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HEMATOPOIESIS
Manipulating the onset of cell cycle withdrawal in differentiated erythroid
cells with cyclin-dependent kinases and inhibitors
Igor Matushansky, Farshid Radparvar, and Arthur I. Skoultchi
Terminal differentiation of erythroid cells
results in terminal cell divisions followed
by irreversible cell cycle withdrawal of
hemoglobinized cells. The mechanisms
leading to cell cycle withdrawal were assessed in stable transfectants of murine
erythroleukemia cells, in which the activities of cyclin-dependent kinases (CDKs)
and CDK inhibitors (CDKIs) could be
tightly regulated during differentiation.
Cell cycle withdrawal of differentiating
cells is mediated by induction of several
CDKIs, thereby leading to inhibition of
CDK2 and CDK4. Manipulation of CDK
activity in differentiating cells demonstrates that the onset of cell cycle withdrawal can be either greatly accelerated
or greatly delayed without affecting hemoglobin levels. Extending the proliferation
of differentiating cells requires the syner-
gistic action of CDK2 and CDK4. Importantly, CDK6 cannot substitute for CDK4
in this role, which demonstrates that the 2
cyclin D–dependent kinases are functionally different. The results show that differentiating hemoglobinized cells can be made
to proliferate far beyond their normal capacity to divide. (Blood. 2000;96:2755-2764)
© 2000 by The American Society of Hematology
Introduction
The process of differentiation of primitive cells into more
specialized cells involves both the expression of new sets of
genes (phenotypic differentiation) and expansion of the differentiating cell population through cell division.1,2 In recent years a
great deal has been learned about the factors controlling gene
expression during differentiation. Much less is known about the
mechanisms that determine the number of cell divisions that a
terminally differentiating cell can make and the mechanisms
that maintain fully mature cells in the state of permanent cell
cycle withdrawal. There is an equally important question to
consider: Once triggered, are phenotypic differentiation and
terminal cell division coupled, or alternatively, do they proceed
independently?
The eukaryotic cell cycle is primarily regulated by a family of
serine/threonine protein kinases that each consist of a catalytic
subunit (cyclin-dependent kinase [CDK]) and a regulatory subunit
(cyclin).3 In mammalian cells, progression through the G1 phase is
controlled by the activities of CDK4 and CDK6, which associate
with 1 of 3 D-cyclins (D1, D2, and D3), and CDK2, which
associates first with cyclin E and then later in S phase with cyclin
A.4 The enzymatic activities of the CDKs are controlled at several
levels: cyclin binding,5 CDK-activating kinase (CAK) activation,
CDK phosphorylation and dephosphorylation, and binding of
cyclin-dependent kinase inhibitors (CDKIs).6,7 To date 2 families
of CDKIs that differ in their specificity and mechanism of
inhibition have been identified. INK4 family members p16INK4A,
p15INK4B, p18INK4C, and p19INK4D inhibit only CDK4 and CDK6 by
interfering with cyclin D binding.8,9 The KIP family of inhibitors,
p21CIP, p27KIP1, and p57KIP2, is thought to inhibit primarily CDK2 in
vivo.10,11 Whereas functional differences are known among the D-,
E-, and A-dependent kinases, differences between the 2 cyclin
D–dependent kinases, CDK4 and CDK6, have not been reported.
We have studied the mechanisms leading to terminal cell
division and irreversible cell cycle withdrawal in differentiating red
blood cells using murine erythroleukemia (MEL) cells.12 MEL
cells are erythroid precursors that are blocked at the proerythroblast
stage. Treatment of the cells with certain chemical inducers relieves
the block to differentiation and allows the cells to resume erythroid
differentiation. In vitro differentiation of MEL cells recapitulates
many aspects of normal red blood cell differentiation including
synthesis of hemoglobin and other erythrocyte-specific proteins as
well as terminal cell division. After committing to terminal
differentiation, the cells undergo a maximum of 5-6 cell divisions
and then permanently withdraw from the cell cycle.13 This process
closely resembles the terminal cell divisions of colony-forming
unit–E (CFU-E), normal, committed erythroid precursors that also
differentiate and divide 5-6 times before arresting.14 We found that
MEL cell differentiation is accompanied by specific, temporally
defined changes in the levels and activities of the CDKs and the
CDK inhibitors including differences in the regulation of CDK4
and CDK6. To investigate the role of these changes in terminal cell
division and cell cycle withdrawal, we generated and analyzed
stable MEL cell transfectants expressing these cell cycle regulators
under an inducible promoter. Our results show that cell cycle
withdrawal of differentiating erythroid cells is mediated by the
induction of CDKIs, which leads to inhibition of CDK2 and
CDK4. The results also imply that once differentiation is triggered, phenotypic differentiation (hemoglobin synthesis) and
terminal cell division need not be tightly coupled, and the 2
programs can proceed independently at different rates without
affecting one another.
From the Department of Cell Biology, Albert Einstein College of Medicine,
Bronx, NY.
Reprints: Arthur I. Skoultchi, Department of Cell Biology, Albert Einstein
College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461; e-mail:
skoultch@aecom.yu.edu.
Submitted February 18, 2000; accepted June 9, 2000.
Supported by grant 2P30CA13330 (A.I.S.) from the National Cancer Institute
Cancer Center, grants 5T32AG00194 (F.R.) and 5R37CA16368 from the
National Institutes of Health (NIH), and grant 5T32GM07288-25 (I.M.) from the
NIH/Medical Scientist Training Program, Bethesda, MD.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
2755
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2756
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
MATUSHANSKY et al
Materials and methods
Cell culture and DNA transfections
Clone DS19 MEL cells were grown, and differentiation was initiated as
previously described.15,16 Benzidine staining and plasma clot assays were
performed as previously described.15 Stable MEL cell transfectants expressing doxycycline (Dox)-inducible cell cycle regulators were generated by
transfecting 1 of 2 MEL cell clones (B1 and C2) that stably express the
reverse tetracycline–controlled transactivator (rtTA).17 Clones B1 and C2
were prepared by cotransfecting DS19 MEL cells with pPGK-neo and
pUHD 172-1.17 MEL cell transfectants p18 and p27 were generated by
cotransfecting clone B1 with pPGK-puro and either pUHD 10-3 p18 or
pUHD 10-3 p27. CDK2-HA (hemagglutinin), CDK4R24C-HA, and
CDK6R31C-HA transfectants were generated by cotransfecting clone C2
with either pUHD 10-3 CDK2-HA, pUHD 10-3 CDK4R24C-HA, or pUHD
10-3 CDK6R31C-HA and with pPGK-puro. Transfectants were selected
and maintained in growth medium containing 1 mg/mL G418 and 5 ␮g/mL
puromycin. CDK(2 ⫹ 4R24C) and CDK(2 ⫹ 6R31C) MEL cell transfectants were generated by cotransfecting pUHD 10-3 CDK2-HA and pPGKhygro into CDK4R24C.24 and CDK6R31C.34 transfectants, respectively.
Transfectant clones were selected and maintained in medium containing 1
mg/mL G418, 5 ␮g/mL puromycin, and 1.1 mg/mL hygromycin. Antibioticresistant clones were expanded, and the cell extracts were analyzed by
immunoblotting.
Plasmids
S-transferase (GST)–tagged carboxy-terminal fragment of Rb (gift of R.
Pestell) as a substrate. For CDK(2 ⫹ 4R24C) and CDK(2 ⫹ 6R31C)
transfectants containing 2 exogenous kinases, the assays were carried out as
described above except that human-specific ␣-CDK2 antibody (nonreactive
with mouse CDK2) was used first to completely immunoprecipitate
exogenous CDK2 activity. Extracts found to be free of exogenous CDK2
were then immunoprecipitated for exogenous CDK4R24C-HA and
CDK6R31C-HA as described above.
Results
Resumption of terminal differentiation by erythroleukemia
cells is accompanied by specific temporal
changes in cell cycle regulators
Treatment of MEL cells with inducers of differentiation, such as
hexamethylene bisacetimide (HMBA), causes the cells to become
irreversibly committed to reinitiate erythroid differentiation.12 By
48 hours most cells are committed and no longer require the
presence of the inducer to complete differentiation.13 As cells
differentiate they continue to undergo cell division and accumulate
hemoglobin (Figure 1A, percent of B⫹) and other red cell markers. By 96-120 hours nearly all cells have undergone terminal
cell division (Figure 1A) and enter permanent growth arrest in
phase G1/G0.20
Complementary DNAs (cDNAs) encoding human p18 cDNA, CDK2-HA,
CDK4R24C-HA, and CDK6R31C-HA (gifts of J. Koh, L. Zhu, D. Franklin,
and Y. Xiong, respectively) were cloned into pUHD 10-3 (pUHD 10-3 p27,
gift of R. Pestell).
Immunoblot and immunoprecipitation assays
Immunoblot assays were performed on 100 ␮g total protein extract as
previously described.18 Immunoblotting for hemoglobin was performed
similarly, except that sonication extracts were used and polyacrylamide gel
electrophoresis (PAGE) gels were run under nondenaturing conditions.
Immunoprecipitation assays were performed on 500 ␮g total protein extract
from cells lysed by sonication, as previously described.18,19
Antibodies
Immunoblot analysis was performed with the following antibodies: polyclonal human-specific (nonmouse cross-reactive) ␣-CDK2 (AB-1, PC44;
CalBiochem, Nottingham, England); polyclonal human ␣-CDK2, ␣-cyclin
D1 and D3, ␣-p15, ␣-p18, and ␣-p27 (all gifts of Y. Xiong); polyclonal
human ␣-CDK4 (C-22), ␣-CDK6 (C-21), ␣-cyclin D1 (R124), ␣-cyclin D1
(HD11), ␣-cyclin D2 (M-20), ␣-cyclin E (M-20), ␣-cyclin A (C-19), and
␣-p16 (M-156) (brand names in parentheses; all from Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal human ␣-p21 (65951A; PharMingen,
San Diego, CA); monoclonal ␣-hemagglutinin (HA) 12CA5 (Boehringer
Mannheim, Mannheim, Germany); and polyclonal ␣-mouse hemoglobin
(ICN-Koeppell). We also used HRP-conjugated antimouse immunoglobulin G (IgG), antirabbit IgG, and enhanced chemiluminescence (ECL)
(Amersham); CDK2 blocking peptide (BP) (gift of Y. Xiong) and CDK4 bp
(C-22) and CDK6 bp (C-21) (Santa Cruz Biotechnology); and for immunoprecipitation-kinase analysis, affinity-purified polyclonal human ␣-CDK2,
␣-CDK4, and ␣-CDK6 (gifts of Y. Xiong).
Immunoprecipitation-kinase assay
We immunoprecipitated 100 ␮g (for CDK2) or 500 ␮g (for CDK4 or
CDK6) of total cellular protein extract with either a mouse-specific
anti-CDK antibody (nonreactive with HA-tagged human CDKs) for
endogenous CDKs or with an anti-HA antibody for exogenous CDKs.
Kinase assays were performed as previously published18 with either histone
H1 (CDK2; Sigma Chemical Co., St Louis, MO) or a glutathione
Figure 1. Changes in CDK activities during erythroleukemia cell terminal
differentiation. Logarithmically growing MEL cells were induced to differentiate with
5 mmol/L HMBA as described in “Materials and methods.” (A) At the indicated times
the cell number (E) and the percentage of benzidine-positive hemoglobinized cells
(percent of B⫹, 䊐) were determined. (B) Total cellular protein extracts were prepared
and analyzed for specific CDK activities by immunoprecipitation (IP)-kinase assays,
and (C) CDK protein levels were determined by immunoblotting as described in
“Materials and methods.” BP indicates blocking peptide.
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
To ascertain the relationship between cell cycle regulatory
proteins and permanent cell cycle withdrawal, we assayed the
levels and activities of the 3 G1/S-phase CDKs (CDK2, CDK4, and
CDK6) throughout differentiation (Figure 1B,C). Both CDK6
activity and CDK6 protein levels are markedly reduced within
24-48 hours after initiating HMBA treatment. The early decline of
CDK6 occurs as cells are committing to differentiation and suggests that
the loss of CDK6 may be important for this first step in the reprogramming of the tumor cells (see “Discussion”). However, the late decline in
CDK2 and CDK4 activities, occurring as the cells undergo terminal cell
division, suggests that these 2 CDKs drive cell division in differentiating
cells and that loss of their activities is responsible for withdrawal of the
cells from the cell cycle. An earlier study21 indicated that CDK4 protein
levels decline rapidly after HMBA treatment. Although contrary to
expectation but in agreement with the data of Figure 1A, this study also
showed that CDK4 activity declined at later times as cells complete
differentiation and undergo cell cycle arrest. It is not clear from this work
how CDK4 protein levels could decline while enzyme activity is
maintained. CDK6 was not investigated in this earlier work.
Given the constant levels of the CDK2 and CDK4 proteins
(Figure 1C), the loss of CDK2 and CDK4 activities at 96-120 hours
could be due to either a decline in the levels of cyclins or an
induction of the CDK inhibitors. The cyclin D2, D3, E, and A levels
were observed to be mostly constant throughout differentiation
(Figure 2A), although cyclin D1 could not be detected in MEL cells
even with the use of several antisera. Interestingly, we observed
that in undifferentiated cells, CDK4 associates primarily with
cyclin D2, whereas CDK6 associates primarily with cyclin D3
(Figure 2B). The loss of CDK6–cyclin D3 complexes was observed
early in the differentiation program and probably reflects the rapid
decline in CDK6 levels induced by HMBA. On the other hand,
CDK4–cyclin D2 complexes dissociate much later, during terminal
cell division. This dissociation occurs despite the fact that CDK4
Figure 2. Levels of cyclins and CDK inhibitors and
their associations with CDKs during MEL cell differentiation. Total cellular protein extracts were prepared at the
indicated times of HMBA treatment, and the levels of (A)
specific cyclins and (C) CDK inhibitors were determined
by immunoblotting. Extracts were immunoprecipitated (IP)
with antibodies specific for the indicated (B) cyclin or (D)
CDK inhibitor as described in “Materials and methods.”
The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) and immunoblotted for the indicated CDK.
MANIPULATING THE ONSET OF CELL CYCLE WITHDRAWAL
2757
(Figure 1C) and cyclin D2 (Figure 2A) protein levels remain
constant during this period. Furthermore, we observed only a minor
reduction in CDK2–cyclin E/A complexes despite the complete
inhibition of CDK2 activity during this late period.
The foregoing observations suggest that inhibition of CDK2
and CDK4 activities is more likely due to induction of CDK
inhibitors. We observed that p15, p18, p21, and p27 levels increase
dramatically during the period of terminal cell division, with the
increase in p27 slightly preceding that of p15, p18, and p21 (Figure
2C). Undifferentiated MEL cells express p16 and p19; however,
their levels decline early in the differentiation program (data not
shown), suggesting that they are not involved in terminal cell
division. Using 2 different commercial antibodies, p57 was not
detected in MEL cells at any stage. We found that during the late
stages of differentiation, the induced p15 and p18 associate with
CDK4, whereas p21 associates primarily with CDK2 (Figure 2D).
However, the induced p27, which as noted above increases slightly
earlier than the other CDKIs, is found initially in complexes with
CDK4, and later, as cells cease dividing, it associates with CDK2.
Note that the earlier increase in p27 compared with the other
inhibitors is accentuated when assayed in complexes with CDK4.
These results suggest that during terminal cell division, p15 and
p18 block cyclin binding to CDK4, which results in the inhibition
of CDK4 activity, whereas p21 and p27 associate with and inhibit
dimeric CDK2–cyclin E/A complexes.
Premature inhibition of CDK2 and CDK4 in differentiating cells
causes early and permanent cell cycle withdrawal without
affecting the formation of hemoglobinized cells
The observed induction of several CDKIs, their association with
CDK2 and CDK4, and the concomitant inhibition of the 2 kinase
activities, all occurring as cells undergo terminal cell division,
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2758
MATUSHANSKY et al
suggest that these events may cause the cells to withdraw permanently from the cell cycle. To test this hypothesis we sought to
cause premature inhibition of CDK activities in differentiating cells
by generating stable MEL cell transfectants containing tetracyclinecontrolled expression vectors17 driving the synthesis of human p18
or p27. For each inhibitor, many transfectants were screened. Two
transfectant clones were chosen that (1) did not exhibit detectable
expression of the exogenous inhibitors in the absence of Dox and
(2) had Dox-induced levels of the exogenous inhibitors equivalent
to the levels of endogenous inhibitors present in fully differentiated
parental MEL cells (Figure 3A).
The biological activity of the transfected gene products was
ascertained by measuring their effect on endogenous kinase
activities and proliferation. Induction of p18 transfectants with Dox
caused a complete inhibition of the CDK4 activity (Figure 3B), but
Figure 3. Characterization of p18 and p27 MEL cell transfectants. (A) MEL cell
transfectant clones expressing either p18 or p27 under control of the tetracycline
inducible promoter in pUHD 10-3 were cultured either in the absence (⫺) or presence
(⫹) of 1 ␮g/mL Dox for 36 hours. Total cellular protein extracts were prepared, and the
levels of p18 (left panel) and p27 (right panel) were determined by immunoblotting.
The parental MEL cells (clone B1) containing only the rtTA regulator were cultured in
the presence of 5 mmol/L HMBA for 120 hours to indicate the levels of endogenous
p18 and p27 present in fully differentiated MEL cells. For further details see “Materials
and methods.” (B) The effect of exogenous p18 and p27 on CDK activities in
undifferentiated and differentiated MEL cell transfectants. Extracts prepared from the
indicated transfectant clones cultured in the presence of 5 mmol/L HMBA and either
in the absence (⫺) or presence (⫹) of Dox were prepared at the indicated times and
assayed for the indicated CDK activity by IP-kinase assays as described in “Materials
and methods.”
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
it was not accompanied by any effect on the doubling time or
accumulation of cells in G1 (Table 1). On the other hand, induction
of p27 transfectants with Dox led to a nearly complete inhibition of
the CDK2 and CDK4 activities (Figure 3B), an almost 4-fold
increase in cell generation time, and a dramatic accumulation of
cells in G1 (Table 1). Unexpectedly, induction of p18 or p27 did not
inhibit CDK6 activity (Figure 3B). Induction of either p18 or p27
did not alter the rate of HMBA-induced differentiation, as measured by the rate of accumulation of benzidine-stained hemoglobinized cells, nor did it cause spontaneous differentiation (data not
shown). We believe that induction of p18 and p27 failed to inhibit
CDK6, whereas it did lead to inhibition of CDK4 because CDK6 is
located in the nucleus of undifferentiated MEL cells, and CDK4 is
found in the cytoplasm (I.M., F.R., A.I.S., unpublished data, 2000).
Perhaps the levels of synthesis of the exogenous p18 and p27 are
not high enough to complex with both CDKs, resulting in
preferential association of the inhibitor with CDK4 located in the
cytoplasm.
The proliferation capacity of differentiating cells can be measured by plasma clot assays in which the size of colonies arising
from individual cells is determined after several days of growth in
the clot in the absence of the differentiation inducer. Cells that have
not committed to differentiate produce very large colonies consisting of hundreds to thousands of undifferentiated cells. On the other
hand, cells that have committed to differentiate by prior exposure to
a differentiation inducer, in this case HMBA, give rise to small
colonies consisting of fewer than 64 cells, all of which stain
positively for hemoglobin with benzidine. If cells are treated with
HMBA for 12 hours and then plated in plasma clots, the average
number of cells in a differentiated colony is 45 after 7 days of
incubation (Figure 4). Thus, once differentiation is triggered, the
differentiating cells usually undergo an average of 5-6 cell divisions before withdrawing permanently from the cell cycle.
To ascertain whether premature induction of p18 or p27 would
cause early cell cycle withdrawal in differentiating cells, we treated
transfectants for 12 hours with HMBA, which is sufficient time to
cause 20% of the cells to become committed to differentiation. We
then removed the HMBA and cultured the cells for 12 hours in the
presence or absence of Dox. Finally, Dox was removed, and the
cells were plated in plasma clots in the absence of both HMBA and
Dox and incubated for 4 days. Despite the fact that induction of
exogenous p18 by Dox in these transfectants caused a complete
inhibition of CDK4 activity in the cells (Figure 3B), it caused only
a moderate decrease in the size of differentiated (benzidinepositive) colonies (Figure 4). On the other hand, treatment of p27
transfectants with Dox caused a dramatic decrease in the size of
differentiated colonies, to an average size of 4 cells per colony
(Figure 4). Even though the colonies were reduced in size, all of the
cells in the colonies stained positive with benzidine and appeared to
have normal levels of hemoglobin.
To determine whether the smaller colonies produced by induction of exogenous p27 had indeed undergone permanent cell cycle
withdrawal or were merely retarded in their growth, we incubated
the plasma clots for an additional 3 days. We observed virtually no
increase in average colony size with further incubation (Figure 4,
white bars). The observed permanent cell cycle withdrawal in
Dox-treated p27 transfectants is in contrast to the behavior of the
parental line and p18 transfectants, in which increased incubation
time in the clots resulted in an increase in colony size (Figure 4,
white bars). These results indicate that induction of p27 in
differentiating cells causes them to withdraw prematurely and
permanently from the cell cycle. This is in contrast to the effect of
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
MANIPULATING THE ONSET OF CELL CYCLE WITHDRAWAL
Table 1. Effect of induction of exogenous p18 and p27 on growth
and cell cycle profiles of MEL cell transfectants
Clone
Dox
T(g), h
Phase
G0-G1, %
Phase S, %
Phase
G2-M, %
6.9
⫺
12.2
31.6
61.4
⫹
12.1
32.3
58.7
9.0
p18.36
⫺
12.2
29.6
58.4
12.0
⫹
11.8
30.3
57.9
11.8
p27.16
⫺
12.1
28.3
52.0
19.7
⫹
42.1
84.9
8.0
7.0
⫺
11.9
27.6
55.4
17.0
⫹
40.2
87.8
6.7
5.5
⫺
11.9
28.8
53.4
17.6
⫹
11.8
29.3
50.4
19.6
p18.5
p27.77
MEL rtTA;
B1
The indicated MEL cell transfectant clones and parental MEL cells (clone B1)
containing only the rtTA regulator were cultured in the absence (⫺) or presence (⫹) of
1 ␮g/mL Dox for 14 days. The cells were subcultured to 2 ⫻ 105 cells per mL each day
by centrifugation and resuspension of the appropriate fraction of cells in fresh
medium. The cell densities were measured daily with a Coulter counter, and the
average generation time, T(g), of each culture was calculated. After 36 hours of
culture, approximately 1⫻106 cells were removed from each culture, centrifuged, and
suspended in 1 mL propidium iodide/sodium citrate buffer. The percentage of cells in
each stage of the cell cycle was then determined by fluorescence-activated cell sorter
(FACS) analysis.
exogenous p27 in undifferentiated MEL cells, in which it causes an
accumulation of cells in G1 (Table 1) that is fully reversible upon
removing Dox (data not shown). Because p27 inhibits both CDK2
and CDK4 activities in differentiating cells, whereas p18 inhibits
only CDK4, the results imply that inhibition of both kinases is
required for cells to undergo terminal cell division. Finally, because
16% of the Dox-treated differentiated colonies in p27 transfectants
consisted of single cells which stained positive for hemoglobin, we
believe that once differentiation is triggered, the cells need not
proliferate in order to differentiate.
2759
the 2 exogenous HA-tagged CDKs, and expression of these
proteins could be maintained throughout differentiation (Figure 5C).
To directly assess the activities of exogenous CDKs and to
compare their activities in the transfectants to that of the endogenous CDKs, we performed in vitro kinase assays for each specific
CDK using extracts from the transfected cells. The specificity of
the immunoprecipitation procedures used for the respective exogenous and endogenous CDKs was demonstrated by several results.
First, CDK activity was not recovered in anti-HA immunoprecipitates of extracts from untransfected MEL cells (Figure 6, lanes 11
and 12 in each panel). Nor was CDK activity recovered in each
anti-CDK immunoprecipitate of transfectants that were treated
with HMBA plus Dox. This occurred because HMBA treatment
leads to the loss of all endogenous CDK activities (Figure 6, lanes 5
and 6 in each panel). However, abundant Dox-induced CDK
activities were recovered from the same cell extracts immunoprecipitated with anti-HA antibody (Figure 6, lane 8 in each panel).
Furthermore, the activity of each exogenous kinase in the transfectants was strongly induced by Dox (Figure 6, compare lanes 3 and 4
in each panel) to levels that are comparable to the endogenous
CDK levels (Figure 6, compare lanes 4 and 1 or 2 in each panel).
Moreover, whereas all endogenous CDK activities are lost during
HMBA-induced differentiation (Figure 6, lanes 5 and 6 in each
panel), when HMBA-treated transfected cells are also treated with
Dox, which induces the transfected CDK genes, the respective
CDK activities are maintained in the treated cells at levels
comparable to the activities present in undifferentiated cells
(Figure 6, compare lanes 1 or 2 and lane 8 in each panel). The
generation time of the CDK transfectants was unaffected by
induction of the exogenous CDKs with Dox, suggesting that CDK
activity is present in excess in the undifferentiated cells.
To determine whether restoration of CDK2 or CDK4 activity in
differentiated cells prevents their withdrawal from the cell cycle,
we measured the size of the colonies arising from differentiated
Synergistic action of CDK2 and CDK4 delays terminal
cell division and causes extensive proliferation of
hemoglobinized cells
An alternative approach for examining the requirement for inhibition of CDK2 and CDK4 activities in terminal cell division is (1) to
introduce expression vectors encoding these CDKs into MEL cells
and (2) to determine whether inducing their synthesis during the
period when the activities of the endogenous kinases decline
promotes extended proliferation of differentiated cells. We initially
generated MEL cell transfectants expressing a tetracyclinecontrolled human CDK4 fusion protein containing a C-terminal
HA tag. However, while these transfectants express readily detectable exogenous CDK4 kinase activity in undifferentiated cells, the
activity of the exogenous CDK4, like that of endogenous CDK4,
was inhibited during differentiation (data not shown), presumably
due to the rise of the endogenous CDKIs during differentiation. To
avoid inhibition of the exogenous CDK4 by the endogenous CDK
inhibitors, we used a mutant form of CDK4 (R24C)22 that is
resistant to inhibition by the INK4 family of CDKIs. We generated
MEL cell lines expressing a tetracycline-controlled human
CDK4R24C fusion protein containing a C-terminal HA tag. We
also generated lines expressing a tetracycline-controlled HAtagged human CDK2 and lines expressing both proteins by
supertransfecting a CDK4R24C transfectant (clone 24) with the
CDK2 expression construct. Two clones of each type that showed
highly inducible expression were chosen for further analysis
(Figure 5A). These clones exhibited comparable protein levels of
Figure 4. Effect of p18 and p27 on proliferation of differentiated cells. We
cultured p18 and p27 MEL cell transfectants in the presence of 5 mmol/L HMBA for 12
hours. HMBA was washed out, and the transfectants were cultured in the presence of
1 ␮g/mL Dox for 12 hours. The cells were plated in plasma clots and incubated for 4
days (solid bars) and 7 days (open bars) at 37°C in the absence of both HMBA and
Dox. The clots were then stained with benzidine and hematoxylin as described in
“Materials and methods.” The proliferative capacity of cells that were committed to
differentiation was determined by counting the number of cells in at least 100 colonies
that stained positive with benzidine.
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MATUSHANSKY et al
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
Figure 5. Inducible expression of exogenous CDK2,
CDK4R24C, and CDK6R31C in MEL cell transfectants. (A, B)
The extracts were prepared from the indicated CDK-HA MEL cell
transfectants cultured in the absence (⫺) or presence (⫹) of Dox
for 36 hours. The levels of exogenous HA-tagged CDKs were
determined by immunoblotting with anti-HA–specific antibody.
(C, D) Extracts were prepared from the indicated CDK-HA MEL
cell transfectants that had been cultured in the presence of Dox
for 24 hours (0 time in the figure) followed by treatment with 5
mmol/L HMBA for the indicated times. The levels of exogenous
HA-tagged CDKs were determined by immunoblotting as in
panels A and B. The upper band in the 2 lower sections of panel C
is CDK4R24C-HA, and the lower band is CDK2-HA. The upper
band in the 2 lower sections of panel D is CDK6R31C-HA, and the
lower band is CDK2-HA.
Figure 6. Comparison of exogenous and endogenous kinase activities in undifferentiated and differentiated MEL cell CDK transfectants. The indicated
MEL cell transfectants were cultured in the absence (0)
or presence (120) of HMBA for 120 hours and then further
cultured in the absence (⫺) or presence (⫹) of Dox for 36
hours. Cell extracts were prepared and immunoprecipitated
with antibodies specific for the endogenous murine CDKs
(K2, K4, and K6) or the exogenous human HA-tagged CDKs
(HA or HuK2). In doubly transfected cells (panels B and C)
assays of exogenous kinase activities were performed by
first immunoprecipitating with an antibody specific for the
exogenous human CDK2 (HuK2), and after checking that
the exogenous CDK2 was completely removed, the supernatant was immunoprecipitated with an anti-HA antibody specific for the exogenous HA-tagged (B) CDK4R24C or (C)
CDK6R31C. The immunoprecipitates were incubated with
␥-32P–ATP (␥-phosphorous 32–adenosine 5⬘-triphosphate)
in the presence of either H1 or the GST-Rb C-terminal
fragment, and the reactions were resolved by SDS-PAGE.
For further details see “Materials and methods.”
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
transfected cells by plasma clot assays. Transfectants expressing
either CDK2 or CDK4R24C or both proteins, were treated for 48
hours in liquid culture with HMBA and then plated in plasma clots
in the presence or absence of Dox. After 48 hours of HMBA
treatment, the average size of benzidine-positive hemoglobinized
colonies produced from the parental MEL rtTA line (as well as
MEL cells) is approximately 4 cells (Figure 7A,B). The small size
of these colonies is due to the differentiated cells undergoing most
of their terminal cell divisions in liquid culture, prior to plating in
plasma clots. The differentiated colonies produced from each type
of transfectant were similarly small in size when the cells were
plated in plasma clots lacking Dox. However, in clots containing
Dox, transfectants CDK2 and CDK4R24C produced differentiated
colonies that were about twice as large, indicating that either CDK
has the capacity to cause a modest increase in the number of cell
divisions that can be undertaken by differentiating cells.
Much more potent, however, are the combined activities of
CDK2 and CDK4R24C. Remarkably, the double transfectants
showed a very large increase in the size of differentiated colonies,
which grew to contain on average about 50 cells (Figure 7A,B).
This effect was strictly dependent on the presence of Dox in the
plasma clots. To determine whether the observed Dox-dependent
expansion of differentiated cells produced from the double transfectants represents a limit, we incubated the clots for an additional 4
days. The differentiated colonies became very large, too large to
effectively count the number of cells in each colony (Figure 7A,B).
Note that within these large colonies produced at 3 days and 7 days
in the clot, most cells in the colony contain hemoglobin, as judged
by benzidine staining (Figure 7B).
Because it was not possible to accurately estimate the extent of
proliferation in plasma clots of Dox-treated differentiated double
transfectants, we measured their growth and differentiation in
liquid cultures containing HMBA and Dox. Figure 7C shows the
cumulative cell densities of the cultures. In the absence of Dox
Figure 7. Synergistic activities of CDK2 and CDK4 in
causing extensive proliferation of fully differentiated
cells. (A) The indicated MEL cell CDK transfectants were
cultured in the presence of 5 mmol/L HMBA for 48 hours and
then plated in plasma clots either in the absence (⫺) or
presence (⫹) of Dox. The plasma clots were analyzed as
described in the legend of Figure 4 after 3 days (■) and
7 days (䊐) of incubation at 37°C. (B) Plasma clots of a doubly
transfected cell line were photographed (original magnification ⫻ 10) after staining with benzidine and hematoxylin as
described in “Materials and methods.” (C) Logarithmically
growing CDK(2 ⫹ 4R24C).32 and CDK(2 ⫹ 4R24C).40 were
treated at 1 ⫻ 105 cells per mL with 5 mmol/L HMBA in the
presence (shaded symbols) or absence (open symbols) of
Dox. The cell densities were maintained at less than 1 ⫻ 106
cells per mL by subculturing to 2 ⫻ 105 cells per mL with the
indicated growth medium every 24 hours as required. The cell
densities were measured daily with a Coulter counter (Coulter
Electronics, Miami, FL), and the cumulative cell densities
were calculated. (D) Total cellular extracts were prepared at
the indicated times from cells treated as described in panel C.
The extracts were analyzed by nondenaturing PAGE and
immunoblotting with an antibody specific for mouse hemoglobin. The lower section shows hemoglobin levels in the rtTA
MEL cell parental line after 5 days of treatment with HMBA, with
and without Dox.
MANIPULATING THE ONSET OF CELL CYCLE WITHDRAWAL
2761
these cells, similar to HMBA-treated MEL cells, grew exponentially, with a doubling time of 11.8 hours for about 2-3 days and
then gradually ceased dividing as cells became terminally differentiated. The final cumulative cell density of these cultures was about
1.2 ⫻ 106 cells per mL. However, when the double transfectants
were treated with both HMBA and Dox, the cells grew logarithmically for up to 7-8 days. The doubling time of these cultures was
14.3 hours, which was very similar to the time in the early phase of
growth in HMBA-treated parental MEL cells, prior to their
undergoing differentiation. The final cumulative cell density of
these cultures was about 1.8 ⫻ 108 cells per mL, at least 100 times
higher than that observed in the absence of Dox. These results
indicate that the maintenance of both CDK activities in differentiating cells allows the cells to undergo at least 6-7 additional cell
divisions while still maintaining their differentiated phenotype, as
judged by the fact that 100% of the cells stained positively with
benzidine (Figure 7C, bottom). Moreover, cell extracts prepared
from these cells contained as much hemoglobin as fully differentiated MEL cells (Figure 7D). We conclude that restoration of CDK2
and CDK4 activities in differentiated cells prevents their withdrawal from the cell cycle and allows them to proliferate extensively. Nevertheless, ultimately even these cells cease dividing
(Figure 7C and “Discussion”).
CDK6 cannot substitute for CDK4 in differentiating cells
To date functional differences have not been established for the 2
cyclin D–dependent kinases,23 and it was of interest to determine
whether or not CDK6 could, like CDK4, synergize with CDK2 to
promote extended proliferation in differentiating cells. To test this
possibility we prepared MEL cell transfectants expressing Doxinducible HA-tagged CDK6. Using a similar rationale to that used
for the CDK4R24C transfections, we used a newly constructed
mutant form of CDK6, designed and characterized by D. Franklin
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2762
MATUSHANSKY et al
and Y. Xiong (University of North Carolina, Chapel Hill), which
does not bind and is resistant to inhibition by the INK4 inhibitors.24
Both single and double (with CDK2) transfectants of CDK6R31C
were prepared and analyzed as described above (Figure 5B,D).
Both types of transfectants exhibited levels of exogenous CDK6
activity comparable to that of endogenous CDK6 activity (Figure
6A,C, compare lanes 4 and 1 or 2 in right panels) as well as to the
exogenous CDK4 activity in undifferentiated and differentiated
transfectants described in the previous section (Figure 6A,C, lane
8, right panels).
Despite the evidence indicating that both single and double
CDK6R31C transfectants had exogenous kinase activities comparable to that of the corresponding single and double CDK4R24C
transfectants, we did not observe any effect of CDK6R31C on
proliferation of differentiating cells, as measured by the size of
differentiated colonies (Figure 7A). The ability of CDK4R24C to
synergize with CDK2 to produce extremely large differentiated
colonies in plasma clot represents a very sensitive assay for the
ability of CDK4, along with CDK2, to promote cell division in
differentiating cells. We found that the size of differentiated
colonies produced by CDK2 and CDK6R31C doubles transfectants
in the presence of Dox was not increased at all above that seen in
the clones transfected with CDK2 alone. We conclude that CDK6
cannot substitute for CDK4 in promoting cell division in differentiating cells.
Discussion
Mechanism of permanent cell cycle withdrawal
The results reported here show that in differentiating MEL cells,
terminal cell division is brought about by induction of 4 CDK
inhibitors, their association with CDK2 and CDK4, and the
resulting inhibition of the activities of the 2 kinases. These studies
provide the most direct support for the view that these changes,
which have been observed in several other in vitro differentiation
systems,25-28 constitute the “clock” that determines the number of
cell divisions which can be undertaken by a differentiating cell. We
also suggest that induction of the CDK inhibitors is essential for
making cell cycle withdrawal irreversible.29 However, we believe
that other events preprogrammed to occur after induction of the
CDKIs may also be needed to ensure that the cell cycle exit is
irreversible. Although maintenance of CDK2 and CDK4 activities
could cause greatly extended proliferation of differentiating cells,
they did not proliferate indefinitely. We think that differentiating
MEL cells may become refractory to induction of exogenous CDKs
due to a decline in cyclin levels at much later stages of differentiation30; reduction in cyclin levels has also been seen in other
differentiating systems.31,32 The combination of induction of CDK
inhibitors and a decline in cyclin levels may serve to ensure that
cell cycle withdrawal is permanent.
CDK4 and CDK6 play different roles at different
stages of differentiation
Although differences in the regulation of CDK4 and CDK6
synthesis have sometimes been observed,33-35 to date functional
differences between the 2 cyclin D kinases have not been reported.36,37 The results reported here show that inhibition of CDK4
in differentiating cells inhibits their proliferation, and conversely,
maintenance of CDK4 activity extends their proliferation. On the
other hand, we found that while both CDK4 and CDK6 are active
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
in undifferentiated MEL cells, only inhibition of CDK6 slows their
proliferation (I.M. et al, unpublished data, 2000). These observations suggested to us that CDK6 in conjunction with CDK2
promotes proliferation in the undifferentiated cells, whereas CDK4
along with CDK2 promotes proliferation in the differentiating
cells. The ability of CDK4R24C to synergize with CDK2 to cause
the differentiating cells to undergo many more cell divisions than
usual provided us with the opportunity to assess possible functional
differences between CDK4 and CDK6. We found that CDK6R31C
could not substitute for CDK4R24C in synergizing with CDK2 to
extend proliferation of the differentiated cells. We are certain that
the CDK6R31C expression vector used in the current work
produces a functionally active form of CDK6 because we were able
to measure its activity in transfectants undergoing differentiation
(Figure 6). Furthermore, we have found that when CDK6R31C
transfectants are treated with Dox before initiating differentiation,
they are blocked from differentiating (I.M. et al, unpublished data,
2000). This effect was not observed in CDK4R24C transfectants.
These results indicate that the early decline in CDK6 plays an
important role in the cell’s decision to differentiate and further
strengthens the view that CDK6 and CDK4 play different roles at
different stages of differentiation.
How might such a restriction in biological activity of 2
otherwise very similar enzymes arise? Although hypophosphorylated forms of Rb accumulate in differentiating MEL cells,38 most
E2F is found associated with p107 in these cells.39,40 Similar
observations have been made in other differentiating cell types.41-43
Thus, although Rb may be a key player in controlling proliferation
in growing cells (eg, undifferentiated MEL cells), p107 or possibly
p130 may assume this role during terminal differentiation.44-46
Preferential substrate specificity of CDK4 and CDK6 among the
Rb-related proteins could explain their different biological activities in differentiating vs undifferentiated MEL cells, respectively.
Alternatively, currently unrecognized substrates of the 2 kinases
may be involved. The system and transfected cell lines described
here should help further our understanding in this area.
Phenotypic differentiation and terminal cell division:
2 independent aspects of cell differentiation
The prevailing view of the relationship of cell division to other
aspects of cell differentiation is that there is an intimate connection.47,48 In principle, the control of cell division and control of cell
differentiation could be coupled at either the time cells make the
decision to differentiate (commitment) or as cells actually execute
their phenotypic differentiation program, or both. Studies in mice
lacking 1 or more specific CDK inhibitors and in cells derived from
such mice have indicated that loss of these inhibitors that normally
promote cell cycle exit does not affect the capacity of cells to
differentiate.49-51 Furthermore, continued proliferation of certain
highly differentiated cell types was observed in such mice indicating that phenotypic differentiation and terminal cell division can be
uncoupled.52-54 However, such studies in live animals do not
address whether the observed additional cell divisions are due to
cell intrinsic vs. cell extrinsic controls, the precise stage of
differentiation at which the additional cell divisions occur, the
exact number of such extra cell divisions, and whether or not the
cellular concentration of gene products characteristic of the differentiated cells is affected. Furthermore, loss of inhibitors may have
effects within the cell beyond liberation of CDK activity.
The temporally well-defined in vitro differentiation process
provided by the MEL cell system permitted us to overcome most of
these limitations. The results obtained with the CDK and CDKI
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
MANIPULATING THE ONSET OF CELL CYCLE WITHDRAWAL
transfectants imply that differentiating erythroid cells do not need
to proliferate in order to differentiate and that differentiating cells
can proliferate further without affecting the synthesis of gene
products that are specific to the mature cells. Thus, our results
imply that there need not be a tight coupling between phenotypic
differentiation and terminal cell division in differentiating erythroid cells. Recent studies in p27-deficient mice suggested that
loss of cell cycle exit controls within the hematopoietic compartment leads to additional cell divisions in progenitors but not in
more highly differentiated cells.51 Differences between these
results and those reported here may be due to homeostatic
mechanisms present in vivo or to compensation by other CDK
inhibitors still present in the p27 deficient mice. Alternatively, it
may be that differentiating erythroleukemia cells, although similar
to normal differentiating erythroblasts, are especially sensitive to
abrogation of terminal cell division by exogenous CDK2 plus
CDK4. Therefore, it will be important to determine whether
2763
proliferation of normal erythroblasts can be extended by maintenance of CDK2 and CDK4 activities in these cells. Successful
manipulation of the terminal cell divisions in normal, differentiating cells could have important implications for cell transplantation therapies.
Acknowledgments
We are grateful to David Franklin and Yue Xiong for providing us
with constructs, antibodies, technical support, and advice that were
essential to the completion of these experiments. We also thank
Richard Pestell and Liang Zhu for providing us with critical
reagents and advice. We also thank Matthew Scharff, Liang Zhu,
Richard Pestell, Stuart Murray, and Natasha Rekhtman for critical
reading of the manuscript.
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2000 96: 2755-2764
Manipulating the onset of cell cycle withdrawal in differentiated
erythroid cells with cyclin-dependent kinases and inhibitors
Igor Matushansky, Farshid Radparvar and Arthur I. Skoultchi
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