急性淋巴细胞白血病患者DNA拓扑异构酶的表达及临床意义

 目的 探讨急性淋巴细胞白血病（ALL）患者DNA拓扑异构酶（Topo）mRNA水平的表达及其临床意义。方法 用半定量反转录-聚合酶链反应（RT-PCR）技术检测了90例ALL患者DNA Topo的表达。结果 DNA Topo各亚型（Topo-Ⅰ,Topo-Ⅱa,Topo-Ⅱb）的表达阳性率初治组（58.1 %,51.2 %,81.5 %）高于复发难治组（33.3 %,44.4 %,77.9 %）和完全缓解（CR）组（14.9 %,23.4 %,34.1 %）,各组比较差异均有统计学意义（P＜0.05）。表达量各组间比较,CR组与初治组差异有统计学意义（P＜0.005）;CR组与复发难治组Topo-Ⅱb差异有统计学意义（P＜0.05）,DNA To各亚型表达量之间有显著相关性（P＜0.001）,且各亚型表达量与WBC数之间亦有显著相关性（P＜0.01）。结论 ALL患者DNA Topo各亚型mRNA水平的表达量可能与WBC数共同作为Topo抑制剂药物个体化合理用药的参考指标,但不宜作为判断患者是否对Topo抑制剂耐药的参考指标。     

DNA拓扑异构酶Ⅰ在小细胞肺癌组织中的表达及其意义

目的探讨DNA拓扑异构酶I(Topo I)在小细胞肺癌(SCLC)组织中的表达及其临床意义。方法应用免疫组织化学S-P法,检测50例SCLC组织标本和12例正常肺组织标本中Topo I的表达情况。结果50例SCLC病理组织标本中,39例呈现Topo I的细胞核阳性表达,阳性率为78.0%(39/50);4个等级( 、 、 、 )的阳性率分别为34.0%(17/50)、18.0%(9/50)、10.0%(5/50)和16.0%(8/50)。12例正常肺组织中,3例出现阳性表达,阳性率为25.0%(3/12)。SCLC组与正常对照组比较,差异有统计学意义(P<0.05)。SCLC临床分期与Topo I表达呈正相关关系,随着临床分期的增高,Topo I阳性表达愈高。淋巴结转移阴性和阳性患者的Topo I表达阳性率分别为44.4%和85.4%,差异有统计学意义(P<0.05);Topo I表达与淋巴结转移相关,但与年龄、性别、吸烟指数、肿瘤大小、肿瘤部位无关(P>0.05)。结论肿瘤组织细胞中Topo I高表达是TopoI抑制剂应用于肿瘤治疗的理论根据,通过检测TopoI在SCLC组织中的表达情况,可预测患者是否对化疗药物敏感,提高疗效。     

Enhanced etoposide sensitivity following adenovirus-mediated human topoisomerase IIalpha gene transfer is independent of topoisomerase IIbeta

The roles that the alpha and beta isoforms of topoisomerase II (topo II) play in anticancer drug action were determined using MDA-VP etoposide-resistant human breast cancer cells and a newly constructed adenoviral vector containing the topo IIalpha gene (Ad-topo IIalpha). MDA-VP cells were more resistant to etoposide than to amsacrine and had more resistance to etoposide than did MDA-parental cells. MDA-VP cells also expressed lower topo IIalpha RNA and protein levels than parental cells but had comparable topo IIbeta levels. After infection with Ad-topo IIalpha, topo IIalpha, RNA and protein levels increased significantly, as did the cells' sensitivity to etoposide. In contrast, topo IIbeta levels remained constant with little alteration in the cells' sensitivity to amsacrine. Band-depletion immunoblotting assays indicated that topo IIalpha was depleted in etoposide-treated, Ad-topo IIalpha-transduced MDA-VP cells but not in amsacrine-treated cells. Topo IIbeta was depleted in amsacrine-treated, Ad-topo IIalpha-MDA-VP cells, with little change in the topo IIalpha levels. These results suggest that topo IIalpha gene transfer does not alter topo IIbeta expression and that enhanced sensitivity to etoposide is therefore secondary to change in topo IIalpha levels. These studies support the theory that etoposide preferentially targets topo IIalpha, while amsacrine targets topo IIbeta.     

In vivo etoposide-resistant C6 glioma cell line: Significance of altered DNA topoisomerase II activity in multi-drug resistance

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase II isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase II in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase II isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.     

Resistance to topoisomerase poisons due to loss of DNA mismatch repair

Sporadic breast carcinomas demonstrate microsatellite instability, reflecting the presence of DNA mismatch repair-deficient cells, in about one fourth of cases at the time of diagnosis. Loss of DNA mismatch repair has been reported to result in resistance not only to cisplatin and alkylating agents but also to the topoisomerase II poison doxorubicin, suggesting an association between DNA mismatch repair and topoisomerase II poison-induced cytotoxicity. Our study investigates the relationship between loss of MSH2 or MLH1 function and sensitivity to the topoisomerase I and II poisons, and to the taxanes, 2 classes of cytotoxic drugs commonly used in breast cancer. Two pairs of cell lines proficient and deficient in mismatch repair due to loss of either MSH2 or MLH1 function were used. Loss of either MSH2 or MLH1 function resulted in resistance to the topoisomerase II poisons doxorubicin, epirubicin and mitoxantrone, whereas only loss of MLH1 function was associated with low-level resistance to the topoisomerase I poisons camptothecin and topotecan. In contrast, there was no resistance to docetaxel and paclitaxel. Our data support the hypothesis that both MSH2 and MLH1 are involved in topoisomerase II poison-mediated cytotoxicity, whereas only MLH1 is involved in topoisomerase I poison-mediated cytotoxicity. Since our study shows that loss of DNA mismatch repair does not result in resistance to the taxanes, these drugs can be recommended for use in breast cancer deficient in mismatch repair.     

Identification of gene polymorphisms of human DNA topoisomerase I in the National Cancer Institute panel of human tumour cell lines

Topoisomerase 1 (Top1), a nuclear enzyme involved in DNA relaxation, is the target of several anticancer drugs. TOP1 mutations occur in camptothecin-resistant tumour cell lines. We explored, in the NCI panel of 60 human tumour cell lines, whether polymorphic variations in the TOP1 gene could explain differences in drug sensitivity. The 21 exons of the gene were fully studied as well as five intronic domains that had previously been shown to harbour single nucleotide polymorphisms (SNPs) or mutations. PCR products covering the whole exonic sequences or the relevant intronic domains were subjected to denaturing high-performance liquid chromatography. Nucleotide variations were then determined by sequencing. Discrimination between intronic common and variant homozygous samples was performed using a restriction fragment length polymorphism technique. Only one exonic mutation was detected, at the heterozygous state; it occurs in exon 19 of a colon cancer cell line (HCT-15) and consists of a G>A transition at position 75, resulting in a Met675Ile change. The intronic sequences studied harboured the SNPs expected with allelic frequencies between 20 and 40%. Three major haplotypes, generating 92% of the 10 genotypes encountered, were defined as containing none of the intronic SNPs, or three of them, or all of them. No significant relationship was evidenced between Top1 expression and the TOP1 polymorphisms studied. However, when comparing the cytotoxicity of 138 drugs as a function of the genotypes, several drug groups, namely Top1 inhibitors, antifolates and taxanes, had significantly different IC(50)s as a function of the distribution of the intronic SNPs of the TOP1 gene.     

Detection of dermatophytes in human nail and skin dust produced during podiatric treatments in people without typical clinical signs of mycoses

Pedicures are the most common cosmetic foot treatment. Many pedicurists and podiatrists suffer from respiratory infections and diseases such as asthma, sinusitis, chronic cough and bronchitis. Skin and nail dust may play an important role in the development of occupational diseases and the transmission of mycosis to other clients. To examine the presence of dermatophytes in nail and skin dust produced during podiatric treatments of people without typical symptoms of mycosis and to assess the epidemiological hazards of tinea pedis for podiatrists as well as other clients. Seventy‐seven samples underwent direct microscopy and culture. The results of direct microscopy were positive in 28/77 samples (36.36%) and doubtful in 3/77 (3.9%). Fungi were cultured from 36/77 samples (46.75%), including 8/77 (10.3%) positive for dermatophytes (Trichophyton rubrum‐6 isolates and Trichophyton mentagrophytes‐2). Material collected during podiatric treatments is potentially infected by pathogenic fungi; thus, there is a need to protect both workers who perform such treatments, as well as other clients, to prevent the transmission of pathogens in the Salon environment. Exposure to this occupational hazard may increase not only the risk of respiratory infections but also increase asthmatic or allergic reactions to Trichophyton.     

Perniosis-like tinea corporis caused by Trichophyton verrucosum in cold-exposed individuals

Trichophyton verrucosum is a zoophilic infectious agent causing 98% of the dermatophytic infections of cattle. Transmission to humans has, until recently, been rare. One reason for an increase of infection in humans and animals seems to be the decrease in immunisation of cattle. We report on three cases of pertinent human infections with disseminated, sharply defined, bluish red, partly oedematous nodules and plaques in particular not only on the thighs, but also on the trunk and arms. Two of our patients work with farm animals. The third one works as an assistant in a butcher shop, but lives on a cow farm. All three patients are often exposed to the cold. In all three cases T. verrucosum was detected by culture. Tinea corporis was histologically confirmed in two patients. Based on the microbiological results, we began a combined systemic and local antimycotic therapy with fluconazole 50 mg day(-1) in two patients, itraconazole 100 mg day(-1) in one patient p.o. combined with topical ciclopiroxolamine. All patients were cured. Dermatophytosis caused by T. verrucosum can, under certain circumstances, such as frequent exposure to cold or a long-term corticosteroid therapy, mimic the characteristic clinical picture of perniosis, as we demonstrate here.     

Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.     

DNA topoisomerase IIalpha predicts progression-free and overall survival in pediatric malignant non-brainstem gliomas

Malignant non-brainstem glioma (MNBG) is a rare pediatric brain tumor. The prognosis for children harboring this lesion remains largely unpredictable. Assessment of histologic features alone only provides a marginal insight into the biologic behavior of these lesions. Hence, the identification of novel molecular markers capable of characterizing these lesions more accurately with respect to their biologic aggressiveness is definitely needed. Our current study examined the expression of nuclear DNA topoisomerase IIalpha (TIIalpha), a novel marker of cell cycle turnover and a determinant of tumor cell resistance to chemotherapy, in a series of 17 archival pediatric MNBGs. TIIalpha expression was found to extend over a wide range in the study cohort (3.9-69.1%). A cutoff labeling index of 12% was found to define 2 prognostic subgroups (TIIalpha <12 vs. >or=12) with profoundly different 5-year progression-free survival (60% vs. 8%; p = 0.0108, log-rank test) and overall survival (100% vs. 8%; p = 0.0038) rates. TIIalpha expression was significantly linked to MIB-1 antibody labeling of the Ki-67 nuclear antigen (R = 0.919, p < 0.001). A high TIIalpha labeling index remained associated with short progression-free survival (p = 0.022) and overall survival (p = 0.022) in multivariate analysis (Cox regression). In conclusion, considering that TIIalpha expression was not related to histopathologic grade, biological characteristics as assessed by TIIalpha labeling may complement the information obtained by tumor morphology as a means of improving the accuracy of patient prognosis prediction.     

Comparison of the in vitro activity of terbinafine and lanoconazole against dermatophytes

The objective of this study was to compare the antifungal activity of terbinafine (TERB) with that of lanoconazole (LAN). Test isolates, which were clinical isolates of Japanese origin, included 10 strains each of Trichophyton rubrum, T. mentagrophytes and Epidermophyton floccosum. The minimum inhibitory concentration (MIC) of TERB and LAN against each dermatophyte isolate was determined according to the Clinical and Laboratory Standards Institute microbroth methodology, M38‐A2. Minimum fungicidal concentrations were determined by subculturing the contents of each visibly clear well from the MIC assay for colony count. All LAN MICs were ≤0.008 μg ml^?1, while the TERB range was 0.008–0.03 μg ml^?1. Moreover, by standard definition, LAN was fungistatic against most strains, whereas TERB was fungicidal. Both LAN and TERB demonstrated potent antifungal activity against dermatophytes; however, the lack of fungicidal activity by LAN needs to be evaluated in terms of potential clinical efficacy.     

Role of Chrysosporium keratinophillum in the parasitic evolution of dermatophytes

Anti-dermatophytic activity of Chrysosporium keratinophillum against species of the genera Trichophyton, Microsporum and Epidermophyton floccosum was tested in vitro. When C. keratinophillum and different species of dermatophytes were inoculated on Sabouraud's dextrose agar plates 2 cm apart, no antagonistic effect of C. keratinophillum on the mycelial growth of dermatophytes was observed. However, conidia production was not observed on the hyphae of Trichophyton rubrum, Trichophyton tonsurans and E. floccosum grown near C. keratinophillum. The secretory substances released by C. keratinophillum inhibited the growth of T. rubrum, T. tonsurans, Trichophyton mentagrophytes var. interdigitale and E. floccosum at a concentration of 2,000 microg ml(-1) when tested by broth dilution technique. No inhibition of the growth was observed for Microsporum gypseum and Microsporum nanum. The anti-fungal activity of secretory substances released by C. keratinophillum was recorded to be heat stable. Results of the present study suggest that the anti-dermatophytic activity of the secretory substances of C. keratinophillum on T. rubrum, T. mentagrophytes var. interdigitale, T. tonsurans and E. floccosum may be responsible in part, for the absence of these dermatophyte species in soil. Considering the global prevalence of C. keratinophillum in soil one may speculate that the anti-dermatophytic activity of C. keratinophillum is one of the early events for the evolutionary divergence of saprophytic archi-dermatophytes to obligate parasitic dermatophyte species.     

Preferential potentiation of topoisomerase I poison cytotoxicity by PARP inhibition in S phase

Background:

Topoisomerase I (Topo I) poisons (e.g., camptothecin (CPT)), used to treat cancer, cause DNA breaks that are most cytotoxic during S phase. PARP-1 promotes DNA repair and PARP inhibitors (PARPi) sensitise cells to Topo I poisons. We aimed to determine whether chemosensitisation is also S phase specific using rucaparib, a potent PARPi in advanced clinical evaluation.

Methods:

The impact of rucaparib, on CPT-induced cytotoxicity was measured in human colon cancer (LoVo) and leukaemic (K562) cells in asynchronous and cell cycle phase-separated cultures. Topoisomerase I and PARP levels and activity and the effect of rucaparib on DNA single-strand breaks (SSBs), double-strand breaks (DSBs) and collapsed replication fork induction and repair were determined in cell cycle phase-separated cells.

Results:

The cytotoxicity of CPT was greatest during S phase, partially attributable to high Topo I activity, and rucaparib preferentially sensitised S-phase cells. Rucaparib increased CPT-induced DNA SSBs in all phases of the cell cycle, and increased DSB and γH2AX foci in S and G2, with γH2AX foci being highest in S-phase cells. Repair of SSBs and DSBs was most rapid during S then G2 phases and was substantially hindered by rucaparib.

Conclusions:

Rucaparib preferentially sensitises S-phase cells by increasing the frequency of collapsed replication forks.     

DNA拓扑异构酶Ⅰ在骨肉瘤中的表达及临床意义

目的 研究骨肉瘤中TopoⅠ的表达情况,探讨其与TopoⅡ、Ki67之间的相互关系和临床意义。方法 采用S-P免疫组化方法,检测TopoⅠ、TopoⅡ和Ki67在67例骨肉瘤、10例骨样骨瘤、15骨软骨瘤及15例正常骨组织中的表达,分析相互间的关系,对其中31例随访资料进行生存分析。结果 67例骨肉瘤中,TopoⅠ、TopoⅡ与和Ki67的表达率分别为22.39％、44.78％和40.30％,均明显增高,同患者术后生存率呈负相关;TopoⅠ、TopoⅡ的表达率与应用于临床的抗DNA拓扑异构酶化疗药物有效率相近。结论骨肉瘤中有小部分患者对于新的以TopoⅠ为靶点的化疗药物敏感,对骨肉瘤组织行免疫组化方法检测TopoⅠ、TopoⅡ可能作为骨肉瘤患者选择化疗方案一个较为简单又有意义的指标。     

Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction

Resveratrol, a stilbene found in grapes and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines. Here we report that resveratrol treatment of human glioblastoma cells induces a delay in cell cycle progression during S phase associated with an increase in histone H2AX phosphorylation. Furthermore, with an in vitro assay of topoisomerase IIα catalytic activity we show that resveratrol is able to inhibit the ability of recombinant human TOPO IIα to decatenate kDNA, so that it could be considered a TOPO II poison.