The Peter MacCallum Cancer Institute, Gene
Regulation Laboratory, Cancer Immunology Division, East Melbourne, 3002 Victoria, Australia,1 and Edward A. Doisy Department of Biochemistry2 and
Department of Molecular Microbiology and
Immunology,3 St. Louis University School of
Medicine, St. Louis, Missouri 63104
Received 11 August 2000/Returned for modification 8 September
2000/Accepted 14 November 2000
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INTRODUCTION |
Many cellular factors involved in
human oncogenesis have been identified as the products of genes at
breakpoints of frequently occuring chromosomal translocations. The
protein products of some of these genes are transcriptional factors
that regulate the general or specific expression of many genes. The
ELL gene was initially identified on chromosome 19p 13.1, which undergoes frequent translocation with the
trithorax-like MLL (ALL-1, HRX) gene on
chromosome 11q23 in acute myeloid leukemia (AML) (47). The
3,968-amino-acid MLL protein contains an N-terminal A-T hook
DNA-binding domain, a methyltransferase-like domain near the center of
the molecule, and a C-terminal domain with several contiguous zinc
fingers (35, 36, 48). Chromosomal translocations involving
the MLL gene occur in approximately 80% of infants with AML
and acute lymphoblastic leukemia (ALL) and approximately 5% of adult
patients with AML as well as up to 10% with ALL (36).
These translocations result in fusion of the N-terminal region of the
MLL gene product to other cellular gene products to produce
chimeric proteins. To date, more than 16 different MLL fusion partner
proteins been identified, with the partner proteins of MLL displaying
seemingly limited structural similarity (3, 7, 13, 16, 17, 25, 29, 30, 33, 34, 37, 43, 44, 47). The molecular function of MLL
is thus far unknown: however, its homology to the Drosophila
trithorax protein TRX indicates that MLL serves to regulate and/or
maintain homeotic gene expression. Indeed, the production of
MLL knockout mice has revealed that MLL is required for
maintenance of gene expression during early embryogenesis (52,
53).
The question of whether MLL translocations are oncogenic because of a
gain of function of the fusion protein or a loss of function of MLL
and/or the partner protein is still unresolved. However, there is
evidence to suggest that loss of MLL function alone is not the
mechanism. For instance, MLL knockout mice do not develop
leukemia, although the development of a number of different tissues,
including the hemopoietic compartment, is compromised (15,
52). Additionally, mutations of the MLL gene in AML
and ALL have not been found. Transgenic expression of the MLL-AF9 fusion protein in mice (10) and retroviral infection of
mouse bone marrow with constructs expressing MLL-ENL and MLL-ELL result in induction of myeloid leukemia (9a, 22). Interestingly, fusion of MLL to the oncogene myc, a chimera not
seen in ALL or AML, did not induce this phenotype, indicating that the
specific fusion protein produced is important for oncogenesis
(8).
While there appears to be little structural similarity between the
different MLL fusion proteins, there is now evidence that the proteins
may be functionally related. For example, CREB-binding protein (CBP)
and p300 are transcriptional coactivators that acetylate histones to
mediate gene expression (18), AF4, AF9, and ENL can
function as transcription factors (34, 37), and ELL is a
transcription factor (38) increasing the catalytic rate of transcription elongation by RNA polymerase II (Pol II). This indicates that altered transcriptional regulation mediated by MLL-containing fusion proteins may be a key molecular event leading to leukemia. Interestingly, it has recently been shown that MLL-ELL, MLL-ENL, and
MLL-AF9 but not wild-type MLL can physically interact with GADD34, a
growth arrest- and DNA damage-inducible protein, and abrogate
GADD34-mediated apoptosis (1). These studies were the
first to conclusively demonstrate a gain-of-function phenotype for MLL
fused to completely different partner proteins compared to wild-type
MLL. While the molecular functions of many of the MLL partner proteins
have been identified, their physiological roles are mostly ill defined.
As loss of physiological function of the partner proteins may be
important for inducing cellular transformation, it is important to
determine what cellular function(s) they might regulate in order to
understand the events leading to tumorigenesis.
We purified ELL as an 80-kDa Pol II transcription factor that can both
increase the catalytic rate of transcription elongation by Pol II and
regulate formation of preinitiation complexes by Pol II (38,
39). The Pol II elongation factors fall into at least three
functional classes (reviewed in reference 40). The first
class prevents pausing or arrest of Pol II and includes P-TEFb, DSIF,
and SII. The second class includes the FACT (facilitates chromatin
transcription) complex and facilitates transcription elongation through
nucleosomes. The third class increases the catalytic rate of
transcription elongation and includes TFIIF, elongin, and members of
the ELL family. It is presently unclear which members of each class of
elongation factors are necessary for correct mRNA synthesis or the
stoichiometry of the proposed large complex involving elongation
factors and Pol II in vivo. The MLL-ELL fusion proteins contain the
first 1,439 amino acids of MLL, containing the A-T hook and
methyltransferase-like domains, fused to amino acids 46 to 621 of ELL.
ELL mutants lacking the first 50 amino acids can still bind Pol II and
are fully active in elongation but do not inhibit promoter-specific
initiation (39). In addition to its role in regulating
transcription elongation and initiation, ELL may also function as a
modulator of gene expression mediated by cellular transcription factors
such as p53. ELL has been shown to physically associate with p53 and
inhibit both sequence-specific transactivation and sequence-independent
transrepression by p53 and to inhibit p53-mediated apoptosis
(41). Importantly, the MLL-ELL fusion protein may also
bind to p53 and similarly affect the transcription regulation function
of p53 (23). Conversely, binding of p53 to ELL results in
inhibition of ELL-mediated transcription elongation (41).
Although transcriptional activities of the ELL protein have been well
characterized in vitro, we know very little about the physiological
role of the ELL within mammalian cells.
A limited number of studies have been performed to determine the
biological role of ELL. Consistent with its putative oncogenic function, constitutive overexpression of human ELL in RAT1 fibroblast cells resulted in an increase in anchorage-independent cell growth and
a decreased dependence on growth factors (20). However, neither morphological change nor focus-forming ability was observed in
ELL-expressing cells, indicating that expression of wild-type ELL is
not sufficient to induce full neoplastic transformation. In an attempt
to determine the effect of expression of human ELL in a human cell
line, we expressed ELL in a tetracycline-inducible manner in 293 cells,
which do not express endogenous ELL. Human 293 cells are an human
embryonic kidney cell line transformed by adenovirus type 5 DNA
(11).
In contrast to the previous experiments by Kanda et al.
(20), expression of ELL resulted in a decrease in cell
growth. However, in support of our observation, it was recently
demonstrated that expression of ELL in primary murine myeloid
progenitor cells did not result in immortalization of these cells
(21a). Analysis of the cell cycle profiles of
ELL-expressing cells revealed that there was a dramatic decrease in the
G1 population commensurate with a reproducible increase in
the percentage of cells in G2/M, followed by a steady
increase in the sub-G1population of cells, indicating that
these cells were undergoing apoptosis. This was confirmed by assays
demonstrating that the ELL-expressing cells underwent morphological
changes consistent with programmed cell death. In addition, caspase
activation was observed, and inhibition of caspase activity with
specific peptide inhibitors or expression of antisense ELL mRNA
significantly inhibited ELL-induced cell death. These data show for the
first time that expression of wild-type ELL in cells results in the
regulation of cell proliferation. It is therefore possible that in
addition to the production of MLL-ELL, which might inhibit apoptosis
mediated by GADD34, the (11;19)(q23;p13.1) (MLL;ELL) chromosomal
translocation may induce tumorigenesis by disrupting in vivo regulatory
activities of wild-type ELL.
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MATERIALS AND METHODS |
Plasmids.
Full-length human ELL was amplified by PCR with
ELL-specific primers. The ELL PCR product was inserted into the pTRE
expression vector (Clontech, Palo Alto, Calif.) digested with
BamHI and EcoRI, and the ELL-pTRE construct was
verified by sequence analysis. An antisense ELL expression plasmid was
produced by excising the ELL cDNA from ELL-pTRE with BamHI
and EcoRI and inserting it into the BamHI and
EcoRI cloning sites of pCDNA (Invitrogen, Carlsbad, Calif.).
Transient transfection into 293 Tet Off cells.
293 Tet Off
cells (Clontech) were cultured in Eagle's minimal essential medium
(EMEM) containing 10% fetal bovine serum (FBS), 2 mM
L-glutamine, 100 µg of penicillin-streptomycin per ml,
and 100 µg of G418 sulfate per ml in the absence of tetracycline. One
day prior to transfection, 3 × 105 cells were plated
on 100 20-mm dishes. For each plate, 2 µg of ELL/pTRE or pTRE alone
was mixed with 12 µl of lipofectamine (Gibco-BRL, Grand Island, N.Y.)
in 600 µl of EMEM. This solution was incubated for 45 min, 2.4 ml of
EMEM was added, and the cells were then overlaid with this complex
solution for 4 h. After 48 h, the cells were harvested.
Stable transfection into 293 Tet Off cells.
293 Tet Off
cells were cotransfected with 500 ng of pTK-Hyg vector (Clontech) and
ELL/pTRE or pTRE alone as above. After 48 h, selection for stable
clones was performed using tetracycline (1 µg/ml) and hygromycin B
(100 µg/ml). Positive clones were selected using cloning cylinders
and plated into individual flasks. ELL 293 cells are cultured in 90%
EMEM with 10% FBS, 2 mM L-glutamine, penicillin-streptomycin (100 µg/ml), G418 sulfate (100 µg/ml) tetracycline (1 µg/ml) and hygromycin B (100 µg/ml). Pooled clones of ELL 293 Tet Off cells were used for functional studies.
Transient transfection of antisense ELL/pCDNA into ELL 293 Tet
Off cells.
ELL 293 Tet Off cells were transiently transfected with
2 µg of pCDNA-ELLas or pCDNA alone as above, and cells were grown in
the presence or absence of tetracycline for various times before harvesting.
Western blot analysis of expressed ELL.
Transiently
transfected 293 Tet Off cells or ELL 293 Tet Off stable cell clones
were grown in the presence or absence of tetracycline and lysed in
lysis buffer (25 mm HEPES [pH 7.0], 0.25 M NaCl, 2.5 mM EDTA, 0.5 mM
dithiothreitol, 10 µg of leupeptin and 1 µg of pepstatin A panel 2 mM phenylmethylsulfonyl fluoride, and 0.1% NP-40) for 30 min on ice.
The cell extracts were boiled for 5 min at 95°C, and proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) on 12% gels and electroblotted onto a nitrocellulose
membrane for 1 h at 450 mA. After blocking for 30 min with 5%
nonfat dry milk in Tris-buffered saline, blots were incubated with ELL
monoclonal antibody or anti poly (ADP ribose) polymerase (PARP,
Boehringer Mannheim, Indianapolis, Ind.) monoclonal antibodies
overnight at 4°C. Blots were incubated with a 1:10,000 dilution of
horseradish peroxidase-coupled goat anti-mouse immunoglobulin antibody
(Dako, Carpinteria, Calif.), and immunoreactive proteins were
visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotechnology, Buckingshire, U.K.).
Cell proliferation and viability assays.
Cells were cultured
at 1 × 105 cells/ml in the presence or absence of
tetracycline for various times. Trypan blue dye exclusion assays were
performed as previously described (19), or cells were
analysed by phase contrast microscopy. In all assays, 150 to 300 cells
were counted for each data point, and data were calculated as the
mean ± standard error of triplicate samples and are
representative of at least three separate assays. The number of
apoptotic or dead cells (blue cells) was expressed as a percentage of
the total cell number. To inhibit the activation of caspases, cells
were treated for 30 min with peptidyl fluoromethylketones (zFA-fmk or
zVAD-fmk; final concentration, 40 µM) (Enzyme System Products, Dublin, Calif.).
Propidium iodide staining.
Cells (2 × 105)
grown in the presence or absence of tetracycline for various lengths of
time were washed in phosphate-buffered saline (PBS) and fixed in 50%
ice-cold ethanol-PBS (50% vol/vol) for 20 min on ice. Cell were
washed with PBS and incubated in propidium iodide solution (69 µM
propidium iodide 388 mM sodium citrate, 100 µg of Rnase A per ml) for
15 min at 37°C. Cells were immediately analyzed with a FACscan flow
cytometer (BD Pharmingen, San Diego, Calif.).
Immunofluorescence microscopy.
Cells were resuspended in PBS
supplemented with 5% bovine serum albumin for slide preparation and
developed as described before using monoclonal antibodies generated to
either ELL or active caspase 3 (49).
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RESULTS |
In an early attempt to study the physiological role of ELL, we set
out to make a stable cell line constitutively expressing full-length
ELL under the control of the Rous sarcoma virus promoter. Although
transiently transfected cells expressed ample amounts of ELL, we were
unable to select stable cell lines that expressed ELL at high levels
(unpublished data). Stable cells selected for constitutive expression
of ELL expressed meager amounts of the protein. Furthermore, the
selected cells grew very poorly in comparison with mock-transfected
cells. We therefore hypothesized that expression of full-length ELL may
inhibit growth due to either loss of cell proliferation or induction of apoptosis.
Regulated expression of ELL.
To test the above hypothesis, we
constructed the ELL cDNA under the minimal cytomegaloviris (CMV)
promoter regulated by the Tet operator and developed ELL 293 Tet Off
cell lines expressing full-length ELL under control of the
Tet-regulated promoter. Removal of tetracycline from the tissue culture
medium for 48 h resulted in the expression of full-length ELL of
approximately 80 kDa, as determined by Western blot using an
ELL-specific monoclonal antibody (Fig.
1A). It is important to note that ELL in
these selected cells is not overexpressed, but rather is produced in amounts that would normally be found in cells expressing ELL. This has
been determined by comparing the amount of ELL expressed in 5 × 106 ELL 293 Tet Off cells expressing ELL to the amount
expressed by 5 × 106 normal T cells (unpublished
data). A time course experiment revealed that expression of ELL in ELL
293 Tet Off cells was detected as little as 6 h following the removal
of the tetracycline from the medium (Fig. 1B). Consistent with the
nuclear expression of endogenous ELL, Tet-regulated ELL was expressed
exclusively in the nucleus of ELL 293 Tet Off cells grown in the
absence of tetracycline, as determined by confocal microscopy and
Western blot using nuclear lysates (data not shown).
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FIG. 1.
ELL expression in the absence of tetracycline. (A)
ELL/pTRE was co transfected with the pTK-Hyg selection marker into 293 Tet Off cells to create ELL 293 Tet Off stable cell lines with
regulated ELL expression. Cells were incubated in the presence or
absence of tetracycline for 48 h. In the presence of tetracycline,
the promoter driving ELL expression is inactive. In the absence of
tetracycline, the promoter driving ELL expression is activated. Western
blot analysis of cell extracts was performed using an anti-ELL
monoclonal antibody. The positions of molecular size markers are shown
on the left (in kilodaltons), and the presence of a band of
approximately 80 kDa in cells grown in the absence of ELL is indicated
by an arrow on the right. (B) Time-dependent expression of ELL in ELL
293 Tet Off cells. ELL 293 Tet Off cells were grown in the presence or
absence of tetracycline for 0, 2, 4, 6, 12, 24, and 48 h. Cells
were harvested, and Western blot analysis was performed using an
anti-ELL monoclonal antibody.
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Effect of ELL expression on cell proliferation.
Having
produced a cell line capable of expressing ELL in a regulated manner,
we next tested the effect of ELL expression on the rate of cell growth.
Incubation of ELL 293 Tet Off cells in the absence of tetracycline
severely retarded cell proliferation over a 144-h time course compared
to cells grown in the presence of tetracycline, as determined by
counting cell numbers over time (Fig.
2A). ELL 293 Tet Off cells grown in the
absence of tetracycline did not proliferate, and morphological changes
were observed, with cells rounding up and detaching from the culture
dish, as shown by phase contrast microscopy (Fig. 2B). By contrast, 293 Tet Off cells transfected with empty pTRE vector grew similarly in the
presence and absence of tetracycline (Fig. 2C and D).
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FIG. 2.
Cell growth in the presence and absence of ELL. ELL 293 Tet Off and 293 Tet Off cells were plated at 10% confluency and
incubated in the presence or absence of tetracycline. Each day, cells
were photographed and counted. (A) Growth curve and (B) phase contrast
microscopy of ELL 293 Tet Off cells grown in the presence and absence
of tetracycline. (C) Growth curve and (D) phase contrast microscopy of
293 Tet Off cells grown in the presence and absence of tetracycline.
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To determine whether ELL expression resulted in altered cell cycle
regulation and/or induction of apoptosis, propidium iodide staining and
flow cytometry were performed on ELL 293 Tet Off cells grown in the
presence or absence of tetracycline (Fig.
3A). Induction of ELL expression resulted
in a decrease in the percentage of cells in the
G1/G0 phase of the cell cycle and a
commensurate increase in the percentage cells in G2/M and
sub-G1 (Fig. 3A and Table 1).
The presence of sub-G1 cells indicated that these cells had
lost or degraded genomic DNA, a phenomenon characteristic of cells
undergoing apoptosis. The induction of cell death following removal of
tetracycline in ELL 293 Tet Off cells was confirmed by trypan blue dye
exclusion assays (Fig. 3B). As can be seen, induction of ELL expression
correlated with a time-dependent increase in trypan-positive cells.
These data indicate that regulated expression of ELL results in a loss
of cell proliferation and induction of apoptosis. It is interesting
that there was a consistent increase in the percentage of cells in
G2/M following ELL induction, indicating that cells in this
phase of the cell cycle were somewhat protected against ELL-mediated
apoptosis or that there may be a cell cycle block.
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FIG. 3.
Altered cell cycle and induction of cell death as a
result of the expression of ELL. (A) Propidium iodide cell cycle
analysis was performed on cells cultured at the same initial density
for 60 h in the presence (top panels) or absence (bottom panels)
of tetracycline. Cell cycle progression of parental 293 Tet Off cells
was unaffected by tetracycline withdrawal. Expression of ELL correlated
with a loss of the G1/G0 population of cells
and an increase in the G2/M and
sub-G1/G0 population. (B) Trypan blue dye
exclusion assays were performed on ELL 293 Tet Off cells grown in the
absence of tetracycline (tet). Induction of ELL resulted in a loss of
cell membrane integrity and induction of cell death.
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To determine whether the inhibition of cell growth and induction of
apoptosis in ELL 293 Tet Off cells grown in the absence of tetracycline
was directly related to the expression of the ELL protein, we
determined whether the expression of an ELL antisense RNA could inhibit
this effect. ELL 293 Tet Off cells were grown in the presence or
absence of tetracycline and transfected with either pCDNA-ELLas,
containing an ELL antisense cDNA under control of the CMV promoter, or
pCDNA alone. Cells were grown for 4 days and then analyzed for the
effect of the ELL antisense RNA on the rate of cell growth. As shown in
Fig. 4, cells proliferated in the
presence of tetracycline (left panel). However, consistent with our
earlier results, ELL 293 Tet Off cells transfected with pCDNA and grown
in the absence of tetracycline did not proliferate (Fig. 4, middle
panel). However, when cells were transfected with the pCDNA-ELLas,
containing ELL antisense cDNA under control of the CMV promoter vector,
it was reproducibly observed that cell proliferation was restored (Fig.
4, right panel). These data indicate that the decrease in cell growth
and alteration of cell viability in the ELL 293 Tet Off cells grown in
the absence of tetracycline is a direct effect of expression of ELL in
these cells.
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FIG. 4.
Expression of ELL antisense mRNA results in reversal of
ELL-dependent cell death. ELL 293 Tet Off cells were transiently
transfected with pCDNA or pCDNA-ELLas, expressing antisense ELL mRNA,
and grown in the absence of tetracycline for 96 h. Transfection of
pCDNA-ELLas correlated with an increase in the number of viable cells
present following tetracycline withdrawal and a loss of apoptotic
morphology. The above picture is a representative colony that survived
in the presence of the antisense mRNA.
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ELL-induced apoptosis is mediated by caspases.
As demonstrated
above, induction of ELL expression results in cell death, as determined
by the loss of DNA content (Fig. 3A and Table 1) and plasma membrane
integrity (Fig. 3B). Apoptosis is mediated by intracellular cysteine
proteases (caspases), which are integral components of a highly
organized and regulated programmed cell death cascade inducing the
morphological changes associated with apoptosis (28). To
determine if the expression of ELL results in caspase-dependent
apoptotic death, ELL 293 Tet Off cells were grown in the presence or
absence of tetracycline for 24 h and analyzed by microscopy for
chromatin condensation (a morphological hallmark of apoptosis) and
caspase activity. Cells were stained with 4',6-diamidino-2-phenylindole
(DAPI) to visualize DNA and either anti-ELL monoclonal antibody or
anti-activated caspase 3 monoclonal antibody. As shown in Fig.
5A, growth of ELL 293 Tet Off cells in
the absence of tetracycline results in the expression of ELL in the
nucleus coincident with chromatin condensation and activation of
caspase 3. In the presence of tetracycline, ELL is not expressed,
caspase 3 is not active, and the cell nuclei appear normal. The DNA
repair enzyme PARP is a cellular caspase substrate specifically cleaved
and inactivated by activated caspase 3 during programmed cell death. We
therefore tested the cleavage and inactivation of PARP in response to
the regulated expression of ELL over 60 h. Cell lysates were
prepared and analyzed by SDS-PAGE and Western blot (Fig. 5B) using
antibodies against PARP (top panel) and ELL (bottom panel). Cleavage of
PARP from the 118-kDa active form to the 89-kDa inactive form
corresponded with induction of ELL expression (Fig. 5B).
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FIG. 5.
Expression of ELL induces chromatin condensation and
results in the activation of caspase 3. (A) ELL 293 Tet Off cells were
grown in the presence or absence of tetracycline for 24 h and
analyzed for ELL expression using an anti-ELL monoclonal antibody (left
panels), changes to the nucleus via DAPI staining (middle panels), and
activation of caspase 3 using a monoclonal antibody specific for the
active caspase 3 fragment (right panels). Induction of ELL expression
in the nucleus correlated with chromatin condensation and caspase 3 activation. (B) Protein lysates were prepared from ELL 293 Tet Off
cells grown in the presence or absence of tetracycline and separated by
SDS-PAGE. Western blots were performed using polyclonal anti-PARP (top
panel) or monoclonal anti-ELL (bottom panel) antibodies. The positions
of ELL and the active 118-kDa and inactive 89-kDa forms of PARP are
indicated.
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To determine whether activation of caspases in response to
expression of ELL is necessary for the induction of apoptosis, we
tested the effect of the caspase inhibitor zVAD-fmk on ELL 293 Tet Off
cells grown in the absence of tetracycline. As shown in Fig.
6A, removal of tetracycline induced cell
death in ELL 293 Tet Off cells, and this death could be significantly
inhibited by preincubation of cells with zVAD-fmk but not with the
control peptide zFA-fmk. ELL-induced DNA damage was also inhibited by addition of zVAD-fmk, as shown in Table
2, with a significant decrease in the
sub-G1 population of cells compared to cells
treated with the zFA-fmk control peptide over a 72-h time course. To
confirm that zVAD-fmk was indeed inhibiting ELL-induced caspase
activation, Western blots to detect PARP cleavage were performed using
ELL 293 Tet Off cells grown in the presence or in the absence of
tetracycline with zVAD-fmk or zFA-fmk (Fig. 6B). Once again, expression
of ELL induced cleavage of PARP, indicating that caspases were
activated. Importantly, addition of zVAD-fmk inhibited ELL-induced PARP
cleavage, while zFA-fmk had no effect. Taken together, these data
demonstrate that expression of ELL induces caspase activation and the
activation of these apoptotic molecules is important to mediate
apoptosis following ELL's expression in these stable cell lines.
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FIG. 6.
Induction of cell death by ELL can be reversed by
inhibition of caspase activation. ELL 293 Tet Off cells were treated
with the caspase inhibitor zVAD-fmk or the control peptide zFA-fmk and
grown in the presence or absence of tetracycline (tet) for 72 h. (A)
Induction of cell death was determined by trypan blue dye exclusion
assays. Apoptosis was induced in ELL 293 Tet Off cells following
tetracycline withdrawal, and this death could be inhibited with
zVAD-fmk but not with the control zFA-fmk peptide. (B) Protein lysates
were prepared from ELL 293 Tet Off cells treated as above and separated
by SDS-PAGE. Western blots were performed using monoclonal anti-ELL
(top panel) or polyclonal anti-PARP (bottom panel) antibodies. The
positions of ELL and the active (118 kDa) and inactive (89 kDa) forms
of PARP are indicated.
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TABLE 2.
Inhibition of apoptotic (sub-G1)
ELL-expressing 293 Tet Off cells by addition of the zVAD-fmk
caspase inhibitor
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To test the hypothesis that the cellular regulatory activity of ELL is
specific and associated with a particular domain of ELL, we generated
several mutations within the ELL protein and developed several clonal
cell lines with regulated expression of ELL mutant proteins. We
reasoned that having such cell lines available, we should be
able to define which domain of ELL is required for its cellular growth
regulatory activity. Our data presented here (Fig.
7) indicate that the elongation
activation domain of ELL is dispensable for the growth-regulatory
activity of ELL. However, clonal cell lines expressing ELL missing the conserved C-terminal domain do not demonstrate any slow-growth phenotype (Fig. 7A). We also tested the level of expression of ELL and
ELL mutants in these cells (Fig. 7B). This observation indicates that the cellular regulatory activity of ELL in these clonal
cell lines is specifically associated with the C-terminal domain of ELL and is not due to a nonspecific ectopic
expression of ELL. We have also recently shown that this domain of ELL
is highly conserved among the three human ELL proteins that we have cloned (26a). The conservation of the C-terminal domain of
ELL is an indication of its physiological importance. We have also demonstrated that this conserved C-terminal domain of ELL is sufficient and essential for the immortalization activity of the MLL-ELL chimera,
which is found in patients with leukemia (9a).
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FIG. 7.
Growth comparison among cells expressing full-length ELL
and those expressing ELL deletion mutants. (A) Cells expressing
full-length ELL, ELL( 150-200), or ELL(374-620) were plated at
106 cells per 25-cm2 flask and incubated in the
absence of tetracycline. Cells were photographed at 24, 72, and 96 h
after plating to record growth. (B) Plating was performed as in panel
A, and cells were incubated in the absence of tetracycline and
harvested at 72 h after plating. Total cell lysates were prepared
and analyzed by SDS-PAGE and Western analysis employing an ELL-specific
monoclonal antibody to detect ELL and ELL mutants.
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DISCUSSION |
Our data demonstrate that expression of ELL results in a loss of
the G1 population of cells, induction of caspase
activation, and cell death. A role for caspases in mediating
ELL-induced cell death was shown by the addition of the broad
caspase-inhibitory peptide zVAD-fmk, which significantly inhibited
death of cells following ELL expression. One question that now remains
and is currently being tested in our laboratories is how the caspases are activated following ELL expression. At present, there are two
well-defined pathways leading to caspase-mediated cell death (12). The first involves activation of the so-called death
receptor pathway, resulting in caspase activation following binding of ligands such as Fas ligand, tumor necrosis factor, or tumor necrosis factor receptor-like apoptosis-inducing ligand to their cell surface receptors (42). Another arm of the caspase-mediated cell
death pathway involves the mitochondria (12). In response
to stress stimuli such as serum starvation and addition of
chemotherapeutic drugs, the mitochondrial membrane potential is
affected, resulting in release of mitochondrial components such as
cytochrome c and caspase activation. The death receptor and
mitochondrial apoptotic pathways are linked by caspase 8, which not
only cleaves and activates caspase 3 but also cleaves and activates
Bid, which in turns inserts into the mitochondrial membrane to induce
cytochrome c release (9).
The 293 cells contain and express the genes for adenovirus proteins
E1A, E1B-19K, and E1B-55K (2, 21). The E1A proteins are
transcriptional adaptors that deregulate the cell cycle and, in the
absence of the E1B-19K and E1B-55K proteins, induce apoptosis (reviewed
in reference 51). The E1B-19K protein is a distant functional homolog of antiapoptotic members of the Bcl-2 family (51). E1B-19K inhibits apoptosis at several levels: it
binds proapoptotic members of the Bcl-2 family (6), it
binds CED-4 (the homolog of Apaf-1) (14), and it inhibits
Fas-mediated apoptosis through Fas-associated death domain
sequestration of procaspase 8 (32). The E1B-55K protein
inhibits apoptosis by forming a complex with the tumor suppressor p53
and repressing p53-responsive promoters (24, 51). It is
not known what effect, if any, these adenovirus proteins have on the
apoptosis observed after expression of ELL. ELL-mediated caspase 3 activation and apoptosis could occur via a pathway that is not blocked
by E1B-19K and E1B-55K. Alternatively, and perhaps more likely,
ELL-induced apoptosis may occur through known pathways, and E1B-19K and
E1B-55K are insufficient to block these pathways.
While caspases may be activated in the absence of de novo protein
synthesis, there is a link between gene regulation and apoptosis. For
example, DNA damage by chemotherapeutic drugs or ionizing radiation
results in activation of the p53 tumor suppressor protein, which in
turn transcriptionally activates Bax (9). The
interferon-responsive transcriptional activator IRF-1 has been shown to
induce cell death by transcriptionally upregulating caspases and the
Fas receptor (46). Recently, it has been shown that
regulation of gene expression on a more general scale by agents such as
trichostatin A, sodium butyrate, and suberoylanilide hydroxamic acid,
which function as histone deacetylase inhibitors, can induce caspase
activation and apoptosis (4, 5, 50). As is the case for
ELL, the molecular mechanism(s) underpinning apoptosis induced by the
above agents has not been dissected, and the specific genes with
altered expression in response to inhibition of histone deacetylases
have yet to be identified.
Our preliminary structure-function studies suggest that removal of the
elongation activation domain of ELL did not induce cell death but did
induce growth suppression (Fig. 7). Furthermore, ELL lacking the C
terminus, which is conserved among the ELL family of proteins and plays
an essential role in the immortalization of myeloid progenitors, fails
to mediate either apoptosis or growth suppression. Comprehensive
structure-function analyses are currently being performed to identify
the molecular functions of ELL that are necessary and sufficient to
induce ELL-mediated growth suppression and/or cell death.
Chromosomal translocations at 11q23 are commonly seen in AML and ALL,
resulting in the formation of fusion proteins containing the product of
the MLL gene. The production of these fusion proteins is
thought to provide a survival or proliferative advantage to myeloid
progenitor cells to mediate tumorigenesis. In both ALL and AML,
MLL has been shown to be fused to at least 16 different cellular genes, many of which are involved in transcription activation or maintenance of gene expression. Indeed, MLL itself has been reported
to be a transcription factor that can regulate the expression of
homeobox genes (36). The molecular function of the MLL-ELL fusion protein has not yet been investigated, and while ELL has been
demonstrated to play a role in transcription elongation, a
physiological function for ELL has not been identified. Our results
showing that ELL itself can regulate cell survival indicates that the
(t11; 19)(q23; p13.1) (MLL-ELL) translocation may contribute to
leukemogenesis in a number ways. First, the loss of function of
wild-type MLL could lead to a loss of transcriptional maintenance during morphogenesis, resulting in aberrant cellular development, as
demonstrated in MLL knockout mice (15, 52, 53). Second, our studies showing that ELL may regulate apoptosis indicate that a
loss of ELL function could result in survival of cells that otherwise
may have been destined to die. Additionally, ELL and MLL-ELL can bind
to and regulate the transcriptional activities of the p53 tumor
suppressor protein (23, 41). Maki and colleagues speculate
that altered expression of MLL-ELL or increased in vivo half-life of
the fusion protein compared to the wild-type ELL protein may be
important for tumor formation, as aberrant expression of MLL-ELL could
affect the antitumor activities of p53. Thus, loss of ELL expression
and formation of the MLL-ELL fusion protein due to the (11; 19)(q23;
p13.1) (MLL; ELL) chromosomal translocation may have at least two
consequences: loss of ELL-induced apoptosis, and altered p53 function
due to overexpression of MLL-ELL. Indeed it has been demonstrated that
p53 mutations occur infrequently in leukemias with 11q23 translocations
(26). Therefore, if, as in many other cancers, disruption
of p53 function is important for the development of ALL and/or AML,
then the overexpression of MLL-ELL may serve this purpose. Finally, the
MLL-ELL fusion protein itself is likely to have oncogenic properties,
similar to that demonstrated for the MLL-AF9 and MLL-ENL fusion
proteins (8, 10, 22). The recent demonstration that MLL
fused to ELL, AF9, or ENL can inhibit apoptosis induced by GADD34 while the wild-type MLL protein does not also points to a gain-of-function process as being important for leukemogenesis. Thus, the combination of
a loss of MLL and ELL functions and the gain of function resulting from
production of the MLL-ELL fusion protein could be sufficient and/or
necessary for induction of leukemia following a (t11; 19)(q23; p13.1)
(MLL-ELL) translocation.
The molecular mechanisms leading to ELL-induced cell death have yet to
be identified and are subject to ongoing experiments in our
laboratories. However, our demonstration that the C-terminal domain of
ELL, which is conserved among the three ELL proteins that we have
cloned (ELL, ELL2, and ELL3), is required for this cellular activity of
ELL focuses our studies on the physiological role of this conserved
domain of ELL. We are currently testing whether the expression of ELL
results in altered expression of genes that are involved in cell death
or whether ELL plays a direct role in the regulation of cell growth and
survival. The absence of genetically altered mice either devoid of
wild-type ELL expression or aberrantly overexpressing ELL with which to
study such a physiological function makes the question regarding the
role of ELL in regulation of cell growth and survival difficult to
answer at present. However, our studies demonstrating that expression
of wild-type ELL can induce cell death, coupled with the finding that
the ELL locus is frequently disrupted in ALL and AML, raises the
possibility that a biological function of ELL may be to regulate cell
growth and survival.
This work is supported in part by a grant from the American Cancer
Society (RPG-99-218-01-MGO) to A.S. A.S. is an Edward
Mallinckrodt, Jr., Young Investigator. R.J. is a Wellcome Trust Senior
Research Fellow.
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Abstract
Introduction
