Combined study on clastogenic, aneugenic and apoptotic properties of doxorubicin in human cells in vitro.

BACKGROUND: Doxorubicin is a widely used anticancer drug due to its broad spectrum of antitumor activity. Various mechanisms have been proposed for its cytostatic activity, including DNA intercalation, topoisomerase II inhibition, generation of free radicals and apoptosis. The present study aims to further clarify the cytostatic activity of doxorubicin by its specific effect on (a) DNA damage, (b) micronucleation and (c) apoptosis, using a combination of different methods and cell systems such as human lymphocytes and HL-60 human leukemic cells. DNA lesions were analyzed by the alkaline comet assay in combination with formamidopyrimidine (Fpg) and human 8-oxoguanine (hOGG1) repair enzymes. Micronucleation was investigated by the Cytokinesis-Block Micronucleus assay (CBMN) in combination with Fluorescence In Situ Hybridization analysis. Impairment on mitotic apparatus was investigated by double immunofluorescence of ß- and γ-tubulin. Apoptotic cell frequency was determined by the CBMN cytome assay. Complementary to the above, caspase-3 level was investigated by Western blot. RESULTS: It was found that doxorubicin generates DNA breakage induced by oxidative damage in DNA bases, which can be repaired by the Fpg and hOGG1 enzymes. Increased micronucleus frequency was identified mainly through chromosome breakage and, at a lesser extent, through chromosome delay. Analysis of mitotic spindle showed disturbance of chromosome orientation and centrosome duplication and/or separation, leading to aneuploidy. Enhanced frequency of apoptotic leukemic cells was also observed. Caspase-3 seems to be involved in the generation of apoptosis. CONCLUSIONS: The aforementioned findings derived from different treatment schedules, doses and time of exposure on primary versus transformed cells extend our knowledge about doxorubicin genotoxicity and contribute to the better understanding of the mechanisms by which doxorubicin induces genotoxic effects on human cells.

Deregulation of methylation of transcribed-ultra conserved regions in colorectal cancer and their value for detection of adenomas and adenocarcinomas.

Expression of Transcribed Ultraconserved Regions (T-UCRs) is often deregulated in cancer. The present study assesses the expression and methylation of three T-UCRs (Uc160, Uc283 and Uc346) in colorectal cancer (CRC) and explores the potential of T-UCR methylation in circulating DNA for the detection of adenomas and adenocarcinomas. Expression levels of Uc160, Uc283 and Uc346 were lower in neoplastic tissues from 64 CRC patients (statistically significant for Uc160, p<0.001), compared to non-malignant tissues, while methylation levels displayed the inverse pattern (p<0.001, p=0.001 and p=0.004 respectively). In colon cancer cell lines, overexpression of Uc160 and Uc346 led to increased proliferation and migration rates. Methylation levels of Uc160 in plasma of 50 CRC, 59 adenoma patients, 40 healthy subjects and 12 patients with colon inflammation or diverticulosis predicted the presence of CRC with 35% sensitivity and 89% specificity (p=0.016), while methylation levels of the combination of all three T-UCRs resulted in 45% sensitivity and 74.3% specificity (p=0.013). In conclusion, studied T-UCRs' expression and methylation status are deregulated in CRC while Uc160 and Uc346 appear to have a complicated role in CRC progression. Moreover their methylation status appears a promising non-invasive screening test for CRC, provided that the sensitivity of the assay is improved.

DNA fragmentation induced by all-trans retinoic acid and its steroidal analogue EA-4 in C2 C12 mouse and HL-60 human leukemic cells in vitro.

We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified.

Comparison of the aneugenic properties of nocodazole, paclitaxel and griseofulvin in vitro. Centrosome defects and alterations in protein expression profiles.

We have comparatively investigated the aneugenic activity of two anticancer drugs, nocodazole (NOC) and paclitaxel (PTX), and the antifungal griseofulvin with promising role in cancer treatment (GF), which affect microtubule dynamics in different ways. The comparison was achieved in HFFF2 human fibroblasts, MCF-7 human breast cancer cells and C2C12 mouse myoblasts, and focused on three issues: (i) induction of chromosome delay by estimation of MN frequency using CREST analysis; (ii) disturbance of spindle organization with Aurora-A/ß-tubulin immunofluorescence; and (iii) alterations in the expression of Aurora-A, ß- and γ-tubulin by western blotting. They induced chromosome delay, provoked metaphase arrest and promoted microtubule disorganization, reflecting their common characteristic of generating aneuploidy. In particular, NOC induced mainly monopolar metaphases, although PTX induced only multipolar metaphases. GF generated different types of abnormal metaphases, exhibiting cell specificity. Additionally, NOC decreased the expression of Aurora-A and ß-tubulin, while the opposite held true for PTX and GF. γ-Tubulin expression was not modulated owing to NOC treatment, whereas PTX and GF increased γ-tubulin expression. Our findings throw a light on the manifestation of the aneugenicity of the studied compounds through centrosome proliferation/separation and protein expression, reflecting their different effects on microtubule dynamics.

Aneugenic potential of the anticancer drugs melphalan and chlorambucil. The involvement of apoptosis and chromosome segregation regulating proteins.

Previous findings showed that the anticancer drugs p-N,N-bis(2-chloroethyl) amino-l-phenylalanine (melphalan, MEL) and p-N,N-bis(2-chloroethyl)aminophenylbutyric acid (chlorambucil, CAB) belonging to the nitrogen mustard group, in addition to their clastogenic activity, also exert aneugenic potential, nondisjunction and chromosome delay. Their aneugenic potential is mainly mediated through centrosome defects. To further investigate their aneugenicity we (a) studied whether apoptosis is a mechanism responsible for the elimination of damaged cells generated by MEL and CAB and (b) investigated if proteins that regulate chromosome segregation are involved in the modulation of their aneugenic potential. Apoptosis was studied by Annexin-V/Propidium Iodide staining and fluorescence microscopy. The involvement of apoptosis on the exclusion of cells with genetic damage and centrosome disturbances was analyzed by DAPI staining and immunofluorescence of ß- and γ-tubulin in the presence of pan-caspase inhibitor. The expressions of Aurora-A, Aurora-B, survivin and γ-tubulin were studied by western blot. We found that (a) apoptosis is not the mechanism of choice for selectively eliminating cells with supernumerary centrosomes, and (b) the proteins Aurora-A, Aurora-B and survivin are involved in the modulation of MEL and CAB aneugenicity. These findings are important for the understanding of the mechanism responsible for the aneugenic activity of the anticancer drugs melphalan and chlorambucil.

Genotoxicity of all-trans retinoic acid (ATRA) and its steroidal analogue EA-4 in human lymphocytes and mouse cells in vitro.

The aim of our study is to: (a) investigate whether ATRA and its steroidal analogue EA-4 enhance micronucleation in human lymphocytes and mouse cells in vitro and clarify the micronucleation mechanism by FISH and CREST analysis respectively, and (b) analyze their effect on spindle organization by immunofluorescence of ß- and γ-tubulin in mouse cells. We found that they: (a) induce micronucleation mainly via chromosome breakage and chromosome delay in a lesser extent, (b) disturb microtubule network, chromosome orientation and centrosome duplication/separation, (c) accumulate cell cycle at ana-telophases, which exert micronucleation, multiple γ-tubulin signals, nucleoplasmic bridges and multinucleation, and (d) generate multinucleated and multimicronucleated interphase cells.

Ehlers-Danlos type IV syndrome in a patient with Down syndrome: simple co-occurrence or true association?

Down syndrome, a common chromosome aneuploidy, has been associated with an increased incidence of cutaneous disorders. The simultaneous occurrence with Ehlers-Danlos syndrome (EDS) is rare. We report here the clinical case of a 19-year-old female patient with Down syndrome (trisomy 21) who was also affected by EDS type IV. She died from spontaneous bleeding due to rupture of nonaneurysmal abdominal aorta. Since the affected chromosomes in these two syndromes are different (21 and 2, respectively), the concomitant appearance of Down syndrome and EDS type IV in our patient, though clinically intriguing, most likely represents a co-occurrence. However, the possibility that a presently unknown link may exist between these syndromes cannot be ruled out.

Genotoxicity of hydrochlorothiazide in cultured human lymphocytes. I. Evaluation of chromosome delay and chromosome breakage.

Hypertension is often treated with diuretics, like hydrochlorothiazide (HCTZ). Previous results on the in vitro genotoxicity of HCTZ are equivocal. In the present study, we have evaluated the genotoxicity of HCTZ in cultured human lymphocytes using the Cytokinesis Blocked Micronucleus (CBMN) assay. In addition, micronucleus (MN) induction was analyzed by Fluorescence In Situ Hybridization (FISH) with an alpha-satellite DNA centromeric probe to distinguish between clastogenic and aneugenic effects. Lymphocyte cultures from 32 healthy adults were exposed to 5 and 40 microg/ml HCTZ. Age, gender, and smoking were evaluated as factors affecting the MN analysis. We found that HCTZ increased MN frequencies. FISH analysis revealed that HCTZ exerts its genotoxicity more strongly at the 40 microg/ml concentration, and principally through chromosome delay (aneugenicity). Multiregression analysis of our results confirmed the known effect of age and gender on MN induction in human lymphocytes. Smoking was also a confounding factor for MN induction, especially for centromere-negative MN frequencies. Under the experimental conditions used, only age had a clear positive effect on the response of lymphocytes to HCTZ. These data indicate that HCTZ produces micronuclei in cultured human lymphocytes by a mechanism that involves chromosome delay and to a lesser extent through chromosome breakage.

Short stature, type E brachydactyly, exostoses, gynecomastia, and cryptorchidism in a patient with 47,XYY/45,X/46,XY mosaicism.

We report a 72-year-old male patient with a 47,XYY/45,X/46,XY mosaicism associated with short stature, exostoses, type E brachydactyly, gynecomastia, cryptorchidism, mild mental retardation, and a paranoid personality and conversion disorder. Since his prevalent cell line was 47,XYY (about 75%), our patient could be karyotypically classified as a case of 47,XYY syndrome. In view of the striking similarity of the clinical features of this case and those of a XYY case previously reported by Ikegawa et al (1992), it seems reasonable to suggest that these patients are representatives of a novel syndrome with a XYY karyotype.

Interactions between CYP1A1 polymorphisms and exposure to environmental tobacco smoke in the modulation of lymphocyte bulky DNA adducts and chromosomal aberrations.

CYP1A1 plays an important role in the metabolic activation of polycyclic aromatic hydrocarbons (PAH), carcinogenic components of air pollution. The influence of CYP1A1 genotype (*2A, *2B and *4) on the levels of lymphocyte bulky DNA adducts and the frequency of cells with aberrant chromosomes was assessed in 194 non-smoking subjects in whom recent exposure to environmental tobacco smoke (ETS) and airborne particulate-associated PAH were measured during two consecutive seasons (winter and summer). While CYP1A1*4 had no consistent effect on either biomarker of genetic damage, the levels of both biomarkers responded in a parallel fashion to changes in exposure/CYP1A1*2A genotype combinations during both seasons. Specifically, the levels of both biomarkers were increased in carriers of at least one CYP1A1*2A allele, as compared with CYP1A1*1 homozygotes, in subjects with ETS exposures >0.8 h/day during the previous 4 days and mean personal exposure to benzo[a]pyrene <0.9 ng/m3 during the previous 24 h (all P < 0.05). Outside these exposure limits the differential effect in CYP1A1*2A variants was lost. Although the numbers of subjects with the CYP1A1*2B polymorphism was small, the same trend appeared to be followed in this case. These effects are interpreted as resulting from differential induction of CYP1A1 expression in CYP1A1*2A and CYP1A1*2A/*2B carriers by components of ETS-polluted air at levels of exposure readily suffered by large segments of the general population and suggest that subjects with these genotypes may have increased susceptibility to the genotoxic effects of ETS.

Genetic effects caused by potent antileukemic steroidal esters of chlorambucil's active metabolite.

Three steroidal esters with a common alkylating agent (chlorambucil's active metabolite, PHE) and PHE were studied with regard to their genetic activity in human lymphocyte cultures treated in vitro. The cytokinesis block micronucleus assay was used in combination with fluorescence in situ hybridization and the cytosine arabinoside method (ARA-C). The aim of this study was (i) to examine if the modified analogs (EA-72 and SOT-19) of the parent compound (ASE) exerted the same genetic activity with ASE and to correlate the genetic activity with the chemical structure, (ii) to investigate whether these steroidal esters are able to induce excision repairable lesions, through the alkylation of DNA, and (iii) to collect data in order to evaluate the exact role of the steroidal skeleton on the expression of the antileukemic activity. We found that PHE and its steroidal esters are cytotoxic for human lymphocyte cultures, as indicated by the reduction of Cytokinesis Blocked Proliferation Index, PHE being the most cytotoxic molecule. All studied compounds are capable of inducing both chromosome breakage and chromosome delay as indicated by the increased CMN and CMN frequencies. The steroidal derivatives gave reduced genetic activity. The conjugate ketone at the B ring of the steroidal skeleton resulted in decreased genetic activity mainly due to decreased chromosome delay. All studied compounds are capable of inducing DNA excision repair.