Incorporation Of Labeled Thymidine Research Articles

Androgens control androgen-binding sites in rat epididymis.

Previous results suggested that the number of androgen-binding sites in rat epididymis was controlled by androgens. Using an exchange technique for receptor determination, we now report that cytoplasmic receptor number decreased to about half the control value after 4 days of castration and reached its lowest level (30% of the control value) after 12 days of castration. The Kd for [3H]methyltrienolone for cytoplasmic receptor varied from 2.8 X 10(-9) M in controls to 0.6 X (10-9) M (P less than 0.001) after 6 or more days of castration. Nuclear binding sites were reduced to 17% of the control value from the second day of castration on, but no change in the affinity for methyltrienolone was noted. The administration of testosterone propionate (400 micrograms/day) to rats castrated for 20 days significantly increased the number of nuclear and cytoplasmic binding sites from the second and third days of treatment, respectively. Labeled thymidine incorporation into DNA rose significantly after a 4-day latency period. The proportion of total binding sites capable of translocation into the nucleus was elevated during the early phase (2-4 days) of androgen treatment. These results also suggest the heterogneity of the cytoplasmic binding site population. Control epididymides were composed of 61.5% receptor-containing epithelial cells, and this proportion was significantly reduced to 53.2% after 20 days of castration. Androgen administration elevated this percentage after an 8-day latency period. Proteolytic activity in cytosol was increased over control values after castration (for 20 days) and until the fourth day after the onset of androgen treatment. However, this activity does not seem to be an important factor in the decrease in binding sites caused by orchidectomy.

Changes in trophic and functional parameters of the rat epididymis during sexual maturation.

We have previously shown that the metabolism of androgens in rat epididymis suffers a transient deviation between Days 20 and 30 of life during which the formation of 3α,17β-dihydroxy-5α androstane is favored over that of 5α-dihydrotestosterone. Searching for the biological significance of this phenomenon, we have studied some trophic and functional parameters of the epididymis in rats 15, 20, 25, 30, 40, 50, and 90 days of age. The protein content of the organ reaches a maximum in the 25-day-old rat. The relative DNA content of the epididymis (μg DNA/mg dry weight) increases fourfold between Days 15 and 25 and decreases thereafter. Labeled thymidine incorporation at Day 25 is double that found at Days 20 and 30 (P<0.05). This phenomenon is not seen in prostate. The number of cells undergoing mitotic division in the epididymal epitheium of the 25-day-old rat was double that found at all other ages studied. The content of glycerylphosphorylcholine was lowest in the 20-day-old rat (P<0.05 vs 15 and 25 days) while zinc was increased threefold at the same age (P<0.01 vs 15 and 25 days). N-Acetyl-neuraminic acid increased by ~50% between Days 20 and 25 (P<0.05). Therefore, the wave of cell replication and the increases in zinc and sialic acid content of the epididymis can be singled out as the most marked changes bearing a temporal coincidence with the shift in androgen metabolism. If the diol shift is the signal to trigger these events, the response can be qualified as atypical since only some parameters of epididymal function are involved.

Mesenchymal cells from the human embryonic palate are highly responsive to epidermal growth factor.

An established line of mesenchymal cells from the human embryonic palate is highly sensitive to the stimulatory effect of epidermal growth factor on growth, labeled thymidine incorporation, and ornithine decarboxylase activity. The results suggest that epidermal growth factor may play a key role in development of various human embryonic and fetal tissues.

Effects of prolactin on DNA synthesis and growth of the accessory sex organs in male mice.

The role of PRL in the development of male accessory sex organs remains unresolved. In the present studies, the influence of PRL and testosterone upon DNA synthesis and cell proliferation was examined in the anterior prostate gland (AP) and the seminal vesicles (SV) of mice. Hormonal effects on DNA synthesis were evaluated by examining the in vitro incorporation of [3H]thymidine into DNA in relation to temporal alterations in tissue DNA content. In intact mice, PRL (300 IU/kg daily) produced peak stimulation of [3H]thymidine incorporation into DNA by SV after 3 days of treatment. In contrast, PRL failed to alter [3H]thymidine incorporation by the AP. Only in SV did PRL injections lead to elevated levels of tissue DNA. Injections of testosterone (0.75 or 7.5 mg/kg daily for 3 days) to castrated mice also produced significant stimulation of labeled thymidine incorporation into DNA by AP and SV. Concomitant injections of PRL (150 IU/kg) and testosterone (0.75 or 7.5 mg/kg) enhanced the stimulatory effects of this androgen on DNA synthesis and DNA content in the SV, but not in the AP. When PRL alone was administered to castrated mice, it failed to affect either [3H]thymidine incorporation into DNA by AP and SV or accessory sex organ weights and DNA contents. The results of these studies suggest that PRL enhances the effects of androgens upon DNA synthesis and growth of the seminal vesicles, but not of the anterior prostate glands of mice.

DNA Synthesis in Generation 1 in X-Irradiated HeLa Cells

HAWKINS, R. B., TOLMACH, L. J., AND GRIFFITHS, T. D. DNA Synthesis in Generation 1 in X-Irradiated HeLa Cells. Radiat. Res. 85, 85-98 (1981). Measurements of DNA replication in a line of HeLa S3 cells during the generation (Generation 1) following that in which the cells are irradiated with 500 rad of 220-kV X rays (Generation 0) were carried out according to a number of different experimental protocols. These involved preirradiation labeling of the cells with low levels of ['4C]thymidine in Generation - 1 to provide a measure of the template DNA, synchronization by mitotic collection in Generation 0, resynchronization by either mitotic recollection or temporary drug-induced blockades in Generation 1, and either labeled-thymidine incorporation or density-label transfer during Generation 1. The results show that those cells that progress through S phase of Generation 1 and divide at the next mitosis replicate a full complement of DNA, within the approximately 10% uncertainty of the experimental methods. However, apparent deficits of as much as 45% are measured if resynchronization in Generation 1 is effected by drug treatment following manipulations of the culture in the presence of particular media and drugs during Generation 0. These are attributed to combined radiation- and drug-induced disturbances in cell progression.

Effects of diethyltindichloride on amino acid and nucleoside transport in suspended rat thymocytes

The effects of diethyltindichloride (DETD) on basic biochemical processes of thymus cells were investigated in order to develop an understanding of the mechanism by which dialkyltins act on the thymus of rodents in vivo. Accumulation of the nonmetabolizable amino acid α-aminoisobutyrate (AIB) was markedly inhibited by the dialkyltins in suspended rat thymocytes. The inhibitory effects on AIB influx were apparent after as little as 5 min of thymocyte preincubation with DETD. Low dialkyltin concentrations, which did not inhibit AIB influx in noninsulin-stimulated cells, abolished the insulin-mediated AIB influx. Addition of lipoic acid (reduced) to the incubation medium prevented the inhibitory action of DETD on AIB influx. DETD markedly inhibited labeled thymidine incorporation into DNA of suspended rat thymocytes, possibly via inhibition of transmembrane transport of this nucleoside precursor. In sharp contrast to its effect on thymidine incorporation, DETD stimulated the incorporation of labeled uridine into RNA.

Inhibition by arabinosylcytosine of DNA synthesis in bone marrows of relapsed AML patients

This study was designed to examine whether the selective pressure of intermittent araC maintenance therapy in AML was sufficient for the selection of cells, in either the myeloid or erythroid lines, in which araC was no longer capable of inhibiting DNA synthesis as evidenced by labelled thymidine incorporation. Nucleated cells from bone marrow aspirations were exposed to araC for 1 hr prior to addition of tritiated thymidine. Grain counts on radioautograms were determined either visually or photometrically. In only 1 of 12 patients examined was there evidence of myeloblasts in which in vitro DNA synthesis was relatively unaffected by araC. In the remaining patients there was no evidence for the emegence of myeloblast or erythroid precursor populations in which araC did not inhibit DNA synthesis. DNA synthesis in the more differentiated myeloid precursors appeared less sensitive to inhibition by araC. It would thus appear that in 11 of the 12 AML patients studied the inability to clinically control the disease with araC is not due to biochemical changes related to araC transport, phosphorylation or the ability of araC triphosphate to inhibit DNA synthesis.

Effect of thymidine and thymidylate analogs on nucleic acid synthesis in tumor cells.

Synthetic 5'-amino-5'-deoxythymidine (5'-AdThd), alpha,beta-methylenethymidine diphosphate (alpha,beta-MTDP), and alpha,beta-methylenethimidine triphosphate (alpha,beta-MTTP) were found to inhibit thymidine kinase. Using thymidine kinase extracted from FM 3A/B cells (a strain of mouse mammary gland tumor cells), the Ki values of 5'-AdThd, alpha,beta-MTDP, and alpha,beta-MTTP against thymidine were calculated to be 9.2 X 10(-5)M, 2.3 X 10(-5) M, and 1.8 X 10(-5) M, respectively. At concentrations above their Ki values alpha,beta-MTDP and alpha,beta-MTTP did not inhibit incorporation of labeled thymidine into DNA of cultured cells, whereas 5'-AdThd did. Under the same conditions all three compounds inhibited TMP incorporation. The inhibitions of thymidine and TMP incorporation were specific, since the incorporation of deoxyguanosine was scarcely inhibired by 5'-AdThd. These results suggested that the specific inhibition of thymidine and TMP incorporation was mostly due to reduction in permeability of the cells to these substrates rather than to inhibition of thymidine kinase activity.

Identification of the inhibitor of labelled thymidine incorporation into HeLa cell DNA present in endometrial extract

An extract of bovine endometrium was found to decrease 3 H-thymidine incorporation into DNA of HeLa cells, but also, although to a lesser extent, of Ehrlich and Novikoff cells. Thymidine in the extract was responsible for about 80% of this inhibition. No inhibitor strictly specific for HeLa cells could be detected. An extract of human endometrium decreased, while an extract of human myometrium increased 3 H-thymidine incorporation into DNA of HeLa cells. The inhibitory effect of the endometrial extract was also observed on Ehrlich and Novikoff cells. The endometrial extract contained thymidine which was absent from the myometrial extract; thymidine in the endometrial extract was, however, a preparation artefact. Both human endometrial and myometrial extracts possessed an enzymic activity that converted thymidine into thymine; at high doses, the myometrial extract also decreased 3 H-thymidine incorporation into DNA of HeLa cells. Addition of unlabelled thymidine to the incubation medium has not the same inhibitory action in different kinds of cells on 3 H-thymidine incorporation into DNA. HeLa cells are very sensitive to this dilution effect. The differences in behavior of various cellular systems can be wrongly interpreted as the result of the action of a tissue-specific inhibitor.

Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid

The short term effect of 11.4 mum indoleacetic acid on the incorporation of (methyl-(3)H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 mum) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 mum indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments.

Prediction of the response to chemotherapy in acute leukemia.

The prognostic value of the in vitro incorporation of labeled thymidine was evaluated in 53 cases of acute leukemia, by means of autoradiography and liquid scintillation (LS) count. A higher rate of incorporation before therapy with higher labeling index and LS- specific activity was observed in patients who later responded to chemotherapy with a complete remission. The in vitro depression of labeled thymidine incorporation after incubation with the two drugs selected for the induction therapy was more marked in those who responded to therapy than in the nonresponders. The in vitro incorporation of 3H-thymidine was also evaluated after 48 hours of chemotherapy ("vivo-vitro" test). A decrease of the incorporation was observed frequently in the responders. On the other hand, in the group of nonresponders, there was generally an increase of the incorporation measured by the liquid scintillation count, often contrasting with a decrease of the labeling index, and corresponding to a higher rate of intracellular incorporation. The prognostic value of these findings appears to be significant, as it allows the response to chemotherapy to be predicted, as well as allowing the selection of the various cytotoxic drugs.

Effect of a protease inhibitor on the adhesion of Ehrlich ascites cells to host cells in vivo.

Ehrlich ascites tumours (EAT) were grown in mice by injecting 1 × 10(6) cells intraperitoneally. In mice which received one or more injections of 30 mg soybean trypsin inhibitor (TI) i.p. during tumour growth, the number of recoverable tumour cells was significantly reduced by up to 92%. Also, the mean size of these cells was significantly smaller.When the rate of labelled thymidine incorporation in vitro was compared in TI-treated and control cells, no significant differences were detected. However, when the population doubling time of EAT cells in vivo was calculated, it was apparent that recoverable TI-treated cells were dividing more rapidly than controls. Consequently, the reduced number of cells recovered from TI-treated mice did not result from a reduced growth rate.Viability, assessed by trypan blue dye exclusion and rate of labelled chromium release, was the same in TI-treated and control cell populations. Thus TI was nontoxic to EAT cells and the reduced number of cells from treated tumours was not therefore due to cytotoxicity.Scanning electron microscopy revealed that normal EAT cells did not adhere to internal host surfaces but that after TI treatment they adhered in large numbers to produce an appearance which resembled a confluent monolayer. This binding to host tissue accounted for the reduction in the number of cells recovered from TI-treated animals. We propose that TI acts as a protease inhibitor to prevent intrinsic proteolytic enzyme activity at the tumour cell surface. This activity would normally destroy the binding sites required for adhesion to host tissue.

Mechanism of inhibition of DNA synthesis in Ehrlich ascites tumour cells by diazoacetyl glycine-amide

Treatment of Ehrlich ascites carcinoma bearing mice with DGA, the amide of diazoacetyl-glycine, leads to an inhibition of labelled thymidine incorporation into DNA of the tumour cells. This inhibition is not due to impairment of the nucleoside transport into the cell or to a modification in the activity of thymidine kinase. A possible explanation of the DGA effects resides in its partial inhibition of DNA polymerase and in its ability to alter the template activity of native DNA.

Effects of 4(5)-(3,3-dimethyl-1-triazeno)-imidazole-5(4)-carboxamide (NSC 45388) in proliferating rat tissues

Some effects of 4(5)-(3,3-dimethyl-1-triazeno)imidazole-5(4)-carboxamide (DIC) on normal proliferating tissues in rats were described. DIC caused marked but transient inhibition of labeled thymidine incorporation into DNA and presumably DNA synthesis of thymus, regenerating liver, spleen and small intestine. The inhibition was reversible and its duration dose dependent. When incorporation of precursors into DNA was markedly inhibited, no consistent concomitant effect on incorporation of precursors into RNA or protein was observed. Karyorrhectic cells appeared in the intestinal crypts as well as signs of necrosis in the thymus after administration of 125 mg/kg of DIC, but no significant pathologic changes were found in the spleen or regenerating liver. The relationship between selective inhibition of DNA synthesis and cytotoxic effect was observed.

Observations on the role of circulating lymphocytes in thyroid auto-immunity.

Peripheral leukocytes from normal subjects and patients with Hashimoto's thyroiditis or Graves' disease were cultured in vitro. Induction of blast transformation and stimulation of tritium labeled thymidine incorporation into DNA were assessed in cultures in the absence of an antigenic stimulus, or after addition of phytohemagglutinin, human thyroid supernatant fraction, thyroid microsomal fraction, and monilia extract. Phytohemagglutinin and monilia extract produced a consistent stimulation of blast transformation and/or thymidine incorporation in cells from all 3 groups of subjects. The degree of response was not related to the metabolic status of the patient. Thyroid antigens did not depress reactivity to phytohemagglutinin. Neither thyroid supernatant fraction nor thyroid microsomal fraction stimulated blast transformation or thymidine incorporation in lymphocytes from normal subjects, or the patients with thyroid disease. The studies suggest that circulating lymphocytes do not play an important etiologic role in the pathogenesis of thyroid auto-immune reactions. Lymphocytes inside the thyroid gland may be more important in this process.

The process of cervical regeneration after electrocauterization. II. Histochemical, autoradiographic and pH study.

summaryDetailed studies were made of certain aspects of regenerating cells produced in response to destruction of cervical erosions by electrocautery. These aspects were local pH variarions by a polarographic technique, autoradiographic studies, studies of the formationof the epithelial basement membrane and phagocyte properties of the regenerating cells.The range of pH environment over the regenerating area was 7‐7.4. There was evidence indicating that lower values induced a keratinproducing epithelium and higher values induced a mucinproducing epithelium. Labelled thymidine incorporation showed desoxyribose nucleic acid sythesis to be active in cells migrating into the wounded area. It was especially active in presumptive squamous epithelium formed by these cells. Ribosenucleic acid synthesis and amino acid uptake was distributed throughout the regenerating tissue but was especially marked in new squamous epithelium.Evidence is presented that the basement membrane of the epithelium is produced by a restriction of the pericellular glycoprotein capsule of the migrating cells to their basal aspect. The capsule beneath each cell then joins its neighbours laterally.Regenerating cells are actively phagocytic toward spermatozoa.

Infants and children with haematological disease

Infants and children with haematological disease

Effects of single and divided doses of radiation on labeled thymidine incorporation into DNA.

Previous studies on the variation of dose rate suggested that the duration of radiation was an important factor in the depression of labeled thymidine incorporation into DNA (desoxyribonucleic acid) (1, 2). Preliminary studies on the effects of fractionated doses of radiation suggest that the critical time for the depression of the labeled thymidine incorporation into DNA was in the order of a few minutes. This work was undertaken to further assess the magnitude of the radiation effects on the depression of thymidine incorporation into DNA by varying the temporal relationship between single and divided doses of radiation administered over periods of time ranging from 0 to 256 minutes. These studies have been carried out on semisynchronous populations of liver cells, following partial hepatectomy, as well as asynchronous populations of intestinal cells in the same animal. It has been possible, therefore, to study the effects of different radiation doses given at different times on two different organ systems which, in general, differ markedly in their radiosensitivity. Materials and Methods In this series, the radiation was given locally to the liver and adjacent intestines through a 3 cm cone at the rate of 300 R∕min. The remainder of the animal was shielded. The recorded time between fractionated doses was the time between the end of irradiation of one dose and the beginning of the next. Thus, in the fractionated (750 R × 2) 4-minute experiment, the 4 minutes were allowed between the end of the first dose and the beginning of the second dose. It required 2.5 minutes to give the first and second dose so that a total of 6.5 minutes elapsed between the end of the first dose and the end of the second dose. To compare the effects of a single dose of 1,500 R × 1 with the fractionated dose of 750 R × 2, 4 minutes apart, the 1,500 R dose was initiated so that it ended at the time the first fractionated dose ended. The termination of the single 1,500 R × 1 dose was, therefore, 6.5 minutes before the termination of the second dose. Comparison of a single dose of 750 R × 1 with a divided dose of 750 R × 2 was carried out in a comparable way. For example, the single dose of 750 R × 1 was compared with a divided dose of 750 R × 2 given 8 minutes apart. The single dose of 750 R was initiated so as to end at the same time as the first of the fractionated doses. This was 8 minutes before the second dose was instituted and 10.5 minutes before the end of the second dose. Two additional series of animals were used to compare the effects of divided doses of radiation (375 R × 2) on liver given between 1 and 256 minutes apart, with a single dose of radiation (750 R × 1) at zero time.