Diploid Chinese Hamster Research Articles

Early chromosome changes in diploid Chinese hamster cells after infection with Simian virus 40.

AbstractPrimary and secondary Chinese hamster (ChH) embryo cells infected with SV40 exhibited chromosomal changes within one cell generation (24 h). The initial change seen is an increase in the number of polyploid metaphases (>10%). The majority of the polyploid cells are tetraploid (8×), however, higher ploidy values were also observed (16 ×, 32 × and 64 ×). In approximately 20–30% of the polyploid metaphases there were chromosome changes such as breaks, abnormal chromosomes and loss and addition of chromosomes. The remaining polyploids were normal in regard to chromosome number and chromosome morphology. A primary mouse embryo cell system also responded to SV40 infection with an increase in the number of polyploid cells within 48 h.

Intermittent DNA synthesis and periodic expression of enzyme activity in the cell cycle of WI-38.

Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. The generation time in synchronous WI-38 is 19.5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period. Lactate dehydrogenase (LDH) displayed maxima in G(1) between 2 and 4 h and again at 10 and 16 h. Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells. Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines.

Relative radiosensitivities of tetraploid and diploid Chinese hamster cells in culture exposed to ionizing radiation

Relative radiosensitivities of tetraploid and diploid Chinese hamster cells in culture exposed to ionizing radiation

Replicative heterogeneity of mammalian DNA

Studies of diploid Chinese hamster cells synchronized by mitotic selection suggest that the very earliest DNA to be replicated is relatively AT-rich. There is then a shift to GC-richness as the bulk of the euchromatin is replicated, followed by a return to relative AT-richness as heterochromatin begins to be synthesized later in the S period. These and other observations suggest that the shift in base composition of DNA replicated during the latter part of the S period is a reflection of the distinct types of DNA present in heterochromatin. The frequent appearance of subcomponents in the curves of pulse-labeled DNA centrifuged in cesium chloride suggest there are families of DNA sequences of similar base composition that tend to replicate at similar times.

Temporal Coordination of DNA Replication with Enzyme Synthesis in Diploid and Heteroploid Cells

The rate of DNA synthesis in the S phase of growth of synchronized diploid Chinese hamster cells shows two maximums, while in heteroploid hamster cells the DNA replication rate is constant. In diploid cells a reciprocal relationship exists between maximum DNA synthetic rates and maximum lactate dyhydrogenase and thymidine kinase enzyme levels. Enzyme activity in heteroploid cells increases continuously through the cell cycle with no evidence of oscillations. It seems possible that these differences in molecular organization may accompany or precede the transition to heteroploidy.

The progression of mammalian cells through the division cycle following ultraviolet irradiation

Exposure of living cells to ultraviolet light in the 254 mμ range leads to photochemical changes in the nucleic acids. The influence of these changes on progression through the division cycle was investigated using two different cell lines: a heteroploid epithelial cell line of human kidney origin (T cell) and a diploid chinese hamster fibroblast (DON cell). The duration of the cell cycle of these lines was 24–26 and 10–12 h respectively. Synchronous and asynchronous cultures were used. Cells irradiated in late G 2 phase with doses of 100 erg/mm 2 or less were not or only slightly delayed in entering mitosis. Mitotic delay increased when the cells were irradiated earlier in G 2. Exposure in S phase was followed by an inhibition of DNA synthesis, leading to a lengthening of S (4–5 h after a dose of 35 erg/mm 2) and delay of cell division. This inhibition increased if irradiation was carried out earlier in S phase. After exposure of T cells in G 1 phase, which has a relatively long duration, the cells started earlier with [ 3H]thymidine incorporation. This incorporation indicates a shortening of G 1 phase rather than an unscheduled synthesis of DNA. In general these data suggest that the UV induced changes in the nucleic acids (at a relatively low dose level) mainly interfere with DNA replication in S phase and do not or only slightly inhibit progression through other parts of the cell cycle.

Replicative behaviour of bovine X-chromosomes during early DNA synthesis.

THE patterns of DNA replication in several mammalian chromosome complements have been investigated by the use of labelled DNA precursors and autoradiography1–5. Such work has established that replication of the sex chromosomes is asynchronous during the terminal stages of DNA synthesis. Several investigations have attempted to elucidate whether or not the chromosomes which terminate their replication later than all other chromosomes in a mammalian complement also start their synthesis late. In cultured cells from male and female Chinese hamsters Taylor1 observed that the late-replicating sex chromosomes duplicate only during the last half of the 6 h synthetic period. Using 5-fluorodeoxyuridine (FUdR) to synchronize cultures of diploid Chinese hamster cells, Hsu6 obtained evidence which suggested that the late-replicating Y and long arm of the X-chromosome begin DNA synthesis approximately 4.5 h after DNA synthesis has commenced in the rest of the complement. Using a comparable technique, Petersen7 independently identified a “late-starting” chromosome in cultured leucocytes from a human female, which he presumed to be the late-replicating X- chromosome. In cultured cells from bovine males, Gartler and Burt8 observed that the “late synthesizing Y- chromosome” begins replication later than the rest of the chromosomes in the complement. These observations suggest that “late-replicating chromosomes” or chromosome segments are correspondingly late in beginning DNA synthesis. Stubblefield9, however, using a modified autoradiographic technique, came to the conclusion that the “late-replicating Y” and the long arm of the X in the male Chinese hamster complement do “incorporate a significant amount of isotope during the first 15 min of synthesis”. Making use of a continuous labelling technique and a human complement with X/iso-X sex chromosome constitution, where the iso-X is known to be “late-replicating”, it has been shown10 that the iso-X chromosome begins its replication along with autosomes of comparable length and is therefore not the last chromosome in the complement to initiate replication.

SEQUENCE OF DNA REPLICATION IN THE ISO-X CHROMOSOME FROM X/ISO-X HUMAN FEMALES DURING THE INITIAL STAGES OF THE SYNTHETIC PERIOD

The asynchronous mode of terminal replication in the sex-chron~osomes of mammalian cells has been well established (Taylor, 1960; German, 1962; A4ultherjee and Sinha, 1963; Galton, 1964) but the pattern of initiation ol' DN.4 synthesis in these chromosomes has remained more of an open question. In his autoradiographic studies on chromosome duplication in cultured cells from Chinese hamsters, Taylor (1960) observed that the long arm of the X-chromosome and the entire Y in the male, and the long arm of one X-chromosome and the entire portion of the other X in a female strain, duplicated only during the last half of the DNA synthetic period. Hsu (1964) examined the initiation of DNL4 synthesis in the late-replicating X and Y chromosomes of cells from a diploid male Chinese hamster strain. By inhibiting DNA synthesis with the application of 5-fluorodeoxyuridine (5-FUdR) to the culture to accumulate a large number of cells at the entry to the DNA synthetic period, and then relieving the inhibition with the addition of tritiated thymidine (H'TdR), he obtained evidence which suggests that the late-replicating Y-chromosome and long arm of the X begin DNA synthesis approximately 4.5 hours later than the remainder of the con~plement. Following a con~parable lal~elling technique, Petersen ( 1964) independently identified one chromosome in the normal human female complement which was late in initiating its DNA synthesis and this chromosome was presumed to be the late-replicating X. In ciltured cells from bovine males, Gartler and Burt (1964) found that the late replicating 1--chromasome begins replication later than all other chromosomes in the complen~ent. These and other investigations (Chang e t nl., 1965; Hsu and Loclthart, 1965; Cave, 1966) have suggested that late-replicating chroniosonies or chron~osome segments are correspondingly late in beginning DNA synthesis. Using the same Chinese hamster cell strain and FudR technique as Hsu, Stubblefield ( 1965) made repeated autoradiographs of the isotope distribution in the chron~osomes of a single cell. After the separate autoradiographs were combined into a single photograph, it became clear that the Y-chromosome and the long arm of the X do incorporate a significant amount of isotope during the early synthetic period, and are therefore not late in beginning DNA replication. The present study, malting use of a continuous labelling technique (Multherjee and Sinha, 1965) applied to human X/iso-X female complen~ents where the iso-X could be clearly identified, was undertaken to deterlllille the esact time of inception of DNA svnthesis in the late-replicating isochroniosome-X. The evidence presented in this paper strongly suggests that the latesynthesizing iso-X chromosome is not correspondingly late in initiating DNA synthesis, but begins replication almost simultaneously with chromosomes of

Viruses and mammalian chromosomes IV. Replication of herpes simplex virus in diploid Chinese hamster cells

Diploid Chinese hamster cells are less susceptible to herpes simplex virus than are primary cells derived from young rabbit kidneys. Study of the most sensitive Chinese hamster cell-virus system revealed a 12-hour latent period; at that time, intranuclear inclusion bodies and both intranuclear and intracytoplasmic virus antigen could be detected. Newly synthesized infectious virus was detected 2 hours later and continued to increase until 24 hours after inoculation of the cultures. A study of the replicative site of virus nucleic acid failed to reveal specific sites of synthesis on the chromosomes. Nevertheless, virus replication is necessary for the induction of the chromosomal aberrations previously observed for noninfective virus was unable to induce such chromosomal changes. However, the aberrations are not caused by synthesis of the viral nucleic acid at or near the broken or uncoiled areas on the chromosomes.

Mammalian Chromosomes <italic>In Vitro</italic>. XVII. Idiogram of the Chinese Hamster<xref ref-type="fn" rid="FN2">2</xref>

From 67 diploid male Chinese hamster cells in vitro, an idiogram was constructed. The chromosomes can be classified into 4 natural groups: group I, 2 pairs of large submetacentrics (1 and 2); group II, 3 pairs of medium-sized submetacentrics (the sex pair, 4 and 5); group III, 3 pairs of medium-sized subtelocentrics (6,7, and 8); group IV, 3 pairs of small metacentrics (9,10, and 11). A system of subdividing each chromosome into regions is also suggested.

X-Ray-Induced Heritable Damage (Small-Colony Formation) in Cultured Mammalian Cells

Colonies of diploid Chinese hamster lung cells (strain V) surviving large doses (1500 r) of 250-kvp x rays have a broad size spectrum which is shown to be due to the variable growth rate of the colonies. The properties of large and small colonies were corapared after isolation and subcultivation. Large colonies give rise to fast-growing populations of high plating efficiency which have the same radiosensitivity as the parent unirradiated cells. Small colonies of 50 cells or more at 12 to 14 days are viable and give rise to sublines which have slow growth rates, poor plating efficiency, and increased radiosensitivity (D/sub 0/ changed from 205 rads to approximates 145 rads, n unaltered). The chromosome number spectrum is broadened for all surviving colonies, severely in some cases to modal tetraploid numbers; but neither this, nor the high frequency of chromosome aberrations, appears to be involved in the radiosensitivity of the survivors. Reirradiation of smallcolony populations appears to produce a further spread in clone size but no further changes in the radiosensitivity of individual clones.

An improved nutrient solution for diploid Chinese hamster and human cell lines

Summary 1. Single cell plating of diploid Chinese hamster ovary cells (Strain CHD-3) in a medium containing a minimum supplement of purified proteins has been used to determine optimum concentrations for the components of a previously described nutrient mixture, F7. 2. The more significant results of these titrations include: (a) a high arginine requirement; (b) a low toxic limit for tryptophane; (c) a requirement for proline; and (d) a narrow optimum range for serine and glycine. 3. A new nutrient mixture, F10, has been prepared in which each component has been added at the experimentally determined optimum concentration. 4. Tests of the new nutrient mixture using strain CHD-3 revealed: (a) improved growth rate and reliability in a fetuin and albumin supplemented medium; (b) clonal growth at total protein concentrations as low as 100 μg/ml.; (c) excellent growth at reduced serum concentrations; (d) satisfactory clonal growth using batches of fetal calf serum which would not support such growth in F7 media; (e) ability to use sera other than fetal calf serum, for clonal growth. 5. Tests of media prepared from the new nutrient mixture on other cell strains revealed: (a) good growth with a fetuin and albumin supplement of strain CHL-1 Chinese hamster lung cells and strain S3-9-IV HeLa cells; (b) rapid growth of strain CHL-1 on a 2 per cent serum medium yielding dense colonies estimated to contain 1000 or more cells in five days' incubation time; (c) improved growth of a human diploid cell line; (d) excellent short term growth of human white blood cells for chromosome preparations.

Clonal growth of diploid Chinese hamster cells in a synthetic medium supplemented with purified protein fractions

1. 1. A nutrient mixture is described which, when supplemented with two purified protein fractions, forms a medium which will support colonial growth of an essentially euploid Chinese hamster cell line (CHBOC1D1). 2. 2. This serum-free, protein-supplemented medium differs from a similar medium previously described for the growth of S3 HeLa cells in the following ways: ( a) the amino acids, vitamins, growth factors and proteins have been reduced in concentration; ( b) folic acid and calcium chloride have been increased in concentration; and ( c) thymidine, pyruvate, cysteine, serine, asparagine and ferric chloride have been added. 3. 3. Data demonstrating specific requirements for a number of components of the medium for colonial growth of strain CHBOC1D1 are presented, together with a discussion of the biochemical and physiological basis of the requirements.