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MICROSCOPY RESEARCH AND TECHNIQUE 36:172–178 (1997)
Increase of Nuclear Phosphatidylinositol 4,5-Bisphosphate
and Phospholipase C b1 Is Not Associated to Variations of
Protein Kinase C in Multidrug-Resistant Saos-2 Cells
NICOLETTA ZINI,1 LUCA M. NERI,1,2 ANDREA OGNIBENE,3 KATIA SCOTLANDI,4 NICOLA BALDINI,4
AND NADIR M. MARALDI1,3,5*
1Istituto
di Citomorfologia Normale e Patologica, CNR, Chieti-Bologna, Sezione di Bologna,
c/o IOR, via di Barbiano 1/10 40136 Bologna, Italy
di Anatomia Umana Normale, Università di Ferrara, 44100, Ferrara, Italy
3Laboratorio di Biologia Cellulare e Microscopia Elettronica, Italy
4Laboratorio di Ricerca Oncologica, IOR, 40136, Bologna, Italy
5Unità Complessa di Scienze Anatomiche Umane e Fisiopatologia dell’Apparato Locomotore, University of Bologna, 40126,
Bologna, Italy
2Istituto
KEY WORDS
human osteosarcoma; P-glycoprotein; polyphosphoinositides; nuclear signal transduction; immunocytochemistry
ABSTRACT
The multidrug resistance (MDR) phenotype that is mediated by an overexpression
of P-glycoprotein, has been suggested to be related also to an increased activity of protein kinase C
(PKC) and to changes in phospholipid pattern. By electron microscope quantitative immunocytochemistry, we investigated whether PKC and other elements of the polyphosphoinositide signal
transduction system are affected in an MDR variant of the human osteosarcoma cell line Saos-2.
These cells, which are characterized by an increased expression of P-glycoprotein not only at the
plasma membrane but also at the nuclear level, showed increased intranuclear amounts of
phosphatidylinositol 4,5-bisphosphate and of phospholipase C b1, while both the amount and
activity of both nuclear and cellular PKC were not modified with respect to sensitive cells. These
results suggest that, in this model, the changes observed in the elements of nuclear signal
transduction could be related to previously reported modifications of the MDR phenotype, but that
P-glycoprotein phosphorylation is not dependent from increased PKC activity. Microsc. Res. Tech.
36:172–178, 1997. r 1997 Wiley-Liss, Inc.
INTRODUCTION
The mechanism of development of multidrug-resistance (MDR) to a broad spectrum of chemotherapeutic
agents is still unclear, although the decrease of intracellular accumulation of the drug appears in many cases
related to an enhanced drug efflux due to the overexpression of the membrane-associated 170 KDa P-glycoprotein (Bradley et al., 1989; Endicott and Ling, 1989;
Gottesman and Pastan, 1988; Greenberger et al., 1988;
Pastan and Gottesman, 1987). However, other mechanisms may also be involved, such as an alteration in
glutathione metabolism (Batist et al., 1986; Cowan et
al., 1986; Hamilton et al., 1985; Somfai-Rene et al.,
1984), a decreased drug metabolism (Mungikar et al.,
1981), and oxygen free-radical susceptibility (Mimnaugh et al., 1989). A great body of evidence indicates
that MDR is also frequently associated with increased
protein kinase C (PKC) activity (Anderson et al., 1991;
Blobe et al., 1993; Chambers et al., 1990; Ferguson and
Cheng, 1987; Fine et al., 1988; Kessel, 1987; O’Brian et
al., 1989). In particular, in MCF-7 MDR human breast
carcinoma cells, elevated levels of PKC a have been
detected in the nucleus, suggesting that altered transcription of this protein can promote MDR (Lee et al.,
1992). MDR has also been found to be associated with
an increased production of inositol phosphates (Fine et
r 1997 WILEY-LISS, INC.
al., 1987) and with changes in phospholipid pattern
(Ramu et al., 1984; Tapiero et al., 1989; Vrignaud et al.,
1986).
Two human osteosarcoma cell lines (U-2 OS/DX and
Saos-2/DX) have been selected for their resistance to
doxorubicin (DX) (Serra et al., 1993). The MDR derived
cell lines, besides an overexpression of the mdr1 gene
product P-glycoprotein at the membrane level, exhibit
an increased amount of the protein within the nucleus
(Baldini et al., 1995; Maraldi et al., 1996), and a
modified phenotype (Zini et al., 1995c). These cells
present a signal transduction system based on polyphosphoinositide hydrolysis by specific phospholipase C
(PLC) isoforms, that are active within the nucleus, as
previously reported in other cell lines (Divecha et al.,
1993; Maraldi et al., 1995; Martelli et al., 1992; Mazzoni et al., 1992; Neri et al., 1993; Zini et al., 1993,
1995a,b). Saos-2 cells present a partitioning of the PLC
isoforms, being the PLC b1 almost exclusively nuclear
and the g1 both cytoplasmic and nuclear (Maraldi et al.,
1993). Moreover, the nuclear PLC b1 isoform can be
activated by interleukin 1a (IL-1a), causing the intra-
*Correspondence to: Nadir M. Maraldi, Istituto di Citomorfologia Normale e
Patologica, CNR, c/o IOR, via di Barbiano 1/10, 40136, Bologna, Italy.
Received 11 January 1996; Accepted in revised form 1 February 1996.
NUCLEAR SIGNAL TRANSDUCTION IN MDR SAOS-2 CELLS
nuclear breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2 ) and the release of the second messenger
diacylglycerol (DG) (Marmiroli et al., 1994), responsible
for a further cascade of events, among which the
activation of some transcription factors has been preliminarly investigated (Ognibene et al., 1995).
In this study, we utilized electron microscope immunocytochemistry to analyze the intracellular distribution and the quantitative variations of some key elements of nuclear signal transduction, such as PIP2,
PLC b1, and PKC, in sensitive and in the MDR variant
of human osteosarcoma Saos-2 cells. In these cells, the
presence of a polyphosphoinositide autonomous signal
transduction system has been demonstrated within the
nucleus (Maraldi et al., 1993; Marmiroli et al., 1994);
moreover, MDR Saos-2 variant presents increased
amount, also at the nuclear level, of P-glycoprotein
(Maraldi et al., 1996), which is a possible substrate of
PKC-dependent phosphorylation (Epand and Stafford,
1993). Therefore, we utilized immunocytochemical
analysis, which appears particularly suitable to detect
possible variations in the intracellular distribution of
all the elements of signal transduction. The activity of
PKC was also tested both in the whole cell and in the
nuclear fraction, in order to determine whether Pglycoprotein activation can be mediated by this enzyme
or by other phosphorylating enzymes.
MATERIALS AND METHODS
Materials
Culture media and fetal calf serum (FCS) were from
ICN Flow (Costa Mesa, CA). Monoclonal antibody
(MoAb) against PLC b1 was from UBI (Lake Placid,
NY). KT 10 MoAb against PIP2 was a generous gift of
Dr. T. Takenawa, Tokyo, Japan). The anti-PKC antibody was obtained by injecting rabbits with a synthetic
peptide derived from the C-terminal sequence of PKC;
this antibody presents a 100% sequence homology to a,
93% to d, 89% to g, and 60% to b PKC isoforms (Zini et
al., 1995b).
Cell Cultures
Human osteosarcoma Saos-2 cells were grown in
Iscove’s modified Dulbecco’s medium supplemented with
10% FCS. MDR variants were obtained by exposing
parental cell line initially to 3 ng/ml DX and then to
stepwise increases in DX concentration (Serra et al.,
1993). Variant continuosly cultured in the presence of
580 ng/ml DX for at least 8 months, referred to as
Saos-2/DX580, has been utilized for this study.
Cell Fractionation
Nuclei were obtained essentially as previosly described (Martelli et al., 1992). The cells, washed in PBS,
were resuspended in 10 mM Tris-Cl, pH 7.8, containing
1% Nonidet P-40, 10 mM b-mercaptoethanol, 0.5 mM
phenylmethylsulphonyl fluoride, 1 µg/ml soybean trypsin inhibitor, 15 µg/ml calpain inhibitor I, and 7 µg/ml
calpain inhibitor II. After 5 minutes at 4°C, swelling
was induced by adding double-distilled water at 0°C for
5 minutes; the cells were then sheared by five passages
through a 22-gauge needle and nuclei were centrifuged
at 300g for 6 minutes. After washings in 10 mM Tris-Cl,
pH 7.4, 2 mM MgCl2, plus protease inhibitors, the
173
nuclear pellet was maintained in the same buffer at
4°C.
Electron Microscopy Immunocytochemistry
The cells were fixed with 1% glutaraldehyde in 0.1 M
phosphate buffer for 30 minutes at 4°C, dehydrated up
to 70% ethanol, and embedded in London Resin White
(LR White) at 0°C (Zini et al., 1993). Thin sections,
carried out on nickel grids, were preincubated with 5%
NGS in 0.05 M Tris-Cl, pH 7.6, 0.14 M NaCl, and 0.1%
BSA and then incubated overnight at 4°C with the
following primary antibodies: anti-PLC b1 MoAb, diluted up to 1:60; anti-PIP2 KT 10 MoAb, diluted up
1:300; multitopic anti-PKC antibody, diluted 1:50 in the
same buffer. The secondary incubation was with a goat
anti-mouse (GAM) for MoAbs and with a goat antirabbit (GAR) for anti-PKC, diluted 1:10 in 0.02 M
Tris-Cl, pH 8.2, 0.14 M NaCl, and 0.1% BSA for 1 hour
at room temperature and then amplified with Silver
Enhancer Kit (Amersham Life Science, Amersham,
UK). The sections were stained with aqueous uranyl
acetate and lead citrate.
Controls consisted of: use of the gold-conjugated
GAM or GAR without the primary antibody; preincubation of KT 10 MoAb with PIP2 multilamellar vesicles
(Mazzotti et al., 1995); preincubation of the anti-PLC b1
MoAb with the protein utilized for immunization (gift of
Dr. S.G. Rhee, Bethesda, MD); preincubation with the
preimmune rabbit serum for the multitopic anti-PKC
antibody (Zini et al., 1995b).
Quantitative Evaluations
A Quantimet-970 image analyzer (Leica-Cambridge
Inst., Cambridge, UK) was used. All the experiments
were done in triplicate; in order to reduce the influence
of efficiency variations among different experiments,
the comparison between parental and MDR cells was
done in samples treated at the same time under identical immunolabeling conditions. Quantitative evaluation of gold particle distribution was according to
Durrenberger et al. (1988). At least 25 micrographs at
the same magnification have been selected for each
sample; the actual labeling amount was obtained by
subtracting the background found in the extracellular
spaces; the density of the labeling (means of gold
particle number/µm2 6 SD) was evaluated in each cell
compartment. The statistical significance of the differences between parental and MDR cells was determined
by the Student’s t-test.
Immunoblotting and Phosphorylation Assay
The protein concentration of total cell homogenates
or nuclear extracts was measured using the Bio-Rad
(Gaithersburg, MD) D/C protein assay kit with bovine
serum albumin as a standard. Proteins were resolved
on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by overnight Western blot. Membranes were immersed in 0.1% PBS/
Tween 20 (PBST) plus 3% milk and 2% BSA for 1 hour
at room temperature to block nonspecific binding. The
multitopic anti-PKC antibody was diluted 1:750 in
PBST and allowed to react for 1 hour at room temperature. After extensive washes, membranes were incubated with a 1:3,000 dilution of mouse anti-rabbit IgG
in PBST for 20 minutes. After washes in PBST, mem-
174
N. ZINI ET AL.
TABLE 1. Quantitative evaluation of the labeling density
in the nucleus1
Saos-2
Saos-2/DX580
PIP2
PLCb1
PKC
40.5 6 3.8
56.7 6 4.1*
63.7 6 5.5
87.9 6 7.6*
35.9 6 2.3
33.8 6 2.8
1Values are the means 6 SD.
*Differences that are significant (P , 0.001).
brane bound antibody was visualized by ECL Western
blotting detection reagents (Amersham Life Science)
and Kodak XS1 films.
Protein kinase C activity was assayed in vitro by
using as substrate a peptide corresponding to the
amino terminal domain of PKC with substitution of
serine for alanine at position 13 (PKC substrate, Santa
Cruz Biotechnology, Santa Cruz, CA). Standard reaction mixtures of 50 µl, incubated for 10 minutes at
37°C, contained 20 mM Tris-Cl, pH 7.5, 5 mM MgCl2, 5
mM Mg acetate, 1 mM DTT, 20 µg peptide, 10 µM
[g-32P] ATP, and 20 µg protein from total cell homogenate or nuclear fraction. Reaction mixtures were spotted onto P81 phosphocellulose paper and washed 4
times in 0.5% phosphoric acid. Incorporation of 32P was
determined by liquid scintillation counting.
RESULTS
Electron Microscopy Immunocytochemistry
The immunocytochemical reactions, the quantitative
evaluations and the control conditions for anti-PIP2 and
PLC b1 MoAbs (Maraldi et al., 1993, 1995; Mazzotti et
al., 1995; Zini et al., 1993), and those for anti-PKC
antibody (Zini et al., 1995b) are similar to those previously reported. The control samples presented a very
low background labeling with all the three antibodies
used (data not shown), in agreement with previous
studies (Zini et al., 1993, 1995b).
The differences between sensitive and MDR cells
were determined by quantitative evaluations; since no
significant variations were observed in the cytoplasm,
the data reported in Table 1 refer exclusively to the
nucleus.
With the KT 10 anti-PIP2 MoAb, the phospholipid
could be detected in parental Saos-2 cells both in the
cytoplasm and in the nucleus (Fig. 1a). It has been
previously reported that, by using the anti-PIP2 MoAb,
the labeling in the cytoplasm is prevailingly associated
with the rough endoplasmic reticulum, where the phospholipid is synthesized (Maraldi et al., 1995; Mazzotti
et al., 1995). In Saos-2 cells, which contain a few
endoplasmic reticulum elements, the labeling was
scarce, being mainly diffused in the cytosol. This phospholipid, in fact, has also been found in association with
cytoskeletal elements (Lee and Rhee, 1995; Mazzotti et
al., 1995). Within the nucleus, the labeling was mainly
present on the heterochromatin domains, as previously
reported (Mazzotti et al., 1995). In Saos-2/DX580 cells,
the labeling was present in the same sites, but appeared to be increased at the nuclear level (Fig. 1b). As
indicated by quantitative evaluations, a 40% increase
occurred in MDR with respect to parental cells (Table 1).
With the anti-PLC b1 MoAb, in parental Saos-2 cells,
the labeling was present mainly at the nuclear level
(Fig. 2a). The labeling scattered in the cytoplasm can be
Fig. 1. Electron microscope immunocytochemistry. Anti-PIP2 MoAb.
a: Saos-2 cells. A scarce labeling is present in the cytoplasm, mainly
diffused in the cytosol. The nuclear labeling is present on the heterochromatin (HC), at the nuclear periphery, and around the nucleolus
(N). b: Saos-2/DX580 cells. No variations are detectable in the labeling
amount in the cytoplasm, while the nuclear labeling appears increased
with respect to Saos-2 cells. 314,000.
due, as previously reported (Maraldi et al., 1993), to the
enzyme synthesized in the cytoplasm before its translocation to the nucleus, where the enzyme activity was
exclusively detectable (Marmiroli et al., 1994). Within
the nucleus, the sites of PLC b1 localization corresponded to the inter-heterochromatin borders, as previously reported (Maraldi et al., 1993). In Saos-2/DX580
cells, the labeling was present in the same subcellular
sites (Fig. 2b), but a significant 38% increase with
respect to parental cells was revealed in the nucleus by
quantitative evaluations (Table 1).
With the anti-PKC antibody, in Saos-2 parental cells
the labeling was present in the cytosol, while the cell
NUCLEAR SIGNAL TRANSDUCTION IN MDR SAOS-2 CELLS
Fig. 2. Electron microscope immunocytochemistry. Anti-PLC b1
MoAb. a: Saos-2 cells. The labeling is more intense in the nucleus than
in the cytoplasm. In the nucleus the labeling appears localized at the
interchromatin domains (IC), at the border of heterochromatin. b:
Saos-2/DX580 cells. No variation of labeling intensity occurs in the
cytoplasm, while the nuclear labeling appears increased with respect
to Saos-2 cells. 313,000.
organelles were weakly labeled (Fig. 3a). Within the
nucleus, the labeling was present in the interchromatin
domains, mainly in correspondence with the interchromatin granules and at the borders of heterochromatin.
The nucleoli presented a low labeling. In Saos-2/DX580
cells (Fig. 3b) the nuclear labeling did not show significant quantitative differences with respect to parental
cells (Table 1).
These data indicate that the MDR phenotype is
characterized by an accumulation within the nucleus of
both the main phospholipid involved in signal transduction and of a phospholipase isozyme involved in its
hydrolysis.
Immunoblotting and Phosphorylation Assay
Since no significant variations occurred in the intranuclear amount of PKC, which could represent a key
enzyme activated by the release of lipid second messengers, we evaluated whether the PKC activity was
affected in MDR cells, at the cytoplasmic or at the
nuclear level.
175
Fig. 3. Electron microscope immunocytochemistry. Anti-PKC Ab.
a: Saos-2 cells. b: Saos/DX580 cells. No variations of the labeling
amount are detectable in the cytoplasm and in the nucleus. 312,500.
Western blotting of total cell homogenate and nuclear
fraction proteins showed a unique band at about 80
KDa corresponding to the native form of PKC. No
significant densitometric differences were detectable in
the samples from parental and MDR cells (data not
shown), in agreement with the quantitative immunocytochemical data (Table 1).
PKC activity on a specific substrate, evaluated on the
same protein fractions, did not show significant changes
in MDR vs. sensitive cells (Table 2). These results
indicate that in Saos-2/DX580 cells the PKC amount and
activity are not directly related to the resistance phenomenon which, on the other hand, involves an increase of the amount of nuclear PIP2 and PLC b1, which
can be related to the changes in nuclear morphology
and to an increased P-glycoprotein expression within
the nucleus (Maraldi et al., 1996).
DISCUSSION
The aim of this study was to determine whether the
phenotype of MDR Saos-2 cells is characterized by
different intracellular amount and distribution of three
176
N. ZINI ET AL.
TABLE 2. Protein kinase C activity assay in sensitive and MDR
Saos-2 cells1
TCH
NF
Saos-2
Saos-2/DX580
79,600 6 9,200
14,700 6 1,900
83,400 6 10,100
15,000 6 1,800
1 32P
incorporation in total cell homogenate (TCH) and nuclear fraction (NF) in
the presence of the PKC substrate. The values are the means 6 SD of three
different experiments.
key elements of nuclear signal transduction (PIP2, PLC
b1, and PKC), with respect to parental cell line. This
was suggested by the following findings: Saos-2/DX580
cells present an increased amount of P-glycoprotein
also within the nucleus (Maraldi et al., 1996); all the
main members of the signal transduction pathway
(PIP2, PLC isoforms, PKC) are present within the
nucleus of parental Saos-2 cell line (Maraldi et al.,
1993; Marmiroli et al., 1994); at least in MCF-7 MDR
cells an increased PKC activity has also been found in
the nucleus, which specifically shows a slightly altered
form of PKCa (Blobe et al., 1993). We have analyzed
whether, in Saos-/DX580, intranuclear P-glycoprotein
phosphorylation occurs via PKC activation by DG derived from PIP2 breakdown through a specific PLC
isoform. Ultrastructural immunocytochemistry allows
the identification of all these elements in the same cell,
while by cell fractionation it is not possible to detect
PIP2 and PLC b1 in the same cell fraction, the procedure requiring either deoxycholate/Triton-washed nuclei, or the absence of detergents (Banfic et al., 1993;
Maraldi et al., 1995).
PKC occupies a key role in signal transduction mechanisms, since its activation results in the phosphorylation of a variety of proteins, also at the nuclear level
(Buchner, 1995). Increased PKC activity may be of
particular relevance in the MDR development because
of its possible role in P-glycoprotein activation (Chambers et al., 1990). In some MDR cells, an increased
activity of PKC (Aquino et al., 1988; Cornwell et al.,
1986; Fine et al., 1988; Hirai et al., 1989; O’Brian et al.,
1989), as well as an increased basal and stimulated
production rate of inositol phosphates (Fine et al., 1987)
or changes in phospholipid pattern (Ramu et al., 1984;
Tapiero et al., 1989; Vrignaud et al., 1986) have been
observed. Moreover, an increased number of phorbol
ester receptors (Niedal et al., 1983) has been observed
in MDR cells, and an increased PKC activity by phorbol
esters is capable of inducing MDR phenotype in MCF-7
parental cell lines (Fine et al., 1988).
However, the protein kinases responsible for Pglycoprotein phosphorylation and the actual role of
phosphorylation in MDR are not well established. In
fact, transfection with the gene of PKCa isoform, but
not of g isoform, enhances drug resistance (Ahmad et
al., 1992; Yu et al., 1991), while clonally selected MDR
cells exhibit overexpression of PKC (Posada et al., 1989;
Schwartz et al., 1991; Ward and O’Brian, 1991), mainly
of a and b isoforms (Blobe et al., 1993; Gollapudi et al.,
1992). However, some findings indicate that the overexpression of the PKC b isoform can itself increase MDR
even in the absence of amplification of P-glycoprotein
expression (Fan et al., 1992). Moreover, the results with
PKC inhibitors do not appear conclusive, since several
inhibitors are very aspecific, possibly acting by PKC-
independent mechanisms (Epand and Stafford, 1993;
Hagiwara et al., 1991; Sato et al., 1990). Other agents,
such as verapamil (Hamada et al., 1987) and a membrane-associated protein kinase P, different from PKC
(Staats et al., 1990), can promote P-glycoprotein phosphorylation. In other MDR cells, such as MOLT-3
human acute lymphoblastic leukemia cell lines, no
differences in PKC activity have been observed
(Schwartz et al., 1991).
MDR Saos-2 cells, which exhibit an increased expression of P-glycoprotein at the membrane and nuclear
level, and typical phenotypic changes of the cell surface
and of nuclear components (Maraldi et al., 1996), also
present modifications of some elements of the signal
transduction system at the nuclear level. These consist
in an increased amount of PIP2 and of PLC b1 isoform,
while the amount of nuclear PKC and the activity of the
enzyme are unaffected with respect to parental Saos-2
cells. The changes observed could be due to the altered
MDR phenotype, which, in some cases has been reported to be associated with changes in phospholipid
pattern (Ramu et al., 1984; Tapiero et al., 1989; Vrignaud et al., 1986). On the other hand, the absence of
significant changes of PKC amount and activity exclude
that, in this case, MDR could be due to an increased
activation of the P-glycoprotein by PKC itself. On the
other hand, other MDR cells, such as MOLT-3, did not
show variations of PKC activity (Schwartz et al., 1991).
In this case, as well as in Saos-2 MDR cells, Pglycoprotein activation could be achieved by other
phosphorylating enzymes.
It seems particularly interesting, however, to observe
that Saos-2 cells present an increased accumulation of
P-glycoprotein in the nucleus (Maraldi et al., 1996),
associated with an increased amount of nuclear PIP2.
Since P-glycoprotein presents several hydrophobic domains, its accumulation within the nucleus could be
favoured by the increased amount of the phospholipid,
mainly detectable in the heterochromatin by immunogold labeling. On the other hand, MDR Saos-2 cells
present modifications of the chromatin arrangement
(Maraldi et al., 1996). These modifications could depend
on an altered organization and composition of nuclear
matrix proteins (Tew et al., 1983), which present PIconsensus binding sequences (Yu et al., 1992). The
possibility that P-glycoprotein could remove DX by
acting as a ‘‘flippase,’’ exchanging the drug with hydrophobic lipid domains (Higgins and Gottesman, 1992),
could agree with the observed intranuclear increase of
the P-glycoprotein (Maraldi et al., 1996) and of a
phospholipid in MDR Saos-2 cells.
ACKNOWLEDGMENTS
The authors thank Mr. A. Valmori for photographic
assistance. This work was supported by the ‘‘Associazione Italiana per la Ricerca sul Cancro’’ (AIRC), the
Consiglio Nazionale delle Ricerche (PF IG/PF ACRO),
40 and 60% grants from the Ministero della Ricerca
Scientifica e Tecnologica (MURST), and the Istituti
Ortopedici Rizzoli (Ricerca Corrente and Finalizzata).
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