Non-cycling Cells Research Articles

Studies on Thymocyte Subpopulations in Guinea Pigs

Thymocytes were separated according to increasing buoyant density into the three subpopulations la (25% of recovered cells), lb (20%) and II (55%), and according to binding to peanut agglutinin (PNA) into PNA<sup>+</sup> (65 %) and PNA<sup>–</sup> cells (35 %). The frequency of PNA<sup>+</sup> was 56% in la, 60% in lb and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for la, 130 fl for lb and 100 fl for population II. PNA<sup>+</sup> and PNA<sup>–</sup> cells were very similar as regards cell volume. Thus, PNA<sup>+</sup> and PNA<sup>–</sup> cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA<sup>+</sup> cells of population la. The mitogen-responsive thymocytes also belonged to population la, but were PNA<sup>-</sup>. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA<sup>+</sup> cells of population II.

Cinemicrographic study of cell proliferation pattern and interdivision times of human keratinocytes in primary culture.

The stratified squamous epithelium of the skin, the epidermis, is a renewing cell population. In order for epidermis to remain the same size, each dividing basal cell must produce on an average, one daughter cell that will remain as a germinative cell and another that will perform terminal differentiation (differential mitosis). In order to investigate cell kinetics in the epidermis, the information obtained from in vivo study is limited, and offers only indirect evidence for the determination of cell cycle time and cell proliferation pattern. Keratinocytes in cell culture are unique in formation of a multilayered cellular sheet in which the keratinocytes form a structure resembling the epidermis in vivo, and keratinize at the top. In the early days of the primary culture of human keratinocytes, when the cells proliferate to form a monolayer sheet, direct access to cell proliferation pattern and measurement of interdivision time can be done using techniques of time-lapse cinemicrography. The primary cultures at 8-20 days of incubation were employed for cinemicrographic observation when small polygonal cells appeared in groups, when numerous mitotic figures were observed, and when stratification of the cells had not yet occurred. The appropriate field was marked and followed for up to 6 days. Photographic prints were made from the 16 mm cine film, and dendrograms were made and analyzed for pattern of cell proliferation and interdivision time. Most cells in the field divided two or three times during the period of observation. Sister-sister pairs of the second and third generations divided after approximately the same interdivision times. However, some cells have never divided. Some of the sister-sister pairs differed considerably in their interdivision times. In some cultures synchronous division was quite evident. The average interdivision time was about 26 hr in the majority of cultures, and it is suggested that the estimated long cell cycle time in vivo might be overestimated due to the existence of non-cycling cells in the germinative population.

Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes.

Human fibroblasts and lymphocytes were grown in bromodeoxyuridine (BrdUrd) containing medium, harvested after 27-71 hours and stained with the fluorochrome Hoechst 33258. Parameters that influence the BrdUrd quenching effect of Hoechst fluorescence at a given pH and ionic strength of the staining solution were determined. The parameters were determined in order to obtain high resolution histograms with differentiation of each cell cycle compartment for noncycling cells and for cycling cells in the first and second cycle. The BrdUrd concentration was found to be the most significant determinant, but cell density, cell type, dye concentration, type of Hoechst stain and incubation temperature during staining also contribute to the modulation of fluorescence of BrdUrd substituted nuclei. Optimization of these factors is required in order to achieve complete and well resolved cell cycle histograms by the BrdUrd-Hoechst flow cytometric technique.

Hydrocortisone stimulation of DNA synthesis in bromodeoxyuridine-selected non-dividing WI-38 cells

Ostensibly noncycling WI-38 cells were selected by incubating growing cultures for 7 days with 10 −5 M bromodeoxyuridine. These cells were then exposed to Hoechst dye 33258, and then visible light which kills cells that incorporated bromodeoxyuridine. Following selection, cultures were refed with medium containing 10% fetal bovine serum. By autoradiography, we determined that less than 10% of these cells could incorporate [ 3H] thymidine during the next 7 days. This value was increased to 25% or more by adding hydrocortisone to the medium. The proliferative response was also increased by refeeding cultures with hydrocortisone-containing medium conditioned 24 h by freshly seeded mid-, but not late, population doubling level (PDL) cultures. Medium conditioned without hydrocortisone did not stimulate incorporation of [ 3H] thymidine beyond the control value. These results show that: (1) cells that might otherwise not initiate DNA synthesis are stimulated to do so by hydrocortisone; (2)hydrocortisone-conditioned medium from mid-PDL culture increases this stimulation; and (3) hydrocortisone-conditioned medium from late PDL cultures is not stimulatory.

Effects of fluence fractionation on UV-induced malignant transformation and cell killing in non-cycling plateau phase mouse cells.

AbstractPlateau phase C3H 10T 1/2 mouse cells were used to measure the response to split fluence UV light irradiation in the absence of any cell cycle effects. It was found that fluence fractionation with up to 24 h between the fluences resulted in no survival enhancement. The frequency of malignant transformation was potentiated 2.5‐fold when the time interval between the fluences was greater than 4h. This potentiation of transformation was attributed to plateau phase holding rather than to fluence fractionation per se.

Viscoelastic properties of native DNA from intact nuclei of mammalian cells: Higher-order DNA packing and cell function

Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B-form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G 1 cycling versus G 0 non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.

Association between colony forming units-granulocyte macrophage expression of Ia-like (HLA-DR) antigen and control of granulocyte and macrophage production. A new role for prostaglandin E.

The expression of Ia-like antigens on human colony forming units-granulocyte macrophage (CFU-GM) is related to S-phase of the cell cycle, and associated with the control of normal granulocyte and macrophage production by prostaglandin E and acidic isoferritins in vitro. Ia-antigen expression by CFU-GM is lost within 3-6 h of culture at 37 degrees C and occurs simultaneously with loss of responsiveness to inhibition by these factors. Culture of bone marrow CFU-GM in a limited exposure suspension culture with 1 microM-1pM prostaglandin E (PGE1 or PGE2), but not prostaglandin F2 alpha or dibutyryl-cyclic-3'-5'-AMP results in the detection of CFU-GM Ia-antigen after 24 h. Antigen expression is associated with an absolute increase in total and S-phase CFU-GM, and restoration of responsiveness to inhibition by prostaglandin E and acidic isoferritins. The detection of Ia-antigen on CFU-GM after suspension culture with prostaglandin E results both from Ia-antigen reexpression as well as stimulation of noncycling cells to enter S-phase, express Ia-antigen and give rise to CFU-GM sensitive to inhibition by prostaglandin E and acidic isoferritins. The sensitivity of CFU-GM to inhibition by these factors after suspension culture with prostaglandin E is identical to that of the same cells tested prior to the suspension culture. These studies provide evidence for a direct regulatory association between Ia-antigen expression and control of myeloid progenitor cell differentiation, and suggest a role for prostaglandin E in the control of CFU-GM cell cycle, Ia-antigen expression, and growth regulation.

Analysis of cell cycle in root meristems

We have analyzed the cell proliferation in a meristem assuming a single file model for root architecture. The meristem file appears to be built up by two clearly separated zones: the first going from the initial cell to the middle of the meristem and the second from the middle to the meristem boundary. The first half of the meristem shows an exponential age distribution for the cell population. In contrast, in the second half of the meristem, the cell kinetics of cycling cells strongly disagree with exponential kinetics and due to the compensation between the observed deviations in both halves, cell supply in the file meristem is in line with linear kinetics. However, we proposed that exponential kinetic equations offer a suitable approach to problems of cell cycle compartments and population age distributions in real meristems, where non-cycling cells cannot be identified inside the meristem, whether we consider the meristem as a whole or study a “window” inside it. Nevertheless, for more exact kinetic analysis, when estimating the proliferative fraction, the width of the “window” and its location along the axis must carefully be taken into account.

Cell Cycle Kinetics in the Primary Root Meristem ofVicia Faba: Influence of Different Ambient Water Levels

SUMMARYCell cycle kinetics were determined for roots grown either in tanks of water from 24 to 144 hr or in a moistened mixture of vermiculite and perlite (V/P) from 24 to 96 hr. For roots grown in tanks of water Tc was 23.4 hr (TG1 = 6.1 hr, TG2—5.5 hr, T2 = 9.6 hr, Tm = 2.3 hr) while roots grown in V/P had a shorter Tc of 17 hr (TG1 = 5.6 hr, TG2 = 4.0 hr, T, = 5.0 hr, Tm = 2.4 hr). Most of the difference in cycle times was due to a disproportionate increase in the duration of S for water grown roots while the duration of G1 and G2 showed relatively smaller changes. This difference in TS was coupled with an increase in L.I. in water grown roots over that seen in V/P grown roots, i.e. 31.0 ± 8.6 cf. 25.9 ± 6.5. Changes in the proportions of fast, slow and non-cycling cells also occurred; i.e. in V/P grown roots there were 83.2% fast, 11.4% slow and 5.4% non-cycling cells, while in tanks of water there were 52.9% fast, 22.7% slow and 24.4% non-cycling cells. The results are evidence of differences both in mean cycle duration and phase duration within the cell cycle for roots grown in different volumes of water. They also show that roots respond to different volumes of water by altering their GF and the proportions of fast, slow and non-cycling cells in their meristems. These changes may be part of a mechanism used by roots to regulate the overall rate of cell proliferation and thus to achieve different TDT values under different environmental conditions.

Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation

A 36K MW polypeptide was previously identified by two-dimensional gel electrophoresis whose relative proportion increases in S phase of HeLa cells, and following viral transformation of mouse 3T3B and hamster BHK21 cells. We report here experiments that show that this polypeptide (isoelectric focusing (IEF 49)) is localized in the nucleus and that its relative proportion decreases dramatically under conditions for which there is a decrease in the rate of cell proliferation, due for example, to the formation of giant HeLa cells by X-ray irradiation or the senescence of human skin fibroblasts. It is present in negligible amounts in non-cycling cells of adult tissues. These studies also revealed and unrelated cytoplasmic polypeptide (IEF 52; tropomyosin related; coordinates 35/1.43) whose relative proportion increased significantly and reproducibly with a decrease in the rate of cell proliferation. It is suggested that IEF 49 may be a useful marker for cycling cells.

Cellular aging: further evidence for the commitment theory.

A large, transient reduction in the population size of human fibroblasts in early passages significantly increases the variability of the life-spans of cultures in comparison to control cultures, as predicted by the commitment theory of cellular aging. The theory also predicts that a constant population of noncycling cells will appear in the later part of the culture life-span. This was confirmed by labeling the cells in culture with tritiated thymidine.

Response of Epidermal Cell Proliferation to Orally Administered Aromatic Retinoid

Hairless albino mice were fed aromatic retinoid in daily doses ranging from 1 to 50 mg/kg body weight. In animals receiving 1, 5, and 10 mg/kg, the thickness of the epidermis (vertical epidermal cross sectional areas) and the 3H-thymidine labeling indices were determined throughout a 3-week observation period. The following additional parameters were determined for animals fed 5 mg/kg; individual and cumulative cross sectional areas of epidermal cell nuclei and cytoplasms, nuclear-cytoplasm ratios, mean epidermal cell volume and mean number of epidermal cells. In animals fed 5 mg/kg for 7 days, an analysis of the cell kinetic parameters was performed including determination of duration of S and G2 phases, mean generation time and the proportion of noncycling (Go) cells. The changes induced by the aromatic retinoid were found to be dose dependent and consisted of a transitory cellular hypertrophy and a persistent numerical hyperplasia linked to increased labeling indices. The mean basal cell generation time was found to be greatly accelerated owing to a shortening of all cell cycle phases investigated.

Effect of tritiated thymidine on the kinetics and viability of 9L cells in vitro.

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 muCi/ml of [3H]TdR. However, when cells were treated with 1 or 10 muCi/ml of [3H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 muCi/ml of [3H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 muCi/ml of [3H]TdR for 72 hr, less survival was observed. The additional cell kill observed may be induced by [3H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.

Réactivation du bourgeon cotylédonnaire du Pois en réponse à la kinétine

For three components (apical meristem, subapical internodes, and foliar apparatus) of Pisum sativum (cv. nain hâtif d'Annonay) cotyledonary buds, DNA microdensitometry, mitotic indices, and release of growth have been used to detect changes produced by kinetin applications (50 μg/mL). One kinetin treatment is sufficient to release the cell cycle, but continuous kinetin supply is necessary to maintain bud elongation. At the apical, subapical, and foliar levels, inhibited cotyledonary buds contain a majority of nuclei with 2C DNA content, in the G1 phase of the cell cycle. Following hormonal treatment, the prereplicative phase of the noncycling cells is at least 6 h in the whole bud. The lag period for the release of bud growth is between 15 and 23 h in subapical regions (first and second internodes) and between 23 and 38 h at the foliar level. At subapical and foliar levels, a few mitoses precede and then follow the elongation process. The first mitoses are from G2 nuclei of the inhibited bud. The following ones, more numerous, are from noncycling cells of inhibited buds, which have entered the S phase in response to kinetin application. The degree of inhibition of an axillary bud, estimated by the distribution of the DNA content in its three components, conditions the events induced by hormonal treatment or decapitation. The discussion shows how the knowledge of the original nuclear states of inhibited buds is necessary to understand axillary bud reactivation.

A cyanine dye distinguishes between cycling and non-cycling fibroblasts.

Cellular proliferative activity has previously been determined by measuring the incorporation of radiolabelled nucleotides or by visual inspection of cellular morphology. Although two flow cytometric methods have recently been developed which can distinguish cycling from non-cycling cells, both have serious disadvantages. One method requires uptake of a substantial amount of BUdR, limiting its usefulness for in vitro systems. The other method utilizes RNA/DNA content differences but its successful application has proved cell-type dependent. We have now used the findings that the cell membrane is more highly polarized in resting than in proliferating cells and that cyanine dyes carrying a delocalized positive charge enter live cells to an extent that depends on the cell membrane potential, to develop a method of distinguishing between cycling and non-cycling cells. The greater the membrane polarization, the greater is the concentration of dye within the cell. At high concentrations, the dye molecules aggregate and their fluorescence is quenched. Thus, for a given external dye concentration, cells of different membrane potential would accumulate different amounts of fluorescent (non-aggregated) dye. Using fibroblasts in culture conditions chosen to provide various models of cycling and non-cycling cells, we found that fluorescence intensity with the dye, 3,3'-diheptyloxycarbocyanine (Di-O-C,(3)) was consistently greater in the former than the latter.

Effect of hydroxyurea on two different types of hematopoietic stem cells (CFU-S and DCPC) of newborn mice.

Pluripotent hematopoietic stem cells from the bone marrow of newborn mice (9--13 days of age) have been studied following the administration of hydroxyurea (HU, 1,000 mg/kg). Following a single injection as well as a regimen of eight injections complete bone marrow reconstitution was achieved after 7 days. During the recovery phase diffusion chamber progenitor cells (DCPC) from pretreated newborn mouse bone marrow proliferated strongly to give significantly higher cell yields than DCPC from control animals. By contrast (colony-forming units-spleen [CFU-S]) levels were reduced at the same time to between 15 and 40% of controls suggesting a small-sized reserve pool of resting CFU-S in newborn mice. Assuming that DCPC are less differentiated than CFU-S and are non-cycling stem cells they may possibly pass through the CFU-S stage rapidly in newborn mice under the stress of hematopoietic reconstitution and proceed directly to myelopoiesis.

Cycling ⇄ Noncycling Cell Transitions in Tissue Aging, Immunological Surveillance, Transformation, and Tumor Growth

Publisher Summary This chapter discusses the model for cell and tissue proliferation, which interrelates the problems of tissue aging, immunological surveillance, transformation, and tumor growth. The chapter illustrates diagrams that depict cycling and noncycling cell proliferative transitions as they apply to the problems of aging, immunological surveillance, transformation, and tumor growth. It also describes the cycling to noncycling cell transitions, which lead to the establishment of normal tissues composed of four major categories of cycling, noncycling G 1 -, G 0 -, and G 2 -blocked cells, which are arrested at different temporal and biochemical points in the G 1 and the G 2 periods of the cell cycle. Tissue proliferative aging and immunosenescence (age-related decline in immune function) are depicted as being caused by impaired release of noncycling cells to the proliferative cycling state. A primary tumor may arise from the immunological release and proliferation of preexisting, previously transformed dormant noncycling tumor cells, which had been held in restraint by immunological suppression.

Characteristics of root apical meristem cell population kinetics: A review of analyses and concepts

Various conclusions have been reached in the literature concerning the organization of root meristems in terms of cell proliferation and quiescence. In this article the methods used in such analyses are examined in order to assess the validity of the results obtained. Cells in root meristems are clearly heterogeneous in terms of cycle time. The existence of quiescent populations of cells outside the quiescent center, however, is more problematic because (i) it is difficult to define clearly the proximal boundary of the root apical meristem and (ii) present techniques used to measure the size of the proliferative cell fraction are likely to be, in many, if not all cases, highly inaccurate. We have concluded that non-cycling cells, other than those of the quiescent center, do not exist within the meristem proper at the apex of the proliferating angiosperm root.

Ultrastructural and cytochemical comparison of cultured normal and cystic fibrosis fibroblasts

Fibroblasts were cultured from skin biopsies of normal donors and cystic fibrosis (CF) patients, and compared morphologically and cytochemically by light and electron microscopy. Normal and CF fibroblasts revealed by light microscopy a comparable number of cytoplasmic bodies which contained acidic complex carbohydrate and acid phosphatase. These increased in prevalence with the number of days in culture, the time in culture between the last passage and assay, and the percentage of noncycling cells. The fibroblasts did not exhibit extensive or intense metachromasia. Normal and CF cells displayed similar fine structural morphologic features, including a comparable population of dense bodies. These contained acid phosphatase and became labeled by ferritin pinocytosed from the medium. The dense bodies were thus identified as secondary lysosomes engaged, at least in part, in the degradation of endocytosed material. The CF and normal cells exhibited no difference by light microscopy in reactivity to stains for neutral, sialic acid-rich, and sulfated complex carbohydrate. By electron microscopy, no difference in the content of acid complex carbohydrate was demonstrable with dialyzed iron. The amount and distribution of antimonate-precipitable cations was observed ultrastructurally and found to be similar in CF and normal cells. By morphologic or cytochemical criteria, it was not possible to distinguish CF from normal cells or to provide a basis for the extensive cellular metachromasia observed by some investigators in CF cells examined by light microscopy.

Reinitiation of DNA Synthesis in Postmitotic Nuclei of Myotubes by Virus-Mediated Fusion with Embryonic Fibroblasts

Myotubes, whose nuclei have stopped DNA synthesis were fused with replicative embryonic fibroblasts. In heterokaryons the postmitotic muscle nuclei resumed DNA synthesis. Incorporation of radioactive thymidine into muscle, and also into fibroblast nuclei was dependent upon the time elapsed between virus-mediated fusion and administration of radioactive thymidine. Whereas incorporation into fibroblast nuclei diminished with time, there was an early increase of labelling into muscle nuclei followed by a decrease of incorporation of 3H thymidine. DNA synthesis was also dependent upon the ratio of noncycling (muscle) to cycling (fibroblast) nuclei. There was a greater incorporation of 3H thymidine into muscle and fibroblast nuclei in myotubes containing larger numbers of fibroblast nuclei. A model is discussed for the control of DNA synthesis in polykaryocytes derived from fusion of cycling and noncycling cells.