Rate Of 3H-thymidine Incorporation Research Articles

Age-associated T-lymphocyte activation through mitogens in patients with multiple sclerosis and blood donors.

In 128 patients with multiple sclerosis (MS) and 204 blood donors (control persons) the in vitro activation of 'Ficoll-purified' peripheral blood T lymphocytes was measured using the 3H-thymidine incorporation rate without (spontaneous proliferation) and with mitogen addition (concanavalin A, phytohemagglutinin). Mitogen responsiveness in control persons and MS patients decreased with age, reflecting a T-lymphocyte inherent mechanism for the diminution of responsiveness. The MS patients, ranging in age between 20 and 30 years, showed decreased mitogen responsiveness and a tendency to increased spontaneous proliferation compared to the control persons. These results could be an expression of a viral infection.

A long term culture of cells from Swarm rat chondrosarcoma in suspension after alginate preculture.

Chondrocytes from Swarm rat chondrosarcoma were successfully maintained in a suspension culture for over 18 months without losing its original phenotypic traits of producing tumor upon transplantation and of synthesizing cartilage-type proteoglycans. The chondrocytes were initially cultured by using a cell-encapsulation technique with alginate gels and were grown in an ordinary Dullbecco's modified Eagle's medium containing fetal bovine serum. The cells thus obtained after 18 months were morphologically indistinguishable from the chondrocytes in situ in the orginal tumor. No fibroblast-like cell was detected in the cultured cell population. When transplanted into rats, they grew at a rate comparable to that of original chondrosarcoma cells. This observation was supported, thought not directly, by measuring the rate of 3H-thymidine incorporation into their DNA fractions, which was also comparable to that of the original tumor cells. Biochemically, the cultured cells were able to synthesize proteoglycans at a similar rate to that by freshly isolated cells from the tumor. The molecular size and subclass of the proteoglycans produced within the cells or secreted to the extracellular medium were the same as those produced by the chondrosarcoma tumor cells and also to those synthesized by normal cartilage. Thus, the present culture system can be used as a suitable model to study the synthesis and assembly of the extracellular matrix in connective tissues, especially, those in cartilage.

Effect of somatostatin on the proliferation of mouse spleen lymphocytes [formula omitted

The effect of somatostatin on the spontaneous proliferation of mouse spleen lymphocytes was investigated in vitro . The rate of 3H-thymidine incorporation was used as an index of lymphocyte proliferation. Somatostatin in a concentration of 10 −7M enhanced the lymphocyte proliferation and abolished the antiproliferative effect of rat hypothalamic extract. Lower concentrations of somatostatin slightly decreased the lymphocyte DNA synthesis.

Cytokinetics of subpopulations in mixed polyploid tumors by television imaging. III. 3H-thymidine incorporation by ploidy subpopulations--control of 3H-absorption and emulsion efficiency in autoradiography.

An approach is presented for determining relative rates of 3H-thymidine incorporation in S-phase for discrete ploidy subpopulations that coexist in heteroploid tumors. The analysis is facilitated by an automated imaging methodology developed with a Quantimet 720 System for simultaneous determination of Feulgen-stained DNA content and grain counts of 3H-thymidine-labeled cells in Feulgen-stained autoradiographs (Sklarew RJ: J Histochem Cytochem 30:35, 30:49, 1982). The experimental design involves 3H- and 14C-thymidine double labeling in the presence of Colcemid. Data from heteroploid rat sarcoma cultures illustrate the profound influence of differential 3H absorption and relative emulsion efficiency. These factors are controlled, respectively, through measurements of mean nuclear optical density and grain density. This is essential for a valid comparison of grain counts between cells that differ in their autoradiographic geometry. Thus, relative DNA specific activity may be assessed in individual cells. The protocol and analysis supports a simultaneous evaluation of the S-phase ploidy composition and S-emptying profile, as presented in a companion article (Sklarew RJ: J Histochem Cytochem 32:413, 1984).

Prediction of the response to chemotherapy in acute leukemia by a short-term test in vitro.

To detect sensitivity or resistance of leukemic cells to chemotherapy prior to treatment, a short-term incubation method was employed. Blast cells from the peripheral blood or bone marrow of adult patients presenting with different forms of acute leukemia were analyzed for in vitro responsiveness to anticancer drugs in terms of suppression of 3H-uridine incorporation into cellular nucleic acids. The following agents were tested over a wide range of concentrations: Adriamycin, cytosine arabinoside, thioguanine, 6-mercaptopurine, prednisone, and 4-hydroperoxycyclophosphamide. Retrospectively, the in vitro data were compared to the clinical response of the patients to polychemotherapy. In the majority of the patients, in vitro cytotoxic effectiveness of Adriamycin (doxorubicin) and cytosine arabinoside reflected the in vivo situation. The levels of in vitro inhibition that could distinguish between drug-sensitive and drug-resistant diseases appeared to be 30% for Adriamycin and 20% for cytosine arabinoside. No correlation was found between the Adriamycin effect in vitro and the proliferative state (rate of 3H-thymidine incorporation) of the leukemic cell population. Serial in vitro sensitivity testing during the course of disease of various patients proved the ability of the test system to detect acquired resistance to chemotherapeutic agents. Therefore, the assay might serve as a reliable tool in the selection of effective chemotherapy in individual patients suffering from acute leukemia.

Comparison of the gastrointestinal growth-promoting effects of rat colostrum and mature milk in newborn rats in vivo

AbstractThe purpose of this study was to compare the effects of rat colostrum and mature milk on newborn rat gastrointestinal growth under conditions that controlled the possible confounding effects of energy intake and mode of feeding. Newborn Sprague-Dawley rat pups were tube-fed equicaloric amounts of rat colostrum or mature rat milk for 40 h before they were killed. Compared to littermates that were killed immediately after birth, both groups of fed rats demonstrated increases in the weights of stomach and intestine, but there was no organ weight difference detected between colostrum-fed and mature milk-fed rat pups. However, both the concentration of DNA and the rate of synthesis of DNA in the intestines were greater in rats fed colostrum than in those at birth or those fed mature milk. Although the pancreas exhibited no detectable increase in weight by 41 h, the DNA concentration and total DNA content increased and RNA/DNA ratio decreased in both fed groups, also without apparent difference between rats fed colostrum and those fed mature milk. The rate of 3H-thymidine incorporation into DNA in pancreas, however, was greater in colostrum-fed pups than in mature milk-fed pups. These differences at 40 h age in intestinal and pancreatic cell replication activity, but not organ weights, can be ascribed to feeding colostrum.

Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes.

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of 3H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominantly in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.

In vitro effects of cyclosporin A (CSA) on human hemopoietic cell lines

The sensitivity of 18 permanent hemopoietic cell lines to Cyclosporin A (CsA) was tested in a 3H-thymidine incorporation rate assay. Two human T cell lines (Molt4 and CEM) were significantly inhibited by a CsA concentration of 0.5 μg/ml. Not affected at all or only inhibited by 10 to 20 times higher CsA concentrations were: three human B cell lines (4413a, Daudi, Raji), a monkey B cell line (B95-8), a mouse plasmocytoma line (X63-Ag8/653), a human non-B T cell line (Reh), four human myeloid lines (HL-60, ML-1, ML-2, ML-3), a human myelomonocytic line (Karpas 230), four human monoblastic lines (U 937, SU-DHL-1, THP-1, Karpas 241) and a human erythroid line (K 562). It therefore seems that among permanently growing hemopoietic cells a cell type specificity for T cells also exists.

Effects of Cholera Toxin on Proliferation of Cultured Human Keratinocytes in Relation to Intracellular Cyclic AMP Levels

In the culture of epidermal keratinocytes, the cellular growth rate is reported to be accelerated by cholera toxin. The mechanism by which cholera toxin exerts biological effects is thought to result from changes in intracellular cyclic AMP concentrations. But in many reports cyclic AMP elevating agents appeared to inhibit growth of keratinocytes in culture. This study was done to clarify the discrepancy of this problem. Determination of cyclic AMP revealed that cholera toxin over a range of 10-14-10-8 M increased the intracellular concentration of cyclic AMP of cultured keratinocytes about 100-fold over the controls after incubation for 6 hr. When a small number (10(5)) of cells were inoculated in a 60 X 15 mm culture dish, cholera toxin strongly stimulated colony growth. When a relatively larger number (8 X 10(5)) of cells were inoculated in a dish, cholera toxin moderately accelerated cell division, and increased DNA and protein levels of the culture during early days of cultivation. But after about 20 days, of cultivation when the culture reached confluence, cholera toxin decreased both DNA and protein content in a culture dish. The cultures were pulse labeled with 3H-thyrmidine at 12 and 24 hr after the addition of 10-10 M cholera toxin, and its uptake into DNA was determined. In the early days of cultivation the uptake of 3H-thymidine increased after treatment with cholera toxin. But in the late days of cultivation, cholera toxin decreased the rate of 3H-thymidine incorporation into DNA. These results indicated that cholera toxin-cyclic AMP has effects on the proliferation of keratinocytes in culture biphasically according to cellular concentrations in culture.

Effects, in tissue culture, of serum from obese mice on the DNA synthesis of the pancreatic B-cell

In order to investigate whether the marked hyperplasia of the endocrine pancreas of obese-hyperglycemic mice (gene symbol ob ) is due to circulating growth factors, the effect on islet DNA synthesis of serum from obese mice and their lean littermates was tested in vitro. Fetal rat islets, which were maintained in tissue culture for three days in media differently supplemented with glucose and sera were subsequently labelled with 3H-thymidine to estimate DNA replication. Omission of serum from the culture medium markedly lowered DNA synthesis. In the presence of 10% fetal calf serum 16.7 mmol/l glucose enhanced replication more than 2.7 mmol/l. Mouse serum supported a high rate of 3H-thymidine incorporation which was not affected by the glucose concentration of the medium. Differences between serum from lean and obese mice were not demonstrated at any of the serum concentrations tested.

Population and developmental changes in thymidine uptake kinetics of Hymenolepis diminuta (Cestoda: cyclophyllidea)

1. 1. Thymidine uptake kinetics in 10-day old crowded (100 worms/rat), 10 day uncrowded (10 worms/rat) and 6-day old H. diminuta were investigated. Succinate, a partial mediator of the “crowding effect” was also tested for its effect on the thymidine uptake of uncrowded worms. 2. 2. Immature (6-day old) H. diminuta have a high affinity transport site for thymidine which persists in the anterior regions of 10-day old worms, but is overshadowed by the proliferation of lower affinity sites in the remainder of the 10-day old strobila. 3. 3. Lower rates of 3H thymidine incorporation into DNA by crowded or succinate-treated worms cannot be accounted for by differences in thymidine uptake characteristics. 4. 4. The inclusion of 14C-PEG in uptake experiments as a non-absorbable marker is of particular use when studying small, young worms with high surface to mass ratios.

IN VITRO CYTOTOXIC PROPERTIES OF PLASMA FROM UREMIC CHILDREN

This study was designed to compare the effects of control and uremic plasma on growth of cultured cells. Cell multiplication was measured by 3H-Thymidine incorporation during one population doubling time of mouse 3T3 fibroblasts. Uremic plasma were taken from severe uremic children (plasma creat. > 45 mM/1) and from hemodialysed (HD) patients: Plasma from HD patients were obtained at the beginning and end of a dialysis session. The 3H-Thymidine incorporation in the presence of uremic plasma was decreased (3520 ± 160 cpm, n = 13) as compared to the control incorporation (8500 ± 520 cpm, n = 14) (p < 0.01). Dose-response experiments showed a regular decrease in the rate of 3H-Thymidine incorporation in the presence of increasing amounts of uremic plasma. The comparison between the pre and post-dialysis incorporation values (3902 ± 664 cpm vs 9430 ± 1540 cpm, n = 8) showed an improvement in the 3H-Thymidine incorporation with postdialysis plasma (paired t test: p < 0.01). To obtain information on the properties of the cytotoxic material, incubations were made with ultrafiltrate (UF) of plasma and UF fractions obtained by G-15 Sephadex filtration. Uremic UF and fractions displayed no cytotoxic properties when added alone in the culture medium. The preincubation of uremic UF and fraction peak 7 (“middle molecules”) with control plasma restored a cytotoxic effect which was not different from that observed with uremic plasma. Conclusion: uremic plasma displays cytotoxic properties related to ultrafiltrable molecules which are no cytotoxic themselves but when mixed with plasma macromolecules.

Radiational basis and practice of the citrovorum factor (Leucovorin) protection after high-dose methotrexate therapy. High-dose methotrexate/leucovorin

The effects of methotrexate on the DNA metabolism of human lymphoblast cultures and bone marrow cells were estimated. The ratio of the 3H-deoxyuridine and 3H-thymidine incorporation rates ia a good parameter for the methotrexate effect. Citrovorum factor (5-formyltetrahydrofolic acid) reverses methotrexate toxicity only when its concentration exceeds that of methotrexate at least ten times. Guidelines for the practice of high-dose methotrexate therapy and the following citrovorum factor rescue, especially for patients with retarded methotrexate elimination are presented. A new simple formula enables the exact calculation of the citrovorum factor dose for individual patients according to their methotrexate serum levels. This formula, derived from the experimental data of the bone marrow and lymphoblast culture cells, gives a rational basis for the high-dose rescue with citrovorum factor in cases with expected or manifest toxicity, and means, moreover, a safety factor for the practice of high-dose methotrexate therapy.

DNA SYNTHESIS IN RANA PIPIENS TADPOLE LIVER DURING TRIIODOTHYRONINE-INDUCED METAMORPHOSIS1.

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during Ts-induced metamorphosis of R. pipiens liver was investigated. Rates of DNA synthesis were estimated by rates of 3H-thymidine incorporation into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. During T3-induced metamorphosis, periods of DNA synthesis and fluctuations in DNA content preceded expression of biochemical differentiation as measured by the enzyme arginase, and fluctuations in synthesis rates preceded corresponding fluctuations in content. The earliest response to T3-, was a 50% decrease in liver DNA, followed by increases in thymidine incorporation at 16 hr, 2 days, and 5-8 days. The size of the endogenous thymine pool was not significantly altered by T3 These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthetic rates and content during metamorphosis. Expression of thyroxin-induced development of the tadpole liver appears to be associated with both proliferation and cellular death, and metamorphosis of the liver cannot be occurring in a "fixed population of cells."

The Role of Auxin and Abscisic Acid in the Induction of Cell Division in Jerusalem Artichoke Tuber Tissue Cultured in vitro

Summary This paper deals with two specific questions with respect to the role of auxin and abscisic acid in the induction and promotion, respectively, of DNA synthesis in tuber tissue of Jerusalem artichoke (Helianthus tuberosus). The first of the question concerns with the need of an auxin during first few hrs of the wound response in the tissue, for induction of DNA synthesis and cell division. The second of the questions relates to the need for the simultaneous presence of NAA and ABA for the promotion of auxin-induced DNA replication. Evidence is presented here to show that a pre-incubation of the explants in the absence of auxin neither affects the duration of the lag period observed when auxin is provided, nor does it affect the number of cells/explant that respond to auxin at the later stage. If the tuber explants are pre-incubated in the absence of a hormone for 12 hrs before transfer to medium containing NAA, the synthesis of DNA does not commence until after the tissue has been in contact with the auxin for a time equal to the total duration of the lag period. The peak of DNA synthesis is also delayed by about 12 hrs in these explants as compared to the controls. While an auxin is required during the whole length of the lag period for the induction of DNA replication, it does not seem to be true of ABA for the promotion of auxin-induced DNA replication. When the explants are pre-incubated in NAA (1 mg/1) for 12 hrs and then transferred to NAA + ABA, the rates of 3H-thymidine incorporation at different times during the S period are quite comparable with those grown in a mixture of NAA + ABA from the very beginning. On the basis of the data presented here, it is suggested that auxin acts on about 60–70% of the cells in the explant to initiate a sequence of events that can eventually lead to DNA synthesis in these cells. But only about half of these cells (about 30–35% of the total cell population) actually undergo DNA synthesis, while the other half are somehow inhibited from doing so. Abscisic acid if given from the beginning or just before the initiation of DNA replication, somehow overcomes this inhibition and as a result all of the auxin-activated cells enter the S phase.

Coordinate control of balb/c3T3 cell survival and multiplication by serum or calcium pyrophosphate complexes

Balb/c3T3 cells in crowded cultures detach from the dish when deprived of serum, and the survivors incorporate 3H-thymidine at a reduced rate. The detachment becomes pronounced two hours after removal of serum, and reaches its maximum rate between two and four hours. Cells in sparse culture are not detached by serum removal, and their rate of 3H-thymidine incorporation is only slightly reduced. As the sparse cultures grow into more crowded cultures, and the serum is depleted, increasing numbers detach. The detached cells are incapable of reattaching when placed in a new dish with ample fresh serum. The cells are leaky to cellular constituents and appear to be dead. Detachment is a consequence rather than the cause of cell death, and can be produced by agents which inhibit cellular energy metabolism. The cells on the dish which survive serum deprivation are fully viable and grow rapidly when serum is added. When they become crowded they are as sensitive to serum deprivation as was the original population. They are therefore not selected for a low serum requirement but apparently survive because they spread into the space vacated by the detaching cells and then behave as sparse cultures in response to serum variations. Insoluble complexes of Ca2+ and pyrophosphate (Ca2+-PPi) show the same concentration dependence in promoting cell survival as in stimulating 3H-thymidine incorporation, showing that a single substance can be responsible for both activities. It is concluded that survival and growth are part of the coordinate response of 3T3 cells to single external effectors. The results are discussed in terms of a simple model in which the coordinate response is regulated by the availability of Mg2+ for transphosphorylation reactions within the cell, and the availability depends on the binding affinity of cellular membranes for Mg2+. The difference between survival and multiplication is postulated to be in the intensity and duration rather than the kind of stimulus.

Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells

Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0-0.1 U insulin/ml resulted in a 17-fold increase in the rate of 3H-thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) AND Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H-thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the centtral agent in the coordinate control of metabolism and growth in animal cells.

Reversible regulation by magnesium of chick embryo fibroblast proliferation.

The rate of 3H-thymidine incorporation and of cell proliferation in chick embryo fibroblast cultures are reduced coordinately when the [Mg2+] of the external medium is reduced below the physiological concentration of about 0.8 mM. These effects of moderately reduced [Mg2+] and the accompanying change in appearance of the cells, resemble the effects produced by lowering the [serum] of the medium. Cells subjected to severe Mg2+ deprivation, especially at low [Ca2+], die and detach from the culture dish. Cells kept at a reduced rate of proliferation for three days by moderate Mg2+ deprivation are quickly restored to rapid proliferation upon restoration of the normal [Mg2+] of the medium. The rate of proliferation of the chick embryo cells is reduced markedly by lowering [Ca2+] about 100-fold, but unlike the case of Mg2+-deprivation this can occur without significant effect on the rate of 3H-thymidine incorporation. More severe Ca2+ deprivation, which does lower the rate of 3H-thymidine incorporation, produces retraction of cells from one another and from the dish, and results in a distinctly abnormal, rounded appearance. The results lend weight to the thesis that free [Mg2+] plays a central role within the cell in the coordinate control of metabolism and growth. They also suggest that the effects produced by varying [Ca2+] in the medium are caused by changes at the external surface of the cell.

Reversible tenfod reduction in mitochondria DNA content of human cells treated with ethidium bromide.

Cells of the human line VA2-B in suspension culture have been treated with very low concentrations of ethidium bromide for the purpose of reducing the amount of mitochondrial DNA (mit-DNA) per cell. Cells maintained in the presence of 5 ng/ml ethidium bromide grew at a normal rate for three days; thereafter, their doubling time gradually increased to a stable value of about 60 h. In these cells, the rate of 3H thymidine incorporation into mit-DNA decreased very rapidly to approximately 60% of the normal, and remained thereafter at this level, while the amount of mit-DNA per cell stabilized around a level of 70--80% of the control. In cells long-term treated with 5 ng/ml ethidium bromide, the rate of mitochondrial protein synthesis was about 35% of the normal, and the cytochrome c oxidase activity about 50% of the control. Cells treated with 20 ng/ml of the drug underwent 3--4 cell doublings at control rates, then gradually stopped growing, and eventually died. In these cells, the rate of incorporation of 3H thymidine into mit-DNA was reduced to 50% of the control value after 10 min treatment with ethidium bromide, and became barely detectable after three cell doublings. At this time, the cells had on the average less than 10% of the control amount of mit-DNA, the rate of mitochondrial protein synthesis was reduced to 3% of the normal, and the specific activities of cytochrome c oxidase and rutamycin-sensitive ATPase were less than 20% of the control values. In spite of these marked changes, the cells exhibited only a 20--30% loss in cell viability, as estimated by cloning efficiency, after three days of exposure to the drug. Cells treated with ethidium bromide at 20 ng/ml for three days, and then transferred to drug-free medium, recovered a near-to-normal growth rate and cloning efficiency and a near-to-normal rate of synthesis and amount of mit-DNA in about five days.

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of3H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication.