Specificity of ribozyme designed for mutated DHFR mRNA

Hiroyuki Kobayashi; Nuna Kim; Marc Eric Halatsch; Takao Ohnuma

doi:10.1016/0006-2952(94)90539-8

Specificity of ribozyme designed for mutated DHFR mRNA

Hiroyuki Kobayashi
, Nuna Kim
, Marc Eric Halatsch
, Takao Ohnuma

Icahn School of Medicine at Mount Sinai

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

18 Scopus citations

Abstract

When MOLT-3 human acute leukemia cells were exposed sequentially to trimetrexate (TMQ) and then to methotrexate (MTX), the cells became resistant to antifolate. We designated this subline MOLT-3/TMQ₈₀₀-MTX_10,000. This cell line was found to contain two point mutations in the dihydrofolate reductase (DHFR) gene: a T → C transition at nucleotide 95 in codon 31, and a T → A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we synthesized a ribozyme which perfectly base-paired with the double-mutated DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleaved the wild-type RNA substrate. This observation suggests proceeding with caution when using a ribozyme against a mutated mRNA of an essential enzyme as a specific means of treatment.

Original language	English
Pages (from-to)	1607-1613
Number of pages	7
Journal	Biochemical Pharmacology
Volume	47
Issue number	9
DOIs	https://doi.org/10.1016/0006-2952(94)90539-8
State	Published - 29 Apr 1994

Keywords

dihydrofolate reductase
drug resistance
methotrexate
mutation
ribozyme
trimetrexate

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10.1016/0006-2952(94)90539-8

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title = "Specificity of ribozyme designed for mutated DHFR mRNA",

abstract = "When MOLT-3 human acute leukemia cells were exposed sequentially to trimetrexate (TMQ) and then to methotrexate (MTX), the cells became resistant to antifolate. We designated this subline MOLT-3/TMQ800-MTX10,000. This cell line was found to contain two point mutations in the dihydrofolate reductase (DHFR) gene: a T → C transition at nucleotide 95 in codon 31, and a T → A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we synthesized a ribozyme which perfectly base-paired with the double-mutated DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleaved the wild-type RNA substrate. This observation suggests proceeding with caution when using a ribozyme against a mutated mRNA of an essential enzyme as a specific means of treatment.",

keywords = "dihydrofolate reductase, drug resistance, methotrexate, mutation, ribozyme, trimetrexate",

author = "Hiroyuki Kobayashi and Nuna Kim and Halatsch, \{Marc Eric\} and Takao Ohnuma",

note = "Funding Information: Acknowledgements--This work was supported, in part, by the T. J. Martell Memorial Foundation for Leukemia, Cancer and AIDS Research, New York, NY; by the Charlotte Geyer Foundation, Sarasota, FL; by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan; and by the United Leukemia Fund, New York, NY. The authors thank Dr. Joseph R. Bertino for providing us with DHFR antibody; Dr, Thambi Dorai for his valuable suggestions; Ms. Lenina Szrajer for performing the MTT assay; and Mr. Larry Brennan for preparing the manuscript.",

year = "1994",

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doi = "10.1016/0006-2952(94)90539-8",

language = "English",

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AU - Kobayashi, Hiroyuki

AU - Kim, Nuna

AU - Halatsch, Marc Eric

AU - Ohnuma, Takao

N1 - Funding Information: Acknowledgements--This work was supported, in part, by the T. J. Martell Memorial Foundation for Leukemia, Cancer and AIDS Research, New York, NY; by the Charlotte Geyer Foundation, Sarasota, FL; by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan; and by the United Leukemia Fund, New York, NY. The authors thank Dr. Joseph R. Bertino for providing us with DHFR antibody; Dr, Thambi Dorai for his valuable suggestions; Ms. Lenina Szrajer for performing the MTT assay; and Mr. Larry Brennan for preparing the manuscript.

PY - 1994/4/29

Y1 - 1994/4/29

N2 - When MOLT-3 human acute leukemia cells were exposed sequentially to trimetrexate (TMQ) and then to methotrexate (MTX), the cells became resistant to antifolate. We designated this subline MOLT-3/TMQ800-MTX10,000. This cell line was found to contain two point mutations in the dihydrofolate reductase (DHFR) gene: a T → C transition at nucleotide 95 in codon 31, and a T → A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we synthesized a ribozyme which perfectly base-paired with the double-mutated DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleaved the wild-type RNA substrate. This observation suggests proceeding with caution when using a ribozyme against a mutated mRNA of an essential enzyme as a specific means of treatment.

AB - When MOLT-3 human acute leukemia cells were exposed sequentially to trimetrexate (TMQ) and then to methotrexate (MTX), the cells became resistant to antifolate. We designated this subline MOLT-3/TMQ800-MTX10,000. This cell line was found to contain two point mutations in the dihydrofolate reductase (DHFR) gene: a T → C transition at nucleotide 95 in codon 31, and a T → A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we synthesized a ribozyme which perfectly base-paired with the double-mutated DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleaved the wild-type RNA substrate. This observation suggests proceeding with caution when using a ribozyme against a mutated mRNA of an essential enzyme as a specific means of treatment.

KW - dihydrofolate reductase

KW - drug resistance

KW - methotrexate

KW - mutation

KW - ribozyme

KW - trimetrexate

UR - https://www.scopus.com/pages/publications/0028226055

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JO - Biochemical Pharmacology

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ER -

Specificity of ribozyme designed for mutated DHFR mRNA

Abstract

Keywords

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