From the
Jurkat T-lymphoblasts were induced to undergo apoptosis by
treatment with either EGTA (5 m
M/24 h) or a high concentration
of lovastatin (100 µ
M/48 h) to identify proteins that
exhibited coordinate regulation between the two treatments and thus
provide candidate proteins in the common apoptotic induction pathway. A
pure population of apoptotic cells, as determined by morphology,
``DNA laddering,'' and flow cytometry, was obtained by
Percoll density gradient centrifugation. Cells of increased buoyant
density were clearly apoptotic by all criteria. Following this gradient
centrifugation, the cells were labeled with
[
The physiological process of apoptosis is a ubiquitous mechanism
for the regulation of tissue homeostasis, embryogenesis, and immune
system maturation
(1, 2, 3, 4) .
Apoptosis can be induced by both physiological and nonphysiological
means and has been shown to require specific gene transcription and new
protein synthesis in many but not all cells. However, in either case,
the apoptotic process is the same. Although there are a few exceptions
( e.g. lack of frequent DNA strand breaks
(5, 6) ), apoptosis results in a series of morphological
and biochemical events characterized by chromatin condensation and
margination, cell shrinkage, internucleosomal cleavage of DNA, and
budding of membrane-bound vesicles
(7, 8, 9, 10) . Furthermore, the kinase
inhibitor staurosporine has been shown to induce apoptosis in a variety
of cell types
(11, 12) . Based on these extensive
similarities, it would appear that there is a common pathway(s) of
apoptotic induction.
There have been an enormous number of
descriptive studies of apoptosis in specific cell types that are
related to the initial triggering events. This is in contrast with the
fewer studies on the downstream effector processes that control the
apoptotic cascade described above. Many studies into the molecular
mechanism of apoptosis have concentrated on either specific gene
products implicated in apoptosis (and their protein-protein
interactions) or enzyme activities (kinases or endonucleases) often in
mixed populations of nonapoptotic and apoptotic cells. Although some
agents do induce apoptosis en masse, the induction is often cell-cycle
phase dependent, and consequently only a fraction of the cell culture
population is undergoing apoptosis synchronously. Several authors have
reported that an early event in apoptosis of cultured cells
(13, 14, 15) and thymocytes
(16) is an
increase in their buoyant density. Thus, for some cells, density
gradient separation provides a convenient means of separating out a
pure population of cells at a similar stage of apoptosis, even when the
culture contains only a low percentage of apoptotic cells.
Two-dimensional polyacrylamide gel electrophoresis (two-dimensional
PAGE)
We are
interested in the putative common apoptotic induction pathway and have
utilized high resolution two-dimensional PAGE to search for changes in
protein synthesis/degradation (turnover) that are common between two
different inducers of apoptosis. We used metabolic labeling as a
sensitive measure of subtle protein turnover changes that are unable to
be revealed by analysis of total protein levels, e.g. by
prelabeling of the cells, immunoblotting, or general protein staining.
Two separate stimuli, both of which have been shown to induce apoptosis
in hematopoietic cell lines, were chosen for these studies. Lovastatin,
a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase
at low concentrations, has been shown to induce apoptosis in both
CEM-C7 cells
(25) and HL-60 cells
(26) . Bazar and Deeg
(27) demonstrated the apoptotic induction of Jurkat cells using
5 m
M EGTA for 24 h. As our interest lies in the putative
common apoptotic induction pathway, these two diverse stimuli were
chosen. The experimental paradigm involved comparison of the protein
turnover between untreated cells and a pure population of apoptotic
cells obtained by density gradient centrifugation.
In this report,
we show that apoptotic Jurkat cells can be metabolically labeled with
methionine/cysteine and that there is no extensive proteolysis in those
cells. In addition, we have identified one of the few proteins that
showed a change in turnover (down-regulation) using quantitative
analysis. The identified protein was numatrin/B23/nucleophosmin (NPM),
which is a ubiquitous nucleolar phosphoprotein that displays a number
of activities. These include a potential role as a positive regulator
of cell proliferation ( i.e. it is dramatically increased in
tumorigenic cells
(28) and decreased upon growth arrest
(29) ) and possibly a role in ribosomal assembly and processing,
based on its localization in the granular region of the nucleolus
(30) , among other identified activities. It is yet to be
ascertained whether NPM plays a role in the induction of apoptosis or
is merely affected as a consequence of apoptotic induction.
Changes in PCNA and
The protein was also analyzed by MALDI-MS following
CNBr cleavage of the spot from a single preparative two-dimensional
PAGE gel. The result of this analysis is shown in Fig. 8. The masses
obtained were consistent with the intact protein as well as several
peptides derived from incomplete cleavage at methionine residues, thus
providing further confirmatory evidence for the identity of this
protein of interest. It should be noted that there was considerable
structure to many of the peaks, reflecting heterogeneity of the NPM
molecules in this protein spot. The intact mass of the protein was
approximately 360 Da higher than predicted, and this mass difference
was further reflected in the masses of the CNBr cleavage fragments.
However, this mass establishes that the NPM protein is the B232 isoform
and not a human homolog of the shorter alternative spliced rat isoform
(B23.1)
(57) .
The MALDI-MS data from CNBr cleaved NPM
isolated from asynchronously growing Jurkat cells showed increases in
masses of the fragments over expected as follows: residues 1-81,
119 Da; 82-294, 226 Da; 66-294, 359 Da; and 252-294,
69 Da. The sum of these differences is 16 Da less than the difference
of the intact protein. This is within the accuracy of the method
(0.04%). NPM is phosphorylated, and given the error of the measurement,
speculation as to the identity of these mass differences could indicate
the presence of 1 phosphate (+80 Da) in the fragments 1-81
and 252-294 and 2 or 3 phosphates in the fragment 82-251.
The major site of phosphorylation of NPM is at Ser
An important finding about NPM related to the present studies was
that of Feuerstein and Mond
(29) , who reported a 68% inhibition
of synthesis of NPM as a result of anti-µ-induced growth arrest.
The WEHI-231 B-cell lymphoma line was treated with anti-µ for 20 h
to induce growth arrest, and lysates from the
[
In light of
the many potential roles of NPM, it is conceivable that it may play a
downstream role in the apoptotic cascade, but considerably more work is
required to determine whether this is the case. It would seem as though
the decrease in NPM labeling does not correspond with a general
decrease in ribosomal assembly and transport, as protein synthesis was
not grossly affected as revealed from our two-dimensional PAGE
analyses. It is also interesting to note that Goldstone and Lavin
(80) , using a double selection subtractive hybridization
strategy from
Although NPM
down-regulation has been demonstrated in a different apoptotic model
system, further studies will be required to confirm whether NPM is part
of the final common pathway of apoptotic induction. It would also seem
that if it is part of the apoptotic pathway, then it is probably a more
distal component, as it has recently been shown that the nucleus is
dispensable for the onset of apoptosis
(11, 82) . Thus,
our studies are continuing to determine whether either of the
cytoplasmic proteins observed to be down-regulated (or maybe
specifically proteolyzed) in this study show similar characteristics in
other apoptotic model systems.
We thank Drs. D. Spector and R. Kobayashi for
conducting the laser confocal microscopy and amino acid sequencing,
respectively, N. Sareen and N. Bizios of the two-dimensional Gel
Laboratory Core Facility for two-dimensional PAGE, P. Burfiend for flow
cytometry, Z. Yu for assistance with preliminary cell culture
experiments, J. Keysor and D. Barron of the Amgen, Inc. Graphics Dept.
for assistance with reproduction of the figures, and Dr. Tony Polverino
for critical reading of the manuscript. Support of this project by Dr.
J. Watson and Dr. B. Stillman is gratefully acknowledged.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S]methionine/cysteine, and lysates were
separated by two-dimensional polyacrylamide gel electrophoresis.
Surprisingly, the two-dimensional polyacrylamide gel electrophoresis
patterns generated from the apoptotic cells did not differ dramatically
from that of control cells. Thus, apoptotic Jurkat cells are able to
synthesize new proteins and do not exhibit extensive proteolysis.
Subsequent quantitative analysis revealed that only five proteins
exhibited decreases in turnover that were common to the two treatments.
No increases in protein turnover were able to be confirmed across the
replicate experiments. One of the proteins that showed decreased
labeling by both apoptotic inductions was an abundant nuclear protein
with a pI of 5.1 and M
40,000. This protein was
identified as numatrin/B23/nucleophosmin (NPM) based on internal amino
acid sequence, and this identity was confirmed by immunoblotting and
mass spectrometry. NPM is implicated in a range of diverse cellular
functions, but its role in apoptosis is unclear.
(
)
has been utilized to search for total
protein changes in both cultured cells
(17, 18, 19) and tissues
(20, 21, 22, 23) , as well as
phosphorylation changes during apoptosis
(24) . In most systems,
mixed non-apoptotic and apoptotic cells were examined, increasing the
complexity of the analysis. However, in only one of these studies
(19) was a pure population of apoptotic cells (those that became
detached from a monolayer) compared with control cells, and in that
case the cells were prelabeled with radioisotope. Extended prelabeling
restricts the analysis to proteins with longer half-lives as they
incorporate relatively more label than those with shorter half-lives
and does not reveal whether any active protein synthesis occurs during
apoptosis. However, Baxter and Lavin
(24) examined two
different cell lines with the same two apoptotic inducers, revealing a
single common apoptotic-dependent dephosphorylation event.
Materials
Cell culture reagents RPMI
1640,
L-methionine-free and sodium phosphate-free RPMI 1640,
dialyzed fetal bovine serum (FBS), and streptomycin/penicillin were
from Life Technologies, Inc., and FBS (characterized) was from Hyclone
Laboratories, Inc. (Logan, UT).
EXPRES
S-[
S]methionine/cysteine
mix (>1000 Ci/m
M), and
[
P]orthophosphate (
9000 Ci/m
M)
were from DuPont NEN. Lovastatin was a gift from A. W. Alberts (Merck)
and was prepared by conversion of the lactone form to the sodium salt
as described
(31) . EGTA and Percoll were from Sigma. All other
chemicals and reagents were of the highest grade available.
Cell Culture
Jurkat T-lymphoblasts (Clone
E6-1, ATCC TIB 152) were cultured in 90% RPMI 1640, 10% FBS
(characterized), supplemented with streptomycin/penicillin, and
maintained at a concentration between 10and 10
cells/ml in a humidified incubator with 95% air, 5% CO
at 37 °C.
Separation of Apoptotic Cells
Following
chelator (EGTA) or drug (lovastatin) treatment, PBS-washed cells were
layered in 30% Percoll RPMI 1640 on top of a discontinuous gradient
with increasing concentrations (10% each step) of Percoll in RPMI to
100% as described
(32) . Healthy cells (normal buoyant density)
banded between the 40 and 50% layers while the apoptotic cells
(increased buoyant density) banded between the 50 and 60% layers.
Following centrifugation at 400 g for 30 min, cells
were washed in PBS and RPMI 1640 prior to metabolic labeling,
preparation for DNA separation, flow cytometry, or laser confocal
microscopy.
DNA Isolation and Separation, Flow Cytometry, and
Confocal Laser Scanning Microscopy
DNA was isolated and
separated according to Lennon et al. (33) . Briefly
this involved lysis of the cells, incubation with RNase A, and followed
by phenol and chloroform:isoamyl alcohol extraction. The high and low
molecular weight DNA was separated by centrifugation, precipitated with
ethanol overnight, and quantitated by spectrofluorimetry. The DNA was
separated on a 1% agarose gel in Tris boric acid EDTA buffer and
visualized by UV fluorescence after staining the gels with ethidium
bromide. Flow cytometry (FACS) was conducted following ethanol
fixation, RNase digestion, and propidium iodide incubation as described
(34) . Fluorescence intensities of the samples were measured by
quantitative flow cytometry using an EPICS C system (Coulter
Electronics Inc., Hialeah, FL). Cells were prepared for microscopy
using a Zeiss confocal laser scanning microscope (Carl Zeiss,
Thornwood, NY) by fixation in 2% formaldehyde, permeabilization with
0.2% Triton X-100, and staining with propidium iodide, with images
being generated as described by O'Keefe et al. (35) .
Metabolic Labeling
Metabolic labeling of
cells was conducted as follows. For
[S]methionine/cysteine, the labeling medium was
90% methionine-free RPMI 1640, 10% dialyzed FBS; for
[
P]orthophosphate labeling, cells were
preincubated for 30 min in 90% phosphate-free RPMI 1640 and 10% FBS
(characterized) prior to addition of the radiolabel for 3 h.
Subcellular Fractionation, Sample Preparation, and
Analytical Two-dimensional PAGE
Metabolically labeled
samples were prepared as described by Garrels and Franza
(36) except as noted
(37) . For nuclei enrichment and
postnuclear supernatant preparation, the PBS-washed cells were
resuspended in buffer (1% Triton X-100, 0.01
M MgCl, 0.03
M Tris-HCl, pH 7.5) in a microfuge
tube on ice. The cells were then passaged through a 28-gauge needle
several times and centrifuged at low speed (325
g) for
2 min to pellet the nuclei. The postnuclear supernatant was removed,
and the pellet was washed once with the same buffer and once with PBS
prior to lysis with a boiling SDS containing Tris buffer and treatment
with DNase/RNase
(38) . High resolution two-dimensional PAGE was
performed according to previously published procedures
(39) ,
with modifications noted
(37) by the Two-Dimensional Gel
Laboratory Core Facility at Cold Spring Harbor Laboratory. For
[
S]methionine/cysteine and
[
P]orthophosphate, 500,000 and 100,000 dpm of
precipitable counts of material was loaded per gel, respectively
(<10 µg total protein). A broad range first-dimensional gel
generating a pH gradient of approximately 3.5-7.5 and a second
dimension gel of 10% acrylamide was used for all experiments reported
here. The gels were dried immediately following electrophoresis and
imaged using a Bio-Imaging Analyzer BAS 2000
(Fuji Medical
Systems, Stamford, CT)
(37) .
Internal Amino Acid Sequence
Analysis
Preparative two-dimensional gels (2D Investigator,
Millipore, MA) loaded with 500-1000 µg of total protein were
transferred to nitrocellulose (0.45 µm) in Tris-glycine/10%
methanol
(40) for 45 min using a Genie electroblotter (Idea
Scientific, MN). Following blotting and rinsing with deionized water,
the blots were briefly stained with Amido Black (0.1% in
methanol/acetic acid) and destained in water. The spots of interest
were excised and stored in water at -20 °C. The collected
spots were then processed for tryptic digestion as described by
Aebersold et al. (41, 42) . The extracted
peptides were fractionated by reversed-phase HPLC on a Ccolumn (300 Å, 5 µm, 2.1
150 mm) using a 1090L
system (Hewlett-Packard, Palo Alto, CA). Column effluent was monitored
at 214 nm, and fractions were collected manually and stored at
-80 °C until sequence analysis. Sequence analysis was
conducted by Dr. R. Kobayashi (Cold Spring Harbor Laboratory) using a
470A sequencer (Applied Biosystems Inc.).
Western Blotting
Micropreparative
two-dimensional PAGE gels were run as for the analytical gels, except
that a larger diameter IEF gel tube of 0.065 inches in diameter was
used, and 50 µg of unlabeled whole cell lysate supplemented with
500,000 dpm of labeled cell lysate was loaded. Transfer to
nitrocellulose was performed as described above. The blot was blocked
and then incubated with appropriately diluted primary antisera and
detected using enzyme-conjugated horse anti-rabbit IgG F(ab)`followed by chemiluminescent detection using the ECL system
according to the manufacturer's instructions (Amersham Corp.).
The monospecific antisera used were raised against
numatrin/B23/nucleophosmin (kindly provided by Dr. M. Olsen, University
of Mississippi Medical Center, MI) and proliferating cell nuclear
antigen (PCNA)
(43) (kindly provided by Drs. S. Brand and M.
Mathews, Cold Spring Harbor Laboratory).
Mass Spectrometry
Protein for
matrix-assisted laser desorption mass spectrometry (MALDI-MS) was
isolated following preparative two-dimensional PAGE as described above
but with blotting to Immobilon-CD and subsequent staining with
QuickStain (Zoion Research, MA)
(44) . Limited cyanogen bromide
cleavage/elution (1
M CNBr in 70% formic acid for 5 min) was
performed on a single protein spot in a volume of 5 µl, sufficient
to cover the diced and five times alkaline methanol (20 m
M Tris, pH 9.0, 50% methanol)-washed membrane pieces. One-third of
this digest was mixed with 10 µl of matrix (5 mg/ml -cyano
4-hydroxycinnamic acid in 0.1% trifluoroacetic
acid/CHCl
/MeOH (2:2:5)), and 1 µl was applied to the
probe. A Kompact MALDI III (Kratos Analytical, Ramsey, NJ) kindly made
available by Kratos Analytical was used for this analysis. The spectra
were obtained in positive ion mode with 20 kV accelerating voltage and
externally calibrated using the dehydrated matrix ion (MH
172.1) and myoglobin (MH
16951).
Quantitative Two-dimensional Gel
Analysis
Analysis of the radiolabeled two-dimensional PAGE
patterns was conducted utilizing the Quest II software system described
by Monardo et al. (45) . The analysis procedure details
have been described elsewhere
(46) . Storage phosphoimages
obtained as described above were used for the quantitative analysis.
These digital images were then background subtracted and smoothed.
Spots were detected and fitted with gaussians, and the spots were then
matched to corresponding spots in images to be compared. The spot
volumes were calculated in parts/million as a fraction of the sample
loaded on the gel. There is then an additional normalization adjustment
in each gel to make the average ratio of spot parts/million values
closer to 1.0. In our case, this normalization was performed with the
highest quality (quality 4) spots of which there were over 250 that
matched on all four gels. There were over 750 valid spots on all four
gels. We considered only spots that were greater than 60 ppm in at
least one of the two gels being compared (the control or the treated)
for both sets of treatments for the original experiment and in both
sets of treatments for the repeat experiment as well.
EGTA and High Concentrations of Lovastatin Induce
Apoptosis in Jurkats
To confirm that both EGTA and
lovastatin induced apoptosis in our Jurkat T-lymphoblasts, the treated
cells were analyzed for characteristic morphological changes,
``DNA laddering,'' and DNA content by flow cytometry
following separation on a Percoll density gradient. The detailed
results of the analysis of lovastatin-treated cells before and after
treatment and following Percoll density gradient fractionation are
shown in Fig. 1. Morphological analysis revealed that some of the
treated cells exhibited characteristic apoptotic features, that is,
condensed chromatin, apparent decreased cell volume, and apoptotic
bodies. Due to the loss of nuclear material and enhanced side scatter
owing to the condensation of the residual chromatin, a sub- or
hypo-diploid DNA content peak was observed by FACS following apoptotic
induction (Fig. 1)
(47) . Fractionation of treated cells
by Percoll density gradient centrifugation yielded a pure population of
apoptotic cells of increased buoyant density (Fig. 1 D).
There were very few cells that appeared morphologically normal in this
apoptotic population, and there was little DNA in an undegraded state,
as revealed by the FACS analysis and agarose gel electrophoresis.
Interestingly, in the population of treated cells of normal buoyant
density, there were some cells that exhibited morphological
characteristics of apoptosis, and some degraded DNA was observed by
FACS and agarose electrophoresis of isolated low molecular weight DNA
(Fig. 1 C). It is possible that either these cells
represent a very early stage of apoptosis prior to increased buoyant
density or they are at a later stage of apoptosis when the cells are
beginning to decrease their buoyant density due to cell membrane
fragility. The apoptotic induction of Jurkats by lovastatin was not
apparent at 12.5 µ
M (the level required to inhibit
prenylation
(38) ) but showed a dose-dependent increase in
apoptotic induction from 50 to 100 µ
M with a further
increase being apparent after 48 h compared with 24 h (data not shown).
This is presumably due to effects on processes other than the
inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.
However, lovastatin has been shown to induce apoptosis at lower
concentrations in both CEM-C7 cells (5 µ
M for 72 h)
(25) and HL-60 cells (10 µ
M for 24 h)
(26) .
Fig. 2 shows that EGTA was able to induce apoptosis in Jurkats,
confirming the results previously obtained by Bazar and Deeg
(27) . In Fig. 2, the sub-diploid peak is apparent by FACS
(compare panels A and B), and the
characteristic DNA ladder is apparent ( lane 2,
morphological data not shown). Fewer cells were induced to undergo
apoptosis with EGTA compared with lovastatin. However, these cells were
also separated by Percoll density gradient centrifugation to yield a
pure apoptotic population at a similar stage of apoptosis to the
lovastatin-induced apoptotic cells (data not shown).
Figure 1:
Morphology, FACS analysis, and DNA
integrity of Jurkat T-lymphoblasts before and after apoptotic induction
with lovastatin. In each panel, the following is shown: fixed
cells stained with propidium iodide examined by laser confocal
microscopy, FACS analysis ( inset), and, to the right of each panel, agarose electrophoresis of DNA isolated
from those cells together with a 1-kilobase ladder ( left lane) (except for panel B). The
panels are as follows: A, control cells; B,
unfractionated cells following 48 h of treatment with 100 µ
M lovastatin; C, and D, same cells treated in
B fractionated by Percoll density gradient centrifugation to
yield cells of either normal buoyant density ( C) or increased
buoyant density ( D). In panel A, the FACS
inset is labeled with the positions of G, S, and
G
/M populations of cells. Note the increasing amounts of
DNA showing sub-diploid (or sub-G
) light scatter from the
apoptotic cells. The white bar in each panel represents 10 µm. All microscopy was at the same
magnification. Equal quantities of approximately 1 µg of DNA was
loaded in each lane of the agarose
gels.
Figure 2:
FACS
and DNA laddering of EGTA-treated Jurkat T-lymphoblasts. FACS data from
control cells ( A) and EGTA (5 m
M for 24 h)-treated
cells ( B). DNA integrity was also assessed by agarose
electrophoresis in the right panel with labeling as
follows: M, 1-kilobase ladder; 1, control cells; and
2, EGTA-treated cells. Equal quantities of approximately 1
µg of DNA were loaded in each
lane.
Apoptotic Cells Can Be Metabolically Labeled With
Methionine/Cysteine
Following separation of control, drug,
and chelator-treated cells by Percoll density gradient centrifugation,
the cells were washed and then incubated with
[S]methionine/cysteine mix for 3 h. Three
populations of cells were obtained in this manner, being the control
and treated cells of normal buoyant density and treated cells of
increased buoyant density. Cell lysates were prepared and separated by
two-dimensional PAGE. Fig. 3 shows the two-dimensional PAGE patterns
obtained from two of the populations, control and apoptotic cells
(those of increased buoyant density), for cells treated with
lovastatin. The treated cells of normal buoyant density showed a
pattern most similar to the control cell but with some features of the
apoptotic cells (data not shown). The most striking feature of these
separations is the similarity of the patterns despite the clearly
apoptotic appearance of the treated cells of increased buoyant density
(see Fig. 1). To our knowledge, this is the first report of
metabolic radiolabeling following separation of apoptotic cells, thus
demonstrating that there is active synthesis in these apoptotic Jurkat
cells. For example, Baxter and Lavin
(24) in their examination
of phosphorylation status prelabeled the cells, as did Robaye et
al. (19) in their study of protein turnover. Previous
studies have mostly used unfractionated populations
(17, 18, 20, 21, 23) or
prelabeled the cells prior to apoptotic induction and enrichment of the
apoptotic population by detachment of the cells
(19) . An in
vivo study of degenerating intersegmental muscles of the tobacco
hornworm also used two-dimensional PAGE of both isolated cellular
protein and in vitro translated mRNA, demonstrating that there
was new protein synthesis in these cells undergoing apoptosis
(22) . However, it has been reported that apoptotic cells have a
decreasing capacity for protein synthesis
(48) and an increased
proteolytic activity
(49, 50, 51) . Yet, our
data clearly show this not to be a general phenomenon. There is also
not a marked decrease in protein synthesis. However, despite the
similarity of the patterns, some proteins were obviously decreased by
both apoptotic inductions ( arrow in Fig. 3for
lovastatin apoptotic induction). It was decided to undertake a
quantitative analysis of the two-dimensional PAGE patterns to determine
how many proteins were coordinately regulated by these two treatments.
Figure 3:
Two-dimensional PAGE phosphorimager
patterns of control and apoptotic Jurkat T-lymphoblasts. Whole cell
lysates were prepared from metabolically labeled control ( A)
and lovastatin-induced apoptotic cells ( B), separated by
two-dimensional PAGE, and subsequently imaged using a storage
phosphorimager. One of the few proteins exhibiting a major reduction in
labeling following lovastatin treatment is shown with an arrow in both panels. The pH gradient generated in the first
dimension gels was approximately 3.5-7.5 (from left to
right), and the second dimension 10% acrylamide SDS-PAGE
separated from approximately M200,000-20,000 ( top to
bottom).
Quantitative Analysis of Two-dimensional PAGE
Patterns from Normal and Apoptotic Cells
To determine how
many proteins displayed a common coordinate regulation (either
increased or decreased levels) between both types of apoptotic
induction, quantitative analysis of both pairs of control/apoptotic
two-dimensional PAGE patterns was undertaken. Two different methods of
apoptotic induction were used so that any changes that were specific to
the particular type of apoptotic induction, and not part of a common
apoptotic dependent effect, could be ruled out. Stringent criteria were
applied to the analysis as described under ``Experimental
Procedures.'' Protein spots were identified that exhibited changes
of greater than 2-fold either up or down between the control/apoptotic
patterns for both pairs of treatments. To confirm that these protein
spots did show coordinate regulation in apoptotic cells, these protein
spots were then quantitated in additional two-dimensional PAGE patterns
from replicate experiments. Following this analysis, only five protein
spots were found to undergo at least 2-fold decreases in intensity, and
no protein spots were found to show reproducible increases in intensity
across all experiments. The five proteins that exhibited reproducible
decreased labeling are shown in Fig. 4. The fold decrease in labeling
is shown graphically in Fig. 5. Fold changes from the replicate
experiment were similar. From these experiments, we were unable to
determine whether the down-regulation was due to decreased synthesis or
increased degradation. The lack of confirmed increases is more likely
due to the stringent criteria applied to this analysis rather than
there being no such changes. Increases required that protein spots were
present in both control patterns at a level of 60 ppm or more and an
increase of 2-fold or more to be scored in all four sets of
control/apoptotic gel patterns.
-tubulin in
apoptotic cells have been quantitated by others using
immunofluorescence staining intensities
(52, 53) .
Gorczyca et al. (53) used topoisomerase inhibitors to
induce apoptosis in HL-60 cells, while Gazitt and Erdos
(52) used an extended thymidine block to induce apoptosis in an
early T-cell line, AGF. PCNA was found to decrease slowly during
apoptosis of the HL-60 cell line (especially compared with some other
nucleolar proteins, not including NPM), whereas it increased slightly
in the AGF cell line during apoptotic induction.
-Tubulin was
essentially unchanged in the AGF cell line and was not examined in the
HL-60 cell line. In this study, there was no consistent change in
either of these proteins (see Fig. 4for qualitative comparison).
Figure 4:
Proteins separated by two-dimensional PAGE
that demonstrated coordinate down-regulation upon two separate
apoptotic inductions. Smaller region of the control ( A) and
lovastatin-induced apoptotic enriched population of Jurkat cells
( B) shown in Fig. 3 and the same regions of the control
( C) and EGTA-induced apoptotic cells ( D) is as shown.
The five protein spots showing coordinate regulation between both forms
of apoptotic induction are circled in all panels and
numbered in panel A. The boxed proteins are labeled as follows: ,
-tubulin (identified
by amino acid sequence analysis of a tryptic peptide, which yielded the
sequence EVDEQMLNV); and P, PCNA (identified using
monospecific antiserum described in text). Gel type and orientation are
as in Fig. 3. The region encompasses approximately pH 4.7-6.0 and
M
80,000-28,000.
Subcellular Localization of Coordinately Regulated
Proteins
In control Jurkat cells, the subcellular
localization of proteins showing coordinate regulation is shown in Fig.
6. Three of the proteins, 2, 3, and 4, were shown to be enriched in the
nuclear fraction over the postnuclear supernatant fraction. Protein
spots 1 and 5 exhibited inverse fractionation characteristics, i.e. were enriched in the postnuclear supernatant fraction over the
nuclei-enriched fraction.
Identification and Partial Characterization of an
Apoptotically Modulated Protein
Peptides were generated
from protein spot 3 (see Figs. 3 and 4) pooled from several preparative
two-dimensional PAGE separations of untreated Jurkat cell lysates. Spot
3 was chosen as it exhibited the most intense labeling and therefore
was probably the most abundant of the five spots of interest. Following
reversed phase-HPLC separation, one peptide fraction was chosen for
amino acid sequence analysis and yielded the sequence MTDQEAIQDL with
an initial yield of 13 pmol. A search of the SwissProt release 29 using
the PepPepSearch component of the Darwin program
(54) available
on the Worldwide Web (http://cbrg.inf.ethz.ch/) found only the
following three matches with 100% identity: B231_HUMAN, B232_HUMAN, and
B23_RAT, thereby uniquely identifying this sequence as being derived
from NPM (numatrin/B23). The human sequence B231 is a partial sequence
entry, whereas B232 is the complete gene of the major B23 isoform of
human cells. The sequence obtained corresponds to residues
278-287 of the human sequence
(55) . B23_RAT is the rat
NPM homolog. To further confirm this identification, antibodies to NPM
were obtained, and a nitrocellulose blot of a two-dimensional PAGE gel
was probed with monospecific anti-B23 antibody. Chemiluminescent
detection confirmed the identity of spot 3 as NPM and two other
isoforms of very low abundance previously reported by others
(56) (Fig. 7). Interestingly, neither of these low abundance
cytoplasmic NPM isoforms corresponded to any of the other coordinately
down-regulated proteins and did not change coordinately with the major
NPM isoform.
(58) , with another reported at Thr
(59) , both in the middle CNBr fragment, 82-251.
Additional mass in the fragment 1-81 could be the result of
oxidation of methionine residues present in this region and/or
acetylation of the N terminus. Oxidation of methionines has been
reported for many proteins following PAGE
(60) . It is also of
interest to note that the intact mass of this protein was shown to be
32,935 Da (MH
32936), yet it migrates as a
M
40,000 protein in SDS-PAGE. This is most likely
due to the high number of aspartic and glutamic acid residues present
in this protein in highly acidic regions causing altered
electrophoretic mobility. Unfortunately, insufficient quantities of
this protein from apoptotic cells were available for MALDI-MS analysis.
A Role for NPM in Apoptosis?
Of the five
proteins that exhibited down-modulation during apoptosis, three were
nuclear and two were cytoplasmic in asynchronously growing cells. One
of the nuclear proteins was identified as NPM, a known major nucleolar
phosphoprotein. Interestingly, NPM also displayed a concomitant
decrease in phosphorylation, as revealed by
[P]orthophosphate labeling of the apoptotic
cells and subsequent two-dimensional PAGE analysis (data not shown).
This analysis measures not only phosphorylation of newly synthesized
NPM but also phosphorylation of any existing NPM in the cell. NPM is
known to be enriched in proliferating cells and thought to be involved
in ribosome assembly and transport due to its localization to the
granular region of the nucleolus
(30) . NPM has been ascribed a
number of diverse properties, including a potential role in
proliferation due to its rapid increase in synthesis during mitogenic
stimulation
(61) ; a role as a cytoplasmic/nuclear shuttle
protein
(62) ; has DNA binding activity
(63, 64) ; relieves transcriptional repression by YY1
(65) ; binds the HIV Type 1 Rev protein
(66) , the human
T-cell leukemia virus-1 Rex protein
(67) , and another cell
cycle-regulated nucleolar protein p120
(68) ; is a nuclear
substrate of protein kinase C
(69) , mitotic cdc2 kinase
(70) , and casein kinase II
(71) ; can be ADP-ribosylated
(72) ; and the N-terminal region has been shown to be a part of
a fusion protein with a tyrosine kinase, a result of a chromosomal
rearrangement, in some non-Hodgkin's lymphomas
(73) .
S]methionine-labeled cells were analyzed by
two-dimensional PAGE. The remainder of the two-dimensional PAGE pattern
was also shown to be relatively unchanged. Anti-µ (membrane-bound
IgM) treatment was subsequently shown by others to induce apoptosis in
this cell line
(74) . Thus, a reduced turnover of NPM in
response to apoptosis appears to have already been shown in another
apoptotic model system. Of further interest is the ability of NPM to
undergo translocation from the nucleolus to the nucleoplasm by agents
that inhibit cell growth. This ``B23 translocation assay''
has been postulated to be indicative of the efficacy of anti-tumor
drugs in both cell culture
(75) and in vivo (76) . It is interesting to note that some of the drugs
used to demonstrate the NPM (or B23) translocation also induce
apoptosis in the cell types used in these studies, e.g. adriamycin and daunomycin, induce apoptosis in HeLa cells
(77) , and induce translocation of NPM
(78) . It has
recently been demonstrated that GTP levels regulate the localization of
NPM in the nucleolus, as depleted GTP levels result in ``B23 (NPM)
translocation''
(79) . We were unable to determine the NPM
subcellular localization in our apoptotic Jurkat cells.
-irradiated and dexamethasone-treated CEM C7 cells,
isolated a cDNA clone encoding the nucleolar ribosomal protein S20 that
was down-regulated 5-fold during the early stages of apoptosis. This
however, is not a reflection of nucleolar disintegration during
apoptosis, as nucleoli are reported to be clearly recognizable until
the late apoptotic stages
(81) . Although the evidence is
circumstantial, there may be some correlation between altered NPM
function and induction of apoptosis. A plausible role could be in the
relocalization by NPM of other critical factors that would result in
more of the downstream nuclear features of apoptosis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.