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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Key Words: ODD-PCR macrophage cell differentiation
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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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-32P]dCTP (
3,000 Ci/mmol),
[
-32P]ATP (
3,000 Ci/mmol), and
[
-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 Dulbeccos modified Eagles 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 manufacturers 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 manufacturers 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 [
-32P]dCTP, using
the random primer-labeling method, and washed according to the
manufacturers instructions.
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![]() View larger version (88K): [in a new window] |
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.
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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
2448 h. There was no specific mRNA detectable for an integrin
6
subunit until 18 h after treatment, when a faint band was
observed. The level of integrin
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.
![]() View larger version (46K): [in a new window] |
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
6 subunit, MCAK, cyclin A, and 18S rRNA cDNA.
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![]() View larger version (42K): [in a new window] |
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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![]() View larger version (53K): [in a new window] |
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.
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![]() View larger version (36K): [in a new window] |
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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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 2448 h
after TPA treatment. Because cell growth arrest as well as induction of
integrin
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.
Received June 2, 2000; revised November 30, 2000; accepted December 1, 2000.
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