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Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells
Mutation Research 431 Ž1999. 133–139 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Apoptosis and catastrophic cell death in benzow axpyrene-transformed human breast epithelial cells q Luıs ´ Fernando Barbisan a, Maria Luıza ´ S. Mello Benedicto de Campos Vidal a a b
a,)
, Jose Russo b,
Department of Cell Biology, Institute of Biology, UNICAMP, 13083-970, Campinas, SP, Brazil Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
Received 23 March 1999; received in revised form 16 September 1999; accepted 17 September 1999
Abstract Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzow axpyrene ŽBP. ŽBP1, BP1-E and BP1-E1 cells. and in BP1 cells transfected with the c-Ha-ras oncogene ŽBP1-Tras cells.. Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Benzow axpyrene; c-Ha-ras; Mitotic death; Apoptosis; Human breast epithelial cells
1. Introduction The regulation of cell population growth depends on a balance between cell proliferation and cell death
q
Part of a thesis presented by L.F.B. to the Institute of Biology, UNICAMP, Campinas, in partial fullfilment of the requirements for the Masters’ degree. ) Corresponding author. Fax: q55-19-7887821. E-mail address: [email protected] ŽM.L.S. Mello.
w1x. In general, cells proliferate using the common process of mitosis, which has been extensively studied in normal and cancer cells w2x. In contrast, cell death occurs in several ways, two of which have been described as apoptosis and mitotic death. Apoptosis is a form of cellular suicide characterized morphologically by progressive cell shrinkage with condensation and margination of chromatin that culminates in cellular collapse and fragmentation into small, membrane-encapsulated apoptotic bodies w3x. Biochemically, apoptosis is characterized by the
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fragmentation of DNA which results in a ladder-like pattern of 180–200 pb fragment multiples in agarose gel electrophoresis w4,5x. In contrast to the relatively rapid apoptotic process w6x, mitotic death is characterized by a slow process of cell enlargement and G2 arrest, followed by mitotic catastrophe and subsequent nuclear fragmentation. This fragmentation apparently results from aberrant mitosis followed by relocation of the nuclear membrane around chromosome fragments or masses w7–9x. Thus, cells committed to mitotic death complete at least one mitosis before their disintegration w8x. Treatment of the spontaneously immortalized human breast epithelial cell line, MCF-10F, with the chemical carcinogen, benzow axpyrene ŽBP., yielded the cell lines BP1, BP1-E and BP1-E1 which express progressive stages of neoplastic transformation w10x. BP1 cells emerged after 248 days in culture and gave rise to the clone BP1-E, which in turn, after several passages in culture, gave rise to the cell line BP1-E1 w10x. A highly aggressive tumorigenic cell line, BP1-Tras, was generated when transformed nontumorigenic BP1 cells were transfected with the c-Ha-ras oncogene w11x. BP-transformed cell lines show a higher rate of proliferation, increased anchorage-independent growth and in vitro chemotactic and chemoinvasive abilities. However, only BP1-E, BP1E1, and BP1-Tras cell lines express the tumorigenic phenotype when inoculated into mice with severe combined immunodeficiency w10,11x. Since BP is capable of inducing apoptosis via a caspase-dependent pathway w12x, BP-transformed MCF-10F cell lines should exhibit this form of cell death. On the other hand, since BP affects the ras genes of MCF-10F cells and transfection with the Ha-ras oncogene enhances the neoplastic changes initiated by BP treatment w11,13x, catastrophic mitoses may result from the abnormal expression of c-Ha-ras in BP1-Tras cells in a manner similar to that reported for HeLa cells w14x. In the present study, the different types of cell death and their relationship to other cellular changes such as bi- and multinucleation and micronucleus formation during the tumorigenic progression were investigated in human breast epithelial cell lines transformed by BP and additionally transfected with the c-Ha-ras oncogene.
2. Material and methods 2.1. Cell lines and culture conditions Nontransformed ŽMCF-10F. and BP-transformed cell lines ŽBP1, BP1-E and BP1-E1. w10x were used. BP1-Tras cells, obtained by transfection of BP1 cells with the plasmid vector pH06T1 containing the T24 Ha-ras oncogene w11x were also used. The MCF-10F, BP1, BP1-E, BP1-E1 and BP1-Tras cells were used at passages 140, 23, 42, 19 and 61, respectively. The cells were grown in Dulbecco’s minimal essential mediumrF-12 medium Ž1:1. supplemented with 5% equine serum, 0.1 mgrml cholera-toxin, 10 mgrml insulin, 100 unitsrml penicillin, 100 mgrml streptomycin, 2.5 mgrml amphotericin B ŽGibco, Grand Island, NY., 0.5 mgrml hydrocortisone ŽSigma. and 0.02 mgrml epidermal growth factor ŽCollaborative Research, Palo Alto, CA. as described previously w15x. 2.2. Cell preparations and staining Cells grown on coverslips were fixed in 4% buffered formalin for 10 min, rinsed in distilled water and air dried at room temperature or in an absolute ethanol–acetic acid mixture Ž3:1, vrv. for 1 min, rinsed in 70% ethanol for 3–5 min and air dried at room temperature. The formalin-fixed preparations were processed for C banding w16x, stained with 8% Giemsa ŽGurr. solution in Sorensen buffer at pH 6.8 for 30 min, and counterstained with a fast green solution at pH 2.7 for 30 s. The preparations fixed in ethanol–acetic acid were subjected to a previously described Ag–NOR staining w17x. The optimal staining time in this case was 9 min at 378C. After staining, the preparations were rinsed in distilled water, air dried, cleared in xylene and mounted in Entellan ŽMerck.. In some cases ethanol–acetic acid-fixed preparations were processed by a variant of the critical electrolyte concentration assay suggested for apoptosis studies w18x. For this, the cells were stained with a 0.025% toluidine blue ŽMerck. solution in McIlvaine buffer at pH 4.1 for 15 min and next treated with a 0.05 M aqueous MgCl 2 solution for 15 min, rapidly rinsed in distilled water, air-dried, cleared in xylene and mounted in Canada balsam.
L.F. Barbisan et al.r Mutation Research 431 (1999) 133–139
2.3. Analysis of cellular parameters The mitotic index, apoptotic index and frequency of bi-, multi- and micronucleate cells as well as of single- or multinucleate giant cells were determined in nearly 2500 cells for each cell line. Micronuclei were identified and analyzed as described by Tolbert et al. w19x and Fenech w20x. Morphological identification of the two types of cell death was based on the following criteria: shrunken cells containing regions of chromatin condensation with or without the formation of apoptotic bodies indicated apoptotic death whereas cells containing multiple nuclear fragments and micronuclei were indicative of ‘‘mitotic or catastrophic death’’ w3,9,14x. Interphase nuclei that stained deeply green after the critical electrolyte concentration assay w18x and exhibited DNA birefringence under polarized light were considered as apoptotic nuclei. This method was used to determine the apoptotic index. 2.4. Statistical analysis Goodman’s test w21x for contrast among multinomial populations was used. 3. Results 3.1. Mitotic abnormality, cell death and cell proliferation Cells with an apoptotic morphology were observed in nontransformed and all transformed cell lines, but were significantly more frequent in BP1-
135
T ras cells ŽTable 1.. Nuclear chromatin and cytoplasm condensation with cellular collapse and fragmentation were well characterized in these cells ŽFig. 1a–i.. RNA was observed to be confined to even bodies positioned laterally to condensed masses of DNA Žgreen. or appeared as heterogeneous coarse granules surrounding the DNA clumps or localized to one side of these clumps in apoptotic cells of the transformed cell lines subjected to the critical electrolyte concentration assay w18x ŽFig. 1c–i.. Some apoptotic nuclei showed metachromatic nucleolar remnants within a condensed chromatin mass, which exhibited the brilliant green color of DNA staining ŽFig. lg.. In most of the apoptotic nuclei there were no structured nucleolar bodies or granules ŽFig. 1h.. When examined under polarized light these nuclei exhibited typical DNA birefringence ŽFig. 1h–i.. RNA relocation in apoptotic cells did not follow a specific pattern of dispersion or disruption, particularly in the cell line transformed by BP and then transfected with the c-Ha-ras oncogene. Abnormal mitotic figures were seen in all cell lines analyzed. The most common mitotic changes were tripolar metaphases, metaphases and anaphases with lagging chromosome fragments, and anaphase bridges ŽFig. 1j,k.. Larger cells containing pale or deeply stained nuclear fragments and more than one micronucleus, characteristic of mitotic death, were present in all cell lines, but were more frequent in BP1-Tras cells ŽFig. 1l–n.. Ag–NOR stained areas were found in most nuclear fragments ŽFig. 1o.. Regardless of the cell line, mononucleate cells seem to be eliminated preferentially through apoptosis
Table 1 Incidence of mitosis and apoptosis in transformed human breast epithelial cells Different symbols Ža, b. mean differences significant at p - 0.05 using Goodman’s w21x test for contrast among multinomial proportions. Cells
MCF-10F BP1 BP1-E BP1-E1 BP1-Tras
No. of cells
Mitotic
Mitoticphase
Apoptotic
Index
counted
index Ž%.
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Ž%.
no. of cells counted
2277 2570 2537 2520 2519
2.61a 3.21a 4.38 b 4.05 b 3.93 b
5.0 7.5 8.1 8.8 10.1
46.7 51.2 45.9 53.9 46.5
6.7 11.3 4.5 12.7 12.1
13.3 7.5 15.3 9.8 9.1
28.3 22.5 26.1 14.7 22.2
0.56 a 0.42 a 0.20 a 0.28 a 2.21b
2503 2590 2521 2505 2531
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L.F. Barbisan et al.r Mutation Research 431 (1999) 133–139
137
Table 2 Frequency of bi-, multi- and micronucleate cells, and giants in transformed human breast epithelial cells Cells
No. of cells counted
MCF-10F BP1 BP1-E BP1-E1 BP1-Tras )
2577 2570 2537 2520 2519
Cell types Ž%. Binucleate
Multinucleate
Giants
Micronucleate
2.09 1.56 1.30 1.23 2.18
0.23 0.35 0.32 0.28 1.39U
0.12 0.16 0.35 0.39 0.32
1.18 1.25 1.43 1.39 2.97U
Significantly different from the other cell lines at p - 0.05 using Goodman’s w21x test.
whereas bi- and multinucleate cells were eliminated through mitotic death. As shown by the mitotic index values, cell lines at late stages of the tumorigenesis process ŽBP1-E, BP1-E1 and BP1-Tras cells. had increased proliferative activity ŽTable 1.. 3.2. Micronuclei, bi- and multinucleation and giant cells The highest frequencies of multinucleate and micronucleate cells, associated with catastrophic mitosis, were found in BP1-Tras cells ŽTable 2.. All cell lines exhibited bi- and single-nucleate giant cells, but there were no significant differences in their frequencies ŽTable 2..
4. Discussion BP is a chemical that elicits apoptosis, by activating ICErCed-3 in the mouse Hepa 1c1c7 hepatoma cell line w12x whereas abnormal expression of Ha-ras induces catastrophic mitotic death in HeLa cells w14x. In the present study, both distinct forms of cell death, apoptosis and mitotic catastrophe, were found in the different cell lines obtained after BP transformation of human breast epithelial MCF-10F cells. The frequency of these phenomena was significantly
higher in BP-transformed cells after transfection with the Ha-ras oncogene. Although an increase in the frequency of apoptosis was expected in the BP-transformed MCF-10F cells, the apoptotic ratios of nontransformed and BP-transformed MCF-10F cells did not differ significantly. It is thus possible that in this sytem BP does not enhance the activation of the apoptosis path described for BP-treated Hepa 1c1c7 hepatoma cells w12x or activation may only occur in the presence of abnormal expression of Ha-ras. Ha-ras oncogene expression alone probably does not activate apoptosis in this case since no increase in apoptosis was seen in MCF-10A cells Žanother human breast epithelial cell line related to MCF-10F w15x. transfected with the Ha-ras oncogene but not treated with BP ŽMCF-10A cells: apoptotic ratio s 1.08, n s 2038 cells; MCF-10neoT cells: apoptotic ratio s 0.44, n s 2248 cells; Mello, M.L.S. — unpublished results.. Mitotic death has been reported in cells exposed to DNA-damaging agents such as ionizing radiation w7x and chemicals w8,9x, or in cells abnormally expressing the c-Ha-ras oncogene w14x. Abnormal expression of c-Ha-ras could disrupt the mitotic machinery and cause genetic instability that could in turn contribute to aberrant mitosis Žmitotic catastrophe., multinucleation and micronucleation w14,22x. BP1-Tras cells showed an increased number of giant multinucleate cells with nuclear fragmentation
Fig. 1. Apoptosis ŽA., aberrant mitosis and mitotic death in BP1-Tras cells with C banding plus fast green staining Ža, b, j–n., CEC test Žc–i. and Ag-NOR staining Žo.. Arrows in panels d–h indicate RNA metachromasy. Panel i is a polarized light view of h, exhibiting DNA birefringence Žarrow.. Bar s 10 mm.
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L.F. Barbisan et al.r Mutation Research 431 (1999) 133–139
and micronucleus formation and of binucleate cells with micronuclei. Mitotic death in these cells was probably a consequence of the altered expression of the transfected oncogene w14x. Indeed, the insertion of the c-Ha-ras oncogene into transformed BP1 cells, giving rise to the cell line BP1-Tras, was sufficient to enhance the neoplastic changes initiated by exposure to BP, including the expression of an aggressive tumorigenic phenotype in mice with severe combined immunodeficiency w11,23x. MCF-10A cells also show a significant increase in multi- and micronucleation when transformed by transfection with the Ha-ras oncogene Žw24x, Mello, M.L.S. — unpublished resultsx. In contrast to apoptosis, cells undergoing mitotic catastrophe showed no chromatin condensation or cytoplasmatic condensation and fragmentation. The weakly stained nuclear fragments indicated that the nuclear remnants were undergoing karyolysis Žnuclear dissolution.. The patterns of DNA fragmentation based on agarose gel electrophoresis were not examined here since both apoptosis and catastrophic death were clearly demonstrated by morphology and cytochemistry in transformed and non-transformed MCF-10F cells. The persistence of nucleoli in some apoptotic nuclei, especially in BP1-Tras cells, has also been reported for other cell systems w25,26x. The high concentration of Ag–NOR positive protein within the nuclear fragments of multinucleated cells may indicate polyploidy, an event demonstrated in BP1T ras cells w23x. This phenomenon may be responsible for the amplification of NOR sites involved in intense rRNA transcription in cells, which could be affected later by death programs. In conclusion, these data are in agreement with the idea that complex and distinct morphophysiological mechanisms are involved in the progressive stages of tumorigenesis in MCF-10F cells. Acknowledgements This research was supported by the Sao ˜ Paulo State Research Foundation ŽFAPESP, grant no. 95r6629-8. and by NCI grant PHS-CA 67238. L.F.B., M.L.S.M. and B.C.V. were recipients of fellowships from the Brazilian National Council for Research and Development ŽCNPq..
References w1x M.C. Raff, Social controls on cell survival and cell death, Nature 356 Ž1992. 397–400. w2x L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 Ž1994. 1821–1828. w3x A.H. Wyllie, J.F.R. Kerr, A.R. Currie, Cell death: the significance of apoptosis, Int. Rev. Cytol. 68 Ž1980. 251–306. w4x A.H. Wyllie, Glucocorticoid induced thymocyte apoptosis is associated with endogenous endonuclease activation, Nature 284 Ž1980. 555–556. w5x M.J. Arends, R.G. Morris, A.H. Wyllie, Apoptosis: the role of the endonuclease, Am. J. Pathol. 136 Ž1990. 593–608. w6x W. Bursh, S. Paff, B. Putz, G. Barthel, R. Schulte-Hermann, Determination of the length of the histological stages of apoptosis in normal liver and altered hepatic foci of rats, Carcinogenesis 11 Ž1990. 847–853. w7x W.P. Chang, J.B. Little, Delayed reproductive death in Xirradiated Chinese hamster ovary cells, Int. J. Radiat. Biol. 60 Ž1991. 483–496. w8x O. Tounekti, G. Pron, J. Belehradek Jr., L.M. Mir, Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized, Cancer Res. 53 Ž1993. 5462–5469. w9x R.B. Lock, O.V. Galperina, R.C. Feldhoff, L.J. Rhodes, Concentration-dependent differences in the mechanisms by which caffeine potentiates etoposide cytotoxicity in HeLa cells, Cancer Res. 54 Ž1994. 4933–4939. w10x G. Calaf, J. Russo, Transformation of human breast epithelial cells by chemical carcinogens, Carcinogenesis 14 Ž1993. 483–492. w11x G. Calaf, P.L. Hang, M.V. Alvarado, S. Estrada, J. Russo, c-Ha-ras enhances the neoplastic transformation of human breast epithelial cells treated with chemical carcinogens, Int. J. Oncol. 6 Ž1995. 5–11. w12x W. Lei, R. Yu, S. Mandlekar, A.N.T. Kong, Induction of apoptosis and activation of interleukin 1 b-converting enzymerCed-3 protease Žcaspase-3. and c-Jun NH 2-terminal kinase 1 by benzoŽ a.pyrene, Cancer Res. 58 Ž1998. 2102– 2106. w13x P.L. Zhang, Y.L. Chai, T.Y. Ho, G. Calaf, J. Russo, Allele loss and point mutation in codons 12 and 61 of the c-Ha-ras oncogene in carcinogen-transformed human breast epithelial cells, Mol. Carcinog. 9 Ž1994. 46–56. w14x E.I. Miranda, C. Santana, E. Rojas, S. Hernandez, P. Os´ trostry-Wegman, A. Garcıa-Carranca, ´ ´ Induced mitotic death of HeLa cells by abnormal expression of c-Ha-ras, Mutat. Res. 349 Ž1996. 173–182. w15x H.D. Soule, T.M. Maloney, S.R. Wolman, W.D. Peterson Jr., R. Brenz, C.M. McGrath, J. Russo, R.J. Pauley, R.F. Jones, S.C. Brooks, Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF10, Cancer Res. 50 Ž1990. 6075–6086. w16x A.T. Sumner, A simple technique for demonstrating heterochromatin, Exp. Cell Res. 75 Ž1972. 304–306. w17x B.C. Vidal, W. Planding, M.L.S. Mello, U. Schenck, Quantitative evaluation of Ag–NOR in liver by high-resolution image cytometry, Anal. Cell Pathol. 7 Ž1994. 27–41.
L.F. Barbisan et al.r Mutation Research 431 (1999) 133–139 w18x B.C. Vidal, L.F. Barbisan, S.S. Maria, J. Russo, M.L.S. Mello, Apoptosis: identification by a critical electrolyte concentration method, Apoptosis 1 Ž1996. 218–221. w19x P.E. Tolbert, C.M. Shy, J.W. Allen, Micronuclei and other nuclear anomalies in buccal smears: methods development, Mutat. Res. 27 Ž1992. 69–77. w20x M. Fenech, The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutat. Res. 285 Ž1993. 25–44. w21x L.A. Goodman, Simultaneous confidence intervals for contrasts among multinomial populations, Ann. Math. Stat. 35 Ž1964. 716–725. w22x N. Denko, J. Stringer, M. Wani, P. Stambrook, Mitotic and post mitotic consequences of genomic instability induced by
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oncogenic Ha-ras, Somatic Cell Mol. Genet. 21 Ž1995. 241– 253. M.L.S. Mello, B.C. Vidal, J. Russo, Ha-ras oncogene effect on chromatin supraorganization of benzowaxpyrene-transformed human breast epithelial cells. Anal. Cell. Pathol., 1999, in press. M.L.S. Mello, T.Y. Lin, J. Russo, Scanning microphotometry image analysis of Ha-ras-transformed human breast epithelial cells, Anal. Cell Pathol. 7 Ž1994. 301–319. E. Falcieri, L. Zamai, S. Santi, C. Cinti, P. Gobbi, D. Bosco, A. Cataldi, C. Betts, M. Vitale, The behaviour of nuclear domains in the course of apoptosis, Histochemistry 102 Ž1994. 221–231. W.C. Earnshaw, Apoptosis: lessons from in vitro systems, Trends Cell Biol. 5 Ž1995. 217–220.
Apoptosis and catastrophic cell death in benzow axpyrene-transformed human breast epithelial cells q Luıs ´ Fernando Barbisan a, Maria Luıza ´ S. Mello Benedicto de Campos Vidal a a b
a,)
, Jose Russo b,
Department of Cell Biology, Institute of Biology, UNICAMP, 13083-970, Campinas, SP, Brazil Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
Received 23 March 1999; received in revised form 16 September 1999; accepted 17 September 1999
Abstract Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzow axpyrene ŽBP. ŽBP1, BP1-E and BP1-E1 cells. and in BP1 cells transfected with the c-Ha-ras oncogene ŽBP1-Tras cells.. Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Benzow axpyrene; c-Ha-ras; Mitotic death; Apoptosis; Human breast epithelial cells
1. Introduction The regulation of cell population growth depends on a balance between cell proliferation and cell death
q
Part of a thesis presented by L.F.B. to the Institute of Biology, UNICAMP, Campinas, in partial fullfilment of the requirements for the Masters’ degree. ) Corresponding author. Fax: q55-19-7887821. E-mail address: [email protected] ŽM.L.S. Mello.
w1x. In general, cells proliferate using the common process of mitosis, which has been extensively studied in normal and cancer cells w2x. In contrast, cell death occurs in several ways, two of which have been described as apoptosis and mitotic death. Apoptosis is a form of cellular suicide characterized morphologically by progressive cell shrinkage with condensation and margination of chromatin that culminates in cellular collapse and fragmentation into small, membrane-encapsulated apoptotic bodies w3x. Biochemically, apoptosis is characterized by the
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 9 3 - 1
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L.F. Barbisan et al.r Mutation Research 431 (1999) 133–139
fragmentation of DNA which results in a ladder-like pattern of 180–200 pb fragment multiples in agarose gel electrophoresis w4,5x. In contrast to the relatively rapid apoptotic process w6x, mitotic death is characterized by a slow process of cell enlargement and G2 arrest, followed by mitotic catastrophe and subsequent nuclear fragmentation. This fragmentation apparently results from aberrant mitosis followed by relocation of the nuclear membrane around chromosome fragments or masses w7–9x. Thus, cells committed to mitotic death complete at least one mitosis before their disintegration w8x. Treatment of the spontaneously immortalized human breast epithelial cell line, MCF-10F, with the chemical carcinogen, benzow axpyrene ŽBP., yielded the cell lines BP1, BP1-E and BP1-E1 which express progressive stages of neoplastic transformation w10x. BP1 cells emerged after 248 days in culture and gave rise to the clone BP1-E, which in turn, after several passages in culture, gave rise to the cell line BP1-E1 w10x. A highly aggressive tumorigenic cell line, BP1-Tras, was generated when transformed nontumorigenic BP1 cells were transfected with the c-Ha-ras oncogene w11x. BP-transformed cell lines show a higher rate of proliferation, increased anchorage-independent growth and in vitro chemotactic and chemoinvasive abilities. However, only BP1-E, BP1E1, and BP1-Tras cell lines express the tumorigenic phenotype when inoculated into mice with severe combined immunodeficiency w10,11x. Since BP is capable of inducing apoptosis via a caspase-dependent pathway w12x, BP-transformed MCF-10F cell lines should exhibit this form of cell death. On the other hand, since BP affects the ras genes of MCF-10F cells and transfection with the Ha-ras oncogene enhances the neoplastic changes initiated by BP treatment w11,13x, catastrophic mitoses may result from the abnormal expression of c-Ha-ras in BP1-Tras cells in a manner similar to that reported for HeLa cells w14x. In the present study, the different types of cell death and their relationship to other cellular changes such as bi- and multinucleation and micronucleus formation during the tumorigenic progression were investigated in human breast epithelial cell lines transformed by BP and additionally transfected with the c-Ha-ras oncogene.
2. Material and methods 2.1. Cell lines and culture conditions Nontransformed ŽMCF-10F. and BP-transformed cell lines ŽBP1, BP1-E and BP1-E1. w10x were used. BP1-Tras cells, obtained by transfection of BP1 cells with the plasmid vector pH06T1 containing the T24 Ha-ras oncogene w11x were also used. The MCF-10F, BP1, BP1-E, BP1-E1 and BP1-Tras cells were used at passages 140, 23, 42, 19 and 61, respectively. The cells were grown in Dulbecco’s minimal essential mediumrF-12 medium Ž1:1. supplemented with 5% equine serum, 0.1 mgrml cholera-toxin, 10 mgrml insulin, 100 unitsrml penicillin, 100 mgrml streptomycin, 2.5 mgrml amphotericin B ŽGibco, Grand Island, NY., 0.5 mgrml hydrocortisone ŽSigma. and 0.02 mgrml epidermal growth factor ŽCollaborative Research, Palo Alto, CA. as described previously w15x. 2.2. Cell preparations and staining Cells grown on coverslips were fixed in 4% buffered formalin for 10 min, rinsed in distilled water and air dried at room temperature or in an absolute ethanol–acetic acid mixture Ž3:1, vrv. for 1 min, rinsed in 70% ethanol for 3–5 min and air dried at room temperature. The formalin-fixed preparations were processed for C banding w16x, stained with 8% Giemsa ŽGurr. solution in Sorensen buffer at pH 6.8 for 30 min, and counterstained with a fast green solution at pH 2.7 for 30 s. The preparations fixed in ethanol–acetic acid were subjected to a previously described Ag–NOR staining w17x. The optimal staining time in this case was 9 min at 378C. After staining, the preparations were rinsed in distilled water, air dried, cleared in xylene and mounted in Entellan ŽMerck.. In some cases ethanol–acetic acid-fixed preparations were processed by a variant of the critical electrolyte concentration assay suggested for apoptosis studies w18x. For this, the cells were stained with a 0.025% toluidine blue ŽMerck. solution in McIlvaine buffer at pH 4.1 for 15 min and next treated with a 0.05 M aqueous MgCl 2 solution for 15 min, rapidly rinsed in distilled water, air-dried, cleared in xylene and mounted in Canada balsam.
L.F. Barbisan et al.r Mutation Research 431 (1999) 133–139
2.3. Analysis of cellular parameters The mitotic index, apoptotic index and frequency of bi-, multi- and micronucleate cells as well as of single- or multinucleate giant cells were determined in nearly 2500 cells for each cell line. Micronuclei were identified and analyzed as described by Tolbert et al. w19x and Fenech w20x. Morphological identification of the two types of cell death was based on the following criteria: shrunken cells containing regions of chromatin condensation with or without the formation of apoptotic bodies indicated apoptotic death whereas cells containing multiple nuclear fragments and micronuclei were indicative of ‘‘mitotic or catastrophic death’’ w3,9,14x. Interphase nuclei that stained deeply green after the critical electrolyte concentration assay w18x and exhibited DNA birefringence under polarized light were considered as apoptotic nuclei. This method was used to determine the apoptotic index. 2.4. Statistical analysis Goodman’s test w21x for contrast among multinomial populations was used. 3. Results 3.1. Mitotic abnormality, cell death and cell proliferation Cells with an apoptotic morphology were observed in nontransformed and all transformed cell lines, but were significantly more frequent in BP1-
135
T ras cells ŽTable 1.. Nuclear chromatin and cytoplasm condensation with cellular collapse and fragmentation were well characterized in these cells ŽFig. 1a–i.. RNA was observed to be confined to even bodies positioned laterally to condensed masses of DNA Žgreen. or appeared as heterogeneous coarse granules surrounding the DNA clumps or localized to one side of these clumps in apoptotic cells of the transformed cell lines subjected to the critical electrolyte concentration assay w18x ŽFig. 1c–i.. Some apoptotic nuclei showed metachromatic nucleolar remnants within a condensed chromatin mass, which exhibited the brilliant green color of DNA staining ŽFig. lg.. In most of the apoptotic nuclei there were no structured nucleolar bodies or granules ŽFig. 1h.. When examined under polarized light these nuclei exhibited typical DNA birefringence ŽFig. 1h–i.. RNA relocation in apoptotic cells did not follow a specific pattern of dispersion or disruption, particularly in the cell line transformed by BP and then transfected with the c-Ha-ras oncogene. Abnormal mitotic figures were seen in all cell lines analyzed. The most common mitotic changes were tripolar metaphases, metaphases and anaphases with lagging chromosome fragments, and anaphase bridges ŽFig. 1j,k.. Larger cells containing pale or deeply stained nuclear fragments and more than one micronucleus, characteristic of mitotic death, were present in all cell lines, but were more frequent in BP1-Tras cells ŽFig. 1l–n.. Ag–NOR stained areas were found in most nuclear fragments ŽFig. 1o.. Regardless of the cell line, mononucleate cells seem to be eliminated preferentially through apoptosis
Table 1 Incidence of mitosis and apoptosis in transformed human breast epithelial cells Different symbols Ža, b. mean differences significant at p - 0.05 using Goodman’s w21x test for contrast among multinomial proportions. Cells
MCF-10F BP1 BP1-E BP1-E1 BP1-Tras
No. of cells
Mitotic
Mitoticphase
Apoptotic
Index
counted
index Ž%.
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Ž%.
no. of cells counted
2277 2570 2537 2520 2519
2.61a 3.21a 4.38 b 4.05 b 3.93 b
5.0 7.5 8.1 8.8 10.1
46.7 51.2 45.9 53.9 46.5
6.7 11.3 4.5 12.7 12.1
13.3 7.5 15.3 9.8 9.1
28.3 22.5 26.1 14.7 22.2
0.56 a 0.42 a 0.20 a 0.28 a 2.21b
2503 2590 2521 2505 2531
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Table 2 Frequency of bi-, multi- and micronucleate cells, and giants in transformed human breast epithelial cells Cells
No. of cells counted
MCF-10F BP1 BP1-E BP1-E1 BP1-Tras )
2577 2570 2537 2520 2519
Cell types Ž%. Binucleate
Multinucleate
Giants
Micronucleate
2.09 1.56 1.30 1.23 2.18
0.23 0.35 0.32 0.28 1.39U
0.12 0.16 0.35 0.39 0.32
1.18 1.25 1.43 1.39 2.97U
Significantly different from the other cell lines at p - 0.05 using Goodman’s w21x test.
whereas bi- and multinucleate cells were eliminated through mitotic death. As shown by the mitotic index values, cell lines at late stages of the tumorigenesis process ŽBP1-E, BP1-E1 and BP1-Tras cells. had increased proliferative activity ŽTable 1.. 3.2. Micronuclei, bi- and multinucleation and giant cells The highest frequencies of multinucleate and micronucleate cells, associated with catastrophic mitosis, were found in BP1-Tras cells ŽTable 2.. All cell lines exhibited bi- and single-nucleate giant cells, but there were no significant differences in their frequencies ŽTable 2..
4. Discussion BP is a chemical that elicits apoptosis, by activating ICErCed-3 in the mouse Hepa 1c1c7 hepatoma cell line w12x whereas abnormal expression of Ha-ras induces catastrophic mitotic death in HeLa cells w14x. In the present study, both distinct forms of cell death, apoptosis and mitotic catastrophe, were found in the different cell lines obtained after BP transformation of human breast epithelial MCF-10F cells. The frequency of these phenomena was significantly
higher in BP-transformed cells after transfection with the Ha-ras oncogene. Although an increase in the frequency of apoptosis was expected in the BP-transformed MCF-10F cells, the apoptotic ratios of nontransformed and BP-transformed MCF-10F cells did not differ significantly. It is thus possible that in this sytem BP does not enhance the activation of the apoptosis path described for BP-treated Hepa 1c1c7 hepatoma cells w12x or activation may only occur in the presence of abnormal expression of Ha-ras. Ha-ras oncogene expression alone probably does not activate apoptosis in this case since no increase in apoptosis was seen in MCF-10A cells Žanother human breast epithelial cell line related to MCF-10F w15x. transfected with the Ha-ras oncogene but not treated with BP ŽMCF-10A cells: apoptotic ratio s 1.08, n s 2038 cells; MCF-10neoT cells: apoptotic ratio s 0.44, n s 2248 cells; Mello, M.L.S. — unpublished results.. Mitotic death has been reported in cells exposed to DNA-damaging agents such as ionizing radiation w7x and chemicals w8,9x, or in cells abnormally expressing the c-Ha-ras oncogene w14x. Abnormal expression of c-Ha-ras could disrupt the mitotic machinery and cause genetic instability that could in turn contribute to aberrant mitosis Žmitotic catastrophe., multinucleation and micronucleation w14,22x. BP1-Tras cells showed an increased number of giant multinucleate cells with nuclear fragmentation
Fig. 1. Apoptosis ŽA., aberrant mitosis and mitotic death in BP1-Tras cells with C banding plus fast green staining Ža, b, j–n., CEC test Žc–i. and Ag-NOR staining Žo.. Arrows in panels d–h indicate RNA metachromasy. Panel i is a polarized light view of h, exhibiting DNA birefringence Žarrow.. Bar s 10 mm.
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and micronucleus formation and of binucleate cells with micronuclei. Mitotic death in these cells was probably a consequence of the altered expression of the transfected oncogene w14x. Indeed, the insertion of the c-Ha-ras oncogene into transformed BP1 cells, giving rise to the cell line BP1-Tras, was sufficient to enhance the neoplastic changes initiated by exposure to BP, including the expression of an aggressive tumorigenic phenotype in mice with severe combined immunodeficiency w11,23x. MCF-10A cells also show a significant increase in multi- and micronucleation when transformed by transfection with the Ha-ras oncogene Žw24x, Mello, M.L.S. — unpublished resultsx. In contrast to apoptosis, cells undergoing mitotic catastrophe showed no chromatin condensation or cytoplasmatic condensation and fragmentation. The weakly stained nuclear fragments indicated that the nuclear remnants were undergoing karyolysis Žnuclear dissolution.. The patterns of DNA fragmentation based on agarose gel electrophoresis were not examined here since both apoptosis and catastrophic death were clearly demonstrated by morphology and cytochemistry in transformed and non-transformed MCF-10F cells. The persistence of nucleoli in some apoptotic nuclei, especially in BP1-Tras cells, has also been reported for other cell systems w25,26x. The high concentration of Ag–NOR positive protein within the nuclear fragments of multinucleated cells may indicate polyploidy, an event demonstrated in BP1T ras cells w23x. This phenomenon may be responsible for the amplification of NOR sites involved in intense rRNA transcription in cells, which could be affected later by death programs. In conclusion, these data are in agreement with the idea that complex and distinct morphophysiological mechanisms are involved in the progressive stages of tumorigenesis in MCF-10F cells. Acknowledgements This research was supported by the Sao ˜ Paulo State Research Foundation ŽFAPESP, grant no. 95r6629-8. and by NCI grant PHS-CA 67238. L.F.B., M.L.S.M. and B.C.V. were recipients of fellowships from the Brazilian National Council for Research and Development ŽCNPq..
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