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Cell Growth & Differentiation Vol. 13, 275-283, June 2002
© 2002 American Association for Cancer Research

The Role of Growth Factors in the Activity of Pharmacological Differentiation Agents1

William H. Matsui, Douglas E. Gladstone2, Milada S. Vala, James P. Barber, Robert A. Brodsky, B. Douglas Smith and Richard J. Jones3

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University, Baltimore, Maryland 21231


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Bryostatin-1 inhibits acute myeloid leukemia (AML) in vitroat doses that stimulate the growth of normal hematopoietic progenitors.Although bryostatin-1 has a number of distinct biological activities, those specifically responsible for its antileukemic activity are unclear. We found that bryostatin-1 (10-8 M) inhibited cell cycling at G1, induced phenotypic evidence of differentiation, and limited the clonogenic growth of both AML cell lines and patient specimens. This activity was markedly enhanced by granulocyte/macrophage-colony stimulating factor, whereas growth factor-neutralizing antibodies completely inhibited both the differentiating and antileukemic activities of bryostatin-1. Cell cycle inhibition and growth factors were also required for the differentiating activities of two unrelated agents, hydroxyurea and phenylbutyrate. These data suggest that many pharmacological differentiating agents require both cell cycle arrest and lineage-specific growth factors for full activity and may explain why these agents have demonstrated only limited clinical efficacy.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Induction therapy with standard cytotoxic chemotherapeutic agents produces complete remissions in the majority of patients diagnosed with AML.4 Unfortunately, most of these patients relapse and eventually die from their disease (1) . Efforts to increase the overall and disease-free survival rates in AML have focused on improving the efficacy of postremission consolidation by administering dose-intensified cytotoxic chemotherapeutic agents alone or combined with autologous or allogeneic stem cell support (2 , 3) . These approaches have improved the clinical outcomes for selected subsets of patients with AML but have also increased the incidence of treatment-related morbidity and mortality; therefore, novel agents and therapeutic modalities are needed.

Bryostatin-1 is a promising agent in the treatment of AML. Numerous in vitro studies have shown that bryostatin-1 has inhibitory effects on both AML cell lines and patient samples, whereas it stimulates the growth of normal hematopoietic cells (4, 5, 6, 7, 8, 9) . The antileukemic activity of bryostatin-1 is also enhanced by a number of compounds, including the cytotoxic chemotherapeutic agent ara-C and the myeloid growth factor GM-CSF (10 , 11) . Although it is not surprising that bryostatin-1 augments the cytotoxic effects of ara-C because each has antitumor activity on its own, the interaction between bryostatin-1 and GM-CSF is somewhat surprising, given that GM-CSF alone may increase the proliferation and survival of AML cells both in vitro and in vivo (12, 13, 14) .

The effects of bryostatin-1 are mediated by PKC. Acute exposure to bryostatin-1 activates PKC, whereas chronic treatment results in the down-regulation of PKC through the ubiquitination and subsequent proteolysis of the cPKC{alpha} subunit (15, 16, 17, 18) . Although its cellular target has been well delineated, bryostatin-1 has a number of distinct biological properties, and the actual mechanism responsible for its antileukemic activity is largely speculative. Similar to the prototypical PKC activator, phorbol ester, bryostatin-1 induces the differentiation of AML cell lines (19, 20, 21) . It also inhibits cell proliferation by blocking cell cycling at G1 or both G1 and G2-M that is mediated by the inhibition of cdk2 and the up-regulation of the cdk inhibitors, p21cip1/waf1 and p27kip1 (20 , 22) . Furthermore, bryostatin-1 has potent immunoregulatory properties. It induces the expression of IL-2 receptors on CD4+ and CD8+ T cells and enhances IL-2-mediated activation of in vivo primed CTLs (23 , 24) . To better define the cellular processes responsible for its antileukemic activity, we studied the effects of bryostatin-1, alone and in combination with GM-CSF, on AML cell lines and patient specimens.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Effects of Bryostatin-1 on AML Cells.
We initially examined the effect of bryostatin-1 on the growth, self-renewal, and differentiation of AML cells. We have demonstrated previously that bryostatin-1 inhibits the growth of myeloid leukemia at concentrations between 10-9 and 10-7 M, with 10-8 M generally being the most active (6) . At this concentration, bryostatin-1 inhibited the proliferation of the human AML cell lines U937 and HL60 associated with cell cycle arrest at G1 (Table 1)Citation but without significant cytotoxicity, as assessed by trypan blue dye exclusion or flow cytometric analysis of apoptosis (data not shown). We also studied the effect of bryostatin-1 on the self-renewal of each cell line by examining clonogenic AML growth in methylcellulose. After 120 h of treatment in liquid culture and before plating, cells were washed to remove bryostatin-1 and ensure that colony formation reflected clonogenic potential at the end of the treatment period in liquid culture, rather than a direct antiproliferative effect of bryostatin-1 during the subsequent growth in methylcellulose. Compared with untreated cells, the clonogenic recovery of both cell lines was modestly inhibited by bryostatin-1 alone (Fig. 1)Citation . Because the inhibition of clonogenic growth without direct cytotoxicity could result from the induction of terminal differentiation, cells were examined for evidence of myeloid differentiation. Previous studies have demonstrated that U937 cells primarily develop monocytic features, whereas HL60 cells can undergo either granulocytic or monocytic differentiation (25 , 26) . Bryostatin-1 modestly induced the phenotypic differentiation of both U937 and HL60 cells, as evidenced by nuclear condensation on morphological examination (data not shown) and enhanced expression of the monocytic cell surface antigens CD11b and CD14 (Fig. 2)Citation .


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Table 1 Cell cycle inhibition by pharmacological differentiating agents

U937 cells were incubated alone or in the presence of byrostatin-1 (10 nM) with or without GM-CSF (200 units/ml), hydroxyurea (100 µg/ml), or phenylbutyrate (1.5 mM) for 48 h and then analyzed for cell cycle distribution. Results are provided as the percentage of total cells and represent the mean ± SE of five separate experiments.

 


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Fig. 1. Effects of bryostatin-1 and GM-CSF on AML cell line clonogenic growth. U937 (A) and HL60 (B) cells were incubated in the absence (Control) or presence of bryostatin-1 (10-8 M) with or without GM-CSF (200 units/ml). Results represent the percentage of clonogenic recovery compared with untreated control samples and are the means of five separate experiments; bars, SE. P < 0.001 was accessed by ANOVA for the comparison of the treatment groups in both the U937 (A) and HL60 (B) cells.

 


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Fig. 2. Effects of bryostatin-1 and GM-CSF on cell surface antigen expression by AML cell lines. U937 (A) and HL60 (B) cells were incubated for 120 h in the absence (Control) or presence of bryostatin-1 (Bryo, 10-8 M) with or without GM-CSF (200 units/ml) and then assessed for the surface expression of CD11b and CD14 by flow cytometry. Results represent means of five separate experiments; bars, SE. Ps were determined by ANOVA for the comparison of all treatment groups for both CD11b and CD14 expression.

 
Although both U937 and HL60 cells express functional GM-CSF receptors (27) , GM-CSF alone had little effect on the growth or differentiation of either cell line as assessed by clonogenic growth (Fig. 1)Citation , morphology (data not shown), or CD11b and CD14 expression (Fig. 2)Citation . However, the addition of GM-CSF to bryostatin-1 further inhibited the clonogenic growth of both U937 and HL60 cells when compared with bryostatin-1 alone (Fig. 1)Citation . This effect was not attributable to enhancement of the antiproliferative activity of bryostatin-1 because the addition of GM-CSF had no effect on the cell cycle profile of U937 cells (Table 1)Citation . This combination also induced further phenotypic evidence of differentiation, with the majority of cells resembling mature monocytes (data not shown) and displaying enhanced CD11b and CD14 expression on U937 cells and CD14 on HL60 cells (Fig. 2)Citation . Furthermore, examination of CD11b expression demonstrated that the entire population of U937 cells underwent phenotypic differentiation when treated with bryostatin-1 and GM-CSF (Fig. 3A)Citation .



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Fig. 3. Flow cytometry histograms of cell surface antigen expression by AML. A, U937 cells were incubated in the presence of GM-CSF (200 units/ml) and bryostatin-1 (10-8 M), hydroxyurea (100 µg/ml), or phenylbutyrate (1.5 mM) for 120 h and analyzed for CD11b expression by flow cytometry. B, AML blasts from patient number 2 were incubated with bryostatin-1 (10-8 M) and GM-CSF (200 units/ml) for 120 h and then evaluated for CD15 expression. Experimental groups are depicted by solid lines, whereas dotted lines represent untreated control cells. Cell numbers of all groups remained constant throughout the 5-day incubation.

 
The combination of GM-CSF and bryostatin-1 was also tested against primary samples derived from seven newly diagnosed patients with AML; patients with AML-M3 (APL) were excluded because this subtype of AML readily differentiates in response to a number of pharmacological differentiating agents (28) . Patient characteristics are described in Table 2Citation . Neither bryostatin-1 nor GM-CSF alone induced significant evidence of differentiation of primary AML cells as assessed by morphological examination (Fig. 4)Citation , surface antigen expression (Fig. 5A)Citation , or inhibition of clonogenic AML growth (Fig. 5B)Citation . However, as with the AML cell lines, the combination of bryostatin-1 and GM-CSF induced morphological evidence of myeloid differentiation in all cases with a decrease in the nuclear:cytoplasmic ratio, nuclear condensation with the loss of nucleoli, and the appearance of cytoplasmic granulation and vacuoles (Fig. 4)Citation . The expression of the monocytic antigens CD11b and CD14 varied considerably among patient specimens, particularly in the samples derived from the four patients with relatively undifferentiated M1-AML; however, all specimens demonstrated evidence of granulocytic differentiation with increased expression of CD15 following the addition of GM-CSF to bryostatin-1 (Fig. 5ACitation ; P = 0.003). As in the cell lines, the entire population of blasts underwent phenotypic differentiation (Fig. 3B)Citation . The combination of bryostatin-1 and GM-CSF also significantly inhibited the clonogenic recovery of primary AML samples (Fig. 5BCitation ; P = 0.002).


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Table 2 Clinical characteristics of seven cases of AML

 


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Fig. 4. Effects of bryostatin-1 and GM-CSF on the morphology of AML blasts. Isolated AML blasts were incubated for 120 h in the absence (Control) or presence of bryostatin-1 (10-8 M) with or without GM-CSF (200 units/ml). Results represent Wright-Giemsa stained cytospin preparations from a representative patient. x400.

 


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Fig. 5. Effects of bryostatin-1 + GM-CSF on primary AML samples. Isolated AML blasts were incubated for 120 h in the absence (Control) or presence of bryostatin-1 (10-8 M) with or without GM-CSF (200 units/ml) and assayed for CD15 expression (A) and clonogenic growth (B). Results represent the means for the seven patient specimens; bars, SE. P = 0.002 was determined by ANOVA for the comparison of all treatment groups for both CD15 expression (A) and clonogenic growth (B).

 
Requirement of Growth Factors for the Activity of Bryostatin-1.
Although its antileukemic activity was significantly enhanced by GM-CSF, bryostatin-1 induced a modest degree of differentiation on its own. To determine whether this activity was dependent on growth factors present in the FBS or resulting from autocrine secretion by the leukemia cells, U937 cells were treated with bryostatin-1 in the presence of neutralizing antibodies directed against GM-CSF and IL-3. Antibodies against both GM-CSF and IL-3 were used because U937 cells express both types of receptors, and there is significant cross-talk between their intracellular signaling pathways (27 , 29 , 30) . Antibodies against human GM-CSF and IL-3 have been shown to block the activity of these growth factors present in FBS (31) . These antibodies significantly, and almost completely, abrogated both the up-regulation of CD11b (Fig. 6ACitation ; P < 0.001) and the inhibition of clonogenic leukemia growth (Fig. 6BCitation ; P < 0.001) induced by bryostatin-1 alone.



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Fig. 6. Role of growth factors in the activity of bryostatin-1. U937 cells were incubated for 120 h in the absence (Control) or presence of bryostatin-1 (10-8 M) along with neutralizing antihuman GM-CSF (0.1 mg/ml) and antihuman IL-3 (0.15 mg/ml) or nonbinding isotypic control antibodies and assayed for CD11b expression (A) and clonogenic growth (B). All results represent the means of three separate experiments; bars, SE. P < 0.001 was determined by the paired Student’s ttest for the comparison of bryostatin-1 + isotypic control antibodies and bryostatin-1 + growth factor blocking antibodies for both CD11b expression (A) and clonogenic growth (B).

 
Effect of GM-CSF on the Activity of Other Differentiating Agents.
Similar to bryostatin-1, many other pharmacological agents that induce tumor cell differentiation in vitro are potent cell cycle inhibitors, regardless of their cellular targets (32, 33, 34) . Standard chemotherapeutic agents, such as ara-C and hydroxyurea, also inhibit cell cycling and promote differentiation of leukemia cells when used at low doses (35, 36, 37, 38) . Furthermore, signals that induce cell cycle arrest often are associated with cellular differentiation, whereas those that promote cell cycle progression usually block cellular differentiation (39 , 40) . To investigate the roles of cell cycle inhibition and growth factors in the activity of other differentiating agents, we examined the effects of cytostatic doses of the chemotherapeutic agent hydroxyurea and the pharmacological differentiating agent phenylbutyrate, alone and in combination with GM-CSF. Hydroxyurea interferes with deoxyribonucleotide synthesis and inhibits the cell cycle at S-phase (41 , 42) , whereas phenylbutyrate inhibits histone deacetylation and induces a G1 arrest (43 , 44) .

Hydroxyurea and butyrates have also been reported to induce the differentiation of U937 cells (45, 46, 47, 48) . Growth curves demonstrated that hydroxyurea at 100 µg/ml and phenylbutyrate at 1.5 mM inhibited the growth of U937 cells without inducing apoptosis (data not shown), and DNA content analysis revealed that cell cycling was inhibited at the expected points (Table 1)Citation . As with bryostatin-1, both hydroxyurea and phenylbutyrate inhibited clonogenic leukemia growth that was enhanced by the addition of GM-CSF (Fig. 7A)Citation . Hydroxyurea and phenylbutyrate also induced phenotypic differentiation of the entire population of U937 cells, as evidenced by the up-regulation of CD11b, that was augmented by GM-CSF (Figs. 3ACitation and 7BCitation ). Similar to bryostatin-1, neither drug affected clonogenic growth or phenotypic differentiation at doses (bryostatin-1, <5 x 10-10 M; hydroxyurea, <50 µg/ml; phenylbutyrate, <1 mM) that failed to inhibit proliferation and induce cell cycle arrest (data not shown). Furthermore, neutralizing antibodies against GM-CSF and IL-3 were effective in limiting the differentiation of U937 cells mediated by both hydroxyurea and phenylbutyrate, as demonstrated by the inhibition of CD11b expression (Fig. 8A)Citation as well as the decrease in clonogenic growth (Fig. 8B)Citation .



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Fig. 7. Effects of cytostatic agents on U937 cells. U937 cells were incubated in the absence (Control) or presence of hydroxyurea (100 µg/ml) or phenylbutyrate (1.5 mM) with or without GM-CSF (200 units/ml) for 120 h and then assessed for clonogenic leukemia growth (A) and CD11b expression (B). Results represent means of five separate experiments; bars, SE. Ps are as determined by ANOVA for the comparison of all treatment groups.

 


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Fig. 8. Role of growth factors on the differentiating activity of pharmacological differentiating agents. U937 cells were incubated for 120 h in the absence (Control) or presence of hydroxyurea (100 µg/ml) or phenylbutyrate (1.5 mM) along with neutralizing antihuman GM-CSF (0.1 mg/ml) and antihuman IL-3 (0.15 mg/ml) or nonbinding isotypic control antibodies and assessed for CD11b expression (A) and clonogenic growth (B). All results represent the means of three separate experiments; bars, SE. Ps compare agents treated with blocking antibodies or control antibodies by the paired Student’s t test.

 
Effect of Cytostatic Agents + GM-CSF on Normal Hematopoietic Progenitors.
Given the potent antileukemic activity of combining any of the pharmacological agents tested with GM-CSF, we examined their effects on normal hematopoietic progenitors. Incubation of normal bone marrow mononuclear cells with any of the pharmacological agents combined with GM-CSF yielded only limited inhibition in CFU-GM growth (Fig. 9)Citation .



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Fig. 9. Effects of combining GM-CSF and cytostatic agents on normal hematopoiesis. Bone marrow mononuclear cells from normal donors were treated for 120 h in the absence (Control) or presence of hydroxyurea (HU, 100 µg/ml), phenylbutyrate (PB, 1.5 mM), or bryostatin-1 (Bryo, 10-8 M) with or without GM-CSF (200 units/ml) and then washed and plated in methylcellulose. Results are the means of three separate experiments; bars, SE. Ps are as determined by ANOVA for the comparison of all treatment groups.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We found that bryostatin-1 inhibited the clonogenic growth of AML cell lines and patient samples. This was not attributable to the induction of cellular apoptosis nor a direct cytostatic effect of bryostatin-1 on AML cells because it was removed before assessing clonogenic growth. The loss of leukemic clonogenic capacity was associated with phenotypic evidence of myeloid maturation, suggesting that cells lost the ability to replicate as a result of terminal differentiation. The addition of GM-CSF markedly enhanced the antileukemic effects of bryostatin-1 as well as two other unrelated agents, hydroxyurea and phenylbutyrate. Importantly, the activity of bryostatin-1 combined with GM-CSF was seen not only in cell lines but also in all clinical AML samples tested. Another report has likewise described the differentiation of AML blasts using a combination of bryostatin-1 and cytokines (49) . Outside of APL, the induction of differentiation in clinical AML samples has generally been unsuccessful (50) , especially in the M1 subtype of AML that constituted the majority of cases in our study. Although enhanced leukemic differentiation resulting from the combined use of growth factors and pharmacological differentiating agents, such as ATRA and vitamin D, has been described (51, 52, 53) , these studies did not examine the relative roles of growth factors and pharmacological differentiating agents. We found that neutralizing antibodies directed against GM-CSF and IL-3 limited the activity of all of the pharmacological agents tested, and in the case of bryostatin-1, completely blocked its antileukemic effects. These data suggest that pharmacological differentiating agents are not sufficient for the induction of tumor cell terminal differentiation but require the activity of growth factors.

Lineage-specific growth factors, such as GM-CSF, have pleiotropic effects on both malignant and normal cells, that include enhancing proliferation, promoting cell survival, and inducing differentiation. The net clinical effect of growth factors on tumor cell growth is determined by the relative contribution of each of these activities. Tumor progression may result from the enhancement of tumor cell proliferation or survival, whereas the preferential induction of terminal differentiation and subsequent loss of self-renewal capacity may lead to the exhaustion or eradication of the neoplastic clone. The stimulatory effects of myeloid growth factors on leukemic cell growth may predominate in most settings; however, we and others have demonstrated previously that GM-CSF preferentially enhances the differentiation, rather than proliferation, of malignant progenitors in CML (54, 55, 56) . Therefore, growth factors may predominantly act as differentiating agents in some circumstances.

In CML, the characteristic fusion protein p210bcr-abl prolongs G2-M (57 , 58) , and this effect on the cell cycle may be responsible for selectively inducing differentiation in response to GM-CSF. The association between cell cycle inhibition and cellular differentiation is well recognized; the induction of differentiation of both normal and malignant cells is associated with cell cycle inhibition that is mediated by the inhibition of cdk activity and the induction of the cdk inhibitors p21cip1/waf1 and p27kip1 (39) . Furthermore, the ectopic expression of p21cip1/waf1 and subsequent induction of G1 cell cycle arrest induces the differentiation of U937 cells (25 , 59) , whereas the inhibition of p21cip1/waf1 blocks differentiation (48) . The inhibition of cell cycling may play an important role the activity of pharmacological differentiating agents because most agents, such as ATRA, vitamin D, and those examined in this study, share this biological property despite interacting with a diverse array of cellular targets (25 , 34 , 60) . Furthermore, standard chemotherapeutic agents, such as ara-C and hydroxyurea, can also induce tumor cell maturation when used at low concentrations that primarily inhibit cell cycling rather than induce cell death, and their ability to induce differentiation can be enhanced by growth factors (35 , 61) . Our data suggest that pharmacological differentiating agents and cytotoxic chemotherapeutic agents used at cytostatic concentrations selectively enhance the differentiating activity of growth factors by inhibiting cell cycling and blocking the effects of growth factors on leukemia cell proliferation. We found that blocking the cell cycle at G1 (bryostatin-1 and phenylbutyrate) or S-phase (hydroxyurea) augmented growth factor-driven differentiation of AML cells; thus, general slowing of cell cycle progression rather than inhibition at a specific phase may be sufficient for this process.

In contrast to its effects on AML cells, previous studies have demonstrated that bryostatin-1 enhances the growth of normal hematopoietic progenitors (7 , 8) . We found that the combination of bryostatin-1 and GM-CSF also enhanced the growth of normal hematopoietic progenitors. Moreover, the other pharmacological differentiating agents tested also had limited effects on normal hematopoietic progenitor growth in combination with GM-CSF. A possible explanation for the lack of normal marrow progenitor inhibition by these combinations may be that progenitors are generally quiescent, residing in G0-G1 (62, 63, 64) , and further delaying the cell cycle may not alter their differentiation programs.

The goal of effective differentiation therapy is to promote the maturation of tumor cells leading to the loss of self-renewal capacity. The success of differentiation therapy in the treatment of APL with ATRA has demonstrated that the induction of tumor cell differentiation is a promising strategy against AML (65) . Unfortunately, the use of ATRA in APL has remained the only clinical example of successful differentiation therapy, although many pharmacological agents are active in vitro (66) . Our data indicate that the full induction of terminal differentiation requires cell cycle inhibition combined with lineage-specific growth factors. Therefore, the clinical inactivity of pharmacological differentiating agents may be attributable to inadequate levels of lineage-specific growth factors. Accordingly, a recent report has described the successful treatment of a patient with APL harboring the atypical t(11;17) chromosomal translocation, which is normally refractory to ATRA, by combining ATRA with granulocyte-CSF (67) . Therefore, combining pharmacological differentiating agents with exogenous growth factors may be effective clinically, because this approach has potent antileukemic activity while sparing normal hematopoiesis in vitro.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
The human AML cell lines, U937 and HL-60, were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 (Life Technologies, Inc., Rockville, MD) supplemented with 7.5% heat-inactivated FBS (Life Technologies, Inc.), 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine in a humidified atmosphere at 37°C and 5.0% CO2. Cells in logarithmic phase were seeded at a density of 1 x 105 cells/ml and incubated for 120 h with various concentrations of bryostatin-1 (National Cancer Institute, Bethesda, MD), hydroxyurea (Bristol Laboratories, Princeton, NJ), or phenylbutyrate (National Cancer Institute) with or without recombinant human GM-CSF (200 units/ml; Immunex, Seattle, WA).

Alternatively, cells were incubated as described above in the presence of rat antihuman GM-CSF IgG2a (0.1 mg/ml) and rat antihuman IL-3 IgG1 (0.15 mg/ml) antibodies (PharMingen, San Diego, CA). Nonbinding rat isotypic IgG2a and IgG1 antibodies were used as controls (PharMingen), and anti-growth factor or control antibodies were readded after 48 h of incubation. Clinical specimens were obtained from patients with newly diagnosed AML or normal donors granting informed consent as approved by the Joint Commission on Clinical Investigation of the Johns Hopkins Hospital. Mononuclear cells were isolated from freshly harvested bone marrow aspirates by density centrifugation (density, <1.078; Ficoll-Paque, Pharmacia, Piscataway, NJ), followed by two washes in {alpha}MEM (Life Technologies, Inc.). For morphological studies and cell surface antigen analysis, AML blasts were further isolated using either antihuman CD34 or antihuman CD33 antibodies coupled to magnetic microbeads (Miltenyi Biotec, Auburn, CA), followed by magnetic column purification (Miltenyi VarioMACS) according to the manufacturer’s instructions (Miltenyi Biotec). Enriched fractions containing >95% leukemic cells (as measured by flow cytometry) were obtained. For leukemia clonogenic assays, bone marrow mononuclear cells were depleted of T cells with an antihuman CD3 antibody coupled to magnetic microbeads (Miltenyi Biotec).

Cell Cycle Analysis.
After 48 h in culture with hydroxyurea, bryostatin-1, or phenylbutyrate, cell cycle distribution was analyzed using BrdUrd and PI staining as described previously (57) . Briefly, cells (1 x 105) were washed with complete medium and incubated at 37°C with 20 µM BrdUrd (Roche Molecular Biochemicals, Indianapolis, IN) for 60 min. Cells were washed with PBS, fixed with ice-cold 70% ethanol, and digested for 30 min at room temperature with 0.2 mg/ml pepsin in 2 N HCl (Sigma, St. Louis, MO). Cells were then labeled for 30 min at room temperature with FITC-conjugated anti-BrdUrd antibody (Becton-Dickinson, Mountain View, CA) in 0.5% Tween 20 (Bio-Rad, Hercules, CA). After the removal of unbound anti-BrdUrd, the cells were incubated in 5 µg/ml DNase-free RNase (Roche Molecular Biochemicals) and 25 µM/ml PI (Sigma) for an additional 30 min at room temperature. Cell cycle distribution was analyzed using a FACScan flow cytometer (Becton Dickinson).

Determination of Apoptosis.
Cells (1 x 105) were washed with PBS, fixed in 70% ethanol, and resuspended in 0.1% Triton X-100 (Sigma) containing 5 µg/ml DNase-free RNase for 15 min at 37°C. Cells were then stained with 50 µg/ml PI for 60 min at 4°C. The fraction of subdiploid cells with oligonucleosomal DNA degradation characteristic of apoptosis was quantified by flow cytometric analysis as described previously (57) .

Clonogenic Assays.
After 120 h of incubation, cells were evaluated for clonogenic growth potential as described previously (54 , 57 , 68) . Briefly, 200-1000 cells (for cell lines), 5 x 104 cells (for bone marrow mononuclear cells from normal donors), or 2 x 105 cells (for clinical AML samples) were washed and placed in 1.0 ml of 1.2% methylcellulose, 30% FBS, 1% BSA, 10-4 M 2-mercaptoethanol, and 2 µM L-glutamine containing 10% lymphocyte conditioned medium. Samples were plated in quadruplicate onto 35-mm2 tissue culture dishes and incubated in a humidified atmosphere at 37°C and 5.0% CO2. Colonies consisting of >40 cells were counted using an inverted microscope at 7 days for leukemia colonies and 14 days for CFU-GM from normal bone marrow. Results are presented as the percentage of colonies relative to the untreated controls ± SE.

Cell Surface Antigen Expression.
After 120 h of incubation, AML cell lines were analyzed for differentiation by examining the expression of the monocytic surface antigens CD11b and CD14 by flow cytometry as described previously (54) CD11b is the {alpha} subunit of the CD11b/CD18 heterodimeric complex involved in monocyte-endothelial cell interaction; CD14 acts a monocyte receptor for lipopolysaccharides (69 , 70) . Briefly, 1 x 105 cells were washed with PBS containing 0.2% BSA. Staining was then performed with FITC-conjugated mouse antihuman CD11b and phycoerythrin-conjugated mouse antihuman CD14 antibodies for 30 min at 4°C (Becton Dickinson). Cells were washed to remove unbound antibody, fixed with 2% paraformaldehyde, and evaluated by flow cytometry. Control studies were performed with nonbinding mouse IgG1 and IgG2a isotype antibodies (Becton Dickinson). Clinical AML specimens were analyzed for evidence of myeloid differentiation by the surface expression of CD15, using a FITC-conjugated mouse antihuman CD15 IgM antibody (Becton Dickinson) or nonbinding FITC-conjugated mouse IgM isotype control antibody as described above. CD15 is a carbohydrate moiety thought to be involved in neutrophil function (71) . Results are presented as the relative mean fluorescence intensity ± SE as described previously (72) .

Statistical Analysis.
Data are expressed as mean ± SE. Comparisons between treatments were performed using a two-tailed, paired Student’s t test and ANOVA as indicated. ANOVA was used to calculate the significance when comparing the means of all of the experimental groups during the evaluation of a single variable (e.g., clonogenic growth or relative mean fluorescence intensity). P < 0.05 was considered significant.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grant P01CA15396. W. H. M. is a fellow of the Leukemia and Lymphoma Society. Back

2 Present address: The Medical College of Pennsylvania-Hahnemann University School of Medicine, Philadelphia, PA 19102. Back

3 To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center, The Bunting-Blaustein Cancer Research Building, Room 207, 1650 Orleans Street, Baltimore, MD 21231. Phone: (410) 955-2813; Fax: (410) 614-7279; E-mail: rjjones{at}jhmi.edu Back

4 The abbreviations used are: AML, acute myeloid leukemia; ara-C, 1-ß-D-arabinofuranosylcytosine; GM-CSF, granulocyte/macrophage-colony stimulating factor; PKC, protein kinase C; cdk, cyclin dependent kinase; IL, interleukin; ATRA, all-trans-retinoic acid; CML, chronic myeloid leukemia; APL, acute promyelocytic leukemia; FBS, fetal bovine serum; BrdUrd, 5-bromo-2-deoxyuridine; PI, propidium iodide; CFU-GM, granulocyte/macrophage colony-forming unit. Back

Received for publication 2/25/02. Revision received 5/16/02. Accepted for publication 5/23/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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