Tumour Cell Recruitment of the Jb-1 and L 1210 Ascites Tumour Determined Directly By Double Labelling With [14C]- and [3H]-Thymidine

Abstract. Tumour cell recruitment of the JB-1 and L 1210 ascites tumour has been demonstrated directly by a double-labelling method with [¹⁴C]- and [³H]-thymidine (TdR). After [¹⁴C]-labelling of all proliferating tumour cells by multiple injections of [¹⁴C]TdR, recruitment of resting cells was stimulated by removal of the majority of tumour cells, i.e. by maximum aspiration of ascitic fluid. the number of recruited resting cells in the remaining tumour that re-enter the cell cycle after stimulation was demonstrated directly by a single injection of [³H]TdR given at different times after stimulation. the increase in the percentage of purely [³H]-labelled cells, i.e. recruited cells, with increasing time after stimulation, shows that recruitment is not a synchronous but a continuous process, the maximum of which occurs earlier in the case of the L 1210 than the JB-1 tumour. This suggests that there seems to be a relationship between the time required for maximum recruitment and the corresponding cell cycle parameters of the unperturbed tumour. There is a transitory increase of the growth fraction to about 100% and a considerable shortening of the cycle time at the maximum of recruitment.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro

Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [3H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G0/G1, S, and G2/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [3H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [3H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G2 tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Effect of cyclophosphamide on the proliferation of L 1210 ascites tumour cells and of jejunal crypt cells in the mouse

Cyclophosphamide (CY) does not act in a cell-cycle specific manner, i.e. exclusively on proliferating cells. It also kills non-proliferating cells, as shown by application of CY to L 1210 ascites tumour-bearing mice during plateau phase growth of the tumour. Moreover, treatment with CY of L 1210 ascites tumour cells, double-labelled with [3H] and [14C]-thymidine, suggests that CY is not cell cycle phase dependent, but kills cells out of all cycle phases. There is also an extensive cytocidal effect of CY (300 mg/kg) on the jejunal crypt cells of the mouse, which is even more pronounced than that of cisplatinum (DDP, 13 mg/kg). However, rapid regeneration of crypt cells occurs after treatment with the drugs.     

Double labelling with bromodeoxyuridine and [3H]-thymidine of proliferative cells in small intestinal epithelium in steady state and after irradiation

The simultaneous immunohistochemical detection of bromodeoxyuridine (BrdU) and [3H]-thymidine ([3H]TdR), by conventional autoradiography, was performed on the mouse small intestine (ileum). Proliferation was studied under normal conditions as well as after 3 Gy of gamma-rays. The BrdU method in conjunction with [3H]TdR autoradiography appears to be reliable and useful for the study of cell kinetics especially in disturbed states, on condition that [3H]TdR is delivered to the animals before BrdU. It has been found that cells in the crypt are delayed by irradiation in their progression through the cell cycle predominantly in late S phase. The cells at the bottom of the crypt are more affected than the more differentiated but proliferating cells in the upper part of the crypt.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

A Method For Measuring the Generation Time and Length of Dna Synthesizing Phase of Clonogenic Cells In A Heterogenous Population

Information on the cell cycle of progenitor cells in haemopoietic tissue is useful for understanding population control under physiological and abnormal conditions. Unfortunately, methods that have been developed for measuring cell cycle parameters are applicable only to cells of homogenous populations and not to morphologically non-recognizable progenitor cells such as colony forming units (CFU) that are present at low frequency in a heterogenous population. to circumvent this difficulty, a method was developed to measure CFU cell cycle parameters based on specific killing of cells in S phase by [³H]thymidine ([³H]TdR). This was done by estimating the number of CFU killed following exposure of the cell suspension to [³H]TdR for various time periods. Since cycling CFU are continuously entering S phase, a linear curve relating the percentage CFU-kill to the length of exposure of the cells to [³H]TdR in culture can be obtained. the slope of the curve (percentage kill/hr) indicates the rate that CFU enter the S phase and travel through the cell cycle. the inverse of this value will then represent a time period for CFU to move through a complete cell cycle (generation time). the length of S phase can then be obtained by multiplying generation time by the fraction of cells in S phase at time zero. This method has been used to measure generation time and length of S phase of three kinds of haemopoietic progenitor cells: mouse granulocyte-macrophage CFU, human T lymphocyte CFU and CFU from regenerating mouse spleens. This method should be applicable to any normal or neoplastic clonogenic cell populations and the latter could be either of haematological or of solid tumour origin.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

The present experiments with [14C]-thymidine (TdR) and [3H]-bromodeoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 microgram g body weight-1 and that of BrdU is about 5.0 micrograms g body weight-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [3H]-TdR doses commonly used in cell kinetic studies this proportion is only 0.1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [3H]-TdR.     

Continuous [3H] thymidine infusion: a method for the study of follicular dynamics

Cycling rats were given continuous infusions of [3H] thymidine [( 3H] TdR) by means of osmotic minipumps, and autoradiographs of ovaries were prepared. Silver grains were distributed in a diffuse and uniform fashion over the granulosa layer of growing follicles. Nearly 100% of granulosa cells in large healthy follicles were labeled within 24 h. This uniform labeling makes possible detailed cell cycle analysis, as well as other kinetic studies. Follicles which were already atretic before the minipumps were inserted remained unlabeled. Follicles which became atretic after the minipumps were inserted were heavily labeled. Thus, with continuous labeling, it is possible to deduce retrospectively the viability of a particular follicle as it had been at the time the minipumps were inserted. Short-term continuous infusion of [3H] TdR, therefore, provides a valuable temporal component to morphometric studies of the ovary and should be useful for the study of other rapidly growing tissues as well.     

Stimulation by tumor necrosis factor of HL-60 thymidine salvage pathway metabolism dissociated from proliferation

The effect of human tumor necrosis factor (TNF) on early-passage HL-60 cells was studied. A transient phase of increased [3H]thymidine (TdR) incorporation was noted at 20-24 hr of exposure to TNF. This increase was disproportionate to the much slighter stimulation of the percentage of S-phase cells, which was measured by flow cytometry. Evidence for increased metabolic trapping of [3H]TdR following TNF treatment was apparent from whole cell uptake experiments. The salvage pathway enzyme TdR kinase was therefore measured and was found to be elevated comparably to [3H]TdR uptake. The mechanism of TNF regulation of TdR kinase was further investigated by a series of combination treatment experiments using other biologic factors and pharmacologic inhibitors of various intracellular steps. The response to TNF was not potentiated or reproduced by IL-1, IL-2, IL-3, IL-4, G-CSF, M-CSF, GM-CSF or alpha- or gamma-interferon. Blockers of early signal transduction steps, including H7, W7, sphingosine, and pertussis toxin, failed to inhibit TNF stimulation of [3H]TdR incorporation. mRNA synthesis inhibition with alpha-amanitin blocked this TNF effect, as did cAMP but not cGMP analogues. A sensitizing effect was noted with amiloride or cytochalasin B, characterized by greater relative increases of [3H]TdR incorporation and TdR kinase activity in response to TNF. In the presence of cytochalasin B, TNF treatment resulted in no change or slight decreases in the percentage of S-phase cells. Regulation of TdR kinase could thereby be dissociated from the usual cell cycle control. This study thus documents a unique example of stimulation of thymidine salvage pathway metabolism by a biologic factor, dissociable from overall cell cycle regulation.     

Retention of labelled DNA precursor by murine cells after a single administration of tritium labelled thymidine

The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [3H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [3H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [3H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [3H]TdR and consequent light labelling of the cells.     

The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [3H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA-versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.     

Effects of [3H]UdR on the cell-cycle progression of L1210 cells

Tritium-labelled uridine [( 3H]UdR) perturbs progression of L1210 cells through the mitotic cycle. The main effect manifests as a slowdown or arrest of a portion of cells in G2 and is already observed 2 hr after addition of 0.5-5.0 microCi/ml of [3H]UdR into cultures. At 2.5-5.0 microCi/ml of [3H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [3H]UdR for 8-24 hr. A pulse of [3H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G2 slowdown) are observed when the amount of the incorporated [3H]UdR is such that, on average there are fewer than thirty-six [3H] decays per cell which corresponds to approximately 12-19 rads of radiation. The S-phase slowdown is seen at a dose of incorporated [3H]UdR twice as high as that inducing G2 effects. The specific localization of [3H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [3H]UdR and [3H]thymidine. Mathematical modelling of the cell-cycle effects of [3H]UdR is provided.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Induction of splenic granulopoiesis in vitro.

Murine spleen cells were cultured in vitro to study the induction of committed granulopoietic stem cell (CFU-C) proliferation and maturation. Marbrook-type diffusion cultures were established with and without the addition of colony-stimulating activity (CSA) and harvested at intervals up to 14 days for viable and differential cell counts, [3H]TdR autoradiography, and quantitation of CFU-C by the agar plate method. Without CSA there was poor cell viability and little proliferative capacity. In CSA-stimulated cultures there was a prominent rise in viable cell counts and [3H]TdR labeling indices rose from a mean of 2% at 0 time to 47% after 5 days in vitro. CFU-C increased by 70-fold in these cultures. Peak numbers of CFU-C, immature cells, and [3H]TdR-labeled cells occurred at about 7 days. Thereafter, mature granulocytes and macrophages predominated in culture. Because the liquid spleen cell culture system begins in a resting state and undergoes a wave of proliferative activity in response to CSA, it can provide a useful model system for studying phenomena associated with stem cell activation and differentiation in vitro.     

Cell kinetic studies of chick brain cells in culture: an autoradiographic study with [3H]- and [14C]-thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [3H]- and/or [14C]-thymidine. The dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. The labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [3H]- and [14C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. The cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). The results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

AN IN VIVO DOUBLE LABELLING STUDY OF THE SUBSEQUENT FATE OF CELLS ARRESTED IN METAPHASE BY VINCRISTINE IN THE JB-1 MOUSE ASCITES TUMOUR

The fate of cells arrested by Vincristine (VCR) in metaphase is of interest because of the wide use of this substance in cancer chemotherapy and, particularly, in relation to its use in so-called ‘synchronization’ therapy. The present study was designed to answer the question of whether cells blocked in metaphase by VCR subsequently proliferate further or whether they become infertile and die. By means of a double labelling technique with [³H] and [¹⁴C]thymidine (TdR) it was shown that all VCR-arrested metaphases in the JB-1 ascites tumour subsequently became necrotic. These cells did not re-enter a viable G₂ phase following arrest and thus could not take part in a wave of synchronous proliferation. In agreement with earlier studies, VCR was found to lead to arrest in metaphase, not only of cells in or shortly prior to mitosis at the time of VCR administration, but also of the majority of cells which had at this time been in the S and G₂ phase.     

Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.