Interaction with fibronectin regulates cytokine gene expression in human melanoma cells

Abstract

Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.

Expression of Basic Fibroblast Growth Factor, Vascular Endothelial Growth Factor, and Thrombospondin‐1 Related to Microvessel Density in Nonaggressive and Aggressive Basal Cell Carcinomas

“…First‐strand cDNA was synthesized by reverse transcription of 5 μg of total RNA using 200 units of Moloney Murine Leukemia Virus Reverse Transcriptase (M‐MLV RT, Promega Co., Maison, WI, USA) and 500 ng of oligo‐dT primer at 42°C for 1 hour. For amplification of cDNAs, oligonucleotide primers for PCR were designed as shown in Table 2 (10–13). β‐actin was employed as a positive internal control for the suitability of the respective specimen for PCR amplification.…”

Relative expression of PTTG and bFGF in oral squamous cell carcinoma and Tca8113

“…The biological function of bFGF depends on the high affinity fibroblast growth factor receptor (FGFR) and the low affinity receptor heparan sulfate proteoglycan (HSPG) on the cell membrane. bFGF has high homology with oncogenes hst, int-2 and hst/k-fgf, which suggests that bFGF participates in tumorigenesis at the molecular level [21,22]. After the cell basement membrane is destroyed due to the necrosis of cancer cells as well as the release of protease, it facilitates bFGF to approach the basement membrane, which furthermore promotes tumor invasion and metastasis.…”

Matricellular proteins and inflammatory cells: A task force to promote or defeat cancer?

“…Indeed, recent work highlighted that SPARC exerts some of its functions, including fibronectin matrix production and assembly, through interaction with integrin-linked kinase [35][36][37][38]. Interestingly, GRO and IL-8 were shown to be induced by fibronectin-mediated integrin activation [39,40]. Thus, it can be hypothesized that PMN recruitment in the melanoma model may be mediated by the modulation of integrin engagement that occurs in the absence of SPARC.…”