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(Journal of Leukocyte Biology. 2002;72:1228-1233.)
© 2002 by Society for Leukocyte Biology

The effect of GPI-anchor deficiency on apoptosis in mice carrying a Piga gene mutation in hematopoietic cells

Shashikant Kulkarni and Monica Bessler

Department of Internal Medicine, Division of Hematology, Washington University School of Medicine, St. Louis, Missouri

Correspondence: Monica Bessler, M.D., Ph.D., Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, Campus Box 8125, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail: mbessler{at}im.wustl.edu


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ABSTRACT
 
Glycosyl phosphatidylinositol (GPI) anchors are used by a variety of proteins to link to the cell surface. GPI-anchored proteins are deficient on a proportion of blood cells from patients with paroxysmal nocturnal hemoglobinuria. This is caused by the expansion of a cell clone that has acquired a mutation in a gene, PIGA, which is essential in the synthesis of GPI anchors. The nature of the growth/survival advantage permitting the expansion of PIGA- cells is unknown. A decreased susceptibility to apoptosis has been found in blood cells from patients, but the contribution of the PIGA gene mutation to this finding remained controversial. Therefore, we investigated apoptosis in mice that harbor a targeted Piga gene mutation in hematopoietic cells. When exposed to a variety of apoptotic stimuli, apoptosis in PIGA- thymocytes, granulocytes, and hematopoietic progenitor cells was similar to apoptosis induced in PIGA+ cells from the same mouse or from wild-type controls. Similarly, whole-body {gamma}-irradiation did not produce an in vivo survival advantage of PIGA- hematopoietic stem cells. Our findings imply that a Piga gene mutation does not alter susceptibility to cell death, indicating that other factors in addition to the PIGA gene mutation are necessary to promote the clonal outgrowth of PIGA- cells.

Key Words: glycosyl phosphatidylinositol • PIGA • paroxysmal nocturnal hemoglobinuria • hematopoiesis


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INTRODUCTION
 
Paroxysmal nocturnal hemoglobinuria (PNH) is caused by the clonal expansion of a hematopoietic progenitor cell that has acquired a mutation in the X-linked PIGA gene [1 , 2 ]. PIGA encodes a subunit of the N-acetyl glucosaminyltransferase, an enzyme essential in the synthesis of glycosyl phosphatidylinositol (GPI)-anchor molecules [3 ]. Therefore, clonally expanded blood cells lack all surface proteins that are attached to the cell membrane by a GPI anchor (PIGA- cells) [4 ]. Although some of the clinical symptoms in patients with PNH are explained by the lack of GPI-linked proteins on blood cells, the cause for the clonal expansion of PIGA- cells is not understood. PNH has a close association with aplastic anemia (AA); all patients with PNH have signs of bone marrow failure, and 30–40% of patients with acquired AA have circulating PIGA- blood cells [5 , 6 ]. It is therefore widely believed that the lack of GPI-linked proteins confers a growth or survival advantage to PIGA- hematopoietic progenitor cells in a bone marrow environment that prevails in AA and inhibits or damages normal hematopoietic stem cells [7 , 8 ]. To explain this phenomenon, a number of recent studies have examined blood cells, bone marrow cells, and cell lines isolated from patients with PNH for their susceptibility to apoptosis [9 10 11 12 ]. The comparison of these studies is difficult, as different cell types, different apoptotic stimuli, and different experimental conditions were used. In addition, limited patient material often restricted the number of stimuli tested, and often only one stimulus and one concentration of a particular stimulus were applied. It is therefore not surprising that the results were controversial and that the role of a PIGA gene mutation in programmed cell death remained unclear. To address this controversy and to investigate the effect of a PIGA gene mutation on cell survival, we studied the susceptibility to programmed cell death in a murine model in which a proportion of hematopoietic cells has a null mutation in the murine homologue Piga [13 ]. Mice with PIGA- blood cells share some of the biological features characteristic for patients with PNH. Similar to PNH patients, PIGA- red cells of these mice are also hypersensitive to complement and have a reduced half-life in circulation [13 ]. However, in contrast to patients, these genetically engineered mice only have the pathology caused by the Piga gene mutation but have no additional bone marrow failure. The use of these mice allowed us to more extensively investigate differences in apoptotic cell death between PIGA+ and PIGA- cells in various blood cell lineages, using several specific, apoptotic stimuli at different concentrations investigating diverse pathways of apoptosis. Our studies show that PIGA- thymocytes, granulocytes, and bone marrow progenitor cells have a similar sensitivity to apoptotic cell death induced by {gamma}-irradiation, etoposide, dexamethasone, anti-Fas antibody, and serum starvation compared with PIGA+ cells isolated from the same mouse. The rate of apoptosis in PIGA- blood cells also was not significantly different from the rate of apoptosis measured in blood cells from wild-type (wt) animals. Furthermore, exposure of mice to graded doses of total body irradiation did not cause an increase in the proportion of circulating PIGA- cells in peripheral blood or in the bone marrow, clearly indicating that a PIGA gene mutation per se does not protect the PNH cell from apoptotic cell death.


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MATERIALS AND METHODS
 
Mice
Two different mouse strains (LF and EL) with PIGA- blood cells were used in this study. In LF mice, Cre expression is controlled by the transcription regulatory sequences of the human c-fes gene, which inactivates the floxed Piga gene in hematopoietic stem cells [14 ]. In EL mice, Cre is expressed under the adenoviral EIIa promoter, causing recombination of the floxed Piga gene in early embryogenesis [15 ]. EL mice are mosaic for PIGA- cells in all blood lineages [13 ]. Mice carrying only the floxed Piga gene or only the Cre transgene were used as wt controls. All experiments were performed with mice in a C57BL/6 background, N >= 10. In this report, "PIGA" refers to the human gene, "Piga" to the murine gene, and "PIGA" to the gene product of either species [16 ].

Induction of apoptosis in thymocytes
Single-cell suspensions of thymocytes were prepared from wt and LF mice and were washed twice in RPMI containing 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). Thymocytes were suspended at 1 x 106/ml in the above medium and plated in a final volume of 1 ml in 24-well flat-bottom tissue-culture plates. The following cell death stimuli were administered: {gamma}-irradiation (100, 250, and 500 cGy, Gamacell, Nordion International, Inc., Ontario, Canada), dexamethasone (10-8, 10-7, and 10-6 M, Sigma Chemical Co., St. Louis, MO), etoposide (25, 50, and 100 µM, Sigma Chemical Co.), and anti-mouse Fas antibody (100, 500, and 1000 ng/ml, Jo2, BD PharMingen, Los Angeles, CA). Treated and untreated thymocytes were cultured at 37°C in 5% CO2. At the indicated time points, the cultured cells were harvested and stained with Annexin V (NeXins Research B.V., The Netherlands) and appropriate monoclonal antibodies (mAb) and were analyzed by fluorescence-activated cell scanner (FACS). Cells were also stained for 7-amino-actinomycin D (7 AAD; Calbiochem, San Diego, CA) and Annexin V. The majority of cells staining for 7 AAD were also positive for Annexin V. In all experiments, less than 2% was positive for 7 AAD only (data not shown). In our experiments, Annexin V-positive cells therefore represent apoptotic cells and the majority of cells that proceeded from apoptosis to necrosis.

Induction of apoptosis in granulocytes
Granulocytes were isolated from the bone marrow of LF mice and wt controls using a discontinuous Percoll gradient as described previously [17 ]. The isolated granulocytes from wt and mutant mice were at least 80% pure, as determined by leukocyte differentials. Cells were counted and suspended in RPMI with or without 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). These were then cultured at 37°C in 5% CO2 and analyzed for cell viability using FACS at 6, 24, and 48 h after culture.

Bone marrow clonogenic assays
Bone marrow was harvested by flushing femurs and tibias with {alpha}-minimum essential medium containing 10% FCS. After counting the bone marrow cells, they were subjected to appropriate apoptotic stimuli such as {gamma}-irradiation (Gamacell) at 100 and 250 cGy and etoposide (1, 10, and 1000 nM) and were plated in methylcellulose supplemented with 50 ng/ml recombinant murine stem-cell factor, 10 ng/ml recombinant murine interleukin-3 (IL-3), 10 ng/ml recombinant human IL-6, 3 U/ml recombinant human erythropoietin, and 10 µg/ml recombinant human insulin (MethocultTM M3434; Stem Cell Technologies, Vancouver, B.C., Canada). These plates were incubated in a humidified chamber with 5% CO2 at 37°C. Colonies were scored on day 7. Cells were harvested from the entire culture plate, washed, and suspended in phosphate-buffered saline containing 0.1% azide, 0.3% bovine serum albumin (BSA), and 2 mM EDTA. The cells were counted and analyzed by FACS.

Flow cytometry
Thymocytes and granulocytes were washed twice in Annexin V-binding buffer (20 mM Hepes, 132 mM NaCl, 1.2 mM potassium phosphate, 5 mM glucose, and 0.5% BSA, pH 7.4) and were incubated in fluorescein isothiocyanate-conjugated Annexin V with appropriate mAb for 30 min at 4°C. Lineage specificity was analyzed by the use of antibodies against: CD11b (M1/70), B220 (RA3–6B2), CD4 (GK1.5), CD8 (53–6.7), and CD71 (C2). mAb against Gr1 (RB6–8C5), CD24 (M1/69), and CD48 (HM48–1) were used to determine the expression of GPI-anchored proteins. Phycoerythrin-conjugated anti-mouse Fas mAb (Jo2) was used to determine the Fas expression on thymocytes. FACS analyses were performed using standard techniques and equipment (FACScanTM flow cytometer, Becton Dickinson, San Jose, CA).

Whole-body irradiation
Sex- and age-matched EL and wt-control mice were irradiated with 450 cGy (n=2), 600 cGy (n=9), 800 cGy (n=3), and 900 cGy (n=9). Eight EL mice were used as nonirradiated controls. Peripheral blood cell parameters including differential white blood cell counts, platelet counts, red cell counts, and hemoglobin were followed over a period of 6 months. The percentage of PIGA- red blood cells, B cells, T cells, and granulocytes was determined at 16 days, 1, 2, 4, and 6 months post-irradiation.

Statistical analyses
All the statistical analyses of the proportion of viable, hematopoietic cells at each period of culture and each dose were tested by Student’s t-test. Values of P <= 0.05 were considered statistically significant. Results are expressed as the mean ± SD.


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RESULTS
 
To investigate whether the resistance to apoptosis is a result of the loss of PIGA function, we studied apoptotic cell death in mice carrying a Piga gene mutation in hematopoietic cells [14 ]. The difference in sensitivity to cell death in response to a number of apoptotic stimuli was measured in PIGA- and PIGA+ cells obtained from the same mouse and compared with the rate of apoptosis in PIGA+ cells from a wt mouse. Figure 1 shows a representative example of the flow cytometric analysis of cell death in PIGA+ thymocytes derived from a wt mouse and in PIGA+ and PIGA- thymocytes from a LF mouse in response to {gamma}-irradiation. On viable cells (Annexin V-), the expression of the GPI-linked antigen CD48 clearly distinguishes PIGA+ from PIGA- cells. In contrast, dead cells (Annexin V+) partially lose the expression of CD48. Thus, in the following experiments, changes in the proportion of viable cells were used to determine the rate of apoptotic cell death in PIGA+ and PIGA- cells.



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  		Figure 1. Flow cytometric analysis of apoptotic cell death in thymocytes. Thymocytes were analyzed by FACS for Annexin V staining and expression of CD48. One representative example of thymocytes from a LF and wt (WT) mouse before (NT) and 6 h after {gamma}-irradiation with 250 cGy is shown. Note that apoptotic cells (Annexin V+) partially lose the expression of GPI-liked proteins; thus, in dead cells, the lack of CD48 does not accurately represent the proportion of PIGA- cells. The numbers indicate the percentage of cells in the individual quadrant (viable cells PIGA-, left lower quadrants; PIGA+ cells, right lower quadrants).

Apoptotic response of PIGA- and PIGA+ thymocytes
The development of the thymus in LF mice appeared largely normal with a comparable ratio of CD4+, CD8+, and CD4+CD8+ thymocytes when compared with wt mice. The proportion of PIGA- and PIGA+ was not significantly different among CD4+CD8+, CD4+, and CD8+ cells. Similarly, there was no difference in the expression of Fas between PIGA+ and PIGA- thymocytes from the LF mice and PIGA+ thymocytes from wt control (data not shown). To assess the sensitivity to apoptotic cell death, thymocytes from LF mice and from wt control mice were challenged with graded doses of {gamma}-irradiation (100, 250, and 500 cGy), different concentrations of dexamethasone (10-8, 10-7, and 10-6 M), etoposide (25, 50, and 100 µM), and anti-Fas antibody (Jo2) at 100, 500, and 1000 ng/ml. The viability of treated and untreated cells was measured by FACS analysis at 6 and 24 h after treatment with dexamethasone and etoposide and after 6, 24, and 48 h after {gamma}-irradiation and anti-Fas antibody treatment. At all time points and at all concentrations of the applied stimuli, PIGA- and PIGA+ thymocytes from LF mice and PIGA+ thymocytes from wt mice showed an almost identical cell viability, indicating that PIGA+ and PIGA- thymocytes are equally susceptible to apoptosis induced by these stimuli. Figure 2 shows representative examples of the viability of thymocytes after 250 cGy {gamma}-irradiation, 500 ng/ml anti-Fas antibody, 10-7 M dexamethasone, and 50 µM etoposide.



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  		Figure 2. Sensitivity of PIGA- and PIGA+ to apoptosis in thymocytes is almost identical. Thymocytes from LF mice (n=3) and wt mice (n=2) were analyzed for viable cells by FACS after treatment with apoptotic stimuli. Each data point represents the average percentage of viable cells normalized to the average of viable cells at the start of each experiment. Error bars represent standard deviation. Black triangle, solid line, PIGA-; black triangle, dashed line, PIGA+ from LF mice; gray triangle, solid line, PIGA+ thymocytes from wt mice.

Sensitivity of PIGA- and PIGA+ bone marrow progenitor cells to specific apoptotic stimuli
Next, we measured the sensitivity to cell death in bone marrow cells. Bone marrow cellularity and the proportion of myeloid and erythroid cells in the bone marrow from LF mice were similar to those of wt mice [14 ]. The mean proportion of PIGA- bone marrow cells in LF mice varied between 50% and 60%. To compare the susceptibility to apoptosis of PIGA- and PIGA+ bone marrow cells, we assessed the colony-forming ability in clonogenic progenitor assays after {gamma}-irradiation and after coculture with various concentrations of etoposide. After 7 days of methylcellulose culture, there was no significant difference (P>0.05) between total number of colony-forming units (CFUs) obtained from bone marrow cells from LF mice and the number of CFUs obtained from wt-control mice after apoptotic stimuli at any dose or concentration (see Fig. 3A ). To determine a possible survival advantage of PIGA- progenitor cells, colonies from culture plates were pooled and analyzed for the lack of GPI-linked proteins. The expression of CD24 (GPI-linked) was determined on erythroid cells, identified by their high level of CD71 expression (CD71high), and on CD11b+ myeloid cells. With the exception of etoposide-treated marrow cells from one LF mouse, whose proportion of PIGA- erythroid cells doubled, no significant increase (P>0.05) in the proportion of PIGA- erythroid cells or PIGA- myeloid cells was found when compared with untreated samples (Fig. 3B and 3C ). The increased proportion of PIGA- CD71high cells in one etoposide-treated bone marrow culture lies far outside the normal range of the remainder of the data. As in the four other bone marrow samples, no statistical significant increase (P>0.05) in PIGA- cells was documented, we conclude that a Piga gene mutation alone does not confer resistance to etoposide-induced cell death. Thus, our analysis of apoptosis in bone marrow progenitor cells reveals a similar sensitivity to apoptotic cell death induced by {gamma}-irradiation and etoposide in PIGA+ and PIGA- bone marrow cells.



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  		Figure 3. Effect of {gamma}-irradiation and etoposide on PIGA+ and PIGA- bone marrow progenitor. (A) Number of colonies (CFU’s) obtained after graded doses of {gamma}-irradiation and different concentration of etoposide. Each data point indicates the number of CFUs obtained in a treated sample normalized to number of CFUs in the untreated sample from the same animal. Note that values from the four LF mice superimpose (black lines). (A) Black solid triangle and black solid line represent total CFUs from LF mice (n=4 for {gamma}-irradiation, and n=5 for etoposide), and wt mice are shown in gray triangles and gray solid lines (n=2). (B and C) Relative proportion of Piga- CD11b+ myeloid cells and Piga- erythroid cells (CD 71high), respectively, as determined by flow cytometry in {gamma}-irradiation-treated bone marrow cultures from LF mice (n=4 and n=5) after etoposide treatment compared with untreated samples. Each line represents values from one LF mouse. For two culture experiments, the results were very similar. To discriminate between the two experiments, one is shown stippled.

Apoptosis in PIGA+ and PIGA- granulocytes
It has previously been shown that granulocytes from PNH patients are more resistant to serum-starvation-induced apoptosis than granulocytes from normal control individuals [9 10 11 ]. To examine the role of a PIGA gene mutation in this finding, we studied the rate of cell death in PIGA+ and PIGA- granulocytes from LF mice challenged by serum starvation. The rate of serum-starvation-induced cell death was also determined in granulocytes from wt control mice. Mice have rather low numbers of circulating granulocytes. Thus, granulocytes isolated from the bone marrow by centrifugation over a discontinuous Percoll gradient were used (see Materials and Methods). Cell survival in PIGA- and PIGA+ granulocytes was assessed by double-staining with Annexin V and a mAb for CD24 (GPI-linked). The decrease in cell viability as a result of serum starvation was not significantly different between PIGA- and PIGA+ granulocytes isolated from LF mice and did not differ from the viability of serum-starved granulocytes obtained from wt mice (P>0.05; Fig. 4 ).



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  		Figure 4. Sensitivity to serum starvation-induced apoptosis in PIGA- and PIGA+ granulocytes. Bone marrow-derived granulocytes from LF mice (n=4) and wt mice (n=4) were cultured in RPMI with or without 10% FCS. The expression of GPI-linked antigen CD24 along with negative staining with Annexin V was measured to distinguish viable PIGA- and PIGA+ granulocytes. The percentage of viable cells was normalized to untreated controls (RPMI+10% FCS). Error bars represent standard deviation from the mean expressed as a percentage.

Effect of graded doses of total body {gamma}-irradiation on PIGA- and PIGA+ blood cells
Finally, to test the in vivo resistance of PIGA+ and PIGA- bone marrow cells and to assess the susceptibility of a more immature hematopoietic precursor cell toward programmed cell death, we subjected mice with PIGA- blood cells and wt-control mice to graded doses of {gamma}-irradiation. For these experiments, we used mice that are mosaic for PIGA- cells (EL mice). We have previously shown that EL mice under laboratory conditions have a stable proportion of PIGA- cells in all blood cell lineages throughout adulthood [13 ]. EL and control mice were {gamma}-irradiated with a single dose of 450, 600, 800, or 900 cGy. Age- and sex-matched syngeneic littermates were used as nonirradiated, control animals. The proportion of circulating PIGA- blood cells and peripheral blood cell parameters, including differential white blood cell counts, platelet counts, red cell counts, and hemoglobin levels, was followed over a period of 6 months. All animals showed a dose-dependent, transient pancytopenia in the first 2 weeks after irradiation (data not shown). Within the group of animals irradiated with 900 cGy, 2 out of 11 EL mice died within the first 2 weeks after irradiation. All other EL and control mice survived. Peripheral blood values of irradiated EL and irradiated wt-control mice were not significantly different during the observation period (data not shown). In EL mice, the proportion of PIGA- blood cells within red cells, granulocytes, B cells, and T cells showed considerable fluctuations when compared with untreated EL mice. However, in none of the animals treated was a persistent increase of PIGA- cells documented, which would imply a resistance of PIGA- progenitor cells to ionizing irradiation. Figure 5 shows as a representative example the changes in the proportion of PIGA- red cells, granulocytes, B cells, and T cells over time in EL mice after {gamma}-irradiation with 900 cGy. Conversely, the proportion of circulating PIGA- red cells, T cells, and particularly B cells decreased after {gamma}-irradiation. In {gamma}-irradiated EL mice, very low numbers of PIGA- B cells were detected in spleen, bone marrow, and lymph nodes 6 months after treatment (data not shown). In vivo, {gamma}-irradiation causes significant changes in the microenvironment in addition to DNA damage to hematopoietic cells. Thus, in our long-term follow-up, the changes in the microenvironment might contribute to the decrease of PIGA- cells. Indeed, a poor repopulation of B cells was also noted by Murakami et al. [18 ] when normal fetal liver cells were transplanted into lethally irradiated, recipient mice. No tumor development or evidence of leukemia/lymphoma was observed in {gamma}-irradiated EL mice or {gamma}-irradiated wt-control mice during the period of observation and at necropsy at the end of the observation period.



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  		Figure 5. Proportion of PIGA- red cells (RBC), polymorphonuclear neutrophils (PMN), B cells, and T cells after total body irradiation with 900 cGy (n=9) and untreated EL mice (n=8) determined over a period of 6 months. Each data point represents the mean of the proportion of PIGA- cells normalized to the proportion of PIGA- cells at base line (before treatment). Asterisks and solid lines represent irradiated EL mice; dashed lines show nonirradiated EL mice.


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DISCUSSION
 
The nature of the growth advantage that allows a hematopoietic progenitor cell deficient in all GPI-linked proteins to clonally expand is a highly debated subject in the field of PNH and is the focus of intense research [19 ]. We and others have previously shown, using genetically engineered mice, that a Piga gene mutation alone does not provide a proliferative advantage to PIGA- hematopoietic stem cells [13 , 18 ]. PNH has a well-known, close relationship with AA [5 ], suggesting that PNH progenitor cells have a survival advantage in the hostile environment of AA [7 , 8 ]. An intrinsic resistance toward apoptotic cell death in an environment of AA when normal hematopoietic progenitor cells die would elegantly explain the association of PNH with AA and explain in this context the growth advantage of PNH cells. Indeed, an increased resistance toward specific apoptosis-inducing stimuli has been documented in a number of studies using blood cells from patients with PNH [9 10 11 ]. However, the contribution of the PIGA gene mutation to this finding remained controversial [9 10 11 ]. In our study, we took advantage of our murine model that, like patients with PNH, has a proportion of hematopoietic cells deficient in GPI-linked proteins. Thus, mice with PIGA- blood cells, genetically, accurately imitate the situation of a patient with an established PNH clone. However, in contrast to patients with the disease, the genetically engineered mice do not have additional bone marrow failure. This provides the unique opportunity to more comprehensively study the effect of the Piga gene mutation on programmed cell death in vitro and in vivo than was previously possible using patient material. For comparison, we also included in our studies apoptotic stimuli and experimental conditions that have previously been used in the assessment of apoptotic cell death in human PNH cells. As in our experiments, PIGA+ and PIGA- cells are derived from the same animal and are exposed to identical apoptosis-inducing conditions, even subtle differences in apoptotic cell death between PIGA+ and PIGA- cells detected are specific for the Piga gene mutation.

Thymocytes have been widely used to assess apoptotic cell death in mutant mice. Therefore, we included the assessment of apoptotic cell death in PIGA+ and PIGA- thymocytes in our studies. PIGA+ and PIGA- thymocytes exposed to graded doses of {gamma}-irradiation or subjected to different concentrations of anti-Fas antibody, etoposide, and dexamethasone showed an almost identical decrease in cell viability over time, which also was not significantly different to that of normal PIGA+ thymocytes from a normal wt-control mouse. A similar sensitivity to apoptosis has previously been shown in immortalized, mutagenized PIGA- Jurkat T cell lines when compared with the parental wt cell line [20 ]. Our findings in primary thymocytes confirm that a mutant Piga gene does not alter the sensitivity to apoptosis toward any stimuli tested, nor does the coexistence of PIGA- cells affect the sensitivity to apoptosis in PIGA+ cells.

PNH is a disease of the hematopoietic progenitor cell, possibly the hematopoietic stem cell. Therefore, we studied the sensitivity of hematopoietic progenitor cells isolated from the bone marrow toward apoptotic cell death. The total number of colonies obtained in 7 days of methylcellulose cultures from bone marrow from LF mice and from wt mice decreased similarly in response to different doses of {gamma}-irradiation or to coculture with different concentrations of etoposide (Fig. 3A) . Likewise, with the exception of the bone marrow sample from one mouse, there was no increase in the proportion of PIGA- erythroid or PIGA- myeloid cells when compared with the untreated control sample, indicating that PIGA- hematopoietic progenitor cells respond equally to the apoptotic stimuli. Two groups have investigated apoptotic cell death in bone marrow cells from patients with PNH; however, their findings and the interpretation of results were contradictory. After 72 h in serum-free medium, Brodsky et al. [9 ] found an increased survival in sorted CD34+CD59- (PNH) bone marrow cells when compared with CD34+CD59+ (normal) from the same patient. Similarly, Horikawa et al. [10 ] described an increased survival in CD34+ marrow cells from PNH patients in comparison to bone marrow cells form normal individuals in response to tumor necrosis factor {alpha} and interferon-{gamma} followed by incubation with antibody to Fas. However, a similar, increased survival was found in CD34+ cells from myelodysplasia patients, suggesting that the difference in cell survival in PNH compared with normal individuals might be caused by the underlying bone marrow failure [10 ]. Our finding that a Piga gene mutation does not alter the response to apoptosis in murine bone marrow cells derived from a neutral environment supports Horikawa and co-workers’ interpretation [10].

The sensitivity to apoptotic cell death in mature granulocytes has been used previously to document the increased resistance to programmed cell death in PNH compared with normal individuals [9 10 11 ]. However, Horikawa et al. [10 ] and Ware et al. [11 ] found a similarly increased resistance of apoptosis in patients with a low percentage of PNH granulocytes, suggesting that the increased resistance occurs independent from the PIGA gene mutation. In contrast, Yamamoto et al. [21 ] found no difference in apoptosis of serum-starved peripheral blood granulocytes from PNH patients compared with healthy individuals. In our study, we subjected granulocytes from our LF mice to serum starvation and compared the rate of apoptosis in PIGA+ with PIGA- granulocytes from the same mouse under identical conditions. No survival advantage was detected in PIGA- compared with PIGA+ granulocytes within the same mouse, nor was the survival different from serum-deprived PIGA+ granulocytes from wt-control mice. These results clearly indicate that a Piga gene mutation has no impact on the rate of apoptosis in serum-starved granulocytes. Thus, our study in mice indicates that the increased resistance to apoptosis reported in some studies using granulocytes and bone marrow cells from PNH patients is not explained by a PIGA gene mutation.

The expansion of the PNH cell clone is thought to occur at the level of a multipotent hematopoietic progenitor cell, possibly the hematopoietic stem cell [22 ]. The sensitivity of the PIGA- hematopoietic stem cell toward apoptotic cell death has not been investigated so far. The use of a mouse model allowed us to investigate the survival of PIGA- hematopoietic progenitor cells in vivo under experimental conditions that lead to apoptosis in normal hematopoietic stem cells. We used graded doses of total body irradiation to induce apoptosis and screened for a possible survival advantage by monitoring the proportion of PIGA- blood cells in all blood cell lineages. During the 6 months of follow-up, there was a considerable fluctuation in the proportion of PIGA- cells in different blood cell lineages in all mice analyzed (Fig. 5) . However, in none of the animals was a persistent increase in the proportion of PIGA- cells documented. In contrast, after {gamma}-irradiation in most blood cell lineages, the proportion of PIGA- cells decreased. This was particularly evident for B cells, which almost completely disappeared from peripheral blood, primary and secondary lymphoid organs, 6 months after 900 cGy irradiation. This clearly demonstrates that a Piga gene mutation does not protect PIGA- hematopoietic progenitor cells from irradiation-induced cell death in vivo.

In summary, our studies of apoptosis in mice carrying a Piga gene mutation in hematopoietic cells unambiguously demonstrate that for all stimuli tested, the Piga gene mutation does not confer an increased resistance to programmed cell death. The increased resistance to apoptosis found in blood cells from patients with PNH in some studies might therefore occur independently from the PIGA gene mutation and is possibly induced by the hostile environment of the underlying bone marrow failure. Cell death of hematopoietic stem cells in most forms of acquired AA is thought to be autoimmune-mediated [23 ]. As a result of their inability to express GPI-linked proteins, PNH cells might escape immune recognition or the autoimmune attack.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grant RO1-CA-89091, the Barnes Jewish Hospital Foundation, Mallinckrodt Foundation, McDonnell Foundation, and American Cancer Society IRG-58–010–41. S. K. is supported by the Lady Tata Foundation, London, U.K. The authors thank J. H. Russell for his expert advice on apoptosis; I. Pantazopoulos, M. Rogers, and P. Sipes for their technical assistance; M. L. McLemore and D. C. Link for valuable discussions; P. Keller and M. Jasinski for helpful suggestions; and P. J. Mason for critical reading of the manuscript. We also thank Amgen (Thousand Oaks, CA) for the kind gift of recombinant human erythropoietin and rat stem-cell factor.

Received June 17, 2002; revised September 5, 2002; accepted September 24, 2002.


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R. A. Brodsky
Paroxysmal Nocturnal Hemoglobinuria: Stem Cells and Clonality
Hematology, January 1, 2008; 2008(1): 111 - 115.
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