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1.
Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [3H]uridine incorporation into RNA and [3H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10−21 M). Insulin stimulated the rate of [3H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [3H]uridine, [3H]thymidine and [3H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.  相似文献   

2.
In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [3H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [3H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [3H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 106 cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.  相似文献   

3.
Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [3H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [3H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [3H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.  相似文献   

4.
Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [3H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [3H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [3H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.  相似文献   

5.
Summary The human fetal adrenal cortex is one of the largest fetal organs and synthesizes precursors for placental estrogen production as part of the feto-placental unit. The factors controlling the rapid growth of the human fetal adrenal cortex during the second and third trimesters are not known. Placental regulation of the growth of human fetal adrenocortical cell cultures from second trimester fetuses was studied. A placental-derived mitogenic factor (PDMF) was detected in tissue homogenates of 14 to 22 week human placentas and stimulated adrenocortical cell number and [3H]thymidine incorporation into DNA 5–8 fold. PDMF has been partially purified by ammonium sulfate precipitation and anion exchange chromatography. PDMF is a heat sensitive protein with disulfide bonds required for activity. The growth stimulation by PDMF was significantly greater than that for basic or acidic fibroblast growth factor by 25–50% and epidermal growth factor by 3–4 fold. The placental hormones, progesterone, estriol, estradiol, placental lactogen and chorionic gonadotropin, either alone or in combination did not stimulate fetal adrenocortical cell growth, except for a 41% cell number increase by progesterone. Platelet-derived growth factor and insulin-like growth factors I and II were not mitogenic for these cells. These results show that the placenta contains a potent growth factor for human fetal adrenocortical cell cultures. This implies a direct role for the placenta in control of this fetal organ’s growth, which would make the human feto-placental unit a bi-directional relationship. This research was supported by Grants HD15882 and HD21798 from the National Institutes of Health, Bethesda, MD. This work was presented in part at the 68th Annual Meeting of The Endocrine Society, Anaheim, CA, June 1986. Editor’s Statement This report describes a potentially new relationship between placenta and the regulation of growth of fetal adrenalcortical cells. Since these cells produce several of the essential hormones influencing fetal development, characterization of this factor(s) could provide important insights into the process of differentiation. David A. Sirbasku  相似文献   

6.
《Phytochemistry》1987,26(2):331-334
Both [3H]thymidine and [3H]deoxyadenosine were found to be incorporated into the nuclear DNA of wheat embryos immediately after dry embryos were allowed to imbibe aqueous solutions of the radioactive precursors. The early labelled DNA sedimented in a manner suggesting that replicative intermediates were already formed within the first 90 min of germination. However, aphidicolin remained without any effect on this early DNA synthesis. Likewise, a cell-free system derived from early embryos incorporated [3H]dCTP into DNA independently of the presence of aphidicolin. On the contrary, dideoxyTTP inhibited the DNA synthesis considerably. It is concluded that a proportion of the resting wheat embryo cells is able to initiate a replicative DNA synthesis immediately upon imbibition. The synthesis seems, however, to proceed with the participation of a γ-like, rather than an α-like, DNA polymerase.  相似文献   

7.
HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE : Growth and DNA Synthesis   总被引:83,自引:15,他引:68       下载免费PDF全文
Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.  相似文献   

8.
Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [35S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [35S]sulfate incorporation but also [3H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [3H]uridine incorporation into rabbit chondrocytes and significantly enhanced [3H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.  相似文献   

9.
Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (3H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (3H)thymidine (10–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (3H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10–8 to 10–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (3H)thymidine supplied at 5 × 10–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.  相似文献   

10.
DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.  相似文献   

11.
We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 M/m2 and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.  相似文献   

12.
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.  相似文献   

13.
In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [3H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [3H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.  相似文献   

14.
Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.  相似文献   

15.
Summary Culture conditions for maintaining first trimester human placenta in organ culture, which enhance the secretion of human chorionic gonadotropin (hCG), are described. Nutrient medium, oxygen tension and Gelfoam support matrix infuence the synthesis of hCG by these cultures. Placental tissue remained viable for the duration of experiments (12 days) as judged by the incorporation of tritiated thymidine into DNA and the lack of release of incorporated [125Iiododeoxyuridine. Optimal conditions for hCG synthesis in placental organ culture included an atmosphere of 95% air and 5% CO2 (approximately 20% O2), CMRL 1066 medium containing fetal human or bovine serum, insulin, hydrocortisone and retinal acetate. Multiple pieces of placenta could be cultured in the same dish with an additive effect on hCG secretion. The functional responsiveness of these placental cultures was demonstrated by modulation of hCG synthesis with theophylline and 3′5′ dibutyryl cyclic AMP. Presented in part at the meeting of the American Association for Cancer Research, April 1978. This work is being submitted in partial fulfillment of the Ph.D. requirements in the Department of Biology, Catholic University of America.  相似文献   

16.
Summary A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37°C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum,l-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmosphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland. This work was supported by the National Cancer Institute Contract N01-CP-75953 and in part by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, under Contract N01-CO-65341 with the International Union Against Cancer.  相似文献   

17.
A direct comparison of [3H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [3H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [3H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [3H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [3H]thymidine incorporation and isotope dilution assays.  相似文献   

18.
Culture conditions for maintaining first trimester human placenta in organ culture, which enhance the secretion of human chorionic gonadotropin (hCG), are described. Nutrient medium, oxygen tension and Gelfoam support matrix influence the synthesis of hCG by these cultures. Placental tissue remained viable for the duration of experiments (12 days) as judged by the incorporation of tritiated thymidine into DNA and the lack of release of incorporated [125I]iododeoxyuridine. Optimal conditions for hCG synthesis in placental organ culture included an atmosphere of 95% air and 5% Co2 (approximately 20% O2), CMRL 1066 medium containing fetal human or bovine serum, insulin, hydrocortisone and retinal acetate. Multiple pieces of placenta could be cultured in the same dish with an additive effect on hCG secretion. The functional responsiveness of these placental cultures was demonstrated by modulation of hCG synthesis with theophylline and 3'5' dibutyryl cyclic AMP.  相似文献   

19.
Summary Arrested BALB/c 3T3 cells were induced to the G0-G1 transition by fetal calf serum (FCS) and S phase entry was measured by [3H]thymidine incorporation as an index of DNA synthesis. [3H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [3H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum  相似文献   

20.
Placental transfer of lactate, glucose and 2-deoxyglucose was examined employing the in situ perfused placenta. Control and streptozotocin induced diabetic Wistar rats were infused with [U14C]-glucose and [3H]-2-deoxyglucose (2DG). The fetal side of the placenta was perfuseci with a cell free medium and glucose uptake was calculated in the adjacent fetuses. Despite the 5-fold higher maternal plasma glucose concentration in the diabetic dams the calculated fetal glucose metabolic index was not significantly different between the 2 groups. Placental blood flow was reduced in the diabetic animals compared with controls but reduction of transfer of [U14C]-glucose and [3H]-2-deoxyglucose and endogenously derived [14C]-Lactate to the fetal compartment, could not be accounted for by reduced placental blood flow alone. There was no significant net production or uptake of lactate into the perfusion medium that had perfused the fetal side of the placenta in either group. The plasma lactate levels in the fetuses adjacent to the perfused placenta were found to be higher than in the maternal plasma and significantly higher in the fetuses of the diabetic group compared with control group. In this model the in situ perfused placenta does not secrete significant quantities of lactate into the fetal compartment in either the control or diabetic group.  相似文献   

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