Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing.

In genetic toxicology, risk assessment has traditionally adopted linear dose-responses for any compound that causes genotoxic effects. Increasing evidence of non-linear dose-responses, however, suggests potential cellular tolerance to low levels of many genotoxicants with diverse modes of action. Such putative non-linear dose-responses need to be substantiated by strong mechanistic data that identifies the mechanisms responsible for the tolerance to low doses. This can be achieved by experimental demonstration of cytoprotective mechanisms and by providing experimental support for the existence of tolerance mechanisms against low dose effects. By highlighting key experiments into low dose mechanisms, this review aims to clarify which mechanistic data are required to support the use of non-linear dose-response models in risk assessment. Such key experiments are presented and discussed for alkylating agents, oxidants, particulate matter, nucleoside analogues, topoisomerase inhibitors and aneugens and exemplify the use of gene knockout models or transgenic models as well as chemical modulators of key effectors of relevant pathways and their impact on dose-response relationships. In vitro studies are particularly valuable to elucidate mechanisms of low-dose protection or lack thereof, while in vivo experiments are most appropriate for deriving a safe dose. In order to evaluate the existence of non-linear dose-response relationships for genotoxicants, we suggest that careful attention should be given to the mode of genotoxic action, relevant biomarkers of exposure, as well as to the existence and impact of potential cytoprotective mechanisms like detoxifying metabolism and DNA repair.

Malignant melanoma cells acquire resistance to DNA interstrand cross-linking chemotherapeutics by p53-triggered upregulation of DDB2/XPC-mediated DNA repair.

Malignant melanoma is a cancer characterized by high chemoresistance although p53 is rarely mutated. Here, we show that p53 wild-type melanoma cells acquire resistance to cell death induced by fotemustine (FM), which is a representative of alkylating DNA interstrand cross-linking agents used in melanoma therapy. We show that drug-induced resistance is a result of p53-dependent upregulation of the nucleotide excision repair (NER) genes xeroderma pigmentosum complementation group C (XPC) and damaged DNA-binding protein 2 (DDB2), which stimulate the repair of DNA interstrand cross-links (ICLs) arising from O(6)-chloroethylguanine. Consequently, TP53 mutated cells are unable to repair ICLs, leading to prolonged ATM, ATR and checkpoint kinase 1 (CHK1) activation, and finally apoptosis. The roles of p53 and NER in ICL-triggered cell death were confirmed by knockdown of p53 and XPC. Upregulation of XPC and DDB2 in p53wt cells following a single drug treatment is a robust and sustained response that lasts for up to 1 week. Pretreatment with an inducing dose followed by a high and toxic dose of FM provoked an adaptive response as the killing outcome of the challenge dose was reduced. Upregulation of XPC and DDB2 was also observed in a melanoma mouse xenograft model following systemic administration of FM. Additionally, XPC and DDB2 induction occurred upon treatment with other cross-linking anticancer drugs, such as cisplatin and mafosfamide, indicating it is a general response of cancer cells to this group of chemotherapeutics. Collectively, the data indicate that p53-dependent upregulation of XPC and DDB2 is a key mechanism upon genotoxic stress, whereby melanoma cells acquire resistance towards DNA cross-linking agents. To our knowledge, this is the first demonstration of upregulation of NER following a single dose of a DNA interstrand cross-linker, which is a robust and long-lasting effect that impacts the killing response of cancer cells to subsequent treatments.

Rac1 signaling protects monocytic AML cells expressing the MLL-AF9 oncogene from caspase-mediated apoptotic death.

We investigated the relevance of signaling mechanisms regulated by the Ras-homologous GTPase Rac1 for survival of acute myeloid leukemia (AML) cells harbouring the MLL-AF9 oncogene due to t(9;11)(p21;q23) translocation. Monocytic MLL-AF9 expressing cells (MM6, THP-1) were hypersensitive to both small-molecule inhibitors targeting Rac1 (EHT 1864, NSC 23766) (IC50EHT ~12.5 µM) and lipid lowering drugs (lovastatin, atorvastatin) (IC50Lova ~7.5 µM) as compared to acute myelocytic leukemia (NOMO-1, HL60) and T cell leukemia (Jurkat) cells (IC50EHT >30 µM; IC50Lova >25 µM). Hypersensitivity of monocytic cells following Rac1 inhibition resulted from caspase-driven apoptosis as shown by profound activation of caspase-8,-9,-7,-3 and substantial (~90 %) decrease in protein expression of pro-survival factors (survivin, XIAP, p-Akt). Apoptotic death was preceded by S139-posphorylation of histone H2AX (γH2AX), a prototypical surrogate marker of DNA double-strand breaks (DSBs). Taken together, abrogation of Rac1 signaling causes DSBs in acute monocytic leukemia cells harbouring the MLL-AF9 oncogene, which, together with downregulation of survivin, XIAP and p-Akt, results in massive induction of caspase-driven apoptotic death. Apparently, Rac1 signaling is required for maintaining genetic stability and maintaining survival in specific subtypes of AML. Hence, targeting of Rac1 is considered a promising novel strategy to induce lethality in MLL-AF9 expressing AML.

Rac1 modulates acute and subacute genotoxin-induced hepatic stress responses, fibrosis and liver aging.

To investigate the importance of the Ras-homologous GTPase Rac1 for the hepatic response to genotoxic insults and liver aging, rac1 was deleted in liver of mice by Mx1-Cre-based recombination. Knockout of rac1 caused complex changes in basal as well as doxorubicin and ionizing radiation-induced mRNA expression of various genotoxic stress response-related genes, including hspa1b, rad51, wrn and xpc. Rac1 deletion protected the liver from acute toxicity following doxorubicin treatment. Moreover, the level of S139 phosphorylated histone H2AX (γH2AX), which is indicative of DNA damage, and mRNA expression of pro-inflammatory (IL-6) and pro-fibrotic (CTGF, TGFß, αSMA) factors were mitigated in rac1 knockout animals. By contrast, lack of rac1 promoted subacute hepatotoxicity, which was determined 3 weeks after injection of multiple low doses of doxorubicin by assaying the γH2AX level, mitotic index and pro-fibrotic gene expression. Regarding ionizing radiation, rac1 deficiency had no major effects on DNA damage induction or acute pro-inflammatory and pro-fibrotic stress responses. Mice lacking hepatic rac1 for extended period of time (15 months) revealed increased mRNA expression of fibrosis-related factors (CTGF, TGFß, collagen, MMP1) and fibrotic tissue remodeling. In addition, protein expression of the senescence marker p16 was enhanced in the absence of rac1. Taken together, the data provide evidence that Rac1 is required for doxorubicin-induced DNA damage induction. It is also involved in both the acute and delayed inflammatory and fibrotic stress response in the liver following doxorubicin, but not ionizing radiation, treatment and, furthermore, protects against endogenous liver aging.

Survivin as a prognostic/predictive marker and molecular target in cancer therapy.

Evasion from apoptotic cell death is reported to be a pivotal mechanism by which tumor cells acquire resistance to therapeutic treatment. Targeting the apoptotic pathways may constitute a promising strategy to counteract therapy resistance and to re-sensitize cancer cells. Expression of survivin, the smallest and structurally unique member of the inhibitor of apoptosis protein (IAP) family, has been shown to be associated with poor clinical outcome, more aggressive clinicopathologic features and resistance to both, conventional chemo and radiation therapy. Moreover, survivin detection in cancer tissue, in circulating tumor cells and in patient's serum has prognostic and predictive relevance and may display a prerequisite for marker based molecular therapies. Indeed, due to its universal over expression in malignant tissue, and its prominent role at disparate networks of cellular division, intracellular signaling, apoptosis and adaption to unfavorable surroundings, survivin has been shown to be a suitable target for a targeted therapy. The applicability of survivindriven strategies in clinical practice is currently under investigation as the first survivin antagonists (small molecule inhibitors, antisense oligonucleotides and immunotherapy) successfully entered phase I/II trials. Taken together, these data provide a rationale for the implementation of both, survivin as a molecular diagnostic tool and survivin targeted therapies, within future clinical practice.

Inhibition of Rac1 signaling by lovastatin protects against anthracycline-induced cardiac toxicity.

Normal tissue damage limits the efficacy of anticancer therapy. For anthracyclines, the clinically most relevant adverse effect is cardiotoxicity. The mechanisms involved are poorly understood and putative cardioprotectants are controversially discussed. Here, we show that the lipid-lowering drug lovastatin protects rat H9c2 cardiomyoblasts from doxorubicin in vitro. Protection by lovastatin is related to inhibition of the Ras-homologous GTPase Rac1. It rests on a reduced formation of DNA double-strand breaks, resulting from the inhibition of topoisomerase II by doxorubicin. Doxorubicin transport and reactive oxygen species are not involved. Protection by lovastatin was confirmed in vivo. In mice, lovastatin mitigated acute doxorubicin-induced heart and liver damage as indicated by reduced mRNA levels of the pro-fibrotic cytokine connective tissue growth factor (CTGF) and pro-inflammatory cytokines, respectively. Lovastatin also protected from doxorubicin-provoked subacute cardiac damage as shown by lowered mRNA levels of CTGF and atrial natriuretic peptide. Increase in the serum concentration of troponin I and cardiac fibrosis following doxorubicin treatment were also reduced by lovastatin. Whereas protecting the heart from harmful doxorubicin effects, lovastatin augmented its anticancer efficacy in a mouse xenograft model with human sarcoma cells. These data show that statins lower the incidence of cardiac tissue injury after anthracycline treatment in a Rac1-dependent manner, without impairing the therapeutic efficacy.

Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53.

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O(6)-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg(-1) protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O(6)-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O(6)-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O(6)-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.

Rechallenge with temozolomide with different scheduling is effective in recurrent malignant gliomas.

Treatment of recurrent malignant glioma, which has a poor patient prognosis, has not been standardised. Moreover, it is unclear whether repeated treatment with temozolomide is effective in patients who received previous temozolomide treatment before developing a recurrence. Here, we present the results of a high-dose individually adapted 21-day regimen demonstrating that rechallenge is effective even in patients expressing O6-methylguanine-DNA methyltransferase (MGMT) in the tumor. Twenty-one patients with recurrent malignant gliomas pre-treated with temozolomide, 18 WHO IV glioblastoma (GBM) and 3 WHO III patients, received 100 mg/m2 temozolomide on days 1-21/28. The GBM patients had a median Karnofsky performance status of 60% and a median age of 54.8 years. Blood counts decreased continuously, enabling a gradual dose adaptation. When blood counts dropped below normal values, temozolomide was applied on days 1-5/7. Dosage was reduced to 50-75 mg/m2 in 11 patients and gradually increased up to 130 mg/m2 in 3 patients. WHO grade 3/4 toxicity was hematological in 3 patients and non-hematological in 3 patients. In GBM patients (n=18), response after >3 months was complete in 3 patients, partial in 1 (22%), stable disease in 7 (39%) and progressive disease in 7 (39%). Progression-free survival at 6 months (PFS-6M) was 39%. Median survival was 9.1 months from relapse and 17.9 months overall. Of the patients with unmethylated MGMT promoter, 2/7 were progression-free for >6 (15 and 19) months. The data indicate that rechallenge with near-continuous, higher-dose temozolomide (100 mg/m2 on days 1-21/28 or days 1-5/7 with individual dose adaptation) is also feasible in patients with critical blood counts. Objective responses can be achieved even after relapse during a conventional 5/28-day regimen. The resistance of tumors characterized by unmethylated MGMT promoter may be overcome by near continuous temozolomide administration, which is probably most effective with a 5/7-day schedule. In spite of the relatively poor clinical prognosis, the data indicate that rechallenge with temozolomide with a dose-dense and long-lasting administration protocol is tolerable and comparable with other reported treatment protocols involving temozolomide.

Degradation of heterocyclic aromatic amines in oil under storage and frying conditions and reduction of their mutagenic potential.

Heterocyclic aromatic amines (HAA) were systematically studied concerning their partition behavior in water/oil-systems and their thermostability in different animal derived fats and vegetable oils. Partitioning of IQx-compounds and PhIP in water/oil systems was found to depend on the polarity defined by the molecular structure and on the pH-value of the aqueous phase. In particular, beta-carbolines norharman and harman showed a significant strong lipophilic character at alkaline pH. After heating in frying fats at 130 degrees C, contents of IQx compounds and PhIP were reduced by more than 40% and after heating at 180 degrees C less than 10% of the HAA initial concentration was recovered. By contrast, norharman and harman were much more stable under equivalent conditions. The present study leads for the first time to the conclusion that degradation of HAA in frying fats strongly correlates to the type of frying fat and is promoted by lipid oxidation products. Firstly, addition of hydroperoxides to model oils lead to a decrease of HAA during storage at 40 degrees C. Secondly, stability of HAA correlated with the content of unsaturated fatty acids in the oil, which is indicative for the oxidative stability of the medium. Degradation of HAA by heat treatment was associated with a reduction of their mutagenic potential towards strain TA98 of Salmonella typhimurium.

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O(6)-methylguanine due to high E2F1 regulated mismatch repair.

Exposure of stem cells to genotoxins may lead to embryonic lethality or teratogenic effects. This can be prevented by efficient DNA repair or by eliminating genetically damaged cells. Using undifferentiated mouse embryonic stem (ES) cells as a pluripotent model system, we compared ES cells with differentiated cells, with regard to apoptosis induction by alkylating agents forming the highly mutagenic and killing DNA adduct O(6)-methylguanine. Upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ES cells undergo apoptosis at much higher frequency than differentiated cells, although they express a high level of the repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Apoptosis induced by MNNG is due to O(6)-methylguanine DNA adducts, since inhibition of MGMT sensitized ES cells. The high sensitivity of ES cells to O(6)-methylating agents is due to high expression of the mismatch repair proteins MSH2 and MSH6 (MutSalpha), which declines during differentiation. High MutSalpha expression in ES cells was related to a high hyperphosphorylated retinoblastoma (ppRb) level and E2F1 activity that upregulates MSH2, causing, in turn, stabilization of MSH6. Non-repaired O(6)-methylguanine adducts were shown to cause DNA double-stranded breaks, stabilization of p53 and upregulation of Fas/CD95/Apo-1 at significantly higher level in ES cells than in fibroblasts. The high apoptotic response of ES cells to O(6)-methylguanine adducts may contribute to reduction of the mutational load in the progenitor population.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.