Study on the Biological Functions of WT1 Gene Isoforms in Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4323-4323
Author(s):  
Hui-ling Shen ◽  
Zi-xing Chen ◽  
Wei Wang ◽  
Shao-yan Hu ◽  
Jian-nong Cen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Leukemia Cell ◽  
Gene Expression Profiles ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Wt1 Gene ◽  
Rt Pcr ◽  
Nb4 Cells ◽  
Gene Isoforms

Abstract It has been well known that WT1 gene is overexpressed in leukemia cells regardless subtypes comparing to normal hematopoietic cells. The precise effect of WT1, whether an oncogenic or tumor suppressive effect, in leukemogenesis remains controversial. Isoforms of WT1 protein products caused by alternative splicing may exert different biological function. To investigate the role of WT1 product composed of four major isoforms in certain ratios in hematopoietic cells, we have established a leukemia cell line NB4 which stably expressed exogenous WT1 gene isoforms, then studied their effects on cell biologic behaviors including proliferation, apoptosis and differentiation and its possible molecular mechanisms. The eukaryotic expression recombinant vectors (pCB6+/WT1) containing 4 clones of full-length human WT1 isoforms (WTA: −17aa/-KTS, WTB:+17aa/-KTS, WTC: −17aa/+KTS, WTD: +17aa/+KTS) cDNA were transduced into the leukemia cell line NB4 by electroporation.The positive stable cell clones (NB4/WT1) were obtained. The integration and expresion of WT1 gene isoforms in NB4 cells were confirmed by PCR. RT-PCR and western blotting. We then mainly concentrated on the effect of WT1 isoform WTA (−17aa/-KTS) since this transgene will markedly change the WT1 isoform ratio in NB4 cells from +17aa/+KTS dominant to −17aa/-KTS dominant. The proliferation ability was measured by trypan blue exclusion assay, MTT assay, colony forming assay and cell cycles analysis. Morphology, NBT reduction and CD11b expression were examined to access the cell differentiation. AnnexinV binding tested by FCM and agarose gel electrophoresis were performed to access the susceptibility to action of apoptosis inducing agents. Expressions of PML/RARα, RbAP46, P21, P53, Bcl-2, Bcl-XL and C-myc genes in NB4/WTA cells were determined by semi-quantitative RT-PCR DNA microarray was used to explore the alteration of gene expression profiles in NB4/WTA cells. The proliferation rate of NB4/WTA significantly decreased as measured by growth curves and colony forming ability, while the NB4/WTA cells arrested in S stage increased. NB4/WTA cells treated with ATRA 0.5μM for 2 days were induce to partially differentiate compared to a much higher morphological differentiation rate and CD11b expression level in the negative control cells in same condition. After exposure to As2O3 at 0.8μM for 48 hours, the NB4/WTA cells,but not the control cells, exhibited features of apoptosis RT-PCR have showed increasing level of PML/RARα, RbAP46, P21 and C-myc gene expression, a decreased level of Bcl-2 and a relative constant expression of P53,Bcl-XL, VEGF, CyclinD1 and CyclinD2 in NB4/WTA cells. The gene expression profiles were found changed in transfected NB4 cells. 89 of 4096(2.17%) genes were found to have a differential expression pattern, most of which (nearly 88.7%) were down-regulated. Our results indicated that overexpression of exogenous WT1 isoform (−17aa/-KTS) gene inhibited the proliferation of leukemia cells by delaying the progression of S into G2/M stage in cell cycle and inducing cell apoptosis by down-regulating the Bcl-2 gene expression. WTA gene could also partially inhibit the differentiation of NB4 cells. The introduction and expression of exogenous WT1 isoform gene can change the gene expression profiles in NB4 cells, leading to inhibition of cell growth, retardation of differentiation and more sensitive to apoptosis inducing agents.

scholarly journals Analysis of ceRNA networks and identification of potential drug targets for drug-resistant leukemia cell K562/ADR

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11429
Author(s):  
Zhaoping Liu ◽  
Yanyan Wang ◽  
Zhenru Xu ◽  
Shunling Yuan ◽  
Yanglin Ou ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Drug Targets ◽  
Regulatory Networks ◽  
Expression Profiles ◽  
Leukemia Cell ◽  
Gene Expression Profiles ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Drug Resistant ◽  
Cerna Network

Background Drug resistance is the main obstacle in the treatment of leukemia. As a member of the competitive endogenous RNA (ceRNA) mechanism, underlying roles of lncRNA are rarely reported in drug-resistant leukemia cells. Methods The gene expression profiles of lncRNAs and mRNAs in doxorubicin-resistant K562/ADR and sensitive K562 cells were established by RNA sequencing (RNA-seq). Expression of differentially expressed lncRNAs (DElncRNAs) and DEmRNAs was validated by qRT-PCR. The potential biological functions of DElncRNAs targets were identified by GO and KEGG pathway enrichment analyses, and the lncRNA-miRNA-mRNA ceRNA network was further constructed. K562/ADR cells were transfected with CCDC26 and LINC01515 siRNAs to detect the mRNA levels of GLRX5 and DICER1, respectively. The cell survival rate after transfection was detected by CCK-8 assay. Results The ceRNA network was composed of 409 lncRNA-miRNA pairs and 306 miRNA-mRNA pairs based on 67 DElncRNAs, 58 DEmiRNAs and 192 DEmRNAs. Knockdown of CCDC26 and LINC01515 increased the sensitivity of K562/ADR cells to doxorubicin and significantly reduced the half-maximal inhibitory concentration (IC50) of doxorubicin. Furthermore, knockdown of GLRX5 and DICER1 increased the sensitivity of K562/ADR cells to doxorubicin and significantly reduced the IC50 of doxorubicin. Conclusions The ceRNA regulatory networks may play important roles in drug resistance of leukemia cells. CCDC26/miR-140-5p/GLRX5 and LINC01515/miR-425-5p/DICER1 may be potential targets for drug resistance in K562/ADR cells. This study provides a promising strategy to overcome drug resistance and deepens the understanding of the ceRNA regulatory mechanism related to drug resistance in CML cells.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

Effect on NB4 Cells Proliferation, Differentiation and Resistance to Chemotherapy Apoptosis by VEGF-C cDNA Transfection.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1529-1529
Author(s):  
Kaiyang Ding ◽  
Xia Bai ◽  
Lan Dai ◽  
Ningzheng Dong ◽  
Changgeng Ruan
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Leukemia Cell ◽  
Eukaryotic Expression ◽  
Leukemia Cell Line ◽  
Apoptotic Cells ◽  
C Cells ◽  
Expression Levels ◽  
Nb4 Cells ◽  
Proliferation Ability

Abstract VEGF-C secreted by kinds of tumor cells play the key role in lymphangiogenesis via activating tyrosine kinase of VEGFR-3, which is one of most important pathway of infiltration and metastasis of tumor cells. In recent years, the effects of VEGF-C on hematological malignant cells are paid attention. In the current study, the recombinant eukaryotic expression plasmid (pcDNA3.1-VEGF-C) and the vacant pcDNA3.1 vector were introduced into the acute promyelocytic leukemia cell line- NB4 cells (VEGFR-3+/VEGFR-2−) by lipofectamine mediation and positive clones were screened by G418. The stable expression of VEGF-C was detected by reverse transcriptase-PCR and Western blotting. The proliferation ability of NB4/VEGF-C cells is analysed by MTT assay. After NB4/VEGF-C cells were induced by ATRA (1μmol/L), the expression levels of C/EBPα gene which can promote myelocytes differentiation and maturation, CD11b on cells surface and morphological variation were analysed by real-time quantitative PCR (RT-QPCR), flow cytometry analysis (FCM), Wright-Giemsa staining respectively. Furthermore, after the NB4/VEGF-C cells were cultured together with Etoposide (200ng/ml) for 48 hours, the numbers of AnnexinV+/PI−cells (apoptotic cells) were determined by FCM to investigate the effect of VEGF-C on the cells apoptosis and expression levels of antiapoptotic bcl-2 gene in these cells were analysed by RT-QPCR. The NB4/pcDNA3.1 cells was used as control during the experiments. From these experiments, NB4/VEGF-C cells can stably secret active protein VEGF-C in the cell supernatants. The cell growth curve shows that the proliferation ability of NB4/VEGF-C cells is stronger compared with NB4/pcDNA3.1cells. The expression levels of C/EBPα gene of NB4/VEGF-C cells after induced by ATRA is only 1/32 that of NB4/pcDNA3.1 cells. Morphological analysis shows that the degree of promyelocytes gradually maturing into myelocytes among NB4/VEGF-C cells is weaker compared with the controls after induced by ATRA. After induced by Etoposide, the percent of apoptotic cells of NB4/VEGF-C cells (7.20±2.52%) is significantly lower than that of NB4/pcDNA3.1 cells (16.07±3.58%)( P=0.005) and the bcl-2 gene expression level of NB4/VEGF-C cells is 2.28 times more than that of NB4/pcDNA3.1 cells. The results implied that the VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of NB4 cells by autocrine pathway and inhibit the NB4cells differentiation and maturation and chemotherapy-induced apoptosis via upregulating bcl-2 gene expression. Thus, VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.

Liver Fibrosis In Myeloid Leukemia of Down Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 874-874
Author(s):  
John Chen ◽  
Yue Li ◽  
Monica Doedens ◽  
John E. Dick ◽  
Johann K. Hitzler
Keyword(s):  
Gene Expression ◽  
Liver Fibrosis ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
U937 Cells ◽  
Recipient Liver ◽  
Life Threatening ◽  
Organ Fibrosis

Abstract Abstract 874 Background. Fibrosis is a common complication of leukemia with a megakaryoblastic blast phenotype. Children with Down syndrome (DS) have an increased risk for two hematological disorders with a predominantly megakaryoblastic phenotype. First, approximately 10% of newborns with DS develop Transient Leukemia (TL; also termed Transient Myeloproliferative disorder, TMD), which is self-resolving in the majority but associated with life-threatening complications in approximately 15% of cases. Liver fibrosis and failure are predominant causes of fatal outcomes in these infants. Second, children with DS develop acute myeloid leukemia, predominantly with a megakaryoblastic phenotype, approximately 150-times more often than the general pediatric population during the first 4 years of life. Somatic mutations of GATA1 are specific for TL/TMD and myeloid leukemia of DS. We found significant liver fibrosis in NOD/SCID recipients of human DS myeloid cells. We are using this model to define the mechanisms that control the development of this potentially life-threatening complication. Methods. Recipients were engrafted with primary human DS myeloid leukemia cells and cells of the human DS myeloid leukemia cell line GRW (established at our institution). U937 human monocytic leukemia cells, which lack trisomy 21, were used in control experiments. Cells were injected into the right femur of 8-week-old irradiated NOD/SCID mice, which had also been injected with anti-NK (anti-CD122) antibody. Phenotypic analysis at 4 to 6 weeks after transplantation used standard cytological, histological and flowcytometric methods (CD45, CD61 and CD34 antibodies were obtained from B&D). Affymetrix human Gene ST1.0 expression arrays were used to compare GRW and U937 cells. Specific gene expression was quantified by TaqMan real time RT-PCR relative to b-actin (Applied Biosystems). Results. Organ fibrosis in recipients of DS myeloid leukemia cells. Primary cells from patients with DS myeloid leukemia engrafted murine recipients and caused moderate bone marrow fibrosis. Transplantation of GRW human DS myeloid cells resulted in significant infiltration of recipient liver and bone marrow (33±10% of mononuclear cells, n=6). The frequency of the leukemia-initiating cells was 1/87,448 by limiting dilution. Marked fibrosis of the liver developed within 6 weeks of transplantation. Fibrosis-associated gene expression in DS myeloid leukemia cells. DS myeloid leukemia cells (GRW) expressed significantly more PDGFD and FGF17 transcripts than non-DS myeloid leukemia control cells (U937). PDGFD transcripts were 4.3-fold more abundant in GRW cells than U937 cells (p=2.67 × 10-7; n=4). FGF17 expression levels were moderately increased (1.24, p=0.07). Quantitative real time RT-PCR analysis confirmed expression of PDGFD in DS myeloid leukemia cells (GRW) (5.55 × 10-4PDFGD/b-actin transcripts; n=4) and did not detect measurable expression in control (U937) cells. FGF17 transcripts were approximately 100-fold more abundant in DS myeloid leukemia cells (GRW) cells compared to U937 controls (4.85 × 10-4 and 4.91 × 10-6 for FGF17/b-actin transcripts, n=4). Analysis of fibrosis-associated gene expression in recipient (murine) liver cells in response to transplanted DS myeloid leukemia cells (GRW) and functional analysis of the role of PDGFD and FGF17 expression in DS myeloid leukemia cells is underway. Conclusion. Organ fibrosis is a significant cause of mortality of children with DS and megakaryoblastic disorders, particularly during the newborn period. Chemotherapy frequently is unable to achieve a timely reversal of this process. Better understanding of the mechanisms controlling the development of organ fibrosis is required to develop better anti-fibrotic intervention. The observed phenotype of marked liver fibrosis in murine recipients of the human DS myeloid leukemia cell line GRW provides an experimental tool to determine the components of the fibrogenic process both in infiltrating leukemia cells and recipient liver tissue. PDGFD and FGF17 expression by DS myeloid leukemia cells has been identified as possible inducer of organ fibrosis. The model can now be used to define tissue responses to infiltrating human DS myeloid leukemia cells in liver tissue of recipients and to determine the functional role of candidate genes in DS myeloid leukemia cells by altering expression levels prior to transplantation. Disclosures: No relevant conflicts of interest to declare.

Functional Somatic and Germline Variants in the NF-κB Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 560-560 ◽  
Author(s):  
Ma. Reina Improgo ◽  
Adam Kiezun ◽  
Yaoyu Wang ◽  
Lillian Werner ◽  
Petar Stojanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Transcriptional Activity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Rt Pcr ◽  
Germline Variants

Abstract Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p<0.0001). We further hypothesized that this increased transcriptional activity would lead to increased expression of NFKB1 downstream target genes. Analysis of gene expression profiles from Affymetrix HG-U133 Plus 2.0 Arrays of 65 CLL patient samples showed that the NFKB1 downstream targets CCL3, CCL4, and CD69 are upregulated in NFKB1 variants. To validate these results, we performed quantitative RT-PCR in patients with (n=3) or without (n=9) NFKB1 variants and confirmed upregulation of CCL3 (p=0.0286), CCL4 (p=0.0384), and CD69 (p=0.0263). Direct transfection of HEK293T cells with NFKB1 variants also resulted in a 3.3-fold upregulation of CCL3 (p=0.05). To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p<0.0001). Quantitative RT-PCR also showed a 59% decrease in expression of CCL3 one hour post-treatment, confirming inhibition of the NF-κB pathway. In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

Age-related changes in the transcriptional profile of mouse RPE/choroid

2003 ◽  
Vol 15 (3) ◽  
pp. 258-262 ◽  
Author(s):  
Hisashi Ida ◽  
Sharon A. Boylan ◽  
Andrea L. Weigel ◽  
Leonard M. Hjelmeland
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Cdna Probe ◽  
Gene Expression Profiles ◽  
Age Related Macular Degeneration ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Mouse Cdna ◽  
Age Related ◽  
Age Related Changes

To evaluate the age-related changes in gene expression occurring in the complex of retinal pigmented epithelium, Bruch’s membrane, and choroid (RPE/choroid), we examined the gene expression profiles of young adult (2 mo) and old (24 mo) male C57BL/6 mice. cDNA probe sets from individual animals were synthesized using total RNA isolated from the RPE/choroid of each animal. Probes were amplified using the Clontech SMART system, radioactively labeled, and hybridized to two different Clontech Atlas mouse cDNA arrays. From each age group, three independent triplicates were hybridized to the arrays. Statistical analyses were performed using the Significance Analysis of Microarrays program (SAM version 1.13; Stanford University). Selected array results were confirmed by semi-quantitative RT-PCR analysis. Of 2,340 genes represented on the arrays, ∼60% were expressed in young and/or old mouse RPE/choroid. A moderate fraction (12%) of all expressed genes exhibited a statistically significant change in expression with age. Of these 150 genes, all but two, HMG14 and carboxypeptidase E, were upregulated with age. Many of these upregulated genes can be grouped into several broad functional categories: immune response, proteases and protease inhibitors, stress response, and neovascularization. RT-PCR results from six of six genes examined confirmed the differential change in expression with age of these genes. Our study provides likely candidate genes to further study their role in the development of age-related macular degeneration and other aging diseases affecting the RPE/choroid.

scholarly journals NB4 cells show bilineage potential and an aberrant pattern of neutrophil secondary granule protein gene expression

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 294-302 ◽  
Author(s):  
A Khanna-Gupta ◽  
K Kolibaba ◽  
TA Zibello ◽  
N Berliner
Keyword(s):  
Gene Expression ◽  
Protein Gene ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Phenotypic Change ◽  
Hl60 Cells ◽  
Nb4 Cells ◽  
Granule Protein ◽  
All Trans Retinoic Acid ◽  
Secondary Granule

NB4 is an acute promyelocytic leukemia cell line that has been shown to be inducible to terminal neutrophil maturation with all-trans retinoic acid (ATRA). HL60 cells are differentially inducible with 12-O- tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide (DMSO) to monocytes or granulocytes, respectively. HL60 cells induced with DMSO undergo defective neutrophil maturation, manifested by a coordinate failure of secondary granule protein gene expression. We observed a similar defect in granulocytic maturation in ATRA-induced NB4 cells. In addition, because normal promyelocytes are known to have bilineage potential, we have investigated differentiation along monocytoid lines induced with TPA. We observed a striking phenotypic change along monocytoid/macrophage lines with TPA induction. Flow cytometry showed a TPA-induced increase in HLA-DR expression, and Northern blot analysis showed induction of expression of CD18, c-fos, and human neutrophil gelatinase (HNG). HNG is unique among the neutrophil secondary granule protein genes in that it is expressed in both the neutrophil and monocyte lineages. This again parallels our findings in TPA-induced HL60 cells, which retain the ability to express HNG. These findings confirm bilineage potential in NB4 cells. They also support the hypothesis of coordinate neutrophil secondary granule protein gene expression and a defect in this control as part of the leukemic phenotype.

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