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D K Perry

Publications and source records attributed to D K Perry.

At least 19 recordsLinked to original sources
PubMed2001

Determination of ceramides and diglycerides by the diglyceride kinase assay.

This review will discuss the utilization of the diglyceride (DG) kinase assay as an analytical method that allows the simultaneous quantitation of DG and ceramide from cell and tissue samples. This enzymatic approach is sensitive, quantitative, and linear over a broad range (20 pmol to 20-25 nmol) for both classes of lipids. It is also practical in that it can be applied to crude lipid extracts and used to process many samples (up to 100) concurrently. However, it has become apparent that this assay has not been conducted optimally, primarily as a result of lack of adherence to its basic principles. The principles illustrated here are also useful to all enzymatic quantitative methods.

Animals↗
PubMed2001

Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death.

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor alpha. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.

Apoptosis↗
PubMed2001

Proteolytic cleavage of epidermal growth factor receptor by caspases.

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)-gamma1, and Bcl-2. We have found that treatment of A431 cells with tumor necrosis factor-alpha in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase-3. Among various caspases, caspase-1, caspase-3 and caspase-7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD-cmk and DEVD-fmk in vitro. We also investigated the effect of caspase-dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC-gamma1. Cleavage of EGFR by caspase-3 significantly impaired the tyrosine phosphorylation of PLC-gamma1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti-apoptotic signaling, to abrogate the activation of EGFR-dependent downstream survival signaling molecules.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.

The de novo pathway of sphingolipid synthesis has been identified recently as a novel means of generating ceramide during apoptosis. Furthermore, it has been suggested that the activation of dihydroceramide synthase is responsible for increased ceramide production through this pathway. In this study, accumulation of ceramide mass in Molt-4 human leukemia cells by the chemotherapy agent etoposide was found to occur primarily due to activation of the de novo pathway. However, when the cells were labeled with a substrate for dihydroceramide synthase in the presence of etoposide, there was no corresponding increase in labeled ceramide. Further investigation using a labeled substrate for serine palmitoyltransferase, the rate-limiting enzyme in the pathway, resulted in an accumulation of label in ceramide upon etoposide treatment. This result suggests that the activation of serine palmitoyltransferase is the event responsible for increased ceramide generation during de novo synthesis initiated by etoposide. Importantly, the ceramide generated from de novo synthesis appears to have a distinct function from that induced by sphingomyelinase action in that it is not involved in caspase-induced poly (ADP-ribose)polymerase proteolysis but does play a role in disrupting membrane integrity in this model system. These results implicate serine palmitoyltransferase as the enzyme controlling de novo ceramide synthesis during apoptosis and begin to define a unique function of ceramide generated from this pathway.

Acyltransferases↗
PubMed2000

Proteolytic cleavage of phospholipase C-gamma1 during apoptosis in Molt-4 cells.

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospholipase C-gamma1 (PLC-gamma1), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leukemic Molt-4 cells with PLC inhibitors such as U-73122 or ET-18-OCH(3) potentiated etoposide-induced apoptosis in these cells. PLC-gamma1 was fragmented when Molt-4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor alpha. Cleavage of PLC-gamma1 was blocked by overexpression of Bcl-2 and by specific inhibitors of caspases such as Z-DEVD-CH(2)F and YVAD-cmk. Purified caspase-3 and caspase-7, group II caspases, cleaved PLC-gamma1 in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC-gamma1 is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala-Glu-Pro-Asp(770) was identified to be a cleavage site within PLC-gamma1. Epidermal growth factor receptor (EGFR) -induced tyrosine phosphorylation of PLC-gamma1 resulted in resistance to cleavage by caspase-3 in vitro. Furthermore, cleaved PLC-gamma1 could not be tyrosine-phosphorylated by EGFR in vitro. In addition, tyrosine-phosphorylated PLC-gamma1 was not significantly cleaved during etoposide-induced apoptosis in Molt-4 cells. This suggests that the growth factor-induced tyrosine phosphorylation may suppress apoptosis-induced fragmentation of PLC-gamma1. We provide evidence for the biochemical relationship between PLC-gamma1-mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC-gamma1-mediated signaling pathway can facilitate an apoptotic progression.

Amino Acid Sequence↗
PubMed2000

The role of de novo ceramide synthesis in chemotherapy-induced apoptosis.

The de novo pathway of sphingolipid synthesis has been implicated as an alternative to sphingomyelinase activation in generating an apoptotic response through ceramide. A chemotherapy agent was used to activate this pathway in a human T-cell line in order to investigate the role of de novo ceramide synthesis in apoptosis. In data obtained from intact cell radiolabeling studies, it was observed that the first and rate-limiting enzyme in de novo synthesis, serine palmitoyltransferase, is activated and controls the production of ceramide through this pathway. Furthermore, using agents that selectively inhibit ceramide production by this pathway, partial protection from cell death was observed that was independent of caspase activation. These results reveal that serine palmitoyltransferase, an enzyme that controls sphingolipid synthesis for housekeeping functions, is activated during apoptosis and serves to mediate events in this process.

Antineoplastic Agents↗
PubMed1999

Phosphatidic acid is a potent and selective inhibitor of protein phosphatase 1 and an inhibitor of ceramide-mediated responses.

In the present study, we report that phosphatidic acid (PA) functions as a novel, potent, and selective inhibitor of protein phosphatase 1 (PP1). The catalytic subunit of PP1alpha was inhibited by PA dose-dependently in a noncompetitive manner with a K(i) value of 80 nM. The inhibition by PA was specific to PP1 as PA failed to inhibit protein phosphatase 2A (PP2A) or PP2B. Furthermore, PA was the most effective and potent inhibitor of PP1 compared with other phospholipids. Because we recently showed that ceramides activated PP1, we next examined the effects of PA on ceramide stimulation of PP1. PA inhibited both basal and ceramide-stimulated PP1 activities, and ceramide showed potent and stereoselective activation of PP1 in the presence of PA. Next, the effects of PA on ceramide-induced responses were examined. Molt-4 cells took up PA dose- and time-dependently such that by 1 and 3 h, uptake of PA was 0.37 and 0. 65% of total PA added, respectively. PA at 30 microM and calyculin A at 10 nM (an inhibitor of PP1 and PP2A at low concentrations), but not okadaic acid at 10 nM (a PP2A inhibitor at low concentrations) prevented poly(ADP-ribose) polymerase proteolysis induced by C(6)-ceramide. Moreover, the combination of PA with okadaic acid prevented retinoblastoma gene product dephosphorylation induced by C(6)-ceramide. These data suggest that PA functions as a specific regulator of PP1 and may reverse or counteract those effects of ceramide that are mediated by PP1, such as apoptosis and retinoblastoma gene product dephosphorylation.

Animals↗
PubMed1999

Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway.

A short period of ischemia and reperfusion, called ischemic preconditioning, protects various tissues against subsequent sustained ischemic insults. We previously showed that apoptosis of hepatocytes and sinusoidal endothelial cells is a critical mechanism of injury in the ischemic liver. Because caspases, calpains, and Bcl-2 have a pivotal role in the regulation of apoptosis, we hypothesized that ischemic preconditioning protects by inhibition of apoptosis through down-regulation of caspase and calpain activities and up-regulation of Bcl-2. A preconditioning period of 10 minutes of ischemia followed by 15 minutes of reperfusion maximally protected livers subjected to prolonged ischemia. After reperfusion, serum aspartate transaminase (AST) levels were reduced up to 3-fold in preconditioned animals. All animals subjected to 75 minutes of ischemia died, whereas all those who received ischemic preconditioning survived. Apoptosis of hepatocytes and sinusoidal endothelial cells, assessed by in situ TUNEL assay and DNA fragmentation by gel electrophoresis, was dramatically reduced with preconditioning. Caspase activity, measured by poly (adenosine diphosphate ribose) polymerase (PARP) proteolysis and a specific caspase-3 fluorometric assay, was inhibited by ischemic preconditioning. The antiapoptotic mechanism did not involve calpain-like activity or Bcl-2 expression because levels were similar in control and preconditioned livers. In conclusion, ischemic preconditioning confers dramatic protection against prolonged ischemia via inhibition of apoptosis through down-regulation of caspase 3 activity, independent of calpain-like activity or Bcl-2 expression.

Animals↗
PubMed1997

Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis.

The prevention of apoptosis by Zn2+ has generally been attributed to its inhibition of an endonuclease acting in the late phase of apoptosis. In this study we investigated the effect of Zn2+ on an earlier event in the apoptotic process, the proteolysis of the "death substrate" poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4 leukemia cells with micromolar concentrations of Zn2+ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a cell-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn2+ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn2+ on PARP, a protein with two zinc finger domains, we used recombinant caspase-3 and a chromogenic tetrapeptide substrate containing the caspase-3 cleavage site. In this system, Zn2+ inhibited caspase-3 with an IC50 of 0.1 microM. These results identify caspase-3 as a novel target of Zn2+ inhibition in apoptosis and suggest a regulatory role for Zn2+ in modulating the upstream apoptotic machinery.

Animals↗