Maximum Rate Of Hydrolysis Research Articles

Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate

Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0–9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10 − 2 M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000 ± 500 and 2800 ± 60 μmol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240 ± 17 and 432 ± 62 U/mg, respectively) on MGDG.

Highly efficient hydrolysis of phosphodiester by a copper(II)-chelated chitosan magnetic nanocarrier

A novel heterogeneous magnetic nanocatalyst was designed by conjugating copper(II) ions with chitosan-bound magnetic nanocarrier. Its hydrolytic activity for phosphodiesters was evaluated by investigating the hydrolysis of secondary ester bis-(nitrophenyl) phosphate (BNPP) in aqueous media. The maximum hydrolysis rate of BNPP was observed around the p K a of nanocatalyst which was found to be 12. The kinetic studies revealed that the binding affinity of BNPP to the copper(II)-chelated chitosan magnetic nanocarrier was low as compared to other catalyst systems. However, the rate constants ( k cat) and maximum reaction rate ( V max) were found to be higher with lower activation energy for the developed nanocatalyst. Moreover, the developed nanocatalyst was magnetically recoverable and also had good reusability.

A kinetic model for the enzymatic action of cellulase.

We develop a mechanochemical model for the dynamics of cellulase, a two-domain enzyme connected by a peptide linker, as it extracts and hydrolyzes a cellulose polymer from a crystalline substrate. We consider two random walkers, representing the catalytic domain (CD) and the carbohydrate binding module (CBM), whose rates for stepping are biased by the coupling through the linker and the energy required to lift the cellulose polymer from the crystalline surface. Our results show that the linker length and stiffness play a critical role in the cooperative action of the CD and CBM domains and that, for a given linker length, the steady-state hydrolysis shows a maximum at some intermediate linker stiffness. The maximum hydrolysis rate corresponds to a transition of the linker from a compressed to an extended conformation, where the system exhibits maximum fluctuation, as measured by the variance of the separation distance between the two domains and the dispersion around the mean hydrolysis speed. In the range of experimentally known values of the parameters of our model, improving the intrinsic hydrolytic activity of the CD leads to a proportional increase in the overall hydrolysis rate.

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ∼456 kDa and p I of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T 1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol −1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg 2+ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate ( V max) and apparent Michaelis–Menten constant ( K m) for sodium phytate were 83 nmol mg −1 s −1 and 0.156 mM, respectively. The catalytic turnover number ( K cat) and catalytic efficiency ( K cat/ K m) of phytase were 37.8 s −1 and 2.4 × 10 5 M −1 s −1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB 1–23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB 1–23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.

Properties and catalytic mechanism of γ-glutamyltranspeptidase from B.subtilis NX-2

Since γ-glutamyltranspeptidase (GGT) especially catalyses the transfer of the γ-glutamyl moiety to a variety of amino acids and short peptides, GGT has important practical value for enzymatic synthesis of γ-glutamyl compounds. In this paper, the GGT produced from Bacillus subtilis NX-2 was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, and the properties of purified GGT were investigated. At the conditions of pH 10.0, D-glutamine (D-Gln)/L-tryptophan (L-Trp) with a molar ratio of 5: 7, a temperature 40°C and a reaction time of 4 h, a higher conversion rate of 42% was obtained. According to the time course, the catalytic mechanism of enzymatic synthesis of γ-D-glutamyl-L-tryptophan (γ-D-Gln-L-Trp) was discussed. It was demonstrated that the GGT can catalyze not only the reaction of transpeptidation, but also the irreversible hydrolysis of the products which results in the decrease of the yield of the products. The affinity parameter of GGT to D-Gln (Km) was 5.08 mmol·L−1 and the maximum reaction rate of transpeptidation (r max) was determined as 0.034 mmol·min−1·L−1, while the affinity parameter of GGT to γ-D-Gln-L-Trp (K’m) was 2.267 mmol·L−1, and the maximum reaction rate of hydrolysis (r’max) was 0.012 mmol·min−1·L−1.

The Kinetics of Ribosomal Peptidyl Transfer Revisited

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

Estimating the magnitude of PDE4 activity near the plasma membrane of HEK‐293 cells

The importance of subcellular localization of phosphodiesterases (PDEs) has been clearly demonstrated. However, with the exception of sensory neurons, little is known about the subcellular concentrations of these enzymes. We have previously used cyclic nucleotide‐gated (CNG) channels as biosensors to measure near‐membrane cAMP signals. We have used this approach to demonstrate that PDE4 is primarily responsible for the hydrolysis of cAMP near the plasma membrane of HEK‐293 cells. Here, we use a similar approach in addition to measurements of total cellular PDE activity and mathematical modeling to estimate the concentration of PDE4 near the plasma membrane. Currents through CNG channels were measured in order to monitor the time‐course and magnitude of cAMP dialysis from the whole‐cell patch pipette to the plasma membrane. We found that PDE4 inhibitors had a profound effect on the magnitude of cAMP‐induced currents at cAMP levels ranging from 2 to 200 μM. In separate experiments, we estimated that PDE4 was responsible for ∼50% of the total cAMP PDE activity, with a maximal hydrolysis rate of ∼0.5 μM/s and a Km of ∼3 μM. We are using these data to constrain our previously described compartmental model (Rich et al., J. Gen. Physiol., 2000, 116:147), and estimate the magnitude of PDE4 activity in the near‐membrane compartment. This work was supported by NIH awards R01HL074278 and K02HL081463.

The effect of mixing pharmaceutical and tannery wastewaters on the biodegradation characteristics of the effluents

This paper evaluated the effect of mixing the effluent of a pharmaceutical plant producing acetylsalicylic acid with tannery wastewater, on the biodegradation of the effluents. The evaluation involved the analysis of the oxygen uptake rate (OUR), profiles of each wastewater and the mixture by respirometry. Model calibration using the experimental OUR data identified major COD fractions and associated process kinetics for all samples analyzed. The tannery sample was a plain-settled effluent having a total COD of around 2200 mg/L with a readily biodegradable fraction of 15%. The same fraction was 57% in the pharmaceutical wastewater sample having a much stronger total COD content of 40,435 mg/L. Consequently, mixing of the pharmaceutical effluent with the tannery wastewater up to 38% of the total COD in the mixture increased the readily biodegradable COD fraction but had an inhibitory effect on the biodegradation kinetics. This effect was relatively lower on growth, but quite significant on the hydrolysis of the slowly biodegradable COD decreasing the maximum hydrolysis rate from 2.0 day −1 to 1.2 day −1. Model evaluation of the respirometric data, as performed in this study sets a workable protocol for the assessment of the compatibility of different wastewater mixtures for biological treatability.

Inhibition of Na+, K+-ATPase in the presence of crown ethers: modulation of ionic composition or pharmacological effects

It is believed that the biological effects of chelating agents such as crown ethers are largely related to their ability to form complexes with ions and/or to facilitate ion transport across membranes. Specific influences are rarely related. Here we present the evidence that even one of the simplest representatives of the crown ether super-family, 1,4,7,10,13,16-hexaoxacyclooctane (18-crown-6), is able to affect the activity of Na+, K+-ATPase directly. Using nonlinear regression fitting to kinetic data we have found that the crown ether diminishes the apparent Michaelis constant, K m , and the maximal rate of ATP hydrolysis, V m , acting as noncompetitive inhibitors. The apparent dissociation constants, K i , for the crown interaction with the free ATPase and with the enzyme-substrate complex were established to be of 77 ± 3 mM and 21 ± 2 mM, respectively. So 18-crown-6 possesses weak but “direct” pharmacological activity on Na+, K+-ATPase hinders the formation of enzyme–substrate complex and detains the enzyme in this state.

Model evaluation of temperature dependency for carbon and nitrogen removal in a full-scale activated sludge plant treating leather-tanning wastewater

Organic carbon and nitrogen removal performance of a full-scale activated sludge plant treating pre-settled leather tanning wastewater was evaluated under dynamic process temperatures. Emphasis was placed upon observed nitrogen removal depicting a highly variable magnitude with changing process temperatures. As the plant was not specifically designed for this purpose, observed nitrogen removal could be largely attributed to simultaneous nitrification and denitrification presumably occurring at increased process temperatures (T > 25°C) and resulting low dissolved oxygen levels (DO < 0.5 mgO2/L). Model evaluation using long-term data revealed that the yearly performance of activated sludge reactor could be successfully calibrated by means of temperature dependent parameters associated with nitrification, hydrolysis, ammonification and endogenous decay parameters. In this context, the Arrhenius coefficients of (i) for the maximum autotrophic growth rate, A, (ii) maximum hydrolysis rate, khs and (iii) endogenous heterotrophic decay rate, bH were found to be 1.045, 1.070 and 1.035, respectively. The ammonification rate (ka) defining the degradation of soluble organic nitrogen could not be characterized however via an Arrhenius-type equation.

Temperature dependent properties of a kinesin-3 motor protein from Thermomyces lanuginosus

Kinesins are cytoskeletal motor proteins that share a common mechanochemical motor domain, and are responsible for trafficking macromolecules. Here we report the cloning and characterization of a monomeric, kinesin-3 (TKIN) from Thermomyces lanuginosus. TKIN displayed a maximum rate of ATP hydrolysis at ∼55 °C; the K m ATP was also significantly greater at 50 °C. Gliding motility rates reached a maximum of 5.5 μm s −1 at 45 °C, which is among the highest rates reported for kinesin. Arrhenius energy barriers were calculated to be ∼103 kJ mol −1, nearly twofold greater than other mesophilic kinesin motors. The enthalpy of activation and entropy activation of TKIN were also significantly greater when compared to other mesophilic kinesins. A thermally induced aggregation of TKIN, which could be moderated by the addition of ATP, was observed at temperatures above 45 °C. Together, these results illustrate the kinetic response and stability of this unique motor protein at elevated temperatures.

Purification of breast cancer resistance protein ABCG2 and role of arginine-482.

Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding.

Kinetics of urothelial ATP release

Recent reports have proposed that the urothelium can sense mechanical stretch and communicate this information to sensory afferent neurons by the release of ATP into the vicinity of P2X-containing neurons. This report investigates the bidirectional release of ATP by in vitro rabbit urothelium. ATP was measured using the luciferin-luciferase assay. Immediately after washing of both sides of the epithelium, there was a linear increase in ATP content in the mucosal compartment with a rate of 23 +/- 6.5 fmol x min(-1) x cm(-2) (n = 18). Serosal ATP content increased as a saturating exponential function, suggesting a constant rate of release and degradation of ATP by ectonucleotidases/exonucleotidases. The presence of a serosal ectonucleotidase/exonucleotidases was demonstrated by the time-dependent decrease in exogenously added ATP. The maximum rate of hydrolysis was 11 pmol x min(-1) x cm(-2) with a K(m) of 0.49 microM. The time course of serosal ATP release was modeled as a constant rate of release (d: mol x min(-1) x cm(-2)) and rate constant of hydrolysis (k(h): min(-)). In control conditions d was 18 fmol x min(-1) x cm(-2) and k(h) of 0.056 +/- 0.01 min(-) (n = 18). Steady-state serosal chamber content is 370 +/- 90 fmol/cm(2), and concentration is 50 +/- 1.2 x 10(-12) M. Stretching the tissue resulted in a transient fivefold increase in the rate of mucosal ATP release and a transient sixfold increase in serosal ATP release. Half-osmotic strength solutions increased mucosal release by 10-fold and serosal release by 5-fold. Tissue damage resulted in a step-increase in mucosal chamber ATP content by 6.6 +/- 1 pmol/cm(2) and serosal chamber ATP by 0.1 +/- 0.06 pmol/cm(2) (n = 5).

The Binding of W7, an Inhibitor of Striated Muscle Contraction, to Cardiac Troponin C

W7 is a well-characterized calmodulin antagonist. It decreases the maximal tension and rate of ATP hydrolysis in cardiac muscle fibers. Cardiac troponin C (cTnC) has been previously implicated as the mechanistically significant target for W7 in the myofilament. Two-dimensional NMR spectra ({1H,15N}- and {1H,13C}-HSQCs) were used to monitor the Ca2+-dependent binding of W7 to cTnC. Titration of cTnC x 3Ca2+ with W7 indicated binding to both domains of the protein. We examined the binding of W7 to the separated domains of cTnC to simplify the spectral analysis. In the titration of the C-terminal domain (cCTnC x 2Ca2+), the spectral peaks originating from a subset of residues changed nonuniformly, and could not be well-described as single-site binding. A global fit of the cCTnC x 2Ca2+ titration data to a two-site, sequential binding model (47 residues simultaneously fit) yielded a dissociation constant (Kd1) of 0.85-0.91 mM for the singly bound state, with the second dissociation constant fit to 3.40-3.65 mM (> or = 4 x Kd1). The titration data for the N-terminal domain (cNTnC x Ca2+) was globally fit to single-site binding model with a Kd of 0.15-0.30 mM (41 residues fit). The data are consistent with W7 binding to each domain's major hydrophobic pocket, coordinating side chains responsible for liganding cTnI. When in muscle fibers, W7 may compete with cTnI for target sites on cTnC.

Switches, Catapults, and Chaperones: Steady-State Kinetic Analysis of Hsp70−Substrate Interactions

Hsp70 chaperones are heterotropic allosteric systems in which ATP and misfolded or aggregated polypeptides are the activating ligands. To gain insight into the mechanism by which ATP and polypeptides regulate Hsp70 chaperone activity, the effect of a short peptide on the K(M) for ATP was analyzed using the Escherichia coli Hsp70 called DnaK. In the absence of peptide, the K(-P)(M) for ATP is 52 +/- 11 nM, whereas this value jumps to 14.6 +/- 1.6 microM in the presence of saturating peptide. This finding supports a mechanism in which ATP binding drives the chaperone in one direction and peptide binding pushes the chaperone back in the opposite direction (and thus increases K(M)), according to ATP + DnaK.P <==> ATP.DnaK.P <==> ATP.DnaK* + P, where ATP.DnaK.P is an intermediate from which competing ATP hydrolysis occurs (ATP.DnaK.P --> ADP.DnaK.P). We show that this branched mechanism can even explain how DnaK hydrolyzes ATP in the absence of peptide and that the true rate constant for DnaK-mediated ATP hydrolysis (k(hy)) in the absence of peptide may be as high as 0.5 s(-)(1) (rather than 5 x 10(-)(4) s(-)(1) as often stated in the literature). What happens is that a conformational equilibrium outcompetes ATP hydrolysis and effectively reduces the concentration of the intermediate by a factor of a thousand, resulting in the following relation: k(cat) = k(hy)/1000 = 5 x 10(-)(4) s(-)(1). How polypeptide substrates and the co-chaperone DnaJ modulate DnaK to achieve its theoretical maximal rate of ATP hydrolysis, which we suggest is 0.5 s(-)(1), is discussed.

Characterization of the penicillin G acylase from Bacillus megaterium ATCC 14945

The purpose of this work was to characterize the enzyme penicillin G acylase (PGA) produced by Bacillus megaterium. Purification of the enzyme by ultra/diafiltration did not allow the detection of the PGA band by SDS-PAGE electrophoresis due to the high content of remaining proteins. However, using the DNA of the microorganism, it was possible to replicate the genes of the two B. megaterium PGA reported in literature, showing that the enzyme consisted of two sub-units, having 245 and 537 amino acids each and an average molecular mass of 26950 and 59070 Da, respectively. The parameters studied were: 1) the influence of temperature in the 25-60(0)C range, 2) pH in the 5-10 range and 3) substrate concentration, this was tested to obtain results on the Penicillin G hydrolysis reaction rate, using the initial velocities approach. The maximum hydrolysis rate was obtained at 37ºC and pH 8.0. The Michaelis-Menten model fitted well, resulting in estimated Km and Vmax parameters values of 1.83 mM and 0.165*10-3 mmol/min/UI, respectively.

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)-1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52-55 degrees C. The enzyme retained 97% activity after a 24-h incubation at 55 degrees C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1-5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5-8 M), significantly affected phytase activity. The maximum hydrolysis rate (Vmax) and apparent Michaelis-Menten constant (Km) were 1,074 IU/mL and 606 microM, respectively, with a catalytic turnover number of 3x10(5) s-1 and catalytic efficiency of 3.69x10(8) M-1 s-1.

Triacylglycerol Is Synthesized in a Specific Subclass of Caveolae in Primary Adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

Silencing of Caspase 8 Expression in Leukemia Cells and Patient Samples.

Gene silencing through hypermethylation is common in cancer cells and may contribute to chemoresistance and poor patient outcomes. To investigate this mechanism in leukemia, OCI-AML2 leukemia cells were screened for methylated genes. Among the genes identified using Cpg arrays was caspase-8. Levels of mRNA and protein of caspase-8 were decreased in OCI-AML2 cells compared to control cell lines (Jurkat cells). In contrast, levels of FLIP, caspase-3 and XIAP did not differ between both cell lines. As further evidence of caspase-8 hypermethylation, treatment of OCI-AML2 cells with the demethylating agent 5-Aza-2′-Deoxycytidine (AzaD) increased expression of caspase-8 protein to levels similar to Jurkat cells. To determine whether the silencing of caspase-8 occurs in primary patient samples, levels of caspase-8 were measured by immunoblots in 16 samples from patients with AML. Compared to normal peripheral blood mononuclear stem cells, caspase-8 was reduced in five of 16 (31%) samples. To determine whether the low caspase 8 was a functional consequence, OCI-AML2 cells were treated with CH-11 anti-FAS antibody (100 ng/mL) for 24 hours. Despite the cells expressing the FAS receptor on the surface, less than 5% of apoptosis occurred. In contrast, an equal concentration of CH-11 induced 80% apoptosis in Jurkat cells. To test the status of the death receptor pathway downstream of caspase-8, cytosolic lysates from OCI-AML2 cells were stimulated with recombinant active caspase-8 to directly activate effector caspases. Stimulation of OCI-AML2 lysates with recombinant active caspase-8 increased the maximal rate of effector caspase hydrolysis of Ac-DEVD-AFC 8 fold above buffer treated control, comparable to treatment of Jurkat lysates. These results indicate that OCI-AML2 cells have defects above the level of the effector casapses that renders them resistant to death receptor ligands. To determine if silencing of casp-8 is sufficient to explain these defects, OCI-AML2 cells were cultured with AzaD and CH-11 anti-FAS antibody. Despite restoration of caspase-8 protein levels, AzaD did not restore sensitivity to CH-11. Likewise, transfection of caspase-8 into OCI-AML2 did not restore sensitivity. Therefore, casp-8 is silenced in OCI-AML2 cells and primary patient samples. However, the events indicate the presence of additional defects in the death receptor pathway that cannot be reversed by demethylation. These results suggest that blockade of apoptosis throughout the death receptor pathway may occur at several points and that in some cases, simple reversal of demethylation may not be sufficient.