| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Brown University School of Medicine and Rhode Island Hospital,
* Division of Surgical Research, and
Center for Surgical Research, Providence, Rhode Island
Correspondence: Dr. H. Hank Simms, MD, Rhode Island Hospital, 593 Eddy Street, Department of Surgery, APC 110, Providence, RI 02903. E-mail: hank_simms{at}brown.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: neutrophil • hypoxia • laminin
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The general mechanisms governing PMN apoptosis are not well understood. The caspase cascade [10 , 11 ], crucial to most apoptotic programs, is active in apoptotic PMN, and PMN also express the proapoptotic protein Bax [12 ]. In contrast, mature PMN lack detectable levels of Bcl-2 [13 ], the prototypic member of a large family of antiapoptotic proteins that are capable of blocking various stages of the caspase cascade. Recently, data have been published that demonstrate the presence of Mcl-1, a Bcl-2 homologue expressed predominantly in cells of the hematopoietic lineage [14 15 16 ], in mature human PMN [12 ]. However, the contribution of Mcl-1 to the regulation of apoptosis in response to stimuli associated with inflammation or the wound environment is not known.
Mcl-1 was first characterized from the myeloblastic ML-1 cell line induced to differentiate along the monocytic lineage [14 ]. It shares homology with the Bcl-2 family, is capable of localizing to membrane-bound organelles, and presumably can be expressed as a long or short form as a result of translation from two start codons within its transcript. Mcl-1 is unique from other Bcl-2 family members in that it has a potential signal-like sequence that has characteristic-charged amino acids flanked by neutral residues at the amino terminus [14 ]. However, genetic analysis shows that the coding region for this sequence lies upstream of the second start codon; therefore, the short form of Mcl-1 would lack this signal-like sequence. Mcl-1 is also a short-lived protein with a half-life of less than 3 h because of its PEST (proline, glutamate, serine, and threonine) motifs, which are targets of ubiquitination [14 ]. Because Mcl-1 is rapidly turned over, it is an ideal protein to regulate apoptosis within the neutrophil. In theory, Mcl-1 could be induced by antiapoptotic stimuli but quickly would be degraded once the stimuli were interrupted or lost, thereby permitting apoptosis. Alternatively, the antiapoptotic stimulus could delay the degradation of Mcl-1.
We hypothesized that Mcl-1 had a functional role in the inhibition of apoptosis and, therefore, wanted to further characterize Mcl-1 within the neutrophil. Because Mcl-1 has been previously shown to insert into membrane-bound organelles, we investigated localization of Mcl-1 by immunohistochemistry and confocal microscopy. Furthermore, total Mcl-1 expression was assessed over a 20-h time course by western blot analysis and by immunofluorescent labeling, including quantification of individual cells. To attenuate Mcl-1 expression and therefore determine its functionality within PMN, Mcl-1 antisense oligonucleotides were used under normal culture conditions and in the presence of the antiapoptotic stimuli of hypoxia and laminin adhesion. Here, we provide data that demonstrate distinct expression patterns of Mcl-1 within human PMN and define its effect on the regulation of apoptosis
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
PMN isolation and culture
PMN were purified from fresh human whole blood using
Ficoll-Hypaque and dextran sedimentation followed by hypotonic lysis,
as previously described [4
, 6
]. PMN purity
and viability were >95%, as assessed by morphology and Trypan blue
exclusion. Freshly purified PMN were incubated in RPMI-1640 media
containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and
17.2 µg/ml adenine. PMN were cultured at 37°C at a concentration of
1.5 x 106 cells/ml for all experiments. For the
appropriate conditions, cells were plated on laminin (10 µg per 60 mm
plate) or plated onto Permanox dishes (Nunc, Naperville, IL) and
circulated with 5% CO2, 2% H2, and 93%
N2 (BOC Gases, Hingham, MA) to achieve a hypoxic
environment. Buffer pO2 during hypoxia was <15 mm Hg,
confirmed using a blood gas analyzer (Ciba Corning Diagnostics Corp.,
Medfield, MA).
Immunofluorescent localization and quantitation
PMN were harvested after 0, 4, 8, and 20 h of culture,
fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100.
Cells were blocked in PBS + 1.5% NGS with 5 µg/ml human IgG for
2 h. Mcl-1 or rabbit IgG primary antibody (3 µg/ml) was added to
cells in PBS + 1.5% NGS and incubated overnight at 4°C. A
secondary antirabbit FITC conjugate was used at a dilution of 1:200 and
incubated for 1 h at room temperature. Controls for staining
included using a primary nonspecific rabbit IgG (3 µg/ml) or a
five-fold excess of Mcl-1 blocking peptide (Santa Cruz Biotechnology),
which was incubated with Mcl-1 antibody for several hours prior to the
primary incubation. PMN were mounted in Vectashield containing
propidium iodide (Vector Laboratories, Burlingame, CA) and viewed by
immunofluorescent and confocal microscopy. To obtain images from
confocal microscopy, dual-wavelength fluorescent images were captured
by scanning one-micron sections through the cell to determine staining
patterns in and around the nucleus. Three-dimensional reconstructed
images from each focal plane were prepared in Adobe Photoshop 4.0.
Relative quantitation of the total fluorescence of each cell was
accomplished by acquiring grayscale images in 16-bit mode using a Spot2
camera, and fluorescence intensities were integrated using NIH Image
1.61. All images were acquired for 3 sec, and 40 cells from three
fields were quantitated for each time point (n=3). Relative fluorescent
intensity units were calculated by multiplying the area of the cell by
its mean fluorescence, minus the background fluorescence of the slide.
Inhibition of Mcl-1 expression by antisense oligonucleotides
Antisense (5'GGGGCTTCCATCTCCTCAA3') and sense
(5'CCCCGAAGGTAGAGGAGTT3') oligonucleotides were designed using GenBank
and BLAST 2.0 search from the National Center for Biotechnology
Information. Oligonucleotide constructs were obtained from Oligos Etc.
(Wilsonville, OR) using their optimized antisense modifications, which
include a partial phosphorothioate backbone to activate RNAse H, and
modified 5' and 3' terminal bases to resist nuclease attack. PMN were
plated in 35-mm tissue culture plates (Fisher, Pittsburgh, PA) ±
laminin in unsupplemented RPMI media ± 5 µM of Mcl-1 antisense
or sense oligonucleotides and were incubated for 3 h at 37°C
under normoxic or hypoxic conditions. Subsequently, the media was
removed, replaced with supplemented RPMI, and incubated for an
additional 5–9 h. Cells were harvested at total incubation times of 0,
3, 8, and 12 h. PMN were prepared by centrifugation onto slides,
fixed in methanol, and stained with GIEMSA stain (Sigma). PMN apoptosis
was assessed by morphology (>1000 cells counted per condition), where
apoptotic PMN displayed darkly stained, condensed nuclei and a loss of
cytoplasm.
As a second method to assess apoptosis, the TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling] In Situ Cell Death Detection Assay was used according to the manufacturer’s directions (Boehringer Mannheim, Indianapolis, IN). PMN were incubated for 8 h in media alone or with 5 µM of the Mcl-1 sense or antisense constructs and then were assayed for percent of positive cells and mean channel fluorescence by fluorescein-activated cell sorter (FACS) analysis.
The efficiency of oligo uptake was assessed using 5' FITC-tagged Mcl-1 oligonucleotides (Oligos Etc.). Cells were cultured for 3 h in the presence of the FITC oligonucleotide, washed in PBS, and analyzed by flow cytometry for the percent of positive cells and mean channel fluorescence.
Western blot analysis
Cells were harvested after 0, 4, 8, and 20 h and washed
twice with cold PBS. Whole cell lysates were prepared using a 3%
sodium dodecyl sulfate (SDS) lysis buffer [10 mM HEPES, pH 8.0, 1.5 mM
MgCl2, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, 0.1% NP-40, 3%
SDS, and Complete protease inhibitors (Boehringer Mannheim)]. After
lysates were boiled for 5 min to solubilize all proteins, protein was
quantitated using the BCA protein assay (Pierce, Rockford, IL).
Proteins (25 µg per sample) were separated by SDS-polyacrylamide gel
electrophoresis (PAGE) using 12% SDS-polyacrylamide gels and
transferred to nitrocellulose according to the manufacturer’s
instructions (MiniProtean II, Bio-Rad, Hercules, CA). Membranes were
blocked for 1 h at room temperature in PBS/Tween 20 + 3%
nonfat dry milk. Mcl-1 antibody was added at a dilution of 1:200 in
PBS + 3% nonfat dry milk and incubated overnight at 4°C. After
washing the membrane for 30 min, an antirabbit-horseradish peroxidase
(HRP) secondary antibody at 1:5000 in PBS was incubated for 1 h at
room temperature. Protein bands were visualized by Enhanced
Chemi-luminescence detection (Amersham, Arlington Heights, IL).
Statistical analysis
Statistical analysis was performed with StatView for Macintosh
with a one-way analysis of varients (ANOVA) and SuperANOVA for
Macintosh with a two-way ANOVA. Statistical significance was
established at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Mcl-1 protein expression
Potentially, Mcl-1 can be expressed as two isoforms, a long form
containing a signal-like sequence and a short form lacking this region.
Although we cannot rule out the possibility that the lower molecular
weight isoform is a degradation product, we chose to examine total
Mcl-1 expression over time in culture. Western blot analysis of Mcl-1
showed that freshly isolated PMN, as well as those cultured through
8 h, express the high molecular-weight form of Mcl-1 (Fig. 2A
, lanes 1–3) predominantly and that this level of expression is
constant. However, by 20 h in culture, there was an apparent
decrease in Mcl-1 expression (Fig. 2A
, lane 4). The lower
molecular-weight form of Mcl-1 was barely detectable in freshly
isolated PMN, but was expressed by 4 h and remained constant
through 20 h of culture. As a control for the specificity of our
antibody, we used a blocking peptide against Mcl-1, which blocked
detection of both forms of Mcl-1 (Fig. 2B)
.
|
|
|
After the 3-h incubation with or without the antisense oligonucleotide, apoptosis was minimal, demonstrating that the construct was not inducing early-cell death (Fig. 5B ). To establish that the oligonucleotide construct was not toxic, PMN were incubated with an Mcl-1 sense oligonucleotide and assessed for apoptosis at each time point with control and antisense samples. After 8 h of culture, control cells and those incubated with antisense or sense constructs were assessed for percent apoptosis by cellular morphology. Figure 5A depicts representative fields of PMN cultured for 8 h, which clearly show viable, multilobed PMN and apoptotic cells with darkly stained, condensed nuclei. Control and sense-treated cultures had similar apoptosis levels (14.7±1.9% and 15.6±2.8%, respectively). In contrast, Mcl-1 antisense-treated cultures had a significant increase in apoptosis (44.6±3.2%, P<0.001) (Fig. 5B , solid bars). A similar trend was observed after 12 h of culture, where there was no difference between control and sense-treated cells (43.1±1.8 and 45.6±0.4, respectively) and increased apoptosis with Mcl-1 antisense-treated cells (64.7±1.5, P<0.001).
|
Mcl-1 antisense in the presence of hypoxia and laminin
We and others have shown that hypoxic conditions and adherence to
laminin will delay PMN apoptosis, which is maximally seen between 8 and
12 h in culture. Because the addition of the Mcl-1 antisense
construct was able to increase apoptosis during normal culture
conditions, we examined the effect of the Mcl-1 antisense construct
under the antiapoptotic conditions of hypoxia and adhesion to laminin
to determine if their antiapoptotic effects were mediated through
Mcl-1. After 8 h of culture under hypoxia, cells treated with the
antisense oligonucleotide exhibited a significant increase in apoptosis
over the hypoxic control (58.9±6.6% and 21.5±2.1%, respectively,
P<0.001) (Fig. 6
). Lysates were prepared from hypoxic cultures to determine if
Mcl-1 expression was attenuated. Figure 4C
demonstrates that Mcl-1
expression was decreased during hypoxia in the presence of the
antisense construct (lanes 3 and 4). These data suggest that Mcl-1 is
required for PMN viability under the antiapoptotic stimulus of hypoxia.
|
Because previously published data suggest that matrix proteins may interfere with oligonucleotide uptake [17 ], we assessed the uptake of the oligonucleotides by PMN adhered to laminin. We found the percentage of PMN that took up the oligonucleotides while adhered to laminin was similar to those cells plated on plastic (80% vs. 88%, respectively); however, the amount of the oligonucleotide taken up per cell was lower as determined by mean channel fluorescence (MCF) but did not reach statistical significance (560±67 vs. 630±107, respectively) (Fig. 4B) . To further confirm that antisense decreases Mcl-1 in the presence of laminin, western blot analysis of lysates made from PMN cultures adhered to laminin showed an attenuation of Mcl-1 expression in those cells treated with the antisense construct (Fig. 4C , lanes 5 and 6).
As an additional experiment to determine if Mcl-1 was attenuated by antisense oligonucleotides during laminin adhesion, we also looked at individual cell expression of Mcl-1 by mean relative fluorescence intensity. After 8 h of incubation, adhesion to laminin as compared with plastic resulted in an increase in Mcl-1 expression (Fig. 7 ), as western blot analysis suggested (Fig. 4C , lane 5 vs. lane 1, respectively). However, addition of the antisense construct significantly decreased Mcl-1 levels >50% (Fig. 7) . Additionally, if Mcl-1 was required for laminin to delay apoptosis, this attenuation of Mcl-1 expression in the presence of the antisense construct should have at least brought apoptosis levels back to that of normoxia, which it did not (Fig. 6) . In fact, apoptosis levels during laminin adherence in the presence of 5 or 10 µM of Mcl-1 antisense never showed any increase whatsoever. Therefore, these data suggest that laminin is capable of exerting its antiapoptotic effect on PMN irrespective of Mcl-1 expression.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previously, Mcl-1 expression has been shown to be contained in the nuclear fraction of PMN by western blot analysis [12 ]. In agreement with this finding, we found Mcl-1 to be associated predominantly within the nucleus with punctate staining prominent through 8 h of incubation (Fig. 1) . On inspection by confocal microscopy, the punctate Mcl-1 staining pattern was shown to be intranuclear, localizing between condensed regions of chromatin. However, as PMN were aged for 20 h, only perinuclear localization of Mcl-1 remained. Therefore, Mcl-1 is lost from within the nucleus as PMN age and is completely absent at end-stage apoptosis. In addition to the loss of Mcl-1 nuclear localization over time, we expected total cellular Mcl-1 expression to be attenuated. Viable cells after 20 h in culture showed a significant decrease in total Mcl-1 expression, as a higher percentage of cells were becoming apoptotic. Although the function of the two isoforms is not known, loss of the higher molecular weight form was associated with PMN aged for 20 h (Fig. 2A) , and the lower molecular weight form remained relatively constant in aging cells. Although Mcl-1 could potentially be expressed as two isoforms, we cannot rule out the possibility that the lower molecular weight form is a product of proteolytic degradation. Because other antiapoptotic mediators such as GM-CSF, IL-1, and vascular endothelial growth factor have been shown to upregulate Mcl-1 [12 , 25 , 26 ], it is possible that hypoxia and/or laminin may function by upregulating Mcl-1 expression as well. Preliminary data by RNAse protection assays suggest that hypoxia increases Mcl-1 transcript levels (unpublished data), and western blot analysis indicates that hypoxia and laminin increase Mcl-1 protein expression (Fig. 4C , lanes 3 and 5). Ongoing studies are aimed to determine whether Mcl-1 localization changes as a result of these two inhibitors of apoptosis.
Although we have shown Mcl-1 localization within and surrounding the nucleus, the question of Mcl-1 function still remained. To relate expression of Mcl-1 to a functional role in the inhibition of apoptosis, we reduced Mcl-1 protein expression through the use of antisense methodology. Because Mcl-1 has a short half-life, we hypothesized that within a relatively short time span, the antisense construct could penetrate through the cell, bind Mcl-1 mRNA, and block further Mcl-1 protein production. Therefore, if Mcl-1 expression was consistently required for PMN viability during normal culture, elimination of the means of Mcl-1 production would allow for the onset of constitutive apoptosis. As expected, addition of the antisense oligonucleotides promoted a significant increase in PMN apoptosis seen as early as 8 h in culture (Fig. 5) . This effect continued through 12 h of incubation, suggesting that Mcl-1 is necessary to impede constitutive apoptosis. We expected that because hypoxia and laminin delay apoptosis and appear to upregulate Mcl-1, their antiapoptotic effect would be lost with Mcl-1 antisense oligonucleotides as well. During incubation under hypoxic conditions, we did find that loss of Mcl-1 expression was sufficient to promote apoptosis. However, adhesion to laminin over 12 h significantly reduced apoptosis, regardless of Mcl-1 expression (Fig. 6) . We considered the possibility that matrix adhesion could physically interfere with oligonucleotide uptake, because receptors such as Mac-1 required for oligonucleotide uptake may localize to the surface of adhesion [17 ]. Although the amount of oligonucleotide present in cells adhered to laminin was decreased as shown by FACS, western blot analysis and relative Mcl-1 quantitation after antisense treatment confirmed the attenuation of Mcl-1 expression. These results clearly demonstrate that although hypoxia and laminin delay apoptosis, the requirement for Mcl-1 differs between them.
A potential explanation for this divergent control of apoptosis is currently under investigation, but we speculate that it may involve two separate pathways: one directly blocking caspase activation and the other leading to Mcl-1 upregulation. With respect to the latter, intracellular signaling stimulated by hypoxia has been shown to activate pathways involving mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), which have been shown to transcriptionally activate Mcl-1 independently [27 , 28 ]. Transcriptional regulation of Mcl-1 is controlled in part by Elk-1, a transcription factor dependent on the MAPK signaling pathway, as well as Sp1 and the serum response factor (SRF) [29 , 30 ]. Therefore, hypoxia could serve as a constant stimulus for the upregulation of Mcl-1, thereby delaying PMN apoptosis. In contrast, laminin adhesion via the integrin receptors activates PI3K and Akt [31 , 32 ], among other signaling intermediates. Akt has been shown to directly phosphorylate and inactivate BAD [33 ] and more recently has been shown to phosphorylate caspase-9, thereby preventing further caspase activation [34 ]. Therefore, laminin has the potential to directly suppress a global apoptotic mechanism such as the caspase cascade, which could delay apoptosis regardless of Mcl-1 expression. Additionally, signaling by integrin adhesion to laminin could upregulate other Bcl-2 family members such as A1 in addition to Mcl-1. Such a mechanism could provide a profound decrease in apoptosis by the upregulation of multiple antiapoptotic Bcl-2 members.
Although we have shown the functional importance of Mcl-1 in the delay of apoptosis, the mechanism(s) by which Mcl-1 functions is not known. The full length form of Mcl-1 contains a localization-like sequence that could permit its entry into the nucleus, and it is also capable of inserting into membrane-bound organelles. A possible role for Mcl-1 within the nucleus could be similar to that of the BAG-1 protein. BAG-1, although not a Bcl-2 homologue, has been implicated in the blockage of apoptosis by interaction with Bcl-2 family proteins, Raf-1, and heat shock proteins [22 , 35 ]. Mcl-1 and BAG-1 have many similarities: They are present in multiple isoforms, including a long form containing a localization sequence; they localize to the cytosol and inside the nucleus; and they have been found to bind with Bcl-2 family proteins and Raf-1 associated with the mitochondrial membrane [22 , 36 ]. Therefore, Mcl-1 may function in the nucleus to regulate transcription, especially because Mcl-1 has been shown to inhibit apoptosis after overexpression of c-Myc [37 ]. Alternatively, Mcl-1 may function in a classic Bcl-2 mechanism through the formation of a heterodimer with Bax or homodimerizing with itself, thereby preventing pore formation in organelles. Additionally, Bcl-2 has recently been shown to disrupt nuclear pore complex formation and therefore prevent granzyme accumulation in the nucleus [38 ]. In a similar role, Mcl-1 may also prevent passage of proteases, including effector caspases, that would initiate apoptosis.
In summary, we have demonstrated in this study that the Mcl-1 expression pattern changes as PMN are aged in culture and that Mcl-1 is necessary in the delay of constitutive PMN apoptosis. Additionally, we have shown that the antiapoptotic effect of hypoxia requires the presence of Mcl-1, where laminin does not. Therefore, Mcl-1 may be a target of regulation in certain, but not all, antiapoptotic signaling in PMN. Further work is needed to dissect out the specific mechanisms of Mcl-1 function within the nucleus and Mcl-1 bound to other organelles.
|
ACKNOWLEDGEMENTS |
|---|
Received November 29, 1999; revised February 7, 2000; accepted February 9, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-mediated apoptosis of human neutrophils Kidney Int 53,84-91[Medline]
Am. J. Respir. Cell Mol. Biol. 18,315-322This article has been cited by other articles:
|
|
 |
|
  I. Dzhagalov, A. St. John, and Y.-W. He The antiapoptotic protein Mcl-1 is essential for the survival of neutrophils but not macrophages Blood, February 15, 2007; 109(4): 1620 - 1626. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Cross, T. Barnes, R. C. Bucknall, S. W. Edwards, and R. J. Moots Neutrophil apoptosis in rheumatoid arthritis is regulated by local oxygen tensions within joints J. Leukoc. Biol., September 1, 2006; 80(3): 521 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Xiao, S. P. Anderson, C. Swanson, R. Bahnemann, K. A. Voss, A. J. Stauber, and J. C. Corton Activation of Peroxisome Proliferator-Activated Receptor Alpha Enhances Apoptosis in the Mouse Liver Toxicol. Sci., August 1, 2006; 92(2): 368 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu, P. Eksarko, V. Temkin, G. K. Haines III, H. Perlman, A. E. Koch, B. Thimmapaya, and R. M. Pope Mcl-1 Is Essential for the Survival of Synovial Fibroblasts in Rheumatoid Arthritis J. Immunol., December 15, 2005; 175(12): 8337 - 8345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-S. Choi, J. T. Park, and J. S. Dumler Anaplasma phagocytophilum Delay of Neutrophil Apoptosis through the p38 Mitogen-Activated Protein Kinase Signal Pathway Infect. Immun., December 1, 2005; 73(12): 8209 - 8218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Wesche, J. L. Lomas-Neira, M. Perl, C.-S. Chung, and A. Ayala Leukocyte apoptosis and its significance in sepsis and shock J. Leukoc. Biol., August 1, 2005; 78(2): 325 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Piret, E. Minet, J.-P. Cosse, N. Ninane, C. Debacq, M. Raes, and C. Michiels Hypoxia-inducible Factor-1-dependent Overexpression of Myeloid Cell Factor-1 Protects Hypoxic Cells against tert-Butyl Hydroperoxide-induced Apoptosis J. Biol. Chem., March 11, 2005; 280(10): 9336 - 9344. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Li, R. Shu, G. Filippatos, and B. D. Uhal Apoptosis in lung injury and remodeling J Appl Physiol, October 1, 2004; 97(4): 1535 - 1542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Derouet, L. Thomas, A. Cross, R. J. Moots, and S. W. Edwards Granulocyte Macrophage Colony-stimulating Factor Signaling and Proteasome Inhibition Delay Neutrophil Apoptosis by Increasing the Stability of Mcl-1 J. Biol. Chem., June 25, 2004; 279(26): 26915 - 26921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Thallinger, M. F. Wolschek, H. Maierhofer, H. Skvara, H. Pehamberger, B. P. Monia, B. Jansen, V. Wacheck, and E. Selzer Mcl-1 Is a Novel Therapeutic Target for Human Sarcoma: Synergistic Inhibition of Human Sarcoma Xenotransplants by a Combination of Mcl-1 Antisense Oligonucleotides with Low-Dose Cyclophosphamide Clin. Cancer Res., June 15, 2004; 10(12): 4185 - 4191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Gardai, D. A. Hildeman, S. K. Frankel, B. B. Whitlock, S. C. Frasch, N. Borregaard, P. Marrack, D. L. Bratton, and P. M. Henson xPhosphorylation of Bax Ser184 by Akt Regulates Its Activity and Apoptosis in Neutrophils J. Biol. Chem., May 14, 2004; 279(20): 21085 - 21095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bouchard, C. Ratthe, and D. Girard Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8 J. Leukoc. Biol., May 1, 2004; 75(5): 893 - 900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 e. Moisan, e. Kouassi, and D. Girard Mechanisms involved in methylmercuric chloride (MeHgCl)-induced suppression of human neutrophil apoptosis Human and Experimental Toxicology, December 1, 2003; 22(12): 629 - 637. [Abstract] [PDF]
|
|
|
|
|
|
H. Liu, Y. Ma, S. M. Cole, C. Zander, K.-H. Chen, J. Karras, and R. M. Pope Serine phosphorylation of STAT3 is essential for Mcl-1 expression and macrophage survival Blood, July 1, 2003; 102(1): 344 - 352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hasegawa, K. Suzuki, C. Sakamoto, K. Ohta, S. Nishiki, M. Hino, N. Tatsumi, and S. Kitagawa Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia Blood, February 1, 2003; 101(3): 1164 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Sly, S. M. Hingley-Wilson, N. E. Reiner, and W. R. McMaster Survival of Mycobacterium tuberculosis in Host Macrophages Involves Resistance to Apoptosis Dependent upon Induction of Antiapoptotic Bcl-2 Family Member Mcl-1 J. Immunol., January 1, 2003; 170(1): 430 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Lavastre, M. Pelletier, R. Saller, K. Hostanska, and |
