Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry.

Assaraf, Yehuda G.; Schimke, Robert T.

doi:10.1073/pnas.84.20.7154

Cited by 68 publications

(39 citation statements)

References 27 publications

(16 reference statements)

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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

280

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

280

223

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“…The most extensively studied of these compounds, fluorescein MTX (F-MTX), has been reported to penetrate cells slowly over several hours and to accumulate to high levels in both MTXsensitive and transport-impaired cells (Assaraf and Schimke, 1987), presumably by a non-RFC uptake process. As F-MTX binds avidly to DHFR, it has frequently been used to detect elevated levels of this enzyme target in MTX-resistant cells by flow cytometry (Kaufman et al, 1978).…”

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confidence: 99%

“…In recent years, fluorescent analogues of MTX have fostered studies of MTX resistance in cultured cells and leukaemic blasts by flow cytometry (Kaufman et al, 1978;Rosowsky et al, 1982; Assaraf and Schimke, 1987;Trippett et al, 1992;Matherly et al, 1995). The most extensively studied of these compounds, fluorescein MTX (F-MTX), has been reported to penetrate cells slowly over several hours and to accumulate to high levels in both MTXsensitive and transport-impaired cells (Assaraf and Schimke, 1987), presumably by a non-RFC uptake process.…”

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confidence: 99%

“…As F-MTX binds avidly to DHFR, it has frequently been used to detect elevated levels of this enzyme target in MTX-resistant cells by flow cytometry (Kaufman et al, 1978). Whereas capacities for MTX membrane transport can also be assayed with F-MTX and flow cytometry, by following the loss of cellular fluorescence due to the competitive displacement of DHFR-bound F-MTX with exogenous MTX (Assaraf and Schimke, 1987), the sensitivity of this indirect assay of RFC function is limited by the range of displacing MTX concentrations used.On this basis, we sought to develop a more direct and sensitive approach for assaying RFC function in intact cells that might be amenable to the study of clinical specimens. In this report, we describe the use of confocal microscopy to visualize directly a RFCmediated uptake of F-MTX that is potently inhibited by RFC-transport substrates and is completely abolished in cultured cells with impaired MTX transport.…”

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Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells

et al. 1997

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Summary Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in human leukaemic cell lines and leukaemic blasts. Cytosolic labelling of wild-type K562 human erythroleukaemia cells was detected during 3-60 min incubations with F-MTX (1 giM) and was completely inhibited by co-exposure to either methotrexate or the thymidylate synthase inhibitor, ZD1694. There was no significant intracellular F-MTX accumulation over this period in a K562 subline (K500E) with a documented defect (approximately 10% of wild type) in membrane transport by the reduced folate carrier (RFC). F-MTX uptake was reestablished in K500E cells transfected with a cDNA to human RFC, establishing a role for RFC in the cellular uptake of this compound. High levels of intracellular labelling were detected in all cell lines after prolonged (24 h) F-MTX incubations, however F-MTX accumulation at this time was not inhibited by ZD1694. F-MTX uptake by RFC was also detected in leukaemic blasts from children with acute lymphoblastic leukaemia and could be blocked with ZD1694. In leukaemic blasts with a documented defect in MTX uptake, F-MTX accumulation was abolished in almost all the cells. These results display the power of confocal microscopy for directly visualizing RFC-mediated anti-folate uptake. Over short intervals, F-MTX uptake is mediated by RFC, however, RFC-independent processes predominate during long drug exposures. Direct assay by confocal microscopy may be better suited than other indirect methods (i.e. flow cytometry) for detecting low levels of RFC transport in leukaemic blasts from patients undergoing chemotherapy with methotrexate.Keywords: folate; fluorescein methotrexate; methotrexate; reduced folate carrier; membrane transportThe folate analogue, methotrexate (MTX) is an important component in the chemotherapy of childhood acute lymphocytic leukaemia (ALL). Although long-term disease-free survival for children with ALL has continued to increase and now approaches 70% (Pizzo and Poplack, 1993), further improvements in ALL treatment will have better results if patients who may benefit from more intensive therapies can be identified.Critical determinants of MTX sensitivity and resistance have been previously described in cultured cells (Jolivet et al, 1983;Goldman and Matherly, 1985). MTX is transported into cells by the reduced folate carrier (RFC) where it binds to dihydrofolate reductase (DHFR) and is metabolized to MTX polyglutamates by folylpolyglutamate synthetase (FPGS). Lymphoblasts from children with ALL also synthesize long-chain MTX polyglutamates (Whitehead et al, 1990), and correlations have been established between accumulation of these metabolites and characteristic patient prognostic features (i.e. lineage, hyperdiploidy, etc.) or MTX dose (Whitehead et al, 1992;Barredo et al, 1994;Synold et al, 1994).In recent years, fluorescent analogues of MTX have fostered studies of MTX resistance in cultured cells and leukaemic blasts by flow cytometry...

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Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2)

Losman

Hansen

Dworak

et al. 1997

Cancer

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Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry.

Cited by 68 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?

Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2)

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