(CANCER RESEARCH 47, 4544-4547, September 1, 1987)
Erythroid Differentiation and Modulation of c-myc Expression Induced by
Antineoplastic Drugs in the Human Leukemic Cell Line K562
G. P. Tonini, D. Radzioch, A. Gronberg, M. Clayton, E. Blasi, G. Benetton, and L. Varesio'
Laboratory of Oncology, Department of Haematology and Oncology, Gaslini Institute, Genova, Italy [G. P. T], and Immunobiology Section, Laboratory of Molecular
Immunology, Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute, NIH, Frederick Cancer Research Facility,
Frederick, Maryland 21701 [D. R., A. G., M. C., E. B.. G. B., L. V.]
The human leukemia cell line K562 expresses constitutively high levels
of I'-myc inK.VA and can be induced to differentiate along the erythroid
lineage. Treatment of k 562 cells with the antineoplastic drugs 1-0-Darabinofuranosylcytosine and daunomycin causes differentiation into
hemoglobin-producing cells. The differentiation process is associated with
an early block of cellular proliferation occurring during the first 24 h of
treatment. RNA synthesis is progressively reduced to 20 to 30% of the
control levels after 3 days of exposure to the drugs. Dot and Northern
blot analyses were performed to evaluate the levels of c-myc or globin
niKNA during the differentiation of KS62. Daunomycin and 1-0-D-arabinofuranosylcytosine, despite their distinct chemical nature, induced sim
ilar modulation of mRNA levels. Globin mRNA did not change during
the first 24 h of culture and began to increase after 48 h of treatment
with drugs, reflecting the kinetic of appearance of hemoglobin-producing
cells. In contrast, a transient decrease of c-myc mRNA was observed
after the first 24 h of drug treatment, followed by a return to normal
levels of c-myc mRNA after 48 h of treatment. Thus, the expression of
r-m.iv mRNA in K562 did not reflect their proliferative activity nor their
stage of differentiation. We speculate that the transient down-regulation
of c-myc mRNA may be an initial event in the erythroid differentiation
of K562.
loid, or could be attributed to a different response of mouse or
human cells to differentiative stimuli. Analysis of c-mycexpres
sion during erythroid differentiation of human cells could pro
vide information on this issue.
Although many agents can promote differentiation of KS62
i/i vitro, we decided to study DNM and ARA-C, since these 2
drugs are currently used in chemotherapy. ARA-C is a purine
antimetabolite (13, 14), extensively used for the treatment of
acute myelogenous leukemias. It has been reported that ARAC can induce erythroid differentiation of K562 (15). DNM is
an alkylating agent (16), effective on different types of tumors,
but its effects on erythroid differentiation are unknown.
We found that both DNM and ARA-C can induce erythroid
differentiation of K562 cells and sequentially modulate c-myc
and globin mRNA expression, c-myc mRNA is transiently
down-regulated during the first 24 h of treatment, whereas
globin mRNA is up-regulated after 48 h of treatment. These
results show that antineoplastic drugs can modulate cell prolif
eration and gene expression and suggest that the initial transient
down-regulation of c-myc mRNA may be linked to the differ
entiation process.
INTRODUCTION
MATERIALS AND METHODS
A cell line (K562) was established by Lozzio and Lozzio (1)
from a patient with chronic myeloid leukemia in acute phase.
These cells can be induced to undergo erythroid differentiation
in vitro by a variety of agents including hemin and butyric acid
(2-6). K562 cells carry a rearrangement and amplification of cabl gene and constitutively express high levels of c-myc mRNA
(7, 8). It has been suggested that amplification and increased
expression of cellular protooncogenes (c-onc) may be involved
in neoplastic transformation (7, 8). However, no information
is available on c-onc expression during differentiation of K562
cells.
In the mouse system, erythroid differentiation of erythroleukemia cells (MEL)2 induced by dimethyl sulfoxide of hypoxanthine is associated with a biphasic change of c-myc expression
occurring during the first two hours after exposure to the
chemicals (9). It has been suggested that the transient downregulation of c-myc mRNA could be an early signal for terminal
differentiation of MEL. In contrast, differentiation of murine
and human leukemias along the myeloid lineage is associated
with a persistent decrease of c-myc expression (10-12). In
myeloid tumors, c-myc may be mainly involved in maintaining
proliferative state and thus its expression may be decreased by
the growth arrest of differentiating cells (10). The transient
versus persistent pattern of c-myc down-regulation could be
related to the lineage of differentiation, erythroid versus mye-
Cells and Treatments. The K562 cell line (1) was maintained as a
suspension culture in KI'Ml 1640 with 5% fetal calf serum (Grand
ABSTRACT
Received 5/1/86; revised 5/21/87; accepted 6/5/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
2The abbreviations used are: MEL, myeloid leukemia; DMN, daunomycin;
ARA-C, 1-/3-u-arabinofuranosylcytosine.
Island Biological Company, Grand Island, NY). The medium was
further supplemented with 2 IHML-glutamine, 100 U 7mI penicillin,
and 100 iig/ml streptomycin. The cells were demonstrated to be free of
Mycoplasma. Treatment of K562 cells was performed in culture me
dium at a starting concentration of 5 x 10s cells/ml. Viable cell counts
were obtained with 0.1 % trypan blue. Cultures were scored for hemo
globin-producing cells by staining with benzidine dihydrochloride
(Sigma Chemical Co., St. Louis, MO), 2 mg/ml in 0.5% acetic acid.
The cells were washed once in phosphate-buffered saline and adjusted
to approximately 10'' cells/m!. The benzidine solution was mixed with
SO ¿tl/mlof a 3% hydrogen peroxide solution and added to an equal
volume of cell suspension. Morphological changes were followed on
Giemsa-stained cytospin preparation of cells. Cell synchronization by
double ihymid ine block was performed as described elsewhere (17).
Briefly, K562 at a concentration of 106/ml where incubated with 2 mM
thymidine for two 24-h periods separated by a 12 h period in the
absence of a synchronizing agent.
Drugs. ARA-C (Upjohn, Puurs, Belgium) and DNM (kindly provided
by the Gaslini Institute, Genova, Italy) were dissolved in water and
further diluted in medium to the desired concentration.
Measurement of DNA and RNA Synthesis. DNA and RNA synthesis
were measured as previously described (18). Briefly, 100 ¿i
I aliquots
were taken from cell cultures at various time points and distributed
into 96-well microtiter plates (Dynatech, Alexandria, VA). The cells
were pulsed with 1 ¿iCi/mlof [methyl-}H]thym\dine or [3H]uridine
(New England Nuclear, Boston, MA) for 3 h, precipitated with cold
trichloroacetic acid (10% solution in water), and transferred to glass
fiber filters using a multichannel harvester (Mash II; Dynatech). The
amount of radioactivity incorporated was measured by liquid scintilla
tion counting.
RNA Extraction and Analysis. RNA was extracted and purified by
sedimentation through a cesium chloride cushion as previously de-
4544
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c-myc EXPRESSION
AND DIFFERENTIATION
scribed (19). Northern and dot blot analyses were performed as de
scribed by Maniatis et al. (20). Total RNA extracted from the HL-60
cell line and purified rabbit globin m RN A were used as standard positive
controls for expression of c-myc and a-globin, respectively. Clal-EcoRl
fragment of the third exon of human c-myc (kindly provided by Dr. D.
Watson, Laboratory of Molecular Oncology, National Cancer Insti
tute), the Pstl fragment of the human a-globin (kindly provided by Dr.
A Nienhius), and "25-46" or "1-19" fragments of 18 or 28S ribosomal
genes, respectively (both kindly provided by Dr. Arnheim), were used
as probes after labeling with }2P by nick translation under standard
conditions (20).
RESULTS
DNM (100 ng/ml) or ARA-C (1 Mg/ml) induced differentia
tion of K562 cells which undergo major morphological changes
including increase in cell size and formation of 2 or more
nucleoli. The differentiation process was preceded by inhibition
of DNA synthesis and cell replication within the first 24 h of
exposure to either DNM or ARA-C (Table 1). In contrast, RNA
synthesis (measured by [3H]uridine uptake) was less affected by
the drugs since it was about 80% of the control levels after the
first 24 h of treatment and slowly decreased to 30 to 40% of
control by Day 3 (Table 1). Cell viability, as assessed by trypan
blue exclusion, remained greater than 85% during the first 4
days of treatment with DNM or ARA-C under our experimental
conditions. Moreover, the cell number in drug-treated cultures
remained constant in this time frame, confirming that the drugs
were not toxic at these concentrations (data not shown).
To establish whether these drugs induced differentiation of
K562 along the erythroid lineage the cells were tested for
hemoglobin production by benzidine reactivity at various time
intervals. Both drugs induced an increase in the frequency of
hen/idiiu'- positive cells which became significant after 2 days
of treatment and reached plateau levels after 4 to 5 days of
treatment with DNM or ARA-C (Table 2). The fact that 2
drugs such as DNM and ARA-C, with very different pharma
cological properties, exerted similar effects on the differentia
tion of K562 cells suggested that arrest of DNA synthesis may
be in itself sufficient to trigger erythroid differentiation. To test
this hypothesis we monitored the benzidine reactivity of K562
following a reversible growth arrest induced by double thymidine block. As shown in Table 2, a reversible growth arrest was
sufficient to induce differentiation of KS62 cells comparable to
that induced by DNM or ARA-C. These results indicate that
the block of DNA synthesis induced by DNM or ARA-C could
be responsible for the induction of K562 differentiation. In
parallel experiments, the levels of globin mRNA in control or
drug-treated cells were quantified by dot blot analysis. As shown
in Fig. 1, accumulation of globin mRNA in K562 cells was
increased after the first 48 h of treatment with DNM or ARAC, reaching about 10-fold increase by Day 3. Similar results
were observed when RNAs were analyzed by Northern blotting
(data not shown). Increase in globin mRNA after 2 to 3 days
of drug treatment was consistently detected and the maximal
augmentation ranged between 8- and 16-fold. These results
showed that both DNM and ARA-C induced accumulation of
globin mRNA and thus promoted differentiation of K562 cells
along the erythroid lineage (21, 22). In conclusion, the accu
mulation of globin mRNA induced by DNM and ARA-C occurs
when the K562 cells are in a nonreplicating state with a reduced,
but still significant, synthesis of total cellular RNA.
K562 cells constitutively express detectable levels of c-myc
mRNA appearing as a major 2.4-kilobase band in Northern
blots (Fig. 2). Upon treatment with DNM or ARA-C for 24 h,
a considerable reduction of 2.4-kilobase c-myc mRNA expres
sion was observed. Dot blot analysis demonstrated about 5- to
10-fold decrease of c-myc mRNA expression in drug-treated
cells (data not shown). However, the levels of c-myc mRNA
returned to normal after 48 h of drug treatment. No major
differences were observed in the pattern of modulation of cmyc mRNA expression by DNM or ARA-C. The possibility of
differential binding of mRNA to nitrocellulose was excluded by
sequential hybridization of the same blot with either globin
followed by myc and again globin or myc followed by globin
and again myc. In 5 independent experiments, we consistently
observed low c-myc mRNA and unchanged globin mRNA levels
in K562 treated with the drugs for 24 h as compared to
untreated controls. On the other hand, augmented globin
expression and c-myc mRNA levels comparable to untreated
controls were found at 48 and 72 h (data not shown). No
Table 1 Nucleic acid synthesis in K562 cells treated with DMN or ARA-C
U
K562 cells were cultured in the presence or absence of the drugs and assayed
daily for ['H]uridine and | '1(jihymittme uptake.
Time of
culture
(h)24
['Hlthymidine up
takeDNM7±r4
O
O
uptakeDNM78
['HJuridine
EC
<
5
±0.85
±5
±6
±0.5
±0.4
55 ±4
65 ±5
5 ±0.4ARA-C6
28 ±IARA-C79
41 ±3
2± 1%
" Mean ±SE of incorporation of quadruplicate cultures of drug-treated K562
relative to untreated controls. Cultures were labeled as described in "Materials
and Methods."
4872%
Table 2 Differentiation ofK562 cells treated with DNM or ARA-C
2.5
J
1.2
g
0.6
75 >a
K562 cells were cultured in the presence or absence of drugs and assayed daily
for benzidine positivity. Cells were scored for benzidine positivity at the indicated
O
^~
times following a double block of proliferation with 2 imi thymidine as detailed
in "Materials and Methods."
Time of
culture
(h)24
Z
O
0.3
0.15
'
Q QJ
withNone5"
benzidine-positive cells after treatment
48
1825
64
72
96%
47ARA-C7
5DNM6
" Calculated after scoring randomly at least 300
0.03
IS
22
3543
31
59ThymidineIO
cells on 2 or 3 different slides.
Fig. 1. Globin mRNA expression in KS62 cells treated with DNM or ARAC. K562 cells were treated for 24 (left), 48 (middle), or 72 (right) h with DNM or
ARA-C. and the total RNA was tested by dot blot analysis for its content of
globin mRNA. Amounts of total RNA/dot bound to nitrocellulose are shown on
the left. Contr, control.
4545
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c-myc EXPRESSION AND DIFFERENTIATION
c-myc mRNA could be detected 4 h after the drug treatment.
No differences were observed in the kinetics of c-myc mRNA
decrease induced by DNM or ARA-C.
In summary, the erythroid differentiation induced in K562
by DNM or ARA-C is associated with an early, transient downregulation of c-myc expression followed by an up-regulation of
globin expression.
2.4 kb-
DISCUSSION
-28S rRNA
-18S rRNA
Fig. 2. c-myc mRNA expression in K562 cells treated with DNM or ARA-C.
K562 cells were treated for 24, 48, and 72 h with DNM or ARA-C, and the total
RNA was tested by Northern blot analysis for its content of c-myc mRNA (lop)
and 28 and 18S ribosomal RNA (middle and bottom, respectively). Total RNA
extracted from the III 60 cell line (right lane) was used as a positive control
(Contr). Ten pg of total RNA were loaded on the gel for Northern blotting, kb,
kilobase.
100
l
at
8.
1
3
6
18
24
Hours of Treatment
Fig. 3. Kinetics of down-regulation of c-myc mRNA expression in K562 cells
treated with DNM (A) or ARA-C (•).K562 cells were treated for 1, 3, 6,12, 18,
and 24 h with DNM or ARA-C, and the total RNA was extracted and tested by
dot blot analysis for its content of c-myc mRNA. Results are expressed as a
percentage of c-myc mRNA expression in drug-treated KS62 relative to untreated
controls. Values represent the average of triplicate samples. Quantification of the
c-myc mRNA was done by liquid scintillation counting of the "I' levels in the
individual dots. SEs were less than 5% of the mean and were not reported.
differences in 28 or 18S ribosomal RNA were observed when
the blots of RNA from control or drug-treated K562 cells were
hybridized with probes specific for the 48 or 28S ribosomal
RNA (Fig. 2).
Studies were performed to determine the kinetics of c-myc
mRNA decrease following ARA-C or DNM treatment of K562.
As shown by the results depicted in Fig. 3, significant decrease
of c-myc mRNA expression was detectable as soon as 6 h after
ARA-C or DNM treatment. In some experiments, decrease in
DNM and ARA-C are currently used in cancer therapy,
including chronic and acute leukemias (35,36). Since the mech
anism of interaction with cellular DNA of these chemically
distinct drugs appears to be different (14-16), it was of interest
to compare their effects on the biology and gene expression of
KS62 cells. KS62 cells were utilized for these studies since little
is known about the modulation of gene expression during
erythroid differentiation, and K562 can differentiate along this
lineage. Insights into the effects of chemotherapeutic agents on
human tumor cells could be of clinical interest (23).
We have shown that DNM is a potent inducer of erythroid
differentiation of K562 cells, comparable to ARA-C. Moreover,
both drugs were very similar in the kinetics of inhibition of
nucleic acids synthesis. Within the first 24 h, DNM and ARAC induce a complete block of DNA synthesis and a limited
inhibition of RNA synthesis, confirming their selectivity in
inhibiting DNA replication rather than transcription. At later
time points inhibition of RNA synthesis becomes progressively
more evident. [3H]uridine uptake measures mainly ribosomal
RNA metabolism and preliminary experiments support the
hypothesis of decreased synthesis of ribosomal RNA following
drug treatment (data not shown). Decrease of ribosomal RNA
synthesis as well as unbalanced accumulation of mature ribo
somal RNA species has been previously reported following
growth arrest of many cell lines (24-26) or in nonproliferating
cells (27-29). Decrease of RNA synthesis in drug-treated K562
may be a direct consequence of the early inhibition of DNA
synthesis.
The inhibitory effects of the drugs on DNA synthesis may be
a signal sufficient to commit K562 cells to terminal differentia
tion. In fact, we observed that K562 cells were induced to
become benzidine positive by a reversible inhibition of DNA
synthesis caused by double thymidine block. In contrast, di
methyl sulfoxide, which is a differentiating agent in MEL (9)
did not induce differentiation of K562 (data not shown), con
firming previously published observations (5).
K562 cells constitutively express c-myc mRNA, but following
treatment with either DNM or ARA-C, c-myc RNA levels
dropped during the first 24 h and returned to normal levels 48
h after treatment. The transient down-regulation of c-myc
mRNA preceded the increase in globin mRNA, and the ap
pearance of ben/idine positive cells. Transient down-regulation
of c-myc expression during the early phases of erythroid differ
entiation induced by dimethyl sulfoxide has also reported in
MEL (9). It is possible that a transient drop of c-myc mRNA
may be one of the initial events that commits the cells to
differentiate (9). Since the half-life of the c-myc protein is in
the 60- to 120-min range (30), a transient decrease of c-myc
mRNA could indeed result in a reduction of the nuclear c-myc
protein, which may affect the expression of other genes regu
lating the differentiation process.
Transient modulation of c-onc may be a more general phe
nomenon associated with the differentiation of many cell types
since transient up-regulation of c-fos expression in the HL-60
4546
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c-myc EXPRESSION
AND DIFFERENTIATION
cell line after induction of differentiation along the monocytic
lineage has been reported (32). However, in differentiating
myeloid cell lines, c-myc mRNA is permanently down-regu
lated, whereas during differentiation of erythroid cell lines, only
a transient inhibition of c-myc in mRNA expression has been
reported. We are tempted to speculate that a distinct pattern of
c-myc mRNA expression may characterize the differentiation
along different lineages. However, more information on gene
expression during differentiation of cells and mostly of normal
stem cells is needed to support this hypothesis.
The transient nature of c-myc mRNA down-regulation in
differentiating K562, returning to normal levels even if the cells
are growth inhibited by the drugs, argues against a direct role
of c-myc in controlling cell cycle or in maintaining cell prolif
eration (33). It has been previously reported that the levels of
c-myc are similar in cells at different stages of the cell cycle
(31, 33, 34). Our results provide direct evidence that c-myc
mRNA can be expressed at the same levels as proliferating
K562 when cellular replication is inhibited by drugs. It is
interesting to note that following the initial decrease, c-myc
mRNA was again highly expressed at the time in which the
globin mRNA becomes evident. This observation is even more
dramatic in view of the decrease in total RNA synthesis that
occurs at the time of myc mRNA reappearance. This finding
suggests that c-myc mRNA may also have a positive effect on
differentiation since the reappearance seems to precede or
coincide with the increase of globin mRNA.
After the submission of this manuscript we learned that
Bianchi-Scarra et al. (37) have reported that ARA-C induces
differentiation of K562 cells and a concomitant inhibition of cmyc mRNA.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
ACKNOWLEDGMENTS
25.
The authors wish to thank Drs. J. J. Oppenheim, H. Young, and D.
Longo for their helpful suggestions and for reviewing the manuscript.
Appreciation is extended to the NCI-FCRF Central Clerical Pool for
typing and editing the manuscript.
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Erythroid Differentiation and Modulation of c-myc Expression
Induced by Antineoplastic Drugs in the Human Leukemic Cell
Line K562
G. P. Tonini, D. Radzioch, A. Gronberg, et al.
Cancer Res 1987;47:4544-4547.
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