Decreased DNA repair capacity of UV-irradiated cells following interferon treatment

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-alpha, IFN-beta and IFN-gamma on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-alpha had a stronger inhibitory effect than IFN-gamma. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.     

The inhibition of DNA repair capacity by stilbene estrogen in Leydig cells: its implications in the induction of instability in the testicular genome

In this study, we examined the effect of stilbene estrogen, diethylstilbestrol (DES), on the DNA repair capacity of mouse Leydig cells using the host cell reactivation assay. Cells transfected with UV-damaged plasmids, undamaged plasmids, or no plasmids (sham treatment) were grown in serum, serum-free, or DES plus serum-free medium. The serum-grown cells which have a shorter cell cycle time (16h) showed a 40% decrease in DNA repair capacity when compared to serum-free cells with a longer cell cycle time (25h). A significant decrease in the DNA repair capacity of the Leydig cells exposed to DES was observed compared to untreated cells grown in a serum-free environment (P<0.05). The effect of DES on DNA repair in Leydig cells was dose dependent. We have recently shown that DES stimulates the growth of Leydig cells. Stimulatory growth of Leydig cells coupled with a decrease in DNA repair capacity by DES may allow the accumulation of mutagenic lesions in DNA. The mutagenic lesions may result from the attack of redox cycling products of DES and/or errors of replication. This, in turn, may produce alterations in the genome of Leydig cells resulting in genetically unstable cells that may serve as precursor cells for testicular carcinogenesis.     

Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

DNA-PKcs Inhibition Sensitizes Cancer Cells to Carbon-Ion Irradiation via Telomere Capping Disruption

Heavy-ion irradiation induces a higher frequency of DNA double strand breaks (DSBs) which must be properly repaired. Critical shortening of telomeres can trigger DNA damage responses such as DSBs. Telomeres are very sensitive to oxidative stress such as ionizing radiation. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the central component in the non-homologous end joining (NHEJ) repair complex and participates in telomere maintenance. Therefore, it is expected to enhance the cell killing effect of heavy-ion irradiation via DNA-PKcs inhibition. To test this hypothesis, cellular radiosensitivity was measured by the clonal genetic assay. DNA damage repair was relatively quantified by long PCR. Apoptosis was quantified by flow-cytometric analysis of annexin V/PI double staining, and senescence was analyzed by galactosidase activity. Telomere length was semi-quantified by real-time PCR. P53 and p21 expression was determined by western blotting. Our data demonstrated that MCF-7 and HeLa cells with DNA-PKcs inhibition were more susceptible to carbon-ion irradiation than Those without DNA-PKcs inhibition. Even though NHEJ was inhibited by the DNA-PKcs specific inhibitor, NU7026, most DNA damage induced by carbon-ion irradiation was repaired within 24 hours after irradiation in both cell lines. However, potential lethal damage repair (PLDR) could not restore cellular inactivation in DNA-PKcs inhibited cells. MCF-7 cells showed extensive senescence and accelerated telomere length reduction, while HeLa cells underwent significant apoptosis after irradiation with NU7026 incubation. In addition, both cell lines with shorter telomere were more susceptible to carbon-ion radiation. Our current data suggested that DNA-PKcs inhibition could enhance cellular sensitivity to carbon-ion radiation via disturbing its functional role in telomere end protection. The combination of DNA-PKcs inhibition and carbon-ion irradiation may be an efficient method of heavy-ion therapy.     

Accumulation of Oxidatively Induced DNA Damage in Human Breast Cancer Cell Lines Following Treatment with Hydrogen Peroxide

Breast cancer is a leading cause of cancer deaths in women. Although the causes of this disease are largely unknown, inefficient repair of oxidatively induced DNA lesions has been thought to play a major role in the transformation of normal breast tissue to malignant breast tissue. Previous studies have revealed higher levels of 8-hydroxyguanine in malignant breast tissue compared to non-malignant breast tissue. Furthermore, some breast cancer cell lines have greatly reduced capacity to repair this lesion suggesting that oxidatively induced DNA lesions may be elevated in breast cancer cells. We used liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of 8-hydroxy-2’-deoxyadenosine, (5’S)-8,5’-cyclo-2’-deoxyadenosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine in MCF-7 and HCC1937 breast cancer cell lines before and after exposure to H2O2 followed by a DNA repair period. We show that H2O2-treated HCC1937 and MCF-7 cell lines accumulate significantly higher levels of these lesions than the untreated cells despite a 1 h repair period. In contrast, the four lesions did not accumulate to any significant level in H2O2-treated non-malignant cell lines, AG11134 and HCC1937BL. Furthermore, MCF-7 and HCC1937 cell lines were deficient in the excision repair of all the four lesions studied. These results suggest that oxidatively induced DNA damage and its repair may be critical in the etiology of breast cancer.     

Modification of DNA repair by human interferons

We have found a new biological function of interferons, namely, their capacity to protect human cells from the action of some physical and chemical mutagens. To evaluate the protective effect of interferons the following criteria were applied: formation of sister chromatid exchanges (SCE) and chromosomal aberrations (CA), as well as viability of cells and intensity of DNA repair synthesis. Pretreatment of cells with natural interferon decreased the number of sister chromatid exchanges and chromosomal aberrations, induced by different mutagens, and increased the intensity of DNA repair synthesis. This is attributed to the ability of interferon to enhance certain phases of DNA repair. In the case of photomutagenic action of 8-methoxypsoralen (8-MOP) on the lymphocytes, when monoadducts (MA) only, or both monoadducts and interstrand cross-links (ICL) are formed, the antimutagenic effect of interferon is exhibited only with respect to ICL. Unlike the natural interferon, the recombinant alpha 2-interferon failed to have any effect on the lymphocytes of clinically healthy donors exposed to gamma-radiation. In the repair- deficient cells (Marfan's syndrome) the protection of natural interferon against the action of 4-nitroquinoline-1'-oxide and gamma- radiation was found to be reduced significantly and that of alpha 2-interferon was not manifested at all. Thus, the capacity of interferons to alter the DNA repair, conceivably, depends on the type of interferon and on the cell genotype.     

The Presence of Human Immunodeficiency Virus Type 1 Vpr Correlates with a Decrease in the Frequency of Mutations in a Plasmid Shuttle Vector

The human immunodeficiency virus type 1 (HIV-1) Vpr protein induces cell cycle arrest at the border of G(2) and M similar to the arrest caused by agents which damage DNA. We determined whether the presence of Vpr would affect the ability of cells to repair DNA. We developed a shuttle vector system to analyze the effect of Vpr upon the repair of UV-damaged DNA. Our results demonstrated that the presence of Vpr decreased the rate of deletions in this system. Of note, cells arrested in G(2) by other genotoxic agents also increased the frequency of DNA repair of UV-damaged shuttle vectors. We did not observe any direct effect of Vpr upon the rate of double-strand break repair and/or nucleotide excision repair of genomic DNA in cells. Our results suggest a role for HIV-1 Vpr in altering the frequency of DNA repair, a property which may have importance for HIV-1 replication and pathogenesis.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant gamma-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, gamma-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.     

Effect of modified DNA and RNA oligonucleotides on telomerase activity and tumor cell survival in vitro

Comparative analysis of the inhibitory effect of modified DNA and RNA oligonucleotides on telomerase activity and tumor cell growth in vitro has been carried out. The study was performed with MCF-7, HeLa, and Mel-10 human cell line extracts. It was shown that PS-TelP5 and PS-TM024 phosphorothioate DNA oligonucleotides inhibited telomerase at nanomolar concentrations and suppressed the growth of tumor cells in vitro.     

Additive and synergistic antitumor effects with toremifene and interferons

MFC-7 cells were exposed to toremifene, human alpha and gamma interferons and combinations of them in vitro. Growth of the cells was followed by ATP bioluminescence method. Rats bearing DMBA-induced tumors were treated with toremifene, rat gamma interferon and their combination daily for five weeks. The growth of the tumors was followed by palpation weekly. Toremifene and interferons inhibited the growth of MCF-7 cells. Interferons alpha and gamma were additive; toremifene and interferons were additive or at the best synergistic. Toremifene inhibited the growth of DMBA-induced tumors. Rat gamma interferon alone had no clear effect on the tumor growth. Combination of toremifene and gamma interferone was the most effective treatment and did not show any detectable toxicity. Toremifene and interferons have interesting interactions. Clinical studies using the combination might be warranted.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.     

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms’ tumor 1 (WT1)

Background

Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis.

Results

In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for “DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer”.

Conclusions

These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways.     

Endogenous production and exogenous exposure to nitric oxide augment doxorubicin cytotoxicity for breast cancer cells but not cardiac myoblasts.

We studied the effect of nitric oxide (*NO) on the anticancer activity of doxorubicin. When MCF-7 human breast cancer cells were exposed to an aqueous solution of *NO delivered as a bolus 30 min prior to doxorubicin, the cytotoxic effect as measured in a clonogenic assay was increased (doxorubicin alone, 40% survival, doxorubicin plus *NO, 5% survival). The *NO donor diethylamine nitric oxide, but not inactivated donor, also yielded an increase in doxorubicin cytotoxicity. The sequence was important since the simultaneous application of *NO with doxorubicin yielded only a small augmentation of effect, and the exposure of the cells to doxorubicin prior to the *NO obliterated the augmentation. Prior depletion of glutathione by incubation of the cells for 24h with D,L-buthionine-S,R-sulfoximine (BSO) further increased the cytotoxicity so that BSO plus *NO plus doxorubicin killed all of the clones. MCF-7 cells transduced with inducible nitric oxide synthase gene (iNOS) through an adenoviral vector overexpressed iNOS and produced increased amounts of nitrite, an indicator of increased *NO production. These iNOS transduced cells were more susceptible to doxorubicin than vector control or wild-type cells. Cell cycle progression of iNOS transduced cells was not different from controls. Likewise, iNOS transduction resulted in no change in cellular glutathione levels. For comparison, we examined the effect of iNOS transduction on the sensitivity of MCF-7 to edelfosine, a membrane-localizing anticancer drug without direct DNA interaction. Insertion of the iNOS had no effect on killing of the MCF-7 cells by this ether lipid class drug. We also tested the effect of iNOS transduction on doxorubicin sensitivity of H9c2 rat heart-derived myoblasts. We found no augmentation of cytotoxicity by *NO, and this observation offers potential therapeutic tumor selectivity by using *NO with doxorubicin. Therefore, we conclude that *NO produced intracellularly by iNOS overexpression or delivered as a bolus sensitizes human breast cancer cells in culture to doxorubicin, but not to a cardiac cell line or to edelfosine. This augmentation is not due to a modulation of cell cycle distribution or measurable cellular glutathione resulting from the transduction.     

The effect of natural and recombinant leukocyte interferons on DNA repair and the formation of chromosome aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine

The anticlastogenic action of natural leukocyte and recombinant (alpha 2) interferons was studied in human lymphocyte cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine. The criteria of cell viability, proliferation, chromosome aberrations, frequency of micronucleus formation, formation and repair of DNA breaks were used for estimation of interferons activity. Reduction of the induced chromosomal aberrations was obtained in cells pretreated with interferons. The protective effect of natural leukocytic interferon was more expressed as compared with the effect of recombinant (alpha 2) interferon. The natural interferon was also more efficient than the recombinant one in DNA breaks formation and repair.     

Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines

Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.     

Role of glutathione in apoptosis induced by radiation as determined by 1H MR spectra of cultured tumor cells

The relationship between apoptosis induced by gamma radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by (1)H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of gamma-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of gamma-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.