Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells

Flintoff, Wayne F.; Davidson, Sandra V.; Siminovitch, Louis

doi:10.1007/bf01538963

Cited by 233 publications

(150 citation statements)

References 27 publications

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“…However, the specific productivity decreased upon further increase of MTX level (Kim et al, 2001). Gene amplification by MTX selection is generally obtained by gradual increase of MTX selection pressure rather than rapid increase as single-step high-level resistance to MTX may result in mechanisms other than dhfr-mediated gene amplification, such as altered MTX transport properties (Assaraf and Schimke, 1987;Kaufman, 1990;Sirotnak et al, 1981) or an altered, MTX-resistant DHFR enzyme (Flintoff and Essani, 1980;Flintoff et al, 1976;Haber and Schimke, 1981). Hence, a gradual increase, instead of a rapid increase, of MTX concentration is more effective for constructing high-producer recombinant CHO cell lines as previously shown by Nakanishi et al (1997) and Yoshikawa et al (2000a).…”

Section: Resultsmentioning

confidence: 99%

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Chusainow

Yang

Yeo

et al. 2008

Biotech & Bioengineering

279

223

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Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell À1 day À1 using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high-and lowproducer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.

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Section: Resultsmentioning

confidence: 99%

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Chusainow

Yang

Yeo

et al. 2008

Biotech & Bioengineering

279

223

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“…These changes in the properties of DHFR suggested that MTX resistance was the result of mutations in the protein (Albrecht et al, 1972;Flintoff et al, 1976;Goldie et al, 1980). This was subsequently confirmed.…”

Section: Alterations In Dhfrmentioning

confidence: 91%

Resistance to antifolates

2003

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“…The approach used was chemical mutagenesis followed by selective pressure with dihydrofolate reductase (DHFR) inhibitors, a technique applied in this and other laboratories (9,35,40). The agents used were MTX, a classical antifolate that forms polyglutamate derivatives in cells, and 5,8-dideaza-N ␣ -(-4-amino-4-deoxypteroyl)-N ␦ -hemiphthaloyl-1-ornithine (PT632), a novel antifolate with 10-fold higher affinity for DHFR and RFC that does not form polyglutamate derivatives (34) and has very low affinity for low-pH transporters in several tumor cell lines (32).…”

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confidence: 99%

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

Wang

Rajgopal

Goldman

et al. 2005

American Journal of Physiology-Cell Physiology

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. Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function. Am J Physiol Cell Physiol 288: C65-C71, 2005. First published September 22, 2004; doi:10.1152/ajpcell. 00307.2004.-Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln 420 . A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser 135 was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC. folic acid; folate absorption; methotrexate

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Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells

Cited by 233 publications

References 27 publications

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Resistance to antifolates

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

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