Identification of a Yeast Artificial Chromosome That Spans the Human Papillary Renal Cell Carcinoma-Associated t(X;1) Breakpoint in Xp11.2

R. F. Suijkerbuijk, A. M. Meloni, R. J. Sinke, B. de Leeuw, M. Wilbrink, H. A. P. Janssen, M. T. Geraghty, A. P. Monaco, A. A. Sandberg, and A. Geurts van Kessel

ABSTRACT: Recently, a specific chromosome abnormality, t(X;1)(p11;q21), was described for a subgroup of human papil lary renal cell carcinomas. The translocation breakpoint in Xp11 is located in the same region as that in t(X;18)(p11;q11)-positive synovial sarcoma. We used f luorescence in situ hybridization (FISH) and somatic cell hybridization techniques to demonstrate 1) that the Xp11 translocation break- point in papil lary renal cell carcinoma differs from that observed in synovial sarcoma and has a more proximal location, and 2) that an ornithine aminotransferase (OAT)L2 containing yeast artificial chromo- some (YAC) spans the X;1 translocation. This YAC provides an ideal starting point from which the break- point i tself and the gene(s) involved can be isolated and characterized.

INTRODUCTION

Renal cell carcinomas represent 85% of all primary renal tumors [1]. Histogenetically, these carcinomas can be sub- divided into two major groups: papillary and nonpapillary tumors. Renal cell carcinomas generally are sporadic, but familial cases have been reported [2]. All these familial cases have chromosome 3 anomalies in common, suggesting a pri- mary role for this chromosome in renal cancer causation, particularly the nonpapillary type. This notion has now been firmly established by molecular analysis of tumor material in both familial and sporadic cases, but the critical chromo- some 3 sequences involved in this group of tumors has not yet been identified completely [3, 4]. At the cytogenetic level, the papillary renal cell carcinomas differ distinctly from

From the Department of Human Genetics, University Hospital, Nijmegen, The Netherlands (R. F. S., R. J. S., B. d. L., M. W, H. A. P. J., A. G. v. K.), The Cancer Center of the Southwest Biomed- ical Research Institute and Genetrix Inc., Scottsdale, Arizona, (A. M. M., A. A. S.), The Department of Pediatrics and The Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland (M. T. G.), and ICRF Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, England (.4. P M.).

Address reprint requests to: R. E Suijkerbuijk, Department of Human Genetics, University Hospital, PO. Box 9101, 6500 lib Nijme- gen, The Netherlands.

Received March 30, 1993; accepted August 3, 1993.

164 Cancer Genet Cytogenet 71:164-169 (1993) 0165-4608/93/$06.00

the nonpapillary tumors in that they do not involve chromo- some 3.

Numerical abnormalities also are common in the various subtypes of renal tumors, particularly + 7 and + 10. Recently, however, several groups of investigators reported that these aneuploidies may also be observed in surrounding (normal) kidney tissue and, as such, may not be tumor specific [5, 6]. In addition, Dal Cin et al. [7] demonstrated that the ob- served trisomies may be a characteristic of infiltrating inflam- matory (T) lymphocytes.

In 1986, de Jong et al. [8] reported a case of trabecular papillary renal adenocarcinoma in a 2-year-old boy. The tu- mor cells were characterized by a unique cytogenetic abnor- mality involving chromosomes X and 1. That this t(X;1) (p11;q21) may actually characterize a subtype of renal tumors with papillary features arose from observation of several ad- ditional cases with the same translocation, sometimes as the sole anomaly (Fig. 1) [9]. This cytogenetic abnormality has not been described in any malignant disorder other than re- nal tumors with papillary characteristics and, as such, should be considered a specific primary change. Occurrence of a very similar breakpoint in band Xp11 was previously reported in t(X;18)-positive synovial sarcomas [10] and more recently in two cases of renal cell carcinoma with a t(X;17)(p11;q23) and a t(X;1)(p11;p34), respectively [11, 12]. In a previous study, using a combination of somatic cell hybrids and fluorescence in situ hybridization techniques (FISH), we noted that the Xp11 breakpoint in synovial sarcomas maps in the ornithine aminotransfemse (OAT)L1 cluster [13, 14]. Using similar tech-

© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

YAC Spans Carcinoma-Associated Breakpoint 165

1 2 3 4 5

i K i 8 a 13 14 15 16

# ti u

11

17 18

12

:ilM Y m 20

i

21 22

Figure 1 GTG-banded karyotype of the renal tumor CL-89-12117 exhibiting the characteristic t(X;1)(p11;q21).

niques, we have now identified a yeast artificial chromosome (YAC) clone that spans the papillary renal cell carcinoma- associated t(X;1) breakpoint. In addition, our results define the relative positions of the different tumor-specific break- points in Xp11 and may contribute to the physical ordering of DNA markers in the proximal Xp region.

MATERIAI~ AND METHODS

Tumor Material and Somatic Cell Hybrids Two primary papillary renal cell carcinoma cultures (CL- 89-12117 and CL-89-17872) were used in the present study. In CL-89-12117, the characteristic t(X;1)(p11;q21) was present as the sole cytogenetic anomaly, whereas in the second tu- mor some numerical changes were observed in addition to the X;1 translocation [9]. Both tumors have been maintained in culture for more than 10 passages, and their cytogenetic constitution appears to be stable.

Tumor CL-89-12117 was used for fusion with the Chinese hamster cell lines A3 (thymidine kinase deficient) and Wg3-h (hypaxanthine phosphoribosyltransfemse deficient). This fu- sion resulted in a panel of somatic cell hybrids in which the translocation chromosomes segregate. For our present study we selected one hybrid cell line (WgRe5) that has retained the der(X) in the absence of other (normal) chromosomes X or 1 material. Fusion and hybrid selection procedures es-

sentially were those reported before, using Sendai virus as fusogen and a combined HAT/ouabain protocol for hybrid selection [15].

Isolation of the hybrid cell line Hlsynsarc, after fusion of t(X;18)(p11;q11)-positive synovial sarcoma cells with the Chinese hamster cell line CH3S, was reported previously [16]. This cell line has retained the X-derivative of the tmnsloca- tion in the absence of a normal X or 18. As reference, a hy- brid cell line (578) containing a complete X chromosome as its sole human constituent was used [17]. Chromosome con- stitution of the somatic cell hybrids was evaluated by cyto- genetic analysis with R-banding after heat denaturation and by FISH with a combination of centromere specific probes and whole chromosome paints [13, 14].

FISH All FISH procedures used were essentially as described pre- viously [18, 20]. Probes were labeled with biotin-14-dATP (GIBCO-BRL Life Technologies, Gaithersburg, MD) or digoxi- genin-11-dUTP (Boehringer, Mannheim, Germany) by stan- dard nick-translation according to the manufacturers' pro- tocols. The labeled DNA was purified through a Sephadex G50 spin column and precipitated in the presence of soni- cared herring sperm DNA (50 ~g-fold). Then, for each incu- bation, a mixture of 200 ng YAC probe DNA and 10 ~g soni-

166 R.F. Suijkerbuijk et al.

cated total human DNA was coprecipitated and subsequently dissolved in 10 ~1 hybridization solution (50% vol/vol deionized formamide, 10% wt/vol dextran sulfate, 2 x SSC, 1% vol/vol Tween-20, pH Z0). Before hybridization, the probe was denatured at 80°C for 10 minutes, chilled on ice, and incubated at 37°C for 4 hours, allowing preannealing. For cen- tromeric probes and chromosome libraries, no preanneal- ing and preannealing for 20 rain (the latter in the presence of 50-fold Cot-1 DNA: GIBCO-BRL Life Technologies) were performed, and the probe concentrations were 20 and 100 ng per reaction, respectively. Metaphase spreads were pre- pared by standard procedures. Before hybridization, the slides were pretreated with RNase A [100 ttg/ml in 2 × SSC at 37°C for I hour). Subsequently, the slides were denatured in 70% formamide, 2 x SSC (final pH Z0) at 70°C for 2-3 minutes and incubated with the probes under an 18 × 18- mm coverslip in a moist chamber for 1-3 nights.

Immunocytochemical detection of the hybridizing probes was achieved using FITC-conjugated avidin (Vector Labora- tories, Burlingame, CA) followed by an amplification step using biotinylated goat anti-avidin [Vector Laboratories) and another FITC-avidin step [20, 21] for biotinylated probes or rhodamine-conjugated sheep anti-digoxigenin (Boehringer Mannheim) and amplification with Texas red-conjugated rab- bit anti-sheep (Jackson Immunoresearch Laboratories, West Grove, PA) for digoxigenin-laheled probes. The slides were mounted in antifade medium (1.4% wt/vol di-azobicyclo- 2,2,2)-octane: DABCO, Merck, Darmstadt, Germany) contain- ing 4,6-diamino-2-phenylindole (DAPI, 0.5 t~g/ml, Sigma, St. Louis, MO) or propidium iodide (0.5 ~g/ml: Sigma) for chro- mosome counterstaining.

Chromosomes were studied under a Zeiss Axiophot epi- fluorescence microscope equipped with appropriate filters. Digital images were recorded using a Photometrics high- performance CH250/A cooled CCD-camera (Photometrics, Tucson, AZ) interfaced to a Macintosh IIci computer, using the image-analysis and -processing program (BDS-Image, Biological Detection Systems, Rockville, MD).

Southern Blot Analysis DNAs of hybrid and parental cell lines were isolated accord- ing to procedures described by Jeffreys and Flavell [22], digested to completion with restriction endonucleases (Life Technologies) and, after agarose gel electrophoresis, blot- ted onto Genescreen Plus membranes (Dupont) using stan- dard protocols. Subsequently, the blots were hybridized with the cDNA probe Huoat-6 {labeled by random-priming) [23] in 0.5 M PO 4, 1 InlX/[ NazElYrA, and 7% sodium dodecyl sul- fate (SDS, wt/vol) at 65°C overnight, washed in 40 and 10 IHIV[ PO4, 0.1% SDS at 65°C and exposed to x-ray films for 1-3 days at - 80°C using intensifier screens as described pre- viously [13].

RESULTS AND DISCUSSION

Xp11.2-Linked DNA Markers and Their Localization Previously, we reported a most likely probe order in the Xp11.2 region based on both literature data [24] and our own analysis of a panel of radiation-reduced cell hybrids [25]. Sub- sequently, this information was used to map the synovial

sarcoma-associated t(X;18) breakpoint between the markers TIMP and DXS266 [13]. This same region was believed to con- tain the ornithine aminotransferase-like 1 (OATL1) cluster [25-27], and further breakpoint analysis showed that this cluster is split as a result of the translocation. All known se- quences contained within a second OAT-like [OATL2) clus- ter appeared to be located proximal to the t(X;18) breakpoint on Xp. Analysis of OATL1- and OATL2-specific YACs and cos- mids on primary tumor material using both FISH and South- ern blotting techniques confirmed and substantiated these preliminary observations [14]. This mapping information on the proximal Xp arm is used and extended further in the pres- ent analysis of a cytogenetically similar breakpoint in Xp11.2 specifically observed in a subgroup of renal cell adenocar- cinomas, i.e., papillary renal cell carcinoma [9].

Analysis of OAT-Specific YACs on tOi;1)-Positive Papillary Renal Cell Carcinomas Essentially, two OAT-specific YACs, one corresponding to the OATL1 (Yac 2; insert size 550 kilobases, kb) and the other corresponding to the OATL2 (YAC 7; insert size 600 kb) cluster on the human X chromosome, were used. These two YACs were selected from a larger pool of OAT-containing YACs [28] based on their correct and respective localization on Xp and their lack of (cross)hybridization to other human chromo- somes. The latter phenomenon is frequently observed and is usually due to the chimeric nature of the YAC concerned [13]. Metaphase spreads of the t(X;1)-positive papillary re- nal cell carcinomas CL-89-12117 and CL-89-17872 were ana- lyzed with YACs 2 and 7 in conjunction with FISH, accord- ing to methods described previously [13, 14]. As shown in Figure 2A, all sequences contained within YAC 2 (i.e., OATL1) are translocated to the der(1)t(X;1)(p11;q21) in tumor CL-89- 12117. In contrast, YAC 7 (containing OATL2) displays hybrid- ization signals on both the der(1) and der(X) of the translo- cation (Fig. 2B), indicating that human sequences contained within YAC 7 are split as a result of the (X;1) translocation. Identical results were obtained with the second tumor (CL- 89-17872, not shown). These findings are in full accordance with the above OATL1 results and also are in agreement with the known, more proximal localization of OATL2 on Xp [27]. In addition, these observations indicate that the OAT-specific YACs 2 and 7 do not show cross-hybridization among each other or with other chromosome regions under the stringency conditions applied in our FISH experiments.

The validity of these FISH results with the OATL2 YAC was tested further using a set of different cosmid clones de- rived from YAC 7 (full details of YAC subcloning into cos- mids will be published elsewhere). Our initial results indi- cate that some of these cosmids map distal, whereas others map proximal to the t(X;1) breakpoint (not shown). These latter findings are in accordance with the above notion that human sequences in YAC 7 are split as a result of the tumor- specific chromosome translocation.

Localization of the t(X;1) Breakpoint Relative to the OATL2 Cluster To establish further the exact position of the t(X;1) breakpoint relative to the OATL2 sequences on Xp, a panel of somatic cell hybrids was generated by fusion of CL-89-12117 tumor

YAC Spans Carcinoma-Associated Breakpoint 167

B

Figure 2 FISH of OATL1 YAC 2 (A) and OATL2 YAC 7 (B) on metaphase spreads of the renal tumor CL-89-12117. In A, a signal is apparent on der(1) only; in B, a split signal is apparent on both der(X) and der(1) (arrows).

cells with the Chinese hamster cell lines A3 and Wg3-h. These cell hybrids were characterized using both cytogenetic (R-banding) analysis of metaphase spreads and FISH with chromosomes X- and 1-specific centromeric probes and whole chromosome libraries ("paints", not shown}. As a re- sult, one hybrid (WgRe5} was selected for our present analy- sis. This hybrid has retained a der(X} and, in addition, three to four other human chromosomes. DNA was extracted from this hybrid and from another cell hybrid (578) containing an intact X chromosome as the sole human component and from the parental hamster cell line Wg3-h. For comparison, hy- brid cell line Hlsynsarc, containing the synovial sarcoma- associated der{X}t(X;18}(p11.2 ;q11.2} [13, 17], was used. The different DNAs were cleaved with HindIII and subjected to Southern blotting. The resulting blot was hybridized to the human OAT-specific cDNA probe Huoat-6 [13, 29]. As shown in Figure 3, several hybridizing bands are detected by Huoat-6 corresponding to both the OATL1 {marked by 1] and OATL2 (marked by 2) clusters. Some of the bands (marked by 1.2} comigrate. A Chinese hamster cross-hybridizing fragment also is apparent {marked by H}. All these bands are present in the control hybrid cell line 578, as expected. In Hlsynsarc, with the der(X}t(X;18)(p11;q11), some of the OATLl-specific

bands are present and others are absent (Fig. 3), exactly as reported previously [13]. In the hybrid WgRe5 [containing the dar(X}t{X;1)(p11;q21}], however, all OATLl-specific frag- ments are absent (arrows}. This result is also in complete ac- cordance with the FISH experiments using YAC 2 (described herein] and indicates that in papillary renal cell carcinomas the breakpoint is proximal to OATL1. In contrast, all OATL2- specific fragments, as detected by Huoat-6, are still present in WgRe5, indicating that the breakpoint maps distal to the OATL2 cluster on Xp.

Our results indicate that the papillary renal cell carci- noma-associated t(X;1} breakpoint in Xp11.2 differs from the one we previously identified in t(X;18}(p11;q11}-positive sy- novial sarcoma and that it has a more proximal localization. Recently, however, Knight et al. [30] reported that in their synovial sarcoma material the breakpoint could be mapped just centmmerically to the OATL2 cluster on Xp. These ap- parently conflicting results may indicate that two different breakpoints in Xp11.2 exist in synovial sarcoma. If so, these breakpoints may involve related sequences present in both the OATL1 and OATL2 regions [14]. The breakpoint we de- scribe for papillary renal cell carcinomas also differs from the second possible synovial sarcoma-associated breakpoint:

168 R . F . Suijkerbuijk et al.

1~ o co

"I- X X" X

I.:

I.-'

Figure 3 Southern blot containing HindIII digested Chinese ham- ster {HAM}, X-only hybrid 578 (X-tot), der(X)-containing synovial sarcoma-derived hybrid Hlsynsarc {X;18) and der(X}-containing papillary renal cell carcinoma-derived hybrid WgRe5 (X;1) DNAs, hybridized to cDNA probe Huoat6. 1, OATLl-specific; 2; OATL2- specific; and H, hamster-specific fragments. OATLl-specific bands partly or completely absent in (X;18) and {X;1), respectively, are marked by arrows.

in t(X;1)-positive cells the break occurs telomerically to the OATL2 cluster. Further analysis of the available breakpoint- spanning YACs in both tumor types and molecular charac- terization of the breakpoints will eventually elucidate the gene(s) involved and their possible interrelation(s} and role(s) in the process of neoplastic transformation in these two differ- ent tumors.

Whether other renal cell carcinoma-associated break- points, such as t(X;17){p11;q23) and t(X;1)(p11;p34} [11, 12], are identical to or different from that observed in the stan- dard t(X;1)(p11;q21) and whether the respective breakpoint positions may have consequences in the clinical behavior of the corresponding tumors is not yet known. With iden- tification of YAC Z we now have the tools available to per- form such studies.

The authors thank Drs. H.H. Ropers, D. Valle, and S. Gilgenkrantz for advice and support and M. Powell, D. Olde Weghuis, M. Bale- mans, and the members of the "Bone Marrow Group" for expert tech- nical assistance. The authors also thank Dr. B. de Jong for referring patient material to them. This work was supported by the Dutch Can- cer Society (Koningin Wilhelmina Fonds), Grant No. Ro 389/15-4 fi-om the Deutsche Forschungsgemeinschaft (to H.H.R.), and National Can- cer Institute Grant No. CA-41185 (to A.A.S.).

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