AbstractThe effect of interferon-α on Daudi lymphoma cells either sensitive or resistant to the action of this cytokine has been analysed in terms of phospholipase C (PLC) and D (PLD) activities. Results have shown a combined modulation of PIP2-specific phospholipase C and phospholipase D. In particular, a decreased activity of PIP2-specific PLC has been found, concomitant to a PLD-mediated phosphatidylcholine hydrolysis, suggesting that the intracellular signaling activated by interferon in Daudi cells involves a phospholipase D/phosphohydrolase pathway

Bareggi, R.

Cataldi, A.

di Primio, R.

Lisio, R.

Miscia, S.

Rana, R.

Toto, N.

Elsevier - Publisher Connector 

Volume 313, number 3~ 210-212 FEBS 11792 
© 1992 Federation of European Biochemical Societies 00145793/92/$5.00 
November 1992 
Interferon-mediated intracellular signalling 
Modulation of different phospholipase activities in Burkitt lyrnphorna cells 
A. Cataldi ~, R. Rana ~, R. di Primio", R. Bareggi ~, R. Lisio ", N. Toto" and S. Mis¢ i# 
"Istituto di Morfologia Umana Normale, Universith di Chieti, Chieti, Italy and ~lstituto di Anatomia Umana Normale, Unh,ersitd 
di Trieste, Trieste, Italy 
Received 1 September 1992; revi~ed version received 5 October 1992 
Tire effect of interferon-0: on Daudi lymphoma cells either sensitive or resistant to the action of this cytokine has been analysed in terms of 
phospholipase C (PLC) and D (PLD) activities. Results have shown a combined modulation of PlP,-speeifie phospholipas¢ C and phospholipase 
D. In particular, a deereased activity of PIP,.speeifie PLC has been found, concomitant to a PLD-mediated plaosphatidyleholi:~e hydrolysis, 
suggesting that the intraeeilular signalling activated by interferon in Daudi cells invohes a phospholipase D/phosphohydrolase pathway. 
Interferon; Phospi~olipase: Daudi cell 
1. INTRODUCTION 
Molecular events controlling signal transduction gen- 
erated by the interaction of different agents, ~ike 
hormones or cytokines, with cell surface receptors are 
an intriguing aspect of the cell metabolism the knowl- 
edge of which has enormously expanded in recent years 
[1-5]. Among the agents able to regulate cell prolifera- 
tion, type-I interferons (~ and ]3) are cytokines known 
to bind to cell-surface receptors inducing resistance to 
the action of different viruses and producing a decrease 
in cell growth rate [6]. Such properties identity interfer- 
ons as potential tumor suppressors. The molecular 
mechanism by which interferon exerts its antiviral and 
antiproliferative effect i~0 anyway~ still unclear. Among 
the molecular changes produced by this cytokine [7-9], 
an early and transient rise of cellular diacylglycerol 
(DAG) has been reported, suggesting the possibility 
that the intracellular cascade of interferon-generated 
signals could be activated by a degradation of cell-sur- 
face phospholipids [10,11]. Recent data on cell metabo- 
lism in response to interferon-a treatment, in different 
cell lines have shown that the interaction of this cyto- 
kine with its cell-surface receptors produces different 
responses of polyphosphoinositide and diacylglycerol 
metabolism in the whole cells as compared to isolated 
nuclei [11-13]. In particular, it has been reported that 
the transient rise of DAG induced by inted'eron is not 
Correspondence address: S. Miseia, lstitato di Morfologia Umana 
Norm.'tle, "Via dei Yestini 6, 66100 Chieti, Italy. 
Abbreviations: PIP..,, phosphatidylinositol-bisplaosphate; IP~, ino~itol- 
trisphosphate; PA, pho~hatidic acid; LPL, Its5 polar lipids. 
accompanied by any modifications in inositol lipid 
plaosphorylation in the cell homogenates, whereas PIP2- 
phosphorylation changes have been found to parallel 
the DAG increase after 90 rain of interferon treatment 
in isolated nuclei [11]. These results allow to speculate 
on the existence of two distinct pathways, for cytoplas- 
mic and ntLclear compartments, involved in the inter- 
feron-mediated signal tr,'msduction into the cell. To fur- 
ther understand this intriguing aspect of interferon ac- 
tion, in this paper we have focused our attention on a 
possible PLD and phosphoinositide-specific PLC 
(phosphoinositidase) involvement in the cascade of mo- 
lecular events generated at the cytoplasmic level by this 
cytokine in Daudi lylnphoma cells. A characterized line 
of Daudi cells selected tbr resistance to the antipro~ifer- 
ative action of interferon-~z provided controls for speci- 
ficity of results. 
2, MATERIALS AND METHODS 
2.1. Ce/[ cult~,'e and #ltelferotl reftlmetll 
D~udi lymphoma cells were grown in sta~.ionary ~uspension culture 
at densities of 4 × 10 ~ to 12 × 10S/ml. RPM1 1640 medium was supple- 
mented with 10% foetal calf serum, glulamia, penicillin-streptomycin 
and neomycin. Viability was determined by the Trypan blue exclusion 
te~t. Cells from Daudi interferon-resistant clones were eontlnuously 
maint~tined in the presence of 300 I U/ml of recombinant interferon-a 
2A (La RochO. The resistant phenotype was stable without any inter- 
feron treatment for up to six culture doublings mid resistant cells were 
passaged for at least three doublings without interferon prior to use 
in experimcntz. For Cxl~rimeat~, cells wer~ prelabelled with [2- 
• ~H]glyceroi (7,uCi/ml per 10 ~ cells) for 40 h. R-IFN-~ A treatment oi' 
the cells (300 IU/ml) was pea tbrmed for pcriod~ up to 90 rain. Whe~ 
required, cells were incubated with 200/.tM propranolol as described 
by Pal 1141 before adding interferon. 
210 Published by Elsevier Science Publishers B. V. 
Volume 313, number  3 FEB$ LETTERS November  1992 
R 
g 
~h 
e-  (3 1 Inln 0 thin O0 rain 
tlmo ol Interleron ~lpha treatmenl 
alin|lnw eQIla ~ realatont colin 
Fig, 1. nmol of [sH]PlP, hydrolyzed in interferon-or (300 IU/ml)- 
treated sensitive and resistant D,'tudi cells. Results are the mean of 3 
separate ¢nperiments -+ S.D, 
LPL 
2.2, Assay for PiP, specific PLC 
PLC activity was assayed using 3 nmol of[-~H]PlP, (90,000 dpm) as 
exogenous substrate, 100~tg protein as enzyme ~ource in the presence 
of 0.06% taurodeoxycholate and by incubating for 30 rain at 37°C. 
For PIPe hydrolysis the buffer used was 100/a M MES pH 6.2 plus 100 
~tM CaCb [15]. 
2.3. Lipid extraction attd thin layer chromatography 
Cells (7 × 10 ~1 were processed for lipid extraction and TLC separa- 
tion using the procedure of Bligh and Dyer [16]. When appropriate, 
a different solvent system (chlorolbrm/metlaanoi/aceti¢ acid: 65:15:2, 
by volume) was used to separate phosphatidie acid (PA). Lipids were 
fluorographed at -80°C and ~pols corresponding to internal stan- 
dards were scraped off and counted in a liquid scintillation counter. 
3. RESULTS AND DISCUSSION 
The effect of interferon on diacylglycerol levels in 
Daudi cells is reported in Table I. Since interferon pro- 
duces a rapid and transient increase of this lipid, it 
appeared of interest o evaluate the possible invoh,e- 
ment of PlP_~-specific phospholipase C in DAG produc- 
tion. This enzyme, in fact, is responsible for P1P2 lay- 
drolysis generating the two well-known second messen- 
gers DAG and IPs [1]. Fig. ! shows the PLC activity on 
PiPe in the samples treated ..'?or up to 90 rain with inter- 
feron-:g with respect to the control, suggesting an inhib- 
itory effect of this cytokine on PIP~_-specific phos- 
pholipase C activity. This result prompts the question 
l¥om which phospholipid source DAG is produced. 
Among the cellular phospholipids believed to exert a 
role on cell metabolism regulation, phosphatidylcholine 
Table I 
Effect of recombinant interferon-~ (300 IU/ml) on DAG levels in 
Daudi sensitive elis (clpm per 7 x 10" cells) 
Control 1 rain 5 rain 90 rain 
DAG 431 ± 16 407 __ 14 791 ," 18 501 • 11 
LPL 2316 +_ 37 2431 +_ 31 2471 ± 42 2936 ± 29 
Data are the mean of 3 separate xperiments ___S.D. 
m g DAG 
@ O Oo,. 
1 2 a 
Fig. 2. Ef f~t of propranolo! on interferon induced DAG production 
in Daudl ~¢nsitive celh. Cells were labelled with [3H]glyeerol as staled 
in section 2. Results represent at least 3 separate xperiments. Lane 
1, control; lane 2, 5 rain of interferon-a; lane 3, 5 rain of interferon-~ 
plus propranolol (200/aM). 
(PC), the principal phospholipid class in mammalian 
tissues, is supposed to be involved in mechanisms which 
reg;.!~-~ :ignal transduction [17]. There is considerable 
evidence ,hat cellular PC is a major sour¢~ of DAG via 
phosphodiesteratic leavage by a PLD-generating 
choline and PA. The latter is converted into DAG by 
a PA-speeific phosphohydrolase [14,17,18]. Since this 
latter enzyme is specifically inhibited by propranolol, a 
compound also reported to be uneffective on phospho- 
inositide-specific phospholipase C [18], we have checked 
the el'feet of interferon, in the presence of this PA 
phosphohydrolase inhibitor, on DAG production. Re- 
suits have disclosed that the rapid DAG increase, usu- 
ally induced by interferon, is dramatically restored to 
Table 1 ! 
Effect of propranolol on PA levels in interferon-lr~ted Daudi cells 
(dpm per 1 x 10 ~ cells) 
Control 5 rain IFN 5 min IFN+propranolol 
PA 577 +_ 13 618 +_ 15 g03 +_ 24 
Data are the mean of 3 separate xperiments ± IS.D, 
211 
Volume 313, number 3 FEBS LETTERS November 1992 
LPL 
lipase D/phosphohydrolase pathway is supposed to 
occur when stimulated by different external agents [17]. 
Interestingly, interferon does not affect both PIP.~-PLC 
and DAG levels in the clone of Daudi cells selected for 
resistance to this c2¢tokine (Figs. 1 and 3). It must be 
noted, anyway, that the addition of propranolol in con- 
trol cells both sensitive and resistant to interferon, re- 
subs in a slight decrease in the recovery of the label in 
the DAG fraction (not shown), strongly indicating that 
the phospholipase D/phosphohydrolase pathway is 
likely to contribute to DAG formation also at basal 
levels, possibly becoming the major route involved in 
the intracellular transduetion system activated by the 
interaction between interferon and its receptors in 
Burkitt lymphoma cells. 
, Or ,  
1 2 3 
Fig. 3. EIT~t of propraaolol n interferon~indueed DAGproduction 
in Daudi resistant cells. Cells were labelled with [~H]glycerol as stated 
in section 2. Results represent a least 3 separate experiments. Lane 
1, control; ane 2, 5 rain of interferoa-~; lane 3, 5 rain of interferon-~ 
plus propranolol (200/~M). 
control levels when cells are incubated in the presence 
of this compound (Fig. 2), indicating that interferon 
may exert its action at the cytoplasmic level through a 
rapid and transient hydrolysis of PC via the phospholip- 
ase D/phosphohydrolase pathway. The reduced hydrol. 
ysis of the PIP~. fraction in the interferon,treated sam- 
ples ~oncomitant with the rise of DAG levels, allows for 
speculation on the possibility that inhibition of PIP, 
specific phospholipase C might be a crucial metabolic 
modulation required by the signalling system activated 
by interferon in the transduction of its molecular sig- 
nals. PA accumulation, observed when propranolol is 
added to the system (Table II), further supports the 
involvement of the PLD-rnediated PC hydrolysis and 
reasonably excludes the possibility that PC hydrolysis 
would occur via a non-specific PLC involvement, in 
which ease DAG would have been produced also in the 
presence of propranolol. The results here reported are 
in accordance with a number of findings in other cell 
systems, in which PC hydrolysis through the phospho- 
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212 


Interferon-mediated intracellular signalling Modulation of different phospholipase activities in Burkitt lymphoma cells 

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Interferon-mediated intracellular signalling Modulation of different phospholipase activities in Burkitt lymphoma cells

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