RESUMEN
The motor protein kinesin is a tetramer consisting of two heavy and two light chains. Expression of an antisense RNA fragment derived from the mouse ubiquitous kinesin heavy chain (uKHC) cDNA is associated with a unique type of multidrug resistance. We analyzed the effects of retroviral transduction of the human uKHC and its derivatives on drug sensitivity of the human fibrosarcoma cell line HT1080. Surprisingly, overexpression of full-length uKHC and its variants that were deficient in the NH2-terminal motor domain produced a phenotype similar to that of antisense RNA, characterized by resistance to etoposide and collateral sensitivity to colchicine. This altered drug response, therefore, appears to be a general consequence of kinesin deregulation. The genetic suppressor element approach was applied to map the determinants of drug response in the kinesin heavy chain. A sense-oriented genetic suppressor element conferring resistance to etoposide was isolated from a retroviral library of randomly fragmented uKHC cDNA. This element encodes the last 55 amino acids of uKHC, suggesting that the COOH-terminal tail domain of uKHC is involved in the cellular drug response.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Colchicina/farmacología , Etopósido/farmacología , Supresores de la Gota/farmacología , Cinesinas/biosíntesis , Cinesinas/fisiología , Células 3T3/metabolismo , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN Complementario/genética , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Humanos , Cinesinas/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Supresión Genética , Transfección , Células Tumorales CultivadasRESUMEN
The tumor suppressor/developmental regulator protein WT1 encoded by the Wilms' tumor gene is a zinc finger-containing transcription factor which binds to the G+C-rich motif 5'-GCGGGGGCG-3' and represses transcription. Alternatively spliced variants of WT1 (termed+KTS) having an insertion in the zinc finger region are defective for binding to and hence for repression of transcription from promoters containing this motif. Due to the known interactions of two other tumor suppressor proteins with the simian virus 40 (SV40) oncoprotein large tumor antigen (TAg) [which in one case (p53) results in inhibition of the replication initiation activity of TAg], and because of the presence of G+C-rich sequences in the SV40 origin region, we tested the effect of WT1 on TAg- and SV40 origin-dependent DNA replication. WT1 and its alternatively spliced variants were found to be potent inhibitors of replication. Inhibition of replication by WT1 required portions of the N-terminal transcription repression domain and the C-terminal DNA binding domain, while other WT1 sequences needed for transcriptional regulation were dispensable. WT1 neither inhibited the synthesis of TAg nor formed a stable complex with it. Studies of the requirement of cis-active origin sequences in vivo and protein-DNA interactions in vitro indicated that WT1 and its alternatively spliced variants might inhibit replication by their novel binding to the GC box promoter motifs of the SV40 21 bp repeat replication-auxiliary sequence.
Asunto(s)
Replicación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Empalme Alternativo , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Secuencia de Bases , Células Cultivadas , ADN/biosíntesis , ADN/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Genes del Tumor de Wilms , Haplorrinos , Humanos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Origen de Réplica , Virus 40 de los Simios/genética , Transcripción Genética , Proteínas WT1 , Dedos de Zinc/genéticaRESUMEN
We describe a general strategy for cloning mammalian genes whose downregulation results in a selectable phenotype. This strategy is based on expression selection of genetic suppressor elements (GSEs), cDNA fragments encoding either specific peptides that act as dominant inhibitors of protein function or antisense RNA segments that efficiently inhibit gene expression. Since GSEs counteract the gene from which they are derived, they can be used as dominant selectable markers for the phenotype associated with downregulation of the corresponding gene. A retroviral library containing random fragments of normalized (uniform abundance) cDNA expressed in mouse NIH 3T3 cells was used to select for GSEs inducing resistance to the anticancer drug etoposide. Three GSEs were isolated, two of which are derived from unknown genes and the third encodes antisense RNA for the heavy chain of a motor protein kinesin. The kinesin-derived GSE induces resistance to several DNA-damaging drugs and immortalizes senescent mouse embryo fibroblasts, indicating that kinesin is involved in the mechanisms of drug sensitivity and in vitro senescence. Expression of the human kinesin heavy-chain gene was decreased in four of four etoposide-resistant HeLa cell lines, derived by conventional drug selection, indicating that downregulation of kinesin represents a natural mechanism of drug resistance in mammalian cells.