Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Evidence for an endothelial cell-derived factor which stimulates the growth of human peripheral blood mononuclear cells

Evidence is provided which demonstrates that conditioned media of cultured endothelial cells derived from human umbilical veins contained a factor which stimulated peripheral blood mononuclear cell [3H]thymidine uptake. A dose-dependent response in peripheral blood mononuclear cell [3H]thymidine uptake was obtained when cells were incubated with increasing concentrations of supernatant of endothelial cell cultures. Studies on temporal kinetics demonstrated that stimulatory activity was evident when mononuclear cells had been incubated with endothelial cell supernatant for 120 hr or more. Preliminary characterization showed the growth immunoregulatory factor to have a molecular weight greater than 100,000 Da.     

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

[³H] DNA fromEscherichia coli and [³H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [³H] thymidine and [³H] DNA treatment which shows that the activity of [³H] DNA is utilized during the S phase. After application of [³H] thymidine, only cell nuclei in S phase were labelled. After the application of [³H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [³H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [³H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [³H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Human urine deoxyribonuclease increases endogenous thymidine in the mouse thymocyte interleukin 1 assay: an artificial interleukin-1 inhibitor

Molecular size chromatography of urine from normal individuals showed two peaks of apparent IL-1 suppressive activities when tested by the murine thymocyte comitogenic assay (mol wt greater than 600 kD and 20 to 60 kD). However, the high molecular weight inhibitory activity disappeared if the concentration of PHA was increased during assay, and the low molecular weight inhibitory activity subsided in the presence of a high concentration of [3H]thymidine. The 20 to 60 kD fractions contained DNase activity which acted on DNA liberated from the considerable number of dying thymocytes during the course of the assay. Thus, incubating the urine fractions with freeze-killed murine T cells, whose DNA was prelabeled with [3H]thymidine, showed the appearance of supernatant [3H]thymidine correlating quantitatively with the DNase activity in the fractions. This indicates that urine DNase together with phosphatase(s) in the thymocyte cultures increase the level of extracellular, unlabeled thymidine, thereby diluting the specific activity of the tracer. These artificial IL-1-inhibitors may explain why urine from both normal and febrile individuals 'inhibits' IL-1 only when tested for thymocyte-activating activity but not when tested for other biological activities.     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats.

Human cells (normal and xeroderma pigmentosum variant) irradiated with ultraviolet light and pulse-labelled with [3H]thymidine underwent transient decline and recovery of molecular weights of newly synthesized DNA and rates of [3H]thymidine incorporation. The ability to synthesize normal-sized DNA recovered more rapidly in both cell types than thymidine incorporation. During recovery cells steadily increased in their ability to replicate normal-sized DNA on damaged templates. The molecular weight versus time curves fitted exponential functions with similar rate constants in normal and heterozygous xeroderma pigmentosum cells, but with a slower rate in two xeroderma pigmentosum variant cell lines. Caffeine added during the post-irradiation period eliminated the recovery of molecular weights in xeroderma pigmentosum variant but not in normal cells. The recovery of the ability to synthesize normal-sized DNA represents a combination of a number of cellular regulatory processes, some of which are constitutive, and one of which is altered in the xeroderma pigmentosum variant such that recovery becomes slow and caffeine sensitive.     

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.     

Partial purification and characterization of an endothelial cell growth regulator from the bovine ovary

An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.     

Calculation of cell production from [h]thymidine incorporation with freshwater bacteria

The conversion factor for the calculation of bacterial production from rates of [H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20 degrees C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 x 10 cells mol of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 x 10; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 x 10 cells mol of thymidine incorporated into TCA precipitate (standard error = 1.72 x 10; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20 degrees C than at 15 and 10 degrees C. A detailed examination of the [H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 x 10 cells mol of thymidine incorporated is used.     

Glial conditioned media inhibit the proliferation of cultured rat cerebellar astrocytes

Conditioned medium (CM) obtained from rat cerebellar astrocytes cultured in a serumcontaining medium was able to inhibit [³H]thymidine incorporation into proliferating astrocytes, when compared to fresh medium. This effect could be attributed to two fractions of the CM with different molecular weights. The low molecular weight fraction (M_r<1,000) inhibited the cellular transport of the labeled precursor, without significantly affecting cell proliferation. The high molecular weight fraction (M_r>10,000) showed a strong inhibitory effect on astrocyte proliferation, which was documented using different assay techniques: i) [³H]thymidine incorporation performed in conditions preventing the effects of CM on transport; ii) [³H]thymidine autoradiography; iii) determination of the DNA content of the cultures. The inhibitory activity was present in media conditioned by non proliferating astrocytes treated with the antimitotic cytosine arabinoside, but not in media conditioned by neuron-enriched cultures nor in a chemically defined (N2) CM. The antiproliferative activity of astrocyte CM could be due either to a rapid depletion of mitogenic factors present in serum, or, to a secretion of growth inhibitory factor(s) by cultured astrocytes.Special Issue Dedicated to Dr. Abel Lajtha.     

Radiotoxicities of [3H]thymidine and of [3H]arginine compared in mouse embryos in vitro

Tritium that is bound to organic molecules is of special risk for living systems, in particular when such molecules are components of the cell nucleus. Therefore, [3H]thymidine and [3H]arginine were studied for radiotoxicity in early mammalian embryo development. Starting with the two-cell stage, mouse embryos were incubated in vitro with [3H]thymidine or [3H]arginine at either 370 Bq/ml (10 nCi/ml) or 925 Bq/ml (25 nCi/ml). Development in vitro was followed up to the formation of the inner cell mass at 192 h postconception (p.c.). There was no difference in radiotoxicity of the two substances with respect to cell proliferation; however, formation of blastocysts, hatching of blastocysts, trophoblast outgrowth, and formation of inner cell mass were impaired more strongly by [3H]arginine than by [3H]thymidine when the external exposure concentrations were the same. Similarly, micronuclei were seen in blastocysts at 96 h p.c. at higher frequency after incubation with [3H]arginine. However, uptake of [3H]arginine by the embryos was considerably faster than that of [3H]thymidine, and this most probably accounts for the apparent difference in radiotoxicity.     

Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Developmental changes in myelin-induced proliferation of cultured Schwann cells

Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated [3H]thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with [14C]thymidine followed by a pulse of [3H]thymidine 24 h later, every Schwann cell labeled with [3H]thymidine was also labeled with [14C]thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.     

Direct cell culture assay of thin-layer-chromatographed material without prior substance elution

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [³H]thymidine incorporation. The reliability of the method was tested with l-alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [³H]thymidine was stimulated with disks containing l-alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l-alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.     

Study of DNA synthesis stimulators in nerve tissue cells

Ontogenetic changes in DNA synthesis in different divisions and regions of the brain occur via different mechanisms, which is conditioned by changes in the composition and properties of corresponding mediators. Tissue extracts obtained from the parts of brain characterized by intensive cell division (cortex of 15-17-day-old embryos or cerebellum of 8-10-day-old animals) can stimulate the incorporation of labeled precursors into brain cell DNA in both neonatal and adult rats. Using desalting at increasing concentrations of ammonium sulfate, gel filtration on sephadexes and isoelectrofocusing, three fractions of DNA synthesis stimulators whose isoelectric points lie at acidic, neutral and alkaline regions of pH, were isolated. A method is described, which can be employed for isolation in a pure state and determination of some physico-chemical properties of an acid activator. The latter is a low molecular weight peptide causing marked stimulation of [3H]thymidine incorporation into nervous tissue cell DNA in an in vitro system, when taken at a concentration of about 1 microgram/ml.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones).

Partially purified lymphocytic factors were obtained from boving spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo: [3H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited; similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked. [3H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced. The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in cluture, as far as thymidine incorporation into DNA is concerned. The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation, fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F'. Both fractions F and F' were further fractionated by chromatography on a Sephadex G75 column: 7 peaks were obtained (F1--F7). Biological activity was found in fraction F1, corresponding to high molecular weight material, and in fraction F6, corresponding to low molecular weight substances. By rechromatography on Sephadex G 75, it is easy to dissociate from F1 a small molecular weight fraction which might be similar to F6 as far as elution volume and biological properties are concerned.