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Results for “Apoptosis”

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PubMed2005

Influenza A virus-induced apoptosis is a multifactorial process: exploiting reverse genetics to elucidate the role of influenza A virus proteins in virus-induced apoptosis.

Three influenza viruses, A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to the herpes simplex virus tegument protein, VP22. However, this did not reflect viral protein-protein-RNA interactions known to occur within infected cells. To explore the role of viral proteins in apoptosis under infection conditions, recombinant viruses with single or triple gene exchanges were generated using A/Victoria or clone 7a as the background virus. Inserting the A/Fiji NS or PB2 gene into A/Victoria or clone 7a significantly reduced the level of apoptosis compared to the parent virus while clone 7a PA or NP genes increased apoptosis. Inserting A/Fiji NA or HA or clone 7a NS, M, NA, or HA genes individually into A/Victoria had no significant effect on apoptosis. Surprisingly, inserting the M, NA, and HA genes of A/Fiji together into clone 7a reduced apoptosis, whereas inserting clone 7a M, NA, and HA together into A/Fiji increased apoptosis. These results suggest that no single virus protein induces apoptosis and that the combination of genes required may be strain specific, highlighting the difficulty of predicting the virulence of new strains that arise in nature. No support for the view that apoptosis is essential for high virus yields was obtained as high virus yields were obtained with viruses that induced both high and low levels of apoptosis.

Amino Acid Sequence↗

PubMed2005

A radiation-induced acute apoptosis involving TP53 and BAX precedes the delayed apoptosis and neoplastic transformation of CGL1 human hybrid cells.

Exposing CGL1 (HeLa x fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of approximately 50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X(L) is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.

Apoptosis↗

PubMedDate not supplied

Apoptosis and anti-apoptosis signalling in glaucomatous retinopathy.

PURPOSE: Recent studies in the post-mortem human retina have suggested that apoptosis contributes to retinal ganglion cell (RGC) loss in glaucoma. If apoptosis contributes significantly to glaucomatous RGC loss, and if the specific apoptosis signalling pathways for glaucomatous apoptosis can be determined, agents that interrupt or oppose the signalling have the potential to slow the progression of glaucoma. METHODS: Recent data in animal models indicate that mitochondrially-dependent apoptosis contributes to RGC loss in glaucoma. Mitochondrially-dependent apoptosis involves proteins like BAX that increase mitochondrial membrane permeability and promote apoptosis and proteins like BCL-2 that decrease mitochondrial membrane permeability and reduce apoptosis. New protein synthesis induced by the alpha-2 agonist, brimonidine, prevents decreases in the levels of BCL-2 and thereby reduces mitochondrially-dependent apoptosis. CONCLUSIONS: Brimonidine appears to maintain BCL-2 levels by supporting the activity of an intrinsic anti-apoptosis signalling system that involves phosphorylation of protein kinase B. Phosphorylated protein kinase B appears to counteract the apoptosis signalling mechanisms which operate in glaucomatous retina.

Adrenergic alpha-Agonists↗

PubMedDate not supplied

Expression of apoptosis and apoptosis-related peptides in various stages of rejection in the human transplanted liver.

BACKGROUND/AIMS: In the transplanted liver, the role of apoptosis and apoptosis-related proteins are largely unknown. This study addresses the question whether hepatocyte or leukocyte apoptosis plays an important role in acute rejection of the transplanted human liver and which pathways are involved. METHODOLOGY: Cryosections from liver biopsies with acute rejection were stained with the TUNEL technique for detection of apoptosis and labeled immunohistochemically with antibodies against CD95, bcl-2, TGF-beta and iNOS. A double-labeling protocol was developed for simultaneous detection of iNOS and apoptosis. Liver tissue with chronic viral hepatitis, with hepatitis reinfection and tissue without pathological findings served as a control. RESULTS: Leukocyte apoptosis was markedly reduced in severe compared to mild or moderate acute rejection. Hepatocyte apoptosis is detected rarely in acute rejection with a slight increase from mild to severe despite a strong expression of CD95 and TGF-beta on hepatocytes. The hepatocyte expression of iNOS is weak in acute rejection but strong in control slides with hepatitis B/C reinfection. In acute rejection, simultaneous expression of iNOS and apoptosis could be demonstrated in Kupffer-cells. CONCLUSIONS: Severe acute rejection in the human transplanted liver is characterized by a lack of apoptosis of infiltrating portal lymphocytes probably caused by a reduced downregulation of lymphocyte function. Secondly, in spite of the strong expression of CD95 and TGF-alpha, hepatocyte apoptosis plays a limited role for liver damage in acute rejection. Finally, Kupffer cell apoptosis is increased in acute rejection and seems to be induced by nitric oxide.

Apoptosis↗

PubMed1996

Spontaneous apoptosis in human colon tumor cell lines and the relation of wt p53 to apoptosis.

OBJECTIVE: To examine spontaneous apoptosis of cultured human colon tumor cell lines in vitro and to investigate the role of wild type (wt) p53 in regulation of apoptosis induced by DNA-damaging treatment. METHODS: A model system of human tumor progression involving three cell lines was used in this study for examination of apoptosis. They were originally established from human colon villous adenoma, including an early passage of non-tumorigenic cell line, V235E; a late passage of weakly tumorigenic cell line, V235L; and a spontaneous progressing highly tumorigenic cell line. V411. All of them maintain wt p53 expression. For identification of apoptosis, two tests were performed: 1. morphology study using acridine orange (AO)/ethidium bromide (EB) stainning by fluorescence microscopy; 2. DNA electrophoresis on agarose gel. P53 and WAF-1 (a downstream gene of p53) expressions were analysed at mRNA level using Northern blot technique. Apoptotic index of cell lines examined was measured by DNA fluorescence assay. RESULTS: Spontaneous apoptosis was demonstrated in cell lines of all stages of progression by both morphology and DNA agarose gel electrophoresis. Apoptosis was further induced in V411 after treatment of cells with 137Cs gamma-irradiation and accompanied by increases in p53 and WAF-1 expression. In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen. CONCLUSIONS: Apoptosis can be maintained in some human tumor cell lines despite transformation and carcinogenesis. Wt p53 and WAF-1 products are two of the potential mediators which effect apoptosis. Additionally, since apoptosis was enhanced by irradiation in V411, but not in sw480, it suggests that wt p53 cancer cells are more sensitive to DNA-damaging treatment than mutant p53 cancer cells. These finding may have implications for cancer therapy.

Adenoma, Villous↗

PubMed1997

Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro.

Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-alpha (TNF-alpha), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-alpha inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4, and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-alpha was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-alpha neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-alpha to accelerate apoptosis was lost if neutrophils were primed with 1 micromol/L PAF or aged for 6 hours before TNF-alpha addition. The TNFR55-selective TNF-alpha mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-alpha. Although the TNFR75-selective mutant (D143F) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-alpha-stimulated apoptosis. Hence, TNF-alpha has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.

Antibodies, Monoclonal↗

PubMed1998

Cytochrome c induces caspase-dependent apoptosis in intact hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-kinase-kinase, and malignant change.

It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3-dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.

Animals↗

PubMed2003

The histone deacetylase inhibitor suberic bishydroxamate: a potential sensitizer of melanoma to TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis.

TRAIL appears to be a promising anticancer agent in that it induces apoptosis in a wide range of cancer cells but not normal tissues. Sensitivity of melanoma cells to TRAIL-induced apoptosis varied considerably because of their development of various resistance mechanisms against apoptosis. We discuss in this report the potential effect of a histone deacetylase inhibitor SBHA on TRAIL-induced apoptosis. Histone deacetylase (HDAC) inhibitors regulate histone acetylation and thereby modulate the transcriptional activity of certain genes leading to cell growth arrest, cellular differentiation, and apoptosis. Suberic bishydroxamate (SBHA) is a relatively new HDAC inhibitor that induced apoptosis in the majority of melanoma cell lines through a mitochondrial and caspase-dependent pathway. This was due to its regulation of the expression of multiple proteins that are involved in either the mitochondrial apoptotic pathway (Bcl-2 family members) or the final phase of apoptosis (caspase-3 and XIAP). Co-treatment with SBHA at nontoxic doses and TRAIL resulted in a marked increase in TRAIL-induced apoptosis of melanoma, but showed no toxicity to melanocytes. SBHA appeared to sensitize melanoma to TRAIL-induced apoptosis by up-regulation of pro-apoptotic proteins in the TRAIL-induced apoptotic pathway such as caspase-8, caspase-3, Bid, Bak, and Bax, and up-regulation of the BH3 domain only protein, Bim. This, together with activated Bid, may have acted synergistically to cause changes in mitochondria. Treatment with SBHA also resulted in down-regulation of antiapoptotic members of the Bcl-2 family, Bcl-X(L) and Mcl-1, and the IAP member, XIAP. These changes would further facilitate apoptotic signaling. SBHA appeared therefore to be a potent agent in overcoming resistance of melanoma to TRAIL-induced apoptosis.

Animals↗

PubMed2004

Bcl-2 inhibitors sensitize tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by uncoupling of mitochondrial respiration in human leukemic CEM cells.

Previous studies have shown that the lymphoblastic leukemia CEM cell line is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis because of a low expression of caspase-8. Bcl-2 inhibitors, BH3I-2' and HA14-1, are small cell-permeable nonpeptide compounds, are able to induce apoptosis by mediating cytochrome c release, and also lead to dissipation of the mitochondrial membrane potential (DeltaPsim). This study aimed to use the Bcl-2 inhibitors to sensitize CEM cells to TRAIL-induced apoptosis by switching on the mitochondrial apoptotic pathway. We found that a low dose of BH3I-2' or HA14-1, which did not induce cytochrome c release, greatly sensitized CEM cells to TRAIL-induced apoptosis. In a similar manner to the classical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), both BH3I-2' and HA14-1 induced a reduction in DeltaPsim, a generation of reactive oxygen species (ROS), an increased mitochondrial respiration, and a decreased ATP synthesis. This uncoupling function of the Bcl-2 inhibitors was responsible for the synergy with TRAIL-induced apoptosis. CCCP per se did not induce apoptosis but again sensitized CEM cells to TRAIL-induced apoptosis by uncoupling mitochondrial respiration. The uncoupling effect facilitated TRAIL-induced Bax conformational change and cytochrome c release from mitochondria. Inhibition of caspases failed to block TRAIL-mediated cell death when mitochondrial respiration was uncoupled. We observed that BH3I-2', HA14-1, or CCCP can overcome resistance to TRAIL-induced apoptosis in TRAIL-resistant cell lines, such as CEM, HL-60, and U937. Our results suggest that the uncoupling of mitochondrial respiration can sensitize leukemic cells to TRAIL-induced apoptosis. However, caspase activation per se does not represent an irreversible point of commitment to TRAIL-induced cell death when mitochondrial respiration is uncoupled.

Apoptosis↗

PubMed2006

Cell cycle- and apoptosis-regulatory protein-1 is involved in apoptosis signaling by epidermal growth factor receptor.

CARP-1, a novel apoptosis inducer, regulates apoptosis signaling by diverse agents, including adriamycin and growth factors. Epidermal growth factor receptor (EGFR)-related protein (ERRP), a pan-ErbB inhibitor, inhibits EGFR and stimulates apoptosis. Treatments of cells with ERRP or Iressa (an EGFR tyrosine kinase inhibitor) results in elevated CARP-1 levels, whereas antisense-dependent depletion of CARP-1 causes inhibition of apoptosis by ERRP. CARP-1 is a tyrosine-phosphorylated protein, and ERRP treatments cause elevated tyrosine phosphorylation of CARP-1. CARP-1 contains multiple, nonoverlapping apoptosis-inducing subdomains; one such subdomain is present within amino acids 1-198. Wild-type or CARP-1-(1-198) proteins that have substitution of tyrosine 192 to phenylalanine abrogate apoptosis by ERRP. In addition, apoptosis mediated by wild type or CARP-1-(1-198), and not CARP-1-(1-198(Y192F)), results in activation of caspase-9 and increased phosphorylation of p38 MAPK. However, the expression of dominant-negative forms of p38 MAPK activators MKK3 or MKK6 proteins inhibits apoptosis induced by both the full-length and truncated (amino acids 1-198) proteins. Together, data demonstrate that tyrosine 192 of CARP-1 is a target of apoptosis signaling, and CARP-1, in turn, promotes apoptosis by activating p38 MAPK and caspase-9.

Apoptosis↗

PubMed2003

Actinomycin D renders cultured synovial fibroblasts susceptible to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis.

OBJECTIVE: To investigate the expression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors in cultured synovial fibroblasts from rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and to examine their susceptibility to TRAIL-induced apoptosis in the presence or absence of metabolic inhibitors. METHODS: The expression of TRAIL receptors in synovial fibroblasts was examined by Western blot and immunohistochemistry. Expression of TRAIL-receptor 1 (TRAIL-R1), FLICE-inhibitory protein (Fas-associating protein with death domain-like interleukin-1-converting enzyme), and Bcl-2 was assessed by Western blot. Synovial cell viability was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay (XTT), and apoptosis was determined both by DNA content analysis after propidium iodide staining and Annexin V stain. RESULTS: TRAIL-R1 was constitutively expressed on cultured synovial fibroblasts from RA and OA, however, expression of TRAIL-R2 and TRAIL-R3 was not observed by immunohistochemistry and Western blot. Cultured synovial fibroblasts were resistant to apoptosis by TRAIL alone, but combined treatment of TRAIL with actinomycin D (ActD: 200 ng/mL), cycloheximide (CHX: 10 microg/mL), or proteasome inhibitor (MG132: 20 microM) induced apoptosis in a dose-dependent manner. The apoptosis was completely or partially inhibited by various caspase inhibitors, implicating an involvement of caspase pathway in TRAIL-induced apoptosis in the presence of these metabolic inhibitors. Expression of TRAIL-R1, FLIPL, and Bcl-2 did not account for the apoptosis by the combined treatment of TRAIL with ActD. CONCLUSIONS: Although TRAIL-R1 was constitutively expressed; cultured synovial fibroblasts were resistant to apoptosis by TRAIL. ActD, CHX, and MG132 rendered cultured synovial fibroblasts susceptible to TRAIL-induced apoptosis by a caspase-dependent mechanism. However, the exact mechanism of sensitization by these metabolic inhibitors remains to be determined.

Apoptosis↗

PubMed2006

Sensitization to gimatecan-induced apoptosis by tumor necrosis factor-related apoptosis inducing ligand in prostate carcinoma cells.

Since the intrinsic resistance of prostate carcinoma likely reflects a low susceptibility to drug-induced apoptosis, in this study we explored the possibility of sensitizing prostate carcinoma cells to apoptosis by combination of TRAIL with camptothecins. Indeed, these agents are known to activate different pathways of apoptosis. Topotecan- and gimatecan induced moderate up-regulation of TRAIL-R1 and -R2 which resulted in a different cell response to the combination in androgen-independent cells (DU-145 and PC-3). In DU-145 cells apoptosis was increased by lower TRAIL concentrations and was earlier than in PC-3 cells, as shown using Annexin V-binding assay. The relative resistance of PC-3 cells to drug-induced apoptosis was associated with constitutive Akt activation, higher levels of cFLIP-L and Bcl-2, and lower levels of Bax. The different expression/activation of apoptosis-related factors appears to influence the sensitization of prostate carcinoma cells by TRAIL. Potentiation of camptothecin-induced apoptosis by TRAIL appears dependent on cooperation between extrinsic and intrinsic pathways, as documented by loss of the sensitization to apoptosis following reduction of caspase 8 after small interfering RNA transfection. The efficacy of the approach may be critically dependent on the intrinsic susceptibility to apoptosis of different tumors. These observations support that the activation of multiple signals could enhance apoptotic response and suggest the therapeutic interest of the TRAIL/camptothecin combination.

Adenocarcinoma↗

PubMed2000

Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand.

Fas (APO-1/CD95) is a transmembrane protein of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily that induces apoptosis in susceptible normal and neoplastic cells upon cross-linking by its ligand (FasL). TNF-related apoptosis-inducing ligand (TRAIL) is a more recently identified member of the TNF superfamily that has been shown to selectively kill neoplastic cells by engaging two cell-surface receptors, DR4 and DR5. Two additional TRAIL receptors (DcR1 and DcR2) do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis. We addressed the expression of Fas, DR4, and DR5 in thyroid carcinoma cell lines and in 31 thyroid carcinoma specimens by Western blot analysis and immunohistochemistry, respectively, and tested the sensitivity of thyroid carcinoma cell lines to Fas- and TRAIL-induced apoptosis. Fas was found to be expressed in most thyroid carcinoma cell lines and tissue specimens. Although cross-linking of Fas did not induce apoptosis in thyroid carcinoma cell lines, Fas-mediated apoptosis did occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting the presence of a short-lived inhibitor of the Fas pathway in these cells. Cross-linking of Fas failed to induce recruitment and activation of caspase 8, whereas transfection of a constitutively active caspase 8 construct effectively killed the SW579 papillary carcinoma cell line, arguing that the action of the putative inhibitor occurs upstream of caspase 8. By contrast, recombinant TRAIL induced apoptosis in 10 of 12 thyroid carcinoma cell lines tested, by activating caspase-10 at the receptor level and triggering a caspase-mediated apoptotic cascade. Resistance to TRAIL did not correlate with DcR1 or DcR2 protein expression and was overcome by protein synthesis inhibition in 50% of the resistant cell lines. One medullary carcinoma cell line was resistant to Fas-and TRAIL-induced apoptosis, even in the presence of cycloheximide, and to transfection of constitutively active caspase-8, suggesting a different regulation of the apoptotic pathway. Our observations indicate that TRAIL effectively kills carcinomas that originate from the follicular epithelium of the thyroid gland, by inducing caspase-mediated apoptosis, and may provide a potentially potent therapeutic reagent against thyroid cancer.

Adult↗

PubMed2003

Apoptosis induction in prostate cancer cells and xenografts by combined treatment with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and CPT-11.

Because apoptosis is deregulated in most cancers, apoptosis-modulating approaches offer an attractive opportunity for clinical therapy of many tumors, including that of the prostate. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to treatment with Apo2 ligand (Apo2L) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), when using a nontagged, Zn-bound recombinant trimeric version that is devoid of any exogeneous sequences and therefore least likely to be immunogenic in human patients and that has been optimized for maximum efficacy and minimum toxicity. When combined with the topoisomerase I inhibitor CPT-11 (irinotecan), Apo2L/TRAIL exhibits enhanced apoptotic activity in C4-2 cells cultured in vitro as well as xenografted as tumors in vivo. Apoptosis both in vitro and in vivo was characterized by two major molecular events. First, apoptosis induction was accompanied by changes in expression levels of the Bcl-2 family genes and their products. However, whereas combination treatment applied to in vitro cell culture was characterized by a significant up-regulation and activation of Bax and down-regulation of Bcl-xL, the treatment applied to tumors induced Bak and Bcl-xS, whereas Bcl-omega and Bcl-xL were down-regulated. Because there are multiple members of the Bcl-2 family (24 members to date), these data indicate that, under different biological conditions, different proteins may be responsible for activating apoptosis and provide evidence for a differential regulation of the multidomain Bcl-2 protein-encoding genes, bax and bak. Increased Bax expression led to its activation, translocation to the mitochondria, and release of cytochrome c. In addition, this combination treatment induced apoptosis through potent activation of caspase-8 and the proapoptotic protein Bid, resulting in activation of effector caspase-3 and cleavage of its cellular target protein, poly(ADP-ribose) polymerase (PARP), events blocked by the pan-caspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoro methylketone (zVAD-fmk). Activation of multiple caspases and PARP cleavage were also observed in the C4-2 tumors treated with doses resulting in effective tumor control at 42 days after Apo2L/TRAIL plus CPT-11 treatment. Down-regulation of Bax by small interference (RNA) (siRNA) in C4-2 cells significantly prevented PARP cleavage and apoptosis. Strikingly, similar experiments in cells stably expressing a dominant-negative death receptor DR5 led to complete ablation of PARP cleavage and apoptosis, indicating the essential role of both mitochondrial and receptor-mediated apoptotic pathways. Our data indicate that the combined treatment of Apo2L/TRAIL and CPT-11 achieves tumor control in prostate cancer tumors through regulation of Bcl-2 family proteins and potent activation of caspases.

Animals↗

PubMed2002

Apoptosis and apoptosis-related factors Bcl-2, Bax, tumor necrosis factor-alpha, and NF-kappaB in human endometrial hyperplasia and carcinoma.

BACKGROUND: Apoptosis controls cell homeostasis in the endometrium during normal menstrual cycles, and morphologic studies have suggested its association with the development of endometrial carcinoma. Apoptosis is regulated by several genes, especially those of the Bcl-2 gene family, but their significance in endometrial pathologies is not well understood. METHODS: To study the role and regulation of apoptosis in endometrial hyperplasia and carcinoma, human endometrial specimens were analyzed using in situ 3'-end labeling of apoptotic cells and in situ hybridization and immunohistochemistry of apoptosis-related factors. RESULTS: Apoptosis was scarce in normal proliferating endometrium as well as in simplex, complex, and atypical hyperplasia and was low in Grade I adenocarcinoma. In Grade II adenocarcinoma a significant increase in the rate of apoptosis was observed. Apoptosis decreased in Grade III adenocarcinoma, but it was still higher than in normal or hyperplastic endometrium. Bcl-2 and Bax were expressed in normal and hyperplastic endometrium, and the Bcl-2/Bax ratio was lower in endometrial carcinoma. Tumor necrosis factor-alpha was expressed in normal endometrium and simplex and complex hyperplasia, but it was down-regulated in atypical hyperplasia and endometrial carcinoma. The transcription factor NF-kappaB was present in proliferating endometrium and in endometrial hyperplasia, but its expression was lower in carcinoma. CONCLUSIONS: In endometrial proliferation and hyperplasia a low rate of apoptosis is present. In Grade I carcinoma the rate of apoptosis is decreased, but the rate is subsequently increased in advanced carcinoma. The decrease in the rate of apoptosis in Grade III adenocarcinoma may reflect loss of control of cell homeostasis, decreased differentiation, and increased malignancy.

Adult↗

PubMed1997

Germinal center B cells rescued from apoptosis by CD40 ligation or attachment to follicular dendritic cells, but not by engagement of surface immunoglobulin or adhesion receptors, become resistant to CD95-induced apoptosis.

Germinal centers (GC) constitute a specialized microenvironment essential for the formation of memory B cells, B cell affinity maturation and isotype switching. Within the GC, the B cells closely interact with follicular dendritic cells (FDC) and T cells, which both provide stimuli to the B cells that prevent their entry into apoptosis and promote their differentiation into memory cells or plasma cells. Cross-linking of B cell immunoglobulin (Ig) receptors by antigen, stimulation of the integrin adhesion molecules LFA-1 and VLA-4 on the B cell through interaction with their counter receptors ICAM-1 and VCAM-1 on the FDC and cross-linking of CD40 on the B cells through interaction with the CD40 ligand (CD40L) on T cells have been shown to prevent entry into apoptosis of GC B cells. Triggering of CD95, on the other hand, has been shown to induce apoptosis. We therefore investigated the interaction between adhesion-mediated signals, Ig, CD40, and CD95. The spontaneous apoptosis of GC B cells was not further increased by adding anti-CD95. However, CD95 stimulation did result in apoptosis of GC B cells in the presence of anti-Ig or adhesion-mediated rescue signals, which indicates that CD95 expressed on GC B cells is functionally active. In contrast, anti-CD95 was unable to induce apoptosis in cells rescued via CD40 stimulation, suggesting an important role for CD40L expressed on GC T cells in apoptosis regulation. We also studied apoptosis of B cells adhering to FDC, and found that B cells that interact with FDC were also rescued from CD95-induced apoptosis. A human CD40.Fc mu fusion protein that blocks CD40 ligation failed to inhibit this effect. Our studies therefore indicate that neither CD40, Ig receptors, nor adhesion receptors mediate rescue from apoptosis by FDC.

Apoptosis↗

PubMed2002

Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells for studies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role.

Previously, we have shown that apoptosis induced by influenza virus was inhibited by an anti-neuraminidase compound [4-guanidino-2, 3-dehydro-N-acetylneuraminic acid (GG167; Relenza; Zanamivir)], which does not enter cells, and acts at the attachment/entry phase of virus replication. Furthermore, a virulent virus, clone 7a, induced greater levels of apoptosis than the attenuated A/Fiji and had greater neuraminidase (NA) activity. To confirm more directly that NA induces apoptosis, the NA of clone 7a and A/Fiji was expressed fused to the Herpes simplex virus tegument coat protein VP22, transfected into HeLa cells and the level of apoptosis determined. VP22 translocates between cells via the medium thus allowing expressed proteins to transfer to a larger number of cells than those originally transfected. Clone 7a NA fused to VP22 induced a significant level of apoptosis whereas A/Fiji NA/VP22 did not, confirming that NA activity is an important determinant of apoptosis acting during fusion protein translocation between cells. Furthermore, the induction of apoptosis was abrogated by antibody to transforming growth factor-beta, which is activated by NA. This approach also showed that VP22/NS1 proteins of both clone 7a and A/Fiji induced apoptosis when expressed alone but inhibited double stranded RNA-induced apoptosis suggesting that this protein may have a dual mode of action. Also, the M1 and M2 proteins of both viruses induced apoptosis but their NP proteins did not.

Apoptosis↗

PubMed2005

The scavenger cell hypothesis of apoptosis: apoptosis redefined as a process by which a cell in living tissue is destroyed by phagocytosis.

Current literature on the definition and description of apoptosis is very confusing and erratic, due to voluminous studies in recent decades using cell culture technique. Apoptosis has evolved as a programmed mechanism of cell demise to get rid of the cells that are no longer needed by the body. The most important reason for a creature to use this mechanism to kill cells is to avoid inflammatory response that causes tissue damage and ensuing scar formation, as seen in necrosis. To reach this aim, the dying cell communicates, at early stages of the dying process, with macrophages or its neighboring cells that have phagocytotic ability, coined collectively as scavenger cells herein. The dying cell is swiftly engulfed by a scavenger cell without leaking any noxious cellular components into the intercellular space to provoke an inflammatory response. Thus, apoptosis is a process involving at least one other cell type and is actually a mechanism occurring in live tissue. Most studies of apoptosis in recent decades neglect this fundamental point and use cell culture system with a single cell type in the medium, in which avoidance of inflammatory response and tissue damage is no longer a reason. In culture, the dying cell has no way to signal scavenger cells to engulf itself and thus needs to demobilize a series of special mechanisms, which have no need in live tissue, to complete the suicidal process and clearance of its own corpse. These "otherwise-no-need" mechanisms seem to involve activation of executor caspases by cytochrome c, and the activated caspases mediate late processes of apoptosis in vitro. However, because the late processes of apoptosis in vivo actually occur in a phagosome of scavenger cell, it may be phagosomal enzymes, but not executor caspases of the apoptotic cell origin, that are really involved in apoptosis. Therefore, I propose a "scavenger cell hypothesis of apoptosis" to redefine apoptosis as an in vivo mechanism of cell death, and suggest that programmed cell death in culture in a third cell demise mechanism besides necrosis and apoptosis that should be defined using other nomenclatures.

Apoptosis↗