©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reduced Numatrin/B23/Nucleophosmin Labeling in Apoptotic Jurkat T-lymphoblasts (*)

Scott D. Patterson (§) , Jill S. Grossman , Peter D'Andrea , Gerald I. Latter

From the (1) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [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 M40,000. 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.


INTRODUCTION

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)() 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.

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.


EXPERIMENTAL PROCEDURES

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). EXPRESS-[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 10cells/ml in a humidified incubator with 95% air, 5% COat 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 (MH172.1) and myoglobin (MH16951).

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.


RESULTS AND DISCUSSION

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.

Changes in PCNA and -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 M80,000-28,000.



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.

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(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 (MH32936), yet it migrates as a M40,000 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) .

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 [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.

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 -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.

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.


FOOTNOTES

*
This work was supported in part by a Long Island Biological Association Award (to S. D. P.), and National Institutes of Health Grants P41-RR02188 and P30-CA45508. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address and to whom correspondence should be addressed: Amgen Inc., Amgen Ctr., mail stop 14-2-A-229, 1840 DeHavilland Dr., Thousand Oaks, CA, 91320-1789. Tel.: 805-447-3692; Fax: 805-499-7464; E-mail: spatters@amgen.com.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; MALDI-MS, matrix-assisted laser-desorption/ionization mass spectrometry; NPM, the protein product of the NPM gene also known as numatrin, B23, NO38, and nucleophosmin; PCNA, proliferating cell nuclear antigen; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

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.


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