Homeostatic mechanisms and the loss of mitotic potential

A model for cell division in mammalian tissues is analyzed. The model treats cells capable of division and cells not so capable as separate populations. In the model homeostasis is achieved by a secretion of the non-dividers which acts upon newly formed cells to convert them into non-dividers. The principal result is given in the form of a theorem: if the rate at which cells divide exceeds the rate at which the non-dividers die, then the ratio of dividers to non-dividers tends to zero with time.     

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.     

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.     

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.     

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.     

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.     

HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import.

Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.     

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.     

Renewal of the epithelium in the descending colon of the mouse. IV. Cell population kinetics of vacuolated-columnar and mucous cells.

Proliferation and migration of cells in the vacuolated-columnar and mucous cell lines were studied in the descending colon of adult female mice given a single injection or a continuous infusion of 3H-thymidine and killed at various intervals from one hour to 12 days. This investigation was carried out using one mum-thick Epon sections which were radioautographed after staining with the periodic acid-Schiff technique and iron-hematoxylin. In the normalized crypts with ten equal segments, labeled vacuolated cells at one hour after injection of 3H-thymidine were encountered in the lower four segments and in decreasing numbers in segments 5 through 7. From the percent labeled cells in segments of the crypt, the birth rate and fluxes of cells were computed. Moreover, it was found that a cell in the vacuolated-columnar cell line would undergo three mitotic cycles on the average from its birth at the cryptal base to its extrusion from the surface; of these three cycles, the last one which took place from segment 3 to segment 7 appeared to be a changeover from dividing cells to non-dividing cells, in accordance with the "slow cut-off" model of Cairnie et al. ('65b). Mucous cells located in segments 1 through 6 of the crypt were capable of incorporating 3H-thymidine and thus capable of undergoing mitosis. However, the rate of turnover of mucous cells based on proliferative rate was found to be much lower than the rate of turnover of mucous cells based on the transit time in the non-dividing segments of the crypt. Since there was a concomitant overproduction of cells in the vacuolated cells and newly formed mucous cells in the lower portion of the crypt, it was concluded that some vacuolated cells would give rise to mucous cells. This putative transformation occurred in the lower four segments of the crypt. Mucous cells which were formed by transformation would migrate upward along the cryptal wall and accumulate more mucus in the theca; in doing so, they would undergo two divisions, on the average, before they became non-dividing mucous cells. In ascending the cryptal walls, both vacuolated-columnar cells and mucous cells appeared to migrate at a similar speed; they moved much slower at the base of the crypt and accelerated toward the upper portion of the crypt, but they migrated at a constant speed in the non-dividing segments of the crypt.     

Biochemical and enzymic changes during erythrocyte differentiation. The significance of the final cell division.

1. The haemoglobin content of developing erythroblasts was shown to increase rapidly when the cells completed the final cell division of erythroid development and passed from the dividing into the non-dividing cell compartment. 2. The activity of carbonic anhydrase was measured and shown to increase continually throughout erythroid differentiation. The activity increased most rapidly in the polychromatic stage. 3. Catalase activity did not increase significantly during erythroid differentiation until the reticulocyte stage. 4. The activity of four enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, adenosine deaminase and nucleoside phosphorylase, exhibited a similar pattern of change during erythroid differentiation. In the dividing cell compartment their activity was relatively high but exhibited a steep decline between the polychromatic stage and the orthochromatic stage, that is, as the cell completed its final cell division and moved from the dividing to the non-dividing compartment. After this the activity of these enzymes was stabilized at a relatively low value, and this activity persisted at such a value until the reticulocyte stage. 5. Lactate dehydrogenase activity also declined after the cell had crossed from the dividing into the non-dividing stage, but in this case the decline was less than in the case of the above four enzymes. 6. Adenylate kinase activity was relatively constant in the dividing cell compartment but exhibited a 60 percent increase when the cell passed from the dividing into the non-dividing compartment. 7. The cessation of cell division appears to coincide with a set of complex biochemical changes.     

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.     

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.     

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.     

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.     

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.     

基于人免疫缺陷病毒-1的慢病毒载体系统研究进展

慢病毒能够感染分裂细胞和非分裂细胞,因而被发展成为重要的转基因载体。慢病毒载体已经发展到了第三代,其安全性已经大为提高。经过结构优化的慢病毒载体已经用于转基因动物生产和基因治疗研究,而稳定包装细胞系的建立使得慢病毒的生产更为简便。     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.