Search PubMed⌕ Search

Biomedical subjects

John C Reed

Publications and source records attributed to John C Reed.

162 records · Page 9Linked to original sources
PubMed2002

Remodeling for demolition: changes in mitochondrial ultrastructure during apoptosis.

The release of Cytochrome c from mitochondria is a pivotal event in the apoptotic process, but the precise mechanisms remain controversial. A recent paper proposes that this involves two steps, one involving permeabilization of the mitochondrial outer membrane and a second in which the structural organization of the cristae changes to make Cytochrome c accessible for release. Both events are induced by proapoptotic members of the Bcl-2 family and appear to be separable.

Animals↗
PubMed2002

Apoptosis-based therapies.

Many of today's medical illnesses can be attributed directly or indirectly to problems with apoptosis--a programmed cell-suicide mechanism. Disorders in which defective regulation of apoptosis contributes to disease pathogenesis or progression can involve either cell accumulation, in which cell eradication or cell turnover is impaired, or cell loss, in which the cell-suicide programme is inappropriately triggered. Identification of the genes and gene products that are responsible for apoptosis, together with emerging information about the mechanisms of action and structures of apoptotic regulatory and effector proteins, has laid a foundation for the discovery of drugs, some of which are now undergoing evaluation in human clinical trials.

Animals↗
PubMed2002

Yeast and apoptosis.

Even though yeast lack much of the molecular machinery that is responsible for apoptosis in metazoans, they can be a powerful tool in apoptosis research. The ectopic expression of several animal apoptosis proteins in yeast can help us to discover new genes -- and chemical compounds -- that modulate the cell-death pathways of higher eukaryotes.

Animals↗
PubMed2002

The anti-apoptotic genes Bcl-X(L) and Bcl-2 are over-expressed and contribute to chemoresistance of non-proliferating leukaemic CD34+ cells.

In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti-apoptotic genes Bcl-2 and Bcl-X(L) have been implicated in cell cycle regulation, we investigated the expression of these genes in non-proliferating (Q) and proliferating (P) AML and normal CD34+ progenitor cells. Using reverse transcription polymerase chain reaction, Bcl-X(L) and Bcl-2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl-XS and Bax expression was found. Furthermore, the Bcl-X(L)/X(S) but not the Bcl-2/Bax ratio was higher in Q AML compared with normal CD34+ Q cells (P = 0.001). An inverse correlation between Bcl-2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all-trans retinoic acid (ATRA) reduced Bcl-2 and Bcl-X(L) expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara-C). These findings demonstrate overexpression of the anti-apoptotic proteins Bcl-X(L) and Bcl-2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl-2 and proliferation suggests a role for Bcl-2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.

Acute Disease↗
PubMed2002

Multiple caspases are activated after traumatic brain injury: evidence for involvement in functional outcome.

Caspase-3 is a cysteine protease that is strongly implicated in neuronal apoptosis. Activation of caspase-3 may be induced by at least two major initiator pathways: a caspase-8-mediated pathway activated through cell surface death receptors (extrinsic pathway), and a caspase-9-mediated pathway activated by signals from the mitochondria that lead to formation of an apoptosomal complex (intrinsic pathway). In the present studies, we compare the activation of caspases-3, -8, and -9 after lateral fluid-percussion traumatic brain injury (TBI) in rats. Immunoblot analysis identified cleaved forms of caspases-3 and -9, but not caspase-8, at 1, 12, and 48 h after injury. Immunocytochemistry specific for cleaved caspases-3 and -9 revealed their expression primarily in neurons. These caspases were also frequently localized in TUNEL-positive cells, some of which demonstrated morphological features of apoptosis. However, caspases-3 and -9 were also found in neurons that were not TUNEL-positive, and other TUNEL-positive cells did not show activated caspases. In contrast to caspases-3 or -9, caspase-8 expression was only minimally changed by injury. An increase in expression of this caspase was undetectable by immunoblotting methods, and appeared as positive immunostaining restricted to a few cells within the injured cortex. Treatment with the pan-caspase inhibitor z-VAD-fmk at 15 min after TBI improved performance on motor and spatial learning tests. These data suggest that several caspases may be involved in the pathophysiology of TBI and that pan-caspase inhibition strategies may improve neurological outcomes.

Animals↗
PubMed2002

Temporal and spatial profile of Bid cleavage after experimental traumatic brain injury.

Apoptosis plays an essential role in the cascade of CNS cell degeneration after traumatic brain injury. However, the underlying mechanisms are poorly understood. The authors examined the temporal profile and cell subtype distribution of the proapoptotic protein Bid from 6 hours to 7 days after cortical impact injury in the rat. Increased protein levels of tBid were seen in the cortex ipsilateral to the injury site from 6 hours to 3 days after trauma. Immunohistologic examinations revealed expression of tBid in neurons, astrocytes, and oligodendrocytes from 6 hours to 3 days after impact injury, and concurrent assessment of DNA damage using TUNEL identified tBid-immunopositive cells with apoptoticlike morphology in the traumatized cortex. Moreover, Bid cleavage and activation of caspase-8 and caspase-9 occurred at similar time points and in similar brain regions (i.e., cortical layers 2 to 5) after impact injury. In contrast, there was no evidence of caspase-8 or caspase-9 processing or Bid cleavage in the ipsilateral hippocampus, contralateral cortex, and hippocampus up to 7 days after the injury. The results provide the first evidence of Bid cleavage in the traumatized cortex after experimental traumatic brain injury in vivo, and demonstrate that tBid is expressed in neurons and glial cells. Further, findings indicate that cleavage of Bid may be associated with the activation of the initiator caspase-8 and caspase-9. Finally, these data support the hypothesis that cleavage of Bid contributes to the apoptotic degeneration of different CNS cells in the injured cortex.

Animals↗
PubMed2002

Role of Bcl-2 family members in caspase-independent apoptosis during Chlamydia infection.

Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Testis-specific cytochrome c-null mice produce functional sperm but undergo early testicular atrophy.

Differentiating male germ cells express a testis-specific form of cytochrome c (Cyt c(T)) that is distinct from the cytochrome c expressed in somatic cells (Cyt c(S)). To examine the role of Cyt c(T) in germ cells, we generated mice null for Cyt c(T). Homozygous Cyt c(T)(-/-) pups were statistically underrepresented (21%) but developed normally and were fertile. However, spermatozoa isolated from the cauda epididymis of Cyt c(T)-null animals were less effective in fertilizing oocytes in vitro and contain reduced levels of ATP compared to wild-type sperm. Sperm from Cyt c(T)-null mice contained a greater number of immotile spermatozoa than did samples from control mice, i.e., 53.1% +/- 13.7% versus 33.2% +/- 10.3% (P < 0.0001) for vas deferens sperm and 40.1% +/- 9.6% versus 33.2% +/- 7.5% (P = 0.0104) for epididymal sperm. Cyt c(T)-null mice often exhibit early atrophy of the testes after 4 months of age, losing germ cells as a result of increased apoptosis. However, no difference in the activation of caspase-3, -8, or -9 was detected between the Cyt c(T)(-/-) testes and controls. Our data indicate that the Cyt c(T)-null testes undergo early atrophy equivalent to that which occurs during aging as a consequence of a reduction in oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed2002

The retinoblastoma protein binds the promoter of the survival gene bcl-2 and regulates its transcription in epithelial cells through transcription factor AP-2.

The retinoblastoma (RB) gene product has been shown to restrict cell proliferation, promote cell differentiation, and inhibit apoptosis. Loss of RB function can induce both p53-dependent apoptosis and p53-independent apoptosis; little is known about the mechanisms of RB-regulated p53-independent apoptosis. Here we show that RB specifically activates transcription of the survival gene bcl-2 in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated by the transcription factor AP-2. By monitoring protein-DNA interactions in living cells using formaldehyde cross-linking and chromatin immunoprecipitation, we show that endogenous RB and AP-2 both bind to the same bcl-2 promoter sequence. In addition, we demonstrate that RB and AP-2 also bind to the E-cadherin gene promoter in vivo, consistent with regulation of this promoter by both AP-2 and RB in epithelial cells. This study provides evidence that RB activates bcl-2 and E-cadherin by binding directly to the respective promoter sequences and not indirectly by repressing an inhibitor. This recruitment is mediated by a transcription factor, in this case AP-2. For the first time, our results suggest a direct molecular mechanism by which RB might inhibit apoptosis independently of p53. The results are discussed in a context where RB and Bcl-2 contribute under nonpathological conditions to the maintenance of cell viability in association with a differentiated phenotype, contributing to the tumor suppressor function of RB and playing important roles in normal development.

Acetylation↗
PubMed2002

Modulating apoptosis pathways in low-grade B-cell malignancies using biological response modifiers.

Collectively, low-grade B-cell malignancies constitute the fifth most common form of potentially lethal cancer in North America and Europe, with chronic lymphocytic leukemia (CLL) and follicular non-Hodgkin's lymphoma (FL) representing the most prevalent of these disorders. Chronic lymphocytic leukemia and FL represent quintessential examples of human malignancies that are caused primarily by defects in programmed cell death (apoptosis). During the early stages of disease, the mature B lymphocytes that comprise most CLLs and FLs are largely quiescent G(0) phase cells, which accumulate not because they are dividing more rapidly than normal but because they survive longer than their normal counterparts because of defects in the normal pathways for apoptosis. Defects in apoptosis pathways contribute to chemoresistance, rendering tumor cells less sensitive to the cytotoxic actions of currently available anticancer drugs, and can also promote resistance to cellular immune responses. Several biological agents or their synthetic derivatives show promise as apoptosis modulators, having the potential to place neoplastic cells into a more susceptible state or activating latent programs for cell suicide. These biological response modifiers include monoclonal antibodies such as rituximab (Rituxan; Genentech, Inc, South San Francisco, CA, and IDEC Pharmaceuticals, San Diego, CA) that alter signal transduction pathways, cytokines such as TRAIL (Apo2 ligand), ligands for retinoid/steroid family nuclear receptors, and small-molecule compounds that bind and inhibit protein kinases. Knowledge about the mechanisms by which these agents influence apoptosis pathways in neoplastic diseases may suggest strategies for more effective and less toxic therapies for patients suffering from CLL, FL, and other malignancies.

Animals↗
PubMed2002

The predictive value of bcl-2, bax, bcl-xL, bag-1, fas, and fasL for chemotherapy response in advanced breast cancer.

PURPOSE: The purpose was to evaluate the utility of some bcl-2 family proteins fas and fasL as predictive indicators for chemotherapy response in advanced breast cancer. EXPERIMENTAL DESIGN: Between October 1994 and October 1997, 283 patients with advanced breast cancer were included in a multicenter randomized study comparing docetaxel (D) to sequential methotrexate and 5-fluorouracil (MF) after anthracycline failure. The response rates (complete response + partial response) were 42 and 21% in the D and MF arms, respectively (P < 0.001). In 126 patients, histological blocks of primary tumors were available for immunohistochemical analysis of bax, bcl-2, bcl-xL, bag-1, fas and fasL. RESULTS: Of the investigated factors, bag-1 correlated positively with bax, bcl-2, and fasL, and fasL correlated positively with fas and bax. None of these apoptosis-related factors was associated with a response to chemotherapy either in the whole patient population or in the D or MF arms. Interestingly, low bcl-2 expression was associated with shorter time to progression (P = 0.02) and shorter overall survival (OS; P = 0.001). High fasL expression showed a trend toward shorter OS. In multivariate backwards stepwise Cox analysis, in which histological grade and estrogen receptor status (ER) were also included, bcl-2 (P = 0.01) and fasL (P = 0.005) remained highly significantly associated with OS, whereas histological grade and ER lost their significance. CONCLUSIONS: None of the investigated apoptosis-related factors of primary tumor could predict the later response to either D or MF treatment. However, fasL and bcl-2 were strong prognostic factors. Patients who had tumors with high fasL and low bcl-2 expression had the shortest OS.

Adolescent↗
PubMed2002

Apoptosis induction by 1alpha,25-dihydroxyvitamin D3 in prostate cancer.

Calcitriol [1alpha,25-dihydroxyvitamin D3] is the natural ligand of the vitamin D receptor (VDR). Using cultured prostate cancer (PC) cell lines, LN-CaP and ALVA-31, we studied the effects of 1alpha,25(OH)2-Vitamin D3 (VD3) on expression of several apoptosis-regulating proteins including: (a) Bcl-2 family proteins (Bcl-2, Bcl-X(L), Mcl-1, Bax, and Bak); (b) the heat shock protein 70-binding protein BAG1L; and (c) IAP family proteins (XIAP, cIAP1, and cIAP2). VD3 induced decreases in levels of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1, BAG1L, XIAP, cIAP1, and cIAP2 (without altering proapoptotic Bax and Bak) in association with increases in apoptosis. In contrast to VDR-expressing LN-CaP and ALVA-31 cells, VDR-deficient prostate cancer line Du-145 demonstrated no changes in apoptosis protein expression after treatment with VD3. In sensitive PC cell lines, VD3 activates downstream effector protease, caspase-3, and upstream initiator protease caspase-9, the apical protease in the mitochondrial ("intrinsic") pathway for apoptosis, but not caspase-8, an initiator caspase linked to an alternative ("extrinsic") apoptosis pathway triggered by cytokine receptors. VD3 induced declines in antiapoptotic proteins and also stimulated cytochrome c release from mitochondria by a caspase-independent mechanism. Moreover, apoptosis induction by VD3 was suppressed by overexpressing Bcl-2, a known blocker of cytochrome c release, whereas the caspase-8 suppressor CrmA afforded little protection. Thus, VD3 is capable of inhibiting expression of multiple antiapoptotic proteins in VDR-expressing prostate cancer cells, leading to activation of the mitochondrial pathway for apoptosis.

Apoptosis↗
PubMed2001

Identification and characterization of DEDD2, a death effector domain-containing protein.

A novel Death Effector Domain-containing protein was identified, DEDD2, which is closest in amino acid sequence homology to death effector domain-containing DNA-binding protein, DEDD. DEDD2 mRNA is expressed widely in adult human tissues with highest levels in liver, kidney, and peripheral blood leukocytes. DEDD2 interacts with FLIP, but not with Fas-associated death domain (FADD) or caspase-8. Overexpression of DEDD2 induces moderate apoptosis and results in substantial sensitization to apoptosis induced by Fas (CD95/APO-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L), or FADD. In contrast, Bax- or staurosporine-mediated cell death is not affected by expression of DEDD2. Fluorescence microscopy showed that overexpressed DEDD2 translocates to the nucleus, which is dependent on the presence of a bipartite nuclear localization signal in the DEDD2 protein. Mutagenesis studies revealed that the translocation of the DED of DEDD2 to the nucleus is essential for its pro-apoptotic activity. These findings suggest that DEDD2 is involved in the regulation of nuclear events mediated by the extrinsic apoptosis pathway.

Active Transport, Cell Nucleus↗
PubMed2001

Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein.

Apoptosis induction through CD95 (APO-1/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-x(L) in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-x(L) transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-x(L) protected cells, raising the question of how Bcl-x(L)-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-x(L) cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIP(L), caspase-8-dependent clustering, and internalization of CD95, as well as processing of pro-caspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-x(L)-expressing cells. Our data suggest that, in Bcl-x(L)-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-x(L).

Breast Neoplasms↗
PubMedDate not supplied

Expression of Bcl-2 family member Bid in normal and malignant tissues.

Bid is the only known Bcl-2 family member that can function as an agonist of proapoptotic Bcl-2-related proteins such as Bax and Bak. Expression of the proapoptotic Bcl-2 family protein Bid was assessed by immunoblotting and immunohistochemical methods in normal murine and human tissues, and in several types of human cancers and tumor cell lines. Bid expression in normal tissues varied widely, with prominent Bid immunostaining occurring in several types of short-lived cells (e.g., germinal center B cells, peripheral blood granulocytes, differentiated keratinocytes) and in apoptosis-sensitive cells (e.g., adult neurons). Analysis of Bid expression by immunostaining of 100 colon, 95 ovarian, and 254 prostate cancers, as well as 59 brain tumors and 50 lymphomas, revealed evidence of altered Bid regulation in some types of cancers. Correlations with clinical outcome data revealed association of higher levels of Bid with longer recurrence-free survival in men with locally advanced (T3 stage) prostate cancer (P=0.04). Immunoblot analysis of Bid protein levels in the NCI's panel of 60 human tumor cell lines revealed a correlation between higher levels of Bid and sensitivity to ribonucleotide reductase (RR)-inhibiting drugs (P<0.0005). Overexpression of Bid in a model tumor cell line by gene transfection resulted in increased sensitivity to apoptosis induction by a RR inhibitor. Taken together, these observations suggest a potential role for Bid in tumor responses to specific chemotherapeutic drugs, and lay a foundation for future investigations of this member of the Bcl-2 family in healthy and diseased tissues.

Animals↗
PubMedDate not supplied

Downregulation of c-FLIP sensitizes DU145 prostate cancer cells to Fas-mediated apoptosis.

Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasL(TET)) [Hyer et al., Molecular Therapy, 2000; 2:348-58 (ref.12)]. The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasL(TET) to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasL(TET) is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasL(TET) treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.

Apoptosis↗