Biomedical subjects

A A Ruefli

Publications and source records attributed to A A Ruefli.

10 recordsLinked to original sources

PubMed2002

P-glycoprotein inhibits caspase-8 activation but not formation of the death inducing signal complex (disc) following Fas ligation.

Previous studies by our laboratory have shown that the drug transporter protein P-glycoprotein, P-gp, can specifically inhibit Fas-induced caspase-3 activation and apoptosis. Importantly, inhibition of both caspase-3 activation and cell death could be reversed by pharmacological and antibody inhibitors of P-gp function. However, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remained unknown. We therefore sought to identify the point(s) within the death receptor pathway at which P-gp exerted its inhibitory effect and to determine whether the ATPase activity of P-gp was required. Structure-function analysis determined that ATP hydrolysis was necessary for P-gp to confer resistance to Fas-induced caspase activation and cell death. Importantly, although both FADD and caspase-8 were recruited to the Death Inducing Signal Complex (DISC) in wild-type P-gp expressing cells following Fas ligation, subsequent activation of caspase-8 at the DISC was inhibited. The ability of P-gp to inhibit caspase-8 activation was also ATP dependent. These studies demonstrate that P-gp inhibits Fas-induced caspase-8 activation but not formation of the DISC and that this activity of P-gp is dependent on ATP hydrolysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species.

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.

Antineoplastic Agents↗

PubMed2001

P-glycoprotein does not protect cells against cytolysis induced by pore-forming proteins.

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance (MDR). In addition to its ability to efflux toxins, P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We therefore hypothesized that P-gp may have additional functions in addition to its role in effluxing xenotoxins that could provide protection to tumor cells against a host response. There have been a number of contradictory reports concerning the role of P-gp in regulating complement activation. Given the disparate results obtained by different laboratories and our published results demonstrating that P-gp does not affect cell death induced by another membranolytic protein, perforin, we decided to assess the role of P-gp in regulating cell lysis induced by a number of different pore-forming proteins. Testing a variety of different P-gp-expressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we found no difference in sensitivity of P-gp(+ve) or P-gp(-ve) cells to the pore-forming proteins complement, perforin, or pneumolysin. Based on these results, we conclude that P-gp does not affect cell lysis induced by pore-forming proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.

The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Equivalent death of P-glycoprotein expressing and nonexpressing cells induced by the protein kinase C inhibitor staurosporine.

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance. In addition to its ability to efflux toxins P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We have previously demonstrated that stimuli including drugs such as hexamethylene bisacetamide (HMBA), the cytotoxic lymphocyte granule protein granzyme B, and pore-forming proteins such as perforin, kill P-gp positive cells in a caspase-independent manner. We therefore hypothesised that drugs that are not effluxed by P-gp and which induce cell death in the absence of caspase activation could induce death of P-gp expressing cells. Staurosporine has been previously shown to kill cells in the absence of caspase activation. Consistent with our hypothesis, we demonstrate here that staurosporine can equivalently kill P-gp(+ve) and P-gp(-ve) tumor cell lines in a caspase-independent manner.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance.

Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein. As well as effluxing xenotoxins, functional P-gp can confer resistance to caspase-dependent apoptosis induced by a range of different stimuli, including Fas ligand, tumor necrosis factor, UV irradiation, and serum starvation. However, P-gp-positive cells remain sensitive to caspase-independent death induced by cytotoxic T-cell granule proteins, perforin, and granzyme B. It is, therefore, possible that agents that induce cell death in a caspase-independent manner might circumvent P-gp-mediated MDR. We demonstrated here that hexamethylene bisacetamide (HMBA) induced equivalent caspase-independent cell death in both P-gp-positive and -negative cell lines at concentrations of 10 mmol/L and above. The HMBA-induced death pathway was marked by release of cytochrome c from the mitochondria and reduction of Bcl-2 protein levels. In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected. These studies greatly enhance our understanding of the molecular cell death events that can be regulated by functional P-gp and highlight the potential clinical use of drugs that function via a caspase-independent pathway for the treatment of MDR tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Multiple physiological functions for multidrug transporter P-glycoprotein?

Multidrug resistance mediated by the drug-efflux protein P-glycoprotein (P-gp) is one mechanism that tumor cells use to escape death induced by chemotherapeutic drugs. Although it is irrefutable that P-gp can efflux xenobiotics out of cells, biological regulatory functions for P-gp in multicellular organisms have yet to be established firmly. Recent observations have challenged the notion that P-gp has evolved merely to efflux xenotoxins out of healthy cells and raised the possibility that P-gp and related transporter molecules might play a fundamental role in regulating cell differentiation, proliferation and survival.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

A role for P-glycoprotein in regulating cell death.

P-glycoprotein (P-gp) is an energy dependent drug pump responsible for multidrug resistance (MDR) in human cancers. While it is irrefutable that P-gp can efflux xenobiotics out of cells, the biological function of P-gp in multicellular organisms has yet to be firmly established. The question of what, if anything, P-gp does when not effluxing drugs has been raised by recent reports indicating that P-gp may regulate apoptosis, chloride channel activity, cholesterol metabolism and immune cell function. There is now a lively debate regarding the possible role of P-gp in regulating cell differentiation, proliferation and survival.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Calcium regulates processing of the Alzheimer amyloid protein precursor in a protein kinase C-independent manner.

Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid beta-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects of calcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.

Amyloid beta-Peptides↗

PubMed1993

Expression of APP in brains of transgenic mice containing the entire human APP gene.

A major component of amyloid deposits found in Alzheimer disease and Down syndrome is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence indicates that increases in APP expression and/or beta/A4 peptide accumulation may underlie the amyloidosis characteristic of these diseases. In the present study, transgenic mice carrying the entire human APP gene were studied for expression of human APP. Significant expression of human APP protein was observed in these animals, and this expression paralleled the expression of endogenous APP. These results, which represent a first demonstration of significant human APP expression in transgenic animals, support the use of such animals to study human APP expression and processing in vivo and possibly as models for the amyloidosis associated with Alzheimer disease.

Amyloid beta-Protein Precursor↗