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Novel Tumor Suppressor Locus in Human Chromosome Region 3p14.2

Knut Jülicher, Guido Marquitan, Nicola Werner, Walter Bardenheuer, Lydia Vieten, Frank Bröcker, Hüsnü Topal, Siegfried Seeber, Bertram Opalka, Jochen Schütte

Affiliation of authors: Innere Klinik und Poliklinik (Tumorforschung), Universitätsklinikum Essen, Germany.

Correspondence to: Bertram Opalka, Ph.D., Innere Klinik und Poliklinik (Tumorforschung), Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany (e-mail: bertram.opalka{at}uni-essen.de).

or
Correspondence to:Jochen Schütte, M.D., Innere Klinik und Poliklinik (Tumorforschung), Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany (e-mail: jochen.schuette{at}uni-essen.de).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. METHODS: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. RESULTS: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. CONCLUSION: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Loss or functional inactivation of tumor suppressor genes is known to play an important role in the development of human malignancies, and recurrent allelic losses are considered to indicate the localization of such genes. Particular interest has focused during the past several years on human chromosome region (HCR) 3p14 and neighboring bands. Deletions within this region are found in preneoplastic tissues, occur with high frequency in many human tumors, and are associated with reversal of the senescence block in human papillomavirus 16-E6/E7-transduced uroepithelial cells (1-16). In addition, HCR 3p14 contains translocation breakpoints for a hereditary renal cell carcinoma (RCC) and for hematologic malignancies, and this region harbors the most fragile site of the human genome, FRA3B (17-20). A homozygous deletion in 3p14.2 led to the identification of the putative tumor suppressor gene, FHIT (fragile histidine triad) (21,22). Another candidate tumor suppressor gene, WNT5A, has been identified in 3p14.3-21.1 (23-25). More recently, a putative telomerase inhibitor has been tentatively mapped by two other groups to 3p12-21.1 and 3p14.2-21.1, respectively (26,27).

Since the role of the aforementioned genes in the pathogenesis of the various tumors associated with 3p14 alterations is still under investigation, and a uniform pattern of aberrations within this chromosomal region has not yet emerged (11-13,28-31), we decided to investigate this chromosomal band for tumor suppressor loci by use of functional complementation assays. This approach became available with the advent of cloned large genomic fragments, e.g., yeast artificial chromosomes (YACs) and has previously proven to be useful in animal systems (32,33) and, more recently, in the human lung cancer cell line A549 for the identification of a tumor suppressor locus on chromosome 11q (34). For this purpose, we, as had others, had constructed a library of YACs (called a contig) containing overlapping sequences of HCR 3p14 (35-38) and established the transfer of YACs into a human RCC cell line (34,39,40).


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Cell Lines and Culture Conditions

The human RCC cell line RCC-1 (alternate name: D-RC-1) has been described earlier (40,41). RCC-1 cells are nonsenescent in vitro under usual culture conditions, do not form colonies in soft agarose, and are tumorigenic in nude mice, forming tumors that correspond histopathologically to the original human clear cell renal carcinoma from which they were derived. Initially, these cells showed an almost tetraploid karyotype with interstitial deletions in 3p13-25 on one copy of chromosome 3, additions at the short arm of chromosome 7, and a deletion in chromosome band 11p11.2 (Ebert T: personal communication). At later passages in our laboratory, these cells showed a mainly triploid karyotype with deletions in 1p, 3p, and 11p and additions on 11p, with the deletion-carrying chromosome 3 being lost on further passaging of the cells. Fluorescence in situ hybridization (FISH) analyses with the use of a chromosome 3 centromere-specific probe showed that the majority (>85%) of these cells retained three copies of chromosome 3, with a deletion on one copy of chromosome 3 for the region corresponding to YAC 145F7 (40). These cells were used for microcell fusions and spheroplast fusion series I. Subsequent FISH analyses on cells that were used in spheroplast fusion series II and III showed a loss of the deletion-carrying copy of chromosome 3 in most metaphases and interphases analyzed. Microsatellite analyses of polymorphic loci on cells from earlier (fusion series I) and later (fusion series II and III) passages by use of four chromosome 3-specific probes (D3S1296, D3S1300, D3S1295, and D3S1289) showed persistent heterozygosity at three loci, suggesting the presence of both parental alleles and chromosomal duplication (data not shown). GM11713 is a mouse A9 cell line that contains a pSV2neo-tagged chromosome 3 derived from normal human fibroblasts (National Institute for Human Genetics Mutant Cell Repository, Coriell Institute for Medical Research, Camden, NJ). All cell lines including the RCC-1 derivatives were maintained as monolayer cultures in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. For selection, cells were grown in medium supplemented with 500 µg/mL active G418 (Life Technologies, Eggenstein, Germany).

In Vitro Growth Assay

To examine the growth rates and saturation densities of parental RCC-1 cells and their derivative clones, cells were plated in 75-cm2 dishes at a density of 2 x 105 cells per dish. Cells were counted in quadruplicate once every 24 hours for 12 days, and the doubling time was calculated from the logarithmic part of the growth curve. The saturation density was defined as the number of cells per centimeter squared at the time of reaching confluence.

Microcell-Mediated Chromosome Transfer

To analyze whether the tumorigenic phenotype of parental RCC-1 cells can be reverted with human chromosome 3, a neomycin resistance gene (neoR)-tagged chromosome 3 from the GM11713 mouse cell line was transferred into RCC-1 cells by microcell fusion with the use of a method described by Fournier and Ruddle (42) with modifications. RCC-1 cells transfected with pSV2Neo plasmid DNA were used as a control. In brief, GM11713 cells were grown to 90% confluence and treated with 0.2 µg/mL Colcemid (Life Technologies) for 48 hours. Micronuclei were harvested by filling the flasks with prewarmed medium containing 10 µg/mL cytochalasin B (Sigma Chemical Co., Deisenhofen, Germany) and centrifuging them in a JA-14 fixed-angle rotor (Beckman Instruments, Düsseldorf, Germany) at 24 000g for 75 minutes at 34 °C. Microcell pellets were resuspended in DMEM and filtered serially through 8-, 5-, and 3-µm polycarbonate membranes (Nucleopore, Tübingen, Germany). Filtered microcells were attached to recipient monolayer cells with 100 µg/mL phytohemagglutinin-P (Boehringer Mannheim GmbH, Mannheim, Germany) and were treated with 50% PEG 1500 (Boehringer Mannheim GmbH) and 10 mM CaCl2 in serum-free medium at 37 °C. Following a 1-minute incubation, cells were diluted 1 : 2 with unsupplemented DMEM. After 5 minutes, cells were collected and washed twice with unsupplemented DMEM, and G418 was added to the medium after 48 hours. Ten independent G418-resistant colonies were isolated after 2-3 weeks of culture from two microcell fusion experiments. Four of these clones were further analyzed. Transfer of the neoR-tagged chromosome 3 into clone MF1-1 was confirmed by double-color FISH analyses by use of a chromosome 3-specific centromere probe and a neoR-gene probe.

Spheroplast Fusion and FIS

Spheroplast fusions and FISH analyses were done as previously reported by use of the nonchimeric CEPH YAC 145F7 and other distantly located YACs from 3p14 (16,36,40). YAC 145F7 has been previously described (40) and contains sequence-tagged sites (STS) 2F2-A92 and 2K3-92 (37), D3S1384, and FHIT exons 9 and 10. An additional eight STS established in our laboratory have been submitted to the GenBank sequence database (accession numbers: G49402-G49409). No expressed sequence tags (ESTs) have yet been unambiguously mapped to YAC 145F7.

Assessment of ß-Galactosidase Activity

An assay for senescence-associated ß-galactosidase activity was performed as described previously (43). In brief, cells were fixed with formalin : glutaraldehyde, rinsed with phosphate-buffered saline (PBS), and then incubated in ß-galactose staining solution for 16 hours. Cells were subsequently rinsed with PBS, and senescent cells showing ß-galactosidase activity were microscopically identified.

Tumorigenicity Assay

For determination of tumorigenicity, 4 x 106 cells from microcell fusion cell lines or cell lines from spheroplast fusion series I were injected into nude mice. To decrease the latency period for tumor formation in the animals given injections of the control cells, 1 x 107 cells were inoculated in spheroplast fusion series II and III. Cells were injected subcutaneously between the shoulder blades or into the right flank of 4- to 6-week-old female NMRI nu/nu mice. Animals were monitored at least weekly for tumor formation. Tumors were removed, weighed, and (in most cases) established in culture. One animal that received spheroplast fusion cell line B8 was considered unevaluable because of formation at the injection site at week 13 of an apparent tumor that, on dissection, was found to be completely cystic and that most likely represented a seroma. Animal care was in accord with institutional guidelines.

Telomerase Assay

Telomerase activity was determined in RCC-1 parental cells, microcell fusion cell line MF1-1 (passage 13), and YAC 145F7-transduced RCC-1 cells, as previously described (44). RCC-1 parental cells and human embryonic kidney cell line 293 (obtained from the American Type Culture Collection, Manassas, VA) were used as controls.

Statistical Analysis

The two-tailed Fisher's exact test was applied for calculation of statistical differences between the numbers of tumors that formed with RCC-1 control cells and with spheroplast fusion cell lines containing YAC 145F7. Two-sided P values were considered to be statistically significant when less than .05. Tumor-free survival of mice was calculated according to the product limit method by use of the Lifetest procedure of the SAS program 6.1 (SAS Inc., Cary, NC). Comparison of survival curves was performed by use of the logrank test (SAS 6.1).


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Introduction of an Additional Copy of Human Chromosome 3 Into RCC-1 Cells

The in vitro growth characteristics of RCC-1 microcell hybrids are shown in Table 1.Go Saturation densities and population doubling times of the hybrid cell lines were lower than those of the RCC-1 parental and pSV2Neo-transduced RCC-1 control cells. Microcell hybrid cells ceased proliferation after 13-15 passages and subsequently died off. About 1 week before growth arrest, microcell hybrids stained positive for senescence-associated ß-galactosidase activity (Table 1Go; Fig. 1Go). In contrast, a revertant subclone, MF1-1del3p—which had lost most of the short arm of the transferred chromosome 3—showed a saturation density and a population doubling time similar to that of the RCC-1 parental cells, continued to proliferate, and did not exhibit ß-galactosidase activity (Fig. 1Go). Two of the hybrid clones were analyzed for tumorigenicity and, in contrast to controls, failed to form tumors within 9 months after cell injection (Table 2Go). These data demonstrate the reversibility of the malignant phenotype of RCC-1 cells by chromosome 3 sequences.


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Table 1. In vitro growth characteristics of human renal cell carcinoma RCC-1 parental cells and derivative cell lines following transfer of human chromosome 3 or a 3p14-specific YAC, 145F7*

 


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Fig. 1. Determination of senescence-associated ß-galactosidase activity in A) RCC-1 (human renal cell carcinoma) parental cells, B) microcell fusion cell line MF1-1 (day 90; passage 15) after transfer of human chromosome 3, C) revertant cell line MF1-1del3p (at 130 days after fusion), and D) spheroplast fusion cell line 20E (at 80 days; passage 13). Senescent cells in panels B and D stain darker; original magnification, x100.

 

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Table 2. Tumorigenicity of human renal cell carcinoma RCC-1 cells and derivative cell lines following introduction of human chromosome 3 or a 3p-specific YAC, 145F7*

 
Since a telomerase inhibitor gene has been tentatively mapped to chromosome 3p, we assessed the telomerase activity in RCC-1 parental cells and in cell line MF1-1 and found no reduction of telomerase activity (data not shown).

YAC Transfer Into RCC-1 Cells

To search within HCR 3p14 for sequences that show tumor-suppressive and senescence-inducing activity, we transferred YACs from our 3p14-specific YAC contig into RCC-1 cells by spheroplast fusion (36,38,40). Screening analyses showed in vitro growth inhibition following transfer of YAC 145F7, which is located within a region commonly deleted during human papillomavirus E6/E7-induced immortalization of human uroepithelial cells (16). In addition, FISH analyses revealed a deletion of sequences corresponding to this YAC on one copy of chromosome 3. Given this, YAC 145F7 was subsequently examined in more detail. Documentation of YAC 145F7 integration has been described previously (40). Overall, three independent spheroplast fusion experiments with YAC 145F7 were performed. Four cell lines were established from the first series (7A, 20E, 11L, and 20R) and analyzed for in vitro growth characteristics (Table 1Go). Three of these clones (7A, 20E, and 20R) as well as two (A1.4 and A33) and three (B1, B8, and B12) clones from fusion series II and III, respectively, were assayed for tumorigenicity in vivo (Table 2Go).

In Vitro Growth Properties of Spheroplast Hybrid Clones

Microscopically detectable alterations of cellular morphology were not observed in any of the cell lines harboring YAC 145F7. The four cell lines examined exhibited a longer doubling time and a lower saturation density than did RCC-1 parental cells and pSV2Neo-transfected RCC-1 control cells (Table 1Go). A revertant cell line (E15-del145F7), which had lost all of YAC 145F7 except the right YAC vector arm on continuous culturing (data not shown), showed a doubling time and a saturation density similar to those of the parental RCC-1 cells (Table 1Go).

Induction of Cellular Senescence by YAC 145F7

Proliferation arrest was observed for all four cell lines from fusion series I after 12-13 passages, corresponding to about 80-90 days after spheroplast fusion (Table 1Go). Subsequently, these cells died off within 1 week. This finding was confirmed in 10 independent experiments for all four cell lines with intermittently frozen cell passages. Similar to the results obtained with chromosome 3-transduced RCC-1 cells (see above), an induction of senescence-associated ß-galactosidase activity was detected about 1 week before cell death. In contrast, no ß-galactosidase activity in these cell lines was observed at day 60 after spheroplast fusion, in the RCC-1 parental controls, and in the E15-del145F7 cell line. Of note, in cell lines obtained from spheroplast fusion series II and III with later passages of the parental RCC-1 cell line, no growth arrest and induction of senescence-associated ß-galactosidase activity occurred.

Tumorigenicity of RCC-1 Cells After Transfer of YAC 145F7

In the first series of experiments comprising 4 x 106 cells per injection, no tumor formation was observed within a follow-up period of 9 months (Table 2Go). In contrast, control mice inoculated with RCC-1 parental cells (n = 2) or revertant cell line E15-del145F7 (n = 2) developed tumors within a median of 64 days (range, 64-77 days; P<.0001; Table 2Go).

In the second series of experiments, after administration of 1 x 107 cells per injection, tumors developed in 19 of 20 control animals given injections of either RCC-1 parental cells (14 of 15) or derivatives harboring 3p14-specific control YACs (five of five; data not shown) within a median of 42 days (i.e., 6 weeks; range, 25-56 days). Fourteen of 15 mice given injections of cell lines A1.4, A33, B1, B8, and B12 were evaluable for tumor growth, with four mice being killed after tumor-free observation periods of 11 (B12), 20 (A1.4), 31 (A1.4), and 42 (A33) weeks because of unrelated diseases. Following an observation period of more than 6 months, seven (50%) mice did not exhibit tumors. Those tumors arising occurred after a median of 13 weeks (range, 8-28 weeks).

In summary, tumors were observed in 23 of 24 control animals given injections of RCC-1 parental cells, cell line E15-del145F7, or RCC-1 cells harboring unrelated YACs. The median tumor-free survival time in this control group was 6.0 weeks (95% confidence interval = 5.4-6.6 weeks; Fig. 2Go). In contrast, 16 (70%) and 13 (57%) of 23 mice given injections of YAC 145F7-transduced cell lines have remained tumor free after latency periods of more than 6 and at least 9 months, respectively (P<.0001). The median tumor-free survival of mice receiving injections of YAC 145F7-transduced cells has not been reached (Fig. 2Go; P<.0001), thus indicating either complete tumor suppression or a sustained retardation of tumor growth.



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Fig. 2. Kaplan-Meier analysis of tumor-free survival of nude mice given injections of RCC-1 (human renal cell carcinoma) cell lines transduced with yeast artificial chromosome (YAC) 145F7 (dashed line; n = 23) or control cell lines (solid line; n = 24) of either RCC-1 parental cells (n = 17), revertant cell line E15-del145F7 (n = 2), or RCC-1 cells transduced with unrelated 3p14-specific YACs (n = 5). Error bars at representative time points indicate the 95% confidence intervals.

 

    DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
This study demonstrates that functional complementation analyses by YAC transfer into human tumor cells represent a valuable tool in the search for tumor suppressor loci within a chromosomal region characterized by increased genomic instability and widespread loss of heterozygosity, as is observed in HCR 3p14 and neighboring chromosomal bands. With the use of this approach, we herein provide evidence for the presence of a novel tumor suppressor locus in HCR 3p14.2 that is confined to the 530-kilobase insert of YAC 145F7. Given the phenotypic consistency of cell lines harboring YAC 145F7 at different chromosomal integration sites (40), this tumor suppressor activity is unlikely to result from inactivation of cellular genes by nonrandom YAC integration. Following the proposed designation of a previously identified tumor suppressor locus, NRC-1 (nonpapillary renal carcinoma 1), within HCR 3p12 (45,46), the novel tumor suppressor locus described in this study was provisionally termed NRC-2. An involvement of the last coding exon of FHIT (exon 9) in the tumor suppressor activity of YAC 145F7—which contains FHIT exons 9 and 10 in its centromeric part—is considered improbable because it would require the presence of a cryptic promoter and a translational start codon upstream of, and in frame with, exon 9. Furthermore, this small, hypothetical peptide would have to have biologic activity. Another candidate tumor suppressor gene, WNT5A, which has been recently mapped to 3p14.3-p21.1, is not located on YAC 145F7, as confirmed by polymerase chain reaction analysis [(23,37); and our own data not shown].

Consistently, throughout the three series of nude mice experiments, YAC 145F7 produced a sustained suppression of tumorigenicity in vivo. Tumor formation was completely suppressed in all mice of series I when a standard number of cells was injected into the animals (23,29,34,47). Even after the inoculations were increased to 1 x 107 cells per injection in mice from series II and III, some animals remained tumor free over the entire observation period, and tumors in the other animals in comparison with the controls occurred with a statistically significantly longer latency. These tumors probably represent the clonal outgrowth of cells harboring YAC 145F7 deletions as demonstrated for cell line E15-del145F7; thus, they may be beneficial in mapping the tumor suppressor locus to a smaller subregion of YAC 145F7 by use of FISH probes derived from a P1-derived artificial chromosome (PAC) contig corresponding to this YAC.

Moreover, we noticed a marked alteration in in vitro growth characteristcs of cell lines derived from spheroplast fusion series I. This alteration included a prolonged generation time, a lower saturation density, the induction of cellular senescence, and subsequent cell death. These findings were in complete accordance with the effects observed after microcell-mediated transfer of an additional chromosome 3. Of interest, cellular senescence was not observed in the following series of experiments, indicating that tumorigenicity in vivo and senescence in vitro are controlled separately. There may be different explanations for the lack of senescence induction in the later series of experiments. One explanation might be the possible occurrence of a mutation during propagation of the YAC in our laboratory in a gene that controls senescence or within a multifunctional gene that affects senescence induction and other proliferation characteristics in vitro. Alternatively, genetic instability of the RCC-1 cell line as indicated by loss of the deleted copy of chromosome 3 in later passages may have led to the loss of a gene cooperating with the putative tumor suppressor gene in a cascade of genes involved in the senescence program.

In conclusion, the data presented here add a novel tumor suppressor activity to those genes/loci already described for chromosome 3p: NRC-1, FHIT, WNT5A, the tumor suppressor locus in 3p21.2-21.3, and VHL (23,46-49). Moreover, a telomerase suppressor locus has been recently identified in HCR 3p12-21.1 and 3p14.2-21.1, respectively, and telomerase inhibition has been associated with growth arrest and senescence induction in vitro(26,27,50). In our experimental system, senescence induction in vitro and suppression of tumorigenicity in vivo are clearly separable from a decrease in telomerase activity because RCC-1 cells did not exhibit telomerase suppression following transfer of the entire chromosome 3 or YAC 145F7. It will be interesting to determine if YAC 145F7, which maps within the telomerase inhibitor region SCDR-2 described by Cuthbert et al. (26), will reveal such activity in other cell systems. Overall, these data suggest that several genes within HCR 3p14 and neighboring bands may be involved in the control of senescence, telomerase activity, and tumorigenesis. Consistent with this notion, putative 3p tumor suppressor genes other than VHL have recently been implicated in the development of RCC (9,51). The isolation of the gene(s) contained in YAC 145F7 in the near future after its conversion into PAC clones and ongoing selection of expressed sequence tags will help to unravel its/their role in the pathogenesis of RCC and other tumors showing 3p aberrations.


    NOTES
 
K. Jülicher and G. Marquitan contributed equally to this work.

Supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany, and Stiftung VerUm, Munich, Germany.

We thank the staff of the Centre d'Etude du Polymorphisme Humain, Paris, France, for continuous yeast artificial chromosome support; Ms. K. Renzing, Department of Biomathematics, and Drs. D. W. Beelen and J. Hense, Universitätsklinikum Essen, for their help with statistical analyses; Ms. Claudia Heyer for her excellent technical assistance; and Ms. C. Wartchow for her help with the manuscript.


    REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received February 9, 1999; revised June 29, 1999; accepted July 21, 1999.


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