Radiothymidine Incorporation Research Articles

Cell cycle effects of nitric oxide on vascular smooth muscle cells.

We characterized the cell cycle block induced by nitric oxide (NO) on smooth muscle cells (SMC). We hypothesized that the inhibition of SMC proliferation by NO was due to a specific block in cell cycle progression. Treatment of cultured rat aortic SMC with the NO donors S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione (0.1 mM for 48 h) resulted in a 50% decrease (P < 0.05) in the fraction of cells in the S and G2 + M phases and a corresponding increase in the G1 fraction, suggesting that NO inhibits entry into S phase, causing accumulation of cells in G1 phase. Application of both NO donors to cycling SMC resulted in a short-term, concentration-dependent (0.06-0.3 mM) inhibition of ongoing DNA synthesis as measured by radiothymidine incorporation, demonstrating that NO causes an S-phase arrest. The S-phase arrest by NO was not mimicked by exogenous guanosine 3',5'-cyclic monophosphate (cGMP, 10 mM) and was associated with, but not due to, a 20% inhibition of RNA synthesis. The S-phase block was completely reversed within 2 h of removal of the NO donors, similar to inhibition by the ribonucleotide reductase inhibitor hydroxyurea. Prolonged treatment of SMC with either NO donor (0.1 mM) did not synchronize cells at the G1-S boundary as expected after a prolonged S-phase arrest, but instead induced a quiescent G0-like state characterized by a 12- to 18-h lag before DNA synthesis returned to normal levels after NO removal. These findings demonstrate that NO inhibition of SMC proliferation is associated with two distinct and reversible cell cycle arrests, an immediate cGMP-independent S-phase block followed by a shift back in the cell cycle from the G1-S boundary to a quiescent G0-like state.

Inhibition of growth by calcitriol in a proximal tubular cell line (OK)

Calcitriol has been shown to inhibit (i) cell proliferation of renal carcinoma cell lines and of cultured adult human mesangial cells in vitro, and (ii) renal compensatory growth in vivo. In the present study we examined the effects of calcitriol on DNA synthesis and cell replication in an immortalized cell line showing the phenotypic characteristics of proximal tubular cells (opossum kidney, OK cells). The viability of OK cells was not affected by calcitriol (Trypan-blue exclusion, LDH and K+ release), but the cells did not convert 3H-25(OH)2D3 to 3H-1,25(OH)2D3. In the log growth phase, calcitriol (but not alternative vitamin D metabolites) caused dose-dependent (10(-12) to 10(-6) M) inhibition of radiothymidine incorporation. Inhibition was calcium dependent, i.e. it was more pronounced at the lower nominal calcium concentration in tissue culture media (0.9 versus 1.8 mmol/l) and amplified by coincubation with nifedipine (1 microM). Inhibition of DNA synthesis was paralleled by inhibition of cell replication (growth curve) under basal conditions and after stimulation with EGF (10 ng/ml). In conclusion, calcitriol inhibits proliferation of proximal tubular cells which normally express 1-alpha-hydroxylase activity.

Intermittent and continuous exposure to 1,25(OH)2D3 have different effects on growth plate chondrocytes in vitro

Intermittent 1,25(OH)2D3 administration is widely used to suppress parathyroid glands in secondary (renal) hyperparathyroidism. It is unknown whether the effects of continuous and intermittent 1,25(OH)2D3 differ on vitamin D target organs other than parathyroids. Using primary cultures of rat chondrocytes (tibia) we compared the effects of continuous versus intermittent exposure to physiologic concentrations of 1 alpha,25(OH)2D3 on proliferation (radiothymidine incorporation), cell count, protein synthesis ([3H]-leucine incorporation), alkaline phosphatase activity (as a marker of differentiation) and 1 alpha,25(OH)2D3 receptor (VDR) regulation. Cells were synchronized and then exposed for variable periods to a medium containing 10% delipidated FCS and 10(-8) M to 10(-12) M 1 alpha,25(OH)2D3 (or 1 beta,25(OH)2D3 as specificity control). Intermittent (8 hr exposure every 48 hr) as well as continuous (sham washing) administration of 1 alpha,25(OH)2D3 had a biphasic effect on proliferation, that is, stimulation at low (10(-12) M) and inhibition at high (10(-8) M) concentrations. At 10(-12) M intermittent 1 alpha,25(OH)2D3 yielded higher cell counts than continuous 1,25(OH)2D3. This was seen in the log phase, which was day 3 (continuous 141 +/- 2.3% of solvent control; intermittent 185 +/- 2.0%) and in the plateau phase of growth, which was day 6 (128 +/- 2.6 vs. 169 +/- 2.7% of solvent control). Dependence on extracellular Ca is suggested by the effects of varying nominal Ca concentrations in the medium and of Ca channel blockers. Even two hours of exposure to 1 alpha,25(OH)2D3 (10(-12) M) yielded maximal activation of AP during postincubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of L-Dopa Methylester and Glutathione Depletion on Murine B16BL6 Melanoma Growth In Vitro

The cytotoxic and growth-inhibitory effect of levodopa methylester (LDME) in murine B16BL6 (BL6) melanoma cells after glutathione (GSH) depletion was studied in vitro. Pretreatment of BL6 cells with 50 microM buthionine sulfoximine (BSO) depleted GSH content by nearly 90% and enhanced the growth-inhibitory effect of even a minimally cytotoxic concentration of LDME. Radiothymidine incorporation into BL6 cells significantly increased compared to untreated controls during the first 4 h of exposure to 0.2 mM LDME. However, pretreatment with BSO prevented this LDME-induced increase in radiothymidine incorporation. Because the percentage of cells in S-phase of the cell cycle was not altered, these results suggest that BSO exposure may be inhibiting unscheduled DNA synthesis, which could contribute to the cytotoxic effects of LDME. In addition, spectrophotometric studies indicated that in a cell-free system, GSH scavenged dopaquinone produced by the tyrosinase-mediated oxidation of LDME, presumably by formation of glutathionyldopa. Thus, enhancement of LDME cytotoxicity by BSO may also involve depleting the amount of GSH available for the nucleophilic addition to the quinone.

1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia

Parathyroid cell proliferation and parathyroid hyperplasia are features of renal secondary hyperparathyroidism. Since parathyroids have recently been recognized as an important target for 1,25(OH)2D3, the effects of administration of variable doses of 1,25(OH)2D3 on ex vivo radiothymidine incorporation in the parathyroid glands, on parathyroid cell mitoses, on parathyroid weight, morphometric indices and on parathyroid protein/DNA ratio were examined in rats with uremia (subtotal nephrectomy; NX) or with calcium deficiency. 3H-thymidine incorporation (3 hr; 37 degrees C; PBS with 10 mmol glucose) was elevated in NX animals, that is, 204 +/- 51 dpm/micrograms DNA versus 96 +/- 28 in controls. In vivo pretreatment with 1,25(OH)2D3, either by intermittent i.p. injection or by osmotic minipump, dose-dependently decreased 3H-thymidine incorporation and parathyroid cell mitoses without affecting morphometric indices of parathyroid cells. Prophylactic administration (i.p.) of 1,25(OH)2D3, starting on the day of nephrectomy, prevented parathyroid hyperplasia (NX + 1,25(OH)2D3 0.84 micrograms tissue/g body wt vs. 1.25 micrograms in untreated NX and 0.54 in ad libitum fed controls), but 10 days of treatment beginning on the 21st day of uremia did not reverse existing hyperplasia (NX + 1,25(OH)2D3 1.5 micrograms/g body wt vs. 1.37 micrograms in untreated NX and 0.56 micrograms in ad libitum fed controls). The inhibitory effect was specific for 1,25(OH)2D3 and not imitated by Dexamethason. However, the effect was not specific for parathyroid hyperplasia of uremia, since similar inhibition of 3H-thymidine incorporation by 1,25(OH)2D3 was also observed in rats on low calcium diet.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Corticosterone on the Growth of Perinatal Rat Lung Fibroblasts in Culture: Modulation by the Extracellular Matrix: (Cell culture/endothelial cells/extracellular matrix/fibroblasts/glucocorticoids).

Third passage lung fibroblasts from rat pups between the ages of fetal day 17 and postnatal day 15 were allowed to attach onto either tissue culture plastic or an endothelial cell-derived matrix, and were then exposed to different concentrations of corticosterone in culture medium containing 1% charcoal-stripped serum. The effect of the hormone on growth of these cells was assessed after 48 hours of exposure by radiothymidine incorporation into DNA. Lung fibroblasts on plastic responded to the hormone in an age-dependent manner; thus, cells which were obtained during late gestation (days 19-21) and after the third day of extrauterine life were consistently growth-inhibited by corticosterone, whereas those which were obtained from fetal donors prior to day 19 and from neonatal donors (days 0-3 after birth) were stimulated to grow in response to the hormone. On the other hand, cells that were plated onto an endothelial cell-derived matrix showed a different age-related response to the hormone. Thus, lung fibroblasts from all fetal donors and from postnatal donors up until 4-5 days of age were stimulated to grow in response to corticosterone. This modulatory effect of the matrix on the fibroblast response to corticosterone was also seen in early passage fibroblasts. In an attempt to identify the modulatory agent(s) in the extracellular matrix, fibroblasts from day 19 fetal rat lung were challenged with corticosterone in the presence of laminin, fibronectin, type IV collagen, type I collagen, heparin, or chondroitin sulfate. None of these agents exerted a modulatory effect resembling that seen with the extracellular matrix. These results suggest the existence during lung development of a fibroblast-endothelial interaction, the nature of which remains to be elucidated, which may serve to modify the effects of circulating hormones.

17 Beta-estradiol-sensitivity of cultured myometrial cells.

The estrogen sensitivity of cells cultured from the rat myometrium was studied by growing the cells in the absence or presence of 1 nM 17 beta-estradiol. Following a time lag of approximately 10 days, exposure to estrogen resulted in increased incorporation of radiothymidine by the cells. Estrogen treatment also decreased isoproterenol-dependent and GTP-dependent adenylate cyclase activity, but had no effect on basal activity. These cultured cells have been shown previously to have some properties of uterine smooth muscle. The effects estrogen has in vitro, therefore, may reflect important properties in vivo that account for the mechanism by which the sex steroid decreases the sensitivity of the myometrium to isoproterenol.

Incorporation of [35S]sulphate and [3H]thymidine into isolated bovine articular chondrocytes in vitro. Effect of autogenous conditioned synovial medium.

The present work is a part of a study series concerned with the metabolic events taking place in the synovial joint tissues. We have previously reported that synovial products not only enhance the degradation of articular cartilage proteoglycans but also inhibit the synthesis of articular cartilage proteoglycans, and that these cellular interactions may be partly modulated by arachidonic acid metabolites. In the experiments reported here we show that the addition of conditioned synovial medium to confluent monolayer cultures of articular chondrocytes not only inhibits the synthesis of proteoglycans, measured as the incorporation of radiosulphate into cartilage proteoglycans but also inhibits the synthesis of DNA, measured as the incorporation of radiothymidine into the chondrocytes, in a dose-dependent manner. In our culture system, calf knee-joint synovial tissue produces PGE, and it has been reported that previous exposure to PGE2 affects the ability of the cell to respond to that same hormone. In these experiments, previous exposure to conditioned synovial medium affected neither the inhibition of the incorporation of radiosulphate nor the inhibition of the incorporation of radiothymidine.

Somatomedin activity in tracheal fluid from the newborn infant.

Somatomedin activity was determined by porcine cartilage bioassay and somatomedin-C RIA in matched samples of tracheal fluid (TF), amniotic fluid (AF), and cord (CP) and maternal plasma collected during the delivery of normal infants of 36-40 weeks gestation. Somatomedin levels determined by both assay systems were significantly greater in TF than in AF [TF, 0.40 +/- 0.04 (+/- SE) U/ml; AF, 0.20 +/- 0.02 U/ml by bioassay in 11 infants; TF, 0.25 +/- 0.04 U/ml; AF, 0.18 +/- 0.03 U/ml, by RIA in 6 infants; P less than 0.005]. When chromatographed on Bio-Gel P10 at pH 3.5, the major part of the somatomedin immunoreactivity from both TF and CP eluted in the same position as iodinated somatomedin-C (mol wt, 7600). Somatomedin activity recovered after chromatography of TF and CP increased the incorporation of radiothymidine by growth-restricted human fetal fibroblasts in vitro. Maternal plasma contained 2.49 U/ml radioimmunoassayable somatomedin-C, a value higher than that in the nonpregnant adult. The results suggest that the human fetal lung contributes somatomedin to TF and raises the possibility that these peptides are involved in lung growth or maturation.

Somatomedin activity in tracheal fluid from the newborn infant

Abstract Somatomedin activity was determined by porcine cartilage bioassay and somatomedin-C RIA in matched samples of tracheal fluid (TF), amniotic fluid (AF), and cord (CP) and maternal plasma collected during the delivery of normal infants of 36–40 weeks gestation. Somatomedin levels determined by both assay systems were significantly greater in TF than in AF [TF, 0.40 ± 0.04 (±SE) U/ml; AF, 0.20 ± 0.02 U/ml by bioassay in 11 infants; TF, 0.25 ± 0.04 U/ml; AF, 0.18 ± 0.03 U/ml, by RIA in 6 infants; P < 0.005]. When chromatographed on Bio-Gel P10 at pH 3.5, the major part of the somatomedin immunoreactivity from both TF and CP eluted in the same position as iodinated somatomedin-C (mol wt, 7600). Somato-medin activity recovered after chromatography of TF and CP increased the incorporation of radiothymidine by growth-restricted human fetal fibroblasts in vitro. Maternal plasma contained 2.49 U/ml radioimmunoassayable somatomedin-C, a value higher than that in the nonpregnant adult. The results suggest th...

Influence of glucocorticoids on postnatal lung development in the rat: possible modulation by thyroid hormone.

Between the 4th and 10th days of postnatal life in the rat, serum corticosterone levels were low and basal, while the rate of [3H]thymidine incorporation into lung DNA was maximal. From day 13, serum corticosterone levels began to rise significantly, and the lung [3H]thymidine incorporation rate began to fall dramatically; however, these events were obtunded by propylthiouracil-induced hypothyroidism. When 6- to 8-day-old euthyroid pups were given a single sc injection of 10 micrograms dexamethasone, the rate of DNA synthesis in the lung fell by 96.7% of the initial rate at 24 h. This steroidal effect was blunted in hypothyroid pups and restored by exogenous thyroid hormone. The thyroid status of the pup did not modify the response patterns of lung phosphodiesterase and cytosolic glucocorticoid receptor levels to dexamethasone treatment, although both parameters were influenced by thyroid hormone availability. Radiocholine incorporation into lung phospholipids, which was altered in hypothyroidism, was unaffected by dexamethasone treatment. An in vivo assessment of radiothymidine incorporation into DNA of various tissues in 5-day-old euthyroid pups given 10 micrograms dexamethasone 24 h earlier revealed that of the several tissues in which inhibition of DNA synthesis was demonstrable, the developing lung was the most sensitive to the anti-mitogenic steroidal effect. When considered in the light of existing evidence, these observations suggest that glucocorticoids play an important role in triggering lung cytodifferentiation during the third postnatal week in the rat, and that preconditioning of the lung by thyroid hormone optimizes this developmental effect of glucocorticoids.

Proliferation kinetics of human endometrium during the normal menstrual cycle

A radioautographic analysis of the number and distribution of labeled nuclei after in vitro incorporation of radiothymidine was carried out in human endometrium during the normal menstrual cycle. The highest rates of proliferation were recorded on cycle days 8 to 10 in the upper one third of the functionalis layer. Coinciding with intraglandular secretory activity on cycle day 19, the rate of proliferation of gland cells decreased near zero levels and remained unchanged thereafter. Midsecretory phase arteriolar coiling and stromal predecidualization were associated with increased isotope incorporation. DNA synthesis in premenstrual endometrium was limited to the surface epithelium, predecidua, and vascular endothelium. The endometrium of the isthmus and cornual regions and of the basalis zone demonstrated relatively constant and comparatively lower rates of proliferation throughout the cycle. Regional and cellular variations in nucleic acid synthesis in cyclic endometrium are likely to reflect differences in physiologic functions in target cells in response to cyclic hormonal stimuli.

The role of H-2 and H-2-like determinants in syngeneic and allogeneic cytostasis.

Specific sensitization against H-2 determinants was affected by immunizing allogeneic mice with spleen and lymph node cells in H-2 congenic combinations. Lymph nodes from the sensitized and non-sensitized mice were respectively cultured together with H-2 syngeneic tumour cell lines. The growth and viability of the tumour cells was subsequently measured by the amount of radiothymidine incorporation. If the tumour cells incorporated less isotope when cultured with the immune cells than with the normal cells this was termed 'cytostasis'. To identify the H-2 genes controlling the sensitization phase in the cytostasis assay, we studied the effect on different transplantable tumour target cells of lymphoid cells from mice sensitized against different congenic spleen cells. The results suggest that the cytostasis assay can can measure an in vitro specific response to H-2-incompatible sensitizing antigens, and that I--B incompatibility, together with K and/or D, is essential to produce effectors. Furthermore, H-2 allogeneic sensitization could induce cytostasis even against tumour cells syngeneic for the H-2 halotype of the responder strain. The implications of these findings are discussed.

Fine structure and replication of the kinetoplast of Trypanosoma lewisi.

The kinetoplastic DNA of Trypanosoma lewisi is described as a filamentous body lying within a mitochondrion, with the filaments oriented parallel to the long axis of the cell. The manner of fixation, the replicative state, and perhaps the physiological state of the cell, may result in slight morphological differences among such bodies. The kinetoplastic DNA replicates to form "left" and "right" rather than "upper" and "lower" members, and both the kinetoplast and nucleus incorporate radiothymidine as shown by radioautography. Radioautographic analyses suggest a random incorporation of radiothymidine by kinetoplasts. Silver grains were occasionally observed over centriolar elements. Finally, the observations are discussed with respect to the sequential replication of the aforementioned organelles by T. lewisi.

X-irradiation of splenic lymphocytes. I. Thymidine pools and radiothymidine incorporation into DNA.

An immediate suppression of radiothymidine incorporation into DNA is found in splenic lymphocytes of the golden hamster when they are irradiated in vitro in the kilorad range (Manasek, Adelstein and Lyman 1966). Although the mechanism of this suppression has not been elucidated, Smets (1966 a) has put forth the hypothesis that at least part of the apparent depression in DNA synthesis is due to changes in the precursor-pool size. A number of investigators, working with a wide range of biological systems, have suggested that the decreased uptake of radiothymidine into DNA often observed after irradiation is due to an expansion of nucleoside and nucleotide pools consequent on DNA degradation and not to a net decrease in DNA synthesis (Hell, Berry and Lajtha 1960, Kelly 1961, Nygaard 1962, Smets 1966b). Several experimental approaches have been utilized to test for such amplification of intracellular pools. In one (Hell et al. 1960, Smets 1966b), the precursor is varied over a wide concentration range in the hope of eliminating pool effects by flooding. In the experiments to be described, the method is applied to x-irradiated splenic lymphocytes. Cell suspensions were prepared from the spleens of mature, male, golden hamsters (Manasek et al. 1966). From radioautographs we have determined that the predominant nucleated cell which is synthesizing DNA is a medium-sized lymphocyte. The minced tissue was passed through stainless-steel screens for dispersion, and the cells were suspended in Hanks' salt solution at pH 7-25. They were subsequently exposed to unfiltered 250kvp x-rays generated at a dose-rate of 300 rads per minute as determined with ferrous ammonium sulphate. The suspensions were maintained at a controlled temperature throughout their irradiation. Immediately thereafter, the cells were incubated with tritiated thymidine (specific activity 2 Ci/mmole) at 37°c for 30 min. DNA was isolated by a modification of Schmidt and Thannhauser's method (Munro and Fleck 1966). In this assay the incorporation of thymidine into DNA is linear for 4 hours in control and up to 1 hours in irradiated suspensions. In figure 1, the uptake of radiothymidine into the DNA of unirradiated splenic lymphocytes is shown for total thymidine concentrations varied from 1 x 10 - 7 M to 35 x 10- M. The 350-fold increase in concentration results in a ten-fold increase in radiothymidine incorporation. The ' S ' shape of the uptake curve suggests that the overall incorporation obeys Michaelis-Menten kinetics. Under

Induction of Aberrations in Meiotic Chromosomes of Wheat by Means of Thymidine-H 3

Embryos removed from wheat seeds which had taken up water for 4 hr were incubated in thymidine-d for 4 hr, and then grown on isotope-free medium for 1 hr. No activity was found in these embryos. Since 16 hr is required for visible germination at 25 deg C, it may be assumed that cell division with consequent incorporation of radiothymidine in DNA must begin sometime during the second half of the pregermination period. Localization of thymidine-d during the periods 1 to 5, 4 to 9, and 8 to 13 hr after germination appeared to be in about the same amounts and indicated that cell division was proceeding at an even rate over these periods. The primary root, both lateral roots, and coleoptile were the most active organs. The first foliage leaf was the next most active component. The high incidence of stunting and retardation in wheat seediings after germination of seeds in thymidine-d was due largely to radiation injury of the early root system. Injury to the seedling root system was also the commonest effect after embryo incubation in thymidine-d; effects in the epicotyl were slightly fewer. This would be expected in view of thymidine localization. During the period 24more » to 48 hr after germination, the first lateral roots surpassed the primary root in uptake of thymidine-d as they enlongated and passed the primary in length. The coleoptile, which doubled in length during this period, had a thymidine-d content slightly less than in the earlier periods. Cell elongation was, therefore, the chief contributor to coleoptile growth during this period. Increase in lengih of the first foilage leaf during this period was also through cell elongation. Thymidine moved from the endosperm to the developing embryo. The low yields of chromosome aberrations in pollen mother cells of adult plants arising from embryos incubated in thymidine-d or from seedlings with epicotyls cut back and then immersed in radiothymidine are accounted for on the basis of the following considerations. There are about 200 cells in the growing point at 24, 48, or 72 hr postgermination. The uptake of thymidine-d in the growing point was limited because oniy a few cells were in division, and the number of cells in DNA synthesis at any one time would therefore be small. Thus, radioautographs of serial sections showed strong label in the growing point in oniy two of fifteen embryos given a 24-hr exposure to thymidine-d, even when coleoptiles were cut back and the embryo was submerged upside down to ensure contact with isotope. The growing point was surrounded by organs which contained many dividing cells. Pronounced label was found in coleoptile, first foliage leaf, second foliage leaf, meristematic area proximal to growing point, third foilage leaf, and bases of roots in radioautographs of serial sections. Although some seedlings died of radiation damage after embryo treatment with thymidine-d, many eventually recovered the normal phenotype. This suggests that enough undamaged or only partially damaged cells survived within the injured organs to proliferate and eventually produce normal structures. The incidence of aberrations in meiotic chromosomes increased when exposure time of the growing point to isotope was increased and isotope was replenished to ensure its availability to the relatively few cells of the growing point undergoing DNA synthesis at any one time. (auth)« less