Cell-Cycle-Dependent and -Independent Damage to Rat Haemopoiesis By Hydroxyurea

The generally accepted cell-killing effect of hydroxyurea (HU) on S-phase cells, as well as its potential to arrest cells at the G₁/S boundary, hardly explain its benefit for application in human chronic myelogenous leukaemia. Studies were therefore performed in rat haemopoiesis in order to quantify the cell-killing effect on various phases of the cell cycle. For this purpose, the [³H]thymidine ([³H]TdR) labelling index and the specific activity of [³H]TdR in the DNA-synthesizing fraction of cells were determined after a non-cytoreductive dose of 25 mg/kg HU, as well as a medium cytoreductive dose of 100 mg/kg. Furthermore, flow cytometric DNA histograms and absolute as well as differential cell counts of femoral bone marrow were performed after 100 mg/kg HU. The results indicate a predominant cell kill in G₁ encompassing almost all 2c cells in the proliferative pool, while the S-phase fraction is not even reduced to half its initial value. the specific activity of [³H]TdR in cells synthesizing DNA, as well as the labelling index after HU show an initial dip and a tendency to recovery, as has been observed in many other cell systems. Instead of a complete restoration, however, there is a second depression of these parameters lasting for at least one cell cycle. the results are interpreted as a partly cell-cycle-dependent and partly independent action of HU in this cell system. the independent component may be attributed to the repeatedly described direct interference of HU with DNA. In rat haemopoiesis, therefore, this direct effect of HU on the DNA strands appears to be much more pronounced than in cell-culture systems and other mammalian tissues. In view of these findings, some caution should be taken in using HU for the determination of the S-phase fraction by way of a suicide experiment.     

EFFECT OF TRITIATED THYMIDINE ON THE KINETICS AND VIABILITY OF 9L CELLS IN VITRO

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 μCi/ml of [³H]TdR. However, when cells were treated with 1 or 10 μCi/ml of [³H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 μCi/ml of [³H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 μCi/ml of [³H]TdR for 72 hr, less survival was observed. the additional cell kill observed may be induced by [³H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.     

CELL POPULATION KINETIC PROFILE OF THE MOUSE THYMUS, AND THE CHANGES INDUCED BY PREDNISOLONE

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [³H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G₁. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [³H]TdR techniques.     

Endogenous Regulation of Endothelial Cell Proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [³H]TdR incorporation as well as their proliferation. the inhibition is dose- and time-dependent; maximum inhibition of [³H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 μg/ml and reaches a maximum at 600 μg/ml. the blockage of [³H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 μg of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)     

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.     

Detection of bacterial populations in oyster tissue homogenates using direct microscopic counts and thymidine incorporation

Acridine orange direct counts and incorporation of [³H]thymidine ([³H]TdR) were used to determine the effectiveness of an antibiotic treatment on reducing bacterial activity in oyster tissue. Cell counts, as well as total [³H]TdR incorporation into the acid insoluble pool, were significantly lower in antibiotically treated oyster tissue homogenates than in untreated controls. However, rates of [³H]TdR incorporation were not significantly different between treatments, indicating increased metabolic activity (on a per cell basis) in the antibiotically treated bacterial population versus the control population.     

Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation

Populations of G1 phase 3T3 and SV40 3T3 mouse fibroblasts have been isolated from exponentially growing cultures by the technique of centrifugal elutriation. Return of the G1 phase cells to growth conditions results in their synchronous passage through the cell cycle, as determined from monitoring of cell number, [³H]thymidine ([³H]TdR) incorporation and fraction of [³H]TdR labeled nuclei. The durations of G1, S and G2 phases are consistent with values obtained by previous investigators using conventional induction techniques for synchronization. The method for isolation of the G1 phase cells is rapid, the yield is high and the process does not appear to alter the temporal aspects of the cell cycle in either cell type.     

THE CELL POPULATION KINETICS AND RESPONSE TO AN S-PHASE SPECIFIC AGENT OF THREE TRANSPLANTABLE COLON TUMOR LINES

The relative cell population kinetics of three transplantable murine colon tumor lines (Colon 26, 36 and 38) with different histological and metastatic characteristics were studied in relation to the response of each line to an S-phase specific agent. The mean doubling times for the three lines between 0·1 and 1·0 g are similar (4·2 days) but marked differences are apparent in times to tumor appearance (0·1 g) and in median days to death. The length of the cell cycle is about one day and the length of the S-phase 10–11 hr for Colon 36 and 38. The length of the cell cycle in Colon 26 is difficult to estimate by conventional methods but probably exceeds 24 hr and the S-phase is 10–11 hr; [³H]TdR pulse labeling indices for Colon 36 and 38 decrease with time and tumor size from about 0·45 in 0·1 to 0·2 g tumors to about 0·33 at 3 g. The decrease in the [³H]TdR labeling index for Colon 26 is more pronounced (from about 0·38 at 0·1 g to 0·21 at 1·0 g). The shapes of the PLM curves and the [³H]TdR labeling index data are consistent with the observed sensitivity to an S-phase specific agent (Palmo-AraC, NSC 135962) in Colon 36 and the minimal response observed in Colon 26. Colon 38 is intermediate between Colon 36 and Colon 26 in kinetic properties and in response to the S-phase agent.     

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.     

Different Epidermal Cell Kinetic Effects of Hydroxyurea When Injected At Two Different Times of the Day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods—[³H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)—were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. the proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. the observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G₂ and in the mitotic rate. Hence the changes in G₂ and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Different Sensitivities of Granulocyte-Macrophage and Erythroid Progenitor Cells of Normal Human Bone Marrow to S-Phase-Specific Agents

The extraordinary sensitivity of early erythroid progenitor cells (BFU-e) of normal human bone marrow to tritiated thymidine ([³H]TdR) was studied. While exposure of bone-marrow cells to [³H]TdR for 1 hr resulted in the death of only 40% of the granulocyte-macrophage progenitor cells (CFU-c), 90% of BFU-e were killed. Experiments in which normal bone-marrow cells were mixed with bone-marrow cells which had been exposed to [³H]TdR demonstrated that the excessive killing of BFU-e by [³H]TdR reflected carry-over of the [³H]TdR by the exposed cells. A carry-over effect was not observed for CFU-c, suggesting the presence of a fundamental difference in the metabolism of TdR between CFU-c and BFU-e. There was a suggestion of a carry-over effect regarding two other S-phase-specific agents, hydroxyurea and 1-β-D-arabinofuranosylcytosine.     

Endogenous regulation of endothelial cell proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

Preparation, characterization, and functions of rabbit lymph node populations. III. Antigen-induced uptake of thymidine and dibutyryl cyclic AMP: inhibition of thymidine uptake by cholera toxin and dibutyryl cyclic AMP.

Keyhole limpet hemocyanin (KLH)-primed lymph node cell (LNC) populations were incubated with various amounts of KLH and the cellular incorporation of tritiated thymidine ([³H]TdR) or tritiated N⁶, O^2′ dibutyryl cyclic AMP ([³H]DbcAMP) was determined. T LNC responded more vigorously than did complement receptor lymphocytes (CRL), i.e., B cells, at all KLH concentrations, during all time intervals examined, and in the presence or absence of normal rabbit serum (NRS). The depletion of adherent cells from KLH-primed LNC resulted in no significant decrease in KLH-induced incorporation of either [³H]TdR or [³H]DbcAMP in any of the LNC populations. Thus it appeared that variation among LNC populations in the incidence of macrophages did not account for the marked variation in their responses. Cultures containing equal numbers of T and CRL were induced to incorporate more [³H]TdR or [³H]DbcAMP than either population cultured separately or the sum of their individual responses. It was concluded that KLH-induced incorporation of these substances into primed, isolated LNC, was primarily manifested in the T-cell population. The synergism seen in cultures containing mixtures of T and CRL suggested that B cells are induced to incorporate [³H]TdR or [³H]DbcAMP in the presence of antigen and T-cell product(s). KLH-induced incorporation of [³H]TdR into KLH-primed LNC was inhibited by cholera enterotoxin (CT) and DbcAMP as previously reported. However, CT or DbcAMP inhibited this incorporation into T LNC to a greater extent than into CRL or unfractionated LNC.     

Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10-⁷M [³H] thymidine (TdR) or 10^?7M [³H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [³H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [³H]BrdU moieties as compared to [³H]TdR. These results suggest a nonrandom incorporation of 10^?7M BrdU into rat cell DNA sequences.     

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.     

The cell kinetics of the adaptation of the human esophagus to organ culture

Summary The adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [³H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [³H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [³H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR=1.44%), but was much lower than predicted by [³H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G₂ and continued to generate cells that remained in premitosis longer than 24 h. These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro. Supported in part by Contract NOI-CP-75909 and NOI-CP-25604-59 from the National Cancer Institute, Bethesda, MD.     

Repair replication and unscheduled DNA synthesis in mammalian cell lines showing differential sensitivity to alkylating agents

Repair replication has been measured by CsCl density gradient centrifugation in cell lines showing differential sensitivity to mono- and bifunctional alkylating agents. A correlation between cellular sensitivity as measured by the D₀ value and amount of repair replication was demonstrated after exposure of Yoshida cells to nitrogen mustard (HN2) and methylene dimethanesulphonate (MDMS). No differences in the amount of repair replication after methyl methanesulphonate (MMS) were observed in two L5178Y cell lines which differed in sensitivity by virtue of the shoulder size only. The Yoshida cell lines showed no difference in sensitivity to MMS and no difference in amount of repair replication. Incorporation of tritiated thymidine 9[³H]TdR) after drug treament was also measured by autoradiography. The qualitative differences observed between the two cell lines were similar to those obtained in density gradient experiments. The temporal pattern of [³H]TdR uptake indicated that the reduced repair replication observed in the sensitive line after HN2 and MDMS is not due to slower synthesis. The kinetics of [^3H]TdR incorporation differed for all three mutagens suggesting that different enzymes may be involved in each case.     

Reentry into the cell cycle of differentiated skeletal myocytes

The activation of muscle-specific myosin synthesis and its relationship to withdrawal from the cell cycle have been examined in cycle-synchronized myoblasts under growth-restrictive, fusion-impermissive (low Ca²⁺) culture conditions. Under these conditions, embryonic quail skeletal myoblasts, collected in mitosis by mechanical shake-off, complete one normal cycle and arrest in G₁. The presence of skeletal muscle myosin is first detected, by indirect immunofluorescence, 8 hr into this protracted G₁. Within the next 10–11 hr the percentage myosin positive (Myo+) cells increases with good synchrony, reaching approximately 95%. Refeeding with a proliferation-stimulating, low Ca²⁺ medium when approximately 50% of the cells are Myo+ induces reentry into S. Applying a 15-min pulse with [³H]TdR immediately preceding fixation at regular intervals following refeeding, cells can be detected which are Myo+ and whose nuclei have incorporated [³H]TdR. The numbers of such doubly labeled cells are small but consistent with the fraction of cells in S (by time-lapse analysis) at the postfeeding times sampled. These cinematographic studies also indicate that progression to mitosis following stimulation occurs slowly and asynchronously. The kinetics of progression of the stimulated cells suggest that they reenter S from a different compartment in G₁ than do log-phase myoblasts. We conclude that in fusion-blocked quail myocytes irreversible withdrawal from the cell cycle is neither an obligate precondition for, nor an immediate consequence of the activation of the muscle-specific contractile gene set.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.