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Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei
Mutation Research, 243 (1990) 127-131
127
Elsevier MUTLET 0289
Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei A. De Sario, P. Vagnarelli and L. De Carli Dipartimento di Genetica e Microbiologia 'A. Buzzati-Traverso', 27100 Pavia ( Baly )
(Accepted22 August 1989)
Keywords." Aneuploidy;Diethylstilboestrol;BiotinylatedDNA probes; Hybridization,in situ; Interphase nuclei
Summary Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.
Diethylstilbestrol (DES) is a synthetic oestrogen known as a carcinogenic agent in humans and rodents (McLachlan and Dixon, 1976; IARC, 19791). Although it has been extensively studied for this property, its mechanism of action is poorly understood. DES is able to induce cancer and to transform Syrian hamster embryo cells in vitro (Barrett et al., 1981), but it is unable to induce both point mutations and chromosome aberrations in mammalian cultured cells. There is evidence of aneuploidy induction by DES in mammalian cell lines (Sawada and Ishidate, 1978) and in yeast, probably attributable to its effects on microtubule Correspondence: Dr. A. De Sario, Dipartimento di Genetica e Microbiologia 'A. Buzzati-Traverso',27100 Pavia (Italy).
assembly. Tsutsui and co-workers reported that DES causes a dose-dependent increase in the incidence of both aneuploidy and in vitro transformation in Syrian hamster embryo cells (Tsutsui et al., 1983). Aneuploidy has been observed also in vivo in displastic but not in metaplastic lesions in the offspring of women treated with DES during their pregnancy (Fu et al., 1979). The overall data suggest that the induction of chromosome number variation can be considered one of the possible mechanisms of carcinogenesis of the compound. This justifies the interest in evaluating the effect of DES on non-disjunction in human cells in culture. We have developed an assay system on lymphocyte cultures to measure chemically induced aneuploidy. The assay is based on in situ hybridiza-
0165-7992/90/$ 03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
128 tion on interphase nuclei with chromosomespecific DNA probes homologous to repetitive sequences located in the centromeric region of the chromosome. Using radioactive probes it is possible to detect the number of copies of a target chromosome by autoradiography on interphase nuclei. Three chemicals (griseofulvin, benomyl and chloral hydrate), known as aneuploidy inducers, have already been tested in this assay using a probe specific either for chromosome Y (Y97) or chromosome 9 (QP23) (Raimondi et al., 1989). All the compounds induced a significant increase in the frequency of hyperdiploid cells. In the present work we have tested DES to evaluate its ability to induce chromosome number variation in human cells in culture and to validate further the method described above. Moreover, we were interested in increasing the sensitivity of the assay and in making the procedure more simple and efficient. To these purposes the techniques based on in situ hybridization of radioactive and biotinylated probes have been compared in order to find the best experimental conditions for the performance of the test. Materials and methods
(a) Cell cultures and drug treatment PHA-stimulated peripheral blood lymphocytes from an adult male were cultured for 48 h in RPMI medium supplemented with 1007o FCS (Flow) and then treated for 24 h with diethylstilbestrol (Sigma) at concentrations of 15, 10 and 3.16/zg/ml. Cells were grown for an additional 48 h in minimal medium and exposed to colcemid (Gibco) (0.03 /~g/ml) during the last 2 h. Cytological preparations were made according to the standard procedure including hypotonic swelling with 0.075 M KC1 and fixation with methanol:acetic acid (3:1).
(b) Probe labelling Y97, a human derived-sequence, cloned in a cosmid, identifies a 5.5-Kb, 100-fold repeated DNA fragment in the pericentromeric region of the Y chromosome (Wolfe et al., 1985). For aH-labelling Y97 was nick-translated (nick-
translation kit No. 5500 Amersham) with 3 nucleotides: [3H]dTTP (93 Ci/mM), [3H]dCTP (50 Ci/mM), [3H]dATP (98 Ci/mM) (Amersham). The DNA was purified from unincorporated nucleotides on a Sephadex G50 column. The probe was then precipitated by 2 vol. of 10007o ethanol and resuspended in the hybridization buffer at 500 ng/ml (Mattei et al., 1985). For nick-translation of Y97 with biotin-lld U T P (BRL) a nick-translation reagent system (BRL No. 8160) was used according to the protocol provided by the supplier. A probe concentration of 3000 ng/ml was used in the final experiment. DNA denaturation was performed at 70°C (10 min).
(c) In situ hybridization In situ hybridization was performed according to the modified method of Mattei et al. (1985). After treatment with RNAase (Sigma) at 37°C for 1 h and denaturation in 70070 formamide/2 × SSC at 70°C (2 min), slides were hybridized with 30 ttl of the denaturated probe and covered with a coverslip. Following an overnight incubation at 42°C, washings were carried out in 5007o formamide/2 × SSC (higher stringency condition) or 4007o formamide/2 × SSC (lower stringency condition) at 39°C (10 min), in 2 × SSC (10 min), in 0.1 × SSC (1 h).
(d) Autoradiography preparations
and
immunofluorescent
Kodak NTB-2 emulsion (1:1 diluted in 207o glycerol) was used for the detection of the 3Hlabelled probe. Following 6 days exposure at 4°C, the slides were developed using Kodak D-19 and fixed in Kodak Unifix. For the detection of the biotinylated probe a double antibody fluorescent system DETEK I-f, supplied by ENZO, was used. Slides were rinsed for 5 min in 1 × PBS, 1 × PBS + 0.1°70 Triton X-100, 1 × PBS. The first antibody, IgG fraction rabbit antibiotin, diluted 1 : 100 in PBS, containing 0.2070 BSA was added to the slides. Incubation was carried out for 1 h at 37°C in a moist chamber. After 3 washings in 1 × PBS 5 min each, the sec-
129
ond antibody FITC-conjugated IgG fraction goat anti-rabbit diluted 1 : 100 was added and the slides were incubated as before. They were then rinsed again 3 times in PBS (5 min) and counterstained with ethidium bromide 1 ttg/ml (5 min). Ektachrome ASA 400 colour slides were used for the photographs.
(e) Scoring criteria and statistical analysis The scoring was done by counting either the autoradiographic grain clusters or the fluorescent spots on samples of 1000-2000 nuclei per dose. Nuclear patterns corresponding to a hyperdiploid constitution were considered indicative of aneuploidy induction. Tetraploid cells were not included in the scoring on the basis of the results of a preliminary analysis of the size distribution of the nuclei. Differences between treated and control samples were compared by means of a G test (Sokal, 1981). Results Preliminary experiments were done to determine
the best stringency conditions and the most effective probe concentration in the assay performed using the non-radioactive technique. At low stringency (40°70 formamide) the probe hybridized aspecifically to the centromeric region of all chromosomes on the metaphases and produced multiple signals on interphase nuclei; at higher stringency (50°70 formamide) only the Y chromosome was labelled (Fig. 1). Probe concentrations of 1000, 1500, 2000, 3000 and 4000 #g/ml were tested. An increase in the intensity of the signal was observed up to 3000 #g/ml which was chosen as the most suitable dose. In situ hybridization with the ¥97 probe labelled with either radioactive or biotinylated nucleotides was carried out on preparations of lymphocytes treated with DES at 3 different doses. Samples of 1000-2000 nuclei were scored for the presence of a double Y (Fig. 2). The results are shown in Tables 1 and 2. With the radioactive probe a statistically significant increase of hyperdiploid nuclei was observed only at 15 #g/ml, whereas with the biotinylated probe the concentration of 10 #g/ml was also active. In this latter case a dose-related effect was evident. Furthermore at the maximum tested dose (15 #g/ml), the increase in the frequency of hyperdiploid nuclei, with respect to controls, was found to be higher for the immunofluorescent technique. Discussion In a previous paper (Raimondi et al., 1989) we have proposed a new quantitative method to measure aneuploidy induction in cultured cells. The TABLE 1 P E R C E N T A G E OF H Y P E R D I P L O I D N U C L E I IN SITU H Y B R I D I Z E D W I T H 3H-LABELLED P R O B E
Fig. 1. Metaphase spread from PHA-stimulated h u m a n lymphocytes after in situ hybridization with the biotinylated probe Y97. The visualization of the hybridization site is obtained by using a rabbit anti-biotin antibody and a FITC-goat anti rabbit IgG fraction.
Doses (/zg/ml)
N u m b e r of scored nuclei
°70 of hyperdiploid nuclei
0 3.16 10 15
2244 2269 2228 2059
0.18 0.13 0.13 0.92 a
a Significantly higher than the control value (G test): p < 0 . 0 0 1 .
130
,a,
-I
Fig. 2. Interphase nuclei of PHA-stimulated human lymphocytes in situ hybridized with 3H-labelled (A) and biotinylated (B) Y97 probe: left, a double Y; right, a single Y. m e t h o d has been applied to c o m p o u n d s differing in their mechanism o f action. One limitation o f this assay is that only hyperdiploid nuclei can be included in the counts, owing to the excess o f missing signals originated by technical artifacts inherent in the hybridization procedure (Raimondi et al., 1987). However, such a limitation, which also exists in the metaphase analysis, is amply c o m p e n sated for by the advantage o f extending the scoring to non-dividing cells and above all o f examining large cell samples. T h e positive response obtained with DES in the present work, beside providing a further contribution to the validation o f the m e t h o d , reinforces the evidence o f the aneugenic activity o f the compound.
As far as the sensitivity o f the assay is concerned a definite i m p r o v e m e n t can be obtained by using biotinylated probes in place o f radioactive probes.
TABLE 2 PERCENTAGE OF HYPERDIPLOID NUCLEI 1N SITU HYBRIDIZED WITH BIOTINYLATED PROBE Doses (#g/ml)
Number of scored nuclei
% of hyperdiploid nuclei
0 3.16 10 15
1068 1101 2032 2015
0.28 0.09 1.28a* 1.98a**
a
Significantly higher than the control value (G test): p<0.005(*); p<0.001 (**).
131
Indeed, the minimum effective dose of DES as determined in our assay was lower when the immunofluorescence system of detection was used. This result can be ascribed to the high specificity of the signal and to the low background level achieved with this procedure in in situ hybridization experiments.
Acknowledgements We are indebted to Dr. Goodfellow (ICRF London) for generously supplying the probe Y97. This work was supported by grants from MPI (40%) and CNR 86.00325.
References Barrett, J.C., A. Wong and J.A. McLachlan (1981) Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci, Science, 212, 1402-1404. Fu, J.S., J.W. Reagan, R.M. Richart and D.E. Townsend (1979) Nuclear DNA and histologic studies of genital lesions in diethylstilbestrol-exposed progeny, I. Intraepithelial squamous abnormalities, Am. J. Clin. Pathol., 72, 503-514. IARC (1979) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 21, Sex Hormones, 11, pp. 173-231.
Mattei, M.G., P.N. Passage, J.P. Moisan, J.L. Mandel and J.F. Mattei (1985) DNA probe, localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome, Hum. Genet., 69, 268-271. McLachlan, J.A., and R.L. Dixon (1976) in: M.A. Mehlman, R.E. Shapiro and H. Blumental (Eds.), Advances in Modern Toxicology, Hemisphere, Washington, DC, p. 423. Raimondi, E., S. Scariolo, P. Vagnarelli, A. De Sario and L. De Carli (1987) Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing, Cytotechnology, 1, 13-17. Raimondi, E., S. Scariolo, A. De Sario and L. De Carli (1989) Aneuploidy assays on interphase nuclei by means of in situ hybridization with DNA probes, Mutagenesis, 4, 165-169. Sawada, M., and M. Ishidate Jr. (1978) Colchicine-like effect of diethylstilbestrol on mammalian cells in vitro, Mutation Res., 57, 175-182. Sokal, R.R., and F.J. Rohlf (1981) Biometry, Freeman, New York, pp. 286-303. Tsutsui, T., H. Maizumi, J.A. McLachlan and J.C. Barrett (1983) Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism of carcinogenesis, Cancer Res., 83, 3814-3821. Wolfe, J., S.M. Darling, R.P. Erickson, I.W. Craig, V.J. Buckle, P.W.J. Rigby, H.F. Willard and P.N. Goodfellow (1985) Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome, J. Mol. Biol., 182, 477-485. Communicated by A. Abbondandolo
127
Elsevier MUTLET 0289
Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei A. De Sario, P. Vagnarelli and L. De Carli Dipartimento di Genetica e Microbiologia 'A. Buzzati-Traverso', 27100 Pavia ( Baly )
(Accepted22 August 1989)
Keywords." Aneuploidy;Diethylstilboestrol;BiotinylatedDNA probes; Hybridization,in situ; Interphase nuclei
Summary Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.
Diethylstilbestrol (DES) is a synthetic oestrogen known as a carcinogenic agent in humans and rodents (McLachlan and Dixon, 1976; IARC, 19791). Although it has been extensively studied for this property, its mechanism of action is poorly understood. DES is able to induce cancer and to transform Syrian hamster embryo cells in vitro (Barrett et al., 1981), but it is unable to induce both point mutations and chromosome aberrations in mammalian cultured cells. There is evidence of aneuploidy induction by DES in mammalian cell lines (Sawada and Ishidate, 1978) and in yeast, probably attributable to its effects on microtubule Correspondence: Dr. A. De Sario, Dipartimento di Genetica e Microbiologia 'A. Buzzati-Traverso',27100 Pavia (Italy).
assembly. Tsutsui and co-workers reported that DES causes a dose-dependent increase in the incidence of both aneuploidy and in vitro transformation in Syrian hamster embryo cells (Tsutsui et al., 1983). Aneuploidy has been observed also in vivo in displastic but not in metaplastic lesions in the offspring of women treated with DES during their pregnancy (Fu et al., 1979). The overall data suggest that the induction of chromosome number variation can be considered one of the possible mechanisms of carcinogenesis of the compound. This justifies the interest in evaluating the effect of DES on non-disjunction in human cells in culture. We have developed an assay system on lymphocyte cultures to measure chemically induced aneuploidy. The assay is based on in situ hybridiza-
0165-7992/90/$ 03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
128 tion on interphase nuclei with chromosomespecific DNA probes homologous to repetitive sequences located in the centromeric region of the chromosome. Using radioactive probes it is possible to detect the number of copies of a target chromosome by autoradiography on interphase nuclei. Three chemicals (griseofulvin, benomyl and chloral hydrate), known as aneuploidy inducers, have already been tested in this assay using a probe specific either for chromosome Y (Y97) or chromosome 9 (QP23) (Raimondi et al., 1989). All the compounds induced a significant increase in the frequency of hyperdiploid cells. In the present work we have tested DES to evaluate its ability to induce chromosome number variation in human cells in culture and to validate further the method described above. Moreover, we were interested in increasing the sensitivity of the assay and in making the procedure more simple and efficient. To these purposes the techniques based on in situ hybridization of radioactive and biotinylated probes have been compared in order to find the best experimental conditions for the performance of the test. Materials and methods
(a) Cell cultures and drug treatment PHA-stimulated peripheral blood lymphocytes from an adult male were cultured for 48 h in RPMI medium supplemented with 1007o FCS (Flow) and then treated for 24 h with diethylstilbestrol (Sigma) at concentrations of 15, 10 and 3.16/zg/ml. Cells were grown for an additional 48 h in minimal medium and exposed to colcemid (Gibco) (0.03 /~g/ml) during the last 2 h. Cytological preparations were made according to the standard procedure including hypotonic swelling with 0.075 M KC1 and fixation with methanol:acetic acid (3:1).
(b) Probe labelling Y97, a human derived-sequence, cloned in a cosmid, identifies a 5.5-Kb, 100-fold repeated DNA fragment in the pericentromeric region of the Y chromosome (Wolfe et al., 1985). For aH-labelling Y97 was nick-translated (nick-
translation kit No. 5500 Amersham) with 3 nucleotides: [3H]dTTP (93 Ci/mM), [3H]dCTP (50 Ci/mM), [3H]dATP (98 Ci/mM) (Amersham). The DNA was purified from unincorporated nucleotides on a Sephadex G50 column. The probe was then precipitated by 2 vol. of 10007o ethanol and resuspended in the hybridization buffer at 500 ng/ml (Mattei et al., 1985). For nick-translation of Y97 with biotin-lld U T P (BRL) a nick-translation reagent system (BRL No. 8160) was used according to the protocol provided by the supplier. A probe concentration of 3000 ng/ml was used in the final experiment. DNA denaturation was performed at 70°C (10 min).
(c) In situ hybridization In situ hybridization was performed according to the modified method of Mattei et al. (1985). After treatment with RNAase (Sigma) at 37°C for 1 h and denaturation in 70070 formamide/2 × SSC at 70°C (2 min), slides were hybridized with 30 ttl of the denaturated probe and covered with a coverslip. Following an overnight incubation at 42°C, washings were carried out in 5007o formamide/2 × SSC (higher stringency condition) or 4007o formamide/2 × SSC (lower stringency condition) at 39°C (10 min), in 2 × SSC (10 min), in 0.1 × SSC (1 h).
(d) Autoradiography preparations
and
immunofluorescent
Kodak NTB-2 emulsion (1:1 diluted in 207o glycerol) was used for the detection of the 3Hlabelled probe. Following 6 days exposure at 4°C, the slides were developed using Kodak D-19 and fixed in Kodak Unifix. For the detection of the biotinylated probe a double antibody fluorescent system DETEK I-f, supplied by ENZO, was used. Slides were rinsed for 5 min in 1 × PBS, 1 × PBS + 0.1°70 Triton X-100, 1 × PBS. The first antibody, IgG fraction rabbit antibiotin, diluted 1 : 100 in PBS, containing 0.2070 BSA was added to the slides. Incubation was carried out for 1 h at 37°C in a moist chamber. After 3 washings in 1 × PBS 5 min each, the sec-
129
ond antibody FITC-conjugated IgG fraction goat anti-rabbit diluted 1 : 100 was added and the slides were incubated as before. They were then rinsed again 3 times in PBS (5 min) and counterstained with ethidium bromide 1 ttg/ml (5 min). Ektachrome ASA 400 colour slides were used for the photographs.
(e) Scoring criteria and statistical analysis The scoring was done by counting either the autoradiographic grain clusters or the fluorescent spots on samples of 1000-2000 nuclei per dose. Nuclear patterns corresponding to a hyperdiploid constitution were considered indicative of aneuploidy induction. Tetraploid cells were not included in the scoring on the basis of the results of a preliminary analysis of the size distribution of the nuclei. Differences between treated and control samples were compared by means of a G test (Sokal, 1981). Results Preliminary experiments were done to determine
the best stringency conditions and the most effective probe concentration in the assay performed using the non-radioactive technique. At low stringency (40°70 formamide) the probe hybridized aspecifically to the centromeric region of all chromosomes on the metaphases and produced multiple signals on interphase nuclei; at higher stringency (50°70 formamide) only the Y chromosome was labelled (Fig. 1). Probe concentrations of 1000, 1500, 2000, 3000 and 4000 #g/ml were tested. An increase in the intensity of the signal was observed up to 3000 #g/ml which was chosen as the most suitable dose. In situ hybridization with the ¥97 probe labelled with either radioactive or biotinylated nucleotides was carried out on preparations of lymphocytes treated with DES at 3 different doses. Samples of 1000-2000 nuclei were scored for the presence of a double Y (Fig. 2). The results are shown in Tables 1 and 2. With the radioactive probe a statistically significant increase of hyperdiploid nuclei was observed only at 15 #g/ml, whereas with the biotinylated probe the concentration of 10 #g/ml was also active. In this latter case a dose-related effect was evident. Furthermore at the maximum tested dose (15 #g/ml), the increase in the frequency of hyperdiploid nuclei, with respect to controls, was found to be higher for the immunofluorescent technique. Discussion In a previous paper (Raimondi et al., 1989) we have proposed a new quantitative method to measure aneuploidy induction in cultured cells. The TABLE 1 P E R C E N T A G E OF H Y P E R D I P L O I D N U C L E I IN SITU H Y B R I D I Z E D W I T H 3H-LABELLED P R O B E
Fig. 1. Metaphase spread from PHA-stimulated h u m a n lymphocytes after in situ hybridization with the biotinylated probe Y97. The visualization of the hybridization site is obtained by using a rabbit anti-biotin antibody and a FITC-goat anti rabbit IgG fraction.
Doses (/zg/ml)
N u m b e r of scored nuclei
°70 of hyperdiploid nuclei
0 3.16 10 15
2244 2269 2228 2059
0.18 0.13 0.13 0.92 a
a Significantly higher than the control value (G test): p < 0 . 0 0 1 .
130
,a,
-I
Fig. 2. Interphase nuclei of PHA-stimulated human lymphocytes in situ hybridized with 3H-labelled (A) and biotinylated (B) Y97 probe: left, a double Y; right, a single Y. m e t h o d has been applied to c o m p o u n d s differing in their mechanism o f action. One limitation o f this assay is that only hyperdiploid nuclei can be included in the counts, owing to the excess o f missing signals originated by technical artifacts inherent in the hybridization procedure (Raimondi et al., 1987). However, such a limitation, which also exists in the metaphase analysis, is amply c o m p e n sated for by the advantage o f extending the scoring to non-dividing cells and above all o f examining large cell samples. T h e positive response obtained with DES in the present work, beside providing a further contribution to the validation o f the m e t h o d , reinforces the evidence o f the aneugenic activity o f the compound.
As far as the sensitivity o f the assay is concerned a definite i m p r o v e m e n t can be obtained by using biotinylated probes in place o f radioactive probes.
TABLE 2 PERCENTAGE OF HYPERDIPLOID NUCLEI 1N SITU HYBRIDIZED WITH BIOTINYLATED PROBE Doses (#g/ml)
Number of scored nuclei
% of hyperdiploid nuclei
0 3.16 10 15
1068 1101 2032 2015
0.28 0.09 1.28a* 1.98a**
a
Significantly higher than the control value (G test): p<0.005(*); p<0.001 (**).
131
Indeed, the minimum effective dose of DES as determined in our assay was lower when the immunofluorescence system of detection was used. This result can be ascribed to the high specificity of the signal and to the low background level achieved with this procedure in in situ hybridization experiments.
Acknowledgements We are indebted to Dr. Goodfellow (ICRF London) for generously supplying the probe Y97. This work was supported by grants from MPI (40%) and CNR 86.00325.
References Barrett, J.C., A. Wong and J.A. McLachlan (1981) Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci, Science, 212, 1402-1404. Fu, J.S., J.W. Reagan, R.M. Richart and D.E. Townsend (1979) Nuclear DNA and histologic studies of genital lesions in diethylstilbestrol-exposed progeny, I. Intraepithelial squamous abnormalities, Am. J. Clin. Pathol., 72, 503-514. IARC (1979) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 21, Sex Hormones, 11, pp. 173-231.
Mattei, M.G., P.N. Passage, J.P. Moisan, J.L. Mandel and J.F. Mattei (1985) DNA probe, localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome, Hum. Genet., 69, 268-271. McLachlan, J.A., and R.L. Dixon (1976) in: M.A. Mehlman, R.E. Shapiro and H. Blumental (Eds.), Advances in Modern Toxicology, Hemisphere, Washington, DC, p. 423. Raimondi, E., S. Scariolo, P. Vagnarelli, A. De Sario and L. De Carli (1987) Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing, Cytotechnology, 1, 13-17. Raimondi, E., S. Scariolo, A. De Sario and L. De Carli (1989) Aneuploidy assays on interphase nuclei by means of in situ hybridization with DNA probes, Mutagenesis, 4, 165-169. Sawada, M., and M. Ishidate Jr. (1978) Colchicine-like effect of diethylstilbestrol on mammalian cells in vitro, Mutation Res., 57, 175-182. Sokal, R.R., and F.J. Rohlf (1981) Biometry, Freeman, New York, pp. 286-303. Tsutsui, T., H. Maizumi, J.A. McLachlan and J.C. Barrett (1983) Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism of carcinogenesis, Cancer Res., 83, 3814-3821. Wolfe, J., S.M. Darling, R.P. Erickson, I.W. Craig, V.J. Buckle, P.W.J. Rigby, H.F. Willard and P.N. Goodfellow (1985) Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome, J. Mol. Biol., 182, 477-485. Communicated by A. Abbondandolo