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Stem Cells, Vol. 19, No. 4, 329-338, July 2001
© 2001 AlphaMed Press

Promegapoietin-1a, an Engineered Chimeric IL-3 and Mpl-L Receptor Agonist, Stimulates Hematopoietic Recovery in Conventional and Abbreviated Schedules Following Radiation-Induced Myelosuppression in Nonhuman Primates

Ann M. Faresea, Walter G. Smithb, Judith G. Girib, Ned Siegelb, John P. McKearnb, Thomas J. MacVittiea

a Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland, USA;
b Pharmacia Corporation, St. Louis, Missouri, USA

Key Words. Promegapoietin-1a • Myelosuppression • Nonhuman primate • Platelet • Hematopoiesis

Ann M. Farese, M.S., Greenebaum Cancer Center, University of Maryland, 655 West Baltimore St., BRB 7-047, Baltimore, Maryland 21201, USA. Telephone: 410-328-5347; Fax: 410-328-5488; e-mail: afarese{at}umaryland.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Clinical Support
 Results
 Discussion
 Acknowledgements
 References
 
Promegapoietin-1a (PMP-1a), a multifunctional agonist for the human interleukin 3 and Mpl receptors, was evaluated for its ability to stimulate hematopoietic reconstitution in nonhuman primates following severe radiation-induced myelosuppression. Animals were total body x-irradiated (250 kVp) to 600 cGy total midline tissue dose. PMP-1a was administered s.c. in several protocols: A) daily (50 µg/kg) for 18 days; B) nine doses (5 µg/kg) every other day for 3 weeks; C) a single high dose (100 µg/kg) at 20 hours, or D) a single high dose (100 µg/kg) at 1 hour following TBI. The irradiation controls received 0.1% autologous serum for 18 consecutive days. Hematopoietic recovery was assessed by bone marrow clonogenic activity, peripheral blood cell nadirs, duration of cytopenias, time to recovery to cellular thresholds, and requirements for clinical support. PMP-1a, irrespective of administration schedule, significantly improved all platelet-related parameters: thrombocytopenia was eliminated, the severity of platelet nadirs was significantly improved, and recovery of platelet counts to >=20,000/µl was significantly reduced in all PMP-1a-treated cohorts. As a consequence, all PMP-1a-treated cohorts were transfusion-independent. Neutrophil regeneration was augmented in all treatment schedules. Additionally, all PMP-1a-treated cohorts showed an improvement in red blood cell nadir and recovery. PMP-1a in conventional or abbreviated schedules induced significant thrombopoietic regeneration relative to the control cohort, whereas significant improvement in neutrophil recovery was schedule-dependent in radiation-myelosuppressed nonhuman primates.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Clinical Support
 Results
 Discussion
 Acknowledgements
 References
 
Realizing the full potential of the intended schedule of cytotoxic therapy may well depend upon controlling the associated toxicities that include neutropenia, thrombocytopenia, and anemia. The growth factors (GFs), G-CSF, GM-CSF, and erythropoietin have provided a measure of relief from the duration of neutropenia and anemia [1-6] while the control of thrombocytopenia awaits the definition of optimal dose and schedule of administration for the thrombopoietic GFs interleukin 11 (IL-11) and thrombopoietin (TPO) [7-10]. The preclinical data base for TPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) established their thrombopoietic and hematopoietic activity when used alone or in combination with other GFs [11-14]. The administration of TPO or PEG-rHuMGDF has been shown to significantly improve platelet nadirs, reduce the duration of thrombocytopenia and effectively render myelosuppressed nonhuman primates transfusion-independent [11, 13]. However, the ineffective results of conventional TPO or PEG-rHuMGDF administration in nonhuman primate models of bone marrow (BM) transplantation-associated thrombocytopenia have suggested that demonstration of clinical benefit may depend upon defining the optimal dose and schedule [15-18].

New GF and/or GF combinations may be required to effectively manage the hematopoietic toxicities associated with protocols that include dose intensification or schedule compression in an effort to optimize therapy. The lineage-dominant GFs TPO and PEG-rHuMGDF have been combined with G-CSF in both rodent and nonhuman primate models of myelosuppression to clearly enhance multilineage recovery without evidence of lineage competition [11, 13]. Furthermore, conventional administration of TPO or PEG-rHuMGDF following myelosuppression is not required in these preclinical models [14, 19, 20]. A single dose of TPO or PEG-rHuMGDF given within 24 hours of irradiation was as effective as consecutive, daily dosing in enhancing recovery of platelets.

In two other studies of GF combinations, the engineered IL-3 receptor agonist, daniplestim was combined with either G-CSF or a truncated form of the Mpl-ligand (L) [21, 22]. In each study, the combination of the IL-3 receptor agonist with G-CSF or Mpl-L further improved hematopoietic recovery and enhanced the stimulatory effect of the respective cytokine monotherapy in nonhuman primates following severe radiation-induced myelosuppression. These combinations also further reduced the clinical support requirements for whole blood transfusions and antibiotics.

Another approach toward enhancing hematopoietic and/or immune recovery, subsequent to myelosuppressive therapy or stem cell transplantation, has been to engineer chimeric GF receptor agonists that possess greater biologic activity than either GF administered as monotherapy or in combination protocols. The combination of an IL-3 receptor agonist, daniplestim, with G-CSF or Mpl-L was based on a consistent amount of in vitro and in vivo evidence suggesting that these combinations would enhance neutrophil and platelet regeneration within a favorable toxicity profile and administration schedule [21, 23-28]. Myelopoietin has recently been shown to enhance all neutrophil and platelet-related parameters consequent to high-dose radiation-induced myelosuppression above that observed with either GF alone or in combination [29]. Furthermore, recent studies have shown that myelopoietin administered in an abbreviated, qod schedule was found to be as effective in enhancing multilineage recovery as qd or bid schedules [30].

In this study we evaluated the therapeutic efficacy of Promegapoietin-1a (PMP-1a), the chimeric IL-3 and Mpl-L receptor agonist, administered in conventional and abbreviated schedules to enhance hematopoietic recovery in a nonhuman primate model of high-dose radiation-induced myelosuppression.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Clinical Support
 Results
 Discussion
 Acknowledgements
 References
 
Animals
Male rhesus monkeys, Macaca mulatta, mean weight 4.35 ± 0.32 kg, were housed in individual stainless steel cages in conventional holding rooms at the Veterinary Resources Department at Greenebaum Cancer Center in animal facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Monkeys were provided 10 air changes/h of 100% fresh air, conditioned to 72° ± 2°F with a relative humidity of 50 ± 20% and maintained on a 12-hour light/dark full spectrum light cycle, with no twilight. Monkeys were provided with commercial primate chow, supplemented with fresh fruit and tap water ad libitum. Research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals [31], prepared by the Institute of Laboratory Animal Resources, National Research Council.

Irradiation
Monkeys, following a prehabituation period, were unilaterally irradiated in Lucite restraining chairs with 250 kVp x-radiation at 13 cGy/minutes in the posterior-anterior position, rotated 180° at the mid-dose (300 cGy) to the anterior-posterior position for completion of the total 600 cGy midline tissue exposure. Dosimetry was performed using paired 0.5 cm3 ionization chambers, with calibration factors traceable to the National Institute of Standards and Technology.

Recombinant PMP-1a
PMP-1a is an engineered, chimeric hematopoietic GF containing IL-3 receptor and c-mpl agonist moieties. PMP-1a was produced in E. coli through the use of a plasmid-based expression vector controlled by a modified E. coli rec A promoter inducible with nalidixic acid. PMP-1a was expressed in insoluble inclusion bodies within the E. coli cells. The washed inclusion bodies were solubilized and the disulfide bonds were formed in an SDS buffer. Refolded PMP-1a was purified by ion exchange chromatographic and filtration steps. Analytical assays used to characterize the purified PMP-1a included amino-acid composition, N-terminal amino acid sequencing, trypsin mapping, reversed-phased HPLC, size-exclusion HPLC, endotoxin levels, residual DNA, residual host cell protein, and SDS-PAGE. Bioactivity of the bulk PMP-1a was measured using TF-1 and BaF3/c-mpl cell proliferation bioassays. Purified PMP-1a bulk protein was stored as a frozen solution in Tris/mannitol/sucrose, pH 8.0. Prior to administration, PMP-1a was diluted to the appropriate concentration in a total volume of 0.1% autologous serum (AS) in sterile, injectable 0.9% sodium chloride (McGaw, Inc.; Irvine, CA).

Study Design
Animals were total body x-irradiated (TBI) (250 kVp), to a midline tissue dose of 600 cGy and randomly assigned to a treatment protocol utilizing PMP-1a or control AS. PMP-1a was s.c. administered in the following four protocols: A) daily for 18 days ([qd 18] n = 4, 50 µg/kg); B) 9 doses every other day for 3 weeks ([qod 9] n = 4, 5 µg/kg); C) a single dose ([qd 20 hours] n = 4, 100 µg/kg) one day (approximately 20 hours) following TBI, or D) a single dose ([qd 1 hour] n = 3, 100 µg/kg) 1 hour following TBI. The irradiation controls (n = 10) received 0.1% AS, daily for 18 days. Complete blood counts were monitored for 60 days following irradiation and the duration of neutropenia (ANC < 500/ul) and thrombocytopenia (platelet [PLT] < 20,000/ul) was assessed. BM-derived clonogenic activity was examined prior to irradiation (baseline) and on days 7, 14, 21, 46 post-TBI.


   CLINICAL SUPPORT
Top
 Abstract
 Introduction
 Materials and Methods
 Clinical Support
Results
 Discussion
 Acknowledgements
 References
 
Clinical support consisting of an antimicrobial regimen and whole blood transfusions were provided to all animals as required. An antibiotic regimen and transfusions of fresh, irradiated (1,500 cGy, Cobalt-60 {gamma}-irradiation) whole blood were administered as previously described [11, 22].

Hematologic Evaluations

CBCs   Peripheral blood was obtained from the saphenous vein to assay complete blood (Sysmex K-4500; Long Grove, IL; http://www.sysmex.com) and differential counts (Wright-Giemsa Stain, Ames Automated Slide Stainer; Elkhart, IN) for 60 days post-TBI.

BM-Derived Clonogenics   Animals were sedated with (Ketaset® [10 mg/kg, i.m.] Fort Dodge Laboratories; Fort Dodge, IA) plus buprenorphine (Buprenex® Injectable [10 µg/kg, i.m.] Reckitt & Colman Pharmaceuticals; Richmond, VA; http://www.reccolpharm.com) and approximately 2 ml of heparinized-BM were aspirated from the humerus. Low-density (<1.077 g/cm3) mononuclear cells (MNC) were separated and cultured as previously described [32]. Granulocyte-macrophage colony-forming cells (GM-CFC) and burst forming units-erythroid (BFU-E)-derived colonies (>50 cells) were expressed as the number of CFC/105 MNC. Megakaryocyte (MK)-CFC (10-50 cells/colony) are not distinguished from MK-burst-forming cells in this study, and are termed MK-CFC.

Pharmacokinetic Analysis
PMP-1a plasma concentrations were determined by enzyme-linked immunosorbent assay utilizing a goat polyclonal anti-c-Mpl ligand capture antibody precoated on a microtiter plate and an anti-IL-3 receptor agonist (rHuIL-3) antibody-peroxidase conjugate. PMP-1a was quantified by the chromogenic reaction of the antibody-peroxidase conjugate with a peroxidase substrate detectable at 650 nm. PMP-1a concentrations were determined by reference to a standard curve prepared with PMP-1a in plasma with a sensitivity of 0.313 ng/ml. Rhesus monkeys were dosed i.v. with 10 µg/kg PMP-1a and plasma samples were obtained at predose, 2, 5, 15, and 30 minutes and 1, 1.5, 2, 3, 5, 8, 24, and 48 hours after administration. Alternatively, a different cohort was dosed with 20 µg/kg s.c. and plasma samples were obtained at predose, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 hours after administration. Pharmacokinetic parameters were calculated using the AUC, NONLIN, and CSTRIP computer programs.

Statistical Analysis
The Normal Scores Test was used to make pair-wise comparisons of the durations of neutropenia and thrombocytopenia and to evaluate the statistical significance between the nadirs. The exact p values were obtained. The test was carried out using the software package StatXact (Cytel Software Corp.; Cambridge, MA; http://www.cytel.com).

Clonogenic Analysis
For each day (d 7-d 46) a Kruskal-Wallis Test was run comparing all treatments including control. When results of this test were significant (p < .05), Dunn's test was used post hoc to compare treatments with control for each day.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Clinical Support
 Results
Discussion
 Acknowledgements
 References
 
Pharmacokinetics of PMP-1a in Normal Primates
Terminal-phase elimination half-life and total plasma clearance of PMP-1a following i.v. administration at 10 µg/kg were 4.11 hours and 2.65 ml/min/kg, respectively. The central compartment volume of distribution and steady-state volume of distribution were less than total blood volume suggesting low uptake into tissues. Systemic availability of PMP-1a following 20 µg/kg s.c. administration was 41.3%. Peak plasma concentration was 8.35 ng/ml and was reached at 1.75 hours (Table 1Go).


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  		Table 1. Plasma pharmacokinetic parameters for recombinant-derived PMP-1a, after i.v. or s.c. administration to normal rhesus monkeys
 
Modulation of Thrombocytopenia: Duration, Nadir, Recovery Time, and Transfusion Requirements
Control animals exposed to 600 cGy TBI experienced thrombocytopenia (PLT < 20,000/µl) characterized by a duration of 10.4 days and a nadir of 3,000/µl. The recovery time to PLT levels >=20,000/µl required 20.7 days. Therapeutic administration of PMP-1a in schedules of either qd for 18 consecutive days (qd 18), qod for nine injections from days 1-17 (qod 9) or single injections at either 20 hours or 1 hour post-TBI significantly improved all platelet-related parameters relative to controls (Table 2Go). The duration of thrombocytopenia was reduced to zero (p <= .003) in all PMP-1a-treated cohorts, platelet nadirs were significantly increased (p <= .003) to 34,250, 40,500, 47,000, and 28,667/µl for the qd 18, qod 9, qd (20 hours), and qd (1 hour) cohorts, respectively. Consequently, recovery times to PLT levels of >= 20,000/µl were reduced significantly (p <= .003) to zero in all cohorts with the exception of qod 9 (3.5 days) relative to the 20.7 days required for the control-treated cohort (Fig. 1Go, Table 2Go). All PMP-1a-treated cohorts were platelet transfusion-independent relative to the average 2.6 transfusions required for the controls (Table 2Go) (p <= .001).


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  		Table 2. Thrombocytopenia and neutropenia in sublethally irradiated and PMP-1a-treated nonhuman primates: duration, nadir, time to recovery, transfusion, and antibiotic requirements; duration, nadir; time to recovery, and clinical support
 


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  		Figure 1. Effects of PMP-1a administration in four different schedules on PLT peripheral blood counts in 600 cGy, x-irradiated nonhuman primates. The PLT were observed prior to and after treatment with control protein (0.1% AS, n = 11), or PMP-1a administered: A) qd for 18 days (qd 18, n = 4); B) qod for 9 injections (qod 9, n = 4); C) qd one time at 20 hours (qd 20 hours, n = 4), and D) qd one time at 1 hour post TBI (qd 1 hour, n = 3) as described in Materials and Methods. Data represent the mean values (± SE) of absolute platelet counts.

 
Modulation of Neutropenia: Duration, Nadir, Recovery Time, and Days on Antibiotics
Consequent to 600 cGy TBI, control-treated animals experienced neutropenia (ANC < 500/µl) of a 16.2-day duration with a mean ANC nadir of 3/µl requiring an average 19.3 days of antibiotic treatment. The recovery time for ANC to >= 500/µl was 21.7 days (Fig. 2Go, Table 2Go). PMP-1a treatment significantly improved (p <= .033) the ANC nadirs in all treatment cohorts to 72/µl, 42/µl, 61/µl, and 32/µl for the qd 18, qod 9, qd 20 hours, and qd 1 hour cohorts, respectively, relative to the control cohort (3/µl). The qd 18 (13.3 days) and qod 9 (13.3 days) but not the single injection qd 20 hours (14.3 days) or qd 1 hour (14.3 days) of PMP-1a administration schedules significantly improved the neutropenic duration (p <= 0.011) relative to the control value (16.2 days). Furthermore, the recovery time of ANC >= 500/µl was significantly shortened in the qd 18 (19.0 days) and qod 9 (19.3 days) cohorts but not the qd 20 hours (21.5 days) or the qd 1 hour (20.7 days) cohorts compared to the AS-treated controls (21.7 days) (p <= .011). The corresponding days on antibiotics were significantly reduced in the qd 18 (16.0 days) and qod 9 (16.5 days) PMP-1a treatment cohorts (p <= .005), whereas the qd 20 hours (18.0 days) and qd at 1 hour (17.7 days) PMP-1a injection did not modify the antibiotic requirements relative to the control cohort (19.2 days) (Table 2Go).



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  		Figure 2. Effects of PMP-1a administration in four different schedules on peripheral blood ANC in 600 cGy, x-irradiated nonhuman primates. The ANCs were observed prior to and after treatment with control protein (0.1% AS, n = 11), or PMP-1a administered: A) qd for 18 days (qd 18, n = 4); B) qod for 9 injections (qod 9, n = 4); C) qd one time at 20 hours (qd 20 hours, n = 4), and D) qd one time at 1 hour post TBI (qd 1 hour, n = 3) as described in Materials and Methods. Data represent the mean values (± SE) of the ANC.

 
Modulation of RBC
The RBC in the irradiated cohort decreased from a baseline preirradiation value of 5.5 x 106/µl to a nadir of 2.8 x 106/µl entering the third week post TBI (Fig 3Go). PMP-1a treatment regardless of administration schedule improved the RBC nadir to values ranging from 3.5-3.8 x 106/µl. The one-time PMP-1a administration, whether at 20 hours or 1 hour post-TBI, was as effective as the conventional qd 18 schedule. It is worth noting that the control cohort received an average 2.6 whole blood transfusions per animal during the nadir period, whereas the PMP-1a-treated cohorts were transfusion-independent (Table 2Go). The transfusions administered to the control cohort undoubtedly influenced the observed RBC nadir and recovery kinetics.



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  		Figure 3. Effects of PMP-1a administration in four different schedules on peripheral RBC counts in 600 cGy, x-irradiated nonhuman primates. The RBCs were observed after treatment with control protein (0.1% AS, n = 11), or PMP-1a administered: A) qd for 18 days (qd 18, n = 4); B) qod for 9 injections (qod 9, n = 4); C) qd one time at 20 hours (qd 20 hours, n = 4), and D) qd one time at 1 hour post TBI (qd 1 hour, n = 3) as described in Materials and Methods. Data represent the mean values (± SE) of the RBC counts.

 
BM-Derived Clonogenic Activity
BM-derived MK-CFC, GM-CFC, and BFU-E clonogenic activity was evaluated before and at days 7, 14, 21, and 46 after TBI. In the control-treated cohort the concentration of MK-CFC required approximately 21 days to return to within preirradiation levels whereas GM-CFC and BFU-E activity remained decreased through day 46 after TBI (Table 3Go). PMP-1a treatment, independent of administration schedule, induced an earlier recovery of MK-CFC, GM-CFC, and BFU-E than that noted in time-matched controls. All three lineage-specific CFCs exhibited a return to preirradiation levels or an overshoot within 21 days post-TBI, (single exception was PMP-1a qod 9 cohort for BFU-E).


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  		Table 3. Recovery of BM-derived clonogenic activity (CFC/105 MNCs as percent of baseline) in animals exposed to 600 cGy x-irradiation and treated with different administration schedules of PMP-1a
 
Comparison of PMP-1a-Induced Hematopoietic Recovery to that of Coadministered Daniplestim and Mpl-L
We previously investigated the relative efficacy of coadministered daniplestim (bid) plus truncated-Mpl-L (qd) to monotherapy with daniplestim (bid) or truncated-Mpl-L (qd) in the same model of radiation-induced myelosuppression [21]. We have added the key parameters in Table 2Go for ready comparison to those generated herein. In that study, the coadministered daniplestim/Mpl-L significantly enhanced recovery of all platelet and neutrophil-related parameters relative to the control-treated cohort (Table 2Go) (p <= 0.12). Also, the coadministered daniplestim/Mpl-L significantly improved platelet nadir (p = .029), thrombocytopenic duration (p = .029), and platelet recovery (p = .043) relative to the effects of daniplestim monotherapy, and further improved all the above parameters relative to that observed with Mpl-L monotherapy. Herein, we show that PMP-1a further improved all platelet-related parameters relative to the coadministered daniplestim and Mpl-L (Table 2Go).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Clinical Support
 Results
 Discussion
Acknowledgements
 References
 
Administration of PMP-1a, an engineered, chimeric IL-3 and Mpl-L receptor agonist, has demonstrated profound platelet regeneration in a nonhuman primate model of severe radiation-induced myelosuppression. Furthermore, PMP-1a, regardless of conventional or abbreviated administration schedules (qd 1-18, qod 9 injections, or single injections within 20 hours or 1 hour of irradiation) improved upon all platelet-related parameters noted previously with the coadministration of its component IL-3 and Mpl-L receptor agonists [21]. PMP-1a stimulated platelet production to such a degree that the resultant improvement in platelet nadirs virtually eliminated thrombocytopenia and the need for whole blood transfusions. Furthermore, in concert with the noted pleiotropic and synergistic activity of the Mpl-L and the multilineage effects of IL-3, the chimeric PMP-1a enhanced both red cell and neutrophil recovery as well as BM-derived MK-CFC, GM-CFC, and BFU-E relative to the preclinical activity of daniplestim or Mpl-L alone or in combination. When compared to the combination of the respective PMP-1a components, daniplestim and truncated-Mpl-L, PMP-1a further improved the time to recovery of platelet counts to >= 20,000/µl, the number of transfusions, the duration of thrombocytopenia, and the platelet nadirs.

TPO has been characterized as the primary physiologic regulator of MK and PLT development [11, 27, 33-37]. Preclinical trials in rodents and nonhuman primates substantiate its ability to promote enhanced MK and PLT regeneration subsequent to radiation and/or chemotherapy-induced myelosuppression [11, 13, 14, 20, 38, 39]. Furthermore, a wide range of studies has indicated that TPO has profound effects on early hematopoiesis. Several in vivo studies showed that TPO administration enhanced erythroid and myeloid recovery in mice and nonhuman primates suggesting an effect on multipotent progenitors [11, 13, 33, 40-43]. In concert, in vitro analysis indicated that TPO had direct and synergistic effects on populations of early hematopoietic progenitors. TPO alone or in combination with GFs such as c-kit-L, IL-3, or Flt3-L stimulated proliferation of primitive hematopoietic progenitors [25, 26, 44-46]. Ku et al. [47] showed that the combination of TPO with IL-3 or c-kit-L accelerated the entry of post-5-fluorouracil, primitive hematopoietic cells into cell cycle and enhanced the generation of multipotent progenitors. The use of TPO in combination with c-kit-L or Flt3-L helped progenitors retain a primitive phenotype and maintain the capacity for multilineage differentiation [48].

These results were substantiated by the targeted gene disruption models of c-mpl and TPO-deficient mice and analysis of receptor expression and function on the hematopoietic repopulating ability of primitive hematopoietic cells. Mice produced with the c-mpl and TPO deficiency showed significant reduction in myeloid, erythroid and multilineage progenitor cells in marrow, spleen and peripheral blood [33, 34, 36, 40]. Kimura et al. [49] further demonstrated that the mpl–/– deficient mice had greatly reduced numbers of colony-forming units (CFUs), and the marrow cells were markedly inferior to their mpl+/+ controls in a competitive repopulation assay. Solar et al. [43] extended these studies by showing that all hematopoietic repopulating ability resides in the mpl+ subset of primitive murine fetal liver or marrow cells and that the CD34+38– mpl+ cells would engraft SCID-hu bone mice more efficiently than the mpl-counterparts.

Furthermore, recent studies have emphasized the ability of TPO to support the viability of hematopoietic progenitor cells (HPCs) [50-55]. TPO can suppress GF withdrawal-induced apoptosis in the MO7e cell line [55] as well as promote clonal growth with suppression of apoptosis in murine primitive Sca+lin– and human CD34+ Thy1+ cells [23, 48, 51, 56]. In this regard, it has been suggested that TPO upregulates the promoter conformation of p53 in MO7e cells which has a diminished ability to mediate cell cycle arrest and apoptosis [54]. Ritchie et al. further suggest that this functional inactivation of p53 allows for developing MKs to undergo endomitosis, thus facilitating maturation and thrombopoiesis [54]. Other studies have suggested that suppression of p53 function facilitates hematopoietic recovery following chemotherapy by delaying exhaustion of the primitive stem cell pool, stimulating production of multipotent HSCs, and increasing the sensitivity of HPCs to GFs such as c-kit-L, TPO, and Flt3-L [53, 57].

IL-3, the other component of PMP-1a, is known for its anti-apoptotic effects in HPCs [58, 59], while several studies have suggested that IL-3 negatively impacts in vitro stem cell expansion [60, 61]. Bryder and Jacobsen [62] have recently shown that long-term repopulating HSCs are not compromised by IL-3 stimulation even after multiple cell divisions. Furthermore, while IL-3 is not required for residual platelet production in c-mpl–/– deficient mice [63], it may be required in emergency states such as after irradiation or chemotherapy. Kaushansky [27] has suggested that although TPO is the primary physiologic regulator of MK development and PLT production, there may be a population of MK progenitors responsive to IL-3 but requiring TPO for full development.

These pleiotropic activities of the PMP-1a component GFs namely, to act alone and in combination to stimulate thrombopoiesis, as well as to act alone and in synergy with other GFs to stimulate self-renewal and proliferation of primitive c-mpl+ HPCs and preserve viability and integrity of primitive HPCs by suppressing p53-mediated apoptosis effects, provide the mechanistic underpinning for its marked hematopoietic effects.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Clinical Support
 Results
 Discussion
 Acknowledgements
References
 
The authors wish to thank Michael Flynn, Lisa B. Lind, Lorelei Dacquel Smith, Daniel Casey, Heather Webster, Mark Abram, Doreen Villani-Price, and Gerald Galluppi for their superb technical assistance. This work was supported by a grant from Searle Research and Discovery, Monsanto Co., St. Louis, MO.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Clinical Support
 Results
 Discussion
 Acknowledgements
 References
 

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Received March 27, 2001; accepted for publication March 28, 2001.




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