基于生物信息学分析丝甘蛋白聚糖对卵巢癌耐药性的影响及作用机制

目的:研究丝甘蛋白聚糖(SRGN)对卵巢癌耐药性的影响及作用机制。方法:采用基因表达谱交互分析工具(GEPIA)提取卵巢癌相关数据集并分析SRGN m RNA在人类正常卵巢组织与卵巢癌组织的表达差异;基于基因表达数据库(GEO)获取SRGN mRNA在顺铂敏感性与顺铂耐药性卵巢癌细胞株(A2780)中的表达差异;采用STRING在线数据库筛选SRGN的互作蛋白(置信度为0.900,联结数为10),然后通过生物学信息注释数据库(DAVID)进行京都基因和基因组百科全书(KEGG)代谢通路分析,预测SRGN调节卵巢癌耐药性的潜在通路;采用医学本体信息检索平台COREMINE挖掘SRGN、卵巢癌、耐药性三者显著关联的生物过程。结果:SRGN mRNA在卵巢癌组织中的表达显著高于正常卵巢组织(P<0.05),在顺铂耐药性卵巢癌细胞株中表达显著高于顺铂敏感性卵巢癌细胞株(P<0.001)。筛选出10个与SRGN互作的蛋白,包括白蛋白、转化生长因子β1、血小板因子4、血纤维蛋白溶酶原、血管内皮生长因子A等;SRGN参与调节卵巢癌耐药性的KEGG代谢通路有缺氧诱导因子1α信号通路、细胞因子-细胞因子受体通路、凝血与补体级联反应信号通路等,生物过程有基因表达、细胞生长、凋亡过程、细胞死亡。结论:SRGN介导卵巢癌耐药可能与缺氧诱导因子1α信号通路、细胞因子-细胞因子受体通路等有关。     

基于生物信息学分析高表达EFNA1对卵巢癌铂类耐药的影响

目的 研究肝配蛋白A1(ephrin-A1,EFNA1)与卵巢癌铂类耐药的关系。方法 运用Oncomine和基因表达谱交互分析在线工具分析EFNA1在全身肿瘤组织和卵巢癌组织中的表达;TISIDB结合TNMplot以及muTarget分析EFNA1与不同卵巢癌病理参数的相关性;Kaplan-Meier Plotter结合PrognoScan进行EFNA1表达量与卵巢癌患者生存率相关性分析;GEO数据库分析EFNA1在铂类敏感性与铂类耐药性的卵巢癌细胞株中的表达差异;采用反转录-聚合酶链反应及Western blot检测EFNA1在顺铂敏感或耐药性卵巢癌细胞系中的表达,CCK8检测顺铂耐药性卵巢癌细胞系A2780-DDP增殖,流式细胞术检测A2780-DDP细胞凋亡,Coremine文本挖掘分析EFNA1介导卵巢癌顺铂耐药的生物过程。结果 EFNA1在卵巢癌中高表达,而且与卵巢癌患者的病理参数、生存率显著相关;EFNA1在铂类耐药性卵巢癌组织或细胞中高表达,并且与卵巢癌铂类化疗患者的不良预后显著相关。EFNA1敲低后抑制了顺铂处理后的A2780-DDP细胞的增殖,促进了凋亡。细胞增殖、凋...     

卵巢癌对顺铂类药物耐药性的相关分析

摘要： 目的 筛选影响卵巢癌顺铂耐药性的靶点基因。方法 从 GEO 数据库中下载对顺铂敏感和产生抗药性 的人类卵巢癌细胞基因表达谱和甲基化谱数据（GSE15709）, 利用 R 的相关工具包筛选 A2780 和 A2780/DDP（顺铂 耐药卵巢癌细胞系）两类卵巢癌细胞之间的差异表达和差异甲基化的基因; 使用 DAVID 数据库对差异表达基因进 行功能富集分析; 对同时发生了差异甲基化、 差异表达且甲基化水平、 表达水平的变化趋势相反的基因, 进一步利用 qRT-PCR 技术检测这些基因在两种卵巢癌细胞中的表达值。结果 研究发现在两种卵巢癌细胞之间发生了 416 个 差异表达和 281 个差异甲基化的基因, 这些差异表达基因主要富集于细胞周期、 核分裂和蛋白修饰负调控等生物过 程。此外, 细胞周期、 DNA 复制和 p53 等通路在这些基因中同样富集。共发现 4 个发生了差异甲基化、 差异表达且 甲基化变化水平、 表达水平变化趋势相反的基因, qRT-PCR 实验验证了这些基因在两种卵巢癌细胞中的表达水平。 结论 利用生物信息学和分子生物学相结合的方法, 可以筛选出部分影响卵巢癌对顺铂抗药性的基因, 为进一步揭 示其中的分子机制提供实验参考。     

基于数据库挖掘分析CKMT1A在非小细胞肺癌化疗耐药性中的作用

目的 探讨线粒体肌酸激酶1A（creatine kinase mitochondrial 1A，CKMT1A）在非小细胞肺癌（non-small cell lung cancer，NSCLC）化疗耐药性中的作用。方法 通过HPA、GEPIA、GEO等数据库或在线分析工具分析CKMT1A在NSCLC及顺铂耐药性NSCLC细胞系的表达，运用非配对t检验分析组间差异；通过STRING结合DAVID 6.8分析CKMT1A互作基因的通路富集，运用Fisher Exact Test计算富集P值；microRNA.org结合Targetscan进行miRNA-mRNA互作分析进一步证明CKMT1A在NSCLC化疗耐药性中的作用；运用COREMINE工具进行文本挖掘分析显著富集的通路与NSCLC化疗耐药性的关系，以及microRNA与NSCLC化疗耐药性的关系；通过Kaplan Meier-plotter运用log-rank检验分析CKMT1A表达量、microRNA表达量与NSCLC患者总生存率（overall survival，OS）的相关性，以及CKMT1A表达量与NSCLC化疗患者OS的相关性。结果 CKMT1A在NSCLC患者及顺铂耐药性的NSCLC细胞系中的表达量显著升高（P=0.000），而且与患者的OS显著相关（P<0.05）；代谢途径，尤其是精氨酸和脯氨酸代谢途径在CKMT1A互作基因中具有显著性富集（P<0.05），而且与NSCLC化疗耐药性密切相关；hsa-miR-103和hsa-miR-107靶向于CKMT1A，而且与NSCLC患者OS和化疗耐药性相关。结论 高表达CKMT1A影响NSCLC的预后及化疗耐药性，而且有可能通过精氨酸和脯氨酸代谢途径或者microRNA转录后调控介导NSCLC化疗耐药性。     

拉帕替尼联合顺铂对人卵巢癌细胞SKOV3生长的影响

目的探讨新型靶向治疗药物拉帕替尼联合顺铂(DDP)对人卵巢腺癌细胞株SKOV3的增殖抑制作用以及对磷酸AKT蛋白表达的影响。方法体外培养人卵巢癌细胞株SKOV3,采用MTT方法检测不同浓度拉帕替尼、DDP单用或者合用对细胞增殖活力的改变;用流式细胞仪进行细胞周期分析;用流式细胞仪检测SKOV3应用拉帕替尼前后磷酸化AKT(P-AKT)蛋白的表达。结果拉帕替尼及顺铂单独使用即对人卵巢癌细胞株SKOV3细胞有增殖抑制作用,不同浓度的拉帕替尼与顺铂联合应用对人卵巢癌细胞株SKOV3细胞的增殖抑制作用较单药组增强(P<0.05),与对照组相比有统计学意义(P<0.05);应用拉帕替尼前后P-AKT蛋白的表达有显著不同(P<0.05)。结论拉帕替尼联合顺铂可显著抑制SKOV3细胞株的增殖,其作用可能与P-AKT的表达有关,为其在卵巢癌上的应用提供了新的靶点和一定的理论基础。     

低表达SLC1A4在卵巢癌顺铂耐药中的作用研究

目的 探讨溶质载体家族1成员4(solute carrier family 1 member 4,SLC1A4)在卵巢癌铂类化疗耐药性中的作用。方法 通过GEO、TCGA数据库分析工具分析SLC1A4在卵巢癌或铂类耐药性卵巢癌中的表达;通过GEO数据库分析SLC1A4在铂类药物处理的卵巢癌细胞系中的表达;通过Kaplan Meier-plotter分析SLC1A4表达量与卵巢癌患者总生存期(overall survival,OS)、无疾病进展生存率(progression free survival,PFS)的相关性;通过DepMap平台分析SLC1A4基因效应与卵巢癌化疗药物敏感相关性;通过流式细胞试验和肿瘤细胞克隆集落形成试验验证低表达SLC1A4介导卵巢癌细胞顺铂耐药;通过TargetScan预测靶向于SLC1A4的微小RNA(miRNA),并在TCGA数据库卵巢癌样本中验证其相关性;运用COREMINE工具进行文本挖掘分析SLC1A4介导卵巢癌化疗耐药性的生物过程。结果 SLC1A4在卵巢癌患者及铂类耐药性的卵巢癌中的显著降低(P<0.05),而且与患者的OS、PFS显著相关(P<0.05);铂类药物处理卵巢癌细胞增加SLC1A4的表达;SLC1A4对卵巢癌的基因效应与铂类药物敏感性正相关;过表达SLC1A4增加顺铂引起的卵巢癌细胞凋亡和减少肿瘤细胞克隆集落形成;hsa-let-7c-5p靶向于SLC1A4并在耐药性卵巢癌患者样本中呈显著负相关。结论 低表达SLC1A4介导卵巢癌铂类耐药性,并且有可能与hsa-let-7c-5p调控有关。     

基于Oncomine数据库及生物信息学方法挖掘UBE2C 基因在卵巢癌中的表达及作用机制

摘要： 目的 通过深度挖掘Oncomine数据库中的基因信息， 分析泛素结合酶E2C （UBE2C） 在卵巢癌中的表达及作用机制。方法 收集Oncomine数据库中关于UBE2C研究的信息， 对其在卵巢癌的表达水平变化进行分析。采用 Kaplan-Meier法分析UBE2C表达水平与卵巢癌患者生存之间的关系， 探讨其临床意义。应用Genecards数据库收集与UBE2C基因相关的蛋白， 并通过STRING绘制UBE2C相关蛋白网络图， 分析蛋白富集的生理过程。结果 在 Oncomine数据库中共收集14项关于卵巢癌和卵巢正常组织UBE2C基因有差异表达的研究， 对其数据进行分析， 发现卵巢癌组织中UBE2C基因的表达显著高于卵巢正常组织 （P＜0.05）。通过Kaplan-Meier生存分析发现高表达 UBE2C基因的卵巢癌患者无病生存期、 总生存期明显短于低表达者， 低表达患者的预后更好 （P＜0.05）。通过 Genecards数据库收集到与UBE2C相关的蛋白有RLIM、 CBX4、 SIAH2等25个， 其相关蛋白富集分析结果显示主要富集在泛素依赖蛋白的分解代谢过程、 蛋白质分解代谢过程、 蛋白质泛素化、 有丝分裂细胞周期中泛素-蛋白连接酶活性的调控、 有丝分裂细胞周期的调节、 细胞周期等生理过程。结论 UBE2C基因可能通过调节蛋白泛素化过程在卵巢癌发生、 发展中发挥作用， 高表达时提示卵巢癌患者预后不良， 靶向UBE2C可能是一个潜在的肿瘤诊断和治疗工具。     

天花粉蛋白、米非司酮和顺铂对卵巢癌细胞HLA-G表达的影响

目的检测HLA-G在卵巢癌细胞中表达情况,探讨天花粉蛋白、米非司酮、顺铂是否能够通过下调HLA-G的表达,达到抑制肿瘤生长的目的。方法采用RT-PCR技术检测卵巢癌卵巢浆液性囊腺癌（SKOV3）、卵巢粘液性囊腺癌（3AO）、卵巢腺癌细胞株（OVCAR3）细胞中HLA-G表达情况,选择出表达HLA-G阳性的细胞株。MTT技术观察不同浓度天花粉蛋白、米非司酮、顺铂对该细胞体外生长增殖能力的影响及对细胞中HLA-GmRNA表达水平的影响。结果卵巢癌OVCAR3细胞株表达HLA-G为阳性,SKOV3和3AO细胞表达均为阴性;天花粉蛋白、米非司酮、顺铂均能抑制OVCAR3细胞生长,并呈浓度依赖性;天花粉蛋白、米非司酮均能明显下调OVCAR3细胞中HLA-GmRNA表达水平（P〈0.05）,而顺铂对OVCAR3细胞中HLA-GmRNA表达无明显影响（P〉0.05）。结论天花粉蛋白、米非司酮和顺铂都具有抑制卵巢癌细胞生长的作用,前二者治疗卵巢癌的作用机制同顺铂有所不同,与顺铂联合应用,可能会取得更好的治疗效果。     

卵巢上皮性癌转录激活因子-2表达与顺铂耐药的关系

目的:探讨卵巢上皮性癌组织转录激活因子-2(ATF-2)表达水平与顺铂耐药的关系.方法:应用免疫组化链霉素抗生物-过氧化物酶连接(SP)法检测56例石蜡包埋卵巢癌标本的ATF-2蛋白表达,逆转录聚合酶链反应(RT-PCR)方法检测其中34例卵巢癌新鲜组织的ATF-2 mRNA表达,分析其与顺铂耐药的关系.结果:不同年龄、组织学类型及有无腹水患者ATF-2 表达差别无统计学意义,而不同手术分期患者表达差异有统计学意义(P<0.05).顺铂化疗耐药组、敏感组ATF-2蛋白表达染色积分值分别为4.88±2.94、2.80±3.10,差异有统计学意义(t=2.568,P<0.05),RT-PCR 结果显示34例卵巢上皮性癌新鲜组织中ATF-2/β-actin mRNA波动于0.181～2.276,化疗耐药者、敏感者ATF-2 mRNA相对表达量分别为1.132±0.645、0.651±0.461,差异有统计学意义(t=2.136,P<0.05).结论:ATF-2表达与卵巢上皮性癌顺铂耐药有关,可能成为预测卵巢癌顺铂敏感性指标.     

Survivin蛋白在卵巢癌组织中的表达及临床意义

目的:运用组织芯片技术结合免疫组化的方法分析Survivin蛋白在正常卵巢、卵巢良性肿瘤、卵巢癌组织中的表达情况,探讨Survivin蛋白在卵巢癌的发生发展、浸润转移和化疗耐药中的作用,为以Survivin作为分子治疗靶点的抗肿瘤基因治疗提供理论依据。方法:运用组织芯片技术联合免疫组化法检测27例卵巢癌、20例正常卵巢组织、30例卵巢良性肿瘤中凋亡抑制基因Survivin的表达情况,分析其与卵巢癌临床病理特征的关系,并进行临床随访分析Survivin基因与卵巢癌化疗耐药的关系。结果:①Survivin蛋白在卵巢癌组织中的阳性表达显著高于卵巢良性肿瘤和正常卵巢,两两比较,Survivin在卵巢癌中的表达率与在正常卵巢组织及卵巢良性肿瘤中的表达率比较有差别,且差别有统计学意义(P<0.05);Survivin蛋白与卵巢癌的临床分期有显著相关性(P<0.05);Survivin蛋白表达与卵巢癌的组织类型、病理学分级、患者年龄、是否绝经、有无腹水形成无关(P>0.05)。②Survivin蛋白在卵巢癌化疗耐药组中高表达。结论:Survivin蛋白高表达在卵巢癌的发生发展中起重要作用,Survivin蛋白在卵巢癌以顺铂和紫杉醇为基础的化疗耐药中起重要作用。     

Increased nucleotide excision repair in cisplatin-resistant ovarian cancer cells: role of ERCC1-XPF

Increased platinum-DNA adduct removal has been shown by several DNA repair assays to be associated with cisplatin resistance in the A2780/C-series human ovarian cancer model system. In the present study, we provide further evidence that the resistance phenotype of these cell lines is due, in part, to enhanced nucleotide excision repair (NER). Cisplatin resistance was found to be associated with increased UV resistance. Northern blot analysis revealed that increased expression of ERCC1 was also associated with cisplatin resistance in this panel. Several other NER genes were found to be constitutively overexpressed in the most resistant cell line, C200, as compared with the parental A2780 cells. A plasmid substrate containing a site-specific cisplatin adduct was used to measure the nucleotide excision activity of cell extracts prepared from cisplatin-sensitive and -resistant cells. Using this in vitro assay, extracts prepared from C200 cells exhibited approximately 3-fold more activity than extracts prepared from A2780 cells, similar to the difference in UV sensitivity. Complementation of A2780 extracts with ERCC1-XPF protein resulted in approximately 2-fold increased activity, but had little effect on excision in C200 extracts. Overall, these results support a role for the ERCC1-XPF endonuclease as a determinant of increased NER in this cisplatin resistance model.     

miR-218抑制卵巢癌细胞A2780对顺铂耐药性的实验研究

目的研究miR-218抑制卵巢癌细胞A2780对顺铂耐药性的影响及可能机制。方法实时定量PCR检测A2780及顺铂耐药细胞株A2780/DDP中miR-218的表达,转染miR-218 inhibitors和mimics改变A2780及A2780/DDP细胞中miR-218的表达,MTT法检测转染前后细胞对顺铂的敏感性,并分析Wnt2B蛋白表达的改变。结果与A2780细胞相比,miR-218在A2780/DDP中的表达明显降低。A2780细胞转染miR-218 inhibitors后,细胞对顺铂的敏感性降低,Wnt2B蛋白表达明显增强;而A2780/DDP细胞转染miR-218mimics后,细胞对顺铂的敏感性增强,Wnt2B蛋白表达明显减少。结论 miR-218可能通过靶向Wnt2B抑制卵巢癌细胞A2780对顺铂的耐药性。     

High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro

[(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand - adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC(50) and IC(90) concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC(50) or IC(90)) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G(2)/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC(50) or IC(90) of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.     

Cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells

OBJECTIVE To investigate the underlying mechanism of drug resistance to cisplatin and increasing the sensitivity to therapeutic drugs are key steps towards the improved treatment of patients with ovarian cancer.Gap junction(GJ)and connexin(Cx)are closely related to tumor formation,but the relationship between cisplatin resistance and GJ or Cx are undetermined.METHODS We established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP over an 11-month period,with the concentration of cisplatin gradually increasing from 0.5 g·L~(-1) to 16 g·L~(-1).To explore the effect of GJ in the process of cisplatin resistance,we investigated GJ using a parachute dyecoupling assay in A2780-RI(1.2),A2780-RI(1.7),A2780-RI(2.9),A2780-RI(4.3)and A2780-CDDP cells.We further explored whether the Cxs responsible for GJ were related to cisplatin resistance.In A2780-RI(1.2),A2780-RI(1.7),A2780-RI(2.9),A2780-RI(4.3)and A2780-CDDP,we used q-PCR to analyze the levels of Cx43,Cx40,Cx37,and Cx32.To confirm the effect of Cx32 on cisplatin resistance,we knocked down Cx32 in A2780-CDDP cells with si RNA-Cx32.As GJ was decreased whereas Cx32 expression was elevated during the cisplatin resistance process,it drove us to explore the underlying mechanism.To resolve this issue,we extracted membrane-bound and cytoplasmic proteins from A2780 and A2780-CDDP cells.RESULTS Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression.Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment.Furthermore,although Cx32 expression was increased in A2780-CDDP cells,it was more localized to the cytoplasm rather than in the membrane,and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment.CONCLUSION In summary,Cx32 is involved in cisplatin resistance,and cytoplasmic Cx32 plays an important role in chemoresistance.     

Different cell cycle modulation following treatment of human ovarian carcinoma cells with a new platinum(IV) complex vs cisplatin

Summary Platinum (IV) derivative with adamantylamine—LA-12—represents a new generation of highly efficient anti-cancer drug derived from cisplatin and is currently in the final stage of phase I clinical trials. Understanding the specific mechanisms of its effects on cell cycle is necessary for defining the mode of action of LA-12. In this study, we characterized the ability of LA-12 to induce cell cycle perturbations in ovarian cancer cell line A2780 as compared to equitoxic cisplatin treatment. LA-12 induced a permanent accumulation of A2780 cells in S phase while cisplatin caused G₂/M arrest at 24-h time point, where we also detected an increased expression of Gadd45α protein. Although both derivatives induced a rapid increase of p53 expression, this was not associated with a down-regulation of Mdm2 protein. Increased expression of p21^Cip1/WAF1 protein and its association with cyclins A and B1 suggested that this cyclin-dependent kinase inhibitor might contribute significantly to the observed perturbations of cell cycle. The results of this study provide insight into the mechanism of action of platinum-based derivative with adamantylamine on cell cycle in ovarian cancer cells. The differences between effects of LA-12 and cisplatin suggest that more attention should be paid to elucidation of modes of action of novel platinum(IV) complexes at cellular level.     

Relevance of drug uptake and efflux for cisplatin sensitivity of tumor cells

Platinum sensitivity and platinum resistance may involve altered activity of transport proteins. In order to assess the role of drug uptake and efflux in this phenomenon, we compared the expression of three copper transporters, intracellular platinum accumulation, DNA platination and cytotoxicity of cisplatin in two cisplatin-sensitive and -resistant tumor cell line pairs (ovarian A2780/A2780cis and cervical HeLa/HeLaCK cells). Gene expression of importer CTR1, and ATP7A and ATP7B efflux transporters (with and without cisplatin treatment) was investigated using quantitative real-time PCR and platinum concentrations were determined by flameless atomic absorption spectrometry. After incubation with cisplatin, DNA platination was significantly lower in the resistant variants compared to the respective sensitive cell lines, whereas no obvious difference in DNA repair was found. Accordingly, the resistant variants exhibited lower intracellular platinum concentrations than their respective parental cells (2.5- and 2.9-fold lower in A2780cis and HeLaCK cells, respectively). No differences in efflux were observed. Resistant cells expressed lower levels of CTR1 (1.5-1.8-fold) than their sensitive counterparts. Expression differences of ATP7A and ATP7B between resistant and sensitive cells were cell type-specific. The results highlight the relevance of CTR1 for cisplatin sensitivity as there is a clear relationship between lower CTR1 expression, intracellular concentration, DNA platination and cytotoxicity of cisplatin in both resistant cell lines. Our data provide the basis for a quantitative understanding of alterations in uptake and efflux processes leading to cisplatin resistance and might hence facilitate the development of ex vivo assays that can predict cisplatin sensitivity in tumor specimens of patients.     

The role of protein kinase C and the phosphatidylinositol cycle in multidrug resistance in human ovarian cancer cells

The present study aimed to investigate the role of protein kinase C (PKC), the phosphatidylinositol pathway (PI) and cytosolic calcium in multidrug resistance (MDR) in human ovarian carcinoma cells. Binding of the phorbol ester 13,14-dibutyrate (PDBu) was 3-fold higher in resistant A2780AD versus sensitive A2780 cells indicating increased PKC activity. However, when inositol phosphate production (IP) was measured in quiescent cells similar total IP release was seen in both lines suggesting no difference in the basal turnover of PI. Non-specific stimulation of the PI pathway was achieved with the calcium ionophore A23187 which increased IP production in a time- and dose-dependent fashion in both cell lines but was significantly less effective in A2780AD. The PI pathway was investigated further using the agonists aluminium fluoride, serum and bombesin but these agents failed to elicit a response. The effect of a wide range of Adriamycin concentrations on the PI cycle and cell growth was also studied. Intracellular calcium was measured with the fluorescent dye fura-2-pentaacetoxymethylester (Fura-2). A23187 produced a rise in cytosolic calcium in A2780 and A2780AD but from a level 3-fold lower in the unstimulated resistant cell line. The dose responsiveness of this effect was greater but irreversible in A2780AD cells. Collectively these results imply that alterations in PI turnover appear not to be responsible for the differences in PDBu binding and calcium handling observed between A2780 and A2780AD and suggests only a minor role for the PI cycle in the maintenance of MDR in human ovarian cancer cell lines.