Differential expression of novH and CTGF in human glioma cell lines
Downloaded from mp.bmj.com on October 4, 2014 - Published by group.bmj.com M92 Table 1 Patient data, tumour histology and grading Cell line Age WI-HO Number of NCE Sex (years) Histology grade passages in vitro G-22 F 56 GM 4 219 G-28 M 66 GS 4 395 G-44 F 64 GM 4 340 G-55 M 65 AA 3 235 G-59 M 66 GM 4 290 G-60 F 68 AO 4 268 G-61 M 58 AA 4 327 G-62 F 4 GM 4 324 G-63 M 52 GM 4 244 G-84 F 47 AA 3 171 G-96 F 9 GM 4 323 G-111 F 47 GM 4 233 G-118 F 54 GM 4 146 G-120 M 59 GM 4 250 G-121 M 53 GM (recurrent) 4 208 G-122 M 50 GM 4 140 G-123 M 68 GM 4 240 G-140 M 59 GM 4 99 G-141 M 52 GM 4 141 G-142 M 58 GM 4 68 G-167 M 50 GM 4 13 G-168 M 71 GM 4 75 G-169 F 71 GM 4 74 G-195 M 59 GM (recurrent) 4 38 GM = glioblastoma multiforme, GS= gliosarcoma; AA = anaplastic astrocytoma; AO = anaplastic oligodendroglioma. In an attempt to determine whether novH or CTGF, or both, play a role in the biology of these tumours, we compared the pattern of their expression in 24 cell lines, mainly established from human glioblastomas.'5 We also characterised the CTGF promoter region and assessed its activity in vitro. Methods CELL CULTURE HBL100-ras,'6 HEL-1'7 and 293 cells (ATCC CRL 1573) were grown in Eagle's minimum essential medium (MEM, Gibco/BRL) supplemented, respectively, with 10% newborn calf serum, 20% heat inactivated newborn calf serum and 10% heat inactivated horse serum. Glioma cell lines were established from fresh tumour specimens,'5 obtained from patients aged from four to 71 years. The tumours were classified according to WHO guidelines. The tumour cell lines analysed in this study (table 1) were derived either from tumours of WHO grade 3 or 4 and are referred to as high grade hereafter.'8 The cell lines were maintained at 37°C in Earle's modification of MEM, containing 10% fetal calf serum in a 8% CO2 humidified atmosphere. GENOMIC CLONES AND PLASMID SUBCLONES The structure of the pBH7 and pS6 clones which contain, respectively, the novH and the CTGF genes have been described previously.5 RNA PURIFICATION AND NORTHERN BLOTTING Procedures for purifying RNA from cell cultures and northern blotting are described elsewhere."' Northern blots were hybridised either to the 3-5 kilobase BglII-BamHI fragment from the pBH7 novH clone (probe pBH7/BB) or to the 700 base pair PstI fragment derived from the pS6 CTGF clone pS6 (probe pS6/PSP07) (fig lA). Xin, Martinerie, Zumkeller, Westphal, Perbal NUCLEOTIDE SEQUENCING Double stranded DNA sequencing was carried out by using the dideoxy-chain termination method20 in the presence of (oc-35S) dATP and T7 polymerase (Pharmacia, St Quentin en Yvelines, France). Compressions were resolved using deaza 7-GTP. Sequence data treatments were carried out using the computer facilities at CITI2 in Paris.2' RNASE PROTECTION ANALYSES The pEP subclone was linearised with EcoRV. The linearised subclone (1 fig), was transcribed for 45 minutes at 37°C in a mixture containing transcription buffer (Promega, Charbonnieres, France), 10 mM DTT, 25 units RNAsin (Promega), 50 mM UTP, 0-3 mM each of ATP, GTP, CTP, 40 pCi (800 Ci/mmol) 32p [UTP], and 15 units T7 RNA polymerase (Promega). The DNA template was digested with RNasefree DNase (1 unit for 15 minutes at 37°C). The antisense riboprobe (3 ng) was hybridised with total RNA (15 ptg) for 24 hours at 50°C in a buffer containing 80% formamide. RNA- RNA hybrids were then digested with RNaseA (40 ,ug/ml) and RNaseT I (1000 U/ml) (Boehringer) and analysed in a denaturing 6% polyacrylamide-urea sequencing gel along with 32p labelled size markers and a nucleotide sequence ladder. CONSTRUCTION OF THE CHLORAMPHENICOL ACETYLTRANSFERASE (CAT) REPORTER PLASMID The p605C-CAT was obtained by fusing a PCR generated fragment to the promoterless pCAT5 vector.22 Oligomer #151 (with a HindIII restriction site at its 5' end) and oligomer #152 (with a BglII restriction site at its 5' end) were used to amplify p605C-CAT. The integrity of the PCR generated fragments was checked by sequencing. TRANSIENT TRANSFECTIONS AND CAT ASSAYS Forty eight hours before transfection, 293 cells were seeded at a density of 5 x 105 cells per 6 cm dish. Cells were co-transfected with reporter plasmids (10 zig), carTier DNA (10 jtg) and the reference RSV-,B-galactosidase plasmid (2 jtg) using the calcium-phosphate procedure. Plasmids pCAT5 and pRSV-CAT were used as negative and positive controls, respectively. Forty eight hours after transfection, cell extracts and CAT assays were carried out as described by Gorman et al.21 CAT activities were quantitated using a phosphoimager. Cell cultures were transfected in duplicate with each reporter plasmid sample. At least two different preparations of each plasmid DNA were used in these transfection experiments. Results Northern blotting experiments carried out on 24 cell lines derived from gliomas (table 1) revealed that novH was expressed as a 2-5 kilobase mRNA (fig 1B), which is in agreement with results from earlier studies. "
Downloaded from mp.bmj.com on October 4, 2014 - Published by group.bmj.com Differential expression of novH and CTGF in human glioma cell lines M93 A H PK P P BgP p P Hc Hc B I~~L l- _n WI El E2 E3 E4 I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I pBH7/BB pBH7 (novH) l J 1 Kb S B E PP P K H P S _i II E1 E2, E3 $4 E5 pS6 (CTGF) pS6/PSP07 B NCE-G 140 60 84 141 169 168 118 111 96 63 167 142 120 55 44 195 121 123 61 59 122 62 28 22 HeLa 9 . e 4 n , -e0 S * *M* t c- 2.5 Kb NOH a" 4- 7 0 Kb p|= 5 Kb CTGF 00 000f* 4* GGAPDH Figure 1 Expression of novH and CTGF in glioma cell lines. A, Schematic representation of the novH and CTGF specific probes. The black boxes represent exons. H=HindIII; P=PstI; K=KpnI; Bg= BglII; Hc =HincII; B=BamHI; S= SacL B, Samples containing total RNA (20 ,ug) were transferred to Nytran + membranes (Schleicher and Schuell) and hybridised either with 32P labelled novH or CTGF specific probes. The amount of RNA in each lane was normalised following hybridisation with a human GAPDH probe (Clontech). Levels of novH mRNA varied from one type of glioma derived cell line to another and within the same type of tumour derived cells. Expression of novH could not be detected in G59, G167, G122, and G120 and barely detected in G169, G142, and G141 cell lines derived from glioblastoma multiforme. Three different CTGF mRNAs species (2-5, 3-5 and 7T0 kilobases) were expressed (fig 1B). The most abundant, 2-5 kilobases, CTGF mRNA species has been described previously in HUVE cells.624 This mRNA species was expressed at various levels in all of the glioma cell lines studied. The 315 kilobase mRNA was observed in a subset of these cell lines. There was no correlation between the expression of the 2-5 and 3-5 kilobase mRNA species. Expression of the 7T0 kilobase mRNA was detected in most of the glioma cell lines except G60, G141 and G120. No obvious correlation with the two other CTGF mRNA species could be established. Both novH and CTGF genes were expressed at either high (for example, G22, G63, or G62) or low (for example, G60) levels in some glioma cell lines, whereas in others (for example, G59 and G140), expression of these two genes was found to be inversely correlated. These ob-