Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells

The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.     

Murine Mammary Tumour Cells In Vitro. I. the Development of A Quiescent State

Abstract Three mouse mammary tumour lines (66, 67, and 68H) derived from a single mouse mammary tumour were investigated for their growth kinetics and development of quiescent cells in unfed monolayer cultures. All three lines develop pure quiescent populations when grown in unfed plateau cultures. A dramatic cell-cycle redistribution accompanied the proliferating (P) to quiescent (Q) transition, with the percentage of cells having a G₁ DNA content increasing from 50% in the P state to <97% in the Q state. As the cultures progressed from exponential to plateau growth, a decrease of 50% in cellular RNA was observed in all three lines. This property enables the clear identification of P v. Q cells by flow cytometry using the two-step acridine orange assay. Autoradiographic data verified that these plateau cells were quiescent since >2.5% of the cells incorporated [³H]TdR when labelled for approximately two doubling times. Further comparison of the P and Q cells showed that: (a) the Coulter volume of Q cells was approximately half that of P cells in all three lines; (b) viability, as measured by dye exclusion was >95% in all cultures regardless of their proliferative state; and (c) colony-forming ability decreased as the cells entered the quiescent state. In each of these cell lines the development of Q-cell populations was marked by similar changes in all measured parameters. These quiescent tumour cells provide a relatively simple model to evaluate what, if any, important differences exist between the response of P v. Q cells to various therapeutic agents.     

Murine mammary tumour cells in vitro. I. The development of a quiescent state

Three mouse mammary tumour lines (66, 67, and 68H) derived from a single mouse mammary tumour were investigated for their growth kinetics and development of quiescent cells in unfed monolayer cultures. All three lines develop pure quiescent populations when grown in unfed plateau cultures. A dramatic cell-cycle redistribution accompanied the proliferating (P) to quiescent (Q) transition, with the percentage of cells having a G1 DNA content increasing from 50% in the P state to greater than 97% in the Q state. As the cultures progressed from exponential to plateau growth, a decrease of greater than or equal to 50% in cellular RNA was observed in all three lines. This property enables the clear identification of P v. Q cells by flow cytometry using the two-step acridine orange assay. Autoradiographic data verified that these plateau cells were quiescent since less than 2.5% of the cells incorporated [3H]TdR when labelled for approximately two doubling times. Further comparison of the P and Q cells showed that: (a) the Coulter volume of Q cells was approximately half that of P cells in all three lines; (b) viability, as measured by dye exclusion was greater than 95% in all cultures regardless of their proliferative state; and (c) colony-forming ability decreased as the cells entered the quiescent state. In each of these cell lines the development of Q-cell populations was marked by similar changes in all measured parameters. These quiescent tumour cells provide a relatively simple model to evaluate what, if any, important differences exist between the response of P v. Q cells to various therapeutic agents.     

Cell resensitization after delivery of a cycle-specific anticancer drug and effect of dose splitting: learning from tumour cords

After a single dose of an anticancer agent, changes due to cell death are expected to occur in the distribution of cells between proliferating and quiescent compartment as well as in the oxygenation and nutritional state of surviving cells. These changes are transient because tumour regrowth tends to restore the pretreatment status. The reoxygenation due to the decrease of oxygen consumption is expected to induce cell recruitment from quiescence into proliferation, and consequently to increase the sensitivity of the cell population to a successive treatment by a cycle-specific drug. In previous papers we proposed a model of the response of tumour cords (cylindrical arrangements of tumour cells growing around a blood vessel of the tumour) to single-dose treatments. The model included the motion of cells and oxygen diffusion and consumption. On the basis of that model suitably extended to better account for the action of anticancer drugs, we study the time course of the oxygenation and of the redistribution of cells between the proliferating and quiescent compartments. By means of simulations of the response to a dose delivered as two spaced equal fractions, we investigate the dependence of tumour response on the spacing between the fractions and on the main parameters of the system. A time window may be found in which the delivery of two fractions is more effective than the delivery of the undivided dose.     

THE KINETICS OF GRANULOSA CELLS IN DEVELOPING FOLLICLES IN THE MOUSE OVARY

This investigation describes the kinetics of the granulosa cells in medium-sized follicles type 3b, 4 and 5a in ovaries of 28-day-old Bagg mice. the method of labelling with ³H-thymidine followed by high resolution autoradiography is used in the experimental work, which consist of determining percentage labelled mitosis (PLM-) and continuous labelling (CL-) curves. In order to analyse the data by computer two alternative hypotheses A and B are set up. Both include the assumptions of no cell loss, exponential growth and a resting compartment Q. In hypothesis A cells from Q re-enter the mitotic cycle via the normal DNA-synthesis compartment S_p. Hypothesis B includes beside compartment S_p a special DNA-synthesis compartment S_q where only cells from Q are synthesizing DNA, and these cells re-enter the mitotic cycle via the G₂ compartment. the mean transit time in S_q is considered to be longer than the mean transit time in S_q. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are obtained, and by means of a computer the theoretical curves are fitted to the experimental values: thereby all relevant cell kinetical parameters are estimated. Hypothesis B seems to give the best fit between the theoretical and experimental curves. the estimated parameters are: mean cycle times, μ_c= (56.1 hr, 56.1 hr and 22.3 hr for type 3b, 4 and 5a respectively), doubling times, T _D= (96.4 hr, 118.6 hr and 59.1 hr) and the proportion of cells in Q, p _Q = (0.60, 0.71 and 0.69).     

In vitro holding and PLD repair

Summary The Stationary or Plateau-Phase of commonly used rodent cell lines like the V79 are often assumed to be quiescent (non-mitotic). An analysis of cell turnover in V79 plateau-phase cultures through BrUdR-incorporation combined with FUdR-block and light exposure (S-phase cytocide) revealed such cultures to be in a state of kinetic equilibrium. Even when the state of maximal permissible density was acquired, at least 50% of the population of cells were cycling within the time for one population doubling. Attempts at holding the cells from cycling (through nutrient-depletion and serum-privation) were unsuccessful, although the turnover-rate was reduced. Our assays for X-irradiated clonogenic survivors after attempted holding combined with delayed plating (DP) showed differences in the survival curves for exponentially growing and confluent cultures. Elimination of cycling cells by S-phase cytocide removed these differences. Since a significant fraction of plateau-phase cells are not mitotically quiescent (Q), one must eliminate the proliferating (P) fraction if one wishes to examine the PLDR of the Q cells. For V79 cells, removal of the P cells eliminates the higher survival (usually interpreted as Q cell PLDR) of plateau-phase cells.     

Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X-irradiation

The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient-deprived quiescence QI; and QI cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol-containing proteins changed whenever the cells were in transition between different physiological states (e.g., when QI cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either QI or P cells). The NPSH and GSH levels were both down significantly in the QI vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P-cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 QI cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation-induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulated 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the QI cells could represent a cause-and-effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.     

Cell cycle effect on the induction of DNA double-strand breaks by X rays

Filter elution was used to compare X-ray-induced DNA single- and double-strand breaks in proliferating (P) and quiescent (Q) cells of the 66 and 67 mouse mammary tumor lines. There was no difference either between cell type or between growth states in the amount of single-strand breaks as defined by elution at pH 12.2. In contrast, Q cells appeared to sustain a much larger amount of double-strand break damage per Gray than P cells, when the damage was measured by elution at either pH 7.2 or pH 9.6. Experiments which combined centrifugal elutriation with pH 7.2 elution demonstrated that G1-P cells were similar to Q (greater than or equal to 95% G1) cells in the induction of elution-detectable double-strand breaks, while the S-phase enriched fractions sustained less damage than G1-P, Q, or asynchronous P populations. Studies in which P populations were pulse labeled with [14C]thymidine confirmed this finding. Mathematical analysis of the elution kinetics of irradiated P, Q, and S-phase cells supports a model in which the complex elution profiles observed for P cells could be explained as the sum of the one-component exponential elution profiles of G1- and S-phase subpopulations. Also, the correlation between damage measured by pH 7.2 elution and cell survival was tested by examining the dose response for stimulated 66 cells (St4), which like Q cells are greater than or equal to 95% in G1 but are more resistant to X-ray-induced cytotoxicity than are the 66 Q cells. However, the induction of double-strand breaks in St4 cells was identical to that in Q cells. Thus we conclude that there is not necessarily a correlation between the amount of elution-detectable X-ray-induced double-strand breaks and cell survival.     

Cell cycle regulation by environmental pH

Purified populations of quiescent human tumour cells were isolated from plateau phase cultures of PMC-22 cells by centrifugal elutriation. Dilution into fresh medium resulted in these quiescent cells entering S phase exponentially with a t1/2 of 12 hr, after a 18-20-hr lag period during which cellular RNA content increased. Subsequent studies showed that recruitment of quiescent cells into the cell cycle could be regulated by extracellular pH. When exponentially growing PMC-22 cells were exposed to acidic extracellular pH levels, three growth patterns were observed: (1) Normal growth between pH 7.2 to pH 6.8; (2) A reduction in growth rate associated with accumulation of cells with a G₁ DNA content between pH 6.7 and 6.4 (this was also shown to occur in a number of other tumour cell lines); (3) Non-cell-cycle-phase-specific arrest of growth at pH levels less than 6.3. Further studies with purified quiescent cell populations showed the possible existence of a pH-dependent restriction point in the G₁ phase of these tumour cells. The implications of these observations to tumour biology are discussed.     

Modelling complex populations formed by proliferating,quiescent and quasi-quiescent cells: application to plant root meristems

A proliferating population of cells may be considered complex when its proliferative or growth fraction P is lower than 1 and/or when it is formed by subpopulations with different mean cycle times. The present paper shows that in such complex populations exponential growth is consistent with a steady-state distribution of cells. Obviously, when P=1 then cell distribution is only a function of cell age. An analytical model has been developed to study complex populations including both quiescent fractions formed by cells with unreplicated genome (G(0) cells) and cells with fully duplicated chromosomes (Q(2) cells). The model also considers those quasi-quiescent cells in their last transit through G(1) and S (Q(1) and Q(s) cells) before becoming quiescent. In order to solve the difficulties of a direct analysis of the whole population, its kinetic parameters have been obtained by studying the negative exponential distribution of two subpopulations: one formed by the proliferating cells and another formed by the quasi-quiescent cells. Additionally, the model could be applied when quiescence is initiated at any other cycle phase different from G(1) and G(2), for instance, cells in the process of replicating their DNA or being at any other mitotic phases. The utility of the method was illustrated in populations which constitute the root meristems of both Allium cepa L. and Pisum sativum L. Three facts should be stressed: (1) the method seems to be rather powerful because it can be carried out from different sets of experimentally measured parameters; (2) the rate of division and, therefore, the population doubling time can be easily estimated by this method; and (3) it also allows the determination of the amount of cells that had become quiescent either before they had replicated their DNA (G(0)) or after having completed their replication (Q(2)), as well as those quasi-quiescent cells which are progressing throughout their last pre-replicative and replicative periods (thus Q(1) and Q(s), respectively).     

Radiation-induced cytotoxicity,DNA damage and DNA repair: Implications for cell survival theory

Summary The radiosensitivities and the kinetics for removal of radiation-induced DNA damage were compared for proliferative (P) and quiescent (Q) cells of the lines 66 and 67 derived from a mouse mammary adenocarcinoma. As determined from cell survival assays, the 66 and 67 Q cells were more radiosensitive than their 66 and 67 P counterparts. The rank order of their radiosensitivity was: 67 Q > 66 Q 67 P > 66 P. Induction of radiation damage in the DNA of these cells, as measured by the alkaline elution technique, was identical for 66 and 67 P and Q cells. The repair of this DNA damage was biphasic for 66 and 67 P and Q cells. The half-times for the fast and slow repair phases in 66 Q cells were identical to those previously measured in 67 Q cells. The half-times of the fast and slow repair phases in 66 P cells were also identical to those previously measured in 67 P cells. However, the half-times for the fast and slow repair phases in 66 and 67 Q cells were longer than those measured in their 66 and 67 P counterparts. The 66 cell data are consistent with our previously published hypothesis that Q cells are more radiosensitive than their corresponding P cells because they repair their radiation-induced DNA damage slower. However, our results are not consistent with hypotheses that attempt to explain the radiosensitivity differences between lines 66 and 67 solely on the basis of measurable induction and repair of DNA damage.     

Experimental chemotherapy and concepts related to the cell cycle

Scheduling of chemotherapy is limited by damage to normal tissues, and tolerated schedules are dependent on normal tissue recovery. Most anticancer drugs are more toxic to proliferating cells and the fall and recovery of granulocyte counts after chemotherapy may be explained by the effect of drugs on rapidly proliferating precursor cells in the bone marrow. It is argued that serious toxicity due to myelosuppression most often occurs because of damage to proliferating precursors that may be recognized in bone marrow rather than to stem cells. In contrast, therapy that is aimed at producing cure or long-term remission of tumours must be directed at killing tumour stem cells. The evidence that tumours contain a limited population of cells which can repopulate the tumour after treatment (and are therefore tumour stem cells) is reviewed critically. While there is quite strong evidence for a limited population of target cells, evidence from studies on metastases suggests that the tumour cells which may express this stem cell property may change with time. The stem cell concept has major implications for predictive assays. Although colony-forming assays appear to have a sound biological background for predicting tumour response, technical problems prevent them from being used routinely in patient management. Cells in tumours are known to be heterogeneous and at least three types of heterogeneity may influence tumour response to drug treatment: the development of subclones with differing properties including drug resistance; variation in cellular properties due to differentiation during clonal expansion; and variation in properties due to nutritional status and micro-anatomy. Heterogeneity in drug distribution within solid tumours may occur because of limited drug penetration from blood vessels, and nutrient-deprived cells in solid tumours may be expected to escape the toxicity of some anticancer drugs as well as being resistant to radiation because of hypoxia. This may occur both because nutrient-deprived cells have a low rate of cell proliferation, and also because of poor drug penetration to them. There is a need for improved understanding of the mechanisms that lead to cell death in tumours. If these mechanisms were understood, it might be possible to simulate them by therapeutic manoeuvres. Recent research from our laboratory suggests that the combination of low extracellular pH and hypoxia may be very toxic to cells in nutrient-deprived regions. Drugs which limit the cell's ability to survive in regions of acid pH may provide strategy for therapy of nutrient-deprived cells.     

Modelling the Cell Cycle and Cell Movement in Multicellular Tumour Spheroids

This paper analyses a recent mathematical model of avascular tumour spheroid growth which accounts for both cell cycle dynamics and chemotactic driven cell movement. The model considers cells to exist in one of two compartments: proliferating and quiescent, as well as accounting for necrosis and apoptosis. One particular focus of this paper is the behaviour created when proliferating and quiescent cells have different chemotactic responses to an extracellular nutrient supply. Two very different steady-state behaviours are identified corresponding to those cases where proliferating cells move either more quickly or more slowly than quiescent cells in response to a gradient in the extracellular nutrient supply. The case where proliferating cells move more rapidly leads to the commonly accepted spheroid structure of a thin layer of proliferating cells surrounding an inner quiescent core. In the case where proliferating cells move more slowly than quiescent cells the model predicts an interesting structure of a thin layer of quiescent cells surrounding an inner core of proliferating and quiescent cells. The sensitivity of this tumour structure to the cell cycle model parameters is also discussed. In particular variations in the steady-state size of the tumour and the types of transient behaviour are explored. The model reveals interesting transient behaviour with sharply delineated regions of proliferating and quiescent cells.     

Matrix simulation of duodenal crypt cell kinetics. I. The steady state.

Steady state crypt cell kinetics have been simulated using matrix algebra. The model crypt cell population is distributed through two proliferation compartments (P1 and P2) and a quiescent state (Q). Under steady state conditions half the daughter cells produced on completion of P1 enter G1 of P2 and half enter G1 of P1. Both P2 daughter cells enter Q. Cells in Q are non-dividing but retain the potential to divide. On completion of Q, cells lose the potential to divide and move up onto the villi. The model has been developed by simultaneously simulating the following biological data: (1) the per cent labeled mitosis (PML) curve, (2) the number of labeled cells per crypt as a function of time following an injection of 3H-thymidine, and (3) the total number of cells per crypt.     

Long-Term Quiescent Fibroblast Cells Transit into Senescence

Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development.     

"Sleeping beauty": quiescence in Saccharomyces cerevisiae.

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.