Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors.

Takebe, N; Nakahara, S; Zhao, S C; Adhikari, D; Ural, A U; Iwamoto, M; Banerjee, D; Bertino, J R

Takebe, N; Nakahara, S; Zhao, S C; Adhikari, D; Ural, A U; Iwamoto, M; Banerjee, D; Bertino, J R.

Afiliación

Takebe N; Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

Cancer Gene Ther ; 7(6): 910-9, 2000 Jun.

Article en En | MEDLINE | ID: mdl-10880023

RESUMEN

We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.

Asunto(s)

Células 3T3/efectos de los fármacos; Médula Ósea/metabolismo; ADN Complementario/genética; Resistencia a Medicamentos/genética; Células Madre Hematopoyéticas/metabolismo; Metotrexato/farmacología; Virus de la Leucemia Murina de Moloney/genética; Tetrahidrofolato Deshidrogenasa/genética; Animales; Northern Blotting; Southern Blotting; Western Blotting; Médula Ósea/efectos de los fármacos; Cartilla de ADN/química; Regulación Enzimológica de la Expresión Génica; Vectores Genéticos; Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis; Células Madre Hematopoyéticas/efectos de los fármacos; Humanos; Leucemia/patología; Masculino; Ratones; Regiones Promotoras Genéticas; Reacción en Cadena de la Polimerasa de Transcriptasa Inversa; Tetrahidrofolato Deshidrogenasa/metabolismo; Transducción Genética; Células Tumorales Cultivadas

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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Tetrahidrofolato Deshidrogenasa / Médula Ósea / Resistencia a Medicamentos / Células Madre Hematopoyéticas / Metotrexato / Células 3T3 / ADN Complementario / Virus de la Leucemia Murina de Moloney Límite: Animals / Humans / Male Idioma: En Revista: Cancer Gene Ther Asunto de la revista: GENETICA MEDICA / NEOPLASIAS / TERAPEUTICA Año: 2000 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido

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