Recurrent alterations of the short arm of chromosome 3 define a tumor suppressor region in rat mammary tumor cells
Nicholas C. Popescu1 and
John W. Greiner
Laboratory of Experimental Carcinogenesis and Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Abstract
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Cytogenetic alterations associated with different stages in carcinogenesis can be distinguished in cultured human or rodent cells transformed by carcinogenic agents. Three tumorigenic rat mammary epithelial cell lines transformed in vitro with 7,12,-dimethylbenz[a]anthracene alone or in combination with 12-O-tetradecanoylphorbol-13-acetate were examined cytogenetically. Non-random alterations consisting of translocations involving the short arm of chromosome 3 and trisomy of chromosomes 14 and X were identified in all three lines. Deletion and inversion of chromosome 1 with the breakpoint at band 1q22 and a duplication 1q 3243 and trisomy of chromosome 2 were observed in two cell lines. The accumulation of structural alterations and chromosome imbalances during the process of cell immortalization and acquisition of tumorigenicity are required for normal rat mammary cells to become malignant. Unbalanced translocations of chromosome 3 resulting in loss of the short arm had the breakpoint at 3p11. This site is a hotspot of breakage and recombination in various rat tumors and may represent a region of tumor suppressor gene critical to the development of rat mammary tumors, as well as other types of tumors.
Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; FBS, fetal bovine serum; LOH, loss of heterozygosity; NMU, N-nitroso-N-methylurea; NOR, nucleolar organizer regions; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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The genetic alterations in carcinogenesis include point mutations, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms (15). Recurrent and specific translocations and inversions have been identified in many cancers, particularly in leukemias, lymphomas and sarcomas (610). In breast cancer, despite a worldwide effort, a hallmark specific chromosomal abnormality has not yet been identified (610).
Carcinogen-induced transformation of human and rodent cells in culture has provided valuable data concerning the identification of potential carcinogens and the cellular and molecular alterations that are involved in carcinogenesis. In a previous study in vitro neoplastic transformation of rat mammary epithelial cells by 7,12,-dimethylbenz[a]anthracene (DMBA) or promotion of DMBA-initiated cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was demonstrated. Three transformed epithelial cell lines that were isolated formed carcinomas in nude mice and were estrogen responsive (11). The cytogenetic analysis of these cell lines presented here provides evidence for non-random numerical and structural alterations related to acquisition of tumorigenicity that are relevant to pathogenesis of breast neoplasia.
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Materials and methods
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The culture conditions, isolation of mammary epithelial transformed cell lines and assessment of tumorigenicity were described in detail previously (11). Briefly, virgin female SpragueDawley rats (42 days old; Charles River Breeding Laboratories, Wilmington, MA) were killed by cervical dislocation. The abdominal and inguinal mammary glands were aseptically removed and transferred to Medium 199Earle's salts (M199-E) containing 1.0% charcoal-extracted fetal bovine serum (FBS) and 0.45% type II collagenase (Worthington Biochemical Corp., Freehold, NJ) and incubated at 37°C for 60 min. The mixture was filtered through 157 mesh nylon (Martin Supply Co., Baltimore, MD) to remove all dissociated tissues. The filtrate was incubated in the presence of 10 000 U/10 ml DNase I at 37°C for 10 min, after which the cellular component was pelleted by centrifugation at 400 g for 10 min. The cells were suspended in M199-E containing 10% charcoal-extracted FBS and layered onto a continuous 28% Ficoll gradient which rested on a cushion of 20% Ficoll. The cells were centrifuged at 70 g for 5 min at 4°C. Mammary epithelial micro-organelles (i.e. cellular aggregates) migrated to the bottom 10 ml of the 90 ml gradient. These aliquots were pooled, counted using trypan blue exclusion and stored in liquid nitrogen.
Mammary epithelial cells were thawed and grown in M199-H containing 5% charcoal-extracted FBS, 0.5 µg insulin, 0.5 µg cortisol, 0.5 µg bovine prolactin (Pituitary Hormone Distribution Program, NIH, Bethesda, MD), 30 ng progesterone and 0.3 ng/ml 17ß-estradiol. Approximately 2.55.0x106 mammary epithelial cells were seeded per 100 mm plastic dish. When the cells reached a confluence of 5065%, DMBA, initially dissolved in acetone, was diluted in serum-containing M199-E medium and added at a final concentration of 0.25 µg/ml. The DMBA-containing medium was removed 24 h later and replaced with fresh growth medium. Selected DMBA-treated mammary cultures were subsequently treated with TPA, resulting in the outgrowth of rat mammary cell lines, ME11CL2 and ME12CL3. TPA (Chemicals for Cancer Research Inc., Eden Prairie, MN) was initially dissolved in acetone and 100 µg/ml, diluted in M199-E containing 5% charcoal-extracted FBS, was added to the cultures at a final concentration of 100 ng/ml TPA every 48 h for 30 days.
Cultures from transformed cell lines were subcultured 1:5 and used 24 h later for chromosome preparation. Colcemid was added 4 h before harvesting. The cells were mechanically detached from the surface of dishes and exposed to 0.075 M KCl for 1520 min. The cells were fixed with a 3:1 mixture of methanol:acetic acid; the fixative was changed three times before preparing slides. Chromosome preparations were stained with Giemsa or Wright stain after trypsin pretreatment to obtain G-bands or were processed for detection of nucleolar organizer regions (NOR) by silver staining as described previously (12). Chromosome number was determined on 50 conventionally stained metaphases and 25 G-band karyotypes.
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Results
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Three rat mammary epithelial cell lines derived from separate pools of primary cultures (ME10CL1, ME11CL2 and ME12CL3) transformed in vitro by chemical carcinogen alone or in combination with a tumor promoter were examined (11). Line ME10CL1 was transformed with DMBA while ME11CL2 and ME12CL3 were transformed with DMBA followed by TPA treatment. The isolation and characterization of the first two lines has been described previously (11), while the third line was generated subsequently. All three lines acquired indefinite lifespans and formed progressively growing carcinomas in nu/nu mice and were cytogenetically analyzed 510 passages after the cells become tumorigenic and after 5060 passages of continuous cultivation (11). All three cell lines have a stable aneuploid karyotype with non-random numerical and structural alterations. Translocations of chromosome 3 and trisomies of chromosomes 14 and X were identified in all three lines. Structural or numerical alterations of chromosomes 1 and 2 were observed in two lines.
The first line, ME10CL1, became highly tumorigenic after 38 days of subculture (11) and had a highly rearranged karyotype. This line was examined at passages 5 and 50 after acquisition of tumorigenicity. ME10CL1 cells have a hyperdiploid chromosome number (4852), consistent trisomies of chromosomes 4, 6, 14 and X, monosomies of chromosomes 10 and 19 and nine abnormal chromosomes (Figure 1
). Numerical deviations of chromosomes 6 and 10 were present in 70 and 80% of the cells, respectively. Structural abnormalities consisted of translocations t(3;13)(p11;q11) and t(7;4)(q44;q24) and a translocation involving the terminus of chromosome X, t(X;?)(q37;?) (Figure 1
and Table I
). The translocation t(3;13) involved the short arm of chromosome 3 and most likely part of the long arm of chromosome 13 (Figure 1
). The short arm of chromosome 3 is one of the four arms in the rat genome harboring multiple copies of genes for the larger fraction (28s) of rRNA that forms and maintains the nucleoli in interphase nuclei. These regions, known as NOR, can be visualized on metaphase chromosomes by silver staining. As a result of the 3;13 rearrangement, the short arm of chromosome 3 was lost as silver staining for NOR regions was detected only on a single copy of chromosome 3, as well as on both copies of chromosomes 11 and 12. Therefore, t(3;13) is an unbalanced translocation with the breakpoint at 3p11, resulting in loss of 3p and gain of 13q. Other translocations identified are probably unbalanced as well. The origin of five abnormal chromosome markers could not be determined (Figure 1
). The analysis of ME10CL1 at passage 50 showed a significant new numerical deviation, as the majority of the cells were trisomic for chromosome 2. Other numerical or structural alterations were random or sporadic. A cell line derived from a tumor developed in nude mice after the inoculation of transformed cells was also examined at passages 310 after its establishment. These cells had a similar karyotype, with the transformed inoculated cells having three copies of chromosome 2.

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Fig. 1. G-band karyotype of a hyperdiploid cell from line ME10CL1 exhibiting structural and numerical alterations. Translocations involving chromosomes 3, 7 and X are indicated by arrows. The derivation of five abnormal chromosomes (M1M5) placed separately was not determined.
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ME11CL2 cells became tumorigenic after 140 days in culture (11). These cells had a hyperdiploid chromosome number (4254), contained six structural abnormalities involving chromosomes 1, 2, 6, 9 and 10, exhibited complete or partial trisomies of chromosomes 2, 6, 9, 13, 14 and X and consistent loss of a copy of chromosomes 8 and 16 (Figure 2
and Table I
). Strikingly, translocation t(3;13)(p11;q11), identified in the majority of the cells, was similar if not identical to that observed in the ME11CL1 line and the breakpoint was most likely at 3p11 (Figure 2
). Two types of alterations of chromosome 1 were detected in >50% of the metaphases: a deletion 1q22 frequently involving a third copy of chromosome 1 and a duplication of 1q 3342 (Figure 2
and Table I
). Chromosome 2 was trisomic in >50% of the cells and one copy had a translocated segment in the telomeric region. A marker chromosome derived from a centric fusion of chromosomes 8 and an abnormal X present in Figure 2
was not a recurrent abnormality.

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Fig 2. G-band karyotype of a hyperdiploid cell from line ME11CL2. Partial duplication of chromosome 1, translocation of chromosome 3 and deletions of chromosomes 6 and 10 are indicated by arrows. The abnormality involving chromosomes 8 and a der(X) was not recurrent.
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ME12CL3 became tumorigenic within 130 days after treatment with DMBA and TPA. This line contained a mixture of cells with 4143 chromosomes and cells with 4452 chromosomes. Cells with 42 chromosomes had either an apparently normal karyotype or an abnormal karyotype with consistent gain of chromosome 14 and random chromosome losses (Figure 3
). Cells with 4452 chromosomes had structural alterations of chromosomes 1 and 3, trisomies 14, 19, 20 and X and monosomy 18 (Figure 4
and Table I
). Chromosome 1 had an apparent inversion 1q1122 and a duplication 1q3243 similar if not identical to that observed in ME11CL2. The abnormality of chromosome 3 was depicted as a translocation t(3;18)(p11;q11). Only one copy of chromosome 3 exhibited silver staining for NOR.

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Fig 3. G-band karyotype of a metaphase with a normal chromosome number of 42 derived from line ME11Cl2, having an extra chromosome 14 and missing a chromosome 20. Structural alterations are not apparent on extended prometaphasic chromosomes with good resolution G-banding. An extra copy of chromosome 14 was the only consistent karyotypic change in this cell population.
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Fig 4. G-band karyotype of a metaphase with a hyperdiploid chromosome number derived from line ME11Cl2. Inversion of chromosome 1 and translocation of chromosome 3 are indicated by arrows.
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Discussion
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The complexity and heterogeneity of the cytogenetic alterations in solid tumors hamper the discrimination of primary changes related to the initiation of the neoplastic process from secondary ones associated with tumor progression and acquisition of cell invasiveness (610). In vitro systems for neoplastic transformation could partially overcome this obstacle as changes associated with early stages of cell immortality and progression to malignancy are discernible. This in vitro analysis of chromosome changes in carcinogen-induced malignant transformation of rat mammary epithelial cells demonstrates non-random changes that include deletion and inversion 1q1122, partial duplication 1q3243, unbalanced translocations and consequential loss of 3p and trisomy of chromosomes 2, 14 and X. Trisomy of the X chromosome resulting in an increase in estrogen receptors explains the inhibitory effect of tamoxifen on cell growth of both transformed and tumor-derived cell lines (11). Apparently, over-representation of chromosomes 2 and 14 are related to different stages of neoplastic transformation. One line contained a mixture of aneuploid and near diploid cells exhibiting trisomy 14 as a sole alteration. Probably trisomy 14 preceded the occurrence of other changes associated with the acquisition of cell tumorigenicity. Meanwhile, trisomy 2, a common alteration in rat neoplasia known to be carcinogen- rather than tumor type-specific, occurred in transformed cells at advanced passages in culture. This finding stresses the importance of trisomy 2 in the propagation of the transformed phenotype (1215).
The identification of unbalanced translocations involving 3p in all lines is of special significance and defines a new region of tumor suppressor gene(s) in rat mammary tumors. The breakpoint in 3p translocations occurred at band 3p11, a site that, independent of the tissue type of the tumor, exhibits by far the highest incidence of structural changes in the rat genome (15). As a result of the translocations, the whole 3p was lost, as demonstrated by G-banding and silver staining for NOR. Frequent participation of chromosomes bearing NOR in chromosomal rearrangements suggested the importance of NOR function in the initiation of neoplastic transformation (1618). Region 3pterq12 was implicated in the induction of aneuploidy, the most common feature of the neoplastic cells, due to an aberrant NOR activity and related mitotic spindle malfunction (19). Furthermore, loss of 3p has been reported in a number of transformed rat cell lines and in vivo hepatic lesions (2023) and a conclusive association between 3p deletion and tumorigenicity in nude mice was demonstrated in rat bladder carcinoma cells (24).
Deletion 1q22, inversion 1q1122 and duplication of 1q 3243, where the HRAS-1 gene is located, were observed in two lines (25). HRAS-1 has been implicated in rat mammary carcinogenesis since early studies with N-nitroso-N-methylurea (NMU)-induced tumors and, more recently, a comprehensive cytogenetic and molecular analysis of rat NMU-induced mammary tumors at different stages of tumor progression demonstrated both allelic loss and an increased HRAS-1 copy number due to direct duplication in region 1q2243 (2628). Significantly, the breakpoint of the deletion and inversion 1q1122 observed in these lines corresponds to the breakpoint in recurrent interstitial deletions and translocations in NMU-induced mammary tumors (28).
Interestingly, the most prevalent structural changes involved chromosomes 1 and 3, both having regions of homology with chromosome 11 in human (2931). The loci of several bona fide and putative tumor suppressor genes for different cancers have been characterized on chromosome 11 (32). Three separate regions of loss of heterozygosity (LOH) on both arms of chromosome 11 consistently detected in breast carcinoma pose a complicated dilemma as to the role of chromosome 11 in the development of breast cancer (32). In this respect, there are similarities between rat and human breast tumors as deletions of rat chromosomes 1 and 3 may be analogous to LOH in both arms of human chromosome 11 (28). Microcell-mediated transfer of region p11.212 of chromosome 11, presumably syntenic with rat 3p, suppressed the tumorigenic phenotype of a rat liver tumor cell line (33). Thus, the short arm of rat chromosome 3 may harbor tumor suppressor genes for mammary tumors as well as for other rat malignancies of epithelial origin.
Overall, this analysis demonstrates that a number of alterations, translocations, inversions, deletions and chromosome copy number imbalances are required for normal rat mammary cells to become malignant.
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Notes
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1 To whom correspondence should be addressed Email: popescun{at}dc37a.nci.nih.gov 
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Received March 23, 1999;
revised June 8, 1999;
accepted June 24, 1999.