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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2863-2870
Altered Expression and Activity of Topoisomerases During
All-Trans Retinoic Acid-Induced Differentiation of
HL-60 Cells
By
Masako Aoyama,
Dale R. Grabowski,
Richard J. Isaacs,
Kim A. Krivacic,
Lisa A. Rybicki,
Ronald M. Bukowski,
Mahrukh K. Ganapathi,
Ian D. Hickson, and
Ram Ganapathi
From the Experimental Therapeutics Program, Taussig Cancer Center,
Cleveland Clinic Foundation, Cleveland, OH; and the Molecular
Oncology Laboratory, ICRF Unit, John Radcliffe Hospital, Oxford,
UK.
 |
ABSTRACT |
Regulation of topoisomerase II (TOPO II) isozymes  and  is
influenced by the growth and transformation state of cells. Using HL-60
cells induced to differentiate by all-trans retinoic acid (RA),
we have investigated the expression and regulation of TOPO II isozymes
as well as the levels of topoisomerase I (TOPO I). During RA-induced
differentiation of human leukemia HL-60 cells, levels of TOPO I
remained unchanged, whereas the levels and phosphorylation of TOPO
II and TOPO II proteins were increased twofold to fourfold and
fourfold to eightfold, respectively. The elevation of TOPO II ( and
) protein levels and phosphorylation was apparent at 48 hours of
treatment with RA and persisted through 96 hours. The increased level
of TOPO II protein was also detected in differentiated cells
subsequently cultured for 96 hours in RA-free medium. Pulse chase
experiments in cells labeled with 35S-methionine showed
that the rate of degradation of TOPO II protein in control cells was
about twofold faster than that in the differentiated RA-treated cells.
The level of decatenation activity of kDNA was comparable in nuclear
extracts from control or RA-treated cells. Whereas etoposide (1 to 10 µmol/L) -induced DNA cleavage was not significantly different,
apoptosis was significantly lower (P = .012) in RA-treated
versus control cells after exposure to 10 µmol/L etoposide.
Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced
DNA cleavage was similar in control or RA-treated cells. However,
apoptosis after exposure to 1 to 10 µmol/L CPT was significantly
lower (P = .003 to P < .001) in RA-treated versus
control cells. Results suggest that TOPO II protein levels are
posttranscriptionally regulated and that degradation of TOPO II is
decreased during RA-induced differentiation. Furthermore, whereas the
total level of TOPO II ( + ) is increased with RA, the level of
TOPO II catalytic activity and etoposide-stabilized DNA cleavage
activity remains unaltered. Thus, TOPO II may have a specific role
in transcription of genes involved in differentiation with RA
treatment.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THE TOPOISOMERASES are key nuclear
enzymes that alter DNA topology for the efficient processing of genetic
material.1,2 This is accomplished by the generation of
single-strand and double-strand breaks in DNA catalyzed by
topoisomerase I (TOPO I) and topoisomerase II (TOPO II), respectively.
Among the topoisomerases,1,2 TOPO I exists as a single
97-kD protein, whereas TOPO II has two distinct isoforms of molecular
weight (Mr) 170,000 () and 180,000 (). A critical
role for TOPO II has been suggested in recombination, replication,
chromosome structure, and segregation.1,2 Although the
topoisomerases are essential enzymes for cell function, they also
represent targets for a number of clinically important agents used in
the chemotherapy of cancer.1-5
The role of TOPO II in cell proliferation and as a target for
antitumor agents has been extensively studied.1-5 However, the precise functional role of TOPO II during cell growth
and/or differentiation has been elusive. Specific alterations
in the phosphorylation state of TOPO II is seen in mitotic cells and this enzyme has also been suggested to be a potential target for the
antitumor agent mitoxantrone.6-9 Woessner et
al10,11 were the first to report the potential role of TOPO
II in cell differentiation. Subsequently, it was demonstrated that
the genes for the and isoforms of TOPO II are expressed in a
varied manner in proliferating versus differentiated
tissue,12 and a specific role for TOPO II in
neutrophil-granulocyte differentiation has been
suggested.13,14 Kaufmann et al,15,16 evaluating
the role of topoisomerases during granulocytic maturation of HL-60
cells induced with dimethyl sulfoxide, suggested that both protein
levels and actvity of TOPO I and TOPO II were reduced in
differentiated cells. Similarly, based on
phorbol-12-myristate-13-acetate-induced differentiation of HL-60
cells, an apparent link between a decrease in topoisomerase II levels
and growth and differentiation has been suggested.17-19
The role of retinoic acid as an inducer of differentiation has received
considerable attention because of the potential role of
retinoid-regulated transcription. Given the complexity of cell differentiation and the potential regulation of these events by retinoids,20 we were prompted to explore the role of
topoisomerases during granulocytic maturation of HL-60 cells induced by
all-trans retinoic acid (RA). Also, the availability of TOPO
II isoform-specific antisera with utility in both immunoblotting and
immunoprecipitation procedures provided an opportunity to specifically
address the differential regulation of TOPO II isoforms.21
Our results demonstrate that, although TOPO I is unaffected, there is
an anomalous relationship between the expression of TOPO II and proteins and activity. Furthermore, during RA-induced differentiation
of HL-60 cells, there is an increased upregulation of TOPO II,
particularly the TOPO II isoform, apparently by a
posttranscriptional mechanism.
| |
MATERIALS AND METHODS |
The parental HL-60 cells were obtained from the American Type Culture
Collection (Rockville, MD). Cultures were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mmol/L
L-glutamine (M.A. Bioproducts, Gaithersburg, MD) at 37°C in a
humidified 5% CO2 plus 95% air atmosphere. Under these
conditions, the doubling time in vitro of the HL-60 cells was 40 to 45 hours.
The HL-60 cells were treated with 1 µmol/L RA for as long as 120 hours, and at various time intervals during this period the control and
RA-treated cells were analyzed for (1) proliferation and
differentiation; (2) protein levels of TOPO I; (3) mRNA, protein, and
phosphorylation of TOPO II and ; and (4) induction of etoposide (VP-16) or camptothecin (CPT)-induced DNA damage and apoptotic response. The effects of RA (1 µmol/L) on cell proliferation and induction of differentiation were determined by counting cells in a
hemacytometer and the ability of cells to reduce nitroblue tetrazolium,
respectively.22
Analysis of mRNA levels of TOPO II and in control and RA-treated
cells was performed using an RNase protection assay.23,24 The human TOPO II and probes were prepared as described
previously and generated a 215-bp (), 228-bp (1), and
292-bp (2) protected fragment.23,24 The
1-globin probe generated a 133-bp fragment.23,24 Briefly, RNase protection analysis was performed on 20 µg of total RNA extracted from cells using the RNeasy kit (Qiagen, Valencia, CA). Total RNA was hybridized with TOPO II and antisense probes and 1 µg of total RNA from K562 cells (which
strongly overexpress 1-globin) was spiked into the reactions as an
exogenous control and detected with 1-globin antisense probe as
described earlier.23,24 All RNase protection assays were
performed as previously described.23,24
TOPO I or TOPO II and protein levels were determined in extracts
from control and treated cells by immunoblotting. Briefly, cells were
lysed in RIPA buffer and protein content was determined. Lysates
containing equal amounts of protein were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose,25 and probed with
antibodies for TOPO I26 and antibodies for TOPO II that
simultaneously15,16 or individually21 recognize
the and isoforms.
Phosphorylation of the and isoforms of TOPO II was determined
in control and RA-treated cells metabolically labeled with [32P]-orthophosphoric acid.25 Briefly,
control and RA cells were labeled with
[32P]-orthophosphoric acid for 2 to 4 hours and lysed in
RIPA buffer supplemented with 0.35 mol/L NaCl, and the TOPO II was
immunoprecipitated25 with an antibody that simultaneously
or individually recognizes the and isoforms of TOPO II. The
immunoprecipitate was resolved by SDS-PAGE, and the signal intensity
was determined with a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA).
Phosphopeptide analysis of the immunoprecipitated 180-kD () TOPO II
was performed as described by Wells et al27 and Boyle et
al.28 Briefly, the band corresponding to the 180-kD ()
TOPO II protein was excised from the dried, unfixed gel and then
eluted with 50 mmol/L ammonium bicarbonate, 0.1% SDS, and
0.5% 2-mercaptoethanol overnight. The protein was precipitated with
15% to 20% trichloroacetic acid and oxidized with performic acid.
Protein samples were digested overnight in TPCK-trypsin, and
phosphopeptides were analyzed by electrophoresis with pH 1.9 buffer in
the horizontal dimension and chromatography in the vertical dimension
using phospho-chromatography buffer.27,28
Synthesis and degradation of TOPO II was determined in control or
RA-treated cells metabolically labeled with
[35S]-L-methionine Tran 35S-Label for 3 hours.29 After labeling, cells were washed twice and
reincubated in complete medium supplemented with 0.1 mmol/L L-methionine. Samples of cells were retrieved at 0, 2, 4, and 6 hours,
and the TOPO II was immunoprecipitated from cell lysates as
described earlier.29 Signal intensity was quantified in a PhosphorImager.
CPT (0.5 to 10 µmol/L) or etoposide (1 to 10 µmol/L)-stabilized DNA
cleavable complex formation and apoptosis in control and RA-treated
cells were determined by the SDS-KCl technique25 and
labeling with fluorescein-12-dUTP,30 respectively, after drug treatment for 1 hour. Control or RA-treated cells after drug treatment were reincubated in drug-free medium for 4 hours before labeling30 for analysis of apoptotic cells.
| |
RESULTS |
The data in Fig 1 outline the growth and differentiation
of control and RA-treated HL-60 cells. It is apparent that, in contrast to the control cells, there is minimal proliferation beyond 48 hours in
the RA-treated cells. Furthermore, differentiation (based on reduction
of nitroblue tetrazolium) progressively increases and is essentially
complete by 96 hours. In cells harvested at 96 hours and washed and
reincubated in fresh medium, the cells maintain the differentiated
phenotype for periods as long as 120 hours (data not shown).
Immunoblot analysis of TOPO I levels in control and RA-treated cells
showed that neither the proliferation nor the differentiation state of
the cells impacted upon the level of TOPO I protein (Fig 2).
The mRNA expression of TOPO II, 1, and
2 (2 differentially spliced forms) in control and
RA-treated cells is shown in Fig 3. Interestingly,
despite the growth cessation by 48 hours, mRNA levels of TOPO II,
1, and 2 in RA-treated cells were
maintained at 60% to 80% of the level in control, untreated cells. A
maximal decrease (50% to 60%) in TOPO II mRNA levels occurs at 96 hours. Furthermore, in cells treated with RA for 120 hours and
reincubated in fresh RA-free medium, levels of TOPO II mRNA were
maintained at 50% to 60% of control levels.
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| Fig 3.
(A) RNase protection analysis of TOPO II,
1, and 2 in control (lanes 2, 4, 6, 8, 10, and 12 at 24, 48, 72, 96, and 120 hours and 120R [recovery in
medium after treatment for 120 hours], respectively) and RA-treated
(lanes 3, 5, 7, 9, 11, and 13 at 24, 48, 72, 96, and 120 hours and 120R
[recovery in medium after treatment for 120 hours], respectively)
HL-60 cells. M is the molecular weight marker, and lane 1 is tRNA.
(B) Phosphorimager analysis of TOPO II isozymes mRNA relative
to globin. The signal intensity of the sample from control cells at 24 hours was set at 100%.
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The protein level and phosphorylation of TOPO II and in control
and RA-treated cells is outlined in Figs 4 and
5, respectively. The data in Fig 4 show that levels
of TOPO II are elevated twofold to fourfold in cells treated with
RA. Also, maximal elevation in levels of TOPO II appears to occur
around 96 hours, which coincides with the state of maximal
differentiation. The levels of TOPO II were also elevated in
response to the differentiated state of the cells, with a twofold to
fourfold elevation between 48 and 96 hours. The immunoprecipitation
data shown in Fig 5 demonstrate that elevated levels of TOPO II or
protein also correspond with an increased phosphorylated state of
the protein. However, whereas the increase in phosphorylation of TOPO
II in RA-treated cells was about twofold higher (compared with the
untreated control), the level of TOPO II phosphorylation in
RA-treated cells was greater than fourfold. Based on this differential
response, a majority of additional experiments were focused on the
regulation of TOPO II.
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| Fig 5.
(A) Phosphorylation of TOPO II and in control (C)
and RA (R) -treated HL-60 cells. Control and RA-treated cells were
metabolically labeled with [32P]-orthophosphoric acid,
and cell lysates were immunoprecipitated with serum IID (15) and
analyzed by SDS-PAGE. (B) Phosphorimager analysis of TOPO II and isozyme phosphorylation.
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To determine mechanisms responsible for the more pronounced increase in
levels of TOPO II in the RA-treated cells, the stability of the
protein in control and RA-treated cells pulse labeled with [35S]-L-methionine was determined. Results from these
experiments ( Fig 6 and Table 1) suggest
that the levels of TOPO II in cells treated with RA are twofold
higher compared with that in untreated cells.
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| Fig 6.
Degradation of TOPO II in control and RA-treated HL-60
cells. Control cells (lanes 1 through 4), 48-hour RA-treated cells
(lanes 5 through 8), and 96-hour RA-treated cells (lanes 9 through 12).
Lanes 1, 5, and 9, protein level immediately after labeling with
35S-methionine; lanes 2, 6, and 10, labeled protein levels
at 2 hours after reincubation in complete medium; lanes 3, 7, and 11, labeled protein levels at 4 hours after reincubation in complete
medium; and lanes 4, 8, and 12, labeled protein levels at 6 hours after
reincubation in complete medium.
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Because the levels and phosphorylation of the TOPO II isozymes were
increased in RA-treated cells, the catalytic activity based on
decatenation of kDNA was determined. Results from these experiments
(Fig 7) demonstrate that, despite the increased protein levels and phosphorylation of the TOPO II isozymes in the RA-treated cells, the total level of decatenating activity is comparable in
extracts from control and RA-treated cells.
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| Fig 7.
Analysis of decatenating activity in nuclear extracts
from control (lanes 1 through 7) and RA-treated (lanes 8 through 14)
HL-60 cells. The substrate 200 ng kDNA (TOPOGEN Inc) was incubated for
30 minutes with nuclear extracts that were serially diluted to contain
the following: lanes 1 and 8 (1:2), lanes 2 and 9 (1:4), lanes 3 and 10 (1:8), lanes 4 and 11 (1:16), lanes 5 and 12 (1:32), lanes 6 and 13 (1:64), and lanes 7 and 14 (1:128). The position of the decatenated
kDNA is indicated by the arrow.
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Based on the more prominent increase in the phosphorylation of TOPO
II in the RA-treated cells, possible alterations in site-specific phosphorylation were determined in complete tryptic digests by two-dimensional mapping. The results shown in Fig 8
indicate that, although a site appears to be hypophosphorylated in the
RA-treated cells, four additional sites are hyperphosphorylated in the
RA-treated versus control cells.
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| Fig 8.
Representative two-dimensional tryptic phosphopeptide
maps of TOPO II protein from control (left) and RA-treated (right)
HL-60 cells. Cells were metabolically labeled with
[32P]-orthophosphoric acid, and lysates in RIPA buffer
were immunoprecipitated with antiserum specific for TOPO II.
Peptides were separated horizontally by electrophoresis at pH 1.9 and
vertically by chromatography as described in Materials and Methods. The
sites that are differentially phosphorylated between control and
RA-treated cells are identified by arrows.
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It is well recognized that CPT and VP-16 are effective in stimulating
DNA cleavable complex formation mediated by TOPO I and TOPO II,
respectively.3-5 To assess the sensitivity of control and
RA-treated differentiating cells to the DNA-damaging effects of CPT or
VP-16, we evaluated drug-stimulated DNA cleavable complex formation by
the SDS-KCl technique.25 Results from these experiments are
outlined in Table 2. The data suggest that, except for a decrease in DNA cleavable complex formation in cells treated with 10 µmol/L VP-16, the DNA cleavable complex formation induced by VP-16 or
CPT in control or RA-treated cells is not significantly different
(P = .6 to .13).
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Table 2.
Effect of Treatment With RA on Drug-Stimulated DNA
Cleavable Complex Formation and Apoptosis in HL-60 Cells
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DNA damage induced by VP-16 or CPT results in an apoptotic response in
treated cells.3,4 To determine the apoptotic response of
control or differentiated HL-60 cells after drug-stabilized topoisomerase-mediated DNA cleavable complex formation, we used a
modification of the TUNEL assay.30 Results on induction of apoptosis based on staining with fluorescein-12-dUTP are shown in Table
2. The data suggest that, after exposure for 1 hour to 1, 5, and 10 µmol/L CPT or 10 µmol/L VP-16, the apoptosis is significantly lower
(P = .012 to P < .001) in RA-treated cells compared
with the control untreated cells.
| |
DISCUSSION |
The retinoids are an important class of agents in their ability to
induce cellular differentiation. In the HL-60 model system, RA induces
maturation in the granulocytic lineage and this process has been well
characterized.31 Accompanying the induction of cellular
differentiation by RA, numerous membrane, cytoplasmic, and nuclear
changes have been identified in HL-60 cells.31,32 Although
the role of TOPO II in cell differentiation has been suggested,12 this is the first report demonstrating a more
pronounced upregulation of the isoform during the differentiation
process in HL-60 cells. Notably, TOPO II and not TOPO I is actively
regulated during RA-induced differentiation. This differential effect
may also reflect the active role of TOPO II versus TOPO I during cell growth and differentiation.
A number of earlier reports focusing on TOPO II have implied that
the downregulation at the mRNA and protein level after treatment with
dimethyl sulfoxide or phorbol-12-myristate-13-acetate reflects
cessation of growth after commitment to differentiate.15-18 The results from this study are in agreement with the downregulation of
TOPO II and mRNA that occurs within 24 hours after exposure to
RA.20 However, unlike treatment with dimethyl sulfoxide or phorbol-12-myristate-13-acetate, our results demonstrate that, whereas
the protein levels of TOPO I remain unchanged, protein levels of both
TOPO II and are elevated in HL-60 cells induced to differentiate
after treatment with RA. It should also be noted that mRNA levels of
both TOPO II isoforms are not only continuously maintained at 50% to
60% of control levels during the process of differentiation, but are
also maintained in the differentiated state with an elevation in TOPO
II and protein levels in RA-treated cells. Also,
upregulation of TOPO II protein levels appear to be more pronounced
than the isoform. The higher levels of TOPO II protein in
RA-treated cells also represents active protein, because the catalytic
activity, measured by decatenation of kDNA, was similar in control and
RA-treated cells. Increased levels of TOPO II protein in the
RA-treated cells appear to be due to slower degradation of the protein.
The TOPO II and in differentiated cells was also
posttranslationally modified by phosphorylation, similar to the control
cells. The level of phosphorylation was higher in the differentiated
versus control cells, in accordance with the higher TOPO II protein
level. These results on increased phosphorylation of TOPO II are
comparable to the results of Chresta et al33 after
treatment with RA for only 1 to 2 hours. Also, the increased
phosphorylation of TOPO II in the RA-treated cells is of interest,
because they are growth arrested in G1 phase of the cell
cycle after differentiation. However, unlike data with mitotic
cells,7-9 no apparent change in the molecular weight of
TOPO II was apparent with the phosphorylated form in RA-treated cells. Interestingly, the data also showed that four peptides in the
TOPO II protein are differentially hyperphosphorylated in RA-treated
versus control cells when complete tryptic digests were analyzed by
two-dimensional peptide mapping. Because there is a much greater degree
of sequence similarity in the ATPase and breakage/reunion domains than
the C-terminal domain between the and isoforms of TOPO II, the
role of site-specific phosphorylation needs to be addressed. Our
ongoing studies are now focused in sequencing the hyperphosphorylated
sites of TOPO II to characterize their functional role in catalytic
and drug-stimulated DNA cleavage activity during growth and
differentiation of RA-treated cells.
Despite the increased levels of TOPO II protein in RA versus control
cells, the overall increase in CPT-stimulated or VP-16-stimulated DNA
cleavable complex formation was comparable and not significantly different. However, apoptosis in response to the DNA damage was significantly lower in RA-treated versus control cells treated with 1, 5, and 10 µmol/L CPT or 10 µmol/L VP-16. The finding that drug-stabilized DNA cleavable complex formation and decatenation of
kDNA are similar in the control and RA-treated cells suggests activity
of the enzyme, but there may be a differential regulation in the
processing of the breaks leading to apoptosis in control (proliferating) versus differentiated (nonproliferating) cells. The
activity of TOPO II in the differentiated, nonproliferating cells is of
interest, because circulating lymphocytes are essentially devoid of
TOPO II-mediated DNA decatenating activity.
In summary, results from this study demonstrate that TOPO II levels are
posttranscriptionally regulated in HL-60 cells induced to differentiate
with RA. These results with RA and its relationship to the levels and
activity of TOPO II isoforms is substantially different from the
reports using dimethyl sulfoxide or phorbol-12-myristate-13-acetate, suggesting that RA-induced effects on cell differentiation may actively
involve a role for TOPO II. Furthermore, among the TOPO II isoforms,
the isoform appears to be more actively regulated in the RA-treated
cells. Further studies delineating the role of TOPO II isoforms,
particularly that of TOPO II in differentiated cells, could aid in
the identification of a specific role for these enzymes in
transcription of genes involved in
differentiation.
| |
FOOTNOTES |
Submitted February 12, 1998;
accepted June 8, 1998.
Supported by US Public Health Services Grant No. CA35531
from the Department of Health and Human Services (M.A., D.R.G, K.A.K, and R.G.) and by the Imperial Cancer Research Fund (R.J.I. and I.D.H.).
Address reprint requests to Ram Ganapathi, PhD, Cancer
Center, M38, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge Dr Scott Kaufmann (Department of
Pharmacology, Mayo Clinic, Rochester, MN) for the gift of
anti-topoisomerase II antibody and Jim Reed of the Art-Medical Illustrations and Photography Department for skillful preparation of
the figures.
| |
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Introduction
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