Chinese Hamster Tissue Research Articles

Optimal recovery of lymphocytes and tissue culture cells following rapid cooling.

CELLS die if cooled either too rapidly or too slowly during freezing in the presence of dimethylsulphoxide (DMSO)1–4, but survive at an intermediate optimal cooling rate5. We have now obtained improved recovery of lymphocytes, lymphoid cell line cells and Chinese hamster tissue culture cells by interrupting rapid cooling with a timed exposure to a single subzero temperature. This method is simple and here we put forward some theoretical reasons for the improved cell recovery.

Mechanisms of chromosomal aberration production I. Aberration induction by ultraviolet light

We have studied chromosomal aberration production in V-79 Chinese hamster tissue culture cells by UV light administered during the post-DNA-synthetic G 2 phase of the cell cycle. The treatment produced achromatic lesions and some chromatid deletions in the first post-irradiation mitosis, but no isochromatid deletions or chromatid exchange aberrations. In contrast, when G 2 UV-irradiated cells were examined in their second post-irradiation mitosis, there were significant yields of chromatid-type aberrations of all types, including isochromatid deletions and chromatid exchanges. We have earlier reported 21 that UV-irradiation during the pre-DNA-synthetic G 1 phase of the cell cycle induces only chromatid aberrations and also that most chromosomal aberration production by UV in G 1 can be photoreactivated in cells possessing the photoreactivating enzyme. We present here a model for chromosomal aberration production by UV. In the model all aberration production is enzymatically mediated, a consequence of the functioning of known molecular repair mechanisms. The important elements in the model are the following: 1. (1) The vertebrate chromosome is mononeme; i.e., contains but a single DNA double helix during the prereplication G 1 phase of the cell cycle. 2. (2) The UV-induced DNA lesion leading to the production of most aberrations is the cyclobutane dimer between adjacent pyrimidines in one polynucleotide strand. 3. (3) Single chain breaks appear at metaphase as achromatic lesions. 4. (4) Dimer removal sometimes leaves unrepaired single chain gaps, possibly as a result of incomplete excision repair. 5. (5) The single-stranded DNA opposite a single chain gap can be cleaved by a single-strand DNAase. 6. (6) Gaps are left in newly synthesized DNA polynucleotide chains opposite defective template chains ( i.e., opposite dimers and chain breaks). 7. (7) Double-strand breaks present following local DNA replication may “spread” to the other chromatid by a recombinational process between template and new polynucleotide chains, one from each of the homologous double helices. The model predicts the occurrence of isoachromatic lesions and of chromatid deletions paired (isolocus) with achromatic lesions. Though often not reported, both do, in fact, occur. In addition, the model accounts for the phenomenon of sister-chromatid exchange as a manifestation of a recombinational, or post-replication, repair mechanism. Finally, the model offers a simple interpretation of chromosomal aberration production by a variety of chemical agents.

Mechanisms of chromosomal aberration production II. Aberrations induced by 5-bromodeoxyuridine and visible light

We have allowed synchronized V79B Chinese hamster tissue culture cells to incorporate 5-bromodeoxyuridine (BUdR) during one DNA synthetic (S) period of the cell cycle and then determined chromosomal aberration yields induced by illumination of the cells with visible light during the succeeding pre- and post-DNA-synthetic (G 1 and G 2) phases of the cell cycle. At the level used, BUdR by itself induces no aberrations. Illumination during the G 1 phase following incorporation induces aberrations of the chromatid type, but none of the chromosome type. All types of chromatid aberrations are induced, including isochromatid deletions and exchange types. In contrast, when cells are illuminated during the immediately following G 2 phase, large numbers of achromatic lesions and chromatic deletions are seen at the first post-illumination mitosis, but no isochromatid deletions and few exchange-type aberrations occur. When G 2-illuminated cells are examined in their second mitosis, however, chromatid aberrations of all types are again seen. These results are interpreted within the “repair” model of chromosomal aberration production by UV light presented earlier 3. The model assumes that the vertebrate chromosome is mononeme, consisting of but a single DNA double helix during the prereplication G 1 phase. The initial lesions induced by illumination of BUdR-containing DNA are believed to be single-chain breaks, and the observation that G 1 illumination produces only chromatid-type aberrations is taken as additional evidence for the mononeme chromosome. Conversion of single-chain breaks into double chain breaks through the action of a single-strand nuclease is postulated to account for the production of chromatid deletions at the first mitosis of G 2-illuminated cells. The action of this enzyme, plus a recombinational or post-replication repair mechanism, are postulated to account for the production of isochromatid deletions in G 1-illuminated cells. A rapid decline in achromatic lesion frequency with increasing time between G 2 illumination and fixation of the cells is considered evidence for rapid rejoining of most of the initial chain breaks.

Cytoplasmic DNA-binding proteins

Cytoplasmic DNA-binding proteins were isolated from Chinese hamster liver, kidney and tissue culture cells by DNA-polyacrylamide chromatography. With homologous Chinese hamster DNA, and with calf thymus DNA, 1.4% of the proteins were bound to the column. With single-stranded DNA and with heterologous Micrococcus lysodeikticus DNA there was only 0.3% binding, suggesting the proteins preferentially bind to double-stranded DNA and show some sequence specificity. By a nitrocellulose filter assay the bound proteins had at least a 4- to 7-fold greater affinity for DNA than bulk cytoplasmic protein. SDS gel electrophoresis showed that specific proteins were being markedly concentrated by the column and it was primarily the high molecular weight proteins of 65 000 D and over which showed sequence specificity. Some proteins appeared in common with different organs, others were unique. These studies thus define a group of high molecular weight, cytoplasmic proteins which bind to native DNA with a degree of sequence specificity. Their possible relationship to gene regulation is discussed.

Photoreactivation of ultraviolet-induced chromosomal aberrations.

Ultraviolet induces only chromatid-type aberrations in synchronized G(1) V-79 Chinese hamster and A8W243 Xenopus tissue culture cells. Posttreatment with white light prevents expression of most potential aberrations in the A8 toad cell, which possesses a photoreactivation enzyme. We conclude that the major ultraviolet-induced DNA lesion leading to chromosomal aberrations is the pyrimidine dimer.

High Oxygen Pressures as a Contributing Factor in Retinal Detachments

Retinal detachment results from a number of causes and has received attention since 1904 when Gonin postulated the entry of vitreous fluid into the pigment area after a hole occurred in the retina. A symposium on retinal detachment held in 1952 summarized the current information and concluded that breaks in the retina are the most essential factor in the production of detachments and listed three main causes for such breaks: l) localized atrophying processes; 2) localized vitreous pull and 3) trauma. Recent work in our laboratory with tissue culture cells and animals exposed to 100% oxygen has suggested another mechanism.Mouse fibroblast L-929 and Don Chinese Hamster tissue culture cells were exposed for 3, 6 and 12 hours to 100% oxygen at one atmosphere while the rats and rabbits were exposed to the same conditions for 12, 24, 36, 48 and 72 hours. The tissue culture cells were fixed in situ to reduce any mechanical damage and the animals were fixed by perfusion to prevent any movement of the retina before fixation. Conventional methods of dehydration, embedding and sectioning were used.

Ultrastructural manifestations of cellular radiation dose.

One approach to understanding initial radiation effects at the cellular level is the study of irradiated cells with the electron microscope. If the pattern of development of ultrastructural alterations following irradiation can be established and distinguished from more general degenerative changes, then it may be possible to better understand intracellular chemical reactions which precede the observed alterations. Tissue culture cells were selected for the initial phase of this investigation, in hopes that information gained might help orient an electron microscopic examination of the sequential patterns of radiation-induced lesions in cells from selected organ systems of irradiated animals. Some of the results of this study are presented here. Chinese hamster tissue culture cells attached to glass cover slips were irradiated to doses between 2,000 and 10,000 rads with 250 kVp, h.v.t. 1.40 mm Cu x rays. A factor of 1.38 was applied to measured dose rates to correct for electron scatter from the glass sur...

Free fatty acid metabolism in Chinese hamsters.

In normal Chinese hamsters (Cricetulus griseus) the mean concentration of free fatty acids (FFA) in serum varied from group to group, but was (i) consistently 4 to 9 times greater than in rats, dogs, or man; (ii) slightly higher than in Syrian hamsters; (iii) two- to four-fold higher than in fasting or alloxan-diabetic rats. The epididymal adipose tissue of the Chinese hamster (i) had initial concentrations of FFA comparable to those in the rat and Syrian hamster; (ii) released, in the same time interval, 8- to 10-fold more FFA in vitro than this tissue of the rat; (iii) had higher concentrations of FFA after incubation than the incubated tissue of the rat. The retroperitoneal (perirenal) adipose tissue of the Chinese hamster was less active in release of fatty acids in vitro than the epididymal, but was, however, more active than the epididymal adipose tissue of the rat. These characteristics of FFA metabolism in the Chinese hamster were apparently attributable to species, not to age, diet, or sex. In the Chinese hamster, the weight of the epididymal adipose tissue per gram of body was relatively high. It appears that in this species the rate of release of fatty acids from adipose tissue is great, leading to high FFA concentrations in serum.In Chinese hamster and rat adipose tissues in vitro, glucose and insulin (separately) reduced the rate of release of FFA and the amount of FFA in the tissues, but glucose and insulin together produced the greatest reductions. The net reduction in FFA release by glucose and insulin in vitro was greater in tissue from the Chinese hamster. Insulin markedly increased glucose uptake by the adipose tissues of both species. The possible relation of the results to spontaneous diabetes in the Chinese hamster is discussed.

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.

The nucleoli in mitotic divisions of mammalian cells in vitro.

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.

Synthesis of RNA and protein during mitosis in mammalian tissue culture cells

Chinese hamster and HeLa tissue culture cells were incubated in 3H uridine or 3H histidine for a few minutes to 1 hr. 3H-uridine incorporation stopped completely during mitosis and 3H-histidine incorporation was severely depressed. RNA synthesis ceased before nucleolar disappearance and before nuclear membrane disintegration. At the time of nuclear membrane breakdown the nucleolus disappeared and a large amount of nuclear RNA was released to the cytoplasm. Cells arrested in metaphase with colchicine for several hours did not synthesize RNA. The capacity of these cells to incorporate 3H histidine appeared to decline as the duration of metaphase arrest increased. During interphase 3H uridine was incorporated only into nuclear RNA for the first 20 min. Radioactive RNA began to appear in the cytoplasm 10 min later. The transfer of RNA from nucleus to cytoplasm was faster for the more rapidly growing hamster cells than for HeLa. The nucleolus of hamster cells incorporated very little 3H histidine into protein.

Biochemical studies on the Chinese hamster (22 chromosomes) and the Syrian hamster (44 chromosomes)

The DNA content, transaminase, succinoxidase, cytochrome oxidase, and free amino acid patterns of tissues of the Chinese hamster (2N = 22) and the Syrian hamster (2N = 44) were compared. The DNA concentration per cell of liver and spleen and the DNA concentration per gram wet weight of liver, kidney, and lung were of the same order of magnitude for the two species. Transaminase activity per gamma DNA was also about the same but succinoxidase and cytochrome oxidase activity per gamma DNA of the Chinese hamster was about twice that of the Syrian hamster. The free amino acid pattern of the Chinese hamster tissue resembled that of the analogous Syrian hamster tissue more than it resembled other tissues of the Chinese hamster. Some free amino acids were present at approximately the same concentration per gamma DNA in the corresponding tissues of the two hamster species. However, in other cases, an amino acid was present at two times the concentration in the Chinese hamster as in the Syrian hamster tissue. The reverse of this was also observed.