Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression

Philipp Mayer-Kuckuk; Debabrata Banerjee; Sundeep Malhotra; Mikhail Doubrovin; Marian Iwamoto; Tim Akhurst; Julius Balatoni; William Bornmann; Ronald Finn; Steven Larson; Yuman Fong; Juri Gelovani Tjuvajev; Ronald Blasberg; Joseph R. Bertino

doi:https://doi.org/10.1073/pnas.062036899

Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression

Philipp Mayer-Kuckuk, Debabrata Banerjee, Sundeep Malhotra, Mikhail Doubrovin, Marian Iwamoto, Tim Akhurst, Julius Balatoni, William Bornmann, Ronald Finn, Steven Larson, Yuman Fong, Juri Gelovani Tjuvajev, Ronald Blasberg, Joseph R. Bertino

Cancer Institute of New Jersey (CINJ), Basic Research

Rutgers, The State University

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Abstract

Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drugmediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.

Original language	American English
Pages (from-to)	3400-3405
Number of pages	6
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	99
Issue number	6
DOIs	https://doi.org/10.1073/pnas.062036899
State	Published - Mar 19 2002

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Mayer-Kuckuk, P., Banerjee, D., Malhotra, S., Doubrovin, M., Iwamoto, M., Akhurst, T., Balatoni, J., Bornmann, W., Finn, R., Larson, S., Fong, Y., Tjuvajev, J. G., Blasberg, R., & Bertino, J. R. (2002). Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression. Proceedings of the National Academy of Sciences of the United States of America, 99(6), 3400-3405. https://doi.org/10.1073/pnas.062036899

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title = "Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression",

abstract = "Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drugmediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.",

author = "Philipp Mayer-Kuckuk and Debabrata Banerjee and Sundeep Malhotra and Mikhail Doubrovin and Marian Iwamoto and Tim Akhurst and Julius Balatoni and William Bornmann and Ronald Finn and Steven Larson and Yuman Fong and Tjuvajev, {Juri Gelovani} and Ronald Blasberg and Bertino, {Joseph R.}",

year = "2002",

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doi = "https://doi.org/10.1073/pnas.062036899",

language = "American English",

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Mayer-Kuckuk, P, Banerjee, D, Malhotra, S, Doubrovin, M, Iwamoto, M, Akhurst, T, Balatoni, J, Bornmann, W, Finn, R, Larson, S, Fong, Y, Tjuvajev, JG, Blasberg, R & Bertino, JR 2002, 'Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression', Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 6, pp. 3400-3405. https://doi.org/10.1073/pnas.062036899

Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression. / Mayer-Kuckuk, Philipp; Banerjee, Debabrata; Malhotra, Sundeep et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 6, 19.03.2002, p. 3400-3405.

Research output: Contribution to journal › Article › peer-review

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T1 - Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase

T2 - A method to modulate gene expression

AU - Mayer-Kuckuk, Philipp

AU - Banerjee, Debabrata

AU - Malhotra, Sundeep

AU - Doubrovin, Mikhail

AU - Iwamoto, Marian

AU - Akhurst, Tim

AU - Balatoni, Julius

AU - Bornmann, William

AU - Finn, Ronald

AU - Larson, Steven

AU - Fong, Yuman

AU - Tjuvajev, Juri Gelovani

AU - Blasberg, Ronald

AU - Bertino, Joseph R.

PY - 2002/3/19

Y1 - 2002/3/19

N2 - Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drugmediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.

AB - Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drugmediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.

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Mayer-Kuckuk P, Banerjee D, Malhotra S, Doubrovin M, Iwamoto M, Akhurst T et al. Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression. Proceedings of the National Academy of Sciences of the United States of America. 2002 Mar 19;99(6):3400-3405. doi: https://doi.org/10.1073/pnas.062036899

Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: A method to modulate gene expression

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