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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 475-482
HEMATOPOIESIS
From the Fels Institute for Cancer Research and Molecular
Biology and Department of Biochemistry, Temple University School
of Medicine; and Cell and Molecular Biology Graduate Group, University
of Pennsylvania, Philadelphia, PA.
The transcription factor E2F-1 has been postulated to play a crucial
role in the control of cell cycle progression because of its ability to
be bound and regulated by the retinoblastoma gene product (pRb).
Exogenous expression of E2F-1, under growth restrictive conditions, was
shown to result in p53-dependent programmed cell death. The
consequences of deregulated expression of E2F-1 on terminal
differentiation of hematopoietic cells in the absence of
E2F-1-mediated apoptosis, as well as mechanistic insights into how
deregulated E2F-1 may affect terminal differentiation, have not been
established. The autonomously proliferating M1 myeloblastic leukemia
cell line, which is null for p53 expression and can be induced by
interleukin-6 (IL-6) to undergo terminal macrophage differentiation
with concomitant loss of leukemogenicity, provides a particularly
attractive model system to address these issues. Deregulated and
continued expression of E2F-1 blocked the IL-6-induced terminal
differentiation program at an early blast stage, giving rise to
immature cells, which continued to proliferate without undergoing
apoptosis and retained their leukemogenic phenotype. Although E2F-1
blocked IL-6-mediated terminal differentiation and its associated
growth arrest, it did not prevent the rapid induction of both
p15INK4B and p16INK4A, inhibition of cdk4
kinase activity, and subsequent hypophosphorylation of pRb. The results
obtained imply that genetic alterations that both impair p53 function
and deregulate E2F-1 expression may render hematopoietic cells
refractory to the induction of differentiation and are, thereby, likely
to play a major role in the progression of leukemias.
(Blood. 2000;96:475-482)
In recent years, transcription factors of the E2F
family have emerged as key regulators of cell cycle progression, which
function by activating the transcription of genes necessary for S-phase entry and progression.1-7 The functional E2F transcription
factor is a heterodimer composed of 1 E2F and 1 DP
subunit.8-14 The transcriptional activity of E2F factors is
negatively regulated by their physical association with products of the
retinoblastoma gene family (pRb, p107, p130) in the G0/G1
phases of the cell cycle.15-21 The ability of Rb
family members to sequester and repress the activity of E2F factors is,
in turn, regulated by cyclin/cdk kinases, including cyclin D/cdk4 and
cyclin E/cdk2. Phosphorylation of pRb by these kinases leads to the
dissociation of E2F from the inhibitory pRb-E2F complex, which results
in transcriptionally active E2F.22-25 The activity of the
cyclin/cdk complexes is itself negatively regulated by specific
cyclin-dependent kinase inhibitors such as p15INK4B and
p16INK4A.26-28
Among the different E2F factors that have been characterized, ample
evidence has accumulated that E2F-1 plays a central role in G1 to
S-phase cell cycle progression.4,29-33 Deregulated
expression of E2F-1 under growth restrictive conditions was observed to
result in programmed cell death.30,31,34-36 Using an in
vivo transgenic mouse model, deregulated expression of E2F-1 from a
megakaryocyte-specific promoter was found to impair maturation of
megakaryocytes and to increase their apoptosis, leading to reduced
platelet formation.37 In addition, deregulated E2F-1, in
conjunction with ectopic expression of Bcl-2 to delay
apoptosis, was observed to prevent granulocytic differentiation of
IL-3-dependent 32Dcl3 hematopoietic progenitor cells, after IL-3
deprivation and treatment with granulocyte colony-stimulating factor
(G-CSF).38 However, the consequence of deregulated
expression of E2F-1 on terminal differentiation in the sustained
absence of E2F-1-induced apoptosis has not been established. In this
study, we have addressed this issue using M1 cells in which the
deregulated expression of E2F-1 does not result in cell death and
therefore does not require ectopic Bcl-2. This allows for an
examination of E2F-1 function in the absence of any possible
contribution from the Bcl-2 transgene.
Autonomously proliferating M1 myeloblastic leukemia cells, which are
null for p53 expression, can be induced by interleukin-6 (IL-6) to
undergo terminal macrophage differentiation and concomitant loss of
leukemogenicity.39,40 Thus, these cells provide a
particularly attractive model system to gain insights into how
deregulated expression of E2F-1 may affect the terminal differentiation
program and loss of the leukemic phenotype, in the absence of
apoptosis. Toward this end, expression of endogenous E2F-1 in M1 cells
induced for terminal differentiation has been analyzed, and M1E2F-1
cell lines, expressing an E2F-1 transgene, have been generated and subjected to IL-6 treatment.
It is shown that, in contrast to the parental cells in which treatment
with IL-6 resulted in suppression of endogenous E2F-1 and
induction of terminal differentiation, deregulated, and continued expression of ectopic E2F-1 blocked the M1 terminal differentiation program at an early blast stage. In the presence of IL-6, the immature
cells continued to proliferate, without undergoing apoptosis or losing
their leukemic phenotype. Furthermore, it is shown that although E2F-1
blocked IL-6-mediated terminal differentiation and prevented growth
arrest, it did not prevent the rapid induction of both
p15INK4B and p16INK4A, inhibition
of cdk4 kinase activity, and subsequent pRb hypophosphorylation. These data suggest that E2F-1 may block terminal differentiation by overriding the p15/p16-Rb-E2F checkpoint in the G1 phase of the cell cycle.
Cells, cell culture, cytokines, and mice
Assays for differentiation-associated properties
RNA extraction, Northern blotting, and hybridization Total RNA was prepared from 3 to 5 × 106 cells using TRIzol reagent (Gibco BRL) as described in the manufacturer's specifications. RNA gel electrophoresis, Northern blotting, hybridization, and stripping of blots were performed as previously described.42-44,48-49 Equal loading of RNA in each lane was confirmed both by visualizing equal intensity of ethidium bromide staining of ribosomal RNA bands and by hybridization of the Northern blots with radiolabeled -actin probe.
Protein extraction and immunoblotting (Western blots) Extracts of total proteins were prepared by resuspending cell pellets at a final concentration of 107 per milliliter in lysis buffer (2 × phosphate buffered saline [PBS], 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mmol/L EDTA,10 µg/mL leupeptin, 10 µg/mL chymostatin, 100 µg/mL phenyl-methylsulfonyl fluoride [PMSF]). After incubation for 15 minutes on ice, DNAse I (50 µg/mL) was added and samples were kept on ice for an additional 30 minutes. Four volumes of cold acetone were added and samples were placed at 20°C. The protein precipitate was pelleted and dissolved in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Total protein concentration was determined using the micro-BCA protein quantitation kit (Pierce, Rockford, IL). Fifty micrograms of each protein extract sample was
fractionated on SDS-PAGE gels. Resolved proteins were transferred to
Immobilon-P membrane (Millipore, Bedford, MA) using a Trans-blot apparatus (Bio-Rad, Hercules, CA) at 50 V for 18 hours. Equal loading
of protein in each lane was verified by staining the Western blots with
0.1% Ponceau S solution (Sigma, St Louis, MO) before incubation with
antibody. Blots were probed with the appropriate primary antibody for 2 hours at room temperature in PBS (pH 7.2), 0.5% bovine serum albumin
(BSA), and 0.5% Tween-20. After rinsing in PBS containing 0.5%
Tween-20, the blots were incubated for 30 minutes at room temperature
in a solution containing secondary antibody conjugated to horseradish
peroxidase (Amersham, Arlington Heights, IL). Signals were developed
using the enhanced chemiluminescence (ECL) Western blotting system
(Amersham). Primary antibody against the retinoblastoma gene product
was obtained from Pharmingen, San Diego, CA (clone G3-245, cat no.
14001A). Antibodies against murine actin (cat no. sc-1616), E2F-1(cat
no. sc-193), cdk-2 (cat no. sc-163), cdk-4 (cat no. sc-260), cyclin E
(cat no. sc-481, p15 (cat no. sc-1429), p16 (cat no. sc-1661), p21 (cat
no. sc-6246), and p27 (cat no. sc-1641), were all from Santa Cruz
Biotechnology (Santa Cruz, CA).
General recombinant DNA techniques, expression vectors, and DNA probes Plasmid preparations, restriction enzyme digestions, DNA fragment preparations, and agarose gel electrophoresis were performed as previously described.45,46 The retroviral plasmid expression vector, MSCV-puromycin, was a gift from Dr Robert G. Hawley.47 To construct the vector, MSCV-puro-E2F1, purified EcoR1 fragment of full-length murine E2F-1 was cloned into the EcoR1 site of the 6.3-kilobase (kb) MSCV-puromycin plasmid. The source of the 2.0-kb murine E2F-1 complementary DNA (cDNA) was the pBS-E2F1 plasmid (P. Farnham, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI).
Establishment of M1E2F-1 cell lines that ectopically express an E2F1 transgene Virus was generated from the plasmid forms of the retroviral vectors, MSCV-puro (as a control) and MSCV-puro-E2F1 and M1 cells were infected as previously descsribed.51 For puromycin-resistant colony selection, infected cells were seeded at 100 cells/mL in growth medium (DMEM + 10% horse serum), containing puromycin at 4 µg/mL and 1 mL aliquots, were dispensed into 24-well trays. After 10 to 15 days, cultures containing surviving cells were expanded. The infectants were continuously maintained in growth media containing 4 µg/mL of puromycin. Several independent M1E2F-1 clones were isolated and characterized as to the level of expression of their transgene. At least 5 independent clones were examined for their response to IL-6; in each case, the results were similar. Moreover, the response of 5 M1/MSCV-puro clones (empty vector controls) to IL-6 was indistinguishable from that of the parental M1 cells.Transient transfection and luciferase assay M1 and M1E2F-1 cells were seeded at a density of 0.5 × 106 cells/mL 1 day before transfection, and 24 hours later, were transfected with the appropriate plasmids using the DEAE/dextran method as described elsewhere.52 Briefly, equimolar ratios of either wt-E2F-Luc or mut-E2F-Luc (luciferase reporter plasmids containing 3 tandem copies of wild-type or mutant E2F-1 binding sites, respectively11; a kind gift of D. M. Livingston, Harvard University, Boston MA) plasmids together with a-gal control vector (pMLV--gal; used to determine transfection
efficiency) were transfected together. pMLV--gal was constructed by
R. T. T. Sjin in our laboratory. The total amount of DNA was adjusted
to 20 µg with pBS-KS plasmid (Stratagene, La Jolla, CA). The cells
were then incubated in the absence or presence of 50 ng/mL IL-6 and
were harvested at 1-day intervals (day 0 to day 3) by centrifugation.
After being washed with PBS, they were lysed with 120 µL reporter
lysis buffer (Promega, Madison, WI). Forty microliters of cell extract
was used to quantitate -gal expression to enable normalizing for
transfection efficiency. Lysate amounts were then adjusted for equal
levels of -gal activity and luciferase activity was measured in a
Lumat LB 9501 luminometer.
Flow cytometric analysis Cells were harvested at different time points by centrifugation, and washed 3 times in PBS containing 1% fetal bovine serum (FBS). Cells were fixed in 70% ice-cold ethanol, spun down, and washed once with PBS/1% FBS, and then treated for 30 minutes with RNase A (180 µg/mL RNase A in PBS). The cells were subsequently stained with propidium iodide (100 µg/mL in 7.6 mmol/L sodium citrate; Sigma) and analyzed using a Coulter Epics Elite system (Coulter, Miami, FL). Cell cycle analysis was performed at least 3 times with similar results.Immunoprecipitation and in vitro kinase assays For cdk2 kinase assay, 2 × 107 cells were lysed in 1 mL of lysis buffer (50 mmol/L Tris-Cl pH 7.4, 5 mmol/L EDTA, 250 mmol/L NaCl, 50 mmol/L NaF, 0.1% Triton X-100, 0.1 mmol/L sodium vanadate), supplemented with the protease inhibitors 1 mmol/L PMSF and 10 µg/mL leupeptin. Clarified supernatants were collected and the protein concentration was determined by using the micro-BCA protein quantitation kit (Pierce). One hundred micrograms of total protein was incubated with 10 µg of anti-cdk-2 antibody (cat no. 163; Santa Cruz Biotech Inc, Santa Cruz, CA) for 2 hours at 4°C. Thirty microliters of Protein A-Sepharose (cat no. 20334; Pierce) was added to each sample and incubated at 4°C for 1 hour. After centrifugation, the samples were washed extensively in lysis buffer, followed by 2 washes in kinase buffer (20 mmol/L HEPES pH 7.4, 10 mmol/L Mg acetate). The pellets were then incubated in 30 µL of kinase reaction buffer (kinase buffer containing 0.5 µg histone H1, 20 mmol/L cold ATP, and 0.185 MBq [5 µCi]] 32P-gamma-ATP [11 100 × 1010 Bq (3000 Ci/mmol) DuPont, Wilmington, DE]) for 30 minutes at 30°C. The reaction was stopped by the addition of Laemmli's buffer, boiled 5 minutes, and the kinase products were resolved by 10% SDS-PAGE, dried, and exposed for autoradiography.
Analysis of endogenous E2F-1 expression in M1 cells and establishment of M1E2F-1 cell lines As a first step toward understanding the contribution of E2F-1 to myeloid cell growth and differentiation, we analyzed the expression of E2F-1 transcripts in M1 leukemic cells in the absence or presence of IL-6. Northern blot analysis (Figure 1A) revealed that the relatively high level of E2F-1 messenger RNA (mRNA) present in proliferating cells was down-regulated in response to growth arrest and differentiation induced by IL-6. Moreover, the down-regulation of the E2F-1 message was mirrored by both a down-regulation of the E2F-1 protein (Figure 1B) and a loss of E2F-1 transcriptional activity (Figure 1C).
Effect of deregulated expression of E2F-1 on terminal differentiation, growth arrest and apoptosis of myeloid cells IL-6 treatment of M1 cells leads to growth arrest within 24 hours, associated with induction of terminal differentiation. Analysis of the growth kinetics of IL-6-treated M1E2F-1 cells showed that they continued to proliferate at the same rate as untreated M1E2F-1 and parental M1 cells (Figure 2A). These data were corroborated by cytofluorometric analysis (Figure 2B), which indicated that IL-6-treated M1E2F-1 cells, unlike similarly treated M1 cells, did not undergo growth arrest in the G0/G1 phase of the cell cycle. Furthermore, in agreement with the unaltered growth kinetics of IL-6-treated M1E2F-1 cells, there was no evidence for apoptosis, determined by cytofluorometric analysis (Figure 2B), and absence of both apoptotic morphology and genomic DNA ladders (data not shown). Taken together, these results indicate that the deregulated expression of E2F-1 prevents the growth inhibition induced by IL-6, without the associated cell death reported for other cell types.30,31,34,35,53-56
Deregulated E2F-1 and the p15/p16-Rb-E2F checkpoint
Effect of deregulated E2F-1 on IL-6-mediated suppression of M1
leukemogenicity
To investigate the role of E2F-1 in myeloid differentiation, M1E2F-1
cell lines were established, which expressed the E2F-1 transgene in a
deregulated mode. When these cells were subjected to IL-6 treatment,
exogenously expressed E2F-1 blocked the normal program of
differentiation at a very early stage. M1E2F-1 cells stimulated with
IL-6 have the same blast-like morphology as untreated controls, and
there is no elevated expression of the differentiation-associated cell
surface Fc and C3 receptors. Moreover, E2F-1 expression also inhibited
the growth arrest that is normally associated with terminal differentiation; the IL-6-treated M1E2F-1 cells continued to
proliferate at the same rate as their untreated counterparts. These
results demonstrate, therefore, that the down-regulation of E2F-1
expression by IL-6 in M1 cells is a necessary event for differentiation
to proceed.
Submitted November 24, 1999; accepted March 2, 2000.
Supported by National Institutes of Health Grants No. lROlCA51162
(B.H.), lROlCA43618 (D.A.L.), Amgen (B.H. and D.A.L.), and the core
program on carcinogenesis (5P30CA12227).
Reprints: Dan A. Liebermann, Temple University School of
Medicine, 3307 N Broad St, Philadelphia, PA 19140; e-mail: lieberma{at}unix.temple.edu.
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.
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Abstract
Introduction
