Changes in RNA-, DNA- and proteinsynthetic activity during the formation of analfin processes in ethisterone-treated females of Oryzias latipes

The incorporations of uridine-³H, thymidine-³H, and leucine-³H were studied in the process-forming regions of the anal-fin rays of the ethisterone-treated females of Oryzias latipes. The activity of alkaline phosphatase was also studied. The increased incorporation of uridine-³H was detected between 12 and 24 hours of ethisterone treatment, attaining the maximum at 24 hours. The percentage of thymidine-³H labeled nuclei increased rapidly between 48 and 84 hours. The incorporation of leucine-³H was found to increase during the first 12 hours, attaining a constant level at 24 hours. An additional increase in incorporation of leucine-³H took place at 60 hours, the incorporation coming up to the maximum at 72 hours. In the horny substance secreted by the scleroblast mass, grains in the autoradiograph were detected at and after 72 hours. Alkaline phosphatase activity was manifested between 48 and 72 hours. These results seem to correspond to the histological changes, such as the appearance of the precursor cells of scleroblasts at 48 hours, the formation of scleroblast mass during the next 24 hours, and the initiation of horny substance secretion at 72 hours.     

PANETH AND GOBLET CELL RENEWAL IN MOUSE DUODENAL CRYPTS

Proliferation of Paneth and goblet cells of mouse duodenal crypts was studied by high resolution light microscope radioautography. In one group of mice, blood levels of thymidine-³H were sustained for up to 12 hr by repeated injections of isotope to facilitate identification of proliferating cells. In these animals, many goblet cell nuclei incorporated thymidine-³H whereas only 1 of 6261 tabulated Paneth cells was labeled. Cells intermediate in structure between undifferentiated and goblet cells and between undifferentiated and Paneth cells were identified and their light and electron microscopic features are described. A significant number of these "intermediate" cells incorporated thymidine-³H into their nuclei. Another group of mice received a single injection of thymidine-³H. These animals were killed 4 hr to 29 days after isotope administration. Goblet cells and intermediate cells with labeled nuclei were identified 4 hr after thymidine-³H but could not be seen after 15 days. In contrast, Paneth cells with labeled nuclei were not observed until 24 hr after thymidine-³H but were still present at 29 days, long after labeled undifferentiated, goblet, and intermediate cells had disappeared. We conclude that differentiated Paneth cells in mouse duodenum do not normally proliferate, but, instead, arise by differentiation from undifferentiated crypt cells or from intermediate cells. Moreover, once formed, Paneth cells persist in crypts for a prolonged period. In contrast, intermediate cells and crypt goblet cells proliferate actively and are less stable cell populations than differentiated Paneth cells. The precise function of the intermediate cells is not known, but they may represent transition forms between undifferentiated cells and the more matrure secretory cells. Damage of crypt epithelial cells, thought to be due to radiation effects, was evident in both groups of mice.     

pH AND POPULATION DENSITY IN THE REGULATION OF ANIMAL CELL MULTIPLICATION

Sparse and dense cultures of chick embryo cells were affected differently by pH. The rates of cell multiplication and of thymidine-³H incorporation into DNA of dense cultures were increased as the pH was increased from 6.6 to 7.6. At pH higher than 7.6 the rate of multiplication decreased slightly in the dense cultures, but the rate of thymidine-³H incorporation continued to increase. The discrepancy was due in part to cell death and detachment at very high pH, and in part to a more rapid uptake of thymidine-³H at very high pH. Sparse cultures were much less sensitive to pH reduction and, when a suitably conditioned medium was used to minimize cell damage, very sparse cultures grew almost as well at pH 6.7 as at higher pH. The rates of cell multiplication and thymidine-³H incorporation at low pH decreased in the initially sparse cultures before they reached confluent cell densities. There was no microscope evidence of direct contact between plasma membranes of cells at these densities although the parallel orientation indicated that the cells were influencing locally each other's behavior. Even at much higher cell densities, electron microscopy revealed large intercellular gaps partly filled with a fragmentary electron-opaque material suspected to be glycoprotein. Wounding experiments showed that pH affected cell migration in a manner similar to its effects on cell multiplication. Low pH inhibited cell migration, but those cells which migrated into the denuded region multiplied as rapidly at low pH as at high pH. The effects of pH on growth were correlated with effects on the uptake of 2-deoxyglucose-³H. Dense populations of cells inhibited by low pH were stimulated to incorporate thymidine-³H by the addition of small amounts of diethylaminoethyl-dextran. Rous sarcoma cells at high cell density were less sensitive to pH than were normal cells at the same density, but were more sensitive than sparse normal cultures. The results suggest that cell growth is inhibited through the combined effects of both lowered pH and high cell density on cell surface permeability.     

DNA synthesis, mitosis and fusion of myocardial cells

Myocardial cells obtained from embryonic chick ventricles have been used to investigate (1) whether differentiated cells can undergo DNA synthesis and mitosis and, (2) whether heart cells when grown in culture can fuse with each other and with chick skeletal myoblasts to form heterokaryon myotubes. Electron microscopic observations have shown that myocardial cells of day 3 and day 20 chick embryos did contain myofibrils with defined sarcomeres; these cells have been observed in mitosis. Cells obtained by tryptic digestion of day 12 chick ventricles when grown in culture continued to replicate their DNA as shown by thymidine-³H radioautography with DNase controls and were observed in all stages of mitosis. Electron microscopy showed that myofibrils were present in some of the cultured cells. Bi-, tri- and tetranucleate cells were observed in the cultures. Thymidine-³H radioautography showed that these cells were formed by karyokinesis without cytokinesis and by the fusion of uninucleate cells. Since the heart cells could fuse with each other, we tested the possibility that they could fuse with skeletal myoblasts to form heterokaryon myotubes. This was accomplished by co-culturing thymidine-³H labeled ventricular cells and unlabeled skeletal myoblasts. Radioautography with DNase controls showed that some of the myotubes consisted of unlabeled skeletal muscle nuclei and labeled heart nuclei in varied proportions. The factors initiating the formation of these heterokaryons have not been elucidated.     

THE FINE STRUCTURE OF PROLIFERATING CELLS IN PRENEOPLASTIC RAT LIVERS DURING AZO-DYE CARCINOGENESIS

The continuous feeding of the carcinogenic aminoazo dye DAB to rats produces hyperbasophilic foci in the preneoplastic livers. After injections of thymidine-³H into the rats, such foci were isolated from the livers and studied by radioautography with the phase-contrast and electron microscopes. In these foci, the only cells found to be proliferating, as determined by the uptake of thymidine-³H into their nuclei, were a poorly differentiated type; well differentiated hepatocytes in the same regions were not labeled with the isotope. The labeled cells had an irregular cell outline and a high nucleocytoplasmic ratio; the cytoplasm had almost completely lost the specialized elements characteristic of hepatocytes; the irregular nuclei with prominent nucleoli, the altered mitochondria, and the increased free ribosomes noted in these cells are features which are characteristic of neoplastic cells induced by DAB. Thus, it seems likely that the hyperbasophilic foci represent the sites of extensive dedifferentiation of hepatocytes followed by rapid cellular proliferation, leading to neoplastic growth.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.     

CELL POPULATION CHANGES IN THE INTESTINAL MUCOSA OF PROTEIN-DEPLETED OR STARVED RATS : II. Changes in Cellular Migration Rates

The effect of protein-free and starvation diets on the migration of cells from the crypts onto and up the villi of the rat ileum was studied. Rats starved for 3, 7, or 10 days or fed a protein-free diet (PFD) for 3, 7, or 11 wk were injected with thymidine-³H and sacrificed at timed intervals. The time required for the labeled cells to first appear on the villi of experimental animals was longer than in the controls. This was the result of an elongated cycle in the protein-depleted animals and a lengthening of the maturation period in both the starved and protein-depleted animals. Determination of the distance which labeled cells had migrated up the villi in control and experimental animals, after thymidine-³H injection, indicated that cells in animals starved for 7 days migrated more rapidly than those in the fed controls, while those of 10-day starved animals moved more slowly. The cells of animals fed PFD for 3 wk migrated up the villi more rapidly, those of animals depleted for 7 wk migrated at the same time rate, and those of 11-wk PFD animals migrated more slowly than the fed controls. There is apparently no correlation between the cell cycle time in the crypt cells and the rate of migration of cells up the villus.     

CYTOPLASMIC LABEL FOLLOWING TRITIATED THYMIDINE TREATMENT OF ALLIUM CEPA L. ROOTS : Cytochemical and Electron Microscope Study

Tritiated thymidine routinely labels onion root cytoplasm during most of the cell cycle. One-third of this label could be cytochemically identified as DNA. The balance of the label was not RNA or a lipid, or attributable to labeled impurities in thymidine-³H. In electron microscope radioautographs one-third of the cytoplasmic silver grains was over organelles, presumably mitochondria and plastids. The other two-thirds of the silver grains in electron micrographs was distributed widely, 41% over ground cytoplasm and 10% over cell walls-cell membranes. Snake venom phosphodiesterase (SVDase) extracted a cytoplasmic fraction not degraded by DNase, and did not appear to extract nuclear DNA. The SVDase-extractable fraction may be DNA or a thymidine 5'-phosphoryl group in an ester linkage with another hydroxylic compound. The nature of the nonextractable fraction is considered. Possibilities discussed are: (1) technical problems such as the binding of an acid-labile nuclear DNA in the cytoplasm; (2) non-DNA, such as breakdown products, and thymine compounds other than DNA; (3) DNA, not extractable because of the nature of its binding to other compounds or because it is a "core" resistant to DNase. Until the chemical nature of this nonextractable fraction is known, cytoplasmic label following thymidine-³H treatment cannot necessarily be considered DNA, nor the assumption made that thymidine-³H exclusively labels DNA.     

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

Summary 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. Supported in part by Contract NOI-CP-75909 and NOI-CP-25604-59 from the National Cancer Institute, Bethesda, MD.     

DNA SYNTHESIS AND MITOSIS IN ERYTHROPOIETIC CELLS

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-³H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.     

Replication of nucleolus-associated DNA during "G2 phase" in Physarum polycephalum

In the myxomycete, Physarum polycephalum, the bulk of nuclear DNA replication occurs during a period of a few hours immediately following upon mitosis. During the remainder of the intermitotic period, incorporation of thymidine-³H continues at a low rate in the region of the nucleolus (radioautographs). A few nuclei incorporated thymidine-³H into the extranucleolar chromatin at a high rate at all times of the intermitotic period. These nuclei were exceptionally large and they frequently contained several small nucleoli of different sizes rather than the one, central nucleolus which is characteristic of a normal interphase nucleus.     

Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia

Essentially normal development of early embryonic pancreatic epithelium occurs only in the presence of mesenchymal tissues (Golosow and Grobstein, 1962), or a particulate fraction (MF) obtained from extracts of chicken embryos (Rutter et al., 1964). We have shown that this fraction also stimulates the incorporation of thymidine-³H into DNA. This stimulatory activity was detected in particulate fractions from homogenates of several mesodermal tissues from rat and chick embryos, as well as in fibroblasts cultured from these tissues, but not in embryonic epithelial tissues. This activity may thus be related to the mesodermal tissue requirement for pancreatic development. MF was solubilized and partially purified from homogenates of chick embryos. It is stable to collagenase, hyaluronidase, and neuraminidase. Activity is lost by heating and by treatment with trypsin. It is presumed, therefore, that the factor is associated with a protein that is not collagen.The effects of the MF upon macromolecular synthesis were tested in pancreatic tissues from 12-day rat embryos. When isolated epithelia were cultured in the absence of mesoderm or MF, the rate of thymidine-³H incorporation into DNA decreased to low levels. The specific activities of DNA polymerase and deoxycytidylate deaminase in epithelial extracts also declined. In contrast, the rate of thymidine-³H incorporation into DNA increased 5- to 8-fold over the initial rates in epithelia cultured with MF. Concurrently DNA polymerase activity in tissue extracts increased by 2- to 3-fold; deoxycytidylate deaminase activity declined slightly.MF also affected RNA and protein synthesis. The rate of leucine-³H incorporation into protein and uridine-¹⁴C incorporation into RNA in isolated pancreatic epithelia was comparable to that of intact rudiments. Cultures in the presence of MF increased these rates severalfold after 20 hr. These results suggest that MF, and by implication, mesoderm, may supply a growth factor for epithelial tissue and thus serves a permissive rather than a determining role in the differentiation process in pancreatic development.     

TUMOR ANGIOGENESIS : Rapid Induction of Endothelial Mitoses Demonstrated by Autoradiography

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-³H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1µ thick Epon sections showed thymidine-³H labeling in endothelial cells of small vessels 1–3 mm from the site of implantation, as early as 6–8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-³H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

IN VIVO SPECIFIC LABELING OF CHLAMYDOMONAS CHLOROPLAST DNA

When Chlamydomonas reinhardi is supplied with (methyl-³H)-thymidine, radioactivity is incorporated specifically into chloroplast DNA Chromatographic analysis of the products of enzymatic hydrolysis of the DNA reveals that only thymidine monophosphate has been labeled. Use of thymidine-6-³H yields an identical result. If thymidine-³H monophosphate is supplied, a small amount of radioactivity is incorporated into both nuclear and chloroplast DNA in proportion to the abundance of these DNA components. These observations are consistent with earlier suggestions that algae lack cytoplasmic thymidine kinase, but that the enzyme is present within their chloroplasts.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

RADIOAUTOGRAPHIC LOCALIZATION OF DEOXYTHYMIDINE TRIPHOSPHATE IN TRADESCANTIA POLLEN GRAINS DURING DNA SYNTHESIS

Tradescantia pollen grains, isolated during the period of DNA synthesis in the generative cell, accumulate deoxythymidine triphosphate (dTTP)-³H after incubation with thymidine-³H in the presence of millimolar deoxyadenosine. Most of this dTTP-³H was found to resist extraction by the fixative, cold ethanol-acetic acid, and its location was investigated by radioautography with thin, dry emulsion. Substantial binding of dTTP-³H occurred as an artifact; but when nuclei were isolated from the fixed pollen grains by sonication, it was found that they were differentially labeled: generative nuclei contained dTTP-³H, vegetative nuclei did not. This observation is discussed and is interpreted as evidence supporting the idea that thymidine is phosphorylated only in the generative cell of the pollen grain.     

INCREASED CELL PROLIFERATION WITH PERSISTENCE OF CIRCADIAN RHYTHMS IN HAMSTER CHEEK POUCH NEOPLASMS

Squamous cell neoplasms induced by repeated topical application of 7,12-dimethyl-benz(a)anthracene in Syrian hamster cheek pouch exhibited circadian rhythms of DNA synthesis and mitotic activity. Fluctuations in the fractions of cells in mitosis and DNA synthesis observed in the tumors were approximately in phase with the circadian rhythms from normal precursor epithelium, indicating that some degree of host physiologic modulation persists during neoplastic growth. The labeling (thymidine-³H) and mitotic indices of neoplasms were considerably higher than normal throughout the 24 hr period. The duration of the neoplastic S phase—measured from the PLM curve—was 30% shorter than normal; G₂ did not show detectable variation. The data demonstrated that chemically induced squamous cell neoplasms had markedly increased rates of cell production. It is postulated that applications of a carcinogen upon a cell-renewing population generate a multicompartmental cytokinetic imbalance in which: (1) a higher proportion of G₀ cells is stimulated to enter the cycle; (2) the duration of the cell cycle is shortened; (3) the regulatory mechanisms fail to stimulate an accelerated rate of differentiation to compensate for the overproduction of cells; and (4) the state of proliferative hyperactivity becomes stable. An oncogenic cytokinetic mechanism based solely on a persistent decrease in cell loss (differentiation) is ruled out by the present investigation, at least for squamous cell neoplasms.     

Measurements of Diel Rates of Bacterial Secondary Production in Aquatic Environments

Measurements of bacterial secondary production were carried out during 13 diel studies at one coastal marine station and in five lakes differing with respect to nutrient concentration and primary production. Bacterial secondary production was measured in situ every 3 to 5 h by [³H]thymidine incorporation into DNA. In some of the diel studies, these results were compared with results obtained from dark ¹⁴CO₂ uptake and frequency of dividing cells. Only minor diel changes were observed. The rate of [³H]thymidine incorporation into DNA and the frequency of dividing cells varied from 23 to 194% of the diel mean. The dark CO₂ uptake rate varied from 12 to 259% of the diel mean. An analysis of variance demonstrated that no specific time periods during 24 h showed significantly different production rates, supporting the idea that bacterial activities in natural assemblages are controlled by a variety of events. The best correction (r² = 0.74) was obtained between the [³H]thymidine incorporation and frequency of dividing cells procedures from the lake water samples. The actual production rates calculated by [³H]thymidine incorporation into DNA were appreciably lower than those obtained by the frequency of dividing cells and the dark CO₂ uptake techniques. Diel rates of bacterial production are discussed in relation to sampling frequency, statistical errors, and choice of method.