The M1 subunit of rat liver ribonucleotide reductase appears to be modified by ubiquitination.

Using a combination of immunoblotting, double immunoprecipitation, immunoglobulin-affinity chromatography, and isoelectrofocusing, we have been able to identify a group of proteins that display CDP-reductase activity and contain antigenic epitopes recognized by anti-ribonucleotide reductase M1 subunit and anti-ubiquitin antibodies. In the cytoplasm of rat liver cells, we could detect a total of five proteins with molecular masses of 92, 89, 56, 45, and 37 kilodaltons which reacted with the anti-M1 subunit serum. All of them, except the 89-kilodalton protein (the nascent unmodified M1), were also recognized by the anti-ubiquitin antibody. In normal liver cells, all of the apparently ubiquitinated species of the M1 protein were found in the cytoplasm, but not in the nuclear envelope associated pool of the enzyme. However, we did not detect ubiquitinated M1 protein fragments in the cytoplasm of Morris hepatoma 5123tc. The level of the apparently ubiquitinated fragments of the M1 subunit increased in parallel to the DNA-synthetic activity of normal liver cells, suggesting that ubiquitination plays a key role in the regulation of the activity of the enzyme during the cell cycle.

Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes.

The effects of topoisomerase II-reactive epipodophyllotoxins etoposide and teniposide as well as amsacrine on the viability of thymocytes in primary culture has been examined. All three drugs were shown to produce DNA cleavage detectable by resolving isolated DNA by pulsed field agarose gel electrophoresis. The DNA cleavage was found to have two components. The first was due to the interaction of the drugs with topoisomerase II, whereas the second component was due to endonuclease cleavage caused by the drug-induced entry of the thymocytes into programmed cell death or apoptosis. This second component of the DNA cleavage was also detected in thymocytes undergoing apoptosis following exposure to the glucocorticoid analogue, dexamethasone. The effect of the drugs on programmed cell death is dependent upon new protein and RNA synthesis, indicating that topoisomerase II has a role in the very first stages of the process. These results are discussed in terms of the use of this class of topoisomerase II-reactive drugs in chemotherapy.

tsLA23-NRK cells need pp60v-src protein-tyrosine kinase activity in G2 phase to initiate mitosis in serum-free medium.

Lowering the temperature from 41 to 36 degrees C stimulates quiescent tsLA23-NRK rat cells (infected with the tsLA23 mutant of the Rous sarcoma virus) in serum-free medium to resume cycling and initiate DNA replication by reactivating the tsLA23-RSV's abnormally thermolabile pp60v-src protein-tyrosine kinase. Inactivating the enzyme in these pp60v-src-stimulated cells by again raising the temperature to 41 degrees C after the cells had initiated DNA replication did not prevent the completion of DNA replication and entry into the G2 phase, but it stopped the initiation of mitosis. Adding serum at the time of the temperature increase replaced the lost pp60v-src activity and the cells were able to continue to mitosis. The G2-arrested cells at 41 degrees C were able to initiate mitosis when pp60v-src was reactivated again by lowering the temperature to 36 degrees C. These observations suggest that protein-tyrosine kinase activity is needed to initiate mitosis and that the tsLA23-NRK cell is a good model for studying the function of this kinase activity in the initiation of mitosis.

Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein.

Epitope-specific antibodies to the M1 and M2 subunits of mammalian ribonucleotide reductase were prepared using peptides predicted to have a high antigenic index. Western blotting demonstrated that the anti-M1 antibody was specific for the 89-kilodalton M1 subunit (and its degradation fragments) and the anti-M2 antibody specifically recognized the 45-kilodalton M2 subunit. Both antibodies inhibited the CDP-reductase activity of the holoenzyme. Using these antibodies, both the M1 and M2 subunits were shown to be localized in the cytoplasm and in the nuclear regions of a number of cell types, including B77 avian sarcoma virus transformed NRK cells, T51B rat liver cells, 5123tc hepatoma cells, and rat liver cells in vivo. In addition, the M1 subunit was found to be localized as a halo around isolated rat liver nuclei. Biochemical analysis of the cytoplasmic fraction of liver cells and a Triton X-100 wash of nuclei from these cells confirmed the location of the enzyme activity in these cellular compartments. The M1 subunit appears to be glycosylated, as indicated by its retention on a Affi-Gel-concanavalin A affinity column. Therefore, in mammalian cells ribonucleotide reductase appears to be not only in the cytoplasm, but is also associated with the nuclear membrane or nuclear lamina. The activity of the enzyme in the membrane fraction changes dynamically during the cell cycle.

Periodicity of DNA folding in higher order chromatin structures.

Each level of DNA folding in cells corresponds to a distinct chromatin structure. The basic chromatin units, nucleosomes, are arranged into solenoids which form chromatin loops. To characterize better the loop organization of chromatin we have assumed that the accessibility of DNA inside these structures is lower than on the outside and examined the size distribution of high mol. wt DNA fragments obtained from cells and isolated nuclei after digestion with endogenous nuclease or topoisomerase II. The largest discrete fragments obtained contain 300 kbp of DNA. Their further degradation proceeds through another discrete size step of 50 kbp. This suggests that chromatin loops contain approximately 50 kbp of DNA and that they are grouped into hexameric rosettes at the next higher level of chromatin structure. Based upon these observations a model by which the 30 nm chromatin fibre can be folded up into compact metaphase chromosomes is also described.

Ribonucleotide reductase--new twists in an old tale.

Although they are proliferatively quiescent, the cells in the intact adult rat liver express the gene coding for the M1 subunit of ribonucleotide reductase. But since they do not need deoxyribonucleotides, they promptly inactivate the 88 to 90 kDa M1 products and degrade them into 40 kDa fragments. Partial hepatectomy signals the remaining cells to start proliferating. Two hours before the onset of DNA replication, around 16 to 18 hr after partial hepatectomy, the cells start accumulating a large pool of functional ribonucleotide reductase M2 subunits. Near the end of the G1 build-up the cells step up M1 gene expression, stop inactivating, and reduce the degradation of the M1 products. The accumulating functional 88 to 90 kDa M1 subunits, each with more than one catalytic site, couple with functional M2 subunits to produce active ribonucleotide reductase holoenzyme which accumulates in the outer nuclear membrane from which they supply deoxyribonucleotide precursors to intranuclear replication enzymes. At the end of the S phase, the cell reduces M1 gene expression and resumes degrading 88 to 90 kDa M1 subunits. At least some of the 40 kDa M1 fragments are still active and can form partially active "holoenzymes" when mixed with a standard preparation of functional M2 subunits. The M1 control mechanism appears not to operate in hepatoma cells and Ehrlich ascites tumor cells, both of which maintain a pool of undegraded 88 to 90 kDa M1 components.

1 alpha,25-Dihydroxyvitamin D3 enables regenerating liver cells to make functional ribonucleotide reductase subunits and replicate DNA in thyroparathyroidectomized rats.

Between 16 and 20 h after partial (70%) hepatectomy (HPX) in normal rats, the remaining liver cells accumulate ribonucleotide reductase subunits, assemble these into active holoenzyme, and initiate DNA replication. These late prereplicative activities did not occur in most of the liver cells remaining after HPX in rats which had been thyroparathyroidectomized (TPTX) 72 h previously. However, one intraperitoneal injection of 400 or 600 ng 1 alpha,25-dihydroxyvitamin D3/100 g body weight at the time of HPX enabled the remaining liver cells in such TPTX rats to make functional ribonucleotide reductase subunits, assemble these subunits into active CDP-reducing holoenzymes, and replicate their DNA, though they started to do so 4 to 16 h later than in normal animals.

Changes in the cytoplasmic and nuclear activities of the ribonucleotide reductase holoenzyme and its subunits in regenerating liver cells in normal and thyroparathyroidectomized rats.

The level of the cytoplasmic ribonucleotide reductase nonheme-iron-containing L2 subunit in regenerating rat liver cells began rising about 2 h before the onset of DNA synthesis, rose sharply to a maximum level about 4 h before the DNA-synthetic activity reached its peak, and then stayed at this high level even after the cells had finished replicating their DNA. The cytoplasmic level of the CDP-specific, effector-binding L1 subunit and the holoenzyme activity began rising together about 2 h after the L2 subunit began increasing and at the same time as the DNA-synthetic activity, but subsequently rose much more slowly than the L2 subunit and continued rising even after the cells had finished making DNA. The nuclear level of the L2 subunit did not rise in the regenerating liver cells, but the nuclear level of the L1 subunit and the holoenzyme activity began rising together about the same time as the DNA-synthetic activity, peaked briefly 4-6 h before the peak DNA-synthetic activity, and dropped sharply back to the basal levels by the time the DNA-synthetic activity reached its peak, but then rose again slowly as the cells finished making DNA. Thyroparathyroidectomy 72 h before partial hepatectomy prevented the cytoplasmic and nuclear subunits and holoenzyme activity from rising and prevented most of the remaining liver cells from initiating DNA synthesis.

Use of radioisotopes in quantitative studies of lung metabolism.

Quantitatively accurate studies of macromolecule and lipid synthesis in lung and other tissues by using radioactive substrates require detailed knowledge of the specific radioactivity of the appropriate pool of precursor molecules serving the synthetic pathway. A brief summary is provided of how considerations of precursor availability, metabolism, and compartmentation, as well as product remodeling, may affect the accuracy with which rates of protein, DNA, RNA, and lipid synthesis can be measured. Where possible, the application of this material to studies of lung metabolism is discussed, along with approaches that may minimize experimental uncertainties.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits.

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 X 10(-4) for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS-polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

REgulation of the primary immune response in vivo by parathyroid hormone.

Injection of sheep red blood cells into rats stimulates splenic DNA synthesis, then increases the proportion of plaque-forming cells, and finally raises the circulating antibody level. Removal of the parathyroid glands, which causes hypocalcemia, 24 hr before antigen injection impairs all of these responses, but is without effect after antigen injection. It is suggested that parathyroid hormone and calcium control the immune response, probably by affecting the proliferative phase of this response.

The positive control of cell proliferation by the interplay on calcium ions and cyclic nucleotides. A review.

Calcium, cyclic AMP, and cyclic GMP do not seem to be involved in proliferative activation of postmitotic differentiated cells. Instead, they are intracycle regulators, and we propose the following working model of their control of the initiation of DNA synthesis. While a role for cyclic GMP cannot yet be defined, a brief postmitotic burst of its synthesis might serve to prevent certain activated cells (e.g. 3T3 mouse cells) from being diverted into a nonproliferating (but still activated) G0 state (Figs. 1 and 17). In a latter part of the G1 phase, something happens to stimulate briefly the synthesis of cyclic AMP which, in turn, drives calcium ions from the mitochondria into the cytosol to activate newly synthesized thymidylate synthetase (or other primed enzymic assemblies) (Fig. 1). Having "turned on" their target enzymes, the accumulated cyclic AMP is destroyed and the excess calcium ions are reaccumulated by the mitochondria to avoid interfering with succeeding reactions. This model predicts that persistent changes in cyclic AMP metabolism and the respiration-linked, calcium-accumulating (ion-buffering) activity of mitochondria may be responsible for the sustained growth of tumors.

Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis.

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with 3H-TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of 3H-TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of 14C-formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of 3H-TdR into DNA. It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous 3H-TdR from accurately reflecting the true level of DNA synthesis.

Roles of calcium, serum, plasma, and folic acid in the control of proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts.

In a culture medium of pH 7.4 and a folic acid concentration of 100 mug/liter that contains 5% heat-inactivated chicken plasma rather than serum, the rate of proliferation of normal chicken fibroblasts is determined by the concentration of calcium. Proliferation, rapid when the calcium concentration is physiological, decreases when the calcium concentration is reduced. At a very low calcium concentration, in this culture medium, normal fibroblasts are maintained without proliferation, whereas those infected with Rous sarcoma virus proliferate rapidly. This proliferative inactivity of normal fibroblasts does not involve contact-inhibition, since the effect is observed at low, as well as higher, culture densities. When a physiological amount of calcium is added to cultures of normal fibroblasts that have been maintained in very low calcium-plasma medium for 3 days, labeled thymidine uptake and protein synthesis are strongly stimulated, and cell division follows. The use of heat-inactivated chicken serum, instead of plasma, in this medium appears to strongly sensitize normal fibroblasts to the mitogenic action of calcium. In a plasma-containing culture medium of physiological calcium concentration and a folate concentration of 5 mug/liter, neither normal nor Rous sarcoma virus-infected fibroblasts proliferate to an appreciable extent. The use of serum, however, instead of plasma results in rapid proliferation of both normal and infected cells, as does increase in the folate concentration of the plasma-containing medium to 100 mug/liter.The fact that while normal fibroblasts are maintained without proliferation in low calcium-plasma medium, Rous sarcoma virus-infected fibroblasts proliferate rapidly, indicates that the effect of calcium is regulatory rather than permissive. These results suggest that the proliferation of normal fibroblasts is initiated by a cellular function involving calcium, and that the autonomous proliferation of the neoplastic fibroblasts results either from increased calcium uptake or from an alteration or a hypass of that function. The results also suggest that serum contains a mitogenic factor(s) not present in plasma, possibly a "wound hormone" for fibroblasts.