A Truncated Isoform of the Protein Phosphatase 2A B56γ Regulatory Subunit May Promote Genetic Instability and Cause Tumor Progression
2003; Elsevier BV; Volume: 162; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63800-x
ISSN1525-2191
AutoresAkihiko Ito, Yu‐ichiro Koma, Kenji Watabe, Teruaki Nagano, Yuichi Endo, Hiroshi Nojima, Yukihiko Kitamura,
Tópico(s)Cell death mechanisms and regulation
ResumoF10, a subline of the B16 mouse melanoma cell line, is itself the parent of the more metastatic BL6 line. BL6 cells differ from F10 cells by an alteration of the gene encoding the B56γ regulatory subunit of protein phosphatase 2A (PP2A), which results in the expression of a truncated variant of the subunit (Δγ1). PP2A is involved in regulating the cell-cycle checkpoint and we found that the checkpoint in BL6 cells is aberrant when the Δγ1 protein is expressed. That is, although Δγ1 protein levels in cultured BL6 cells are low and these cells do not show an altered checkpoint on γ-irradiation, irradiated footpad BL6 tumor cells show both a marked increase in Δγ1 levels and more extensive polyploidy and less apoptosis than F10 cells. These observations were reproduced with Δγ1 gene-transfected F10 cells (F10Δγ1). Δγ1 expression and an aberrant checkpoint are also associated with a higher metastatic ability because irradiated F10Δγ1 tumors metastasized much more frequently than F10 tumors, which rarely metastasized whether irradiated or not. Nonirradiated F10Δγ1 tumors, which do not express Δγ1 protein, had similarly low rates of metastasis. The greater metastatic ability of irradiated F10Δγ1 tumors also correlated with the acquisition of many more genomic alterations. Thus, it seems that Δγ1 expression may damage the checkpoint, which may then allow the acquisition of genetic alterations that promote metastasis. These observations support the notion that mechanisms promoting the genetic instability of tumors could also aid tumor progression from the nonmetastatic to the metastatic state. F10, a subline of the B16 mouse melanoma cell line, is itself the parent of the more metastatic BL6 line. BL6 cells differ from F10 cells by an alteration of the gene encoding the B56γ regulatory subunit of protein phosphatase 2A (PP2A), which results in the expression of a truncated variant of the subunit (Δγ1). PP2A is involved in regulating the cell-cycle checkpoint and we found that the checkpoint in BL6 cells is aberrant when the Δγ1 protein is expressed. That is, although Δγ1 protein levels in cultured BL6 cells are low and these cells do not show an altered checkpoint on γ-irradiation, irradiated footpad BL6 tumor cells show both a marked increase in Δγ1 levels and more extensive polyploidy and less apoptosis than F10 cells. These observations were reproduced with Δγ1 gene-transfected F10 cells (F10Δγ1). Δγ1 expression and an aberrant checkpoint are also associated with a higher metastatic ability because irradiated F10Δγ1 tumors metastasized much more frequently than F10 tumors, which rarely metastasized whether irradiated or not. Nonirradiated F10Δγ1 tumors, which do not express Δγ1 protein, had similarly low rates of metastasis. The greater metastatic ability of irradiated F10Δγ1 tumors also correlated with the acquisition of many more genomic alterations. Thus, it seems that Δγ1 expression may damage the checkpoint, which may then allow the acquisition of genetic alterations that promote metastasis. These observations support the notion that mechanisms promoting the genetic instability of tumors could also aid tumor progression from the nonmetastatic to the metastatic state. Two well-known properties of tumor cells are that they are genetically unstable1Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3341) Google Scholar, 2Cahill DP Kinzler KW Vogelstein B Lengauer C Genetic instability and Darwinian selection in tumours.Trends Cell Biol. 1999; 9: 57-60Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar and tend to progress toward increasing malignancy.3Chambers AF Hill RP Tumor progression and metastasis.in: Tannock IF Hill RP The Basic Science of Oncology. McGraw-Hill, New York1992: 219-239Google Scholar As early as 1986, Nowell4Nowell PC The clonal evolution of tumor cell populations.Science. 1976; 194: 23-28Crossref PubMed Scopus (4763) Google Scholar drew a link between these two characteristics when he predicted that the genetic instability of tumor cells could contribute to disease progression by generating subclones with different properties. Consistent with this hypothesis, human tumor cells often have mutations in the genes that play essential roles in the cell-cycle checkpoint, an intracellular event that protects the integrity of the genome against genotoxic stress.1Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3341) Google Scholar, 5Sherr CJ Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4927) Google Scholar For example, the p53 tumor suppressor gene, which plays a pivotal role in the checkpoint, is mutated in more than half of a wide variety of sporadically occurring human tumors.1Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3341) Google Scholar, 5Sherr CJ Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4927) Google Scholar An intact cell-cycle checkpoint responds to DNA damage by either arresting cell-cycle progression, thereby facilitating DNA repair, or by initiating an apoptotic pathway that eliminates the damaged cell.6Paulovich AG Toczyski DP Hartwell LH When checkpoints fail.Cell. 1997; 88: 315-321Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar However, when the checkpoint fails, the cell-cycle progresses despite the DNA damage and can occasionally result in chromosomal aberration,6Paulovich AG Toczyski DP Hartwell LH When checkpoints fail.Cell. 1997; 88: 315-321Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar extensive polyploidy,7Illidge TM Cragg MS Fringes B Olive P Erenpreisa JA Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage.Cell Biol Int. 2000; 24: 621-633Crossref PubMed Scopus (129) Google Scholar and reduced apoptosis.8Brown JM Wouters BG Apoptosis, p53, and tumor cell sensitivity to anticancer agents.Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar Consequently, checkpoint failure is postulated to contribute to malignant progression by generating multiple subclones in a tumor.1Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers.Nature. 1998; 396: 643-649Crossref PubMed Scopus (3341) Google Scholar, 2Cahill DP Kinzler KW Vogelstein B Lengauer C Genetic instability and Darwinian selection in tumours.Trends Cell Biol. 1999; 9: 57-60Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar This is supported by the work of Cifone and Fidler,9Cifone MA Fidler IJ Increasing metastatic potential is associated with increasing genetic instability of clones isolated from murine neoplasms.Proc Natl Acad Sci USA. 1981; 78: 6949-6952Crossref PubMed Scopus (256) Google Scholar who showed that the increasing metastatic potential of murine fibrosarcoma cells is associated with an increased genetic instability. Metastasis is the most malignant characteristic of a tumor and can be considered as the final stage of tumor progression.3Chambers AF Hill RP Tumor progression and metastasis.in: Tannock IF Hill RP The Basic Science of Oncology. McGraw-Hill, New York1992: 219-239Google Scholar It is difficult to show clearly that genetic instability is directly responsible for the metastasis of clinical tumor cells, but studies with experimental tumors that can be transplanted into animals may be useful in investigating this issue. This is because it is often possible to select sublines with increased metastatic ability from a parental tumor population,3Chambers AF Hill RP Tumor progression and metastasis.in: Tannock IF Hill RP The Basic Science of Oncology. McGraw-Hill, New York1992: 219-239Google Scholar whose heterogeneity seems to arise from its genetic instability.3Chambers AF Hill RP Tumor progression and metastasis.in: Tannock IF Hill RP The Basic Science of Oncology. McGraw-Hill, New York1992: 219-239Google Scholar, 10Shapiro JR Yung WK Shapiro WR Isolation, karyotype, and clonal growth of heterogeneous subpopulations of human malignant gliomas.Cancer Res. 1981; 41: 2349-2359PubMed Google Scholar One of the best known examples of this is the selection of the F10 and BL6 sublines from B16 mouse melanoma cells.11Hart IR The selection and characterization of an invasive variant of the B16 melanoma.Am J Pathol. 1979; 97: 587-600PubMed Google Scholar, 12Poste G Doll J Hart IR Fidler IJ In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res. 1980; 40: 1636-1644PubMed Google Scholar Although the BL6 subline was derived from F10 cells by six rounds of in vitro selection, it is markedly more metastatic than its parent. That is, while both cells metastasize to the lungs after being injected intravenously into mice, BL6 cells will metastasize to the lungs even after being injected subcutaneously.12Poste G Doll J Hart IR Fidler IJ In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res. 1980; 40: 1636-1644PubMed Google Scholar According to the hypothesis proposed by Nowell,4Nowell PC The clonal evolution of tumor cell populations.Science. 1976; 194: 23-28Crossref PubMed Scopus (4763) Google Scholar it may be that BL6 cells are genetically more unstable than F10 cells and that this has led to the accumulation of a number of genomic alterations that increased their metastatic ability. We addressed this issue by examining the difference in gene expression between the F10 and BL6 sublines.13Nakaji T Kataoka TR Watabe K Nishiyama K Nojima H Shimada Y Sato F Matsushima H Endo Y Kuroda Y Kitamura Y Ito A Maeda S A new member of the GTPase superfamily that is upregulated in highly metastatic cells.Cancer Lett. 1999; 147: 139-147Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Kataoka TR Ito A Asada H Watabe K Nishiyama K Nakamoto K Itami S Yoshikawa K Ito M Nojima H Kitamura Y Annexin VII as a novel marker for invasive phenotype of malignant melanoma.Jpn J Cancer Res. 2000; 91: 75-83Crossref PubMed Scopus (33) Google Scholar, 15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar, 16Ito A Katoh F Kataoka TR Okada M Tsubota N Asada H Yoshikawa K Maeda S Kitamura Y Yamasaki H Nojima H A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.J Clin Invest. 2000; 105: 1189-1197Crossref PubMed Scopus (162) Google Scholar, 17Watabe K Ito A Asada H Endo Y Kobayashi T Nakamoto K Itami S Takao S Shinomura Y Aikou T Yoshikawa K Matsuzawa Y Kitamura Y Nojima H Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.Jpn J Cancer Res. 2001; 92: 140-151Crossref PubMed Scopus (70) Google Scholar, 18Nakamoto K Ito A Watabe K Koma Y Asada H Yoshikawa K Shinomura Y Matsuzawa Y Nojima H Kitamura Y Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.Am J Pathol. 2001; 159: 1363-1374Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar We found that in BL6 cells, a type of retrotransposon had been inserted into an intronic region of the gene encoding the B56γ regulatory subunit of protein phosphatase type 2A (PP2A).15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar PP2A consists of a series of serine/threonine phosphatase holoenzymes that are composed of a common dimeric core of invariable catalytic (C) and structural (A) subunits associated with a variable regulatory (B) subunit.19Usui H Imazu M Maeta K Tsukamoto H Azuma K Takeda M Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol.J Biol Chem. 1988; 263: 3752-3761Abstract Full Text PDF PubMed Google Scholar The regulatory subunit is extremely diverse because it is constituted by members from at least three unrelated families, namely, PR55 (or simply B), B56 (B′), and PR72 (B″).20Virshup DM Protein phosphatase 2A: a panoply of enzymes.Curr Opin Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (289) Google Scholar Each of these families in turn consists of several subfamilies, each of which contains several proteins resembling each other structurally. For example, the B56γ subfamily belongs to the B56 family and consists of three alternative splicing isoforms, B56γ1, -γ2, and -γ3. In BL6 cells, the rearrangement of the gene encoding the B56γ regulatory subunit results in the abundant expression of a chimeric mRNA in which the 5′ part of the original B56γ subunit mRNA is replaced with the retrotransposon sequence.15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar The chimeric mRNA encodes a mutant protein, termed Δγ1, that lacks the N-terminal 65 amino acid residues of B56γ1. Δγ1 protein expression is undetectable in in vitro-grown BL6 cells. However, when BL6 cells are transplanted into mice and undergo metastasis, Δγ1 expression is detectable in the metastatic foci but not in the transplantation site.15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar The diversity of the regulatory subunit of PP2A results in different PP2A holoenzymes with distinct substrate specificities and subcellular localizations.20Virshup DM Protein phosphatase 2A: a panoply of enzymes.Curr Opin Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (289) Google Scholar This allows PP2A to regulate a variety of cellular processes. Studies with yeast,21Kinoshita N Ohkura H Yanagida M Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle.Cell. 1990; 63: 405-415Abstract Full Text PDF PubMed Scopus (242) Google Scholar Xenopus oocytes,22Millward TA Zolnierowicz S Hemmings BA Regulation of protein kinase cascades by protein phosphatase 2A.Trends Biochem Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar and mammalian cells23Yamashita K Yasuda H Pines J Yasumoto K Nishitani H Ohtsubo M Hunter T Sugimura T Nishimoto T Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells.EMBO J. 1990; 9: 4331-4338Crossref PubMed Scopus (245) Google Scholar, 24Kawabe T Muslin AJ Korsmeyer SJ HOX11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2/M cell-cycle checkpoint.Nature. 1997; 385: 454-458Crossref PubMed Scopus (176) Google Scholar, 25Okamoto K Li H Jensen RM Zhang T Taya Y Thorgeirsson SS Prives C Cyclin G recruits PP2A to dephosphorylate Mdm2.Mol Cell. 2002; 9: 761-771Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar reveal that one of the most important functions of PP2A is to participate in the regulation of the cell-cycle checkpoint. Our previous work showed that Δγ1 prevents the B56γ1-containing PP2A heterotrimer from dephosphorylating its specific substrates.15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar Consistent with this inhibitory effect of Δγ1, NIH3T3 cells that stably express Δγ1 are defective in cell-cycle checkpoint regulation when exposed to γ-radiation.15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar This suggests that BL6 cells may also be defective in checkpoint regulation when Δγ1 is expressed. In the present study, we found that the ability of the cell-cycle checkpoint in BL6 cells to respond appropriately to γ-radiation is indeed inferior to that in F10 cells when the cells are grown in vivo and express Δγ1. These characteristics of BL6 cells were reproduced in F10 cells transfected with Δγ1 (F10Δγ1). F10 cells transfected with Δγ1 also showed an increased metastatic ability after being grown in mouse footpads and then irradiated. The metastatic colonies of these cells were found to have acquired considerable numbers of genomic alterations relative to the genomes of the primary lesions. Such alterations were infrequently detected in the more rarely occurring metastatic colonies of F10 cells. These observations suggest that Δγ1 expression could have caused the checkpoint of BL6 cells to degenerate, thereby helping BL6 cells to acquire multiple genetic alterations that promote their metastatic ability. B16 melanoma sublines F10 and BL6 were kindly provided by Dr. I. J. Fidler (University of Texas). NIH3T3 mouse fibroblastic cells were purchased from the American Type Culture Collection (Manassas, VA). All cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Construction of the plasmid vector encoding Δγ1 and transfection of F10 and NIH3T3 cells with this plasmid or the empty vector have been described previously.15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar After the transfection, subclones of F10 and NIH3T3 cells were selected with 1.5 and 1.0 mg/ml of G-418, respectively. The cell-doubling time was determined by counting cells in triplicate samples at 24-, 48-, and 72-hour intervals as described previously.18Nakamoto K Ito A Watabe K Koma Y Asada H Yoshikawa K Shinomura Y Matsuzawa Y Nojima H Kitamura Y Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.Am J Pathol. 2001; 159: 1363-1374Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Cells growing exponentially in culture dishes were γ-irradiated using a 137Cs AECL Gamma cell 40 γ-irradiator (Nordion, Ottawa, Ontario, Canada) at a dose rate of 1.1 Gy/minute. Cells were subsequently cultured for the indicated time periods, fixed in 70% ethanol at −20°C, treated with RNase, stained with propidium iodide (PI; Sigma, St. Louis, MO), and analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ). Cells (1 × 105) were injected subcutaneously into the right footpads of 4-week-old male C57BL/6 mice. Tumor size was monitored by measuring the anteroposterior diameter with calipers three or more times a week. Tumor-bearing mice were anesthetized, placed in a lead box that shields the entire trunk and head, and the right foot was extended outside through a hole in the side of the box. Radiation was delivered over the box with a collimated beam in a Radioflex 350 irradiator (Rigaku Co. Ltd., Osaka, Japan) at a dose rate of 1.5 Gy/minute. Control mice were anesthetized and placed in a lead box but received no γ-irradiation. Tumor cell masses were removed from the footpads by cutting out the skin tissue and single tumor-cell suspensions were obtained as described previously.26Ito A Morii E Maeyama K Jippo T Kim DK Lee YM Ogihara H Hashimoto K Kitamura Y Nojima H Systematic method to obtain novel genes that are regulated by mi transcription factor: impaired expression of granzyme B and tryptophan hydroxylase in mi/mi cultured mast cells.Blood. 1998; 91: 3210-3221Crossref PubMed Google Scholar, 27Ito A Kataoka TR Kim DK Koma Y Lee YM Kitamura Y Inhibitory effect on natural killer activity of microphthalmia transcription factor encoded by the mutant mi allele of mice.Blood. 2001; 97: 2075-2083Crossref PubMed Scopus (22) Google Scholar Briefly, the tumor mass was minced with sharp scissors and passed first through a 23-gauge needle and then a nylon net (pore size 40 μm). During the latter procedure, phosphate-buffered saline (PBS) was poured continuously over the net. The tumor-cell suspension was washed twice with PBS by centrifugation and resuspension. The cells were fixed in 70% ethanol at −20°C, treated with RNase, stained with PI (Sigma) and analyzed by FACScan (Becton Dickinson). Mice were inoculated subcutaneously with F10, 10Δγ1, F10Vec, or BL6 cells, and half received γ-irradiation locally to the footpads. On the day the footpad tumors reached a diameter of 6 mm, they were excised along with the popliteal lymph node. The mice were allowed to survive an additional 4 weeks after which autopsies were performed to macroscopically count the metastatic colonies that had formed in the bilateral lungs. All experiments included five to seven mice per group and were reproduced twice. Tumor cell masses were removed from the footpads by cutting out the skin tissue which was then fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick slices. DNA fragmentation was detected with the fluorescein-based terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay using the DeadEnd Fluorometric TUNEL system (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, sections were stained with the TUNEL regent and PI and observed through a ×40 objective of a confocal laser-scanning microscope (LSM510; Carl Zeiss, OberKochen, Germany). The high-power field contained ∼100 tumor cells. After images were created on the computer monitor of the LSM510 system, sections were washed with distilled water and stained with hematoxylin and eosin (H&E). To quantify apoptosis of tumor cells, the areas where tumor cells grew compact were observed. Tumor samples were obtained from two individual mice before and after irradiation, and 10 randomly selected high-power fields were examined in each sample. The proportions of TUNEL-positive cells to the total cell number were pooled and the mean and SE were calculated. All experiments were reproduced once. The production and specificity of the anti-B56γ antibody has been described previously.15Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (142) Google Scholar Tumor cells growing in culture, in the footpads and in the lungs were lysed in a buffer containing 10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, 1 mmol/L phenylmethylsulfonyl fluoride. The resulting lysates were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred to Immobilon (Millipore, Bedford, MA), and reacted with the anti-B56γ antibody. After washing, the blots were incubated with peroxidase-labeled anti-rabbit IgG antibody (MBL Co. Ltd., Nagoya, Japan) and then reacted with Renaissance reagents (NEN, Boston, MA) before exposure. After stripping, the blot was reacted with the anti-α-tubulin monoclonal antibody (Sigma), whose binding was detected with a peroxidase-labeled anti-mouse IgG antibody (MBL Co. Ltd.). Cells (n = 3000) were plated in a 10-cm culture dish. The following day, after the cells had attached to the plate, they were γ-irradiated with single doses ranging from 2 to 10 Gy. The cells were grown for another 10 days without changing the medium and then stained with Giemsa solution (Sigma). Colonies containing >50 cells were counted. The number of colonies in the plate receiving γ-irradiation was divided by the number not receiving γ-irradiation and expressed as survival fraction. Each experiment was done in triplicate and repeated three times. Mice were treated with the protocol of the spontaneous metastasis assay combined with in vivo γ-irradiation. Footpad tumors were isolated aseptically by amputation from the mice who were not γ-irradiated, and lung metastatic colonies were isolated at autopsy from the mice who were γ-irradiated. A single metastatic colony was isolated from each mouse. Tumor cell masses were minced with sharp scissors in Dulbecco's modified Eagle's medium/10% fetal calf serum supplemented with G-418 (1.5 mg/ml) and proliferating adherent cells from these preparations were used for cytogenetic chromosome analysis and DNA extraction. For cytogenetic analysis, the chromosomes were stained with Giemsa solution as described previously.14Kataoka TR Ito A Asada H Watabe K Nishiyama K Nakamoto K Itami S Yoshikawa K Ito M Nojima H Kitamura Y Annexin VII as a novel marker for invasive phenotype of malignant melanoma.Jpn J Cancer Res. 2000; 91: 75-83Crossref PubMed Scopus (33) Google Scholar, 17Watabe K Ito A Asada H Endo Y Kobayashi T Nakamoto K Itami S Takao S Shinomura Y Aikou T Yoshikawa K Matsuzawa Y Kitamura Y Nojima H Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.Jpn J Cancer Res. 2001; 92: 140-151Crossref PubMed Scopus (70) Google Scholar To extract genomic DNA, cells were digested with 1 mg/ml of proteinase K for 3 hours at 55°C, followed by 0.5 mg/ml of RNase treatment for 1 hour and then phenol:chloroform extraction and ethanol precipitation. DNA was subjected to intersimple sequence repeat polymerase chain reaction (inter-SSR PCR) analysis according to the procedure of Basik and colleagues28Basik M Stoler DL Kontzoglou KC Rodriguez-Bigas MA Petrelli NJ Anderson GR Genomic instability in sporadic colorectal cancer quantitated by inter-simple sequence repeat PCR analysis.Genes Chromosom Cancer. 1997; 18: 19-29Crossref PubMed Scopus (59) Google Scholar but with the minor modification described by Jackson and Matthews.29Jackson JA Matthews D Modified inter-simple sequence repeat PCR protocol for use in conjunction with the LI-COR Gene ImagIR2 DNA analyzer.Biotechniques. 2000; 28: 914-916PubMed Google Scholar (CA)8AG and (CA)8GG primers were synthesized and the 5′ ends of a proportion of the primers were labeled with an infrared fluorescence dye (IRD 800; Nisshinbo, Chiba, Japan). PCR amplification was performed in 20-μl volumes containing 200 nmol/L of labeled primer [a 1:1 mixture of (CA)8AG and (CA)8GG] and 800 nmol/L of unlabeled primer of the same mixture. In addition, the reaction contained 50 ng of genomic DNA and 0.2 μl of Ex Taq polymerase (Takara, Ohtsu, Japan) in PCR buffer (10 mmol/L Tris-HCl, pH 9.0, 2% formamide, 50 mmol/L KCl, 0.2 mmol/L dNTPs, 1.5 mmol/L MgCl2, 0.01% gelatin, and 0.01% Triton X-100). The thermal profile consisted of an initial denaturation for 3 minutes at 94°C followed by 30 cycles of 30 seconds at 94°C, 45 seconds at 52°C, and 2 minutes at 72°C, and a final extension for 7 minutes at 72°C. After denaturing the samples, a 2-μl aliquot of each PCR mixture was loaded onto a 3% polyacrylamide gel containing 5.2 mol/L of urea and visualized on the Base ImagIR DNA analyzer (model 4000L; Li-Cor, Lincoln, NE). Experiments were reproduced at least twice. Four primary tumors were examined for each cell type and all yielded a similar electrophoretic band pattern. Electrophoretic bands of the primary tumor and metastatic colony of the same cell type were compared. A genomic alteration index28Basik M Stoler DL Kontzoglou KC Rodriguez-Bigas MA Petrelli NJ Anderson GR Genomic instability in sporadic colorectal cancer quantitated by inter-simple sequence repeat PCR analysis.Genes Chromosom Cancer. 1997; 18: 19-29Crossref PubMed Scopus (59) Google Scholar was determined by dividing the number of altered and lost bands in the metastatic colony DNA by the total number of bands in the primary tumor DNA. The t-test, the Mann-Whitney U-test, and the chi-square test were performed using StatView (Abacus Concepts Inc., Cary, NC) software on a Macintosh computer. Data are given as mean ± SE. A P value of <0.05 was considered to be significant. To examine the integrity of the DNA damage checkpoints in F10 and BL6 cells, we monitored the cell-cycle profile of both cells after they were cultured and then γ-irradiated (Figure 1). Before γ-irradiation, most of the cells had a 2N DNA content (G1 phase) while a smaller but significant fraction contained 4N DNA (G2/M phase). A few cells had a DNA content greater than 2N but less than 4N (S phase). A day (24 hours) after γ-irradiation, both the F10 and BL6 cells showed a decrease in 2N DNA cells and an increase in 4N DNA cells, indicating cell-cycle arrest in the G2/M phase but not in G1. The next day (48 hours), considerable numbers of F10 and BL6 cells had an 8N DNA content. These polyploid cells had probably exited mitosis and initiated a new round of DNA replication without cytokinesis. After 2 more days, the polyploid fraction decreased and cells with a sub-G1 DNA content appeared, indicating that the polyploid cells were proceeding to apoptosis. By 7 days after irradiation, the 8N- and sub-G1 DNA-containing cells had become much less frequent and both F10 and BL6 cells had returned to their original diploid state. Obvious differences between F10 and BL6 cells in this pattern were not observed. We next examined the response to γ-irradiation by F10 and BL6 cells grown in vivo. Two wee
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