Abstract
Transforming growth factor-alpha (TGF-alpha)-mediated autocrine regulation in human non-small-cell lung cancer (NSCLC) cells NCI-H226 and its brain metastatic variant H226Br were compared. An enhanced TGF-alpha-induced dose-dependent mitogenic responsiveness in H226Br cells was observed. Neutralising antibody that binds TGF-alpha inhibits H226Br cell growth more effectively than NCI-H226 cell growth. Binding assay with 125I-labelled epidermal growth factor (EGF) revealed that H226Br has two types of EGF receptors (EGFRs), whereas the parental cell line, NCI-H226, has only one. H226Br cells contain twice as many EGFRs as H226 cells, as proved by Scatchard analysis and immune kinase assay. Northern analysis indicated that there is more EGFR transcript in H226Br than in NCI-H226, indicating a transcriptional EGFR gene elevation during metastasis progression. The level of accumulated immunoactive TGF-alpha is lower in the conditioned medium of H226Br than in that of NCI-H226. demonstrating down-regulation of TGF-alpha transcript. The accumulated data suggest an elevated and sensitive autocrine modulation by TGF-alpha and EGFR in immortalising the brain metastatic variant cells that were derived from a human NSCLC squamous cell line.
Highlights
We found that EGF receptors (EGFRs) expression of H226Br cells is elevated to varying extents, whereas ligand TGF-a expression is decreased compared with the parental cells NCI-H226
Scatchard analysis indicated that the parental cell line, NCI-H226, has one type of epidermal growth factor (EGF) binding site (4.5 x 104 per cell) with a dissociation constant, Kd, of 12.5 nM
The presence of EGFR in cell suggests that TGF-a may act as autocrine regulator for both cell lines
Summary
Cell linesHuman lung squamous cell carcinoma cell lines NCI-H226, NCI-H460 and NCI-H322 were obtained from Dr A Gazdar (Southwestern Medical Centre, Dallas, TX, USA). Cell line H226Br was developed by Dr IJ Fidler and was cultured in RPMI-1640 supplemented with 5% heat-inactivated fetal calf serum. The medium was aspirated and replaced with serum-free medium after 3 h incubation. After determination of cell numbers per well, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) supplemented with 0.2% bovine serum albumin (BSA). After 2 h incubation at 4°C, the cells were washed in ice-cold PBS three times, and the bound radioactivity was determined after the cells were lysed in a 50 mM sodium hydroxide and 10% sodium dodecyl sulphate (SDS) mixture. The non-specific binding was determined and contained a 100-fold molar excess of native EGF. The cells were labelled with [35S]methionine in methionine-free medium and incubated at 37°C for 4h. The pellets after Staphylococcus aureus precipitation were resolved by 7.5%
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