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(Journal of Leukocyte Biology. 2001;69:645-650.)
© 2001 by Society for Leukocyte Biology

Expression of the apolipoprotein C-II gene during myelomonocytic differentiation of human leukemic cells

Eun Mi Chun, Young Jae Park, Hong Soon Kang, Hyun Min Cho, Do Youn Jun and Young Ho Kim

Department of Microbiology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea

Correspondence: Young Ho Kim, Department of Microbiology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea. E-mail: ykim{at}kyungpook.ac.kr


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ABSTRACT
 
Apolipoprotein C-II (apoC-II), which is known to activate lipoprotein lipase (LPL), was identified by ordered differential display (ODD)-polymerase chain reaction (PCR) as a cDNA fragment exhibiting a distinct increase in expression during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of promonocytic U937 cells into monocytes and macrophages. The amount of apoC-II mRNA expression detectable in U937 cells significantly increased and reached a maximum 24–48 h after treatment with 32 nM TPA. apoC-II mRNA was also detected in monocytic THP-1 cells but was not detected in promyelocytic HL-60 cells. In healthy human tissues, the most significant expression of apoC-II mRNA was in the liver. Although apoC-II mRNA expression was markedly up-regulated during the induced differentiation of HL-60 cells into monocytes and macrophages with 32 nM TPA, such expression was not induced during the differentiation of HL-60 cells into granulocytes with 1.25% dimethyl sulfoxide. These results suggest that human apoC-II expression is induced at the transcription level during myelomonocytic differentiation and may confer an important role to macrophages involved in normal lipid metabolism and atherosclerosis.

Key Words: ODD-PCR • macrophage • cell differentiation


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INTRODUCTION
 
Macrophages are unique differentiated progeny of the pluripotent bone marrow stem cells. In vivo, macrophages mature from circulating monocytes and then migrate from the circulatory system into tissues. Development of bone marrow progenitors into monocytes proceeds through monoblast and promonocyte stages in the bone marrow. It is generally accepted that, during this development, precise regulation of essential gene expression occurs, resulting in timely production of cell surface molecules as well as cytokines; thus, macrophages confer orderly progression on cell differentiation and other appropriate physiological functions [1 2 3 ]. However, the mechanisms involved in the development of macrophages are not fully understood.

Human promonocytic leukemia U937 cells differentiate into monocytes and macrophages by use of various agents such as retinoic acids, 1,25-dihydroxyvitamin D3 (VD3), and 12-O-tetradecanoylphorbol-13-acetate (TPA) [4 5 6 ]. Differentiation of human promyelocytic leukemia HL-60 cells into granulocytes can be induced by exposure to dimethyl sulfoxide (DMSO) or retinoic acid [7 8 ], whereas these cells differentiate into a monocyte/macrophage lineage after exposure to VD3 or TPA [9 ]. Since the induction of terminal differentiation of both U937 and HL-60 cells arrests cell growth in the G0/G1 phase of the cell cycle and results in expression of functional differentiation markers as well as acquisition of specific cell morphology, these leukemia cells have been used as the experimental model to elucidate the mechanisms of monocyte and macrophage differentiation.

Recently we initiated an ordered differential display (ODD)-polymerase chain reaction (PCR), a method for displaying 3'-end RsaI restriction fragments of cDNAs [10 ] to isolate the genes that show significantly up-regulated expression during TPA-induced differentiation of U937 cells into monocytes and macrophages. It is likely that this identification of genes differentially expressed during induced differentiation of U937 is an important step towards understanding apparent molecular mechanisms that regulate differentiation. One of 62 cDNA clones that appeared to be significantly up-regulated by TPA-induced differentiation of U937 cells was the human apolipoprotein C-II (apoC-II) gene. apoC-II acts as a cofactor required for efficient actions of lipoprotein lipase (LPL), which is responsible for the hydrolysis of triglycerides in very-low-density lipoproteins (VLDLs) and chylomicrons [11 12 13 ]. LPLs secreted by macrophages are believed to stimulate the uptake of low-density lipoproteins (LDLs) and VLDLs in macrophages located in developing arterial wall lesions, which results in transformation into form cells [14 15 16 ], suggesting that LPL and apoC-II may enable macrophages to exert unique roles during normal lipid metabolism as well as atherosclerosis. Because it has been demonstrated that apoC-II mRNA is mainly expressed in the liver and at much lower levels in the intestine and pancreas, these tissues are believed to be the main sites of apoC-II synthesis [17 , 18 ]. However, the mode of expression of apoC-II in macrophages has not been elucidated.

In this study, we show that apoC-II mRNA is detectable in promonocytic U937 cells as well as monocytic THP-1 cells but is not detectable in promyelocytic HL-60 cells. We also show that the expression of apoC-II mRNA is significantly up-regulated after TPA-induced differentiation of both U937 and HL-60 cells into monocytes and macrophages, whereas apoC-II expression is not detected along with DMSO-induced differentiation of HL-60 cells into granulocytes. This demonstrates that apoC-II gene expression is up-regulated at the transcription level during differentiation from the promyelocytic stage into the monocyte and macrophage lineage.


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MATERIALS AND METHODS
 
Kits, enzymes, reagents, media, and cells
The SuperScripTM system for cDNA synthesis was purchased from Life Technologies, Gaithersburg, MD. All restriction enzymes and DNA-modifying enzymes including T4 DNA ligase, RNase, and T4 polynucleotide kinase were purchased from Boehringer Mannheim, Indianapolis, IN. A DNA-sequencing kit (OmnibaseTM), Taq DNA polymerase, and a pGEM-T Easy Vector System I were purchased from Promega, Madison, WI. TaqStartTM antibody for PCR amplification and human multiple-tissue Northern blot and Express HybTM hybridization solution for Northern blot analysis were purchased from Clontech, Palo Alto, CA. Radioactive materials including [{alpha}-32P]dCTP (~3,000 Ci/mmol), [{gamma}-32P]ATP (~3,000 Ci/mmol), and [{alpha}-35S]dATP (~1,000 Ci/mmol) and a random primer labeling kit were from Amersham, Arlington Heights, IL. [3H]Thymidine deoxyribose ([3H]TdR) (2 Ci/mmol) and nylon membrane (GeneScreen PlusTM) were from NEN Biotechnology System, Boston, MA. A Geneclean II kit was obtained from Bio 101, Vista, CA. The host strain used for cDNA cloning was Escherichia coli JM 109. All components of bacterial media were from Difco, Detroit, MI. To prepare human peripheral T cells, heparinized blood obtained by vein puncture from healthy laboratory personnel was centrifuged at 800 g for 20 min over Histopaque-1077 (Sigma Chemical Co., St. Louis, MO). T cells were isolated from mononuclear cells with a human T-cell enrichment column kit (R&D Systems, Minneapolis, MN). Human peripheral T cells, leukemia cells (Jurkat, MOLT-3, K562, HL-60, U937, and THP-1 cells), and lymphoma (Sup-T1) and COLO 320DM cells were maintained in RPMI 1640 (Bethesda Research Laboratories, Gaithersburg, MD) containing 10% fetal bovine serum (FBS) (Upstate Biotechnology, Lake Placid, NY), 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES [pH 7.0]), 5 x 10-5 M ß-MeOH, and 100 µg/mL of gentamycin. The culture medium used for murine NIH 3T3 and BW5147.G.1.4 cells was Dulbecco’s modified Eagle’s medium (Bethesda Research Laboratories) supplemented with 10% FBS, 20 mM HEPES (pH 7.0), 1 mM sodium pyruvate, 5 x 10-5 M ß-MeOH, and 100 µg/mL of gentamycin. Oligonucleotides used as primers and adapters for ODD-PCR were synthesized by Bio-Synthesis, Lewisville, TX.

Induction of differentiation of U937 and HL-60 cells
Differentiation of U937 cells was induced by adding TPA to a final concentration of 32 nM in the culture media and incubating the cells for 48 h as previously described [5 , 6 , 19 ]. Differentiation of HL-60 cells into the monocyte/macrophage stage was induced by adding 32 nM TPA to the culture media and incubating the cells for 60 h. Differentiation of HL-60 cells into granulocytes was induced in the presence of 1.25% DMSO under the same conditions [20 ]. Incorporation of [3H]TdR into the DNA of TPA- or DMSO-treated cells was used to assess growth arrest of cells during induced differentiation. Approximately 5 x 104 cells were added to each well of a 96-well plate with 32 nM TPA or 1.25% DMSO and pulsed for 4 h with 1 µCi of [3H]TdR at the times indicated. The cells were harvested and assayed by liquid scintillation for the incorporation of [3H]TdR.

ODD-PCR
ODD-PCR was performed essentially as reported by Matz et al. [10 ]. Total RNA from U937 and U937 cells was treated with TPA for 18 or 48 h, and extracted, double-stranded cDNA was synthesized with the T-primer 5'-GCGAGTCGACCG(T)13, using the SuperScriptTM system. The synthesized cDNA samples were digested with RsaI, and half were then used for ligation with a pseudo-double-stranded adapter (a long oligo 5'-GCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT and a short oligo 5'-ACCGCCCTCCGC). Ligation was performed overnight at 16°C in a 10-µL volume with a 2 µM adapter. Then 1 µL of a 1:5 dilution of the ligation mixture was used for PCR with an adapter-specific primer (5'-TGTAGCGTGAAGACGACAGAA) and the T-primer. Amplification was carried out in a 20-µL mixture containing 1x reaction buffer (5 mM KCl, 10 mM Tris-HCl [pH 9.0], 0.1% Triton X-100, 1.5 mM MgCl2, and 15 µM ammonium sulfate), with 250 µM dNTPs, 0.3 µM primers, and 2.5 U of Taq DNA polymerase mixed with TaqStartTM antibody. Polymerase was added to the PCR mixtures at 72°C and incubated for 10 min before the first denaturation stage. The amplification profile included 20 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 1.5 min. One microliter of a 1:20 dilution (~1 ng/µL) of this PCR product in water served as initial material for the amplification of the simplified 3'-end cDNA subsets. For the amplification, individual AdE primers (adapter-specific Extended; 5'-AGGGCGTGGTGCGGAGGGCGGTCCNN, where NN is GC or AG) were 32P-labeled by T4 polynucleotide kinase (Boehringer Mannheim) according to the manufacturer’s instructions. The reaction was conducted for 30 min at 37°C and stopped by heating the tube for 1 min at 100°C. Then 2 µL of this mixture was added to 8 µL of PCR mixture containing 5 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl2, 15 µM ammonium sulfate, 250 µM dNTPs, 2.5 U of Taq DNA polymerase mixed with TaqStartTM antibody, 0.2 µM nonlabeled TE-primer (T-Extended; 5'-GCGAGTCGACCG(T)13NN, where NN is AG, GG, GA, GT, or GC), and 1 ng of the representative 3'-end cDNA fragment sample under investigation. PCR was performed with the following conditions: 23 cycles of 95°C for 30 s, 69°C for 30 s, and 72°C for 1.5 min. To resolve PCR products, 2 µL of each reaction mixture were electrophoresed on 6% polyacrylamide sequencing gel. Detection of the amplified cDNA fragments was visualized by autoradiography after the gel was dried and exposed to X-ray film at -70°C. Differentially displayed cDNAs on the dried sequencing gel were eluted into 20 µL of TE buffer (pH 8.0) at 70°C for 2 h. Two microliters of the eluant were reamplified with T-primer 5'-GCGAGTCGACCG(T)13 and nonextended adapter-specific primer (5'-AGGGCGTGGTGCGGAGGGCGGT) for 20 cycles. Reamplified DNA fragment was electrophoresed on 2% agarose gel, purified using the Geneclean II kit, and then used for cloning and sequence analysis.

DNA sequence analysis and homology search
The cDNA fragment differentially expressed was cloned using a pGEM-T Easy Vector System I (Promega) and was sequenced using the OmnibaseTM DNA cycle sequencing system (Promega) according to the manufacturer’s instructions. The sequence information of the cDNA fragment was compared with GenBank and European Molecular Biology Laboratory (EMBL) databases with the BLAST search program of the National Center for Biotechnology Information (NCBI), National Institutes of Health (NIH), Bethesda, MD.

Northern blot analysis
Total RNA was extracted and isolated by solubilization in guanidine thiocyanate as described elsewhere [21 ]. Fifteen micrograms of total RNA were electrophoresed on 1% formaldehyde-agarose gels and transferred to GeneScreen Plus membranes. The nylon membrane as well as human multiple-tissue Northern blot solution was hybridized in ExpressHyb solution at 68°C for 2 h with a cDNA probe radiolabeled with [{alpha}-32P]dCTP, using the random primer-labeling method, and washed according to the manufacturer’s instructions.


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RESULTS
 
Identification of human apoC-II cDNA
We initiated this ODD-PCR project to investigate the expression profiles of U937 cells during TPA-induced differentiation into monocytes and macrophages and to identify novel, previously uncharacterized genes. Since U937 cells are known to stop cell proliferation and to differentiate into monocytes and macrophages in the presence of 32 nM TPA [5 , 6 , 19 ], the ODD-PCR was performed using total RNA extracted from continuously growing U937 cells and U937 cells treated with 32 nM TPA for 18 or 48 h. A representative example of ODD-PCR for five 3' cDNA fragment subsets compared in three samples is shown in Figure 1 . Many bands representing PCR-amplified 3'-end RsaI-restriction fragments of cDNAs appeared with different patterns in abundance, suggesting that their corresponding mRNA might be differentially expressed in U937 cells after TPA treatment. Various cDNA fragments exhibiting a distinctive increase in expression level during the ODD-PCR picture were eluted from the gel and cloned and analyzed for their nucleotide sequences. The sequence for each cDNA fragment was compared with those in the GenBank database, using the BLAST search program. One of 62 cDNA clones that appeared to be significantly up-regulated upon TPA-induced differentiation of U937 cells was 237 bp in size and showed 100% similarity with the 3'-end of the human apoC-II gene (GenBank X00568). This nucleotide sequence has been submitted to the GenBank database under the accession number AF113884.



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  		Figure 1. ODD-PCR images for five different 3' cDNA fragment subsets compared in three samples for each subset. The individual combination of extended primers used for subset generation is indicated at the top of each set of three lanes. Total RNAs from U937 cells and U937 cells treated with TPA for 18 or 48 h were reverse transcribed, and subsequently the obtained 3'-end RsaI restriction fragments of cDNAs were amplified by PCR as described in Materials and Methods. The PCR products were electrophoresed on 6% polyacrylamide sequencing gel, and detection of the amplified cDNA fragments was visualized by autoradiography after the gel was dried and exposed to X-ray film.

Expression of apoC-II mRNA during differentiation of U937 cells
To confirm that the expression level of apoC-II detected in the ODD-PCR image reflects the real expression pattern of apoC-II mRNA regulated along with TPA-induced differentiation of U937 into monocytes and macrophages, the expression of apoC-II was examined by Northern blot analysis in U937 cells during induced differentiation. After U937 cells were treated with 32 nM TPA for 36 h, the cells no longer incorporated [3H]TdR (Fig. 2A ). Under these conditions, the expression of ~700-bp apoC-II mRNA detected in continuously growing U937 cells began to increase by 12 h after treatment with TPA and reached a maximum level in 24–48 h. There was no specific mRNA detectable for an integrin {alpha}6 subunit until 18 h after treatment, when a faint band was observed. The level of integrin {alpha}6 subunit mRNA increased markedly and reached a maximum after 48 h. However, the expression of cyclin A and mitotic centromere-associated kinesin, both of which are known to accumulate in S phase and support cellular replication [19 , 22 ], appeared to decline and was undetectable by the time that the cells no longer incorporated [3H]TdR (Fig. 2B) . These results demonstrated that, consistent with the results for ODD-PCR, the expression level of apoC-II mRNA was significantly up-regulated along with TPA-induced differentiation of U937 cells into monocytes and macrophages.



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  		Figure 2. Kinetic analysis of [3H]thymidine incorporation (A) and expression of the human apoC-II gene during TPA-induced differentiation of U937 cells into monocytes and macrophages (B). For the proliferation assay, U937 cells (105/well) were treated with 32 nM TPA in 96-well plates and pulsed for 4 h with 1 µM [3H]thymidine at times indicated. The SE determined on six replicate samples was <10%. Equivalent cultures were incubated, and the cells were harvested at the indicated times for RNA extraction. Ten micrograms of total RNA were electrophoresed, transferred, and probed with 32P-labeled apoC-II, integrin {alpha}6 subunit, MCAK, cyclin A, and 18S rRNA cDNA.

Cell and tissue distribution of apoC-II mRNA
Based on protein quantitation techniques, two organs—the liver and intestines—were initially thought to be the sites of plasma apolipoprotein synthesis in mammalian species. Subsequently, the availability of apoC-II cDNA allowed to determine that apoC-II mRNA was mainly expressed at significantly high levels in liver and at low levels in the small intestine and pancreas [17 , 18 ]. Since our data indicate that apoC-II mRNA is detectable in promonocytic U937 cells and is significantly up-regulated at the transcription level during TPA-induced terminal differentiation of U937 cells into monocytes and macrophages, it seems likely that expression of the apoC-II gene is regulated to confer an appropriate function to macrophages during their development. To test this prediction and examine the abundance of apoC-II mRNA in different cell types, several human and murine cells were analyzed for apoC-II mRNA expression by Northern blot analysis. As shown in Figure 3 , apoC-II mRNA was detected only in promonocytic U937 and monocytic THP-1 cells. However, under the same conditions, apoC-II mRNA was not detected in most continuously growing tumor cells tested, including those from the human leukemias in the myeloid lineage (cell lines K562 and HL-60), the human leukemias and lymphoma in the lymphoid lineage (cell lines Jurkat, MOLT-3, and Sup-T1), the human colon adenocarcinoma COLO 320DM cell line, the human epitheloid carcinoma HeLa S3 cell line, murine lymphoma BW5147.G.1.4 cells, and murine fibroblast NIH 3T3 cells. In addition, both human MCAK and human cyclin A appeared to be highly expressed in all of the continuously growing human tumor cells. When the tissue distribution of apoC-II mRNA was analyzed using a human multiple-tissue Northern blot, apoC-II mRNA was mainly expressed at significantly high levels in liver and at low levels in the small intestine, brain, and lung (Fig. 4 ). These results indicate that the expression of apoC-II is regulated at the transcription level depending on cell and tissue types and also possibly depending on the differentiation status during the development of macrophages.



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  		Figure 3. Northern blot analysis of apoC-II-specific mRNA in various human tumor cell lines. The Northern blot containing 15 µg of total RNA in each lane was sequentially hybridized with 32P-labeled apoC-II, MCAK, cyclin A, and 18S rRNA cDNA.



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  		Figure 4. Northern analysis of tissue distribution of apoC-II-specific mRNA. Multiple-tissue Northern membranes (MTNTM I and II; Clontech), each containing 2 µg of poly(A)+ RNA per lane, were sequentially hybridized using 32P-labeled apoC-II and ß-actin cDNA probes.

Lineage-specific expression of apoC-II mRNA during differentiation of HL-60 cells into the terminal stage
HL-60 cells differentiate into granulocytes upon exposure to DMSO or retinoic acid [7 , 8 ], but the same cells differentiate into monocytes and macrophages in the presence of TPA or VD3 [9 ]. We further investigated the lineage-specific expression of apoC-II mRNA during the terminal differentiation of cell line HL-60. As shown in Figure 5 , cell growth arrest rapidly occurred during 32 nM TPA-induced terminal differentiation of HL-60 cells into monocytes and macrophages, and [3H]TdR incorporation was not detected 48 h after treatment of TPA. apoC-II mRNA expression, which was undetectable in HL-60 cells, was markedly up-regulated and was first detectable within 24 h of treatment with 32 nM TPA to induce differentiation of cells into a monocyte and macrophage lineage. However, neither the rapid growth arrest nor apoC-II mRNA expression occurred during 1.25% DMSO-induced terminal differentiation of HL-60 cells into granulocytes. Since it has been previously demonstrated that apoC-II is required for efficient actions of LPL [11 12 13 ], these and previous results together indicate that, although apoC-II mRNA expression is undetectable in promyelocytic HL-60 cells, it is specifically up-regulated at transcription level along with terminal differentiation into a monocyte and macrophage lineage, and this expression may confer an important role to macrophages involved in the catabolism of triglyceride-rich lipoprotein as well as atherosclerosis.



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  		Figure 5. Kinetic analysis of [3H]thymidine-incorporation (A), and expression of apoC-II gene (B) during the induced differentiation of HL-60 cells into terminal stages. HL-60 cells were induced to differentiate into monocyte/macrophage lineage with 32 nM TPA or to differentiate into granulocytes with 1.25% DMSO. For proliferation assay, HL-60 cells (105/well) were treated with 32 nM TPA or 1.25% DMSO in 96-well plates and pulsed for 4 h with 1 µCi of [3H]thymidine at times indicated. Equivalent cultures were incubated, and the cells were harvested at the indicated times for RNA extraction. Ten micrograms of total RNA were electrophoresed, transferred, and probed with 32P-labeled apoC-II and 18S rRNA cDNA.


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DISCUSSION
 
Macrophages play a critical role in early nonadaptive phases of innate immunity as migratory phagocytic cells as well as in humoral and cell-mediated immunity as antigen-presenting cells and effector cells. Macrophages have also been suggested to play a pivotal role in the development of atherosclerosis, since they are precursors of arterial wall foam cells that are considered typical features of the formation of atherosclerotic lesions [23 –25 ]. The mechanism responsible for the transformation of macrophages into foam cells centers around the role of LPL that is produced by macrophages and is required for hydrolysis of triglyceride in VLDLs and chylomicrons. It has been suggested that LPL-mediated hydrolysis of triglyceride plays a role in the uptake of triglyceride-rich lipoproteins in macrophages, because LPL increases the association of lipoproteins with the surface of macrophages and thus augments the uptake and degradation of lipoproteins [15 ]. LPL has separate domains within the protein structure, which can bridge between lipoproteins and the heparin sulfate proteoglycans that are present on plasma membranes [25 ].

apoC-II has been previously known to be produced mainly in the liver and to act as a cofactor which activates LPL [11 , 12 ]. apoC-II in this activation process is believed to increase the catalytic rate constant of LPL by inducing conformational changes in LPL [26 ]. The importance of apoC-II as a protein supporting the role of LPL in triglyceride hydrolysis has been illustrated by human disorders with genetic defects in the structure or production of this protein. Patients with these disorders have high levels of circulating triglyceride and are phenotypically indistinguishable from patients with LPL deficiency [12 ]. The role of LPL in the uptake of VLDL into macrophages could also be enhanced by the presence of apoC-II [13 ].

The data presented here show first that the expression of human apoC-II mRNA is not detectable in promyelocytic HL-60 cells but is detectable in promonocytic U937 cells as well as monocytic THP-1 cells and that expression is markedly up-regulated at the transcription level during the induced differentiation of HL-60 or U937 cells into the monocyte/macrophage stage. In these studies, the apoC-II gene was initially identified by ODD-PCR as a transcript exhibiting a remarkable increase during 32 nM TPA-induced differentiation of U937 cells. Since it is generally accepted that the level of a transcript represented by an ODD-PCR image sometimes does not reflect the real expression pattern of the gene, the expression of apoC-II mRNA was sequentially confirmed by Northern blot analysis. In accordance with the result of ODD-PCR, the expression level of mRNA specific for apoC-II was significantly up-regulated along with the TPA-induced differentiation of U937 cells into monocytes and macrophages and reached a maximum level 24–48 h after TPA treatment. Because cell growth arrest as well as induction of integrin {alpha}6 subunit-specific mRNA expression as a differentiation marker was also accompanied by the up-regulation of apoC-II expression, the expression of the apoC-II gene is likely to be up-regulated at the transcription level during the terminal differentiation of U937 cells.

To determine the abundance of apoC-II mRNA in different cell types and to examine whether the detectable level of apoC-II mRNA expression is restricted to the developmental stage between promonocytic U937 cells and macrophages, several human cells including myelogenous K562, promyelocytic HL-60, and monocytic THP-1 cells were analyzed for apoC-II mRNA expression by Northern blot analysis. Only U937 and THP-1 cells appeared to express a detectable level of apoC-II mRNA, but none of the other cells tested expressed apoC-II mRNA. Northern analysis using a human multiple-tissue Northern blot revealed that apoC-II mRNA was mainly expressed at significantly high levels in liver and at low levels in the small intestine, brain, and lung. Since the promyelocytic HL-60 cells involved in the development of bone marrow progenitors that proceed toward macrophages are known to be upstream of promonocytic U937 cells [4 ], these results show that the expression of apoC-II is regulated at the transcription level depending on tissue as well as cell types and suggest that this expression might also be regulated during monocytic differentiation of HL-60 cells. By using HL-60 cells that differentiate into either monocytes and macrophages or granulocytes, depending on the choice of chemical inducers [7 8 9 ], we further investigated whether the expression of apoC-II mRNA is inducible during the terminal differentiation of HL-60 cells into monocytes and macrophages or granulocytes. Although apoC-II mRNA expression was significantly up-regulated along with the induced differentiation of HL-60 cells into monocytes and macrophages in the presence of 32 nM TPA, this expression was not induced during differentiation of HL-60 cells into granulocytes with the addition of 1.25% DMSO. These results indicate that apoC-II mRNA expression that is undetectable in the promyelocytic HL-60 stage is specifically up-regulated along with terminal differentiation into the monocyte/macrophage stage.

In summary, we have demonstrated that apoC-II mRNA expression, which is not detectable in the promyelocytic stage of HL-60 cells, can be up-regulated at the transcription level along with terminal differentiation into monocytes and macrophages. In addition, we suggest that expressed apolipoprotein C-II may confer an important role on macrophages involved in normal lipid metabolism and atherosclerosis by acting as a cofactor that was previously known to activate LPL.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Korean Ministry of Health and Welfare for cancer research during 1999–2000.

Received June 2, 2000; revised November 30, 2000; accepted December 1, 2000.


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