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Fibroblast Growth Factor Messenger Ribonucleic Acid Expression in a Human Astrocytoma Cell Line: Regulation by Serum and Cell Density

Department of Physiology, Faculty of Medicine, University of Manitoba Winnipeg, Manitoba, Canada R3E OW3

Address requests for reprints to: Dr. Paul R. Murphy, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3.

Abstract

A human astrocytoma cell line, U87-MG, synthesizes a growth factor which is structurally related to basic fibroblast growth factor (bFGF) by several criteria: 1) it binds to heparin-Sepharose and elutes at 2 M NaCI; 2) it cross-reacts with N-terminal specific anti-bFGF antibodies; 3) it is a potent mitogen for rabbit fetal chondrocytes. Northern blotting analysis of total RNA reveals that the cells express high levels of two bFGF mRNA transcripts of 7 and 3.7 kilobase in size. The levels of both transcripts rise rapidly (within 3 h) after addition of serum to serum-deprived cultures, reach a maximum within 6–12 h and remain elevated for at least 24 h. Basic FGF mRNA expression was low in confluent cultures but was increased after replating at sparse density. Transcript levels began to increase 4 h after plating, reaching a maximum (7-fold above confluent cells) within 24 h. The rise in bFGF mRNA levels was preceded by a rapid rise in c-myc expression which peaked 8 h after plating and then declined. The level of bFGF mRNA expression began to decline by 48 h, before any detectable increase in cell number, and returned to control levels when the cells reached confluence after 10 days. The level of transforming growth factor β mRNA, which is also expressed in these cells, was not affected by cell density. The effects of density on bFGF mRNA levels were not duplicated by culturing low density cells in conditioned medium from confluent cultures or in medium containing 10 ng/ml bFGF.

These findings indicate that bFGF expression is tightly regulated by cell density, and may be directly controlled by cell-cell contact or by factors present in the extracellular matrix. The transient peak of expression shortly after plating suggests that bFGF expression is closely associated with factors regulating cell cycle progression, and may help to explain the low levels of expression detected in differentiated normal tissues.

FOOTNOTES

This work was supported by grants from MRC and National Cancer Institute of Canada.

We are grateful to Drs. Catherine Too and R. P. C. Shiu for critical reading of the manuscript, and to Dr. Leigh Murphy for helpful technical suggestions. The secretarial assistance of A. Macintosh is greatly appreciated.

Received for publication December 23, 1987. Accepted for publication March 17, 1988.

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