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).
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ABSTRACT |
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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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).
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RESULTS |
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The in vitro growth characteristics of RCC-1 microcell
hybrids are shown in Table 1. 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 1
; Fig. 1
). In contrast, a revertant
subclone, MF1-1del3pwhich had lost most of the short arm of the
transferred chromosome 3showed 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. 1
).
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 2
). These data demonstrate the
reversibility of the malignant phenotype of RCC-1 cells by chromosome 3
sequences.
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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 1). 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 2
).
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 1). 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 1
).
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 1). 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 2). 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 2
).
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. 2). 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. 2
; P<.0001), thus indicating either complete tumor suppression or a sustained retardation of tumor
growth.
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DISCUSSION |
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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.
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NOTES |
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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.
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Manuscript received February 9, 1999; revised June 29, 1999; accepted July 21, 1999.
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