Changes in cellularity induced by radiation in a solid tumour.

The growth, and cellular responses of Morris hepatoma 3924 A to a locally-administered dose of 3750 R X-rays were studied using the following parameters; (1) relative tumour volume changes; (2) tritiated thymidine (3H-TdR) incorporation into DNA; (3) tumour DNA content and (4) cellular analysis, including 3H-TdR labelling index, mitotic index, aberrant mitotic frequency and relative cell density. Before depression of tumour growth, cell proliferation is temporarily interuppted. As proliferation is reinitiated, a short-lived synhcrony and prolongation of cell-cycle traverse are reflected in (a) the labelling index and mitotic index, (b) the relative cell density, and (c) the rate of incorporation of 3H-TdR into DNA. Within 4 days after radiation, cell proliferation and 3H-TdR incorporation are significantly depressed. Simultaneously there are reductions in both the relative cell density and tumour DNA contents, and these remain depressed as the tumours initiate regression. From these studies, it is apparent that the cellular responses to radiation insult occur well in advance of measurable volume changes and are observed both in tumours that continue to regress and in those that initiate regrowth.     

Unscheduled DNA synthesis in growing roots and stored embryos of Vicia faba after the action of maleic hydrazide and methyl methanesulphonate

Growing roots of Vicia faba were treated with MH for 5 h, washed for 2 h and exposed to 3H-thymidine (3H-TdR) for additional 2-h periods at 7 h, 24 h and 32 h after the onset of MH treatment, to label DNA. As the replicative DNA synthesis was suppressed by HU, an enhancement of 3H-TdR incorporation into nuclear DNA above the control, as determined by microautoradiography, was considered to be due to unscheduled DNA synthesis induced by the mutagen. A significantly higher incorporation of 3H-TdR into DNA of MH-treated roots occurred, when labelling was applied 7 h after the MH action, whereas at 24 h only slight and at 32 h no enhancement of DNA labelling above control was registered. A 3-14-day storage with 50% water content of V. faba seeds exposed to MH or MMS resulted in a recovery from mutagen-induced chromosomal damage and a significantly higher incorporation of 3H-TdR into nuclear DNA. This supports the hypothesis that recovery from MH- and MMS-induced chromosomal damage is mediated by excision repair during seed storage.     

Ehrlich ascites tumor (EAT) chalone effects on nascent DNA synthesis and DNA polymerase alpha and beta.

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of 3H-thymidine (3H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50-200 mug/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude alpha- and beta-polymerase activities were inhibited. Crude DNA polymerase for C3H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or 'gapped' DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting alpha- and beta-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating the DNA ligase is not inhibited.     

Differential incorporation of 3H-thymidine into DNA in cultured skin fibroblasts derived from patients with cystic fibrosis and controls.

Tritiated thymidine (³H-TdR) incorporation into DNA of the fibroblasts derived from subjects with cystic fibrosis (CF) and their controls was studied with scintillation counting and autoradiography. ³H-TdR incorporation at 24 hours postseeding was significantly less (p<0.005) in CF strains in comparison with cells from controls. The percentage of labeled fibroblasts was not significantly different between the two strains (p>0.1). The cell cycle time and the duration of each phase were studied by a mitotic selection and scintillation counting technique. There was no difference in cell cycle time between CF and control fibroblasts, however, the duration of the synthetic phase was significantly (p<0.005) longer in CF subjects.     

Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum)

The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.     

缺氧对新生小牛肺血管平滑肌细胞增殖的影响及764—3的抑制作用

本研究应用细胞培养、~3H—TdR参入技术,探讨缺氧对新生小牛肺血管平滑肌细胞(PASMC)DNA合成和细胞增殖的影响及746—3和川芎嗪抗细胞增殖效果。结果表明:缺氧的肺血管内皮细胞(PAEC)培养液可以明显增加PASMC中~3H—TdR的参入;缺氧24h亦可直接刺激PASMC,使其中~3H—TdR的参入显著增加(P<0.001);在缺氧的PASMC培养基中加入764—3,~3H—TdR参入值与单纯缺氧组相比显著下降,并使细胞数减少36.3％,764—3还可以显著抑制常氧PASMC的增殖;川芎嗪的作用较小。实验结果提示,缺氧不仅可以刺激内皮细胞(EC)产生促分裂素使PASMC增殖,而且还可以直接刺激PASMC的增殖,764—3可抑制缺氧及血清刺激的PASMC的增殖。     

EHRLICH ASCITES TUMOR (EAT) CHALONE EFFECTS ON NASCENT DNA SYNTHESIS AND DNA POLYMERASE ALPHA AND BETA

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of ³H-thymidine (³H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50–200 μg/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude α and β-polymerase activities were inhibited. Crude DNA polymerase from C₃H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or ‘gapped’ DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting α- and β-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating that DNA ligase is not inhibited.     

INCORPORATION OF TRITIATED THYMIDINE BY EUCARYOTIC MICROALGAE1

The uptake and incorporation of tritiated thymidine (³H-TdR) by axenic laboratory cultures of marine diatoms and dinoflagellates was measured. ³H-TdR was incorporated into nucleic acids by all four algae examined during a two to six hour period prior to cytokinesis and not during other times of the cell cycle. Between 90-95% of the ³H label incorporated into (cold trichloroacetic acid insoluble) nucleic acids was recovered from DNA. Incorporation of ³H-TdR appears to accurately indicate the timing of DNA synthesis. The incorporation of ³H-TdR by eucaryotic algae during long term (24 h) incubations does not generally preclude using ³H-TdR uptake to estimate bacterial production and growth during short term incubations.     

Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with ³H-TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of ³H-TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of ¹⁴C-formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of ³H-TdR into DNA. It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous ³H-TdR from accurately reflecting the true level of DNA synthesis.     

黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Influence of irradiation or thymidine (TdR) on the pattern of 3H-TdR incorporation at each cell position in the crypts of the small intestine of the mouse

Using autoradiographic methods it was noted that S phase cells at the bottom of the crypts in the small intestine were the most efficient scavengers of exogenous injected thymidine. The efficiency of the incorporation of 3H-TdR (salvage pathway of DNA synthesis) by cells at the crypt base (stem cell zone) was twice as high as for the S phase cells at the top of the crypt (maturing proliferative cells). There were no such position-dependent differences in incorporation of 3H-UdR (de novo pathway of DNA synthesis). Radiation (0.75-5.0 Gy 137Cs gamma-rays) inhibited the incorporation of 3H-TdR very rapidly and this was also cell-position dependent. The cells at the bottom of the crypt were the most affected. The injection of cold thymidine before 3H-TdR changed the pattern of the incorporation of 3H-TdR along the side of the crypt in a very similar way to radiation, and the grain number was decreased predominantly in the cells at lower positions. The possibility of the existence of a regional gradient of endogenous thymidine (reutilization from intestinal sources), and the influence of irradiation on the gradient of thymidine incorporation resulting from direct and abscopal effects of whole body exposure, are discussed.     

中药固真方对ROS17/2.8细胞增殖分化的影响

中药固真方具有补肾益精、延缓衰老等作用。本文主要研究固真方对~3H-TdR参入DNA及碱性磷酸酶活性的影响。实验结果表明,当大鼠成骨肉瘤细胞ROS17/2.8经固真方药处理24h后,其~3H-TdR的参入比对照组增高13.9％至319.2％,而其碱性磷酸酶活性在经固真方药处理2—3天后则增高4.4％至52.8％(平均30.2％),提示固真方药在成骨样细胞中既有增殖作用,也有促分化作用。     

转化细胞及腹水癌细胞对生长调节因子敏感性的研究

 本文~3H-TdR参入细胞DNA为指标研究了EGF等生长调节因子对小鼠腹水癌细胞DNA合成的影响,发现不同癌细胞对EGF等生长因子的敏感性有所差异,考虑到这也许与肿瘤细胞自身特性如恶性度有关。为了进一步探讨恶性度与这一敏感性是否相关,我们观察并比较了C_3H10T1/2CL_8(一种来源于鼠胚的正常成纤维细胞,简称NC_3H_(10)及转化的C_3H_(10)T1/2CL_8(用~3H-TdR转化的上述细胞,简称TC_3H_(10))对EGF等生长因子的敏感性。实验证明,细胞恶性转化后,对EGF的敏感性明显降低,~3H-TdR参入率降至原先的1/4以下。用DBcAMP作用于NC_3H_(10)和TC_3H_(10)均能抑制~3H-TdR参入DNA并可抑制EGF诱导的~3H-TdR参入作用。因此,我们认为,有关物理的致癌因素如放射性同位素,像生物、化学的致癌因素一样,亦能引起其转化细胞对外源性生长调节因子敏感性的改变。     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on stimulated DNA synthesis in the liver and kidney of the rat

The administration of the toxic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) to rats at 0, 24 or 72 h before 70% partial hepatectomy had no significant effect on DNA synthesis, measured by [³H]thymidine incorporation 24 h after the operation, in the remaining liver lobes. However, dioxin depressed DNA synthesis in the folate-stimulated rat kidney when given from 0–9 days before the folic acid. A similar effect was observed using inorganic lead to stimulate kidney mitosis. This effect does not appear to be due to direct interaction of dioxin with DNA, inhibition of the protein synthesis required for DNA synthesis, alteration of the time course of events in the mitotic cycle of the kidney or the depression of food intake of the rats caused by dosage with dioxin. Experiments with methylcholanthrene suggest that levels of drug-metabolising enzymes in the liver or kidney might possibly have an indirect effect on folate-stimulated kidney mitosis.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Stimulation of growth of serum-deprived chick embryo fibroblasts by 3',5'-phosphodiesterase.

In chick embryo fibroblasts (CEF) deprived of serum, DNA synthesis is reduced to a basal level in about 12 h, cell division ceases after 24–36 h and their morphology changes to a rounded, less refringent form. During several days without serum the cAMP content of the cells showed a slow increase or a maintenance of the level found before serum was removed. When CEF deprived of serum for 24 h were treated with beef heart 3′,5′-phosphodiesterase (PHD) the cAMP level fell about 40% after 3 h, ³H-thymidine incorporation into DNA was strongly stimulated with a peak of incorporation at 12 h after the start of PHD treatment, cell morphology returned to that observed before serum deprivation, and at 24 h there was an evident growth in cell population, with a parallel increase in protein content. The growth stimulation by PHD is transitory: after cells had been deprived of serum for 4 days the PHD effect was no longer noticeable on the above parameters. Theophylline (1 mM and 4 mM) inhibited the PHD-mediated stimulation of ³H-TdR incorporation, this could well have been due to its general toxic effect on the cells (see Discussion).     

AUTOSYNCHRONIZATION OF RAT LIVER CELLS WITH ENDOGENOUS CORTICOSTERONE AFTER PARTIAL HEPATECTOMY

After adrenalectomy in adult male rats ³H-TdR incorporation into the liver parenchymal cells is increased 4–8 times and the mitotic index rises from 0–31 % to 1–3%; this is inhibited by corticosterone. After hepatectomy the serum corticosterone level increases from 18 μg/100 ml to 57 μg/100 ml. The corticosterone binding capacity of the serum declines from 2–06 to 0–17. The activity of tyrosine transaminase doubles, whereas the incorporation of ³H-TdR into the liver cells is decreased by a factor of 5–7. Thereafter the binding capacity increases again and reaches, 24 hr after operation, a value of 3–82. The tyrosine transaminase activity and the serum corticosterone content return to normal. ³H-TdR incorporation, however, increases by a factor of 7-7 of the initial value. We concluded that in the first few hours after partial hepatectomy corticosterone blocks the liver cells in G₁ and an accumulation of the cells occurs at this cell cycle phase. Folio wing the binding of the corticosterone by serum proteins a little later the liver cells enter the S-phase synchronously.     

Rates of incorporation of radioactive molecules during the cell cycle

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (³H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) ³H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) ³H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing ³H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of ³H-TdR and ³⁵S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, ³HH-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside ³H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of ³⁵S-methionine increased continuously with cell size and DNA content. Incorporation of ³H-TdR in CHO cells was proportional to DNA synthesis.     

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.     

RNA synthesis: a requirement for hormone-induced DNA amplification in Rhynchosciara americana

Injection of beta-ecdysone into mid fourth instar larvae of Rhynchosciara americana induced within 23-28 hours after injection a rise in the percentage of 3H-thymidine (3H-TdR) incorporating nuclei in salivary gland region S1 from about 10-20% in the controls to 80-90% in the injected larvae. The 3H-TdR incorporating nuclei displayed a weak continuous labeling pattern or a band-labeling pattern with grains over the vast majority of the bands. The majority of nuclei with a band labeling pattern displayed DNA amplification at the DNA-puff regions.--Injection of actinomycin D at different times after ecdysone injection abolished the higher incorporation rate at the amplifying regions within 15 hours after the injection. However, the percentage of nuclei incorporating 3H-TdR and the frequency of the two labeling patterns remained essentially the same when RNA synthesis was inhibited. Only the over-all rate of 3H-TdR incorporation seemed to be reduced.--These data suggest that in the DNA puff regions the rate of DNA chain elongation is higher when amplification occurs than during a normal replication cycle. It, further, seems that the higher rate during amplification is dependent upon de novo RNA synthesis.