Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines

The multidrug-resistance phenotype expressed in mammalian cell lines is complex. Cells selected with a single agent can acquire cross-resistance to a remarkably wide range of compounds which have no obvious structural or functional similarities. The basis for cross-resistance seems to be a decreased net cellular accumulation of the drug involved, and has been attributed to alterations in the plasma membrane. An over-expressed plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (P-glycoprotein) is consistently found in different multidrug-resistant human and animal cell lines, and in transplantable tumours. Consequently, it has been postulated that P-glycoprotein directly or indirectly mediates multidrug resistance. Here we report the cloning of a complementary DNA encoding P-glycoprotein. Southern blot analysis of hamster, mouse and human DNA using this cDNA as a probe showed that P-glycoprotein is conserved and is probably encoded by a gene family, and that members of this putative family are amplified in multidrug-resistant cells.     

Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum

The lethal form of human malaria caused by Plasmodium falciparum is virtually uncontrollable in many areas because of the development of drug resistance, in particular chloroquine resistance (CQR). CQR is biologically similar to the multiple drug resistance phenotype (MDR) of mammalian tumour cells, as both involve expulsion of drug from the cell and both can be reversed by calcium channel antagonists. A homologue (pfmdr1) of the mammalian multidrug resistance gene has been implicated in CQR because it is amplified in some CQR isolates of P. falciparum as is an mdr gene in MDR tumour cells. We show here that the complete sequences of pfmdr1 genes from 2 CQ sensitive (CQS) P. falciparum isolates are identical. In 5 CQR isolates, 1-4 key nucleotide differences resulted in amino acid substitutions. On the basis of these substitutions, we have correctly predicted the CQS/CQR status of a further 34 out of 36 isolates. This is a paradox as CQR arises much less frequently than would be predicted if single point mutations were sufficient. We conclude that a mutated pfmdr1 gene is one of at least two mutated genes required for CQR.     

Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross

Chloroquine is thought to act against falciparum malaria by accumulating in the acid vesicles of the parasite and interfering with their function. Parasites resistant to chloroquine expel the drug rapidly in an unaltered form, thereby reducing levels of accumulation in the vesicles. The discovery that verapamil partially reverses chloroquine resistance in vitro led to the proposal that efflux may involve an ATP-driven P-glycoprotein pump similar to that in mammalian multidrug-resistant (mdr) tumor cell lines. Indeed, Plasmodium falciparum contains at least two mdr-like genes, one of which has been suggested to confer the chloroquine resistant (CQR) phenotype. To determine if either of these genes is linked to chloroquine resistance, we performed a genetic cross between CQR and chloroquine-susceptible (CQS) clones of P. falciparum. Examination of 16 independent recombinant progeny indicated that the rapid efflux phenotype is controlled by a single gene or a closely linked group of genes. But, there was no linkage between the rapid efflux, CQR phenotype and either of the mdr-like P. falciparum genes or amplification of those genes. These data indicate that the genetic locus governing chloroquine efflux and resistance is independent of the known mdr-like genes.     

横纹肌肉瘤多药耐药细胞系RD/VCR的建立

横纹肌肉瘤是一种高度恶性的软组织肿瘤，化疗是最主要的辅助治疗手段．然而，肿瘤细胞对药物产生多药耐药常常导致治疗失败．本实验以长春新碱为诱导剂，在体外成功建立了耐药细胞系DD／VCR，为进一步研究横纹肌肉瘤的耐药机理制作了模型．耐药前后细胞超微结构发生显变化，mdr1及mrp基因，P-糖蛋白及多药相关蛋白的表达与横纹肌肉瘤多药耐药的发生有关。     

Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies

One reason for the failure of chemotherapy in the treatment of advanced cancers may be the outgrowth of multidrug-resistant tumour cells. Multidrug resistance has been modelled in numerous mammalian cell lines in which the phenotype is characterized by a pleiotropic cross-resistance to unrelated drugs. In the study reported here, we have produced monoclonal antibodies whose binding to plasma membranes of different multidrug-resistant mammalian cells correlates with the degree of drug resistance. All these antibodies are specific for P-glycoprotein, a cell surface component of relative molecular mass (Mr) 170,000 (170K) that has been described previously, and are directed against three spatially distinct epitopes which define a conserved cytoplasmic domain in the C-terminal region of the P-glycoprotein polypeptide. The conserved nature of P-glycoprotein and its low-level expression is drug-sensitive cells suggest that it has an important function at the cell surface. The monoclonal antibodies against P-glycoprotein described here might serve as diagnostic reagents for clinically unresponsive tumours.     

靶向肿瘤多药耐药基因mdr1的siRNA的构建与鉴定

mdr 1基因及其表达产物P-gp是引起肿瘤细胞多药耐药(MDR)的主要原因,抑制mdr 1基因的表达可用于逆转MDR.RNAi可用于特异抑制靶基因的表达,本研究的目的是构建获得可特异有效靶向mdr 1基因的siRNA元件.应用siRNA设计软件与mRNA结构分析软件设计构建了3个分别靶向mdr 1基因mRNA环结构和茎结构的siRNA元件,同时构建了携带mdr1基因序列的luc报告质粒,通过siRNA表达质粒与携带靶序列的报告质粒的共转染抑制实验检测不同siRNA的抑制效率,结果显示靶向环结构siMDR1B具有较好的抑制效率和特异性.进一步将siMDR1B表达载体与mdr1基因表达载体共转染细胞,应用免疫流式细胞术检测显示,相比对照细胞,siMDR1B可显著抑制其转染后mdr1基因产物P-gp蛋白的表达活性.同时采用CCK-8细胞活性检测试剂评价了siMDR1B对细胞活性的影响,结果显示siMDR1B不会影响细胞活性,具有良好的特异性.本研究获得的可有效靶向mdr 1基因的siRNA元件可为进一步开展逆转MDR研究提供重要基础.     

Amplification of specific DNA sequences correlates with multi-drug resistance in Chinese hamster cells

Mammalian cells selected for resistance to certain cytotoxic drugs frequently develop cross-resistance to a broad spectrum of other drugs unrelated in structure to the original selective agent. This phenomenon constitutes a major problem in cancer chemotherapy. Multi-drug resistance arises from decreased intracellular drug accumulation, apparently due to an alteration of the plasma membrane. The observation of double minute chromosomes or homogeneously staining regions in some of the multi-drug-resistant cell lines suggests that gene amplification underlies this phenomenon. We have used the technique of DNA renaturation in agarose gels to detect, compare and clone amplified DNA sequences in Adriamycin- and colchicine-resistant sublines of Chinese hamster cells. We show that both Adriamycin- and colchicine-resistant cells contain amplified DNA fragments, some of which are amplified in both of these independently derived cell lines. Furthermore, loss of the multi-drug resistance phenotype on growth in the absence of drugs correlates with the loss of amplified DNA. These results strongly suggest that the DNA sequences which are amplified in common in multi-drug-resistant cell lines include the gene(s) responsible for a common mechanism of multi-drug resistance in these cells. We have cloned one of the commonly amplified DNA fragments and show that the degree of amplification of this fragment in the cells correlates with the degree of their drug resistance.     

mdr1介导的获得性多药耐药性在胃癌细胞SGC7901的自然逆转

目的:探讨mdr1介导的获得性多药耐药在胃癌细胞SGC7901的自然逆转情况.方法:将培养细胞分为SGC7901、加长春新碱(VCR)的SGC7901/VCR和停VCR 6个月的SGC7901/VCR(设为SGC7901/VCR-)3组,用RT-PCR和原位杂交检测mdr1 mRNA的表达,免疫印迹和免疫组化检测P-gp的表达,流式细胞仪检测阿霉素在细胞内的蓄积和潴留,MTT法检测细胞对化疗药物的敏感性.结果:SGC7901/VCR-组mdr1 mRNA和P-gp表达水平介于SGC7901和SGC7901/VCR之间.SGC7901/VCR-阿霉素蓄积和潴留均高于SGC7901/VCR,低于SGC7901(P<0.05).SGC7901/VCR-对化疗药物的IC50小于SGC7901/VCR,大于SGC7901(P<0.01);对DDP和ADR的耐药指数低于SGC7901/VCR(P<0.05).结论:停化疗药物6个月后,胃癌耐药细胞亚系SGC7901/VCR的多药耐药性降低,但未降到亲本SGC7901水平.     

Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain

Xeroderma pigmentosum (XP) is an autosomal recessive disease, characterized by a high incidence of sunlight-induced skin cancer. Cells from people with this condition are hypersensitive to ultraviolet because of a defect in DNA repair. There are nine genetic complementation groups of XP, groups A-H and a variant. We have cloned the mouse DNA repair gene that complements the defect of group A, the XPAC gene. Here we report molecular cloning of human and mouse XPAC complementary DNAs. Expression of XPAC cDNA confers ultraviolet-resistance on several group A cell lines, but not on lines of other XP groups. Almost all group A lines tested showed abnormality or absence of XPAC messenger RNAs. These results indicate that a defective XPAC gene causes group A XP. The human and mouse XPAC genes are located on chromosome 9q34.1 and chromosome 4C2, respectively. Human XPAC cDNA encodes a protein of 273 amino acids with a zinc-finger motif.     

Transforming ras genes from human melanoma: a manifestation of tumour heterogeneity?

Variability in the phenotype of cells comprising individual tumours is a striking feature of animal and human cancer and is generally referred to as tumour heterogeneity. Studies of clonally derived cell populations from tumours that originated presumably from a single transformed cell have shown that tumours are made up of cells that differ in a variety of traits, including drug resistance, antigen expression and metastatic potential. The origin and maintenance of tumour heterogeneity are unclear, but mutational and epigenetic mechanisms are thought to be involved. Here we report the results of a search for transforming genes in human melanoma which have raised the possibility that ras gene activation follows the same variable pattern as other traits involved in tumour heterogeneity. DNA from 4 of 30 melanoma cell lines yielded transforming ras genes in the NIH/3T3 assay. Of five cell lines originating from separate metastatic deposits of a single patient, only one contained activated ras, indicating heterogeneity in ras activation in this case and suggesting that ras activation was not involved in tumour initiation or maintenance in this patient.     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSD1-like zinc finger domains regulate disease resistance and programmed cell death(PCD). We cloned a rice OsLOL2 gene, orthologous to LSDI of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci (Pst). RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related (PR) protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR pro-teins and hypersensitive response-like reaction.     

用流式细胞术研究PKC及VRP，CsA对多药抗性细胞的调制与逆转

利用流式细胞术检测抗药性细胞内Ｒｈｏ－１２３的荧光强度作为检测抗性细胞抗性程度的指标，研究了蛋白激酶Ｃ（ＰＫＣ）抑制剂Ｈ７，肿瘤促进剂佛波酯（ＴＰＡ）对多药抗性细胞的调节；钙离子通道阻断剂维拉帕米（ＶＲＰ），免疫抑制剂（ＣｓＡ）对多药抗性的逆转作用．测定了抗性细胞细胞膜和胞浆ＰＫＣ活性水平，指出ＰＫＣ可能通过磷酸化细胞膜上的Ｐ－糖蛋白（Ｐ－ｇｐ）来调节多药抗性细胞的抗性水平，并提供了一种筛选多药抗性逆转药物的简便方法．     

Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-ras oncogene

Several groups have shown that the malignant phenotype can be transferred to NIH/3T3 fibroblasts by incorporation of DNA isolated from tumour cell lines. These studies have demonstrated that the transforming activity of DNA isolated from human bladder, lung and colon carcinoma cell lines is related to an alteration of the cellular homologues of the ras genes of Harvey or Kirsten murine sarcoma viruses. It is, however, unclear what relevance these observations have to the multi-stage nature of tumorigenesis in vivo, in which several independent events are required in both humans and experimental animals. The activation of a cellular oncogene in a defined experimental system for the progressive induction of solid tumours has not yet been demonstrated. We report here that high molecular weight DNA from transplanted squamous cell carcinomas induced by sequential treatment of mouse skin with initiators and promoters of carcinogenesis causes morphological transformation of NIH/3T3 fibroblasts at high frequency. The transforming properties are due to the transfer of an activated cellular homologue of the Harvey-ras (rasH) oncogene.     

Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter.

In mammalian cells, short peptides derived from intracellular proteins are displayed on the cell membrane associated with class I molecules of the major histocompatibility complex (MHC). The surface presentation of class I-peptide complexes presumably alerts the immune system to intracellular viral protein synthesis. Peptides derived from the cytosol must reach the cisternae of the endoplasmic reticulum where they are required for the assembly of stable class I molecules, and it has been proposed that the products of the two MHC-encoded ATP-binding cassette (ABC) transporter genes function to deliver the peptides across the membrane of the endoplasmic reticulum. This idea is supported by experiments in which transfection of a human cell line defective in class I expression with a complementary DNA of one of these genes restored cell surface expression levels. Here we show that the complete phenotype of the mouse mutant cell line RMA-S, in which lack of surface expression of stable class I molecules correlates with an inability to present viral peptides originating in the cytosol, is repaired by the cDNA of the other transporter gene. These results are consistent with the possibility that the two transporter polypeptides form a heterodimer.     

Failure of wild-type or a mutant form of protein kinase C-alpha to transform fibroblasts

A mutant form of the alpha-isoform of protein kinase C (PKC) was recently isolated from an ultraviolet radiation-induced murine fibrosarcoma cell line and reported to transform mouse BALB/c 3T3 fibroblasts on transfection. Four point mutations in the regulatory domain were assumed to be responsible for its oncogenicity and unusual preference for membrane localization. Here, we report that overexpression of the reported mutant PKC alpha complementary DNA in three fibroblast cell lines, including BALB/c 3T3, does not enable these cells to grow in soft agar or nude mice. In addition, this mutant PKC alpha form seems to be indistinguishable from the wild-type PKC alpha with respect to its dependence on cofactors, phorbol ester binding, subcellular distribution and its effects on growth and morphology. These results fail to confirm the previous study and indicate that overexpression of either the wild-type or the reported mutant form of PKC alpha does not transform rodent fibroblasts.     

Isolation and characterization of rice lesion mimic mutants from a T-DNA tagged population

A rice ( Oryza sativa L. ssp. japonica cv. Nipponbare) T-DNA tagged population consisting of about 7000 individual lines was generated and screened for rice lesion mimic mutants in the T1 generation. Ten lines were found to develop spontaneous lesions in the absence of pathogen infection and displayed distinct lesion phenotypes. These mutants were tentatively designated as lm1 -lm10 (for lesion mimic), respectively. Lesion formation of lm mutants was developmentally regulated, and all the mutants showed stunted growth and reduced fertility. Genetic analysis demonstrated that all the mutations were recessive, and five partially fertile mutants (lm4-lm8) were derived from different loci. Mimic lesions occurring on the leaves of lm mutants resulted from cell death as revealed by trypan blue staining. Six of them ( lm3 -lm8 ) exhibited enhanced resistance to five bacterial blight isolates, indicating their wide-spectrum resistance to this pathogen. These results imply that some lesion mimic mutations of rice might be involved in disease resistance signaling pathways,and that isolation of these mutated genes may be useful for elucidating molecular mechanisms of plant disease resistance. Among the mutants, only one mutant, lm6, was preliminarily shown to cosegregate with the inserted T-DNA in its T1 generation, making it feasible to isolate the gene responsible for the phenotype of this mutant.     

Prediction of multiple drug resistance phenotype in cancer cell lines using gene expression profiles and phylogenetic trees

When microarray gene expression data are used to predict multiple drug resistance (MDR) phenotypes for anticancer drugs, the normalization strategy and the quality of the selected signature genes are usually the main causes of inconsistency among different experiments. A stable statistical drug response prediction model is urgently required in oncology. In this study, the microarray gene expression data of multiple cancer cell lines with MDR was analyzed. For each probe-set, the expression value was defined as present/absent (1/0) and was classified into a gene set defined with protein domain organization (PDO). After employing the gene content method of phylogenetic analysis, a phylogenetic model (cell tree) for MDR phenotype prediction was built at the PDO gene set level. The results indicate that classification of cancer cell lines is predominantly affected by both the histopathological features and the MDR phenotype (paclitaxel and vinblastine). When applying this model to predict the MDR phenotype of independent samples, the phylogenetic model performs better than signature gene models. Although the utility of our procedure is limited due to sample heterogeneity, it still has potential application in MDR research, especially for hematological tumors or established cell lines.     

A complementary DNA clone for a macrophage-lymphocyte Fc receptor

Macrophages, granulocytes and many lymphocytes express or secrete receptors for the Fc domain of immunoglobulins (Ig). These Fc receptors (FcRs) are heterogeneous and can be distinguished on the basis of their cellular distribution and specificities for different immunoglobulin isotypes. Although their functions are not completely understood, FcRs are known to be involved in triggering various effector cell functions and in regulating differentiation and development of B-cells. One of the best characterized is the mouse macrophage-lymphocyte receptor for IgG1 and IgG2b (ref. 5). On macrophages, this FcR mediates the endocytosis of antibody-antigen complexes via coated pits and coated vesicles, the phagocytosis of Ig-coated particles, and the release of various inflammatory and cytotoxic agents. It is possible that the receptor possesses an intrinsic ligand-activated ion channel activity responsible for some of these functions. The IgG1/IgG2b FcR has been isolated and shown to be a transmembrane glycoprotein of relative molecular mass (Mr) 47,000-60,000 (47-60 K) containing four N-linked oligosaccharide chains and a large (greater than 10K) cytoplasmic domain. It is also immunologically indistinguishable from the murine Ly-17 alloantigen which, in turn, is tightly linked to the Mls lymphocyte activation locus. Here we describe the isolation and characterisation of a complementary DNA clone encoding the whole of the IgG1/IgG2b FcR expressed by the mouse macrophage-like cell line P388D1. The receptor is a member of the immunoglobulin superfamily and, like Ly-17, maps to the distal portion of chromosome 1. cDNA probes detect one or two mRNA species in FcR+ macrophage and B-cell lines, but not in FcR- cells or a receptor-deficient variant derived from a FcR+ B-cell line. Finally, DNA hybridization analysis indicates the receptor gene is partially deleted or rearranged in the FcR- variant.