Small molecules targeted to the microtubule-Hec1 interaction inhibit cancer cell growth through microtubule stabilization.

Highly expressed in cancer protein 1 (Hec1) is a subunit of the kinetochore (KT)-associated Ndc80 complex, which ensures proper segregation of sister chromatids at mitosis by mediating the interaction between KTs and microtubules (MTs). HEC1 mRNA and protein are highly expressed in many malignancies as part of a signature of chromosome instability. These properties render Hec1 a promising molecular target for developing therapeutic drugs that exert their anticancer activities by producing massive chromosome aneuploidy. A virtual screening study aimed at identifying small molecules able to bind at the Hec1-MT interaction domain identified one positive hit compound and two analogs of the hit with high cytotoxic, pro-apoptotic and anti-mitotic activities. The most cytotoxic analog (SM15) was shown to produce chromosome segregation defects in cancer cells by inhibiting the correction of erroneous KT-MT interactions. Live cell imaging of treated cells demonstrated that mitotic arrest and segregation abnormalities lead to cell death through mitotic catastrophe and that cell death occurred also from interphase. Importantly, SM15 was shown to be more effective in inducing apoptotic cell death in cancer cells as compared to normal ones and effectively reduced tumor growth in a mouse xenograft model. Mechanistically, cold-induced MT depolymerization experiments demonstrated a hyper-stabilization of both mitotic and interphase MTs. Molecular dynamics simulations corroborate this finding by showing that SM15 can bind the MT surface independently from Hec1 and acts as a stabilizer of both MTs and KT-MT interactions. Overall, our studies represent a clear proof of principle that MT-Hec1-interacting compounds may represent novel powerful anticancer agents.

N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy.

Incomplete sister chromatid separation of long chromosome arms.

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.

Genotoxic damage in free-living Algerian mouse (Mus spretus) after the Coto Doñana ecological disaster.

The Doñana National Park (Spain), one of the most important wildlife sites in the West of Europe, was affected (25 April 1998) by the spill of acidic waste rich in toxic metals (mainly zinc, lead, copper, etc.), arsenic and aromatic amines from the Aznalcollar mine accident. Micronuclei test with May Grunwald-Giemsa and with CREST-antikinetocore staining using DAPI as counter-staining were performed on peripheral blood erythrocytes from Algerian mice to evaluate genotoxic damage. Animals were collected in four locations each differently affected by the disaster. Higher frequencies of micronuclei and CREST-positive micronuclei were observed in the sites, which were reached by toxic sludge and contaminated water in comparison with those located within the park. The results obtained applying the two methods indicate that DAPI staining is more sensitive in detecting micronuclei. Genotoxic biomonitoring should be further carried out in the area to control the mutagenetic level in natural populations.

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells.

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells.

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.

Changes in microtubule organization after exposure to a benzimidazole derivative in Chinese hamster cells.

Many aneugenic compounds are known to affect one or several components of the mitotic apparatus. The mechanisms and targets of the aneuploidy-inducing activity of the benzimidazole derivative thiabendazole remain uninvestigated. In our experiments we found that thiabendazole-treated Chinese hamster cells (Cl-1) exhibited low levels of newly synthesized tubulin, indicating microtubule poisoning. In addition, microtubule growth and organization were substantially affected at mitosis. This was revealed by the reduced length of both interpolar and astral microtubules. Furthermore, thiabendazole strongly induced multipolar and asymmetric alpha-tubulin-positive metaphase spindles, characterized, however, by the absence of fragmentation of centrosome material as evaluated by anti-gamma-tubulin antibody staining. Interestingly, we found that microtubule poisoning induced by thiabendazole was qualitatively different from that of colchicine, the best known microtubule depolymerizing agent. In fact, in interphase cells colchicine was comparatively more effective than thiabendazole in promoting depolymerization of cytoplasmic microtubules. However, colchicine could not depolymerize a sub-population of stable, acetylated microtubules, which were however significantly reduced after thiabendazole exposure. In conclusion, the capability of thiabendazole to promote chromosomal malsegregation could be related to an effect on microtubule polymerization that specifically promotes formation of aberrant spindles.

Micronuclei, CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality.

PURPOSE: To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. MATERIALS AND METHODS: Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/microm and 27.6 keV/microm protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. RESULTS: MN yield and the frequency of BN cells carrying multiple MN (> or =2) were significantly higher after exposure to 27.6 keV/microm protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/microm protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6 keV/microm protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3 Gy). CONCLUSIONS: Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.

Direct and indirect non-disjunction in the origin of trisomy in cultured human lymphocytes.

The aim of the present work was to investigate the processes involved in the origin of trisomic karyotypes, i.e. co-migration of sister chromatids (mitotic non-disjunction, MND) and recovery of micronuclei (MN) originating from lagging chromosomes/chromatids at anaphase (mitotic indirect non-disjunction, MIND), and to evaluate their relative contribution to aneuploidy in human lymphocytes mitotically activated in vitro. Therefore, phytohaemagglutinin-stimulated human lymphocytes from one donor were treated with 10 and 25 nM colchicine and analysed through two cell cycles by means of both molecular (FISH with centromeric DNA probes specific for chromosomes 7 and 11) and classical cytogenetic techniques. The following events were analysed: (i) chromosome/chromatid loss (a MN-generating event) in M(1) bipolar ana-telophases; (ii) MN recovery in M(2+) prophases; (iii) non-disjunction and loss of chromosomes 7 and 11 by FISH analysis in cytochalasin B-induced binucleate cells; (iv) spontaneous frequency of trisomic cells by chromosome counting and FISH analysis in M(1) c-metaphases; (v) induced frequency of trisomic cells by chromosome counting and FISH analysis in M(2) c-metaphases. Our results indicate that MND plays a major role compared with MIND in the origin of trisomic karyotypes, being approximately 4- to 5-fold higher in colchicine-treated cells. Moreover, remarkable reductions in the observed frequencies of trisomic cells were recorded in comparison with the expected ones, with an observed/expected frequency ratio of trisomic M(2) c-metaphases ranging between 1/3 and 1/6.

Differences in malsegregation rates obtained by scoring ana-telophases or binucleate cells.

In this work we have applied in situ hybridization with alphoid centromeric probes specific to chromosomes 7 and 11 to ana-telophase cells from human primary fibroblasts. The aim was to visualize the events leading to aneuploidy directly during anaphase, analyse the induction of aneuploidy during this mitotic stage and compare the frequencies of chromosome malsegregation observed in ana-telophases with the estimated malsegregation obtained in binucleate cells after a short cytochalasin B treatment. Significantly higher frequencies of chromosome loss and chromosome non-disjunction were observed in fibroblasts undergoing ana-telophase during recovery from a nocodazole-induced mitotic arrest compared with binucleate cells obtained by a further 30 min incubation with cytochalasin B. Using the same experimental schedule, analysis of hybridization signals in mononucleate cells showed higher frequencies of polyploid nuclei in cytochalasin B-treated cultures, indicating that part of the ana-telophases observed after release from the nocodazole-induced mitotic arrest may give rise to polyploid mononucleate cells instead of binucleate ones. A reduced distance between spindle poles was also measured in cells undergoing ana-telophase in the presence of cytochalasin B. Our study suggests that in nocodazole and cytochalasin B-treated cultures the shorter pole-to-pole distance may favour the reformation of a single membrane around telophase chromosomes, especially when several lagging chromosomes lie between the two future daughter nuclei. This would give rise to polyploid mononucleate cells at the ensuing interphase.

A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA.

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of 'active' chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.

Analysis of chromosome loss and non-disjunction in cytokinesis-blocked lymphocytes of 24 male subjects.

Chromosome malsegregation in peripheral blood lymphocytes of 24 healthy male subjects was analysed by means of fluorescence in situ hybridization with centromeric probes of chromosomes 7, 11, 18 and X. On the basis of the distribution of centromeric signals in cytokinesis-blocked cells, both loss (leading to centromere-positive micronuclei) and non-disjunction (resulting in an unbalanced distribution of signals in the main nuclei) of the hybridized chromosomes in vitro were identified. In addition, the incidence of binucleated cells with two hyperploid nuclei, possibly arising from mitotic division of trisomic types, was determined. In this way, the incidence of chromosome malsegregation in vivo and in vitro could be compared in the same cell samples. The results obtained show that ageing is positively correlated with the incidence of malsegregation of chromosome X in peripheral lymphocytes of male subjects and confirm the higher susceptibility of chromosome X to malsegregation in comparison with autosomes. A positive correlation between in vitro and in vivo malsegregation rates was observed for both chromosome X and for autosomes. Finally, relatively high frequencies of multiple malsegregation events, greater than expected for independent events, were recorded for both chromosome X and for autosomes, indicating that the abnormal segregation of chromosomes may be connected to a general dysfunction of the mitotic apparatus. The correlation observed between in vitro and in vivo malsegregation frequencies and the association of both parameters with ageing suggest that analysis of chromosome malsegregation in binucleated cells is a useful tool in the study of genomic instability in human populations.

Effect of cytochalasin B on the induction of chromosome missegregation by colchicine at low concentrations in human lymphocytes.

The aim of the present work was to investigate the possible interference of cytochalasin B (cyt B) with low concentration treatment with colchicine in the induction of chromosome/chromatid loss and micronuclei in human lymphocytes mitotically activated in vitro. Thus, cells from a single female donor were treated with colchicine (10 or 25 nM, from 24 h after PHA addition to fixation at 66 h) either in the presence or absence of cyt B. Single lagging chromosomes/chromatids were scored in bipolar ana-telophases and greater damage (disrupted and c-anaphases) was scored in cells at anaphase. Micronuclei were scored in the first 4000 nuclei observed in both cyt B-treated (in mononucleate and binucleate cells) and untreated cultures. With the same criterion, FISH analysis was performed on 2000 nuclei where chromosome 7 and 11 centromeric DNA probes were used in pairs. Our results showed that: (i) the frequency of laggards and of micronuclei increased with colchicine concentration but in the presence of cyt B there was a lower frequency of both (with a mean reduction of approximately 49%); (ii) FISH analysis showed a colchicine concentration-dependent increase in nuclei with three spots for chromosome 7; (iii) a colchicine concentration-dependent increase in tetraploid cells was observed. This increase was particularly remarkable (5-fold) in cells grown in the presence of cyt B compared with cyt B-untreated cells. The observed 'cyt B effects' can be explained if it is assumed that in cytokinesis-blocked cells there is a shorter distance between the poles. As a consequence: (i) laggards would be engulfed in the nearest daughter nucleus with a consequent lower induction of micronuclei; (ii) segregating sister chromatids in heavily impaired anaphases would not travel a sufficient distance to give rise to two daughter nuclei, leading to an increased frequency of polyploid nuclei.

Dimethyl sulfoxide restores contact inhibition-induced growth arrest and inhibits cell density-dependent apoptosis in hamster cells.

Most nontransformed cell lines respond to confluence by arresting the cell cycle in a viable G(1) phase, whereas immortalized cell lines growing in monolayer do not stop cell cycle progression in response to high cell density and are subjected to density-dependent apoptosis. We have examined the effects, in terms of cell growth, apoptosis, and expression of adhesion molecules of culturing contact inhibition-deficient hamster cells in the presence of dimethyl sulfoxide (DMSO). Addition of 1.5% DMSO to the growth medium for 96 h arrested Chinese hamster ovary (CHO) cells in the G(1) phase as a confluent monolayer, associated with a remarkable increase in the expression of the cyclin-dependent kinase inhibitor p27. Cells cultured in DMSO-containing medium showed increased levels of cadherins and alpha5beta1 and beta1 integrin complexes. Cell exposure to DMSO also reduced both cell density-dependent apoptosis and necrosis and resulted in increased Bcl-2 expression. These results converge to indicate that DMSO restores contact inhibition-induced growth arrest and prevents high-density-dependent apoptosis and suggest that the effect of DMSO may be mediated by intracellular signaling triggered by cell-extracellular matrix and cell-cell interactions. Both p27 and bcl-2 appear to be involved in the resumption of growth control accompanying cell adhesion in DMSO-exposed CHO cells.

CREST staining of micronuclei from free-living rodents to detect environmental contamination in situ.

In this work immunofluorescent antikinetochore (CREST) staining was used to analyse bone marrow micronuclei (MN) from free-living animals belonging to four different rodent species. Yellow-necked mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus) were trapped in the Czech Republic, Algerian mice (Mus spretus) in Spain and house mice (Mus musculus domesticus) in Italy. Animals were collected in areas displaying low or high environmental pollution in order to investigate the sensitivity of CREST analysis on bone marrow MN as a biomarker of environmental stress in situ. Differences in total MN frequencies between animals collected in control or contaminated areas were statistically significant for two species, whereas the differences in CREST+ MN were statistically significant for three species. Interestingly, the percentages of CREST+ MN in animals collected in the control areas were very low (3. 2-8.7%), suggesting that activities inducing alterations in the distribution of chromosomes are very rare in natural conditions. The increased frequencies of CREST+ MN observed in areas with high environmental impact indicate that activities producing loss of chromosomes at mitosis may be characteristic of anthropogenic environments such as industrial settlements around petrochemical factories. Our data suggest that the analysis of CREST+ MN may represent a sensitive end-point for the detection of environmental contamination by genotoxic xenobiotics, offering the advantage of providing information on the mechanism of action of environmental contaminants.

Immunofluorescence analysis of diazepam-induced mitotic apparatus anomalies and chromosome loss in Chinese hamster cells.

To study the mechanisms leading to diazepam (DZ)-induced chromosome loss, we evaluated the effect of the drug on the distribution of cytoplasmic and mitotic apparatus proteins using specific antibodies. The use of antibodies directed against dynein and kinetochores (CREST staining) suggested that chromosomes arranged in monopolar spindles were interacting with short fibers originating from the monopole. Interestingly, nearly 50% of DZ-induced monopolar mitoses showed a punctate staining of centrosomes when evaluated with an anti-gamma-tubulin antibody. The extent of phosphorylation of mitotic proteins was not affected by drug treatment, as shown by staining the cells with an antibody against mitotic phosphorylated proteins (MPM-2). After recovery of DZ, nearly 20% of anaphases were abnormal and mainly consisted of multipolar anaphases and lagging chromosomes; this was consistent with a high frequency of kinetochore-containing micronuclei as evaluated by CREST antibody staining in cells that had divided only once after drug removal, i.e. binucleate cells obtained by cytochalasin-B treatment. Our data confirmed that DZ is a powerful inducer of chromosome loss in cultured rodent cells. Moreover, our results indicate that DZ interfered with the correct assembly of centrosomes.

Genetic effects of petroleum fuels: II. Analysis of chromosome loss and hyperploidy in peripheral lymphocytes of gasoline station attendants.

Molecular cytogenetic methods were applied to investigate the effect of the occupational exposure to low concentrations of benzene and petroleum fuels on genomic stability. Twelve male gasoline station attendants (average benzene exposure of 0.32 mg/m3 as 8h TWA) and 12 age- and smoking-matched unexposed controls were selected for the study. The incidence of hyperploidy and polyploidy in peripheral lymphocytes was evaluated through in situ hybridization of interphase cells, harvested 24 hr after stimulation, with centromeric probes of chromosomes 7, 11, 18, and X. For half of the subjects, metaphases harvested 24 hr later were analyzed. The incidence of chromosome loss in vitro was determined in cytokinesis-blocked cells, harvested at 66 hr, through the hybridization of micronuclei with a pancentromeric probe. Ten thousand chromosomes (more than 200 metaphases equivalent) and 2,000 binucleated cells/person were scored for hyperploidy and micronucleus analysis, respectively. The results obtained did not show any exposure-related excess of hyperploidy or micronucleus formation. Conversely, the age of the subjects was significantly correlated with several markers of genomic instability, such as the incidence of chromosome X and chromosome 18 hyperploidy, total hyperploidy and polyploidy, and close to statistical significance with chromosome loss. Smoking habits did not appear to contribute significantly to the effects measured. The parallel analysis of hyperploidy and polyploidy in interphase nuclei in 24-hr cultures and in metaphase cells harvested 24 hr later showed basically similar incidences of aneuploid cells, indicating that no significant selection against hyperploid and polyploid types occurred during the first cell cycle in vitro.

DNA damage and cytotoxicity induced by beta-lapachone: relation to poly(ADP-ribose) polymerase inhibition.

beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5, 6-dione) was previously shown to enhance the lethality of X-rays and radiomimetic agents and its radiosensitizing role in mammalian cells was attributed to a possible interference with topoisomerase I activity. Furthermore, beta-lapachone alone was found to induce chromosomal damage in Chinese hamster ovary (CHO) cells. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its genotoxic action in cultured mammalian cells. Flow cytometry analysis of beta-lapachone-treated CHO cells indicated a selective cytotoxic effect upon S phase of the cell cycle. beta-lapachone produced DNA strand breaks as determined by alkaline elution assay; alkaline elution profiles from treated cells showed a bimodal dose-response pattern, with a threshold dose above which a massive dose-independent DNA degradation was observed. Furthermore, beta-lapachone increased the capacity of crude CHO cellular extracts to unwind supercoiled plasmid DNA, while significantly inhibiting in vitro poly(ADP-ribose) polymerase (PARP). These results suggest that damage induction is probably mediated by the interaction between beta-lapachone and cellular enzymatic function(s), rather than reflecting a direct action on the DNA. We suggest that the inhibition of PARP plays a central role in the complex biological effects induced by beta-lapachone in CHO cells.