Normal Human Myeloid Progenitor Research Articles

Pharmacologic Restoration of PP2A Activity and Interference with the SET-PP2A Interplay by FTY720 and Its Non-Immunosuppressive Derivative as a Novel and Efficient Therapy for Ph-Negative Myeloproliferative Disorders

AbstractAbstract 775We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2).Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients.To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells.Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min).Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures:Verstovsek:Incyte Corporation: Research Funding.

Effects of 5-aza-2′-deoxycytidine on proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells

The demethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) have been shown to induce differentiation and inhibit growth of leukemic myeloid cells at low concentrations. However, the effect of DAC in changing the differentiation and proliferation behavior of normal human myeloid progenitors has rarely been investigated. Therefore, we established an in vitro model of normal hematopoietic differentiation, using CD34+ cells from mobilized peripheral blood, to study proliferation and colony formation, expression of several myeloid maturation markers and of the inhibitor of cyclin-dependent kinases p15/INK4b. Upon DAC treatment, cell growth was significantly decreased in a dose-dependent manner, without an increase in cytotoxicity. DAC treatment also resulted in a substantial increase of lysozyme-positive cells, which could be enhanced by G-CSF, a modest increase of myeloperoxidase+ and CD15+ cells, as well as an increase of colony-forming cells (CFU-GM) compared to control cells. p15/INK4b protein expression was strongly upregulated upon myeloid maturation, and additional DAC treatment did not change p15 expression or the methylation status of the p15 promoter at the noncytotoxic concentrations used. Taken together, these data indicate a role of DAC in changing myeloid progenitor cell expansion and differentiation. This model appears suitable also for global analyses of multiple differentially methylated genes.

IL-3 Inhibits Terminal Granulocytic Differentiation by Inducing the Binding of Stabilizing Proteins to a 300 Base Pair Region within p21cip1/waf1 Messenger RNA.

Elevated levels of the molecular adaptor protein p21cip1/waf1 (p21) and of the IL-3 receptor α chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demo−nstrated that p21 undergoes dynamic changes in expression levels and subcellular compartmentalization during cytokine-induced granulocytic differentiation, suggesting that p21 may play an important role in myeloid development. Based on our observation that p21 protein levels decrease during granulocytic differentiation of CD34+ human progenitor cells, we hypothesized that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 is required to maintain the undifferentiated state in murine 32Dcl3 cells and has been shown to slow the kinetics of differentiation of normal human myeloid progenitors (Hevehan DL, 2000). Given that 32Dcl3 myeloblasts express high basal levels of p21, we also hypothesized that IL-3 inhibition of 32Dcl3 differentiation is mediated in part by p21. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3 that are normally not abundant in cells maintained as myeloblasts by IL-3. Rescue of p21 knockdown myeloblasts with human p21 suppressed aberrant expression of granule proteins. The upregulation of myeloperoxidase and proteinase-3 occurred at a posttranscriptional level. These findings indicated that p21 prevented premature expression of primary granule proteins and may contribute to maintenance of the myeloblast phenotype. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF, indicating that p21 antagonized the entire differentiation process rather than only suppressing primary granule proteins. We then determined how IL-3 maintains p21 expression in myeloblast cells. We demonstrated that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3′ untranslated region (UTR) of the p21 transcript. IL-3 also rescued the decrease in p21 mRNA stability noted during G-CSF-induced differentiation. This has been shown to coincide with differentiation blockade. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3′ UTR and are regulated by IL-3 and G-CSF signaling. Association of a ~60–65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a ~40–42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis.

Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development

We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34+ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34+ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34+ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2′-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.

Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development.

We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34+ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34+ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34+ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2'-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.

All-trans-retinoic acid effects the growth, differentiation and apoptosis of normal human myeloid progenitors derived from purified CD34+ bone marrow cells.

We have previously shown that all-trans retinoic acid (ATRA) increases the number of CFU-GM colonies grown from unseparated human bone marrow cells with crude sources of colony stimulating factors. In this study, we further characterized the effect of ATRA on the growth of CFU-GM stimulated by individual cytokines from multiple samples of CD34+ enriched or purified human bone marrow cells. The number of IL-3- or GM-CSF-induced CFU-GM with 3 x 10(-7) M ATRA was 3.25+/-1.13, and 2.17+/-0.8-fold greater respectively, compared to controls without ATRA, while G-CSF had no effect and the ratio of colony-induced with or without ATRA was 1.06+/-0.17 (P = 0.00012). No colonies grew with ATRA + IL-6 or ATRA without a cytokine. Maximum enhancing effect on IL-3-induced CFU-GM occurred when ATRA was added on day 2, gradually diminished when delaying ATRA, and in cultures of day 9 or older adding ATRA had no effect. In 14 days liquid cultures of purified CD34+ cells with IL-3, ATRA increased the number of myeloid differentiated cells to 91-95%, compared to 37-70% with IL-3 alone. In addition, the number of apoptotic cells using the annexin V method increased after 14 days from 5.1% with IL-3 to 17.1% with IL-3 + ATRA and by the TUNEL in situ method from 10-26% to 60-95%, respectively. This study demonstrates that ATRA consistently enhances the growth of myeloid progenitors from CD34+ cells. This effect is dependent on the stimulating cytokine, suggesting the myeloid cells responding to ATRA are the less mature CFU-GMs that are targets of IL-3 and GM-CSF and not the G-CSF-responding mature progenitors. The growth stimulation by ATRA and IL-3 is also associated with granulocyte differentiation and increased apoptosis. These studies further suggest a potential role of pharmacological doses of ATRA on the development of normal human hematopoietic cells.

Modulation by bryostatin 1 of the in vitro radioprotective effects of the GM-CSF/IL-3 fusion protein, PIXY 321, on normal human myeloid progenitors

We have examined the effect of the macrocyclic lactone PK-C activator, bryostatin 1, on the in vitro radioprotective capacity of the GM-CSF/IL-3 fusion protein, PIXY 321, toward normal committed myeloid progenitors (day 14 CFU-GM). Preincubation of CD 34 + cells for 24 h with 10ng/ml PIXY 321 exerted significant radioprotective effects on these progenitors, (D̄ = 1.403 vs 0.715 for controls), which were at least as great as those previously reported for higher concentration (e.g., 50 ng/ml) or rGM-CSF. In contrast to the results of earlier studies involving rGM-CSF, preincubation of cells with both PIXY 321 and 10nM bryostatin 1 did not lead to an increase in radioprotective effect when the total number of day 14 colonies was assessed. However, combinations of PIXY 321 and bryostatin 1 (or the tumour-promoting PK-C activator, PDBu) significantly increased the relative percentage and absolute number of surviving non-eosinophilic colonies (e.g., pure neutrophil, pure monocyte-macrophage, or mixed neutrophil-macrophage) at each radiation dose level. A similar pattern of response was noted in cells irradiated without a preconditioning interval, and in cells exposed to divided radiation doses. These results indicate that the GM-CSF/IL-3 fusion protein PIXY 321 exhibits significant in vitro radioprotective effects toward normal human bone marrow myeloid progenitors, and that co-administration of PK-C activators such as bryostatin 1 of PDBu selectively augments the radioprotective capacity of this hybrid cytokine toward non-eosinophilic elements.

Expression of CD7 on normal human myeloid progenitors.

Existence of biphenotypic leukemias co-expressing CD7 and CD34 has prompted the question of whether a similar population of cells is present in normal human bone marrow. As CD7 is considered to be a T cell-restricted Ag, the co-expression of CD7 with the "human stem cell Ag" CD34 may identify a bipotent stage within hemopoietic differentiation. Cells with this phenotype have previously been isolated from human thymus. In this report we provide evidence that human marrow mononuclear cells also contain a minor subpopulation of cells co-expressing CD7 and CD34. The CD7+/CD34+ cells were found to contain committed myeloid progenitors assayed both as CFU in semi-solid media and by their ability to produce granulocytes in long term marrow cultures. Expression of CD7 on myeloid committed progenitors was further confirmed in a C-mediated cytotoxic assay. We conclude that CD7 expression is not restricted to T cells but is also expressed during early stages of myeloid differentiation.

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells.

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.

Low concentrations of cytosine arabinoside, 6-thioguanine, actinomycin-D and aclacinomycin A stimulates the differentiation of normal human marrow myeloid progenitor cells.

Myelosuppression is the major side effect of most anticancer cytotoxic drugs. Low concentrations of cytosine arabinoside (Ara-C), 6-thioguanine (6-Th), actinomycin-D (Act-D) and aclacinomycin A (ACM) have been reported to induce differentiation of leukaemic cell lines. With the proposed clinical trials of low-dosage of these four drugs, their effect on the differentiation of normal human myeloid progenitor marrow cells was studied. The four cytotoxic anticancer drugs at low concentrations stimulated normal human myeloid differentiation. Low dosage Ara-C has been used with great success in several clinical trials. The results suggest a similar therapeutic role for the other three anticancer drugs.

Molecular cloning, expression, and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen

Monoclonal antibodies of the CD33 cluster group recognize a 67- kilodalton (Kd) protein, designated p67, expressed on the surface of normal human myeloid progenitors and leukemic cells from most patients with acute myelogenous leukemia. The human gene encoding p67 was isolated in a mouse genetic background after DNA-mediated gene transfer and fluorescence-activated cell sorting (FACS) for transformants that bound the monoclonal antibody MY9. After three serial rounds of gene transfer and cell sorting, multiple independently derived tertiary mouse cell transformants were obtained that expressed p67. Southern blot analysis revealed that these transformants shared restriction fragments containing highly reiterated human DNA sequences. Two shared EcoRI fragments of 3.3-kilobase (kb) and 9.5-kb pairs were molecularly cloned into bacteriophage vectors. A subsegment of the 3.3-kb fragment lacking repeated sequences was then used as a unique sequence probe to isolate two independent cosmid clones. Cells transfected with DNA from both cosmid clones bound MY9, and the human p67 protein was demonstrated by immunoprecipitation. NFS mice inoculated with a mouse cell transformant coexpressing p67 and the v-fms oncogene product produced antisera that specifically immunoprecipitated p67 from human leukemic cell lines, mouse cell transformants, and mouse cells transfected with the biologically active cosmid clones. The human p67 locus was previously assigned to chromosome 19 by screening a panel of rodent X human somatic cell hybrids with the unique sequence probe. The gene was sublocalized to the q13.3 region of chromosome 19 by in situ hybridization. RNA transcripts of approximately 1.6 kb and 1.4 kb were identified in polyadenylated RNA from human myeloid leukemia cell lines using a probe from the genomic locus. Manipulation of the cloned p67 gene may provide insight into the function of its product and mechanisms regulating its expression.

Molecular Cloning, Expression, and Chromosomal Localization of a Human Gene Encoding the CD33 Myeloid Differentiation Antigen

Monoclonal antibodies of the CD33 cluster group recognize a 67-kilodalton (Kd) protein, designated p67, expressed on the surface of normal human myeloid progenitors and leukemic cells from most patients with acute myelogenous leukemia. The human gene encoding p67 was isolated in a mouse genetic background after DNA-mediated gene transfer and fluorescence-activated cell sorting (FACS) for transformants that bound the monoclonal antibody MY9. After three serial rounds of gene transfer and cell sorting, multiple independently derived tertiary mouse cell transformants were obtained that expressed p67. Southern blot analysis revealed that these transformants shared restriction fragments containing highly reiterated human DNA sequences. Two shared EcoRI fragments of 3.3-kilobase (kb) and 9.5-kb pairs were molecularly cloned into bacteriophage vectors. A subsegment of the 3.3-kb fragment lacking repeated sequences was then used as a unique sequence probe to isolate two independent cosmid clones. Cells transfected with DNA from both cosmid clones bound MY9, and the human p67 protein was demonstrated by immunoprecipitation. NFS mice inoculated with a mouse cell transformant coexpressing p67 and the v-fms oncogene product produced antisera that specifically immunoprecipitated p67 from human leukemic cell lines, mouse cell transformants, and mouse cells transfected with the biologically active cosmid clones. The human p67 locus was previously assigned to chromosome 19 by screening a panel of rodent X human somatic cell hybrids with the unique sequence probe. The gene was sublocalized to the q13.3 region of chromosome 19 by in situ hybridization. RNA transcripts of approximately 1.6 kb and 1.4 kb were identified in polyadenylated RNA from human myeloid leukemia cell lines using a probe from the genomic locus. Manipulation of the cloned p67 gene may provide insight into the function of its product and mechanisms regulating its expression.

Interaction of deoxycytidine and deoxycytidine analogs in normal and leukemic human myeloid progenitor cells

The inhibitory effects of three deoxycytidine analogs, 1-B-D-arabinofuranosylcytosine (Ara-C), 5-aza-2′-deoxycytidine, (DAZ) and Ara-5-azacytosine (AAC) were compared with respect to the clonogenic behavior of human promyelocytic leukemic cells (HL-60), a deoxycytidine kinase deficient subvariant (HL-60/Ara-C), and normal human myeloid progenitor cells (CFU-GM). When cells were continuously exposed to each agent for 7 days, Ara-C was the most inhibitory, DAZ slightly less effective and AAC the least inhibitory on a molar basis. HL-60/Ara-C were also highly cross-resistant to both DAZ and AAC. In the absence of deoxycytidine, all three agents were either equally inhibitory or slightly more inhibitory to the growth of CFU-GM than to HL-60, whereas administration of deoxycytidine in ten to one-hundred fold excess protected CFU-GM to a greater extent than HL-60. In contrast, administration of high concentrations of drugs, e.g. 10 −5−10 −4 M, in conjunction with excess deoxycytidine exhibited greater toxicity toward CFU-GM than toward HL-60/Ara-C. Coadministration of deoxycytidine in ten-fold excess reduced the total intracellular accumulation and DNA incorporation of each analog in HL-60 cells by approx. 50%, whereas a hundred fold excess was associated with greater than a 90% reduction in these values. These studies demonstrate that deoxycytidine may antagonize the effects of Ara-C, DAZ and AAC in both normal and leukemic human myeloid cells, and that at low drug concentrations the degree of protection may be greater for normal elements. However, regimens employing high drug concentrations in conjunction with deoxycytidine do not appear to exert a selective inhibitory effect toward a highly resistant leukemic subvariant. These data suggest that alternative deoxycytidine/deoxycytidine analog dose relationships and schedules must be sought which are capable of selectively eradicating resistant cells.

In-vitro effects of antineoplastic prostaglandins on human leukemic cell growth and normal myelopoiesis

The effects of prostaglandin (PG)E 1, PGD 2 and 9-deoxy-Δ 9-PGD 2 (PGJ 2) on the clonogenic growth of six kinds of human leukemic cell lines (K562, KG1, HL60, U937, THP1 and Molt4) and normal human myeloid progenitor cells (CFU-GM) were studied using semisolid agar cultures. While the degree of suppression of leukemic growth by PGE 1 varied from cell line to cell line, PGD 2 and PGJ 2 equally suppressed the growth of all leukemic cell lines. The potency of growth inhibition was as follows: PGJ 2 > PGD 2 > PGE 1. The increase of cellular cAMP level induced by prostaglandin treatment did not parallel their cytotoxic potency. Normal myeloid colony formation was also suppressed by PGE 1, PGD 2 or PGJ 2. In contrast to the preferential inhibition of macrophage colony formation by PGE 1 such lineage-selective suppression was not observed for PGD 2 or PGJ 2. These findings suggest that PGD 2 and PGJ 2 potently inhibit the leukemic growth by a different mechanism from that of PGE 1 and by a cAMP-independent mechanism. These prostaglandins seem to be promising chemotherapeutic agents for acute leukemia.

Effect of 4-hydroperoxycyclophosphamide on leukemic and normal human myeloid progenitor cells

The sensitivity of myeloid progenitor cells from normal subjects (N-CFU-GM) and from leukemic patients in complete remission (LR-CFU-GM) to 4-hydroperoxycyclophosphamide (4-HC) were compared to the sensitivity of leukemic progenitor cells (L-CFU) to this drug. The results were expressed as the dose of 4-HC needed to kill 90% (TD 90) of the progenitor cells. The mean TD 90 were respectively for N-CFU-GM : 59(± 11 S.E.M.) nM ml −1 and for L-CFU 79(± 6 S.E.M.) nM ml −1. Thus, L-CFU were equally sensitive to 4-HC as N-CFU-GM. Moreover, the mean TD 90 for LR-CFU-GM was 87 (± 5 S.E.M.) nM ml −1. Thus, the sensitivity of N-CFU-GM and LR-CFU-GM did not differ significantly from that of L-CFU. These results are not encouraging for the use of 4-HC in vitro to eliminate the residual leukemic cells from autologous bone marrow of AML patients in complete remission. The sensitivity of L-CFU was modified neither by previous cytoreductive therapy (different from cyclophosphamide) nor by the time elapsed since diagnosis of AML.

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10 −8 to 3 × 10 −6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10 −7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.