Stem Cells, Vol. 14, No. 3, 330-336,
May 1996
© 1996 AlphaMed Press
Thrombopoietin Suppresses Apoptosis and Behaves as a Survival Factor for the Human Growth Factor-Dependent Cell Line, M07e
Alec Ritchiea,
Saroj Vadhan-Rajd,
Hal E. Broxmeyera,b,c
a Departments of Microbiology/Immunology,
b Medicine, and
c the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA, and
d the MD Anderson Cancer Center, Houston, TX, USA
Key Words. M07e • Thrombopoietin • Survival factors • Growth factors • Apoptosis
Dr. Hal E. Broxmeyer, Walther Oncology Center, Indiana University School of Medicine, 975 W. Walnut Street, Indianapolis, IN 46202-5121, USA.
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Abstract
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Thrombopoietin (TPO) has been demonstrated to have proliferative effects on hematopoietic progenitor cells and maturational effects on more committed populations which express a megakaryocyte lineage-specific phenotype. M07e is a GM-CSF or interleukin 3 (IL-3)-dependent human leukemic cell line having surface markers characteristic of both myeloid progenitors and megakaryocytes. The effects of TPO on the proliferation and survival of M07e cells were investigated. Following an 18-h factor starvation period to remove residual growth factor signals and phase the cells in G0/G1, TPO provides a weak proliferative signal to M07e compared to IL-3 or GM-CSF treatment under the same conditions. However, TPO synergizes with both GM-CSF and IL-3, and to a greater extent with steel factor, a competence factor for M07e, in the induction of cellular proliferation. TPO sustains cellular integrity of M07e during prolonged (18 days) growth factor withdrawal and also protects M07e cells in serum-free conditions. In addition, preincubation of M07e for 72 h in TPO maintains its survival for subsequent cytokine-induced proliferation, while control media do not. TPO suppresses growth factor withdrawal-induced apoptosis as evaluated by flow cytometric detection of both in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and cellular DNA content via propidium iodide staining. These results suggest a role for TPO as a survival factor for M07e cells.
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Introduction
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Thrombopoietin (TPO), also referred to as megakaryocyte growth and development factor or the c-Mpl ligand, is a recently purified and cloned hematopoietic cytokine whose biological significance has been described by a number of groups [1–5 ]. TPO has been shown to act in the stimulation of progenitor cell proliferation and in the induction of megakaryocyte maturation into platelets in vitro [1– 3,5– 7 ]. Furthermore, mice treated with TPO show markedly expanded megakaryocyte pools and elevated platelet production [2, 3 ]. TPO is therefore believed to be a critical thrombopoietic and megakaryocytopoietic regulator. However, TPO is not the only hematopoietic growth factor that can support megakaryocytopoiesis, as interleukin 3 (IL-3), and to a lesser extent steel factor (SLF), can also induce the stimulation of megakaryocyte colony formation [8–11 ]. SLF is a potent costimulatory molecule which can augment the effects of IL-3 in the induction of megakaryocyte-progenitor colony formation, but has little effect on this lineage when used alone [2, 8, 12 ]. M07e is an IL-3- or GM-CSF-dependent human leukemic cell line that expresses both early progenitor and platelet-restricted surface markers [13]. SLF alone does not induce a high level of proliferation in M07e but does synergize with both GM-CSF and IL-3 in this regard [14]. Since M07e has been shown to respond to TPO previously [15,16], and also responds to IL-3 and SLF in a manner similar to that described for the in vitro megakaryocytopoiesis mentioned above, we decided to evaluate the effects of TPO on M07e proliferation alone or in conjunction with cytokines known to be either progression or competence factors for M07e, and the maintenance of cell integrity during growth factor and serum deprivation.
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Materials and Methods
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Cytokines and Reagents
Recombinant human (rHu)GM-CSF, rHuIL-3 and rHuSLF were kind gifts from Immunex Corporation, Seattle, WA. rHuTPO was kindly provided by Zymogenetics, Seattle, WA. Concentrations used in these assays were 100 U/ml for GM-CSF, 500 U/ml for IL-3, 100 ng/ml for SLF and 500 U/ml for TPO. The in situ cell death detection kit employing the dUTP nicked end-labeling (TUNEL) method of labeling fragmented DNA was purchased from Boehringer Mannheim, Indianapolis, IN. Dilipidated, deionized and dialyzed bovine serum albumin (BSA), insulin, saturated human transferrin, low density lipoprotein (LDL) and propidium iodide (PI) were purchased from Sigma Chemical Co., St. Louis, MO.
Cells
The human growth factor-dependent subline, M07e, was obtained from Genetics Institute, Boston, MA. The M07e subline, and its parental line M07, have been biologically characterized previously [13,14,17]. The culture and factor-starvation conditions for M07e have also been described in detail [14,18]. Serum-free media consisted of RPMI 1640 (Bio Whittaker; Walkersville, MD) with 2% dilipidated, deionized and dialyzed BSA, 10µg/ml insulin, 200 µg/ml saturated human transferrin, 50 µM beta-mercaptoethanol and 40 µg/ml LDL.
Short-Term Proliferation Assay in Suspension Culture
The M07e proliferation assay has been described previously [14,17]. Cell viability assessment for the initiation of cultures and experimental endpoint determination of cell viability was made by trypan blue exclusion in hemocytometric counts. Preincubation studies were carried out in 25 cm2 culture flasks at 1 x 106 cells/ml in RPMI 1640 plus 20% fetal bovine serum (FBS) (Hyclone Laboratories; Logan, UT) either with 500 U/ml TPO or control media alone. After 72 h, the cells were collected, washed twice in RPMI 1640, and viable cell counts were determined to initiate short-term proliferation assays.
DNA Fragmentation Analysis
Cells treated with either TPO or control media were subjected to PI staining or in situ TUNEL. Briefly, half of the cells were gently resuspended in a hypotonic PI solution of 50 µg/ml PI in 0.1% sodium citrate with 0.1% Triton-X for 20 min, and the other half of the cells was washed, fixed in paraformaldehyde, permeabilized and subjected to in situ terminal deoxyribonucleotide transferase-mediated TUNEL. Both preparations were then analyzed by flow cytometry.
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Results and Discussion
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M07e Provides a Weak Growth and Proliferative Signal to M07e Cells but Synergizes with GM-CSF, IL-3 and SLF in Stimulating Proliferation of M07e
We evaluated the ability of TPO to stimulate proliferation of M07e cells in liquid culture. M07e cells responded to TPO in a dose-dependent manner, reaching a maximal effect at ~500 U/ml of TPO as measured by tritiated thymidine incorporation (Fig. 1
); therefore, 500 U/ml of TPO was chosen as the working concentration in our further assays. When used alone, TPO did not induce a time-dependent change in cell viability (Fig. 2A) or 3H-Thymidine uptake (Fig. 2B), compared to IL-3 or GM-CSF treatment under the same conditions. The positive effect that TPO did have on M07e cell growth was transient (24 h), thereafter viable cell counts gradually returned to the initial inoculum concentration (Fig. 2A). When TPO was combined with GM-CSF (100 U/ml) or IL-3 (500 U/ml), a greater than additive effect on proliferation of M07e was observed (Fig. 3). SLF alone did not induce a high level of proliferation, but when combined with TPO it greatly enhanced the proliferation of M07e, synergizing with TPO to a degree higher than that observed with GM-CSF+TPO or IL-3+TPO combinations. To evaluate the notion that TPO could be lowering the degree of growth factor dependency in M07e, cultures were incubated in increasing amounts of GM-CSF supplemented with a constant amount of TPO. Figure 4 illustrates that TPO lowers the amount of GM-CSF required to induce a given amount of proliferation in M07e throughout the 0.1 to 100 U/ml range of concentrations tested (p 
0.006, Student's t-test). Although others have reported that TPO can induce a state of proliferation in M07e comparable to that induced by IL-3 [16], our lesser proliferative effects with TPO could reflect different subsets of M07e used.
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Figure 1. Dose-response curve of M07e to TPO. Cells were factor starved for 18 h then adjusted to 5x104cells/ml in RPMI 1640 with incremental amounts of TPO or control media alone, and 200µl of cell suspensions were deposited in triplicate wells of microtitre plates. After a 68-h incubation, cultures were pulsed for 4 h with tritiated thymidine (0.5µCi/well), harvested onto filter paper, immersed in scintillation fluid and counts per minute (CPM) were determined. Each data point was sampled in triplicate. This figure represents data from one of two experiments which yielded similar results.
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Figure 2. Time-related observations of M07e cell growth in response to TPO or other growth factors. A) Viable cell counts of M07e during a 72-h incubation in either TPO (500 U/ml), GM-CSF (100 U/ml), IL-3 (500 U/ml), SLF (100 ng/ml) or control media. At each time point, 100µl of cell suspension were added to 100µl of trypan blue solution and cell viability was assessed by hemocytometric counts. B) Proliferation measurement of M07e cells during a 72-h incubation in either TPO, GM-CSF, IL-3, SLF or control media. For each experimental point in part A, the tritiated thymidine incorporation was measured as described for experiments in Figure 1 . Each data point was sampled in duplicate for the viability assay, and in triplicate for the short-term proliferation assay. These figures represent data from one of three experiments which yielded similar results.
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Figure 3. Measurement of tritiated thymidine incorporation of M07e cells in response to a 72-h treatment with TPO (500 U/ml), GM-CSF (100 U/ml), IL-3 (500 U/ml), SLF (100 ng/ml), GM-CSF + TPO, IL-3 + TPO, SLF + TPO or control medium alone. Each data point was sampled in triplicate. This figure represents data from one of three experiments which yielded similar results.
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Figure 4. Measurement of tritiated thymidine incorporation of M07e cells in response to a 72-h treatment with TPO (500 U/ml) or control medium and increasing amounts of GM-CSF. Each data point was sampled in triplicate. This figure represents data from one of three experiments which yielded similar results.
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TPO Preincubation Enhances Survival of M07e Cells for Subsequent Cytokine-Induced Proliferation
We then evaluated the ability of TPO to maintain the growth factor responsiveness of M07e cells after a three-day growth factor deprivation period. M07e cells were cultured for 72 h in the presence of either TPO or control medium containing 20% FBS. After this three-day incubation period, the cells were washed in RPMI 1640 to remove residual TPO in TPO-containing cultures. After cell adjustment, either TPO, GM-CSF, IL-3, SLF or control medium was added. Preincubation with TPO resulted in the subsequent proliferation of M07e cells when treated with cytokines while control medium preincubation did not (Fig. 5). Moreover, the degree of proliferation seen when the growth factors were added at day 3 after TPO preincubation was very similar to that seen when the growth factors are added at day 0 (Fig. 2A). This not only suggests that cellular membrane integrity is being maintained, as evaluated by trypan blue exclusion, but also that the integrity of intracellular subsystems including metabolic pathways and signaling cascades are preserved throughout the preincubation period.
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Figure 5. Preincubation of M07e in TPO sustains viability for subsequent cytokine treatment. After 72-h incubation in either TPO or control medium in the presence of serum, the samples were washed and resuspended in either TPO (500 U/ml), GM-CSF (100 U/ml), IL-3 (500 U/ml), SLF (100 ng/ml) or control medium and a subsequent 72-h incubation followed. Tritiated thymidine incorporation was then measured as described for Figure 1. Each data point was sampled in triplicate. This figure represents data from one of three experiments which yielded similar results.
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TPO Provides a Survival-Promoting Effect for M07e Cells During Serum Withdrawal and Long-Term Growth Factor Deprivation
We next evaluated the ability of TPO to protect M07e cells under serum-free conditions. After a 72-h incubation, tritiated thymidine incorporation in M07e cells treated with TPO was 255 ±17% of control media cultures (Fig. 6). In an attempt to discern what length of time TPO could provide a survival-promoting effect on M07e cells, cultures were initiated with either TPO, GM-CSF or control medium, and allowed to incubate for three-day intervals (Fig. 7A). Every three days, cell viability was assessed, the cultures were split 1:5 with fresh media and allowed to incubate an additional three days. GM-CSF-treated cells rapidly proliferated, TPO-treated cells remained static, while control cultures quickly died off. M07e cells were also cultured for an 18-day period, during which the medium was not replenished (Fig. 7B). Viable cell counts in TPO-containing cultures remained relatively constant for one week, after which cell numbers transiently rose and returned to baseline before the end of the 18-day period. GM-CSF-stimulated cells reached a maximum viable cell density at one week, after which cell viability fell precipitously and reached zero by the end of the 18-day period. Control medium provided neither a proliferative stimulus nor protected M07e against cell death, as cell viability reached zero within three days in control media cultures. The failure of GM-CSF-treated cultures to thrive for extended periods of time could be attributed to the high density of cells reached, and concomitant depletion of growth factor and nutrients. In comparison, viability is sustained in the presence of TPO, which could reflect a lack of nutrient and cytokine depletion, in turn due to a lack of an appreciable induced amount of metabolic and/or proliferative activity.
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Figure 6. TPO protects M07e during serum deprivation. M07e was incubated in RPMI 1640 with 20% FBS or in serum-free media supplemented with either TPO (500 U/ml), GM-CSF (100 U/ml), IL-3 (500 U/ml), SLF (100 ng/ml) or control medium alone for 72 h. Tritiated thymidine incorporation was measured following a 4-h pulse as described for Figure 1. Each data point was sampled in triplicate. This figure represents data from one of three experiments which yielded similar results.
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Figure 7. TPO maintains cell viability over extended periods of time. A) M07e cells were cultured with TPO, GM-CSF or control medium for three days, after which time cell viability was assessed via trypan blue exclusion. The cultures were diluted 1:5 with fresh medium and allowed to incubate an additional three days. The viability assessment/subculturing was repeated again at six days and a final assessment was made on day 9. B) M07e cells were cultured with TPO, GM-CSF or control medium for 18 days without medium renewal. Cell viability assessment was made at the days indicated, in duplicate. These figures represent data from one of two experiments which yielded similar results.
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TPO Suppresses Growth Factor Withdrawal-Induced Apoptosis
To support the observation that TPO does not induce substantial proliferation but rather maintains cell viability, the ability of TPO to retard the apoptotic process was evaluated. TPO-containing and control medium cultures were incubated for 24 h, after which they were subjected to TUNEL (Fig. 8A, B) and PI (Fig. 8C, D) staining and examined by flow cytometry. TPO-treated cells had a markedly decreased population of apoptotic cells (4.5%) compared to control media cells (37.8%). This proportion of apoptotic cells in the control media culture reflects the fraction of nonviable cells in similar control medium cultures as detected by trypan blue exclusion after 24 h (Fig. 2A). By failing to provide a proliferative stimulus, but instead protecting M07e from growth factor withdrawal-induced apoptosis, these data further support a primarily survival-enhancing role for TPO in M07e cells. The mechanistic components of the suppressive activity of TPO for apoptosis remain to be elucidated at this point, but further studies probing the biochemical and biological relationship between TPO signals and the suppression of apoptosis in M07e are underway.
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Figure 8. TPO protects M07e from growth factor withdrawal-induced apoptosis. A) M07e treated with TPO (A) or control medium (B) for 24 h was subjected to TUNEL as described in Materials and Methods. The higher level of fluorescence seen in control media cultures reflects the increased availability of DNA fragment ends for labeling due to endonuclear degradation during the apoptotic process. B) M07e treated with control media (C) or TPO (D) for 24 h was stained with PI and the cellular DNA content was measured by flow cytometry. During apoptosis, the cellular DNA content becomes subdiploid due to nuclear degradation and subsequent cytoplasmic shedding, and as a result a shift to the left in fluorescent intensity is seen when the amount of PI per cell is quantitated.
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Acknowledgments
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This study was supported by Public Health Service Grants R37 CA36464, RO1 HL-56416, RO1 HL-49202, RO1 54037 and by a project in PO1 HL-53586 to H.E.B.
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Footnotes
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Provisionally accepted March 8, 1996.
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Received January 8, 1996;
accepted for publication March 18, 1996.
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Abstract
Introduction
