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HEMATOPOIESIS
From the Division of Experimental Hematology and the
Department of Biochemistry, St Jude Children's Research Hospital,
Memphis, TN.
A single dose of Mpl ligand (Mpl-L) given immediately after lethal
DNA-damaging regimens prevents the death of mice. However, the
mechanism of this myeloprotection is unknown. The induction of
p53-dependent apoptosis in response to DNA damage signals suggests that
immediate administration of Mpl-L may inhibit p53-dependent apoptosis.
This hypothesis was tested by administering a single injection of
pegylated murine Megakaryocyte Growth and Development Factor
(PEG-rmMGDF, a truncated recombinant Mpl-L) to
p53 Recent studies indicate that Mpl ligand (Mpl-L)
reduces hematopoietic toxicity much more effectively when administered
immediately after a myelosuppressive insult than when given beforehand
or at later times.1-5 However, the mechanism of this
multilineage myeloprotection has not been defined.
The involvement of Mpl-L (also known as thrombopoietin or TPO) and its
receptor Mpl in early hematopoiesis is consistent with the following
observations: (1) Mpl is expressed on the AA4+
Sca+ subpopulation of murine hematopoietic stem
cells6; (2) Mpl-L stimulates the expansion of very
immature precursors both in vitro and in vivo; and (3) Mpl and Mpl-L
are required for optimal multilineage hematopoietic development, as
well as for thrombocytopoietic differentiation. This latter point is
evident by marked reductions in granulocyte-macrophage, erythroid, and
multipotential progenitor cells in Tpo Recent studies indicate that p53 mediates apoptosis of hematopoietic
progenitors exposed to DNA-damaging drugs or
Here we report that administration of a single dose of polyethylene
glycol-conjugated recombinant murine Megakaryocyte Growth and
Development Factor (PEG-rmMGDF), a truncated form of Mpl-L, is
sufficient to prevent p53-dependent apoptosis in a murine model of
lethal myelosuppression. These findings suggest that Mpl-L may be
clinically useful in reducing neutropenia, thrombocytopenia, and anemia
caused by myelosuppressive treatments, such as radiation or
chemotherapy, and by preparatory regimens for hematopoietic stem cell transplantation.
Reagents and animals
p53-knockout mice (C57Bl/6) were purchased from The Jackson
Laboratory (Bar Harbor, ME), and were studied at 2.5 months of age,
prior to the development of tumors.
Atm Myelosuppressive regimen
Bone marrow transplantation Bone marrow was collected from femurs and tibias of appropriate donor mice and suspended in Dulbecco modified Eagle medium (DMEM) with 10% fetal calf serum. Recipients were exposed to 11.25 Gy and given donor cells intravenously within 2 hours after irradiation. Each recipient received the marrow from 1 to 2 bones. Mice were used in experiments 2 to 4 months after transplantation, after blood cell indices had returned to the normal range.Administration of PEG-rmMGDF A single dose of PEG-rmMGDF (50 or 65 µg/kg) was injected into a lateral tail vein after vasodilation was induced by warming under an examination lamp. An equivalent volume of PBS with 1% isologous mouse serum (carrier) was injected intravenously into control mice.Blood cell counts Blood was collected into ethylenediaminetetraacetic acid (EDTA)-coated 20-µL microcapillary tubes (CDC Technologies, Oxford, CT) and immediately diluted in buffered diluent. Platelet counts, white blood cell (WBC) counts, and hemoglobin concentration were measured by a MASCOT Automated Hematology System (CDC Technologies).Bone marrow colony assays Mice were injected intravenously with 80 mg/kg carboplatin and irradiated with 7.5 Gy. Immediately afterward, half of the animals were injected intravenously with 65 µg/kg PEG-rmMGDF. Bone marrow cells were aseptically harvested from mouse femurs and tibias by flushing the bone marrow cavity with 2 mL -MEM (minimum essential medium: with
Earle salts and with L-glutamine; Life Technologies, Grand Island, NY)
containing 2% fetal bovine serum (FBS). A cell suspension was prepared
by passing the bone marrow through a 25-gauge needle. The concentration
of nucleated cells in the bone marrow cell suspension was determined
after dilution in 3% acetic acid to lyse erythrocytes. Diluted cell suspensions and recombinant mouse interleukin 3 (rmIL-3) were mixed
with methylcellulose (MethoCult M3230; Stem Cell Technologies, Vancouver, BC, Canada). The cell suspensions were plated in duplicate in 35-mm culture dishes in medium that contained a final concentration of 0.9% methycellulose, 30% FBS, 1% bovine serum albumin, 0.1 mM
2-mercaptoethanol, and 2 mM L-glutamine, and were cultured at 37°C
with 5% CO2. For day 1 assays, 1 to 2 × 106
cells per dish were cultured in 20 U/mL rmIL-3. For day 2 assays, due
to the lower bone marrow cellularity at this time after treatment, fewer cells (0.4 × 106 per dish) were plated. Colonies
were scored on day 13, and colony sizes were classified as large
(> 500 cells/colony), medium (200-400 cells/colony), or small (15-150 cells/colony). Results were expressed as the numbers of colonies per
106 cells plated.
Immunoblot analyses Bone marrow was collected from mouse femurs and tibias by flushing the bone marrow cavity with 1X DPBS (Dulbecco phosphate-buffered saline, Life Technologies). Bone marrow cells were washed twice in EHS buffer (1 mM Na2EDTA, 10 mM HEPES pH 7.6, and 150 mM NaCl) and were collected by centrifugation at 900g, 4°C for 10 minutes. Cell pellets were suspended in 60 volumes of EHS buffer and cells were lysed by boiling after addition of one-third volume of Laemmli gel sample buffer (125 mM Tris-HCL pH 6.8, 40% glycerol, 8% SDS, 160 mM dithiothreitol, and 0.01% bromphenol blue). Proteins (50-150 µg per lane) were electrophoretically separated in 7.5% to 15% linear polyacrylamide gradients or 12% polyacrylamide gels containing sodium dodecyl sulfate (SDS), transferred to nitrocellulose membranes (Protran; Schleicher & Schuell, Dassel, Germany) and blotted with antibodies specific for p53 (AB-7, Calbiochem, La Jolla, CA), Bax and p21Cip1 (Santa Cruz Biotechnology, Santa Cruz, CA), and actin (Boehringer-Mannheim, Indianapolis, IN). Bound immune complexes were detected by enhanced chemiluminescence (ECL reagent; Amersham, Arlington Heights, IL, or Supersignal, Pierce, Rockford, IL).
A single dose of Mpl-L or loss of p53 protects mice from lethal myelosuppression Recent studies using mouse models have established that administration of a single high dose of Mpl-L immediately after a lethal myelosuppressive regimen promotes hematopoietic recovery and prevents death.1-5 The p53 tumor suppressor protein is an important mediator of apoptosis in response to chemotherapeutic DNA-damaging agents and -irradiation4,14 and therefore
may contribute to the pathogenicity associated with myelosuppression. The rationale and hypothesis of our studies was that if Mpl-L was
acting to prevent cell death through a p53-dependent pathway, then
Mpl-L administration to p53 / mice given a
myelosuppressive regimen should provide no further hematopoietic
protection over carrier-treated p53-null animals subjected to the same
DNA damage. Alternatively, if the cytokine blocks cell death via
another pathway, p53/mice treated with
DNA-damaging agents and Mpl-L should display less myelosuppression and
faster hematopoietic recovery than p53/mice
given myelosuppression and carrier alone. In this case, the Mpl-L
protective effect should be additive to the loss of p53 function.
To determine the role of p53 in this response, and whether the
protective effect of Mpl-L is due to its ability to inhibit p53-dependent apoptosis, we exposed p53-deficient mice to a
myelosuppressive regimen that is lethal to wild-type (WT) mice. The WT
and p53
p53 promotes survival of bone marrow-derived progenitor cells
exposed to carboplatin and / mice treated with PEG-rmMGDF, as
well as p53/ animals injected with carrier
alone, experienced nearly identical courses of platelet suppression and
recovery following carboplatin and -irradiation (-IR), whereas
platelet counts of carrier-treated WT mice failed to recover (Figure
2A). These results were confirmed in 2 repeat experiments. In contrast, the declines in WBC and hemoglobin
values were significantly less severe in p53/
mice, with or without PEG-rmMGDF treatment, as compared with WT
animals given PEG-rmMGDF (Figure 2B-C). Wild-type mice treated with
PEG-rmMGDF had appreciably lower WBC counts than did the 2 p53/ groups between days 8 and 22 after
treatment (P < .02-.001 in 3 experiments). Similarly,
decreases in hemoglobin concentration were significantly greater in WT
mice given PEG-rmMGDF than in both groups of p53/
mice on day 14 (P < .005-.001 in 3 experiments). The
hemoglobin levels of WT mice treated with PEG-rmMGDF did recover to a
level equal to that of p53/ mice by day 22 (Figure 2B). By contrast, PEG-rmMGDF administration did not enhance
hematopoietic recovery in p53/ mice.
Collectively, these findings suggest that thrombopoiesis is protected
to the same extent in PEG-rmMGDF-treated WT mice and in
p53/ mice by conferring survival to
c-Mpl-expressing multipotential hematopoietic precursors and committed
megakaryocyte progenitors. In contrast, granulopoiesis and
erythropoiesis are protected less efficiently by PEG-rmMGDF in WT mice
than in p53/ mice because committed
granulocyte and erythroid precursors do not express c-Mpl.
To demonstrate that p53-knockout mice survive the lethal
myelosuppression regimen through increased hematopoietic cell survival, we examined WT mice transplanted with p53 Rescue of bone marrow progenitors by administration of Mpl-L To determine whether the beneficial effects of PEG-rmMGDF targeted progenitor cells, we compared the in vitro colony-forming ability of bone marrow cells harvested on days 1 and 2 after the myelosuppressive regimen to that of marrow from WT untreated mice. Bone marrow obtained from these mice was plated in methylcellulose containing IL-3, and colony number and size were scored after 13 days. Colony formation from marrow of mice treated with PEG-rmMGDF or carrier and collected on day 1 and day 2 after treatment was markedly decreased compared with that of untreated marrow (Figure 3). However, bone marrow cells collected at 24 and 48 hours generated significantly greater colony numbers and sizes when derived from mice treated with PEG-rmMGDF as compared with carrier-treated mice (P < .05) (Figure 3, middle and bottom panels). Furthermore, bone marrow from mice given the myelosuppression regimen and PEG-rmMGDF yielded a full spectrum of colony sizes, whereas bone marrow from those mice exposed to the myelosuppressive regimen without PEG-rmMGDF formed predominantly small colonies (Figure 3, middle and bottom panels). Therefore, Mpl-L promotes hematopoietic recovery by protecting bone marrow progenitors from DNA-damaging insults. Colony formation by untreated p53/ marrow cells was not
significantly different than that of untreated WT marrow (Figure
4, top panel). As in Figure 3, colony
formation from WT mice treated with PEG-rmMGDF at day 2 after
treatment was significantly higher than that of carrier-treated
p53+/+ mice (P < .005) (Figure 4,
middle panel). In contrast, there was no additional protective effect
of Mpl-L on the colony formation by bone marrow cells of
p53/ mice collected at 48 hours after the
myelosuppressive regimen as the number of colonies derived from the
Mpl-L- and carrier-treated p53/ mice were
not different (Figure 4, bottom panel). This trend was confirmed in a
repeat experiment.
Effect of Mpl-L administration on the p53 pathway in bone marrow cells p53 is an important regulator of the DNA damage response in mammalian cells. DNA damage is followed by rapid accumulation and activation of p53 that induces the expression of a number of downstream target genes, resulting in cell cycle arrest or apoptosis.18 To assess the function of the p53 pathway in response to our myelosuppression regimen, we analyzed the expression of p53 and 2 of its transcription targets (p21Cip1 and Bax) in bone marrow cells at specific time intervals following carboplatin and-IR exposure by immunoblotting (Figure
5). In response to this regimen, levels
of p53 protein increased rapidly and were sustained for at least 6 hours before declining; PEG-rmMGDF administration did not significantly
alter the magnitude or the kinetics of p53 induction (Figure 5,
repeated in 5 separate experiments). Bax, a proapoptotic factor that is
up-regulated by p53 in some cell types,19-23 was
constitutively expressed in bone marrow cells exposed to the
DNA-damaging regimen and unaffected by the administration of
PEG-rmMGDF. By contrast, expression of p21Cip1,
a cyclin kinase inhibitor,24 was markedly induced
following carboplatin and -IR and this response paralleled the
induction of p53 (Figure 5). PEG-rmMGDF had no consistent differential
effect on the induction of p21 during DNA damage, indicating
that the beneficial effect of Mpl-L on hematopoietic cells is not due
to a direct disruption of the p53 response; instead, Mpl-L appears to
act downstream of p53 to prevent apoptosis (see "Discussion").
Bax influences hematopoietic recovery following lethal myelosuppression Previous studies reported that Bax expression is induced in radiosensitive tissues following irradiation.25 By contrast, we observed that carboplatin and-IR, with or without
administration of PEG-rmMGDF, did not affect the steady-state level of
Bax protein in bone marrow cells. These results, however, did not rule
out the possibility that small changes in Bax expression and/or
posttranslational modifications could significantly regulate the
apoptotic response to this myelosuppressive regimen. To assess the role
of Bax in p53-mediated apoptosis in our myelosuppression model, we
measured the survival and the hematopoietic recovery of
Bax/ mice exposed to carboplatin and -IR,
and whether PEG-rmMGDF affects this response. Similar to WT littermate
animals, Bax/ mice were highly susceptible
to the lethal effects of this DNA-damaging regimen. Assessment of
platelet counts, WBC counts, and hemoglobin levels indicated that
Bax/ mice died due to hematopoietic failure
(Figure 6A-C) (confirmed in 2 independent
experiments). Interestingly, Bax/ mice
treated with PEG-rmMGDF exhibited a more rapid recovery of hemoglobin
levels (on day 14, P < .005; days 18 and 22, P < .001), platelet counts (day 14, P < .01
and day 18, P < .001) and WBC counts (day 10, P < .025 and day 18, P < .001) as compared with those of wild-type mice (Figure 6A-C). Thus, although clearly not
required, Bax appears to play some role in p53-dependent apoptosis in
this myelosuppression model.
Loss of Atm compromises Mpl-L rescue from lethal myelosuppression Atm is a member of the PI-3' serine/threonine kinase family that phosphorylates and activates p53 in response to certain DNA damage signals.25,26 Loss of Atm in mice and humans is typically associated with attenuated p53 responsiveness and impaired cell cycle arrest. Paradoxically, Atm/ mice
display remarkable susceptibility to DNA-damaging
insults,27,28 which appears to be cell-type specific and
largely attributable to increased gastrointestinal
sensitivity.29 Since p53-null bone marrow
progenitors are resistant to DNA damage, we tested whether loss of
Atm would also influence the response of bone marrow-derived progenitors to carboplatin and -IR. To this end, we
assessed the responses of WT mice receiving
Atm/, p53/,
p53+/, or WT bone marrow transplants to DNA damage.
Animals transplanted with Atm/ marrow and
treated with PEG-rmMGDF succumbed earlier to the myelosuppressive regimen than did similarly treated mice transplanted with
p53+/ and WT bone marrow, whereas all mice receiving
p53/ transplants survived without PEG-rmMGDF
treatment (Figure 7). All
Atm/, p53+/, or WT bone
marrow-transplanted mice treated with carrier after exposure to the
myelosuppressive regimen died (data not shown). The inability of
Atm/ bone marrow cells to properly arrest in
response to DNA damage presumably overrode the protective effects of
Mpl-L, resulting in dramatically earlier hematopoietic death of all
Atm/ mice.
The mechanism by which Mpl-L protects animals from a lethal
myelosuppressive regimen has not been previously established. Myelosuppressive agents such as chemotherapy and The increased resistance of p53-deficient mice to the carboplatin and
We observed interesting differences between the hematopoietic responses of p53-deficient mice given PEG-rmMGDF or carrier versus WT mice given PEG-rmMGDF after the myelosuppressive regimen. Hemoglobin concentration and WBC counts were reduced to a greater extent in WT mice given PEG-rmMGDF than in p53-deficient mice given PEG-rmMGDF or carrier alone. By contrast, the degree of thrombocytopenia in these 3 groups was virtually identical. We speculate that this difference reflects the disparate patterns of Mpl expression on the various hematopoietic progenitors.9,10 In that case, all p53-null multipotential hematopoietic progenitors and committed precursors would be protected from p53-dependent apoptosis, whereas in the normal phenotype, only hematopoietic cells that express Mpl would be protected by Mpl-L. If this view is correct, then concomitant administration of Mpl-L in combination with erythropoietin and granulocyte-colony stimulating factor (G-CSF) immediately after a myelosuppressive insult might optimally reduce the degree of suppression of all 3 committed hematopoietic lineages. The number of bone marrow-derived colonies generated by cultures with
IL-3 was substantially higher in mice that received PEG-rmMGDF after
the myelosuppression regimen than carrier-only controls; this finding
suggests that Mpl-L protects hematopoietic progenitors. In contrast,
the assays of bone marrow-derived colonies generated from
myelosuppressed, Mpl-L-treated p53 Our analysis of protein expression provides insight into how Mpl-L
prevents p53-dependent apoptosis after the myelosuppressive regimen.
The increase in p53 protein levels in bone marrow cells in response to
carboplatin and Because Bax is directly transactivated by p53 and promotes
apoptosis,21,34 we also tested the role of this
gene by using Bax-deficient mice in our myelosuppression model. We
found that Bax is constitutively expressed in mouse bone marrow cells
and that Bax levels are not affected by carboplatin plus The increased radiosensitivity of ATM In summary, our observations indicate that Mpl-L administered immediately after a lethal myelosuppressive regimen promotes the survival of hematopoietic progenitor cells in vivo by attenuating p53-dependent apoptosis. Myelosuppression is a toxicity-limiting component of chemotherapy and/or radiation therapy, and a major clinical problem in autologous bone marrow transplantation regimens. Administration of Mpl-L during these DNA-damaging treatments should suppress the p53 response, thereby alleviating the severity of neutropenia, thrombocytopenia, and anemia and the need for platelet transfusions.
The authors thank Shirley Steward, Nancy Hutson, Jinling Wang, Dr
Christine Eischen, Charles M. Strain, and Joseph M. Emmons for
outstanding technical assistance; the staff of the St Jude Animal
Resources Center for their support; Dr Stanley Korsmeyer for providing
Bax
Submitted November 20, 2000; accepted May 24, 2001.
Supported in part by grants CA76379, DK44158 (J.L.C.), CA63230 (G.P.Z.), and CA21765 from National Institutes of Health; by the Assisi Foundation of Memphis; and by the American Lebanese Syrian Associated Charities.
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: Tamara I. Pestina, Division of Experimental Hematology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794; e-mail: tamara.pestina{at}stjude.org.
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© 2001 by The American Society of Hematology.
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J.-M. Paulus, N. Debili, F. Larbret, J. Levin, and W. Vainchenker Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors Blood, October 15, 2004; 104(8): 2291 - 2298. [Abstract] [Full Text] [PDF]
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M. Drouet, F. Mourcin, N. Grenier, V. Leroux, J. Denis, J.-F. Mayol, P. Thullier, J.-J. Lataillade, and F. Herodin Single administration of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination soon after irradiation prevents nonhuman primates from myelosuppression: long-term follow-up of hematopoiesis Blood, February 1, 2004; 103(3): 878 - 885. [Abstract] [Full Text] [PDF]
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D. D. Dahlen, V. C. Broudy, and J. G. Drachman Internalization of the thrombopoietin receptor is regulated by 2 cytoplasmic motifs Blood, July 1, 2003; 102(1): 102 - 108. [Abstract] [Full Text] [PDF]
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F. Herodin, P. Bourin, J.-F. Mayol, J.-J. Lataillade, and M. Drouet Short-term injection of antiapoptotic cytokine combinations soon after lethal gamma -irradiation promotes survival Blood, April 1, 2003; 101(7): 2609 - 2616. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
