Effect of ABCB1 and ABCC3 Polymorphisms on Osteosarcoma Survival after Chemotherapy: A Pharmacogenetic Study

Background: Standard treatment for osteosarcoma patients consists of a combination of cisplatin, adriamycin, and methotrexate before surgical resection of the primary tumour, followed by postoperative chemotherapy including vincristine and cyclophosphamide. Unfortunately, many patients still relapse or suffer adverse events. We examined whether common germline polymorphisms in chemotherapeutic transporter and metabolic pathway genes of the drugs used in standard osteosarcoma treatment may predict treatment response. Methodology/Principal Findings: In this study we screened 102 osteosarcoma patients for 346 Single Nucleotide Polymorphisms (SNPs) and 2 Copy Number Variants (CNVs) in 24 genes involved in the metabolism or transport of cisplatin, adriamycin, methotrexate, vincristine, and cyclophosphamide. We studied the association of the genotypes with tumour response and overall survival. We found that four SNPs in two ATP-binding cassette genes were significantly associated with overall survival: rs4148416 in ABCC3 (per-allele HR = 8.14, 95%CI = 2.73-20.2, p-value = 5.1×10 -5), and three SNPs in ABCB1, rs4148737 (per-allele HR = 3.66, 95%CI = 1.85-6.11, p-value = 6.9×10 -5), rs1128503 and rs10276036 (r 2 = 1, per-allele HR = 0.24, 95%CI = 0.11-0.47 p-value = 7.9×10 -5). Associations with these SNPs remained statistically significant after correction for multiple testing (all corrected p-values [permutation test] ≤0.03). Conclusions: Our findings suggest that these polymorphisms may affect osteosarcoma treatment efficacy. If these associations are independently validated, these variants could be used as genetic predictors of clinical outcome in the treatment of osteosarcoma, helping in the design of individualized therapyThis work was supported by the AECC (Asociación Española contra el Cáncer), FIS (Fondo de Investigación Sanitaria-Instituto de Salud Carlos III) and the ‘‘Inocente Inocente’’ Foundatio

Pharmacological Properties and Physiological Function of a P2X-Like Current in Single Proximal Tubule Cells Isolated from Frog Kidney

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2Xf) that was also Ca2+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2Xf was inhibited by suramin, PPADS and TNP-ATP. Activation of P2Xf attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2Xf plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2Xf inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells

Membrane stretch affects gating modes of a skeletal muscle sodium channel.

The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension

Stretch-activation and stretch-inactivation of Shaker-IR, a voltage-gated K+ channel.

Mechanosensitive (MS) ion channels are ubiquitous in eukaryotic cell types but baffling because of their contentious physiologies and diverse molecular identities. In some cellular contexts mechanically responsive ion channels are undoubtedly mechanosensory transducers, but it does not follow that all MS channels are mechanotransducers. Here we demonstrate, for an archetypical voltage-gated channel (Shaker-IR; inactivation-removed), robust MS channel behavior. In oocyte patches subjected to stretch, Shaker-IR exhibits both stretch-activation (SA) and stretch-inactivation (SI). SA is seen when prestretch P(open) (set by voltage) is low, and SI is seen when it is high. The stretch effects occur in cell-attached and excised patches at both macroscopic and single-channel levels. Were one ignorant of this particular MS channel's identity, one might propose it had been designed as a sophisticated reporter of bilayer tension. Knowing Shaker-IR's provenance and biology, however, such a suggestion would be absurd. We argue that the MS responses of Shaker-IR reflect not overlooked "mechano-gating" specializations of Shaker, but a common property of multiconformation membrane proteins: inherent susceptibility to bilayer tension. The molecular diversity of MS channels indicates that susceptibility to bilayer tension is hard to design out of dynamic membrane proteins. Presumably the cost of being insusceptible to bilayer tension often outweighs the benefits, especially where the in situ milieu of channels can provide mechanoprotection

Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1.

We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F). The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E. coli. A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that beta-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function