Monoblastic Differentiation Research Articles

Subtype-specific patterns of molecular mutations in acute myeloid leukemia.

Acute myeloid leukemia (AML) can be grouped into morphologically or genetically defined subtypes. Today, the AML phenotype-genotype associations, that is, FAB/WHO (French-American-British/World Health Organization) definitions and recurrent molecular mutations, are not fully understood. Therefore, we evaluated the impact of molecular mutations on the AML differentiation stage by molecular profiling of 4373 adult de novo AML patients in 7 cytomorphological subtypes. We investigated mutations in 20 genes, including myeloid transcription factors (CEBPA, RUNX1), tumor suppressors (TP53, WT1), DNA modifiers (DNMT3A, IDH1/2, TET2), chromatin modifiers (ASXL1, MLL), signal transduction genes (FLT3, KRAS, NRAS) and NPM1. The most frequently mutated genes per cytomorphological subtype were RUNX1 in M0 (43%), NPM1 in M1 (42%), DNMT3A in M2 (26%), NPM1 in M4 (57%), M5a (49%) and M5b (70%) and TP53 in M6 (36%). Although some gene mutations were frequent in several cytomorphological subtypes, a series of associations of co-occurring mutations with distinct phenotypes were identified for molecularly defined subcohorts. FLT3, NPM1 and WT1 mutations were associated with an immature phenotype in myeloblastic AML, whereas other combinations involving ASXL1, RUNX1, MLL-PTD, CEBPA or KRAS were more frequent in myeloblastic AML with maturation. Within the NPM1 mutated subcohort, ASXL1 mutations were significantly associated with a monoblastic differentiation and DNMT3A mutations with a monocytic phenotype.

The leukemogenic fusion gene MLL-AF9 alters microRNA expression pattern and inhibits monoblastic differentiation via miR-511 repression.

BackgroundIn this study we explored the role of microRNAs (miRNAs) as mediators of leukemogenic effects of the fusion gene MLL-AF9, which results from a frequent chromosomal translocation in infant and monoblastic acute myeloid leukemia (AML).MethodsWe performed a specific and efficient knockdown of endogenous MLL-AF9 in the human monoblastic AML cell line THP1.ResultsThe knockdown associated miRNA expression profile revealed 21 MLL-AF9 dependently expressed miRNAs. Gene ontology analyses of target genes suggested an impact of these miRNAs on downstream gene regulation via targeting of transcriptional modulators as well as involvement in many functions important for leukemia maintenance as e.g. myeloid differentiation, cell cycle and stem cell maintenance. Furthermore, we identified one of the most intensely repressed miRNAs, miR-511, to raise CCL2 expression (a chemokine ligand important for immunosurveillance), directly target cyclin D1, inhibit cell cycle progression, increase cellular migration and promote monoblastic differentiation. With these effects, miR-511 may have a therapeutic potential as a pro-differentiation agent as well as in leukemia vaccination approaches.ConclusionsOur study provides new insights into the understanding of miRNAs as functional mediators of the leukemogenic fusion gene MLL-AF9 and opens new opportunities to further investigate specific therapeutic options for AML via the miRNA level.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0283-5) contains supplementary material, which is available to authorized users.

De Novo Acute Myeloid Leukemia in Adults: Suppression of MicroRNA-223 is Independent of LMO2 Protein Expression BUT Associate With Adverse Cytogenetic Profile and Undifferentiated Blast Morphology.

MicroRNA (MIR) signatures are critical to pathobiology and prognosis of acute myeloid leukemia (AML). MIR223 is expressed at low levels in progenitor cells, whereas high expression is induced by granulocytic differentiation. Novel-targeted therapies through epigenetic manipulation of MIR223 regulators are being explored in AML but correlative data between established clinical prognostic markers and MIR223 expression in AML is lacking. MIR223 has inverse relationship with LMO2 protein expression and our group has recently reported a close association between LMO2 protein expression and chromosomal findings in AML patients. In this study, we examined the expression of MIR223 in a large cohort of AML patients and correlated it with LMO2 protein expression, cytogenetic data, degree of differentiation [French-American and British (FAB)/World Health Organization classifications], and overall survival. MIR223 expression was upregulated in only a subset of patients (37%). Suppression of MIR223 was more frequent among patients with aneuploid karyotype compared with diploid karyotype (P=0.005). In AML, not otherwise specified category, AML with maturation (FAB-M2) showed higher levels of MIR223 when compared with either AML without maturation (FAB M0/M1) (P=0.001); AML with monoblastic differentiation (FAB M4/M5) (P=0.004) or AML with myelodysplasia-related changes (P=0.011). Among cytogenetic risk groups, suppression of MIR223 was universal (>95%) in high-risk group when compared with intermediate-risk group (P=0.004). No correlation between MIR223 and LMO2 protein expression was identified. In conclusion, we have shown that suppression of MIR223 expression, as compared with controls, is associated with lack of differentiation and adverse cytogenetic profile, but unrelated with LMO2 protein expression or overall survival.

Myeloid sarcomas: a clinicopathologic study of 20 cases.

Objective:Myeloid sarcoma is a tumoral mass of mature or immature myeloid blasts in extramedullary anatomic locations. It can be seen de novo or in association with acute myeloid leukemia, myeloproliferative neoplasias, or myelodysplastic syndrome. Isolated myeloid sarcoma can be seen as a relapse in cases with allogenic bone marrow transplantation. Although it may involve any tissue in the body, the most common locations are skin, soft tissues, lymph nodes, and the gastrointestinal tract. Immunohistochemically, most cases show myelomonocytic or pure monoblastic differentiation. We reviewed the clinicopathological features of 20 cases of myeloid sarcoma diagnosed in our institute in view of the literature.Materials and Methods:The cases diagnosed between 2005 and 2012 at the Ankara University Faculty of Medicine, Department of Pathology, were selected. Clinicopathological findings including the age and sex of the patients; symptoms; anatomic location; accompanying hematological disease; and the morphological, immunohistochemical, and cytogenetic features of the cases were noted.Results:Sixteen of the patients were male and 4 were female. The median age at diagnosis was 47 years. The most commonly involved locations were the lymph nodes and skin. Immunohistochemically, eleven cases were of the myelomonocytic and 7 cases were of the myeloid phenotype, whereas 2 cases showed pure monoblastic differentiation. The median follow-up period for the 18 cases with known clinical data was 33 weeks. Five patients died of the disease in an average of 36 weeks.Conclusion:Myeloid sarcoma is a rare presentation of leukemias, myeloproliferative neoplasias, or myelodysplastic syndrome, composed of immature myelomonocytic cells in extramedullary tissues. It may present with variable morphological and phenotypic features, always creating a challenge in pathological diagnosis.

Progressive course of isolated splenic myeloid sarcoma

Abstract A previously healthy 40-year-old male presented with a 2-week history of fever and abdominal discomfort that was resistant to empirical broad-spectrum antibiotic treatment. The patient’s blood cell count and complete biochemical panel was normal, except for an increased lactate dehydrogenase level. Ultrasonography and computed tomography of the abdomen showed a large, soft tissue mass had infiltrated superior part of the spleen. Splenectomy with total tumor mass removal were performed. The pathological examination of the tumor tissue confirmed diagnosis of isolated myeloid sarcoma with monoblastic differentiation. Despite intensive antileukemic therapy, patient died four months after diagnosis was established.

Nuclear factor of activated T-cells 1 increases sensitivity of v-myb transformed monoblasts to all-trans retinoic acid

Nuclear factors of activated T-cells (NFATs) are important regulators of the cytokine gene expression in activated T-cells. In the last decade, NFATs have been shown to regulate cell cycle, differentiation and apoptosis in cells of various origins revealing their importance for cell homeostasis. In this study, we investigated the effects of NFAT1 on proliferation and differentiation of v-myb-transformed BM2 monoblasts. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once overexpressing NFAT1, they became sensitive to all-trans retinoic acid (ATRA). The ATRA-treated BM2NFAT1 cells differentiated along monocyte/macrophage pathway as evidenced by changes in cell morphology, adherence, phagocytic and non-specific esterase activities, reactive oxygen species production, and vimentin expression. Furthermore, overexpressed NFAT1 either alone or in combination with the ATRA-driven signalling pathway deregulated cyclin A and retinoic acid receptor proteins in BM2 cells. Data presented in this study indicate that the NFAT1 and ATRA signalling pathways synergize in control of proliferation and differentiation of BM2 monoblasts.

Philadelphie chromosome—Positive <i>de novo</i> acute myeloid leukemia. Isolated meningeal relapse in a patient treated with imatinib mesylate

Acute myeloid leukemia philadelphie positive (Ph+ AML) is a rare aggressive acute leukemia with poor prognosis. We report a patient with ph positive AML (FAB5), the transcript bcr/abl was not performed at diagnosis. She achieved complete remission after conventional induction chemotherapy. The consolidation therapy was based on Imatinib only due to infectious complications. She was in complete hematologic and cytogenetic remission for 19 months, and after she exhibited an isolated meningeal relapse. A second remission was achieved with intrathecal chemotherapy and cranial irradiation. Imatinib was switched to second generation Tyrosine kinase Inhibitor which had better diffusion into cerebrospinal fluid. She is in complete hematologic, cytogenetic and meningeal remission after 14 months of treatment. Imatinib monotherapy affords insufficient protection from CNS relapse. Second generation Tyrosine kinase Inhibitor seems to have better efficiency. Ph+ AML with monoblastic differentiation should be considered, like Ph+ ALL, at high risk of meningeal leukemia and should receive central nervous system prophylaxis.

AML with Translocation T(8;16) Shows Unique Cytomorphological, Cytogenetic, Molecular, and Prognostic Features and Therefore Qualifies as An Own Entity According to WHO Criteria.

Balanced chromosomal rearrangements define distinct biological subsets in acute myeloid leukemia (AML). It is recognized that recurrent balanced aberrations, such as t(15;17), t(8;21), inv(16), and 11q23/MLL translocations, show a close correlation to cytomorphology and also harbor specific gene expression signatures. We here present a cohort of 13 AML cases with t(8;16)(p11;p13). This translocation is rare with only 13 cases (6 males, 7 females) diagnosed from our overall cohort of 6124 cases of AML over recent years, and is more frequently found in therapy-related AML than in de novo AML (7/438 t-AML, and 6/5686 de novo, p=0.00001). Prognosis was poor with median overall survival of 4.7 months. Five patients deceased within the first month after diagnosis. AML with t(8;16) is characterized by striking cytomorphologic features: In all 13 cases the positivity for myeloperoxidase (MPO) on bone marrow smears was >30% (median: 85%) and intriguingly, in parallel also >40% (median: 88%) of blast cells stained strongly positive for non-specific esterase (NSE) in the same cell, suggesting that AML with t(8;16) arise from a very early stem cell with both myeloid and monoblastic differentiation potential. Therefore, AML with t(8;16) cases can not be classified according to standard FAB categories. Morphologically we also detected erythrophagocytosis in 7/13 cases, a specific feature in AML with t(8;16) that was previously described. With respect to cytogenetics, 6/13 patients had t(8;16)(p11;p13) as sole abnormality. 7/13 patients demonstrated additional non-recurrent abnormalities, 4 cases with single additional aberrations, and 3 cases with two or more additional aberrations. Molecular analyses detected the MYST3- CREBBP fusion transcript in all cases tested (12/12). We then compared gene expression patterns in 7 cases of AML with t(8;16) to: (i) AML FAB subtypes M1 and M4/5 with strong MPO or NSE with normal karyotype and to (ii) distinct AML subtypes with balanced chromosomal aberrations according to WHO classification. In a first series using Affymetrix HG-U133A+B microarrays 4 cases of AML with t(8;16) were compared to FAB M1 (n=46), M4 (n=41), M5a (n=9), and M5b (n=16). Hierarchical clustering and principal component analyses revealed that AML with t(8;16) were intercalating rather with FAB subtypes M4 and M5b and did not cluster near to FAB M1, although strong positivity for MPO was seen in all t(8;16) cases. Thus, monocytic characteristics influence the gene expression pattern stronger than myeloid features. When further compared to AML WHO subtypes t(15;17) (n=43), t(8;21) (n=43), inv(16) (n=49), and 11q23/MLL (n=50), AML with t(8;16) samples were repeatedly grouped in the vicinity of the 11q23/MLL cases. This can be explained by a similar expression of genes such as EAF2, HOXA9, HOXA10, PRKCD, or HNMT. Yet, in a subsequent pairwise comparison AML with t(8;16) could also be clearly discriminated from 11q23/MLL with differentially expressed genes including CAPRIN1, RAN, SMARCD2, LRRC41, or H2BFS, higher expressed in AML with t(8;16) and SOCS2, PRAME, RUNX3, or TPT1, lower expressed in AML with t(8;16), respectively. Moreover, the respective FAB-type or WHO-type signatures were validated on a separate cohort of patients (n=3 AML with t(8;16); n=107 other AML subtypes as above), all prospectively analyzed with the successor HG-U133 Plus 2.0 microarray. Again, in direct comparison to FAB-type or WHO-type cases, dominant and unique gene expression patterns were seen for AML with t(8;16), confirming the molecular distinctiveness of this rare AML entity. Using a classification algorithm we were able to correctly predict all AML with t(8;16) cases by their gene expression pattern. This accuracy was observed not only for both FAB-type and WHO-type signatures, but also correctly classified the cases across the different patient cohorts and microarray designs. In conclusion, AML with t(8;16) is a specific subtype of AML with very poor prognosis that often presents as treatment-related AML and with particular characteristics not only in morphology and clinical profile, but also on a molecular level. Due to these unique features, it qualifies as a specific recurrent entity according to WHO criteria.

Retinoic acid enhances differentiation of v- myb-transformed monoblasts induced by okadaic acid

Differentiation of various leukemic cells can be induced by liganded retinoic acid receptors and protein phosphatase inhibitors. In this study, we explored the effects of okadaic acid (OA), the phosphatase inhibitor, and retinoic acid (RA) in v- myb-transformed monoblasts BM2. OA induced differentiation of BM2 monoblasts into macrophage-like cells, as documented by analyses of cell morphology, cell cycle, phagocytic activity, non-specific esterase activity, production of reactive oxygen species and expression of vimentin and Mo-1. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once exposed to OA and RA simultaneously, BM2 cells differentiate along monocyte/macrophage pathway more efficiently. We conclude that RA enhances differentiation of v- myb-transformed monoblasts induced by protein phosphorylation.

Myeloid sarcomas: a histologic, immunohistochemical, and cytogenetic study

Context. -Myeloid sarcoma (MS) is a neoplasm of immature granulocytes, monocytes, or both involving any extramedullary site. The correct diagnosis of MS is important for adequate therapy, which is often delayed because of a high misdiagnosis rate.Objective. -To evaluate the lineage differentiation of neoplastic cells in MS by immunohistochemistry, and to correlate the results with clinicopathologic findings and cytogenetic studies.Design. -Histologic and immunohistochemical examinations were performed on formalin-fixed paraffin-embedded tissue samples from 13 cases of MS. They were classified according to the World Health Organization criteria. Chromosomal analysis data were available in 11 cases. Clinical, pathological, and cytogenetic findings were analyzed.Results. -The study included six male and seven female patients with an age range of 25 to 72 years (mean, 49.3 years) and a male to female ratio of 1:1.2. MS de novo occurred in 4/13 (31%) of cases examined. The most sensitive immunohistochemical markers were CD43 and lysozyme present in all cases with MS (13/13, 100%). All de novo MS showed a normal karyotype, monoblastic differentiation, and lack of CD34. The most common chromosomal abnormalities in MS associated with a hematopoietic disorder were trisomy 8 and inv(16) (2/11, 18%).Conclusion. -An immunohistochemical panel including CD43, lysozyme, myeloperoxidase (MPO), CD68 (or CD163), CD117, CD3 and CD20 can successfully identify the vast majority of MS variants in formalin-fixed paraffin-embedded tissue sections. The present report expands the spectrum of our knowledge showing that de novo MS has frequent monoblastic differentiation and frequently carries a normal karyotype.

Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.

Oncoprotein v-Myb and Retinoic Acid Receptor α Are Mutual Antagonists

ABSTRACT: The process of hematopoiesis is critically dependent on correct interactions of multiple regulatory molecules and transcription factors. We have studied the interactions of the v-Myb and retinoic acid receptor proteins which have opposing effects on hematopoiesis. While v-mybacts as a transforming oncogene preventing differentiation of monoblasts to macrophages, RARα functions as an anti-oncogene arresting the growth of v-myb-transformed cells and allowing their final myeloid differentiation steps to occur. We found that the retinoic acid receptor α inhibits v-Myb transformation by suppressing the expression of v-Myb target genes typified by themim-1 gene. Conversely, v-Myb protein interferes with RARα transactivation function as well as with retinoic acid-induced apoptosis of HL-60 cells. These results demonstrate that retinoic acid receptor and v-Myb proteins act in antagonistic ways and reciprocally modify each other's functions.

Stable Transfection of U937 Cells with Sense or Antisense RXR-α cDNA Suggests a Role for RXR-α in the Control of Monoblastic Differentiation Induced by Retinoic Acid and Vitamin D

Although retinoic acid (RA) has been known for many years to be a modulating agent that plays a role in generating both granulocytes and monocytes, the molecular mechanism underlying this role has not been defined in the monoblast lineage. In particular, the part played by the retinoid X receptors (RXRs), which are members of the steroid/thyroid hormone nuclear receptor family, has not been explored. In this study, therefore, the human monoblastic leukemia cell line U937 has been used as a model system to investigate the role of one of the RXRs, RXR-α, in monoblast differentiation. RXR-α mRNA was present in untreated U937 cells, and levels increased after induction of differentiation with phorbol ester. The same was found for RXR-β mRNA. Using plasmids containing sense or antisense RXR-α sequences under the control of an inducible promoter, we generated stably transfected cell lines which expressed either increased or decreased levels of RXR-α, respectively. The sense cell lines (UαS and its clonal derivative αG2S) showed increased sensitivity to RA, while the antisense cell lines (UαA and its clonal derivative αB5A) showed decreased sensitivity to RA, as demonstrated by growth inhibition and by regulation of an RA-responsive reporter gene. Both UαA and αB5A also failed to respond to another modulating agent, 1α,25-dihydroxycholecalciferol (DHCC), but only UαS and not αG2S showed an enhanced response to DHCC. The combination of RA and DHCC together inhibited growth of both sense and antisense cell lines. In addition, αG2S exhibited increased expression of CD11b and CD54, while αB5A cells showed increased expression of CD102, suggesting that RXR-α has a role in regulating expression of cell adhesion molecules in U937 cells. These results demonstrate that RXR-α has a role in mediating growth inhibition and cell adhesion during myelomonocytic differentiation, and suggest that different species of heterodimers involving RXR-α may control the acquisition of different features of mature monocyte/macrophage function.

Analysis of the DNA-binding activities of Myc/Max/Mad network complexes during induced differentiation of U-937 monoblasts and F9 teratocarcinoma cells.

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Myc:Max or Max:Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.

The Max transcription factor network: involvement of Mad in differentiation and an approach to identification of target genes.

The small bHLHZip protein, Max, was originally identified through its interaction with Myc family proteins and appears to be an obligate partner for Myc function. Max has now been found to interact with at least two other proteins, Mad and Mxi1. These also belong to the bHLHZip class but are otherwise unrelated to Myc. Mad has been shown to abrogate the positive transcriptional activity of Myc and to inhibit Myc in co-transformation assays. This suggests that Mad may antagonize Myc function. Mad is rapidly induced upon differentiation, a time when Myc is frequently down-regulated. We show here evidence for Mad expression upon differentiation of myeloblasts, monoblasts, and keratinocytes. Mad:Max complexes are detected during differentiation and appear to replace the Myc:Max complexes present in proliferating cell populations. Since these complexes appear to form even in the presence of Myc, there may exist mechanisms that act to inhibit Myc:Max, or to promote Mad:Max, complex formation. We speculate that Max complex switching causes a change in the transcriptional activity of groups of target genes. Mad is not induced in all differentiating cell types, suggesting that other, possibly tissue-restricted, proteins might act in similar switch mechanisms to effect changes in transcriptional programs. We have also developed an approach to identification of the gene targets for Myc:Max complexes. By employing an immunoisolation procedure, we have begun characterization of several clones whose expression levels correlate with those of c-myc. Further identification of Myc-regulated genes may allow us to determine the molecular mechanism by which Myc governs cell proliferation and differentiation.

CAMP promotion of osteoclast-like cell development from mouse bone marrow cells requires a permissive action of 1,25-(OH)2D3.

Recruitment of osteoclasts from monocytic precursors is modulated by local signals. We previously showed that monoblastic differentiation in U937 cells is stimulated by 1,25-(OH)2D3 and cAMP in series. We investigate here the combined effects of these agents to stimulate differentiation of osteoclast-like cells from mouse marrow. Cells from mouse marrow were harvested and cultured in alpha-MEM with 10% fetal bovine serum. The appearance of tartrate-resistant acid phosphatase-containing multinuclear cells was measured after 8 days in culture by cytochemical staining. Continuous exposure of cultures to 10 nM 1,25-(OH)2D3 positively stimulated development of these cells after 8 days (101 +/- 3 cells per well, n = 74). No osteoclast-like cells were found when 1,25-(OH)2D3 was added for the first 4 days followed by 4 days more with no treatment. PGE2 (1 microM) as a single agent added during the last 4 days of culture was not able to recruit osteoclast-like cells. However, cultures exposed to 1,25-(OH)2D3 during the first 4 days and 1 microM PGE2 during the second 4 days developed osteoclast-like cells at 8 days [66 +/- 8% of the formation seen with 1,25-(OH)2D3 alone, p less than 0.05]. Dibutyryl cAMP (1 microM to 3 mM) was also not effective used as a single agent, but was able to stimulate formation of TRAP-positive multinuclear cells when 1,25-(OH)2D3 preceded its addition to culture medium. cAMP analogs therefore mimicked the effect of 1 microM PGE2, but these experiments do not allow us to assign the PGE2 action entirely to activation of cAMP second messenger.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E 2 induction of monoblastic differentiation utilizes both cAMP and non-cAMP dependent signalling systems

U937 cells can be induced to express receptor for complement 5a (C5aR) by sequential 2 day treatments of cells with dihydroxyvitamin D-3 (1,25(OH) 2D 3) followed by prostaglandin E 2. We asked whether the action of prostaglandin E 2 to cause maximal C5aR expression required only activation of the cAMP-dependent protein kinase (PKA). Prostaglandin E 2 dose dependently activated PKA in control and 1,25(OH) 2D 3 treated cells; by 4 h the PKA did not respond to further prostaglandin E 2 challenge. We hypothesized that prostaglandin E 2 actions transduced via PKA should be complete by 4 h; i.e., C5aR induction should be equivalent in cells treated with prostaglandin E 2 for 4 h and for 2 days. All cells were treated for the first 2 days with 1,25(OH) 2D 3 and the second 2 days with prostaglandin E 2 or cAMP analogs. C5aR number was measured after 4 days total culture. 4 h pulse treatments with agents were given at the end of the 1,25(OH) 2D 3 treatment. Cells exposed to a 4 h pulse of prostaglandin E 2 had only 68.2 ± 4.4% the amount of C5aR seen in cells continuously exposed to prostaglandin E 2. Continuous culture with a cAMP analog pair (50 μM each of 8-thiomethyl-cAMP + N 6-benzoyl-cAMP), which caused a 41.7% ± 10.8% increase PKA activation above basal, resulted in only 51% ± 16% of the C5aR numbers seen in cells cultured for 2 days with prostaglandin E 2, where PKA remained at basal activity. We therefore concluded that C5aR expression caused by prostaglandin E 2 could not be ascribed entirely to duration or degree of activation of cAMP-dependent signalling pathways. We investigated the possibility that the calcium sensitive protein kinase C was involved. Cytoplasmic protein kinase C was increased 154% ± 14% above control in cells treated with sequential 2 days treatments of 1,25(OH) 2D 3 and prostaglandin E 2. A 147% ± 2% increase in membrane associated protein kinase C was also seen 10 min after phorbol myristate acetate stimulation in the above treatment group. Finally, phorbol myristate acetate augmented the C5aR induction caused by cAMP analog. We propose that the mechanism of prostaglandin E 2 synergism with 1,25(OH) 2D 3 in causing C5aR induction in U937 cells includes signal transduction not only by the cAMP cascade, but also via protein kinase C modulated pathways.

Adult Acute Nonlymphocytic Leukemia in Saudi Arabia: Experience at King Khalid University Hospital in Riyadh

Twenty-two adult patients with acute nonlymphocytic leukemia (ANLL) were analyzed. There were 15 males (68%) and 7 females (32%). Their median age was 42 years. Thirty-six percent had acute myelomo...