Energy metabolism in respiration-deficient and wild type chinese hamster fibroblasts in culture

The extracellular Ca2+-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR−/−) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [Exon5(−)CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR−/− mice express Exon5(−)CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of Exon5(−)CaR in growth plates from CaR−/− mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human Exon5(−)CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human Exon5(−)CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR−/− GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR−/− mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably Exon5(−)CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR−/− mice, high extracellular [Ca2+] ([Ca2+]e) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca2+]e also promoted the differentiation of CaR−/− GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of Exon5(−)CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca2+]e in GPCs in CaR−/− mice.

Download Full-text

Actin-dependent regulation of the cardiac Na+/Ca2+ exchanger

AJP Cell Physiology ◽

10.1152/ajpcell.00232.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. C691-C701 ◽

Cited By ~ 23

Author(s):

Madalina Condrescu ◽

John P. Reeves

Keyword(s):

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Cytochalasin D ◽

Lag Phase ◽

Cell Contact ◽

Wild Type ◽

Filamentous Actin ◽

Surface Distribution ◽

Reverse Mode ◽

Hydrophilic Domain

In the present study, the bovine cardiac Na+/Ca2+ exchanger (NCX1.1) was expressed in Chinese hamster ovary cells. The surface distribution of the exchanger protein, externally tagged with the hemagglutinin (HA) epitope, was associated with underlying actin filaments in regions of cell-to-cell contact and also along stress fibers. After we treated cells with cytochalasin D, NCX1.1 protein colocalized with patches of fragmented filamentous actin (F-actin). In contrast, an HA-tagged deletion mutant of NCX1.1 that was missing much of the exchanger's central hydrophilic domain Δ(241–680) did not associate with F-actin. In cells expressing the wild-type exchanger, cytochalasin D inhibited allosteric Ca2+ activation of NCX activity as shown by prolongation of the lag phase of low Ca2+ uptake after initiation of the reverse (i.e., Ca2+ influx) mode of NCX activity. Other agents that perturbed F-actin structure (methyl-β-cyclodextrin, latrunculin B, and jasplakinolide) also increased the duration of the lag phase. In contrast, when reverse-mode activity was initiated after allosteric Ca2+ activation, both cytochalasin D and methyl-β-cyclodextrin (Me-β-CD) stimulated NCX activity by ∼70%. The activity of the Δ(241–680) mutant, which does not require allosteric Ca2+ activation, was also stimulated by cytochalasin D and Me-β-CD. The increased activity after these treatments appeared to reflect an increased amount of exchanger protein at the cell surface. We conclude that wild-type NCX1.1 associates with the F-actin cytoskeleton, probably through interactions involving the exchanger's central hydrophilic domain, and that this association interferes with allosteric Ca2+ activation.

Download Full-text

Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3020355 ◽

1994 ◽

Vol 302 (2) ◽

pp. 355-361 ◽

Cited By ~ 31

Author(s):

K Inukai ◽

T Asano ◽

H Katagiri ◽

M Anai ◽

M Funaki ◽

...

Keyword(s):

Glucose Transporter ◽

Cytochalasin B ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Site Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

In The Wild ◽

Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1754-1758.1989 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1754-1758

Author(s):

T M Underhill ◽

W F Flintoff

Keyword(s):

Gene Transfer ◽

Dna Sequences ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Ovary Cell ◽

Wild Type ◽

Ovary Cells ◽

Ovary Cell Line ◽

Human Specific

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text