title: Point mutations in human guanylate kinase account for acquired resistance to anticancer nucleotide analogue PMEG.
abstract: Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S(35)N V(168)F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague. kaiserova@uochb.cas.cz
authors.forename: Helena
authors.initials: H
authors.lastname: Mertlíková-Kaiserová
authors.forename: Michaela
authors.initials: M
authors.lastname: Rumlová
authors.forename: Eva
authors.initials: E
authors.lastname: Tloušťová
authors.forename: Eliška
authors.initials: E
authors.lastname: Procházková
authors.forename: Antonín
authors.initials: A
authors.lastname: Holý
authors.forename: Ivan
authors.initials: I
authors.lastname: Votruba
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D000263:Adenylate Kinase / Q000235:genetics; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D019008:Drug Resistance, Neoplasm; D006147:Guanine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D051528:Guanylate Kinases / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000493:pharmacokinetics / Q000494:pharmacology*; D010766:Phosphorylation; D017354:Point Mutation
chemical_list: D000970:Antineoplastic Agents; D009943:Organophosphorus Compounds; C063380:9-((2-phosphonylmethoxy)ethyl)guanine; C059499:9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine; D006147:Guanine; D000263:Adenylate Kinase; C110938:adenylate kinase 2; D051528:Guanylate Kinases; D000225:Adenine