Effect of O6-benzylguanine on nitrogen mustard-induced toxicity,apoptosis, and mutagenicity in Chinese hamster ovary cells

O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 microM BG results in enhancement in the cytotoxicity of PM (300 microM), chlorambucil (40 microM), and melphalan (10 microM) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 microM PM is decreased from 259 mutants/10(6) cells to 22 mutants/10(6) cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a monoalkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72-94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57% of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards.     

Potentiation of antitumor drug action by centrophenoxine: specificity.

The cytotoxic action of certain antitumor agents is potentiated by centrophenoxine although centrophenoxine itself is not an antitumor agent. Previous investigations have suggested that centrophenoxine might potentiate the cytotoxicity produced by antitumor drugs that alkylate, and other modalities that damage, DNA, but that it would not potentiate the cytotoxicity produced by antitumor drugs that inflict cellular damage in other ways. To test this hypothesis, the antitumor effects of X-irradiation UV-irradiation, alkylating agents and antitumor drugs that are not ordinarily considered to be alkylating agents were determined in the presence and absence of centrophenoxine. Mouse P388 lymphoma cells growing in static suspension culture were used as the experimental tumor. The cytotoxic action of most alkylating agents was found to be potentiated by centrophenoxine; Included in this group were several difunctional nitrogen mustards, two ethylenimines, a nitrosourea and mitomycin C. Greatest enhancement, 7-fold, was of chlorambucil antitumor activity. Centrophenoxine did not potentiate the lethality of X- or UV-irradiation or the cytotoxicity of several antineoplastic drugs that are not alkylating agents.     

Therapeutic targeting the loss of the birt-hogg-dube suppressor gene

Brit-Hogg-Dubé (BHD) syndrome, an autosomal dominant familial cancer, is associated with increased risk of kidney cancer. BHD syndrome is caused by loss-of-function mutations in the folliculin (FLCN) protein. To develop therapeutic approaches for renal cell carcinoma (RCC) in BHD syndrome, we adopted a strategy to identify tumor-selective growth inhibition in a RCC cell line with FLCN inactivation. The COMPARE algorithm was used to identify candidate anticancer drugs tested against the NCI-60 cell lines that showed preferential toxicity to low FLCN expressing cell lines. Fifteen compounds were selected and detailed growth inhibition (SRB) assays were done in paired BHD RCC cell lines (UOK257 derived from a patient with BHD). Selective sensitivity of FLCN-null over FLCN-wt UOK257 cells was observed in seven compounds. The most selective growth-inhibitory sensitivity was induced by mithramycin, which showed an approximately 10-fold difference in GI(50) values between FLCN-null (64.2 ± 7.9 nmol/L, n = 3) and FLCN-wt UOK257 cells (634.3 ± 147.9 nmol/L, n = 4). Differential ability to induce caspase 3/7 activity by mithramycin was also detected in a dose-dependent manner. Clonogenic survival studies showed mithramycin to be approximately 10-fold more cytotoxic to FLCN-null than FLCN-wt UOK257 cells (200 nmol/L). Following mithramycin exposure, UOK257-FLCN-null cells were mainly arrested and blocked in S and G(2)-M phases of the cell cycle and low dose of rapamycin (1 nmol/L) potentiated mithramycin sensitivity (1.5-fold in G(2)-M population and 2-fold in G(2)-M period time, 2xGI(50), 48 hours). These results provide a basis for further evaluation of mithramycin as a potential therapeutic drug for RCC associated with BHD.     

Cidal and subcidal effect of triethylene thiophosphoramide on cell kinetics of human bladder carcinoma cells in vitro.

In view of increasing topical use of various chemotherapeutic agents for bladder carcinoma, an experimental study concerning the effect of triethylene thiophosphoramide (Thio-Tepa) on cell kinetics of bladder carcinoma cells was performed, working on an established cell line of human bladder carcinoma. This polyfunctional alkylating agent, which is most widely used for instillation chemotherapy of bladder carcinoma, revealed a concentration dependent cytotoxicity against the cells. DNA precursor incorporation suggested that repair mechanism occurred following subcidal dose of this compound and faulty repair took place following cidal dose. The cell cycle was prolonged after subcidal treatment, the main effect being seen in DNA synthetic phase, and the changes in the cell cycle parameters returned to the normal within 2 cell cycle time. Repeated treatment with subcidal dose at an interval of 48 hr led to more extensive changes in the cell cycle as compared with that of single dose. Repeated exposures to subcidal dose, however, did not show any differences in the growth curves from those of the controls.     

Generation of a drug resistance profile by quantitation of mdr-1/P-glycoprotein in the cell lines of the National Cancer Institute Anticancer Drug Screen.

Identifying new chemotherapeutic agents and characterizing mechanisms of resistance may improve cancer treatment. The Anticancer Drug Screen of the National Cancer Institute uses 60 cell lines to identify new agents. Expression of mdr-1/P-glycoprotein was measured by quantitative PCR. Expression was detected in 39 cell lines; the highest levels were in renal and colon carcinomas. Expression was also detected in all melanomas and central nervous system tumors, but in only one ovarian carcinoma and one leukemia cell line. Using a modified version of the COMPARE program, a high correlation was found between expression of mdr-1 and cellular resistance to a large number of compounds. Evidence that these compounds are P-glycoprotein substrates includes: (a) enhancement of cytotoxicity by verapamil; (b) demonstration of cross-resistance in a multidrug-resistant cell line, (c) ability to antagonize P-glycoprotein, increasing vinblastine accumulation by decreasing efflux; and (d) inhibition of photoaffinity labeling by azidopine. Identification of many heretofore unrecognized compounds as substrates indicates that P-glycoprotein has a broader substrate specificity than previously recognized. This study confirms the validity of this novel approach and provides the basis for similar studies examining a diverse group of gene products, including other resistance mechanisms, putative drug targets, and genes involved in the cell cycle and apoptosis.     

CDH11基因在肾细胞癌中的甲基化状态及其临床意义研究

目的检测CDH11基因启动子在肾癌中的甲基化情况,并分析甲基化与临床病理特征的关系。方法通过甲基化特异性PCR（MSP）检测CDH11基因在5株肾细胞癌细胞系、1株正常肾细胞系、46例肾细胞癌组织、23例癌旁肾组织,以及10例非肾实质肿瘤正常。肾组织中的甲基化状态。将甲基化情况与患者I临床资料联系,进行统计学分析。结果CDH11在5株肾癌细胞系中,有3株出现甲基化,甲基化率为60％;在人正常肾细胞系中未检测到甲基化。CDH11在肾癌组织中的甲基化率为45．7％（21／46）,显著高于癌旁肾组织（26．1％,6／23）及非肾实质肿瘤正常肾组织（0,（3／10）,差异具有统计学意义（P〈0．05）。肾细胞癌各分期间及各分级间,癌组织中CDH11基因甲基化检出率无统计学意义（P〉0．05）;左侧肾癌与右侧肾癌相比,癌组织CDH11基因甲基化率差异无统计学意义（P〉0．05）;男性肾癌患者与女性肾癌患者相比,肾癌组织中CDH1基因甲基化率差异无统计学意义（P〉0．05）。结论肾细胞癌组织中CDH11基因甲基化率显著高于癌旁正常肾组织及非肾实质肿瘤正常肾组织,且癌组织中CDH11基因甲基化率与临床病理资料如肿瘤分期及分级无显著相关性,提示CDH11基因甲基化是。肾细胞癌发生中的早期频发事件,可能是。肾细胞癌独特的基因甲基化谱成员之一,并在肾细胞癌的早期诊断上发挥作用。     

Different DNA lesions trigger distinct cell death responses in HCT116 colon carcinoma cells

The pleiotrophic cellular response to DNA damage includes activation of cell cycle checkpoints, induction of DNA repair pathways, and initiation of programmed cell death among others. The fate of cells with damaged DNA depends on the coordination of these different responses. The clinical efficacy of genotoxic therapies is influenced by cell fate and thus by how the DNA damage response is coordinated. While a great deal has been learned about how different DNA lesions activate distinct cell cycle checkpoints and DNA repair pathways, less is known about whether the type of DNA lesion influences the qualitative and quantitative nature of the cell death response. To address this question, HCT116 colon carcinoma cells have been treated with equally cytotoxic doses of the antitumor DNA alkylating agents adozelesin or bizelesin or the DNA strand scission agent C-1027. The relative contribution of cell cycle arrest and cell death to measured cytotoxicity varied among the three drugs. Apoptotic cell death accounts for most C-1027 cytotoxicity while cell cycle arrest and cell death both contribute to the cytotoxicity of the alkylating agents. Each of the drugs induces a distinct but overlapping pattern of caspase activation. In addition, the cell death response to these drugs is differentially dependent on p53 and p21. These observations suggest that the type of DNA lesion influences not only the relative extent of apoptotic cell death at a given cytotoxic dose but also the qualitative nature of that response.     

MicroRNA-199a-3p在肾癌中的表达及作用

目的检测microRNA-199a-3p（miR-199a-3p）在肾癌细胞株和组织中的表达情况并探究miR-199a-3p在肾癌细胞中的作用。方法利用实时定量RT-PCR检测miR-199a-3p在肾癌细胞和组织中的表达水平;利用miR-199a-3p模拟物转染肾癌细胞786-0上调miR-199a-3p后,通过CCK-8、克隆形成、Transwell以及细胞周期检测来探究其在肾癌细胞中的作用。结果 miR-199a-3p在肾癌细胞中明显低表达,在78%（14/18）的肾癌组织中亦明显低表达;上调miR-199a-3p可显著抑制肾癌细胞的增殖、存活和侵袭并能诱导细胞周期G1期阻滞。结论我们的研究显示在肾癌中miR-199a-3p明显低表达并参与肾癌的发生、发展,这表明miR-199a-3p具有作为肾癌诊断和治疗靶点的潜能。     

Alchemix: a novel alkylating anthraquinone with potent activity against anthracycline- and cisplatin-resistant ovarian cancer

Chloroethylaminoanthraquinones are described with intercalating and alkylating capacity that potentially covalently cross-link topoisomerase II (topo II) to DNA. These compounds have potent cytotoxic activity (IC(50) = 0.9-7.6 nM) against the A2780 human ovarian carcinoma cell line. Hydroxyethylaminoanthraquinones also reported in this paper have similar IC(50) values (0.7-1.7 nM) in the same cell line. Alchemix (ZP281M, 1-(2-[N,N-bis(2-chloroethyl)amino]ethylamino)-4-(2-[N,N-(dimethyl)amino]ethylamino)-5,8-dihydroxy-9,10-anthracenedione), an alkylating anthraquinone, retains excellent antitumor activity in Adriamycin-resistant (2780AD) and cisplatin-resistant (2780/cp70) cell lines in vitro and in vivo. This indicates that Alchemix can evade both P-glycoprotein efflux pump and DNA mismatch repair-mediated resistance. In treated cells, Alchemix was shown to preferentially induce drug-stabilized covalent bound topo IIalpha-DNA complexes over topo IIbeta-DNA complexes.     

Unusual renal cell carcinomas: a pictorial essay

Renal cell carcinoma (RCC) is the most common solid renal neoplasm. Clear cell (conventional) carcinoma is the most common pathologic subtype of RCC. Usually RCC is a hypervascular, solid, solitary mass with contour bulging. However, RCC can manifest different features according to the pathologic tumor subtypes. Preoperative diagnosis of cyst-associated RCC is very difficult, especially in cases of RCC originating in a cyst. Multiple or bilateral presentation of RCC occurs in fewer than 5% of cases. In addition, RCCs may demonstrate unusual findings such as infiltrative growth mimicking transitional cell carcinoma, fatty component mimicking angiomyolipoma, severe perinephric infiltration, and extensive calcifications mimicking inflammation or other tumor. RCCs can be associated with hereditary diseases such as von Hippel-Lindau disease. Familiarity with these radiologic features of unusual RCCs can help ensure correct diagnosis and proper management. Award winning poster at ESUR’04, Santiago de Compostella, Spain     

Molecular targeted therapy for renal cell carcinoma and other urological cancers

In Japan, therapeutic strategy for metastatic renal cell carcinoma(RCC) has been markedly changed since the recent introduction of multiple tyrosine kinase inhibitors, including sorafenib and sunitinib, into the clinical practice. In addition to these agents, inhibitors of mTOR(mammalian target of rapamycin) are scheduled to be available in patients with metastatic RCC near future. In this review, we would like to summarize the current status of these molecular targeted agents for the treatment of RCC, and subsequently describe the important issues associated with the administration of these agents, such as the effects on quality of life and the possible use as neoadjuvant setting. Finally, the prospects for the use of molecular targeted agents against urological cancers other than RCC are mentioned.     

低浓度活性氧逆转肾癌先天性多药耐药的实验研究

目的探讨低浓度活性氧对肾癌786-O细胞先天性多药耐药的逆转作用及相关机制。方法采用WST-1细胞增殖及细胞毒性检测试剂盒确定活性氧H2O2的非细胞毒性剂量,以及对786-O细胞药物敏感性的影响。罗丹明123实验检测细胞P糖蛋白（P-gp）功能,Western blot方法检测经典多药耐药基因（mdr1）产物P-gp的表达。结果在0.00001～0.1mmol/L浓度范围内,H2O2具有明显的促细胞生长作用,且显著增加了阿霉素及长春新碱的细胞毒性,0.02mmol/LH2O2孵育786-O细胞72h后其对阿霉素及长春新碱的药物敏感性分别增加至对照组的5.43及4.47倍,荧光染料罗丹明123的蓄积量显著增加,Western blot检测结果显示0.02mmol/LH2O2可抑制肾癌786-O细胞P-gp的表达。结论低浓度活性氧H2O2可部分逆转人肾癌786-O细胞先天性多药耐药对阿霉素的耐药性,其逆转机制与增加细胞内化疗药物浓度、抑制P-gp的表达有关。     

乳头状肾细胞癌的临床病理特征--附5例报告

目的 探讨乳头状肾细胞癌的临床病理特征及诊断标准。方法 收集５例该肿瘤,作常规ＨＥ及免疫组织化学染色并与７０例非乳头状肾细胞癌比较。结果 乳头状肾细胞癌是一种特殊类型的肾细胞癌,临床经过长,生长缓慢,肾动脉造影表现为少血或无血管性,预后较好。巨检肿瘤边界清楚,有出血囊性变。镜检肿瘤组织呈乳头状或管状乳头状排列,乳状轴心及间人有泡沫状巨噬细胞浸润,有明显出血坏血。结论 乳头状肾细胞癌在临床,血管造影     

Identification of pro-MMP-7 as a serum marker for renal cell carcinoma by use of proteomic analysis

BACKGROUND: No validated renal cell carcinoma (RCC) marker is known for detection of asymptomatic disease in selected populations or for prognostic purposes or treatment monitoring. We identified immunogenic proteins as tumor markers for RCC by combining conventional proteome analysis with serological screening, and we investigated the diagnostic clinical value of such markers in serum. METHODS: We studied the immunogenic protein expression profile of CAL 54, a human RCC cell line, by 2-dimensional electrophoresis combined with immunoblotting using sera from healthy donors compared with RCC patients. We developed a homogeneous, fluorescent, dual-monoclonal immunoassay for metalloproteinase 7 (MMP-7) and used it to measure MMP-7 in sera from 30 healthy donors, 30 RCC patients, and 40 control patients. RESULTS: Pro-MMP-7 (29 kDa; pI 7.7) in the CAL 54 cell line secretome was an immunogenic protein reactive with RCC patient sera but not with control sera. The concentrations of pro-MMP-7 were increased (P <0.0001) in sera of RCC patients (median 7.56 microg/L; range 3.12-30.5 microg/L) compared with healthy controls (median 2.13 microg/L; range 0.17-3.5 microg/L). Serum pro-MMP-7 had a sensitivity of 93% (95% CI 78%-99%) at a specificity of 75% (59%-87%) for RCC in the samples tested. CONCLUSION: Proteomics technology combined with serology led to the identification of serum pro-MMP-7 as a marker of RCC and represents a powerful tool in searching for candidate proteins as biomarkers.     

Epidermal growth factor receptor inhibition sensitizes renal cell carcinoma cells to the cytotoxic effects of bortezomib

In renal cell carcinoma (RCC) models, maximal cytotoxicity of the proteasome inhibitor bortezomib is dependent on efficient blockade of constitutive nuclear factor kappaB (NF-kappaB) activity. Signaling through the epidermal growth factor receptor (EGFR) has been shown to result in NF-kappaB activation. Thus, we sought to investigate whether inhibition of the EGFR sensitizes RCC cells to the cytotoxic effects of bortezomib. We first established that constitutive NF-kappaB activity is dependent on signaling through the EGFR in RCC cells. Indeed, blockade of EGFR signaling with an EGFR tyrosine kinase inhibitor (TKI) resulted in inhibition of NF-kappaB activity. Using pharmacologic and genetic approaches, we also showed that EGFR-mediated NF-kappaB activation occurs through the phosphotidylinositol-3-OH kinase/AKT pathway. Combinations of the EGFR-TKI and bortezomib resulted in synergistic cytotoxic effects when RCC cells were pretreated with the EGFR-TKI, but an antagonistic interaction was observed with bortezomib pretreatment. Evaluation of the effects of drug sequencing on inhibition of NF-kappaB activity revealed that EGFR-TKI pretreatment markedly augmented the NF-kappaB inhibitory effect of bortezomib, whereas bortezomib preexposure resulted in suboptimal NF-kappaB blockade and thus provides a biochemical explanation for the drug interaction results. We conclude that the constitutive NF-kappaB activity observed in RCC cells is mediated, at least in part, through an EGFR/phosphotidylinositol-3-OH kinase/AKT signaling cascade. Pretreatment with an EGFR-TKI sensitizes to bortezomib-mediated cytotoxicity by inhibiting constitutive NF-kappaB activity. The combination of bortezomib and a currently approved EGFR inhibitor warrants clinical investigation.     

Renal cell carcinoma: Evolving and emerging subtypes

Our knowledge of renal cell carcinoma (RCC) is rapidly expanding. For those who diagnose and treat RCC, it is important to understand the new developments. In recent years, many new renal tumors have been described and defined, and our understanding of the biology and clinical correlates of these tumors is changing. Evolving concepts in Xp11 translocation carcinoma, mucinous tubular and spindle cell carcinoma, multilocular cystic clear cell RCC, and carcinoma associated with neuroblastoma are addressed within this review. Tubulocystic carcinoma, thyroid-like follicular carcinoma of kidney, acquired cystic disease-associated RCC, and clear cell papillary RCC are also described. Finally, candidate entities, including RCC with t(6;11) translocation, hybrid oncocytoma/chromophobe RCC, hereditary leiomyomatosis and RCC syndrome, and renal angiomyoadenomatous tumor are reviewed. Knowledge of these new entities is important for diagnosis, treatment and subsequent prognosis. This review provides a targeted summary of new developments in RCC.     

Concentrations of polyamines in renal cell carcinoma

In order to obtain biochemical indicators for histopathological, biological and clinical malignancy of renal cell carcinoma (RCC), concentrations of protein, RNA, DNA and polyamines in 14 samples of RCC were measured and examined on their histopathological properties and clinical malignancies. The concentration of spermidine in RCC was significantly higher than that in normal renal tissue and a statistically significant difference was not detected between the concentrations of the other compounds in RCC and normal tissue. The concentration of spermidine and the ratio of spermidine/spermine were found to increase in the order of normal tissue, the well-differentiated type and the poorly-differentiated type of RCC while no significant difference was detected between the concentrations of the other compounds in these types. There was no difference in the concentrations of the compounds examined among the non-metastasis and istant-metastasis groups of RCC.     

碳酸酐酶Ⅸ在肾癌中的表达及临床意义

目的 探讨碳酸酐酶Ⅸ(CAⅨ/G250)在肾癌组织和血液中的表达情况和临床意义.方法 采用RT-PCR技术检测62例肾癌患者癌组织和外周血液中碳酸酐酶Ⅸ(G250)mRNA的表达,以32例正常肾组织和非肾癌患者外周血作为对照.结果 62例肾癌患者碳酸酐酶Ⅸ(G250)mRNA阳性率在癌组织中为82.3%(51/62),在外周血中为54.8%(34/62),各对照组均无阳性表达,差异有统计学意义(P<0.05);在肾透明细胞癌患者癌组织和外周血中碳酸酐酶Ⅸ(G250)mRNA阳性率分别为98%(49/50)和66%(33/50),显著高于其他类型肾癌(P<0.05).结论 碳酸酐酶Ⅸ(G250)在肾癌尤其是透明细胞癌中有特异性的高表达,可以用于肾癌以及肾癌微转移的诊断和预后评价.     

Metastatic renal cell carcinoma imaging evaluation in the era of anti-angiogenic therapies

During the last decade, the arsenal of anti-angiogenic (AAG) agents used to treat metastatic renal cell carcinoma (RCC) has grown and revolutionized the treatment of metastatic RCC, leading to improved overall survival compared to conventional chemotherapy and traditional immunotherapy agents. AAG agents include inhibitors of vascular endothelial growth factor receptor signaling pathways and mammalian target of rapamycin inhibitors. Both of these classes of targeted agents are considered cytostatic rather than cytotoxic, inducing tumor stabilization rather than marked tumor shrinkage. As a result, decreases in tumor size alone are often minimal and/or occur late in the course of successful AAG therapy, while tumor devascularization is a distinct feature of AAG therapy. In successful AAG therapy, tumor devascularization manifests on computed tomography images as a composite of a decrease in tumor size, a decrease in tumor attenuation, and the development of tumor necrosis. In this article, we review Response Evaluation Criteria in Solid Tumors (RECIST)—the current standard of care for tumor treatment response assessment which is based merely on changes in tumor length—and its assessment of metastatic RCC tumor response in the era of AAG therapies. We then review the features of an ideal tumor imaging biomarker for predicting metastatic RCC response to a particular AAG agent and serving as a longitudinal tumor response assessment tool. Finally, a discussion of the more recently proposed imaging response criteria and new imaging trends in metastatic RCC response assessment will be reviewed.     

PET/CT用于肾细胞癌进展

肾细胞癌（RCC）是泌尿系统最常见的恶性肿瘤之一,约占肾癌的90%。PET/CT已广泛用于RCC的诊断、分级、疗效评价及预后评估等。¹⁸F-FDG为PET最常用显像剂之一,其他新型显像剂也在不断研发中。本文对PET/CT在RCC的应用进展进行综述。