TdR Labeling Index Research Articles

Angiogenesis and its Hormonal Control in the Corpus Luteum of the Pregnant Rat1

Beginning on Day 8 of pregnancy (Day 1 = sperm in vaginal smear), rats were injected i.p. with [3H] thymidine (TDR), killed 3 h later, and corpora lutea (CL) were dissected and saved for determining radioactivity in the acid-insoluble fraction or for autoradiography to determine labeling index (LI) of luteal and endothelial cells. An approximate doubling in DNA content in CL occurred between Days 13 and 14, with a high level maintained through Day 23. This was reflected in an abrupt increase in [3H] TDR incorporation on Day 13, with the peak reached on Day 14 and a subsequent decline to baseline values on Day 18. Autoradiography revealed that the LI of luteal endothelial cells rose from 2.1% on Day 12 to 10.0% on Day 14, and the LI of luteal cells correspondingly increased from 0.3% to 2.3%. Hypophysectomy (H) on Day 12 resulted, by Day 14, in no change in serum progesterone (P4) and TDR incorporation and LI of endothelial cells. However, after H and hysterectomy (HS) on Day 12, by Day 14, animals had low values for LI of endothelial and luteal cells, [3H] TDR incorporation and serum P4. After H + HS at Day 12, animals injected daily with estradiol cyclopentylpropionate (200 micrograms/day) on Days 12-14 had serum P4, [3H] TDR incorporation and LI of endothelial cells comparable to intact controls but not to luteal cells. However, similar treatment with testosterone cypionate (200 micrograms/day) or P4 (10 mg/day) did not maintain [3H] TDR incorporation or LI of either cell type, although serum P4 and estradiol levels were restored to normal values.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell fusion studies in the Werner syndrome.

The mechanism of retarded DNA synthesis was investigated by the cell fusion method. The [3H] TdR labeling index of young cells from patients with Werner syndrome (WS cells) X young normal human diploid fibroblast cells (NH cells) was considerably lower than that of NH homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of old WS X young NH heterodikaryons was as low as that of WS homodikaryons. The labeling index of WS X HeLa heterodikaryons was the same as that of HeLa homodikaryons. These results indicate that WS cells are somewhat similar to senescent NH cells. The labeling indices in both WS karyoplast X NH heterodikaryons and WS cytoplast X NH cybrids were lower than those in normal karyoplast X NH heterodikaryons and normal cytoplast X NH cybrids, respectively. These results indicate that both nuclear and cytoplasmic environments are involved in the retarded DNA synthesis in WS cells. The most plausible interpretation of all our data is that the retarded DNA synthesis in WS cells could be caused by either the 'senescent factor(s)' or the deficiency of gene product(s) that is necessary for DNA synthesis and could be a secondary consequence of the genetic defect.

Receptor-mediated antiproliferative effects of corticosteroids in Lewis lung tumors

Dextran-coated charcoal competitive binding assays and Scatchard analysis revealed the presence of high-affinity, low capacity binding sites for dexamethasone in cytosol preparations from Lewis lung tumors. In vitro studies with live cells indicated approximately 9000 nuclear binding sites/cell for the ligand-receptor complex. In vivo inhibition of cell proliferation by dexamethasone, methylprednisolone and triamcinolone acetonide was found to be dose-dependent. Changes in the [ 3 H]-TdR labeling index, mitotic index and saturable cytosol receptor sites after dexamethasone treatment in vivo suggested a dose-dependent G 1 progression delay which, after cessation of dexamethasone treatments, was apparently reversible. Resumption of cell-cycle progression was characterized by synchronous progression through S-phase and correlated temporally with receptor site desaturation. In vivo studies indicated that the effectiveness of vincristine given after dexamethasone was highly sequence-dependent, with the most effective sequence interval being coincident with the interval of maximal S-phase cellularity. Other studies indicated sequential chemotherapy with dexamethasone, vincristine and 5-Fu could be effectively employed, following primary tumor excision, to increase animal survival.

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

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.

Proliferation-dependent effect of skin extracts (chalone) on mouse epidermal cell flux at the G1-S, S-G2 and G2-M transitions.

Epidermal cell proliferation in mice was studied from 4 to 11 h following a single intraperitoneal (IP) injection of a crude skin extract. Cell cycle flux parameters were evaluated by a combination of several methods. The G1-S and S-G2 transit rates were estimated by means of a 3(H)TdR double labelling technique, and the mitotic rate by use of colcemid. The 3(H)TdR labelling index was also measured. To examine the possible influence of the circadian rhythm, all experiments were performed at two different times of the day with high or low rate, respectively, of epidermal cell proliferation. All flux parameters were altered for the whole 7-h period. The relative inhibitory effect of the skin extract was related to the proliferative state of the epidermal cell population. Cell flux at the G1-S transition showed only minor circadian variations, and treatment with skin extract was followed by a relative reduction of cell flux at this transition that was similar at the two times of the day investigated. In contrast, cell flux at the S-G2 transition showed pronounced circadian variations. The effect of the skin extract on cells at this transition was different at the two times in the 24-h period. In general, inhibition expressed as percent of the controls was stronger when the skin extract was given at times when the cell flux was low or decreasing, and vice versa. In spite of the changes in flux values following administration of a skin extract, the 3(H)TdR labelling indices were reduced only after a delay of 10 h, confirming previous results.

Kinetics of protein and DNA synthesis studied by mathematical modelling of flow cytometric protein and DNA histograms.

Mathematical models for histograms of cellular protein content as measured by flow cytometry were developed, based on theoretical protein distributions. These were derived from the age distribution of cells and the accumulation function for cellular protein content as a function of age within the cell cycle. A model assuming an exponential age distribution and an exponential protein accumulation function was found to give the best representation of protein histograms of exponentially growing NHIK 3025 cells. This is in good agreement with the known kinetic behaviour of such cells. By the combined use of the protein histogram model and a similar model for DNA content, and assuming linear DNA accumulation during S, the fraction of cells in S, as a function of cellular protein content, was simulated. This function showed good agreement with values of the [3H]TdR labelling index scored in cells sorted by flow cytometry from 5-channel intervals of the protein histogram. The protein and DNA histogram models were combined into a two-dimensional model for correlated protein/DNA measurements. Comparison between simulated data and experimentally derived two-dimensional protein/DNA histograms gave further support to the cell kinetic assumptions underlying the models, but also identified some minor deviations which could not be recognized in the analysis of the one-dimensional histograms.

Proliferation and Differentiation in Diffusion Chambers of Marrow, Blood and Spleen Cells from Patients with Chronic Myeloid Leukaemia during Chronic Phase and Blastic Transformation

The proliferative and differentiating capacity was compared between immature normal marrow cells and marrow, blood or spleen cells from patients with chronic myeloid leukaemia (CML) in chronic and blastic phases. Low density cells highly enriched in myeloblasts and promyelocytes were cultured in diffusion chambers (DC) and implanted into the peritoneal cavity of mice pretreasted with cyclophosphamide. In CML of chronic phase cell production was higher than normal with the exception of spleen cells. Total cell production was lower in blastic phase than in CML chronic phase. The [3H]‐TdR labelling index of myeloblasts of blastic phase CML increased considerably upon implantation in DC consistent with re‐entry into active proliferation. Immature normal cells give rise mostly to macrophages while the corresponding CML cells of chronic phase give a much higher PMN production with peaks after 12–18 d. Also in CML of blastic crisis the blasts differentiate into mature PMNs, but the maturation time is shortened with peaks of PMNs after 5–10 d. DC cultures of spleen cells from patients with CML showed a less differentiating capacity with a very low PMN production compared to cultures of marrow or blood cells. Our results indicate intrinsic differences in growth and maturation capacity between normal immature granulopoietic cells and cells from patients with CML of chronic phase or blastic crisis. The results may also indicate intrinsic differences between spleen, blood or marrow cells in CML.

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.

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

ABSTRACTThe strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [3H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application.Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G2 phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G1, S and G2 phases seems to be reduced initially in rapidly proliferating epidermis.

Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro

The stability of the differentiated state of cardiac myocytes in vitro was examined under culture conditions which selectively stimulated or inhibited proliferation of fibroblasts. Regulation of fibroblast proliferation in cultures of myocardial cells from 8-day embryonic chicks was achieved by adjustment of the glutamine (Gln) concentration in the culture medium (Ham's F-12 medium containing 2 × amino acids and 5% fetal calf serum). Myocardial cells, when plated at 80 cells/mm 2 in Gln − medium, maintained a stable density of approximately 40% of the plating density for more than 30 days. When Gln was added to the medium (292 μg/ml) fibroblast proliferation was stimulated, and by 5–6 days after this addition cell densities had increased to confluency. The selective action of glutamine on fibroblast proliferation was determined by labeling cultures with tritiated thymidine ([ 3H]TdR) and scoring its incorporation into myocytes and fibroblasts by radioautography. After 2 weeks in Gln − medium, the mitotic index was 0.3% and the [ 3H]TdR-labeling index (1.5-hr pulse) was 6.4%. In addition, the proportion of myocytes in the population was constant at 64.2% for at least 30 days in vitro, and contractile activity was observed for up to 6 months. After 5 days of Gln replacement, the cells exhibited a labeling index of 25%, the proportion of myocytes decreased to less than 10% and contractile activity was rarely observed. Although the [ 3H]TdR-labeling index of fibroblasts and myocytes was nearly identical in Gln − medium, the addition of Gln produced a fivefold stimulation in the fibroblast labeling index, but did not affect myocyte proliferation or DNA synthesis. A unique phenomenon of myocyte congregation was observed only in Gln − medium which resulted in the formation of myocyte colonies from which fibroblasts were largely absent. It is suggested that this process with the resultant establishment of a functional electrical syncytium plays a significant role in the development and stabilization of myocyte differentiation in vitro.

Tritiated thymidine labeling index of benign and malignant human breast epithelium.

38 specimens of benign breast tissue from young women and 92 primary breast carcinomas were evaluated for tritiated thymidine ([3H]TdR) labeling index (TLI) by in vitro pulse labeling. The TLI on nonneoplastic, terminal breast ducts was significantly higher during the latter half than during the first half of the menstrual cycle (P less than 0.001), and a geometric mean TLI of 1.5 during the latter half of the cycle is consistent with approximately 47% turnover of these cells during the menstrual cycle. Ducts of fibroadenomas showed similar menstrual variation TLI. The range of TLI observed in the carcinomas was 0.04-18.6, arithmetic mean 3.7, geometric mean 2.1. The TLI of carcinomas was not significantly correlated with size of the tumor, but tended to be higher in women less than 50 yr old than in women older than 50 yr (P less than 0.05), and in the presence of two or more metastatic axillary lymph nodes than when fewer than two nodes were involved (P less than 0.02).