THE JOURNAL OF BIOLOGICAL CHEMISTRY (c: 1985 by The American Society of Biological Chemists, Inc Yo]. 260, No. 1, Issue of January 10, ~ p : 604-609, 1985

ranted En U.S.A.

Regulation of Protein Synthesis and Accumulation during Murine Erythroleukemia Cell Differentiation*

(Received for publication, April 2, 1984)

David Parker$ and David Housman From the Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

We have examined the repertoire of cytoplasmic pro- teins present at different times during murine eryth- roleukemia (MEL) cell differentiation. Our laboratory has developed an improved differentiation system in which the use of rapidly inducing MEL subclones and culture conditions which stabilize terminally differ- entiated cells results in highly synchronous differen- tiation and the accumulation of large numbers of cells in the end stages of differentiation. Using two-dimen- sional gel electrophoresis, the proteins of MEL cell cytoplasm have been fractionated at different times of induction in the improved system. The protein compo- sition of MEL cell cytoplasm changes dramatically dur- ing the differentiation program, in contrast to previ- ously reported results. We observe patterns of changes that are consistent with alterations in the relative deg- radative rates as well as the relative synthetic rates of the different proteins. We find that the rate of incor- poration of labeled amino acid into protein is reduced in induced cultures of MEL cells. We demonstrate that the contribution of uninduced cells to the protein pat- terns observed late in differentiation is minor in our system, and argue that the results previously obtained for differentiating MEL cells were influenced by the heterogeneity of the induced populations,

MEL’ cells can be induced to undergo erythroid differentia- tion by a variety of chemical stimuli. The system is widely used as a model for erythroid differentiation (1). Both the biochemical and morphological aspects of differentiating MEL cells have been characterized previously in some detail. Characteristics of the differentiation process include signifi- cant increases in levels of hemoglobin in the cytoplasm and pronounced condensation of chromatin (2) and reductions in cell volume of between 5 and 10-fold (3). However, despite the use of MEL cell cultures in the study of biochemical events in the terminal differentiation program, cultures de- scribed in most previous studies have been quite heteroge- neous in morphology, containing many cells in the early stages of the erythropoietic pathway. The factors which have led to this heterogeneity in the cell population include asynchrony

* This work was supported by NationaLInstitutes of Health Grant CA 17575 and in part by National Cancer Institute Grant CA 14051. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Current address: Department of Cell Biology and Anatomy, Northwestern University Medical School, 303 E. Chicago Ave., Chi- cago, IL 60657.

The abbreviations used are: MEL, murine erythroleukemia; BSA, bovine serum albumin; Me2S0, dimethyl sulfoxide.

60

in the entry of cells into the differentiation pathway and physical destruction of the end stage cells (3). Recently, we have developed an improved MEL system which results in differentiated cultures which are more homogeneous and closer to the end of the erythropoietic pathway (3). The two requirements of the improved system are a cloned population of rapidly and completely inducing MEL cells and their phys- ical stabilization during differentiation. This cellular stabili- zation has been shown to have a profound effect on the measurement of globin mRNA half-life in differentiated MEL cells (4).

The observation that MEL cells appear to complete the differentiation pathway only in the improved induction sys- tem raises the question of whether other previously obscured biochemical changes in differentiating populations may now be observed. The protein composition of uninduced and in- duced MEL cells has previously been examined in a nonsta- bilizing system ( 5 ) . They found that approximately 98% of the detectable total cellular proteins were unchanged in abun- dance, and less than 1% of the cytoplasmic proteins were found to have changed qualitatively or quantitatively. This result contrasts sharply with results obtained from similar studies of other differentiating systems, where in general the spectrum of protein changes has been broad (6-8). We have readdressed this question, using rapidly inducing cells cul- tured and induced under stabilizing conditions. We find that during induction under these culture conditions approxi- mately 50% of the cytoplasmic proteins have changed either rate of synthesis, rate of turnover, or extent of post-transla- tional modification. The implications of these results for the use of the MEL system as a model for in vivo erythroid differentiation are that a larger number of control mecha- nisms affecting a broader spectrum of polypeptide chains may operate during MEL cell differentiation than had been ex- pected based on previous results.

MATERIALS AND METHODS

Cells and Culture Conditions-The MEL cell line D-2 was sub- cloned from the 745-PC4 line, screening for the ability to induce rapidly and completely as measured by the extent and uniformity of hemoglobinization of the subclone when challenged with MezSO. The best subclones were expanded and aliquots were frozen a t a rate that ensured recovery of more than 50% of the frozen cells. Cells from the same aliquot were used for all cultures in a given experiment. Exper- iments were completed within 2 weeks of thawing an aliquot of cells.

Cells were grown in Dulbecco’s modified Eagle medium with 15% fetal bovine serum (Reheis) and penicillin/streptomycin (100 units/ 1OOpg per ml). Induction was in the same medium, with 1.8% MeZSO, and with or without 5% detoxified BSA (Reheis). Cells were accli- matized to BSA immediately before induction by 12 h of growth in media with 2.5% BSA, followed by 12 h in media with 5% BSA. Individual cultures were set up for each experimental condition or time point and not disturbed until harvest.

Determination of Morphological Characteristics-Modal cell vol- umes were determined on a Coulter Counter Model ZBI, equipped

14

Protein Regulation duril

with Channelyzer and X-Y Plotter (Coulter Electronics) after cali- bration with latex beads.

Cell morphologies were examined by mixing equal volumes of cell cultures and fetal bovine serum and air drying aliquots of the mixtures on glass slides. The slides were then fixed in 10% glutaraldehyde in phosphate-buffered saline, stained with benzidine, and counter- stained with hematoxylin. Slides were scored using the 40 X oil objective of a Zeiss microscope. One thousand cells of each population were scored.

P5S]Methionine Labeling and Preparation of Cytoplasmic Ex- tracts-Cells growing exponentially a t cell densities between 2 X 10‘ and 5 X lo5 cells/ml were collected by centrifugation a t 20 X g for 15 min at. room temperature. The growth or induction medium was aspirated and the cells were resuspended in labeling medium consist- ing of Dulbecco’s modified Eagle’s medium without methionine, 15% fetal bovine serum, 250 pCi/ml of [35S]methionine (New England Nuclear), and 1.8% Me2S0, and/or 5% BSA as indicated. Cells were incubated a t 37 C, 5% CO, during labeling at a density of 5-10 X lo6 cells/ml. Seventy-two-h labelings were done by adding [35S] methionine to a final concentration of 5 pCi/ml directly to growth or induction medium.

Incorporation of [%]methionine was stopped by quickly cooling the cells to 4 “C, after which they were collected by centrifugation at 20 X g for 15 minutes a t 4 “C, washed once with cold Hank’s balanced salts, and collected again by centrifugation a t 20 X g for 15 min at 4 “C. The cells were then resuspended a t a concentration of 1-5 X lo7 cells/ml in lysis buffer (10 mM Tris, pH 7.2 150 mM NaC1, 2 mM EDTA, 0.02 TIU/ml aprotinin (Sigma), and 1 bg/ml phenylmethyl- sulfonyl fluoride. Triton X-100 and sodium deoxycholate were then added to a final concentration of 0.05% each, the cell suspension was pipetted vigorously up and down 20 times and then incubated on ice for 10 min. Nuclei were removed by centrifugation a t 1500 X g for 3 min. Supernatants were made 5% 2-mercaptoethanol and 1% sodium dodecyl sulfate, boiled for 5 min, quick frozen in a dry ice/ethanol bath, and stored a t -30 “C. Samples stored in this manner appeared to be stable for a t least 3 months.

Two-Dimensional Electrophoresis-Two-dimensional electropho- resis was performed basically as described by O’Farrell (9). Sample counts per minute were determined by mixing 5 PI of cytoplasmic lysate with 10 pg of carrier BSA and precipitating on ice for 10 min with 10 ml of cold 5% trichloroacetic acid. Precipitated samples were filtered onto 25-mm nitrocellulose discs (Millipore), washed 3 X with 10 ml of cold 5% trichloroacetic acid, dried, and counted in 2,5-diphenyloxazole/1,4-bis[2-(5-phenyloxazolyl)]benzene/toluene. Briefly, the method is as follows. Samples of 2.5 X lo5, trichloroacetic acid-precipitable counts per minute were focused for 14,000 V-h in 3% acrylamide gels containing 8 M urea and 5% pH 3.5-10 Ampho- lines (LKB). After focusing, gels were soaked in equilibration buffer for 1 h and frozen a t -30 “C. The pH gradient was measured in a blank focusing gel run in parallel with the experimental gels. Second dimension gels were 7.5% acrylamide Laemmli gels, with molecular weight standards (Bio-Rad) run on each gel. All eight focusing gels for an experiment were run simultaneously, as were the second dimension gels that were to be directly compared. Gels were fixed

Volume, p3 x 10” FIG. 1. Modal volumes of MEL cells induced in 5% BSA.

-e-, uninduced; - - -, 2 days; -, 5 days. Cells were grown and induced as desdribed under “Materials and Methods.”

Tg MEL Cell Differentiation 605

and stained with Coomassie Blue in methanol/acetic acid/water (5:1:4), and destained with methanol/water (1:l). Gels were then fluorographed with ENHANCE (New England Nuclear), dried, and exposed to Kodak XAR-5 film at -70 ”C. Gels to be compared were treated in parallel.

Gel Scoring-Fluorograms of the two-dimensional gels were com- pared side by side on a light box, and a comparison map for each was prepared, indicating the location of each polypeptide spot and its intensity relative to the corresponding spot on the other fluorogram. The maps for each of a fluorogram pair were prepared independently, and not compared until after completion.

RESULTS

Cell Morphology-Upon treatment with 1.8% MezSO in a stabilization protocol, the D-2 clone follows a rapid course of induction. Thirty per cent of the cells are benzidine-positive by 48 h post-induction, and on the fourth day after induction, the cell pellet is deep red. During this period, cells in 5% BSA underwent a reduction in modal volume from 1300 U‘ to 140 u3 (Fig. l), while control cells without BSA decreased in volume from 1500 u3 to 140 u3. When stabilized cultures were examined by benzidine and hematoxylin staining at the time of preparation of cytoplasmic extracts (5 days), 29% of the cells resembled polychromatophilic normoblasts, 41% resem- bled orthochromatic normoblasts, 5% resembled reticulocytes or erythrocytes, and the remainder appeared to be lysed differentiated cells. Less than 0.1% of the cells had uninduced morphology, which is characterized by large size, an uncon- densed purpIe nucleus and Iight blue cytopIasm. At the same point in a nonstabilization protocol, the majority of cells were found to be polychromatophilic normoblasts, with proportion- ally fewer orthochromatic normoblasts and almost no reticu- locytes. However, approximately the same number of lysed cells were found (Fig. 2).

0 a3 E a 0 u

c

c 0

s

Days of Induction FIG. 2. Distribution of cell types in inducing MEL popula-

tions. a, uninduced cells; b, basophilic normoblasts; c, polychroma- tophilic normoblasts; d , orthochromatic normoblasts: e, burst cells. Cells were grown, induced, and scored as described under “Materials and Methods.”

606 Protein Regulation during MEL Cell Differentiation

FIG. :1. Ten-day exposures of two- dimensional gel fluorograms of cy- toplasmic proteins from MEL cells labeled for different lengths of time during induction. n: uninduced, 2-h 1at)eI: h : 5 days. 2-h label: c: uninduced, 72-h lahel: d 5 days, 72-h latwl. I1orc.n- word pointing nrrorrs indicate polypep- tide spots which are less intense at 5 days of induction. Ilpwnrd pointing or - rows indicate polypeptide spots which are more intense at 5 days o f induction. Horizontal nrrorcs indicate pol-ypeptide spots which are unchanged in intensity during induction, o-T, 1j"I'and A repre- sent cr-tut)ulin, I+tut)ulin, and actin, re- spectively. Samples were prepared, elec- trophoresed, and Iluorographed as de- scrit)ecl under "Materials and Methods."

4.0 5.2 5.7 6.4 6.9 4.0 5.2 5.7 64 6.9 1 I I I 1 I 1 1 I I 1

"ii 66.2

i - C

116.3 92.5

..

0

21

- D

- ? : !

*

- ' ,

'"S Laheling of Soluble Cytoplasmic Proteins-We extracted soluble cytoplasmic proteins as detailed under "Materials and Methods" from cells which had been labeled with [%]methi- onine for 2 or 72 h at either 0 or 5 days of induction, with or without 5% RSA. Proteins were separated in one dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 17.5% gel, and in two dimensions by isoelectric focusing on a broad, pH 3-10, pH gradient, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 7.5% gel. As many as 350 proteins were resolved in the two-dimensional system, and gels run simultaneously were virtually superim- posable. Equal numbers of counts were loaded on each gel. Pairs of gels for comparison were fluorographed under the same conditions and exposed for the same length of time under identical conditions. The sets of gels were exposed for different lengths of time to permit comparisons for proteins of widely varying abundances, and a map of spot location and relative intensity was constructed for each exposure of each gel. Polypeptide spots were placed in abundance classes on the basis of the minimum length of exposure required to render them visible. High-abundance pol-ypeptides could be distinguished on 14-h exposures, middle-abundance polypep- tides on 48-h exposures, and low-abundance polypeptides only on 10-day exposures.

Fig. 3 shows the 10-day exposures of the gels of extracts of 2- and 72-h-labeled MEL cells induced in the presence of 5% RSA for 0 and 5 days. Upward and downward pointing arrows, respectively, indicate representative proteins which are in- creased and decreased in 5-day-induced cells. Horizontal ar- rows indicate representative proteins which are unchanged in intensity from 0 to 5 days of induction. In all four panels, for reference, tu-T &T, and A represent a- and /$tubulin, and

200

116 3

92 5

66 2

45

200

I16 3

32 5

56.2

15

actin, respectively. This figure illustrates the general pattern of protein changes observed during induction. Casual inspec- tion reveals numerous quantitative changes in pol-ypeptide spot intensity. In Fig. 4 are corresponding regions of the flurograms shown in Fig. 3, C and I), and their difference maps, illustrating the scoring of changes in proteins from all abundance classes. Similar maps were constructed for 14 h, 48 h, and 10-day exposures. Maps were constructed by visual comparison of pairs of fluorograms. Examinations of test exposures of [:'"S]methionine indicated that differences in spot intensity resulting from 50% differences in the amount of radioactivity could be reliablv detected. Maps were con- structed for each member of a pair of fluorograms, and com- pared for discrepancies in complementation. The same poly- peptide spot was never scored as being more intense or less intense on both members of a pair of fluorograms when compared to each other. This indicates that spots of very similar intensity were in fact being scored as unchanged, and not arbitrarily placed in one category or the other. To ensure this, i f a spot was scored as unchanged in intensity when scoring one fluorogram, any other score obtained when ana- lyzing the other fluorogram of the pair was disregarded. In this way, uncertainties about changes in spot intensity at the limit of detectability were eliminated, and the percentage of the total number of proteins that changed intensity is there- fore a lower limit.

Table I shows the percentage of polypeptide species of each abundance class that changed intensity with induction, for cells induced with or without RSA, labeled for 2 or 72 h. Approximately 330 spots could be distinguished in the IO-day exposures, although fewer are apparent in the photographic reproductions. For all abundance classes, from 19 to 5 2 5 of

Protein Regulation during MEL Cell Differentiation 607

the proteins were unchanged in intensity as a result of induc- tion. Twenty-five to fifty-nine per cent of the proteins were reduced in intensity, and 10-29'3; were more intense, indicat- ing that a reduction in protein complexity was occurring in the cytoplasm during induction. For cells induced without RSA, the proportion of polypeptide spots that were increased or decreased in intensity upon induction remains relatively constant for all abundance classes. In contrast, in cells in- duced in the presence of BSA, the low and middle-abundance classes exhibit a relatively greater proportion of polypeptides that increase upon induction, than is found for high-abun- dance proteins. This result is consistent with the ohservation that i t is precisely the low-abundance polypeptides whose increased synthesis or accumulation would he most obscured by the lysis of terminally differentiated cells. These results are therefore consistent with a stabilizing effect of BSA. Although hoth 2- and 72-h labelings show similar percentages of proteins in each category, a careful inspection of the maps reveals that the sets of proteins represented in each category,

a

C O A _ _ o n A

d

FIG. 4. Difference maps of IO-day exposures of MEL cyto- plasmic protein fluorograms. Cells were laheled for 72 h and cvtoplasmic extracts were prepared at: a. 0 h of induction orb. 5 days ol induction. a and b are the same region of the two fluorograms. c is the difference map of the fluorogram in a, compared with h. d is the dilference map of'the fluorogram in h, compared with a. A, a, and 0. are used on the maps to indicate the position of spots with intensities that are higher, lower. or unchanged, respectively. relative to the intensity on the complementary fluorogram of the pair. The prepa- rat ion o f t he fluorograms and difference maps was as described under "Materials and Methods."

while overlapping, are not identical. Thus, the process of induction in MEL cells affects not only the rate of synthesis, but also the rate of turnover or of post-translational modifi- cation of a number of cytoplasmic proteins. One such protein is shown in Fig. 5 . This pol-ypeptide spot is slightly reduced in intensity during induction with a 2-h laheling pulse, yet is more intense in 5-day-induced cells than in uninduced cells that have heen labeled for 72 h. This ohservation is consistent with the expected behavior of a protein whose half-life had increased during differentiation.

In each pair of fluorograms, there were a certain percentage of spots which changed particularly dramatically during in- duction. T o systematically identify such spots, we cornpared 10-day and 48-h exposures of pairs of gels. If a pol-ypeptide spot was more intense on the shorter of the two exposures, it was classified as having exhibited a large change. The spots were further classified as having first appeared on the 14-h exposure (high abundance), or on the 48-h exposure (middle abundance). As shown in Table 11, several of these proteins are found for each experimental condition, in hoth abundance classes. In this analysis, cells induced without, as well as with RSA both exhibited a greater proportion of middle-than high- abundance proteins that increased in intensity with induction. This is not surprising, since more dramatic changes in poly- peptide abundance are less easily ohscured by the loss of end stage cells than are subtle changes. Additionally, examination of Fig. 3 reveals several proteins that exhihit large changes in both short and long labeled extracts. Two of them (240,000 daltons, pI 5.3 and 35,000 daltons, pI 6 5 ) increase during induction, and one, (54,000 daltons, pl 6.4) decreases during induction. The 240-kDa protein is notable as one of the most prominent high-molecular mass proteins resolved in our study. The 54- and 35-kDa proteins are notahle for their high abundance and the magnitude of their change in expression. These characteristics suggest that these three proteins may he particularly interesting candidates for further study in this system.

An important issue which must he addressed in a study of this t-ype is the possihility that a small numher of uninduced cells can make a disproportionate contribution to the proteins synthesized by an induced culture. In fact, we have ohserved in our experiments that uninduced cells incorporate laheled amino acids into protein five times more actively than highly differentiated cells. However, the results shown in Fig. 6 argue against such a disproportionate contribution in our study. The virtual disappearance of one or more pol.ypeptide chains during differentiation sets an upper limit on the contribution occasional uninduced cells can make to the protein synthetic pattern of the induced culture. This result agrees with our ohservation of fewer than 0.1% uninduced cells in induced cultures.

During erythropoiesis, globin accumulates to hecome the most abundant protein in erythrocytes. Its behavior during differentiation in the improved MEL system is therefore of

608 Protein Regulation during M E L Cell Differentiation

a b

C d

0s- c

-t - - t - -

FIG. 5. Fluorograms of the same region of two-dimensional gels of cytoplasmic extracts labeled for different lengths of time during differentiation. n: uninduced, 2-h label; h: 5 days, 2- h lahel: r : uninduced, 72-h label; d 5 days. 72-h lahel. In each panel, the arrow indicates the location of the same polypeptide spot. All panels are regions o f IO-day exposures of Iluorograms. Fluorograms were prepared as descrihed under "Materials and Methods."

TAME I1 Chnngrs in pol.vprptidc spot intrnsities rruralrd bv rompnrison of 2-

nnd IO-day rxposurps of 0 - and .5-day-inducrd extracts Extracts were prepared, electrophoresed, and fluorographed as

descrihed under "Materials and Methods." Fluorograms were com- pared as descrihed under "Materials and Methods," except that in each case, a 48-h exposure of one of a pair was compared to a 10-dav exposure o f t he other, and only spots which were more intense on the 48-h exposure were tallied. ~~ ~.

1,aheling conaitions (+/- HSA; h laheled) Number of' spots

intensities with indirtltrd High al,undanre Middle ahundanre

-: L' -: 7 2 +: 2 +: 72 -; 2 -: 7 2 +: 2 +: 7 2 ~

Odavs > 6 days 4 6 9 4 :1 3 3 4 5days >Odavs :1 :1 4 2 3 4 6 3

"

great interest. As measured on one-dimensional gels, globin synthesis increases dramatically during induction in stabilized cells, reaching 13% of the pulse-labeled protein at 5 days, and 30% of the t,otal cytoplasmic protein by the same time.

DISCUSSION

In the present study we have re-examined protein synthesis and accumulation in stabilized, rapidly inducing MEL cells. Our results contrast sharply with previously published studies on this quest.ion in the MEL cell system. In contrast to Peterson and McConkey (5), who found virtually no change in the polypeptide pattern revealed by two-dimensional gel electrophoresis of induced and uninduced MEL cells, we find numerous and dramatic changes in these patterns. As many a s 81% of the polypeptide spots in an abundance class showed detectable changes, of which a significant number were sub- stantially above the limits of detection. In addition, the dif- ferent behavior with short and long labeling times of some polypeptide spots during induction is evidence for changes in the stability, or in post-translational modification, of some proteins.

The contrast of these results with those of Peterson and McConkey ( 5 ) indicates that the hiochernical characteristics of the two MEL cell populations are quite different. We believe that the primary reason for this contrast is the differ- ence in the percentage of uninduced cells in the induced cultures. In the present study, induced cultures were laheled with ["'Sjmethionine for 2 h at 5 days of induction. o r for 7 2 h from 2 to 5 days of induction. In each case, cytoplasmic extracts were prepared from cell populations which were shown morphologically to have fewer than 0.1'; uninduced (LC. benzidine-negative) cells. In contrast, Peterson and McConkey labeled cells from the fourth to the seventh day of induction, in populations which were only 8-55 benzidine- positive on the sixth day. Furthermore, the ohsen.ations on culture morphology presented in Fig. 2 suggest that the num- ber of lysed end stage cells increases rapidly after 5 days of induction. In view of our observations that end stage .VEL cells are 5-fold less metabolically active than uninduced cells, it is likely that the proteins labeled in the overgrowing unin- duced cells of previous cultures ohscured the protein pattern produced by the remaining differentiated cells. In addition, it is likely that the high degree of synchrony ol induction exhibited by the MEI, cell suhclone used in this study further facilitates the observation of differences in protein synthesis patterns of uninduced MEI, cells and terminally differen- tiated cells. The subclone that was selected for the present study shows nearly .iO% of the intact cells in the last two stages of the erythropoietic pathway at the time of induced lysate preparation. While the previous study does not include data on the morphological content of the induced cultures, our experience with nonrapidly inducing MEI, cells has heen that few cells reach the end stages of differentiation in these cultures before significant overgrowth of noninducihle cells occurs. It is therefore probable that both the completeness of the induction and the greater synchrony resulting from the rapidity of induction contrihute to the protein changes ob- served in stabilized inducing MEI, cells.

While our data on morphology indicate that cells induced in the presence of RSA proceed farther down the erythropoi- etic pathway than cells induced without HSA, the proportions of changed and unchanged polypeptide spots with and wit hout RSA are essentially similar. This is an indication that the ohserved changes in protein levels are not specific effects o f the RSA t reatment . The fact that induction without HSA gives substantially similar results to cultures induced with

FIC.. t i . Fluorograms of the same region of two-dimensional gels of cytoplasmic extracts prepared at different times of induction. (1, 0 d a y s o! indurt ion; h. r) (lays 0 1 induction. I n rach panel, the nrrorr's indicate the lorat i o n o f poly1)ept idv spots which decrease i n intensity trom n t o h. Both panels arc regions I ) I I O ~ ; I ~ exposures o f Iluorograms o f cytoplasmic extract5 prt.parc.tl Iron1 cell\ Ial)eled for 72 h. Fluorograms were prc*l);lred ; I> clcwrit)c*d ~ l n t l v r "Materials and Methods."

Protein Regulation during MEL Cell Differentiation 609

BSA suggests that a substantial portion of the improvement in the system should be attributed to the selection of a rapidly and completely inducing MEL subclone.

A number of differentiating systems have been examined by two-dimensional gel electrophoresis. Devlin and Emerson (6) found coordinate regulation of contractile proteins during myoblast differentiation. In another study, it was found that approximately 10% each of cytoplasmic, nonhistone chromo- somal, and plasma membrane proteins were altered in the course of preadipocyte differentiation (7). During granulocytic differentiation of mouse myeloid leukemic cells, 73 proteins were found to have altered expression (8). In general, the observation is that the spectrum of proteins expressed in differentiating cells is shifted significantly in the course of differentiation. Whereas the result previously obtained in the MEL system was exceptional, our results are in agreement with the general observation.

Actin is a protein whose expression is often altered during differentiation. An increase has been reported during granu- locytic differentiation (8). Decreases in actin mRNA and/or protein levels have been reported during adipocyte differen- tiation (7, lo), and in Dictyostelium differentiation (11). In both cerebral and cerebellar tissues of developing rat brain, actin mRNA increases in relative proportion up to postnatal day 8 and then decreases into adulthood (12). We have found that actin represents a constant proportion of the cytoplasmic proteins synthesized and accumulated in uninduced and ter- minally induced MEL cells.

The refinements that have been made in the MEL system have greatly changed the extent of biochemical regulation that can be observed in this system. Whereas the response of MEL cells to induction had seemed surprisingly specific, it is now apparent that more numerous changes occur, suggesting that the MEL system is a realistic model of many processes of in vivo erythroid differentiation. In addition, the resolution of many proteins with altered expression in the MEL system will allow the correlation of regulatory changes at the DNA or RNA level with the actual levels of gene product, for gene products which could previously be assayed only indirectly.

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6. Devlin, R. B., and Emerson, C. P. (1978) Cell 13, 599-611 7. Sidhu, R. S. (1979) J. Biol. Chem. 2 5 4 , 11111-11118 8. Hoffman-Lieberman, B., and Sachs, L. (1978) Cell 14,825-834 9. O’Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021

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