Overexpression of the 18 kDa and 22/24 kDa FGF-2 isoforms results in differential drug resistance and amplification potential.
We investigated the role of low molecular weight (LMW) and high molecular weight (HMW) isoforms of basic fibroblast growth factor 2 (FGF-2) in the expression of transformation-related phenotypic alterations, drug sensitivity modulation, and gene amplification potential. For this purpose, we used NIH 3T3 and A31 cells transfected with different cDNA FGF-2 constructs allowing expression of the different proteins. Both cell lines showed marked phenotypic alterations when expressing the LMW FGF-2 or the four HMW FGF-2 isoforms: they acquired a transformed morphology, grew at higher saturation densities in 10% serum, and exhibited anchorage-independent growth and increased invasive potential. However, HMW FGF-2-expressing cells also grew in 1% serum and their invasive potential was lower than in cells expressing all FGF-2 forms or LMW FGF-2 alone. We have grown the different cell lines under a selective pressure of N-(phosphonacetyl)-l-aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional carbamyl-P-synthetase/aspartate transcarbamylase/dihydro-orotase genes (CAD) enzyme (and thus inhibits de novo pyrimidine biosynthesis) and selects for cells with amplified copies of the CAD gene. Our results demonstrate that aberrant expression of the LMW FGF-2 and/or HMW FGF-2 isoforms differently modulates drug resistance and gene amplification properties in the NIH 3T3 and A31 cell lines by differential amplification of the CAD gene. Coexpression of all isoforms appears to be necessary to obtain cumulative effects and nuclear-targeted HMW FGF-2 has a pivotal role in such a cooperation.
Dini
Germana
G
Department of Experimental Pathology and Oncology of Florence University, Florence, Italy. gdini@unifi.it
Funghini
Silvia
S
Witort
Ewa
E
Magnelli
Lucia
L
Fanti
Elena
E
Rifkin
Daniel B
DB
Del Rosso
Mario
M
D016475:3T3 Cells; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D002999:Clone Cells; D015194:Diffusion Chambers, Culture; D004080:Dihydroorotase / Q000235:genetics; D004351:Drug Resistance / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005784:Gene Amplification / Q000502:physiology*; D015870:Gene Expression; D015151:Immunoblotting; D051379:Mice; D008970:Molecular Weight; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D016222:Fibroblast Growth Factor 2; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D010746:Phosphonoacetic Acid