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Changes in nuclear inositol phospholipids induced in intact cells by insulin-like growth factor I
Vol. 159, No. 2, 1989 March 15, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 720-725
CHANGES IN NUCLEARINDSITOL PHGSPRGLIPIDS INDUCRDIN INTACTCXLLS
BY INSULIN-LIKETGRWITiFACIORI
Lucia Coccal ’ 2 , Albert0 M. Wartelli2, R. Stewart Gilmu?, Andrea Ggnibene4 , Francesco A. Wanzoli2 and Robin F. Irvine3 ‘Istituto
di Norfologia
‘Istituto
di
Anatomia
UmanaNormale, university Umana Nomale,
Irnerio,
University
of Chieti, of
Italy
Bologna,
via
48, 40126 Bologna, Italy
‘Department of Biochemistry,
AFRC Institute
of Animal Physiology
and
Genetics Research, Babraham, Cambridge CR2 4AT, U.K. 41stituto Received
di Citomorfologia
January
16,
CNR, c/o 1st. Rizzoli,
Bologna, Italy
1989
Swiss 3T3 cells were labelled for 36 hours with high levels of 3H-myo-inosi to1 and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 minutes, but not for 4 hours, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a slight decrease in PtdIns and mre marked decreases in PtdInsP and PtdInsP . These effects were not seen if isolated nuclei were -incubated ;sfth IGF-I and bombesin. We interpret these results to mean that rapid mass changes occur in nuclear inositol phospholipids in the early stages of the mitotic response. 0 1989Academic Press,Inc.
We have observed that isolated nuclei can incorporate ATP into inositol phospholipids in vitro and that changes occur in this incorporation during cell growth and differentiation (1,3). For example, in Swiss-mouse 3T3 cells we have shown that IGF-I pretreatment of the cells, results in a transient decrease in incorporation from [’ ‘PI-y-ATP into PtdInsP and PtdIns_P, when isolated nuclei are studied in vitro. No significant parallel change is seen in whole cell homogenates. However, these experiments do not establish that any changes actually occur in the levels of nuclear inositol lipids in the intact cells. We have therefore labelled intact cells with Abbreviations: PtdcXI; Phosphatidic acid; Phosphatidylinositolphosphate; inositolbisphosphate; IGF-I, calf serum
PtdIns, Phosphatidylinositol; PtdInsP, PtdInsPl, PhosphatidylInsulin-Tike growth factor I; FCS, Foetal
0006-291X/89$1.50 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
720
Vol. 159, No. 2, 1989
@!&myo-inositol
BIOCHEMICAL
to
a level
nuclear inositol lipids levels of incorporation. MATERIALS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sufficient
to
detect
and have studied the effect
radioactivity of IGF-I
in on the
AND NETHODS
Cell culture and in vivo labelling with 3BSwiss 3T3 fibroblasts were grown in sified Du minimum essential medium (D-MEM) supplemented with 10% fetal calf serum and containing 100 U&l penicillin, 0.1 n@nl streptonrycin and 2.5 q/nil amphotericin B. Confluent cells were washed with FCS free medium containing 1% bovin erum albmin and were labelled in FCS free medium for 36 hours with PQ myo-inositol (10 fli) ml; specific activity 80 Ci/m mol, Amersham, U.K.). Growth factors were added as follows: (i) IGF-I 20 rig/ml; (ii) bombesin 1nM; (iii) bombesin 1 nW plus IGF-I 20 rig/ml. The incubation was at 37O. Isolation of 3T3 fibroblast nuclei I”Ifmyo-inositol incorporation was stopped by washing the cells twice with cold buffered saline pH 7.4, and cells detached by mild trypsinisation were processed for nuclear purification as described by Cocco et al. (3). The highly pure nuclei were free of nuclear envelope and cytoplasmic markers according to criteria described previously (3). Lipid extraction and t.1.c. separation Nuclei and intact cells were quenched with 10% trichloroacetic acid and the resulting pellets were washed twice with distilled water and extracted with tcidic chlorofornmethanol as described in Uawson and Eichberg (4). H labelled lipids were analysed by t.1.c. on 1% oxalate-sprayed plates developed with chloroform-n&hanol-wateramonia (45:35:8:2, by vol.) (5). T.1.c. plates wre sprayed with En-hancer (Du Pont, NEW, W. Germany) and fluorographed at -8OV. Spots corresponding to internal lipid standards were scraped off, extracted with 1.5 mls 0.6~ HCl-Methanol (60:40 by vol.) for 48 hours with gentle stirring and counted with a liquid scintillation counter using 9 mls of Packard Pica-Fluor 40 scintillation cocktail. Protein assay Protein concentration (6).
was measured as described by Lowry et al.
HESULTS After detected identified
36 hours incubation with~i$my o-inositol,
radioactivity
was
in three phospholipids found in nuclei of 3T3 cells by co-chromatography with standards as PtdIns, PtdInsE and
PtdInsP, (Fig. lA) . When cells were treated for 2 mins with IGF-I and bombesin there was a small decrease in counts in PtdIns and a larger These results are consistent with decrease in PtdInsP and PtdInsgz. in vitro found the effects of IGF-I on 132PI-y-ATP nuclear labellihg previously (3). With both experimental approaches the effect disappears after 4 hours treatment with mitogen. That these inositol phospholipids are truly in the nucleus and not due to contamination, is supported by the observations that (a) intact cells do not show the 721
BIOCHEMICAL
Vol. 159, No. 2, 1989
Fig. 1
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4h 2min 4h C 2min C Incorporation of P&nyc-inositol into pclyphosphoinositides from SWISS3T3 nuclei and intact cells.
Panels A and B show the fluorography of 3H-labelled inositol lipids from purified nuclei and total cell extracts respectively. Padiolabelled compoundswere identified as 1, PtdIns; 2, PtdInsP; 3, PtdInsEr . The spot above PtdIns_P is unidentified, but pro&My represents
lyso-Ptdhs.
same changes (Fig. 1B) , and (b) the ratio of PtdInsg and PtdIns_P, is consistently different between nuclei and whole cell homogenate (Fig. IA, B and Table 1); in nuclei the PtdInszP,:PtdInsP ratio is 3.91, Also in agreement with previous whilst in intact cells it is 0.71. data is the observation that IGF-I can do this alone but it is more effective in combination with bombesin (Table 1). That this change is not a trivial effect of growth factors directly on the nucleus is demonstrated by the experiments showing that incubation of isolated nuclei in vitro with IGF-I and/or bombesin in the presence of [ 32P]-y-ATP does not affect the polyphosphoinositide labelling (Fig. These data suggest to us that these effects arise indirectly 2). from receptor binding on the cell surface , and not by a direct effect on the nucleus of internalised mitogen as has been suggested from observations on other polypeptide growth’factors (7). When nuclei are isolated from cells which have been treated with IGF-I and bombesin, and are then incubated in vitro with [ 32P]-rATP as described previously (3) there is a consistent decrease in incorporation into PtdIns_P and PtdInsE2 (Fig. 2B). These differences can be largely abolished by the addition of appropriate substrate to the incubation mixture. This indicates that it is probably the lipid substrates that are changing (as shown in Fig. IA) rather than the 722
Vol. 159, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 In vivo labelling of polyphosphoinositides frcm purifed nuclei&d intact Swiss 3T3 cells stimulated with growth factors: IGF-I (20 ng/tp1) and bcmbesin (1 molar) (dp@ng protein x 10m3) TWI!ALCELLEXTRACT Experimental Conditions
PtdIns
PtdInsP
Control 2 min +Bcmbesin2 min +IGF-I 2 min +IGF-I +bcnnbasin2 min Control 4 h +Bombesin4 h +IGF-I 4 h +IGF-I +bc&esin 4 h
15973 15530 15240 15625 23253 23535 23957 24915
658 645 631 681 1092 1076 1238 1111
PtdInsEP, 470 475 464 465 547 590 542 620
NUCLEAR EXTRACT (dpq/mgprotein x lo-') PtdIns Control 2 min +Bombasin2 min +IGF-I 2 min +IGF-I +bcmbasin2 min Control 4 h +Bombasin4 h +IGF-I 4 h +IGF-I +bcmbasin4 h -
3385 4273 2590 2520 3352 5141 5181 5149
PtdInsP 116 142 2:
129 165 164 131
Cells were labelled with 3H-myo-inositol and Methods for 36 hrs or 40 hrs in the tion experiments, respectively. Similar in an identical experiment.
PtdInsg, 454 480 362 179 438 620 398 474
as detailed in Materials 2 mins and 4 h stimularesults were obtained
kinases, a result consistent with our previous observations on uudifferentiated Friend cells (1). If that is so, and thus PtdIns as well as the polyphosphoinositides is indeed decreasing, then a phospholipase C-catalysed hydrolysis must be considered as a likely mechanism. DISCUSSION Our present data demonstrate that in Swiss 3T3 cells a mitogenic combination of IGF-I and bombesin (81, induces a very rapid decrease of r $labelled nuclear PtdInsE and PtdInsgP, . Although we cannot demonstrate that isotopic equilibria has been reached in these experiments, the rapidity of the effect after 36 hours labelling leads us to suggest that these decreases in radiolabelling reflect changes in mass. Thus we can suggest that the changes in 3‘P-incorporation from [32P]-yATP that we previously observed in nuclei incubated -in vitro (l-3) are indeed reflections of changes in inositol lipids in the nucleus in vivo. In summary, we have inferred from previous experiments that isolated nuclei contain inositol phospholipids and here we have sought 723
Vol. 159, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a Fig.
2
Incorporation of j2P into nuclei mcubated in vitro.
b cd phospholipids
e in
f Swiss
3T3 cell
Panel A shms the autoradiogram of t.1.c. sfpration of lipid extracts obtained from in vitro incubation with [ PI-y-ATP of nuclei from confluent 3T3 c(a7. in the Presence of bcmbesin 1nM (b). IGF-I 20 ns/ml (c), IGF-I 20 ng&l plus lknbesin lnh (d). The phdsphorylation conditions were the same as described previously (3). Radioactivity proteins in PtdGH, PtdInsP and PtdInsP, expressed as cpr@g nuclear was respectively (a) 411 + 50, 18021 + 1509, 1626 f 160: (b) 498 f 55, 19074 f 1520, 1709 + 165; (c) 504 f 48, 19543 f 1851, 1780 f 167; (d) ccmpounds were 499 + 47, 21041 + 1933, 1824 + 175. Radiolabelled as 1, PtdofI; 2, PtdInsEP; 3, PtdInsEz. l?ie results are the ide&fied mean of 3 different experiments + 8.D. In this particular experiment nuclei frcfa control (a) and Panel 8. with IGF-I 20 rig/ml plus bo&esin 1hM (d) were also cells stimulated incubated in the presence of 100 PM PtdInsP (b and e) or 100 ~JM PtdIns (c and f). Radioactivity expressed as ?psyhq nuclear proteins in (a) 726 + 69, 20786 + PtdCH, PtdInsP and PtdInsP was respectively:
1980, 3456 +300; (b) 369a'f 310, 17636 + 1801, 171495-+ 17201; (c) 526 f 51, 68911 + 5800, 7496 ?: 955; (d) 7i9 f 62, 7659 k-791, 1066 + 908; (e) 3869 f 375, 12467 f 1195, 175458 f 16995; (f) 3796 f 3825 56597 + 6020, 4179 + 605. Numbering of radiolabelled lipids are as in
panel
A.
The results
are the mean of 3 different
experiments
f 6.~.
to establish this by more direct means. Unfortunately, to attain sufficiently high levels of incorporated radioactivity requires numbers of cells and amounts of labelled inositol which preclude routine use of this method. Nevertheless, having once established in these present experiments that inositol lipids are in the nucleus and that their mass levels probably change following mitogenic stimulation, we can now use more convenient and indirect methods to study this intriguing phencmenon. ACKNOWLEDGMENTS We wish to thank lair A. Fantazzini for his skilled technical assistance. This work was supported by a CM grant PF Biotechuol. 724
Vol. 159, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCE
1. 2. 3. 4. 5. 6. 7. 8.
cocco, L., Gilmour, R.S., Ggnibene, A., Letcher, A.J., Manzoli, F.A. and Irvine, R.F. (1987). Biochem. J. 248, 765-770. Manzoli, F.A., Cocco, L., Capita&, S., Marai, NM., Mazzotti, G Barnabei, 0. (1988). Adv. Enzyme Reg. &7, 83-91. &co, L., Martelli., A.M., Gilmour, R.S., Ognibene, A., Manzoli, Biochem. Biophys. Res. Commun. F.A. and Irvine, R.F. (1988). 154, 1266-1272. Dawson, R.M.C. and Eichberg, J. (1965). Biochem. J. 96, 639-643. Irvine, R.F. (1986). In Phosphoinositides d Receptor Mechanisms (Putney, J.W., Jr., ea.)., pp 89-107, Alan Liss, New York. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chem. 193, 265-275. Rakowicz-Szulczynsica, m., Rodeck, U., Herlyn, M. and Koprowski, H. (1986). Proc. Natl. Acad. Sci. USA, 83, 3728-3732. Corps, A.N. and Brcwn, K.D. (1988). Biochem. J. 252, 119-125.
725
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 720-725
CHANGES IN NUCLEARINDSITOL PHGSPRGLIPIDS INDUCRDIN INTACTCXLLS
BY INSULIN-LIKETGRWITiFACIORI
Lucia Coccal ’ 2 , Albert0 M. Wartelli2, R. Stewart Gilmu?, Andrea Ggnibene4 , Francesco A. Wanzoli2 and Robin F. Irvine3 ‘Istituto
di Norfologia
‘Istituto
di
Anatomia
UmanaNormale, university Umana Nomale,
Irnerio,
University
of Chieti, of
Italy
Bologna,
via
48, 40126 Bologna, Italy
‘Department of Biochemistry,
AFRC Institute
of Animal Physiology
and
Genetics Research, Babraham, Cambridge CR2 4AT, U.K. 41stituto Received
di Citomorfologia
January
16,
CNR, c/o 1st. Rizzoli,
Bologna, Italy
1989
Swiss 3T3 cells were labelled for 36 hours with high levels of 3H-myo-inosi to1 and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 minutes, but not for 4 hours, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a slight decrease in PtdIns and mre marked decreases in PtdInsP and PtdInsP . These effects were not seen if isolated nuclei were -incubated ;sfth IGF-I and bombesin. We interpret these results to mean that rapid mass changes occur in nuclear inositol phospholipids in the early stages of the mitotic response. 0 1989Academic Press,Inc.
We have observed that isolated nuclei can incorporate ATP into inositol phospholipids in vitro and that changes occur in this incorporation during cell growth and differentiation (1,3). For example, in Swiss-mouse 3T3 cells we have shown that IGF-I pretreatment of the cells, results in a transient decrease in incorporation from [’ ‘PI-y-ATP into PtdInsP and PtdIns_P, when isolated nuclei are studied in vitro. No significant parallel change is seen in whole cell homogenates. However, these experiments do not establish that any changes actually occur in the levels of nuclear inositol lipids in the intact cells. We have therefore labelled intact cells with Abbreviations: PtdcXI; Phosphatidic acid; Phosphatidylinositolphosphate; inositolbisphosphate; IGF-I, calf serum
PtdIns, Phosphatidylinositol; PtdInsP, PtdInsPl, PhosphatidylInsulin-Tike growth factor I; FCS, Foetal
0006-291X/89$1.50 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
720
Vol. 159, No. 2, 1989
@!&myo-inositol
BIOCHEMICAL
to
a level
nuclear inositol lipids levels of incorporation. MATERIALS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sufficient
to
detect
and have studied the effect
radioactivity of IGF-I
in on the
AND NETHODS
Cell culture and in vivo labelling with 3BSwiss 3T3 fibroblasts were grown in sified Du minimum essential medium (D-MEM) supplemented with 10% fetal calf serum and containing 100 U&l penicillin, 0.1 n@nl streptonrycin and 2.5 q/nil amphotericin B. Confluent cells were washed with FCS free medium containing 1% bovin erum albmin and were labelled in FCS free medium for 36 hours with PQ myo-inositol (10 fli) ml; specific activity 80 Ci/m mol, Amersham, U.K.). Growth factors were added as follows: (i) IGF-I 20 rig/ml; (ii) bombesin 1nM; (iii) bombesin 1 nW plus IGF-I 20 rig/ml. The incubation was at 37O. Isolation of 3T3 fibroblast nuclei I”Ifmyo-inositol incorporation was stopped by washing the cells twice with cold buffered saline pH 7.4, and cells detached by mild trypsinisation were processed for nuclear purification as described by Cocco et al. (3). The highly pure nuclei were free of nuclear envelope and cytoplasmic markers according to criteria described previously (3). Lipid extraction and t.1.c. separation Nuclei and intact cells were quenched with 10% trichloroacetic acid and the resulting pellets were washed twice with distilled water and extracted with tcidic chlorofornmethanol as described in Uawson and Eichberg (4). H labelled lipids were analysed by t.1.c. on 1% oxalate-sprayed plates developed with chloroform-n&hanol-wateramonia (45:35:8:2, by vol.) (5). T.1.c. plates wre sprayed with En-hancer (Du Pont, NEW, W. Germany) and fluorographed at -8OV. Spots corresponding to internal lipid standards were scraped off, extracted with 1.5 mls 0.6~ HCl-Methanol (60:40 by vol.) for 48 hours with gentle stirring and counted with a liquid scintillation counter using 9 mls of Packard Pica-Fluor 40 scintillation cocktail. Protein assay Protein concentration (6).
was measured as described by Lowry et al.
HESULTS After detected identified
36 hours incubation with~i$my o-inositol,
radioactivity
was
in three phospholipids found in nuclei of 3T3 cells by co-chromatography with standards as PtdIns, PtdInsE and
PtdInsP, (Fig. lA) . When cells were treated for 2 mins with IGF-I and bombesin there was a small decrease in counts in PtdIns and a larger These results are consistent with decrease in PtdInsP and PtdInsgz. in vitro found the effects of IGF-I on 132PI-y-ATP nuclear labellihg previously (3). With both experimental approaches the effect disappears after 4 hours treatment with mitogen. That these inositol phospholipids are truly in the nucleus and not due to contamination, is supported by the observations that (a) intact cells do not show the 721
BIOCHEMICAL
Vol. 159, No. 2, 1989
Fig. 1
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4h 2min 4h C 2min C Incorporation of P&nyc-inositol into pclyphosphoinositides from SWISS3T3 nuclei and intact cells.
Panels A and B show the fluorography of 3H-labelled inositol lipids from purified nuclei and total cell extracts respectively. Padiolabelled compoundswere identified as 1, PtdIns; 2, PtdInsP; 3, PtdInsEr . The spot above PtdIns_P is unidentified, but pro&My represents
lyso-Ptdhs.
same changes (Fig. 1B) , and (b) the ratio of PtdInsg and PtdIns_P, is consistently different between nuclei and whole cell homogenate (Fig. IA, B and Table 1); in nuclei the PtdInszP,:PtdInsP ratio is 3.91, Also in agreement with previous whilst in intact cells it is 0.71. data is the observation that IGF-I can do this alone but it is more effective in combination with bombesin (Table 1). That this change is not a trivial effect of growth factors directly on the nucleus is demonstrated by the experiments showing that incubation of isolated nuclei in vitro with IGF-I and/or bombesin in the presence of [ 32P]-y-ATP does not affect the polyphosphoinositide labelling (Fig. These data suggest to us that these effects arise indirectly 2). from receptor binding on the cell surface , and not by a direct effect on the nucleus of internalised mitogen as has been suggested from observations on other polypeptide growth’factors (7). When nuclei are isolated from cells which have been treated with IGF-I and bombesin, and are then incubated in vitro with [ 32P]-rATP as described previously (3) there is a consistent decrease in incorporation into PtdIns_P and PtdInsE2 (Fig. 2B). These differences can be largely abolished by the addition of appropriate substrate to the incubation mixture. This indicates that it is probably the lipid substrates that are changing (as shown in Fig. IA) rather than the 722
Vol. 159, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 In vivo labelling of polyphosphoinositides frcm purifed nuclei&d intact Swiss 3T3 cells stimulated with growth factors: IGF-I (20 ng/tp1) and bcmbesin (1 molar) (dp@ng protein x 10m3) TWI!ALCELLEXTRACT Experimental Conditions
PtdIns
PtdInsP
Control 2 min +Bcmbesin2 min +IGF-I 2 min +IGF-I +bcnnbasin2 min Control 4 h +Bombesin4 h +IGF-I 4 h +IGF-I +bc&esin 4 h
15973 15530 15240 15625 23253 23535 23957 24915
658 645 631 681 1092 1076 1238 1111
PtdInsEP, 470 475 464 465 547 590 542 620
NUCLEAR EXTRACT (dpq/mgprotein x lo-') PtdIns Control 2 min +Bombasin2 min +IGF-I 2 min +IGF-I +bcmbasin2 min Control 4 h +Bombasin4 h +IGF-I 4 h +IGF-I +bcmbasin4 h -
3385 4273 2590 2520 3352 5141 5181 5149
PtdInsP 116 142 2:
129 165 164 131
Cells were labelled with 3H-myo-inositol and Methods for 36 hrs or 40 hrs in the tion experiments, respectively. Similar in an identical experiment.
PtdInsg, 454 480 362 179 438 620 398 474
as detailed in Materials 2 mins and 4 h stimularesults were obtained
kinases, a result consistent with our previous observations on uudifferentiated Friend cells (1). If that is so, and thus PtdIns as well as the polyphosphoinositides is indeed decreasing, then a phospholipase C-catalysed hydrolysis must be considered as a likely mechanism. DISCUSSION Our present data demonstrate that in Swiss 3T3 cells a mitogenic combination of IGF-I and bombesin (81, induces a very rapid decrease of r $labelled nuclear PtdInsE and PtdInsgP, . Although we cannot demonstrate that isotopic equilibria has been reached in these experiments, the rapidity of the effect after 36 hours labelling leads us to suggest that these decreases in radiolabelling reflect changes in mass. Thus we can suggest that the changes in 3‘P-incorporation from [32P]-yATP that we previously observed in nuclei incubated -in vitro (l-3) are indeed reflections of changes in inositol lipids in the nucleus in vivo. In summary, we have inferred from previous experiments that isolated nuclei contain inositol phospholipids and here we have sought 723
Vol. 159, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a Fig.
2
Incorporation of j2P into nuclei mcubated in vitro.
b cd phospholipids
e in
f Swiss
3T3 cell
Panel A shms the autoradiogram of t.1.c. sfpration of lipid extracts obtained from in vitro incubation with [ PI-y-ATP of nuclei from confluent 3T3 c(a7. in the Presence of bcmbesin 1nM (b). IGF-I 20 ns/ml (c), IGF-I 20 ng&l plus lknbesin lnh (d). The phdsphorylation conditions were the same as described previously (3). Radioactivity proteins in PtdGH, PtdInsP and PtdInsP, expressed as cpr@g nuclear was respectively (a) 411 + 50, 18021 + 1509, 1626 f 160: (b) 498 f 55, 19074 f 1520, 1709 + 165; (c) 504 f 48, 19543 f 1851, 1780 f 167; (d) ccmpounds were 499 + 47, 21041 + 1933, 1824 + 175. Radiolabelled as 1, PtdofI; 2, PtdInsEP; 3, PtdInsEz. l?ie results are the ide&fied mean of 3 different experiments + 8.D. In this particular experiment nuclei frcfa control (a) and Panel 8. with IGF-I 20 rig/ml plus bo&esin 1hM (d) were also cells stimulated incubated in the presence of 100 PM PtdInsP (b and e) or 100 ~JM PtdIns (c and f). Radioactivity expressed as ?psyhq nuclear proteins in (a) 726 + 69, 20786 + PtdCH, PtdInsP and PtdInsP was respectively:
1980, 3456 +300; (b) 369a'f 310, 17636 + 1801, 171495-+ 17201; (c) 526 f 51, 68911 + 5800, 7496 ?: 955; (d) 7i9 f 62, 7659 k-791, 1066 + 908; (e) 3869 f 375, 12467 f 1195, 175458 f 16995; (f) 3796 f 3825 56597 + 6020, 4179 + 605. Numbering of radiolabelled lipids are as in
panel
A.
The results
are the mean of 3 different
experiments
f 6.~.
to establish this by more direct means. Unfortunately, to attain sufficiently high levels of incorporated radioactivity requires numbers of cells and amounts of labelled inositol which preclude routine use of this method. Nevertheless, having once established in these present experiments that inositol lipids are in the nucleus and that their mass levels probably change following mitogenic stimulation, we can now use more convenient and indirect methods to study this intriguing phencmenon. ACKNOWLEDGMENTS We wish to thank lair A. Fantazzini for his skilled technical assistance. This work was supported by a CM grant PF Biotechuol. 724
Vol. 159, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCE
1. 2. 3. 4. 5. 6. 7. 8.
cocco, L., Gilmour, R.S., Ggnibene, A., Letcher, A.J., Manzoli, F.A. and Irvine, R.F. (1987). Biochem. J. 248, 765-770. Manzoli, F.A., Cocco, L., Capita&, S., Marai, NM., Mazzotti, G Barnabei, 0. (1988). Adv. Enzyme Reg. &7, 83-91. &co, L., Martelli., A.M., Gilmour, R.S., Ognibene, A., Manzoli, Biochem. Biophys. Res. Commun. F.A. and Irvine, R.F. (1988). 154, 1266-1272. Dawson, R.M.C. and Eichberg, J. (1965). Biochem. J. 96, 639-643. Irvine, R.F. (1986). In Phosphoinositides d Receptor Mechanisms (Putney, J.W., Jr., ea.)., pp 89-107, Alan Liss, New York. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chem. 193, 265-275. Rakowicz-Szulczynsica, m., Rodeck, U., Herlyn, M. and Koprowski, H. (1986). Proc. Natl. Acad. Sci. USA, 83, 3728-3732. Corps, A.N. and Brcwn, K.D. (1988). Biochem. J. 252, 119-125.
725