| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Hematology, St. Bartholomew’s and Royal London School of Medicine and Dentistry, University of London, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is a pleiotropic cytokine that acts as a
host defense factor in immunologic and inflammatory responses (1). TNF
causes cytotoxicity in certain tumor cell lines in vitro, such as the
murine fibroblast cell line L929 and the human leukemic cell
lines U937 and HL60 (2, 3). However, the majority of cell lines are
resistant to TNF-mediated cytotoxicity (4, 5). TNF binds to two distinct but structurally related cell surface receptors, TNFR-P55 and TNFR-P75 (5, 6). TNFR-P55 directly mediates cytotoxicity in a wide variety of cell types (7), but the number of TNFR does not appear to be associated with susceptibility to TNF cytotoxicity (6, 8), suggesting that postreceptor intracellular events, such as second messenger generation, are important.
Several signaling mediators, including oxygen radicals (9, 10),
phospholipase A2
(PLA2)2 (4, 11),
ceramide (3, 12), and caspases (13), are involved in TNF-induced
cytotoxicity. It has been demonstrated that TNF-induced cytotoxicity is
accompanied by activation of PLA2, and thereby the release
of arachidonic acid from membrane phospholipids. The metabolism of
arachidonic acid, via lipoxygenase and cyclooxygenase enzymes, produces
radical oxygen species that are thought to be ultimately responsible
for cytolysis (4). In addition, cPLA2 is involved in
TNF-induced ceramide generation in L929 cells (14). Ceramide is an
important contender in the apoptotic pathways activated in response to
TNF (3, 12). Resistance to TNF may involve defects in the
signal-transduction pathway linking TNFR to the apoptotic mechanism.
Moreover, there is evidence to suggest that inhibitors of this system
may exist that confer protection. The importance of the transcription
factor nuclear factor-
B in protection against apoptotic killing by
TNF has been demonstrated recently (15, 16). In addition, the
mitochondrial O2--scavenging enzyme, manganous
superoxide dismutase (17), major heat-shock protein hsp70 (18), the
TNF-inducible zinc finger protein A20 (19), and endogenous TNF (20)
have been shown to contribute to resistance to TNF cytotoxicity.
Previous studies have shown that the sensitivity of TNF-resistant cell
lines to TNF and cPLA2 activity increased following
exposure to the protein synthesis inhibitors cycloheximide (CHI) or
actinomycin D (21). These studies suggest that resistance to TNF
killing may be due to the constitutive expression of protective
proteins that regulate cPLA2 activity (22).
We therefore studied the role of cPLA2 in TNF-induced apoptosis in a series of human leukemic cell lines. Our results confirm that cPLA2 activity is required for optimal TNF activity. However, two separate mechanisms of resistance to TNF are identified. One mechanism represents a constitutive failure of cPLA2 to be fully activated by stimuli that normally result in arachidonic acid release. The other is a putative protein inhibitor that interrupts the signal-transduction pathway linking the TNFR-P55 to cPLA2 activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human rTNF-, 3-[4, 5-dimethylthiazol-2-yl]-2,
5-diphenyltetrazolium bromide (MTT), cycloheximide (CHI),
4-bromophenacyl bromide (BPB), mellitin, and saponin were all purchased
from Sigma (Dorset, U.K.). Ionophore A23187 was purchased from
Calbiochem (Nottingham, U.K.).
[5,6,8,9,11,12,14,15-3H]arachidonic acid (1 mCi/ml)
was purchased from Amersham (Amersham, U.K.).
Cell line and culture methods
Cell lines were cultured in RPMI 1640 with 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a 5% CO2 incubator.
Cytotoxicity assay
TNF-induced cytotoxicity was determined by MTT assay, as previously described (23). Resuspended cells were plated in a volume of 90 µl at 1.5 x 104/well in the presence or absence of CHI or BPB. After incubation with different concentrations of TNF at 37°C for 48 h, 10 µl MTT (5 mg/ml in PBS, pH 7.2) was added to each well. Following a further 4-h incubation at 37°C, 150 µl of 0.04 N HCl in isopropanol was added to each well. Once the dark blue formazan had been dissolved, the absorbance of each well was measured with a Titertek Multiskan MCC 340 microplate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. Cytotoxicity by MTT conversion at each concentration of TNF was compared with untreated (control) cells (shown as 100%). The ID50 value was taken as the concentration of drug required to produce a 50% inhibition of cell growth. This was calculated from a logarithmic regression curve of the results for at least five separate experiments.
Measurement of [3H]arachidonic acid release
Cells were labeled with 0.3 µCi/ml [5,6,8,9,11,12,14,15-3H]arachidonic acid and incubated at 37°C for 18 h. The unincorporated [3H]arachidonic acid was removed by washing three times with HBSS. Cells were then resuspended in fresh media in 24-well plates in triplicate. For experiments using the protein synthesis inhibitor CHI, the inhibitor was incubated with cells for 3 h before the addition of TNF. For experiments involving mellitin, the agent was added 3 h before the addition of TNF, such that cells were exposed to the agent for 27 h in total by the time of assay. After centrifugation at 4000 x g for 15 min, 0.4 ml of the supernatant was collected, mixed with 3.6 ml of scintillation fluid, and counted by liquid scintillation. To control for the nonenzymatic release of radiolabeled material from lysed cells, cells that had incorporated [3H]arachidonic acid were incubated with media for 18 h, then rapidly frozen at -70°C and thawed at 37°C. After thawing, the media were removed and processed for liquid scintillation counting, as described above. In all experiments, the average release of [3H]arachidonic acid from cells incubated with media only was less than 7% of the total. To confirm that the increase in [3H]arachidonic acid in the supernatant of TNF-treated cells was not due to the failure of dying cells to take up [3H]arachidonic acid, cells were treated with or without TNF for 8 h and then labeled with [3H]arachidonic acid for a further 6 h before both the supernatant and the pellet were counted by liquid scintillation. The amounts of [3H]arachidonic acid in both supernatant and pellet fractions were identical for both untreated and TNF-treated cells, indicating that re-uptake of [3H]arachidonic acid from supernatant, once released, is negligible.
Detection of TNFR expression by flow cytometry
Cell surface TNFR expression was assessed by indirect immunofluorescence using the mouse anti-human TNFR-P55 mAb (Genzyme, Cambridge, MA). Briefly, cells were collected, washed twice with PBS containing 1% BSA (1% BSA-PBS), then incubated with 10 µg/ml mouse anti-human TNFR-P55 mAb at 4°C for 45 min. Cells were then fixed with 1% paraformaldehyde at 4°C for 20 min to prevent receptor internalization. Nonspecific mouse IgG1 was used as a negative control. After washing twice with 1% BSA-PBS, cells were incubated with mouse phycoerythrin-conjugated rabbit anti-mouse F(ab')2 fragments (Dako, Glostrup, Denmark) at 4°C for 30 min. Cells were analyzed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA) and the results are expressed as mean fluorescence intensity and percentage of positive cells.
Preparation of membrane proteins and analysis by Western blot
Cells were washed three times with cold HBSS and rinsed in 0.5 ml homogenization buffer (50 mM HEPES, pH 8, 1 mM EDTA, 1 mM EGTA, 50 mg/ml leupeptin, 1 mM DTT, 0.5 mM PMSF, 10 mM phosphoramidon, 10 mg/ml soybean trypsin inhibitor, and 100 mg/ml aprotinin). Cells were homogenized with a dounce tissue grinder (Jencons, Leighton Buzzard, U.K.). The homogenates were centrifuged at 1,000 x g for 10 min to discard the nuclei and debris, followed by centrifugation at 8,000 x g for 30 min to remove mitochondria. The supernatants were finally centrifuged at 100,000 x g for 60 min to produce a soluble fraction (crude cytosol) and a particulate fraction (pellet). The membrane pellets were resuspended in homogenization buffer and stored at -70°C. Forty micrograms of crude cytosol and membrane proteins were separated on 8% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The blots were stained with ponseau-S solution (Sigma) to monitor the equal loading proteins for each lane. After destaining, the blots were incubated in blocking solution consisting of 5% fat-free dry milk in PBST (PBS containing 0.1% Tween-20) at room temperature for 1 h, followed by incubation at room temperature for 2 h with a 1:100 mouse anti-human cPLA2 mAb (100 µg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoblots were then incubated with goat anti-mouse IgG conjugated to peroxidase for 60 min and visualized with enhanced chemoluminescence detection reagents (Amersham).
Detection of cPLA2 mRNA by RT-PCR
Total cellular RNA was extracted by RNAzol B single step guanidinium thiocyanate-phenol-chloroform extraction method and reverse transcribed into cDNA at 42°C for 60 min using AMV reverse transcriptase and random hexamer primers. The cDNA was amplified via PCR using Taq DNA polymerase. The PCR reaction for cPLA2 was conducted for 35 cycles in 25 µl of reaction mixture using a step program (94°C, 45 s; 56°C, 45 s; 72°C, 1.5 min), followed by a 10-min final extension at 72°C. The PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The cPLA2 primer pair amplified a 554-bp PCR product and was composed of the following sequences: 5'-TTG CAA ACT GCC TCA GCA TCA G-3'; 5'-CTC TAG TCC TCC GTT CAA GGA AC-3'. The ß-actin primer pair amplified a 400-bp product and was composed of the following sequences: 5'-GAT GGA GTT GAA GGT AGT TT-3'; 5'-TGC TAT CCA GGC TGT GCT AT-3'. The reverse transcription and PCR experimental conditions were identical for cPLA2 and ß-actin.
Statistical analysis
Results are expressed as mean ± SD obtained from multiple experiments. Statistical analysis was performed using two-sided paired Student’s t test for grouped data. In all circumstances, a p value less than 0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
TNF-mediated cytotoxicity was determined in a panel of human
leukemic cell lines, the myeloid cell lines U937, HL60, and KG1a, and
the T lymphoblastic cell line CCRF-CEM and its vinblastine-resistant
subline CEM/VLB100 (Fig. 1
). U937 and HL60 cells were relatively
sensitive to the cytotoxic action of TNF (23), previously confirmed to
be apoptotic, whereas KG1a, CEM, and CEM/VLB100 cells were
relatively resistant. The ID50 values for U937 and HL60
cells were 1 to 5 ng/ml of TNF, whereas the TNF ID50 values
were greater than 100 ng/ml for KG1a, CEM, and CEM/VLB100
cells.
|
), although to a varying
degree. No association between TNFR-P55 expression and sensitivity to
TNF was observed.
|
We tested whether cPLA2 activation was associated
with TNF-mediated cytotoxicity by measuring cPLA2-mediated
arachidonic acid release from cell membrane. TNF at 2 ng/ml
significantly enhanced [3H]arachidonic acid release in
TNF-sensitive cells after 24 h (Fig. 2A), whereas no
significant increase in [3H]arachidonic acid release was
observed in KG1a, CEM, and CEM/VLB100 cell lines. A strong
positive correlation was observed between cell death and
cPLA2 activity after exposure to TNF
(r = 0.95, p < 0.01). Some
[3H]arachidonic acid release was observed when cells were
exposed to high concentrations of TNF. TNF at 100 ng/ml increased
[3H]arachidonic acid release by 12, 25, and 35%,
respectively, in the KG1a, CEM, and CEM/VLB100 cell
lines.
|
B). The ID50 values for U937 and HL60
cells treated with BPB increased by approximately 3.5- and 2.1-fold
(p < 0.01), suggesting that cPLA2
activity is, at least in part, required for TNF-mediated cytotoxicity
in these cells. This has been confirmed using an antisense
oligonucleotide to cPLA2 in U937 cells (24). Expression of cPLA2 protein and mRNA by leukemic cell lines
We next set out to establish whether activation of
cPLA2 by TNF was related to constitutive levels of
this protein. cPLA2 protein expression in both the cytosol
and membrane fractions was assessed by Western blot. cPLA2
protein was detectable in the cytosol of U937, HL60, and KG1a cells
(Fig. 3, A and
B). CEM and CEM/VLB100 cells, however,
expressed small but nevertheless detectable amounts of
cPLA2 protein in their cytosol. No cPLA2 was
detected in the membrane of CEM or CEM/VLB100 cells by
Western blot. However, small amounts were detected by flow cytometry in
permeabilized (for cytosol cPLA2) or nonpermeabilized cells
(for membrane cPLA2) (data not shown). Using
reverse-transcriptase PCR, we detected cPLA2 mRNA in all
cell lines studied. However, U937 and HL60 cells expressed relatively
high levels of cPLA2 mRNA, whereas expression levels were
lower in both CEM and CEM/VLB100 cells (Fig. 4). KG1a cells expressed high levels of
cPLA2 mRNA, consistent with protein expression, although
these cells were relatively TNF resistant.
|
|
Clearly, cPLA2 was detectable in the KG1a cell
line, but this was not activated by TNF. By contrast, CEM and
CEM/VLB100 cells have low levels of cPLA2
expression, possibly resulting in the relative resistance of these cell
lines to TNF-mediated apoptosis. To further explore the mechanisms by
which TNF-associated cPLA2 activity in these cell lines is
regulated, cells were exposed to TNF after a 3-h preincubation with the
protein synthesis inhibitor CHI. Our results showed that TNF alone (2
ng/ml) did not produce a significant increase in
[3H]arachidonic acid release in KG1a, CEM, and
CEM/VLB100 cell lines, as shown in Figure 2A.
When these cells were treated with both TNF and CHI, however, a
significant increase in the release of [3H]arachidonic
acid was observed in both CEM and CEM/VLB100 cell lines
(Fig. 5A). By contrast,
only a marginal increase was observed in the KG1a cell line after CHI
exposure. These data suggest the presence of a protein that inhibits
TNF-induced cPLA2 activation in the CEM and
CEM/VLB100 cell lines, but not in KG1a cells.
|
B). By contrast, the viability of the KG1a
cell line was not altered significantly even when CHI was used at
concentrations as high as 300 ng/ml. Our results demonstrate that
inhibition of protein synthesis by CHI significantly potentiates
TNF-mediated cPLA2 activation and cytotoxicity in both CEM
and CEM/VLB100 cell lines, but not in KG1a cells. The data
indicate the existence of cellular proteins that may protect both CEM
and CEM/VLB100 cells against the cytotoxic effects of TNF
in these cell lines, possibly by directly inhibiting the activation of
cPLA2. Mellitin and the calcium ionophore A23187 can directly activate cPLA2 in KG1a cells
To study whether the inactivation of cPLA2 in
KG1a, CEM, and CEM/VLB100 cells is specific to TNF or
whether it is a general phenomenon, we studied the actions of two other
agents known to induce activation of cPLA2, the calmodulin
antagonist, mellitin, and the calcium ionophore A23187. Mellitin alone
induced some activation of cPLA2 in all three cell lines
and had an additive effect with TNF in the CEM and
CEM/VLB100 cell lines (Fig. 6). The combined effect with TNF in KG1a
cells, although not dramatic, was synergistic.
|
, A23187 alone could
stimulate [3H]arachidonic acid release from the KG1a cell
line, and an additive effect of A23187 and TNF (100 ng/ml) was
observed. However, A23187 had no effect in the CEM and
CEM/VLB100 cell lines. These data suggest that at least
some of the cPLA2 protein detectable in KG1a cells is able
to be activated by agents other than TNF, although the level of
activity is low and not sufficient in itself to result in apoptosis
(data not shown). The levels of activation were lower than those that
would have been expected given the relatively high level of
cPLA2 protein. This suggests that there is a constitutive
failure of cPLA2 activation in these cells. The data also
suggest that mellitin, and possibly the calcium ionophore A23187,
permit some activation of cPLA2 by TNF in the KG1a cell
line, but only to a minor degree. Neither mellitin or A23187 had any
significant effect on TNF-induced cPLA2 activation in CEM
and CEM/VLB100 cells. The inhibitory factor preventing
TNF-induced activation of cPLA2 in these cell lines appears
to be independent of calcium mobilization.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Whereas cPLA2 activation and apoptosis were associated in U937 and HL60 cells, the KG1a, CEM, and CEM/VLB100 cell lines were relatively resistant to TNF. In addition, TNF failed to stimulate [3H]arachidonic acid release from these cell lines. We therefore set out to establish the mechanisms that regulate cPLA2 activity in these cell lines. Variations in the amount of [3H]arachidonic acid release could be dependent on the constitutive expression or activity of cPLA2 itself. Overexpression of cPLA2 has been shown to increase the sensitivity of a TNF-resistant L929 subline to TNF (11). Both cPLA2 mRNA and protein were expressed in all of the cell lines studied, although considerably less cPLA2 was found in the CEM and CEM/VLB100 cell lines. These observations may explain, in part, the low level of inducible cPLA2 activity in these cell lines. By contrast, the KG1a cell line, which was more resistant to TNF, expressed more cPLA2 than both CEM and CEM/VLB100 cell lines. This suggests that a mechanism exists whereby cPLA2 in the KG1a cell line is protected from activation by TNF.
Interestingly, prior exposure to the protein synthesis inhibitor, CHI, sensitized the CEM and CEM/VLB100 cell lines to TNF killing, and resulted in greater [3H]arachidonic acid release in response to TNF. These observations suggest that cellular inhibitory proteins exist at or upstream of cPLA2 activation (4). In the presence of CHI, TNF produced a significant increase in [3H]arachidonic acid release, suggesting that, although small amounts of cPLA2 protein are expressed in the CEM and CEM/VLB100 cell lines, cPLA2 is efficiently and actively involved in the TNF-signaling pathway. By contrast, inhibiting protein synthesis had no effect on KG1a cells, suggesting either a constitutive defect of cPLA2 itself or in the signaling cascade linking cPLA2 to the TNFR.
Increases in intracellular calcium can translocate cPLA2 to the membrane (25, 26) and enhance its activity. Using the model described above, we would predict that cPLA2 in the CEM and CEM/VLB100 cell lines would be activated by direct stimuli, such as mellitin or the calcium ionophore A23187, whereas cPLA2 in the KG1a cell line would be unaffected. In contrast, both mellitin and A23187 induced low levels of cPLA2 activity in the KG1a cell line, although to nothing like the same degree as could be optimally induced in CEM and CEM/VLB100 cells with TNF and CHI. In fact, mellitin, which acts as a calmodulin inhibitor (28), and A23187 had no more than an additive effect with TNF in CEM and CEM/VLB100 cells, whereas a small synergistic effect was observed in KG1a cells. These data suggest that cPLA2 in KG1a cells is not completely dysfunctional, and some activation may be induced by TNF when calcium mobilization is altered. As anticipated, the ability of TNF to induce cPLA2 activity in CEM and CEM/VLB100 cells is unchanged by disturbing calcium flux.
Some studies have shown that resistance to TNF killing is due to the
constitutive expression of cellular proteins that inhibit the
TNF-associated lytic pathway in resistant cells. Such proteins
include manganous superoxide dismutase (17), major heat-shock protein
70 (18), and the zinc finger protein A20 (19). It is known that TNF
activates the nuclear factor-B family of transcription factors that
in turn regulates synthesis of a number of proteins, including TNF
itself (15, 16). More recent results suggest a novel caspase-dependent
activation pathway for cPLA2 during apoptosis and identify
cPLA2 as a mediator of TNF-induced cell death acting
downstream of caspases (29).
We propose that cPLA2 activity is involved in TNF-induced cytotoxicity in the human leukemic cell lines used. Resistance to TNF-induced cytotoxicity may involve either protein inhibitors that act upstream of cPLA2 in the TNF-signaling pathway or a constitutive defect of cPLA2 itself, possibly involving calcium utilization.
|
Footnotes |
|---|
2 Abbreviations used in this paper: PLA2, phospholipase A2; BPB, 4-bromophenacyl bromide; CHI, cycloheximide; cPLA2, cytosolic phospholipase A2; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide.
Received for publication November 11, 1997. Accepted for publication February 10, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-interferon. J. Biol. Chem. 266:484. in malignant gynecological cell lines is associated with the expression of protein(s) that prevents the activation of phospholipids A2 by tumor necrosis factor . Cancer Res. 52:866.-induced ceramide generation in L929 cells. J. Biol. Chem. 272:17196.B in preventing TNF--induced cell death. Science 274:782.B. Science 274:784.This article has been cited by other articles:
|
|
 |
|
  M. Chen, M. Divangahi, H. Gan, D. S.J. Shin, S. Hong, D. M. Lee, C. N. Serhan, S. M. Behar, and H. G. Remold Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death J. Exp. Med., November 24, 2008; 205(12): 2791 - 2801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Pedersen, K. A. Poulsen, and I. H. Lambert Roles of phospholipase A2 isoforms in swelling- and melittin-induced arachidonic acid release and taurine efflux in NIH3T3 fibroblasts Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1286 - C1296. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xie, L. Liu, X. Huang, Y. Guo, and L. Lou Scalaradial Inhibition of Epidermal Growth Factor Receptor-Mediated Akt Phosphorylation Is Independent of Secretory Phospholipase A2 J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1210 - 1217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Tommasini and O. Cantoni Dexamethasone Promotes Toxicity in U937 Cells Exposed to Otherwise Nontoxic Concentrations of Peroxynitrite: Pivotal Role for Lipocortin 1-Mediated Inhibition of Cytosolic Phospholipase A2 Mol. Pharmacol., April 1, 2004; 65(4): 964 - 972. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Jiang, B. L. Neubauer, J. R. Graff, M. Chedid, J. E. Thomas, N. W. Roehm, S. Zhang, G. J. Eckert, M. O. Koch, J. N. Eble, et al. Expression of Group IIA Secretory Phospholipase A2 Is Elevated in Prostatic Intraepithelial Neoplasia and Adenocarcinoma Am. J. Pathol., February 1, 2002; 160(2): 667 - 671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Fonteh, C. R. Marion, B. J. Barham, M. B. Edens, G.-i. Atsumi, J. M. Samet, K. P. High, and F. H. Chilton Enhancement of Mast Cell Survival: A Novel Function of Some Secretory Phospholipase A2 Isotypes J. Immunol., October 15, 2001; 167(8): 4161 - 4171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Sheridan, T. Force, H.-J. Yoon, E. O'Leary, G. Choukroun, M. R. Taheri, and J. V. Bonventre PLIP, a Novel Splice Variant of Tip60, Interacts with Group IV Cytosolic Phospholipase A2, Induces Apoptosis, and Potentiates Prostaglandin Production Mol. Cell. Biol., July 15, 2001; 21(14): 4470 - 4481. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Duan, H. Gan, J. Arm, and H. G. Remold Cytosolic Phospholipase A2 Participates with TNF-{{alpha}} in the Induction of Apoptosis of Human Macrophages Infected with Mycobacterium tuberculosis H37Ra J. Immunol., June 15, 2001; 166(12): 7469 - 7476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Vrana and S. Grant Synergistic induction of apoptosis in human leukemia cells (U937) exposed to bryostatin 1 and the proteasome inhibitor lactacystin involves dysregulation of the PKC/MAPK cascade Blood, April 1, 2001; 97(7): 2105 - 2114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N.-E. El Mahdani, M. Ameyar, Z. Cai, O. Colard, J. Masliah, and S. Chouaib Resistance to TNF-Induced Cytotoxicity Correlates with an Abnormal Cleavage of Cytosolic Phospholipase A2 J. Immunol., December 15, 2000; 165(12): 6756 - 6761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yano, E. Kishida, M. Iwasaki, S. Kojo, and Y. Masuzawa Docosahexaenoic Acid and Vitamin E Can Reduce Human Monocytic U937 Cell Apoptosis Induced by Tumor Necrosis Factor J. Nutr., May 1, 2000; 130(5): 1095 - 1101. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
