A growth factor for hepatocytes is inactivated by sub-lethal gamma-irradiation in vivo or in vitro.

Serum from partially hepatectomized rats promoted DNA synthesis in primary adult rat hepatocyte cultures. If the rats had been exposed to sub-lethal gamma-irradiation immediately following operation or if their serum, collected at 3 h, was exposed to irradiation in vitro, the growth-promoting activity was destroyed. Prostaglandin E2 also stimulated DNA synthesis in the cultures; if PGE2 was irradiated in serum from intact or partially hepatectomized rats its growth-promoting activity was markedly diminished.

Serum from partially hepatectomized rats induces primary hepatocytes to enter S phase: a role for prostaglandins?

Serum obtained from partially hepatectomized rats 1, 3 or 24 h after operation was more effective in stimulating DNA synthesis in primary adult rat hepatocytes than serum from sham-operated rats; exposure to the serum for 2 h was sufficient to promote growth. Serum from the partially hepatectomized rats contained elevated levels of PGE2 and PGF2 alpha; it promoted hepatocytes to release prostaglandins into their culture medium. Growth-promoting effects of the serum and its capacity to elicit prostaglandin release into the culture medium were inhibited by 0.1 mM-indomethacin or 1 mM-aspirin. 0.1 mM-indomethacin also prevented DNA synthesis if the inhibitor were added 4 h after growth had been initiated by serum from partially hepatectomized rats, suggesting that prostaglandins continue to be important for the maintenance of hepatocyte growth for at least 6 h.

Regulation of the proliferation of primary rat hepatocytes by eicosanoids.

DNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2 alpha into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.

The radiosensitivity of rat thymocytes.

gamma-Irradiation in vitro apparently blocked a plasma-membrane associated, superoxide-producing, NADPH oxidase in rat thymocytes. Differential centrifugation of the mixed thymocytes indicated the smaller lymphocytes (approx. 6 microns diameter) to be the radiosensitive population. The oxidase system co-isolated in part with thymus nuclei and could be solubilized by detergent treatment [Bellavite, Jones, Cross, Papini & Rossi (1984) Biochem. J. 223, 639-648]. Endogenous NADPH was the rate-limiting component for superoxide formation in vitro. The level of NADPH was lowered by gamma-irradiation, an effect mimicked by GSSG in the presence of 50 microM-ZnCl2 to inhibit GSSG reductase. These findings are suggested as the metabolic basis for interphase death of small lymphocytes exposed to ionizing radiation.

Protein kinase activities in rat liver nuclei: effects of age and partial hepatectomy.

Selective substrates and inhibitors have been used to measure kinases phosphorylating endogenous proteins in rat liver nuclei during growth and regeneration after partial hepatectomy. Peaks in activity were found at 5, 22, and 29 hours after partial hepatectomy. Administration of alpha 1 and beta adrenergic blockers suggested that the Be2+ sensitive and cyclic AMP-dependent protein kinases were interdependently regulated by Ca2+ and cyclic AMP.

Beryllium toxicity. The selective inhibition of casein kinase 1.

1. Cyclic AMP-independent casein kinase 1 in liver cytoplasm and nuclei was inhibited by Be2+ in vitro (Ki 2.5 microM and 29 microM respectively). Casein kinase 2 (phosvitin kinase) and cyclic AMP-dependent protein kinase were unaffected. 2. The inhibition of casein kinase 1 by Be2+ was competitive with respect to the protein substrate; at non-saturating concentrations of casein, inhibition was non-competitive with respect to ATP. 3. In rats given LD50 doses of Be2+ 24 h before death, the activities of cytoplasmic and nuclear casein kinase 1 in livers from partially hepatectomized animals were diminished approx. 50%; with intact rats, nuclear casein kinase 1 was inhibited at concentrations of casein less than the Km.

Enzyme induction in rat liver: the effects of Be2+ in vivo.

Rats given an LD50 dose of Be2+ showed reduced activities of ornithine decarboxylase and tyrosine aminotransferase in liver in response to dexamethasone induction. Control fed animals showed 'superinduction'. Be2+ also inhibited the uptake of [3H]orotic acid into rapidly labelled RNA of ribonucleoprotein particles extracted from liver nuclei in isomolar solutions at pH 8.0. Consistent with inhibition of cytoplasmic protein kinase reported previously (Kaser et al., 1980), the uptake of [32P]Pi into proteins in the ribonucleoprotein particles was also diminished.

Nucleosomes from normal and regenerating rat liver.

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.

Initiation of transcription in normal and phosphorylated rat liver nucleosomes.

Initiation sites were enumerated in rat liver nucleosomes with Echerichia coli RNA polymerase. One site was present in approx. 3.5 mononucleosomes and in a 1200-base-pair length of polynucleosomes. S-phase nuclei or normal nuclei phosphorylated in vitro showed increased numbers of sites; the elongation rates were unchanged.

Histones in the first cleavage cycle of fertilised sea urchin eggs.

Nuclei were isolated from Echinus eggs through the first cleavage cycle by modification of existing techniques. When these nuclei were extracted with 2 M NaCl and the supernatant diluted to 0.15 M, large amounts of non-histone proteins remained in solution. The precipitated nucleoprotein contained expected amounts of DNA and a protein analogous to mammalian histone H1. Extrachromosomal histone H1 was eliminated by the modified isolation procedure. Amounts of nuclear proteins soluble in 0.15 M NaCl reached a peak in G2. Histones and non-histone proteins were phosphorylated postfertilization, in early prophase and in telophase.

Chromatin structure through the cell cycle. Studies with regeneration rat liver.

Liver nuclei were prepared through the first cell cycle in partially hepatectomized young rats showing 30% parenchymal cell synchrony. To determine if nucleosome structure altered during this period, liver nuclei from sham-operated rats were compared with nuclei isolated at various times after partial hepatectomy. These nuclei were exposed to deoxyribonuclease I (EC 3.1.4.5), deoxyribonuclease II (EC 3.1.4.6) or micrococcal nuclease (EC 3.1.4.7) and the nucleosome-associated DNA length was ascertained. In no case was a difference in the DNA lengths associated with nucleosome structure observed. Differences were observed with regard to the histones and their relative association with nuclear material. When nuclei from normal rat livers were incubated in hypo-osmolar medium 9% of histone 1 and 4% of the other histones were released. These released histones, unlike those remaining bound to the nuclei, showed high [3H]adenosine and [3H]acetate uptakes in vivo. [32P]P1 uptake was also much greater into released than bound histones 1 and 3, but was not different for histone2A. At 3.5-4.5 h after partial hepatectomy, the release of histone 1 was trebled and that of histone 4 doubled. By 13.5 h, when phosphorylation of the bound forms of histones 2A and especially 1 was increased, no further changes in histone release in hypo-osmolar medium were found. The released histones from partially hepatectomized livers had indistinguishable [3H]adenosine uptakes from controls. The roles are discussed of phosphorylation and ADP-ribosylation in labilizing histone binding.

Adenosine diphosphate ribosylated histones.

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].

Phosphorylation of rat thymus histones, its control and the effects thereon of gamma-irradiation.

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.