S-phase Duration Research Articles

Cell-Kinetic Characteristics of Human Brain Tumors

The proliferative potential of human brain tumors was investigated, in vivo, using bromodeoxyuridine (BUDR) incorporation and flow cytometry (FCM). Patients with a variety of human brain tumors were preoperatively injected with 250 mg of BUDR intravenously. The cell cycle parameters of most of the specimen were measured within 24 h of sampling. The results show that this method is very practical, fast, and feasible for determining the cell-kinetic parameters of human brain tumors. In this study important kinetic parameters such as the duration of S-phase and the potential doubling time of each tumor were calculated in some samples. Our results show a significant difference in labelling index between meningiomas and gliomas. No difference between benign and anaplastic meningiomas was demonstrated in this limited number of cases. The correlation between the labelling index and the clinical and radiological follow-up of each patient should make the assessment of the prognostic significance of the kinetic evaluation, possible. This method could be a useful aid in the selection of new treatment schedules.

DNA replication fork progression rate and temporal organization of S phase in normal epidermis and in basal cell carcinoma.

The double-pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S-phase duration in BCC, compared with normal skin. The mean RU size was 76 +/- 4 micron in BCC, not significantly different from the 68 +/- 6 micron value found in normal skin, so the mean of those two values (i.e., 72 micron), was used in further calculations. The rate of replication fork progression was 0.59 +/- 0.005 micron/min in the normal epidermis and 0.33 +/- 0.03 micron/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8-fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8-fold increase in S-phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined "sets". These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S-phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10.

Measurement of cell kinetics in human tumours in vivo using bromodeoxyuridine incorporation and flow cytometry

The proliferative potential of human solid tumours, in vivo, was investigated using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry (FCM). Patients with solid tumours from a variety of sites were injected with 500 mg BrdUrd, intravenously, several hours prior to biopsy or surgical excision. The labelling index (LI), duration of S-phase (Ts) and thus the potential doubling time (Tpot) could be measured within 24 h of sampling. The results show that both the LI and Ts vary greatly between tumours (Ts ranges from 5.8 to 30.7 h). However, within this study of 26 evaluable patients, tumours of the same tissue origin tended to have similar Ts values. Melanomas had the shortest Ts (8.8 h), nine patients with head and neck cancer had Ts values ranging from 5.8 to 18.8 h (median 12.5 h). The longest Ts values (24 h) were found in lung and rectum. The estimates of Tpot ranged from only 3.2 days in an oat cell carcinoma to 23.2 days in a lymphoma. The striking feature of the study was that 38% of the tumours had a potential doubling time of 5 days or less. We found no relationship between proliferation and histopathological differentiation or DNA ploidy. It should now be possible to assess the prognostic significance of pretreatment cell kinetic measurements which may, in the future, aid in the selection of treatment schedules for the individual patient.

A comparison of the rate of DNA synthesis in myeloblasts from peripheral blood and bone marrows of patients with acute nonlymphocytic leukemia

Durations of S-phase ( T s) and total cell cycle times ( T c) were measured from the peripheral blood (PB) and bone marrow aspirates (BM) of five patients with acute nonlym-phocytic leukemia (ANLL). Intravenous bromodeoxyuridine (BrdU) was used as the first label for S-phase cells and a monoclonal anti-BrdU antibody was used to detect the positive cells. Tritiated thymidine ([ 3H]Tdr) was used as a second label in vitro, and the T s was calculated by counting the number of cells labeled either by BrdU or by [ 3H]Tdr or by both. Our data demonstrate that the duration of S-phase in myeloblasts obtained from BM is quite similar to that of circulating leukemic cells. Finally, the most accurate assessment of percentage of myeloblasts actively engaged in DNA synthesis can be obtained only from bone marrow biopsies following in vivo labeling.

Paternal influence on timing of pronuclear DNA synthesis in naturally ovulated and fertilized mouse eggs.

Fertilized oocytes of the inbred genotypes AKR (AK), C57BL/6 (B6), DBA/2 (D2), and CBA (CB) and the hybrid genotypes F1 (female AK X male B6) and F1 (female B6 X male AK) were collected by flushing the oviducts of female mice every 2 h from 2 until 26 h post coitum. Developmental stages of the embryos and DNA content of the pronuclei were estimated by morphological criteria and cytofluorometric measurement of the pronuclei (ethidium bromide-stained DNA), respectively. In all genotypes, S-phase started about 4 h post conception (h.p.c.). The duration of S-phase amounted to 5.9 h (F1 [female B6 X male AK]), 6.4 h (AK), 8.5 h (B6), 9.4 h (F1 [female AK X male B6]), 9.8 h (D2), and 11.4 h (CB). In each of the reciprocal F1 hybrids, the length of S-phase differed from the maternal genotype (p less than 0.01) and resembled closely the paternal genotype (p greater than 0.25). Cleavage from one-cell stage to two-cell stage occurred between 16 and 21 h.p.c.

Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

Differences in Cell Cycle Characteristics Among Patients With Acute Nonlymphocytic Leukemia

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post-BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

A new method for studying cell cycle characteristics in ANLL using double labeling with BrdU and 3HTdr

Ten patients with acute nonlymphocytic leukemia (ANLL) received bromodeoxyuridine (BrdU) at 100 mg/M 2 intravenously over 1 h. BrdU is incorporated into the DNA by S-phase cells and was detected by using a monoclonal anti-BrdU antibody in the bone marrow aspirate (BM) and biopsy specimens obtained at the end of the infusion. Additionally, BM was incubated in vitro with tritiated thymidine ( 3HTdr) and processed by our previously described double-label method. This allowed us to measure the duration of S-phase ( T s ) and total cell cycle time ( T c ) of myeloblasts. Data revealed a higher number of S-phase cells from biopsies (21%) than BM (5%). The T s ranged from 9 to 35 h and T c ranged between 36 and 152 h in different patients. Using this method, data are available within 48 h and if shown to be clinically relevant, may be useful for prospective planning of therapy in individual patients.

Establishment and growth kinetics of the mutant Ehrlich-Lettré ascites cell strain HD33 in permanent suspension culture.

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 X 10(-5) of the explanted cells continued to grow in vitro. The resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. The corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. The duration of the G1 phase was effectively zero. Both PLM data and [3H]/[14C] thymidine double-labelling measurements revealed an S-phase duration of between 11 and 12 hr. The G2 phase lasted 3-5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.

Cell kinetic indicators of premalignant stages of colorectal cancer.

Using an in vitro double labeling technique with two different levels of 3H-thymidine, the duration of the phase of DNA synthesis (S) and the labeling index (LI) were measured in the colorectal mucosa of three groups of patients: patients with colorectal neoplasms (adenomas and/or adenocarcinomas), patients with inflammatory bowel disease, and a control group of patients without gastrointestinal pathology. In those patients with colorectal neoplasms, samples were obtained from both the neoplastic mucosa and from the normal appearing mucosa at various distances from the lesions. One-way analyses of variance were used to test the equality of mean S-phase duration and LI in the various types of tissues. S-phase duration was significantly longer in the tumor than in the unaffected mucosa of patients with adenocarcinoma (18.65 hours +/- 2.3 versus 10.13 hours +/- 1.26 P less than 0.0001). However, S-phase duration was significantly longer in the unaffected mucosa of cancer patients than in the mucosa of patients without gastrointestinal pathology (10.58 hours +/- 1.84 versus 7.91 hours +/- 0.46, P = 0.013). Similarly, LI was significantly higher in the unaffected mucosa of patients with adenoma and adenocarcinoma than in the mucosa of patients without gastrointestinal pathology (19.1% +/- 3.0 versus 9.5% +/- 2.2, P less than 0.0001). There was a highly significant trend to a progressive increase of LI from flat histologically normal appearing mucosas to inflammatory mucosas, adenomas, and adenocarcinomas (P less than 0.0001). These results suggest that increased S-phase duration is specifically related to cancer. In mucosa without histologic sign of malignancy, an increased S-phase duration would indicate that the malignant process has started. An increased LI would appear to relate to the selective advantage that rapidly proliferating cells hold over less proliferating ones.

Cell Population Kinetics and Dna Content During Thyroid Carcinogenesis

The proliferation kinetics and DNA content of thyroid follicular cells in rats were studied by autoradiography and cytophotometry. Continuous treatment of animals with methylthiouracil (MTU) results in hyperplasia followed by tumour growth in the thyroid gland. The mitotic index (MI) increases from 0.006 +/- 0.002% in controls to 0.13 +/- 0.06% in hyperplasia and to 0.09 +/- 0.03% in malignant cells. The same is true for the labelling index (LI) which rises from 0.08 +/- 0.003% in controls to 1.4 +/- 1.1% in hyperplasia and to 1.0 +/- 0.6% in follicular adenomas. The S-phase duration (TS) is shortened from 8.0 +/- 1.2 hr in controls to 6.0 +/- 1.4 hr in animals treated for 9 months with MTU and prolonged to 15.4 +/- 2.1 hr in papillary carcinomas. In all MTU-treated animals a decrease in the value of the potential population doubling time (TPD) and thyroid weight doubling time (TD) was observed. The cell loss factor (phi) decreases in animals treated for 3 months with MTU and increases during the stage of tumour growth in the gland (animals treated 12-15 months with MTU). DNA measurements in the nuclei of hyperplastic and neoplastic thyroid tissues reveal cells with values exceeding that in control animals. However, no difference was found in the DNA content between thyroid adenomas and carcinomas, nor between thyroid hyperplasia and neoplasia.

Enhancement of mitogen response and surface marker analysis of lymphocytes from yuong and old donors after preliminary incubation in vitro

We have examined the detailed kinetics of PHA induced proliferation of freshly isolated mononuclear cells from young and old human donors. Our studies confirm that donors over the age of 60 years may have a decreased number of PHA responsive cells and that these cells have a significantly diminished rate of entry into the first cell cycle. Age of the donor does not affect the time at which significant numbers of cells appear in first S-phase, the duration of first S-phase, the doubling time in exponential growth, and thymidine uptake per cell. Cells from young and old donors were also cultured in medium supplemented with pooled human serum for 1 or 2 days prior to the PHA response assay. After 2 days of preliminary culture, the PHA response in both young and old is significantly enhanced, with a greater enhancement in the old. The basis of this enhancement appears to be a significant increase in rate of entry into the first cell cycle. None of the other kinetic parameters were significantly altered. The magnitude of the rate enhancement in old donors' cells eliminated the difference in entry rate betwee young and old after 2 days of preliminary culture. The same rate enhancing effects were seen in the presence of serum pooled from young or old donors. There is no statistically significant change in the number of responding cells after preliminary culture. We had previously suggested, based on lactate dehydrogenase (LD) subunit ratio and patterns of T cell associated surface markers, that less differentiated subpopulations of T cells exist in elderly donors. After preliminary culture, no significant change was seen in the proportions of cells positive for T3, T4, T8, T10 and Ia surface antigens and the unusual pattern of surface marker distribution was still present on the old donors' cells. It appears that the greater rate enhancement in old donors' cells after preliminary culture may not be due to induced maturation of the possibly less differentiated T cell populations described previously. The results do suggest that these subpopulations may be non-responsive to PHA.

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

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

Cell kinetics of the K-562 cell line in microculture.

The cell kinetic parameters of K-562 leukemia cells were studied using microwell cultures in which growth was initiated from a single cell. Total population growth was studied by direct enumeration, 3H-thymidine labelling, and flow cytometry. Clonogenic cell growth was studied by replating and 3H-thymidine suicide. In 7-day clones of K-562 cells, durations of the total cell cycle, G1, S, G2, and M phases were 20.8 h, 3.5 h, 12.9 h, 3.3 h, and 1.1 h, respectively; the growth fraction was 0.92 and the cell loss factor was 0.084. Study of colony-forming cells by replating indicated that clonogenic cells comprised 40% of total cells. 3H-Thymidine suicide showed that cell-cycle duration for these cells was 22.5 h and that S-phase duration was 11.7 h.

Analysis of DNA synthesis rate of cultured cells from flow cytometric data.

The rate of DNA synthesis along S phase is estimated from flow cytometric histograms on the basis of a mathematical model of a cell population. In the absence of loss, the model expresses the population kinetics in terms of DNA synthesis rate, S-phase influx, and population size. A single histogram is sufficient to determine the DNA synthesis rate when the population is in balanced exponential growth. Two suitably chosen histograms are necessary if the S-phase influx is exponential in a time interval longer than the S-phase duration. The analysis procedure was tested on published autoradiographic data and applied to three cultured cell lines (CM-S, 3LL, and M14 cells) that show various patterns of DNA distribution. In each case the cell-cycle fractions, the DNA synthesis rate, and the S-phase duration were obtained.

A study of cell proliferation kinetics in the small intestinal epithelium of psoriasis patients

Summary Small intestinal biopsies from psoriasis patients and from nonnal controls were obtained with a multipurpose suction tube. The tissue was incubated with tritiated thymidine and processed for autoradiography. Labelling indices (LI) were calculated by counting labelled cells in all crypt cross-sections through the entire proliferative compartment. LI was increased in psoriatics as compared to controls indicating alterations in small intestinal epithelial cell kinetics in psoriasis. A continuous labelling allowing us to calculate the S-phase duration and the total cell turnover time was probably not obtained within an experimental period.

Self-renewal of stem cells and differentiation of nerve cells in the developing chick retina

Data on proliferation and self-renewal of stem cells in the developing chick retina were obtained on the basis of measurements of cells labeled by [3H]thymidine pulses in conjunction with the rate of increase in total cell number. Duration of S-phase was found to be about 4 h between stages 4 and 9 days. Self-renewal drops below the critical value of 50% (implying a transition from increase to depletion of absolute number of stem cells in the tissue) around day 7.6. The spatial order of cell proliferation was studied by measurements taken on subregions of retinas at various stages of development. Proliferating cells forming the ventricular layer increase in all regions of the retina up to day 7, though the proportion of proliferating cells is lowest in the center. From day 5 on it is higher in the nasal as compared to the temporal part of the tissue. After day 7 self-renewal of stem cells drops below 50%, stem cells become depleted and withdraw gradually from mitosis. The process is initiated in the center slightly temporal to the dorsal end of the optic fissure and then spreads rapidly towards the periphery, reaching the temporal margin first. The findings imply that while cells at the periphery are younger on the average, those cells which have become postmitotic at an early stage are not confined to a small central core of the fully developed retina, because the tissue continues to grow and produce postmitotic cells in all regions of the retina up to day 7.

Cell kinetic studies in the epidermis of the mouse. I. Changes in labeling index with time after tritiated thymidine administration.

The changes in the labeling index (LI) with time after a single injection of tritiated thymidine (3HTdR) at each of 4 different times of the day have been studied. Slight differences occur in the shape of these LI curves, (e.g. in the timing of the peaks) depending on the time of day when the initial injection was given. Thus, the time of day influences not only the number of cells in DNA synthesis but also determines the subsequent behavior of the labeled cells. The curves show 3 distinct peaks from which estimates of the cell cycle time can be made. The technique permits the cell cycle time to be estimated. From the data as a whole a minimum cell cycle time of 90 h for basal cells in the epidermis on the back of a mouse is obtained. The technique also provides estimates for the duration of S + G2 + M which varies depending on the time of day that the label is given. The LI curves can best be understood if the basal layer is assumed to contain 2 cell populations with differing cell cycle times; one having a long cell cycle (about 180 h) but short S-phase and containing the stem cells, the other having a short cell cycle (about 90 h) and a long S-phase duration and consisting of transit cells.

Cytodynamics of endometrial hyperplasia and neoplasia, part II: In vitro DNA histoautoradiography

The cytokinetics of normal, hyperplastic, and neoplastic human endometrium were evaluated by in-vitro double labeling with low- and high-dose radiothymidine. Relative to cyclic, proliferative endometrium, the mean S-phase duration was shorter in anovulatory, persistent proliferative endometrium, cystic glandular hyperplasia, and adenomatous hyperplasia without atypia, similar in adenomatous hyperplasia with severe atypia (AAH), and significantly prolonged in carcinoma in situ and invasive carcinoma. The S-phase patterns suggest a pathogenetic relationship between hyperplasias of increasing atypia as well as between hyperplasia and carcinoma, with carcinoma in situ as a transitional state between AAH and invasive carcinoma.

Effect of irradiation on the proliferation kinetics of thyroid follicular cells in infant rats

Changes in the 3H-thymidine labeling index (LI), cell cycle time (TC) and growth fraction (GF) of the thyroid follicular cells were studied in 10 days old rats irradiated in the neck region with a single dose of 3 Gy X-rays. During the first 2 days after irradiation the number of proliferating follicular cells sharply increased, followed between the 2nd and 6th days by a phase of sharp decrease. Later, between the 10th and 16th days again an increase in the LI was observed. During the cell proliferation phase, the cell cycle was shortened from 79 hours in the control animals to 30.9 hours in irradiated animals. The GF was decreased from 40% in the former to 25% in the latter. S-phase duration (TS) was 1.6 hours longer in irradiated animals than in controls. The shortening of the cell cycle of irradiated animals is predominantly a result of diminishing G1-phase duration.