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.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Cloning and characterization of small circular DNA from Chinese hamster ovary cells

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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

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.

scholarly journals Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.

1977 ◽  
Vol 73 (1) ◽  
pp. 200-205 ◽  
Author(s):  
A S Weissfeld ◽  
H Rouse
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Multiplication ◽  
Ovary Cells ◽  
Cell Cycle Phases

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.

scholarly journals Comparison of the mRNA sequences for Pi class glutathione transferases in different hamster species and the corresponding enzyme activities with anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide

1996 ◽  
Vol 318 (2) ◽  
pp. 533-538 ◽  
Author(s):  
Stellan SWEDMARK ◽  
Bengt JERNSTRÖM ◽  
Dag JENSSEN
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Cho Cell ◽  
Glutathione S Transferase ◽  
V79 Cells ◽  
Ovary Cells ◽  
Cdna Sequences ◽  
Chinese Hamster V79 Cells

Glutathione S-transferase (GST) of class Pi (GST Pi) is known to detoxify the mutagenic and carcinogenic (+)-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by conjugation with glutathione. Previously, we have shown that Chinese hamster V79 cells contain GST Pi, but seem to lack the capacity to conjugate (+)-anti-BPDE, although these cells do conjugate other substrates with GSH [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701–1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719–1723; Swedmark and Jenssen (1994) Gene 139, 251–256]. In the present study we have compared four cell lines derived from different hamster species with respect to GST cDNA sequences and capacity to conjugate (+)- or (-)-anti-BPDE. The cell lines were V79 and Chinese hamster ovary cells (CHO), Armenian hamster lung (AHL) cells and baby hamster kidney (BHK) cells. The sequencing revealed a complete homology between the V79 and CHO cDNA for GST Pi, whereas the corresponding amino acid sequences predicted from the corresponding AHL and BHK cDNAs differed by six and nine amino acids, respectively, from the predicted V79 sequence. None of these changes alone was found to influence the xenobiotic substrate-binding site. The cytosolic fractions from BHK and AHL cells were found to catalyse conjugation of (+)-anti-BPDE with GSH, whereas the corresponding activity in CHO cells was non-detectable. As shown previously, V79 cells were devoid of activity towards (+)-anti-BPDE. All the cell lines studied demonstrated appreciable GST activity towards 1-chloro-2,4-dinitrobenzene, but no activity with (-)-anti-BPDE. The latter result suggests that GST Pi is the sole or predominant GST in these cell lines. This was confirmed by HPLC analysis of purified enzymes obtained by affinity chromatography. However, when the catalytic activities of the pure enzymes were determined, all four different GST Pi enzymes were found to be highly capable of conjugating (+)-anti-BPDE with GSH. This observation indicates the existence of an intracellular factor that selectively inhibits conjugation of (+)-anti-BPDE, but not of 1-chloro-2,4-dinitrobenzene in the V79 and CHO cell lines. This new phenomenon seems to be specific for Chinese hamster, since both these cell lines originate from this species.

scholarly journals The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

1977 ◽  
Vol 73 (3) ◽  
pp. 601-615 ◽  
Author(s):  
RR Gould ◽  
GG Borisy
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Carbon Coated ◽  
Pericentriolar Material ◽  
And Function

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.

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