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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2541-2550
The PIG-A Mutation and Absence of Glycosylphosphatidylinositol-Linked
Proteins Do Not Confer Resistance to Apoptosis in Paroxysmal
Nocturnal Hemoglobinuria
By
Russell E. Ware,
Jun-ichi Nishimura,
M. Anthony Moody,
Clay Smith,
Wendell F. Rosse, and
Thad A. Howard
From the Division of Hematology/Oncology, Department of Pediatrics;
the Division of Hematology; and the Division of Medical Oncology and
Transplantation, Department of Medicine, Duke University Medical
Center, Durham, NC.
 |
ABSTRACT |
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell
disorder characterized by complement-mediated hemolysis and deficient
hematopoiesis. The development of PNH involves an acquired mutation in
the X-linked PIG-A gene, which leads to incomplete bioassembly of
glycosylphosphatidylinositol (GPI) anchors and absent or reduced
surface expression of GPI-linked proteins. The origin and mechanisms by
which the PNH clone becomes dominant are not well understood, but
recently resistance to apoptosis has been postulated. To test the
hypothesis that the PIG-A mutation and absence of GPI-linked surface
proteins directly confer resistance to apoptosis, we isolated
peripheral granulocytes from 26 patients with PNH and 20 normal
controls and measured apoptosis induced by serum starvation.
Granulocytes from patients with PNH were relatively resistant to
apoptosis (38.8% ± 14.1%) as compared with granulocytes from
controls (55.0% ± 12.0%, P < .001). However, this
resistance to apoptosis was not related to the dominance of the PNH
clone because patients with a low percentage of GPI-deficient granulocytes had a similar rate of apoptosis as those with a high percentage of GPI-deficient granulocytes. Similarly, the resistance to
granulocyte apoptosis was not influenced by the degree of neutropenia or a prior history of aplastic anemia. To investigate formally the
importance of GPI-linked surface proteins in apoptosis, we introduced
the PIG-A cDNA sequence into the JY5 GPI-negative B-lymphoblastoid cell
line using two different methods: (1) stable transfection of a plasmid
containing PIG-A, and (2) stable transduction of a retroviral vector
containing PIG-A. We then measured rates of apoptosis induced either by
Fas antibody, serum starvation, or  -irradiation. With each stimulus,
apoptosis of JY5 with stable surface expression of GPI-linked proteins
was not statistically different from the parent JY5 cell line or the
JY25 (GPI-positive) cell line. Our data confirm that granulocytes from
patients with PNH have a relative resistance to apoptosis as compared
with normal granulocytes. However, this resistance does not vary with
the level of expression of GPI-linked proteins, and stable introduction of PIG-A cDNA with correction of GPI-linked surface expression does not
change the rate of apoptosis. Taken together, our data do not support
the hypothesis that the PIG-A mutation and absence of GPI-linked
surface proteins directly confer resistance to apoptosis in PNH. We
conclude that the resistance to apoptosis in PNH is not related to the
PIG-A mutation, indicating that other factors must be important in the
origin of this phenomenon and the clonal dominance observed in PNH.
| |
INTRODUCTION |
PAROXYSMAL NOCTURNAL hemoglobinuria (PNH)
is an acquired clonal hematologic disorder that is characterized by a
wide variety of clinical manifestations, including episodic hemolysis,
venous thrombosis, deficient hematopoiesis, and occasionally,
leukemia.1,2 The biochemical defect in PNH involves the
defective synthesis of glycosylphosphatidylinositol (GPI) anchors
that are used by certain surface proteins on hematopoietic
cells.3-6 As a result of this defect, affected cells are
missing all surface proteins that use GPI linkage, including proteins
involved in complement regulation, immunologic receptors, enzymes, and
several with unknown function.7,8 All blood cells are
affected, including erythrocytes, granulocytes, monocytes, platelets,
and lymphocytes.9-11 The molecular defect found in PNH is
one or more acquired (nongermline) mutations in PIG-A, an X-linked gene
involved in the first step of GPI anchor biosynthesis.12-16
Patient mutations are predominantly of the frameshift type, and PIG-A
mutations leading to partial or complete loss of PIG-A protein function
have been identifed in all patients with PNH reported to
date.17
Despite the elucidation of the biochemical and molecular defects in
PNH, several unanswered questions remain. The association between
aplastic anemia and PNH is poorly understood,18,19 especially the evolution of aplastic anemia into PNH after treatment with immunomodulatory agents such as antithymocyte globulin or cyclosporine A.20-22 Equally puzzling is the observation
that abnormal PNH cells typically achieve clonal dominance within the
bone marrow and peripheral blood. In many patients with PNH, greater
than 80% of their circulating granulocytes and erythrocytes are
GPI-deficient,11,23 suggesting that the abnormal PNH clone
has a distinct growth advantage over normal hematopoietic progenitors.
Bessler et al24 have suggested that in
patients with aplastic anemia, an acquired PIG-A mutation in a
totipotent stem cell provides a growth advantage that allows marrow
recovery from the aplasia. The nature of this growth advantage, ie, a
proliferative versus a survival advantage, has not been elucidated to
date. Brodsky et al25 recently reported that granulocytes
from four patients with PNH had a relative resistance to apoptosis.
Further, they reported that replacement of the PIG-A sequence into a
deficient B-cell line reversed this resistance to apoptosis. The
authors concluded that the PIG-A mutation and GPI-linked surface
proteins are critical for the regulation of apoptosis, and that
resistance to apoptosis in PNH leads to clonal dominance and possibly
transformation into leukemia.25
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| Fig 1.
Granulocytes from PNH patients resist apoptosis as
compared with normal granulocytes. Freshly purified granulocytes were
serum starved and then analyzed for evidence of apoptosis. (A) PNH
granulocytes at 12 hours were stained with propidium iodide and
analyzed by flow cytometry. The narrow, tall peak on each histogram
represents cells with a normal (2n) amount of DNA, whereas the broader
peak represents apoptotic cells with a sub-2n amount of DNA. The
granulocytes from a PNH patient have less apoptosis (34% sub-2n PI
staining) than those from a normal control (58%). (B) Cells at 6 hours
were stained with annexin V-FITC and PI to identify apoptotic cells
that are positive for annexin binding but exclude PI. The granulocytes
from a PNH patient have less apoptosis (26% annexin V-FITC staining)
than those from a normal control (59%). (C) Genomic DNA was analyzed
for fragmentation. At time zero no laddering was observed, but after 12 hours of serum starvation, DNA from PNH granulocytes had less laddering
than the DNA from the granulocytes of a normal control.
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To test the hypothesis that the PIG-A mutation and absence of
GPI-linked surface proteins directly confer resistance to apoptosis, we
analyzed GPI-deficient cells for the ability to resist apoptosis after
stimulation with apoptotic signals. Peripheral granulocytes from 26 patients with PNH had significantly less apoptosis after serum
starvation than granulocytes from normal controls. However, stable
introduction of PIG-A cDNA into the GPI-deficient JY5 cell line with
correction of surface expression of GPI-linked proteins did not change
the rate of apoptosis. Our data provide direct evidence that resistance
to apoptosis in PNH is not related to the underlying PIG-A mutation or
the lack of GPI-linked surface protein expression. Because the
resistance to apoptosis in PNH is not directly related to the PIG-A
mutation, other factors are necessary to explain this resistance to
apoptosis and the clonal dominance observed in patients with PNH.
| |
MATERIALS AND METHODS |
Cells.
Venous blood was collected in EDTA from patients with PNH after
informed consent using an institutional review
board-approved protocol, and granulocytes were purified
as previously described.6 JY5, a GPI-negative Epstein-Barr
virus-transformed B lymphoblastoid cell line, and its normal
GPI-positive counterpart JY25 (gifts from Dr Timothy Springer,
Harvard) were maintained in RPMI 1640 with 10% fetal calf
serum (FCS; GIBCO, Gaithersburg, MD). The T-cell line Jurkat, also
grown in RPMI with 10% FCS, was used as a positive control for
apoptosis.
Antibodies and reagents.
Monoclonal antibodies (MoAbs) included anti-Fas.3 (IgG1) for the
detection of CD95 (Fas) antigen (Calbiochem, Cambridge, MA), anti-Fas
CH-11 (IgM) for the induction of apoptosis (Upstate Biotech Inc, Lake
Placid, NY), P3 as a negative isotype control (gift from Dr Barton
Haynes, Duke), CD55 ascites and CD59 ascites for detection
of GPI-deficient cells,23 and goat-anti-mouse-fluorescein isothiocyanate (FITC) (Kierkegaard, Gaithersburg, MD) for
secondary amplification in immunophenotype assays. MoAbs CD59-FITC
(Pharmingen, San Diego, CA) and NGFR-PE (Chromoprobe, Mountain View,
CA) were used to document transduction of cells with retroviral
vectors. Hygromycin-B (GIBCO) was used to select transfected cells.
Propidium iodide (PI; Boehringer Mannheim, Indianapolis, IN) was used
to assess cell viability and identify apoptotic cells. Annexin V-FITC (Caltag, Burlingame, CA) also was used to identify apoptotic cells.
Plasmids.
The pEB plasmid (mock control) and the plasmid containing the PIG-A
cDNA sequence (pEBPIG-A) were generously provided by Dr Taroh Kinoshita
(Osaka University, Osaka, Japan).12,26 These plasmids were
used to stably transfect the GPI-deficient JY5 cell line by
electroporation and allow hygromycin-B selection (400 µg/mL) as
previously described.12,26
Retroviral vectors.
The retroviral vectors used for stable transduction of the JY5 cell
line included a negative (mock) vector called MN27-30 and a
vector containing the PIG-A cDNA sequence termed MPIN.31 The MPIN vector was constructed using the MN vector and adding the
PIG-A cDNA sequence and an internal ribosome entry site
sequence.32 The MN and MPIN amphotropic vector packaging
lines were generated by transfection of the ecotropic retroviral vector
producer line E86, followed by infection of the amphotropic producer
AM12.30,31,33
For retroviral vector gene transfer, JY5 cells (2 × 106) were centrifuged with 1 mL of freshly thawed MN or
MPIN supernatant and 8 µg/mL of polybrene (Sigma, St Louis,
MO) at 2,500 rpm for 90 minutes at room temperature
(RT), and then incubated overnight at
37°C. The transduction procedure was repeated two
additional times, and cells were resuspended in fresh
media.31 After transduction with MN supernatant,
NGFR-expressing JY5 cells (termed JY5/MN) were isolated by
one round of fluorescence-activated cell sorting (FACS), and stable
expression (>95%) of surface NGFR was confirmed. After transduction
with MPIN supernatant, NGFR- and CD59-expressing JY5 cells (termed
JY5/MPIN) were isolated by two rounds of FACS sorting, and stable
expression (>98%) of both surface markers was
confirmed.31
Induction of apoptosis.
All apoptosis experiments using freshly isolated peripheral
granulocytes were performed with triplicate data points, using serum
starvation for exactly 12 hours as the apoptotic
stimulus.34 After the granulocytes were purified, aliquots
of 1.0 × 106 cells in 1 mL of RPMI were placed in 12 × 75-mm polypropylene tubes (Becton Dickinson Labware, Lincoln
Park, NJ) and incubated in 5% CO2 at 37°C. All
apoptosis experiments using cell lines also were performed using
triplicate data points, and six to eight independent experiments were
performed for each apoptotic stimulus. Cell lines were grown to log
phase in RPMI 10% FCS at 37°C with 5% CO2 before
induction of apoptosis. Aliquots of 1.0 × 106 cells
in 0.5 mL of media were placed into 12 × 75-mm polystyrene tubes
(Becton Dickinson) with various apoptotic signals and incubated for 24 hours (Fas MoAb CH-11 at 500 ng/mL) or 96 hours (serum starvation or
4,000 cGy -irradiation from a 62Cs source) in 5%
CO2 at 37°C.
Measurement of apoptosis.
Cells were obtained at the specified time points and analyzed for
apoptosis using flow cytometry. Cell membranes were permeabilized with
50% ethanol and incubated with PI to stain DNA35 and then
analyzed on a FACSCAN using LYSIS II software (Becton Dickinson, San
Jose, CA). Cells with a hypodiploid amount of DNA (<2n,
pre-G0G1) were considered to be apoptotic due
to loss of fragmented DNA.36 Alternatively, cells were
stained with annexin V-FITC and PI and analyzed by flow
cytometry.37,38 Genomic DNA was extracted from 2.0 × 106 cells using a commercial kit (Gentra Systems,
Minneapolis, MN) and resolved using a 2.5% agarose gel with ethidium
bromide staining to identify the characteristic "laddering" of
DNA fragments indicative of apoptosis.
Statistics.
The statistical analysis was performed using the Primer of
Biostatistics (McGraw-Hill, New York, NY). Statistical difference between two groups, eg, PNH patients versus controls, was performed using the Student's t-test. Differences among more than two
groups, eg, degree of clonal dominance, was performed using analysis of variance.
| |
RESULTS |
Patient characteristics.
A total of 26 patients with PNH were studied, including 17 women and 9 men, with a median age of 33 years (range, 17 to 66). The percentage of
GPI-deficient circulating granulocytes, as measured by surface CD55
expression, ranged from 3.1% to 98.9%, with a mean of 72.8% and a
median of 91.1%. Eight patients had an absolute neutrophil count (ANC)
of  2,000/µL, 10 had an ANC between 2,000 and 4,000/µL, and 8 had
an ANC of  4,000/µL. Nine of the patients had a documented prior
history of aplastic anemia. A total of 20 normal adult controls were
also studied, with a median age of 32 years (range, 17 to
55).
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| Fig 2.
Granulocytes from PNH patients are relatively resistant
to apoptosis as compared with granulocytes from normal controls. Cells
were tested in triplicate and the mean is plotted for each patient
( ). The mean for each group is shown as a horizontal line. The
amount of apoptosis as measured by flow cytometry was significantly
less for PNH granulocytes (38.8% ± 14.1%, n = 26) as compared
with normal granulocytes (55.0% ± 12.0%, n = 20), P < .001. There was some overlap for individuals within each group.
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| Fig 3.
Stable transfection with the PIG-A cDNA sequence. The
GPI-negative JY5 cell line was transfected by electroporation with
either the PEB (mock control) plasmid or the PEB/PIGA plasmid. After
hygromycin selection, cells were analyzed for the surface expression of
CD59, which depends on a functioning PIG-A gene for complete assembly
of GPI anchors. The left panel shows results after transfection with
PEB, with negative staining observed with both the control antibody
(white) and CD59 antibody (grey). The right panel shows results after
transfection with PEB/PIGA, with high levels of surface CD59 observed
as compared with the control antibody.
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| Fig 4.
Stable transduction with the PIG-A cDNA sequence. The
GPI-negative JY5 cell line was transduced with supernatants from either
the MN (mock control) vector or the MPIN vector. After sorting, cells
were analyzed for the surface expression of NGFR, which indicates
successful transduction of the retroviral sequence, and CD59, which
indicates a functioning PIG-A sequence. Upper left panel: JY5 with MN
vector, control antibody. Upper right panel: JY5 with MN vector,
NGFR-PE antibody, showing 98% surface expression of NGFR. Lower left
panel: JY5 with MPIN vector, control antibodies. Lower right panel:
JY% with MPIN vector, CD59-FITC and NGFR-PE antibodies, showing 98%
surface expression of both NGFR and CD59, indicating successful
transduction of the vector and a functioning PIG-A sequence.
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| Fig 5.
Apoptosis of cell lines. Cells were grown to log phase
and subjected to apoptotic stimuli, and then genomic DNA was extracted
and analyzed for DNA fragmentation. (A) Fas antibody, showing DNA
laddering in all cell lines. (B) -irradiation, showing DNA laddering
in all cell lines. MW, molecular weight.
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| Fig 6.
Stable expression of GPI-linked surface proteins does not
affect the rate of apoptosis. The PIG-A cDNA sequence was stably
introduced as described in Materials and Methods, and apoptosis was
measured by annexin V-FITC binding. There was no difference in the rate
of apoptosis 24 hours after -irradiation between (A) cells stably
transfected with PEB versus PEB/PIGA, or (B) cells stably transduced
with MN versus MPIN. Background levels of annexin V-FITC binding were
higher for JY5 than for granulocytes.
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| Fig 7.
Apoptosis of cell lines does not depend on a functioning
PIG-A gene sequence or levels of GPI-linked surface expression. Cells
were subjected to stimuli and analyzed for apoptosis by PI staining and
flow cytometry. All data points were performed in triplicate for each
experiment, and the displayed results represent six to eight
independent experiments (mean ± 1 SD) for each stimulus. Upper left
panel: Control antibody, with minimal apoptosis detected. Lower left
panel: Fas antibody, with apoptosis ranging from 37.5% ± 10.1% to
46.8% ± 10.3%. Upper right panel: Serum starvation, with apoptosis
ranging from 8.5% ± 2.6% to 22.2% ± 8.0%. Lower right panel:
-irradiation, with apoptosis ranging from 27.9% ± 7.4% to 48.8% ± 18.8%. There were no statistically significant differences among
the various cell lines, according to the presence of a functioning
PIG-A gene or surface expression of GPI-linked proteins.
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Apoptosis of circulating granulocytes.
Freshly purified granulocytes had less that 2% apoptosis at time zero
as detected by propidium iodide staining (data not shown). After serum
starvation, apoptosis was induced in both PNH and normal granulocytes
(Fig 1). Using two different methods for measuring apoptosis (Fig 1A and B), granulocytes from patients with PNH had less
apoptosis than granulocytes from normal controls. This difference in
apoptosis was confirmed by analysis of DNA fragmentation (Fig 1C). When
analyzed as a group ( Fig 2), the rate of apoptosis after serum starvation (mean ± 1 standard deviation) was
significantly less for PNH granulocytes (38.8% ± 14.1%) as
compared with normal granulocytes (55.0% ± 12.0%), P < .001. This resistance to apoptosis was not due to differences in the
surface expression of the CD95 (Fas) antigen on the granulocytes at
time zero or after serum starvation (data not shown).
We then analyzed our data according to several clinical and laboratory
parameters ( Table 1). Patients with a prior history of
aplastic anemia had a similar rate of apoptosis (37.6% ± 17.5%) as those with no history of aplasia (39.2% ± 12.5%),
P = not significant (NS). Similarly, the resistance
to apoptosis did not depend on the degree of neutropenia, because
patients with an ANC 2,000/µL had a similar rate (40.7% ± 17.9%) as those with an ANC 4,000/µL (37.7% ± 15.3%),
P = NS. Finally, the dominance of the PNH clone did not
influence the resistance to apoptosis, because patients with 90%
CD55-negative granulocytes had a similar rate (37.2% ± 9.3%) as
those with 40% CD55-negative granulocytes (43.8% ± 18.6%),
P = NS.
Apoptosis of cell lines.
To determine the importance of normal PIG-A gene function and surface
expression of GPI-linked proteins on the rate of apoptosis, we
introduced the PIG-A cDNA sequence into the GPI-deficient JY5 cell line
using two different experimental strategies. As shown in
Fig 3, stable transfection of JY5 with the PEB/PIGA
plasmid documented greater than 98% surface expression of CD59,
whereas cells transfected with the PEB (mock control) plasmid had no
expression of CD59. Similarly, stable transduction of JY5 with the MPIN
retroviral vector documented greater than 98% surface expression of
both NGFR and CD59, whereas cells transduced with the MN (mock control) vector had only expression of NGFR (Fig 4).
Each of the cell lines, including the GPI-positive JY25 cell line, was
then subjected to a variety of stimuli and analyzed for evidence of
apoptosis. DNA laddering was observed in all cell lines after
incubation with Fas antibody (Fig 5A) and surface expression of the Fas antigen was similar for all cell lines (data not
shown). Similarly, DNA laddering was observed in all cell lines after
exposure to -irradiation (Fig 5B) or serum starvation (data not
shown). When analyzed by annexin V-FITC binding, there were no
differences in the rate of apoptosis between cells transfected with PEB
vs PEB/PIGA (Fig 6A), or cells transduced with MN versus MPIN (Fig 6B). These results were confirmed by PI staining, with no
statistically significant differences identified among the cell lines
with any of these apoptotic stimuli ( Fig 7).
| |
DISCUSSION |
An abundance of experimental data recently has emerged regarding the
cellular, biochemical, and molecular defects in PNH. It is now
generally accepted that in the majority of cases of PNH, an acquired
PIG-A gene mutation occurs within a self-renewing totipotent stem cell
and all of its progeny harbor this same PIG-A mutation. The absence of
a functioning PIG-A gene leads to incomplete bioassembly of
glycosylphosphatidylinositol anchors and reduced or absent surface
expression of GPI-linked surface proteins. The lack of these surface
proteins presumably leads to all of the clinical manifestations of
PNH.17
In contrast to the abundance of information regarding the defects in
PNH, the events (or series of events) that lead to the pathogenesis of
PNH are poorly understood. The etiology of the acquired PIG-A gene
mutation is unknown, although in a minority of cases an overall genetic
instability has been postulated.39 The clinical observation
that PNH can evolve from aplastic anemia,18 particularly
after treatment with immunosuppressive therapy,20-22 has
suggested that the absence of GPI-linked surface proteins may be
advantageous for marrow recovery,24 possibly by evasion of
endogenous immune suppression.19 This hypothesis is
supported by the reports of patients with more than one independent
PIG-A mutation,40,41 which suggests that at least some
patients have a strong in vivo selection pressure to acquire PIG-A
mutations.
The presence of a PIG-A mutation does not seem sufficient to explain
all of the findings that occur in PNH. For example, in most patients
with PNH the abnormal cells become clonally dominant, with greater than
80% of circulating erythrocytes and granulocytes commonly
affected.11,23 This observation supports the hypothesis that the PIG-A mutant stem cell has an intrinsic proliferative advantage and can generate hematopoietic cells faster than normal stem
cells within the marrow.42 However, several pieces of
experimental data do not support this hypothesis. Our in vitro studies
with PNH lymphocytes showed no difference in the proliferative capacity between purified GPI-deficient and normal peripheral T lymphocytes in
response to mitogenic or antigenic stimulation.10 Studies using marrow progenitor cells from PNH patients have shown decreased in
vitro colony growth as compared with that of normal
volunteers.43-46 Finally, chimeric mice generated using
PIG-A-disrupted embryonic stem cells did not develop an increase in
the number of GPI-deficient blood cells,47,48 suggesting
that the PIG-A mutation is not sufficient for clonal proliferation and
dominance.
Brodsky et al25 recently reported that PNH is characterized
by a resistance to apoptosis, and that the expression of GPI-linked proteins influences the rate of apoptosis. They measured apoptosis induced by serum starvation in granulocytes from 4 patients with PNH
and found significantly less apoptosis than granulocytes from 5 controls. Although our experimental design was slightly different, our
data for 26 patients and 20 controls are in agreement with these
findings (Figs 1 and 2). Our average rate of apoptosis for PNH
granulocytes as a group was significantly less than that for normal
granulocytes, although there was some overlap for individuals in both
groups (Fig 2). Importantly, we found no difference in the rate of
apoptosis for granulocytes depending on the dominance of the PNH clone
(Table 1). If the surface expression of GPI-linked surface proteins
were important for resistance to apoptosis, then patients with a
dominant PNH clone would be expected to have less apoptosis than those
with a smaller clone. However, our data showed that patients with a
dominant clone (90% GPI-negative granulocytes) had a similar rate of
apoptosis as those with a smaller clone (40% GPI-negative
granulocytes). These data are supported by the recent report of
resistance to apoptosis in a variety of bone marrow failure syndromes
including PNH, myelodysplasia, and aplastic anemia.49
To investigate formally the contribution of a functioning PIG-A gene
and surface expression of GPI-linked surface proteins to the rate of
apoptosis, we stably introduced the PIG-A cDNA sequence into the
GPI-negative JY5 cell line by two different independent vectors using
appropriate mock controls. We first electroporated the PEB/PIGA plasmid
into JY5, selected with hygromycin, and established long-term
transfectants that had high levels of surface CD59 expression (Fig 3).
We also transduced the MPIN retroviral vector into JY5 with resulting
high levels of surface CD59 expression (Fig 4). When apoptosis was
induced either by Fas antibody, serum starvation, or -irradiation,
there were no differences in the rates of apoptosis according to the
presence of a functioning PIG-A gene sequence. The expression of
GPI-linked surface proteins did not influence apoptosis as measured by
DNA laddering (Fig 5), annexin V binding (Fig 6), or PI staining (Fig
7). These results do not support the hypothesis that the PIG-A mutation
and absence of GPI-linked surface proteins directly confer resistance
to apoptosis in PNH.
In summary, our data confirm that granulocytes from PNH patients have
reduced rates of apoptosis as compared with normal granulocytes. Our
observations that the dominance of the PNH clone does not affect
granulocyte apoptosis, coupled with the failure of a functioning PIG-A
gene sequence to affect apoptosis of the JY5 cell line, provide
evidence that this resistance to apoptosis is not due to the PIG-A
mutation and the absence of GPI-linked protein expression. Although it
is possible that the PIG-A mutation directly influences the apoptosis
of hematopoietic progenitor cells within the bone marrow
microenvironment, we hypothesize that other events influence the rate
of granulocyte apoptosis in PNH, such as circulating levels of
cytokines and other soluble factors,50-53 abnormal
intercellular interactions within the bone marrow
microenvironment,54,55 expression of bcl-2 family
members,56,57 or other changes in gene and protein
expression.58-60 Further studies on granulocyte apoptosis
in PNH and other bone marrow disorders including aplastic anemia,
inherited bone marrow failure syndromes, and
myelodysplasia49,61,62 will help to explain the cause and
significance of the resistance to apoptosis observed in PNH.
| |
FOOTNOTES |
Submitted October 1, 1997;
accepted May 21, 1998.
Supported by Grant No. HL-205691 from the National Heart, Lung, and
Blood Institute, National Institutes of Health (R.E.W.).
Address correspondence to Russell E. Ware, MD, PhD, PO Box 2916, DUMC,
Durham, NC 27710; e-mail: ware0005{at}mc.duke.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Sharon Hall for her assistance with blood samples.
| |
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Abstract
Introduction
Abstract