P-glycoproteins in pathology: the multidrug resistance gene family in humans

Many cancers do not respond to chemotherapy on primary exposure to drugs, thus manifesting intrinsic drug resistance. Other cancers that do initially respond subsequently become resistant to the same drugs and simultaneously to other drugs to which the patient has had no previous exposure. This is a form of acquired drug resistance. There is a pressing need to better understand the mechanisms of drug resistance and to use this information to develop strategies for the chemosensitization of drug-resistant tumors. A goal of the pathology laboratory is to offer chemosensitivity tests that identify intrinsic or acquired resistance of tumors to specific drugs or classes of drugs to enable the clinician to tailor therapy to the biology of cancers in individual patients. Multidrug resistance is one type of drug resistance. It can be present in either an intrinsic or acquired form. The human gene that confers human multidrug resistance, the MDR1 gene, has been cloned and classified as a member of the MDR gene family. Its encoded protein, called Mdr1, is an energy-driven membrane efflux transporter that maintains intracellular concentrations of certain chemotherapeutic drugs at nontoxic levels. Useful model systems for studying multidrug resistance have been developed in several research laboratories. Applying selection pressure by exposing cultured cancer cells to escalating doses of natural product anti-cancer drugs allows cross-resistant cell lines to be produced which share patterns of drug resistance with human cancers. A common feature of these drug-resistant lines is the expression of Mdr1. Using techniques of genetic engineering, molecular probes have been developed that can be used to measure MDR1 mRNA and MDR1 gene amplification. Mdr can be measured by immunochemistry methods. Currently, such measurements are being used to stratify patients in clinical trials designed to determine if chemosensitization by inhibition of the pump function of Mdr is a clinically useful therapeutic strategy. If successful, Mdr/MDR1 mRNA laboratory testing might significantly increase the clinical laboratory's role in cancer patient management.     

Biochemical and genetic analysis of toxic effect of HOE 15030 in mammalian cells

HOE 15030 inhibited the growth of BHK cells at concentrations that did not inhibit their nuclear DNA and RNA syntheses. When BHK cells were cultured in the presence of 30 g/ml of HOE 15030, cells were arrested in the G₁ phase after one or two cell divisions. After removal of the drug, cells progressed through the G₁ to the S phase. HOE 15030 inhibited the activities of both topoisomerases I and II in vitro. To determine the target molecule of HOE 15030 in cells, we isolated a HOE 15030-resistant (HOE^r) mutant of BHK cells. The HOE^r cells exhibited cross-resistance to ethidium bromide, acriflavine, and rhodamine 123, and slight cross-resistance to 4-dimethylepipodophyllotoxin-4-(4,6-O -ethylidine--d-glucopyranoside) (VP-16) and adriamycin, but not to chloramphenicol, oligomycin, novobiocin, colchicine, or vinblastine. The uptake and retention of rhodamine 123 by HOE^r cells were lower than those by BHK cells. Mitochondrial DNA synthesis of HOE^r cells was more resistant to HOE 15030 and ethidium bromide than that of wild-type cells. These results indicate that the resistance of HOE^r cells to drugs is due to reduced uptake or accumulation of the drugs by mitochondria and suggest that the mitochondria are the main target of HOE 15030 in cells.     

Mutational resistance to 4-quinolone antibacterial agents

The activity of ten 4-quinolone drugs was tested against fiveEscherichia coli mutants. Mutational resistance was found to reduce the activity of all ten drugs, indicating that they display biochemical cross-resistance with each other. However, ciprofloxacin and, to a lesser extent, ofloxacin and norfloxacin were so highly active that the most resistance exhibited by any mutant fell well within the serum drug concentration ranges attainable in humans. Hence, clinical cross-resistance inEscherichia coli at least, need not necessarily apply to such highly active 4-quinolone antibacterial agents.     

Development and characterization of five cell models for chemoresistance studies of human ovarian carcinoma

Platinum agents and paclitaxel (taxol) are among the most effective drugs currently available for treatment of ovarian cancer. One of the hurdles with taxol and platinum- based therapy is the clinical development of resistance to these agents. To investigate the mechanism of drug resistance in human ovarian cancer, we developed and characterized 5 cell models for chemoresistance studies of cisplatin, carboplatin and taxol. We report in this study that these human ovarian carcinoma cell model systems include 2 models for cisplatin resistance, 2 models for carboplatin resistance, and 1 model for taxol resistance. The biological and biochemical characteristics of the models showed that (i), the IC50 values of the drugs for all these resistant cell models were 3 times (or more) higher than those for the parental tumor cells. There also exist varying degrees of cross-resistance to several other chemotherapeutic agents in these systems. Moreover, the intracellular drug accumulations in these cells were significantly reduced as compared to those in the parental cells. (ii), The proliferation rates of these resistant cells were markedly decreased. However, there were no obvious changes in cell cycle distribution in these model systems. (iii), Our results for the expression of a few major drug resistance-related genes revealed that the expression of p53, lrp-1 and mrp-1 was decreased, while the expression of pkc, topo I and topo II beta was increased in the resistant tumor cells as compared with the parental cells. In contrast, no significant alterations in gst-pi and topo II alpha expression were found. Interestingly, the levels of mdr-1 expression were elevated in some models, but were reduced in others, thus suggesting that different pathways are involved in the formation of drug resistance in different cell model systems, and that different mechanisms are responsible for the development of different drug resistances in tumor cells. Taken together, our findings indicate that these models may be potentially used to assess the biochemical and genetic mechanisms of drug resistance in human ovarian cancer and to identify new drug resistance-related genes.     

Molecular and genetic characterization of human cell lines resistant tol-asparaginase and albizziin

Human cell lines resistant tol-asparaginase or albizziin were isolated by multistep selection of HT1080 fibrosarcoma and MIA PaCa-2 pancreatic carcinoma cells. Mutants were cross-resistant to both drugs, but more resistant to the drug used for selection. The drug-resistant cell lines expressed elevated levels of asparagine synthetase activity and protein, up to 17-fold over that of the parental cells. Enzyme overproduction was due to gene amplification in the albizziin-resistant cells, whereas increased expression without amplification was observed inl-asparaginase-resistant cells.     

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5–30 times the IC₅₀ (30 μg mg ml⁻¹) of parental cells. The vinblastine-resistant parasites were also resistant to promycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug effiux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdrl, and demonstrate that this gene is amplified on an extrachromosomal circle of 35–40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr 1 and 83% identity with the L. donovani 1mdr 1 gene. The lemdr 1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.     

Molecular mechanisms of drug resistance

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Tumours may be intrinsically drug-resistant or develop resistance to chemotherapy during treatment. Acquired resistance is a particular problem, as tumours not only become resistant to the drugs originally used to treat them, but may also become cross-resistant to other drugs with different mechanisms of action. Resistance to chemotherapy is believed to cause treatment failure in over 90% of patients with metastatic cancer, and resistant micrometastic tumour cells may also reduce the effectiveness of chemotherapy in the adjuvant setting. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanisms of drug resistance that operate to reduce drug sensitivity in cancer cells. Drug resistance can occur at many levels, including increased drug efflux, drug inactivation, alterations in drug target, processing of drug-induced damage, and evasion of apoptosis. Advances in DNA microarray and proteomic technology, and the ongoing development of new targeted therapies have opened up new opportunities to combat drug resistance. We are now able to characterize the signalling pathways involved in regulating tumour cell response to chemotherapy more completely than ever before. This will facilitate the future development of rational combined chemotherapy regimens, in which the newer targeted therapies are used in combination with cytotoxic drugs to enhance chemotherapy activity. The ability to predict response to chemotherapy and to modulate this response with targeted therapies will permit selection of the best treatment for individual patients.     

Overexpression of asparagine synthetase in albizziin-resistant murine diploid embryonic stem cells

Gene amplification is commonly observed in primary tumors and established drug-resistant cell lines, both of which are generally aneuploid. However, this process is undetectable (frequency <10^–9) in normal diploid mammalian cell lines. To investigate whether gene amplification can occur in pluripotent diploid cells, we have selected drug-resistant mutants of mouse embryonic stem (ES) cells. We had previously found that Chinese hamster ovary (CHO) and human cell lines selected in albizziin (Alb), an amino acid analog ofl-glutamine, overexpress asparagine synthetase (AS) due to gene amplification. The same drug selection system was applied to ES cells to isolate single-step and multistep drug-resistant mutants. Albizziin-resistant ES cells exhibited elevated levels of AS; however, drug resistance in ES cells was associated with mRNA overexpression without gene amplification. As gene amplification was observed in only one drug-resistant cell line and was preceded by AS mRNA overexpression. Gene amplification in the latter coincided with the loss of the pluripotent nature of the ES cells.     

Some drug-resistant models for cancer chemotherapy Part 1: Cycle-nonspecific drugs

The pioneering mathematical analyses for describing the phenomenonof clonal selection, whereby cancer cells mutate and becomeresistant to treatment, were carried out by Goldie & Coldman(1979, 1983). The present analysis yields more generalized drug-resistantmodels arising in chemotherapy treatment when cycle-nonspecific(CNS) drugs (i.e. drugs which are toxic to cells irrespectiveof their position in the cell cycle) are deployed. This is accomplishedby modifying models developed by Wheldon (1988) and Usher (1994)for investigating the effect of chemotherapy drugs on homogeneouscell populations (i.e. in the absence of drug resistance); bothtypes of migration, of drug-sensitive cells to drug-resistantcells, and the possibility of a migration of drug-resistantcells to drug-sensitive cells, are incorporated into the models.Subsequently, drug concentrations are sought such that boththe drug-resistant cell subpopulation and the drug-sensitivecell subpopulation either simultaneously decay or remain ofconstant size. The analysis reveals that the role played bythe migration of drug-resistant cells to drug-sensitive cellsis significant, if it occurs, when seeking appropriate drugconcentrations. The qualitative results from these models providefurther insight into the effective treatment of cancer by chemotherapyin the presence of drug resistance.     

Patterns of resistance and sensitivity to antiviral compounds of drug-resistant strains of human cytomegalovirus selected in vitro

Drug-resistant human Cytomegalovirus (HCMV) strains were selected in human embryonic lung (HEL) fibroblasts under pressure of the (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of cytosine (HPMPC) and adenine (HPMPA), the 2-phosphonylmethoxyethyl (PME) derivative of 2,6-diaminopurine (PMEDAP), ganciclovir (GCV), acyclovir (ACV), and foscarnet (PFA). Drug susceptibility profiles of the different drug-resistant (i.e., GCV^r, HPMPC^r, HPMPA^r, PFA^r, ACV^r, and PMEDAP^r) strains were determined in HEL cells. A considerable degree of cross-resistance against GCV, HPMPC, and HPMPA occurred with the GCV^r, HPMPC^r, and HPMPA^r strains. No changes in susceptibility to 9-(2-phosphonylmethoxyethyl)adenine (PMEA), PMEDAP, ACV, or PFA were detected for the HPMPC^r, HPMPA^r, and GCV^r strains when compared to the wild-type virus. On the other hand, a significant degree of cross-resistance was noted with the PMEDAP^r, PFA^r, and ACV^r strains against PMEA, PMEDAP, PFA, and ACV. No differences in susceptibility to HPMPC, HPMPA and GCV were observed for the ACV^r, PFA^r, and PMEDAP^r strains relative to the wild type. The drug susceptibility profiles of the different resistant strains point to a common mechanism of HCMV resistance to PFA and the PME derivatives that is different from the mechanism of HCMV resistance to the HPMP derivatives.     

Non-infectious fluorimetric assay for phenotyping of drug-resistant HIV proteinase mutants.

BACKGROUND: The introduction of HIV proteinase inhibitors (PIs) as anti-AIDS drugs resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, rapid selection of drug-resistant HIV variants is a common complication in patients undergoing highly active anti-retroviral therapy (HAART). Thus, monitoring of clinical resistance development is indispensable for rational pharmacotherapy. OBJECTIVE: We present a non-infectious cell-based assay for drug resistance quantification of HIV proteinase (PR) - an important target of HAART. STUDY DESIGN: Previously, we showed [Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001;45:2616-22] that the expression of a fusion protein (GFP-PR), comprised of HIV-1 proteinase wild-type artificial precursor (PR) and green fluorescent protein (GFP), in transiently transfected tissue culture cells depends on the presence of PR-specific inhibitors (PIs). Here we show that in the GFP-PR reporter the HIV wild-type PR can be replaced by a drug-resistant HIV PR mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PR mutants susceptibility to HIV proteinase inhibitors. RESULTS: We cloned a set of GFP-PR reporters, some of which possess a simple, well-defined drug-resistant PR mutant (G48V L90M, V82A, A71V V82T I84V, D30N, K45I); another four complex PR mutants were obtained from patients undergoing HAART. The results were compared with genotyping and enzyme kinetics data. Furthermore, we designed a single inhibitor concentration experiment setup for easy evaluation of drug resistance profiles for mutants of interest. The resistance profiles clearly demonstrate the importance of succession of individual drugs during the treatment for drug resistance development. CONCLUSION: We show that the GFP-PR assay might serve as a non-infectious, rapid, cheap, and reliable alternative to the currently used phenotypic assays.     

肿瘤细胞通过激活MAPK途径的耐药相关机制

丝裂原活化蛋白激酶（ mitogen－activated protein kinase, MAPK）信号途径是调节细胞增殖、生长、分化、凋亡、黏附和迁移的细胞内信号转导的重要通路。近年来的研究发现MAPK信号转导途径不仅与肿瘤的发生发展有关,激活的MAPK可能与肿瘤细胞耐药相关。文章围绕MAPK3条主要通路： ERK1／2、JNK、 p38从影响细胞药物外排,凋亡通路的异常,以及DNA损伤修复能力增强等方面阐述了MAPK调控肿瘤细胞的耐药机制。对于肿瘤细胞通过激活MAPK信号转导途径调控耐药发生发展机制的了解,将对临床上肿瘤靶向治疗药物的选择起到理论指导作用。     

靶向Tim-3药物研发进展

肿瘤微环境可诱导一些免疫检查点分子的高表达,以利于肿瘤细胞逃避免疫系统的识别和攻击.目前基于程序性死亡受体1(programmed cell death protein 1,PD-1)、细胞毒性T淋巴细胞相关抗原4(cytotoxic T-lymphocyte associated protein 4,CTLA-4)以及T细胞免疫球蛋白黏蛋白3(T-cell immunoglobulin and mucin domain-containing protein 3,Tim-3)等免疫检查点分子的靶向抗体药物研发已经取得了极大的进展,其中多种靶向PD-1和CTLA-4的抗体药物已被美国食品药品监督管理局(Food andDrug Administration,FDA)批准上市,虽然有部分患者经以上药物治疗后出现完全缓解或生存期延长,但尚存在较多患者出现耐药或没有缓解病情的状况.最新的研究发现,靶向PD-1治疗的耐药患者Tim-3表达上调,Tim-3也被认为是潜在的新一代肿瘤治疗靶点,Tim-3与PD-1的靶向联合治疗有可能成为研究的热点.现就国内外靶向Tim-3药物的研究现状做一综述.     

Cross-resistance of novobiocin-resistant BHK cell line to topoisomerase II inhibitors

A novobiocin-resistant BHK cell line, designated as Nov^r A2, was found to exhibit cross-resistance to other topoisomerase II inhibitors such as 4-dimethylepipodophyllotoxin-4-(4,6-O-ethylidine--d-glucopyranoside) (VP-16), adriamycin, and 4-(9-acridinyl-ami-no)methanesulfon-m-anisidide (m-AMSA), and also to different types of drugs such as vinblastine and arabinocytidine. Nalidixic acid-resistant cells (A2Nal^r) of the Nov^rA2 cell line were phenotypically reverted to novobiocin sensitivity like wild-type cells and were also partially reverted to sensitivity to VP-16 and adriamycin, but not to vinblastine and arabinocytidine. When VP-16 was added to cell culture, the drug-induced DNA strand breaks were much fewer in Nov^rA2 cells than in BHK cells. This reduced level of strand breaks in Nov^rA2 cells was not due to reduced drug uptake, because the two cell lines accumulated similar levels of radiolabeled VP-16. VP-16 also induced fewer DNA breaks in isolated nuclei of Nov^rA2 cells than in those of BHK cells. There was no significant difference in the VP-16-induced DNA cleavage activities of partially purified topoisomerase II from BHK and Nov^r cells. These results show that the resistance of Nov^rA2 cells to various drugs is not acquired by a defense mechanism related to membrane permeability and suggest that the resistance of the Nov^rA2 cells to topoisomerase II inhibitors might be due in part to alteration in a topoisomerase II associated factor(s).     

Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-Mediated drug resistance

Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC₅₀ 6.3 μm vs. 36.3 μm) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy.     

PI3K-AKT-mTOR通路对骨肉瘤细胞顺铂耐药性的影响及其机制

目的:探讨磷脂酰肌醇3-激酶(PI3K)-蛋白激酶B(AKT)-哺乳动物雷帕霉素靶蛋白(mTOR)通路对骨肉瘤细胞顺铂耐药性的影响及其机制,为骨肉瘤的靶向治疗提供新的思路。方法:体外培养MG-63细胞,予2 μg/mL顺铂重复给药建立MG-63耐药细胞株,向MG-63耐药细胞株中转染miRNA-22高表达质粒建立miR...