CELL BIOCHEMISTRY AND FUNCTION

VOL. 9: 135-145 (1991)

Inositol Lipids in Friend Erythroleukemia Cells: Evidence for Changes in Nuclear Metabolism After Differentiation S. CAPITANIT, A. M. BILLIS, V. BERTAGNOLO?, M. PREVIATIT, M. MAZZONIT, L. M. NERITg AND F. A. MANZOLIS Istituti di Anatomia Umana Normale, TUniversita di Ferrara e di $Bologna, Italy $lstituto di Citonlorfoloyia clel CNR, c/o IOR, Bologna, Italy

The incorporation of 32Piinto phospholipids was studied in Friend erythroleukemia cells either induced or not to erythroid differentiation with 4 mM hexamethylenebisacetamide (HMBA). The effect of the differentiating agent on the recovery of radiolabelled phospholipids was compared in whole cells, isolated nuclei and nuclear matrix after in vivo labelling for 1 hr. The procedure employed for the isolation of nuclei was demonstrated to allow only negligible lipid redistribution caused by cell manipulations. Among the lipids extractable from nuclei, acidic phospholipids, and particularly polyphosphoinositides, were more represented than in whole cells, while small differences were found in the other phospholipid classes examined. The comparison between the uninduced and induced condition showed that the relative amounts of nuclear inositol lipids were modified by HMBA treatment of the cells, with a decreased recovery of phosphatidylinositol 4,s bisphosphate. These results indicate that phosphatidylinositol and its phosphorylation products synthesized in vivo show a different metabolism in nuclei and whole cells. They appear to be tightly bound nuclear components, also present in membrane-deprived nuclei and nuclear matrix, and are probably related to the nuclear events involved in erythroid differentiation. KEY WORDS-

Inositol lipids; differentiation; nucleus; Friend erythroleukemia cells.

INTRODUCTION Friend erythroleukemia cells (FELC) is a cell line inducible to erythroid differentiation by a number of agents,' among which some planar-polar compounds show high potency and effect numerous cell changes during the early steps of the lag phase that precedes commitment.2s3The key regulating steps of the differentiation process have not been identified yet in a priority scale, and much uncertainty exists concerning the molecular mechanism responsible for commitment and differentiation. Conflicting results have been reported about the lipid composition of the cell, after chemically induced differentiation. The changes appear to involve phospholipids, e.g. phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and triacylglycerol~.~.~ Studies focused on inositol liAddressee ror correspondence: Silvano Capitani, Istituto di Anatomia Umana Normale, Via Fossato di Mortara, 66,44100 Ferrara, Italy. 0263-6484/91/020135-11 $05.50 1991 by John Wiley & Sons, Ltd.

pids have indicated that this lipid class appears modified after treatment of cultured cells with dimethylsulfoxide (DMSO), and represents one of the early targets of the differentiating agent.' Differentiation-linked changes of inositol lipid metabolism have also been found at the nuclear level, as monitored by in oitro phosphorylation of isolated FELC nuclei: suggesting that the phosphoinositide cycle, which plays a crucial role in the generation of intracellular second messengers, is involved in the control of the differentiation pathway at the nuclear level. On the basis of these data and of previous evidence that phospholipids are involved in the regulation of gene expre~sion,~ can stimulate RNA synthesis' and are components of the nuclear we have studied the phospholipid metabolism in nuclei of Friend cells either induced or not to erythroid differentiation with HMBA. Particular attention has been focused on the synthesis and subcellular distribution of acidic phospholipids and mainly of inositol lipids.

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MATERIALS AND METHODS Source of Materials

Hexamethylenebisacetamide (HMBA) and DNase I were purchased from Sigma Chemical Co, St. Louis, MO; culture medium and fetal calf serum (FCS) from Biochrom Beteiligungs GmbH & Co, Berlin, FRG. Silica gel plates were from Merck, Darmstadt, FRG. [3H]-myo-inositol, carrier free 32Piwas obtained from Amersham Laboratories, England. All other reagents and chemicals were analytical grade. Cell Culture and Differentiation FEL cells were collected by centrifugation, washed in PBS and resuspended in TM-2 (10 mM Tris-HC1 pH 7.4,2 mM MgCl, and 0.5 mM phenylml- penicillin and 100 pg ml- streptomycin. The cells were routinely diluted at 2 x lo5 ml-' with fresh medium, and the inducer (4 mM HMBA) was added during the log phase of growth (i.e. 24 h later). The level of erythroid differentiation was scored by benzidine staining. For times of induction shorter than 96 h, aliquots of the cell culture were allowed to reach the final step of differentiation for evaluation of the hemoglobin content.

'

''

Cell Labelling

'

Carrier-free 3zPi was added to 50 pCi ml- after substitution of the medium with phosphate-free medium. Labelling was for 1 h, except when otherwise indicated. Cells were washed twice with phosphate buffered saline (PBS) and then either processed for extraction of lipids or for obtaining isolated nuclei and matrices. Nuclei and Nuclear Matrix Isolation FEL cells were collected by centrifugation, washed in PBS and resuspended in TM-2 (10 mM Tris-HC1 pH 7.4,2 mM MgCl, and 0.5 mM phenylmethylsulfonylfluoride (PMSF)). The hypotonic shock was followed by cooling at O"C, 0.3 per cent Triton X-100 was added and the suspension was then passed through a syringe (22 G needle). The concentration of MgC1, was adjusted to 5 mM, and the nuclear pellet was centrifuged at 7 0 0 g for 5 min. Nuclei were washed twice in TM-5 (10 mM Tris-HC1 pH 7.4, 5 mM MgCl, and 0.5 mM PMSF).

For nuclear matrix extraction, nuclei were digested with DNase I (30 U mg-' DNA) at 4°C for 45 min, twice extracted with LS buffer (10 mM TrisHC1 pH 7.4, 0.2 mM MgCl,, 1 mM PMSF) and twice with HS buffer (10 mM Tris-HC1 pH 7-4, 0.2 mM MgCl,, 2 M NaCl, 1 mM PMSF). The final matrices were washed in LS buffer. Determinations of protein, nucleic acid and phospholipids were as previously described.8 Criteria of Nuclear Purity

For ultrastructural analysis, the isolated nuclei were fixed with 2 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and postfixed in 1 per cent osmium tetroxide. After embedding in Epon, thin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss EM 109 electron microscope.' For cytoplasmic enzyme markers, S-nucleotidase and glucose-6-phosphatase activity was non significant, as previously To further rule out the possibility of cytoplasmic lipid cross-contamination, we evaluated the transfer to nuclei of radioactivity from microsomes containing [3H]-phosphatidylinositol (PI). The microsomal fraction from rat liver was labelled with C3H]-PI by incubation with 0.01 mCi mg-' protein of [3H]-myo-inositol (98 Ci mmol- ').14 Additional experiments were devised to assess contamination by polyphosphoinositides. Red blood cell (RBC) membranes were used as a source of phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP,), by in uitro labelling as described by Wells et aE." After analysis by thin layer chromatography (TLC), [32P]-labelled lipids were shown to be only PIP and PIP,. Either whole membranes washed free of unincorporated label, or acid-extracted lipids were used to mimic adventitious redistribution. In all cases, the radioactive contaminants were added to unlabelled homogenate at the time of syringe processing and the nuclei were purified as described. The nuclei were then extracted for inositol lipids, and the recovery of each single class was determined by TLC separation and liquid scintillation counting. Extraction and Characterization a/ Total Phospholipids Four ml of chloroform/methanol (2:1, v/v) were added to the samples and mixed overnight at 4°C.

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INOSITOL LIPIDS AND DIFFERENTIATION

The organic phases were separated and the pellets were extracted again with chloroform/methanol/ HCl (200:100:0~75,v/v) for 1 h at 4°C. The two organic phases were collected and evaporated under N,. The lipids, solubilized in 500 pl of chloroform, were washed three times in chloroform/methanol/H,O (3:48:47, v/v) and evaporated under N2. The run was carried out in chloroform/ methanol/NH,OH (65:25:4, v/v) on silica gel plates. Extraction and Characterization of Phosphoinositides

Acid-organic phases were separated according to Shaikh and Palmer.16 Briefly, 4 m l of chloroform/methanol/HC1(200: 1000-75, v/v) were added to 2501.11 samples and mixed thoroughly. Phase separation was obtained by adding 1 ml of 0.6 N HCl. Organic phases were washed twice with chloroform/methanol/0.6 N HCl (3:48:47, v/v), and evaporated under N,. The dry residue was dissolved in chloroform/methanol/H20 (75:25:2, v/v), and run on oxalate-impregnated plates with chloroform/ methanol/acetone/acetic acid/H,O (651 5:13:12:8, v/v). Autoradiography was on Kodak X-OMAT S film, and after identification by iodine staining' and comparison with authentic standards, the spots were scraped off the plates and counted by liquid scintillation. All values were corrected for background levels, determined on blank areas of surface equivalent to each spot.

RESULTS Contamination of nuclei and nuclear matrix by organelles and by plasma and cytoplasmic membranes was ruled out by different criteria. These included assay for marker enzymes, morphological analysis by phase-contrast and electron microscopy, and evaluation of cross contamination with labelled inositol lipids. According to the ultrastructural data, and as previously reported,6 the nuclear preparation was very pure and completely deprived of nuclear membranes (data not shown). The results in Table 1 indicate that with labelled membranes, either microsomes or red blood cell ghosts, low levels of inositol lipids were adventitiously redistributed on isolated nuclei. The contamination increased when purified mixtures of PIP and PIP, were used, but attained at most 0.8 per cent of the input. Since cell labelling yielded radioactive lipids in natural cell membranes, the contamination experiments with microsomes or red blood cell ghosts best mimicked the lipid environment present in the cells. Indeed the contribution of extranuclear material to the recovery of inositol lipids in the nuclei was likely to be far less than 0-8 per cent, largely below the levels found in the experiments described. The 32Piuptake into a TCA precipitable form by FELC is shown in Figure 1, which indicates that the labelled phosphate incorporation reached a plateau after 1 h, a time that should ensure the equilibration of the 32Piwith the endogenous ATP pool'* and likely corresponded to a steady-state in

Table 1. Estimate of contamination by inositol lipids of FELC nuclei occurring during the cell fractionation.

['HI-PI microsomes 3ZP-labelledRBC ghosts* 'ZP-PIP? 32P-PIP2t

Input added to cells cpm mg-' DNA

Recovery in nuclei cpm mg-' DNA

% of input

228 571 172 615 100 215 29 157

298 601 705 233

0-13 0.35 0-70 0.80

Microsomes, RBC ghosts and polyphosphoinositides were included in the cell suspension at the beginning of the nuclear extraction procedure. The experiments were performed with 300 x lo6 cells, i.e. an amount comparable to that routinely employed in in uiuo labelling. *Whole RBC membranes. bearing approximately 60 per cent of radioactivity as PIP and PIP,. t Polyphosphoinositides extracted from RBC ghosts, showing a PIP/PIP, ratio of 3.5.

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S. CAPlTANl ET AL.

0

20

60

40

80

100

120

time (min)

Figure 1. Time-course incorporation of 32Piinto acid-precipitable form by FEL cells. Cells were incubated with 5OpCi ml-' of isotope and aliquots were stopped with 20 per cent TCA, 5 per cent Na pyrophosphate at the indicated times. Precipitated radioactivity was determined by liquid scintillation counting. Mean values obtained from four separate experiments, with S.D. < 12 per cent.

terms of ATP availability, even though this reflected total incorporation and could not be related to lipid synthesis. To select labelling conditions suitable for optimal nuclear recovery of inositol lipids, cells were incubated with 32Pifor 1 h and 24 h. After 24 h, the phospholipid pattern was quite different from that at 1 h. Of the lipid extractable under acidic conditions, PE, PC, phosphatidic acid (PA) and PI were largely prevalent in comparison to polyphosphoinositides. Considering inositol lipids, after 1 h the nucleus contained a greater amount of radioactivity in PIP and PIP, than in PI, suggesting that phosphorylation events in the isolated nuclei were much more effective than in whole cells. After 24 h of incubation with "Pi more PI was present in the nucleus, whereas the recovery of labelled PIP and PIP, deeply decreased: this was essentially the case also,for whole cells (Figures 2 and 3). Since we were interested in fast changes of inositol lipid metabolism mostly related to polyphosphoinositide phosphorylation, we selected the 1 h labelling condition

after various differentiation times of cells cultured in the presence of 4 mM HMBA. The 32Pi distribution in FELC and purified nuclei indicates that after 1 h of labelling the nucleus incorporated about 50 per cent of the whole cell TCA-precipitable radioactivity (Table 2). As demonstrated by the total phospholipid recovery of Table 3, most of the cell radioactivity ( > 96 per cent) was obviously incorporated by non-lipid molecules. The amount of phospholipids extractable from isolated nuclei was only a few percent of that of whole cells. After HMBA induction, the labelled phospholipid recovery was reduced in whole cells, according to other findings." The presence of HMBA produced slight changes in the labelling of various phospholipids, in partial agreement with results obtained after differentiation induced by DMS0.4 However, the most striking feature was that nuclei were enriched, with respect to cells, in the polar phospholipid classes, such as PA, phosphatidylserine (PS) and PI.

139

INOSITOL LIPIDS AND DIFFERENTIATION

-

1

PA-

P€

-

PC

-

2

3

4

Figure 2. Representative autoradiogram of acid-organic extractable phospholipids separated on oxalate-impregnated silica gel plate. Cells were labelled with 32Pifor 1 h and 24 h and processed as described in Materials and Methods for nuclear purification and extraction of lipids. The amount ofcpm loaded was approximately 60 OOO for each lane. Conditions: 1 h of labelling, whole cells (lane 1) and nuclei (lane 2); 24 h of labelling, whole cells (lane 3) and nuclei (lane 4).

By comparing all lipid spots identifiable in acidic extracts, i.e. the procedure for extraction of phosphoinositides, it was further demonstrated that the labelled inositol lipids were a class highly represented in nuclei (Table 4). The actual recovery of polyphosphoinositides indicated that PIP and PIP, in nuclei reached even higher levels (Table 5). The relative ratios of inositides, determined after separation on TLC plates and spot scraping, are reported in Table 6. By considering the differences between control and HMBA-induced cells, it can

be observed that the differentiated state corresponds to a reduction of PIP, in nuclei. To verify the presence of these changes also at different times after treatment with HMBA, the percent relative composition of phosphoinositides was studied at 48 h, at 1 h (early stage), at 24 h (when commitment had already occurred) and at 96 h (terminal differentiation stage). The results, plotted in Figure 4, indicated that PI labelling increased for all times explored after HMBA treatment, except for the terminal differentiation stage

140

S . CAPITANI ET AL.

CELLS 280

2 0 240

220 200 180 160

140 120 100

40 20 0

12 11 10 0

7 6

4

3 2 1

0

Figure 3. Recovery of single inositol lipid classes in whole cells and isolated nuclei after in uiuo labelling as described in Figure 2.

141

INOSITOL LIPIDS AND DIFFERENTIATION Table 2. Total TCA-precipitable incorporation of 32Pi in FEL cells and nuclei. Cell mg-' DNA)

(cpm x Control HMBA

Nucleus mg-' DNA)

(cpm x

5303 f 514 5598 f 776

Ratio cell/nucleus

2984 f 720 2902 f 623

1.78 1.92

Control and 48-h induced cells were incubated with 50pCi ml-' of carrier-free 32Pi in phosphate-deprived medium and processed for isolation of nuclei. Acid-insoluble radioactivity was determined by adjusting cell and nuclear suspension to 20 per cent TCA, 5 per cent pyrophosphate and washing repeatedly. Values are mean of three determinations f S.D.

Table 3. Effect of HMBA on "Pi incorporation in total phospholipids and relative percentage distribution of lahel among phospholipid classes in whole FEL cells and isolated nuclei. Cells

"Pi incorporation in total phospholipids cpm x lo-' mg-' DNA 0 ,'o1

Nuclei

Control

HMBA

Control

HMBA

174.2 & 23.4 100

11 1.3 f 18.3

63.9*

4.4 f 0.6 100 2.52

4.2 f 0.6 93.8 3.77

40.2 f 4.1 5.3 f 0.6 8.6 f 1.2 10.0 f 2.3 35.8 +_ 3.8

42 f 2.2 6.3 f 0.9 8.8 +_ 1.8 7.4 f 1.9 35.4 & 1.2

29.6 f 3.9 7.4 f 2.2 7.6 f 1.0 5.2 f 2.2 50.3 f 1.6

31.4 5.6 7.8 f 2.5 9.8 f 2.8 4.9 f 1.3 45.8 f 1.0

"/o of nucleus in respect to cell RelativeA/: of phospholipid classes PE PG PC SM PA + PS + PI ~

~

~~

~~~~

Cells were labelled with 3 2 Pfor ~ 1 h in phosphate-free medium after an induction time of 48 h. Values are means of three experiments 5 S.D. * Paired t-test versus control is significant for cells ( p < 0.01).

Table 4. Effect of erythroid differentiation on 32Pi incorporation in acid-organic extractable phospholipids from FEL cel!s and nuclei, and comparison of polyphosphoinositides and other phospholipids recovered. Cells

"Pi incorporation in acid-extractable lipids cpm x 1 O - j mg-' DNA

x

Nuclei

Control

HMBA

Control

HMBA

232.1 f 34.8 100

157.8 f 20.5 63*

8.8 f 1.2 100 3.79

6.3 f 0.8 71 3.99

56-1 & 9.9 43.9 & 9.9

52.8 f 10.5 47.2 f 11.6

784.6 f 10.8 $15.4 4.1

781.3 f 12.3 $18.7 f 3.7

of nucleus in respect to cell

Relative of inositol lipids and other acid extractable lipids PI + PIP + PIP, PA + PE + PC

Values are mean of three and five experiments, respectively, & S.D. Statistical significance was determined with a paired t-test. Significant differences were: * percentage of label recovery of induced versus uninduced cells (p < 0005, n = 3); t,$percentage of polyphosphoinositides (PI + PIP + PIP,) and other lipids (PA + PE + PC) of nuclei versus cells for both control and induced conditions (p < 0.025, n = 5).

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S. CAPITANI ET AL.

Table 5. Effect of erythroid differentiation on 32Pi incorporation into polyphosphoinositides from FEL cells and isolated nuclei. 32Pi incorporation in PIP and PIP, Cells

cpm x mg-' DNA

% % of nucleus in respect to cell

Nuclei

Control

HMBA

Control

HMBA

64 816 100

36951 *61

3375 100 5.2

2204 67 5.9

Statistical significance was determined with a paired t-test. Significant difference was: * percentage of label recovery of induced versus uninduced cells ( p < 0.01, n = 3).

Table 6. Relative percentage of PI, PIP and PIP, in whole FEL cells and isolated nuclei. Cells

PI PIP PIP,

Nuclei

Control

HMBA

Control

HMBA

16.2 & 5.5 64.0 f 22.3 20.7 2 5.9

19.4 f 7.3 67.7 & 24.0 14.0 f 5.1

7.3 & 3.9 61.4 f 7.8 31.0 f 4.3

10.5 f 4.2 72.3 f 8.4 *18.2 f 6.0

Values are mean of five experiments f S.D. * Paired t-test versus control is significant for nuclei ( p < 0.01).

> -

180

u1 0,

160

0,

140

-0

120

?

100

OP 20 ~

1

24

48

96 Hours

1

24

after

48 HMBA

96

1

24

48

~~

96

treatment

Figure 4. Effect of HMBA on inositol lipid levels in FEL cells and nuclei. Cells were treated with 4 mM HMBA for the indicated times, and then incubated in phosphate free-medium with carrier-free 32Pifor 1 h. Data are mean values from two or more separate experiments carried out at the various times. The levels of control undifferentiated cells and nuclei were given the value of 100. The difference between control and HMBA is statistically significant in the case of PIP, extracted from nuclei as determined by t-test ( p < 0.01) for all indicated differentiation times. Nuclei (0-0) Cells (.---.).

143

INOSITOL LIPIDS AND DIFFERENTIATION

when compared to the uninduced cells. PIP labelling underwent a slight increase, more evident in nuclei after longer induction times, while PIP, labelling was always reduced. For isolated nuclei, in particular, this drop of PIP, was higher than for cells.

Table 7. Relative percentage of PI, P I P and PIP, in FEL nuclear matrix.

PI PIP PIP,

Control

HMBA

22.7 & 4.4 48.0 0.9 29.3 k 4.0

31.1 & 5.8 49.9 & 3.0 *19.0 & 2.2

PA

Relative percentage of PI, P I P and PIP, in FEL nuclear matrix. Values are mean of four experiments S.D. * Paired f-test versus control matrix was significant ( p < 0.025).

PI

To further ascertain the actual presence of inositol-derived substrates and related enzymes at the nuclear level, we examined the inositide composition of the nuclear matrix obtained from FELC nuclei. The nuclear matrix is a non-chromatin structure obtained by nuclease digestion, salt extraction and detergent treatment, retaining the nuclear shape and size after depletion of more than 90 per cent of nuclear proteins and 99 per cent of DNA. The nuclear matrix preparations from FELC nuclei showed the following composition: 93.2 per cent protein, 0-9 per cent DNA, 0.1 per cent RNA and 5.8 per cent phospholipid. Again, in this protein and nucleic-acid depleted subnuclear structure, the qualitative recovery of phosphorylated lipis and the changes after HMBA treatment paralleled those observed in whole nuclei (Figure 5 and Table 7). Furthermore, the recovery of these lipids was much higher in matrices than in nuclei in terms of specific labelling/protein unit (data not shown), suggesting that inositides are tightly bound nuclear components regardless of the presence or absence of membranes, polycationic histones and most of the nuclear proteins.

pIp p I p2

DISCUSSION

OR

1

2

3

4

Figure 5. Autoradiogram of TLC-separated phospholipids, from nuclei and nuclear matrices obtained from uninduced and 48 h-HMBA-induced FEL cells. The amount of nuclei and matricvs extracted were: 1 mg of protein for lane 1 (uninduced nuclei) and lane 2 (induced nuclei); 0.18 mg for lane 3 (uninduccd matrix) and 0.21 mg lor lane 4 (induced matrix).

The comparison of phospholipid pattern after 1 h and 24h of 32Pi labelling indicated that more labelled PI was recovered from the nucleus after 24 h, when lipid metabolism was likely to be in a steady-state condition. This indicates that PI could enter the nucleus and gives a clue to the translocation of cytoplasmic lipids presumably mediated by transfer proteins, as shown by the nuclear PI enrichement mediated by PI transfer protein that has been observed in vitro.14 According to the fractionation studies, the results obtained with 32Piindicate that the nucleus takes

144 up about half of the total cell labelling. This is only partly explained by the relatively high nucleuscytoplasm volume ratio, which is near to 0-5 according to the results of image analysis (not shown). The nucleus appears to be a cell compartment deeply involved in the processing of phosphate groups, with a specific labelling higher than that of the cytoplasm in terms of radioactivity/ volume units. In HMBA-induced cells, total 32Pi incorporation does not change, while the incorporation into whole phospholipids and acid-organic soluble lipids is reduced. Lipid synthesis and phosphorylation seems to be a crucial aspect of the differentiation events, compared to the apparent insensitivity of the overall 32Piincorporation that is largely distributed on non-lipid molecules. The drop in lipid labelling after induction can be related to the reduced metabolic activity that parallels differentiation, but might also be of significance in terms of phosphorylatable molecules carrying informational content, such as polyphosphoinositides. The qualitative lipid composition differs strikingly between cells and nuclei, indicating that anionic phospholipids prevail in the nucleus and confirming previous results we have obtained on the involvement of acidic lipids in the nuclear function.8*20-2 Regarding the polyphosphoinositides, particularly significant is the reduction of PIP, after induction to differentiation. In considering the origin of the PI cycle components found at the nuclear level, PI synthesis can be reasonably ruled out in the nucleus,23 so that it can be assumed that nuclear PI is transported from the cytoplasmic compartment and that the 1-phosphate labelling is totally extranuclear. As to the PIP and PIP,, two possibilities can be envisaged: (i) a cytoplasmic synthesis followed by translocation to the nucleus and (ii) an intranuclear phosphorylation of PI. Our results cannot discriminate between these two possibilities. However, in uitro experiments demonstrated that inositol lipid phosphorylation occurs in rat liver nuclear envelope^,'^ isolated nuclei and subnuclear fractions” and in nuclei obtained from FEL cells and 3T3 fibroblast^.^.^^*^' The reduced PIP, phosphorylation observed in both cells and nuclei after short-term labelling studies with 32Pi can be considered a marker of the differentiated state, being true for all the times explored after treatment with HMBA, from 1 to 96 h. Interference with the PI cycle might be a very early event in FELC differentiation. With a poly-

S. CAPITANI ET AL.

peptide inducer, it has been suggested that PIP, breakdown in the entire cell takes place within seconds after treatment.28 When nuclei have to be isolated, rapid changes due to differentiating agents cannot be detected, at least on the time scale usually employed to study rapid PIP, breakdown in response to agonists. Additional drawbacks to looking at the PIP, hydrolysis come from the difficulty of detecting the production of inositol phosphates by 32Plabelling, and from the fact that they are lost as water-soluble molecules during the isolation of nuclei. The bulk of these results indicates that inositol lipids are also components of the nucleus, that their metabolism differs in whole cells and isolated nu;lei, and that these metabolites are involved in the nuclear differentiation events related to erythroid induction. In addition, they allow us to speculate that HMBA affects the inositol cycle to a much higher extent in the nucleus than in the whole cell. This suggests a mechanism of action different from that of many cell agonists, such as peptide hormones and neurotransmitters, that seem primarily to interact with the plasma membrane inositol lipid cycle. ACKNOWLEDGEMENTS This work was supported by C.N.R. (grants n. 88.01803.04, 89.04123.04 and n. 89.02470.04) and M.P.I. (40 per cent and 60 per cent). REFERENCES 1. Marks, P. A. and Rifkind, R. S. (1978). Erythroleukemic differentiation. Ann. Rev. Biochem., 47, 419-448. 2. Faletto, D. L., Arrow, A. S. and Macara, I. G. (1985). An early decrease in phosphatidylinositol turnover occurs on induction of Friend cell differentiation and precedes the decrease in c-myc expression. Cell., 43, 315-325. 3. Lannigan, 0. A. and Knauf, P. A. (1985). Decreased intracellular Na’ concentrations in an early event in murine erythroleukemia cell differentiation. J. Biol. Chem., 260,7322-7324. 4. Rittmann, L. S., Jelsema, C. L., Schwartz, E. L., Tsiftsoglou, A. S. and Sartorelli, A. C. (1982). Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide. J . Cell. Physiol., 110, 50-55. 5. Fallani, A., Arcangeli, A. and Ruggeri, S. (1988). Lipid characteristics of Friend erythroleukemia cells differentiated by dimethylsulfoxide or hexamethylenebisacetamide, and of non inducible clones treated with the inducers. Biochem. J., 252,917-920. 6. Cocco, L., Gilmour, R. S., Ognibene, A., Letcher, A., Manzoli, F. A. and Irvine, R. F. (1987). Synthesis of

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lNOSITOL LIPIDS AND DIFFERENTIATION

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Received in revised form 28 October 1990 Accepted 9 January 1991