TY - JOUR
T1 - Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro
AU - Ronen, A.
AU - Gingerich, J. D.
AU - Duncan, A. M.V.
AU - Heddle, J. A.
PY - 1984
Y1 - 1984
N2 - Diphtheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. We report here that resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [3H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to γ-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication, which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [3H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium.
AB - Diphtheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. We report here that resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [3H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to γ-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication, which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [3H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium.
UR - http://www.scopus.com/inward/record.url?scp=0344563009&partnerID=8YFLogxK
U2 - 10.1073/pnas.81.19.6124
DO - 10.1073/pnas.81.19.6124
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
C2 - 6592605
AN - SCOPUS:0344563009
SN - 0027-8424
VL - 81
SP - 6124
EP - 6128
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19 I
ER -