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Fibroblast growth factor mediated alterations in drug resistance, and evidence of gene amplification
1Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
Abstract:
We have investigated the drug resistance and gene amplification potential of NIH3T3 cells transfected with sequences coding for K-FGF, a known oncogene product, or bFGF, a non-oncogene member of the fibroblast growth factor family. Resistance to methotrexate, N-(phosphonacetyl)-L-aspartate and hydroxyurea was observed with K-fgf transfectants, due to amplification of dihydrofolate reductase, CAD or ribonucleotide reductase R2 genes, respectively. In keeping with the increase in gene amplification frequency, cells transfected with the K-fgf gene also exhibited a marked increase in CAD gene amplification rate, as determined by fluctuation analysis in the presence of N-(phosphonacetyl)-L-aspartate. Cells transfected with bFGF encoding cDNA also exhibited a significant elevation in N-(phosphonacetyl)-L-aspartate resistance, and CAD gene amplification. Treatment with suramin, which interferes with the interaction of fibroblast growth factors with their cell surface receptors, did not decrease the drug resistance properties of K-fgf transfected cells. These observations with suramin and the findings with bFGF, which lacks a conventional signal sequence for secretion, suggests that the growth factor-mediated effects on drug resistance and gene amplification occur through an intracellular as opposed to autocrine mode of action. The finding that aberrant growth factor expression regulates gene amplification opens up new possibilities for investigating intracellular mechanisms relevant to this process and also describes new functions for the altered expression of K-FGF and bFGF, which are relevant to mechanisms of malignant progression.
Insights
Aberrant expression of fibroblast growth factors (FGFs) enhances drug resistance and gene amplification in NIH3T3 cells. These effects, mediated intracellularly, suggest new mechanisms for malignant progression.
Area of Science:
- Molecular Biology
- Oncology
Background:
- Fibroblast growth factors (FGFs) play roles in cellular processes.
- K-FGF is an oncogene product, while bFGF is a non-oncogene member of the FGF family.
- Gene amplification is a mechanism implicated in drug resistance and cancer progression.
Purpose of the Study:
- To investigate the role of K-FGF and bFGF in drug resistance and gene amplification.
- To explore the mechanism of action for FGF-mediated effects on gene amplification.
- To determine if FGF-mediated effects on drug resistance occur intracellularly or via autocrine signaling.
Main Methods:
- NIH3T3 cells were transfected with sequences encoding K-FGF or bFGF.
- Drug resistance assays were performed using methotrexate, N-(phosphonacetyl)-L-aspartate, and hydroxyurea.
- Gene amplification was assessed by measuring the amplification of dihydrofolate reductase, CAD, and ribonucleotide reductase R2 genes.
- Fluctuation analysis was used to determine CAD gene amplification rates.
- Suramin was used to investigate the role of cell surface receptors.
Main Results:
- K-FGF transfectants showed resistance to methotrexate, N-(phosphonacetyl)-L-aspartate, and hydroxyurea due to amplification of DHFR, CAD, and RNR-R2 genes, respectively.
- K-FGF transfectants exhibited increased CAD gene amplification rates.
- bFGF transfectants also showed increased N-(phosphonacetyl)-L-aspartate resistance and CAD gene amplification.
- Suramin treatment did not affect the drug resistance of K-FGF transfected cells.
- FGF-mediated effects on drug resistance and gene amplification appear to be intracellular.
Conclusions:
- Aberrant expression of K-FGF and bFGF can lead to drug resistance and increased gene amplification.
- The observed effects are mediated through an intracellular mechanism, not autocrine signaling.
- These findings provide insights into intracellular mechanisms of gene amplification and potential roles in malignant progression.