Oncogene (2000) 19, 754 ± 761 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene
Liubov Glukhova1, Christian Lavialle2, Didier Fauvet1, Ilse Chudoba3, GiseÁ le Danglot1, Eric Angevin4, Alain Bernheim1 and Anne-FrancËoise Goguel*,1
1Laboratoire de CytogeÂneÂtique et GeÂneÂtique Oncologiques, UMR 1599, PR2, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif cedex, France; 2Laboratoire de GeÂneÂtique Oncologique, Unite Mixte de Recherche 1599 du Centre National de la Recherche Scienti®que, Institut Gustave Roussy, 94805 Villejuif cedex, France; 3Institut fur Humangenetik und Anthropologie, D-07740 Jena, Germany; 4Unite d'ImmunotheÂrapie, Institut Gustave Roussy, 94805 Villejuif cedex, France
Molecular cytogenetic analysis of several sporadic throughout tumour progression (Kovacs, 1993b). papillary renal cell carcinomas and of their xenografts Indeed, the most common non papillary subtype, clear in immunode®cient mice had previously allowed us to cell renal carcinoma, is characterized by deletions in delimit a minimal overrepresented region of chromo- chromosome arm 3p, whereas the 3p region is intact in some 7 shared by all of them to band 7q31. We have papillary RCC. In addition, the latter exhibits an re®ned the location of the overlapping region to the increase in chromosome number typically including junction of the subbands 7q31.2 and 7q31.3 by reverse chromosomes 7 and 17. These anomalies are also painting with two dierently labelled probes prepared found in early renal lesions such as adenomas, from the small chromosome 7 derivatives microdissected suggesting that they could be involved in tumour from the cells of two distinct tumours. This small region initiation. The evolution of papillary tumours is was shown to contain the MET proto-oncogene, present characterized by additional polysomy of chromo- at three to four copies per cell as determined by somes 8, 12, 16, and 20 (Kovacs et al., 1991; Kovacs, Southern blot analysis. The increased copy number of 1993b). the MET gene was found to be associated with its Trisomy 7 is the most frequent cytogenetic abnorm- overexpression at the mRNA level. However, no change ality in early as well as advanced stages of papillary in MET copy number or expression level was observed in RCCs, suggesting that a gene (or genes) located on the cells from two xenografted tumours serially chromosome 7 plays a role in the pathogenesis of these transplanted into immunode®cient mice, as compared to tumours. One approach to pinpoint such a gene those from the corresponding initial tumours. Our results consists in seeking tumours that have retained only a indicate that expression of the MET proto-oncogene limited region of chromosome 7 as the sole over- above a critical threshold is required for the maintenance represented genetic material from this chromosome. of the tumorigenic phenotype of at least some papillary Reduction of the size of the chromosomal region renal cell carcinomas, but does not further increase bearing a candidate gene is more likely to occur in during tumour progression. Oncogene (2000) 19, 754 ± advanced tumours as a consequence of chromosome 761. rearrangements during tumour progression. We have previously reported the molecular cytogenetic charac- Keywords: chromosome 7 polysomy; 7q31 chromoso- terization of four sporadic metastatic papillary RCCs mal region; papillary renal cell carcinoma; MET and of their counterparts xenografted in severe receptor tyrosine kinase combined immunode®cient (SCID) mice. Among them, two tumours and their corresponding xenografts were found to harbour one to four small derivative Introduction chromosomes (hereafter designated `minichromo- somes'). In both cases, all the minichromosomes were The papillary subtype of renal cell carcinoma (RCC) composed of small fragments derived solely from accounts for about 10% of all kidney tumours. In chromosome 7. Moreover, the der(7) minichromo- general, the diagnosis of papillary RCC is based on somes from both tumours appeared to only have in cytological and growth pattern characteristics (Weiss et common a small region located within band 7q31 al., 1995; Amin et al., 1997). However, renal tumours (Glukhova et al., 1998). exhibit a heterogeneous morphology and their histolo- In the present work, we have re®ned the location of gical features can change during tumour progression the unique region of overlap between the two types of (Kovacs, 1993a; Lager et al., 1995; Wilhelm et al., minichromosomes to the junction of bands 7q31.2 and 1995). Molecular cytogenetic analysis allows the 7q31.3 and shown that it contains the MET proto- distinction of papillary RCC from other RCCs based oncogene. Tumour cell DNA and RNA were analysed on speci®c chromosomal anomalies that persist by Southern and Northern blotting to assess the MET gene copy number and mRNA accumulation level, respectively. In order to determine whether these two *Correspondence: A-F Goguel parameters might evolve during tumour progression, a Received 1 June 1999; revised 15 October 1999; accepted comparison was made between initial tumours and 8 December 1999 their xenografted counterparts serially transplanted MET gene in minichromosomes from two papillary RCCs L Glukhova et al 755 into SCID mice, the latter exhibiting a higher nuclear grade as de®ned by Fuhrman et al. (1982).
Results
Characterization of the 7q31 region carried on minichromosomes We have previously reported that the karyotype of the cells from the three tumours RCC-1, RCC-43 and RCC-47 (nuclear grade 1 ± 2) and from the correspond- ing xenografted tumours RCC-1 P6, RCC-43 P5 and RCC-47 P3 (grade 3 ± 4), serially transplanted into SCID mice for six, ®ve or three passages, respectively, exhibited a total or partial polysomy of chromosome 7 (Glukhova et al., 1998; see Materials and methods for tumour nomenclature). RCC-1 and RCC-1 P6 ex- hibited one or two t(7;2;7) chromosomes per cell and this derivative chromosome was shown to contain a duplicated 7q21-qter region. RCC-43 and RCC-47, as well as RCC-43 P5 and RCC-47 P3, had additional material from chromosome 7 associated with mini- chromosomes. Molecular cytogenetic analysis indicated that a chromosomal region centred on band 7q31 was shared by the der(7) minichromosomes from both tumours (Glukhova et al., 1998). To con®rm these observations, reverse painting on Figure 1 Detection of an overlapping 7q31 subregion common chromosomes from normal cells was carried out by to the minichromosomes of RCC-43 P5 and RCC-47 P3. Reverse painting was carried out by simultaneous hybridization to normal simultaneous hybridization with two probes prepared cell metaphases of two probes generated from the microdissected from the microdissected minichromosomes of the two minichromosomes of the xenografted tumours RCC-43 P5 and xenografted tumours RCC-43 P5 and RCC-47 P3 and RCC-47 P3 and stained with two dierent ¯uorochromes: stained with rhodamine and ¯uorescein, respectively rhodamine and ¯uorescein, respectively. Photographs of the same (see Materials and methods). Fluorescence microscopic normal chromosome 7, taken with dierent UV-light ®lters, are shown. From top to bottom: 4',6-diamidino-2-phenylindole examination showed the speci®c regional staining of (DAPI) counterstaining; double-staining with the two minichro- only chromosome 7 previously observed with each mosome-speci®c probes showing the region of overlap at the individual probe (Glukhova et al., 1998), but disclosed, border of bands 7q31.2 and 7q31.3 (yellow signal); single-staining in addition, the presence of a small overlapping area with the RCC-43 P5 minichromosome-speci®c probe (red signals); single-staining with the RCC-47 P3 minichromosome-speci®c (yellow signal) at the junction of one of the RCC-43 probe (green signals); diagram of chromosome 7 showing the P5-speci®c red signals (7q31.2 ± q31.3) and of one of location of the overlapping region the RCC-47 P3-speci®c green signals (7q31.1 ± q31.3) (Figure 1).
is not present in the minichromosomes from RCC-43 Refined mapping of the overlapping 7q31 subregion and RCC-47 P3. associated with the minichromosomes With the aim of mapping more precisely the region of Southern blot determination of the MET gene copy overlap, ¯uorescent in situ hybridization (FISH) number in RCC tumour cells analyses were carried out, using as probes two nonchimeric and nonoverlapping yeast arti®cial chro- In view of the presence of MET sequences in YAC mosomes (YACs), 746H5 and 880B6 that target the 746H5 (see Materials and methods), we chose to 7q31.2 ± q31.3 and 7q31.3 ± q32.1 regions, respectively. measure directly the copy number of the MET gene As a control, they were ®rst hybridized to the RCC-1 by Southern blot analysis of the DNA extracted from tumour cells. The expected number of ¯uorescent the cells of both the RCC-43 and RCC-47 tumours and signals per metaphase were observed with each of of the RCC47 P3 xenografted tumour, using two them: one twin-spot on each of the two normal dierent probes. (Cells from the RCC-43 P5 xeno- chromosomes 7 and two twin-spots on each t(7;2;7) grafted tumour could not be studied because of their derivative chromosome (data not shown). For both inability to grow into mass culture). In order to RCC-43 and RCC-47 P3, 746H5-speci®c spots were validate the results obtained, DNA from the cells of found, besides the two normal chromosomes 7, on all the RCC-1 tumour and of the RCC-1 P6 xenografted of the minichromosomes. The number of associated tumour (which harbour a number of MET-bearing signals indicated that each minichromosome contrib- chromosome 7 long arms that can be precisely uted about two copies of the 746H5-hybridizing counted) was analysed in parallel, together with DNA region per cell (Figure 2). The 880B6 probe was only from RCC-41 P2 cells, derived from a xenografted able to label the two normal chromosomes 7, undierentiated RCC that contains, on average, close indicating that the region this YAC corresponds to to two normal chromosomes 7. DNA from ICIG-7
Oncogene MET gene in minichromosomes from two papillary RCCs L Glukhova et al 756
Figure 2 Detection of YAC 746H5 sequences in the minichromosomes from both RCC-43 and RCC-47 P3. Fluorescence in situ hybridization (FISH) was performed on metaphases of the RCC-43 (left panel) and RCC-47 P3 (right panel) tumours, using a YAC 746H5-speci®c probe labelled with ¯uorescein (green signals) and a chromosome 7 centromere-speci®c probe labelled with rhodamine (red signals). In each of the two representative metaphases shown, the two normal chromosomes 7 can be identi®ed by the presence of a centromere-speci®c red spot and a pair of green signals corresponding to YAC 746H5 (7q31.2 ± q31.3). All the minichromosomes display red-stained centromeric sequences and, generally, two pairs of green signals
cells, normal human ®broblasts that are perfectly three probes were in close agreement for each of the diploid, was used as a standard to calculate the relative tumours examined. Moreover, the results were con- copy number of the MET gene. Each Southern blot sistent with the average copy number of the 7q31 contained duplicate samples of each cell DNA to check region estimated from the number of spots per cell for any variation in the eciency of DNA transfer detected in the ®ve tumour cell populations analysed and/or hybridization. Two genomic DNA fragments by FISH, using YAC 746H5 as a probe (Table 1). from the human MET locus were used as probes: These data indicate that both the MET and WNT-2 pmetD and pmetH (see Materials and methods). Since genes belong to the short overrepresented 7q31 region all of the cells examined were disomic for chromo- shared by the minichromosomes from RCC-43 and some 11, the BK4 genomic probe speci®c for the FGF- RCC-47. It has to be noted that gene copy numbers 3 gene located at chromosomal band 11q13 was used were not found to be signi®cantly dierent between the to yield positive signals that could serve as a reference two xenografted tumours RCC-1 P6 and RCC-47 P3 to correct for variations in DNA loading. Labelling of and their corresponding initial tumours RCC-1 and the probes was carried out on a mixture of equivalent RCC-47, respectively, despite their higher nuclear amounts of either MET fragment and of the FGF-3 grade. BK4 fragment. Indeed, both genes could be simulta- neously analysed on the same blot due to the large Expression of the MET gene in RCC tumour cells dierence in size of the EcoRI fragments hybridizing to the MET probes (1.2 kb for pmetD and 2.3 kb for On the basis of the increased number of copies of the pmetH) and that hybridizing to the FGF-3 probe MET gene demonstrated above, it was of interest to (9.8 kb). An illustration of the results obtained with analyse its expression by Northern blotting in the cells probe pmetD is shown in Figure 3. For the purpose of from our panel of papillary RCCs. As cultured con®rming the data obtained with the two MET epithelial cells derived from normal kidney proximal probes, the analysis was extended to another gene tubules (from which papillary renal tumours are belonging to the 7q31 region, in the vicinity of the presumed to originate) were not available to us, we MET locus. The blot formerly probed with pmetD was used, as a control, RNA prepared from RCC-41 P2 stripped and rehybridized with the genomic probe cells which have, on average, close to two normal H2.3A speci®c for the WNT-2 gene (Figure 3), which is chromosomes 7 (see Table 1). Hybridization was located at a distance of about 500 kb, telomeric to the carried out with a MET cDNA fragment probe. A MET gene (see Lin et al., 1996). major mRNA species of about 8 kb was detected in the Table 1 summarizes the results obtained from the cells from all of the tumours analysed (Figure 4 and quantitation of the signal intensities of the fragments data not shown). The size of this mRNA is in hybridizing with the two MET probes, as well as the agreement with that of the transcript previously shown WNT-2 probe. The copy numbers determined with the to encode the 190 kDa MET heterodimeric receptor
Oncogene MET gene in minichromosomes from two papillary RCCs L Glukhova et al 757 tumours RCC-1 P6 and RCC-47 P3 compared with those from the corresponding initial tumours RCC-1 and RCC-47, in agreement with the stability of the MET gene copy number noted above.
Discussion
Previous cytogenetic studies have led to the observa- tion that papillary RCCs display a recurrent combina- tion of alterations of three chromosomes: trisomy or tetrasomy 7 as the most frequent genetic event followed by trisomy 17 and loss of the Y chromosome (Kovacs et al., 1991; Kovacs, 1993b; Bentz et al., 1996). Polysomy of a chromosome may cause an imbalance in a gene (or genes), leading to its over- expression and playing an early role in tumour development. Delineation of the minimal chromosomal region required for the maintenance of the tumorigenic phenotype aords a means to identify the gene involved. However, in most cases described to date, the whole chromosome 7 is found duplicated. A partial duplication of the chromosomal region 7q21 ± q35 had been previously described in one out of four tumours from a patient with hereditary papillary RCC (Bernues et al., 1995). The tumours investigated here also exhibit a gain of genetic material derived from dierent regions of chromosome 7. RCC-1 and RCC-1 P6 cells have additional copies of the 7q21-qter region carried in duplicate on a t(7;2;7) derivative chromosome. RCC-43 and RCC-47 P3 harbour one to three der(7) minichromosomes as the sole overexpressed material from chromosome 7. Previous molecular cytogenetic analysis had shown that the minimal overrepresented genomic region common to the minichromosomes from Figure 3 Determination of MET and WNT-2 gene copy both tumours was centred on band 7q31 (Glukhova et numbers in RCC tumour cells. DNA (10 mg) was digested with the restriction endonuclease EcoRI, electrophoresed in a 0.7% al., 1998). agarose gel and transferred to a nylon membrane. The Southern In the present work, we have re®ned the location of blot was ®rst hybridized simultaneously with probes pmetD and the overlapping region to the junction of bands 7q31.2 BK4, speci®c for the MET and FGF-3 genes, respectively (top and 7q31.3 by means of simultaneous reverse painting panel). After stripping, the blot was rehybridized with probe H2.3A, speci®c for WNT-2 (bottom panel). The gel was loaded using two probes prepared from the microdissected with two successive series of duplicate samples and only the left minichromosomes of each tumour and detected with half of the autoradiographs is shown here. DNA sizes (in dierent ¯uorochromes. Of the two YACs chosen as kilobases) were determined by comparison with a DNA marker probes in FISH analyses based on their map location run in a parallel lane, from a photograph of the ethidium to bands 7q31 ± q32, 746H5 was found to target the bromide-stained gel taken prior to the transfer (not shown). DNA from diploid ICIG-7 human ®broblasts, was included as a region of overlap. In agreement with the contig standard physical map published by Lin et al. (1996) on which this YAC is shown to overlap the MET proto- oncogene locus, the presence of MET sequences was con®rmed by PCR with exon 1-speci®c primers (see (Lin et al., 1998). Quantitation of the signals indicated Materials and methods). We have recently observed that the MET gene was overexpressed at a 9 ± 12-fold that the 172-kb BAC genomic clone RG253B13 higher level in the cells that harbour extra-copies of the (Genbank accession no. AC002080) that contains gene, as compared to the cells from the control RCC- exons 1 through 9 of the MET gene can also stain 41 P2 tumour (see Figure 4, bottom of the upper the minichromosomes, yielding smaller, more de®ned panel). Only a low level of MET expression, similar to signals that are superimposable with the spots that in RCC-41 P2, was also detected in several generated by YAC 746H5 (data not shown). tumorigenic and non-tumorigenic clones derived from Southern blotting analyses established the presence the unrelated SW613-S human colon carcinoma cell of one to two additional copies of the MET and WNT- line (Galdemard et al., 1995; data not shown). 2 genes, on average, in whole genomic DNA from the Altogether, these results indicate that MET is the gene papillary RCC tumour cells examined. This technique selected for in the short 7q31 region common to both turned out to be suciently sensitive to measure the RCC-43 and RCC-47 minichromosomes and whose relatively small variations in gene copy numbers, overexpression played a role in the development of provided that signal intensity values are expressed as these tumours. The mRNA accumulation level does the mean of duplicate samples for each cell DNA and not change in the cells from the two xenografted corrected with reference to an internal standard present
Oncogene MET gene in minichromosomes from two papillary RCCs L Glukhova et al 758 Table 1 Comparison of MET and WNT-2 gene copy number determined by Southern blotting with the number of 7q31 region-speci®c signals detected by FISHa Copy number per cell Tumor cells pmetD pmetH H2.3A Mean YAC 746H5 RCC-1b 3.62+0.08 3.52+0.18 3.86+0.28 3.67+0.17 ND RCC-1 P6c 4.24+0.32 4.20+0.16 4.24+0.20 4.23+0.02 4.83+0.99 RCC-43d 3.02+0.20 3.32+0.02 3.40+0.12 3.25+0.20 4.48+1.16 RCC-47e 3.22+0.02 3.50+0.20 3.56+0.06 3.43+0.18 4.08+1.98 RCC-47 P3f 2.98+0.10 3.24+0.18 3.12+0.02 3.11+0.13 3.84+1.68 RCC-41 P2g 1.58+0.04 1.60+0.02 1.76+0.02 1.65+0.10 1.61+0.79 ICIG-7h 2.00+0.03 2.00+0.06 2.00+0.01 2.00 aSouthern blots such as the one shown in Figure 3 were scanned with the aid of a Bio-imaging Analyser. Signal intensities of the fragments detected with each of the three indicated probes (pmetD, pmetH and H2.3A) were corrected for variations in DNA loading, using as a reference the FGF-3-speci®c signal in each corresponding lane. The results are expressed as the average values (+s.d.) of the MET and WNT-2 copy number calculated from duplicate cell DNA samples for each tumour, after normalization to the DNA from ICIG-7 diploid ®broblasts used as an internal standard. The mean of the values obtained with the three probes is also shown. The last column displays the copy number of the 7q31 region (mean+s.d.), estimated from the number of signals detected by FISH on 100 metaphases from ®ve out of the six RCC tumours, using YAC 746H5 as a probe. ND: not determined. bAt the time of analysis, the RCC-1 cell culture exhibited 66% tumour cells and 34% contaminating ®broblasts. This explains why the mean copy number of the MET and WNT-2 genes is lower than that determined for RCC-1 P6, from which contaminating normal cells have been eliminated during xenografting. cIn the RCC-1 P6 tumour cell culture, 59% of the cells had a single t(7;2;7) derivative and 41% had two such derivatives, in addition to the two normal chromosomes 7 (i.e. four and six copies of the 7q31 region, respectively). dIn the RCC-43 tumour cell culture, 93% of the cells had 1 ± 2 der(7) minichromosomes, in addition to two normal chromosomes 7. eThe RCC-47 tumour cell culture was composed of a complex mixture of cell populations: at the time of analysis, 31% of the cells had two normal chromosomes 7 (29% contaminating normal ®broblasts plus 2% tumour cells), 6% had three and 31% had four, without any minimichrosome. In addition, 5% of the cells had two chromsomes 7 and 1 ± 4 der(7) minichromosomes, 23% had three chromosomes 7 and 1 ± 3 minichromosomes and 3% had four chromosomes 7 and 1 ± 3 minichromosomes. The minor tumour cell clone with two normal chromosomes 7 and 1 ± 4 der(7) minichromosomes was selected for during xenografting leading to the more homogeneous RCC-47 P3 cell population. fIn the RCC-47 P3 tumour cell culture, 65% of the cells had 1 ± 4 der(7) minichromosomes, in addition to two normal chromosomes 7. gThe control RCC-41 P2 cell culture derived from an undierentiated renal tumour was aneuploid. 15% of the cells had one normal chromosome 7, 70% had two, 2% had four and 13% had none. hICIG-7 normal human ®broblasts, that are perfectly diploid, were used as a reference to calculate gene copy numbers
at two copies per cell. By comparison with the perfectly anaphase and result in an uneven distribution between diploid ICIG-7 ®broblast line, the copy number of the daughter cells. Loss of the minichromosomes would 7q31 region determined for the control RCC-41 P2 ensue, unless the maintenance of the expression of a tumour cells was quite close to that obtained by direct gene (such as MET) above a minimal threshold be counting of FISH signals on 100 metaphases (1.65 mandatory for proliferation of the cells to proceed. A versus 1.61; see Table 1). Although the dierence is not dynamic equilibrium would follow with, as observed, a statistically signi®cant taking into account the magni- random distribution of the minichromosomes, centred tude of the standard deviations, the mean value of on a number corresponding to the optimal gene FISH signal numbers was always found to be slightly expression level at which the cells will continue to higher than that of the copy numbers determined by grow. Actually, this number (leading to the main- Southern blotting in the cells from the four tumours tenance of, on average, three to four copies of the RCC-1 P6 (4.83 versus 4.23), RCC-43 (4.48 versus MET gene per cell) is in good agreement with the 3.25), RCC-47 (4.08 versus 3.43) and RCC-47 P3 (3.84 presence of a trisomy or tetrasomy 7 previously versus 3.11). This discrepancy could be a consequence reported for the vast majority of primary papillary of the heterogenous distribution of the t(7;2;7) renal carcinomas (Kovacs et al., 1991; Kovacs, 1993b; derivative chromosomes in the case of RCC-1 P6 and Zbar and Lerman, 1998). of the minichromosomes in the case of the three other Interestingly, the MET gene was found to be tumours (see footnotes to Table 1). Thus, one overexpressed by Northern blotting in the cells from explanation for this discrepancy could be that, since all of the tumours with additional copies of the 7q31 FISH signals are counted on metaphasic (i.e. actively region, as compared to those from the control RCC-41 dividing) cells, their number may have been over- P2 tumour. The level of expression was essentially the estimated, if one considers the possibility that the same for all of them, suggesting that a critical presence of additional chromosome 7 derivatives, each threshold has to be reached and maintained for these bearing on average two copies of the MET gene, cells to proliferate. Notably, the cells from the two confers a proliferative advantage on the cells that xenografted tumours (nuclear grade 3 ± 4; see (Glukho- harbour them. va et al., 1998) had a MET gene copy number and a The heterogeneity of the minichromosome distribu- steady-state mRNA level similar to those of the cells tion could be related to a peculiarity of their structure. from the corresponding initial tumours (grade 1 ± 2). Indeed, although these minichromosomes appear to be Thus, although the MET gene is likely required for the endowed with a centromere as illustrated in Figure 2, maintenance of the tumorigenic phenotype, it does not they were found devoid of any detectable telomeric participate in tumour progression by a further increase sequences upon FISH analysis with a probe speci®c to in expression. Tumour progression may actually the telomeric region of all human chromosomes (data involve other genetic loci, as suggested by the presence not shown). Since telomeres are necessary to insure the of additional cytogenetic abnormalities in the cells integrity of chromosome ends, it is possible that the from xenografted tumours (Glukhova et al., 1998). minichromosomes have adopted a ring structure The MET proto-oncogene encodes the transmem- (Blackburn, 1991; Haaf et al., 1992). Such a structure brane tyrosine kinase receptor for the hepatocyte might lead to segregation anomalies at the time of growth factor/scatter factor (HGF/SF) and transduces
Oncogene MET gene in minichromosomes from two papillary RCCs L Glukhova et al 759 sporadic papillary RCCs (Schmidt et al., 1997; Fischer et al., 1998; Zbar and Lerman, 1998). Moreover, it has been shown that the mutant allele was duplicated and overexpressed (with regard to the normal allele) in the tumours with a trisomy 7 that have been analysed (Fischer et al., 1998; Zhuang et al., 1998). Somatic mutations in MET appear to be infrequent, having been detected in the tumours of only three out of 60 patients with sporadic papillary RCC studied by Schmidt et al. (1997). Thus, the probability that the MET gene is mutated in all of our tumours ought to be low. Future work will be aimed at elucidating the role played by MET in our sporadic papillary RCCs, ®rst by looking for the possible presence of mutations in the tyrosine kinase domain. This cellular material, cytogenetically well characterized and that can be easily manipulated experimentally, should be useful to investigate the mechanisms of activation of the MET gene. Other molecular events occurring during tumour initiation and/or progression should also be amenable to analysis by comparing the cells from the xeno- grafted tumours with those from the corresponding initial tumours.
Materials and methods
Cell cultures Figure 4 Northern blot analysis of MET mRNA in RCC tumour cells. A 32P-labelled probe prepared from a MET cDNA The isolation and growth in short-term culture of cells from fragment was hybridized to 10 mg of total cell RNA extracted the metastatic sporadic papillary tumours RCC-1, RCC-43 from the cells of the indicated RCCs (top panel). After stripping, and RCC-47 and from their xenografts serially transplanted the blot was rehybridized, under low stringency conditions, with a into severe combined immunode®cient (SCID) mice (Angevin 32P-labeled probe prepared from a mouse b-actin cDNA fragment et al., 1999) has been described previously (Glukhova et al., (bottom panel). RNA sizes (in kilobases) were determined by 1998). RCC-1 and RCC-43 cells were isolated from comparison with the positions of the 45S, 28S, 18S, 5.8S rRNAs metastatic lesions and RCC-47 cells from a primary tumour. and 4S tRNA, from a photograph of the ethidium bromide- All three tumours exhibited typical cytogenetic characteristics stained nylon membrane taken immediately after transfer. The of the papillary subtype irrespective of their histologic accumulation level of the 8-kb MET mRNA species, normalized to the actin signal and expressed relatively to that of the control features (Glukhova et al., 1998). RCC-1 P6, RCC-43 P5 RCC-41 P2 cells, is indicated below each lane of the upper panel and RCC-47 P3 cells were isolated from the corresponding xenografted tumours, Pn indicating the number of serial passages in SCID mice. RCC-41 P2 cells, used as a control, were isolated from a xenografted, undierentiated renal morphogenic, motility and proliferation signals in tumour. All these cells were used at early in vitro passages epithelial cells (for a recent review, see Bardelli et al., (three to 12) after their isolation in culture. ICIG-7 cells are 1997). HGF/SF is normally expressed in ®broblasts human diploid ®broblasts with a limited in vitro life-span that from mesenchymal tissues, but anomalous derepression were derived from embryonic lung (Macieira-Coelho and of the gene in epithelial tumour cells cannot be Azzarone, 1982). excluded. We have looked for the expression of HGF/SF at the mRNA level by Northern blotting in Microdissection and reverse painting with the cells from our panel of RCC tumours, but could minichromosome-specific probes not detect any (data not shown). Thus, activation of Microdissection of the minichromosomes from cells of the the MET receptor does not occur through an autocrine RCC-43 P5 and RCC-47 P3 tumours and generation of loop. Accumulation of the normal receptor at the cell probes for reverse painting were carried out essentially as membrane due to overexpression of the gene could be previously described (Chudoba et al., 1996). In order to sucient to chronically trigger the downstream de®ne the smallest region of chromosome 7 common to the signalling cascade leading to cell proliferation and minichromosomes from both tumours, two minichromosome- tumorigenicity. Under such conditions, activation of speci®c painting probes detectable with dierent ¯uoro- the accumulated receptor may be further stimulated in chromes were prepared: the RCC-43-speci®c probe was vivo by the endogenous HGF/SF secreted by stromal labelled with digoxigenin-11-dUTP (Boehringer Mannheim cells surrounding the tumour. Finally, constitutive GmbH, Mannheim, Germany) and detected with antidigox- activation of the MET receptor could be the igenin-rhodamine (Dianova GmbH, Hamburg, Germany), whereas the RCC-47 speci®c one was labelled with biotin-16- consequence of a mutagenic event. Indeed, point dUTP (Boehringer Mannheim) and detected with avidin- mutations located in the tyrosine kinase domain of FITC (Vector Lab, Burlingame, CA, USA). Reverse painting the MET gene have recently been identi®ed in the was carried out by hybridizing simultaneously both mini- germline of aected members of families with chromosome-speci®c probes to chromosomes from normal hereditary papillary renal tumours and in a subset of cells.
Oncogene MET gene in minichromosomes from two papillary RCCs L Glukhova et al 760 YAC probes of 10% sodium dodecyl sulphate (SDS) were successively In order to locate more precisely the site of the overlapping added dropwise. The resulting viscous lysate was incubated at region, two nonchimeric YACs mapping to the 7q31 region, 378C overnight. The SDS and residual protein were 746H5 (7q31.2 ± 31.3, D7S486-D7S522) and 880B6 (7q31.3 ± precipitated by adding 1 ml of a saturated NaCl solution 32.1, D7S680 ± D7S686) were obtained from the library of the and vigorously shaking the tube by hand for 15 ± 20 s. After Centre d'Etude du Polymorphisme Humain (CEPH, Paris, centrifugation at 3000 r.p.m. for 15 min at room tempera- France). YAC DNA was prepared and labelled with biotin- ture, the supernatant was supplemented with 2 volumes of 16-dUTP as described (Goguel et al., 1996). YAC 965F1 is an ethanol and the DNA was precipitated by gently inverting unrelated YAC used as a negative control for PCR. the tube several times. The precipitate was collected with a plastic tip, rinsed in 70% ethanol, dried under vacuum and redissolved in 2.8 ml of 10 mM Tris-HCl, pH 8.0, 1 mM Fluorescence in situ hybridization (FISH) with centromeric and EDTA (TE) containing 50 mg/ml of RNAse A. After YAC probes incubation at 378C for 6 h, the salt concentration was A chromosome 7 centromeric probe was obtained from adjusted to 0.15 M by adding 0.24 mlofa5M NaCl solution Vysis, Inc. (Downers Grove, IL, USA). Signal detection and the proteinase K-SDS treatment and
Oncogene MET gene in minichromosomes from two papillary RCCs L Glukhova et al 761 used for Northern blotting were: a 1.3-kb EcoRI fragment of and critical reading of the manuscript. We thank Dr MET cDNA from plasmid pHOS-1 (Park et al., 1987) and a Horacio G Sua rez (Villejuif) for the pmetD, pmetH and 1.1-kb SmaI±BamHI fragment from the mouse b-actin pHOS-1 probes, Dr Francis Collins and Michael R Erdos cDNA insert of plasmid pAL41 (Minty et al., 1983). The (Bethesda, MD, USA) for the H2.3A probe, Drs Gordon relative intensity of the positive signals detected on the blots Peters and Clive Dickson (London) for the BK4 probe, Dr was determined using a Bio-imaging Analyser BAS 1000 and Walter Birchmeier and Martin Sachs (Berlin) for plasmid the MacBas 2.2 software (Fuji Photo Film Co., Ltd., Japan). pBAT-SFtag containing an HGF cDNA fragment probe and Dr Serge Alonso (Paris) for plasmid pAL41. This work was supported by the Centre National de la Recherche Scienti®que, the Ligue Nationale Contre le Cancer (Comite Acknowledgments du Val de Marne) and the Institut Gustave Roussy. L. We are very grateful to Drs Berton Zbar (Frederick, MD, Glukhova was the recipient of a fellowship from the USA) and Olivier Brison (Villejuif) for helpful comments Association pour la Recherche sur le Cancer.
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