Ras-mediated deregulation of the circadian clock in cancer.

Circadian rhythms are essential to the temporal regulation of molecular processes in living systems and as such to life itself. Deregulation of these rhythms leads to failures in biological processes and eventually to the manifestation of pathological phenotypes including cancer. To address the questions as to what are the elicitors of a disrupted clock in cancer, we applied a systems biology approach to correlate experimental, bioinformatics and modelling data from several cell line models for colorectal and skin cancer. We found strong and weak circadian oscillators within the same type of cancer and identified a set of genes, which allows the discrimination between the two oscillator-types. Among those genes are IFNGR2, PITX2, RFWD2, PPARγ, LOXL2, Rab6 and SPARC, all involved in cancer-related pathways. Using a bioinformatics approach, we extended the core-clock network and present its interconnection to the discriminative set of genes. Interestingly, such gene signatures link the clock to oncogenic pathways like the RAS/MAPK pathway. To investigate the potential impact of the RAS/MAPK pathway - a major driver of colorectal carcinogenesis - on the circadian clock, we used a computational model which predicted that perturbation of BMAL1-mediated transcription can generate the circadian phenotypes similar to those observed in metastatic cell lines. Using an inducible RAS expression system, we show that overexpression of RAS disrupts the circadian clock and leads to an increase of the circadian period while RAS inhibition causes a shortening of period length, as predicted by our mathematical simulations. Together, our data demonstrate that perturbations induced by a single oncogene are sufficient to deregulate the mammalian circadian clock.

[Estimating the efficiency of DNA isolation methods in semen, blood and saliva stains using the QuantiBlot system]. / Ocena wydajnosci róznych metod izolacji DNA w plamach nasienia, krwi i sliny metoda QuantiBlot.

The aim of this study was to compare and select the optimal method of DNA isolation from blood, semen and saliva stains, as well as to determine appropriate conditions for employing amplification kits for identification of individual persons [brak w polskim tekscie]. The materials analyzed in this study consisted of stains of blood, semen and saliva samples stored for a year, and stains of blood stored for a month. Seven various methods of isolation were compared: the Fast DNA kit (Qbiogene), phenol/chloroform extraction, Sherlock (DNA II Gdansk), Dneasy (Qiagen), Wizard Genomic Purification Kit (Promega), Chelex 100 (Biorad) and salting out proteins method. After the isolation, the quantity of DNA was measured with QuantiBlot [brak w polskim tekscie]. The highest DNA concentration in bloodstains stored for one year and one month was observed employing the salting out proteins method. The phenol-chloroform extraction method was also found to produce reasonably good results. Isolation from blood and semen with salting-out method appeared to be the most effective. The phenol/chloroform method was dependent on the age and origin of the materials [brak w polskim tekscie]. The Sherlock kit was proven to be effective in blood samples stored for one year. DNA concentration values obtained in semen and saliva samples were very low and characterized by a low repeatability.

[A case of mutation at locus TH01--sequence analysis]. / Przypadek mutacji w locus TH01--analiza sekwencji.

In a paternity test with 21 short tandem repeats (STRs) an isolated exclusion for the TH01 locus was observed. The probability of paternity or maternity in this case turned out greater than 99,999%. The analysis of sequence indicates only the deletion character of the observed mutation event without any changes in the conservative sequence.

[Oxidative stress parameter analysis in rat brains during long-term ethanol intoxication]. / Ocena parametrów stresu oksydacyjnego w mózgach szczurów przewlekle intoksykowanych etanolem.

The aim of this research was to define the influence of long-term ethanol intoxication on the changes in end products of lipid peroxidation, reacting with thiobarbituric acid (TBA-reactive products) and free SH-groups concentrations in rat brains. The experiment was conducted on male Lewis rats. The experimental groups received a 1-molar ethanol solution and the control group received tap water. The animals were killed after 4, 8 and 12 weeks of intoxication and their brains were collected for further examination, encompassing measurement of the concentration of TBA-reactive products and free SH-groups. A statistically significant increase in the concentration of TBA-reactive products in 4th week of intoxication and decrease in the concentration of free SH-groups in 8th and 12th week of intoxication--compared to the control group--was noticed.

[Changes of the concentration of lipid peroxidation products and thiol groups in rat brains with long-term intoxication with methanol]. / Zmiany stezen produktów peroksydacji lipidów i grup tiolowych w mózgach szczurów w przebiegu przewleklej intoksykacji metanolem.

The aim of this paper was to define the influence of long-term methanol intoxication on the changes of end products of lipid peroxidation, reacting with thiobarbituric acid (TBA-reactive products) and free SH-group concentration in rat brains. The experiment was conducted on male Lewis rats. The experimental groups received a 1-molar methanol solution and the control group received tap water. The animals were killed after 4, 8 and 12 weeks of intoxication and their brains were collected for further examination, which encompassed measurement of the concentration of TBA-reactive products and free SH-groups. The revealed changes of TBA-reactive products and free SH-group concentration were not statistically significant.

A comparison of the action of amifostine and melatonin on DNA-damaging effects and apoptosis induced by idarubicin in normal and cancer cells.

Amifostine is a well-known cell protector and its actions involve free radical scavenging, which is also considered as a mechanism underlying the protective actions of melatonin, a secretory product of the pineal gland. In this work we compared the action of 14 mM amifostine and 50 microM melatonin on DNA damage and apoptosis induced by idarubicin in normal human lymphocytes, leukemic K562 cells and HeLa cancer cells. We employed the alkaline comet assay and pulse-field gel electrophoresis to estimate DNA damage. Apoptosis was evaluated by caspase 3 activity assay assisted by the comet assay to evaluate DNA fragmentation and DAPI staining for detection of morphological changes in chromatin. We found that idarubicin induced apoptosis in normal and cancer cells and its level was correlated with the extent of DNA strand breaks. Amifostine reduced apoptosis and DNA damage in normal cells, but it potentiated these effects in cancer cells in this in vitro study. Melatonin protected both normal and cancer cells against genotoxic treatment and apoptosis induced by idarubicin. We conclude that despite its recognized potential as an antioxidant, melatonin should be considered with caution when used in combination with cancer chemotherapy agents, especially in the case of leukemias.

Genotoxicity of acrylamide in human lymphocytes.

Acrylamide is used in the industry and can be a by-product in a high-temperature food processing. It is reported to interact with DNA, but the mechanism of this interaction is not fully understood. In the present study, we investigated the DNA-damaging potential of acrylamide (ACM) in normal human lymphocytes using the alkaline-, neutral- and 12.1 versions of the comet assay and pulsed-field gel electrophoresis. We also investigated effect of acrylamide on caspase-3 activity as well as its influence on the repair process of hydrogen peroxide-induced DNA damage. Acrylamide at 0.5-50 microM induced mainly alkali-labile sites. This damage was repaired during a 60-min repair incubation. Post-treatment of the damaged DNA with repair enzymes: thymine glycol DNA N-glycosylase (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II (Alk A), recognizing alkylated bases, caused an increase in the extent of DNA damage, indicating the induction of oxidative and alkylative DNA base modifications by acrylamide. Pre-treatment of the lymphocytes with N-tert-butyl-alpha-phenylnitrone (PBN), a spin trap, as well as vitamins C and E decreased the DNA-damaging effect of acrylamide, which suggest that free radicals/reactive oxygen species may be involved in this effect. Acrylamide impaired the repair of DNA damaged by hydrogen peroxide and increased the activity of caspase-3, which may indicate its potential to induce apoptosis. Our results suggest that acrylamide may exert a wide spectrum of diverse effects on DNA of normal cells, including mostly DNA base modifications and apoptosis. Acrylamide may also impair DNA repair. Free radicals may underline these effects and some dietary antioxidants can be considered as protective agents against genotoxic action of acrylamide. As normal lymphocytes contain cyp2e1 and P450, engaged in the bioactivation of ACM to glicidamide it is uncertain whether acrylamide causes all of measured effect per se or this is the result of the action of its metabolites.

BCR/ABL oncogenic kinase promotes unfaithful repair of the reactive oxygen species-dependent DNA double-strand breaks.

The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph)-positive leukemia cells. We find that BCR/ABL-induced reactive oxygen species (ROSs) cause chronic oxidative DNA damage resulting in double-strand breaks (DSBs) in S and G(2)/M cell cycle phases. These lesions are repaired by BCR/ABL-stimulated homologous recombination repair (HRR) and nonhomologous end-joining (NHEJ) mechanisms. A high mutation rate is detected in HRR products in BCR/ABL-positive cells, but not in the normal counterparts. In addition, large deletions are found in NHEJ products exclusively in BCR/ABL cells. We propose that the following series of events may contribute to genomic instability of Ph-positive leukemias: BCR/ABL --> ROSs --> oxidative DNA damage --> DSBs in proliferating cells --> unfaithful HRR and NHEJ repair.

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells.

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.

DNA damage and repair in BCR/ABL-expressing cells after combined action of idarubicin, STI571 and amifostine.

STI571 is a specific ABL family tyrosine kinases inhibitor approved for treatment of leukemias. It can differentially modulate the action of other antileukemic drugs. We have recently shown that deregulation of the mechanisms of DNA damage and repair in BCR/ABL-positive cells may be involved in drug resistance of these cells, and thus determine the response of cancer cells to therapy. In the present work we investigated DNA damage and repair induced by idarubicin in the presence of STI571 and amifostine, a normal cell protector, in the BCR/ABL fusion tyrosine kinase-expressing cell line. Amifostine increased the viability of both kinds of cells in the absence of STI571, but had no effect in the presence of the inhibitor. STI571 did not change the response of both BCR/ABL-expressing cells and their control counterparts to idarubicin in terms of DNA damage and repair. However, the presence of amifostine modulated the response of the cells. In the absence of STI571 amifostine decreased the DNA-damaging effect of idarubicin in normal cells and increased it in BCR/ABL-positive cells. STI571 at 2 M abolished the protective effect of amifostine against idarubicin in normal cells and diminished the magnitude of the amifostine-induce increase in cancer cells. These results suggest that amifostine should be applied with special caution in idarubicin-based chemotherapies of BCR/ABL-positive leukemias involving STI571 inhibitor.

Amifostine differentially modulates DNA damage evoked by idarubicin in normal and leukemic cells.

Human lymphocytes, p53 protein-deficient acute promyelocytic leukemia cell line HL-60, murine pro-B lymphoid cell line BaF3 and its TEL/ABL-transformed clone cells were exposed to idarubicin with and without pre-treatment with amifostine. Idarubicin at 0.5-5 microM evoked DNA damage measured by the Comet assay. Amifostine at 14 mM decreased DNA-damaging effect of idarubicin in human lymphocytes and BaF3 cells, but increased the effect in TEL/ABL-transformed cells. Amifostine had no influence on the action of idarubicin in HL-60 cells. Our results suggest that the reaction of the cell to DNA damage may contribute to its diverse response to amifostine combined with anticancer drugs and that p53 and fusion tyrosine kinases may be involved in this diversity.

TEL/JAK2 tyrosine kinase inhibits DNA repair in the presence of amifostine.

The TEL/JAK2 chromosomal translocation (t(9;12)(p24;p13)) is associated with T cell childhood acute lymphoblastic leukemia. The TEL/JAK2 fusion protein contains the JAK2 catalytic domain and the TEL-specific oligomerization domain. TEL-mediated oligomerization of the TEL/JAK2 proteins results in the constitutive activation of the tyrosine kinase activity. Leukemia cells expressing TEL/JAK2 tyrosine kinase become resistant to anti-neoplastic drugs. Amifostine is a pro-drug which can selectively protect normal tissues against the toxicity of anticancer drugs and radiation. We investigated the effects of amifostine on idarubicin-induced DNA damage and repair in murine pro-B lymphoid BaF3 cells and BaF3-TEL/JAK2-transformed cells using alkaline single cell gel electrophoresis (comet assay). Idarubicin induced DNA damage in both cell types but amifostine reduced its extent in control non-transformed BaF3 cells and enhanced it in TEL/JAK2-transformed cells. The transformed cells did not show measurable DNA repair after exposure to amifostine and idarubicin, but cells treated only with idarubicin were able to recover within a 60-min incubation. Because TEL/JAK2-transformed cells can be considered as model cells for certain human leukemias and lymphomas we anticipate an enhancement of idarubicin cytotoxicity by amifostine in these diseases. Moreover, TEL/JAK2 tyrosine kinase might be involved in cellular response to DNA damage. Amifostine could promote apoptosis or lower the threshold for apoptosis induction dependent on TEL/JAK2 activation.

A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone.

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.

Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine.

Idarubicin is an anthracycline anticancer drug used in haematological malignancies. The main side effect of idarubicin is free-radicals based cardiotoxicity. Using the comet assay we showed that the drug at concentrations from the range 0.001 to 10 microM induced DNA damage in normal human lymphocytes, measured as the increase in percentage of DNA in the tail (% tail DNA). The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Recognised cell protector, amifostine at 14 mM decreased the mean % tail DNA of the cells exposed to idarubicin at all tested concentrations of the drug. So did vitamin C at 10 microM, but vitamin E (alpha-tocopherol) at 50 microM increased the % tail DNA. Lymphocytes exposed to idarubicin and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Pretreatment of lymphocytes with nitrone spin traps, N-tert-butyl-alpha-phenylnitrone and alpha-(4-pyridil-1-oxide)-N-tert-butylnitrone decreased the extent of DNA damage evoked by idarubicin. To discuss the influence of vitamins and amifostine in cancer cells we used also murine pro-B lymphoid BaF3 transformed with BCR/ABL oncogene. These cells can be treated as model cells of human acute myelogenous leukemia. The response of these cells to vitamin E was quantitatively the same as human lymphocytes. However, vitamin C did not exert any effect on DNA damage and amifostine, in spite to normal lymphocytes, potentiated this effect. The results obtained suggest that reactive oxygen species, including free radicals, may be involved in the formation of DNA lesions induced by idarubicin. The drug can also methylate DNA bases. Our results indicate that not only cardiotoxicity but also genotoxicity and in consequence induction of secondary malignancies should be taken into account as diverse side effects of idarubicin. Amifostine may potentate DNA-damage effect of idarubicin in cancer cells and decrease this effect in normal cells. Vitamin C can be considered as protective agents against DNA damage in normal cells in persons receiving idarubicin-based chemotherapy, but the use of vitamin E cannot be recommended and at least needs further research.