A comparative kinetic analysis of proliferation in vitro of Con-A-treated splenocytes and syngeneic leukaemia cells

Abstract. The growth kinetics of Con-A-treated mouse splenocytes and syngeneic leukaemia cells cultured in vitro were compared with respect to (i) the total cell number, (ii) the rate of [¹⁴C]thymidine incorporation (measured by pulse-labelling the cells at various times of incubation), and (iii) the labelling index of the cell populations. By correlating the thymidine incorporation, labelling index and cell number data, it has been established that, for both types of cells, the rate of [¹⁴C]thymidine incorporation is directly proportional to the number of cells synthesizing DNA. A new approach to cytokinetic analysis has been developed, showing that important information can be obtained by determining the cumulative kinetics of [¹⁴C]thymidine incorporation. The latter has been calculated by integrating the area underneath the time course of the rate of thymidine incorporation, and was directly proportional to the overall growth of both leukaemia cells and Con-A-stimulated splenocytes. Based on this proportionality, an estimate of the average duration of the S phase for both types of cells was calculated, suggesting that normal and neoplastic blasts maintain this parameter at a constant value (7.6 and 5.9 hr, respectively) throughout different stages of growth. The percentage of Con-A-responsive cells within the initial splenocyte population and their overall proliferation in vitro have been determined by a procedure which measures the cumulative kinetics of thymidine incorporation and the kinetics of cell total number in the presence or in the absence of the lectin, as well as in the presence of Con-A plus colcemid. A minor fraction (11%) of the initial splenocytes is recruited into cycle by Con-A, proliferating with similar kinetics to that of leukaemia cells in the same conditions. The great majority of the initial splenocyte population is unaffected by Con-A, decaying exponentially throughout the incubation with the same half-life (28 hr), both in the presence or in the absence of the lectin.     

Establishment and Growth Kinetics of the Mutant Ehrlich-Lettré Ascites Cell Strain Hd33 In Permanent Suspension Culture1,2

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 × 10^-5 of the explanted cells continued to grow in vitro. the resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. the corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. the duration of the G₁ phase was effectively zero. Both PLM data and [³H]/[¹⁴C] thymidine double-labetling measurements revealed an S-phase duration of between 11 and 12 hr. the G₂ phase lasted 3–5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Studies on the Growth in Culture of Plant Cells: XVII. ANALYSIS OF THE CELL CYCLE OF ASYNCHRONOUSLY DIVIDING ACER PSEUDOPLATANVS L. CELLS IN SUSPENSION CULTURE

Three methods of cell cycle analysis, involving the use of tritiatedthymidine, have been applied to asynchronously dividing suspensioncultures of sycamore. Conditions for an effective chase of unlabelledthymidine were established from a study of the kinetics of entryand incorporation of tritiated thymidine into the cells. Thelevels of thymidine used did not affect the rate of cell divisionor the duration of the phases of the cell cycle. The analyses of the cell cycle based upon pulse labelling, continuouslabelling, and a combination of densitometry and autoradiographywere in good agreement and showed that the phases S (mean 7.0h), G2 (mean 8.5 h) and mitosis (mean 3.0 h), were of relativelyconstant duration, whereas G1 was of variable duration. No relation between nuclear DNA content and mitotic-cycle timeor the duration of S-phase could be inferred from the data presented.     

BISACK ANALYSIS OF THE PHYTOHAEMAGGLUTININ-INDUCED PROLIFERATION OF HUMAN PERIPHERAL LYMPHOCYTES

The rate of stimulation as well as subsequent cell cycle duration was examined in phytohaemagglutinin-stimulated human peripheral lymphocytes grown in vitro in the presence of non-inhibitory concentrations of bromodeoxyuridine. After incorporation of this heavy atom analogue of thymidine into replicating cellular DNA, it was possible to identify unequivocally metaphase cells which had replicated for one, two and three or more cell cycles. Utilizing this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, were analysed by a computer simulation model, and the rate of stimulation (4.5% per hr of the remaining unstimulated population) and cell cycle duration (12·3 hr) were determined. The results were compared with those obtained by autoradiography and the possible relationship to the ‘transition probability’ model for cellular proliferation is 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.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.     

THE DISCRETE–TIME KINETIC MODEL ANALYSIS OF DNA CONTENT DISTRIBUTIONS IN EXPERIMENTAL TUMOUR CELLS

A method was developed to analyse and characterize FMF measurements of DNA content distribution, utilizing the discrete time kinetic (DTK) model for cell kinetics analysis. The DTK model determines the time sequence of the cell age distribution during the proliferation of a tumor cell population and simulates the distribution pattern of the DNA content of cells in each age compartment of the cell cycle. The cells in one age compartment are distributed and spread into several compartments of the DNA content distribution to allow for different rates of DNA synthesis and instrument dispersion effects. It is assumed that the DNA content of cells in each age compartment has a Gaussian distribution. Thus, for a given cell age distribution the DNA content distribution depends on two parameters of the cells in each age compartment: the average DNA content and its coefficient of variation. As the DTK model generates the best fit DNA content distribution to the FMF measurement data, it enables one to estimate specific values of these two parameters in each stage of the cell cycle and to determine the fraction of cells in each cycle phase. The method was utilized to fit FMF measurements of DNA content distributions and to analyse their relationship to the cell kinetic parameters, namely cell loss rate, cell cycle times and growth fraction of exponentially growing Chinese hamster ovary cells in vitro and, also, with a wide range of coefficients of variation, of the L1210 ascites tumour during the growth period.     

Establishment and growth kinetics of the mutant Ehrlich-Lettré ascites cell strain HD33 in permanent suspension culture

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 X 10(-5) of the explanted cells continued to grow in vitro. The resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. The corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. The duration of the G1 phase was effectively zero. Both PLM data and [3H]/[14C] thymidine double-labelling measurements revealed an S-phase duration of between 11 and 12 hr. The G2 phase lasted 3-5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

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.     

ANALYSIS OF THE PROLIFERATION KINETICS OF BURKITT'S LYMPHOMA CELLS

The in vitro proliferation kinetics of a cell line derived from a patient with American Burkitt's lymphoma were investigated at three different growth phases: lag (day 1), exponential (day 3) and plateau (day 5). The growth curve, labeling and mitotic indices, percentage labeled mitosis (PLM) curves and DNA content distributions were determined. The data obtained have been analysed by the previously developed discrete-time kinetic (DTK) model by which a time course of DNA distributions during a 10-day growth period was characterized in terms of other cell kinetic parameters. The mean cell cycle times, initially estimated from PLM curves on days 1, 3 and 5, were further analysed by the DTK model of DNA distributions and subsequently the mean cell cycle times with respect to DNA distributions during the entire growth period were determined. The doubling times were 39·6, 31·2 and 67·2 hr, respectively, at days 1, 3 and 5. The mean cell cycle time increased from 23·0 to 37·7 hr from day 3 to day 5 mainly due to an elongation of the G₁ and G₂ phases. A slight increase in the cell loss rate from 0·0077 to 0·0081 fraction/hr was accompanied by a decrease in the cell production rate from 0·0299 to 0·0184 fraction/hr. This calculated cell loss rate correlated significantly with the number of dead cells determined by trypan blue exclusion. Analysis of the number of dead cells in relation to the cell cycle stage revealed that a majority of cell death occurred in G₁ (r= 0·908; P < 0·0001). There was a good correlation between the in vitro proliferation kinetics at plateau phase of this Burkitt's lymphoma derived cell line and the in vivo proliferation kinetics of African Burkitt's lymphoma (Iversen et al., 1974), suggesting the potential utility of information obtained by in vitro kinetic studies.     

Chromosomes and DNA-replication of rat kangaroo cells (PtK2)

The karyotype, chromosomal measurements, and the time course of DNA replication during the S-phase were determined in metaphase chromosomes of non-synchronized monolayer cultures of PtK₂ cells (CCL 56) derived from Potorous tridactylis. The karotype was the same as originally determined for this cell line. Chromosomal measurements differed from data for primary bone marrow cells of this species published by Shaw and Krooth. PtK₂ cells and chromosomes showed maximal incorporation of tritiated thymidine (³H-TdR) halfway through the S-phase. Chromosome Y₁ showed a second peak of ³H-TdR-incorporation at the end of the S-phase in addition to the peak halfway through S. Comparison of grain densities for chromosomal arms showed late replication of the short arms of chromosomes 1, 3, and X. The time course of incorporation of ³H-TdR was changed when cells were treated for 1 h with fluorodeoxyuridine (FUdR) prior to the ³H-TdR-pulse. FUdR-treated cells showed maximum incorporation of ³H-TdR immediately after the beginning of the S-phase, which was followed by a second peak halfway through the S-phase. This indicated that ³H-TdR-incorporation was partially synchronized by treatment of cells with FUdR. Total radioactivity of FUdR-treated cells had increased by 77% in comparison to cells not treated with FUdR, which indicates that approximately 44% of the TdR-precursors of the latter cells may have originated from cellular precursor pools.     

In vivo bromodeoxyuridine incorporation in human gastric cancer: A study on formalin-fixed and paraffin-embedded sections

Summary Bromodeoxyuridine (BUDR) is a non-radioactive thymidine analogue which is incorporated into the DNA of proliferating cells. This allows evaluation of the size of the S-phase as the BUDR labelling index (BUDR-LI) not onlyin vitro but alsoin vivo, since BUDR is not toxic at the doses needed to label cells. To ascertain whetherin vivo BUDR incorporation can be detected on routine histological material we tested several different procedures prior to immunoperoxidase staining, on formalin-fixed, paraffin-embedded sections from five patients with gastric cancer, who received BUDR (250 mg m^–2, intravenous) 4 h before surgery. To determine the optimal conditions for detecting BUDR in formalin-fixed tissues, immunohistochemical testing for BUDR was performed simultaneously on duplicate sections fixed with 70% ethanol. It was found that hydrolysis with 3N HCl at 37° C for 30 min and digestion with 0.5% in at 37° C for 30 min were sufficient to detect BUDR immunoreactivity in formalin-fixed sections.The method presented extends the range of applications of thein vivo BUDR technique for cell kinetics studies in human neoplasms because it can be used on routinely fixed archival material, with the advantage of correlating the kinetic data with histopathological characters.     

KINETICS OF CELL PROLIFERATION IN THE PRE-IMPLANTED MOUSE EMBRYO IN VIVO AND IN VITRO

The cell proliferation of pre-implanted mouse embryos was investigated after development in vivo and in vitro. The studies were started at the pronuclear stage, 2 h post conception (p.c.) and continued until the hatching of blastocysts, 120–144 h p.c. The number of cell nuclei, the DNA content of each nucleus, the mitotic index and the labelling index were determined. From these data it was possible to calculate the length of the cell generation cycle and its various phases. With the exception of the first cell cycle the S-phase was constant. The G₁- as well as the G₂-phase varied in length during the different cell cycles. From 31–72 h p.c. the increase in cell number was exponential. After cultivation in vitro this increase was smaller than in vivo. At later periods the proliferation rate decreased with proceeding development. In late blastocysts most of the cells were in the G₁-phase. The development of the embryos was somewhat faster in vivo than in vitro. But in principle conditions were comparable.     

Rates of incorporation of radioactive molecules during the cell cycle

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (³H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) ³H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) ³H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing ³H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of ³H-TdR and ³⁵S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, ³HH-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside ³H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of ³⁵S-methionine increased continuously with cell size and DNA content. Incorporation of ³H-TdR in CHO cells was proportional to DNA synthesis.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

COMPARATIVE PROLIFERATIVE KINETICS OF CELLS FROM NORMAL HUMAN EPIDERMIS AND BENIGN EPIDERMAL HYPERPLASIA (PSORIASIS) IN VITRO

Proliferation kinetics of epidermal cells from normal human skin and lesions of psoriasis (benign epidermal hyperplasia) were studied in vitro. Epithelial out-growths were obtained from skin explants and the cell cycle studied using the conventional method of following two successive curves of labeled mitoses after an initial pulse with ³H thymidine. The length of T_c was 59 hr and 53.5 hr respectively for normal and psoriatic cells. The shorter T_c for psoriasis was due to a shorter duration of S. The growth fraction was 66% and 74% for normal and psoriatic cells respectively as determined by continuous labeling with ³H thymidine. Under the conditions of the present experiments, therefore, normal and psoriatic epidermal cells showed no significant difference in proliferative capacity.     

THE EFFECT OF POLYCYTHAEMIC SERUM ON THE PROLIFERATION OF RAT BONE MARROW CELLS IN VITRO

The effect of experimental polycythaemia on the rate of proliferation of erythrocytic precursor cells was investigated by means of an in vitro technique. The serum obtained from polycythaemic rats was found to inhibit significantly ³H-thymidine incorporation in normal rat bone marrow cells in vitro, as compared with normal serum. Autoradiographic analysis revealed that this inhibition resulted from a reduction in the number of labelled bone marrow cells. The inhibition proved to be specific to the erythrocyte precursor cells; the labelling index was reduced in the erythrocytic cell population by 21–50% (P < 0.001) at different incubation times, while the effect on the granulocytic cell population was negligible. It is deduced that an inhibitor substance responsible for the effects observed is present in polycythaemic serum. It is proposed that this factor is the ‘erythrocytic chalone'. The results support the general view that triggering of stem cells is not the only mode of regulation of erythropoiesis, but that the rate of proliferation of the precursor cells in the erythron is also regulated.     

THE EFFECT OF CYTOSINE ARABINOSIDE IN VITRO ON AGAR COLONY FORMING CELLS AND SPLEEN COLONY FORMING CELLS OF C57BL MOUSE BONE MARROW

This study reports the effect of cytosine arabinoside in culture on two classes of bone marrow progenitor cells in C57BL mice, agar colony forming cells (ACU) and spleen colony forming cells (CFU). Both normal cells and rapidly proliferating cells were studied. The results show that in normal mice, 23 % of ACU but only 7 % of CFU are killed following 1 hr incubation with the drug. With longer periods of incubation, the survival of ACU in the controls is poor, and the results for the drug-treated cultures suggest that the cells are held up in cycle. In continuously irradiated mice, the proportion of ACU and CFU killed after 1 hr incubation with drug is increased to 43–54%, confirming previous results that these cells are proliferating more rapidly than in normal mice. In mice treated with myerlan, 54 % of ACU are killed by 1 hr in vitro exposure to cytosine arabinoside, again confirming that ACU are rapidly proliferating. However, the proportion of CFU killed is lower (23 %). These results are compared with other studies of the effect of cytosine arabinoside in vivo and also with thymidine suicide in the same strain of mice. The results show that cytosine arabinoside has the same effect as tritiated thymidine, and also that the proportion of CFU killed by these agents in vitro is lower than when the agents are injected in vivo. It is suggested that the conditions in culture have an adverse effect on CFU, which cease DNA synthesis, and are protected from the killing effect of cytosine arabinoside and tritiated thymidine. Since cytosine arabinoside in vitro has an effect similar to tritiated thymidine in vitro on bone marrow progenitor cells in C57BL mice, in vitro incubation with cytosine arabinoside could be an alternative method to thymidine suicide for measuring differences in cell proliferation rate.     

Growth of rat pancreatic acinar cells quantitated with a monoclonal antibody against the proliferating cell nuclear antigen

The monoclonal antibody PC10 raised against the proliferating cell nuclear antigen (PCNA) was used to study acinar cell replication in the pancreas of rats under different functional conditions. In Western blots, the antibody recognized a single band of 37 kDa in pancreatic homogenates indicating its specificity in this particular species and organ. Three conditions of growth were chosen for immunohistochemical analysis: pancreatic preand postnatal development, pancreatic regeneration after injury, and cholecystokinin-stimulated acinar cell proliferation. The time course of acinar cell replication under each condition was the same as that obtained after tritiated thymidine incorporation with subsequent autoradiography, indicating that the percentage of PCNA-positive cells reflects the pool of cycling cells in the models investigated. However, the absolute number of PCNA-positive cells was two to ten times higher than comparable labeling indices from ³H-thymidine autoradiography. This finding might reflect the half life of PCNA, which exceeds the duration of the S-phase. Thus, PCNA-positive cells not only represent S-phase cells, but also cells that have recently completed the cell cycle.