用双参数流式细胞光度术(FCM)研究阿克拉霉素对L_(1210)白血病细胞周期的影响

本文用双参数FCM技术,对同一个细胞的DNA和RNA含量进行相关测量,比较了ACM B对小鼠L_(1210)白血病细胞周期和RNA含量的影响.结果发现在一次给药后8小时可导致早、中期S的积累,并抑制S期细胞的DNA合成;到24小时DNA合成恢复正常,并进入G_2期,但由于G_2期细胞进入M期受阻,造成G_2期细胞的积累,这时被阻断在G_2期的细胞RNA含量显著增加,形成正不平衡生长,而给药剂量较大的实验组(1/1.5LD_(50))S期细胞的RNA含量不随着DNA含量的增加而增加,形成负不平衡生长,ACM A和ACM B对体内Li_(210)细胞周期作用相同.     

Interrelationships in experiments in vitro between the migration activity of leukocytes and RNA and DNA synthesis]

The following correlations were revealed in the parallel study of leukocyte migration in vitro in the presence of a specific antigen and of spontaneous RNA and DNA synthesis in the cultured lymphocytes: 1) a direct correlation between the RNA and DNA synthesis in lymphocytes; 2) a close correlation between the antigen-induced migration and the levels of RNA and DNA synthesis. The effect of the antigen was evidenced by the inhibition or stimulation of leukocyte migration. A high ratio of RNA synthesis to DNA synthesis corresponded to the migration inhibition and a low one--to the migration stimulation. The ratio value varied mainly on account of the changes in the level of DNA synthesis. Participation of T and B cells in the regulation of the antigen-induced leukocyte mobility is discussed.     

定量细胞化学分析大蒜油抗癌的作用

本文应用细胞化学和显微分光光度计对癌细胞的 DNA,ACP,ANAE、SDH 和G6PDH 进行定量测定,又用流式细胞计分析癌细胞 RNA 含量的变化.实验证明大蒜油能明显抑制癌细胞 DNA 与 RNA 合成,并减低五种酶的活性。表明大蒜油能影响癌细胞的核酸代谢、能量代谢及功能活动、抑制癌细胞的分裂增殖,从而起到抗癌效应.     

Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.     

Cell cycle distribution in-vivo of mononuclear blood cells of renal allograft recipients

In 47 healthy blood donors (controls) and 29 renal allograft recipients (patients) the relative contents of RNA and DNA of peripheral blood mononuclear (PBM) cell populations were estimated from the intensities of green and red fluorescences emitted by complexes that form with DNA and RNA, respectively, after staining the cells with the metachromatic dye, acridine orange. Based on the correlated DNA and RNA estimates for large numbers of cells, the percentages and the relative RNA contents of cells in particular compartments of the cell cycle were determined. PBM cell populations of controls contained less than 0.5% proliferating (SG2M) cells with highly variable relative RNA contents. Among controls, neither percentages nor relative RNA contents of SG2M-cells were correlated with percentages or relative RNA contents of G1-cells with an RNA content 2 (2SD) or 3 (3SD) standard deviations above the mean of the entire G0/1-cell population. Unlike controls, PBM cell populations of patients contained significantly higher percentages of SG2M-cells which were significantly correlated with the relative coefficients of variation (rCV) of the F530 histograms of G0/1-cells; the rCV represents the ratio of a patient's CV to a control's CV. Moreover, significant statistical correlations existed between percentages and relative RNA contents of 2SD-, 3SD-, SG2M- and G0/1-cells, suggesting a well-orchestrated progression of cells through the cell cycle. Different pairs of correlated parameters characterized clinically stable, acutely rejecting, and infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of RNA synthesis in DNA replication of synchronized populations of cultured mammalian cells

Using the harvesting method of synchronizing L cells, the relationship of RNA synthesis of DNA replication was studied by the use of selective inhibitors of RNA synthesis such as actinomycin D and chromomycin succinate. The synthesis of the early replicating DNA fraction is a process sensitive to the inhibition of RNA synthesis during the G1 period. The synthesis of early replicating DNA was inhibited by chromomycin succinate without affecting the initation of DNA synthesis. However, actinomycin D inhibited the synthesis of early replicating DNA and prevented the initiation of DNA synthesis in 50% of the synchronized cells. However, it was found that the continued synthesis of RNA during the S period is not essential for the synthesis of late replicating DNA. In addition to this specific response of DNA synthesis to the inhibitors of RNA synthesis, another function of early and late replicating DNA was determined relative to the cell viability. Cells synthesizing early replicating DNA were killed more efficiently by chromomycin than at other stages of the cell cycle. This indicates that the early replicating DNA unit plays a more important role in cell reproduction than the late replicating DNA unit.     

Sequence of activation of template biosynthesis in normal and transformed human cells after synchronization by a double thymidine block

The sequence of matrix biosyntheses of DNA, RNA and various proteins in normal and transformed human fibroblasts in the first mitotic cycle after synchronization of cells by double thymidine block was studied. Two important regularities of synthesis of acid-soluble histone-like and acid-insoluble proteins in normal and transformed cells were established. In normal fibroblasts, the synthesis of both acid-soluble and acid-insoluble proteins is minimal before DNA replication and maximal in the G2-phase; that in transformed cells is maximal after removal of the thymidine block and decreased in the G2-phase. In normal fibroblasts, the synthesis of acid-insoluble proteins is maximal before, while that of acid-soluble ones--after the maximum of DNA synthesis. In transformed cells the situation is opposite. RNA synthesis in normal and transformed cells is stimulated at the end of the G2-phase. In normal cells, protein synthesis is coupled with the activation of RNA synthesis, whereas in transformed fibroblasts protein synthesis occurs, in all probability, in the next mitotic cycle. These differences are especially well-pronounced in the expression of some LMG proteins. It is concluded that in transformed cells the regulatory control over the coupling of matrix biosyntheses is impaired.     

A quantitative method for evaluating bivariate flow cytometric data obtained using monoclonal antibodies to bromodeoxyuridine.

A method is presented for analyzing data from bivariate analysis of cell populations exposed to bromodeoxyuridine and subsequently examined both for the presence of BrdUrd and for the cellular DNA content. It is shown that certain features may be defined in the bivariate data which are constant independent both of cell type and, within limits, experimental variability. These landmark features include the ratio of red, DNA, fluorescence of G2 + M cells to G1 cells, the ratio of green fluorescence corresponding to the non-specific binding of unlabeled G2 + M cells to unlabeled G1 cells, and the distribution of green fluorescence in unlabeled cells. The landmarks make it possible to standardize rules for establishing the separation line between-labeled and unlabeled cells as required in these experiments to obtain estimates of cytokinetic parameters. Values obtained for the DNA synthesis time and the potential doubling time which result from different decision rules for distinguishing labeled from unlabeled are compared in two murine tumor lines. The potential doubling time, but not the DNA synthesis time is shown to depend sensitively on the separation line. Suggestions are presented for analyzing clinical data with this procedure.     

Retinoblastoma cell cycling

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material. Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesis-phase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48). DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.     

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.     

Escape from X-ray-induced arrest for lens cells stimulated from quiescence: time relationship to RNA, protein, and DNA synthesis

Quiescent cells of the central zone region of the rat lens epithelium were stimulated to enter the proliferation cycle by wounding. RNA synthesis and a corresponding increase in poly(A)+/total RNA reached a peak by Hour 4. Cells progressed into the G1B compartment by Hour 10. A rise in protein synthesis began at Hour 8, and onset of DNA synthesis occurred by Hour 14. The timing of cell cycle progression that allowed escape from a dose of X irradiation that completely inhibited DNA synthesis was investigated. A growth-arrest point was identified at Hour 9 where 10 GY of X irradiation given before, but not after, completely inhibited earliest responding cells from entering DNA synthesis on schedule. Increased quantities of cells entered DNA synthesis on schedule as timing of the X irradiation was moved closer to the end of G1. Based on time relationships, the rise in protein synthesis is correlated with the "sufficient" event for the escape.     

Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.     

Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine

Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.     

Mutants of Escherichia coli Defective in Membrane Phospholipid Synthesis: Macromolecular Synthesis in an sn-Glycerol 3-Phosphate Acyltransferase Km Mutant

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.     

Differential effects of cyclosporin A on diverse B cell activation phenomena triggered by crosslinking of membrane IgM.

Cyclosporin A (CsA) was tested for its modulatory effects on the mIgM-mediated signaling of G0*-associated increases in class II MHC expression, G1-related RNA synthesis, and S phase-related DNA synthesis in human B cells. While CsA at concentrations as low as 10-100 ng/ml could completely ablate anti-IgM-induced DNA synthesis, earlier G1-associated RNA synthesis was only partially inhibited, and signaling of increased membrane class II MHC expression was unaffected by up to 1000 ng/ml of CsA. Similar phenomena were observed in a clonal population of leukemic B lymphocytes susceptible to anti-IgM-mediated activation in the absence of T cells and T cell factors indicating (a) that the inhibitory effects are not due to CsA-mediated suppression of cytokine production by contaminating T cells, and (b) that the varying effects of CsA on the diverse activation phenomena do not reflect B cell subpopulation diversity. Pulsing studies revealed that while maximal suppression of anti-IgM-induced G1-associated RNA synthesis required CsA at culture initiation, near maximal suppression of DNA synthesis occurred when CsA, or soluble human IgM, was added up to 30 hr after the initial exposure of resting B cells to the anti-IgM ligand. These latter findings are consistent with the possibility that the CsA-mediated suppression of S phase entry is due to the inhibition of a signaling event proximal to mIgM ligation which must be repeatedly initiated throughout the first 30 hr of activation.