scholarly journals DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions

2018 ◽  
Vol 19 (8) ◽  
pp. 2228 ◽  
Author(s):  
Ian M. Cartwright ◽  
Cathy Su ◽  
Jeremy S. Haskins ◽  
Victoria A. Salinas ◽  
Shigeaki Sunada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Differential Sensitivity ◽  
Iron Ions ◽  
Carbon Ions ◽  
Hypoxic Conditions ◽  
High Let

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/μm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage.

scholarly journals Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

1987 ◽  
Vol 105 (6) ◽  
pp. 2713-2721 ◽  
Author(s):  
D J Yamashiro ◽  
F R Maxfield
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Ph Value ◽  
Ph Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Cell Biol ◽  
Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

scholarly journals Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

1993 ◽  
Vol 13 (9) ◽  
pp. 5175-5185 ◽  
Author(s):  
M J Evans ◽  
J E Metherall
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cho Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Hmg Coa Reductase ◽  
Low Levels ◽  
Coa Reductase

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

scholarly journals Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells

1996 ◽  
Vol 313 (3) ◽  
pp. 991-996 ◽  
Author(s):  
Michael R. NARKEWICZ ◽  
S. David SAULS ◽  
Susan S. TJOA ◽  
Cecilia TENG ◽  
Paul V. FENNESSEY
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Serine Hydroxymethyltransferase ◽  
Cho Cell ◽  
Ovary Cells ◽  
Glycine Metabolism

Serine hydroxymethyltransferase (SHMT) is the primary enzyme in the interconversion of serine and glycine. The roles of mitochondrial and cytosolic SHMT in the interconversion of serine and glycine were determined in two Chinese hamster ovary (CHO) cell lines that both contain cytosolic SHMT but either have (CHOm+) or lack (CHOm-) mitochondrial SHMT. Mitochondrial SHMT activity was significantly reduced in CHOm- (0.24±0.11 nmol/min per mg of mitochondrial protein) compared with CHOm+ (3.21±0.66 nmol/min per mg of mitochondrial protein; P = 0.02) cells, whereas cytosolic SHMT activity was similar in CHOm- and CHOm+ cells (1.09±0.31 and 1.53±0.12 nmol/min per mg of cytosolic protein respectively; P = 0.57). In CHOm+ and CHOm- cells, the relative flux of glycine to serine measured with either [1-13C]- or [2-13C]-glycine was similar (CHOm-: 538±82 nmol/24 per mg of DNA; CHOm+: 616±88 nmol/24 h per mg of DNA; P = 0.42). In contrast, the relative flux of serine to glycine measured with [1-13C]serine was low in CHOm- cells (80±28 nmol/24 h per mg of DNA) compared with CHOm+ cells (3080±320 nmol/24 h per mg of DNA; P = 0.0001). The rate of glycine production determined by UA-2[1-13C]glycine dilution was lower in CHOm- (1200±200 nmol/24 h per mg of DNA) than CHOm+ (10200±1800 nmol/24 h per mg of DNA; P = 0.03) cells, whereas glycine utilization was similar in the two cell lines. Serine production was similar in the two cell lines but serine utilization was lower in CHOm- (3800±1200 μmol/24 h per mg of DNA) than CHOm+ (6600±1000 nmol/24 h per mg of DNA; P = 0.0002) cells. Increasing the serine concentration in the medium resulted in an increase in glycine production in CHOm+ but not in CHOm- cells. Intracellular studies with [1-13C]serine confirm the findings of decreased glycine production from serine. In CHO cells there is partitioning of intracellular serine and glycine metabolism. Our data support the hypothesis that mitochondrial SHMT is the primary pathway for serine into glycine interconversion.

Revertants of a Chinese Hamster Ovary Cell Mutant with an Altered β-Tubulin: Evidence that the Altered Tubulin Confers Both Colcemid Resistance and Temperature Sensitivity on the Cell

1982 ◽  
Vol 2 (6) ◽  
pp. 720-729
Author(s):  
Fernando Cabral ◽  
Irene Abraham ◽  
Michael M. Gottesman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Temperature Sensitivity ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Β Tubulin

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20 :29-36, 1980). This mutant carries an altered β-tubulin but still grows normally at 37°C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3°C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered β-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant β-tubulin polypeptide. This second alteration appears to make the mutant β-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in β-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered β-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

scholarly journals Overexpression of phosphatidylethanolamine N-methyltransferase 2 in CHO-K1 cells does not attenuate the activity of the CDP-choline pathway for phosphatidylcholine biosynthesis

1996 ◽  
Vol 320 (3) ◽  
pp. 905-910 ◽  
Author(s):  
Mark W. LEE ◽  
Marica BAKOVIC ◽  
Dennis E. VANCE
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Protein Level ◽  
Chinese Hamster ◽  
Immunoblot Analysis ◽  
Phospholipid Metabolism ◽  
Enzymic Activity ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Phosphatidylcholine Biosynthesis

Chinese hamster ovary (CHO) cells express only a trace amount of phosphatidylethanolamine N-methyltransferase (PEMT) activity. CHO cells make their phosphatidylcholine (PC) via the CDP-choline pathway. We investigated whether or not overexpression of PEMT2, an isoform of PEMT, in these cells would down-regulate the activity of the CDP-choline pathway. Transfection of CHO cells with PEMT2 cDNA behind the cytomegalovirus promoter resulted in a series of cell lines that overexpressed PEMT2. Phospholipid metabolism was characterized in cell lines that expressed a medium (281 pmol/min per mg of protein) and a high (1300 pmol/min per mg of protein) level of PEMT activity. The expression of the regulated enzyme (CTP:phosphocholine cytidylyltransferase) in the CDP-choline pathway was increased, not decreased, in these cell lines as judged by immunoblot analysis and enzymic activity. Conversion of phosphatidylethanolamine to PC was enhanced in CHO cells that expressed PEMT2 activity. PC mass was not increased in the transfected compared with the control cells. The rate of PC catabolism made by either the CDP-choline or methylation pathways was unaffected by PEMT2 expression. We conclude that expression of PEMT2 in CHO cells does not down-regulate, but rather enhances, the expression of CTP:phosphocholine cytidylyltransferase.

scholarly journals Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells

1993 ◽  
Vol 13 (9) ◽  
pp. 5175-5185
Author(s):  
M J Evans ◽  
J E Metherall
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cho Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Hmg Coa Reductase ◽  
Low Levels ◽  
Coa Reductase

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

scholarly journals Expression of a human gene for polyamine transport in Chinese-hamster ovary cells

1989 ◽  
Vol 263 (3) ◽  
pp. 745-752 ◽  
Author(s):  
T L Byers ◽  
R Wechter ◽  
M E Nuttall ◽  
A E Pegg
Keyword(s):  
Transport System ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Molecular Genetic ◽  
Chinese Hamster ◽  
Transport Systems ◽  
Polyamine Transport System ◽  
Polyamine Transport

A molecular-genetic approach towards isolating mammalian polyamine-transport genes and their encoded proteins was devised involving the production of Chinese-hamster ovary (CHO) cells expressing a human polyamine-transport protein. CHO cells and a polyamine-transport-deficient CHO mutant cell line (CHOMG) were equally sensitive to the antiproliferative effects of alpha-difluoromethylornithine (DFMO), which blocked endogenous polyamine synthesis. Exposure to exogenous polyamines increased intracellular polyamine levels and reversed this DFMO-induced cytostasis in the CHO cells, but not in the CHOMG cells. CHOMG cells were therefore transfected with human DNA (isolated from HT-29 colon carcinoma cells) and cells expressing the human polyamine-transport system were identified by the ability of these cells to grow in a medium containing DFMO and polyamines. A number of different positive clones were identified and shown to have the capacity for polyamine uptake and an increased sensitivity to the toxic effects of the polyamine analogue methylglyoxal bis(guanylhydrazone). Differences in these properties between the clones are consistent with a multiplicity of polyamine-transport systems. Some clones also showed a change in growth characteristics, which may indicate a relationship between genes involved in the polyamine-transport system and in cell proliferation.

scholarly journals Synthesis and biochemical properties of a new photoactivatable cholesterol analog 7,7-azocholestanol and its linoleate ester in Chinese hamster ovary cell lines

2002 ◽  
Vol 43 (8) ◽  
pp. 1341-1347 ◽  
Author(s):  
Jonathan C. Cruz ◽  
Matthew Thomas ◽  
Edmund Wong ◽  
Nobutaka Ohgami ◽  
Shigeki Sugii ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cholesterol Metabolism ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Biochemical Properties ◽  
Ovary Cell ◽  
Cyclodextrin Complex ◽  
Mutant Cell Line

We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1.These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.

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