Localization of a tumor suppressor gene in 11p15.5 using the G401 Wilms' tumor assay
Localization of a tumor suppressor gene in 11p15.5 using the G401 Wilms' tumor assayLaura Hink Reid1, Ande West1, Daniel G. Gioeli1, Karen K. Phillips1, Kevin F. Kelleher1, Diana Araujo2, Eric J. Stanbridge2, Steven F. Dowdy2,+, Daniela S. Gerhard3 and Bernard E. Weissman1,*
1Department of Pathology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA, 2Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92717, USA and 3Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA
Received September 25, 1995;Revised and Accepted November 21, 1995
Multiple studies have underscored the importance of loss of tumor suppressor genes in the development of human cancer. To identify these genes, we used somatic cell hybrids in a functional assay for tumor suppression in vivo. A tumor suppressor gene in 11p15.5 was detected by transferring single human chromosomes into the G401 Wilms' tumor cell line. In order to better map this gene, we created a series of radiation-reduced t(X;11) chromosomes and characterized them at 24 loci between H-RAS and [beta]-globin. Interestingly, three of the chromosomes were indistinguishable as determined by genomic and cytogenetic analyses. Each contains an interstitial deletion with one breakpoint in 11p14.1 and the other breakpoint between the D11S601 and D11S648 loci in 11p15.5. PFGE analysis localized the 11p15.5 breakpoints to a 175 kb MluI fragment that hybridized to D11S601 and D11S648 probes. Genomic fragments from this 175 kb region were hybridized to DNA from mouse hybrid lines containing the [Delta]t(X;11) chromosomes. This analysis detected the identical 11p15.5 breakpoint which disrupts a 7.8 kb EcoRI fragment in all three of the [Delta]t(X;11) chromosomes, suggesting they are subclones of the same parent colony. Upon transfer into G401 cells, one of the chromosomes suppressed tumor formation in nude mice, while the other two chromosomes lacked this ability. Thus, our mapping data indicate that the gene in 11p15.5 which suppresses tumor formation in G401 cells must lie telomeric to the D11S601 locus. Koi et al. (Science 260: 361-364, 1993) have used a similar functional assay to localize a growth suppressor gene for the RD cell line centromeric to the D11S724 locus. The combination of functional studies by our lab and theirs significantly narrows the location of the tumor suppressor gene in 11p15.5 to the ~500 kb region between D11S601 and D11S724.
Wilms' tumor (WT), a childhood kidney cancer, is believed to develop after the loss or inactivation of one or more tumor suppressor genes (2 ). Cytogenetic deletions observed in tumors from sporadic WT patients and individuals with the related WT/aniridia/genitourinary abnormalities/mental retardation (WAGR) syndrome led to the isolation of one WT gene, WT1, in human band 11p13 (3 ,4 ). This gene encodes a transcription factor expressed in developing kidneys which can suppress growth when introduced into some WT lines (5 ). Mutations in WT1, however, have been detected in only ~5% of sporadic WT cases (6 ) suggesting that additional genes are involved in the development of WT. A second WT locus on chromosome 11 has been proposed based on molecular analyses that demonstrated loss of heterozygosity (LOH) at several 11p15.5 loci in sporadic WT with no detectable alteration in 11p13 (7 -9 ). The location of the second gene, designated WT2, coincides well with the 11p15.5 assignment of Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with a predisposition to pediatric tumors including WT (10 ,11 ). Deletions in the 11p15.5 band have also been observed in rhabdomyosarcoma, hepatoblastoma and carcinomas of the breast, bladder, lung, adrenal cortex and testicles (12 ). These results suggest that defects in the same tumor suppressor gene located in 11p15.5 may be contributing to the development of many types of neoplasia.
We have used somatic cell hybrids in a functional assay to identify regions of chromosome 11 that suppress tumorigenicity in the G401 WT cell line. These cells were isolated from a WT explanted from a 3-month-old male (13 ). They grow indefinitely in culture, form tumors when injected subcutaneously into nude mice and possess a pseudo-diploid karyotype with a marker chromosome 12 and two cytogenetically normal copies of chromosome 11. In our assay, we introduced single human chromosomes into G401 cells via microcell-mediated chromosome transfer (MMCT) (14 ,15 ) and analyzed the tumorigenic potential of the hybrid lines in nude mice. Early experiments by Weissman et al. (16 ) demonstrated that introduction of a t(X;11) (11pter -> 11q23::Xq26 -> Xqter) chromosome suppressed the tumorigenic potential of G401 cells, while transfer of either a t(X;13) chromosome or an intact X chromosome had no influence on tumorigenicity.
In order to better localize the G401 tumor suppressor element, Dowdy et al. (17 ) generated a series of deletions in a t(X;11) (11pter -> 11q13::Xq21 -> Xqter) chromosome by [gamma]-radiation. Cytogenetic analysis of the mouse hybrid lines carrying the radiation-reduced chromosomes [[Delta]t(X;11)] identified clones that contain a single human chromosome with deletions in 11p. Interestingly, several of the hybrid lines, including XMCH 708.24, XMCH 708.25 and XMCH 708.26, have similar interstitial deletions eliminating part of 11p15.5 through 11p14.1 in the [Delta]t(X;11) chromosome. Another hybrid line, XMCH 708.20, contains a [Delta]t(X;11) chromosome with complex rearrangements including an interstitial deletion encompassing 11p12-p13. Dowdy et al. (18 ) determined that the [Delta]t(X;11) chromosome in the XMCH 708.20 cell line suppressed tumorigenicity when transferred into G401 cells, while the chromosome in the XMCH 708.24 cell line did not influence the tumorigenic potential. These experiments provided functional evidence for the existence of a second WT locus, distinct from the WT1 gene.
In this report, we continue our analysis of tumorigenicity control by examining the [Delta]t(X;11) chromosomes in the XMCH 708.25 and XMCH 708.26 lines which have similar genetic alterations. As with the chromosome in the XMCH 708.24 line, introduction of the [Delta]t(X;11) chromosome from the XMCH 708.26 line did not influence tumorigenicity. However, introduction of the [Delta]t(X;11) chromosome from the XMCH 708.25 line suppressed the tumorigenic potential of G401 cells. We then characterized the [Delta]t(X;11) chromosomes by Southern blot, PCR and PFGE analyses. These results identified the 11p15.5 breakpoint in each of the [Delta]t(X;11) chromosomes and localized the WT2 gene to a region of 11p15.5 telomeric to the D11S601 locus.
Genomic analysis of the [Delta]t(X;11) chromosomes
Previous analyses had indicated that each of the XMCH 708 hybrid lines contains the insulin (INS) locus, but lacks the human [beta]-globin (HBB) gene (17 ). This region of 11p15.5 frequently exhibits LOH in tumor DNA from WT and BWS patients, suggesting the presence of a tumor suppressor gene whose loss is associated with the disease development (7 -9 ,19 -22 ). In an attempt to better characterize the [Delta]t(X;11) chromosomes, we examined the microcell hybrids for the presence of 24 11p15.5 loci by Southern blot and/or PCR analysis. The status of these loci in each cell line is summarized in Table 1 . PFGE maps of 11p15.5 predict the following order of probes surrounding the [Delta]t(X;11) chromosome breakpoints: centromere-RRM1-(D11S25/D11S26/D11S470/ D11S1193) - (D11S517/D11S601)-D11S648-telomere (23 ,24 ). The XMCH 708.20 line lacked the ribosomal reductase M1 subunit (RRM1) and H-RAS genes, but retained 11p15.5 loci between them. These results indicate that one breakpoint in the [Delta]t(X;11) chromosome in the XMCH 708.20 line lies between the RRM1 gene and the D11S25/D1S26/D11S470/D11S1193 cluster. No differences were observed among the regions of 11p15.5 DNA retained in the chromosomes from the XMCH 708.24, XMCH 708.25 and XMCH 708.26 hybrid lines. Each of these microcell hybrids contained probes from D11S648 to H-RAS, but lacked the more centromeric 11p15.5 loci, including D11S517 and D11S601. These microcell lines also displayed identical hybridization patterns with 11p13 and 11p14 probes (17 ). These data indicate that the XMCH 708.24, XMCH 708.25 and XMCH 708.26 hybrid lines contain similar [Delta]t(X;11) chromosomes with one deletion breakpoint between the D11S648 probe and the D11S517/D11S601 cluster.
In order to determine their influence on tumorigenicity, we transferred via MMCT the [Delta]t(X;11) chromosomes from the XMCH 708.25 and XMCH 708.26 cell lines into an HPRT-deficient variant of G401 cells, G401.6TG.c6. Since the [Delta]t(X;11) chromosomes contain a functional HPRT gene, G401 hybrid lines were selected in hypoxanthine/aminopterin/thymidine (HAT) medium and expanded for further characterization. Cytogenetics performed at the time of inoculation indicated that each microcell hybrid contained at least one [Delta]t(X;11) chromosome (data not shown). Clones MCH 486.1 and MCH 486.5 contained one copy of the [Delta]t(X;11) chromosome from the XMCH 708.25 line. Clones MCH 486.2G, MCH 486.2J and MCH 486.2L are subclones of the same microcell hybrid and contained two copies of the [Delta]t(X;11) chromosome from the XMCH 708.25 line. Clone 485.2A contained one copy of the [Delta]t(X;11) chromosome from the XMCH 708.26 line. The marker chromosome 12 that is typical of G401 cells was present in all hybrid lines. In addition, MCH 486.5 cells contained an extra copy of human chromosome 2.
The tumorigenic potential of the G401 hybrid lines was analyzed by subcutaneous inoculations into nude mice (Table 2 ). The parental G401.6TG.c6 line formed large tumors at all inoculation sites within 2 weeks of injection. Tumors were also observed from inoculations of the MCH 485.2A hybrid line and from inoculations of the MCH 369.18 hybrid line which contains the [Delta]t(X;11) chromosome from the XMCH 708.24 line, previously demonstrated to be tumorigenic (18 ). Cytogenetic analyses performed on explants of these tumors confirmed the presence of the introduced [Delta]t(X;11) chromosome, indicating that tumor formation cannot be attributed to loss of the [Delta]t(X;11) chromosome after mouse inoculation. In contrast, we observed no tumors in animals inoculated with any of the five MCH 486 hybrid lines that contain the [Delta]t(X;11) chromosome from the XMCH 708.25 cells. These results indicate that introduction of the [Delta]t(X;11) chromosome from the XMCH 708.25 line suppresses the tumorigenic potential of G401 WT cells, while the [Delta]t(X;11) chromosomes from the XMCH 708.24 and XMCH 708.26 cells do not affect tumorigenicity.
Genome analysis of microcell hybrids containing [Delta]t(X;11) chromosomes
11p15.5
Cell line
Method
locus
Mousea
Humanb
MCH
XMCH
XMCH
XMCH
XMCH
of analysisc
701.8
708.20
708.24
708.25
708.26
H-RAS
-
+
+
-
+
+
+
PCR
MUC2
-
+
+
+
+
+
+
SB
CTSD
-
+
+
+
+
+
+
SB
H19
-
+
+
+
+
+
+
PCR
IGF2
-
+
+
+
+
+
+
PCR
INS
-
+
+
+
+
+
+
SB
D11S887
-
+
+
+
+
+
+
SB
D11S19
-
+
+
+
+
+
+
SB
D11S724
-
+
+
+
+
+
nd
SB
D11S679
-
+
+
+
+
+
+
SB+PCR
D11S1
-
+
+
+
+
+
+
SB+PCR
D11S648
-
+
+
+
+
+
+
SB+PCR
D11S601
-
+
+
+
-
-
-
SB
D11S517
-
+
+
+
-
-
-
PCR
D11S26
-
+
+
+
-
-
-
SB+PCR
D11S25
-
+
+
+
-
-
-
SB+PCR
D11S470
-
+
+
+
-
-
-
SB
D11S1193
-
+
+
+
-
-
-
PCR
D11S1044
-
+
+
-
-
-
-
PCR
RRM1
-
+
+
-
-
-
-
SB+PCR
D11S12
-
+
+
-
-
-
nd
SB
D11S191
-
+
+
-
-
-
nd
SB
D11S30
-
+
+
-
-
-
nd
SB
HBB
-
+
+
-
-
-
-
PCR
aMouse DNA was isolated from the GM346A9 cell line.bHuman DNA was isolated from either placental tissue, normal human fibroblasts, or the IMR90 cell line.cSouthern blots (SB) and/or polymerase chain reactions (PCR) were used for genome analysis.MUC2, intestinal mucin II; CTSD, cathepsin D; IGF2, insulin-like growth factor II; INS, insulin; RRM1, ribonucleotide reductase M1 subunit; HBB, [beta]-globin; nd, experiment not done.
Tumorigenicity of G401 microcell hybrids containing [Delta]t(X;11) chromosomes
Cell line
Source of introduced
Karyotype of
Tumorigenicityb
Karyotype of tumor
chromosome
injected cellsa
reconstitutea
G401.6TG.c6
none
46
11/11
MCH 369.18
XMCH 708.24
46 + 1 [Delta]t(X;11)
8/8
46 + 1 [Delta]t(X;11)
MCH 486.1
XMCH 708.25
46 + 1 [Delta]t(X;11)
0/11
MCH 486.2G
XMCH 708.25
46 + 2 [Delta]t(X;11)
0/8
MCH 486.2J
XMCH 708.25
46 + 2 [Delta]t(X;11)
0/8
MCH 486.2L
XMCH 708.25
46 + 2 [Delta]t(X;11)
0/8
MCH 486.5
XMCH 708.25
47+ 1 [Delta]t(X;11)c
0/9
MCH 485.2A
XMCH 708.26
46 + 1 [Delta]t(X;11)
4/4
46 + 1 [Delta]t(X;11)d
aKaryotypes are based on an examination of at least 10 metaphase spreads.bTumorigenicity is the ratio of the number of tumors observed to the number of sites inoculated.cCell line MCH 486.5 contains three copies of human chromosome 2.dThe introduced chromosome was observed in only 60% of the cells from the MCH 485.2A tumor reconstitute.
In order to better characterize the 11p15.5 breakpoint in each of the [Delta]t(X;11) chromosomes, we began PFGE experiments using J1-4a cells, a hamster line that contains human 11p15.5 DNA (25 ), as a control. Southern blots of J1-4a DNA digested with rare-cutting enzymes and separated by PFGE were hybridized to the D11S517, D11S601 and D11S648 probes. As shown in Table 3 , the sizes of PFGE fragments detected by the probes are consistent with other PFGE maps of this region (23 ,24 ). After each digestion, the D11S517 and D11S601 probes hybridized to the same size fragment; therefore the relative order of these probes could not be established. However, the map of a P1, PAC and cosmid contig in this region indicates that the D11S517 probe is centromeric to the D11S601 locus (L.H.R., B.E.W., E.J.S., Tom Shows and Michael Higgins, unpublished data). To determine the distance between D11S601 and D11S648, we looked for PFGE fragments that hybridized to both probes. As shown in Figure 1 A, D11S601 and D11S648 each detected the same 175 kb fragment after hybridization to MluI-digested J1-4a DNA. The 175 kb MluI fragment was also observed in DNA from the XMCH 708.20 cell line and from the MCH 701.8 line, which contains the intact t(X;11) chromosome. This fragment was not detected in identical digests of DNA from the XMCH 708.24, XMCH 708.25 and XMCH 708.26 lines. Instead, the D11S648 probe hybridized to a ~1700 kb MluI fragment in these cell lines (Fig. 1 B). These results indicate that the 175 kb MluI fragment contains D11S601, D11S648 and the [Delta]t(X;11) chromosome breakpoints. Similar results were observed after AscI digestion. The D11S648 probe hybridized to a 300 kb AscI fragment in XMCH 708.20, MCH 701.8 and J1-4a DNA, but a ~2500 kb fragment was detected in DNA from XMCH 708.24, XMCH 708.25 and XMCH 708.26 (data not shown).
The 11p15.5 band contains two imprinted genes: H19 and insulin-like growth factor II (IGF2) which are expressed during fetal development (26 ,27 ). The IGF2 gene is maternally imprinted (28 ,29 ) and its overexpression has been associated with the development of WT (30 -33 ). In contrast, the H19 gene is paternally imprinted (34 ,35 ) and has reduced expression in many WT (32 ,33 ). Although the function of the H19 gene is not known, Hao et al. (36 ) have reported that a G401 clone transfected with an H19 construct no longer forms tumors in nude mice. This ability to abrogate the tumorigenic potential of G401 cells makes H19 an excellent candidate for the tumor suppressor gene identified in our assay. We therefore examined the expression of these two genes in the G401 cell line and the panel of microcell hybrids.
Each of the G401 hybrid lines contains two endogenous copies of the H19 and IGF2 genes and has received an additional H19/IGF2 cluster on each introduced [Delta]t(X;11) chromosome. RNA isolated from either hybrid lines that were suppressed for tumorigenicity or from mouse tumors that developed after inoculation of lines that were not suppressed, was transferred to Northern blots and hybridized to an H19 probe. If H19 modulated the tumorigenic potential of these hybrid cells, we would expect to detect H19 mRNA in all of the nontumorigenic lines, but not in the tumor samples. As shown in Figure 3 , only half of the nontumorigenic lines contained significant levels of H19 mRNA, while most of the tumorigenic lines expressed the gene. Whether these H19 transcripts are derived from the endogenous G401 alleles and/or from the transferred [Delta]t(X;11) chromosome could not be determined as the available polymorphic markers proved uninformative. H19 expression was not detected in the XMCH 708.25 and XMCH 708.26 donor cell lines (data not shown). Similar analysis with an IGF2 cDNA probe detected transcripts in fetal kidney and fetal fibroblast mRNA controls, but not in G401 cells or in any of the hybrid lines (data not shown). These results indicate that neither H19 nor IGF2 expression correlates with tumor suppression in the G401 hybrids.
We have used somatic cell hybrids to localize a genetic element on chromosome 11 that suppresses tumorigenicity in G401 WT cells. This assay provides direct, in vivo evidence for the existence of a tumor suppressor gene and so complements other methods of gene localization which lack functional support. Previous experiments generated a series of mouse hybrid lines that contain radiation-reduced human t(X;11) chromosomes (17 ). In this paper, we demonstrate that the [Delta]t(X;11) chromosomes from two hybrid lines, XMCH 708.20 and XMCH 708.25, suppress tumor formation when transferred into G401 cells, while the [Delta]t(X;11) chromosomes from two other lines, XMCH 708.24 and XMCH 708.26, lack this activity. The chromosomes were originally examined at only three 11p15.5 markers. We have extended this analysis by characterizing the XMCH 708 lines with 24 11p15.5 loci using Southern blot, PCR and PFGE techniques. These results allow us to identify the 11p15.5 breakpoint in each of the [Delta]t(X;11) chromosomes and to localize the tumor suppressor gene to a region of 11p15.5 telomeric to the D11S601 locus.
Interestingly, the [Delta]t(X;11) chromosomes in the XMCH 708.24, XMCH 708.25 and XMCH 708.26 lines contain apparently identical [Delta]t(X;11) chromosomes with an interstitial deletion extending from 11p14.1 into 11p15.5. The proximal breakpoint of this deletion is between the p72 locus and the [beta]-subunit of the follicle stimulating hormone in 11p14.1 (17 ). The distal breakpoint for each of these chromosomes disrupts a 7.8 kb EcoRI fragment between D11S601 and D11S648 in 11p15.5. No differences were detected when these chromosomes were analyzed with 24 11p15.5 probes or with six probes from other regions of 11p (17 ). In addition, each cell line contains the same 12 kb EcoRI fragment that spans the deletion in the [Delta]t(X;11) chromosomes. These data, as well as the sequential numbers of the clones, suggest that the XMCH 708.24, XMCH 708.25 and XMCH 708.26 lines are subclones of the same parent colony.
Assuming the XMCH 708.24, XMCH 708.25 and XMCH 708.26 lines are related, then each would have originally contained on their [Delta]t(X;11) chromosomes an intact copy of the gene that suppresses tumor formation in G401 cells. This genetic element is still functional in the XMCH 708.25 line, but has been inactivated in the XMCH 708.24 and XMCH 708.26 lines. The gene inactivations may result from point mutations or small deletions acquired during in vitro propagation. Such delayed physical alterations have been reported in other cell lines after radiation exposure (37 ). However, independent mutations in the same tumor suppressor gene would occur at a very low frequency, inconsistent with the high frequency of the inactivation event [two of the three related [Delta]t(X;11) chromosomes] and PFGE analysis did not detect small deletions in the breakpoint region (see NotI, BssHII and ClaI digests in Table 3 ). Interestingly, the XMCH 708.20 chromosome which has undergone extensive rearrangements after irradiation retains tumor suppressor activity.
Alternatively, epigenetic modifications, such as DNA methylation, may account for the gene inactivations in the XMCH 708.24 and XMCH 708.26 hybrid lines. Recent evidence suggests that hypermethylation can contribute to the inactivation of some tumor suppressor genes (39 ). In addition, epigenetic modifications in donor chromosomes have been observed in previous somatic cell hybrid experiments. For example, Klein et al. (38 ) reported that the activity of a cellular senescence gene on an introduced chromosome was maximal at early passages of the donor line and reduced when transferred from later passage cells. This alteration was apparently due to hypermethylation in the later passage lines as the senescence activity could be reactivated by treatment with the methylation-altering drug, 5-azacytidine. Further studies are needed in order to determine whether similar differential methylation patterns are present in the XMCH 708 cell lines and G401 hybrids.
Further support for this proposal comes from several lines of evidence that suggest epigenetic factors may regulate the WT2 gene. LOH studies have detected a preferential loss of the maternal allele in BWS and sporadic WT cases (8 ,40 ,41 ). In addition, methylation and expression studies at the H19 and IGF2 loci have demonstrated that ~30% of WT have undergone loss of imprinting (LOI) so that both copies of chromosome 11 have the `paternal' appearance (29 ,32 ,33 ,42 ,43 ). These genetic alterations suggest that the WT2 gene in 11p15.5 is imprinted so that only the maternal allele is expressed in normal tissue.
Hao et al. (36 ) demonstrated that introduction of an H19 cDNA construct into G401 cells or RD rhabdomyosarcoma cells caused morphological changes and growth retardation. These investigators also noted that one H19-transfected G401 clone no longer formed tumors when injected into nude mice and that many clones had reduced growth in soft agar. This ability to abrogate the tumorigenic potential of G401 cells, along with its paternally imprinted expression (34 ,35 ) which is reduced in many WT (32 ,33 ), makes the H19 gene an attractive candidate for the tumor suppressor gene identified in our functional assays. However, H19 expression did not correlate with tumor suppression in our G401 clones. While Northern analysis detected very low levels of the H19 transcript in G401 cells, other tumorigenic lines showed significant H19 expression. In addition, only two of the five nontumorigenic lines expressed H19. Thus, despite the presence of the H19 gene on each of the [Delta]t(X;11) chromosomes, it does not act as the operative tumor suppressor gene in our somatic cell hybrid assay.
Some investigators have postulated that G401 cells are derived from a rhabdoid tumor, rather than a WT (44 ). This reclassification is based in part on Northern and Western blot analyses, which failed to detect WT1 expression in G401 cells. These data vary when tumors derived from renal injections, rather than subcutaneous injections, of G401 are examined (P. Gasque-Carter, J. Garvin, M. K. Howett and B.E.W, unpublished data). Yet, whatever the tumor origin, our assay provides direct, in vivo evidence that the tumorigenic potential of the G401 cell line can be suppressed by a genetic element in 11p15.5.
Table 4 Primer sequences and amplicon sizes used for PCR analysis
11p 15.2 locus
Primer sequences
Amplicon size (ref)
H-RAS
5' -GCT GTG GAT GAA TTC ATG ACG GAA TAT AAG CTG GTG-3'
120 bp
CAC GAG GAC GAA TTC CTC TAT AGT GGG GTC GTA TTC
H19
5' -AAG GAG CAC CTT GGA CAT CTG GA-3'
230 bp (1)
AAC CAG CTG CCA CGT CCT GTA AC
IGF2
5' -CGA CCG TGC TTC CGG TGA GG-3'
300 bp
GGT ATC TGG GGA AGT TGT CC
D11S679
5' -CCT ATC TGA GtC CTT CTG GT-3'
1200 bp
GAT TGG CTG TGG CAC AAG AG
D11S1
5' -CCG CTT GGG ATT CTG GTT CT-3'
196 bp
GCT GCC TAA CTT TCT GAT GC
D11S648
5' -CCC AGG CTC CAG TGA AAA TG-3'
281 bp
GGT GAT GTC CCA GTG CTG TC
D11S517
5' -GGC TCG GCA GGG ACA AAT ACA GAC TC-3'
350 bp (1)
GTG TCA AGT GGG GAT GGC ATC TTC AGT
D11S26
5' -GCC CCT TAC AAT CTC ACA TGC CTG C-3'
402 bp
CAC CCA TTA CTA GAT TCC AGC CCT G
D11S25
5' -CCG GAT CAA GCT ATC AGA ACC TTG C-3'
300 bp
ATC CTG GCA CCC TAT CTG CCC TGG G
D11S1193
5' -CCT GGG TTA AAA CAC TCA ATT G-3'
289 bp (47)
AGG AAG CTG ATT TTC TCC TGC
D11S1044
5' -CGG GAG AAA CTA GAG GCA GA-3'
276 bp (47)
AAC TCT GAG GGG AGC AGT CA
RRM1
5' -CCT CAT CTT TGC TGG TGT ACT CCA C-3'
203 bp
TTA TGA GAA ACA AGG TCG TGT CCG C
HBB
5' -GAA GGG CCT TGA GCA TCT GG-3'
600 bp
GAT AGT TCC GGG AGA CTA GC
Regardless of whether the gene inactivations in the XMCH 708.24 and XMCH 708.26 lines are the result of physical mutations or epigenetic alterations, our results map a tumor suppressor gene for the G401 cell line to a region of 11p15.5 DNA that is present on the [Delta]t(X;11) chromosomes in the XMCH 708.20 and XMCH 708.25 lines. Koi et al. (1 ) have used a similar functional assay to localize in 11p15.5 a growth suppressor gene for the RD cell line. These investigators observed growth suppression after introducing a subchromosomal fragment that contains a 3 Mb region in 11p15.5 between HBB and D11S724. If the same genetic element suppresses both the G401 and RD cell lines, one could limit its location to the ~500 kb area of overlap between D11S601 and D11S724 present in the subchromosomal fragments transferred by Koi et al. and our [Delta]t(X;11) chromosomes. The fact that many of the breakpoints for chromosomal alterations observed in BWS patients map to this same region of 11p15.5 (45 ,46 ) significantly strengthens the notion that one common tumor suppressor gene may reside in this area. Therefore, the combination of functional analyses performed by our laboratory (16 ,18 ) and others (1 ) significantly narrows the location of the WT2 gene.
The XMCH 708 cell lines are mouse cells with a single human [Delta]t(X;11) chromosome. They were generated by exposing microcells from the MCH 701.8 line, which contains an intact t(X;11) (11pter -> 11q13::Xq21 -> Xqter) chromosome, to [gamma]-radiation prior to fusion with HPRT-deficient mouse A9 cells (17 ). The [Delta]t(X;11) chromosomes in the XMCH 708.24, XMCH 708.25 and XMCH 708.26 cells were transferred into HPRT-deficient G401.6TG.c6 cells by MMCT as previously described (15 ,17 ) to generate the MCH 369, MCH 486 and MCH 485 lines, respectively. Cells were grown at 37oC in 5% CO2 in RPMI medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Hybrid lines containing the [Delta]t(X;11) chromosomes were selected and maintained in growth medium with 100 mM hypoxanthine, 0.4 mM aminopterin and 1.6 mM thymidine. All lines were routinely monitored for Mycoplasma infection.
Female nude mice (Harlan laboratories) were inoculated subcutaneously with 1×107 cells resuspended in 100 µl of PBS. The mice were 4-6 weeks old at the time of injection and two sites were inoculated per animal. Animals were examined weekly for 6 months or until tumors developed. In order to determine the karyotype of the tumorigenic cells, reconstitute cell lines were established by growing tumor explants in vitro without selection.
PCR reactions contained 400 ng of genomic DNA, 1 U Taq polymerase, 10 mM Tris, pH 9, 50 mM KCl, 1-2 mM MgCl2 and 0.2 mM each deoxyribonucleoside triphosphate. Generally, the DNA was amplified by 30 cycles of 30 s at 94oC; 1 min at 56-72oC; and 1 min at 72oC. PCR products were visualized in EtBr-stained 2% agarose gels. Many of the PCR primers were based on DNA sequence generated in our laboratory from the genomic clones used in the Southern blot analysis. Primer sequences and amplicon sizes are listed in Table 4.
Genomic DNA (5 µg) was isolated from cultured cells, digested with restriction enzymes, separated by electrophoresis through 1.0% agarose gels and transferred to nylon membranes using standard protocols. DNA probes were 32P-labeled using a random-primed kit (Boehringer Mannheim). Hybridizations were done at 42oC in a 50% formamide solution. Repetitive sequences in cosmid probes were blocked by preannealing with Cot-1 DNA (Bethesda Research Laboratories) according to the manufacturer's directions. Inserts from the following plasmids and cosmids were used as probes: MUC2, SMUC41 (48 ); CTSD, pCD2.1Ex (49 ); INS, phins214 (50 ); D11S887, CR147 (51 ); D11S19, pCDS2.7Z (52 ); D11S724, cCI11-555 (53 ); D11S679, cCI11-469 (53 ); D11S1, pCS1 (54 ); D11S648, cCI11-395 (53 ); D11S601, cCI11-565 (53 ); D11S26, pGGE0.9A (54 ); D11S25, p[gamma]-6 (54 ); D11S470, cCI11-289 (55 ); RRM1, p[epsilon]RED (56 ); D11S12, pADJ762 (57 ); D11S191, LL1030 (58 ); D11S30, pJ1.1 (54 ); HBB, p1.55[theta]1Sst (59 ).
High molecular weight DNA was isolated from cells (107 cells/ml) embedded in SeaPlaque agarose (FMC Bioproducts) as described (60 ). DNA plugs (6×6*1 mm) were digested overnight in buffers supplied by the manufacturer (New England Biolabs). To ensure complete digestion, the plugs were equilibrated in restriction buffer overnight at room temperature, prior to the addition of BSA and enzyme. Generally, DNA was separated in 1% SeaKem LE agarose (FMC Bioproducts)/0.5* TBE gels in a CHEF-DR III apparatus (Biorad) with an included angle of 120o, at 6 V/cm. Pulse conditions were: 24 h with an initial switch time of 58 s and a final switch time of 115 s to resolve 100-1000 kb fragments; or 24 h with an initial switch time of 5 s and a final switch time of 25 s to resolve 50-250 kb fragments. DNA fragments greater than 1 Mb were resolved in 0.8% chromosomal grade agarose (Biorad)/1*TAE gels, with an included angle of 106o, at 3 V/cm, for 48 h with a switch time of 500 s. Gels were nicked with 60 mJ of ultraviolet light prior to denaturation and transferred to positively charged nylon membranes (Boehringer-Mannheim) in alkaline solution.
Polyadenylated RNA (2 µg) was isolated with an oligo-dT kit (Collaborative Research), separated on 1% agarose-formaldehyde/MOPS gel and transferred to nylon membranes in 10* SSC. Northern blots were hybridized using the same protocol as described for Southern blot analysis. The H19 probe was either a cDNA clone or a 230 bp, exon 1 fragment generated by PCR. The IGF2 probe was a human cDNA variant, hIGF2v. The [gamma]-actin probe was a mouse clone, pHF1.
We thank James Gum, Marc Hansen, Peter Little, Ingrid Caras, Daniel Haber, Shirley Tilghman, A. Joseph D'Ercole, Genentech and the Japanese Cancer Research Resources Bank for DNA probes; Cliff Rinehart, John Quakenbush and Ron Shehee for PCR primers; Jennifer Byrne and Peter Smith for RRM1 sequence; Perry Gasque-Carter, A. Julian Garvin and Mary Kay Howett for unpublished data; Carol Jones for the J1 hybrid lines; Leigh Briley and Clare Fasching for technical assistance; as well as Eric Lai for PFGE instruction. In particular, we are indebted to Michael Higgins and David Munroe for 11p15.5 probes and unpublished mapping data. L.H.R. was an American Cancer Society (ACS) Postdoctoral Fellow. This research was supported by NIH grants to B.E.W. (CA4470, CA63176) and E.J.S. (CA19104) and by an ACS grant to D.S.G. (CN64).
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*To whom correspondence should be addressed
+Present address: Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110, USA
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