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Cytogenetic characterization of the murine bladder cancer model MB49 and the derived invasive line MB49-I
Cancer Genetics 205 (2012) 168e176
Cytogenetic characterization of the murine bladder cancer model MB49 and the derived invasive line MB49-I Victoria T. Fabris a,*, Catalina Lodillinsky b, Marı´a Betina Pampena a, n b Denise Belgorosky b, Claudia Lanari a, Ana Marı´a Eija a
Laboratory of Hormonal Carcinogenesis, Instituto de Biologı´a y Medicina Experimental (Consejo Nacional de Investigaciones Cientı´ficas y Te cnicasdCONICET), Buenos Aires, Argentina; b Research Area, Instituto de Oncologı´a Angel H. Roffo, Universidad de Buenos Aires, Buenos Aires, Argentina Bladder cancer is frequently associated with chromosomal abnormalities, and the complexity of karyotypes increases with tumor progression. The murine model MB49 is one of the most widely studied models of bladder cancer. We developed the invasive cell line MB49-I by successive in vivo passages of MB49 primary tumors. Because little is known about the chromosomal alterations of this model, our goal was to perform cytogenetic analyses of the MB49 and MB49-I lines. The karyotypes of both lines were analyzed by G-banding and fluorescence in situ hybridization techniques. Both lines were composed of two cell subpopulations, a diploid population, which was found mainly in the MB49 line, and the tetraploid population, which was found mainly in the MB49-I line. A translocation between chromosomes 5 and 9 and an isochromosome of chromosome 19 were observed in the subpopulations of both lines. New structural abnormalities and additional chromosomal imbalances were detected in the MB49-I line. Tumor progression in the MB49/MB49-I model was associated with a selection of polyploid cells with accompanying chromosomal abnormalities. This model may be advantageous for the study of the genetic changes associated with the progression of bladder cancer. Keywords Bladder cancer, MB49, MB49-I, cytogenetics, FISH ª 2012 Elsevier Inc. All rights reserved.
Bladder cancer is the seventh most common cancer in men worldwide (1). In 2006, there were approximately 61,240 diagnosed cases of bladder cancer, and an estimated 13,060 deaths were attributed to this disease (2,3). Urinary bladder cancer originates mainly from epithelial cells of the urothelium (4,5). Urothelial carcinomas, which account for 90% of the malignant tumors that arise in the urinary bladder (6), are frequently associated with chromosomal abnormalities; these include gains of chromosomes 1, 3, 7, 9, 11, and 17 and deletions of a part of chromosome 9p (locus 9p21). Cytogenetic changes have been useful in the diagnosis and prognosis of bladder cancer and are currently studied by fluorescence in situ hybridization (FISH) using chromosomespecific probes. Most of the urothelial carcinomas show
Received September 13, 2011; received in revised form January 30, 2012; accepted February 3, 2012. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2012.02.002
polysomy of chromosomes 3, 7, and 17 and a deletion of 9p21, which can be detected in urine samples by FISH analysis for the diagnosis of urothelial carcinomas (7). It has been proposed that a loss of material from chromosome 9 is an early event in bladder cancer (8). Nonmuscle invasive (NMI) bladder tumors often present a near-diploid karyotype with few chromosomal aberrations; the only and recurrent alterations that have been detected are the deletion of chromosomes 9p and 9q, together with mutations in the fibroblastic growth factor receptor 3 (FGFR3) gene (9). Muscleinvasive tumors are predominantly polyploid with complex karyotypes: pT2-T4 tumors frequently contain gains of chromosomes 1q, 5p, 6p, 8q, 10p, 17q, and 20q, and losses of chromosomes 2q, 3p, 5q, 8p, 10q, 11q, 13q, 17p, and 18q (9). The number of chromosomal aberrations has been shown to increase with the progression, grade, and stage of the tumors (10). However, chromosomal instability does not predict the risk of progression of bladder tumors (11). The mouse urothelial carcinoma cell line MB49 is one of the in vitro and in vivo models most widely used to study
Cytogenetics of bladder cancer model MB49-MB49-I bladder cancer biology and therapeutic strategies (12). In 1979, Summerhayes et al. derived the MB49 cell line from C57BL/lcrf-a’ mouse bladder epithelial cells that were transformed by 24 hours of treatment with 7,12-dimethylbenz [a]anthracene (DMBA) on the second day of a long-term primary culture. The transformed cells generated carcinomas after transplant into syngeneic mice (13). We have previously generated and described a new invasive bladder tumor cell line, MB49-I, which was developed by successive in vivo passages of primary tumors obtained by inoculation with the MB49 cell line. The MB49-I line showed heterogeneity in cell morphology and was more invasive and metastatic than the parental MB49 line (14). To better understand this murine model, we performed a cytogenetic study of the parental MB49 cell line and the derived MB49-I cell line. Karyotype analysis revealed subpopulations in both of the cell lines: a diploid population with two marker chromosomes, and a tetraploid cell population that retained the same markers. Moreover, the MB49-I line contained a higher percentage of tetraploid cells with new chromosomal abnormalities. These data suggest that during the disease progression in the MB49/ MB49-I bladder cancer model, the selection of polyploid cells carrying new chromosomal abnormalities may be responsible for the switch to the invasive phenotype observed in the MB49-I cells.
Materials and methods Cell lines The murine bladder cancer cell line MB49 and the new invasive cell line MB49-I were cultured in RPMI1640 medium (Sigma-Aldrich, St. Loius, MO) supplemented with glutamine (2 mM), gentamicin (80 mg/mL), and 10% fetal calf serum in a humidified atmosphere with 5% carbon dioxide. The MB49-I cell line samples were obtained as previously described (14). A single cell suspension of MB49 (5 105 cells) was injected subcutaneously in the left flank of syngeneic C57BL/6J male mice. After 24 days, the tumor was surgically removed, and 2-mm tumor pieces were transplanted by trocar into the left flank of mice. After 13 consecutive in vivo transplants, mechanical primary culture of the tumor was done and 5 105 viable cells per mL was cultured in T25 tissue culture flasks. Adhered cells obtained after 24 hours post culture were named MB49-I. Only low in vitro subcultures (1e10) were used in subsequent studies. The MB49 cell line was used in passage numbers between 100 and 180 for the cytogenetic studies. Two different passages of MB49 (passages 100 and 140) and MB49-I lines (passages 2 and 9) were studied by G-banding, and three different passages of MB49 (passages 100, 160, and 180) and four cell passages of MB49-I (passages 2, 5, and 9) were used in FISH analysis. The cells were cultured in P35 petri dishes and 4 105 cells/ mL were harvested of both cell lines for each study.
Cytogenetics Cells in culture were treated with 0.5 mg/mL colcemid (Life Technologies, Carlsbad, CA ) for 2 hours at 37 C and
169 detached with 0.25% trypsin. Hypotonic treatment was performed in 0.075 mol/L potassium chloride for 20 minutes at 37 C, and the cells were fixed with cold 3:1 methanoleglacial acetic acid. The metaphase spreads were analyzed using the G-banding technique (15). The chromosome number was expressed as the modal number, which is the number of chromosomes most frequently found after analyzing at least 100 metaphases. Chromosomes were identified based on their banding pattern according to the Committee on Standardized Genetic Nomenclature for Mice (16).
FISH Mouse paint chromosome biotinylated DNA probes (Cambio, Cambridge, UK) and a bacterial artificial chromosome (BAC) clone from the RPCI-23 mouse library, RP23-339P9 for chromosome 4C7 (Roswell Park Cancer Institute, Buffalo, NY), were used. The BAC clone DNA was labeled with biotin using the Invitrogen BioPrime DNA labeling kit (Life Technologies). The labeled DNA was resuspended in Triseethylenediaminetetraacetic acid (TE) buffer, and 400 ng of labeled DNA was then precipitated with 5 mg of mouse Cot-1 DNA (Life Technologies) and 3 mg of salmon sperm DNA (Sigma-Aldrich). Then the metaphase chromosomes were denatured in 70% formamide/2 standard salineecitrate (SSC) (2XSSC: 0.3 mol/L sodium chloride, 0.03 mol/L sodium citrate) at 70 C for 3 minutes, and the probes were denatured at 70 C for 10 minutes. The commercial probes and the BAC probe were pre-annealed at 37 C for 50 minutes and 10 minutes, respectively. The hybridization was performed overnight at 37 C. After hybridization, the slides were washed once in 2XSSC, three times in 50% formamide/2XSSC and three times in 0.1 SSC; each wash was performed for 5 minutes at 45 C. The probes were detected with fluorescein isothiocyanateeavidin (Vector Laboratories, Burlingame, CA), and the chromosomes were counterstained with propidium iodide. The signal was viewed in a Nikon eclipse E800 confocal microscope.
Results A tetraploid cell subpopulation is enriched in the more invasive cell line The study of ploidy of the MB49 cell line revealed the presence of two cell subpopulations. The near-diploid population had a modal chromosome number of 40, with metaphases ranging from 37 to 45 chromosomes, and represented 71% of the total metaphases analyzed. The tetraploid subpopulation represented 11% of the total metaphases and had a modal number of 80 chromosomes, with metaphases ranging from 70 to 85 chromosomes (Figure 1A). The derived invasive MB49-I line contained both cell subpopulations. However, only 24% of the cells had a near-diploid range, with a modal number of 40 chromosomes; 76% of the cells were in the tetraploid range, with a modal number of 80 chromosomes (Figure 1B).
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Figure 1 range.
V.T. Fabris et al.
Distribution of the percentage of metaphases for the cell lines MB49 (A) and MB49-I (B) for each chromosome number
An increased number of chromosomal abnormalities in the invasive MB49-I line The G-banding analysis of the metaphases from the MB49 line and the derived invasive MB49-I line revealed abnormalities in the number and structure of the chromosomes. The structural aberrations (marker chromosomes) were also identified using
FISH. A G-banded karyotype of the MB49 diploid cells is shown in Figure 2A. All of the metaphases analyzed demonstrated a reciprocal translocation between chromosomes 5 and 9 (t(5;9), Figure 2B), which was identified by G-banding and FISH with a specific probe for chromosome 9 (arrows, Figure 3A). The translocation (5;9) breakpoint involved chromosome bands 5E and 9A. The diploid cells also contained an
Figure 2 (A) G-banded karyotype of the MB49 cell line. The arrows indicate chromosome rearrangements. (B) Structural abnormalities identified with G-banding: t(5;9) is a reciprocal translocation between chromosomes 5 and 9, and i(19) is an isochromosome of chromosome 19. (C) Metaphase spread of the tetraploid subpopulation of MB49 line. The arrows indicate the chromosome abnormalities that were identified.
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Figure 3 Results of FISH analyses on the cells in metaphase from the MB49 and MB49-I cell lines. (A) Results of FISH using a paint probe for chromosome 9 on a metaphase spread from MB49 cells. The arrows indicate the derivative chromosomes as a result of the translocation between chromosomes 5 and 9 (a large acrocentric chromosome and a small chromosome). (B) Results of FISH using a paint probe for chromosome 5 on a metaphase spread from MB49-I cells. The arrows indicate the derivative chromosomes resulting from the translocation between chromosomes 5 and 9 (two large acrocentric chromosomes and two small chromosomes). The arrowheads indicate two acrocentric chromosomes derived from a translocation of the terminal portion of chromosome 5. (C) Results of FISH using a paint probe for chromosome 3 on a metaphase spread from MB49-I cells. The arrows indicate the derivative chromosomes resulting from the rearrangement of chromosome 3. (D) Results of FISH using a BAC probe for chromosome 4 (RP23-339P9 specific for chromosome 4C7) on a metaphase spread from MB49-I cells. The arrow indicates the derivative chromosome from the duplication of chromosome bands 4C3eC7. Insert: Chr4 indicates the normal chromosome 4 and Dp(4) indicates a duplication of bands C3eC7 on chromosome 4.
isochromosome (i) of chromosome 19 (i(19), Figure 2B). In addition, Robertsonian translocations (Rb) involving chromosome 19 and a chromosome not yet identified were observed
in some of the metaphases analyzed (data not shown). However, multiple abnormalities of chromosome 19 were not observed together in the same metaphase. In fact, i(19) was
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the most frequently detected abnormality (80% of the metaphases). The t(5;9) and i(19) were also found in the tetraploid cell subpopulation (arrows, Figure 2C), suggesting that these cells were derived from the diploid cells by duplication of the chromosome set, as two copies of the marker chromosomes were observed. Trisomy of chromosome 6 was found in 100% of the diploid metaphases analyzed, suggesting that it was a clonal numerical abnormality. A gain of chromosome 19 as the result of the structural aberrations was also observed in all of the MB49 cells. In addition, 50% of the metaphases demonstrated gains of chromosome 9. Copy number losses of chromosome 13 were present in 44% of the cells (Table 1). Chromosomes 7, 8, 10, and 14 were lost in less than 30% of the cells. Although the MB49 cell line was derived from a bladder tumor induced in a male mouse, the karyotype analysis revealed the loss of chromosome Y in 100% of the cells (Figure 2A). Moreover, gains of chromosomes 6 and 19 were also observed in 54% and 100% of the tetraploid cells, respectively. Other numerical abnormalities detected were gains of chromosomes 12 and 14 in 38% and 23% of the cells, respectively, and losses of chromosomes 4, 7, 8, 10, and 13 in more than 70% of the cells. Losses of chromosomes 11 and 18 were observed in 46% and 30% of the cells, respectively (Table 1). In addition, chromosome 3 was lost in less than 30% of the cells. A G-banded karyotype of the tetraploid cells of the invasive MB49-I cell line is shown in Figure 4A. Both cell subpopulations of the MB49-I line retained the structural abnormalities, t(5;9) and i(19), observed in the parental MB49 cell line (Figures 2B and 4B). These data confirmed that the tetraploid cells of the invasive line were derived from the MB49 cell line. FISH analysis using a painting probe for Table 1
chromosome 5 detected the derived chromosomes of the translocation (5;9) (arrows, Figure 3B). Two acrocentric chromosomes derived from a rearrangement of the terminal portion of chromosome 5 were also observed (arrowheads, Figure 3B). In addition, new structural chromosome abnormalities were observed in the derived MB49-I line (Figure 4B). A translocation involving chromosome 3 was identified by FISH using a specific painting probe (arrows, Figure 3C). This translocation involves the entire chromosome 3 and the terminal portion of chromosome 5 (t(3;5), Figure 4B). A duplication of bands C3eC7 on a chromosome 4, (Dp(4), Figure 4B) was identified by FISH using a specific probe for chromosome 4C7 (Figure 3D). The tetraploid MB49-I cells also contained an isochromosome of chromosome 17 (i(17), Figure 4B). Gains of chromosomes 6, 9, and 19 and losses of chromosomes 3, 8, and 18 were detected in the diploid subpopulation of the MB39-I line in more than 30% of the cells (Table 1). Gains of chromosomes 14 and 15 and losses of chromosomes 2, 10, 11, and 16 were detected in less than 30% of the cells. Gains of chromosomes 6, 12, 14, and 19 and losses of chromosomes 4, 7, 8, 10, 11, 13, 16, and 18 were most frequently observed in the tetraploid cells (>40% of the cells, Table 1). Other abnormalities observed were gains of chromosome 17 and losses of chromosome 2 (<40% of the cells, Table 1). Gains of chromosomes 2 and 9 were observed in less than 30% of the tetraploid cells.
Discussion The MB49 murine bladder cancer model is widely used to investigate different aspects of the biology of bladder cancer
Ploidy and chromosome abnormalities found in the murine bladder cancer MB49 cell line and the invasive MB49-I line Numerical abnormalities
Structural abnormalities
Cell line
MN, no. (range)
Gains, no.
Cells, % (no./total)
Losses, no.
MB49
40 (37e45)
6 9 19 6 12 19
100 50 100 54 38 100
(87/87) (43/87) (87/87) (7/13) (5/13) (13/13)
13
6 9 19 6 12 14 17 19
67 50 100 45 41 65 31 57
(8/12) (6/12) (12/12) (40/88) (35/88) (57/88) (27/88) (50/88)
80 (70e85)
MB49-I
40 (32e48)
80 (50e87)
Abbreviation: MN, modal number.
4 7 8 10 11 13 18 3 8 18 2 4 7 8 10 11 13 16 18
Cells, % (no./total) 44 (38/87)
100 77 92 77 46 77 30 33 50 33 35 100 75 43 62 54 73 66 59
(13/13) (10/13) (12/13) (10/13) (6/13) (10/13) (4/13) (4/12) (6/12) (4/12) (31/88) (88/88) (66/88) (38/88) (55/88) (48/88) (64/88) (58/88) (52/88)
Type
% (no./total)
t(5;9) i(19)
100 (87/87) 80 (75/87)
t(5;9) i(19)
100 (13/13) 100 (13/13)
t(5;9) i(19)
100 (13/13) 100 (13/13)
t(3;5) Dp(4C3-C7) t(5;9) i(17) i(19)
100 100 100 100 100
(88/88) (88/88) (88/88) (88/88) (88/88)
Cytogenetics of bladder cancer model MB49-MB49-I
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Figure 4 (A) G-banded karyotype of the MB49-I cell line. The arrows indicate chromosome rearrangements. (B) Chromosomal abnormalities identified by G-banding: t(5;9) is a reciprocal translocation involving chromosomes 5 and 9, t(3;5) is a translocation between chromosomes 3 and 5, Dp(4) is a derivative chromosome 4 by duplication of bands 4C3-C7, i(17) is an isochromosome of chromosome 17, and i(19) is an isochromosome of chromosome 19.
rin (BCG) therapy (12). However, and bacille CalmetteeGue little is known about the cytogenetic and genetic changes that might play a role in the biology of the MB49 line. We analyzed the karyotype of the MB49 cell line and the recently derived more invasive MB49-I line. The cytogenetic study of MB49 and MB49-I revealed the presence of two cell subpopulations. The diploid cell population made up 70% of the metaphases of the MB49 line, and a limited number of chromosomal abnormalities were detected. However, in the invasive line, 76% of
the total cell population was tetraploid, suggesting that there was a selection for polyploid MB49 cells. A balanced translocation between chromosomes 5 and 9, an isochromosome of chromosome 19, and trisomies of chromosomes 6 and 19 were the most frequent abnormalities found in the diploid MB49 cells. The tetraploid cell subpopulation observed in the MB49 line presented the same structural abnormalities, suggesting that they were derived from the diploid cells. The mechanism by which the number of chromosomes was
174 duplicated was not evaluated in this study. However, we propose that this population may be derived from the diploid cells either by fusion of two diploid cells or by a failure at some checkpoints during mitosis. Polyploidy is a common occurrence during in vitro culture of cells, and the tetraploid subpopulation of MB49 cell line could be an artifact of this process. However, the increase in the percentage of the tetraploid cells observed in the invasive MB49-I line occurred during the in vivo passages of the MB49 cells and may recapitulate the selection of cell subpopulations that occurs during the metastatic process. The metastatic MB49-I cells conserved the chromosome markers t(5;9) and i(19) observed in the parental line. In addition, they acquired new chromosome numerical and structural alterations. A cytogenetic study comparing a parental and the derived metastatic cell lines was previously performed in human bladder cancer cell lines. Karyotype analysis of the 253J line revealed aneuploidy, with multiple copies of several chromosomes and marker chromosomes as the result of translocations. The derived metastatic lines retained some of the markers of the parental line and acquired new translocations (17). The karyotype of the T24 human cell line of bladder cancer was studied to compare the parental line, and two metastatic cell lines derived in vivo by inoculation of T24 cells (18). The T24 model and the derived metastatic T24T line were found to share marker chromosomes, and the metastatic T24T line contained new structural changes that were associated with the progression of human bladder cancer (18). In addition, loss of heterozygosity on chromosomes 3p, 4, 8p, 10q, 13q, and 18q in the T24T line was associated with tumor progression, as these cytogenetic changes were also observed in the more aggressive bladder carcinomas (19). The other metastatic cell line, T24M, which was derived from T24, presented a karyotype in the near-tetraploid range and shared chromosomal alterations and unique abnormalities with the parental T24 line (20). Our data obtained in the murine MB49 model and the invasive MB49-I line are in agreement with the reported observations of human bladder cancer paired cell lines. The parental and the derived metastatic lines shared chromosome markers, and new chromosomal abnormalities were detected in the more invasive lines. Several chromosomes have been found to be involved in the numerical abnormalities in human bladder cancer. These abnormalities appear progressively during tumor development and are associated with tumor stage (6). Moreover, gains of chromosomes 3, 7, and 17 and loss of 9p21 detected by the UroVysion detection kit (Abbott Molecular, Abbott Park, IL) are used to diagnose bladder cancer in urine human samples, as well as to monitor follow-up therapy (7,21). Similar to the gains observed in human bladder carcinomas, the murine MB49/ MB49-I model demonstrated mostly gain of mouse chromosome 6, which is orthologous to human chromosomes 3 and 7. Mouse chromosome 6 contains the Met proto-oncogene. Met amplification has been observed in pancreatic adenocarcinoma (22) and lung cancer (23). To date, there are no reports of amplification of this oncogene in bladder cancer, although overexpression of MET was related to progression of urothelial carcinomas (24). The oncogenes Ret, Kras, Raf1, and Braf are also located on mouse chromosome 6. Deregulation in the expression of these oncogenes may occur as a consequence of the increased copy number of chromosome 6 in the MB49/ MB49-I lines. Amplification of KRAS and BRAF has been
V.T. Fabris et al. observed in human colorectal cancer cell lines that were resistant to MEK1/2 inhibitors (25). Amplification of KRAS is frequently observed in nonesmall cell lung caner (26), and RAF1 amplification was associated with tumor progression in human bladder cancer (27). Although chromosome abnormalities found in human bladder cancer mostly involve gains and losses of chromosome material, chromosome translocations have been reported in human bladder tumors involving chromosomes 11p15, 14q32, and 19q13 (28), and 8p (29). The murine MB49 cell line showed a translocation involving chromosomes 5 and 9, whereas the invasive line also contained a translocation between chromosomes 3 and 5. Interestingly, mouse chromosome 5 contains the Fgfr3 gene, and mutations in this gene are frequently found in human bladder carcinomas (6). Although this gene maps to band 5B and is not located near the breakpoint of the translocations, on band 5E for t(5;9) and the terminal portion of the chromosome for t(3;5), we cannot rule out that these translocations may affect genes far from the breakpoint. In urothelial carcinomas, the Egfr gene (which maps to mouse chromosome 11A1-A4) and the Hras1 gene (which maps to the terminal portion of the mouse chromosome 7) were found to be overexpressed (6). However, both the MB49 and MB49-I lines contain losses of chromosomes 7 and 11. The tumor suppressor genes Rb1 (on mouse chromosome 14) and Trp53 (on mouse chromosome 11) were deleted in human bladder cancer. The MB49 murine model showed loss of chromosome 11 in the tetraploid subpopulation. However, the Trp53 gene was not evaluated in this line to determine whether the chromosome loss affects the expression of this gene. In contrast, a gain of copy number of chromosome 14 was observed; the oncogenes located on this chromosome could be overexpressed. Moreover, the genes Plau (Plasminogen activator, urokinase) and Ctsb (Cathepsin B), which are associated with invasion and metastasis, map to mouse chromosome 14. Interestingly, the more invasive MB49-I line shows an increase in the activity of the proteolytic enzymes urokinase plasminogen activator (uPA) and Cathepsin B (14). Cytogenetic studies of murine models of bladder cancer are limited. Mickey et al. conducted cytogenetic analysis of the murine MBT bladder cancer model, which is induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide in C3H/He mice. Three transitional cell carcinoma lines and a squamous cell carcinoma line were derived from the induced tumors, and all of the lines were polyploid with several marker chromosomes (30). In contrast, the MB49 cell line derived from tumors induced by DMBA were largely near-diploid and contained two marker chromosomes, with only a small polyploid cell subpopulation. The karyotype of the MBT lines was not studied by G-banding; therefore, the chromosomes involved in the translocations could not be identified. Thus, it was not possible to compare the cytogenetic data of MBT lines with our findings. Although the MB49 cell line originated from a male mouse, loss of chromosome Y was observed in the 100% of the cells analyzed of both the MB49 and MB49-I cell lines. These data demonstrate consistency with other mouse cancer cell lines, because chromosome Y is lost in most mouse tumor cell lines. This abnormality is also a frequent early event in human bladder cancer (31).
Cytogenetics of bladder cancer model MB49-MB49-I Cancer progression has been associated with an increase in chromosome aberrations (10,32). In our murine bladder cancer model, the new invasive cell line derived from the MB49 line contained new chromosomal aberrations and an increase in the chromosome number as a consequence of the selection of the polyploid MB49 cells. The chromosomal abnormalities involving chromosomes 5, 6, 9, and 19 were observed in the 100% of the MB49 cells. These clonal alterations may be an early event that occurred in the MB49 diploid population. A subclone also contained trisomy of chromosome 9 and monosomy of chromosome 13 (50% of the MB49 diploid cells).Of the MB49 tetraploid cells, 100% retained the anomalies of chromosomes 5, 9, and 19. However, the trisomy of chromosome 6 was observed in only 50% of the cells. In addition, more than 70% of MB49 tetraploid cells showed losses of chromosomes 4, 7, 8, and 10. These new abnormalities were accompanied by a gain of chromosome 12 and losses of chromosomes 11 and 18 in a subclone of the MB49 cell line (approximately 30e40% of the tetraploid cells). The numerical and structural abnormalities of the MB49 tetraploid cells were observed in the invasive MB49-I tetraploid cells. However, only loss of chromosome 4 and the structural rearrangements were observed in the 100% of the invasive MB49-I tetraploid cells. In addition, new abnormalities derived of chromosomes 3, 4, and 17 were observed in the 100% of the tetraploid MB49-I cells. These rearrangements were accompanied by the acquisition of gain of chromosome 14 and loss of chromosome 16 in the 60% of the cells. The high rate of cell division observed in MB49-I cells may contribute to the failure of chromosome segregation during mitosis, resulting in the high frequency of aneuploidy, gains, and losses of certain chromosomes observed in these cells. In conclusion, the invasive MB49-I cells demonstrate the same markers as those of the parental MB49 line, indicating that this line was indeed derived from the MB49 cells. Both diploid and tetraploid cell subpopulations are present in both the parental line and the invasive cell line. However, during the successive passages in vivo, there was a selection of the tetraploid cells in the more invasive line. These MB49-I tetraploid cells acquired new chromosome aberrations during this process. Interestingly, we observed a gain of chromosome 14, which contains some of the proteolytic enzymes that are associated with invasion and likely contributed to the high enzymatic activity of MB49-I cells. These data suggest that the selection of the tetraploid cells in the MB49-I line, with an increase in aneusomies and chromosomal instability, are associated with a more invasive phenotype. Moreover, these findings are in agreement with previous results demonstrating that there is an increase in chromosomal alterations and instability during tumor progression (11,12). Thus, the MB49/MB49-I model is a good model to study gene regulation during bladder cancer progression.
Acknowledgments We thank Pablo DoCampo for the excellent technical assistance and Dr. Marı´a Susana Merani for revising the G-banded karyotypes. This work was supported by UBACyT MO17, CONICET (PIP 2010-2012 193, PIP 2010-2012 692) and stamo BID PICT 2005 05-38300, PICT 2007 932). SECYT (Pre
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176 22. Hill KS, Lorinczi M, Elferink LA. Altered down regulation of the receptor tyrosine kinase met in pancreatic adenocarcinoma cells. J Exp Ther Oncol 2010;8:297e312. 23. Toschi L, Cappuzzo F. Clinical implications of MET gene copy number in lung cancer. Future Oncol 2010;6:239e247. 24. Cheng HL, Trink B, Tzai TS, et al. Overexpression of c-met as a prognostic indicator for transitional cell carcinoma of the urinary bladder: a comparison with p53 nuclear accumulation. J Clin Oncol 2002;20:1544e1550. 25. Little AS, Balmanno K, Sale MJ, et al. Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells. Sci Signal 2011;4: ra17. 26. Wagner PL, Stiedl AC, Wilbertz T, et al. Frequency and clinicopathologic correlates of KRAS amplification in non-small cell lung carcinoma. Lung Cancer 2011;74:118e123. 27. Simon R, Richter J, Wagner U, et al. High-throughput tissue microarray analysis of 3p25 (RAF1) and 8p12 (FGFR1) copy
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Cytogenetic characterization of the murine bladder cancer model MB49 and the derived invasive line MB49-I Victoria T. Fabris a,*, Catalina Lodillinsky b, Marı´a Betina Pampena a, n b Denise Belgorosky b, Claudia Lanari a, Ana Marı´a Eija a
Laboratory of Hormonal Carcinogenesis, Instituto de Biologı´a y Medicina Experimental (Consejo Nacional de Investigaciones Cientı´ficas y Te cnicasdCONICET), Buenos Aires, Argentina; b Research Area, Instituto de Oncologı´a Angel H. Roffo, Universidad de Buenos Aires, Buenos Aires, Argentina Bladder cancer is frequently associated with chromosomal abnormalities, and the complexity of karyotypes increases with tumor progression. The murine model MB49 is one of the most widely studied models of bladder cancer. We developed the invasive cell line MB49-I by successive in vivo passages of MB49 primary tumors. Because little is known about the chromosomal alterations of this model, our goal was to perform cytogenetic analyses of the MB49 and MB49-I lines. The karyotypes of both lines were analyzed by G-banding and fluorescence in situ hybridization techniques. Both lines were composed of two cell subpopulations, a diploid population, which was found mainly in the MB49 line, and the tetraploid population, which was found mainly in the MB49-I line. A translocation between chromosomes 5 and 9 and an isochromosome of chromosome 19 were observed in the subpopulations of both lines. New structural abnormalities and additional chromosomal imbalances were detected in the MB49-I line. Tumor progression in the MB49/MB49-I model was associated with a selection of polyploid cells with accompanying chromosomal abnormalities. This model may be advantageous for the study of the genetic changes associated with the progression of bladder cancer. Keywords Bladder cancer, MB49, MB49-I, cytogenetics, FISH ª 2012 Elsevier Inc. All rights reserved.
Bladder cancer is the seventh most common cancer in men worldwide (1). In 2006, there were approximately 61,240 diagnosed cases of bladder cancer, and an estimated 13,060 deaths were attributed to this disease (2,3). Urinary bladder cancer originates mainly from epithelial cells of the urothelium (4,5). Urothelial carcinomas, which account for 90% of the malignant tumors that arise in the urinary bladder (6), are frequently associated with chromosomal abnormalities; these include gains of chromosomes 1, 3, 7, 9, 11, and 17 and deletions of a part of chromosome 9p (locus 9p21). Cytogenetic changes have been useful in the diagnosis and prognosis of bladder cancer and are currently studied by fluorescence in situ hybridization (FISH) using chromosomespecific probes. Most of the urothelial carcinomas show
Received September 13, 2011; received in revised form January 30, 2012; accepted February 3, 2012. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergen.2012.02.002
polysomy of chromosomes 3, 7, and 17 and a deletion of 9p21, which can be detected in urine samples by FISH analysis for the diagnosis of urothelial carcinomas (7). It has been proposed that a loss of material from chromosome 9 is an early event in bladder cancer (8). Nonmuscle invasive (NMI) bladder tumors often present a near-diploid karyotype with few chromosomal aberrations; the only and recurrent alterations that have been detected are the deletion of chromosomes 9p and 9q, together with mutations in the fibroblastic growth factor receptor 3 (FGFR3) gene (9). Muscleinvasive tumors are predominantly polyploid with complex karyotypes: pT2-T4 tumors frequently contain gains of chromosomes 1q, 5p, 6p, 8q, 10p, 17q, and 20q, and losses of chromosomes 2q, 3p, 5q, 8p, 10q, 11q, 13q, 17p, and 18q (9). The number of chromosomal aberrations has been shown to increase with the progression, grade, and stage of the tumors (10). However, chromosomal instability does not predict the risk of progression of bladder tumors (11). The mouse urothelial carcinoma cell line MB49 is one of the in vitro and in vivo models most widely used to study
Cytogenetics of bladder cancer model MB49-MB49-I bladder cancer biology and therapeutic strategies (12). In 1979, Summerhayes et al. derived the MB49 cell line from C57BL/lcrf-a’ mouse bladder epithelial cells that were transformed by 24 hours of treatment with 7,12-dimethylbenz [a]anthracene (DMBA) on the second day of a long-term primary culture. The transformed cells generated carcinomas after transplant into syngeneic mice (13). We have previously generated and described a new invasive bladder tumor cell line, MB49-I, which was developed by successive in vivo passages of primary tumors obtained by inoculation with the MB49 cell line. The MB49-I line showed heterogeneity in cell morphology and was more invasive and metastatic than the parental MB49 line (14). To better understand this murine model, we performed a cytogenetic study of the parental MB49 cell line and the derived MB49-I cell line. Karyotype analysis revealed subpopulations in both of the cell lines: a diploid population with two marker chromosomes, and a tetraploid cell population that retained the same markers. Moreover, the MB49-I line contained a higher percentage of tetraploid cells with new chromosomal abnormalities. These data suggest that during the disease progression in the MB49/ MB49-I bladder cancer model, the selection of polyploid cells carrying new chromosomal abnormalities may be responsible for the switch to the invasive phenotype observed in the MB49-I cells.
Materials and methods Cell lines The murine bladder cancer cell line MB49 and the new invasive cell line MB49-I were cultured in RPMI1640 medium (Sigma-Aldrich, St. Loius, MO) supplemented with glutamine (2 mM), gentamicin (80 mg/mL), and 10% fetal calf serum in a humidified atmosphere with 5% carbon dioxide. The MB49-I cell line samples were obtained as previously described (14). A single cell suspension of MB49 (5 105 cells) was injected subcutaneously in the left flank of syngeneic C57BL/6J male mice. After 24 days, the tumor was surgically removed, and 2-mm tumor pieces were transplanted by trocar into the left flank of mice. After 13 consecutive in vivo transplants, mechanical primary culture of the tumor was done and 5 105 viable cells per mL was cultured in T25 tissue culture flasks. Adhered cells obtained after 24 hours post culture were named MB49-I. Only low in vitro subcultures (1e10) were used in subsequent studies. The MB49 cell line was used in passage numbers between 100 and 180 for the cytogenetic studies. Two different passages of MB49 (passages 100 and 140) and MB49-I lines (passages 2 and 9) were studied by G-banding, and three different passages of MB49 (passages 100, 160, and 180) and four cell passages of MB49-I (passages 2, 5, and 9) were used in FISH analysis. The cells were cultured in P35 petri dishes and 4 105 cells/ mL were harvested of both cell lines for each study.
Cytogenetics Cells in culture were treated with 0.5 mg/mL colcemid (Life Technologies, Carlsbad, CA ) for 2 hours at 37 C and
169 detached with 0.25% trypsin. Hypotonic treatment was performed in 0.075 mol/L potassium chloride for 20 minutes at 37 C, and the cells were fixed with cold 3:1 methanoleglacial acetic acid. The metaphase spreads were analyzed using the G-banding technique (15). The chromosome number was expressed as the modal number, which is the number of chromosomes most frequently found after analyzing at least 100 metaphases. Chromosomes were identified based on their banding pattern according to the Committee on Standardized Genetic Nomenclature for Mice (16).
FISH Mouse paint chromosome biotinylated DNA probes (Cambio, Cambridge, UK) and a bacterial artificial chromosome (BAC) clone from the RPCI-23 mouse library, RP23-339P9 for chromosome 4C7 (Roswell Park Cancer Institute, Buffalo, NY), were used. The BAC clone DNA was labeled with biotin using the Invitrogen BioPrime DNA labeling kit (Life Technologies). The labeled DNA was resuspended in Triseethylenediaminetetraacetic acid (TE) buffer, and 400 ng of labeled DNA was then precipitated with 5 mg of mouse Cot-1 DNA (Life Technologies) and 3 mg of salmon sperm DNA (Sigma-Aldrich). Then the metaphase chromosomes were denatured in 70% formamide/2 standard salineecitrate (SSC) (2XSSC: 0.3 mol/L sodium chloride, 0.03 mol/L sodium citrate) at 70 C for 3 minutes, and the probes were denatured at 70 C for 10 minutes. The commercial probes and the BAC probe were pre-annealed at 37 C for 50 minutes and 10 minutes, respectively. The hybridization was performed overnight at 37 C. After hybridization, the slides were washed once in 2XSSC, three times in 50% formamide/2XSSC and three times in 0.1 SSC; each wash was performed for 5 minutes at 45 C. The probes were detected with fluorescein isothiocyanateeavidin (Vector Laboratories, Burlingame, CA), and the chromosomes were counterstained with propidium iodide. The signal was viewed in a Nikon eclipse E800 confocal microscope.
Results A tetraploid cell subpopulation is enriched in the more invasive cell line The study of ploidy of the MB49 cell line revealed the presence of two cell subpopulations. The near-diploid population had a modal chromosome number of 40, with metaphases ranging from 37 to 45 chromosomes, and represented 71% of the total metaphases analyzed. The tetraploid subpopulation represented 11% of the total metaphases and had a modal number of 80 chromosomes, with metaphases ranging from 70 to 85 chromosomes (Figure 1A). The derived invasive MB49-I line contained both cell subpopulations. However, only 24% of the cells had a near-diploid range, with a modal number of 40 chromosomes; 76% of the cells were in the tetraploid range, with a modal number of 80 chromosomes (Figure 1B).
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Figure 1 range.
V.T. Fabris et al.
Distribution of the percentage of metaphases for the cell lines MB49 (A) and MB49-I (B) for each chromosome number
An increased number of chromosomal abnormalities in the invasive MB49-I line The G-banding analysis of the metaphases from the MB49 line and the derived invasive MB49-I line revealed abnormalities in the number and structure of the chromosomes. The structural aberrations (marker chromosomes) were also identified using
FISH. A G-banded karyotype of the MB49 diploid cells is shown in Figure 2A. All of the metaphases analyzed demonstrated a reciprocal translocation between chromosomes 5 and 9 (t(5;9), Figure 2B), which was identified by G-banding and FISH with a specific probe for chromosome 9 (arrows, Figure 3A). The translocation (5;9) breakpoint involved chromosome bands 5E and 9A. The diploid cells also contained an
Figure 2 (A) G-banded karyotype of the MB49 cell line. The arrows indicate chromosome rearrangements. (B) Structural abnormalities identified with G-banding: t(5;9) is a reciprocal translocation between chromosomes 5 and 9, and i(19) is an isochromosome of chromosome 19. (C) Metaphase spread of the tetraploid subpopulation of MB49 line. The arrows indicate the chromosome abnormalities that were identified.
Cytogenetics of bladder cancer model MB49-MB49-I
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Figure 3 Results of FISH analyses on the cells in metaphase from the MB49 and MB49-I cell lines. (A) Results of FISH using a paint probe for chromosome 9 on a metaphase spread from MB49 cells. The arrows indicate the derivative chromosomes as a result of the translocation between chromosomes 5 and 9 (a large acrocentric chromosome and a small chromosome). (B) Results of FISH using a paint probe for chromosome 5 on a metaphase spread from MB49-I cells. The arrows indicate the derivative chromosomes resulting from the translocation between chromosomes 5 and 9 (two large acrocentric chromosomes and two small chromosomes). The arrowheads indicate two acrocentric chromosomes derived from a translocation of the terminal portion of chromosome 5. (C) Results of FISH using a paint probe for chromosome 3 on a metaphase spread from MB49-I cells. The arrows indicate the derivative chromosomes resulting from the rearrangement of chromosome 3. (D) Results of FISH using a BAC probe for chromosome 4 (RP23-339P9 specific for chromosome 4C7) on a metaphase spread from MB49-I cells. The arrow indicates the derivative chromosome from the duplication of chromosome bands 4C3eC7. Insert: Chr4 indicates the normal chromosome 4 and Dp(4) indicates a duplication of bands C3eC7 on chromosome 4.
isochromosome (i) of chromosome 19 (i(19), Figure 2B). In addition, Robertsonian translocations (Rb) involving chromosome 19 and a chromosome not yet identified were observed
in some of the metaphases analyzed (data not shown). However, multiple abnormalities of chromosome 19 were not observed together in the same metaphase. In fact, i(19) was
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the most frequently detected abnormality (80% of the metaphases). The t(5;9) and i(19) were also found in the tetraploid cell subpopulation (arrows, Figure 2C), suggesting that these cells were derived from the diploid cells by duplication of the chromosome set, as two copies of the marker chromosomes were observed. Trisomy of chromosome 6 was found in 100% of the diploid metaphases analyzed, suggesting that it was a clonal numerical abnormality. A gain of chromosome 19 as the result of the structural aberrations was also observed in all of the MB49 cells. In addition, 50% of the metaphases demonstrated gains of chromosome 9. Copy number losses of chromosome 13 were present in 44% of the cells (Table 1). Chromosomes 7, 8, 10, and 14 were lost in less than 30% of the cells. Although the MB49 cell line was derived from a bladder tumor induced in a male mouse, the karyotype analysis revealed the loss of chromosome Y in 100% of the cells (Figure 2A). Moreover, gains of chromosomes 6 and 19 were also observed in 54% and 100% of the tetraploid cells, respectively. Other numerical abnormalities detected were gains of chromosomes 12 and 14 in 38% and 23% of the cells, respectively, and losses of chromosomes 4, 7, 8, 10, and 13 in more than 70% of the cells. Losses of chromosomes 11 and 18 were observed in 46% and 30% of the cells, respectively (Table 1). In addition, chromosome 3 was lost in less than 30% of the cells. A G-banded karyotype of the tetraploid cells of the invasive MB49-I cell line is shown in Figure 4A. Both cell subpopulations of the MB49-I line retained the structural abnormalities, t(5;9) and i(19), observed in the parental MB49 cell line (Figures 2B and 4B). These data confirmed that the tetraploid cells of the invasive line were derived from the MB49 cell line. FISH analysis using a painting probe for Table 1
chromosome 5 detected the derived chromosomes of the translocation (5;9) (arrows, Figure 3B). Two acrocentric chromosomes derived from a rearrangement of the terminal portion of chromosome 5 were also observed (arrowheads, Figure 3B). In addition, new structural chromosome abnormalities were observed in the derived MB49-I line (Figure 4B). A translocation involving chromosome 3 was identified by FISH using a specific painting probe (arrows, Figure 3C). This translocation involves the entire chromosome 3 and the terminal portion of chromosome 5 (t(3;5), Figure 4B). A duplication of bands C3eC7 on a chromosome 4, (Dp(4), Figure 4B) was identified by FISH using a specific probe for chromosome 4C7 (Figure 3D). The tetraploid MB49-I cells also contained an isochromosome of chromosome 17 (i(17), Figure 4B). Gains of chromosomes 6, 9, and 19 and losses of chromosomes 3, 8, and 18 were detected in the diploid subpopulation of the MB39-I line in more than 30% of the cells (Table 1). Gains of chromosomes 14 and 15 and losses of chromosomes 2, 10, 11, and 16 were detected in less than 30% of the cells. Gains of chromosomes 6, 12, 14, and 19 and losses of chromosomes 4, 7, 8, 10, 11, 13, 16, and 18 were most frequently observed in the tetraploid cells (>40% of the cells, Table 1). Other abnormalities observed were gains of chromosome 17 and losses of chromosome 2 (<40% of the cells, Table 1). Gains of chromosomes 2 and 9 were observed in less than 30% of the tetraploid cells.
Discussion The MB49 murine bladder cancer model is widely used to investigate different aspects of the biology of bladder cancer
Ploidy and chromosome abnormalities found in the murine bladder cancer MB49 cell line and the invasive MB49-I line Numerical abnormalities
Structural abnormalities
Cell line
MN, no. (range)
Gains, no.
Cells, % (no./total)
Losses, no.
MB49
40 (37e45)
6 9 19 6 12 19
100 50 100 54 38 100
(87/87) (43/87) (87/87) (7/13) (5/13) (13/13)
13
6 9 19 6 12 14 17 19
67 50 100 45 41 65 31 57
(8/12) (6/12) (12/12) (40/88) (35/88) (57/88) (27/88) (50/88)
80 (70e85)
MB49-I
40 (32e48)
80 (50e87)
Abbreviation: MN, modal number.
4 7 8 10 11 13 18 3 8 18 2 4 7 8 10 11 13 16 18
Cells, % (no./total) 44 (38/87)
100 77 92 77 46 77 30 33 50 33 35 100 75 43 62 54 73 66 59
(13/13) (10/13) (12/13) (10/13) (6/13) (10/13) (4/13) (4/12) (6/12) (4/12) (31/88) (88/88) (66/88) (38/88) (55/88) (48/88) (64/88) (58/88) (52/88)
Type
% (no./total)
t(5;9) i(19)
100 (87/87) 80 (75/87)
t(5;9) i(19)
100 (13/13) 100 (13/13)
t(5;9) i(19)
100 (13/13) 100 (13/13)
t(3;5) Dp(4C3-C7) t(5;9) i(17) i(19)
100 100 100 100 100
(88/88) (88/88) (88/88) (88/88) (88/88)
Cytogenetics of bladder cancer model MB49-MB49-I
173
Figure 4 (A) G-banded karyotype of the MB49-I cell line. The arrows indicate chromosome rearrangements. (B) Chromosomal abnormalities identified by G-banding: t(5;9) is a reciprocal translocation involving chromosomes 5 and 9, t(3;5) is a translocation between chromosomes 3 and 5, Dp(4) is a derivative chromosome 4 by duplication of bands 4C3-C7, i(17) is an isochromosome of chromosome 17, and i(19) is an isochromosome of chromosome 19.
rin (BCG) therapy (12). However, and bacille CalmetteeGue little is known about the cytogenetic and genetic changes that might play a role in the biology of the MB49 line. We analyzed the karyotype of the MB49 cell line and the recently derived more invasive MB49-I line. The cytogenetic study of MB49 and MB49-I revealed the presence of two cell subpopulations. The diploid cell population made up 70% of the metaphases of the MB49 line, and a limited number of chromosomal abnormalities were detected. However, in the invasive line, 76% of
the total cell population was tetraploid, suggesting that there was a selection for polyploid MB49 cells. A balanced translocation between chromosomes 5 and 9, an isochromosome of chromosome 19, and trisomies of chromosomes 6 and 19 were the most frequent abnormalities found in the diploid MB49 cells. The tetraploid cell subpopulation observed in the MB49 line presented the same structural abnormalities, suggesting that they were derived from the diploid cells. The mechanism by which the number of chromosomes was
174 duplicated was not evaluated in this study. However, we propose that this population may be derived from the diploid cells either by fusion of two diploid cells or by a failure at some checkpoints during mitosis. Polyploidy is a common occurrence during in vitro culture of cells, and the tetraploid subpopulation of MB49 cell line could be an artifact of this process. However, the increase in the percentage of the tetraploid cells observed in the invasive MB49-I line occurred during the in vivo passages of the MB49 cells and may recapitulate the selection of cell subpopulations that occurs during the metastatic process. The metastatic MB49-I cells conserved the chromosome markers t(5;9) and i(19) observed in the parental line. In addition, they acquired new chromosome numerical and structural alterations. A cytogenetic study comparing a parental and the derived metastatic cell lines was previously performed in human bladder cancer cell lines. Karyotype analysis of the 253J line revealed aneuploidy, with multiple copies of several chromosomes and marker chromosomes as the result of translocations. The derived metastatic lines retained some of the markers of the parental line and acquired new translocations (17). The karyotype of the T24 human cell line of bladder cancer was studied to compare the parental line, and two metastatic cell lines derived in vivo by inoculation of T24 cells (18). The T24 model and the derived metastatic T24T line were found to share marker chromosomes, and the metastatic T24T line contained new structural changes that were associated with the progression of human bladder cancer (18). In addition, loss of heterozygosity on chromosomes 3p, 4, 8p, 10q, 13q, and 18q in the T24T line was associated with tumor progression, as these cytogenetic changes were also observed in the more aggressive bladder carcinomas (19). The other metastatic cell line, T24M, which was derived from T24, presented a karyotype in the near-tetraploid range and shared chromosomal alterations and unique abnormalities with the parental T24 line (20). Our data obtained in the murine MB49 model and the invasive MB49-I line are in agreement with the reported observations of human bladder cancer paired cell lines. The parental and the derived metastatic lines shared chromosome markers, and new chromosomal abnormalities were detected in the more invasive lines. Several chromosomes have been found to be involved in the numerical abnormalities in human bladder cancer. These abnormalities appear progressively during tumor development and are associated with tumor stage (6). Moreover, gains of chromosomes 3, 7, and 17 and loss of 9p21 detected by the UroVysion detection kit (Abbott Molecular, Abbott Park, IL) are used to diagnose bladder cancer in urine human samples, as well as to monitor follow-up therapy (7,21). Similar to the gains observed in human bladder carcinomas, the murine MB49/ MB49-I model demonstrated mostly gain of mouse chromosome 6, which is orthologous to human chromosomes 3 and 7. Mouse chromosome 6 contains the Met proto-oncogene. Met amplification has been observed in pancreatic adenocarcinoma (22) and lung cancer (23). To date, there are no reports of amplification of this oncogene in bladder cancer, although overexpression of MET was related to progression of urothelial carcinomas (24). The oncogenes Ret, Kras, Raf1, and Braf are also located on mouse chromosome 6. Deregulation in the expression of these oncogenes may occur as a consequence of the increased copy number of chromosome 6 in the MB49/ MB49-I lines. Amplification of KRAS and BRAF has been
V.T. Fabris et al. observed in human colorectal cancer cell lines that were resistant to MEK1/2 inhibitors (25). Amplification of KRAS is frequently observed in nonesmall cell lung caner (26), and RAF1 amplification was associated with tumor progression in human bladder cancer (27). Although chromosome abnormalities found in human bladder cancer mostly involve gains and losses of chromosome material, chromosome translocations have been reported in human bladder tumors involving chromosomes 11p15, 14q32, and 19q13 (28), and 8p (29). The murine MB49 cell line showed a translocation involving chromosomes 5 and 9, whereas the invasive line also contained a translocation between chromosomes 3 and 5. Interestingly, mouse chromosome 5 contains the Fgfr3 gene, and mutations in this gene are frequently found in human bladder carcinomas (6). Although this gene maps to band 5B and is not located near the breakpoint of the translocations, on band 5E for t(5;9) and the terminal portion of the chromosome for t(3;5), we cannot rule out that these translocations may affect genes far from the breakpoint. In urothelial carcinomas, the Egfr gene (which maps to mouse chromosome 11A1-A4) and the Hras1 gene (which maps to the terminal portion of the mouse chromosome 7) were found to be overexpressed (6). However, both the MB49 and MB49-I lines contain losses of chromosomes 7 and 11. The tumor suppressor genes Rb1 (on mouse chromosome 14) and Trp53 (on mouse chromosome 11) were deleted in human bladder cancer. The MB49 murine model showed loss of chromosome 11 in the tetraploid subpopulation. However, the Trp53 gene was not evaluated in this line to determine whether the chromosome loss affects the expression of this gene. In contrast, a gain of copy number of chromosome 14 was observed; the oncogenes located on this chromosome could be overexpressed. Moreover, the genes Plau (Plasminogen activator, urokinase) and Ctsb (Cathepsin B), which are associated with invasion and metastasis, map to mouse chromosome 14. Interestingly, the more invasive MB49-I line shows an increase in the activity of the proteolytic enzymes urokinase plasminogen activator (uPA) and Cathepsin B (14). Cytogenetic studies of murine models of bladder cancer are limited. Mickey et al. conducted cytogenetic analysis of the murine MBT bladder cancer model, which is induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide in C3H/He mice. Three transitional cell carcinoma lines and a squamous cell carcinoma line were derived from the induced tumors, and all of the lines were polyploid with several marker chromosomes (30). In contrast, the MB49 cell line derived from tumors induced by DMBA were largely near-diploid and contained two marker chromosomes, with only a small polyploid cell subpopulation. The karyotype of the MBT lines was not studied by G-banding; therefore, the chromosomes involved in the translocations could not be identified. Thus, it was not possible to compare the cytogenetic data of MBT lines with our findings. Although the MB49 cell line originated from a male mouse, loss of chromosome Y was observed in the 100% of the cells analyzed of both the MB49 and MB49-I cell lines. These data demonstrate consistency with other mouse cancer cell lines, because chromosome Y is lost in most mouse tumor cell lines. This abnormality is also a frequent early event in human bladder cancer (31).
Cytogenetics of bladder cancer model MB49-MB49-I Cancer progression has been associated with an increase in chromosome aberrations (10,32). In our murine bladder cancer model, the new invasive cell line derived from the MB49 line contained new chromosomal aberrations and an increase in the chromosome number as a consequence of the selection of the polyploid MB49 cells. The chromosomal abnormalities involving chromosomes 5, 6, 9, and 19 were observed in the 100% of the MB49 cells. These clonal alterations may be an early event that occurred in the MB49 diploid population. A subclone also contained trisomy of chromosome 9 and monosomy of chromosome 13 (50% of the MB49 diploid cells).Of the MB49 tetraploid cells, 100% retained the anomalies of chromosomes 5, 9, and 19. However, the trisomy of chromosome 6 was observed in only 50% of the cells. In addition, more than 70% of MB49 tetraploid cells showed losses of chromosomes 4, 7, 8, and 10. These new abnormalities were accompanied by a gain of chromosome 12 and losses of chromosomes 11 and 18 in a subclone of the MB49 cell line (approximately 30e40% of the tetraploid cells). The numerical and structural abnormalities of the MB49 tetraploid cells were observed in the invasive MB49-I tetraploid cells. However, only loss of chromosome 4 and the structural rearrangements were observed in the 100% of the invasive MB49-I tetraploid cells. In addition, new abnormalities derived of chromosomes 3, 4, and 17 were observed in the 100% of the tetraploid MB49-I cells. These rearrangements were accompanied by the acquisition of gain of chromosome 14 and loss of chromosome 16 in the 60% of the cells. The high rate of cell division observed in MB49-I cells may contribute to the failure of chromosome segregation during mitosis, resulting in the high frequency of aneuploidy, gains, and losses of certain chromosomes observed in these cells. In conclusion, the invasive MB49-I cells demonstrate the same markers as those of the parental MB49 line, indicating that this line was indeed derived from the MB49 cells. Both diploid and tetraploid cell subpopulations are present in both the parental line and the invasive cell line. However, during the successive passages in vivo, there was a selection of the tetraploid cells in the more invasive line. These MB49-I tetraploid cells acquired new chromosome aberrations during this process. Interestingly, we observed a gain of chromosome 14, which contains some of the proteolytic enzymes that are associated with invasion and likely contributed to the high enzymatic activity of MB49-I cells. These data suggest that the selection of the tetraploid cells in the MB49-I line, with an increase in aneusomies and chromosomal instability, are associated with a more invasive phenotype. Moreover, these findings are in agreement with previous results demonstrating that there is an increase in chromosomal alterations and instability during tumor progression (11,12). Thus, the MB49/MB49-I model is a good model to study gene regulation during bladder cancer progression.
Acknowledgments We thank Pablo DoCampo for the excellent technical assistance and Dr. Marı´a Susana Merani for revising the G-banded karyotypes. This work was supported by UBACyT MO17, CONICET (PIP 2010-2012 193, PIP 2010-2012 692) and stamo BID PICT 2005 05-38300, PICT 2007 932). SECYT (Pre
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