Mechanisms and strategies to overcome multiple drug resistance in cancer

One of the major problems in chemotherapy is multidrug resistance (MDR) against anticancer drugs. ATP-binding cassette (ABC) transporters are a family of proteins that mediate MDR via ATP-dependent drug efflux pumps. Many MDR inhibitors have been identified, but none of them have been proven clinically useful without side effects. Efforts continue to discover not toxic MDR inhibitors which lack pharmacokinetic interactions with anticancer drugs. Novel approaches have also been designed to inhibit or circumvent MDR. In this review, the structure and function of ABC transporters and development of MDR inhibitors are described briefly including various approaches to suppress MDR mechanisms.     

The evaluations of 99mTc cyclopentadienyl tricarbonyl triphenyl phosphonium cation for multidrug resistance

A triphenylphosphonium cation, [^99mTc]Technetium cyclopentadienyltricarbonyl-6-hexanoyl-triphenylphosphonium cation ([^99mTc]3) was prepared to target multidrug resistance (MDR). The radiotracer was evaluated in the MDR-negative MCF-7 and MDR-positive MCF-7/ADR cell lines in vitro, as well as animal models in vivo. [^99mTc]3 was proofed to be a substrate of P-glycoprotein and multidrug resistant protein 1, and showed a higher accumulation in the MDR-negative MCF-7 cells compared to ^99mTc-sestamibi in vitro. The MCF-7 tumor-to-MCF-7/ADR tumor ratio of [^99mTc]3 was ～3 at 1 h p.i. in the biodistribution study. These results demonstrated the capability of the radiotracer to detect multidrug resistance in tumor cells.     

光神霉素A抑制人肺腺癌A549／DDP细胞MRP1基因表达

该研究旨在探讨Sp1抑制剂光神霉素A（MithramycinA）对人肺腺癌A549／DDP细胞MRPI表达的影响。不同浓度光神霉素A作用A549／DDP细胞48h后,采用MTT法检测细胞存活率,RealtimeRT-PcR检测印,和MRPI基因表达水平,Westernblotg检测NSp1和MRP1蛋白表达水平。结果显示,300nmol／L光神霉素A作用A549／DDP细胞48h后印,和MRP1mRNA表达水平分别降低31．22％和85．44％,Sp1和MRP1蛋白表达水平分别降低53．27％和40．42％。提示光神霉素A能够通过抑制勋,表达,从而抑制MRP1表达。     

Secondary structure probing of the human RNase MRP RNA reveals the potential for MRP RNA subsets

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses an RNA subunit, structurally related to that of RNase P RNA, that is thought to be catalytic. RNase MRP RNA sequences from Saccharomycetaceae species are structurally well defined through detailed phylogenetic and structural analysis. In contrast, higher eukaryote MRP RNA structure models are based on comparative sequence analysis of only five sequences and limited probing data. Detailed structural analysis of the Homo sapiens MRP RNA, entailing enzymatic and chemical probing, is reported. The data are consistent with the phylogenetic secondary structure model and demonstrate unequivocally that higher eukaryote MRP RNA structure differs significantly from that reported for Saccharomycetaceae species. Neither model can account for all of the known MRP RNAs and we thus propose the evolution of at least two subsets of RNase MRP secondary structure, differing predominantly in the predicted specificity domain.     

Structural and functional similarities between MRP and RNase P

RNase P, the enzyme responsible for 5-end processing of tRNAs and 4.5S RNA, has been extensively characterized fromE. coli. The RNA component ofE. coli RNase P, without the protein, has the enzymatic activity and is the first true RNA enzyme to be characterized. RNase P and MRP are two distinct nuclear ribonucleoprotein (RNP) particles characterized in many eukaryotic cells including human, yeast and plant cells. There are many similarities between RNase P and MRP. These include: (1) sequence specific endonuclease activity; (2) homology at the primary and secondary structure levels; and (3) common proteins in both the RNPs. It is likely that RNase P and MRP originated from a common ancestor.     

A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C4 into membrane vesicles

This study investigated a high-throughput assay to measure multidrug resistance-associated protein (MRP1)-mediated uptake into membrane vesicles. Typically, a rapid filtration technique using a 12-filter vacuum manifold is used. We report here the development of a 96-well microtiter dish assay. MRP1-transfected HeLa cells (HeLa-T5) were used for the membrane vesicle preparations. The uptake of 50nM [3H]leukotriene C(4) (LTC(4)) was measured in a 96-well microtiter dish with rapid filtration onto a Perkin Elmer unifilter GF/B plate using a Perkin Elmer Filtermate 196. Counting of the isotype was conducted with a Perkin Elmer Top Count NXT. Uptake was adenosine 5'-triphosphate-dependent and linear over a 120-s time course. Uptake was inhibited by the leukotriene D(4) antagonist, MK 571, with a k(i) of 0.67 microM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-beta-glucuronide was 35-fold greater than inhibition by estradiol-3-beta-glucuronide. The kinetic parameters for LTC(4) uptake were determined to be a K(m) of 157nM with a V(max) of 344pmol/min/mg protein. The properties of MRP1-mediated transport of LTC(4) are consistent with those previously reported. The microtiter dish assay is a more expedient method for measuring transport into membrane vesicles and will have applications to other transporters.     

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain

Ribonuclease (RNase) P is a site‐specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix‐loop‐helix protein‐binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 Å. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.     

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.     

Challenges and issues in scaling commercial production of conifer somatic embryogenesis

Summary Significant progress has been made toward commercial implementation of a large-scale somatic embryogenesis (SE) production process for spruce somatic embryos. The major emphasis was to overlay the established principles and disciplines of “Manufacturing Resource Planning” (MRP) onto the original laboratory-scale protocols used to produce spruce SE and SE products. This approach required extensive and precise characterization of the inputs, actions, labor requirements, and quantification of the resulting outputs for each process step. In addition, computerized process and inventory tracking programs were developed and incorporated. The information collected enabled identification and clear understanding of the biological and physical constraints within the process, and enabled optimization of SE production planning, costing and scheduling activities. Standard operating procedures (SOPs) for each step of the SE process were developed and implemented. The net effect was the productivity of the scaled laboratory spruce SE process was increased by more than 300% without any additional labor inputs, while the direct cost-of-production for a “finished” somatic seedling was reduced by more than 70%.