Host Factor SAMHD1 Restricts DNA Viruses in Non-Dividing Myeloid Cells

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells.     

Cell division arrest by gamma-irradiation in photoautotrophic suspension culture of Euphorbia characias: Maintenance of photosynthetic capacity and overaccumulation of sucrose

Gamma-irradiation (250 Gy) applied to photoautotrophic cell suspensions of Euphorbia characias L. in the exponential growth phase led to the arrest of cell division and to a subsequent overaccumulation of sucrose and dry matter. From the fourth day of culture, the chlorophyll content and gross photosynthesis were not depressed by gamma-treatment nor by sugar accumulation. In both cultures, no difference was observed between oxygen uptake in the light at CO₂ saturating concentration and in the dark, suggesting that no change in energy-dissipative reactions took place after irradiation. A slight increase in oxygen uptake in both light and dark was observed in irradiated cells during the first four days. However, in the absence of limiting factors, the photosynthetic capacities of the dividing and irradiated non-dividing photoautotrophic cells were identical but higher than that of the non-dividing cells in the stationary growth phase. This suggests that gamma-irradiation arrests cell division by a mechanism different to that occuring in stationary-phase cultures. This may be of value in investigating the metabolism of secondary products.     

Giant cells fromSaccharomyces uvarum grown after X-irradiation

Yeast cells, Saccharomyces uvarum, were irradiated with X-rays and grown in liquid suspension. Glucose as the only carbon source was limited to 12.5 mM. Under these conditions giant cells are formed. Cell number, glucose utilization, ethanol production and oxygen consumption are measured during the time of growth. The mean weight of single cells in the stationary phase increases up to 75 krad and is not due to an uptake of water. In irradiated cultures oxygen consumption and glucose utilization per cell are higher than in control cells. The data demonstrate that synthesis- and energy-metabolism during the formation of non-dividing, radiation-induced giant cells is increased.     

Development of irradiated tunicate buds: Is cell division cycle required for morphallaxis?

In the tunicate, Polyandrocarpa misakiensis , transdifferentiation occurs in the multipotent atrial epithelium during morphallactic bud development. Irradiation (10–80 Gy) or aphidicolin (10 μg/mL) blocked this process severely, although the atrial epithelium could form organ placodes. The placodes consisted of cuboidal cells with a high nucleus : cytoplasm ratio and were lacking the alkaline phosphatase antigen from the cell surface, suggesting that the atrial epithelium might undergo dedifferentiation without initiating cell cycling. Irradiated buds could resume organogenesis in temporal accordance with the restoration of mitotic activity. Bud pieces irradiated at 40 Gy were juxtaposed with unirradiated counterparts. In the operated buds, irradiated, non-dividing cells participated in organogenesis at the site of juxtaposition in cooperation with the unirradiated, dividing cells. These results have shown that in P. misakiensis the cell division cycle, probably DNA replication, is indispensable for transdifferentiation of the atrial epithelium, although every cell in the organ rudiment need not enter cell cycling. We suggest that homoiogenetic induction occurs between dividing cells and non-dividing cells.     

Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.     

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.     

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 two-dimensional polypeptide profile of terminally non-dividing human diploid cells

We have used high resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) to compare the polypeptides synthesized by normal human diploid fibroblasts cells which have significant proliferative capacity with those of cells which are terminally non-dividing. Normal cells that are terminally non-dividing (i.e., have no remaining proliferative potential) synthesize two polypeptides that are not detected in young cells which are either actively proliferating or growth arrested by incubation in medium containing a low concentration of serum (0.3%) for 72 h. Continued maintenance of young cells in the growth-arrested state ultimately leads to the detectable synthesis of one of the polypeptides synthesized by terminally non-dividing cells. Some preliminary biochemical properties of these two polypeptides are examined.     

RADIATION EFFECTS ON THE GROWTH RATE AND CELL POPULATION KINETICS OF ACTIVELY GROWING AND DORMANT ROOTS OF TRADESCANTIA PALUDOSA

Actively growing and dormant roots of Tradescantia paludosa were exposed to x-rays to compare the radiosensitivity of an actively proliferating tissue with that of one which is not active but is potentially proliferative. The level of effect was ascertained by the degree of change in the rate of root growth 4 days after exposure. Cell population kinetics were measured in control and in irradiated roots to determine whether or not a change was produced either in the number of proliferating cells or in the mitotic cycle duration which was sufficient to explain the altered rate of root growth. Nuclear volumes were also measured to provide an estimate of the relative total target size in actively growing vs. dormant roots. Tritiated thymidine was used to measure the cycle duration and the proportion of cells synthesizing DNA. The results showed that 184 and 305 r respectively were required to reduce the linear root growth rate to 37 per cent of that of the control for actively growing and dormant roots. Mitotic cycle duration, measured 4 days after x-ray exposure, was the same as in the control. The number of proliferating cells, however, was reduced. The rate of cell production in the irradiated roots was reduced to approximately one-half that of the controls. The average nuclear volumes of active and dormant roots were 733 and 491 µ³ respectively; thus the difference in the number of roentgens required to reduce growth to 37 per cent of that of the control can be attributed to the different average nuclear volumes. Therefore, the experiments suggest that part if not most of the differences in sensitivity between an actively dividing and an essentially non-dividing meristematic cell population resides in their different average nuclear volumes. Thus the law of Bergonie and Tribondeau needs to be reinterpreted, since the basic reason for the differences is secondary to whether or not the meristematic cells are proliferating.     

Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes.

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.     

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.     

Variable association of DNA polymerase with the cytoskeletal fractions of resting and dividing cells

A DNA polymerase activity associated with the detergent insoluble cytoskeletal fraction has been identified in dividing and non-dividing rat hepatocytes and a hepatoma (the Zajdela Ascitic Hepatoma). About 35 % of the enzyme is found associated with the cytoskeletal fraction of non-dividing cells as compared to about 3–6 % of the enzyme in dividing cells even though the dividing cells contain larger amounts of the extranuclear enzyme. The properties of the enzyme are similar to those of DNA polymerase-v. It is suggested that the association of the enzyme with the cytoskeletal fraction has functional significance.     

Unstable Chromosome Aberrations Do Not Accumulate in Normal Human Fibroblast after Fractionated X-Irradiation

We determined the frequencies of dicentric chromosomes per cell in non-dividing confluent normal human fibroblasts (MRC-5) irradiated with a single 1 Gy dose or a fractionated 1 Gy dose (10X0.1 Gy, 5X0.2 Gy, and 2X0.5 Gy). The interval between fractions was between 1 min to 1440 min. After the completion of X-irradiation, the cells were incubated for 24 hours before re-plating at a low density. Then, demecolcine was administrated at 6 hours, and the first mitotic cells were collected for 42 hours. Our study demonstrated that frequencies of dicentric chromosomes in cells irradiated with a 1 Gy dose at different fractions were significantly reduced if the fraction interval was increased from 1 min to 5 min (p<0.05, χ²-test). Further increasing the fraction interval from 5 up to 1440 min did not significantly affect the frequency of dicentric chromosomes. Since misrejoining of two independent chromosome breaks introduced in close proximity gives rise to dicentric chromosome, our results indicated that such circumstances might be quite infrequent in cells exposed to fractionated X-irradiation with prolonged fraction intervals. Our findings should contribute to improve current estimation of cancer risk from chronic low-dose-rate exposure, or intermittent exposure of low-dose radiation by medical exposure.     

Changes in heparan sulfate structure during transition from the proliferating to the non-dividing state of cultured arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize a cell-associated heparan sulfate proteoglycan which consists of a 92 kDa core protein with 3 to 4 heparan sulfate side chains covalently attached. Biosynthesis of the cell-associated heparan sulfate proteoglycan was compared in proliferating and in non-dividing vascular smooth muscle cells which are preincubated in the presence of [35]sulfate or a combination of [35S]methionine and [3H]glucosamine. The Mr of the core protein was identical in either growth state, but changes in the structure of the heparan sulfate side chains were observed. Non-dividing (postconfluent) arterial smooth muscle cells form longer heparan sulfate chains with a higher proportion of hydrophobic (N-acetyl) groups than proliferating (preconfluent) cells as judged from gel filtration experiments, hydrophobic interaction chromatography and heparitinase degradation. An enzyme preparation from proliferating cells catalyzes deacetylation and N-sulfation of heparan sulfate at a 5-fold higher activity than from non-dividing cells. Cell density-dependent structural differences of heparan sulfate are related to the finding that heparan sulfate isolated from non-dividing cells has a 10-fold higher antiproliferative potency than heparan sulfate from proliferating (preconfluent) cells.     

A radiation resistance factor in cultured Cloudman S91 mouse melanoma cells.

The gamma-ray survival of a radiation-sensitive amelanotic subclone of Cloudman S91 mouse melanoma, S91/amel, is increased by the presence in the tissue culture dishes of heavily irradiated cells from the same cell line (amel-HRCells) and from clonally related melanotic S91/I3 radioresistant cells (I3-HRCells). The D0 of the target S91/amel cells increases from 1.25 to 2.08 Gy in the presence of 60,000 heavily irradiated S91/amel cells per dish. The presence of I3-HRCells in dishes of target S91/I3 cells does not increase their radioresistance. Comparable numbers of I3-HRCells are more effective than amel-HRCells at increasing survival of target S91/amel cells irradiated with 3 Gy of gamma rays. Conditioned medium from the S91 melanoma cells also increases the radioresistance of S91/amel, but is not as effective as the HRCells. Unirradiated cells can condition the medium as effectively as irradiated cells. It is concluded that the radiosensitive mouse melanoma cell line is made significantly more resistant by a diffusible cellular factor(s) elaborated more proficiently by radiation-resistant cells.     

Migration of newly-synthesized RNA during mitosis. IV. Content of labelled RNA in nuclei before and after mitosis in Amoeba proteus

Amoeba proteus were incubated with ³H-uracil for 3 h. Thereupon RNA synthesis was blocked by actinomycin D and the population separated into dividing and non-dividing cells. Nuclei were isolated from cells of both groups and their RNA radioactivity was measured by means of autoradiography. The amount of label in the nuclei of non-dividing (interphase) cells was found to be equal to the sum of labels in both nuclei of daughter cells shortly after division. It is concluded that labelled RNA leaves the nucleus at the onset of mitosis and returns to the nuclei of daughter cells immediately after its termination.     

Time-lapse microscopic observation of non-dividing cells in cultured human osteosarcoma MG-63 cell line

Cancer stem cells resemble normal tissue-specific stem cells in many aspects, such as self-renewal and plasticity. Like their non-malignant counterparts, cancer stem cells are suggested to exhibit a relative quiescence. The established cancer cell lines reportedly harbor slow-proliferating cells that are positive for some cancer stem cells markers. However, the fate of these cells and their progeny remains unknown. We used time-lapse microscopy and the contrast-based segmentation algorithm to identify and monitor actively dividing and non-dividing cells in human osteosarcoma MG-63 cell line. Within the monitored field of view the non-dividing cells were represented by three cells that never divided, and one cell that attempted to divide, but failed cytokinesis, and later, after significantly prolonged division, produced the progeny with enlarged segmented nuclei, thus pointing to a possible mitotic catastrophe. Together, these cells initially constituted about 6.2% of the total number of seeded cells, yet only 0.02% of all cells at the end of the observation period when cells became confluent. Non-dividing cells were characterized by rounded shape, dark nuclei, random cytoplasmic streaming and subtle oscillatory movement, however, they did not migrate and rarely formed cell-cell contacts as compared to actively dividing cells. Our data indicate that the observed non-dividing MG-63 cells do not have a growth advantage over other cells and, therefore, they do not contribute to the cancer stem cells pool.     

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.