Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Schwartz, Stephen A.; Horio, David; Kirsten, W. H.

doi:10.1016/0006-291x(74)90244-7

Cited by 13 publications

(13 citation statements)

References 8 publications

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“…Secondary cultures of 16-day old Wistar/ Furth (W/Fu) rat embryo cells were maintained as described (1)(2)(3) (4)(5)(6). Subconfluent roller cultures were treated with 2 mM dT (Calbiochem).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were rinsed and 2 mM dT was added for an additional 16 hr. Cultures were again rinsed and released into synchrony with 10,MM dG, dA, and dC as before (5,6 (2,3). The specific activity of each DNA sample was determined.…”

Section: Methodsmentioning

confidence: 99%

“…Each DNA sample was made 0.40 M in sodium phosphate buffer, pH 6.8, and 3 mM in EDTA (2,3,7,8). The initial A260 of the reaction mixture was determined, and the sample was heat sealed in 5 ml glass ampoules and immersed completely in boiling water at 1000 for 10-15 min (2, 3, 7).…”

Section: Introductionmentioning

confidence: 99%

“…Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H] (2,3). The specific activity of each DNA sample was determined.…”

mentioning

confidence: 99%

“…Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H]BrdU was incorporated predominantly into the repetitive and intermediate repetitive DNA sequences when the concentration of the analog was reduced to 0.1 pAM (2,3).…”

mentioning

confidence: 99%

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Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

Schwartz¹,

Panem²,

Kirsten³

1975

Proc. Natl. Acad. Sci. U.S.A.

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Rat embryo cell cultures were synchronized by a double thymidine block. The DNA replication phase (S) was divided into an early, middle, and late period. Cell cultures in the early, middle, or late S phase were pulsed with 0.1 JM 5-bromo[3H]deoxyuridine (BrdU) or equimolar [3HldT. DNA The thymidine analog 5-bromodeoxyuridine (BrdU) induces the synthesis of a latent type C virus in secondary cultures of normal rat embryo cells (1). Release of type C particles into the culture fluids was first detected 2 days after BrdU removal, peaked 8-10 days later, and disappeared 14-16 days after removal of the analog. Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H] (2,3). The specific activity of each DNA sample was determined. Aliquots of radiolabeled DNA were centrifuged to equilibrium in gradients of neutral CsCl (2, 3). Gradient fractions were assayed for radioactivity, absorbance, and refractive index as described (2, 3).Renaturation Kinetics. Each DNA sample was made 0.40 M in sodium phosphate buffer, pH 6.8, and 3 mM in EDTA (2,3,7,8). The initial A260 of the reaction mixture was determined, and the sample was heat sealed in 5 ml glass ampoules and immersed completely in boiling water at 1000 for 10-15 min (2, 3, 7). After thermal denaturation, each ampoule was transferred to a 680 water bath and incubated until the product of initial DNA concentration (mol of 1829 Abbreviations: S, DNA replication phase of cell cycle; p30, group-specific antigen of Friend leukemia virus; BrdU, 5-bromodeoxyuridine; Cot, initial DNA concentration time.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

“…Density gradient centrifugation and DNA -DNA reassociation experiments revealed extensive and uniform base substitutions throughout the DNA sequences of rat embryo cells at the optimal virogenic BrdU dose (0.1 mM) (2). However, [3H]BrdU was incorporated predominantly into the repetitive and intermediate repetitive DNA sequences when the concentration of the analog was reduced to 0.1 pAM (2,3).…”

mentioning

confidence: 99%

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Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

Schwartz¹,

Panem²,

Kirsten³

1975

Proc. Natl. Acad. Sci. U.S.A.

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The differential susceptibility of early chick embryos to 5-bromodeoxyuridine

Lee

Kalmus

1978

Experientia

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Modulation of the cytotoxic mechanism of 6-thioguanine by 4-amino-5-imidazolecarboxamide

Maybaum

Morgans

Ting

et al. 1990

Cancer Chemother Pharmacol

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Previous evidence has indicated that either purine starvation or incorporation into DNA may be the dominant biochemical effect of the antileukemic agent 6-thioguanine (TG), depending on exposure conditions. Furthermore, it has been suggested that the paradoxical decrease in TG-induced cytotoxicity at high drug concentrations may be due to an antagonistic interaction between these two mechanisms, in which purine starvation inhibits DNA synthesis and, therefore, incorporation of TG into DNA. In this report we test the hypothesis that by concurrent treatment of L1210 cells with TG and the purine precursor 4-amino-5-imidazolecarboxamide (AIC) it is possible to alleviate DNA synthesis inhibition caused by high concentrations of TG, thus enhancing TG incorporation into DNA and TG-induced cell kill. Both the cytotoxic and cytokinetic results presented support this hypothesis. However, gross incorporation of TG into DNA was not increased by AIC under conditions in which a significant enhancement of cytotoxicity (i.e., 1 log) was observed. These findings suggest that the potentiating effect of AIC may be most prominent on the subpopulation of cells that are resistant to treatment with TG alone, and they demonstrate that the cytotoxic effects of TG treatments are more accurately reflected by observing specific cytokinetic changes (delayed late S/G2 arrest) than by measuring the average extent of TG incorporation into DNA within a given population. Finally, we propose that it may be possible to select conditions for administration of TG that favor one or the other cytotoxic mechanism, depending on whether the clinical objective is induction of remission (where rapid cell lysis due to purine starvation would be desired) or eradication of subclinical disease during remission (where proliferation-dependent cytotoxicity due to DNA incorporation should be more effective.

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Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Cited by 13 publications

References 8 publications

Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.

The differential susceptibility of early chick embryos to 5-bromodeoxyuridine

Modulation of the cytotoxic mechanism of 6-thioguanine by 4-amino-5-imidazolecarboxamide

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