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HEMATOPOIESIS
From the Division of Hematology and Medical Oncology,
Oregon Health Sciences University, Portland, OR, and the Molecular
Hematopoiesis Laboratory, NW VA Cancer Research Center, Portland, OR.
Hematopoietic progenitor cells (HPC) from mice nullizygous at the
Fanconi anemia (FA) group C locus and children with Fanconi anemia
group C (FA-C) are hypersensitive to interferon-gamma (IFN- Fanconi anemia (FA) is an autosomal recessive
disorder characterized by cellular hypersensitivity to chemical
cross-linking agents, bone marrow failure, diverse congenital
anomalies, and a marked increase in the incidence of acute myelogenous
leukemia.1-4 Phenotypically, the sine qua non of this
disorder is hypersensitivity of FA cells to DNA cross-linking agents
such as diepoxybutane (DEB) and mitomycin C (MMC).5,6 The
disease is genetically heterogeneous, with at least 7 different
complementation groups having been identified by somatic cell hybrid
analysis.7-10 The genes encoding the A, C, D, F, and G
groups have been cloned and have been mapped to chromosomes 16q24.3,
9q22.3, 3p25.3, 11p15, and 9p13, respectively.9,11-14
The FANCC gene is constitutively expressed in most
cells15 and encodes a 63-kd protein16,17 that
has no strong amino acid sequence homology with any known gene family
and is of unknown function. Although it is hypothesized that the
protein plays some role in either facilitating repair of cross-linked
DNA or resisting the effects of cross-linking agents on nuclear DNA,
and although some of the FANCC protein is found in the
nucleus,18,19 much of the protein is
cytoplasmic.16,17 At least some critical function of the
gene product appears to require cytoplasmic
localization.20
The product of the FANCC gene clearly plays a supportive
role in growth or differentiation (or both) of hematopoietic progenitor cells (HPC),21,22 and progenitor cells from FA-C mice are
suboptimally responsive to erythropoietin and Steel
factor.23 FA-C progenitor cells are also hypersensitive to
the apoptosis-inducing effects of interferon- The active forms of the caspases are multimers the subunits of which
are cleaved from the same proenzyme. The caspase family has been
categorized into 3 subfamilies as determined by their substrate
specificity and function. The ICE subfamily consists of caspases 1, 4, and 5.30 Caspase 1 has recently been reported to play a
significant role in inflammation,31,32 and its role in
fas-induced apoptosis in most cells has now been called into question.33,34 The caspase 3 subfamily consists of
caspases 3, 6, 7, 8, 9, and 10. Of these, those with large prodomains
(ie, caspases 8, 9, and 10) are considered initiator caspases and those with a small prodomain (ie, caspases 3, 6, and 7) are considered effector or executioner caspases.35 A recent model
proposes a branched pathway of caspase activation. In this model,
caspase 8 activates caspases 3 and 7. Caspase 3 then activates caspase 6, which in turn may feed back on procaspase 3, resulting in a protease
amplification cycle.36,37 The third caspase subfamily, ICH-1/Nedd2, consists of caspase 2 and its murine counterpart. It is
predicted to act either as an apoptosis effector protein based on its
substrate specificity,38 or as an initiator caspase, due
to its large prodomain.39
Because fas-mediated apoptosis in a wide variety of cells
involves the ordered activation of the caspases30,40-44
followed by cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP),45 lamin,46,47 GATA-1,48
VAV1,49 and other critical substrates for hematopoietic
cells,30,50-52 we sought to test the notion that the
caspases are also involved in the excessive apoptotic activity of FA
cells. The ordered activation of caspases and their linkage with the
fas pathway are not fully defined in hematopoietic
progenitor cells exposed to IFN- FANCC nullizygous mice
Murine bone marrow, human CD34+ marrow cells, and
Epstein-Barr virus (EBV)-transformed cell lines
The EBV-transformed lymphoblast cell line HSC536N (a gift from Manuel Buchwald) was derived from peripheral blood cells of a child with FA-C. In the cells of this compound heterozygote, one FANCC allele is deleted and the other carries a L-to-P substitution at amino acid position 554 (single-letter amino acid code). The EBV-transformed cell line JY was derived from EBV-infected normal peripheral blood mononuclear leukocytes. Retroviral-mediated gene transfer of FANCC complementary DNA (cDNA) Plasmid construction, packaging, transduction, selection, and complementation analysis were performed as previously described.24Immunoblotting Immunoblot analyses were performed using cell lysates from human EBV-transformed lymphoblasts from normal volunteers and FA-C patients. An agonistic antihuman fas antibody (100 ng/mL, Upstate Biotechnology, Inc [UBI], Lake Placid, NY) and recombinant human IFN- (1 ng/mL, R&D Systems, Minneapolis, MN) were added for the intervals indicated. In some experiments, inhibitors to caspases 1, 3, and 8 (ac-YVAD-cho, ac-DEVD-cho, and Z-IETD-FMK, respectively) were
added to a final concentration of 50 µmol/L for the times indicated.
The cells were harvested, washed 3 times with phosphate-buffered saline
(PBS), and the cell pellets were solubilized in RIPA (10 mmol/L
Tris-Cl, pH 7.6, 150 mmol/L NaCl, 1% sodium deoxycholate, 1% Triton
X-100, 0.1% sodium dodecyl sulfate [SDS], and freshly added 1%
aprotinin, 2 mmol/L Na3VO4, 1 µg/mL
leupeptin, 1 mmol/L pepstatin A, and 1 mmol/L phenylmethylsulfonyl
fluoride [PMSF]). Lysates were centrifuged at 16 000g for
15 minutes at 4°C. Protein concentrations were determined on
supernatants using a protein microassay of the Bradford method
(Bio-Rad, Hercules, CA). Cell lysates were heated at 94°C for 5 minutes in the presence of SDS and Caspase 1 and caspase 10 were detected by incubating blots with a rabbit polyclonal antihuman caspase 1 (ICE) antibody, or a goat polyclonal antihuman caspase 10 (MCH-4) antibody (no. sc-515 and no. sc-6185, respectively, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 in 5% nonfat dry milk. Caspase 3 (CPP32), caspase 7 (MCH-3), and FADD were detected with monoclonal antibodies (no. C31720, no. M64620, and no. F36620, respectively, Transduction Laboratories, Lexington, KY), each diluted 1:1000 in 5% milk. PARP was detected by incubating blots with a monoclonal anti-PARP antibody (no. SA-250; BIOMOL) diluted 1:3000. Caspase 8 was detected with a monoclonal antihuman caspase 8 (FLICE) antibody (no. 66231A; PharMingen, San Diego, CA) diluted 1:1000 in 5% milk. Caspase 9 was detected with a rabbit polyclonal antihuman caspase 9 antibody (no. AAP-109, Stressgen, Victoria, British Colombia, Canada) diluted 1:2000 in 5% milk. All primary antibody incubations were 1 hour except for anticaspase 9, which was for 2 hours. These were followed by 6 (5 minutes) washes with Tris-buffered saline containing 0.005% Tween-20 (TBS-T). The blots were incubated with secondary antibodies (goat antirabbit IgG-horseradish peroxidase [HRP] conjugate, goat antimouse IgG-HRP conjugate [Bio-Rad], or donkey antigoat IgG-HRP conjugate [Santa Cruz]) for 30 minutes at 1:10 000, 1:5000, or 1:4000 dilutions, respectively, then washed as above with TBS-T. Antibody-reactive proteins were detected using enhanced chemiluminescence (ECL) reagents (Amersham, Piscataway, NJ). Fluorescence assays A microfluorescence assay was adapted from a protocol established by Enari and colleagues42,55; 2 × 106 lymphoblasts were washed 2 times in 4°C PBS after timed exposure to human agonistic anti-fas antibody (100 ng/mL, UBI) and human IFN- (1 ng/mL, R&D Systems). In some
experiments, inhibitors to caspases 1 (Z-WEHD-FMK), 1 and 4 (ac-YVAD-cho, Cell Permeable [CP]-YVAD-cho, Z-YVAD-FMK), 2 (Z-VDVAD-FMK), 3 (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK), 6 (Z-VEID-FMK), and 8 (ac-IETD-cho and ac-IETD-FMK) were added for the
times and final concentrations indicated (aldehyde inhibitors were
obtained from BIOMOL, FMK inhibitors were obtained from Enzyme
Systems). Cytosolic extracts were prepared by resuspending cell pellets
in 50 µL extraction buffer (50 mmol/L PIPES-NaOH, pH 7.0, 50 mmol/L
KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L DTT, 20 µmol/L cytochalasin B, 1 mmol/L PMSF, 1 µg/mL leupeptin, 1 µg/mL
pepstatin A, 50 µg/mL antipain, and 10 µg/mL chymopapain). Cells
were disrupted by 5 cycles of freezing and thawing, followed by
centrifugation at 10 000g for 12 minutes at 4°C. Protein
concentrations of the supernatants were determined by a microassay of
the Bradford method (Bio-Rad). A total reaction volume of 50 µL
included 6.25 to 25 µg cell lysate, either
N-acetyl-YVAD-MCA, or N-acetyl-DEVD-MCA fluorogenic substrate (10 µmol/L, BIOMOL), and assay buffer (100 mmol/L HEPES-KOH buffer, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mmol/L DTT, 0.1 mg/mL ovalbumin). Reactions were incubated at 30°C for 60 minutes in 96-well microtiter plates (Falcon) and enzyme activity detected by a Cytofluor II (PerSeptive Biosystems, Framingham, MA) at
an excitation  of 360 nm and an emission of 460 nm. Additional
experiments used fluorogenic substrates for caspases 1, 2, 4, 6, and 7 (N-acetyl-WEHD-MCA, MCA-VDVAD, MCA-LEVDGW[K-DNP]-NH2, N-acetyl-VEID-MCA, and MCA-VDOVDGW[K-DNP]-NH2,
respectively; Enzyme Systems).
Flow cytometry Lymphoblasts (1 × 106/mL) were plated in 24-well plates. IFN- (1 ng/mL, R&D Systems) and activating
anti-fas antibody (100 ng/mL, UBI) were added individually
or in combination for 3 or 48 hours, then washed twice with 2 mL
staining buffer (PBS, 2% fetal bovine serum [FBS], 0.1% sodium
azide). Cells were resuspended in 400 µL staining buffer and 400 µL
cytofix/cytoperm (PharMingen) and incubated on ice for 20 minutes, were
washed with 2 mL perm/wash buffer (PharMingen) and were resuspended in
100 µL perm/wash buffer for staining. Normal rabbit IgG (20 µL,
Caltag) was added for 20 minutes on ice. Twenty microliters
phycoerythrin (PE)-conjugated polyclonal rabbit antiactive caspase 3 antibody (no. 67345x, PharMingen) was then added and cells were
incubated 30 minutes on ice in the dark. Cells were then washed twice
with 2 mL perm/wash buffer and resuspended in 500 µL staining buffer
for analysis. Cells were analyzed using a FACSCalibur (Becton
Dickinson, Franklin Lakes, NJ) flow cytometer.
Quantification of apoptotic cells A portion of the cells cultured under various conditions were quantified for apoptosis using fluorescence microscopy and the TUNEL assay (ApopTag in situ Apoptosis Detection Kit; Oncor, Gaithersburg, MD). Cells with nuclear fragmentation or green fluorescence or both were scored as apoptotic.
IFN- (1 ng/mL) for 0, 10, 20, 30, 60, 120, and 180 minutes. Immunoblots performed on lysates of these cells
demonstrated procaspase 3 cleavage at 60 to 120 minutes and PARP
cleavage by 180 minutes (Figure 1A-B).
Immunoblots also detected the cleaved form of caspase 7 by 180 minutes
(not shown). Activation of caspases 1 (Figure 1C), 9, and 10 (not
shown) was not detected at any time point. A cell-free fluorogenic
assay confirmed activation of caspase 3 by 60 minutes and nearly
maximal activity by 120 minutes, whereas no activation of caspase 1 was detected at any time point (Figure 1D). Flow cytometric analysis of
lymphoblasts detected constitutive caspase 3 activation in FA-C cells,
and a profound increase in this activation in response to IFN- and
anti-fas antibody treatment (Figure 1E). In both cases, the
number of cells containing active caspase 3 was increased from 2- to
4-fold in FA-C cells. A fluorogenic assay for caspase 6 using
ac-VEID-MCA as a substrate detected enzyme activity at 180 minutes (not
shown). Activation of caspases 2 and 4 was not detected (not
shown).
We sought to rule out caspase 1 involvement in these cells by
inhibiting caspase 1 with the inhibitor ac-YVAD-cho and quantifying caspase 3 activation. Such treatment prior to a 120-minute exposure to
IFN-
Because we detected no caspase 1 activation and observed no effect of caspase 1 inhibitors on caspase 3 activation, we sought to confirm that the inhibitors were functional. The addition of ac-YVAD-cho (5-50 µmol/L) or YVAD-FMK (0.1 µmol/L) to a fluorogenic assay with N-ac-WEHD-MCA as substrate, in the presence of recombinant human caspase 1 (R&D Systems), reduced substrate cleavage to 16.3% or 9.3%, respectively, of caspase 1 alone. A negative control (Z-FA-FMK) had no effect (data not shown). Apoptosis increased from 4% to 33% in mutant lymphoblasts (as
analyzed by TUNEL assay) after exposure to IFN-
An inhibitor of caspase 3 blocks IFN- , whereas the caspase 1 (ac-YVAD-cho) inhibitor did not. We have previously shown that
FAC / mice are sensitive to doses of IFN- too low to
have an effect on FAC/+ mice.24 Because we
have previously reported that Fas-ligand is expressed on
CD34+, and also demonstrated that an anti-fas
blocking antibody reduced IFN--triggered inhibition of clonal growth
in FA-C cells,24 we did not add the agonistic
anti-fas antibody to the colony assays performed
here.
In a series of 7 experiments we have found that IFN- An inhibitor of caspase 3, but not caspase 1, suppresses IFN-
on colony-forming units-granulocyte/macrophage (CFU-GM; Figure
5A) and BFU-E (Figure 5B) when compared
to those of the control. The low IFN dose, 0.1 ng/mL, was selected to
maximize the differences between FA and normal progenitor cells. The
caspase 3 inhibitor (ac-DEVD-cho) augmented colony formation in
the IFN--treated cells of the FA-C patient, but the caspase 1 inhibitor (ac-YVAD-cho) had no effect (Figure 5).
Caspase 3 activation is caspase 8 dependent Seeking to clarify the role of caspase 8 in the activation of caspase 3, we first measured caspase 8 activation in cells of the FA-C EBV-transformed lymphoblast lines in response to a 120-minute exposure to IFN- and anti-fas antibody. This
activation is shown in FA-C cells as well as isogenic cells corrected
by introduction of FANCC cDNA (Figure
6A). We then treated these cells with an inhibitor of caspase 8 (ac-IETD-FMK) before IFN- and
anti-fas antibody exposure. The caspase 8 inhibitor
prevented cleavage of caspase 3 as detected by fluorogenic (Figure 6B)
and immunoblot assays (Figure 6C). No induction or inhibition of
activation of caspase 1 was observed as a result of treatment with
ac-IETD-FMK. The caspase 8 inhibitor also blocked caspase 8 activation
in an immunoblot assay, whereas inhibitors of caspases 1 and 3 did not (data not shown). These findings indicate a role for caspase 8, either
directly or through another molecular intermediate (not caspase 1) in
caspase 3 activation. Experiments using the aldehyde inhibitor of
caspase 8, ac-IETD-cho (BIOMOL), resulted in the inhibition of cleavage
of caspase 3 as well (data not shown).
FADD is unaffected by inhibitors of caspases 1, 3, and 8 The FADD is required to link fas ligation with caspase 8 activation. Because this is a proximal event in the apoptotic activation pathway, we did not expect the caspase inhibitors to affect FADD protein levels, but needed to rule this out in the cell types we studied. FADD levels were not influenced by inhibitors in anti-fas antibody-stimulated IFN--primed cells (data
not shown).
Specificity of caspase inhibitors Three types of caspase inhibitors were used in these experiments: tetrapeptide aldehyde inhibitors (cho), cell-permeable tetrapeptide aldehyde inhibitors (CP-cho), and fluoromethyl ketone inhibitors (FMK). Because of the reported overlap of activity for some of these inhibitors,38 particularly those for YVAD (caspases 1 and 4) and DEVD (caspase 3), we carefully determined concentrations that were high enough to inhibit apoptosis but low enough to reveal differential specificities. Cytosolic extracts were made from lymphoblasts that had been treated with IFN- and anti-fas
antibody for 180 minutes after pretreatment with either a dose from 0.1 to 5 µmol/L of CP-cho inhibitors of caspases 1 and 3 (Figure
7A), or from 0.1 to 50 µmol/L range of
FMK inhibitors of caspases 1, 3, 1 and 4, or 8 (Figure 7B).
Fluorogenic assays demonstrated that 1.0 µmol/L CP-DEVD-cho was
sufficient to inhibit caspase 3 activation in these cells, whereas 5 µmol/L CP-YVAD-cho had no effect. YVAD-cho in control experiments did
inhibit the activity of caspase 1 in fluorogenic assays (not shown).
Similarly, a dose of 0.1 µmol/L Z-DEVD-FMK had the most suppressive
effect of all FMK inhibitors tested on IFN/fas-induced
activation of caspase 3. These results suggest the inhibition of
IFN-- and anti-fas antibody-induced apoptosis by caspase
3 inhibitors is indeed selective and specific.
Hematopoietic progenitor cells from children with FA-C
are apoptotic.56,57 Cells are hypersensitive to IFN- Immunoblots (Figure 1) and cell-free fluorescence assays performed on
lysates from the isogenic EBV-transformed cell lines (Figure 2) treated
with IFN- Caspases 6 and 7 (CPP32 family members) were also activated by
treatment with IFN- Considering the mutability of somatic cells of children with FA, we speculate that the apoptotic phenotype in hematopoietic progenitor cells and stem cells creates a perfect selective pressure for the emergence of mutant clones fully resistant to one or more mitogenic inhibitors. Such somatic mutants may have taken the first genetic steps in the conversion of a normal stem cell to one set on a course toward myelodysplasia and acute myeloblastic leukemia, clinical disorders for which children with FA are at substantial risk.59,60 We speculate that at least some of these mutations might result in the inactivation of proteins in signaling pathways that ordinarily control caspase activation.
We thank Dr Manuel Buchwald for providing cells and FANCC cDNA for our studies. The authors thank Dr Michael Heinrich and David and Lynn Frohnmayer for helpful discussions. We also thank Tara Koretsky for invaluable technical assistance and Markus Grompe for providing FA-C knockout mice for our use. Markus Grompe and Robb Moses provided valuable advice.
Submitted September 11, 1998; accepted August 28, 2000.
Supported by grants from the National Institutes of Health (HL48546), Leukemia Society of America, and the Department of Veterans Affairs Merit Review Grant.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Grover C. Bagby Jr, Oregon Cancer Center, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Mail Code CR145, Portland, Oregon 97201-3098.
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activation of caspase 3 family members
Top
Abstract
Introduction
. This hypersensitivity results, in part, from
the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells
involves sequential activation of specific caspases, we tested the
hypothesis that programmed cell death in FA HPC involves the ordered
activation of specific caspase molecules. Lysates from lymphoblasts
treated with both agonistic anti-fas antibody and IFN-
-mercaptoethanol, and subjected
to SDS-polyacrylamide gel electrophoresis (PAGE). Separated proteins
were electroblotted onto Bio-Blot nitrocellulose (Costar, Cambridge,
MA) as previously described.
