Kinetics of telencephalic neural cell proliferation during the fetal regeneration period following a single X-irradiation at the late organogenesis stage

Summary A sequential study of the acute effects of prenatal X-irradiation on telencephalic cell population dynamics was performed by combining a pathological evaluation with autoradiographic methods. This study confirmed that a dose of 0,95 Gy affects nearly all neuroblasts in the G₁-, G₂-, and M-stage lethally. Within ventricular cells staying in the S-phase at the time of X-irradiation, lethal effects are in the range between 50% and 75%. The surviving remainder of these neuroblasts is responsible for subsequent attempts for regeneration of the telencephalic roof. Two subpopulations of the surviving S-phase cells were observed: The first subpopulation which seems to be restricted to the rudiments of the ventricular zone shows a S/G₂-blockade for 8–12 h after X-irradiation. Thereafter these cells start again with mitotic activity. The second subpopulation is morphologically manifested by the formation of heterotopic cell nests, i.e., so-called rosettes. These cells continue with an intense DNA-replication after the 12th h post irradiation and proceed to the mitosis stage only after about another 4–8 h, i.e., the 16th to 20th hour following X-irradiation. These findings indicate the existence of two different pools of regenerative cells within the telencephalic roof, giving rise to either orthotopic or heterotopic growth processes after irradiation injury.Dedicated to Prof. Dr. H. Kriegel on occasion of his 60th anniversary     

Molecular mechanisms involved in the production of cromosomal aberrations II. Utilization of neurospora endonuclease for the study of aberration production by X-rays in G1 and G2 stages of the cell cycle

Chinese hamster ovary cells (CHO) were X-irradiated in G₁ and G₂ stages of the cell cycle and subsequently Neurospora endonuclease (NE) (E.C.3.1.4), an enzyme which is specific in cleaving single-stranded DNA, was introduced into the cells, after making the cells permeable by treatment with inactivated Sendai virus. With this treatment all classes of X-ray-induced chromatid aberrations increased in G₂ cells, whereas in G₁ cells an increase in cromosome type of aberrations was found, associated with a profound induction of chromatid type of aberrations as well. Duration of the availability of single-strand gaps for the action of NE has been studied in G₂ cells following X-irradiation and the influence of different parts of the G₂ stage on the type and frequencies of chromatid aberrations was discerned. While the increase in chromosome type of aberrations by NE in X-irradiated G₁ cells has been interpreted as due to the conversion of DNA single-strand breaks or gaps to double-strand breaks by NE, the induction of chromatid aberrations in G₁ has been assumed to be due to conversion of some of the damaged bases strand breaks by NE. Biochemical evidence is presented for the conversion by NE of DNA single-strand breaks induced by X-rays into double-strand breaks using neutral sucrose gradient centrifugation.     

Effects of X-irradiation and adriamycin on quiescent and proliferating cells of the seminal vesicle in the castrated mouse

The sensitivity of resting and proliferating cells of the seminal vesicle to X-irradiation and adriamycin has been investigated. Stimulation with testosterone propionate (250 μg/day) was started 11 days after castration in BALB/c mice. X-rays (2.5–7.5 Gy total body irradiation) and intraperitoneal injections of adriamycin (4–16 mg/kg body weight) were administered at various times before or after induction of proliferation by testosterone injection. The DNA contents and the weights of the seminal vesicles were determined at 4 days after the start of stimulation. A D₀ for X-rays of about 10 Gy was found for the seminal vesicle epithelium. For both X-irradiation and adriamycin no significant differences in sensitivity were observed between quiescent (G₀) and proliferating (G₁; S) seminal vesicle cells.     

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

Radiosensitivity of Mammalian Cells: IV. Effects of X-Irradiation on the DNA Synthetic Period in Synchronized Cells

The effect of X-irradiation on the timing of DNA synthesis in the Chinese hamster ovary cells has been investigated. Mitotically synchronized cells irradiated in mitosis or early G₁ exhibited a fixed, dose-independent (150-2000 rad) delay of 1.6 hr in entry into S, while the duration of S was unaffected. Cells irradiated during late G₁ or the first 0.8 hr of S were not affected either in time of initiation or duration of S. However, when cells 0.8 hr or more into S were irradiated, completion but not initiation of DNA synthesis was delayed, indicating a very precise separation of X-ray effects upon initiation and replication. There was no indication of a re-ordering of cells following irradiation and recovery, since cells in G₂ at the time of irradiation always divided before cells irradiated in S. The results suggest that two separate functions required for initiation and continued replication of DNA may be differentially sensitive to X-irradiation.     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours

We analysed the effects of nitrogen mustard (HN₂) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN₂ produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN₂, the host cells in the treated tumours showed a significantly decreased G₀/G₁ peak and an increased G₂/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G₀/G₁ peak coupled with relatively decreased proportions of S and G₂/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN₂ administration (P<0.01). Thirty-two days after treatment with HN₂, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P<0.01). These results show that the administration in vivo of HN₂ can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN₂ treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.     

Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV,LC/MS/MS and ESR

To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron–sulphur (Fe?S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24?h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF_2α and 5-iPF_2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24?h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC)?=?[ATP?+?0.5?×?ADP]/[ATP?+?ADP?+?AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g?=?2.004 and Fe–S cluster at g?=?1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe–S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe–S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe–S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.     

Effects of 1000 R whole-body X-irradiation on DNA synthesis and mitosis in the duodenal crypts of the BCF1 mouse

Summary Effects of 1000 R, whole-body X-irradiation on the proliferative cells of the mouse duodenal crypts, in the four phases of the generation cycle; namely, the DNA synthesis phase, S; the pre-mitotic gap, G ₂; the division phase or mitosis, M; and the pre-synthesis gap, G ₁. As pointed out by Whitmore and Till (1964) G₁ and G₂ are characterized only by the fact that no DNA synthesis is taking place in these phases.In the intestinal crypts of BCF₁ mice, a 1000 R whole-body X-ray exposure blocks cells in G₂ for approximately 18 hours, and reduces the number of cells in S to less than 1/2 that observed in control animals during the first 12 hours after exposure. Cells synthesizing DNA, and undergoing division, remain few in number for more than 48 hours. Between 48 and 72 hours a compensatory reaction begins, and the number of cells in M and S increases from 28 at 48 hours to 150 at 72 hours and reaches a mean value of 482 at 96 hours.Work supported under the auspices of the US Atomic Energy Commission.     

The repair of x-ray-induced chromosome aberrations in stimulated and unstimulated human lymphocytes

The repair time for chromosome breaks induced by X-irradiation of unstimulated (G₀) and stimulated (G₁) human lymphocytes has been determined by dose fractionation studies. In both types of cells repair time was approx. 4–5 h. Treatment with hydroxyurea, a DNA synthesis inhibitor, did not prevent or delay the rejoining of broken chromosomes, whereas treatment with cycloheximide, a potent protein synthesis inhibitor, did. Thus, the repair of radiation-induced chromosome breaks in human lymphocytes is similar to the repair observed with plant cells.     

CELL KILLING BY ACTINOMYCIN D IN RELATION TO THE GROWTH CYCLE OF CHINESE HAMSTER CELLS

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G₂ (or G₂-mitosis) they are maximally resistant; at 1.0 µg/ml, for example, the survival in G₂ is 30-fold greater than it is in G₁. These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.     

Basal-Cell Subpopulations and Cell-Cycle Kinetics In Human Epidermal Explant Cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different ³H-thymidine (³H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that ³H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G₂-phase cells were cycling, whereas some 20% of the cells stayed in G₁-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G₁- and G₂-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G₂-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated ³H-dThd at a higher rate than cells with high RNA contents.     

Combination of photodynamic therapy with aspirin in human-derived lung adenocarcinoma cells affects proteasome activity and induces apoptosis

Objectives: Photodynamic treatment (PDT) of human lung carcinoma cells A549 (p53^+/+) and H1299 (p53^?/?) induces fast but transient stalling of proteasome activity. We have explored the possibility of prolonging this effect by combining PDT with drugs capable of sustaining the stall, and promote apoptosis of surviving cells. We show that aspirin can be used to accomplish this. Materials and methods: Cells were irradiated at doses ranging from 0.54 to 1.10 J cm^?2, and subsequently were incubated with aspirin at either high (10 and 5 mm ) or low concentration (2.5 and 1.5 mm ). Photofrin concentration and incubation time were constant (2.5 μg/ml and 16 h). Under these conditions, we analysed cell viability, colony‐forming efficiency, cycle profile, expression patterns of specific proteins and ubiquitination state, after individual or combined administration. Results: Treatment with either PDT or aspirin, rapidly induced proteasome malfunction and accumulation of cells in G₂M, but did not induce apoptosis. However, when aspirin was added to cells (even at low concentrations) after PDT, the proteasome block was sustained. Moreover, significant cytotoxic effects, including apoptosis, were observed along with cytostatic effects (G₂M accumulation/decreased colony formation). Conclusions: Combination of PDT and low‐toxicity drugs (such as aspirin) resulted in protracted inhibition of proteasome activity and induced apoptosis even in apoptosis‐resistant cancer cells.     

Discrepancy between the initial DNA damage and cell survival after camptothecin treatment in two murine lymphoma L5178Y sublines

Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID₅₀) after 48 h of incubation were 0·54μM for LY-R cells and 1·25 μM for LY-S cells. Initial numbers of DNA–protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5·6 Gray-equivalents) than in LY-S cells (3·1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT.^1,2 Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4·2 Gray-equivalent SSBs and 5·8 Gray equivalent DSBs) than in LY-S cells (8·0 Gray-equivalent SSBs and 12·0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells.³ Release from G₂ block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G₂, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.     

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

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

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G₀/G₁ phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G₂/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G₂, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of “decoy, trap and shoot” chemotherapy.     

P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε

Abstract: Incubation of Neuro 2A mouse neuroblastoma cells with UTP and UDP results in a concentration-dependent increase in the accumulation of inositol phosphates with equal potency and maximal effect; ATP, ADP, and 2-methylthioadenosine 5′-triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of UTP and ATP were not affected by pretreatment of cells with pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to pertussis toxin-insensitive G_q protein. Short-term (10 min) treatment of cells with 1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the inhibition of the UTP and ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5–6 h) and was not seen after long-term (24 h) treatment. Western blot analysis showed the expression of protein kinase C (PKC) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down-regulation of both membrane PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA-induced translocation of PKCε was maintained after 3–6 h of treatment, and almost complete down-regulation occurred only after a 24-h treatment. The observed TPA-induced inhibition of UTP- or ATP-stimulated phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of PKCε. Phosphoinositide hydrolysis induced by AlF₄^?, but not Ca²⁺ ionophores, was inhibited by a 10-min treatment with TPA. This was not seen after a 24-h treatment, indicating that the site of action of PKCε in the P2Y receptor/G_q protein/phospholipase Cβ pathway might be the G_q protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor-mediated phosphoinositide turnover.     

Induction of c-fos and c-jun mRNA at the M/G1 border is required for cell cycle progression

The proto-oncogenes c-fos and c-jun have been shown in numerous model systems to be induced within minutes of growth factor stimulation, during the G₀/G₁ transition. In this report we use the mitotic shake-off procedure to generate a population of highly synchronized Swiss 3T3 cells. We show that both of these immediate-early, competence genes are also induced during the M/G₁ transition, immediately after completion of mitosis. While c-fos mRNA levels drop to undetectable levels within 2 hr after division, c-jun mRNA levels are maintained at a basal level which is ～ 30% maximum throughout the remainder of G₁. In order to access the functional significance of these patterns of c-fos and c-jun expression, antisense oligodeoxynucleotides specific to c-fos or c-jun were added to either actively growing Swiss 3T3 cells or mitotically synchronized cells, and their ability to inhibit DNA synthesis and cell division determined. Our results show that treatment of Swiss 3T3 cells with either c-fos or c-jun antisense oligodeoxynucleotides, while actively growing, during mitosis, or in early G₁, results in a reduction in ability to enter S and subsequently divide. This was also true if Swiss 3T3 cells were treated during mid-G₁ with c-jun antisense oligodeoxynucleotides. These results demonstrate that the regulation of G₁ progression following mitosis is dependent upon the expression and function of the immediate-early, competence proto-oncogenes c-fos and c-jun. © 1994 Wiley-Liss, Inc.     

Analysis of Cell Flow and Cell Loss Following X-Irradiation Using Sequential Investigation of the Total Number of Cells In the Various Parts of the Cell Cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. the generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G₂ blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. the experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.