Instability of mRNA expression signatures of drug transporters in chronic myeloid leukemia patients resistant to imatinib.

Despite the success of imatinib mesylate (IM) in the treatment of chronic myeloid leukemia (CML), approximately 30% of patients are resistant to therapy, mostly due to unknown causes. To profile the expression signatures of drug transporters throughout IM therapy and correlate them with resistance, we quantified mRNA expression levels of the SLC22A12, ABCB1, ABCC1, ABCG2 and MVP genes in consecutive samples from peripheral blood or bone marrow of CML patients who were either responsive or resistant to IM. Additionally we identified and quantified BCR-ABL1 transcript variants and analyzed 1236T>C ABCB1 and 480G>C SLC22A1 polymorphisms. A relationship between the type of BCR-ABL1 transcript or ABCB1 or SLC22A1 genotype and response to treatment was not discovered. However, the studied genes had higher expression levels in follow-up compared to the diagnostic samples, demonstrating a possible induction in expression. IM-sensitive patients presented significantly higher values of SLC22A1 expression, suggesting higher drug influx. Most importantly, while responding patients demonstrated stable expression signatures in consecutive samples, there was considerable variation in IM-resistant patients, indicating that single point sampling expression signatures are not reliable in predicting clinical outcomes or prognostic features in these patients. Studies that assessed consecutive samples from CML patients in order to evaluate the variation in expression levels of transporter genes are limited yet our study emphasizes the importance of such approaches.

Estragole: a weak direct-acting food-borne genotoxin and potential carcinogen.

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000µM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.

DNA damage response in imatinib resistant chronic myeloid leukemia K562 cells.

Resistance to imatinib in patients with chronic myeloid leukemia can lead to advanced disease and blast crisis. Conventional chemotherapy with DNA damaging agents is then used, alone or in combination with other tyrosine kinase inhibitors (TKIs). Our aim was to assess whether imatinib resistant K562 cells were also resistant to DNA damaging agents. After treatment with H(2)O(2) and doxorubicin, but not camptothecin, cell survival was higher in imatinib resistant cells compared to parental cells. DNA damage, measured by comet and γ-H2AX assays, was lower in imatinib resistant cells. mRNA expression levels of 50 genes of the DNA damage response pathway showed increased expression of the base excision repair (BER) genes MBD4 and NTHL1. Knockdown of MBD4 and NTHL1 expression in resistant cells using siRNA decreased cell survival after treatment with H(2)O(2) and doxorubicin. Our results indicate that imatinib resistant cells display cross-resistance to oxidative agents, partly through up-regulation of BER genes. Expression of these genes in imatinib resistant patients was not significantly different compared to sensitive patients. However, the strategy followed in this study could help identify chemotherapeutic agents that are more effective as alternative agents in cases of resistance to TKIs.

Genomics and cancer drug resistance.

Cellular drug resistance is a major obstacle in cancer therapy. Mechanisms of resistance can be associated with altered expression of ATP-binding cassette (ABC) family of transporters on cell membrane transporters, the most common cause of multi-drug resistance (MDR), but can also include alterations of DNA repair pathways, resistance to apoptosis and target modifications. Anti-cancer treatments may be divided into different categories based on their purpose and action: chemotherapeutic agents damage and kill dividing cells; hormonal treatments prevent cancer cells from receiving signals essential for their growth; targeted drugs are a relatively new cancer treatment that targets specific proteins and pathways that are limited primarily to cancer cells or that are much more prevalent in cancer cells; and antibodies function by either depriving the cancer cells of necessary signals or by causing their direct death. In any case, resistance to anticancer therapies leads to poor prognosis of patients. Thus, identification of novel molecular targets is critical in development of new, efficient and specific cancer drugs. The aim of this review is to describe the impact of genomics in studying some of the most critical pathways involved in cancer drug resistance and in improving drug development. We shall also focus on the emerging role of microRNAs, as key gene expression regulators, in drug resistance. Finally, we shall address the specific mechanisms involved in resistance to tyrosine kinase inhibitors in chronic myeloid leukemia.

Development of imatinib and dasatinib resistance: dynamics of expression of drug transporters ABCB1, ABCC1, ABCG2, MVP, and SLC22A1.

About 20% of patients with chronic myeloid leukemia (CML) do not respond to treatment with imatinib either initially or because of acquired resistance. To study the development of CML drug resistance, an in vitro experimental system comprising cell lines with different resistance levels was established by exposing K562 cells to increasing concentrations of imatinib and dasatinib anticancer agents. The mRNA levels of BCR- ABL1 and of genes involved in drug transport or redistribution (ABCB1, ABCC1, ABCC3, ABCG2, MVP, and SLC22A1) were measured and the ABL1 kinase domain sequenced. Results excluded BCR- ABL1 overexpression and mutations as relevant resistance mechanisms. Most studied transporters were overexpressed in the majority of resistant cell lines. Their expression pattern was dynamic: varying with resistance level and chronic drug exposure. Studied efflux transporters may have an important role at the initial stages of resistance, but after prolonged exposure and for higher doses of drugs other mechanisms might take place.

Genotoxic and apoptotic activities of the food flavourings myristicin and eugenol in AA8 and XRCC1 deficient EM9 cells.

Some food flavourings, such as safrole and methyleugenol, are known for their genotoxic and hepatocarcinogenic properties whereas for others, such as myristicin, there is less data. Myristicin and eugenol are both alkenylbenzenes, and we compared their direct genotoxicity in repair proficient (AA8) and repair deficient XRCC(-) (EM9) Chinese hamster ovary cells. Cell viability was assessed by the MTT assay. The comet assay was used to evaluate DNA breaks, and the γ-H2AX assay to evaluate induction of double strand breaks. We assessed apoptosis by measuring caspases activation, and the TUNEL assay. Reduction of cell viability was similar in AA8 and EM9 cells, for both compounds. After 1h eugenol produced DNA strand breaks in the comet assay and induced double strand breaks in the γ-H2AX assay in AA8 cells, while myristicin was not genotoxic in both the comet and the γ-H2AX assays. Both flavourings were negative in EM9 cells. After 24h eugenol and myristicin induced DNA fragmentation detected by TUNEL in both cell lines, but only myristicin activated caspases. Myristicin was more apoptotic than eugenol, in both cell lines. The XRCC1 protein does not influence the apoptotic activity of either compound.

Naturally contaminated shellfish samples: quantification of diarrhetic shellfish poisoning toxins in unhydrolysed and hydrolysed extracts and cytotoxicity assessment.

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin-2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1-100 nM). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC50 of 27 nM. The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2.

The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450-mediated biotransformation (a corrigendum report on Duarte et al. (2005) Mutagenesis 20, 93-100).

This corrigendum report describes the study of the comparison of human cytochrome b(5) (b(5)) with rat b(5) when coupled with human cytochrome P450 CYP1A2, 2A6 or 2E1. Results indicate a role of the N-terminal part of b(5) in the coupling with CYP. Indeed, the plasmid pLCM-b(5)-RED used in our former study on b(5) [Duarte et al. (2005) Mutagenesis, 20(2), 193-100] erroneously contained rat b(5). Plasmid pLCM-b(5)-RED was corrected with human b(5) and subsequently all experimental work was repeated as was described for the rat b(5) plasmid. Although absolute values of contents and activities were lower, all key-findings as found for rat b(5) could be confirmed using human b(5). The physiological relevant co-expression of the members of the cytochrome P450 complex, CYP, NADPH-cytochrome P450 oxidoreductase (RED) and human b(5) could be demonstrated in the different BTC strains, as was found before. The stimulatory effect of human b(5) on the activity of CYP1A2, CYP2A6 and CYP2E1 was in general similar, when compared with rat b(5), though less quantitatively pronounced. This was both the case when using membrane preparations as well as by the bioactivation of procarcinogens using the bacterial mutagenicity assay. Human b(5) stimulated the bioactivation of all compounds as described for rat b(5), except for CYP1A2 mediated bioactivation of 2-aminoanthracene (2AA), which was not stimulated by human b(5). All other main findings of the effect of rat b(5) were confirmed with human b(5), i.e. for CYP2A6: N-nitrosodiethylamine (NNdEA): approximately 14-fold increase ( approximately 23-fold with rat b(5)) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): approximately 3-fold ( approximately 9-fold with rat b(5)); for CYP2E1: NNdEA: approximately 1.5-fold increase ( approximately 3-fold with rat b(5)); NNK: no mutagenicity with or without human b(5). Both CYP2A6 and CYP2E1 demonstrated total dependence on the presence of human b(5) for N-nitrosodi-n-propylamine (NNdPA) mutagenicity, as was shown before with rat b(5).

Genotoxicity and endoreduplication inducing activity of the food flavouring eugenol.

Eugenol (1-allyl-3-methoxy-4-hydroxybenzene; CAS No. 97-53-0), a compound extracted from clove oil and marjoram, is widely used as a food flavouring substance and is present in spices such as basil, cinnamon and nutmeg. It is also used in dentistry as an antiseptic and analgesic. Structural similarities with the class IIB IARC carcinogen safrole raises questions on its putative carcinogenicity. We evaluated the genotoxicity of eugenol in V79 cells using chromosomal aberrations (CAs), with and without rat liver biotransformation (S9). Eugenol induced CAs, with significant increases (3.5% aberrant cells) at 2500 microM, demonstrating cytotoxicity at higher doses. S9 increased the induction of CAs in a dose-dependent manner to 15% at 2500 microM, with a high frequency of chromatid exchanges. In particular, an increase of endoreduplicated cells was observed, from 0% at control levels to 2.3 and 5% at 2000 microM, without and with S9, respectively. Since endoreduplication has been linked to inhibition of topoisomerase II, the topoisomerase II inhibitor ICRF-193 was used as a control inducer of endoreduplication (0.1-0.5 microM), increasing the number of endoreduplicated cells from 0% (control) to 3.5% (0.5 microM). S9 did not influence endoreduplication by ICRF-193. Both eugenol and ICRF-193 were also assayed for inhibition of topoisomerase II, and both showed a dose-dependent inhibitory effect, with ICRF-193 being a more potent inhibitor. Our results confirm that eugenol is genotoxic and raises the possibility of it having topoisomerase II inhibiting activity.

Escherichia coli BTC, a human cytochrome P450 competent tester strain with a high sensitivity towards alkylating agents: involvement of alkyltransferases in the repair of DNA damage induced by aromatic amines.

We report here on strain BTC, a new Escherichia coli mutagenicity tester strain for the expression of human cytochrome P450 (CYP) with an enhanced sensitivity for the detection of alkylating agents. This strain was developed first through knocking out of the genes ada and ogt in our previously developed strain BMX100, resulting in PD1000. Strain PD1000 demonstrated a significantly higher detection sensitivity towards several alkylating agents such as N-nitrosodiethylamine (NNdEA), N-nitrosodi-n-propylamine (NNdPA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Unexpectedly, this strain also showed an enhanced sensitivity towards 2-aminoanthracene (2AA), 4-aminobiphenyl (4AbPh), 2-aminofluorene (2AF) and 2-nitroanthracene (2NA) mutagenicity. Subsequently, our previously developed bi-plasmid system for the co-expression of a specific human CYP form (CYP1A2, 2A6 or 2E1) with human NADPH-cytochrome P450 reductase (RED) was introduced in strain PD1000, resulting in strains BTC1A2, BTC2A6 and BTC2E1, respectively. The mutagenicity of NNdEA and NNK was successfully detected with strains BTC2A6 and BTC2E1 and with strains BTC1A2 and BTC2A6, respectively, in contrast to the corresponding MTC (ada+ ogt+) CYP strains. The (ada- ogt-) deficient strain BTC1A2 also showed an enhanced sensitivity towards the detection of 2AA mutagenicity, when compared with the proficient repair strain MTC1A2. This enhancement was much more pronounced with strain PD1000 using the rat liver S9 fraction than with strain BTC1A2.

The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformation.

Cytochrome b(5) (b(5)) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b(5)/CYP-competent mutagenicity tester bacteria to study the role of b(5) in the bioactivation activity of human CYP. These new tester bacteria were derived from the previously engineered human CYP-competent Escherichia coli K12 tester strain MTC, containing a bi-plasmid system for the co-expression of a specific CYP form (CYP1A2, 2A6 or 2E1) with human b(5), and human NADPH cytochrome P450 reductase (RED), resulting in the strain BTC-b(5)-1A2, BTC-b(5)-2A6 and BTC-b(5)-2E1, respectively. The relative content of b(5) with CYP and RED in these three BTC-b(5)-CYP strains demonstrated physiologically relevant co-expression levels and typical CYP-specific activities could be determined with their specific chemical probes. These strains were applied in mutagenicity assays along with their corresponding b(5)-void strains to determine the effect of b(5) on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivation of several promutagens. For CYP1A2, of the 5 compounds tested [2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3-methylimidazo(4,5-f)quinoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], only the mutagenicity of 2AA was slightly increased ( approximately 1.5-fold) in the presence of b(5). The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased approximately 3- and 23-fold, respectively when the bacteria contained b(5). The CYP2A6-mediated mutagenicity of NNK increased approximately 9-fold when co-expressed with b(5). The stimulatory effect of b(5) on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b(5) with CYP2A6 or CYP2E1. This demonstrates the prominent role of b(5) in the bioactivation of this carcinogen.

Possible transient adaptive response to mitomycin C in peripheral lymphocytes from thyroid cancer patients after iodine-131 therapy.

Our study attempted to assess the possible induction and persistence of an adaptive response in lymphocytes of thyroidectomized thyroid cancer patients treated with 131I (2,590 MBq, corresponding to whole body doses in the range of 200-300 mGy), to a testing dose of mitomycin C (MMC) in vitro. The cytogenetic endpoint studied was the induction of micronuclei in cytokinesis-blocked peripheral blood lymphocytes, immediately before treatment and 1, 6 and 24 months after therapy. One month after therapy, induction of micronucleated cytokinesis-blocked lymphocytes ( per thousand ) by MMC was lower (34.6 +/- 7.7) than before therapy (52.1 +/- 5.0). In 7 of 11 patients this reduction was significant. However, at 6 months, induction of micronuclei was markedly higher (133.1 +/- 13.6). This significant increase was observed regardless of the decrease at 1 month. At 24 months, the frequency of micronucleated cells decreased (84.8 +/- 5.5), but remained higher than before treatment. The results obtained 1 month after therapy could reflect adaptation due to radiation, or a higher rate of early apoptosis or cell death, with bone marrow suppression, visible as a lower response in vitro towards MMC. At 6 months, recovery of the lymphocyte population may occur, and higher responses to MMC in vitro could reflect higher chromosomal instability in the previously irradiated stem cells with a concomitant disappearance of adaptation, whereas at 24 months the results show a tendency to return to pretherapy values.