DNA synthesis rate and unlabeled cells with S-phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting

DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S-phase fractions during DNA flow cytometry. During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the "salvage" and the "de novo" DNA synthesis pathways were blocked in most S-phase cells. After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.     

In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging

Cellular labeling with ferumoxides (Feridex IV) superparamagnetic iron oxide nanoparticles can be used to monitor cells in vivo by MRI. The objective of this study was to use histology and MRI to evaluate an in vivo, as opposed to in vitro, technique for labeling of mononuclear leukocytes as a means of tracking inflammatory processes in the brain. Long-Evans rats were intravenously injected with 20 mg/kg ferumoxides, ferumoxtran-10, or ferumoxytol with or without protamine sulfate. Leukocytes and splenocytes were evaluated by cell sorting and iron histochemistry or were implanted into the brain for MRI. Injection of ferumoxides/protamine sulfate complex IV resulted in iron labeling of leukocytes (ranging from 7.4 +/- 0.5% to 12.5 +/- 0.9% with average 9.2 +/- 0.8%) compared with ferumoxides (ranging from 3.9 +/- 0.4% to 6.3 +/- 0.5% with average 5.0 +/- 0.5%) or protamine sulfate alone (ranging from 0% to 0.9 +/- 0.7% with average 0.3 +/- 0.3%). Cell sorting analysis indicated that iron-labeled cells were enriched for cell types positive for the myelomonocytic marker (CD11b/c) and the B lymphocyte marker (CD45RA) and depleted in the T cell marker (CD3). Neither ferumoxtran-10 nor ferumoxytol with protamine sulfate labeled leukocytes. In vivo ferumoxides/protamine sulfate-loaded leukocytes and splenocytes were detected by MRI after intracerebral injection. Ferumoxides/protamine complex labeled CD45RA-positive and CD11b/c-positive leukocytes in vivo without immediate toxicity. The dose of feumoxides in this report is much higher than the approved human dose, so additional animal studies are required before this approach could be translated to the clinic. These results might provide useful information for monitoring leukocyte trafficking into the brain.     

The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Accurate determination of the time of cell birth using a sequential labeling technique with [3H]-thymidine and bromodeoxyuridine ("window labeling").

During tissue embryogenesis, precursor cells divide actively and eventually withdraw from the mitotic cycle before differentiation. Accurate information about the time of terminal mitosis ("birthdate") of precursors is of vital importance for studying relationships between cell proliferation and differentiation. Methods presently available for birthdate determination, based on "pulse" or "cumulative" labeling with either tritiated thymidine (3HT) or bromodeoxyuridine (BrDU) incorporated into DNA during the mitotic cycle, allow only the approximate timing of terminal mitosis. To overcome this limitation, we have developed a "window labeling" technique based on the sequential administration of 3HT and BrDU. Chick retinal precursor cell cultures were first exposed to 3HT and, after a specified time interval, also to BrDU. After 6 days the cultures were fixed and processed for BrDU immunocytochemistry and 3HT autoradiography. Three populations of cells could be easily identified: (a) unlabeled cells, representing post-mitotic cells before label exposure; (b) BrDU-labeled cells [either 3HT (+)/BrDU (+) or 3HT (-)/BrDU (+)], representing those that continue dividing after the addition of BrDU; and (c) "window-labeled" cells, 3HT (+)/BrDU (-), which are those undergoing their last round of DNA synthesis during the interval between 3HT and BrDU administration. Control experiments demonstrated that this method allows birthdate determinations with a resolution of hours or minutes and is essentially free of deleterious effects on precursor cell survival and differentiation.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

Role of the pituitary in controlling oogenesis in the rabbit

The involvement of the pituitary and mesonephros in oogenesis was examined with the rabbit used as an experimental model. Fetal and neonatal ovaries were cultured for 8 days with and without their corresponding pituitaries or as half cranial or caudal portions. Progression of meiosis was followed histologically and autoradiographically by the uptake of tritiated thymidine. Pulse labeling for 24 h occurred on Days 0, 2, 4 and 7 of culture. No difference was noted in labeling or progression of meiosis in neonatal ovaries with or without the pituitary or in sectioned ovaries. By contrast, fetal ovaries with pituitary that were labeled on Day 4 had a labeling index (positive cells/1000 microns 2 of cortex) of 282.1 +/- 32.9 (mean +/- SEM) compared to 107.1 +/- 24.9 for ovaries without pituitary. Those labeled on Day 7 showed the same differences (263.7 +/- 46.0 vs. 160.2 +/- 20.8). Histologically, there was a threefold difference in percentage of germ cells with pituitary undergoing meiosis (21.2 +/- 2.9% compared to 7.1 +/- 2.8% without). These data suggest that the fetal pituitary has an important role in inducing meiotic prophase in the rabbit.     

Kinetics of megakaryocyte progenitor cells in idiopathic thrombocytopenic purpura

The number and proliferative state of megakaryocyte progenitor cells (CFU-Meg) were compared between 13 patients with idiopathic thrombocytopenic purpura (ITP) and hematologically normal controls. The mean frequency of CFU-Meg assayed by the plasma clot method was 27.8 +/- 12.2 (+/- SD)/2 x 10(5) bone marrow light-density cells for the ITP patients, which did not differ significantly from the control value of 31.9 +/- 16.1. The percentage of CFU-Meg in DNA synthesis estimated by the 3H-thymidine suicide technique was 41.3% +/- 9.2% in ITP, which was significantly greater than the control value of 27.1% +/- 7.4% (P less than 0.01). The megakaryocyte counts for histological sections prepared from bone marrow aspirates from the ITP patients and controls were 34.5 +/- 8.5/mm2 and 11.2 +/- 5.8/mm2, respectively, with the difference being highly significant (P less than 0.001). These results suggest that increased cycling activity in a quantitatively unchanged CFU-Meg pool may lead to increased megakaryocytes in the bone marrow of ITP patients.     

Oestradiol is effective in stimulating 3H-Thymidine Incorporation But Not On Proliferation of Breast Cancer Cultured Cells

The growth of numerous human oestrogen target cell lines is said to have been stimulated by oestradiol. We studied the action of this hormone on the growth of two human cancer cell lines originating from endometrium (GUS), and from breast (FAM). Oestradiol was inactive on endometrial cell multiplication as well as on their tritiated thymidine uptake, but in FAM breast cancer cells, we noticed a discrepancy between tritiated thymidine uptake and actual cell proliferation: there was a 40% increase in DNA precursor uptake, but no change in either the number of cells or in their DNA content, both of which were verified by two different methods. Therefore, an actual increased nuclear (autoradiographic) uptake of thymidine did take place in oestrogenized cells, associated with an increase of incorporation into DNA (a rise of radioactivity in the acid-insoluble materials), but finally there was no greater total DNA increase in the whole treated population than in control cells. Then we examined the metabolism of tritiated thymidine in oestradiol-treated FAM cells. We extracted the radioactive thymine nucleotides and characterized them chromatographically: the oestradiol caused an increase in the labelling of deoxythymine monophosphate (TMP). How these results are consistent with both unmodified cell count and whole DNA content is discussed.     

Iododeoxyuridine administered to mice is de-iodinated and incorporated into DNA primarily as thymidylate.

Iododeoxyuridylic acid, a structural analog of thymidylic acid, is extensively de-iodinated in vivo by the enzyme thymidylate synthetase. Substantial amounts of the deoxyuridylic acid formed by this process are subsequently methylated and incorporated into DNA as thymidine. As a result, when mice are given tritiated iododeoxyuridine, most of the tritium incorporated into their DNA is present in thymidine rather than in iododeoxyuridine. Some, but not nearly as much, tritium from tritiated bromodeoxyuridine is also incorporated into DNA thymidine.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine [( 3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5.8 +/- 0.3 h), a high S phase efflux and an initially low influx of cells from G1 into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 +/- 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15-16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300-1400 h, efflux rate falls from high (19-20 cells %/h) to low (4-8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.     

An in vitro nucleoside analog screening method for cancer gene therapy

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC₅₀ on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.Abbreviations ACV acyclovir - ACV-MP acyclovir monophosphate - ACV-DP acyclovir diphosphate - ACV-TP acyclovir triphosphate - BDU bromodeoxyuridine - BVDU bromovinyldeoxyuridine - EDU ethyldeoxyuridine - FDU fluorodeoxyuridine - GCV ganciclovir - HSV-TK herpes simplex virus thymidine kinase gene - IDU iododeoxyuridine - NA nucleoside analog     

Oestradiol is effective in stimulating 3H-thymidine incorporation but not on proliferation of breast cancer cultured cells

The growth of numerous human oestrogen target cell lines is said to have been stimulated by oestradiol. We studied the action of this hormone on the growth of two human cancer cell lines originating from endometrium (GUS), and from breast (FAM). Oestradiol was inactive on endometrial cell multiplication as well as on their tritiated thymidine uptake, but in FAM breast cancer cells, we noticed a discrepancy between tritiated thymidine uptake and actual cell proliferation: there was a 40% increase in DNA precursor uptake, but no change in either the number of cells or in their DNA content, both of which were verified by two different methods. Therefore, an actual increased nuclear (autoradiographic) uptake of thymidine did take place in oestrogenized cells, associated with an increase of incorporation into DNA (a rise of radioactivity in the acid-insoluble materials), but finally there was no greater total DNA increase in the whole treated population than in control cells. Then we examined the metabolism of tritiated thymidine in oestradiol-treated FAM cells. We extracted the radioactive thymine nucleotides and characterized them chromatographically: the oestradiol caused an increase in the labelling of deoxythymine monophosphate (TMP). How these results are consistent with both unmodified cell count and whole DNA content is discussed.     

Serum and insulin initiation of DNA synthesis in mammary gland epithelium in vitro

These experiments demonstrate that serum, like insulin, can initiate DNA synthesis in mouse mammary gland epithelium, resulting in a three- to four-fold increase in the rate of DNA synthesis and number of cells synthesizing DNA. Both serum and insulin also increase the tritiated thymidine counts in the acid soluble material of these cells, suggesting that each alters thymidine transport. When combined at any concentration, these agents produce an additive effect on DNA synthesis, number of cells synthesizing DNA and thymidine transport. The factor(s) in serum responsible for these effects is associated with high molecular weight material. These experiments suggest that DNA synthesis and thymidine transport are affected independently by serum and insulin in mammary gland epithelium.     

In vivo labeling of fission yeast DNA with thymidine and thymidine analogs

In vivo labeling of DNA with thymidine and thymidine analogs has long been a cornerstone of replication studies. Unfortunately, yeast lack a thymidine salvage pathway and thus do not incorporate exogenous thymidine. Specifically, yeast neither efficiently take up exogenous thymidine from their growth media nor phosphorylate it to thymidylate, the precursor of dTTP. We have overcome these problems in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) along with herpes simplex virus thymidine kinase (tk). hENT1 tk cells are healthy and efficiently incorporate exogenous thymidine and thymidine analogs. We present protocols for labeling DNA with tritiated thymidine, for in situ detection of incorporated BrdU by immunofluorescence, for double labeling with CldU and IdU, for CsCl gradient separation of IdU-labeled DNA, and for using hENT1 and tk as both positive and negative selection markers.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

Utility and sensitivity of anti BrdU antibodies in assessing S-phase cells compared to autoradiography

A rapid and convenient method for estimating S-phase cells in a population was developed which detects bromodeoxyuridine (BrdU) incorporation into DNA by means of monoclonal anti-BrdU antibodies. This immunofluorescence technique (RPMB technique) was compared to autoradiographic (ARG) detection of tritiated thymidine (3HTdr) grains incorporated into the DNA. Using incubation periods for BrdU and 3HTdr ranging from one minute to one hour and detecting their incorporation by ARG and RPMB techniques, it became apparent that the RPMB technique was far more sensitive than ARG in addition to being extremely easy to perform. Some possible utilities of the RPMB technique are discussed.