Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
Received January 23, 2002; accepted April 19, 2002
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ABSTRACT |
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Key Words: asbestos fibers; mesothelioma; p53-deficient mice; apoptosis; tumor progression; crocidolite asbestos; genetic instability.
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INTRODUCTION |
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Asbestos fibers induce malignant mesotheliomas in chronic rodent-inhalation assays, as well as after direct intrapleural or intraperitoneal injection (reviewed in Kane, 1996). In vitro models have provided evidence that asbestos fibers have properties of genotoxic as well as nongenotoxic carcinogens. The geometry of long, thin fibers has been associated with genotoxicity in target cells in vitro. The surface reactivity of asbestos fibers and their ability to catalyze iron-dependent generation of oxygen- and nitrogen-derived species has been linked to production of oxidized bases, DNA breaks, mutations, and deletions (reviewed in Kane, 1996
). Asbestos fibers have also been shown to be mitogenic, both by binding to specific surface receptors and by activating intracellular signaling pathways (reviewed by Robledo and Mossman, 1999
). Asbestos fibers also cause apoptosis that may trigger compensatory hyperplasia (Broaddus, 1997
). Activation of signal transduction pathways and induction of apoptosis by asbestos fibers in pleural mesothelial cells are dependent on the generation of reactive oxygen species (Robledo and Mossman, 1999
).
The p53 protein plays a major role in coordinating cell-cycle arrest and DNA repair or apoptosis induced by oxidants or ionizing radiation. Cells that lack a functional p53 protein as a result of gene deletion, point mutation, or complex formation with viral proteins are defective in cell-cycle arrest and resistant to induction of apoptosis by DNA-damaging agents (Ko and Prives, 1996). We have shown that a murine mesothelial cell line that spontaneously acquired a point mutation in the p53 gene during passaging in vitro is defective in the G1 cell cycle checkpoint induced by ionizing radiation. This cell line also shows increased sensitivity to induction of micronuclei by direct exposure to crocidolite asbestos fibers in vitro (Cistulli et al., 1996
). Therefore, we hypothesized that p53-deficient mice would show increased sensitivity to the genotoxic and carcinogenic effects of crocidolite asbestos fibers in vivo. We have reported previously that heterozygous p53+/- mice show an increased incidence and reduced latency of malignant mesotheliomas induced by crocidolite asbestos fibers (Marsella et al., 1997
). In this study, we report that 50% of the tumors induced in p53+/- mice show loss of heterozygosity (LOH) at the p53 tumor suppressor gene locus, increased tumor size, and extensive invasion. The accelerated progression of malignant mesothelioma in heterozygous p53+/- mice could be explained by three mechanisms. First, p53-deficient mesothelioma cells could have a selective growth advantage over wild-type or heterozygous p53+/- mesothelioma cells. Second, p53-deficient mesothelial cells may acquire increased genetic instability secondary to loss of cell-cycle checkpoints and accumulation of oxidant-induced DNA damage. Third, p53-deficient mesothelial cells may be resistant to apoptosis. Evidence for or against each of these three mechanisms was evaluated in vitro and in vivo.
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MATERIALS AND METHODS |
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Histopathology.
Complete necropsies were performed on all mice and histopathologic diagnosis of malignant mesotheliomas was carried out as described previously (Goodglick et al., 1997). For analysis of cell proliferation, mice were injected with BrdC (1 mg/1ml PBS, ip), which is more soluble than BrdU, one h before sacrifice. BrdC is converted to BrdU in vivo. Paraffin-embedded sections of tumors were processed for immunohistochemistry as described previously (Macdonald and Kane, 1997
). The thickness of tumors growing on the serosal surfaces was measured at a magnification of 100x using an eyepiece equipped with a micrometer. An in situ assay was developed to visualize micronuclei in mesothelial cells using whole mounts of the diaphragm. In order to distinguish between micronuclei associated with mesothelial cells and debris from inflammatory cells, proliferating mesothelial cells were labeled with BrdU as described above. After 14 h (the cell cycle time of mesothelial cells is 1824 h), mice were sacrificed and whole mounts of the diaphragm were prepared for BrdU immunohistochemistry. Micronuclei were counted at 40x magnification; at least 1000 proliferating mesothelial cells were counted per diaphragm. Micronuclei were counted if they were in the same focal plane as the nucleus and less than 25% of the diameter of the parent nucleus.
Isolation of mesothelioma cell lines.
Cell lines were isolated from heterozygous p53+/- or wild-type mice after development of ascites or weight loss following weekly intraperitoneal injections of crocidolite asbestos fibers, as described previously (Goodglick et al., 1997). Cell lines were established from ascites or peritoneal lavage fluid and analyzed after 26 passages in vitro.
Tissue microdissection and DNA extraction.
Fixed, paraffin-embedded tumors were cut into 15 µm sections, mounted on glass microscope slides, and dissected with a #11 surgical blade under an inverted phase-contrast microscope at 10x magnification. Tissue samples were deparaffinized in xylene, followed by 100% ethanol, air-dried, and digested in 100 mM NaCl, 10 mM Tris, pH 8.0, 25 mM EDTA, 0.5% SDS, and proteinase K at 100 µg/ml for 4 h at 370C. DNA was extracted in phenol/chloroform/isoamyl alcohol (25:24:1). The aqueous phase was collected and the salt concentration adjusted to 250 mM NaCl. DNA was ethanol-precipitated overnight at 200C, washed, resuspended in water, and stored at 800C.
PCR and RT-PCR analyses.
Genomic DNA was isolated from microdissected tumors, tail biopsies, or cultured mesothelioma cell lines, as described above. For PCR amplification, 200 ng of genomic DNA was amplified using the following primers: X7: 5`-tatactcagagccggcct-3` and X6.5: 5`-acagcgtggtggtaccttat-3` to amplify the wild-type p53 gene and X7 and neo 19: 5`-ctatcaggdcatagcgttgg-3` to amplify the disrupted p53 allele. For microdissected tumors, only the wild-type p53 allele was amplified, due to limited amounts of DNA. Sufficient tumor samples free of stromal cells were available from only 3 mice. PCR amplification was performed at 950C for 5 min.; 950C, 1 min; 600C, 2 min; and 720C, 3 min for 3035 cycles, followed by 720C for 10 min. PCR products were separated on a 3% agarose (TAE) gel. Total RNA was isolated from subconfluent cultures of mesothelioma cell lines using RNazolTM, and used for RT-PCR analysis as described previously (Goodglick et al., 1997). The following primers were used for p53 RT-PCR: 5`-acaggaccctgtcaccgagacc-3` and 5`-gacctccgtcatgtgctgtgac-3`. Oligonucleotide primers for murine ß-actin were purchased from Clontech Laboratories (Palo Alto, CA).
p53 Genomic DNA sequencing.
Genomic DNA, obtained from early passages of mesothelial cell lines isolated from one wild-type and 4 p53+/- mice that retained the wild-type allele, was PCR-amplified for exons 58 of the p53 gene, using Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) and primers derived from intron 4 (forward) and exon 11 (reverse). These products were cloned into a pCRII-Topo vector (Invitrogen) and cycle sequenced using p53 intron-specific forward and reverse primers.
Metaphase chromosome spreads.
Mesothelioma cell lines were maintained for 26 passages in vitro and exposed to colcemid (0.2 µg/ml for 1.5 h.). Metaphase spreads were prepared as described previously (Marsella et al., 1997); at least 20 metaphase spreads were counted to determine chromosome numbers in each cell line.
Apoptosis assays.
Tumor samples from heterozygous p53+/- mice were fixed in Omnifix, embedded in plastic, sectioned at 1 µm, and stained with toluidine blue for histologic analysis of necrosis or apoptosis. Paraffin-embedded tumor tissues were sectioned and analyzed for incorporation of biotinylated dUTP (TUNEL assay kit, Oncor, Inc., Gaithersburg, MD). Apoptotic cells were identified using avidin-peroxidase immunohistochemistry as described previously (Marsella et al., 1997).
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RESULTS |
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Four of the 5 cell lines derived from p53+/- mice that retained the wild-type allele were analyzed for mutations in exons 58 of the p53 gene. No missense mutations or deletions were detected.
Growth of p53-Deficient and Wild-Type Mesothelioma Cells in Vivo
If p53-mutant- or -deficient cells have a selective growth advantage in vivo, we would predict increased mesothelial-cell proliferation in tumors induced by asbestos in p53-deficient mice. Mesothelial-cell proliferation was assessed by BrdU labeling using immunohistochemistry. Sections of mesotheliomas were studied from p53+/+ and +/- mice, including tumors that had lost the wild-type allele. The BrdU labeling index ranged from 69%, with no statistically significant difference between p53+/+, +/-, or -/- tumors.
Genetic Instability of Mesothelioma Cell Lines Derived from p53-Deficient Mice
Tumors that develop in p53-/- mice are characterized by aneuploid and subtetraploid populations and abnormal karyotypes (Donehower et al., 1995). Therefore, we hypothesized that malignant mesotheliomas induced by asbestos in p53-deficient mice would show genetic instability. Cell lines were established from ascites or peritoneal lavage fluid collected from mice as described in Cistulli et al.(1996). Metaphase spreads were prepared from cells at passages 26. Normal p53+/+ mesothelial cells are nearly diploid (2n = 40). Cell lines derived from all of the p53+/- mice that had lost the wild-type allele were tetraploid (mean chromosome number = 81 ± 3.6). This observation is consistent with a p53-mediated spindle checkpoint (Cross et al., 1995
). In contrast, cell lines derived from p53+/+ and p53+/- mice that retained the wild-type allele were aneuploid (mean chromosome number = 55 ± 11). The difference between p53+/+ and p53-/- mesothelioma cell lines is statistically significant (p = 0.01). The p53-/- mesotheliomas showed nuclear pleomorphism with binucleate and multinucleated tumor cells and abnormal mitotic figures (Fig. 5
).
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DISCUSSION |
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The most striking pathologic finding in this chronic carcinogenicity study in heterozygous p53-deficient mice was accelerated tumor progression. Half of these mice had large tumor masses with central areas of necrosis, extensive local invasion, and penetration of lymphatics; these features are rarely seen in wild-type rodent tumors produced by direct intraperitoneal injection of asbestos fibers (Craighead and Kane, 1994). Cell lines were successfully established from 8 of the heterozygous p53+/- mice; 50% of these cell lines showed loss of p53 expression associated with loss of the wild-type allele. LOH at the p53 tumor suppressor gene locus occurs in approximately 50% of the tumors that arise in Li-Fraumeni families, as well as in lymphomas induced by ionizing radiation (Kemp et al., 1994
) or skin tumors induced by DMBA (Kemp et al., 1993
). However, not all tumors induced by carcinogens in heterozygous p53-deficient mice develop LOH or other mutations at the p53 tumor suppressor gene locus. For example, neither mammary tumors induced by DMBA (Jones et al., 1997
) nor lymphomas induced by uv irradiation of the skin (Jiang et al., 2001
) have any apparent mutations in the remaining wild-type p53 allele. Only 50% of the mesotheliomas induced by asbestos fibers had LOH; therefore, LOH is not essential for the development of mesotheliomas. However, LOH does appear to be associated with accelerated progression of these tumors.
Potential mechanisms responsible for accelerated tumor progression in p53-deficient mice have been explored in other model systems. Increased tumor size could be due to increased cell proliferation, reduced apoptosis, or both. In a mouse mammary tumor model (Wnt1-1TG mice crossed with p53+/+, +/-, or -/- mice), increased tumor size occurred in the Wnt-1TG p53+/- mice and was accompanied by loss of the wild-type allele in 50% of the tumors. These tumors had an increased mitotic index but no change in the level of apoptosis in comparison with the Wnt-1 TG p53+/+ or Wnt-1 TG p53-/- tumors (Jones et al., 1997). Although we were unable to measure tumor size directly in this intraperitoneal model because malignant mesotheliomas spread diffusely over the peritoneal surfaces (Craighead and Kane, 1994
), larger tumor masses and increased tumor thickness were seen in half of the heterozygous p53+/- mice. In contrast to the mouse mammary tumor model, no statistically significant differences in the BrdU labeling index were observed in p53+/+, p53+/-, or p53+/- tumors that had LOH. Instead, a statistically significant decrease in the apoptotic index was observed in p53+/- tumors that had lost the wild-type allele. This observation confirms previous studies in p53-deficient mice; for example, increased growth of choroid plexus tumors in p53-/- transgenic mice expressing a truncated SV40 large-T antigen has been attributed to a reduction in the apoptotic index (Symonds et al., 1994
).
In addition to induction of apoptosis, wild-type p53 protein is essential for cell-cycle arrest triggered by DNA damage and maintenance of genomic stability. Induction of the G1 cell cycle checkpoint depends on post-translational modification, stabilization, and translocation of the p53 protein to the nucleus where it activates transcription in conjunction with other co-activators (Prives and Hall, 1999). Activated transcription of p21 inhibits cyclin-dependent kinases resulting in hypophosphorylated Rb and G1 arrest. Other transcriptional targets of p53 include mdm-2, which binds to p53 itself in an autoregulatory fashion, and GADD45, which interacts with PCNA and prevents entry into S phase (Ko and Prives, 1996
). In addition to the G1 cell cycle checkpoint, p53 also regulates a G2 checkpoint, duplication of centrosomes, and a spindle checkpoint during mitosis (Fukasawa et al., 1996
). Therefore, inactivation of p53 function in response to DNA damage would result in unrepaired DNA breaks leading to aneuploidy and multiple rounds of DNA synthesis in the absence of chromosome segregation, resulting in tetraploidy (Cross et al., 1995
; Ko and Prives, 1996
).
Histologic examination of mesotheliomas in heterozygous p53-deficient mice that had lost the wild-type allele showed multinucleated tumor cells and abnormal mitotic figures. Mesothelioma cell lines isolated from these mice showed a subteraploid or tetraploid number of chromosomes. Tetraploid or nearly tetraploid tumor cell populations have been reported previously in mammary tumors induced in transgenic Wnt-1 TG p53-/- mice; these tumor cell populations also had evidence of genetic instability as revealed by karyotyping and comparative genomic hybridization (Donehower et al., 1995).
It is hypothesized that p53 haplo-insufficiency sensitizes mice to the clastogenic or aneuploidogenic effects of crocidolite asbestos fibers, resulting in a shorter latent period. In solid tumors, spontaneous loss of the wild-type allele, accompanied by decreased apoptosis and accumulation of additional mutations, accelerates tumor growth, invasion, and lymphatic dissemination, as suggested by Macleod and Jacks (1999).
This genetically engineered murine model is highly relevant to the pathogenesis of human malignant mesotheliomas. Although p53 mutations, rearrangements, or deletions are rare in either human or rodent malignant mesotheliomas (Lechner et al., 1997; Murthy and Testa, 1999
), some investigators have demonstrated SV40 T-antigen and viral sequences in up to 80% of human mesothelioma tissues examined (reviewed in Carbone et al., 1999
). Antisense constructs directed against large T-antigen have been shown to inhibit proliferation of human mesothelioma cell lines (Waheed et al., 1999
). Although these findings are still controversial (Pipas and Levine, 2001
), this murine model demonstrates a potential role for inactivation of p53 function in the development of asbestos-induced malignant mesotheliomas. This murine model provides the opportunity for additional mechanistic studies to investigate whether SV40 virus and asbestos fibers are cocarcinogens.
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ACKNOWLEDGMENTS |
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NOTES |
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