Preparation and characterization of a murine monoclonal antibody (MDR3M) reactive with mdr3 gene product.

A murine monoclonal antibody (MDR3M) (isotype: IgM) reactive with mdr3 gene product was generated by immunizing mice with mdr3-specific peptide (H2N-12WRPTSAEGDFELGISSKQKRKKTKTVKMI41G-COOH) and hybridizing the primed mouse splenic B cells with X63-Ag8,6.5.3 mouse plasmacytoma cells. MDR3M did not cross-react with mdr1 gene product. This monoclonal antibody may be useful for analyzing the role of mdr3 gene product in cells and tissues.     

mdr1基因表达与人肺腺癌多药抗药细胞系LC-3／CDDP抗药性的关系

目的:探讨多药抗药基因(mdr_1基因)表达与人肺腺癌多药抗药细胞系LC-3/CDDP抗药性产生的关系.方法:应用地高辛标记的mdr_1 cDNA探针,采用斑点杂交方法,检测该细胞系中的mdr_1mRNA含量,同时以免疫细胞化学染色方法,检测P-糖蛋白(P-gP)表达,并与亲代(LC-3)细胞比较.结果:LC-3/CDDP细胞存在mdr_1基因过表达.结论:mdr_1基因过表达是LC—3/CDDP细胞系产抗药性的机制之一.     

Curcumin down-regulates the multidrug-resistance mdr1b gene by inhibiting the PI3K/Akt/NF kappa B pathway

Curcumin, a constituent of turmeric, has anti-inflammatory, anti-carcinogenic, and chemopreventive effects in several animal tumor models. The expression of P-glycoprotein (P-gp), encoded by the mdr gene, is often associated with multidrug resistance (MDR) to unrelated chemotherapeutic drugs in cancer cells. Here, we demonstrate that curcumin down-regulates P-gp expression in multidrug-resistant L1210/Adr cells. Transfection with a series of 5'-deleted constructs of the mdr1b gene promoter indicated that a proximal region between -205 and +42 of the sequence was responsible for the suppression of promoter activity by curcumin. This response might be associated with the inhibition of the phosphatidyinositol 3-kinase (PI3K)/Akt/nuclear factor-kappa B (NF-kappa B) signaling pathway by curcumin. Moreover, curcumin reversed the MDR of the L1210/Adr cells. Thus, curcumin can contribute to the reversal of the MDR phenotype, probably due to the suppression of P-gp expression via the inhibition of the PI3K/Akt/NF-kappa B signaling pathway.     

Multidrug Resistance Phenotype in the RMS-GR Human Rhabdomyosarcoma Cell Line Obtained after Polychemotherapy

Classical cytotoxic treatment of rhabdomyosarcoma (RMS), the most common soft tissue malignancy in children, is often accompanied by significant morbidity and poor response. Chemotherapy may induce multidrug resistance (MDR) associated with the expression of P-glycoprotein, a drug efflux pump which modifies the sensitivity of tumoral cells to drugs. To analyze MDR in RMS we used the RMS-GR cell line, obtained from an embryonal rhabdomyosarcoma treated in vivo with polychemotherapy. The RMS-GR cells showed cross-resistance to vincristine, doxorubicin and actinomycin D, the drugs of choice in the conventional treatment of RMS. Polymerase chain reaction (PCR) analysis showed that these RMS cells overexpressed mdr1 /P-glycoprotein. The pattern of resistance and the level of P-glycoprotein expression were similar to those found in the resistant RMS TE.32.7.DAC cell line obtained in vitro . Southern blot analysis showed that mdr1 overexpression was not due to amplification of the gene. Our results showed that the in vivo treatment of embryonal RMS may induce an MDR phenotype mediated by mdr1 /P-glycoprotein in RMS cells.     

Characterization of an Etoposide-resistant Human K562 Cell Line, K/eto

An etoposide-resistant K562 cell line (K/eto) was obtained by stepwise exposure, in culture, to increasing concentrations of etoposide, without the use of mutagens. This cell line was resistant to etoposide, and slightly resistant to adriamycin, but sensitive to anti-cancer drugs such as camptothecin, vincristine, actinomycin D and so on. P-GIycoprotein, the mdr 1 gene product, was not detected in this cell line, as assessed by immunocytochemistry, itnmunoprecipitation and flow cytometry. Overexpression of mdr 1 mRNA was also not found. Interestingly, expression of 85 kD protein recognized by MRK 20 monoclonal antibody was noted. The level of DNA topoisomerase II protein, detected by antibody staining, decreased concomitantly with a general decrease in DNA topoisomerase II unknotting activity, while DNA topoisomerase I activity was not affected. Cellular accumulation of [³H]etoposide was reduced by 75% in the resistant line compared with parental K562. Karyotype analysis showed that the number of chromosomes in K/eto was 55 and neither a homogeneous staining region nor double-minute chromosomes were detected. These results indicate that this resistance is not due to an altered interaction between the drug and cellular transport machinery, i.e. MDR1, associated with the "classic" multiple drug resistance phenotype, but rather is due to the existence of other mechanism(s) of resistance, decreased transport of the drug and decreased target enzyme, DNA topoisomerase II.     

两种鼻咽癌细胞X线照射前后多药耐药基因的表达

目的检测鼻咽癌CNE1和CNE2细胞在X线照射前后多药耐药基因（mdr1基因）及其编码产物P糖蛋白（P—glycopro—tein，P-gp）表达情况，探讨鼻咽癌细胞射线照射与诱导多药耐药的相关性。方法利用RT—PCR和Western blot方法检测CNE1和CNE2细胞射线照射前后的mdrl基因和P—gP的表达。结果鼻咽癌CNE1和CNE2细胞射线照射前mdr1基因、P-gp不表达；射线照射后较长时间内mdr1基因及P—gP长时间表达。结论鼻咽癌CNE1和CNE2细胞经X线照射有可能诱导多药耐药。     

Mouse myeloma cell line Sp2/0 multidrug-resistant variant as parental cell line for hybridoma construction

The use of multidrug-resistant variant of Sp2/0 mouse myeloma cell line SpEBr-5 as a partner for making mouse hybridoma producing monoclonal antibodies is described here. The resulting hybridoma cell line 1F7 was characterized with a high level of monoclonal antibody production and karyotype containing all normal mouse chromosomes. 1F7 cells were separately selected for resistance to ethidium bromide (EBr) and adriamycin (ADR) and different mechanisms of drug resistance were found in these cell variants. The resistance in ADR-selected 1F7 cells was due to amplification and overexpression of mdr genes. In EBr-resistant 1F7 cells, mdr genes were overexpressed without amplification. Substantially decreased level of Topo II activity in both cell lines also suggests the existence of additional mechanisms for MDR phenotype of hybridoma cells. Finally, adriamycin-resistant 1F7 hybridoma cell variant was found to produce higher level of specific immunoglobulins due to the increased level of Iggamma(2b) heavy chain mRNA.     

Coordinate activation of multidrug-resistance (P-glycoprotein) genes mdr2 and mdr3 during mouse liver regeneration.

We previously reported that only one of the three mouse multidrug-resistance (mdr) genes, mdr3, is activated in hepatocellular carcinomas (HCCs). The present study examined the expression of mdr family members during mouse liver regeneration after partial hepatectomy to determine whether the regeneration that occurs during hepatic tumorigenesis is responsible for mdr3 elevation in HCC. We demonstrated that in both C3H/HeN and B6C3/F1 mice strains, the levels of both mdr2 and mdr3 mRNAs coordinately increased five- to sevenfold 24 h after partial hepatectomy, whereas the levels of mdr1 mRNA were not statistically different from those in the controls. Forty-eight hours after partial hepatectomy, mdr mRNA levels decreased and in most cases returned to normal levels after 72 h. These results indicate that mdr3 induction during hepatocarcinogenesis is not due to liver regeneration alone.     

Reversal of Adriamycin Resistance of Hepatocellular Carcinoma by Targeting with Recombined Adenovirus Caning Antisense mdr1 RNA

OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma (HCC). However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance (MDR) is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS pCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdrl gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-Scel/l-Ceu l, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL 1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1 -mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase (P<0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM asmdr1 group compared to the ADM group at the 4th week (P<0.05). Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.     

Establishment and characterization of new cellular lymphoma model expressing transgenic human MDR1

Multidrug resistance (MDR) due to the expression of the MDR1 gene and its P-glycoprotein (Pgp) product is a major factor in the prognosis and clinical outcome of patients with refractory lymphomas and other malignancies. The aim of our study was to establish a lymphoma, cellular system where a de novo acquisition of multidrug resistance is specifically related to overexpression of a transgenic, human MDR1. A multidrug sensitive lymphoma cell line (LM1) was established from a sporadic T-cell lymphoma of BALB/c mouse and was transduced by a retroviral vector containing the human MDR1 cDNA. The resultant cell variant (LM1/MDR) was characterized in comparison to the parental LM1 cells. The LM1/MDR cell variant is cross-resistant to DOX, COL, ACT D and VBL. This cell variant expresses the human MDR1 and exhibits de novo functional Pgp activity that can be blocked by the Pgp-modulators VRP and KT-5720. The acquired MDR of LM1/MDR is not accompanied with gene amplification, alternative splicing or up-regulation of the murine endogenous mdr1a, mdr1b, mrp1, mrp2 and mrp3 transporter-genes. Therefore, the acquired MDR is, specifically, human MDR1-dependent as it has been found in malignant cells of most lymphoma patients. Moreover, this system can be used as a model to study MDR and the efficacy of drugs and modulators on malignant cells where human Pgp is a major factor of multidrug resistance.     

A new monoclonal antibody that specifically recognises the MDR‐3‐encoded gene product

The MDR‐3‐encoded P‐glycoprotein (Pgp) is highly expressed in liver and is thought to function as a hepatic transporter of phospholipids into bile. However its role, if any, in other tissues remains undefined. Although transfection experiments have indicated that it may be unable to confer drug resistance, there is evidence that it may be involved in drug resistance in certain B‐cell leukaemias. To date, most work on clinical samples has been performed at the mRNA level; limited work has been performed using polyclonal antibodies raised to MDR‐3 and mdr‐2 (the murine equivalent of MDR‐3). We have generated a new monoclonal antibody, termed 6/1G, which specifically recognises the human MDR‐3 gene‐encoded product. Antibody 6/1G was produced by in vitro immunisation of spleen cells from BALB/c mice with a synthetic 12‐amino acid peptide. Cells from MDR‐3 transgenic mice showed consistent membranous staining with antibody 6/1G. Immunoblotting with 6/1G identifed a band at 170 kDa on lysates of MDR‐3 transgenic cells. Preliminary results with a range of B‐cell leukaemias suggest that MDR‐3 Pgp positivity may be a marker for a more malignant phenotype in B‐CLL. Antibody 6/1G may be useful in defining a role for MDR‐3 in malignancy and drug resistance, as well as in certain liver diseases such as progressive familial intracholeostasis. Int. J. Cancer 80:265–271, 1999. © 1999 Wiley‐Liss, Inc.     

P-glycoprotein expression in hepatocellular carcinoma

In hepatocellular carcinoma cell lines, the intensity of staining with the monoclonal antibody C-219 to the multidrug-resistant gene (mdr1) product P-glycoprotein and the intensity of the band at a molecular weight of 170 KDa on Western blot were associated closely with resistance to Adriamycin but not with the resistance to cis-dichlorodiamine platinum (CDDP). In clinical specimens, noncancerous liver tissue was regularly stained with this antibody on the biliary canalicular front of the hepatocyte cell membrane. In liver cancer tissue, however, regular staining as in the noncancerous regions of the liver was observed in only 16% of the patients, irregular staining was seen in only 24%, and no staining was seen at all in 60%. Staining of P-glycoprotein with the C-219 antibody is technically simple and is useful for studying the role of P-glycoprotein in drug-resistant hepatocellular carcinoma. © 1993 Wiley-Liss, Inc.     

Induction of human MDR1 gene expression by 2-acetylaminofluorene is mediated by effectors of the phosphoinositide 3-kinase pathway that activate NF-kappaB signaling

The expression of P-glycoprotein encoded by the multidrug resistance (MDR1) gene is associated with the emergence of the MDR phenotype in cancer cells. Human MDR1 and its rodent homolog mdr1a and mdr1b are frequently overexpressed in liver cancers. However, the underlying mechanisms are largely unknown. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression in cultured cells and in Fisher 344 rats. We recently reported that activation of rat mdr1b in cultured cells by 2-AAF involves a cis-activating element containing a NF-kappaB binding site located -167 to -158 of the rat mdr1b promoter. 2-AAF activates IkappaB kinase (IKK), resulting in degradation of IkappaBbeta and activation of NF-kappaB. In this study, we report that 2-AAF could also activate the human MDR1 gene in human hepatoma and embryonic fibroblast 293 cells. Induction of MDR1 by AAF was mediated by DNA sequence located at -6092 which contains a NF-kappaB binding site. Treating hepatoma cells with 2-AAF activated phosphoinositide 3-kinase (PI3K) and its downstream effectors Rac1, and NAD(P)H oxidase. Transient transfection assays demonstrated that constitutively activated PI3K and Rac1 enhanced the activation of the MDR1 promoter by 2-AAF. Treatment of hepatoma cells with 2-AAF also activated another PI3K downstream effector Akt. Transfection of recombinant encoding a dominant activated Akt also enhanced the activation of MDR1 promoter activation by 2-AAF. These results demonstrated that 2-AAF up-regulates MDR1 expression is mediated by the multiple effectors of the PI3K signaling pathway.     

Augmentation by Bispecific F(ab')2 Reactive with P-Glycoprotein and CD3 of Cytotoxicity of Human Effector Cells on P-Glycoprotein Positive Human Renal Cancer Cells

A bispecific F(ba')₂ was constructed that was composed of two Fab fragments, one derived from anti-CD3 monoclonal antibody (mAb) (OKT3) and the other from anti P-glycoprotein mAb (MRK 16). This bispecific F(ab')₂ enhanced the binding and cytotoxicity of human peripheral blood mononuclear cells (PBMCs) on P-glycoprotein-positive human kidney cancer cells (ADMHK/E). It had no effect on the cytotoxicity of PBMCs on P-glycoprotein-negative HK/E cells [long-term cultured HK/E (LCHK/E)]. Control F(ab')₂ composed of OKT3 or MRK16 alone did not influence the cytotoxicity of PBMCs on ADMHK/E cells. These findings suggest that the MRK16-OKT3 bispecific F(ab')₂ may be therapeutically beneficial in treatment of human multidrug-resistant cancers.     

Characterization by somatic cell genetics of a monoclonal antibody to the MDR1 gene product (P-glycoprotein): determination of P-glycoprotein expression in multi-drug-resistant KB and CEM cell variants.

We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.     

Bispecific Antibody-directed Antitumor Activity of Human CD4+ Helper/Killer T Cells Induced by Anti–CD3 Monoclonal Antibody plus Interleukin 2

Freshly isolated human CD4⁺ T cells can not respond to recombinant interlenkin 2 (rIL–2) because of their lack of p75 IL–2 receptor expression. However, we succeeded In inducing a marked proliferation of purified CD4⁺ T cells by activation with rIL–2 plus anti–CD3 monoclonal antibody (mAb) cross–linked to a plastic plate. The proliferated CD4⁺ T cells produced a significant amount of IL–2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4⁺ T cells activated with anti–CD3 mAb plus rIL–2 revealed a strong cytotoxic activity against Fc receptor (FcR)–positive tumor cells in the presence of anti–CD3 mAb. Moreover, the CD4⁺ T cells could lyse FcR–negative glioma cells by targeting with bispecific mAb containing anti–CD3 mAb and anti–glioma mAb. Thus, we demonstrated that rIL–2 and immobilized anti–CD3 mAb allowed the rapid generation of human CD4⁺ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.