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). REFERENCES Ahmad, S., Trepel, J., Ohno, S., Suzuki, T., Tsuruo, T., and Glazer, R. 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