Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

We describe a reproducible method for combining tritiated thymidine ([H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [3H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [3H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified. The inhibition of uptake into DNA of [3H]TdR from 0.23 to 1.85 MBq (6.25 to 50 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [3H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis

Treatment of normal human (WI-38) cells with exogenous heparan sulfate (HS) reduced cell growth and incorporation of radio-isotope-labeled thymidine (TdR) into DNA. In spite that growth of their transformants (WI-38 CT-1) was enhanced by HS treatment, transformed cells also decreased in TdR incorporation thereby. This peculiar observation was explained by a reduction of TdR uptake, leading to a decrease in specific radioactivity of newly synthesized DNA. The changes in cell growth and TdR incorporation by HS treatment were revealed to be similar to the changes with increasing cell density rather than by serum starvation.     

Double labeling with [3H]thymidine and [125I]iododeoxyuridine as a method for determining the fate of injected DNA and cells in vivo

Mice were injected intravenously and intraperitoneally with preparations of intestinal nucleoprotein, spleen nuclei, mouse thymus cells, or human kidney T cells whose DNA had been labeled with both [3H]thymidine (TdR) and [125I]-iododeoxyuridine (IUdR). Since free TdR is reutilized more efficiently than free IUdR produced by enzymic hydrolysis of the exogenous DNA, the ratio of [3H]TdR/[125I]IUdR in the DNA fraction of the tissues of the recipient mice provides a measure of the amount of intact exogenous DNA in the tissue. In most instances, the doubly labeled exogenous DNA was almost completely hydrolyzed within 1 day injection, but survival of the DNA from whole cells could be demonstrated in some cases.     

A correction: inhibitory activity in the conditioned medium of embryonic chick bones is due to thymidine

Earlier studies from this laboratory suggested that embryonic chick bones in organ culture released into the culture medium a specific inhibitor of bone cell proliferation as defined by inhibition of [3H]TdR incorporation into DNA. Dialysis and membrane ultrafiltration experiments suggested that the inhibitory substance (IS) had a molecular weight between 6000 and 14,000. However, subsequent studies on the purification of IS have revealed that the inhibitory activity in bone-conditioned medium is of lower molecular weight and has several properties in common with thymidine (TdR): (1) IS coeluted with [3H]TdR upon gel filtration chromatography on Sephadex G-10. (2) IS bound to charcoal but not to cation or anion exchange resins. (3) Bone-conditioned medium decreased incorporation of [3H]TdR into the free [3H]TdR pool of cells in monolayer culture. (4) Conditioned medium inhibited [3H]TdR incorporation into [3H]thymidine monophosphate in a reaction catalyzed by thymidine kinase. The equivalent concentration of TdR in conditioned medium as estimated by thymidine kinase assay was sufficient to account for the reduction in [3H]TdR incorporation into bone cell DNA. No evidence was found for a specific inhibitor of bone cell proliferation other than TdR. Hence we conclude that the inhibitory effect of IS is due to dilution of [3H]TdR by nonradioactive TdR. Furthermore, media conditioned by several tumor cell lines also contained a low-molecular-weight component which inhibited [3H]TdR incorporation. The results suggest that organ- and cell-conditioned media can contain significant concentrations of TdR which can artifactually inhibit [3H]TdR incorporation in cell proliferation assays.     

Prolactin and growth hormone synthesis and thymidine incorporation in dissociated rat pituitary tumor cells

The effect of in vivo diethylstilbestrol (DES) treatment on the MtT/W15 transplantable pituitary tumor was examined in dissociated pituitary cells by measuring the rate of incorporation of [3H]thymidine into DNA and the synthesis of prolactin (PRL) and growth hormone (GH) as assessed by the rate of incorporation of [3H]leucine. MtT/W15 transplantable pituitary tumors from rats treated for 3 weeks with DES showed significant reduction in the extent of [3H]thymidine incorporation compared with tumor cells from untreated rats (2231 +/- 182 vs 172 +/- 17 dpm/10(5) cells; n = 3). In addition, tumor cells from DES-treated rats showed a significant increase in GH synthesis compared with tumor cells from untreated rats. In contrast to these findings, dissociated pituitary cells from non-tumor-bearing rats given 10 mg DES in Silastic tubing for 3 weeks showed a three-fold increase in PRL synthesis compared to cells from untreated control rats (29.3 +/- 1.5 vs 10.0 +/- 0.9% of total radioactivity in gel; n = 3. There was also a four-fold increase in the rate of [3H]thymidine incorporation after DES-treatment in non-tumor-bearing rats (695 +/- 114 vs 178 +/- 13.9 dpm/10(5) cells; n = 3). These results indicate that DES inhibits MtT/W15 pituitary tumor cell proliferation, while stimulating synthesis of GH.     

Cell position dependence of labelling thymidine nucleotides using the de novo and salvage pathways in the crypt of small intestine

About twice as much tritiated thymidine ([3H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([3H]UdR) is even throughout the crypt. Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway. Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [3H]UdR, but has no effect on [3H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [3H]TdR into DNA. The increased efficiency of thymidine utilization by crypt base cells is not attributable to differences in accessibility of thymidine; differences in the rate of DNA synthesis or the size of the nuclei. It appears that crypt base cells (which include the putative stem cells) are efficient scavengers of [3H]TdR, and this might be related to the level of thymidine kinase activity within the cells, and/or to changes in the availability of endogenous thymidine (break-down products) which compete with exogenous [3H]TdR.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA synthesis in Langerhans' cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

The proportion of Langerhans' cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [3H]TdR autoradiography with enzyme histochemistry. After injection of [3H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (+/- SE mean) for cytospins, 40 min after injecting [3H]TdR, was 1.6 +/- 0.15%, doubling to 3-4% from 7-17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans' cells rising from 18% at 40 min after injection, to approximately 45%, on days 1-4 after injection. These results suggest that the proliferation of Langerhans' cells in normal mouse skin might be higher than was previously thought to be the case.     

Culture of intact Sertoli/germ cell units and isolated Sertoli cells from Squalus testis: I. Evidence of stage-related functions in vitro

As part of an ongoing program of research using the testis of the dogfish shark (Squalus acanthias) to characterize morphologic and functional changes during spermatogenesis, we have developed procedures for culturing intact spermatocysts (germ cell/Sertoli cell clones) and isolated Sertoli cells from premeiotic, meiotic, and postmeiotic stages of development. Phase contrast and light microscopy confirmed the stage and cellular composition of spermatocysts and showed that they retained their closed, spherical configuration for at least 15 d in culture. Stage-related variations in [3H]thymidine incorporation (premeiotic much greater than meiotic = postmeiotic) were observed, a pattern that was the same quantitatively and qualitatively after one or seven days of culture. [3H]Leucine-labeled protein synthesis was twofold greater in cultures with premeiotic spermatocysts than in cultures with more mature stages, whether medium or cysts were analyzed. Sertoli cells isolated from spermatocysts of different stages differed in size, shape, cytological appearance, ability to form flattened monolayers, and rate of DNA synthesis. One day after seeding, [3H]thymidine labeling of Sertoli cells corresponded to the pattern obtained with intact spermatocysts (premeiotic much greater than meiotic = postmeiotic); however, 7 days in culture effected a 40- to 200-fold increase in this parameter and altered the stage-dependent pattern (premeiotic = meiotic greater than postmeiotic). Also, when [3H]leucine-labeled macromolecules secreted by Sertoli cells from premeiotic versus meiotic stages were analyzed by polyacrylamide gel electrophoresis (PAGE), banding patterns differed. Initial results demonstrate the feasibility and potential of this in vitro system for studying qualitative and quantitative changes during spermatogenesis.     

Biosynthesis of rat sperm outer dense fibers during spermiogenesis. In vivo incorporation of [3H]leucine into the fibrillar complex

The temporal incorporation profile of [3H]leucine into the outer dense fiber polypeptides was determined after the intratesticular injection of the radioisotope. Groups of four rats were killed on alternate days after injection, and the outer dense fibers were isolated from the caput epididymal sperm. The radioactivity incorporated into the whole sperm and into the isolated fibers showed a sharp peak at 10 days after injection. Therefore, considering the known kinetics of spermatogenesis in the rat, the maximal incorporation of radioactivity into the fibers occurred during the second half of spermiogenesis. The radioactivity incorporated into the six major polypeptides of the fibers separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate accounted for 95 percent of the total radioactivity associated with the isolated fibrillar complex. Furthermore, analysis of the time-course incorporation of [3H]leucine into the polypeptides of the fibers indicated that the maximal incorporation into each of the six major components took place within the same period of time. Using two different procedures, the specific activity of each major polypeptide was determined at the time of maximal incorporation. It was found that the specific activity of the most abundant components (molecular weights of 30,400 plus 26,000) was approximately twice that of the other polypeptides.     

beta-Momorcharin, a plant glycoprotein, inhibits synthesis of macromolecules in embryos, splenocytes and tumor cells.

1. beta-Momorcharin, a glycoprotein isolated from seeds of the bitter gourd, inhibited incorporation of [3H]leucine, [3H]uridine and [3H]thymidine into trichloroacetic acid-precipitable radioactivity in peri-implantation mouse embryos, mouse splenocytes with or without activation by concanavalin A, and human squamous carcinoma of the tongue and larynx, but did not affect incorporation of the aforementioned radioisotopes into mouse liver cells. 2. The results suggest that inhibition of protein, RNA and DNA biosynthesis in embryos, splenocytes and tumor cells may represent the mechanism of embryotoxic, immunosuppressive and antitumor actions of beta-momorcharin. 3. The results also suggest that in mouse liver cells biosynthesis of protein, RNA and DNA was not affected by beta-momorcharin.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Proliferation of cells undergoing chondrogenesis in vitro

Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [3H] thymidine thymidine [(3H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [3H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [3H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [3H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype. We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

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.     

白细胞介素对大鼠离体垂体前叶细胞增殖的影响

本工作采用大鼠垂体前叶（ＡＰ）细胞原代培养方法,以３ＨＴｄＲ掺入率反映细胞增殖水平,研究了ＩＬ１和ＩＬ６对ＡＰ细胞增殖的影响。结果表明：（１）ＩＬ１（１－１００ｎｇ／ｍｌ）促进雄性大鼠和雌性大鼠ＡＰ细胞的增殖。（２）低浓度的ＩＬ６（０．１ｎｇ／ｍｌ）抑制雄性大鼠的ＡＰ细胞的增殖,而较高浓度的ＩＬ６（１－１０ｎｇ／ｍｌ）则表现为刺激作用。（３）ＩＬ６（０．１－１０ｎｇ／ｍｌ）促进雌性大鼠ＡＰ细胞的增殖。上述结果说明ＩＬ１和ＩＬ６除直接调控ＡＰ细胞的分泌外,也参与调节ＡＰ细胞增殖活动。     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA

Thymidine (TdR) incorporation into DNA as a measure of bacterial production in environmental samples relies on assumptions about what organisms incorporate exogenous thymidine, extent of dilution of labelled thymidine by internal and external pools, and analytical methods for recovery and purification of bacterial DNA. We have examined these assumptions with regard to the feasibility of using [³H]TdR incorporation in the water column and sediments of a blackwater river. The extent of dilution of added [³H]TdR may be determined with isotope dilution plots (Moriarty and Pollard, 1981 and 1982) and these indicate a wide range of degree of participation of added [³H]TdR. Previously described methods for extracting DNA from sediment bacteria may lead to underestimates and we described a more efficient recovery scheme.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Reactivation of rat peritoneal leukocyte and mast cell nuclei in heterokaryons with actively growing mouse cells

Leukocytes and mast cells of rat peritoneal exudate (PE) were fused in vitro with actively growing mouse cells. Segmented ring-shaped nuclei of granulocytes undergo drastic changes which result in dispersion of tightly condensed chromatin and gradual disappearance of the opening in the centre of the nucleus. These changes are paralleled by a resumption of RNA and DNA synthesis, as shown by autoradiography with [3H]uridine and [3H]thymidine. Solid inactive nuclei of mast cells, lymphocytes, monocytes and macrophages also resume DNA replication and high level of RNA synthesis. Fusion of thymidine kinase-deficient 3T3-4E cells with PE cells results in the incorporation of [3H]thymidine into the nuclei of heterokaryons. This may be considered evidence of the phenotypic expression of rat thymidine kinase gene in heterokaryons. A similar way in which segmented and non-segmented dormant nuclei undergo reactivation suggests that the reversibility of nuclear inactivation is a common feature of differentiated somatic cells.     

The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.