Messenger RNA synthesis in synchronized Chinese hamster ovary cells

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.     

Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.     

Selection of mitotic Chinese hamster ovary cells from microcarriers

We describe a method of collecting large quantities of mitotic cells from a population of Chinese hamster ovary cells which were exponentially growing on positively charged dextran microcarriers in suspension culture. These cells were treated for 2.5 h with colcemid, and mitotic cells were harvested from the oicrocarriers by increasing spinner velocity. A yield of 2–3% of the total population was obtained using this method; of the cells collected, 85–95% were in metaphase as determined by microscopic inspection. Both synchrony and cell viability were excellent in the selected population.     

Laser flow cytofluorometric analysis of HTC cells synchronized with hydroxyurea, nocodazole and aphidicolin

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.     

Kinetics of micronucleus expression in synchronized irradiated Chinese hamster ovary cells

The kinetics of expression of radiation-induced micronuclei (MN) in synchronized Chinese hamster cells (CHO) was examined. The purpose of the study was to determine if the cell cycle distribution of a population significantly influences the levels of radiation induced MN, thereby obscuring the exact quantification of the radiation effect. Cells were synchronized by centrifugal elutriation, irradiated, and then different phases of the cell cycle were examined for: cell cycle progression, division probability, and temporal expression of MN. The results demonstrate that the time interval for maximal MN expression is long enough that the position of cells in the cell cycle and radiation induced division delays do not prevent the majority of cells from completing their first post-irradiation mitosis, therefore, expressing MN. By following the progression of synchronized cell populations by flow cytometry and also examining the time of division of individual cells for 24 hr after irradiation, we observed that the maximum number of cells from all phases of the cell cycle are in their first post-irradiation interphase at that time, thus explaining the MN results.     

A Chinese hamster ovary cell mutant resistant to aphidicolin

A triple (aphr ara-Ar and araCr) mutant (AP7) of Chinese hamster ovary cells resistant to DNA polymerase inhibitors is described. The aphidicolin-resistance of the mutant was stable and inherited as a dominant genetic trait. The DNA polymerase alpha from the wild type (aphs) and the mutant (aphr) cells differed in their elution profiles on DEAE-cellulose chromatography and in their molecular weights which were 192,000 for the wild type (CHO-K-1, AC6a) and 165,000 for the mutant (AP7) enzymes.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

Fibronectin receptors are functional on mitotic Chinese hamster ovary cells.

In this paper, evidence is provided indicating that the blockade of presynchronized CHO 15B cells in prometaphase by nocodazole is fully reversible and efficient enough to allow us to analyze the function of the integrin receptors. Flow cytometry analysis using a specific antibody raised against the fibronectin receptor, and binding studies of the radiolabeled fibronectin on the cell membrane, indicated a stable number of receptors at the cell surface during mitosis. Furthermore, in the mean time, only a slight increase in the Kd value of the fibronectin-receptor interaction was detected. A binding assay designed to test the affinity of the receptor for its extracellular ligand in an insoluble form was used. No difference was observed between mitotic and interphasic cells. Taken together, these results indicate that the rounding up of the cells observed during mitosis is not due to a loss of the receptor affinity for its extracellular ligand.     

Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly

In the accompanying paper (Cabral, F., 1982, J. Cell. Biol., 97:22-29) we described the isolation and properties of taxol-requiring mutants of Chinese hamster ovary cells. We now show that at least one of these mutants, Tax-18, has an impaired ability to form a spindle apparatus. Immunofluorescence studies using antibodies to tubulin demonstrate that, when incubated in the absence of taxol, Tax-18 forms only a rudimentary spindle with few and shortened microtubules associated with the spindle poles. Furthermore, midbodies were not observed, consistent with an absence of cytokinesis. Essentially normal spindles and midbodies are seen in the presence of taxol. Electron microscopic examination indicates that centrioles and kinetochores are morphologically normal in the mutant strain. Pole-to-kinetochore microtubules were seen but interpolar microtubules were not. Taxol-deprived mutant cells stained with anti-centrosome serum show an elevated centriole content, indicating that the defect in Tax-18 does not affect centriole replication or prevent progression through the cell cycle. Although Tax-18 cells do not form a complete spindle in the absence of taxol, cytoplasmic microtubule assembly occurs in association with microtubule-organizing centers, and microtubules with apparently normal morphology exist throughout the cytoplasm. Observation of chromosome movement indicates that the defect in these cells occurs after prometaphase. These studies demonstrate that the formation of spindle microtubules requires cellular conditions that are different from those required for cytoplasmic microtubule formation. They further show that a normal spindle may be necessary for cytokinesis but not for progress of the cells through the cell cycle.     

Resistance to the mitotic inhibitor NY 3170 in Chinese hamster ovary cells

Mutant CHO cells have been isolated which are resistant to NY 3170, a member of the metahalone group of antimitotic drugs. One NY 3170 resistant mutant is hypersensitive to the microtubule-stabilising drug, taxol, but there is not a reciprocal relationship between levels of resistance to these drugs in CHO cells, since it was found that sixteen other mutants isolated on the basis of taxol resistance had wild type levels of NY 3170 resistance. Our NY 3170 resistant mutants are cross-resistant to vincristine and other mutants, selected on the basis of vincristine resistance, are cross-resistant to NY 3170.     

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.     

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.     

Protection of Chinese hamster ovary cells from hyperthermic killing by cycloheximide or puromycin

Cycloheximide (CHM) and puromycin (PUR) were used at various concentrations up to maxima of 10 micrograms/ml and 100 micrograms/ml, respectively, which inhibited protein synthesis by 95% without any cytotoxicity. The drugs were added to the cells for a maximum period of 7 h, with various combinations for treatment before, during, and after heating. Maximum protection, i.e., a 10,000-fold increase in survival from 5 X 10(-6) to 5 X 10(-2) after 4 h at 43 degrees C, required both 1-2 h of treatment before heating and 1-2 h of treatment during heating. For treatments at 45.5 degrees C, the protection was less, i.e., a 100-fold increase in survival from 10(-5) to 10(-3). Little or no protection was observed if after treatment, the drug was removed before heating, or if the drug was added at the start of heating and left on for 5 min to 3 h after heating. For both drugs, the amount of protection increased as inhibition of protein synthesis increased. However, the amount of protection from the drugs was the same only at about 95% inhibition; at 60-85% inhibition, CHM afforded more protection than PUR. Therefore, the modes of action of the drugs might be common at high drug concentrations, but different when intermediate concentrations are used.     

Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding

A regulatory role for cytoplasmically derived proteins in chloroplast translation in organello was examined by analyzing protein synthesis in plastids isolated from cells of Euglena gracilis which had been treated with cycloheximide (CHI). Incorporation of [35S]methionine by chloroplasts from CHI-inhibited Euglena was reduced approximately 40 and 90% by exposure of the cells to the antibiotic for 2 and 4 h, respectively. The chloroplast translation products were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of polypeptides in the soluble compartment of the plastid was substantially diminished by as little as 15 min of CHI pretreatment. No qualitative alterations of the polypeptide pattern were detected. Qualitative changes were seen in the thylakoid fraction, however. Comparison of the stainable polypeptides and fluorographs of thylakoid membranes from CHI-treated cells with those of controls showed several instances in which the more slowly migrating member of a doublet accumulated with a concomitant depletion of a more rapidly migrating component. A pair of polypeptides at 28 and 30 kDa, which we believe are the Euglena homologs of the photogene product and its precursor, respectively, are representative of this phenomenon. Additionally, thylakoids from cells pretreated with CHI sometimes synthesized novel polypeptides larger than 65 kDa. Finally, when intact chloroplasts from CHI-inhibited Euglena were incubated with a postchloroplast supernatant from normal cells, there was a partial reversion of the anomalies seen in the fluorographs. These data are interpreted to indicate the cytoplasmic origin of one or more proteins whose function is to process chloroplast translation products.     

Exocytosis of pinocytic contents by Chinese hamster ovary cells

The extent of exocytosis of pinocytic vesicle contents was studied in suspension-cultured Chinese hamster ovary (CHO) cells using horseradish peroxidase (HRP) as a pinocytic content marker. HRP was shown to be internalized via fluid-phase pinocytosis in CHO cells. After an HRP pulse of 2.5-10 min a rapid decrease of 30-50% in cell-associated HRP activity was observed within 10-20 min at 37 degrees C. During this time the loss of cell-associated HRP was accompanied by an equivalent increase in extracellular HRP. After this rapid exocytosis of HRP, the remaining peroxidase activity decreased with a t1/2 of 6-8 h, the known lysosomal half-life of HRP. In pulse-chase experiments HRP was chased into a nonexocytic compartment. Based on cell fractionation and electron microscopic experiments, this nonexocytic compartment was identified as a lysosome and the compartment from which exocytosis occurs as a pinosome. The occurrence of pinocytic content exocytosis in cultured fibroblasts suggests that exocytosis of pinocytic vesicle contents is a general phenomenon.     

Effect of neon ions on synchronized Chinese hamster cells

The variation in radiosensitivity across the cell cycle after exposure to neon ions and 60Co gamma-rays is reported for cultured hamster cells. The cells were first synchronized by mitotic selection, then resynchronized in the region of the G1/S boundary by treatment with 10(-3)M hydroxyurea. Although the use of hydroxyurea improves the synchrony, it does sensitize cells at the G1/S boundary to some degree. The cells were exposed at the plateau and the distal peak position of a neon ion beam modified by a 10 cm wide ridge filter. The results indicate that the variation (ratio of maximum to minimum survival after fixed doses of radiation that are approximately matched to produce similar cell killing) was approximately 80 to 100-fold for 60Co gamma-rays and neon ions at the plateau, and 25-fold for distal peak neon ions. While the r.b.e. of distal peak neon ions decreased rapidly with increasing dose for cells in late S-phase, the r.b.e. is independent of dose for cells at the G1/S boundary.