Evidence that incorporation of inorganic [ 32P]phosphate into genetic and/or metabolic DNA of regenerating rat liver is stimulated 6 hours earlier than similar incorporation of labeled thymidine

Abstract

The onset of stimulated DNA synthesis in regenerating rat liver was studied by pulsing either [ Me- 3H]thymidine (dThd) or 32P i into rats at various intervals post-partial hepatectomy. Controls were rats subjected to laparotomy. After isolating DNA by 3 different methods, hydrolyzing the DNA enzymatically and chromatographically separating the constituent nucleotides, it was concluded that incorporation of 32P i into DNA was significantly stimulated 12 h post-partial hepatectomy, and that similar stimulation of [ Me- 3H]dThd incorporation occurred 6 h later, i.e., 18 h post-partial hepatectomy. The difference in the entry-time into DNA by the 2 precursors was not associated with mitochondrial DNA synthesis, perturbations of intracellular pools or unique juxtaposition of diurnal rhythms. Incorporation of 32P i into DNA 12 h post-partial hepatectomy was inhibited by 4 of 5 drugs known to inhibit, in other cell or tissue systems, the incorporation of labeled dThd into DNA and/or cell multiplication. Nuclei from 12 h regenerating rat liver incorporated [Me- 3 H]dThd-5′-P 3 into DNA in vitro at more than twice the rate determined for nuclei from 12-h laparotomized rats and after centrifugation through neutral sucrose density gradients, isotope from [Me- 3 H]dThd-5′-P 3 was distributed among all sedimentation species detectable by 260 nm absorbancy. Liver DNA was labeled 12 or 24 h post-partial hepatectomy with a 1 h pulse of 32P i or [ Me- 3H]dThd, respectively. After purification of the DNA and centrifugation through neutral sucrose density gradients, about 70% of 32P-labeled DNA was associated with fractions that absorbed light at 260 nm, while essentially all the 3H-labeled DNA was associated with such fractions. Both 32P-labeled DNA and 3H-labeled DNA were labile to DNAase hydrolysis. Liver DNA also was labeled with [ Me- 3H]dThd the first 20 days post-partum; 10 weeks later, these rats were subjected to partial hepatectomy; 12, 18 and 24 h post-operation, they were pulsed 1 h with 32P i. When purified DNA from these rats was centrifuged through neutral sucrose density gradients, profiles for 3H-radioactivity and absorbancy at 260 nm were superimposable, irrespective of time after operation. At 12, 18 and 24 h post-partial hepatectomy, 20, 80 and 90%, respectively, of 32P-radioactivity was associated with fractions that absorbed light at 260 nm and the specific radioactivity of this 32P-labeled DNA increased; the balance of the 32P-radioactivity was associated with fractions that sedimented more slowly than those showing 260 nm absorbancy. The distribution of 32P-radioactivity, incorporated 12 h post-partial hepatectomy, among pyrimidine isostichs was similar to the distribution of radioactivity from [ Me- 3H]dThd incorporated into DNA during the first 20 days post-partum. When hepatic cells from 12-h laparotomized or partially hepatectomized rats were lysed over and centrifuged through alkaline sucrose density gradients, about twice as much radioactivity was located in high molecular weight fractions from 12-h regenerating cells, and the high molecular weight DNA from either source was about equally labile to hydrolysis by DNAase or hot acid. It was concluded that the synthesis of genetic and/or metabolic DNA was stimulated 12 h post-partial hepatectomy and this stimulation occurred in the absence of demonstrable participation by exogenous [ Me- 3H]dThd.