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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5175-5185.1993 ◽

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

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

The Journal of Cell Biology ◽

10.1083/jcb.105.6.2713 ◽

1987 ◽

Vol 105 (6) ◽

pp. 2713-2721 ◽

Cited By ~ 64

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.

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A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line

Genome ◽

10.1139/g03-046 ◽

2003 ◽

Vol 46 (4) ◽

pp. 707-715 ◽

Cited By ~ 4

Author(s):

K Neumann ◽

K M Al-Batayneh ◽

M J Kuiper ◽

J Parsons-Sheldrake ◽

M G Tyshenko ◽

...

Keyword(s):

Cell Line ◽

Point Mutation ◽

Cell Lines ◽

Dihydrofolate Reductase ◽

Cho Cells ◽

Chinese Hamster ◽

Single Point Mutation ◽

Parental Line ◽

Wild Type ◽

Drosophila Cell

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were ~1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing ~400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.Key words: dihydrofolate reductase, methotrexate, drug resistance, point mutation.

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

International Journal of Molecular Sciences ◽

10.3390/ijms19082228 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2228 ◽

Cited By ~ 7

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.

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Model Identifies Genetic Predisposition of Alzheimer’s Disease as Key Decider in Cell Susceptibility to Stress

International Journal of Molecular Sciences ◽

10.3390/ijms222112001 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12001

Author(s):

Ioanna C. Stefani ◽

François-Xavier Blaudin de Thé ◽

Cleo Kontoravdi ◽

Karen M. Polizzi

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cell Lines ◽

Transcriptional Activation ◽

Mutant Cell ◽

Human Neuroblastoma Cell ◽

Protein Accumulation ◽

Wild Type ◽

Human Neuroblastoma ◽

Unfolded Protein

Accumulation of unfolded/misfolded proteins in neuronal cells perturbs endoplasmic reticulum homeostasis, triggering a stress cascade called unfolded protein response (UPR), markers of which are upregulated in Alzheimer’s disease (AD) brain specimens. We measured the UPR dynamic response in three human neuroblastoma cell lines overexpressing the wild-type and two familial AD (FAD)-associated mutant forms of amyloid precursor protein (APP), the Swedish and Swedish-Indiana mutations, using gene expression analysis. The results reveal a differential response to subsequent environmental stress depending on the genetic background, with cells overexpressing the Swedish variant of APP exhibiting the highest global response. We further developed a dynamic mathematical model of the UPR that describes the activation of the three branches of this stress response in response to unfolded protein accumulation. Model-based analysis of the experimental data suggests that the mutant cell lines experienced a higher protein load and subsequent magnitude of transcriptional activation compared to the cells overexpressing wild-type APP, pointing to higher susceptibility of mutation-carrying cells to stress. The model was then used to understand the effect of therapeutic agents salubrinal, lithium, and valproate on signalling through different UPR branches. This study proposes a novel integrated platform to support the development of therapeutics for AD.

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Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

International Journal of Molecular Sciences ◽

10.3390/ijms22105218 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5218

Author(s):

Tomu Kamijo ◽

Takahiro Kaido ◽

Masahiro Yoda ◽

Shinpei Arai ◽

Kazuyoshi Yamauchi ◽

...

Keyword(s):

Cell Lines ◽

Cho Cells ◽

Fibrinogen Level ◽

Culture Media ◽

Chinese Hamster ◽

Plasma Fibrinogen ◽

Enzyme Linked Immunosorbent Assay ◽

Cho Cell ◽

Accelerated Degradation ◽

Heterozygous Patient

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient’s hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

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Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication

Journal of Virology ◽

10.1128/jvi.79.20.12969-12978.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 12969-12978 ◽

Cited By ~ 16

Author(s):

James W. Bruce ◽

Kenneth A. Bradley ◽

Paul Ahlquist ◽

John A. T. Young

Keyword(s):

Cell Lines ◽

Reverse Transcription ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Mutant Cell ◽

Chinese Hamster ◽

High Volume ◽

Early Stages ◽

Retroviral Replication ◽

Murine Leukemia

ABSTRACT In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

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Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

Molecular and Cellular Biology ◽

10.1128/mcb.3.7.1172-1181.1983 ◽

1983 ◽

Vol 3 (7) ◽

pp. 1172-1181

Author(s):

W E Bradley

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary ◽

High Frequency ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Low Frequency ◽

Class Ii ◽

Class I ◽

Wild Type ◽

Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

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Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.6.l598 ◽

1993 ◽

Vol 264 (6) ◽

pp. L598-L605

Author(s):

B. Warner ◽

R. Papes ◽

M. Heile ◽

D. Spitz ◽

J. Wispe

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Lethal Dose ◽

Chinese Hamster ◽

Eukaryotic Expression ◽

Wild Type ◽

Oxidant Injury ◽

Sod Activity ◽

Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

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