|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2541-2550
By
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.
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
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
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.
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
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 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.
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
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).
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).
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
Submitted October 1, 1997;
accepted May 21, 1998.
The authors thank Sharon Hall for her assistance with blood samples.
1.
Rosse WF,
Parker CJ:
Paroxysmal nocturnal hemoglobinuria.
Clin Haematol
14:105,
1985[Medline]
[Order article via Infotrieve]
2.
Ware RE,
Hall SE,
Rosse WF:
Paroxysmal nocturnal hemoglobinuria with onset in childhood and adolescence.
N Engl J Med
325:991,
1991[Abstract]
3.
Armstrong C,
Schubert J,
Ueda E,
Knez JJ,
Gelperin D,
Hirose S,
Silber R,
Hollan S,
Schmidt RE,
Medof ME:
Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1-murine lymphoma mutants.
J Biol Chem
267:25347,
1992
4.
Takahaski M,
Takeda J,
Hirose S,
Hyman R,
Inoue N,
Miyata T,
Ueda E,
Kitani T,
Medof ME,
Kinoshita T:
Deficient biosynthesis of N-Acetylglucosaminyl-phosphatidyl-inositol, the first intermediate of glycosyl phosphatidyl-inositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.
J Exp Med
177:517,
1993
5.
Hillmen P,
Bessler M,
Mason PJ,
Watkins WM,
Luzzatto L:
Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.
Proc Natl Acad Sci USA
90:5272,
1993
6.
Norris J,
Hall S,
Ware RE,
Kamitani T,
Chang H-M,
Yeh E,
Rosse WF:
Glycosyl phosphatidylinositol anchor synthesis in paroxysmal nocturnal hemoglobinuria: Partial or complete defect in an early step.
Blood
83:816,
1994
7.
Rosse WF:
Phosphatidylinositol-linked proteins and paroxysmal nocturnal hemoglobinuria.
Blood
75:1595,
1990
8.
Rosse WF:
The glycolipid anchor of membrane surface proteins.
Semin Hematol
30:219,
1993[Medline]
[Order article via Infotrieve]
9.
Mahoney JF,
Urakaze M,
Hall S,
DeGasperi R,
Chang H-M,
Sugiyama E,
Warren CD,
Borowitz M,
Nicholson-Weller A,
Rosse WF,
Yeh ETH:
Defective glycosylphosphatidyl-inositol anchor synthesis in paroxysmal nocturnal hemoglobinuria granulocytes.
Blood
79:1400,
1992
10.
Tseng JE,
Hall SE,
Howard TA,
Ware RE:
Phenotypic and functional analysis of lymphocytes in paroxysmal nocturnal hemoglobinuria.
Am J Hematol
50:224,
1995
11.
Ware RE,
Rosse WF,
Hall SE:
Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria.
Blood
86:1586,
1995
12.
Takeda J,
Miyata T,
Kawagoe K,
Iida Y,
Endo Y,
Fujita T,
Takahashi M,
Kitani T,
Kinoshita T:
Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria.
Cell
73:703,
1993[Medline]
[Order article via Infotrieve]
13.
Miyata T,
Yamada N,
Iida Y,
Nishimura J,
Takeda J,
Kitani T,
Kinoshita T:
Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.
N Engl J Med
330:249,
1994
14.
Bessler M,
Mason PJ,
Hillmen P,
Miyata T,
Yamada N,
Takeda J,
Luzzatto L,
Kinoshita T:
Paroxysmal nocturnal hemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.
EMBO J
13:110,
1994[Medline]
[Order article via Infotrieve]
15.
Ware RE,
Rosse WF,
Howard TA:
Mutations within the PIG-A gene in patients with paroxysmal nocturnal hemoglobinuria.
Blood
83:4218,
1994
16.
Bessler M,
Mason PJ,
Hillmen P,
Luzzatto L:
Mutations in the PIG-A gene causing partial deficiency of GPI-linked surface proteins (PNH II) in patients with paroxysmal nocturnal hemoglobinuria.
Br J Haematol
87:863,
1994[Medline]
[Order article via Infotrieve]
17.
Rosse WF,
Ware RE:
The molecular basis of paroxysmal nocturnal hemoglobinuria.
Blood
86:3277,
1995
18.
Lewis SM,
Dacie JV:
The aplastic anaemia-paroxysmal nocturnal haemoglobinuria syndrome.
Br J Haematol
13:236,
1967[Medline]
[Order article via Infotrieve]
19.
Young NS:
The problem of clonality in aplastic anemia: Dr Dameshek's riddle, restated.
Blood
79:1385,
1992
20.
de Planque MM,
Bacigalup A,
Wursch A,
Hows JM,
Devergie A,
Frickhofen N,
Brand A,
Nissen C:
Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin.
Br J Haematol
73:121,
1989[Medline]
[Order article via Infotrieve]
21.
Griscelli-Bennaceur A,
Gluckman E,
Scrobohaci ML,
Jonveaux P,
Vu T,
Bazarbachi A,
Carosella ED,
Sigaux F,
Socie G:
Aplastic anemia and paroxysmal nocturnal hemoglobinuria: Search for a pathogenetic link.
Blood
85:1354,
1995
22.
Nakakuma H,
Nagakura S,
Iwamoto N,
Kawaguchi T,
Hidaka M,
Horikawa K,
Kagimoto T,
Shido T,
Takatsuki K:
Paroxysmal nocturnal hemoglobinuria clone in bone marrow of patients with pancytopenia.
Blood
85:1371,
1995
23.
Hall SE,
Rosse WF:
The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria.
Blood
87:5332,
1996
24.
Bessler M,
Mason P,
Hillmen P,
Luzzatto L:
Somatic mutations and cellular selection in paroxysmal nocturnal hemoglobinuria.
Lancet
343:951,
1994[Medline]
[Order article via Infotrieve]
25.
Brodsky RA,
Vala MS,
Barber JP,
Medof ME,
Jones RJ:
Resistance to apoptosis caused by PIG-A gene mutations in paroxysmal nocturnal hemoglobinuria.
Proc Natl Acad Sci USA
94:8756,
1997
26.
Miyata T,
Takeda J,
Iida Y,
Yamada N,
Inoue N,
Takahashi M,
Maeda K,
Kitani T,
Kinoshita T:
Cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science
259:1318,
1993
27.
Mavilio F,
Ferrari G,
Rossini S,
Nobili N,
Bonini C,
Casorati G,
Traversari C,
Bordignon C:
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood
83:1988,
1994
28.
Sadelain M,
Wang CH,
Antoniou M,
Grosveld F,
Mulligan RC:
Generation of a high-titer retroviral capable of expressing high levels of the human beta-globin gene.
Proc Natl Acad Sci USA
92:6728,
1995
29.
Dwarki V,
Belloni P,
Nijjar T,
Smith J,
Couto L,
Mireille R,
Clift S,
Berns A,
Cohen L:
Gene therapy for hemophilia: Production of therapeutic levels of human factor VIII in vivo in mice.
Proc Natl Acad Sci USA
92:1023,
1995
30.
McCowage GB,
Phillips KL,
Gentry TL,
Hull S,
Kurtzberg J,
Gilboa E,
Smith C:
Multiparameter fluorescence activated cell sorting analysis of retroviral vector gene transfer into primitive umbilical cord blood cells.
Exp Hematol
26:288,
1998[Medline]
[Order article via Infotrieve]
31. (abstr, suppl 1)
Nishimura J,
Phillips KL,
Ware RE,
Hall S,
Howard TA,
Wilson L,
Gentry TL,
Howrey R,
Galardi C,
Kinoshita T,
Gilboa E,
Rosse WF,
Smith CA:
Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in PNH.
Blood
90:274a,
1997
32.
Kim DG,
Kang HM,
Jang SK,
Shin HS:
Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.
Mol Cell Biol
12:3636,
1992
33.
Rudoll T,
Phillips K,
Lee SW,
Hull S,
Gasper O,
Sucgang N,
Gilboa E,
Smith C:
High efficiency retroviral vector mediated gene transfer into human peripheral blood CD4+ T-lymphocytes.
Gene Ther
3:695,
1996[Medline]
[Order article via Infotrieve]
34.
Dransfield I,
Buckle A-M,
Savill JS,
McDowall A,
Haslett C,
Hogg N:
Neutrophil apoptosis is associated with a reduction in CD16 (Fc
35.
Nicoletti I,
Migliorati G,
Pagliacci MC,
Grignani,
Riccardi C:
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J Immunol Methods
139:271,
1991[Medline]
[Order article via Infotrieve]
36.
Darzynkiewicz Z,
Bruno S,
del Bino G,
Corczyca W,
Hotz MA,
Lassota P,
Traganos F:
Features of apoptotic cells measured by flow cytometry.
Cytometry
13:795,
1992[Medline]
[Order article via Infotrieve]
37.
Koopman G,
Reutlingsperger CPM,
Kuijten GAM,
Keehnen RMJ,
Pals ST,
van Oers MHJ:
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
Blood
84:1415,
1994
38.
Vermes I,
Haanen C,
Steffens-Nakken H,
Reutlingsperger CPM:
A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using labelled annexin V.
J Immunol Methods
184:39,
1995[Medline]
[Order article via Infotrieve]
39. (abstr, suppl 1)
Purow DB,
Howard TA,
Heumayer S,
Ware RE:
Genetic instability as the etiology of acquired somatic Piga mutations in paroxysmal nocturnal hemoglobinuria.
Blood
88:304a,
1996
40.
Endo M,
Ware RE,
Vreeke TM,
Singh SP,
Howard TA,
Tomita A,
Holguin MH,
Parker CJ:
Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria.
Blood
87:2546,
1996
41.
Nishimura J,
Inoue N,
Wada H,
Ueda E,
Pramoonjago P,
Hirota T,
Machii T,
Kageyama T,
Kanamaru A,
Takeda J,
Kinoshita T,
Kitani T:
A patient with paroxysmal nocturnal hemoglobinuria bearing four independent PIG-A mutant clones.
Blood
89:3470,
1997
42.
Iwamoto N,
Kawaguchi T,
Horikawa K,
Nagakura S,
Kagimoto T,
Suda T,
Takatsuki K,
Nakakuma H:
Preferential hematopoiesis by paroxysmal nocturnal hemoglobinuria clone engrafted in SCID mice.
Blood
87:4944,
1996
43.
Moore JG,
Frank MM,
Muller-Eberhard JH,
Young NS:
Decay-accelerating factor is present on paroxysmal nocturnal hemoglobinuria erythroid progenitors and lost during erythropoiesis in vitro.
J Exp Med
162:1182,
1985
44.
Nishimura T,
Kanamaru A,
Kakishita E,
Nagai K:
Flow cytometric analysis of homologous restriction factor 20KD (HRF20) expression on progeny cells during differentiation from haemopoietic progenitors in paroxysmal nocturnal haemoglobinuria.
Br J Haematol
90:293,
1995[Medline]
[Order article via Infotrieve]
45. (abstr, suppl 1)
Pendleton AL,
Ware RE,
Kurtzberg J,
Burnett A,
Rosse WF:
The hematopoietic defect in paroxysmal nocturnal hemoglobinuria assessed by long-term bone marrow culture.
Blood
86:131a,
1995
46.
Maciejewski JP,
Sloand EM,
Sato T,
Anderson S,
Young NS:
Impaired hematopoiesis in paroxysmal nocturnal hemoglobinuria/aplastic anemia is not associated with a selective proliferative defect in the glycosylphosphatidylinositol-anchored protein-deficient clone.
Blood
89:1173,
1997
47.
Kawagoe K,
Kitamura D,
Okabe M,
Taniuchi I,
Ikawa M,
Watanabe T,
Kinoshita T,
Takeda J:
Glycosylphosphatidylinositol-anchor-deficient mice: Implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria.
Blood
87:3600,
1996
48.
Rosti V,
Tremml G,
Soares V,
Pandolfi PP,
Luzzatto L,
Bessler M:
Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.
J Clin Invest
100:1028,
1997[Medline]
[Order article via Infotrieve]
49.
Horikawa K,
Nakakuma H,
Kawaguchi T,
Iwamoto N,
Nagakura S,
Kagimoto T,
Takatsuke K:
Apoptosis resistance of blood cells from patients with paroxysmal nocturnal hemoglobinuria, aplastic anemia, and myelodysplastic syndrome.
Blood
90:2716,
1997
50.
Adachi S,
Kubota M,
Lin YW,
Okuda A,
Matsubara K,
Wakazono Y,
Hirota H,
Kuwakado K,
Akiyama Y:
In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro.
Eur J Haematol
53:129,
1994[Medline]
[Order article via Infotrieve]
51.
Pericle F,
Kiu JH,
Diaz JI,
Blanchard DK,
Wei S,
Forni G,
Djeu JY:
Interleukin-2 prevention of apoptosis in human neutrophils.
Eur J Immunol
24:440,
1994[Medline]
[Order article via Infotrieve]
52.
Cox G:
Glucocorticoid treatment inhibits apoptosis in human neutrophils.
J Immunol
154:4719,
1995[Abstract]
53.
Rafiee P,
Lee JK,
Leung C-C,
Raffin TA:
TNF-
54.
Boise LH,
Minn AJ,
Noel PJ,
June CH,
Accavitti MA,
Lindsten T,
Thompson CB:
CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL.
Immunity
3:87,
1995[Medline]
[Order article via Infotrieve]
55.
Gribben JG,
Freeman GJ,
Boussiotis VA,
Rennert P,
Jellis CL,
Greenfield E,
Barber M,
Restivo VA,
Ke X,
Gray GS,
Nadler LM:
CTLA4 mediates antigen-specific apoptosis of human T cells.
Proc Natl Acad Sci USA
92:811,
1995
56.
Iwai K,
Miyawaki T,
Takizawa T,
Konno A,
Ohta K,
Yachie A,
Seki H,
Taniguchi N:
Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils.
Blood
84:1201,
1994
57.
Lagasse E,
Weissman IL:
bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages.
J Exp Med
179:1047,
1994
58.
Yousefi S,
Green DR,
Blaser K,
Simon H-U:
Protein-tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils.
Proc Natl Acad Sci USA
91:10868,
1994 |
Abstract
Introduction
-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.
Abstract
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).
). 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.
induces tyrosine phosphorylation of mitogen-activated protein kinase in adherent human neutrophils.
J Immunol
154:4785,
1995