Study of cell death in Friend leukaemia.

Cell death of splenic Friend leukaemic cells has been studied in vivo, using 125I-UdR and 3H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of 3H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8% of the splenic cellular DNA being renewed per day. Those results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

Thymidine metabolism and the monitoring of DNA synthesis in insects

The thymidine degradation pathway established for other organisms is confirmed in insects. When ³H-TdR is used as a marker for DNA synthesis in developing silkmoths, some is incorporated into DNA and some degraded to compounds not incorporated into DNA. After a single injection, ³H-TdR is rapidly cleared from haemolymph and other tissue, resulting in, at most, a 4 hr pulse. In wing tissue, detection of DNA synthesis is possible for a maximum of 4 hr after injection of precursor and for 6 hr in vitro. Continuous monitoring of DNA synthesis can be attained by perfusion, which maintains high levels of circulating ³H-TdR.     

CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE:

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse ³H-TdR, and (2) nigrosin-dye exclusion combined with ³H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after ³H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of ³H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated ³H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration.     

RECYCLING OF RESTING CELLS IN THE JB-1 ASCITES TUMOUR AFTER TREATMENT WITH 1-β-D-ARABINOFURANOSYLCYTOSINE (Ara-C)

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of growth of the murine JB-1 ascites tumour (i.e. 10 days after 2–5 × 10⁶ cells i.p.) large fractions of non-cycling cells with G₁ and G₂ DNA content (Q₁ and Q₂ cells) are present, and the fate of these resting cells was investigated after treatment with l-β-d-arabinofuranosylcytosine (Ara-C). The experimental work consisted of growth curves, percentage of labelled mitoses curves after continuous labelling with ³H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with ³H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6–8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with ³H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with ³H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q_t cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. These results indicate recycling of resting cells first with G₂ and later with G_x DNA content, which contribute to the regrowth of the tumours.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

PERIODONTAL LIGAMENT CELL KINETICS FOLLOWING ORTHODONTIC TOOTH MOVEMENT

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (³H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with ³H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. ³H-TdR-Iabeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bone-forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G₂ block by the orthodontic stimulation.     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

Cell Proliferation In Emt6 Tumors Treated With Single Doses of X-Rays Or Hydroxyurea. I. Experimental Results

EMT6 mouse mammary tumors were treated in vivo with 5 mg/mouse of hydroxyurea (HU) or 300 rads of X-rays. the proliferation of the tumor cells was followed for 28 hr after treatment. Changes in the ³H-TdR labeling index, the mitotic index, the specific activity of the ³H-TdR-labeled DNA, and the proportion of suspended, clonogenic cells in the S phase of the cell cycle were examined and compared. Evidence was found for reassortment of the surviving cells in treated tumors into partially synchronous cohorts. the partial synchrony in the proliferation of the surviving cells was not accurately predicted by the changes in the labeling index and the mitotic index. the changes in DNA specific activity proved unacceptable as an indicator of cell proliferation in solid EMT6 tumors treated with low doses of radiation or HU.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

Growth Fraction and Cycle Duration of Hepatocytes In the Three-Week-Old Rat

The proliferation of hepatocytes in the liver of 3-week-old rats has been investigated by autoradiographic methods. This investigation is a continuation of earlier work on the same topic (Schultze & Maurer, 1972; 1973). 21 days after birth, 102 rats received a single injection of ³H-TdR. the percentage of labelled mitoses was then determined 1 hr later and at various times throughout the interval up to 12 days after application of ³H-TdR. In agreement with earlier work, a first peak of labelled mitoses was found 7 hr after ³H-TdR injection. the area under the peak indicates an S phase duration of 8 hr. In addition a second very broad peak of labelled mitoses was found between 2 and 12 days after pulse labelling. the analysis of the results leads to the conclusion that the hepatocytes of the 3-week-old rat have a growth fraction close to 1 and a doubling time of 6–7 days. This is at variance with earlier results of Post, Huang & Hoffman (1963) and Grisham (1969) who had derived a value of 21.5 hr for the duration of the cell cycle and a value of only 0. 1–0.2 for the growth fraction of the hepatocytes.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

A TECHNIQUE FOR DETERMINING THE PROPORTION OF THE CLONOGENIC CELLS IN S PHASE IN EMT6 CELL CULTURES AND TUMORS

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (³H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by ³H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of ³H-TdR. Under these conditions, either ³H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or ³H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by ³H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogenic tumor cells in S phase. Incubation of tumor cell suspensions with ³H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of ³H-TdR during the incubation for colony formation.     

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.     

Transforming growth factor-β, osteogenin,and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues—processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-β) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-β and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-β for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by ³H-thymidine (³H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of ³H-TdR at doses of 50–800 ng/ml. Similarly, TGF-β inhibited uptake of ³H-TdR at doses of 2–32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 μg/ml) or higher dose of TGF-β (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-β on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-β (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-β on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-β at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC₅₀) calculated for BMP-2 and TGF-β at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-β. The observation may suggest that TGF-β may have effects upon cytoskeletal elements in osseous tissues. © 1996 Wiley-Liss, Inc.     

CLEARANCE RATE OF EXOGENOUS 3H-THYMIDINE FROM THE PLASMA OF PREGNANT RHESUS MONKEYS

The plasma clearance rate of ³H-TdR was determined by thin-layer chromatography of samples from pregnant rhesus monkeys who had received a single intravenous injection of ³H-TdR. At 10 min after the injection the circulating levels of ³H-TdR had fallen to less than 0.2% of the initial theoretical distribution of the label. In general the plasma clearance rate can be described by a ‘double exponential’curve with half-lives of 1 min and 20 min. Thus, ³H-TdR disappears from the circulation of the pregnant monkey more rapidly than it does from the bloodstream of rodents, which correlates with previously detected interspecies differences in autoradiographic labeling of neurons. This means (1) that higher doses of ³H-TdR must be used for autoradiographic labeling of monkey tissues and (2) that the monkey may be useful for studying cell kinetics because it has a short pulse labeling of the DNA.