Biomedical subjects

B Kaina

Publications and source records attributed to B Kaina.

At least 19 recordsLinked to original sources

PubMed2006

Lovastatin protects human endothelial cells from the genotoxic and cytotoxic effects of the anticancer drugs doxorubicin and etoposide.

BACKGROUND AND PURPOSE: 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are frequently used lipid-lowering drugs. Moreover, they exert pleiotropic effects on cellular stress responses and death. Here, we analysed whether lovastatin affects the sensitivity of primary human endothelial cells (HUVEC) to the anticancer drug doxorubicin. EXPERIMENTAL APPROACH: We investigated whether pretreatment of HUVEC with low dose of lovastatin influences the cellular sensitivity to doxorubicin. To this end, cell viability, proliferation and apoptosis as well as DNA damage-triggered stress response were analysed. KEY RESULTS: Lovastatin reduced the cytotoxic potency of doxorubicin in HUVEC. Lovastatin attenuated the doxorubicin-induced increase in p53 as well as activation of checkpoint kinase (Chk-1) and stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK). Acquired doxorubicin resistance was independent of alterations in doxorubicin efflux and cell cycle progression. Also, doxorubicin-triggered production of reactive oxygen species (ROS) and formation of oxidative DNA lesions remained unaffected by lovastatin. However, lovastatin impaired DNA strand break formation induced by doxorubicin. Notably, lovastatin also conferred cross-resistance to the cytotoxic and genotoxic effects of etoposide, indicating that lovastatin shields topoisomerase II against poisons. CONCLUSIONS AND IMPLICATIONS: Based on these data, we suggest that lovastatin-mediated resistance to topoisomerase II inhibitors is due to a reduction in DNA damage and, hence, it attenuates stress responses leading to cell death that are triggered by DNA damage. Therefore, lovastatin might be useful clinically for alleviating side-effects of anticancer therapies that include topoisomerase II inhibitors.

Antibiotics, Antineoplastic↗

PubMed2006

Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine.

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O(6)MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX formation. Glioma cells mutated in DNA-PK(cs), which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.

Antineoplastic Agents, Alkylating↗

PubMed2005

Apoptosis in UV-C light irradiated p53 wild-type, apaf-1 and p53 knockout mouse embryonic fibroblasts: interplay of receptor and mitochondrial pathway.

Mouse embryonic fibroblasts (MEFs) deficient for the transcription factor p53 are hypersensitive to UV-C light. They also show a reduced recovery from UV-C induced replication blockage and are unable to repair UV-C photoproducts. In this study, we utilized wild-type (wt), Apaf-1 deficient (apaf-1(-/-)) and p53 deficient (p53(-/-)) MEFs in order to elucidate the role of non-repaired UV-C lesions in apoptotic signalling. Corresponding with the cellular sensitivity determined by the WST assay, p53(-/-) cells displayed the highest level of apoptosis, whereas wt cells showed moderate apoptosis after UV-C irradiation. Apaf1(-/-) cells were most resistant. In wt cells apoptosis was executed both via the mitochondrial and the receptor-mediated pathway, as shown by Bcl-2 decline, induction of fasR and activation of caspases-3,8,9. In apaf-1(-/-) (p53(+/+)) cells, the mitochondrial pathway was blocked downstream of Bcl-2, indicating that in this case apoptosis was mediated via the induction of fasR and caspase-3,8 activation. In p53 deficient cells, non-repaired UV-C induced DNA lesions triggered sustained up-regulation of fas ligand (fasL) mRNA, which was not seen in wt and apaf-1(-/-) cells. Therefore, in p53(-/-) MEFs, the receptor/ligand triggered pathway appeared to be dominant. This was confirmed by significant reduction of apoptosis after DN-FADD transfection. As opposed to wt and apaf-1(-/-) cells, p53 deficient MEFs showed no induction of Fas receptor and no Bcl-2 decline. Nevertheless, the resulting caspase-8 and -3 activation was stronger compared to wt and apaf-1(-/-) cells. The data indicate that UV-C light activates in MEFs both the Fas (CD95, Apo-1) receptor and the mitochondrial damage pathways. In p53(-/-) cells, however, the high level of non-repaired DNA damage forces signalling by fasL upregulation, leading to enhanced UV-C-induced apoptosis.

Animals↗

PubMed2004

Cytogenetic detection of a trans-species bystander effect: induction of sister chromatid exchanges in murine 3T3 cells by ganciclovir metabolized in HSV thymidine kinase gene-transfected Chinese hamster ovary cells.

Due to the very limited transduction capacity of hitherto available vectors, the success of gene therapy of tumours by means of suicide genes has so far essentially depended on the transfer of toxic drug metabolites from transduced (metabolizing) cells to adjacent non-transduced cells via gap junctions (bystander effect). Most experimental systems for the detection of a bystander effect yield net data of cell losses and cannot differentiate between killed transduced versus killed bystander cells. Here we report on metabolic cooperation in vitro between CHO cells stably transfected with the thymidine kinase gene of herpes simplex virus type-1 (HSVtk) (metabolizing cells) and Swiss albino 3T3 cells (bystander cells). Both cell lines are readily distinguishable by single cell and colony morphology and by their chromosomal constitution. While 3T3 cells cultured alone were refractory to the antiviral drug ganciclovir (GCV), co-culture with CHO-HSVtk(+) cells led to a dramatic reduction in plating efficiency as well as to a 4-fold increase in sister chromatid exchange rates induced by very low GCV concentrations in the 3T3 bystander cells. The modulator of gap junctional cooperation, all-trans retinoic acid, caused a strong augmentation of the bystander effect, while 18alpha-glycyrrhetinic acid, an inhibitor of gap junctional communication, drastically diminished the toxicity of GCV in the bystander cells. Whereas CHO-HSVtk(+) cells showed a distinct immunoreactivity for connexin43 in the cell membranes, 3T3 cells were almost negative. The co-culture system described here allows unequivocal distinction between metabolizing and bystander cells. In this way, mechanistic aspects of the transfer of genotoxic/cytotoxic metabolites to cells, which per se are unable to form them, become accessible to investigation.

3T3 Cells↗

PubMed2004

Mechanisms and consequences of methylating agent-induced SCEs and chromosomal aberrations: a long road traveled and still a far way to go.

Since the milestone work of Evans and Scott, demonstrating the replication dependence of alkylation-induced aberrations, and Obe and Natarajan, pointing to the critical role of DNA double-strand breaks (DSBs) as the ultimate trigger of aberrations, the field has grown extensively. A notable example is the identification of DNA methylation lesions provoking chromosome breakage (clastogenic) effects, which made it possible to model clastogenic pathways evoked by genotoxins. Experiments with repair-deficient mutants and transgenic cell lines revealed both O6-methylguanine (O6MeG) and N- methylpurines as critical lesions. For S(N)2 agents such as methyl- methanesulfonate (MMS), base N-methylation lesions are most critical, likely because of the formation of apurinic sites blocking replication. For S(N)1 agents, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O6-methylguanine (O6MeG) plays the major role both in recombination and clastogenicity in the post-treatment cell cycle, provided the lesion is not pre-replicatively repaired by O6-methylguanine-DNA methyltransferase (MGMT). The conversion probability of O6MeG into SCEs and chromosomal aberrations is estimated to be about 30:1 and >10,000:1 respectively, indicating this mispairing pro-mutagenic lesion to be highly potent in inducing recombination giving rise to SCEs. O6MeG needs replication and mismatch repair to become converted into a critical secondary genotoxic lesion. Here it is proposed that this secondary lesion can be tolerated by a process termed recombination bypass. This process is supposed to be important in the tolerance of lesions that can not be processed by translesion synthesis accomplished by low-fidelity DNA polymerases. Recombination bypass results in SCEs and might represent an alternative pathway of tolerance of non-instructive lesions. In the case of O6MeG-derived secondary lesions, recombination bypass appears to protect against cell killing since SCEs are already induced with low, non-toxic doses of MNNG. Saturation of lesion tolerance by recombination bypass or translesion synthesis may cause block of DNA replication leading to DSBs at stalled replication forks, which result in chromatid-type aberrations. Along with this model, several putative consequences of methylation-induced aberrations will be discussed such as cell death by apoptosis as well its role in tumor promotion and progression.

Alkylating Agents↗

PubMed2003

Lovastatin causes sensitization of HeLa cells to ionizing radiation-induced apoptosis by the abrogation of G2 blockage.

PURPOSE: To investigate the effect of inhibition of Ras/Rho-regulated signalling by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on radiation-induced cell killing and apoptosis. MATERIALS AND METHODS: Different human cell lines were pretreated or not with lovastatin before exposure to gamma-rays. Afterwards, radiation-induced cell killing, formation and repair of double-strand breaks, activation of radiation-inducible signal mechanisms (i.e. p53, p21, extracellular-signal-related kinase (ERK), NF-kappaB), changes in cell cycle progression and apoptosis were analysed. RESULTS: As shown by a colony formation assay, lovastatin sensitized HeLa cells to gamma-radiation-induced cell killing. The lovastatin effect was cell-type specific. Neither the level of gamma-ray-induced double-strand breaks nor its repair were affected by lovastatin. Sensitization was independent of p53/p21Waf1- and NF-kappaB-related mechanisms. Radiation-stimulated activation of ERKs was attenuated by lovastatin. Cell cycle analyses revealed that the level of gamma-ray-induced G2 blockage was not affected by lovastatin. However, as analysed up to 72 h after irradiation, lovastatin pretreated cells showed an accelerated abrogation of G2 blockage as compared with the control. G2 abrogation is paralleled by an increase in the frequency of apoptotic and necrotic cells. CONCLUSIONS: The data show that lovastatin can render human cells more sensitive to the cytotoxic effect of gamma-rays. This is related to abrogation of G2 blockage and a concomitant increase in apoptotic/necrotic cell death.

Apoptosis↗

PubMed2002

Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters.

In the present study, we addressed the question of a putative relevance of Rho proteins in tumour progression by analysing their expression on protein and mRNA level in breast tumours. We show that the level of RhoA, RhoB, Rac1 and Cdc42 protein is largely enhanced in all tumour samples analysed (n=15) as compared to normal tissues originating from the same individual. The same is true for (32)P-ADP-ribosylation of Rho proteins which is catalysed by Clostridium botulinum exoenzyme C3. Also the amount of Rho-GDI and ERK2 as well as the level of overall (32)P-GTP binding activity was tumour-specific elevated, yet to a lower extent than Rho proteins. Although the amount of Rho proteins was enhanced in tumours, most of them did not show changes in rho mRNA expression as compared to the corresponding normal tissue. Thus, elevated gene expression seems not to be the underlying mechanism of tumour-specific overexpression of Rho proteins. Sequence analysis of RhoA, RhoB, RhoC and Rac1 failed to detect any mutations in both the GTP-binding site and effector binding region. By analysing >50 tumour samples, the amount of RhoA-like proteins (i.e. RhoA, B, C), but not of Rac1, was found to significantly increase with histological grade and proliferation index. Rho protein expression was neither related to p53 nor to HER-2/neu oncogene status. Expression of rho mRNAs did not show a significant increase with histological grade. Overall the data show that (1) Rho proteins are overexpressed in breast tumours (2) overexpression is not regulated on the mRNA level (3) the expression level of RhoA-like proteins correlates with malignancy and (4) Rho proteins are not altered by mutation in breast tumours.

ADP Ribose Transferases↗

PubMed2002

Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard.

To study molecular aspects of cytotoxicity of the anticancer drug beta-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since beta-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D(10) values) to the cytotoxic effects of beta-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

Animals↗

PubMed2002

DNA repair in resistance to alkylating anticancer drugs.

The major critical target of alkylating antineoplastic drugs belonging to the group of methylating and chloroethylating agents is DNA. DNA alkylation lesions can be repaired by the action of alkyltransferase (MGMT) and base excision repair enzymes. The major cell killing and apoptotic alkylation lesions are O6-methylguanine (O6MeG) and O6-chloroethylguanine. O6MeG causes mispairing with thymine which is erroneously processed by mismatch repair (MMR), leading to secondary lesions that potently trigger the mitochondrial apoptotic pathway. Apoptosis induced by O6MeG is a late cellular response that requires cell proliferation to occur. Data are available indicating that DNA double-strand breaks are actively involved as the ultimate trigger of apoptosis. O6MeG and O6-chloroethylguanine are repaired by the specific action of MGMT thus counteracting the killing effects of the lesions. The expression of MGMT is highly variable and is often increased in tumors compared to normal tissue. Determination of MGMT activity in various tumors showed low expression in brain, pancreas and skin and high expression in testicle, breast, colorectal, lung and ovarian tumors. Distribution profiles of MGMT revealed non-random distribution indicating the existence of subpopulations exhibiting low and high activity. Since MGMT is one of the most important factors determining drug resistance to alkylation, strategies have been developed to inhibit MGMT in tumors with the aid of MGMT inhibitors and overexpression of MGMT in healthy, non-target tissue (e.g. blood stem cells) by transferring a mutated form of MGMT inaccessible to inhibition. Targeting MGMT inhibitors to tumors may further enhance the antineoplastic efficiency of alkylating agents. The role of base excision repair, Fos and p53 in drug resistance to alkylation is also discussed.

Alkyl and Aryl Transferases↗

PubMed2001

Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

Cell-Free System↗

PubMed2001

Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins.

Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.

Animals↗

PubMed2001

DNA polymerase beta mediates protection of mammalian cells against ganciclovir-induced cytotoxicity and DNA breakage.

The efficacy of suicide herpes simplex virus-1 thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy is often limited by intrinsic resistance of tumor cells. Here we show that repair of GCV incorporated in DNA is a factor involved in GCV resistance. A protective role of DNA repair in GCV-induced cell killing is supported by the following findings: (a) GCV-exposed Chinese hamster ovary-HSVtk cells exhibited both reduced repair of GCV and cloning efficiency in the presence of a specific polymerase beta (beta-pol) inhibitor, prunasin; (b) DNA beta-pol-deficient mouse fibroblasts were more sensitive to the cytotoxic, apoptosis-inducing, and genotoxic (DNA breakage and chromosomal aberration-inducing) effects of GCV as compared with wild-type and beta-pol-complemented cell lines; (c) methoxyamine, an inhibitor of beta-pol-dependent short-patch base excision repair, sensitized wild-type and complemented beta-pol cells to GCV, whereas it had no effect on the sensitivity of beta-pol-null cells to GCV. Because methoxyamine-mediated sensitization of beta-pol wild-type and beta-pol-complemented cells to GCV did not reach the level of null cells, we suggest that both beta-pol-dependent short- and long-patch base excision repair are involved in protection of cells to GCV. Some implications for HSVtk/GCV gene therapy are being discussed.

Animals↗

PubMed2001

Ultraviolet light-induced DNA damage triggers apoptosis in nucleotide excision repair-deficient cells via Bcl-2 decline and caspase-3/-8 activation.

Ultraviolet (UV) light is a potent mutagenic and genotoxic agent. Whereas DNA damage induced by UV light is known to be responsible for UV-induced genotoxicity, its role in triggering apoptosis is still unclear. We addressed this issue by comparing nucleotide excision repair (NER) deficient 27-1 and 43-3B Chinese hamster (CHO) cells with the corresponding wild-type and ERCC-1 complemented cells. It is shown that NER deficient cells are dramatically hypersensitive to UV-C induced apoptosis, indicating that DNA damage is the major stimulus for the apoptotic response. Apoptosis triggered by UV-C induced DNA damage is related to caspase- and proteosome-dependent degradation of Bcl-2 protein. The expression of other members of the Bcl-2 family such as Bax, Bcl-x(L) and Bak were not affected. Bcl-2 decline is causally involved in UV-C induced apoptosis since overexpression of Bcl-2 protected NER deficient cells against apoptosis. We also demonstrate that caspase-8, caspase-9 and caspase-3 are activated and PARP is cleaved in response to unrepaired UV-C induced DNA damage. Caspase-8 activation occurred independently of CD95 receptor activation since CD95R/FasR and CD95L/FasL were not altered in expression, and transfection of transdominant negative FADD failed to block apoptosis. Overall, the data demonstrate that UV-C induced non-repaired DNA damage triggers apoptosis in NER deficient fibroblasts involving components of the intrinsic mitochondrial damage pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Cisplatin-induced apoptosis in 43-3B and 27-1 cells defective in nucleotide excision repair.

Cisplatin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. Its cytotoxic effect is supposed to be due to the induction of intra- and interstrand DNA cross-links which are repaired via the nucleotide excision repair (NER) pathway. Here, we elucidated the mechanism of cisplatin-induced cytotoxicity in mutants derived from CHO-9 cells defective in NER. We compared 43-3B and 27-1 cells deficient for ERCC1 and ERCC3, respectively, with the corresponding wild-type and ERCC1 complemented 43-3B cells. It is shown that cells defective in ERCC1 are more sensitive than cells defective in ERCC3 with regard to cisplatin-induced reproductive cell death. ERCC1 and ERCC3 mutants showed a higher frequency of apoptosis and, to a lesser degree, necrosis compared to repair proficient cells. Induction of apoptosis in both ERCC1 and ERCC3 defective cells was accompanied by decline in Bcl-2 protein level, activation of caspases 8, 9 and 3 and poly(ADP-ribose)polymerase (PARP) cleavage. Since the mutant cells are defective in the repair of cisplatin-induced DNA lesions, the data demonstrate that non-repaired cisplatin-induced DNA adducts act as a trigger of the mitochondrial apoptotic pathway by down-regulation of Bcl-2 followed by caspase activation.

Animals↗

PubMed2001

Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells.

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.

Antineoplastic Agents↗

PubMed2001

DNA repair activity of 8-oxoguanine DNA glycosylase 1 (OGG1) in human lymphocytes is not dependent on genetic polymorphism Ser326/Cys326.

8-oxoguanine DNA glycosylase 1 (OGG1) is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8oxoG) from DNA. Since 8oxoG is a highly mispairing lesion, decreased OGG1 expression level could lead to a higher background mutation frequency and could possibly increase the cancer risk of an individual under oxidative stress. In order to analyse the natural variation of OGG1, we measured the DNA repair activity in human lymphocytes of healthy individuals by means of an 8oxoG-containing oligonucleotide assay. The data obtained revealed a two fold interindividual variation of OGG1 activity in lymphocytes. There was no difference in OGG1 activity due to gender and smoking behaviour. Transcriptional analyses of OGG1 showed the expression of two isoforms, 1a and b, in lymphocytes. Structural analysis of the human OGG1 (hOGG1) gene revealed a Ser326/Cys326 polymorphism in the Caucasian population with allele frequencies of 75% for Ser326 and 25% for Cys326. This polymorphism was not associated with altered OGG1 activity. The described routine test system for measuring OGG1 activity in cryopreserved lymphocytes provided highly reproducible results and is a useful tool for risk assessment associated with alterations in the repair of oxidative DNA damage.

Alleles↗

PubMed2001

Inactivation of O(6)-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors.

The DNA-repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a decisive determinant of resistance of tumor cells to methylating and chloroethylating anti-cancer drugs. Therefore, selective inhibition of MGMT in tumors is expected to cause tumor sensitization. Several inhibitors of MGMT have been developed which function in both tumors and normal tissue. To deplete MGMT preferentially in tumors, strategies to target the inhibitor to the tumor tissue need to be developed. Here, we report on the properties of glucose-conjugated MGMT inhibitors that might be useful for tumor targeting since tumor cells frequently over-express glucose transporter. O(6)-Benzylguanine (O6BG), 8-aza-O(6)-benzylguanine, O(6)-(4-bromothenyl)-guanine (O6BTG) and the corresponding spacer-linked beta-D-glucose conjugates were analyzed comparatively for MGMT-inhibitory activity. Substitution at the N9 position of the purine moiety resulted generally in a reduction in the efficiency with which the inhibitors blocked MGMT. However, the inhibitory activity of the O6BTG conjugates increased with increasing spacer length, and O6BTG conjugated with a C8 spacer with beta-D-glucose was nearly as effective as O6BTG on its own. MGMT was inhibited by the conjugates both in crude cell extracts and upon treatment of intact HeLa cells, indicating efficient uptake of the glucose conjugates into cells. Since the O6BTG-C8-D-glucose conjugate 8-[O(6)-(4-bromothenyl)-guan-9-yl]-octyl-beta-D-glucoside was highly efficient at MGMT inhibition in a non-toxic concentration range, the drug might be a useful tool for specific tumor sensitization.

Enzyme Inhibitors↗

PubMed2001

Rho GTPases are involved in the regulation of NF-kappaB by genotoxic stress.

A common cellular response to genotoxic agents and inflammatory cytokines is the activation of NF-kappaB. Here, we addressed the question of whether small GTPases of the Rho family are involved in the stimulation of NF-kappaB signaling by genotoxic agents or TNFalpha in HeLa cells. Inhibition of isoprenylation of Rho proteins by use of the HMG-CoA reductase inhibitor lovastatin attenuated UV-, doxorubicin-, and TNFalpha-induced degradation of IkappaBalpha as well as drug-stimulated DNA binding activity of NF-kappaB. Furthermore, NF-kappaB-regulated gene expression stimulated by either UV irradiation or treatment with TNFalpha was abrogated by lovastatin pretreatment. This indicates that isoprenylated regulatory proteins participate in the regulation of NF-kappaB by DNA-damaging agents as well as by TNFalpha. Specific blockage of Rho signaling by Clostridium difficile toxin B attenuated UV- and doxorubicin-induced activation of NF-kappaB, but did not affect stimulation of NF-kappaB by TNFalpha. Obviously, signaling to NF-kappaB by genotoxic and nongenotoxic stimuli occurs via different molecular mechanisms, either involving Rho GTPases or not. Based on the data, we suggest Rho GTPases to be essentially required for genotoxic stress-induced signaling to NF-kappaB.

Bacterial Proteins↗