Associations of single nucleotide polymorphisms in precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and prognosis of autoimmune thyroid diseases.

It is important to search the biomarker to predict the development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto's disease (HD) and Graves' disease (GD). MicroRNA (miR) bind directly to the 3' untranslated region of specific target mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-ß; however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 155 patients with GD, 151 patients with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and the rs12976445 C/T polymorphism were associated with AITD development and prognosis.

Bosutinib, an SRC inhibitor, induces caspase-independent cell death associated with permeabilization of lysosomal membranes in melanoma cells.

BACKGROUND: SRC kinase (SRC proto-oncogene, non-receptor tyrosine kinase) is a promising target for the treatment of solid cancers including human melanoma. Bosutinib (Bosu), a SRC inhibitor, has already been applied to the treatment of human chronic myelogenous leukemia and also has been assessed its safety in dogs. AIM: The aim of this study was to clarify a novel anti-tumour mechanism of Bosu in canine and human melanoma cells. MATERIALS AND METHODS: The canine and human melanoma cells were treated with Bosu and its effects were evaluated by the cell viability, the protein expression levels such as caspase-3 and LC3, Annexin V/Propidium iodide staining, and confocal immunostaining. RESULTS: Bosu induced the massive caspase-independent cell death, and blocked autophagy flux, which resulted from lysosomal dysfunction. Lysosomal dysfunction caused by Bosu was due to lysosomal membrane permeabilization (LMP), which resulted in the release of lysosomal hydrolases including cathepsin B. CONCLUSION: Our data suggest that Bosu induces the cell death through induction of LMP in melanoma cells and is a promising therapeutic agent for treatment of melanoma in both dogs and humans.

Translesional synthesis on a DNA template containing N2-methyl-2'-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I.

Formaldehyde is produced in most living systems and is present in the environment. Evidence that formaldehyde causes cancer in experimental animals infers that it may be a carcinogenic hazard to humans. Formaldehyde reacts with the exocyclic amino group of deoxyguanosine, resulting in the formation of N2-methyl-2'-deoxyguanosine (N2-Me-dG) via reduction of the Schiff base. The same reaction is likely to occur in living cells, because cells contain endogenous reductants such as ascorbic acid and gluthathione. To explore the miscoding properties of formaldehyde-derived DNA adducts a site-specifically modified oligodeoxynucleotide containing a N2-Me-dG was prepared and used as the template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer extension reaction was slightly stalled one base before the N2-Me-dG lesion, but DNA synthesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N2-Me-dG. Preferential incorporation of dCMP, the correct base, opposite the lesion was observed, along with small amounts of misincorporation of dTMP (9.4%). No deletions were detected. Steady-state kinetic studies indicated that the frequency of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3'-terminus of a dT:N2-Me-dG pair was only 2.1 times lower than from a dC:N2-Me-dG pair. We conclude that N2-Me-dG is a miscoding lesion capable of generating G-->A transition mutations.

3,N(4)-ethano-2'-deoxycytidine: chemistry of incorporation into oligomeric DNA and reassessment of miscoding potential.

3,N(4)-Ethano-2'-deoxycytidine (ethano-dC) may be incorporated successfully into synthetic oligodeoxynucleotides by omitting the capping procedure used in the automated DNA synthetic protocols immediately after inserting the lesion and in all iterations thereafter. Ethano-dC is sensitive to acetic anhydride found in the capping reagent, and multiple oligomeric products are formed. These products were identified by examining the reaction of ethano-dC with the capping reagent, and several acetylated, ring-opened products were characterized by electrospray mass spectrometry and collision induced dissociation experiments on a tandem quadrupole mass spectrometer. A scheme for the formation of the acetylated products is proposed. In addition, the mutagenic profile of ethano-dC was re-examined and compared to that for etheno-dC. Ethano-dC is principally a blocking lesion; however, when encountered by the exo(-)Klenow fragment of DNA polymerase, dAMP (22%), TMP (16%), dGMP (5.3%) and dCMP (1.2%) were all incorporated opposite ethano-dC, along with an oligomer containing a one-base deletion (0.6%).

Mutagenic potential of alpha-(N2-deoxyguanosinyl)tamoxifen lesions, the major DNA adducts detected in endometrial tissues of patients treated with tamoxifen.

Breast cancer patients treated with the antiestrogen tamoxifen (TAM) show an increased risk of developing endometrial cancer. We have recently detected TAM-DNA adducts in endometrium obtained from patients treated with TAM and identified them as trans- and cis-forms of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM). To explore the mutagenic properties of these TAM-DNA adducts, we prepared site-specifically modified oligodeoxynucleotides containing a single isomer of dG-N2-TAM by reacting a 15-mer oligodeoxynucleotide containing a single dG (5'-TCCTCCTCGCCTCTC) with tamoxifen alpha-sulfate. These modified oligodeoxynucleotides were inserted into a single-stranded shuttle vector to investigate mutagenic specificities of the adducts in simian kidney (COS-7) cells. An epimer of dG-N2-trans-TAM showed targeted mutations ranging from 0.7 to 1.5%. The other dG-N2-trans-TAM adduct showed 9.6% G-->T transversions, accompanied by 2.8% G-->A transitions. Both dG-N2-cis-TAM adducts showed similar mutation spectra, where G-->T transversions (11-12%) predominated, along with a small number of G-->A transitions and G-->C transversions. Thus, dG-N2-TAMs are mutagenic lesions in mammalian cells. The tamoxifen-DNA adducts detected in patient endometrium may cause mutations and initiate endometrial cancer.

Excision of tamoxifen-DNA adducts by the human nucleotide excision repair system.

The antiestrogen tamoxifen is used in the treatment of breast cancer and has recently been recommended as a chemopreventive drug for women at high risk for breast cancer. However, women treated with the drug have an increased incidence of endometrial cancer. It has been suggested that this endometrial cancer might result from mutagenic DNA adducts, which are formed by electrophilic tamoxifen species generated by metabolic activation of the drug. Because the frequency of damage-induced mutations is strongly dependent on the repairability of the lesion, we investigated the repair of the major tamoxifen-DNA adducts by the human nucleotide excision repair system. Using the reconstituted human excision repair system and synthetic DNA substrates, we found that the four types of tamoxifen-DNA adducts detected in the endometrium were repaired with moderate to poor efficiency by nucleotide excision repair. It is concluded that individual variations in repair capacity may play a role in the development of tamoxifen-induced endometrial cancer.

Alpha-hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts.

When alpha-hydroxytamoxifen (alpha-OHTAM) was incubated with rat liver hydroxysteroid (alcohol) sulfotransferase a (STa) and 3'-phosphoadenosine 5'-phosphosulfate, (E)-alpha-OHTAM was found to be a better substrate for STa than (Z)-alpha-OHTAM. To explore the formation of tamoxifen (TAM)-derived DNA adducts, DNA was incubated with STa and either (E)-alpha-OHTAM or (Z)-alpha-OHTAM in the presence of 3'-phosphoadenosine 5'-phosphosulfate. Using 32P-postlabeling analysis, the amount of TAM-DNA adducts resulting from (E)-alpha-OHTAM was 29 times higher than that observed with (E)-alpha-OHTAM alone. Using (Z)-alpha-OHTAM and STa, some TAM-DNA adducts were also detected but at levels 6.5 times lower than that observed with (E)-alpha-OHTAM and STa. When compared with standards of stereoisomers of 2'-deoxyguanosine 3'-monophosphate-N2-tamoxifen, the major tamoxifen adduct was identified chromatographically as an epimer of the trans form of alpha-(N2-deoxyguanosinyl)tamoxifen, and the minor adduct was identified as an epimer of the cis form. In the reaction mixture, a conversion from (E)-alpha-OHTAM to (Z)-alpha-OHTAM through the carbocation intermediate was also detected. These results show that sulfation of alpha-OHTAM catalyzed by STa results in the formation of TAM-DNA adducts.

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Miscoding during DNA synthesis on damaged DNA templates catalysed by mammalian cell extracts.

Oligodeoxynucleotides, modified site-specifically with 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG), 7,8-dihydro-8-oxoadenosine (8-oxodA) and 6-O-methyldeoxyguanosine (O6medG), were used as templates for DNA synthesis in primer-extension reactions catalysed by extracts prepared from human (HeLa) cells, simian kidney (COS-7) cells and various mouse tissues. Fully-extended reaction products were analysed by two-phase polyacrylamide gel electrophoresis (Shibutani, Chem. Res. Toxicol. 6, 625, 1993). Using extracts prepared from HeLa or COS-7 cells, dAMP was preferentially incorporated opposite 8-oxodG; dTMP was incorporated opposite 8-oxodA and dTMP, accompanied by small amounts of dCMP, was incorporated opposite O6medG. Translesional synthesis was strongly inhibited by N-ethylmaleimide and partially inhibited by N-butylphenyl-dGTP. This model system can be used to predict the mutagenic potential of selectively-damaged DNA in mammalian cells.

Molecular mechanisms of mutagenesis by aromatic amines and amides.

The mutagenic properties of 2-acetylaminofluorene-derived DNA adducts, including N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, N-(deoxyguanosin-8-yl)-2-aminofluorene, N-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, and several minor oxidation products have been explored, using site-specific techniques. Oligodeoxynucleotides containing a single AAF-derived DNA adduct were prepared by postsynthetic modification and used as templates in primer extension reactions catalyzed by bacterial and mammalian DNA polymerases. Base substitutions and deletions occurring during DNA synthesis were quantified. dG-C8-AAF promoted one- and two-base deletions and small amounts of incorporation of dCMP, dAMP, and/or dTMP opposite the lesion in reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of DNA polymerase 1 (exo-) and polymerase alpha. dG-C8-AF did not miscode in reactions catalyzed by exo-; however, base misincorporation and deletions were observed in reactions with pol alpha. dG-N2-AAF promoted small amounts of dAMP incorporation in reactions catalyzed by exo-. The miscoding potential of minor oxidation products of dG-C8-AF was much higher than that of other adducts. Steady-state kinetics were used to measure frequencies of nucleotide insertion opposite the lesion and chain extension from the 3' terminus. Kinetic data were consistent with the results of primer extension studies. A mutation 'hot spot' was constructed and the influence of sequence context on the frequency of deletions generated by dG-C8-AAF was explored systematically in reactions catalyzed by exo-. Based on our results with aminofluorene DNA adducts, we propose a general mechanism for frameshift deletion mutagenesis. Site-specific methods also were used to establish the mutagenic potential of AAF-derived DNA adducts in mammalian cells. dG-C8-AAF and dG-C8-AF exhibited similar mutagenic specificities, predicting the occurrence of G-->T transversions and G-->A transitions in mammalian cells.

Differences in detecting blasts between ADVIA 2120 and Beckman-Coulter LH750 hematology analyzers.

The complete blood count is often used as a screening tool to detect hematologic abnormalities in the peripheral blood. In patients with suspected or known leukemia, blast cells serve as an indicator of disease pathology. We compared the analytical performance of the Siemens ADVIA 2120 to the Beckman-Coulter LH750 in the detection of blasts. In the study, 390 blood samples were analyzed from a general hospital population, which included oncology patients. The presence of blasts, as indicated by the analyzers' flags, was compared with the results of a manual differential (regarded as the reference method). The ADVIA 2120 demonstrated 100% sensitivity at detecting blasts compared with 62% using the LH750. This improved sensitivity came at the expense of a lower specificity (49% vs. 86%). The effect of the false-positive and false-negative samples on the laboratory's manual review was partially (false-positive) or completely (false-negative) mitigated by triggering of other criteria. Detecting blasts in the peripheral blood depends on the performance characteristics of the hematology analyzer, in conjunction with the stringency of the laboratory's manual review criteria.

Quantitation of base substitutions and deletions induced by chemical mutagens during DNA synthesis in vitro.

An experimental system has been developed by which base substitutions and frameshift deletions can be quantitated in vitro, using two-phase 20% polyacrylamide gel electrophoresis. Oligodeoxynucleotides, modified site-specifically, were used as templates in primer extension reactions catalyzed by DNA polymerase alpha, polymerase beta, and the Klenow fragment of Escherichia coli DNA polymerase I, with and without 3'-->5' exonuclease activity. Lesions studied included 7,8-dihydro-8-oxodeoxyguanosine, 7,8-dihydro-8-oxodeoxyadenosine, O6-methyldeoxyguanosine, N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene, and N-(deoxyguanosin-8-yl)-2- aminofluorene. Products of translesional synthesis contained dC, dA, dG, or dT opposite the lesion or one- and two-base deletions and were separated using a two-phase polyacrylamide gel system. When a template containing 8-oxoguanine was used, dAMP and/or dCAMP was incorporated opposite the lesion, the relative amounts depending on the DNA polymerase used. In contrast, the nonmutagenic base, dTMP, was incorporated exclusively opposite 8-oxodA in reactions catalyzed by Klenow fragment and pol alpha. The improved resolution provided by the two-phase gel system revealed misincorporation of dGMP opposite 8-oxodA in reactions catalyzed by pol beta. dTMP and small amounts of dCMP were incorporated opposite the lesion on an O6MedG-modified template. The bulky adduct, dG-C8-AAF, principally produced deletions; in contrast, dG-C8-AF promoted incorporation of dCMP, a nonmutagenic base. This experimental system should prove useful for establishing the miscoding potential of defined lesions in DNA templates and in correlating this information with the mutagenic properties of DNA adducts observed in cells.