and transient event in apoptosis that may be difficult to distinguish from the necrotic cell fraction. Studies utilizing the AV assay without including PI in the analysis run the risk of overestimating the level of apoptosis and incorrectly diagnosing necrosis as apoptotic cell death (43,48). Consequently, the level of EA cell induction by TNF␣ at 24 h was a significant parameter that we had consistently highlighted, and inhibited by an overexpression of COX-2.…”

Section: Discussionmentioning

confidence: 89%

“…Because there is no phagocytic disposal mechanism in vitro apoptotic cells accumulate and continue to undergo degradation and membrane lysis. It was crucial, therefore, to include PI in the reaction, and highlight PS exposure on the surface of EA cells, and distinguish them from N cell populations, which may or may not have transited the process of apoptosis (43). By this analysis it was possible to both qualitatively determine viable, V (AV(Ϫ)/PI(Ϫ)), EA (AV(ϩ)/PI(Ϫ)), and N (AV(Ϫ and ϩ)/PI(ϩ)) cell fractions (Fig.…”

Section: Characterization Of Cox-2 Overexpression Bymentioning

confidence: 99%

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Cyclooxygenase-2 Inhibits Tumor Necrosis Factor α-mediated Apoptosis in Renal Glomerular Mesangial Cells

Ishaque

Dünn

Sorokin

2003

Journal of Biological Chemistry

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Renal mesangial cell apoptosis is a crucial repair mechanism in glomerular nephritis (GN). These cells express receptors to tumor necrosis factor ␣ (TNF␣), a cytokine with proapoptotic properties implicated in the resolution of GN. Progression to proliferative GN is accompanied by cyclooxygenase-mediated formation of prostaglandins and inefficient apoptosis of mesangial cells. The aims of this study were to quantify TNF␣-mediated apoptosis in renal mesangial cells and to determine whether expression of the inducible form of cyclooxygenase, cylooxygenase-2 (COX-2), inhibits this apoptosis. By 24 h significant levels of apoptosis were induced by TNF␣ (100 ng/ml) or etoposide control (100 M), as shown by phosphatidylserine externalization, caspase-3 activation, development of a sub-G 0 /G 1 region, and distinct chromatin condensation. Using adenoviralmediated delivery of the COX-2 gene (AdCOX-2) apoptotic features were prevented from appearing in AdCOX-2 cells treated with TNF␣, whereas etoposidetreated AdCOX-2 cells were not protected. Furthermore, COX-2 expression, induced by the vasoconstrictor peptide ET-1 or the cytokine interleukin-1␤ also inhibited TNF␣-mediated but not etoposide-mediated apoptosis, to an extent, similar to adenoviral COX-2 infection. Selective COX-2 inhibition by NS-398 restored TNF␣-mediated apoptosis. Prostaglandin (PG) E 2 and PGI 2 were shown to be the major prostaglandin metabolites in Ad-COX-2 cells. The addition of PGE 2 and PGI 2 protected against TNF␣-mediated apoptosis. These results demonstrate COX-2 anti-apoptotic activity via a death receptor route and suggest that selective COX-2 inhibition may augment TNF␣ apoptosis in chronic inflammatory conditions.

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Section: Discussionmentioning

confidence: 89%

Section: Characterization Of Cox-2 Overexpression Bymentioning

confidence: 99%

Cyclooxygenase-2 Inhibits Tumor Necrosis Factor α-mediated Apoptosis in Renal Glomerular Mesangial Cells

Ishaque

Dünn

Sorokin

2003

Journal of Biological Chemistry

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“…To analyze the fate of BAF-B03 cells following ␥c blockade, cells were stimulated with IL-2 or IL-15 in the presence of anti-␥c mAbs and examined for apoptotic cell death by annexin V staining (20). Annexin V binds to cell membrane-associated phosphatidylserine, which is restricted to the interior side of the cell membrane in living cells and is rapidly exposed to the exterior side in the early stage of apoptotic cell death.…”

Section: Resultsmentioning

confidence: 99%

Blocking the Common γ-Chain of Cytokine Receptors Induces T Cell Apoptosis and Long-Term Islet Allograft Survival

Ima

et al. 2000

The Journal of Immunology

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The common γc-chain is an essential signaling component shared by all known T cell growth factor (TCGF) receptors (i.e., IL-2, IL-4, IL-7, IL-9, and IL-15). In the present study, we have studied the effect of γc-chain blockade on T cell activation and allograft rejection. Treatment of B6AF1 (H-2b/d.k) recipient mice with anti-γc mAbs induced long-term survival of DBA/2 (H-2d) islet allografts (>150 days, n = 8), whereas control Ab-treated mice rejected the islet allografts within 17 days (n = 6). The state of engraftment induced by the anti-γc mAbs was remarkably stable, as recipient mice bearing the primary islet allografts accepted a second DBA/2 islet allograft without further immunosuppression and systemic administration of high doses of IL-2Ig fusion protein failed to provoke rejection. Blocking the γc-chain inhibited T cell proliferation and induced T cell apoptosis by repressing expression of Bcl-2. Our data suggest that one means of inducing T cell apoptosis and stable allograft survival can be achieved via γc-chain blockade.

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“…Apoptosis, a phenomenon controlled by a tight equilibrium between inducer and suppressor genes (Evan et al, 1992;Farrow et al, 1995;Oltvai et al, 1993;Yang et al, 1995;Yonish-Rouach et al, 1991), can be inhibited by transfection of antiapoptotic genes such as bcl-2-related genes (Boise et al, 1993;Reed, 1994) or the adenovirus E1B-19K gene (Teodoro and Branton, 1997). Indeed, constitutive overexpression of Bcl-2, Bcl-X L , or E1B-19K in B-cell hybridomas has been shown to prolong viability of cultured hybridomas (Charbonneau and Gauthier, 2000;Fassnacht et al, 1998;Gauthier et al, 1996;Ishaque and Al-Rubeai, 1998;Mercille et al, 1999;Minn et al, 1995;Murray et al, 1996;Singh et al, 1996;Terada et al, 1997). However, constitutive overexpression of bcl-2-related genes has been shown to have detrimental eects on genomic stability of other cell types by preventing p53-induced apoptotic death of cells bearing genetic abnormalities such as mutations or mitotic damage (Cherbonnel-Lasserre et al, 1996;Minn et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Inducible expression of Bcl‐X_L restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures

Jung¹,

Côté²,

Drouin³

et al. 2002

Biotech & Bioengineering

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B-cell hybridomas are widely used to produce monoclonal antibodies via large-scale cell culture. Unfortunately, these cells are highly sensitive to apoptotic death under conditions of nutrient deprivation observed at the plateau phase of batch cultures. Previous work has indicated that constitutive high-level expression of antiapoptotic genes in hybridoma cells could delay apoptosis, resulting in higher cell densities and prolonged viability. However, the constitutive high-level expression of antiapoptotic genes has been shown to have detrimental effects on genomic stability of other types of cultured cells. Inducible gene expression may be used to avoid this problem. In the present study, we first constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drives the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in D5 hybridoma cells. Our data show that stably transfected D5 cells (4G1.D9) expressed high levels of Bcl-X(L) following overnight exposure to ZnSO(4) concentrations (50 to 100 microM) that did not affect control cells. The level of Bcl-X(L) expressed after ZnSO(4) induction was sufficient to prevent apoptosis experimentally induced by cycloheximide and allowed 4G1.D9 cells to grow at higher densities and remain viable for prolonged periods in suboptimal culture conditions. The use of inducible bcl-XL expression permits extension of the viability of cultured B-cell hybridomas during the antibody secretion phase without the adverse genetic effects associated with constitutive long-term bcl-XL expression.

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Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture

Cited by 116 publications

References 24 publications

Cyclooxygenase-2 Inhibits Tumor Necrosis Factor α-mediated Apoptosis in Renal Glomerular Mesangial Cells

Cyclooxygenase-2 Inhibits Tumor Necrosis Factor α-mediated Apoptosis in Renal Glomerular Mesangial Cells

Blocking the Common γ-Chain of Cytokine Receptors Induces T Cell Apoptosis and Long-Term Islet Allograft Survival

Inducible expression of Bcl‐X_L restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures

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Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture

Cited by 116 publications

References 24 publications

Cyclooxygenase-2 Inhibits Tumor Necrosis Factor α-mediated Apoptosis in Renal Glomerular Mesangial Cells

Cyclooxygenase-2 Inhibits Tumor Necrosis Factor α-mediated Apoptosis in Renal Glomerular Mesangial Cells

Blocking the Common γ-Chain of Cytokine Receptors Induces T Cell Apoptosis and Long-Term Islet Allograft Survival

Inducible expression of Bcl‐XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures

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Inducible expression of Bcl‐X_L restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures