Cell proliferation kinetics of six xenografted human cervix carcinomas: comparison of autoradiography and bromodeoxyuridine labelling methods

Abstract. Cell kinetic and histologic parameters of six xenografted tumours with volume doubling times ranging from 6 to 43 d were investigated in order to obtain kinetic information on a panel of tumours to be used in radiobiological studies. The six tumours covered a range of histologies and their DNA indices varied from 2–7 to 1–4. The length of the cell cycle (T_c), potential doubling time (T_pot) and labelling index (LI) were determined by continuous labelling with [³H]TdR and autoradiography in three tumours. T_c varied from 30 to 40 h. Determinations of the length of the S phase (T_s) were found to be less reliable by this method. Data on T_s and LI were also determined in all six tumours using bromodeoxyuridine (BrdU) labelling and the single sample method; values of T_pot were slightly longer than those obtained via the autoradiographic method. In addition, multiple samples were taken after BrdU labelling. T_c was determined by fitting the data obtained from mid-S, mid-G₂ and mid-G₁ windows to curves described by a damped oscillator. Data obtained via the mid-S window were found to be most reliable. Generally, cell cycle times obtained by the BrdU method were longer than those observed with the autoradiographic method. Differences between the two methods could be explained by inaccuracies in the determination of T_s, LI and T_c and differences in the experimental approach. We consider the BrdU labelling method to be a suitable alternative for the time-consuming autoradiography, if data on T_s or T_pot are sufficient. Due to difficulties in the reproducibility of the immunofluorescence staining and asynchronization of cells approximately 10 h after labelling, the method of windows analysis was affected by similar problems to those observed in interpretation of percentage labelled mitosis (PLM) curves. However, the method may serve as an alternative to determine cell cycle times in vitro and, if improved technically, in vivo. Careful comparison of the data obtained from mid-S, mid-G₁ and mid-G₂ windows may increase the reliability of the determination of cell kinetic parameters.     

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

EVIDENCE OF RAPID AND SLOW PROGRESSION OF CELLS THROUGH G2 PHASE IN MOUSE EPIDERMIS: A COMPARISON BETWEEN PHASE DURATIONS MEASURED BY DIFFERENT METHODS

Percentage labelled mitosis (PLM) measurements were initiated at four different times during a 24-hr period and continued for 24 hr in hairless mouse epidermis. Estimates of G₂ and S phase durations (mean T_G2 and mean T_S) were calculated. A significant number of labelled mitoses (10–20%) was seen after 30 min in all four PLM measurements and the estimated mean T_G2 varied from 1.4 to 2.5 hr and was in agreement with values from PLM measurements in other epithelial tissues. These mean T_G2 values were much shorter than expected from [³H]TdR double labelling experiments and from a multiparameter cell kinetic study in hairless mouse epidermis and did not reflect the circadian variations seen in these studies. the differences in estimates of phase durations can be explained by postulating two G₂ cell populations; one with a rapid and another with a slow rate of cell cycle progression. the cells with the higher rate are mainly registered by the PLM method, whereas those with the lower rate largely escape detection by this method. T_G2 estimates from PLM measurements in mouse epidermis therefore do not reflect the phase duration of the entire G₂ population. It is also concluded that circadian variations in T_S can not be accurately registered by the PLM method.     

CELL CYCLE STUDY UP TO THE TIME OF HATCHING IN THE EMBRYOS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Cell cycles have been analyzed in 10 divisions up to the time of hatching in the embryos of the sea urchin, Hemicentrotus pulcherrimus. In the first 5 cleavages, division synchrony is very high. On the average, T_GC= 55.4 min, T_G1= 0 min, T_s= 12 min, T_G2=±0 min, T_M= 42 min. In the remaining 5 cleavages, T_GC becomes longer: 70 min for the 7th to 246 min for the 10th cleavage. G₁ and G₂ become definitely recognizable and become longer along with T_s. T_M stays more or less constant. Plots of the changing lengths of the four compartments (G₁, S, G₂, M) on the Y-axis against T_GC (X-axis) can be fitted to the following 4 regression equations; T_G1= 0.28T_GC - 19.7, T_s= 0.609T_GC - 15.2, T_G2= 0.104T_GC - 4.72 and T_M= 0.007T_GC+ 39.6.     

ANALYSIS OF THE KINETICS OF GRANULOSA CELL POPULATIONS IN THE MOUSE OVARY

The kinetics of granulosa cell populations in two types of follicles in ovaries of 28-day-old Bagg mice are investigated. The analysis includes estimations of mean values and standard deviations of the transit times (T_G1, T_S, T_G2 and T_C), the doubling time T_D, and the proliferative fraction p. First the percentage of labelled mitosis curve (PLM-curve) and the continuous labelling curve (CL-curve) are estimated. Then a hypothesis concerning the cell kinetics of the granulosa cells in the two follicle types is set up. The normal distribution is chosen to simulate the probability density functions of the transit times. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are worked out. By fitting the calculated PLM-curve to the experimental one it is possible to estimate mean values and standard deviations of T_G1, T_S>, and T_G2. As a test of the hypothesis the CL-curve is calculated by means of the estimated parameter values and compared to the experimental one. The calculated PLM- and CL-curves are found to be in good agreement with the experimental data as far as both follicle types are concerned. It is concluded that the method is a useful procedure. The choice of a normal distribution does not imply a significant limitation of the method in these investigations. Moreover it is concluded that the hypothesis is plausible. This means, e.g., that the proliferative fraction is unity in the two follicle types and that there is no cell loss from the cell systems.     

ACTIVITY OF TUBULOSINE ON THE KINETICS OF ROOT MERISTEM CELL POPULATION

The action of tubulosine on the mitotic cycle was studied using continuous labelling with tritiated thymidine. This alkaloid provokes a lengthening of the G₁ and S phases and a blocking of G₂ is totally reversible when the treatment is followed by recovery in normal medium. At a dose of tubulosine which induces a reversible mitostasis in the shortest possible time the lengthening of the phases of the cell cycle was estimated by three different techniques: labelled mitoses for the determination of G₂; labelling intensity for the determination of S; binucleate cells for the determination of T, and an original technique using labelling index of binucleate cells for the determination of G₁. The limits of the technique of labelled mitosis together with the interest of the technique aiming at the direct determination of G₁ in the case of a perturbed cycle are then discussed.     

THE PROLIFERATION KINETICS OF NHIK 1922 CELLS IN VITRO AND IN SOLID TUMOURS IN ATHYMIC MICE

The proliferation kinetics of cells of the line NHIK 1922 grown in vitro and as solid tumours in the athymic mutant nude mouse has been studied. In vitro, growth curves were determined for exponentially growing populations and for populations synchronized by mitotic selection. The phase durations for these populations were determined by flow cytofluorometric measurements of DNA-histograms and pulsed incorporation of [³H]TdR respectively. The generation time and the phase durations for synchronized populations were found to be about equal to those for exponentially growing populations. The duration of the phases G₁, S and G₂+ M was found to be 8·5–9·5, 11·0–12·0 and 6·0–6·5 hr respectively, i.e. the generation time was 26·5–27·0 hr. The proliferation kinetics in vivo were studied by flow cytofluorometry and by the technique of percentage labelled mitoses. The median duration of S-phase and (G₂+ M)-phase in vivo was found to be approximately the same as that observed in vitro, while the median duration of G₁-phase was found to be approximately 5 hr longer in vivo than under the present in vitro growth conditions. The growth fraction in vivo was estimated to be approximately 50%. The non-proliferative compartment of the tumour cells was found to consist mainly of cells with the DNA-content of cells in G₁-phase. It is concluded that the reduced rate of proliferation of NHIK 1922 cells in vivo is correlated with alterations in the duration of G₁-phase and, hence, the proportion of cells in G₁-phase.     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase     

EFFECTS OF UTERINE DISTENSION AND OESTRADIOL ON CELL KINETICS IN THE ENDOMETRIAL EPITHELIUM OF OVARIECTOMIZED RATS

Cell kinetics in the uterine epithelium of ovariectomized rats were studied after uterine distension and/or an oestradiol injection, by cumulative ³H-TdR labelling and percentage of labelled mitoses (PLM). With both methods it was found that distension shortens the total cell cycle at the expense of G₁ more than does oestradiol. Both treatments act in a cumulative manner since the greatest reduction in T_c is observed after distension plus oestradiol. PLM curves showed that distension and/or oestradiol induce a 30% reduction in S phase duration. The evolution of percentages of labelled cells and colchicine-blocked mitoses after these treatments confirms their additive effects and indicates that the mitogenic action of oestradiol is delayed compared to that of distension. It is suggested that these factors stimulate epithelial cell division in the uterus through partly different metabolic channels.     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

Uptake of phosphate from soils and fertilizers as affected by soil P availability and solubility of phosphorus fertilizers

Fertilizers labelled with ³²P were used to measure amounts of phosphorus, P_s and P_F, taken up by Lolium perenne from available soil P and from P fertilizer respectively, when applied at a rate of 66 mg P·(kg soil^–1) in greenhouse experiments. The quantity P_s of phosphorus taken up from soil in the presence of P fertilizer was compared to the quantity P_o taken up from soil without P fertilizer. The quantity (P_s–P_o) is positive for low P_o values, i.e. in soils poor in available phosphorus, but is negative for high P_o values indicating that an input of P fertilizer can induce a decrease in the utilization of available soil phosphorus. Moreover, for a given soil, the quantity (P_s–P_o) depends on the chemical form of the fertilizer. The standard method of evaluation of P fertilizer efficiency is based on the assumption that P_s=P_o, but P_s can differ from P_o. This result can explain the contradictory data published from field experiments about the efficiency of the various P fertilizers.     

ANALYSIS OF INTESTINAL CELL PROLIFERATION AFTER GUANETHIDINE-INDUCED SYMPATHECTOMY

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (T_C) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G₁, S and G₂ phases of the crypt cell cycle are altered by sympathectomy, G₁ accounting for most of the increase in T_C. In addition, the percentage of [³H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

GRAIN COUNT THRESHOLD DEPENDENCE OF FLM CURVE PATTERN AND CELL CYCLE PARAMETERS OF HEPATOCYTES IN VIVO

FLM curves from hepatocytes of regenerating rat liver in vivo were compared at different grain count thresholds. Estimates of cell cycle phases derived from curves with thresholds decreasing from 15 to 1 grain (background 0.2 grains per nuclear area) revealed a prolongation of t_s from 6.6 to 9.5 hr, at the expense of t_G2M, and t_G1, whereas t_c remained constant. A similar pattern was observed in FLM curves at various threshold levels for hepatocytes localized in subunits of the liver lobule along the vascular axis from afferent to efferent pole. The shapes of these FLM curves indicated an intralobular gradient of reutilizable labelled material. The use of two different threshold levels is crucial for proper selection of FLM curves to evaluate cell cycle phases in regenerating rat liver: first, a threshold to exclude the autoradiographic background, and a second one to avoid errors due to reutilization of labelled DNA precursors. Each threshold has its own implications for the estimation of cell cycle phases.     

THE CELL CYCLE TIME IN THE RAT JEJUNAL MUCOSA

The regional variation of the duration of cell cycle parameters was studied by constructing fraction of labelled mitoses curves at several levels in the jejunal crypt column of male Wistar rats. Prolonged T_c and T_s values were apparent only in the bottom eight cell positions, and these differences were shown to be significant compared with the remaining cell positions by analysing the data by the method of Gilbert (1972). Above cell position 8 the proliferating crypt cells showed effectively the same phase durations. For the whole crypt column T_c was 11.32 ± 0.14 (SE) and T_s 6.49 ± 0.10. Although variation in phase durations was confined to the basal portion of the crypt, the results essentially confirm the findings of Cairnie, Lamerton & Steel (1965a), and may be interpreted in terms of the slow cut-off model. The demonstration of prolonged T_c values in basal cell positions confirms the presence of a longer cycling subpopulation of cells at the bottom of the crypt.     

STUDIES ON LYMPHOCYTES

Some of the time parameters of the cell cycle in bovine thoracic duct lymphocytes have been estimated by analysing labeled mitoses curves, and by double labeling. The two methods gave similar estimates of T_s. Thus, T_s measured directly from labeled mitoses curves varied from 4 to 6 hr, while the estimates from double labeling were 4.8 and 4.5 hr. T _G measured directly from labeled mitoses curves was 5 hr, and estimates of T_G from the values of T_s ranged from 6.3 to 7.7 hr. The present data confirm the short generative cycle of large thoracic duct lymphocytes. Extracorporeal irradiation of the lymph (ECIL) had no detectable effect on the cell cycle or the fractional production rate of the lymphocytes. However, the calculated absolute production was reduced following ECIL, due to a decrease in the absolute number of cells present. The grain count over mitoses after ECIL was approximately one-half that found before ECIL.     

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G₁-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G₁-phase. The role of ste9⁺ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G₁-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G₁/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10^ts, cells arrested in G₁-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G₁-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G₁ progression possibly by controlling the amount of the mitotic cyclin in the G₁-phase.     

Immunocytochemical localization of callose in root cortical cells parasitized by the ring nematodeCriconemella xenoplax

Summary Under hypoxia (10 and 5% partial oxygen tension) meristematic cells ofAllium cepa L. roots acquired new cycle kinetics, characterized by reduced but constant rates of root growth. Under these conditions, there was preferential lengthening of G₁ and of the last third of the S period, S₃. Since hyperoxygenation shortened S₃ but not G₁ in these cells, the high sensitivity of late replication to environmental oxygen is demonstrated. The preferential depression of the replication rate when those cells replicated the last third of their DNA was not associated with diminished cell size. Rather, the lower the oxygen level the larger the mean size of the cycling cells. Under anoxia (0% oxygen tension) the rate of growth slowed, accompanied by preferential accumulation of cells in G₁. However, steady state kinetics of root growth was not achieved under these extreme conditions.Abbreviations Mean cell length - LI labelling index or frequency of cells with labelled nuclei after [³H]thymidine - G₁, S, G₂ pre-replicative, replicative, and post-replicative periods of the interphase of cycling cells - M mitosis     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.