Induction of resistance to Aplidin in a human ovarian cancer cell line related to MDR expression

Aplidin-resistant IGROV-1/APL cells were derived from the human ovarian cancer IGROV-1 cell line by exposing the cells to increasing concentration of Aplidin for eight months, starting from a concentration of 10 nM to a final concentration of 4 microM. IGROV-1/APL cell line possesses five fold relative resistance to Aplidin. IGROV-1/APL resistant cell line shows the typical MDR phenotype: (1) increased expression of membrane-associated P-glycoprotein, (2) cross-resistance to drugs like etoposide, doxorubicin, vinblastine, vincristine, taxol, colchicin and the novel anticancer drug Yondelis (ET-743). The Pgp inhibitor cyclosporin-A restored the sensitivity of IGROV-1/APL cells to Aplidin by increasing the drug intracellular concentration. The resistance to Aplidin was not due to the other proteins, such as LPR-1 and MRP-1, being expressed at the same level in resistant and parental cell line. The finding that cells over-expressing Pgp are resistant to Aplidin was confirmed in CEM/VLB 100 cells, that was found to be 5-fold resistant to Aplidin compared to the CEM parental cell line.     

The anthracycline resistance-associated (ara) gene, a novel gene associated with multidrug resistance in a human leukaemia cell line.

Multidrug resistance (MDR) in cancer cells is a major contributor to the failure of chemotherapy treatment. This paper describes a novel protein named the anthracycline resistance associated (ARA) protein. The ara gene is amplified in the MDR leukaemia line CCRF-CEM/E1000 and its mRNA is overexpressed. ARA belongs to the ATP binding cassette (ABC) family of proteins. Another ABC protein, the multidrug resistance-associated protein (MRP), has previously been reported to be overexpressed in the CEM/E1000 subline. The primary amino acid sequence of ARA indicates that it is 49.5 kDa without glycosylation, and that it has one potential glycosylation site. ARA has one ATP binding site and associated transmembrane regions. This is in contrast to MRP (190 kDa, 172 kDa deglycosylated) and most other higher eukaryote ABC proteins, which consist of two similar halves, each having one ATP binding site. In addition to ARA being coexpressed with MRP, comparison of amino acid sequences showed that, among known proteins, ARA is most similar to the C-terminal half of MRP.     

Induction of human herpesvirus 8 gene expression in a posttransplantation primary effusion lymphoma cell line

Human herpesvirus 8 (HHV-8 or Kaposi's sarcoma herpesvirus) is a gamma herpesvirus that is most likely the etiologic agent of both Kaposi's sarcoma and primary effusion lymphoma (PEL), a rare HIV-associated lymphoma. The role of HHV-8 in post-transplant lymphoma is less well characterized. We demonstrate that HHV-8 is constitutively present in LH5-21 cells, an atypical patient derived posttransplant PEL cell line. LH5-21 cells lack detectable Epstein-Barr virus, express T cell-associated surface markers and have undergone immunoglobulin heavy chain gene rearrangement. Incubation with 12-O-tetradecanoyl-phorbol- 13-acetate or butyrate induces high levels of several HHV-8 encoded genes that are associated with lytic replication. The patient from whom this cell line was derived demonstrated a dramatic clinical response to withdrawal of immunosuppressive therapy. While HHV-8 associated PELs in the post-transplant setting are rare, this study suggests that improvement in the host immunologic function might help in the management of some PELs.     

Lentivirus-mediated RNA interference reversing the drug-resistance in MDR1 single-factor resistant cell line K562/MDR1

Multidrug-resistance (MDR) is a major hindrance to successful chemotherapy. The emergence of MDR is multi-factorial. Among them, the MDR1 gene/P-glycoprotein (P-gp) is a popular and important reason. In our study, an MDR1 single-factorial drug-resistant leukemia cell line K562/MDR1 was constructed via transferring full-length human MDR1 cDNA into drug-sensitive K562 cells. The short-hairpin RNA (shRNA) targeting MDR1 gene was transfected into K562/MDR1 cell lines by the replication-defective lentiviral vector derived from HIV-1. The efficiency of RNA interference (RNAi) to silence the MDR1 gene and reverse multidrug-resistance in the MDR1 single-factor drug-resistance cell line K562/MDR1 was evaluated. The multi-factor resistant cell line K562/A02, induced by doxorubicin exposure, was used as a control. After RNA interference, the expression of the MDR1 gene and P-gp in K562/MDR1 was markedly down-regulated and the drug sensitivity was restored as IC₅₀ values became similar to the K562 sensitive cell line. The expression of the MDR1 gene and P-gp in K562/A02 was markedly down-regulated too, and drug-resistance to anticancer drug is reduced to some extent but the IC₅₀ was significantly higher than that of the sensitive cell line. These results demonstrated that lentivirus-mediated RNAi could efficiently down-regulate the expression of MDR1 and Pgp, and successfully reverse a cell's resistance to chemotherapeutic. Due to only MDR1 resistance, the K562/MDR1 cell showed much high specificity and thus is a better cell model for MDR1/P-gp research.     

Induction of differentiation by wild-type p53 gene in a human glioma cell line

Wild-type human p53 gene was transfected into the human glioma cell line T-98G. Transfectants were then isolated and characterizedfor growth potential and differentiation phenotype. Growth suppression,overexpression of GFAP, and accumulation in G₁phase were more commonly observed in transfectants than inT-98G cells. p21_WAF1/CIP1 wasoverexpressed in transfectants, and the binding of PCNA and CDK 2 top21_WAF1/CIP1 were increased in transfectants. These results suggested the roles of p21_WAF1/CIP1, PCNA,and CDK 2 in regulation of differentiation in glioma cells and the gene transfer of wild-type p53 may be effective forthe control of glial differentiation in glioma cells.     

As2O3对人膀胱癌细胞凋亡和MDR1表达及细胞周期的影响

目的 :观察三氧化二砷 (As2 O3 )对人膀胱癌细胞株BIU 87的凋亡诱导作用及对多药耐药基因 (MDR1)蛋白表达、细胞周期的影响。方法 :采用四氮唑蓝 (MTT)法检测As2 O3 不同浓度、不同作用时间对BIU 87细胞的生长抑制率 ;流式细胞术(FCM )检测不同浓度As2 O3 作用 72h后细胞中与凋亡有关蛋白Fas、bcl 2及MDR1蛋白表达、细胞周期变化。结果 :As2 O3 可有效抑制BIU 87细胞的生长增殖 ,与浓度、时间相关 ,P <0 0 5 ;Fas、bcl 2的表达分别与浓度增高呈正、负相关 ,P <0 0 5 ,且二者随浓度增高呈负相关 ,P <0 0 5 ;MDR1蛋白表达与对照组相比As2 O3 1μmol/L作用后升高 ,P <0 0 5 ,2、5 μmol/L作用后降低 ,P <0 0 5 ;随As2 O3 浓度升高 ,细胞周期被阻滞在G0 /G1期。结论 :As2 O3 可有效抑制人膀胱癌细胞的生长增殖 ,诱导细胞凋亡及阻滞细胞周期可能起了重要作用     

Chemosensitivity patterns and expression of human multidrug resistance-associated MDR1 gene by human gastric and colorectal carcinoma cell lines

We compared the in vitro sensitivity patterns to cytotoxic drugs and expression of the multidrug resistance-associated MDR1 gene (also known as PGY1 gene) in four gastric carcinoma cell lines with those obtained in a panel of 11 colorectal carcinoma cell lines. In addition, we tested the effects of leucovorin on enhancement of fluorinated pyrimidine-induced cytotoxicity. We used a semiautomated tetrazolium dye assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (tetrazolyl blue)] (MTT) to compare the drug sensitivity of the gastric carcinoma cell lines with that of the colorectal carcinoma cell lines. The gastric carcinoma cell lines were more sensitive to some drugs, including doxorubicin and cisplatin, but not to the fluorinated pyrimidines. Addition of leucovorin at a clinically achievable concentration enhanced the cytotoxic effects of both fluorouracil and floxuridine in colorectal carcinoma cell lines, but it enhanced the effects of only floxuridine in gastric carcinoma cell lines. With the use of a slot blot assay, relatively low levels of MDR1 RNA were present in all four gastric carcinoma cell lines, while intermediate or high levels were present in most of the colorectal carcinoma cell lines. In general, our findings reflect clinical experience and may help in the design of clinical trials.     

中性鞘糖脂在KBv200细胞的表达及其与多药耐药性的关系

目的：研究肿瘤细胞mdr1与其细胞表面中性鞘糖脂（N－GSLs)表达的关系。方法利用特异性切割mdr1的核酶（ribozyme)为工具,以表达mdr1的耐药细胞KBv200为靶细胞,采用脂质体转染技术,将含核酶的质粒pH βApr-1neo/5mR3及空载体pHβApr-1neo导入KBv200其亲本KB细胞内,运用Northern-blotting、免疫组化方法观察核酶对mdr1 mRNA及P－gp的影响,用改良的Hakamori方法提取、纯化耐药逆转前后及KB细胞的N－GSLs,以高效薄层层析（HPTLC）分析不同细胞亚株N－GSLs的表达,采用MTT法分析糖脂合成抑剂DL－PPMP对肿瘤细胞多耐药性（MDR）的逆转作用。结果pHβApr-1neo/5mR3及pHβApr-1neo可以在KB、KBv200细胞中稳定表达,mdr1-核酶可以特异性地切割mdr1,导致KBv200/5mR3的mdr1 mRNA含量下降,P－gp表达减低。KB和KBv200的N－GSLs表达有差异,KBv200的单乙糖神经鞘氨醇（monohexosy lceramide,CMH)、二乙糖神经鞘氨醇（dihexosylceramide,CDH)表达较KB强,核酶逆转MDR后,KBv200/5mR3的CMH、CDH表达降低以CMH为著。DL－PPMP可通过抑制CMH的合成,逆转KBv200／5mR3的CMH、CDH表达降低,以CMH为著。DL－PPMP可通过抑制CMH的合成,逆转KBv200对称春新碱（VCR）的耐药。结论肿瘤细胞mdr1与其细胞表面N－GSLs表达相关,CMH为耐药相关N－GSLs,抑制耐药细胞CMH的合成可能是逆转肿瘤多药耐药的一种新方法。     

Establishment of a murine leukaemia cell line resistant to the growth-inhibitory effect of bryostatin 1.

Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.     

Vindesine receptors in cells of a human leukaemia cell line

To determine whether vindesine receptors are present in human leukaemic cells, K562 cells (established from chronic myelogenous leukaemia in blastic crisis) were incubated with 3H-vindesine. Binding of 3H-vindesine increased with incubation time and with increase in number of K562 cells. However, when excessive amounts of nonradioactive vindesine were added, the 3H-vindesine was displaced. Binding of 3H-vindesine was only inhibited by vinblastine, vincristine and vindesine. These results suggest that K562 cells have receptors for vindesine and that these receptors are common to vinca alkaloids. Scatchard analysis showed that the number of vindesine receptors differed according to the kind of cells tested. K562 and a T-cell leukaemia-derived cell line, MOLT-4, had more receptors than an acute promyelocytic leukaemia-derived cell line, HL-60, and normal blood lymphocytes. The degree of vindesine affinity to receptors did not differ markedly among the above-mentioned cells.     

Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69

Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.     

MDR1 gene expression in lung cancer

The MDR1 gene (also known as PGY1) is frequently overexpressed in multidrug-resistant cell lines. We investigated the role of MDR1 gene expression in lung cancer by performing RNA slot blot analysis in samples from a panel of 24 lung cancers, 10 corresponding nontumorous lung tissues, and 67 tumor cell lines of several histologic types. Almost all of the tumors, nontumorous lung tissues, and cell lines expressed low levels of MDR1 RNA. Relatively higher levels were found in only one type of lung cancer, a subgroup of non-small cell lung cancers expressing neuroendocrine markers. No evidence of MDR1 gene amplification or rearrangements was detected. We found no correlation between MDR1 gene expression in cell lines and (a) in vitro chemosensitivity of the cells, (b) prior therapy status of the patients, or (c) clinical response to therapy. We conclude that the clinical multidrug resistance of many lung cancers cannot be explained solely on the basis of expression of the MDR1 gene.     

小分子干扰RNA抑制TNC基因的表达并逆转卵巢癌紫杉醇耐药

目的 研究小分子干扰RNA(siRNA)介导的RNA干扰(RNAi)对卵巢癌紫杉醇耐药细胞TNC基因表达的影响,探讨TNC基因在紫杉醇耐药中的功能.方法 用脂质体转染剂LipofectamineTM2000将针对TNC基因特异性合成的siRNA转染SKOV3/TAX30细胞,分别在转染后24、48、72 h收集细胞,检测各组细胞TNC mRNA和TNC蛋白的表达水平.siRNA转染SKOV3/TAX30细胞后,采用MTT法检测细胞对紫杉醇的半数抑制浓度(IC50).Western Blot检测SKOV3/TAX30及SKOV3/TAX300两种耐药细胞Wnt信号通路中关键蛋白β-catenin及下游Cyclin D、E-cadherin蛋白的表达.结果 siRNA转染SKOV3/TAX30细胞24、48、72 h后,TNC mRNA和TNC蛋白表达水平均下降,SKOV3/TAX30细胞对紫杉醇药物的IC50下降.TNC表达升高的耐药细胞中,Wnt信号通路中关键蛋白β-catenin及下游Cyclin D蛋白表达上调,E-cadherin蛋白表达下调.结论 针对TNC基因特异性合成的siRNA可激发RNAi介导的TNC沉默,并且逆转了紫杉醇化疗耐药,TNC表达上调与紫杉醇耐药相关,TNC可能通过Wnt信号通路的激活参与了化疗耐药.     

Anti-Cripto Mab inhibit tumour growth and overcome MDR in a human leukaemia MDR cell line by inhibition of Akt and activation of JNK/SAPK and bad death pathways

Doxorubicin (DOX) selection of CCRF-CEM leukaemia cell line resulted in multidrug resistance (MDR) CEM/A7R cell line, which overexpresses MDR, 1 coded P-glycoprotein (Pgp). Here, we report for the first time that oncoprotein Cripto, a founding member of epidermal growth factor-Cripto-FRL, 1-Criptic family is overexpressed in the CEM/A7R cells, and anti-Cripto monoclonal antibodies (Mab) inhibited CEM/A7R cell growth both in vitro and in an established xenograft tumour in severe combined immunodeficiency mice. Cripto Mab synergistically enhanced sensitivity of the MDR cells to Pgp substrates epirubicin (EPI), daunorubicin (DAU) and non-Pgp substrates nucleoside analogue cytosine arabinoside (AraC). In particular, the combination of anti-Cripto Mab at less than 50% of inhibition concentrations with noncytotoxic concentrations of EPI or DAU inhibited more than 90% of CEM/A7R cell growth. Cripto Mab slightly inhibited Pgp expression, and had little effect on Pgp function, indicating that a mechanism independent of Pgp was involved in overcoming MDR. We demonstrated that anti-Cripto Mab-induced CEM/A7R cell apoptosis, which was associated with an enhanced activity of the c-Jun N-terminal kinase/stress-activated protein kinase and inhibition of Akt phosphorylation, resulting in an activation of mitochondrial apoptosis pathway as evidenced by dephosphorylation of Bad at Ser136, Bcl-2 at Ser70 and a cleaved caspase-9.