Estudio de mosaicismo y seudomosaicismo en diagnóstico prenatal citogenético. Experiencias en Ciudad de La Habana / Mosaicism and pseudomosaicism in prenatal diagnosis. Experience in Havana City

Introducción. Se hace un estudio de la incidencia y repercusión fenotípica del mosaicismo y seudomosaicismo cromosómico en diagnóstico prenatal en 18 años de labor en el laboratorio de Ciudad de La Habana.Métodos. Se incluyeron 10.676 amniocentesis cultivadas principalmente en frascos. El seguimiento de los casos se hace a través de especialistas y enfermeras en genética.Resultados y conclusiones. Se halló un 0,33 por ciento de mosaicismo en el muestreo, 4,50 por ciento de seudomosaicismo tipo I y 0,40 por ciento seudomosaicismo tipo II. Se detectaron 36 casos de mosaicos cromosómicos. El 70 por ciento del mosaicismo correspondió a aberraciones numéricas con paridad de aparición para los autosomas y los cromosomas sexuales; los mosaicos de aberraciones estructurales se hallaron en un 13,8 por ciento y los de marcadores cromosómicos supernumerarios en un 16,2 por ciento. El 42,2 por ciento de los fetos abortados o recién nacidos vivos con mosaicos cromosómicos fueron fenotípicamente normales a grosso modo. Se alcanzó un 63 por ciento de seguimiento citogenético en los casos de mosaicos cromosómicos. Las aberraciones cromosómicas mayormente involucradas en mosaicismo fueron la trisomía 21, la monosomía de la X y el cromosoma marcador supernumerario. Se hallaron 459 aberraciones cromosómicas asociadas al seudomosaicismo tipo I (327 aberraciones estructurales y 132 aberraciones numéricas). Fueron encontradas 48 aberraciones cromosómicas en el seudomosacismo tipo II, de las cuales 25 eran estructurales, 21 numéricas y 2 casos 46,XX/46,XY. Los cromosomas más frecuentemente hallados en seudomosaico fueron el 2, el 13, el 17, el 18, el 20 y el 21. La mayoría de los casos de seudomosaico el recién nacido fue normal. (AU)

Actividad genotóxica de la terapia con I131 en pacientes tratados por carcinomas de tiroides / Genotoxic activity of the I 131 therapy in patients treated for thyroid carcinoma

- Introducción: El test de micronúcleos (MN) sobre linfocitos humanos irradiados con bloqueo citocinético (CB) se utiliza para valorar el daño cromosómico y genotóxico de diferentes agentes físicos y químicos.- Objetivo: Determinar un posible efecto genotóxico inducido por la terapia con I131 en pacientes con cáncer de tiroides y determinar la dosis equivalente corporal total (DECT) de radiación ionizante que supone dicho tratamiento.- Material y métodos: Se ha determinado la frecuencia de aparición de MN en cultivos de linfocitos CB en tres grupos de individuos diferentes: 1) en 35 voluntarios sanos para establecer la frecuencia espontánea de MN; 2) en 9 voluntarios supuestamente sanos para realizar las curvas dosisrespuesta "in vitro" con radiación gamma; y 3) en 25 pacientes que han recibido una dosis ablativa de I131 en el tratamiento de un carcinoma de tiroides. Se ha determinado el número de MN/500 células CB previo al tratamiento y tres días después de la administración de I131. La DECT de la terapia se ha calculado por el número de MN en linfocitos obtenido a los tres días de la administración de I131 comparada con la frecuencia de MN expuestas "in vitro" a radiación gamma que produciría una idéntica frecuencia de MN.- Resultados: Se ha obtenido una relación lineal entre la frecuencia de MN y la dosis de radiación ionizante administradas "in vitro" con radiación gamma. La frecuencia de MN tras el tratamiento con I131(8´89 MN/500CB) es significativamente mayor (p<0.01), duplicando la frecuencia espontánea (4´08/500MN) basal.- Conclusión: La terapia con I131 induce un incremento significativo del daño cromosómico en los pacientes irradiados por carcinoma de tiroides, equivalente a una dosis corporal total de 13 cGy durante los tres primeros días desde la administración terapéutica de I131 (AU)

Responsabilidad por lesiones prenatales. Fundamento, Wrongful Life y tendencias. (Especial atención al derecho suizo) / Responsibility for prenatal injuries. Bases, wrongful life and trends

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Relations of the c-myc gene and chromosome 8 in non-small cell lung cancer: analysis by fluorescence in situ hybridization.

BACKGROUND: Amplification of the c-myc gene has been reported in non-small cell lung cancer (NSCLC). We investigated the c-myc gene amplification and the numerical aberration of chromosome 8 by dual color fluorescence in situ hybridization (FISH) to evaluate the relation between possible genetic abnormalities, pathological factors and prognosis. METHODS: Tumor tissue samples were obtained from 31 patients with NSCLC who underwent lobectomy with mediastinal lymph node dissection. Samples were analyzed by FISH using 8 alpha satellite DNA probe and c-myc gene cosmid probe. The relation between genetic abnormalities, pathological factors (T factor, tumor size, and N factor), and prognostic factors was evaluated by univariate and multivariate analysis, and by the Kaplan-Meier method and log-rank analysis. RESULTS: Chromosome 8 aberrations were T1 (n=3), 44.0%; T2 (n=18), 35.7%; T3 (n=7), 40.0%; T4 (n=3), 39.7% (p=NS). The c-myc gene amplifications were T1, 54.3%; T2, 51.1%; T3, 51.0%; T4, 66.3% (p=NS). There was no difference between patients whose tumor was more than 5 cm (n=16), and 5 cm or less (n=15) in the rate of chromosome 8 aberration (39.3%: 36.3%), or the rate of the c-myc gene amplification (52.1%: 53.7%). N factors for chromosome 8 aberrations were N0 (n=18), 35.9%; and N2 (n=11), 44.9% (p=NS). In the c-myc gene amplification, there was a significant difference between N0 and N2 (48.6%, 61.3%, p=0.040). In univariate and multivariate analysis, chromosome 8 aberrations correlated with a poor prognosis (p=0.037 and p=0.041). The 5-year survival rate was 15.4% in patients whose rate of chromosome 8 aberrations was 40% or more (n=13), which was significantly less than that in patients with an aberration rate of less than 40% (n=19, 57.9%, p=0.014). CONCLUSION: The c-myc gene amplification correlates with lymph node metastasis. Although there was no significant link between the amplification of the c-myc gene and clinical outcome, the numerical chromosome 8 aberrations was considered to be a factor for survival.

Is G1 arrest in plant seeds induced by a p53-related pathway?

In mammals, p53 is crucial for inducing the genes that lead to G1 arrest following DNA damage, enabling DNA repair. However, the possibility that such a system exists in plants has attracted little attention. Even though some plant cDNA sequences with partial homology to p53 have been reported recently, there has been little analysis of how these molecules might relate to DNA damage. The lack of investigation into whether a DNA-damage-induced, p53-mediated G1-arrest pathway might exist in plants is remarkable given that plant DNA, like that of all organisms, is continually under the threat of attack.

Replication and G2 checkpoints: their response to caffeine.

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Biparametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G2 checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G2 checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G2 block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine.

Low doses of UVB or UVA induce chromosomal aberrations in cultured human skin cells.

Chromosomal defects are frequently present in malignant and premalignant skin disorders; however, it is not known whether ultraviolet radiation from sunlight plays a role in their induction. To obtain information on the ability of ultraviolet A and ultraviolet B to induce chromosomal aberrations, cultured melanocytes and fibroblasts were exposed to physiologic doses of ultraviolet A or ultraviolet B and, for comparison, to gamma rays. As a measure of chromosomal aberrations, the formation of micronuclei was determined. To obtain sufficient statistical data on induced micronuclei and cell kinetics, a flow cytometry method has been modified and applied. The flow cytometry method analysis is based on staining the DNA with ethidium bromide and the cell membranes with 1,6-diphenyl-1,3,5,-hexatriene. We observed dose-dependent micronuclei formation after gamma or ultraviolet B irradiation in both cell types and also for ultraviolet A in fibroblasts. The yield of micronuclei induced in fibroblasts by ultraviolet A was only a factor 15 smaller than that induced by ultraviolet B (313 nm). The results indicate that 10 kJ per m2 (equivalent to 1 minimal erythema dose) of ultraviolet B and 150 kJ per m2 of ultraviolet A (0.2 minimal erythema dose) can induce 1% of micronuclei in fibroblasts, equivalent to the induction due to 0.6 Gy of gamma radiation. In conclusion, physiologic doses of sunlight can induce chromosomal aberrations at a level comparable with that observed after exposure to approximately 1 Gy of ionizing radiation. Therefore, sunlight can be considered a potential inducer of chromosomal aberrations in skin cells, which may contribute to skin carcinogenesis.

Biopsia corial transcervical versus amniocentesis: evaluación de la pérdida fetal en un estudio randomizado / Transcervical chorial biopsy versus amniocentesis: evaluación of fetal loss in a randomized study

Objetivo: Evaluar el riesgo de pérdida fetal después de la biopsia corial transcervical. Sujetos y métodos: Se randomizó una serie consecutiva de 1.011 gestantes que consultaron antes de las 12 semanas para estudio citogenético en base a su edad materna avanzada, en dos grupos: amniocentesis convencional de segundo trimestre y biopsia corial (BC) de primer trimestre mediante pinza transcervical.Resultados: En las 672 gestantes que completaron el estudio (tratadas según la asignación y con seguimiento completo) las tasas de pérdida fetal espontánea post-procedimiento hasta la primera semana post-parto en ambos grupos no difirieron significativamente, con unos valores de 2,7 por 100 (10/365) para la amniocentesis y 2,2 por 100 (8/357) para la BC.Conclusión: La biopsia corial mediante pinza transcervical presenta un riesgo de pérdida fetal post-procedimiento no superior a la obtenida en el grupo randomizado de amniocentesis en el segundo trimestre (AU)

A follow-up study on chromosomal aberrations in lymphocytes of alcoholics during early, medium, and long-term abstinence.

The frequencies of structural chromosomal aberrations were analyzed in peripheral blood lymphocytes of 31 chronic alcoholics at the beginning of an intensive outpatient treatment program at a neuropsychiatric clinic and were compared with 31 controls matched for gender, age, smoking habits, and nondrinkers. A statistically significant difference was observed in the level of chromosomal aberrations in somatic cells from alcoholics when compared with controls (3.01% vs. 1.28%, p < or = 0.001). A follow-up study was carried out for a subset of the patients after 3 months (8 subjects) and 12 months (14 subjects) of controlled abstinence. A statistically significant increase in the mean frequency of cells with aberrations was observed in the group of 14 subjects reinvestigated after 12 months of abstinence when compared with the mean value of the first blood samples immediately after hospitalization (4.61% vs. 3.01%; p < or = 0.001). An excessive increase in cigarette consumption during alcohol abstinence, reflected by a dramatic elevation of CO-hemoglobin levels, may, at least in part, account for this finding. In conclusion, chronic alcoholism leads to genotoxic effects that, instead of recovering after 1 year of alcohol abstinence, are even enhanced, most likely due to the "shift in addictive behavior."

Chromosomal aberrations in human lymphocytes exposed in vitro to enrofloxacin and ciprofloxacin.

Chromosomal aberrations were evaluated in cultures of human peripheral lymphocytes from eight healthy donors, exposed to the antimicrobial enrofloxacin (EFX) or to its major metabolite ciprofloxacin (CFX). In both treatments cultures revealed an increase in the chromosomal aberration level, detected as chromatid and chromosome breaks and gaps. Control cultures analysis revealed 3.6 +/- 0.6 chromosomal aberrations per 100 cells while treated cultures exhibited 8.3 +/- 0.8 and 9.6 +/- 1.2 aberrations at 5 and 50 microg/ml of EFX respectively. In CFX treated cultures it was found 5.6 +/- 1.3 and 7.7 +/- 3.5 aberrations/100 cells at 5 and 25 microg/ml antimicrobial concentration. These results suggested a genotoxic effect of EFX and CFX in the system used (P < 0.001). A reduction in the mitotic index and fuzzy metaphases were observed at 50 microg/ml of CFX, indicating a cytotoxic effect produced by this antimicrobial.

Cytogenetic investigations in flight personnel.

During long-distance flights at high altitudes flight personnel are exposed to cosmic radiation. In order to determine whether there are biological effects of such low dose radiation exposure in aircrew, chromosomal aberrations were investigated in 59 female cabin attendants and a matched control group of 31 members of station personnel. The mean number of dicentric chromosomes amounts to 1.3 (95% CI 1.0-1.6) per 1000 cells in cabin attendants and 1.4 (95% CI 1.0-1.9) per 1000 cells in controls. In an additional control group of 56 female clerks from Berlin the mean frequency of dicentric chromosomes was 1.3 (95% CI 1.0-1.6). Neither in dicentric frequency and distribution nor in other aberrations was a significant difference between the groups of flight and station personnel found. The high frequency of multi-aberrant cells was remarkable in flight personnel as well as in station personnel. The reason for this phenomenon is unknown and needs further investigation.

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation.

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.

Evaluation of the genotoxic potential of the isocoumarin paepalantine in in vivo and in vitro mammalian systems.

Paepalantine is an isocoumarin isolated from Paepalanthus vellozioides which showed antimicrobial activity in in vitro experiments. In the present study, paepalantine was tested for possible clastogenic and cytotoxic action. Cultures from different individuals were treated with paepalantine at concentrations of 20, 40 and 80 microg/ml. The effect of isocoumarin was also tested in an in vivo assay using Wistar rat bone marrow cells. Paepalantine was administered intraperitoneally at concentrations of 6.25, 12.5 and 25 mg/kg body weight. Under these conditions paepalantine did not have a clastogenic effect, but was significantly cytotoxic in the in vitro and in vivo mammalian cell systems tested in the present work.

Genotoxicity of adriamycin in human cell lines differing in antioxidant enzymes activity.

Genotoxicity of adriamycin in human cell lines was investigated by using a micronucleus assay. The result obtained was negative: the cells treated showed no increase in micronuclei. The chromosome aberration study with adriamycin indicated that the number of aberrant cells, high immediately after treatment, decreased to nearly the control level in 24 h of postincubation, probably as a result of the DNA repair process. Experiments with caffeine--a DNA repair inhibitor--indicated an increase micronuclei in the cells treated with adriamycin and caffeine together. The results obtained suggest that, in a human cells, adriamycin--induced DNA damages are quickly repaired to prevent micronuclei formation.

Reduction of radiation-induced chromosome aberration and apoptosis by dithiothreitol.

We have examined in vitro and in vivo radioprotective effects of a well-known thiol-containing compound, dithiothreitol (DTT). The treatment of both 0.5 and 1 mM of DTT significantly increased clonogenic survival of gamma-ray irradiated Chinese hamster (V79-4) cells. In order to investigate the possible radioprotective mechanism of DTT, we measured gamma-ray induced chromosome aberration by micronucleus assay. In the presence of 0.5 mM or 1 mM DTT, the frequencies of micronuclei were greatly reduced in all dose range examined (1.5-8 Gy). Slightly higher reduction in micronucleus formation was observed in 1 mM DTT-treated cells than in 0.5 mM DTT-treated cells. In addition, incubation with both 0.5 and 1 mM of DTT prior to gamma-ray irradiation reduced nucleosomal DNA fragmentation at about same extent, this result suggests that treatment of DTT at concentrations of 0.5 and 1 mM reduced radiation-induced apoptosis. In vivo experiments, we also observed that DTT treatment reduced the incidence of apoptotic cells in mouse small intestine crypts. In irradiated control group 4.4 +/- 0.5 apoptotic cells per crypt were observed. In DTT-administered and irradiated mice, only 2.1 +/- 0.4 apoptotic cells per crypt was observed. In vitro and in vivo data obtained in this study showed that DTT reduced radiation-induced damages and it seems that the possible radioprotective mechanisms of action of DTT are prevention of chromosome aberration.

Alteraciones cromosómicas en anestésistas del Hospital Universitario de Maracaibo agosto 1996-julio 1997 / Chromosomic alterations in female anesthetist working at the Maracaibo University Hospital (HUM)

Las anestesiólogas que trabajan en los quirófano del HUM (Hospital Universitario de Maracaibo), están expuestas a agentes mutagénicos, gases anestésicos como óxido nitroso, halotano, enflurano y óxido de etileno utilizado en la esterilización de equipos quirúrgicos. Estos gases influyen en la división celular prolongando las fases G1 y G2, inhiben la síntesis de ácido nucleicos, intervienen en la mitosis entre la profase y la anafase. El óxido de etileno, en el hombre induce aberraciones cromosómicas e intercambio de cromátidas hermanas en linfocitos y es considerada como posible carcinógeno, en estudios epidemiológicos realizado con humanos expuestos. En los últimos cinco años se presentó un aumento en el número de abortos y nacidos vivos con malformaciones congénitas en las profesionales anestesistas en el HUM. El objetivo del estudio fue el de demostrar que los agentes anestésicos utilizados determinan daños genotóxicos en las anestesistas expuestas cronicamente, empleando como punto final a las alteraciones cromosómicas. La muestra la constituyeron 21 anestesiólogas, con edad promedio de 40 años, el 50 por ciento tiene más de 5 años de exposición, el 30 por ciento presentó antecedentes de abortos y el 15 por ciento hijos con malformaciones congénitas. Se les práctico estudio cromosómico utilizando la técnica de cultivo en linfocitos humanos con Bandas G. Los resultados mostraron un 55 por ciento de rupturas cromosómicas a nivel de los cromosomas 2 y 3 con mayor frecuecia, y un 100 por ciento de la muestra presentó endorreduplicaciones, con un promedio de 3 por cariotipo. El grupo control presentó un 6 por ciento de endorreduplicaciones, con un promedio de por cariotipo. Esto evidencia daño genotóxico en el grupo expuesto crónicamente a los agentes antes señalados y va depender del mecanismo de reparación del ADN en cada individuo

Biological consequences of the heterogeneous irradiation of lymphocytes during technetium-99m hexamethylpropylene amine oxime white blood cell labelling.

Technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO) labelling of white blood cells, routinely used for the detection of infection, results in the incorporation of radioactivity by polymorphonuclear leucocytes and also lymphocytes and can induce cell lesions in the latter case. The aim of this study was therefore to acquire data on the morphological and functional status of labelled lymphocytes present in the 99mTc-HMPAO leucocyte mixture and to determine the cellular consequences of labelling. The mean radioactivity associated with lymphocytes was 325 +/- 10.8 kBq/10(6) lymphocytes under standard labelling conditions. Microautoradiographic studies showed that labelling was heterogeneous (4% intensely labelled cells), which prevented calculation of the mean absorbed dose. The frequency of chromosomal aberrations (dicentrics and rings) in the labelled lymphocytes for 380 kBq/10(6) cells was 1.08 +/- 0.09 but no abnormality was observed in the unlabelled control lymphocytes. The plating efficiency of labelled lymphocytes was reduced, as compared with that for control cells, but some lymphocytes were still able to form clones and were still "alive" by radiobiological definition. It is therefore suggested that lymphocytes should be removed from 99mTc-HMPAO cell preparations before administration to patients.

Induction of asymmetrical type of chromosomal aberrations in cultured human lymphocytes by ion beams of different energies at varying LET from HIMAC and RRC.

Frequencies of asymmetrical type of chromosome aberration were scored in cultured human blood lymphocytes irradiated with carbon and neon beams. Blood cells were irradiated with various doses to establish dose response curves for chromosome aberration frequency vs. dose, and chromosome preparation was made by conventional method. Dose response curves for the per cell frequencies of the dicentrics and centric rings as well as the excess amount of acentric fragments were described for 7 different qualities (LET = 22.4, 40.0, 41.5, 69.9, 70.0, 100.0 and 150 KeV/micrometer) of carbon and neon beams with three different energies, 135, 290 and 400 MeV/u. From the analysis of those dose response curves, the maximum effect was found in the region of LET value at near 70 KeV/micrometer together with linear expression in the response from all endpoints examined. The 135 MeV/u of carbons (69.9 KeV/micrometer) and neons(70.0 KeV/micrometer) showed linear response. The 290 MeV/u of carbons (100 KeV/m) and neons (150 KeV/micrometer) showed medium effects with different shape of response, linear with a plateau and upward concavity. The 2 carbon beams (41.5 and 40 KeV/micrometer) from 2 different accelerators showed much discrepancy in the response. RBE-LET relationship was also described by comparing the coefficient alpha of the 7 different dose responses. The peak (near 70 KeV/m) was localized close to that (80 KeV/m) for the survivals of dsb repair deficient cells (Eguchi-Kasai et al. 1998), but in different position from that previously reported in many other studies (100-200 KeV/mm). Identification of the RBEmax in the present study has yet to be definitive.

Induction of diplochromosomes in mammalian cells by inhibitors of topoisomerase II.

Diplochromosomes, consisting of four chromatids lying side-by-side, instead of the normal two, are produced when cells go through two rounds of DNA replication without separation of chromatids. They are thus an indication of the failure of the normal chromosome separation mechanism. In the present experiments, induction of diplochromosomes by inhibitors of topoisomerase II (Topo II) was used to provide further evidence that Topo II is required for separation of daughter chromosomes. Actively growing cultures of CHO cells were treated with Colcemid, and separated into metaphase and interphase fractions, each of which was treated for 2 h with the Topo II inhibitor being tested. The cells were then cultivated in fresh medium without inhibitor for periods of between 18 and 44 h, and metaphase cells once again accumulated by treatment with Colcemid. Chromosome preparations were made in the standard way and stained with Giemsa. Up to 2,000 metaphases were counted from each culture, and the proportion with diplochromosomes calculated. At appropriate concentrations, the Topo II inhibitors etoposide and mitoxantrone induced substantial levels of metaphases with diplochromosomes in cultures that had been treated when the cells were in interphase (up to 30% and 11%, respectively). Amsacrine, however, only produced a smaller proportion (4.7%) of metaphases with diplochromosomes after a much longer culture period following treatment. All the inhibitors caused severe chromosome damage. When used to treat metaphase cells, mitoxantrone and amsacrine only induced diplochromosomes after prolonged culture, although a small number of diplochromosomes were seen after etoposide treatment and a shorter period of culture. Results with cells treated in metaphase might indicate that Topo II is, in fact, not required for anaphase chromosome separation, although it is clearly important for segregation of newly replicated DNA.