CHANGES IN SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS DURING THE CELL CYCLE

Synchronized populations of Chinese hamster ovary (CHO) cells in confluent culture have been examined by scanning electron microscopy and their surface changes noted as the cells progress through the cycle. During G₁ it is characteristic for cells to show large numbers of microvilli, blebs, and ruffles. Except for the ruffles, these tend to diminish in prominence during S and the cells become relatively smooth as they spread thinly over the substrate. During G₂ microvilli increase in number and the cells thicken in anticipation of rounding up for mitosis. It appears that the changes observed here reflect the changing capacity of CHO cells during the cycle to respond to contact with other cells in the population, because, as noted in the succeeding paper (Rubin and Everhart), CHO cells in sparse nonconfluent cultures do not show the same wide range of changes during the cell cycle. Normal, nontransformed cells of equivalent type in confluent culture are essentially devoid of microvilli, blebs, and ruffles. The relation of these surface configurations to the internal structure of the cell is discussed.     

CELL KILLING BY ACTINOMYCIN D IN RELATION TO THE GROWTH CYCLE OF CHINESE HAMSTER CELLS

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G₂ (or G₂-mitosis) they are maximally resistant; at 1.0 µg/ml, for example, the survival in G₂ is 30-fold greater than it is in G₁. These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).     

SITES OF NUCLEOLUS PRODUCTION IN CULTURED CHINESE HAMSTER CELLS

Chinese hamster cell strains in the early passages in culture display wide variation in number of nucleolus-like bodies per cell, though such strains are characteristically euploid. A variety of criteria indicate that the nucleolus-like bodies are true nucleoli. Their Azure B- and fast green-staining properties indicate the presence of RNA and protein; they have typical nucleolar fine structure, including both fibrous and granular components; radioautography reveals that their patterns of uptake of uridine-³H into RNA are similar to those reported for nucleoli of other cell types; actinomycin D, at a level which selectively inhibits ribosomal RNA synthesis, greatly reduces their RNA synthesis and also causes segregation of fibrous and granular nucleolar components. Colchicine was used to experimentally fragment the nuclei of these cells into a number of separate karyomeres, each presumably containing some, or only one, of the chromosomes of the complement. Almost all the karyomeres contain nucleolus-like bodies which, by the same criteria applied to the multiple nucleolus-like bodies of uninuclear cells, appear to be true nucleoli. The nucleoli of individual karyomeres of the same cell often differ from each other in fine structure while the multiple nucleoli of a uninuclear cell generally resemble each other. The evidence presented in this study indicates that Chinese hamster cells contain many nucleolus-producing sites scattered through the genome.     

CORRELATION BETWEEN CELL SIZE AND POSITION WITHIN THE DIVISION CYCLE IN SUSPENSION CULTURES OF CHANG LIVER CELLS

Chang liver cells from exponentially growing suspension cultures have been separated by sedimentation at unit gravity. Determinations of the protein content per cell showed that the fractionation procedure resulted in good separation of cells of different size. On the other hand, the DNA content of individual cells from the fractions, as determined cytofluorimetrically, indicated considerable heterogeneity in the size of cells from the same stage of the division cycle. On the basis of earlier results on intermitotic growth and the variation in the length of the cell cycle in homogeneous cell populations, a mathematical model has been constructed and tested using a computer program. The present results on the size distribution of cells from the different stages of the mitotic cycle are consistent with a regeneration of size heterogeneity in each cell generation, as a result of the dispersion of intermitotic times. The variation in cell cycle times may be related to a probabilistic event in the G₁ period. In the mathematical model it was necessary to include a mechanism by which the regeneration of abnormally large cells is prevented. The experimental data are compatible with a gradually increasing inhibition of growth in cells larger than a certain size (circa 400 pg protein per cell).     

LZ—410盼生安对肝癌细胞细胞周期时相的影响及对DNA损伤效应的观察

LZ-410盼生安是一种广谱抗肿瘤药物。本文以5-Fu作为阳性对照药物从对BEL-7402肝癌细胞周期时相的影响及对DNA损伤效应两个方面进行了初步探讨。结果表明,LZ-410能将细胞阻滞在G0/G1期,抑制了细胞的生长增殖作用。并且在本实验条件下,LZ-410并不引起DNA的损伤。这表明LZ-410抑制肝癌细胞生长增殖不是通过对DNA损伤引起的,而主要是通过阻滞肿瘤细胞于G0/G1期实现的。     

RELATION OF PROTEIN SYNTHESIS TO THE DIVISION CYCLE IN MAMMALIAN CELL CULTURES

The distribution of the reaction product of a staining method for adenosine triphosphatase (ATPase) in rat small intestine, kidney, and liver was studied with electron microscopy. Several procedures were tried but the best results were obtained from tissue that had been quenched in liquid nitrogen, sectioned at 25 µ in a cryostat, fixed for 30 to 90 minutes at 4°C in formalin-sucrose buffered to pH 7.2, incubated with substrate, and then osmicated and prepared for electron microscopy in the usual way. This procedure enabled the localization of mitochondrial ATPase to be studied. In tissue fixed in small blocks in osmium tetroxide for 3 minutes prior to incubation with substrate, good preservation was noted, and the reaction product for ATPase was localized on the cell membrane and nuclei. The reaction product was present in abundant amount in the nuclei, and particularly within nucleoli, of all tissues studied. Because the histochemical localization of nuclear enzymes poses numerous interpretative problems at the present time, the significance of this nuclear localization is uncertain. Cell (plasma) membranes were the site of localization, especially at areas where it has been proposed that active transport mechanisms may occur, namely, on the microvilli of intestinal epithelium, endothelial lining of capillaries, glomerular epithelial cell membranes, basal infoldings of the cell membrane of renal tubules, on the microvilli of bile canaliculi, and on the microvilli of proximal convoluted tubular epithelial cells. ATPase localization on the cristae mitochondriales was also demonstrated.     

A TECHNIQUE FOR DETERMINING THE PROPORTION OF THE CLONOGENIC CELLS IN S PHASE IN EMT6 CELL CULTURES AND TUMORS

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (³H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by ³H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of ³H-TdR. Under these conditions, either ³H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or ³H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by ³H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogenic tumor cells in S phase. Incubation of tumor cell suspensions with ³H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of ³H-TdR during the incubation for colony formation.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

NEW METHODS OF CELL CYCLE ANALYSIS BASED ON THE SELECTIVE TAGGING OF CELLS EITHER AT THE BEGINNING OR END OF S PHASE

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [³H]-labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [³H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the ‘early S cohort’ cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G₂, S + G₂+ M and T. It should theoretically be possible to follow ‘late S cohort’ cells in a similar manner, allowing a determination of G₂, G₂+ M and G₂+ M + G₁. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log-normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.     

视黄酸对胃癌细胞周期的调控

视黄酸(RA)能够抑制许多类型癌细胞生长、诱导细胞凋亡和调节细胞周期。本文研究了全反式视黄酸(ATRA)对人胃癌细胞的作用机理。结果表明,ATRA通过诱导细胞滞留在G_0/G_1期而显著抑制胃癌细胞生长,但ATRA不能诱导胃癌细胞凋亡;ATRA调控细胞周期与c-myc、磷酸化Rb水平的下调和p21~(WAF1/CIP1)、p53水平的上调有关,而cyclinD_1和CDK_4水平没有明显变化。在RA抗性细胞中,ATRA不能调节这些基因表达。结果证实,ATRA对胃癌细胞生长抑制与其诱导细胞滞留在G_0/G_1期有关,而与细胞凋亡的诱导无关,许多重要的、与周期相关的分子,包括cmyc、p21~(WAF1/CIP1、p53和Rb等参与细胞周期的调控。     

THE EFFECT OF THYMIDINE ON THE DURATION OF G1 IN CHINESE HAMSTER CELLS

The generation time of a Chinese hamster cell line was varied by the use of different lots of sera in the culture media. Analysis of the division waves following thymidine synchronization showed that lengthening of the generation time was a result of an increase in duration of the G₁ phase and that thymidine treatment reduced the duration of G₁ back to its minimum value.     

AGE-DEPENDENT TOXIC PROPERTIES OF ACTINOMYCIN D AND X-RAYS IN CULTURED CHINESE HAMSTER CELLS

A comparative study was made of the toxic properties of actinomycin D and X-rays using synchronized populations of Chinese hamster cells cultured in vitro. X-irradiated cells are most resistant in the latter half of the DNA synthetic period (late S). While cells treated with actinomycin D appear to go through a survival maximum at the same age, they are most resistant after the completion of DNA synthesis; i.e. in G₂ (or G₂-mitosis). In spite of these differences, we found that actinomycin D damage in late S cells interacts with X-ray damage. Thus, a common locus for the site of actions of both agents is suggested which may be in or around the genome of a cell in view of the well-known DNA binding properties of actinomycin D.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

QUANTIFICATION OF ENTRY INTO AND EXIT FROM THE CELL CYCLE IN HUMAN LYMPHOCYTE CULTURES

A mathematical model is presented for the analysis of transition between cycling and non-cycling compartments by cells responding to a growth stimulus. the cellular age distribution as a function of time is derived from sequential [³H]thymidine pulse labeling indices. Rates of entry into and exit from the cycling compartment are determined on the basis of labeling indices obtained after instantaneous and long duration [³H]thymidine pulses. Analysis of an experiment involving sequential measurements over the whole lifespan of a human lymphocyte culture stimulated by phytohemagglutinin is presented as an example of the application of this method.     

RATE AND TIME OF DNA SYNTHESIS OF INDIVIDUAL CHINESE HAMSTER CELLS

The duration of DNA synthesis of a diploid cell line of Chinese hamster fibroblasts was determined in a comparative study by the FLM technique, and also by a new technique for measuring the rate of DNA synthesis of individual cells. These methods produced comparable results when applied during exponential growth of the cells. The rate of DNA synthesis was measured by means of quantitative autoradiography following a short-term incubation of the cells with 5 × 10^-6 M FUdR and 10^-5 M ¹⁴C-TdR. The choice of the medium for this purpose did not seem to be critical. The autoradiographic silver grains over cells and ¹⁴C-standard sources are counted by microphotometry using incident light bright-field. The direct measurements of DNA synthesis rate are ‘compartment’ statistics which have been converted into ‘flux’ parameters for comparison with the FLM method and applicability in cell-kinetic calculations. Frequency distributions of the rate of DNA synthesis of individual cells thus obtained may resemble normal distributions quite closely. They result from several factors: differences in the rate of synthesis in different parts of the S-phase, the density distribution of cells within the S-phase, the variation in the time of DNA synthesis among individual cells, and the experimental error. In the case of a pronounced partial synchronization as probably has been present in one experiment performed in the lag phase, an incorrect time of DNA synthesis may result from the rate values. Due to the variation in DNA synthesis rate in different parts of the S-phase it is not possible to determine the duration of DNA synthesis of an individual cell. However, the mean values of DNA synthesis time are reliable. The new method will be preferentially applied for determining the duration of DNA synthesis of human cells in as far as difficulties are encountered with the classical methods. In addition, it may be used to advantage for studying cells which make up low percentages in mixed populations. It finally permits a safer morphological classification of the cells under study than is possible with the classical methods.     

丝裂霉素C诱发中国仓鼠体外培养细胞姐妹染色单体互换的研究

本文对不同年龄中国仓鼠各种组织的体外培养细胞姐妹染色单体互换(SCE)频率进行了比较研究。实验结果表明,体外培养细胞的自发SCE频率与动物年龄无关,相同组织细胞经MMC处理后,老龄仓鼠SCE频率比幼龄仓鼠SCE频率明显低。心脏和皮肤细胸的SCE频率高于肺和尾的SCE频率。