Biomedical subjects

R S Knee

Publications and source records attributed to R S Knee.

4 recordsLinked to original sources

PubMed1995

Basic fibroblast growth factor binding and processing by human glioma cells.

Tumor cells of glial origin express high levels of basic fibroblast growth factor (bFGF) which stimulates their proliferation in an autocrine manner. In the present study we examined bFGF receptor (FGFR) expression and 125I-bFGF binding and processing in a human glioma cell line. RT-PCR demonstrated the co-expression of bFGF and FGFR mRNAs in five glioma cell lines examined. The high-affinity FGFR was visualized in U87-MG glioma cells by crosslinking with 125I-bFGF and by Western blotting with anti-receptor antisera. Both techniques identified a discrete 110-kDa moiety associated with the cell membrane, consistent with the reported size of one of the FGFR-1 isoforms. Western blotting also identified an intracellular receptor pool which was not accessible with exogenous 125I-bFGF. Suramin treatment induced a 2-fold increase in immunoreactive FGFR and a 1.5-fold increase in 125I-bFGF binding sites, indicating that FGFRs are chronically down-regulated by endogenous bFGF in U87-MG cells. Removal of extracellular bFGF with heparin resulted in a rapid, cycloheximide-sensitive increase in high-affinity bFGF binding sites. At 37 degrees C, receptor-bound 125I-bFGF was internalized and subjected to limited proteolytic cleavage over 12 h. U87-MG cells also contained abundant low-affinity bFGF binding sites which were removed by digestion with heparinase III but not by chondroitinase ABC. The presence of heparin (25 micrograms/ml) in the binding reaction eliminated the association of 125I-bFGF with the heparin-like sites but did not prevent binding to the high-affinity receptor. Scatchard binding analysis in the presence of heparin revealed a single class of high-affinity sites in U87-MG cells (Kd = 4.9 +/- 0.9 pM; 10-12 x 10(3) sites per cell). Neither heparin nor heparinase digestion prevented the binding of 125I-bFGF to the detergent-extractable high-affinity receptor, although both treatments significantly reduced the extent of 125I-bFGF association with the receptor. These findings indicate that in U87-MG cells, heparan sulfate proteoglycans may be involved in presentation of bFGF to the high-affinity receptor, but are not essential for high-affinity binding to occur.

Animals↗

PubMed1994

Basic fibroblast growth factor sense (FGF) and antisense (gfg) RNA transcripts are expressed in unfertilized human oocytes and in differentiated adult tissues.

Basic fibroblast growth factor (bFGF) is a highly conserved and ubiquitously distributed mitogen. In amphibian oocytes bFGF mRNA is regulated post-transcriptionally by interaction with an antisense RNA transcript. We used reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridization to determine the presence of bFGF and its antisense RNA (gfg) in unfertilized human oocytes and postnatal differentiated tissues. BFGF and gfg transcripts were co-expressed in many tissues, with bFGF transcripts (7, 3.7 and 1.8 kb) being more abundant than the gfg transcript (1.5 kb) in 8 of 16 tissues examined. Sense and antisense expression was approximately equal in kidney and colon, while in heart, liver, skeletal muscle and testis gfg transcripts predominated. RT-PCR demonstrated the presence of bFGF and gfg transcripts in unfertilized oocytes where the antisense transcript was present in excess of the sense transcript. These findings suggest a role for gfg in regulation of bFGF expression.

Adult↗

PubMed1994

Identification and characterization of an antisense RNA transcript (gfg) from the human basic fibroblast growth factor gene.

Basic fibroblast growth factor (bFGF) is an autocrine growth factor that is overexpressed in glial tumor cells and promotes their unregulated proliferation. We have previously reported that increased messenger RNA (mRNA) stability contributes to the elevated steady state levels of bFGF mRNA in human U87-MG glioma cells. Stability of bFGF mRNA is regulated by a natural antisense transcript in Xenopus oocytes, but the mammalian equivalent of this transcript has not previously been described. We were interested in identifying the human equivalent of this antisense transcript in order to study its role in bFGF mRNA stability. Analysis of the 3'-untranslated region of the 6.7-kilobase human bFGF mRNA revealed two areas of greater than 75% homology to exons 3 and 4 of the Xenopus antisense transcript, separated by 4300 basepairs of nonhomologous sequence. We used reverse transcription-polymerase chain reaction to amplify, clone, and sequence a 301-basepair fragment of the antisense splice variant from U87-MG cells. The clone (gfg-1) is 73% identical to the Xenopus sequence, with a conserved splice junction and an open reading frame. Strand-specific gfg-1 complementary RNA probes detect a 1.5-kilobase mRNA transcript in normal rat tissues and human T47D breast cancer cells, which contain very low levels of bFGF mRNA. In contrast, antisense transcript expression was undetectable by Northern hybridization in U87-MG cells, which overexpress the bFGF sense mRNA. The reciprocal relationship between bFGF sense and antisense expression suggests that antisense transcripts may regulate bFGF expression in mammalian cells, and that disruption of normal sense/antisense mRNA ratios may lead to overexpression of bFGF in some tumors.

Animals↗

PubMed1992

Phosphorothioate antisense oligonucleotides against basic fibroblast growth factor inhibit anchorage-dependent and anchorage-independent growth of a malignant glioblastoma cell line.

Basic fibroblast growth factor (bFGF) is a broad spectrum mitogen for many cells of neuroectodermal origin, including glial cells. The human malignant glioblastoma cell line U87-MG expresses high steady state levels of the bFGF mRNA and contains abundant stores of biologically active bFGF protein. In the present study we have examined the contribution of endogenous bFGF to the autocrine growth of these cells. Using reverse transcription-polymerase chain reaction, U87-MG cells were shown to express the mRNAs for both bFGF and the bFGF receptor, confirming the existence of the basic requirements for an autocrine loop. Addition of 5 microM bFGF-specific antisense oligonucleotide to U87-MG cultures significantly inhibited the growth rate of these cells within 48 h and blocked proliferation beyond 2 days. The corresponding bFGF-specific sense oligonucleotide did not significantly inhibit cell proliferation over the course of these experiments. Similarly, antisense oligonucleotides significantly inhibited colony formation in soft agar, while the sense sequence was without effect. Western blotting with antihuman bFGF revealed that U87-MG cells synthesize three isoforms of bFGF, approximately 18, 23, and 25 kilodaltons (kDa) in size. The 23- and 25-kDa isoforms together comprise approximately 80% of the total cellular stores of bFGF. Antisense treatment for 4 days reduced the abundance of the 23- and 25-kDa isoforms by 64-74%, but had little effect on the 18-kDa isoform. The inhibitory effect of the antisense oligonucleotides on anchorage-dependent proliferation was reversed by the addition of recombinant 18-kDa human bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)

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