Thermally Enhanced Radiosensitivity of Cultured Chinese Hamster Cells

X-IRRADIATION of mammalian cells in culture yields a survival curve of the threshold type (for review see ref. 1). It isjnter-esting to ask how one can enhance the radiation response by small changes of the physical environment of the cells, as can be done chemically, for example, by incorporation of 5-bromo-deoxyuridine into DNA^1,2. Elevation of the temperature is a likely prospect for enhancement of radiosensitivity for the following reasons. It is known that proteins are heat labile and that temperature sensitive mutants of bacteria and phage can be obtained for many different enzymes³ which are operative at 37° C but not at 42° or 43°C. For example⁴, DNA polymerase is reversibly temperature sensitive; it is rendered inoperative above 42°C, but will be functional again when the temperature is lowered. It is not unreasonable to expect that temperature sensitive mutations for many enzymes occur frequently and that the use of temperatures somewhat higher than the normal range at which the cells grow might disclose sensitivities for specific enzymes in normal cells of higher organisms.     

Density Invariance of Cultured Chinese Hamster Cells with Stage of the Mitotic Cycle

Isopycnic banding of Chinese hamster line CHO cells in Ficoll gradients shows that a population in balanced, exponential growth is very homogeneous with respect to density, the coefficient of variation of the density distribution spectrum being less than 5% of the mean reduced density (i.e. density minus one). Similar measurements on synchronized cultures indicate that reduced density varies by less than 2% around the life cycle. The mean density of CHO cells in F-10 growth medium is calculated to be 1.051 after correction for osmotic effects of the Ficoll gradient.     

Forssman Reactivity and Cell Contacts in Cultured Hamster Cells

THE net synthesis of Forssman hapten glycolipid is enhanced in contact-inhibited NIL cells, particularly in the early stages of contact inhibition^1,2. We have now found that the surface reactivity of cells to anti-Forssman glycolipid antiserum is, however, significantly decreased when they are contact inhibited.     

Progesterone facilitates Implantation of Xenogenic Cultured Cells in Hamster Uterus

ATTEMPTS to explain the phenomenon of nidation on the basis of maternal immunological tolerance have recently made progress^1–4 through pre-sensitization of the mother by the tissue of the father-to-be, followed by studies of the antigenicity of fertilized ova and evaluation of the mother's antibody production. Other workers have stressed the importance of chorionic cells in nidation, based on the occurrence of intraabdominal pregnancy not involving the uterus. Chambon⁵ and Cochrane and Meyer⁶ recognized the influence of oestrogen and progesterone on uterine tissue during nidation and Psychoyos⁷ emphasized the importance of gonadal hormones in the preparation of endometrium for implantation based on delayed nidation. The role of progesterone in the formation of the decidual reaction as a prerequisite to nidation has been clearly established.     

Histone H4 Acetylation and Replication Timing in Chinese Hamster Chromosomes

The distribution of acetylated isoforms of histone H4 along Chinese hamster chromosomes has been studied by immunostaining with antibodies recognizing H4 acetylated at defined lysines in its N-terminal domain. The heterochromatic long arm of the X chromosome in both female (CHO) and male (DON) cell lines is underacetylated at three out of four lysines (5, 8, and 12). In contrast, the level of acetylation at lysine 16, which is the first to be acetylated in mammals, was similar in X chromosomes and autosomes. Labeling of the cells with bromodeoxyuridine (BrdU) to mark late-replicating chromosome domains, followed by double immunostaining with antibodies to BrdU and acetylated H4, showed a close, though not perfect, correlation between late replication and low levels of H4 acetylation. The results show that levels of histone acetylation are associated with the replication timing of defined domains on both the X chromosome and autosomes, but the exceptions we observe suggest that this link is not absolute or essential.     

改造中国仓鼠卵巢细胞

原核细胞、酵母细胞以及昆虫细胞相比,中国仓鼠卵巢细胞（CHO）作为宿主细胞表达的外源蛋白最接近其天然构象,因而CHO细胞表达系统是生物工程制药最为理想的表达系统。但这种系统也存在诸多缺点。如在大规模培养中CHO细胞会面临着对无血清培养基的适应性差、细胞无限度增殖以及细胞凋亡等很多难题。所以除了在培养基、培养条件和表达载体方面下功夫优化该系统外,对CHO细胞本身进行改造已成为优化CHO表达系统的另一热点。      

Non-linkage of Induced Mutations in Chinese Hamster Cells

The relatively rapid loss of human chromosomes from human-rodent somatic cell hybrids has allowed the determination of linkage relationships between several human genes^1–4. Cells that have segregated out most of the human chromosomes are analysed for the presence or absence of particular human gene products; when two gene products are always found to be retained together, they are assumed to be linked. Little has been done to extend these genetic techniques to cell hybrids formed between two different mutants of the same cell line. A linkage analysis would provide a valuable means of interpreting the gene function altered in such mutants. The principal obstacle to such an approach has been the fact that homospecific cell hybrids are rather stable, losing chromosomes at only a low rate^5–7. Nevertheless, by using suitably marked strains, it is possible to select rare segregants from a homospecific hybrid population^7,8. I have applied such a system to test for linkage between several chemically induced mutations in a Chinese hamster cell line.     

Deoxyribonucleic Acid Synthesis in Ultraviolet-Light-Irradiated Chinese Hamster Cells

Two mammalian cell lines, Chinese hamster ovary (CHO) which can recover colony-forming ability between fractionated doses of ultraviolet light (UV), and Chinese hamster B-14FAF28 which cannot recover, were tested for the ability to bypass UV-induced photoproducts in DNA during postirradiation DNA synthesis. The molecular weight distributions of newly synthesized DNA in UV-irradiated populations of both cell lines showed evidence for photoproduct bypass. Hence, the bypass mechanism does not correlate with recovery after UV.     

Growth Dynamics of Mitochondria in Synchronized Chinese Hamster Cells

The increase in cell volume (from electronic cell sizing) and the apportionment of this volume amongst the nuclear, cytoplasmic, and mitochondrial subcellular compartments (from electron microscopy) were studied throughout the cell division cycle in partially synchronized cultures of Chinese hamster V79-S171 cells. Average whole cell volume was found to increase smoothly, consistent with the doubling in one generation of individual cell volume. Nuclear size increased in like fashion. Mean total mitochondrial volume and number of mitochondria per cell both showed a different kind of variation, most notably a significant decrease in G₁ and G₂ as compared with mid S. These results are therefore counter to a model of simple doubling of mitochondria either synchronously with the cell division cycle or asynchronously. Absolute mean values per cell for log phase Chinese hamster cells were also determined, as follows: whole cell volume, 710 μ³; nuclear volume, 190 μ³; total mitochondrial volume, 37.5 μ³; number of mitochondria per cell, 90.     

Sedimentation of DNA Released from Chinese Hamster Cells

Under high pH and high salt conditions, Chinese hamster cells lyse and release a DNA-containing material of large molecular weight. With increasing lysis time, a smaller material is resolved from the large one. Relative to T4 DNA, the smaller is estimated to be ~2 × 10⁸ daltons (number average). From a comparison of radiation data, the target size of the larger is about 15 times that of the smaller (probably a lower limit estimate). In addition to concentration of alkali, temperature, and time of lysis, the resolution of the smaller from the larger material is shown to be affected by other factors. Three of these are: fluorescent light exposure during lysis, X-irradiation before lysis, and incorporation of actinomycin D before lysis. All of these treatments result in degradation of the smaller molecules if large enough exposures are used. The sedimentation patterns of both DNA materials are strongly speed dependent. This probably results from changes in molecular conformation and concomitant increases in viscous drag with speed. The speed dependence differs qualitatively for the two materials, an observation which suggests that they differ in ways in addition to size.     

Ultraviolet Light-Induced Division Delay in Synchronized Chinese Hamster Cells

The age-dependent, ultraviolet light (UVL) (254 nm)-induced division delay of surviving and nonsurviving Chinese hamster cells was studied. The response was examined after UVL exposures adjusted to yield approximately the same survival levels at different stages of the cell cycle, 60% or 30% survival. Cells irradiated in the middle of S suffered the longest division delay, and cells exposed in mitosis or in G₁ had about the same smaller delay in division. Cells irradiated in G₂, however, were not delayed at either survival level. It was further established, after exposures that yielded about 30% survivors at various stages of the cycle, that surviving cells had shorter delays than nonsurvivors. This difference was not observed for cells in G₂ at the time of exposure; i.e., neither surviving nor nonsurviving G₂ cells were delayed in division. The examination of mitotic index vs. time revealed that most cells reach mitosis, but all of the increase in the number of cells in the population can be accounted for by the increase of the viable cell fraction. These observations suggest strongly that nonsurviving cells, although present during most of the experiment, are stopped at mitosis and do not divide. Cells in mitosis at the time of irradiation complete their division, and in the same length of time as unirradiated controls. Division and mitotic delays after UVL are relatively much larger than after X-ray doses that reduce survival to about the same level.     

红细胞生成素在中国仓鼠卵巢细胞中稳定表达

红细胞生成素是调节体内血液生成的一种重要体液因子,对红细胞以及血小板发生都有调节作用,具有非常巨大的临床应用价值。由于该蛋白在正常体内含量甚微,目前国外已研制出体外高表达红细胞生成素的细胞株,临床应用表明其分泌产物对多种贫血尤其是由肾衰引起的贫血具有十分显著的疗效,而且安全可靠。国外几年前就有批量生产,但我国至今无自己的重组产品问市。为此,我们进     

红细胞生成素cDNA在中国仓鼠卵巢细胞中的稳定表达

将(?)红细胞生成素(EPO)cDNA构建的重组表达质粒用电穿孔法引入COS-7细胞,ELISA和红系集落测活结果表明,该重组质粒在哺乳动物细胞中能够表达有生物活性的红细胞生成素。进一步将其转染CHO-dhfr~-细胞,经氨甲喋呤(MTX)加压扩增,混合细胞中各克隆表达水平比较一致,细胞的平均表达水平为2-3μg/10~6 Cells/24hr。细胞冻存后复苏其表达水平与冻存前一致,表明外源基因整合稳定。     

中国仓鼠卵巢细胞的悬浮适应及代谢特征

目的：通过悬浮适应,使中国仓鼠卵巢细胞（CHO细胞）获得悬浮生长的特性,并可在悬浮培养条件下较快地生长。方法：将CHO细胞以3×10^5／mL接种于100mL的三角瓶内,培养时加入1％小牛血清、1g/LPIuronic F-68、25μg／mL硫酸葡聚糖,培养体积35mL,摇床转速90r／min,每24h离心换液,当细胞增殖为2×10^6／mL时传代。结果：经过悬浮适应,细胞的平均比生长速率由适应最初的0．27／d提高为适应后的0．48／d,最大总细胞密度由适应初期的2．5×10^6／mL提高为适应后的6．3×10^6／mL,目的蛋白活性也由适应前的2781U／mL提高为适应后的8878U／mL,适应后细胞的葡萄糖平均比消耗率为1．42μmol／（10^6细胞·d）,低于适应前的2．16μmol／（10^6细胞·d）。结论：贴壁生长的CHO细胞经过悬浮适应,不仅可以在悬浮培养条件下快速生长,而且细胞对葡萄糖的利用率也得到提高。     

Sensitivity of Synchronized Chinese Hamster Cells to Ultraviolet Light

The age response for lethality of Chinese hamster cells to ultraviolet light shows that they are resistant in G(1), sensitive as they move into and through the S phase and resistant again in G(2) and mitosis. Survival curves determined at different times in the cycle reveal that mitotic cells are the most resistant fraction, much more resistant than S cells, and more resistant than either G(1) or G(2) cells. The extent to which the age response is ilfluenced by nucleic acid and protein synthesis was investigated by using inhibitors of these processes. In the presence of inhibitors of DNA or protein synthesis added to G(1) cells before exposure, cell survival neither declines to the minimum survival of S cells nor rises subsequently to the resistance of G(2) cells. If, before exposure, DNA synthesis is arrested in the middle of S, when survival is at a minimum, the subsequent rise in survival during G(2) is not prevented. However when cycloheximide is added before exposure, during the middle of S, this rise is prevented. When actinomycin D, an inhibitor of RNA synthesis is added prior to exposure the age response is affected only slightly. Postirradiation treatment of G(1) and mid-S cells with inhibitors of DNA or protein synthesis maintains survival at a level characteristic of the age of the cells.     

Infection of Chinese Hamster Ovary Cells by Pseudorabies Virus

Chinese hamster ovary (CHO) cells have recently been used for identification of receptors for several alphaherpesviruses, including pseudorabies virus (PrV) (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998). The experiments were based on the fact that CHO cells are inefficient target cells for PrV. However, a detailed analysis of the interaction between PrV and CHO wild-type and recombinant PrV-receptor bearing cells has not been performed. We show here that PrV has a growth defect on CHO cells which leads to a ca. 100-fold reduction in plating efficiency, strongly delayed penetration kinetics, and a 10(4)-fold reduction in one-step growth. Entry of PrV into CHO cells is significantly delayed but is not affected by inhibitors of endocytosis, suggesting that the mechanism of penetration resembles that on permissive cells. The defects in plating efficiency and penetration could be corrected by expression of herpesvirus entry mediators B (HveB), HveC, or HveD, with HveC being the most effective. However, the defects in one-step growth and plaque formation were not corrected by expression of PrV receptors, indicating an additional restriction in viral replication after entry. Surprisingly, PrV infection of CHO cells was sensitive to neutralization by a gB-specific monoclonal antibody, which does not inhibit PrV infection of other host cells. Moreover, the same monoclonal antibody neutralized PrV infectivity on cells displaying the interference phenomenon by overexpression of gD and subsequent intracellular sequestration of gD receptors. Thus, absence of gD receptors on two different host cells leads to an increased sensitivity of PrV toward gB neutralization. We hypothesize that this is due to the increased requirement for interaction of gB with a cellular surface protein in the absence of the gD-gD receptor interaction. As expected, CHO cells are as susceptible as other host cells to infection by PrV gD(-) Pass, an infectious gD-negative PrV mutant. However, PrV gD(-) Pass was also not able to form plaques on CHO cells.     

中国仓鼠卵巢细胞表达新技术

中国仓鼠卵巢细胞（CHO细胞）是基因工程药物生产的最佳表达系统之一,在生物制药中被广泛应用。传统的获得高表达CHO细胞株的方法费时、费力。近年来出现了一些CHO细胞高效表达新技术,它们从克服位置效应,提高基因转录效率、mRNA翻译效率及稳定性、筛选高表达细胞的效率等不同层次调控外源基因在CHO细胞中的高效表达。与MTX加压扩增基因获得高效表达外源基因的方法比较,能够节约时间、减少工作量,不易丢失高表达细胞株。