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P T Daniel

Publications and source records attributed to P T Daniel.

At least 37 records · Page 2Linked to original sources
PubMed2001

The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis.

Defective cytochrome c release and the resulting loss of caspase-3 activation was recently shown to be essential for the susceptibility of human melanoma cells to CD95/Fas-induced apoptosis. Cytochrome c release from mitochondria is regulated by the relative amounts of apoptosis-promoting and apoptosis-inhibiting Bcl-2 proteins in the outer membrane of these organelles. The assignment of Bax/Bcl-2 ratios by quantitative Western blotting in 11 melanoma cell populations revealed a relation to the susceptibility to CD95-mediated apoptosis. We could show that a low Bax/Bcl-2 ratio was characteristic for resistant cells and a high Bax/Bcl-2 ratio was characteristic for sensitive cells. Low Bax expression was not a consequence of mutations in the p53 coding sequence. The Bax/Bcl-2 ratio was also in clear correlation with sensitivity to another cell death inducer, N-acetylsphingosine. Furthermore, Bcl-2 overexpression abolished apoptosis triggered by both apoptotic stimuli, confirming the critical role of the Bax/Bcl-2 ratio as a rheostat that determines the susceptibility to apoptosis in melanoma cells by regulating mitochondrial function. Interestingly, some chemotherapeutics lead to the activation of death pathways by CD95L upregulation, ceramide generation, direct activation of upstream caspases, or upregulation of proapoptotic genes. Taken together, these signals enter the apoptotic pathway upstream of mitochondria, resulting in activation of this central checkpoint. We therefore assumed that apoptosis deficiency of malignant melanoma can be circumvented by drugs directly influencing mitochondrial functions. For this purpose we used betulinic acid, a cytotoxic agent selective for melanoma, straightly perturbing mitochondrial functions. In fact, betulinic acid induced mitochondrial cytochrome c release and DNA fragmentation in both CD95-resistant and CD95-sensitive melanoma cell populations, independent of the Bax/Bcl-2 ratio.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Relevance of Ki-67 antigen expression and K-ras mutation in colorectal liver metastases.

AIMS: The liver is a frequent site of metastases from colorectal cancer. While these lesions are potentially amenable to surgical resection, they are usually very aggressive, and recurrence is frequent. Mutations of the proto-oncogene K- ras are thought to impart a strong growth signal to tumour cells and are closely associated with the development of malignancies of the colon and rectum. Hepatic metastases from colorectal cancer have notably elevated proliferative rates. The present study was performed to investigate the relationship between proliferation or K- ras mutation and prognosis following curative resection of colorectal liver metastases. METHODS: Colorectal liver metastases from 41 patients undergoing curative hepatic resection were examined for proliferation status and presence of K- ras mutations. The proliferative activity was assessed by Ki-67 immunohistochemistry. DNA from the same tissue samples was screened for point mutations in codon 12 of the K- ras gene using a novel microplate-based allelic-specific hybridization assay. Ki-67 scores and K- ras status were then related with patient survival as determined through retrospective analysis. RESULTS: Median survival was 40 months. Patients with high Ki-67 scores (> or = 50%) had significantly shorter median survival compared with those with low scores (30 vs 44 months, log-rank P=0.02). A high Ki-67 score was an independent negative prognostic factor by multivariate regression analysis (relative risk=3.04, P=0.036). K- ras point mutations were detected in 6/41 patients (15%), but mutational status did not correlate with Ki-67 score or survival. CONCLUSIONS: These findings suggest that the tumour proliferative index is a useful predictor of aggressive tumour behaviour and an indicator of patient survival. The presence of K- ras mutations does not appear to correlate with tumour proliferation status or patient survival.

Adult↗
PubMed2000

Impaired BAX protein expression in breast cancer: mutational analysis of the BAX and the p53 gene.

We have previously shown that the pro-apoptotic BAX protein is differentially expressed in breast cancer and in other epithelial tumors. In this line, a reduced BAX protein expression is a negative prognostic factor in various carcinomas including breast cancer. For p53, a trancriptional activator of BAX in apoptosis, mutations in the coding sequence were shown to modulate BAX protein expression in cell line models on the transcriptional level. We therefore investigated the BAX gene in 68 breast cancer specimens for the presence of mutations in the coding sequence by single-strand conformation polymorphism (SSCP)-PCR and direct sequencing. The expression of BAX protein was assessed by immunohistochemistry. In addition, we screened for mutations in the exons 5-8 of the p53 gene by SSCP-PCR to assess whether mutations in the DNA-binding domain of this upstream regulator of BAX gene transcription are responsible for differences in BAX protein expression. As previously observed, BAX was differentially expressed in the breast cancer samples, but no mutations in the coding sequence of the BAX gene were found besides a polymorphism in exon 6 at the position 552 (G->A) and additional intronic polymorphisms. In contrast, we identified 16 of 68 (23.5%) tumors to bear mutations in the p53 gene. In the subset of BAX-expressing tumors, the mutational inactivation of p53 did result in a reduced BAX protein expression (Fisher exact test, p = 0. 047). Nevertheless, we identified a subset of BAX-negative tumors lacking BAX or p53 mutations. Thus, additional, not yet identified regulators, apart from p53, appear to be involved in the regulation of BAX protein expression.

Base Sequence↗
PubMed2000

GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis.

Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.

Amino Acid Sequence↗
PubMed2000

A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes.

Although bispecific antibodies directed against malignant lymphoma have been shown to be effective in vitro and in vivo, extended clinical trials so far have been hampered by the fact that conventional approaches to produce these antibodies suffer from low yields, ill-defined byproducts, or laborious purification procedures. To overcome this problem, we have generated a small, recombinant, lymphoma-directed, bispecific single-chain (bsc) antibody according to a novel technique recently described. The antibody consists of 2 different single-chain Fv fragments joined by a glycine-serine linker. One specificity is directed against the CD3 antigen of human T cells, and the other antigen-binding site engages the pan-B-cell marker CD19, uniformly expressed on the vast majority of B-cell malignancies. The construct was expressed in Chinese hamster ovary cells and purified by its C-terminal histioline tag. Specific binding to CD19 and CD3 was demonstrated by fluorescence-activated cell sorter analysis. By redirecting unstimulated primary human T cells derived from the peripheral blood against CD19-positive lymphoma cells, the bscCD19 x CD3 antibody showed significant cytotoxic activity at very low concentrations of 10 to 100 pg/mL and at effector to target cell ratios as low as 2:1. Moreover, strong lymphoma-directed cytotoxicity at low antibody concentrations was rapidly induced during 4 hours even in experiments without any T-cell prestimulation. Thus, this particular antibody proves to be much more efficacious than the bispecific antibodies described until now. Therefore, the described bscCD19 x CD3 molecule should be a suitable candidate to prove the therapeutic benefit of bispecific antibodies in the treatment of non-Hodgkin lymphoma. (Blood. 2000;95:2098-2103)

Antibodies, Bispecific↗
PubMed2000

Relapse in childhood acute lymphoblastic leukemia is associated with a decrease of the Bax/Bcl-2 ratio and loss of spontaneous caspase-3 processing in vivo.

Dysfunction of the p53/Bax/caspase-3 apoptosis signaling pathway has been shown to play a role in tumorigenesis and tumor progression, ie the development of acquired drug resistance. Low expression of the apoptosis inducer Bax correlates with poor response to therapy and shorter overall survival in solid tumors. In the present study, we analyzed the p53/Bax/caspase-3 pathway in a paired and an unpaired sample series of children with acute lymphoblastic leukemia (ALL) at initial diagnosis and relapse. The data demonstrate that both Bax expression levels and the Bax/Bcl-2 ratio are significantly lower in samples at relapse as compared with samples at initial diagnosis (P=0.013, Wilcoxon signed rank test (paired samples); P=0.0039, Mann-Whitney U test (unpaired samples)). The loss of Bax protein expression was not a consequence of Bax frameshift mutations of the G8 tract and could not be attributed to mutations of the p53 coding sequence (exons 5 to 8) which were detected to a similar extent in de novo ALL samples and at relapse. Analysis of the downstream effector caspase-3 showed loss of spontaneous caspase-3 processing at relapse. Whereas nine out of 14 (64%, paired samples) or 37 out of 77 (48%, unpaired samples) ALL patients at initial diagnosis displayed spontaneous in vivo processing of caspase-3, this was completely absent in patients at relapse (paired samples) or detected in only one out of 34 patients at relapse (2.9%, unpaired samples). We therefore conclude that in ALL relapse a severe disturbance of apoptotic pathways occurs, both at the level of Bax expression and caspase-3 activation.

Adolescent↗
PubMed2000

Dissecting the pathways to death.

This report summarizes recent findings in the field of basic and translational apoptosis research which were presented at the 1st Conference on 'Mechanisms of Cell Death and Disease: Advances in Therapeutic Intervention' organized by the European School of Hematology and the University of Texas MD Anderson Cancer Center, 13-17 May, in Dublin, Ireland, and puts them in the context of the literature. Recent discoveries have significantly advanced the understanding of biochemical and genetic requirements of distinct apoptosis pathways (ie mitochondrial, death-receptor and endoplasmic reticulum-mediated apoptosis) and their dysregulation in disease. Progress has been made especially in the elucidation of the mechanisms of action of the Bcl-2 family members, in detail the formation of channels and their regulation in the mitochondrial membranes, conformational changes in Bax and Bak, and crosstalk of death receptor-triggered apoptosis to the mitochondria by activation of Bax via Bid. In addition, novel insights have been gained about the regulation of caspases and novel caspase signaling pathways, such as activation of caspase-12 by the endoplasmic reticulum stress response. Therapeutic applications of apoptosis manipulation include (1) the inhibition of caspases in acute and chronic neurodegenerative diseases, ie stroke, Alzheimer's or Huntington's disease by drugs and (2) sensitization of cancer cells for drug/radiation-induced apoptosis by modulation of survival signals and viral transfer of apoptosis promoting genes.

Animals↗
PubMed1999

Expression of the death gene Bik/Nbk promotes sensitivity to drug-induced apoptosis in corticosteroid-resistant T-cell lymphoma and prevents tumor growth in severe combined immunodeficient mice.

Members of the Bcl-2 gene family have been implicated in the regulation of cell death induced by cytostatic drugs. In some malignancies such as B-cell lymphoma, there is evidence that high expression of Bcl-2 is an independent negative prognostic marker and the overexpression of Bcl-2 has been shown to confer resistance to cytotoxic drugs by preventing drug-induced apoptosis. This function of Bcl-2 can be antagonized by apoptosis-promoting members of the Bcl-2 family. We previously showed that overexpression of Bax restores the chemosensitivity of Bax-deficient breast cancer cell lines. Therefore, we investigated whether the death-promoting Bcl-2 homologue Bik/Nbk can enhance cytostatic drug-induced apoptosis. As a model, we used the T-cell leukemia H9 (CD3(+) and CD4(+)CD8(-)), which is resistant to corticosteroid-induced cell death and does not express endogenous Bik/Nbk. Sensitivity for drug-induced apoptosis was increased 10- to 39-fold in cells transfected with the full-length coding sequence of Bik/Nbk. In addition, apoptosis induced via CD95/Fas or heat shock was increased to a similar extent. These data show that Bik/Nbk, which, unlike Bax, carries only a BH3 but no BH1 or BH2 domain may be a target to enhance chemosensitivity. The complete suppression of tumor growth in a severe combined immunodeficient mouse xenotransplant model suggests that, in analogy to Bax, Bik/Nbk may function as a tumor suppressor gene.

Adrenal Cortex Hormones↗
PubMed1999

Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor.

Adsorption↗
PubMed1999

CD95/Fas-triggered apoptosis of activated T lymphocytes is prevented by dendritic cells through a CD58-dependent mechanism.

T-cell apoptosis is a mechanism regulating T-cell homeostasis. Activation renders T cells susceptible to activation-induced cell death, a process mediated through CD95 ligand/CD95 (Apo-1/Fas) ligation. The aim of this study was to test whether antigen-presenting cells can inhibit CD95/Fas-triggered activation-induced cell death. Dendritic cells (DC), which are highly effective antigen-presenting cells, were generated in vitro from human peripheral blood monocytes by culture in granulocyte-macrophage colony-stimulating factor and interleukin 4. Subsequently, DC were cocultured with activated T cells and the effect of DC on CD95/Fas-mediated apoptosis was determined. Coculture with increasing amounts of DC prevented CD95/Fas-triggered apoptosis in a dose-dependent fashion by inhibiting activation of caspase 8 and caspase 3. This protective effect of the DC on T-cell death could be blocked by 50% by adding an anti-CD58 antibody, whereas further addition of anti-CD80 (B7.1) and anti-CD86 (B7.2) led to an even more pronounced effect. Our findings suggest that DC can protect T cells from activation-induced cell death, with CD58 ligation playing a key role.

Antibodies, Monoclonal↗
PubMed1999

Analysis of the p53/BAX pathway in colorectal cancer: low BAX is a negative prognostic factor in patients with resected liver metastases.

PURPOSE: To determine the prognostic value of the central downstream apoptosis effector BAX in relation to its upstream regulator p53 in R0-resected hepatic metastases of colorectal cancer. PATIENTS AND METHODS: Retrospective analysis of 41 patients who underwent potentially curative resection of liver metastases from colarectal cancer was performed. Tumor DNA was screened for p53 mutations by single-stranded conformational polymorphism polymerase chain reaction and for BAX frameshift mutations by fragment length analysis. Protein expression of BAX, p21, and p53 was investigated by immunohistochemistry. RESULTS: Overall median survival was 40.2 months. Tumors with BAX frameshift mutations were considered microsatellite mutator phenotype-positive and were excluded from further prognostic analyses. Patients with high BAX protein expression had a median survival of 53.6 months compared with 35.4 months for patients with low BAX expression (P < .05). The negative prognostic value of low BAX expression was more evident in those patients with wild-type p53 (median survival, 54.0 v 23.3 months for BAX-negative tumors; P < .01). Low BAX expression was an independent negative prognostic marker in multivariate regression analysis for all patients independent of the p53 status (relative risk, 3.03, P = .03), especially for p53 wild-type tumors (relative risk, 8.21; P = .0095). CONCLUSION: We conclude that low BAX expression is an independent negative prognostic marker in patients with hepatic metastases of colorectal cancer. The best survival was seen in patients with an intact p53-to-BAX pathway; ie, wild-type p53- and BAX-positive tumors. Thus, analysis of apoptosis signaling pathways (here, p53 in concert with its downstream death effector, BAX) might yield more prognostic power in future studies as compared with analysis of single genes such as p53 alone.

Adult↗
PubMed1998

Immunotherapy of B-cell lymphoma with CD3x19 bispecific antibodies: costimulation via CD28 prevents "veto" apoptosis of antibody-targeted cytotoxic T cells.

Bispecific antibodies (CD3x19) against the CD3epsilon-chain of the T-cell-receptor/CD3 complex and the CD19 antigen on B cells can target polyclonal, nontumor-specific T cells to B lymphoma cells. This induces T-cell activation, and generation of cytotoxic T cells (CTLs). These polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse CD19(+) B-lymphoma cells. In a xenotransplant model in severe combined immunodeficiency deficient (SCID) mice, we and others observed that CD28 triggering is required for efficient elimination of B-lymphoma cells and cure from the tumor in addition to CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab, induces T-cell death by apoptosis. In blocking experiments we showed that this "veto" apoptosis is mediated by the CD95/Fas ligand. Addition of anti-CD28 (signal 2) renders the T cells resistant for veto apoptosis both in vitro and in vivo. We therefore conclude that the role of costimulation in immunotherapy with bispecific antibodies or other T-cell-based immune strategies is not only to facilitate T-cell activation but also to prevent T-cell deletion by apoptosis.

Animals↗
PubMed1998

Dendritic cells prevent CD95 mediated T lymphocyte death through costimulatory signals.

T cell apoptosis is a mechanism regulating T cell homeostasis. Prolonged stimulation renders T cells susceptible to activation induced cell death (AICD), a process mediated through CD95 (Apo-1/Fas). While under some circumstances AICD can be prevented, little is known about molecules involved. Here, we wanted to assess whether dendritic cells (DC) have the capacity to prevent CD95-dependent AICD. T cells activated with PHA/PMA or anti-CD3 monoclonal antibody (mAb) were cocultured with increasing amounts of DC. While spontaneous T cell apoptosis amounted to 25%, the presence of an agonistic anti-CD95 antibody increased cell death to 64%. Addition of scalar amounts of DC prevented T cell apoptosis in a dose dependent fashion, where coculture of 10(5) DC/ml with 10(6) T cells/ml reduced apoptosis almost to baseline level (33%). Further addition of an anti-CD58 antibody partially abolished this protective effect. This was even more pronounced if anti-CD80 and anti-CD86 antibodies were added. Our findings suggest that dendritic cells are able to rescue T cells from AICD, with CD58 ligation playing a key role.

Antigens, CD↗
PubMed1998

Raltitrexed (Tomudex): an alternative drug for patients with colorectal cancer and 5-fluorouracil associated cardiotoxicity.

Two patients with proven 5-fluorouracil (5-FU)-associated cardiotoxicity were treated with the specific thymidylate synthase inhibitor raltitrexed safely, without evidence of cardiotoxicity. Raltitrexed might be an alternative for patients with advanced colorectal cancer and 5-FU-associated cardiotoxicity. 5-FU cardiotoxicity is not due to the antineoplastic mechanisms via thymidilate synthase.

Adenocarcinoma↗
PubMed1997

Costimulatory signals through B7.1/CD28 prevent T cell apoptosis during target cell lysis.

Expression of B7 on tumor cells can circumvent T cell tolerance and lead to the generation of tumor cell-specific T cell immunity. The effect of B7 expression on the generation of protective antitumor immunity has been attributed primarily to 1) more efficient T cell activation and 2) better generation of tumor-specific killer T cells. We have investigated the role of costimulation through B7.1 and its receptor, the CD28 molecule, in the generation of allogeneic human CTLs against MCF-7 breast cancer cells. In this setting, we describe how activated CTLs undergo activation-induced cell death upon killing the target cell. Instead of proliferation and clonal expansion, the majority of the CTLs underwent apoptotic cell death. CTL apoptosis could be blocked by 50% when binding of the Fas ligand to its receptor, the CD95 (APO-1/Fas) molecule, was prevented. Fas ligand was detected in the activated T cells, but not in MCF-7 or a panel of other breast cancer cell lines. This excludes an active role for MCF-7 during CTL death and indicates that the CTL apoptosis is due to an autocrine production of the Fas ligand by CTLs. Costimulation of CTLs by retrovirally B7.1-transfected MCF-7 drastically reduced the sensitivity of the CTLs to apoptosis during target contact. Thus, in tumor cell vaccination, B7.1 might play a major role in preventing T cell death by altering T cell susceptibility for apoptosis.

Apoptosis↗
PubMed1997

Activation and activation-induced death of human tonsillar B cells and Burkitt lymphoma cells: lack of CD95 (Fas/APO-1) ligand expression and function.

Activation of T cells was shown to up-regulate the Fas ligand (FasL) which binds to the CD95 (APO-1/Fas) antigen and mediates activation-induced cell death (AICD) of activated T cells and T lymphoma cells. A recent report showed that mouse B cells express the FasL upon activation with lipopolysaccharide (LPS). We therefore asked whether activation of human B cells induces expression of FasL and whether AICD is mediated, as in T cells, through autocrine production of the FasL. We used human tonsillar B cells and Burkitt lymphoma cell lines which were activated by CD40 ligand, surface (s)IgM cross-linking, or LPS. Northern and Western blot analysis failed to detect FasL during B cell activation or AICD of both normal and malignant B cells. Low-level expression of FasL was detected by reverse transcriptase-polymerase chain reaction. Functional experiments, however, showed that FasL is not functionally expressed upon activation. IgM-mediated AICD in the tonsillar or Burkitt lymphoma B cells could not be inhibited by FasL blocking. Thus, our data show that, in contrast to T cells, activation of normal or malignant human B cells does not lead to functional FasL expression.

Apoptosis↗
PubMed1996

Induction of the death-promoting gene bax-alpha sensitizes cultured breast-cancer cells to drug-induced apoptosis.

Resistance to apoptosis plays an important role in malignancies that are refractory to chemotherapy treatment. Recently we have shown that the expression of bax-alpha, a death-promoting member of the bcl-2 family, is down-regulated in breast cancer and have provided evidence that low bax expression might contribute to the pathogenesis of breast cancer. In this study we were able to demonstrate the role of this gene in chemotherapy-induced apoptosis. We transfected bax-alpha into the breast-cancer cell lines R30C and MCF-7 under the control of an inducible tetracycline-dependent expression system. Induction of bax-alpha expression did not affect viability by itself but strongly increased chemosensitivity to epirubicin. We were able to demonstrate that this sensitization is due to apoptosis. These data might explain the recently published observation that reduced expression of bax is associated with poor response rates to chemotherapy in breast cancer.

Apoptosis↗