Effects of beta-all-trans retinoic acid on growth, proliferation, and cell death in a multicellular tumor spheroid model for squamous carcinomas

The growth of multicellular tumor spheroids, MTSs, from squamous carcinoma line MDA 886Ln was inhibited by beta-all-trans retinoic acid (RA). Inhibition occurred within 3 to 5 days of treatment, and MTS size then remained static for up to 2 weeks. Although their growth stopped, 10-day-treated MTSs incorporated [3H]thymidine into trichloroacetic acid-precipitable material, and the [3H]thymidine labeling index, determined by autoradiography, was equivalent between control and RA-treated MTSs. Bivariate flow cytometric analysis of bromodeoxyuridine-labeled MTSs showed equivalent S phase progression of labeled cells over an 8-hour chase. MTS growth stasis was not related to RA-induced cell cycle effects. Monitoring of MTSs for cell sloughing showed no significant cell shedding that could account for stasis. Quantitation of cell number and DNA content per MTS showed an RA-induced decrease. This was confirmed by histological analysis, which demonstrated the temporal appearance of acellular areas. MTS growth statis is thus related to an RA-induced cell loss in this MTS model for squamous carcinomas.     

COMPARATIVE PROLIFERATIVE KINETICS OF CELLS FROM NORMAL HUMAN EPIDERMIS AND BENIGN EPIDERMAL HYPERPLASIA (PSORIASIS) IN VITRO

Proliferation kinetics of epidermal cells from normal human skin and lesions of psoriasis (benign epidermal hyperplasia) were studied in vitro. Epithelial out-growths were obtained from skin explants and the cell cycle studied using the conventional method of following two successive curves of labeled mitoses after an initial pulse with ³H thymidine. The length of T_c was 59 hr and 53.5 hr respectively for normal and psoriatic cells. The shorter T_c for psoriasis was due to a shorter duration of S. The growth fraction was 66% and 74% for normal and psoriatic cells respectively as determined by continuous labeling with ³H thymidine. Under the conditions of the present experiments, therefore, normal and psoriatic epidermal cells showed no significant difference in proliferative capacity.     

AUTORADIOGRAPHIC EVIDENCE FOR MANY SEGREGATING DNA MOLECULES IN THE CHLOROPLAST OF OCHROMONAS DANICA

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-³H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.     

THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE

The effect of a goitrogenic stimulus on thyroid weight and thyroid cell ³HTdR labeling of Sprague-Dawley rats varying from 2 to 40 weeks of age was determined. Propylthiouracil ad libitum in drinking water produced a spurt in follicle cell labeling index and thyroid weight evident after 24 hr for all age groups. The increase in labeling index reached a peak at 5–7 days and then decreased to a level a few times greater than that of the normal unstimulated thyroid. The tritiated thymidine labeling index for thyroid follicle cells and the effect of PTU thereon was determined for August male rats of 3 days to 12 weeks of age. In the older rats, the follicle cell labeling index rose to 5–6% after 4–5 days of PTU treatment and then slowly fell to about 1%, For the unstimulated control rat of comparable age, the labeling index was about 0.1%. At all ages the thyroid showed a rapid response to PTU. Examination of the time sequence of mitotic labeling showed that the DNA synthesis period was 7.5 hr for normal 2-week-old rats and for 10–12-week-old rats that had received PTU for 4 days. There was no second wave of labeled mitoses in either group during the 48-hr interval studied. From the curve of thyroid weight vs time on PTU and from the labeled mitoses curve, inferences regarding the minimum fraction of proliferating follicle cells in the stimulated ‘adult’rat thyroid were made.     

Rat C-6 Glioma Cells Maintained In an Organ Culture System: A Study of Kinetic Parameters

Rat C-6 glioma cells were grown on a sponge foam matrix in an organ culture system and the cell cycle parameters, including the growth fraction (GF), were assessed after autoradiography. the zones of growth consisted of a compact upper layer (UL) at the gaseous interface, a central necrotic layer and a deeper lower layer (LL) which invaded the matrix. the fraction of continuously labeled mitoses (FCLM) was similar in both the UL and LL cells. the derivatives of the FCLM curves obtained in three experiments gave an average modal T_G2 of 5 hr. A mathematical model relating GF, T_G2, T_C and labeling index as a function of time, LI(t), was devised for cells in a steady state exposed continuously to tritiated thymidine and was applied to data obtained from UL cells. A mean GF of 9% (range: 8–10%) and a mean cell cycle time (T_C') of 27 hr (range: 13–47 hr) were obtained. the mean T_S was calculated to be 11 hr (range: 8–16 hr) by the method of grain counts per mitotic figure or grain index (GI). Knowledge of T_S permitted alternative calculation of the cell cycle time from the equation T_S/T_C= LI(0)/GF: this gave a mean cell cycle time (T_C) of 29 hr (range: 20–45 hr). Except for the GF, the cell kinetics were comparable to those of the same cell line grown in monolayer culture. the GF in the in vitro system described is in the lower range reported in some human malignant gliomas in vivo.     

Position specific growth regulation of 3T3 cells in vivo.

In this study we have investigated the mechanism by which spatial growth is regulated by monitoring 3T3 cells, introduced into the developing mouse limb using exo utero surgery. The 3T3 cells were labeled with a human cell surface glycoprotein, CD8, and injected into stage 7-9 mouse limbs. At 24 and 48 hr after injection embryos were labeled with [3H]thymidine and processed for immunohistochemistry and autoradiography. The labeling index of CD8 positive cells was compared to that of neighboring limb bud cells and also to the position of the injection site within the limb. We find that the labeling index of 3T3 cells is in accord with that of the limb cells that immediately surround them; 3T3 cells display a high labeling index in limb regions of high growth and a low labeling index in limb regions of low growth. In addition, we find that both limb bud cells and injected 3T3 cells display a general proximal (low) to distal (high) gradient of growth at the stages analyzed. We conclude from these results that position-specific regulation of growth occurs in a non-cell autonomous manner and is likely to be mediated by mitogenic signals that are localized within the limb environment. In addition, our results demonstrate the usefulness of utilizing established cell lines as in vivo probes to monitor developmental mechanisms.     

Effects of growth factors on proliferation of basal and luminal cells in human breast epithelial explants in serum-free culture

Summary A method of culturing human breast epithelium is described in which viable explants can be maintained in protein-free medium while retaining the capacity of responding to added hormones and growth factors for at least 7 days. Culture parameters were chosen to provide maximum sensitivity of detection of proliferative responses by autoradiography. Under basal conditions, the mean thymidine labeling index of the explants was 0.08%. After stimulation with insulin, hydrocortisone, and cholera toxin (I,H,CT), a combination known to stimulate proliferation in human breast epithelium in vitro, the mean labeling index was 15.7%. Stimulation of explants with epidermal growth factor (EGF) and transforming growth factor (TGF)-α resulted in mean labeling indices of 6.6 and 10.8%, respectively. Autoradiography at the ultrastructural level demonstrated that in I,H,CT-stimulated explants the majority of the labeled cells were luminal, with only 1.5% being basal cells. In contrast, after EGF and TGF-α basal cells accounted for 11.5 and 18.5% of the labeled population. These results indicate that this system provides an in vitro assay of proliferative activity in the normal human breast that enables comparisons to be made between both the luminal and the basal cells in the explants and their counterparts in monolayer culture prepared from flow sorted cells. Thus, growth responses dependent on cell-to-cell interactions or stromal modulation can be identified.     

KINETICS OF PHYTOHEMAGGLUTININ STIMULATED LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKEMIA

The DNA synthesis pattern and several kinetic parameters of in vitro PHA stimulated normal and CLL lymphocytes were determined. The DNA synthesis peak of CLL lymphocytes occurred 2–3 days later than that of normal lymphocytes. The generation time, estimated by the labeled mitoses method, was found to be 28 hr and 20 hr for CLL and normal lymphocytes respectively. This difference was mainly due to longer S and G_t periods. It was also shown that both CLL and normal lymphocytes divide several times. These data were confirmed by the chromatid labeling pattern and by the halving of the grains and the double labeling techniques. By combining continuous and pulse labeling the growth fraction of CLL lymphocytes was found to be progressively increasing, because of the recruitment of new cells in cycle, from the third day of culture. Therefore the delayed peak of DNA synthesis of CLL lymphocytes was caused by a longer cell cycle and by a longer pre-replicative phase.     

Proliferative activity of gastric and duodenal endocrine cells in the rat

Summary The replicative activity and migration of gastrin, somatostatin and serotonin cells in rat stomach and doudenum was studied using combined immunocytochemistry and autoradiography after ³H thymidine pulse-labeling. Our results show that a small proportion of gastrin, somatostatin and serotonin immunoreactive cells displays proliferative activity. The overall labeling index ranged from 1.3% for gastric endocrine cells to 3.2% for duodenal endocrine cells.In a pulse chase experiment, labeling indices of immunoreactive cells were estimated at several time intervals after ³H thymidine administration. Significant differences in labeling index were not found. Migration of ³H thymidine labeled endocrine cells towards the luminal surface was not found in the stomach nor in the doudenum.It is concluded that 1) these endocrine cells have replicating activity; 2) the replicative activity of endocrine cells is higher in the duodenum than in the stomach; 3) the various cell types do not show significant differences in replicating activity and 4) endocrine cells did not seem to migrate to the luminal surface of the mucosa along with the other epithelial cells.     

CELL POPULATION KINETICS IN THE RAT JEJUNAL CRYPT

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50 % peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1.43 ± 0.14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1.78 cell positions per hour. The crypt column length was 32.9 ± 0.2 cells and the column count was 22.3 ± 0.2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 ± 6 from direct observation of squashed, microdissected crypts. In each crypt 22.5 ± 0.5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0.62. A value of 0.61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF-BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF-treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

The Time of DNA Synthesis During the Interdivision Growth Cycle of Tetrahymena pyriformis Fed on Living Bacteria

SYNOPSIS. It has been possible to obtain selective labeling of the macronucleus of Tetrahymena pyriformis fed on living Escherichia coli. The bacteria themselves, a thymidine requiring mutant, were labeled by exposure to tritiated thymidine in a lettuce infusion medium supplemented with trypticase broth. Various patterns of labeling were seen in synchronized Tetrahymena when the radioactive bacteria were given at particular times during the growth cycle. These patterns have been interpreted as indicating the duration of the G₁, S, and G₂ periods; they also suggest that a soluble pool of thymine exists in this animal from one S period to the next.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

Cell proliferation and DNA replication in the imaginal wing disc of Drosophila melanogaster

Cell proliferation in the imaginal wing disc of Drosophila has been analyzed by both pulse and chronic labeling with [3H]thymidine. We find neither spatial nor temporal variation in the fraction of S phase cells during the third instar. At or near the time of white prepupae formation the fraction of S phase cells falls sharply. Our chronic labeling experiments have demonstrated that almost all (and perhaps all) of the cells in a mid third instar wing disc are cycling. By examining sectioned material from such experiments we have found that the collumnar epithelial cell and the adepithetial cell populations become labeled with similar kinetics. The peripodial membrane cell population becomes labeled more slowly. We have also obtained estimates of cell cycle parameters for the imaginal wing disc cells.     

Novel derivation of total cell cycle time in malignant cells using two DNA-specific labels.

Cell cycle kinetic analysis in vitro has conventionally been accomplished by labeling S-phase cells using two DNA specific labels given sequentially and separated from each other by a certain time interval. By counting the cells labeled by both versus those labeled by either one of the two labels, and using the formulas described by Wimber and Quastler, approximate values for durations of S-phase (Ts) and the total cell cycle (Tc) can be determined. More recently, instead of radio-isotope labeled thymidine, two thymidine analogues have been used to label S-phase cells in vivo in a variety of human tumors based on the same principles. In the present report, new formulas are proposed for the calculation of Ts and Tc which are simpler to apply since only one type of labeled cells (those exiting S-phase as identified by containing only the first label) need to be differentiated from the remaining population for Tc calculations.     

DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

Using pulse labeling techniques with [³H]thymidine or [³H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [³H]thymidine of high specific activity, followed by [³H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.     

Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.     

STUDIES ON THE KINETICS OF INITIAL CYCLE PROGRESSION IN VITRO OF ASCITES TUMOUR CELLS SUBSEQUENT TO ISOLATION FROM ASCITES FLUID

A method is described for determining the duration of cell cycle phases traversed by cells responding to release from proliferation restraint. Experiments have been performed with arrested Yoshida ascites hepatoma cells allowed to re-enter the growing stage after transfer of cells from the late stage of ascites into an in vitro incubation system. Experimentally, this method requires information on the rate of incorporation of labelled thymidine and on the rate of increase in cell number. The rate of [¹⁴C]thymidine incorporation in vitro was shown to be directly proportional to the number of cells synthesizing DNA. This was shown by correlating data from measurements of the rate of thymidine incorporation with those from measurements of the labelling index of the cell population. Theoretically, the method is based on analysis of the region limited by two integral curves, one corresponding to the kinetics of cell entry into and the other to the kinetics of exit from the S-phase. From data on the actual rate of increase in the total number of cells and data on the S-phase duration it is possible to obtain information on the cytokinetics of growth resumption by the ascites cell population.