Preferential down-regulation of phospholipase C- β in Ewing's sarcoma cells transfected with antisense EWS-Fli-1

EWS-Fli-1, a fusion gene found in Ewing's sarcoma and primitive neuro-ectodermal tumour (PNET), encodes a transcriptional activator and promotes cellular transformation. We have made stable Ewing's sarcoma cells expressing antisense EWS-Fli-1 transcripts by transfecting the antisense EWS-Fli-1 expression plasmid. These cells showed partial loss of endogenous EWS-Fli-1 proteins and suppression of the cell growth. To elucidate the molecular mechanisms underlying the growth inhibition, we examined the changes of signal transducing proteins by immunoblot analysis in Ewing's sarcoma cells stably expressing antisense EWS-Fli-1 transcripts. Western blotting of the cell proteins revealed that expressions of phospholipase Cβ2 and β3 (PLCβ2, PLCβ3), and also protein kinase C α and β (PKCα, β) were significantly reduced by transfecting with antisense EWS-Fli-1. The inositol phosphates production by bradykinin (BK), but not platelet-derived growth factor (PDGF), was suppressed in these cells. These results suggest that the PLCβ2 and PLCβ3 may play a role in tumour proliferation in Ewing's sarcoma cells. © 2000 Cancer Research Campaign

Karyotype analysis of Ewing family of tumours, which includes Ewing's sarcoma, primitive neuroectodermal tumours and Askin tumours, revealed characteristic translocations t(11;22) or t(21;22) (Whang-Peng et al, 1984;Sorensen et al, 1994), and such chromosomal rearrangement resulted in the expression of the aberrant fusion products which may be responsible for malignancy (Delattre et al, 1992;Rabbitts et al, 1994). Molecular analysis of these translocations revealed that 5′-region of EWS (from band 22q12) is fused to the 3′-region of Fli-1 gene (from band 11q24) or erg gene (from band 21q22). Functional characterization of the EWS-Fli-1 and EWS-erg chimaeric proteins suggested that they function as transcriptional activators (May et al, 1993;Ohno et al, 1993;Bailly et al, 1994). The extreme carboxyl terminal region of Fli-1 and erg protein is responsible for sequence specific DNA binding (Reddy et al, 1991;Rao et al, 1993). EWS protein was shown to be an RNA binding protein (Ohno et al, 1994), and the amino-terminal region of EWS protein was shown to function as a regulatory domain or as a transactivator domain depending on the target sequences used (May et al, 1993;Ohno et al, 1993;Bailly et al, 1994). Recently, some of the target genes modulated by the EWS-Fli-1 protein have been identified (Braun et al, 1995;May et al, 1997). Ewing's sarcoma cells transfected with antisense EWS-fusion expression plasmids were severely impaired in growth, colony formation, and tumorigenicity in nude mice (Ouchida et al, 1995). Antisense EWS-Fli-1 oligodeoxynucleotides against the fusion RNA were also shown to reduce the growth of the tumour cells significantly both in vitro and in vivo (Tanaka et al, 1997). Antisense DNA has been considered to inhibit the expression of protein products of the transcripts through several mechanisms (Haeuptle et al, 1986;Munroe, 1988;Walder and Walder, 1988).
In the present study, for the better understanding of the EWS-Fli-1 function in terms of cellular signal transduction mechanism, we have examined the phosphatidylinositol signalling in Ewing's sarcoma cells stably transfected with antisense EWS-Fli-1 expression plasmid.

Cell culture and cell labelling
Ewing's sarcoma cell line (TC 135) kindly gifted from Dr Triche (Univ. Southern California, CA, USA) were transfected with pcDNA expression vector carrying antisense EWS-Fli-1 and with empty pcDNA vector for control and were selected for G-418resistant permanent cell lines as described previously (Ouchida et al, 1995). For the measurement of inositol phosphate, cells were Preferential down-regulation of phospholipase C-β in EwingÕs sarcoma cells transfected with antisense EWS-Fli-1 labelled with [ 3 H]inositol (1 mCi ml -1 ) in inositol-free minimum Eagle's medium containing 0.3% bovine serum albumin (BSA) for 36 h.

Measurement of inositol phosphates
The [ 3 H]inositol-labelled cells were washed two times with modified Krebs-Ringer buffer consisting of 125 mM NaCl, 5 mM potassium chloride (KCl), 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 2 mM calcium chloride (CaCl 2 ), 6 mM glucose, 0.1% BSA, 25 mM Hepes (pH 7.4), and 20 mM LiCl. Experiments were conducted by the adding agonists to the labelled cells, and the reaction was terminated by the addition of 0.5 ml of 10% perchloric acid to each well. Inositol phosphates were separated using Dowex AG1-X8 anion exchange resin (200-400 mesh, formate form, Bio-Rad Laboratories) as described elsewhere (Banno et al, 1994). Protein was determined with the Bio-Rad protein assay kit using BSA as standard.

RESULTS AND DISCUSSION
We have transfected Ewing's sarcoma cell lines (TC135) with pcDNA expression vector carrying antisense EWS-Fli-1 cDNA and obtained ten G-418 resistant clones. These clones were characterized for the expression of antisense transcripts of EWS-Fli-1 by RT-PCR as we described before (Ouchida et al, 1995), with specific primers for the cytomegalovirus promotor in pcDNA and EWS, and selected three cell lines that highly expressed antisense EWS-Fli-1 transcripts confirmed by Northern blotting with RNA probe synthesized by in vitro transcription with EWS-Fli-1 expression plasmid (data not shown). In all of three cell lines, morphological changes were observed, e.g. elliptic shape and longer processes compared with the control small round cells. Western blotting analyses were carried out in these cell lines. These results revealed that there was a significant loss of expression of EWS-Fli-1 protein in Ewing's sarcoma cells transfected with antisense EWS-Fli-1 cDNA ( Figure 1A). We have also compared the growth of these cell lines with TC 135 cells transfected with empty vector. The growth of Ewing's sarcoma cells expressing antisense EWS-Fli-1 was partially inhibited compared to the parent Ewing's sarcoma cells ( Figure 1B).
Next we studied the production of inositol phosphates (IPs) in these cells. As shown in Figure 2, in Ewing's sarcoma cells transfected with or without antisense EWS-Fli-1, IPs production was stimulated by PDGF or BK. No significant difference in IPs production was observed in both cell types stimulated by PDGF. In contrast, IPs production induced by BK was considerably suppressed in the antisense EWS-Fli-1-transfected cells. These results suggest that the suppression of cell growth by transfecting antisense EWS-Fli-1 may be at least in part due to impairment of inositol lipid turnover mediated via G-protein coupled receptor activation rather than via tyrosine phosphorylation.
To examine further the mechanisms of reduced IPs production, immunoblotting was carried out in both cell types. Western blot analysis with antibodies for PLC isozymes, β (1, 2, 3, 4), γ (1, 2), δ (1, 2) revealed that PLCβ1, β2, β3, γ1, δ1, and δ2 were present in both cells (Figures 3 and 4), but that PLC-β4 and γ2 were not or antisense EWS-Fli-1 (cell lines no. 1: s s, no. 2: q and no. 3: s) were plated at a concentration of 2 × 10 5 cells in 60 mm dishes, and incubated for the indicated times as described in Materials and methods. Cell numbers counted one day after plating were taken as zero time. Points represent as mean ± s.d. from duplicates determinations of two different experiments detectable (data not shown). No apparent di fferences were observed in PLC -β1, γ1, δ1, an d δ2 ( Figure 4A). In contrast, to be of great interest, PLC -β2 an d β3 were markedly down-regulated in the antisense-transfected cells ( Figure 3) compared with the control cells. The quantitative determination of PLC isozymes showed that PL C β2 and PL C β3 in the antisense-transfected cells were reduced by 73% and 38% respectivel y. PLC -βs are known to be activated by two G-protein families, one is Gi/o which is inhibited by pertussis toxins (PTX), and the other is Gq which is resistant to PTX. PL C β1 and PL C β3 are activated by a subunits of Gq, whereas PLC -β2 is activated mainly by βα subunits of Gi/o (Rhee, 1994;Rhee and Bae, 1997;Singer et al, 1997). No significant di fferences in expression of G q α and Gi2α were observed between both types of cells ( Figure 4B). These results lead us to consider that suppression of inositol phosphates production in EWS-Fli-1 antisense-transfected cells may be due to the decrease of PL C β2 an d β3.
In addition, PKC isozymes were also examined, PKC isozymes were expressed in various levels in Ewing 's sarcoma cells. Western blot analysis showed quantitative di fferences in PK C α, β1 an d β2 between both cells ( Figure 4C). The levels of PK C α, β1 an d β2 were reduced by 44%, 57% and 30% in the transfected cells respectivel y, as compared with the control cells.
These results indicate that suppression of proliferation caused by transfecting the antisense EWS-Fli-1 could be at least in part due to impairment of the signalling pathway mediated by PL C β2 and PLCβ3 activation and also by the subsequent activation of PKCα, β1, β2. Numerous studies have shown that PLCγ which is activated by tyrosine phosphorylation is involved in cell growth and carcinogenesis (Ji et al, 1997). On the other hand, it has been demonstrated that PLCγ activation is not essential for FGF receptor-mediating cell growth (Mohammadi, 1992;Peters et al, 1992). Furthermore, recent study has indicated essential role of PLCγ1 in mammalian growth (Ji et al, 1997). There have been some reports describing reduction or loss of expression of PLCβ isozymes in human diseases, e.g. loss of PLCβ3 gene expression in MEN1 disease (multiple endocrine neoplasia type 1) (Weber et al, 1994) and decreased PLCβ2 expression in abnormal platelet aggregation (Lee et al, 1996). Furthermore, recent study has demonstrated that expression of catalytically inactive PLCβ inhibits growth of small-cell lung cancer, indicating that signalling through Gq and PLCβ is a dominant pathway involved in the transformed growth of cancer cells (Beekman et al, 1998). In our study, we have shown that in Ewing's sarcoma cells expressing the antisense EWS-Fli-1 transcripts, the levels of PLCβ2 and PLCβ3 were much more decreased than the level of PLCγ1, suggesting that PLCβ2 and PLCβ3 may play an important role in cell proliferation in Ewing's sarcoma cells.