Duration Of DNA Synthesis Research Articles

In vitro examination of cell proliferation in dermatofibroma and in the overlying epidermis.

In nine typical cases of dermatofibroma autoradiographic methods were used to examine the proliferative activity in the tumor and in the overlying epidermis. The number of DNA-synthesizing cells in the dermal nodular lesions was extremely low (0 -- 0.1%). In seven of nine skin lesions acanthosis was to be seen in the overlying epidermis. The quantity of DNA-synthesizing cells in the epidermis overlying the dermatofibroma was not related to the thickness of the epidermis and remained independent, whether the predominant element in the tumor is fibrillar or cellular. In this study we were able to demonstrate the existence of a direct relation between the degree of epidermal proliferation and the distance between the tumor and the epidermis. The duration of DNA-synthesis was constant.

Replicon properties of chromosomal DNA fibers and the duration of DNA synthesis of sunflower root-tip meristem cells at different temperatures

Chromosomal DNA fiber autoradiography was used to examine the replicon properties of root-tip meristem cells of Helianthus annuus intact seedlings grown at temperatures from 10 to 38° C and those of root-tip cells grown in vitro at 23°. The average replicon size was approximately 22 μm and it did not change with temperature nor when the roots were grown in culture. The average fork rate was 6 μm/h at 10° and it rose gradually to 12 μm/h at 38°. The responses of replication fork movement and of the duration of S to temperature were of three types: those in which change in fork rate was primarily (more than 90%) responsible for change in the duration of S, those in which the fork rate remained constant while S increased nearly twofold, and those in which the duration of S increased even though the replication forks were moving faster. The first type of response listed was observed at temperatures from 20 to 35°, the second type listed was observed at 10 to 15°, and the third, was produced at 38°.

Participation of the intracellular enzymes in the control of the mutation process

The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive (3)H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10-11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication - hydroxyurea - had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.

Determination of Cell Cycle and DNA Synthesis Duration in the Shoot Apex of Chrysanthemum segetum L. by Double-labelling Autoradiographic Techniques

The duration of the cell cycle and the DNA synthetic phase were estimated in various zones of the shoot apex of Chrysanthemum segetum according to two different double labelling techniques. In plants growing in a 16-hour photoperiod on which the sixth leaf had been initiated, the cell cycle duration was about 130 hours in the axial zone, 50 hours in the lateral zone and 70 hours in the rib meristem. The mean duration of the synthetic phase, which was between 7.7 and 10.3 hours according to the zone, did not increase in proportion to a lengthening of the cycle either in the axial zone or in the rib meristem. These findings are discussed in relation to the estimation of the DNA content in various zones as obtained by cytophotometric measurements. They support the hypothesis of a particular lengthening of the duration of the G 1 phase in the axial zone, where the cycle is in fact most prolonged.

Qualitative and Quantitative Studies on DNA and RNA Synthesis in Loxodes striatus Nuclei*

In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei and differentiated morphologically into a clearly recoginzable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a 3H-labeled precursor ([3H]thymidine for DNA and [3H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [3H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [3H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macronucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components synthesis can occur in adult macronuclei. The possible explantion of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.

S phase synchrony in monolayer CHO cultures

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [ 3H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [ 3H]uridine or [ 3H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [ 3H]uridine (but not [ 3H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.

Ileal Crypt Cell Population Kinetics After 40% Small Bowel Resection

Ileal crypt cell population kinetics were investigated by autoradiography with tritiated thymidine in rats 60 days after resection of a jejunal segment equivalent to 40% of the length of the small bowel. Controls were subjected to transection and anastomosis. The mean total number of cells and the mean number of labeled cells per crypt cell column were estimated. The distribution of proliferating cells was obtained by determining the percentage of labeled cells at each cell position in the crypt cell columns. The cell cycle was analyzed by the method of percentage-labeled metaphases. The mean total number of cells was increased from 30.5 to 36.6 (P less than 0.002) and the mean number of labeled cells per crypt cell column was increased from 10.2 to 12.4 (P less than 0.001). Labeling index was unaltered. The mean sizes of the proliferative and maturation zones were increased (P less than 0.002). The estimated median duration of the cell cycle was 11.5 hr in the control group and 10.5 hr in the resection group. The duration of DNA synthesis was 6.4 hr in both groups. It is concluded that 40% small bowel resection increases cell renewal by expanding the size of the proliferative zone of the crypt cell population.

Prolongation of replication time after doublings of the DNA content of polytene chromosome bands of Chironomus.

Using 3H-thymidine autoradiography, labeling frequency of homologous asynapsed chromosome bands of the hybrid of Chironomus th. thummi and Chironomus th. piger has been studied. In a number of these bands the DNA content of the thummi bands if 2, 4, 8 or 16 times as large as that of the homologous piger bands (Keyl, 1965). Those bands of CH. TH. thummi which show one doubling of their DNA content in comparison with the homologous piger bands are also labeled two times more frequently than piger. In contrast to this such a correlation between increase of labeling frequency (i.e. prolongation of replication time) and doubling of the DNA content is not observed, when thummi bands have 4, 8 or 16 times more DNA than their homologues in piger. In these cases replication time is also prolonged after each doubling. Duration of DNA synthesis increases linearly but always by a smaller factor as the corresponding DNA content is increased.

Cell population kinetics in the mouse jejunal crypt.

The time parameters of the cell cycle were determined in the jejunal crypts of male Balb/c mice by an FLM experiment. The cell cycle time was 12.42 +/- 0.11 h, and the duration of DNA synthesis was 7.61 +/- 0.99 (mean values +/- S.E.). A stathmokinetic technique using vincristine gave a value of Tc of 11.8 h. The growth fraction (Ip) calculated from cycle parameters and the labelling index was 0.61, while a value of Ip estimated from a labelling index distribution curve was 0.65. For the whole crypt a value of 0.86 h was obtained for the duration of mitosis, longer than that in the rat. The mitotic duration was found to vary with cell position, but values of between 0.8 and 1.2 h prevailed throughout the proliferative compartment; values in the basal cell positions appeared shorter. Apparent cell cycle times were longest in the basal cell positions. A value for crypt migration rate calculated from a cumulative birth rate curve was 1.48 cell positions per h, compared with a value of 1.8 +/- 0.27 cell positions per h as measured from movement of the 50% peak value on the labelling index distribution curve with time after tritiated thymidine. The crypt cell production rate was calculated from microdissected and squashed crypts to be about 14 cells per crypt per h. There was a total crypt population of 282 +/- 65 (S.E.) cells, of which 172 were proliferative.

Cell kinetics of urethane induced murine pulmonary adenomata: I. The growth rate.

A single injection of urethane into adult male A2G mice produced an increase in the proliferative rate of alveolar wall cells, reaching a peak at 2 weeks post urethan (PU) and declining to control levels by 2 months PU. During this urethane induced proliferative response the single and double labelling indices and the native metaphase index were all elevated although there was no corresponding alteration in the arrested metaphase index. This proliferative response may not be restricted to hyperplasia of potentially neoplastic cells, such as type II epithelium, but may also include type I epithelial cells and alveolar macrophage precursors. However, it was impossible to identify individual cell populations by methods used. The growth rate of adenomata decrease with time and cell kinetic techniques showed that the rates of entry of adenoma cells into DNA synthesis and into metaphase were decreasing concurrently with the growth rate. Thus the rate of cell production falls as adenomata age but how much cell loss contributes to the decrease in growth rate is not yet known. Decreasing cell production could be due to an increased cell cycle time and/or a decreased growth fraction. The duration of DNA synthesis in adenomata increased markedly as the mice survived, suggesting that the cell cycle time might be increased, but further experiments are required to determine whether the growth fraction changes. Attention is drawn to a complication that Colcemid introduces into kinetic studies on alveolar wall cells.

Autoradiographic analysis of hormone-independent development of the mouse vaginal epithelium in organ culture.

Epithelial development was studied in organ cultures of vaginas from ovariectomized mice. In defined medium without estrogen, basal cell proliferation and formation of a stratified squamous epithelium occurred spontaneously in a high percentage of cases. During this “spontaneous” development, the length of the cell cycle and duration of DNA synthesis were the same as those occurring in vivo in response to estrogen. RNA synthesis was also stimulated. Thus, spontaneous development in vitro appears to resemble estrogen-induced development in vivo both qualitatively and quantitatively. In vitro, estrogen was found to have no detectable effect on the atrophic epithelium. In addition, estrogen was unable to halt degeneration of the fully developed epithelium on explants that had been estrogen-primed in vivo. The results suggest that both hormone-dependence and ability of hormones to induce a response are dependent on cell environmental factors.

Changes in cell proliferation rate in mouse uterine epithelium during continuous oestrogen treatment.

SUMMARY Fraction of labelled mitoses (FLM) curves were constructed for mouse uterine luminal epithelium during oestradiol treatment; on day 2 when mitosis was high, and on days 4 and 9 when mitosis was low. No difference was found between the duration of DNA synthesis on these 3 days. The distance between the first and second peaks, usually taken as an estimate of the mean cell cycle time, did not change significantly between days 2 and 4, although the labelling index fell from 38 to 8%. The second peaks of the FLM curves became progressively lower on the three days examined, which was consistent with the interpretation that there was a reduction in the probability of transition of cells from G1 (the post-mitotic period) into the replicative phase of the cell cycle, resulting in the observed fall in mitotic index.

On the determination of intestinal epithelial cell generation time from labeling index and DNA synthesis duration.

Dans l'epithelium de la souris, la duree des phases de generation fut determinee theoriquement par la relation Tg=Ts/LI. Les resultats, sans tenir compte des cellules caliciformes, furent compares aux donnees obtenues par les courbes PLM.

Duration of DNA synthesis in granulocytes during acute hyperplasia

<h2>Summary</h2> Acute granulocytic hyperplasia was produced in mice by injection of endotoxin and, using the labelled mitosis method, the duration of DNA synthesis was compared to that in mormal mice. No difference was found. Taken in conjunction with previous studies using labelling indices, the results indicate that in mice showing acute granulocytic hyperplasia, there is only a moderate increase in proliferation of mitotable granulocytes. Thus the major factor responsible for hyperplasia is increased differentiation of precursor cells.

An evaluation of various double labelling and autoradiographic techniques for the measurement of DNA synthesis time in leukaemic cells*

SUMMARYBefore applying the double labelling technique to the measurement of DNA synthesis time in leukaemic cells, various methods of labelling and of autoradiography were compared.The use of low and high doses of 3H‐thymidine proved to be impracticable because, owing to a wide range of uptake per cell, it was impossible to make a clear distinction between ‘lightly’ and ‘heavily’ labelled cells. Two consecutive doses of 3H‐thymidine resulted in only a small increase in labelling index because of the low percentage of cells in the S‐phase and the long duration of DNA synthesis. Consequently, it was not possible to establish the DNA synthesis time with any degree of accuracy by this method.The remaining method employs 3H‐thymidine and 14C‐thymidine. Auto‐radiographs made with a single emulsion proved difficult to interpret since some 14C‐labelled cells give the appearance of being labelled with 3H‐thymidine only. A double emulsion autoradiographic technique is to be preferred, but even then, more than one interval between the two isotopes is necessary in order to eliminate an error in the recognition of 14C‐labelled cells.

Androgen-induced cell proliferation and differentiation in the seminal vesicle and coagulating gland of the castrated mouse.

SUMMARY Castrated male mice were injected with 250 μg testosterone propionate on 3 consecutive days. With the third injection, 1 μCi tritiated thymidine/g body weight was given intraperitoneally. A pulse-chase analysis in the seminal vesicle and coagulating gland was performed during the next 50 h and labelling and mitotic indices were obtained. One hour after the injection of tritiated thymidine the labelling index was 19% in the seminal vesicle and 13% in the coagulating gland. The mitotic index was 1·7% in the seminal vesicle and 1·4% in the coagulating gland. The observed cell cycle (Tc) duration in the seminal vesicle was 25 h, and the duration of DNA synthesis (ts) was 9 h. The calculated duration of mitosis (tm) was 0·6 h, the presynthetic duration (tG1) was 14·2 h, and the postsynthetic duration (tG2) 1·2 h. Forty-eight hours after testosterone propionate the growth fraction was 0·62 and a decycling ratio of 0·8 was calculated. In the coagulating gland Tc was 52·0 h, ts 9·0 h, tm 1·0 h, tg1 41·0 h and tG2 1·0 h.

DNA synthesis in heterokaryons formed by fusion of mammalian cells from different species

Initiation and duration of DNA synthesis was studied in heterokaryons produced by virus-mediated fusion of cultured mammalian cells from different species. Synchronized HeLa cells were fused with synchronized Chinese hamster or mouse lines (whose phase times differed from those of HeLa cells), and DNA synthesis was studied in the nuclei of the heterokaryons. Initiation of DNA synthesis occurred apparently synchronously in the nuclei of HeLa-hamster and HeLa-mouse heterokaryons, and at a time corresponding to the shorter G1 period of the mouse or hamster parent, several hours before mononucleate HeLa cells entered the S phase. The duration of DNA synthesis, however, was found to be unchanged by association of nuclei having S periods of different lengths. Thus there is evidence for the operation of cytoplasmic factors in the control of initiation of DNA synthesis, but not in the regulation of the rate of chromosome replication.

Cell cycle analysis and stimulation of rat kangaroo cells (PtK 2 ) after pulse labeling.

The phases of the cell cycle of Potorous tridactylus (PtK2) cells were determined in vitro by analysis of labeled mitoses for 37-½ hours after a tritiated thymidine pulse. The mean duration of DNA synthesis (tS) was 8 h. The mean duration of the gap phase before appearance of labeled mitoses was 5 h. Whereas the duration of the cell cycle (tC) based on analysis of labeled mitoses was 30 h, the doubling time (tD) derived from cell counts in the same cultures was only about 23 h. The analysis of the indices of labeled nuclei and mitoses suggests a stimulation of cells at the time of pulse labeling, which was maximal after beginning of the gap phase before DNA-synthesis, and possibly caused the observed difference between tC and tD.

Duration of premeiotic deoxyribonucleic acid synthesis and the stages of prophase I in rabbit oocytes.

To estimate the duration of oocyte DNA synthesis 36, 3-day-old female rabbits received 3, 6, 9, 12, 15, or 18 injections of tritiated thymidine (thy-(3)H) at hourly intervals. The ovaries, removed at 1, 10, or 20 days after the first injection, were radioautographed. Counts made of the number of silver grains associated with oocyte nuclei in meiotic Prophase I indicate that the duration of DNA synthesis is between 9 and 12 hr. To determine the length of the stages of meiotic Prophase I, a group of 2-3-day-old rabbits was given a single sub-cutaneous injection of thy-(3)H, and the ovaries were removed at hourly and/or daily intervals after treatment. The minimum duration of leptotene was 3 hr and the maximum duration probably was less than 8 hr. The maximum durations of zygotene, pachytene, and diplotene were estimated to be 44, 216, and 96 hr, respectively. The interval from the end of oogonial DNA synthesis to the beginning ofpremeiotic DNA synthesis (G(2) + Mitosis + G(1)) appeared to be less than 6 hr.

Effect of somatotropic hormone on duration of DNA synthesis during the mitotic cycle

Effect of somatotropic hormone on duration of DNA synthesis during the mitotic cycle