Differentiation Of Promyelocytes Research Articles

All-Trans Retinoic Acid Delays the Differentiation of Primitive Hematopoietic Precursors (lin−c-kit+Sca-1+) While Enhancing the Terminal Maturation of Committed Granulocyte/Monocyte Progenitors

AbstractAll-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin−c-kit+Sca-1+) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin−cells: 31.5% at day 7 and 4% at day 14; Sca-1+: 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin−c-kit+Sca-1+hematopoietic precursors, ATRA did not exert the same effects on the more committed lin−c-kit+Sca-1−progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.

Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation.

The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 microM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development.

Differential expression of the RET gene in human acute myeloid leukemia.

The RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multi-subunit membrane receptor complex for at least two different types of transforming growth factor (TGF)-beta-related neurotrophic factors. We have previously shown that RET gene expression in acute myeloid leukemia (AML) occurs more frequently in AMLs displaying either a monocytic (FAB M4/M5) or intermediate-mature myeloid phenotype (FAB M2/M3) than in leukemias reflecting an earlier stage of myeloid differentiation (FAB M0/M1). To further verify the association between RET expression and the relative maturation stage of AML cells, we have performed a quantitative estimation of relative abundances of RET transcripts among various FAB subtypes of AMLs. By analyzing 13 AML samples and normal hematopoietic cells through a competitive-quantitative RT-PCR approach, we were able to show that the relative levels of RET-specific mRNAs continuously increase with blast cell maturation in human AML, i.e., the amounts of RET gene-specific transcripts differ among RET-expressing AMLs, being higher in the more differentiated FAB phenotypes. In addition, we provide evidence that the relative amounts of RET transcripts increase upon in vitro and in vivo differentiation of leukemic promyelocytes from FAB M3 AML patients, becoming overall comparable to those found in normal granulocytes. These results indicate that RET expression in human AMLs is maturation-associated, probably mirroring the developmental regulation of this gene during differentiation of normal hematopoietic cells.

Opposing effects of protein kinase A and C on capacitative calcium entry into HL-60 promyelocytes

Treatment of HL-60 cells with thapsigargin, a microsomal Ca 2+/ATPase inhibitor, led to depletion of intracellular calcium stores followed by capacitative calcium entry. Stimulation of adenylyl cyclase with forskolin enhanced thapsigargin-induced Ca 2+ influx. The forskolin effect was confirmed by enhanced fluorescence quenching induced by Mn 2+ entry into fura-2 loaded cells. 1,9-Dideoxy-forskolin, an inactive analog of forskolin, did not affect capacitative calcium entry. On the other hand, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, inhibited thapsigargin-induced Ca 2+ entry. Histamine and prostaglandin E 2 (PGE 2) elevated intracellular adenosine 3′:5′-cyclic monophosphate (cAMP) levels and enhanced the thapsigargin-induced capacitative calcium entry. Incubation with N-[2-( p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), an inhibitor of protein kinase A (PKA), blocked the forskolin effect, and GF109203X, an inhibitor of protein kinase C (PKC), blocked the phorbol 12-myristate 13-acetate effect. The results suggest that protein kinase A regulates capacitative calcium entry positively, but that protein kinase C regulates Ca 2+ influx negatively. Furthermore, after differentiation of HL-60 promyelocytes with dimethylsulfoxide to granulocytes, the inhibitory effect of phorbol 12-myristate 13-acetate became more pronounced, whereas the stimulatory effect of prostaglandin E 2 did not change. This result suggests that the regulation of capacitative calcium entry by protein kinase C and protein kinase A develops differently during differentiation.

The Catalytic Domain of Protein Kinase C-δ in Reciprocal δ and ∊ Chimeras Mediates Phorbol Ester-induced Macrophage Differentiation of Mouse Promyelocytes

The overexpression of protein kinase C-delta (PKC-delta), but not PKC-epsilon, enables the mouse myeloid cell line 32D to differentiate into macrophages when treated with phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine the domain of PKC-delta that is responsible for this isotype-specific function, cDNAs that encode reciprocal chimeras of PKC-delta and -epsilon (PKC-delta epsilon and PKC-epsilon delta) were constructed by exchanging regulatory and kinase domains using polymerase chain reaction technology. Both chimeras were stably expressed in 32D cells using the pLTR expression vector and displayed protein kinase activity upon TPA treatment. TPA treatment of L epsilon delta, cells that overexpressed the PKC-epsilon delta chimera, induced a dramatically increased cell volume, surface adherence, surface expression of Mac-1 and Mac-3, lysozyme production, and phagocytosis. These are the characteristics of the macrophage phenotype found in TPA-treated 32D cells that overexpressed PKC-delta. In contrast, little effect was seen in L delta epsilon, 32D cells that overexpressed PKC-delta epsilon, with or without TPA treatment. A PKC inhibitor directed toward the catalytic domain of PKC, GF109203X, and a selective inhibitor of PKC-delta, Rottlerin, blocked the TPA-induced differentiation of PKC-epsilon delta-overexpressing 32D cells. These results demonstrate that the catalytic domain of PKC-delta contains the primary determinants for its activity in phorbol ester-induced macrophage differentiation.

Transmembrane potential responses during HL‐60 promyelocyte differentiation

Transmembrane potential responses during HL‐60 promyelocyte differentiation

PL48: A novel gene associated with cytotrophoblast and lineage-specific HL-60 cell differentiation

Abstract The cDNA for a novel gene, PL48 , isolated by subtractive hybridization between undifferentiated human term cytotrophoblast and differentiating cytotrophoblast, has been cloned and sequenced. PL48 contains an open reading frame coding for a 537-amino-acid protein, has multiple potential PKC, casein kinase II, and cAMP/cGMP-dependent kinase phosphorylation sites, and N-linked glycosylation sites. It is not present in a wide variety of proliferating cancer cells, but PL48 mRNA shows marked expression during cytotrophoblast and granulocyte lineage-specific HL-60 promyelocytic cell differentiation induced by DMSO.

Transmembrane potential responses during HL-60 promyelocyte differentiation.

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/ macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC2-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by KCa channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl- conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions.

Retinol Is Sequestered in the Bone Marrow of Vitamin A-Deficient Rats

Retinoic acid bound to the nuclear retinoic acid receptor-α is required for the differentiation of promyelocytes to mature neutrophils. However, severely vitamin A-deficient rats have normal numbers of neutrophils in the blood and inflamed tissues. This paradox was explored using four dietary groups of rats: 1) vitamin A-deficient rats; 2) vitamin A-deficient rats subsequently receiving vitamin A; 3) weight-matched pair-fed rats; and 4) nonrestricted, vitamin A-complete diet-fed rats. Plasma and liver retinol concentrations of the vitamin A-deficient rats were <1% of those of the other three groups. In contrast, the bone marrow retinol concentrations of the vitamin A-deficient rats were fourfold higher than those in the other three groups. The distribution of myeloid-derived cells in the bone marrow was similar in all four groups of rats with the exception of a significantly greater (P < 0.05) occurrence of hypersegmented neutrophils (six or more lobes) in the vitamin A-deficient rats (2.1%) relative to the control groups (0–0.1%). The blood of the vitamin A-deficient rats also contained significantly higher numbers (P < 0.01) of hypersegmented neutrophils (67%) relative to those in the control groups (2–7%). The hypersegmentation of the neutrophils in this group of rats was not due to a concurrent deficiency of vitamin B-12 or folate. The importance of bone marrow-derived cells to the survival of the animal is suggested by retinol sequestration in the bone marrow of vitamin A-deficient rats, allowing the differentiation of myeloid cells to neutrophils.

Reinforcement effect of histamine on the differentiation of murine myeloblasts and promyelocytes induced by granulocyte colony stimulating factor

Reinforcement effect of histamine on the differentiation of murine myeloblasts and promyelocytes induced by granulocyte colony stimulating factor

Construction of a Novel Bifunctional Biogenic Amine Receptor by Two Point Mutations of the H2-Histamine Receptor

H2-histamine receptors mediate a wide range of physiological functions extending from stimulation of gastric acid secretion to induction of human promyelocyte differentiation. We have previously cloned the H2-histamine receptor gene and noted that only three amino acids on the receptor were sufficient to define its specificity and selectivity. Despite only modest overall amino acid homology (34% amino acid identity and 57.5% similarity) between the H2-histamine receptor and the receptor for another monoamine, the beta 2-adrenergic receptor, there is remarkable similarity at their critical ligand binding sites. We hypothesized that, if the specificity and selectivity of both receptors are invested in just three amino acids, it should be possible to convert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites. We explored the effect of two single mutations in the fifth transmembrane domain of the H2-histamine receptor, which encompasses the sites that determine H2 selectivity. The canine H2 receptor gene was mutated at Asp186 and Gly187 (Asp186 to Ala186 and Gly187 to Ser187) by oligonuceotide directed mutagenesis. The coding region of both the wild-type and mutated H2 receptors was subcloned into the eukaryotic expression vector, CMVneo, and stably transfected into Hepa cells and L cells. The biological activity of histamine and epinephrine on the expressed receptor was examined by measurement of cellular cAMP production and inositol trisphosphate formation. Hepa cells transfected with the Ala186-Ser187 mutant H2 receptor demonstrated a biphasic rise in cAMP in response to epinephrine with an early phase (ED50 approximately 10(-11) M) that could be inhibited by both propranolol and cimetidine. Epinephrine also induced IP3 generation in the same cells, a biological response that is characteristic of activation of the wild-type H2 but not of the beta-adrenergic receptor. L cells transfected with the Ala186-Ser187 mutant H2 receptor also responded to epinephrine in a cimetidine and propranolol inhibitable manner. We converted the H2-histamine receptor into a bifunctional one that has characteristics of both histamine and adrenergic receptors by two simple mutations. These results support the hypothesis that ligand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. Our studies have important implications on the design of pharmacological agents targeted for action at physiological receptors.

Activation of beta-isozyme of protein kinase C (PKC beta) is necessary and sufficient for phorbol ester-induced differentiation of HL-60 promyelocytes. Studies with PKC beta-defective PET mutant.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and differentiation of a number of leukemia cell lines including HL-60 human promyelocytic leukemia. We investigated the involvement of protein kinase C (PKC) isotypes in phorbol ester-induced differentiation using the phorbol ester-tolerant PET mutant of HL-60 cells, which (in contrast to the parent phorbol ester-sensitive (wild-type) S variant of HL-60 cells) does not growth-arrest, become adherent, or undergo apoptosis when exposed to TPA (Macfarlane, D. E., Gailani, D., and Vann, K. (1988) Br. J. Haematol. 68, 291-302). In comparison to S cells, we found that proliferating PET cells markedly underexpress mRNA for PKC beta, but do express PKC alpha and PKC delta. The PKC beta-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate induces growth arrest, adherence, surface expression of CD11a, and apoptosis in S cells, but not in PET cells. Expression of PKC beta in PET cells can be restored by exposing them to dihydroxyvitamin D3, and this treatment restores the ability of subsequently added 12-deoxyphorbol 13-phenylacetate 20-acetate or TPA to induce immediate cell adherence and growth arrest of PET cells. These data led us to conclude that activation of PKC beta is both necessary and sufficient for phorbol ester-induced growth arrest and adherence in these myeloid cells.

Human Immunodeficiency Virus-1 Infection of the Human Promyelocytic Cell Line HL-60: High Frequency of Low-Level Infection and Effect of Subsequent Cell Differentiation

AbstractAs a model system to study the infection of early myeloid cells by human immunodeficiency virus-1 (HIV-1), we have infected the human promyelocytic cell line, HL-60, with a recombinant selectable HIV-1 clone. A fully infected population showed a relatively high frequency of low-level infection, with 40% of subcloned cells being negative by reverse transcriptase and p24 indirect immunofluorescence analysis and displaying only low levels of supernatant p24. The same treatment of a T-lymphoid cell line produced 100% productive infections. HIV-1 infection of HL-60 did not appear to alter the state of differentiation of the cells, as assessed by surface antigen expression, regardless of the level of viral expression. Furthermore, infected cells were able to respond normally to chemical inducers of differentiation. Induction of differentiation towards monocyte/macrophages by phorbol myristate acetate activated the HIV-1 long terminal repeat in a transient transfection system, and there was a corresponding increase in viral production from the infected subclones. Granulocytic differentiation, as stimulated by dimethyl sulfoxide or retinoic acid, had no effect on long terminal repeat activity and did not stimulate viral replication. These data suggest that low-level HIV-1 infections may be established at a relatively high frequency in myeloid precursor cells, and that different pathways of promyelocytic differentiation vary in their ability to stimulate HIV-1 replication.

Human immunodeficiency virus-1 infection of the human promyelocytic cell line HL-60: high frequency of low-level infection and effect of subsequent cell differentiation

As a model system to study the infection of early myeloid cells by human immunodeficiency virus-1 (HIV-1), we have infected the human promyelocytic cell line, HL-60, with a recombinant selectable HIV-1 clone. A fully infected population showed a relatively high frequency of low-level infection, with 40% of subcloned cells being negative by reverse transcriptase and p24 indirect immunofluorescence analysis and displaying only low levels of supernatant p24. The same treatment of a T-lymphoid cell line produced 100% productive infections. HIV-1 infection of HL-60 did not appear to alter the state of differentiation of the cells, as assessed by surface antigen expression, regardless of the level of viral expression. Furthermore, infected cells were able to respond normally to chemical inducers of differentiation. Induction of differentiation towards monocyte/macrophages by phorbol myristate acetate activated the HIV-1 long terminal repeat in a transient transfection system, and there was a corresponding increase in viral production from the infected subclones. Granulocytic differentiation, as stimulated by dimethyl sulfoxide or retinoic acid, had no effect on long terminal repeat activity and did not stimulate viral replication. These data suggest that low-level HIV-1 infections may be established at a relatively high frequency in myeloid precursor cells, and that different pathways of promyelocytic differentiation vary in their ability to stimulate HIV-1 replication.

New insights into the pathogenesis of coagulation dysfunction in acute promyelocytic leukemia.

Patients with acute promyelocytic leukemia (APL) are at high risk for the development of life-threatening thrombotic and hemorrhagic complications, particularly during induction chemotherapy. This propensity has been attributed to the release of tissue factor (TF)-like procoagulants from the leukemic cells leading to disseminated intravascular coagulation (DIC). However, recent data suggest that the pathogenesis of the coagulopathy is more complicated and may involve activation of the generalized proteolytic cascade resulting in either clotting and/or excessive fibrinolysis. Furthermore, controversy exists regarding the mechanism(s) responsible for the activation of either clotting or fibrinolysis. The malignant promyelocyte may act directly to activate coagulation and/or fibrinolysis. Alternatively, reactive inflammatory cells, which express procoagulant and/or profibrinolytic activities may play an essential role. A third possibility may involve endothelial cell expression of mediators with procoagulant/profibrinolytic properties. Putative profibrinolytic mechanisms include the release of urokinase-type and tissue-type plasminogen activators, decreases in plasminogen activator inhibitor-1 and 2, and decreases in alpha-2 plasmin inhibitor. Putative procoagulant mechanisms include the release of tissue factor, Cancer Procoagulant, or cytokines such as interleukin-1, tumor necrosis factor and vascular permeability factor. Putative anticoagulant mediators include annexins, a group of proteins in human tissue which bind phospholipids and have anticoagulant activity, which have been reported in patients with APL. The current treatment of APL is rapidly evolving because of the efficacy of all-trans retinoic acid (ATRA). All-trans retinoic acid promotes terminal differentiation of leukemic promyelocytes leading to complete remission in the majority of patients with APL with rapid resolution of the coagulopathy. Although the mechanism by which this occurs has not been established, preliminary data suggest that ATRA blocks the downregulation of the thrombomodulin gene and the up-regulation of the tissue factor gene induced by tumor necrosis factor. Since APL is a relatively uncommon disorder, the collaboration of cooperative oncology groups will be important to study patients receiving ATRA or conventional chemotherapy to further elucidate the mechanism(s) of the coagulopathy.

The PML-RARα fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR

We have previously shown that the t(15;17) translocation specifically associated with acute promyelocytic leukemia (APL) fuses the retinoic acid receptor α (RARα) locus to an as yet unknown gene, initially called myl and now renamed PML. We report here that this gene product contains a novel zinc finger motif common to several DNA-binding proteins. The PML-RARα mRNA encodes a predicted 106 kd chimeric protein containing most of the PML sequences fused to a large part of RARα, including its DNA- and hormone-binding domains. In transient expression assays, the hybrid protein exhibits altered transactivating properties if compared with the wild-type RARα progenitor. Identical PML-RARα fusion points are found in several patients. These observations suggest that in APL, the t(15;17) translocation generates an RAR mutant that could contribute to leukemogenesis through interference with promyelocytic differentiation.

Post-transcriptional regulation of thymidine kinase gene expression during monocytic differentiation of HL60 promyelocytes

The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were performed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation.

Osteoporosis and the metabolites of vitamin D

Vitamin D is metabolized by sequential steps in the liver and kidney to its active form, a process that is strongly feedback-regulated. In old age, the activity of the enzyme, 25-hydroxyvitamin D 1 hydroxylase, which produces the vitamin D hormone, is diminished. The activities of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) extend beyond increasing intestinal absorption of calcium. The vitamin D hormone (or its analogs) is useful in the treatment of osteoporosis because it not only stimulates intestinal calcium absorption, but also is required for the stimulation of osteoblasts and many other cells in the body. Recent work demonstrates that it is possible to chemically synthesize analogs selective for specific actions of the vitamin D hormone, especially in inducing differentiation of promyelocytes and keratinocytes.

Human peripheral lymphocyte growth regulation and response to phorbol esters is linked to synthesis and phosphorylation of the cytosolic protein, prosolin.

Prosolin is a major cytosolic protein (Mr 18400, isoelectric point 5.9) first reported in HL-60 promyelocytic leukemia cells. It is rapidly phosphorylated (15 to 30 min) in response to TPA treatment as an early event in a sequence that leads to cessation of cell proliferation and to differentiation of promyelocytes into monocytes. In our study we examined the expression of prosolin in human peripheral lymphocytes and investigated the effects of TPA treatment on prosolin phosphorylation and on lymphocyte proliferation. Prosolin was not expressed in resting PBL but was induced after 24 to 36 h of PHA stimulation, simultaneously with induction of DNA synthesis. In rapidly proliferating (IL-2 dependent) PBL prosolin was a major cytosolic component, comprising 0.5% of total cytosolic protein, of which approximately 28% was phosphorylated. Expression of prosolin decreased again when either mitogen-induced or IL-2-dependent proliferation diminished during extended periods in culture. Thus, expression of prosolin is correlated with periods when PBL are cycling through S-phase. TPA treatment of IL-2-dependent PBL at the peak of their growth caused phosphorylation of about two-thirds of preexisting unphosphorylated prosolin within 1 h. This was accompanied by cessation of cell proliferation, as indicated by measurements of TdR incorporation. Although TPA has well known mitogenic effects in lymphocytes during initial activation, this result shows that it exerts an antiproliferative effect in rapidly dividing PBL. It is suggested that increased phosphorylation of prosolin may be an initiating event in the antiproliferative response to TPA, which would occur only in proliferating lymphocytes expressing prosolin.

Novel Vitamin D Analogs That Modulate Leukemic Cell Growth and Differentiation With Little Effect on Either Intestinal Calcium Absorption or Bone Calcium Mobilization

AbstractInduction of terminal differentiation of leukemic and pre- leukemic cells is a therapeutic approach to leukemia and preleukemia. The 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause lifethreatening hypercalcemia. Seven new analogs of 1,25(OH)2 D3 were discovered to be either equivalent or more potent than 1,25(OH)2 D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells; and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne- vitaminD3, was about fourfold more potent than 1,25(OH)2 D3 . This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2 D3 . To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2 -16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(0H)2 D3 for 1,25(OH)2 D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2 D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2 D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2 D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.