Studies in the applicant's laboratory have demonstrated the presence of a novel mechanism capable of transporting both anionic glutathione-electrophile thioester conjugates (GS-E) as well as the weakly cationic chemotherapeutic agent, doxorubicin in erythrocyte membranes. The applicant hypothesizes that the mechanisms capable of shared transport of DOX and GS-E should be useful in modulating resistance to DOX as well as to other natural product chemotherapeutic agents. Preliminary studies of the kinetic properties of a purified and reconstituted 38KD erythrocyte membrane proteins support the hypothesis that this protein is capable of DOX and GS-E transport. A preliminary sequence analysis suggests that it belongs to the ABC family of transport proteins but does not match any of 33 ABC family transporter genes including MRP and related proteins. Studies in a H-69 human small cell lung cancer cell line and derived DOX resistant sublines indicate heterogeneity in GS-E inhibitable DOX transport mechanisms. The 38KD proteins was not detected, but two immunologically related proteins of molecular weights 64 and 109kD were found in H69 cells. A hypothesis is presented that DNP-SG ATPase represents a novel GS-E inhibitable DOX transport mechanisms with related proteins expressed in mammalian cells and that expression of this protein can determine susceptibility to chemotherapeutic agents. Studies are proposed in this application to: 1) isolate and characterize membrane transporters immunologically related to DNP-SGATPase in membrane fraction from several human cancer cell lines; proteins will be isolated using a novel DNP-SG affinity procedure and activity determined using a substrate stimulated ATPase assay. Kinetic properties, temperature and pH dependence and effects of substrates and inhibitors on the protein will be determined followed by peptide fragment analysis and amino acid sequencing. 2) determine the ability of each protein to mediate ATP-dependent active transport of GS-E and chemotherapeutic drugs against a concentration gradient in crude membrane vesicles and in artificial vesicles reconstituted with purified protein ; 3) determine the effects of differential levels of expression of DNP-SG ATPase and related transporters in different cells on drug sensitivity and on cellular drug transport using a cell flow system developed by the applicant and 4) study the interrelationship between transporters through molecular cloning of their cDNA using degenerate oligonucleotides based on the peptide sequence.
| Awasthi, Sanjay; Tompkins, Joshua; Singhal, Jyotsana et al. (2018) Rlip depletion prevents spontaneous neoplasia in TP53 null mice. Proc Natl Acad Sci U S A 115:3918-3923 |
| Wurtzel, Jeremy G T; Lee, Seunghyung; Singhal, Sharad S et al. (2015) RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes. Biochem Biophys Res Commun 467:785-91 |
| Singhal, Sharad S; Singhal, Jyotsana; Figarola, James et al. (2015) RLIP76 Targeted Therapy for Kidney Cancer. Pharm Res 32:3123-36 |
| Singhal, Sharad S; Singhal, Jyotsana; Figarola, James L et al. (2015) 2'-Hydroxyflavanone: A promising molecule for kidney cancer prevention. Biochem Pharmacol 96:151-8 |
| Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti et al. (2015) Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling. Toxicol Appl Pharmacol 289:361-70 |
| Figarola, James L; Singhal, Jyotsana; Rahbar, Samuel et al. (2014) LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells. Apoptosis 19:776-88 |
| Sahu, Mukesh; Sharma, Rajendra; Yadav, Sushma et al. (2014) Lens specific RLIP76 transgenic mice show a phenotype similar to microphthalmia. Exp Eye Res 118:125-34 |
| Sehrawat, Archana; Yadav, Sushma; Awasthi, Yogesh C et al. (2013) P300 regulates the human RLIP76 promoter activity and gene expression. Biochem Pharmacol 85:1203-11 |
| Vatsyayan, Rit; Singhal, Jyotsana; Nagaprashantha, Lokesh Dalasanur et al. (2013) Nutlin-3 enhances sorafenib efficacy in renal cell carcinoma. Mol Carcinog 52:39-48 |
| Chaudhary, Pankaj; Sharma, Rajendra; Sahu, Mukesh et al. (2013) 4-Hydroxynonenal induces G2/M phase cell cycle arrest by activation of the ataxia telangiectasia mutated and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) signaling pathway. J Biol Chem 288:20532-46 |
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