KPT-330 has antitumour activity against non-small cell lung cancer.

BACKGROUND: We investigated the biologic and pharmacologic activities of a chromosome region maintenance 1 (CRM1) inhibitor against human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. METHODS: The in vitro and in vivo effects of a novel CRM1 inhibitor (KPT-330) for a large number of anticancer parameters were evaluated using a large panel of 11 NSCLC cell lines containing different key driver mutations. Mice bearing human NSCLC xenografts were treated with KPT-330, and tumour growth was assessed. RESULTS: KPT-330 inhibited proliferation and induced cell cycle arrest and apoptosis-related proteins in 11 NSCLC cells lines. Moreover, the combination of KPT-330 with cisplatin synergistically enhanced the cell kill of the NSCLC cells in vitro. Human NSCLC tumours growing in immunodeficient mice were markedly inhibited by KPT-330. Also, KPT-330 was effective even against NSCLC cells with a transforming mutation of either exon 20 of EGFR, TP53, phosphatase and tensin homologue, RAS or PIK3CA, suggesting the drug might be effective against a variety of lung cancers irrespective of their driver mutation. CONCLUSIONS: Our results support clinical testing of KPT-330 as a novel therapeutic strategy for NSCLC.

PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells.

BACKGROUND: The PIAS4 protein belongs to the family of protein inhibitors of activated STAT, but has since been implicated in various biological activities including the post-translational modification known as sumoylation. In this study, we explored the roles of PIAS4 in pancreatic tumourigenesis. METHODS: The expression levels of PIAS4 in pancreatic cancer cells were examined. Cell proliferation and invasion was studied after overexpression and gene silencing of PIAS4. The effect of PIAS4 on hypoxia signalling was investigated. RESULTS: The protein was overexpressed in pancreatic cancer cells compared with the normal pancreas. Gene silencing by PIAS4 small interfering RNA (siRNA) suppressed pancreatic cancer cell growth and overexpression of PIAS4 induced expression of genes related to cell growth. The overexpression of PIAS4 is essential for the regulation of the hypoxia signalling pathway. PIAS4 interacts with the tumour suppressor von Hippel-Lindau (VHL) and leads to VHL sumoylation, oligomerization, and impaired function. Pancreatic cancer cells (Panc0327, MiaPaCa2) treated with PIAS4 siRNA suppressed expression of the hypoxia-inducible factor hypoxia-inducible factor 1 alpha and its target genes JMJD1A, VEGF, and STAT3. CONCLUSION: Our study elucidates the role of PIAS4 in the regulation of pancreatic cancer cell growth, where the suppression of its activity represents a novel therapeutic target for pancreatic cancers.

p53 in chronic myelogenous leukemia. Study of mechanisms of differential expression.

The p53 is a nuclear protein that is associated with normal cellular proliferation and can cooperate with Ha-ras in causing cellular transformation in vitro. Lineage association is known to exist between p53 expression and normal lymphopoiesis, but not myelopoiesis. We studied the expression of p53 using chronic myelogenous leukemia (CML) cell lines, somatic hybrids of these cells, and leukemic cells from CML patients. Lymphoid CML lines expressed both p53 mRNA and protein. We also analyzed p53 synthesis by two B-lymphoid lines from the same CML patient; cells of one line were derived from the neoplastic clone, cells of the other were derived from the normal clone. Both synthesized equal amounts of a phosphorylated p53 protein. None of the myeloid CML lines expressed detectable p53 protein and two of four expressed negligible p53 mRNA. Two other myeloid CML lines and myeloid cells from three of four patients expressed p53 mRNA. These findings suggest that expression of the gene is not regulated normally in CML. Several approaches were pursued to explore the differential expression of p53. Southern blot analyses showed no gross alterations in the p53 gene from cells of either the expressing or the nonexpressing lines. No difference in the pattern of demethylated CpG sites was noted in the region of the p53 gene in cells from K562 (myeloid p53 nonexpressor) and in BV173 (lymphoid p53 expressor). The sites of demethylation clustered in and around the p53 promoter in both cell lines. Somatic hybrids formed between a p53 mRNA nonexpressor myeloid line (K562) and the parental p53 expressor lymphoid lines (Daudi, PUT) produced p53 mRNA and protein, suggesting that p53 is a dominantly expressed protein and that lack of expression in myeloid cells is not mediated by a trans-acting negative regulatory protein. DNA transfection experiments performed using the indicator gene chloramphenicol acetyltransferase attached to promoter sequences of p53 showed that these constructs were equally activated in BV173 (p53 expressor) and K562 (p53 mRNA nonexpressor). The mechanism of p53 regulation in CML remains unclear.

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells.

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.

Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity.

A permanent human cell line that maintains the granulocytic characteristics of acute myelogenous leukemia cells has been established. The cells of this line form myeloid colonies in soft gel culture in the presence of human colony-stimulating activity. The cell line may be useful for studying human acute myelogenous leukemia and the mechanism of response to colony-stimulating activity.

Induction of sodium iodide symporter gene and molecular characterisation of HNF3 beta/FoxA2, TTF-1 and C/EBP beta in thyroid carcinoma cells.

Thyroid carcinoma cells often do not express thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). Treatment of thyroid carcinoma cells (four papillary and two anaplastic cell lines) with histone deacetylase inhibitors (SAHA or VPA) modestly induced the expression of the NIS gene. The promoter regions of the thyroid-specific genes contained binding sites for hepatocyte nuclear factor 3 beta (HNF3 beta)/forkhead box A2 (FoxA2), thyroid transcription factor 1 (TTF-1), and CCAAT/enhancer binding protein (C/EBP beta). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed decreased expression of HNF3 beta/FoxA2 and TTF-1 mRNA in papillary thyroid carcinoma cell lines, when compared with normal thyroid cells. Forced expression of these genes in papillary thyroid carcinoma cells inhibited their growth. Furthermore, the CpG island in the promoter region of HNF3 beta/FoxA2 was aberrantly methylated; and treatment with 5-aza-2-deoxycytidine (5-Az) induced its expression. Immunohistochemical staining showed that C/EBP beta was localised in the nucleus in normal thyroid cells but was detected in the cytoplasm in papillary thyroid carcinoma cells. Subcellular fractionation of papillary thyroid carcinoma cell lines also demonstrated high levels of expression of C/EBP beta in the cytoplasm, suggesting that a large proportion of C/EBP beta protein is inappropriately localised in the cytoplasm. In summary, these findings reveal novel abnormalities in thyroid carcinoma cells.

Fludarabine induces apoptosis of human T-cell leukemia virus type 1-infected T cells via inhibition of the nuclear factor-kappaB signal pathway.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease in which the human T-cell lymphotropic virus type I (HTLV-I) has been recognized as the etiologic agent. Fludarabine is a purine analog that has demonstrated significant activity in B-cell malignancies, including chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma. This study explored the effects of fludarabine on HTLV-1-infected T cells (MT-1, -2, -4 and HUT102). Fludarabine induced growth arrest and apoptosis of these cells, as measured by 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell cycle analysis and annexin V staining. Moreover, exposure of HTLV-1-infected T cells to fludarabine decreased the levels of X-inhibitor of apoptosis protein in conjunction with inhibition of nuclear factor kappaB (NF-kappaB)/DNA-binding activity, as measured by Western blot analysis and electrophoretic mobility shift and reporter gene assays, respectively. Further studies found that fludarabine accumulated NF-kappaB and inhibitory subunit of NF-kappaB in cytosole in conjunction with downregulation of NF-kappaB in nucleus, suggesting that fludarabine blocked nuclear translocation of NF-kappaB. Taken together, fludarabine may be useful for treatment of individuals with ATL and other types of cancer in which NF-kappaB plays a role.

Common deleted genes in the 5q- syndrome: thrombocytopenia and reduced erythroid colony formation in SPARC null mice.

The commonly deleted region (CDR) for the 5q- syndrome has been identified as a 1.5-megabase interval on human chromosome 5q32. We studied, by real-time reverse-transcription (RT)-PCR, the expression of 33 genes within the CDR that are known to be expressed in CD34+ hematopoietic stem cells. Genes in the 5q- samples that showed the most pronounced decrease in expression compared to non-5q- samples were: solute carrier family 36, member 1 (SLC36A1; 89% downregulated), Ras-GTPase-activating protein SH3 domain-binding (G3BP; 79%), antioxidant protein 1 (ATOX1; 76%), colony-stimulating factor-1 receptor precursor (CSF1R; 76%), ribosomal protein S14 (RPS14; 74%), platelet-derived growth factor receptor-beta (PDGFRB; 73%), Nef-associated factor 1 (TNIP1; 72%), secreted protein, acidic and rich in cysteine (SPARC; 71%), annexin VI (ANAX6; 69%), NSDT (66%) and TIGD (60%). We further studied the hematopoietic system in SPARC-null mice. These mice showed significantly lower platelet counts compared to wild-type animals (P=0.008). Although hemoglobin, hematocrit and mean corpuscular volume (MCV) were lower in mice lacking SPARC, differences were not statistically significant. SPARC-null mice showed a significantly impaired ability to form erythroid burst-forming units (BFU-E). However, no significant differences were found in the formation of erythroid colony-forming units (CFU-E), granulocyte/monocyte colony-forming units (CFU-GM) or megakaryocyte colony-forming units (CFU-Mk) in these animals. We conclude that many of the genes within the CDR associated with the 5q- syndrome exhibit significantly decreased expression and that SPARC, as a potential tumor suppressor gene, may play a role in the pathogenesis of this disease.

Discovery of epigenetically silenced genes in acute myeloid leukemias.

The demethylating 5-aza-2'deoxycytidine (DAC) and the histone deacetylase inhibitor (HDACi) suberoyl anilide bishydroxamide (SAHA) possess potent antitumorigenic properties in myeloid disorders. However, the transcriptome alterations mediated by these drugs are poorly understood. We analyzed the transcriptional effects of DAC and SAHA in the AML cell line KG-1. Microarray analyses revealed 76 genes expressed in normal CD34+ cells, absent in KG-1 cells but whose expression was induced after drug treatment. A total of 39 of these genes harbored CpG islands in their promoters. We examined the expression level of these genes in 120 AML patient samples representing diverse karyotpyes. Gas2l1, tfIIs, ehd3, enolase 2, mx1, dral, astml and pxdn were diminished across all AML karyotypes examined. Ehd3 was methylated in 63% of AML patients examined. This methylation was lost upon complete remission, and not observed in normal CD34+ cells. CD34+ cells expressed ehd3 at approximately 10-fold higher levels than AML samples. Another highlighted gene, alpha-catenin, is located at q31 of chromosome 5. Analyses of 29 5q- AML/myelodysplastic syndrome (MDS) samples revealed marked decreases in expression of alpha-catenin, compared to non-5q- MDS samples (6.6+/-9-fold). However, no methylation was detected, suggesting indirect effects of these drugs on the expression of alpha-catenin.

Antiproliferative activity of RAD001 (everolimus) as a single agent and combined with other agents in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive form of B-cell non-Hodgkin's lymphoma, with a mean survival of only 3-5 years and suboptimal therapeutic options. MCL is characterized by a balanced translocation t(11;14)(q13;q32), resulting in overexpression of cyclin D1, a G(1) cyclin regulated by the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway. As improved therapy for MCL is required and the mTOR pathway may be involved in its pathophysiology, the antiproliferative effects of RAD001 (everolimus), an mTOR inhibitor, against three MCL cell lines were investigated. As a single agent, RAD001 inhibited proliferation in MCL cell lines (Jeko1, SP49 and NCEB1) approximately 40-65% compared to diluent control cells. This was associated with G(1) cell-cycle arrest and reduced phosphorylation of the mTOR downstream target, 4E-BP1. Furthermore, combination drug studies revealed predominantly synergistic cytotoxicity with RAD001 and several secondary agents, including doxorubicin, vincristine or rituximab (components of the standard MCL regimen), as well as paclitaxel, vorinostat and bortezomib. These data indicate that single agent RAD001 is effective in inhibiting growth of MCL cells in vitro and combination studies with secondary agents further demonstrate synergistic cytotoxicity. Thus, these findings support future clinical studies of RAD001 in the treatment of MCL.

Targeting the vulnerability to NAD+ depletion in B-cell acute lymphoblastic leukemia.

Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.

BRCA1 and FOXA1 proteins coregulate the expression of the cell cycle-dependent kinase inhibitor p27(Kip1).

We have previously shown that the breast cancer susceptibility gene, BRCA1, can transcriptionally activate the p27(Kip1) promoter. The BRCA1-responsive element was defined as a 35 bp region from position -545 to -511. We next determined that within this region is also a potential binding site for the transcription factor Forkhead box (FOX)A1. RNA and protein analysis as well as immunohistochemistry showed that expression of FOXA1 correlated with the expression of the estrogen receptor in a panel of breast cancer cell lines and tissues. In transient transfection reporter assays, FOXA1 could activate the p27(Kip1) promoter. Cotransfection of BRCA1 and FOXA1 resulted in a synergistic activation of the p27(Kip1) promoter. Mutation of the FOXA1 DNA-binding site in the p27(Kip1) promoter-luciferase construct significantly diminished the activity of FOXA1 alone or in combination with BRCA1. Cotransfection of FOXA1 and BRCA1 resulted in a greater amount of each protein compared to transfection of each expression vector alone. The half-life of FOXA1 was increased when coexpressed with BRCA1. Electrophoretic mobility shift assay analysis demonstrated that FOXA1 could bind to a wild-type oligonucleotide containing the FOXA1 binding site in the p27(Kip1) promoter, but this binding was lost upon mutation of this FOXA1 binding site. The protein-DNA binding complex could be supershifted with an antibody directed against FOXA1. The activity of the p27(Kip1) promoter as well as FOXA1 expression was reduced in cells treated with BRCA1 siRNA, thus silencing the expression of BRCA1 protein. In summary, we identified a FOXA1 binding site within the BRCA1-responsive element of the p27(Kip1) promoter and showed that FOXA1 activated the promoter alone and in conjunction with BRCA1. Furthermore, we identified high expression of FOXA1 in breast cancer cell lines and tissues, discovered a role for BRCA1 in the regulation of p27(Kip1) transcription and a possible interaction with BRCA1.

DLK1: increased expression in gliomas and associated with oncogenic activities.

DLK1 (delta-like) is a transmembrane and secreted protein in the epidermal growth factor-like homeotic family. Although expressed widely during embryonic development, only a few tissues retain the expression in adults. Neuroendocrine tumors often highly express this protein; therefore, we hypothesized that brain tumors might also express it. This study found that the expression of DLK1 in gliomas was higher than that in normal brain (P < 0.05). After stable transfection of a DLK1 cDNA expression vector into GBM cell lines, their proliferation was increased. Furthermore, they lost contact inhibition, had enhanced anchorage-independent growth in soft agar, and had significantly greater capacity to migrate. Western blot studies showed that expression of cyclin D1, CDK2, and E2F4 were increased, and Rb levels were decreased in these cells. DLK1 was found on the cell surface and secreted in the medium from the transfected GBM cells. DLK1-enriched condition medium stimulated the growth of glioblastoma multiforme cell lines and explants. DLK1 antibody blocked cell growth stimulated by DLK1. In summary, these results suggest that DLK1 may play a role in the formation or progression of gliomas.

Vitamin D compounds. Effect on clonal proliferation and differentiation of human myeloid cells.

We examined the effect of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and a variety of vitamin D analogs on proliferation and differentiation of normal and leukemic myeloid clonogenic cells. Only cells from myeloid leukemic lines that contained relatively mature cells (HL-60, U937, THP, HEL, M1) were induced to differentiate and were inhibited in their clonal growth by exposure to 1 alpha,25(OH)2D3 (50% inhibition, 3 X 10(-8)-8 X 10(-10) M). A fluorinated analog of vitamin D was 5-10-fold more potent than 1 alpha,25(OH)2D3. Cells from a human myeloblast line (KG-1) and normal human granulocyte-monocyte stem cells (GM-CFC), both of which depend on colony-stimulating factor (CSF) for clonal growth, were stimulated in their clonal proliferation by 1 alpha,25(OH)2D3 in the presence of suboptimal concentrations of CSF. Leukemic cells from 10 of 14 patients with myeloid leukemia, but not normal GM-CFC from 12 patients in remission, were markedly inhibited in their clonal proliferation by 1 alpha,25(OH)2D3. Our results suggest that 1 alpha,25(OH)2D3 may be a cofactor in hematopoiesis and that vitamin D analogs may have a differential effect on normal versus leukemic growth.

Retinoic acid enhances growth of human early erythroid progenitor cells in vitro.

We studied the effect of retinoic acid on the clonal proliferation of normal human early erythroid progenitor cells in vitro. Normal peripheral blood cells were cultured in methylcellulose with erythropoietin and the number of burst-forming units-erythroid (BFU-E) colonies were scored on day 12 of culture. All-trans retinoic acid increased the number of colonies in a dose-response fashion. Maximal stimulation occurred at 30 nM retinoic acid, which increased the number of BFU-E by a mean of 225 +/- 25% (+/- SE) over plates containing erythropoietin alone. Colony formation increased even in the presence of maximally stimulating concentrations of erythropoietin. The 13-cis retinoic acid stimulated BFU-E proliferation in a parallel manner as the trans analogue, while retinol (vitamin A) did not affect clonal growth. This data supports further the thesis that retinoic acid, in addition to its known effect on epithelial cells, may be involved in the growth of normal hematopoietic cells.

Retinoic acid. Inhibition of the clonal growth of human myeloid leukemia cells.

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.

Retinoids. Structure-function relationship in normal and leukemic hematopoiesis in vitro.

Retinoids were studied both to identify what skeletal components are important in the modulation of normal and leukemic human myeloid clonal proliferation and differentiation in vitro and to elucidate the mechanism by which retinoids modulate proliferation of hematopoietic cells. Retinoids with a derivatized terminal carboxyl group were significantly less active than all-trans-retinoic acid, and those with the addition of two methyl groups to the cyclohexenyl ring of retinoic acid or substitution of its beta-cyclogeranylidene group with a 1,1,3,3-5-indanyl ring system were markedly more active than all-trans-retinoic acid. Five of the retinoids strongly inhibited clonal growth of the HL-60 and KG-1 human leukemic cell lines (50% inhibition in the range of 3 X 10(-10)-1 X 10(-8) M) and markedly stimulated normal human myeloid colony formation (granulocyte-macrophage colony-forming cells [GM-CFC] 150% stimulation in the range of 3 X 10(-9)-3 X 10(-8) M). Further studies suggested that: Common structural requirements of the retinoids were important in the modulation of both normal and leukemic hematopoiesis. The retinoids were able to inhibit leukemic proliferation without induction of differentiation of the neoplastic cells. Studies on normal human GM-CFC suggested that the retinoids did not act by themselves as a colony-stimulating factor (CSF), or by stimulating accessory cells to produce CSF, but either required earlier progenitor cells to become GM-CFC or enhanced the sensitivity of GM-CFC to the action of CSF.

Phorbol diester-induced macrophage differentiation of leukemic blasts from patients with human myelogenous leukemia.

Phorbol esters, including 12-O-tetradecanoylphorbol 13-acetate (TPA), induce terminal macrophagelike differentiation of cells from human acute myelogenous leukemia lines. We report that myelogenous leukemia cells obtained from patients undergo macrophagelike differentiation after exposure to TPA. The myeloid leukemic cell cultured with TPA became adherent to charged surfaces with long filamentous pseudopodia; developed positive staining for alpha-napthyl acetate esterase, increased lysozyme secretion, reduced nitroblue tetrazolium, and acquired the ability to phagocytose candida. Cells from patients with lymphocytic leukemia did not become macrophagelike when cultured with TPA.

1,25-Dihydroxyvitamin D3 modulates the expression of a lymphokine (granulocyte-macrophage colony-stimulating factor) posttranscriptionally.

We recently showed that 1,25(OH)2D3 sensitively inhibited the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in normal human mitogen-activated peripheral blood lymphocytes and in the human T lymphotropic virus I immortalized T cell line known as S-LB1 at the levels of both mRNA and protein. Using S-LB1 cells as a model system the present paper identifies at least in part the mechanisms by which 1,25(OH)2D3 regulates the expression of GM-CSF. Time-course studies demonstrated that by 6 and 48 h of exposure of S-LB1 cells to 1,25(OH)2D3 (10(-8) M) the GM-CSF mRNA levels were reduced by 50 and 90%, respectively. Studies using cycloheximide as a protein synthesis inhibitor showed that the inhibitory action of 1,25(OH)2D3 on GM-CSF expression was dependent on new protein synthesis. In vitro nuclear run-on assays demonstrated that 1,25(OH)2D3 (10(-8) M) did not change the rate of transcription of the GM-CSF gene. The t1/2 of GM-CSF mRNA, however, was profoundly reduced by 1,25(OH)2D3 when transcription was blocked by actinomycin D compared with the half-life of GM-CSF in the presence of actinomycin D alone (t1/2, less than 0.5 and 4 h, respectively). Taken together, these results demonstrate that 1,25(OH)2D3 regulates expression of the lymphokine GM-CSF posttranscriptionally by influencing the stability of GM-CSF mRNA.

Granulocyte-macrophage colony-stimulating factor. Sensitive and receptor-mediated regulation by 1,25-dihydroxyvitamin D3 in normal human peripheral blood lymphocytes.

We show that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), the most hormonally active metabolite of vitamin D3, modulates sensitively and specifically both the protein and messenger RNA accumulation of the multilineage growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The regulation of GM-CSF expression is seen in both normal human mitogen-activated T lymphocytes and T lymphocytes from a line (S-LB1) transformed with human T cell lymphotropic virus 1 (HTLV-1). In contrast, cells from a HTLV-1 transformed T lymphocyte line (Ab-VDR) established from a patient with vitamin D-resistant rickets type II with undetectable 1,25(OH)2D3 cellular receptors are resistant to the action of 1,25(OH)2D3. Inhibition of GM-CSF expression by 1,25(OH)2D3 can occur independently of interleukin 2 regulation and is probably mediated through cellular 1,25(OH)2D3 receptors. We conclude that 1,25(OH)2D3 may be important in the physiology of hematopoiesis.