Prematurely condensed chromosomes of dividing and non-dividing cells in aging human cell cultures.

Chromosomes of dividing and non-dividing aging cells were examined by fusing senescent WI38 cells with mitotic HeLa cells to induce premature chromosome condensation (PCC). Exposure of the WI38 cells to ³H-thymidine 48 h prior to fusion allowed autoradiographic identification of cells that did not synthesize DNA (non-dividing cells). Ninety-six percent of the non-dividing cells, diploid or tetraploid, induced into PCC had single chromatids and were therefore blocked in the G1 phase of the cell cycle. Anomalous centromeric pairing of chromatids was noted in the remaining 4% of the non-dividing cells. Typical G2 configurations (double chromatids) were observed only among labeled (dividing) cells. The efficiency of PCC induction was independent of culture age. In addition, the efficiency of PCC induction was independent of phase in the cell cycle, as shown by comparison of observed frequencies with expected frequencies.     

Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells

Ornithine decarboxylase activity increases at least 4–5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G₁ and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G₁-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.     

A general method for modeling cell populations undergoing G1----G0 transitions during development.

The transition from the dividing state to a non-dividing, terminally differentiated state is common to the history of most populations of cells during development. Quantifying such transitions and events related to them is often difficult, even in those cases for which there is a good tissue culture model, because the process is asynchronous and occurs against a background of continued extensive growth. A general model for analyzing these complex population changes is presented here. In the absence of definitive data, the model provides projections of the possible range, under a given set of boundary values, for the rate of terminal differentiation, the overall growth rate, and the degree of cell death. On the other hand, given data on the rate of DNA accumulation, dividing cell fraction, and generation time, the model provides the effective partitioning coefficient between the dividing and non-dividing states averaged over the population, at a given time. These data also allow for an assessment of the degree of actual cell death against a background in which significant numbers of cells are withdrawing from the cell cycle. The types of data required with respect to the model's ability to resolve the nature of a G0 transition "window" within the cell cycle are also discussed.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Pyruvate kinase catalyzed phosphorylation of glycolate

Adult mammalian erythroblasts from an anaemic rabbit were separated into fractions of cells at different stages of development using the velocity sedimentation technique. The iron content of the stroma and cytoplasm of the cells in each fraction was determined by atomic absorption spectroscopy. A high proportion of the iron in the dividing erythroblasts was found to be associated with the cell stroma. After the final cell division the proportion of stromal iron rapidly declines and it appears that stromal iron is mobilised at this stage and utilised by the non-dividing erythroblasts for haemoglobin synthesis.     

RNA Accumulation in Relation to DNA and Protein Accumulation in Jerusalem Artichoke Callus Cultures

RNA accumulation during the synchronous early development ofJerusalem artichoke callus cultures follows a pattern of threestepwise increases in RNA per dividing cell during the firstdivision cycle. Little accumulation occurs in non-dividing cellsduring this time. These data are compared with data availablefor DNA replication, which occurs only in dividing cells, andfor protein accumulation which follows a similar pattern tothat of RNA accumulation in dividing cells, both in dividingcells and in some non-dividing cells.     

Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.     

Cell cycle regulation by the pro-apoptotic gene Scotin

Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in a p53-dependent manner. In this report, we have used Drosophila as a model system to obtain a preliminary insight into the molecular mechanism of Scotin function, which was validated using the mammalian system. Targeted expression of Scotin in developing Drosophila induced apoptosis and developmental defects in wings and eyes. Co-expression of Scotin with the anti-apoptotic protein P35, while inhibited the apoptosis in both dividing and non-dividing cells, rescued adult wing or eye phenotypes only when Scotin was expressed in non-dividing cells. This suggests that mechanisms of Scotin-induced apoptosis in dividing and non-dividing cells may vary. Suppressor-enhancer screen using cell cycle regulators suggested that Scotin may mediate cell cycle arrest at both G1/S and G2/M phases. Over-expression of Scotin in mammalian cells resulted in mitotic arrest and subsequently apoptosis. Furthermore, a larger proportion of cells over-expressing Scotin showed sequestration of Cyclin B1 in the cytoplasm. These results suggest that one of the ways by which Scotin induces apoptosis is by causing cell-cycle arrest.     

Alternative mechanisms for homeostatic regulation of mammalian cell populations

A model for homeostatic regulation in mammalian tissues is analyzed. The model treats resting and active dividing cells, immature and mature non-dividing cells as separate populations. In the model, regulation is accomplished through control of the proportion of newly-formed cells that will become non-dividers. Four possible regulating substances, produced by dividing cells, non-dividing cells, mature non-dividing cells, and newly-formed cells respectively, are considered. Stability theorems are provided. System behavior in each instance depends on the relative values of the rate at which cells divide and the rate at which non-dividers die.     

The use of a cell-cycle phase-marker may decrease the percentage of errors when using FISH in PGD

Fluorescent DNA probes are used to characterise the chromosome constitution of preimplantation embryos. FISH is used to select normal or balanced embryos in carriers of balanced chromosomal rearrangements, for embryo sexing or for aneuploidy screening in women of advanced age, who have had recurrent abortions or IVF failures. In most cases, FISH is performed on interphase blastomeres which are asynchronously dividing cells, that can be in G1, S or G2. However, a correct interpretation of a double FISH signal, which may correspond to a split signal, to a replicated chromosome region or to the presence of an extra chromosome is essential to establish an accurate diagnosis. To determine if the cell stage could influence the interpretation of FISH results, we compared the signal characteristics of one locus-specific probe, two different subtelomere region probes, and a centromere region probe in non-dividing Sertoli cells and in proliferating lymphocytes. Most cells had two signals per chromosome pair (i.e., a situation corresponding to G0 in Sertoli cells and to G1 or to a prereplication stage in lymphocytes). Nevertheless, in proliferating cells the percentage of nuclei with a number of signals different from the expected (two unreplicated chromosomes per pair) was different from that found in non-dividing cells (P < 0.05). It was estimated that 10.8% of double dots in dividing cells resulted from DNA replication. The sequence of replication was first the locus-specific region, second a telomere region, and third the centromere. In conclusion, the DNA replication process could result in errors of interpretation (misdiagnosis) in 7% of proliferating cells. Thus, the use of a cell cycle phase-specific marker could avoid errors by indicating the cell stage in which the nucleus analysed is found.     

Mammalian DNA ligases. Serological evidence for two separate enzymes.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

Binary fission of Pneumocystis carinii trophozoites grown in vitro

Trophozoites grown in vitro were shown to undergo binary fission by transmission electron microscopy (TEM). Standard fixation with subsequent embedding in Spurr was employed using 3% glutaraldehyde and 1% osmium tetroxide with 5% sucrose added to both fixatives and 0.1 M cacodylate buffer washes. Trophozoites were grown on WI-38 cells in vitro. Trophozoites were found in various stages of fission. The dividing trophozoite has daughter cells that are rounder than the pleomorphic, non-dividing trophozoites. Tubular forms external to the dividing trophozoites were decreased in number; tubular forms when present were concentrated around the forming septa. Nuclear material was sometimes, but not always, well defined in both daughter cells. There was no concentration of nuclear material at the poles. Vacuoles without membrane were present in the dividing forms. Separate nuclear regions were sometimes found in the dividing trophozoites. These observations suggest that binary fission does occur in culture; however, the significance of binary fission to the life cycle of Pneumocystis carinii (Pc) is not yet clear.     

Possibility of using irradiated non-dividing target cells for evaluating the cytolytic activity of leukocytes in vitro]

Immune allogeneic cells of lymph nodes, spleen and peritoneal exudate lyse in vitro dividing and irradiated non-dividing target cells with the same intensity. The number of target cells lysed during the immune attack are more precisely registered in irradiated non-dividing cells.     

Binary Fission of Pneumocystis carinii Trophozoites Grown in Vitro

Trophozoites grown in vitro were shown to undergo binary fission by transmission electron microscopy (TEM). Standard fixation with subsequent embedding in Spurr was employed using 3% gluuraldehydc and 1% osmium tetroxide with 5% sucrose added to bom fixatives and 0.1 M cacodylate buffer washes. Trophozoites were grown on WI-38 cells in vitro. Trophozoites were found in various stages of fission. The dividing trophozoite has daughter cells that arc rounder than the pleomorphic, non-dividing trophozoites. Tubular forms external to the dividing trophozoites were decreased in number; tubular forms when present were concentrated around the forming septa. Nuclear material was sometimes, but not always, well defined in both daughter cells. There was no concentration of nuclear material at the poles. Vacuoles without membrane were present in the dividing forms. Separate nuclear regions were sometimes found in the dividing trophozoites. These observations suggest that binary fission does occur in culture; however, the significance of binary fission to the life cycle of Pneumocystis carinii (Pc) is not yet clear.     

Turning anti-ageing genes against cancer

Recent studies in diverse organisms implicate proto-oncogenic pathways, including insulin-like growth factor-I (IGF-I), Ras and AKT/protein kinase B in the ageing process. Although IGF-I is thought to contribute to cancer by promoting growth and preventing apoptosis, evidence from model organisms suggests that proto-oncogene homologues might contribute to the DNA mutations and chromosomal damage that are observed in tumour cells by increasing DNA damage, in both dividing and non-dividing cells, and involving error-prone systems in DNA repair. This raises the possibility that cancer can be reduced by chronic downregulation of pro-ageing pathways.