Cell kinetics of irradiated experimental tumors: relationship between the proliferating and the nonproliferating pool.

The role of nonproliferating cells in tumor regeneration has been studied after subcurative doses of low L.E.T. irradiation. Radiation was applied in a single dose at three different levels; 0-47, 0-94 and 1-88 krad. Studies included estimation of the absolute number of cells per tumor, differential cell counts, and autoradiographic determination of kinetic variables, employing transplantable mouse mammary adenocarcinoma DBAH. Quantitative changes of morphologically defined proliferating and nonproliferating cell pools were followed at different time intervals after irradiation. Irradiation resulted in reduction of the number of cells in both pools, with apparent sparing of nonproliferating cells. The regenerative period started with a gradual increase in the number of cells in the proliferating pool, whereas the number of cells in the nonproliferating pool continued to fall in tumors irradiated with 0-94 and 1-88 krad. In the late phase of tumor regrowth, the increasing number of cells in the non proliferating pool corresponded to its replenishment by cell transition from the proliferating pool. In an effort to clarify whether cell transition from the nonproliferating to the proliferating pool may take place during the regrowth of radiation perturbed tumors, cell loss rates from both pools were estimated using experimental data. In addition to cell loses from the tumor as a whole, the 'net loss rate' of the nonproliferating pool reflects the rate of cell transition from the nonproliferating to the proliferating pool, minus the rate of transition in the opposite direction. A similar definition applies to cell loss rates from the proliferating pool. The results showed: (1) high losses in both pools, with excess losses in the proliferating during the early phase after irradiation; (2) in the early stage of regrowth after irradiation, the cell net loss rate f-or the nonproliferating pool increased, in contrast to the behavior of cell loss rate for the proliferating pool and the average cell loss rate for the tumor as a whole; (3) in the late stage of regrowth a decrease in net loss rate for the nonproliferating pool reflects the excess production of nonproliferating cells over control tumors. These results suggest that cell transition from the nonproliferating to the proliferating pool takes place at the beginning of tumor regrowth after subcurative single-dose irradiation.     

CELL KINETICS OF IRRADIATED EXPERIMENTAL TUMORS: CELL TRANSITION FROM THE NON-PROLIFERATING TO THE PROLIFERATING POOL

Parenchymal tumor cells of murine mammary carcinomas can be divided into two pools, using nucleoli as morphological ‘markers’. Cells with dense nucleoli traverse the cell cycle and divide, thus constituting the proliferating pool. Cells with trabeculate or ring-shaped nucleoli either proceed slowly through G₁ phase or are arrested in it. The role of these non-proliferating, G₁ phase-confined cells in tumor regeneration was studied in vivo after a subcurative dose of X-irradiation in two transplantable tumor lines. Tumor-bearing mice were continuously injected with methyl[³H]thymidine before and after irradiation. Finally, the labeling was discontinued, mice injected with vincristine sulfate and cells arrested in metaphase were accumulated over a 10-hr period. Two clearly delineated groups of vincristinearrested mitoses emerged in autoradiograms prepared from tumor tissue at the time of starting tumor regrowth: one group with the silver-grain counts corresponding to the background level, the other with heavily labeled mitoses. As the only source of unlabeled mitoses was unlabeled G₁ phase-confined cells persisting in the tumor, this observation indicated cell transition from the non-proliferating to the proliferating pool, which took place in the initial phase of the tumor regrowth. Unlabeled progenitors have apparently remained in G₁ phase for at least 5–12 days after irradiation.     

A mathematical model of canine granulocytopoiesis

Summary The granulocyte cell renewal system of the dog is represented by a mathematical model consisting of the following compartments: The pool of pluripotential stem cells, the committed stem cell pool, divided into a blood and a bone marrow compartment, the proliferation pool, the maturation pool, the reserve pool and the blood pool of functional granulocytes. This chain of compartments is described by a system of non-linear differential equations. Cell losses anyplace in the system provoke increased production in all pools containing cells capable to divide. A reduced number of granulocytes in the blood pool stimulates production of a granulocyte releasing factor which mobilizes a rising number of cells to transit from the marrow reserve into the blood pool.The model was simulated on a digital computer. It was found to be capable to reproduce the steady state conditions and it also fits the data of two distinct experimental perturbations of the system both equally well. These perturbations are a loss of proliferating cells as it occurs after the administration of cytostatic drugs and losses of functional cells as they are induced by leukapheresis experiments of differing leukapheresis rates.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 112)     

Combining Gompertzian growth and cell population dynamics

A two-compartment model of cancer cells population dynamics proposed by Gyllenberg and Webb includes transition rates between proliferating and quiescent cells as non-specified functions of the total population, N. We define the net inter-compartmental transition rate function: Phi(N). We assume that the total cell population follows the Gompertz growth model, as it is most often empirically found and derive Phi(N). The Gyllenberg-Webb transition functions are shown to be characteristically related through Phi(N). Effectively, this leads to a hybrid model for which we find the explicit analytical solutions for proliferating and quiescent cell populations, and the relations among model parameters. Several classes of solutions are examined. Our model predicts that the number of proliferating cells may increase along with the total number of cells, but the proliferating fraction appears to be a continuously decreasing function. The net transition rate of cells is shown to retain direction from the proliferating into the quiescent compartment. The death rate parameter for quiescent cell population is shown to be a factor in determining the proliferation level for a particular Gompertz growth curve.     

Distinction between the G0 and the late G1 phase of rat cells in vivo and in vitro with the nuclear QDH-fluorescence pattern

The quinacrine dihydrochloride (QDH) staining and the [3H]thymidine incorporation patterns were simultaneously analyzed in nuclei of rat cells from a proliferating (granulation tissue) and a nonproliferating tissue (liver). Nuclei from freshly isolated and cultured cells of the rapidly proliferating subcutaneous granulation tissue showed a cell cycle-related pattern similar to that previously described with growing fibroblast-like cells in vitro. Nuclei of liver cells in smears from biopsies and in histological sections showed a fluorescence pattern similar to that of serum-deprived arrested G0 cells from established cell lines. Treatment of primary cultured rat hepatocytes with phenobarbital altered their degree of chromatin condensation similar to that seen after treatment of rats in vivo. The data indicate that the QDH staining pattern is an early marker, suitable for detecting the cell cycle-promoting activity of chemicals (e.g., of tumor promoters) in nonproliferating cells from various tissues in vivo and in vitro.     

Expression of the differentiation antigen of activated T and B lymphocytes (ACA-1) on cells of lymphoid and nonlymphoid tumors

In cytotoxicity and indirect immunofluorescence tests an antiserum to ACA-1 (activated cell antigen) reacted with 58–100% of actively proliferating cells from tumors of lymphoid (EL-4 T lymphoma, MOPC 104E plasmacytoma) and nonlymphoid origin (AH-22 hepatoma, Sa-1 and MCh-11 sarcomas, F2 mammary cancer). Absorption of anti-ACA-1 serum with tumor cells sharply reduced its activity both against the cells of all these neoplasms and against normal activated T and B lymphocytes. Absorption with proliferating murine cells from the brain of embryos and the retina of neonates or with similar (nonproliferating) cells from adult mice did not affect the activity of the antiserum. It is concluded that ACA-1 is expressed on actively proliferating cells of the tumors studied.     

Different growth patterns of a cancer cell population as a function of its starting growth characteristics: analysis by mathematical modelling

The growth kinetics of a cancer cell population as a function of the total number of cells and the proportion of proliferating and resting cells at the beginning of the growth has been analysed by a mathematical model. The model takes into account the processes of cell division, death and transition from proliferation to rest and backwards. It is shown that a single cell population growing under the same environmental conditions has an extremely broad spectrum of growth patterns. The whole multiplicity of possible growth patterns has been determined by the inherent cellular growth characteristics of the population, while the growth pattern actually realized of the variety of growth curves depends on the total number of cells and the proportion of proliferating and resting cells at the initial moment of growth. The model is shown to provide a good prediction of experimentally measured kinetics of regrowth of tumour cells subcultured after various times of the growth in unfed cultures, and the kinetics of tumour cell growth after severe hypoxia. The role of cell transitions between proliferating and resting stages in the problem of growth control is discussed.     

Regulating the balance between symmetric and asymmetric stem cell division in the developing brain

Stem cells proliferate through symmetric division or self-renew through asymmetric division whilst generating differentiating cell types. The balance between symmetric and asymmetric division requires tight control to either expand a stem cell pool or to generate cell diversity. In the Drosophila optic lobe, symmetrically dividing neuroepithelial cells transform into asymmetrically dividing neuroblasts. The switch from neuroepithelial cells to neuroblasts is triggered by a proneural wave that sweeps across the neuroepithelium. Here we review recent findings showing that the orchestrated action of the Notch, EGFR, Fat-Hippo, and JAK/STAT signalling pathways controls the progression of the proneural wave and the sequential transition from symmetric to asymmetric division. The neuroepithelial to neuroblast transition in the optic lobe bears many similarities to the switch from neuroepithelial cell to radial glial cell in the developing mammalian cerebral cortex. The Notch signalling pathway has a similar role in the transition from proliferating to differentiating stem cell pools in the developing vertebrate retina and in the neural tube. Therefore, findings in the Drosophila optic lobe provide insights into the transitions between proliferative and differentiative division in the stem cell pools of higher organisms.     

Regulating the balance between symmetric and asymmetric stem cell division in the developing brain

Stem cells proliferate through symmetric division or self-renew through asymmetric division whilst generating differentiating cell types. The balance between symmetric and asymmetric division requires tight control to either expand a stem cell pool or to generate cell diversity. In the Drosophila optic lobe, symmetrically dividing neuroepithelial cells transform into asymmetrically dividing neuroblasts. The switch from neuroepithelial cells to neuroblasts is triggered by a proneural wave that sweeps across the neuroepithelium. Here we review recent findings showing that the orchestrated action of the Notch, EGFR, Fat-Hippo, and JAK/STAT signalling pathways controls the progression of the proneural wave and the sequential transition from symmetric to asymmetric division. The neuroepithelial to neuroblast transition in the optic lobe bears many similarities to the switch from neuroepithelial cell to radial glial cell in the developing mammalian cerebral cortex. The Notch signalling pathway has a similar role in the transition from proliferating to differentiating stem cell pools in the developing vertebrate retina and in the neural tube. Therefore, findings in the Drosophila optic lobe provide insights into the transitions between proliferative and differentiative division in the stem cell pools of higher organisms.     

The disappearance of a cyclin-like protein and the appearance of statin is correlated with the onset of differentiation during myogenesis in vitro

We have used monoclonal antibodies to statin (S-44) and a cyclin-like protein (S-132) to examine the distribution of these two antigens in proliferating and in nonproliferating populations of cells. We have found that this cyclin-like protein is present in proliferating fibroblasts, whereas statin is absent from these same cell populations; in contrast, in senescent populations of fibroblasts the cyclin-like antigen disappears and statin labeling of nuclei appears. During myogenesis in rat muscle cell cultures, S-132 labeling is present in proliferating myoblasts and disappears after cells fuse and differentiate as multinucleated myotubes. In contrast, statin is absent from proliferating myoblasts, but appears when these cells become postmitotic and begin to differentiate. Similar results were seen during chick myogenesis. We have also found similar results during serum-starvation-induced differentiation in neuroblastoma cells. These results indicate that the cyclin-like protein disappears and statin appears upon commitment to differentiation in vitro, and the presence or the absence of these proteins appears to provide cellular markers for the transition from the proliferative to the nonproliferative state during differentiation.     

Adenine metabolism of Ehrlich mouse ascites cells in proliferating and resting phase of tumor growth.

The incorporation of 14C from [U-14C]adenine into the pools of purine nucleotides, nucleosides and bases in Ehrlich mouse ascites cells (EMAC1) during the proliferating and resting phases of tumor growth was compared. In the proliferating phase the total 14C incorporation into purine pools is much faster than in the resting phase. The ATP turnover as well as the purine breakdown to hypoxanthine and uric acid are increased in the proliferating phase. That corresponds to previous findings on higher nucleotide pool sizes and higher ATP yield and ATP-consuming processes in this growth period.     

Histone synthesis in the cells of proliferating and nonproliferating tissues during gametogenesis and early embryogenesis

A review of available data on the replication-dependent and replication-independent histone synthesis in the proliferating and nonproliferating (quiescent) cells during gametogenesis and early embryogenesis. In each of the considered models the replication-dependent and replication-independent histone synthesis play different roles in the chromatin organization and metabolism. The transition from replication-dependent to replication-independent histone synthesis during gametogenesis is a regular process directed to the formation of a highly compacted metabolically inert chromatin (males) and to the formation of histone protein pool in order to provide the chromatin nucleosome structure in the sperm nucleus during fertilization, as well as the nuclear chromatin in zygotes and blastomeres (females). A suggestion is put forward that the coupling of histone and DNA syntheses should arise not simultaneously in all cells of the embryo but have a regional pattern, due, possibly, to the asynchrony of cell cycle in the early embryos.     

DNA synthesis and Bcl-2 expression during development of mucous cell metaplasia in airway epithelium of rats exposed to LPS

Exposure of pulmonary airways to environmental toxins and allergens may cause proliferation of airway epithelial cells and mucous cell metaplasia (MCM); however, it is unclear to what extent proliferating cells differentiate into mucus-storing cells and contribute to MCM. Our previous studies demonstrated that Bcl-2, an inhibitor of apoptosis with cell cycle regulatory functions, is expressed in metaplastic mucous cells. The purpose of the present study was to investigate the number of metaplastic mucous cells that are derived from proliferating epithelial cells and whether Bcl-2 has a role in cell cycle entry in these cells. Rats were intratracheally instilled with 100 microg of LPS from Pseudomonas aeruginosa in 500 microl of saline, and proliferating airway cells were labeled with bromodeoxyuridine (BrdU) by implanting a subcutaneous osmotic pump 24 h before instillation. The volume of stored mucosubstance and the number of mucous cells were increased 10- and 3-fold, respectively, from 24-48 h after instillation. The number of total epithelial cells per millimeter of basal lamina increased, and the number of serous cells per millimeter of basal lamina decreased during this time. Approximately 50% of Alcian blue-periodic acid Schiff-stained mucous cells were labeled with BrdU at 48 h after instillation, suggesting that one-half of the secretory cells were derived from proliferating cells. Furthermore, 50% of the Bcl-2-positive mucous cells were BrdU negative and therefore derived from nonproliferating, preexisting cells. Our findings demonstrate that preexisting and proliferating cells differentiate into mucous cells and compose LPS-induced metaplasia and that Bcl-2 does not have cell cycle regulatory function in these cells.     

Nucleolar morphology and cell proliferation kinetics of thymic lymphocytes

Proliferation kinetics of cells of the lymphocytic series were studied in mouse thymuses using ³H-methyl-thymidine (³H-TdR) as a tracer of DNA synthesis and employing autoradiographic technique. Cells were allocated arbitrarily to several compartments according to their degree of maturity and nucleolar morphology. Serial sampling after continuous ³H-TdR injection showed that thymic cells of the lymphocytic series constitute a small highly proliferating pool that feeds into a large nonproliferating pool. Lymphoblasts with dense and trabeculate nucleoli and prolymphocytes with trabeculate nucleoli represent multiplicative compartments and belong to the proliferating pool. A fraction of multiplicative precursors enters a ‘dormant’ state and these immature lymphocytes are morphologically characterized by ring-shaped nucleoli. Multiplicative compartments and non-dividing compartments of immature lymphocytes differ significantly in labeling indices, kinetics of labeling in serial samples and in other kinetic parameters, namely in the efflux from the unlabeled pool, labeling increment and efflux from the labeled pool. Serially connected compartments of lymphoblasts with dense and trabeculate nucleoli, prolymphocytes with trabeculate nucleoli and mature lymphocytes, represent the main stream of cell differentiation and maturation. At least a portion of mature lymphocytes proceeds during maturation from the compartment of cells with trabeculate nucleoli to the compartment of cells with ring-shaped nucleoli. The presence of proliferating and ‘dormant’ precursors suggests that lymphopoiesis in thymuses may correspond to the advantaged logarithmic system of multiplication.     

Gamma-ray-enhanced reactivation of UV-irradiated adenovirus in normal human fibroblasts.

An enhanced reactivation of UV-irradiated adenovirus type 2 (Ad 2) was detected following irradiation of the host cells with γ-rays prior to infection. Non-irradiated and γ-irradiated normal human fibroblasts were infected immediately after irradiation with either non-irradiated or UV-irradiated Ad 2. At 48h after infection, cultures were examined by indirect immunofluorescence to determine the number cells in which the viral function of viral structural antigen (Vag) was expressed. Pre-irradiation of cells with 1 krad resulted in a 2–3-fold increase in the survival of this viral function following different UV doses to the virus up to 1.75 × 10³ J/m². For a fixed UV dose of 1.0 × 10³ J/m² to the virus this enhancement increased with preirradiation dose to the cells up to a maximum factor of 2–3 for a dose of 1 krad. An examination of Vag expression at various times after infection indicates that pre-irradiation of the cells with γ-rays prior to infection with UV-irradiated virus leads to an earlier onset and/or increased rate of Vag synthesis. This enhancement of Vag production from a UV-damaged template may result from an inducible DNA-repair mechanism in human fibroblasts which may or may not be error-prone.     

Effect of 8-azaguanine on the transition from vegetative growth to presporulation in Bacillus cereus

Stahly, D. P. (University of Illinois, Urbana), V. R. Srinivasan, and H. Orin Halvorson. Effect of 8-azaguanine on the transition from vegetative growth to presporulation in Bacillus cereus. J. Bacteriol. 91:1875-1882. 1966.-The guanine analogue, 8-azaguanine (azaG), was found to inhibit sporulation of Bacillus cereus strain T when added to proliferating cells, but not to inhibit when added after the transition to presporulation. When azaG was added to vegetative cells, the growth rate was reduced, but no immediate bactericidal effect was demonstrated. Azaguanine was shown to be incorporated solely into ribonucleic acid (RNA). All of the natural purine bases and nucleosides were found to prevent azaG inhibition by blocking incorporation of the analogue into the RNA. Addition of a subinhibitory level of C(14)-azaG to proliferating cells resulted in an increase in incorporation paralleling the increase in number of cells. At the time of transition from growth to presporulation, a rapid removal of the azaG label from the cells occurred in the absence of net RNA breakdown. If differentiation was inhibited by increasing the concentration of azaG, then no expulsion took place. Instead, at the end of growth, net incorporation ceased, and a steady-state condition was established in which incorporation equaled breakdown. No azaG degradative enzymes are present in presporulating cells. The possibility is discussed that an increase in the ratio of natural purines to azaG occurred at the time of transition, and that the natural purine derivatives then were reincorporated into RNA preferentially to azaG. The data are consistent with the hypothesis than an increased rate of RNA turnover occurs at the time of transition from vegetative growth to presporulation. Addition of phosphate buffer (pH 7.0, 0.1 m) to azaG-inhibited vegetative cells caused reversal of inhibition, the reversal being accompanied by expulsion of the azaG. At least a partial explanation of this effect is that phosphate causes a decrease in the azaG intracellular pool size.     

The relationship between radiosensitivity and cell kinetic effects after low- and high-dose-rate irradiation in five human tumors in nude mice

Radiation-induced synchronization of cells in the radiosensitive G2 phase can, theoretically, be applied to individual tailoring of fractionation schemes, possibly rendering radiotherapy more effective. For that purpose, cell cycle perturbations were studied in five xenografts by flow cytometry. A dose-dependent increase of cells in G2 phase was noticed in all five tumor cell lines after high-dose-rate irradiation, and in four tumor cell lines after low-dose-rate irradiation. The timing of maximum accumulation was not related to dose, but coincided with the cell cycle time of the respective tumors. Furthermore, the increase in the number of cells in G2 phase correlated with the radiosensitivity of the tumors as assessed by measurements of regrowth delays. The observed synchronization provides a basis for further investigations on the relevance of radiation-induced cell cycle synchrony to the effectiveness of fractionated radiotherapy.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.