Abstract
We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of “cellular sipping” changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6hrs after TNF-α stimulation, but this was lost by 18hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms.
Highlights
Malignant neoplasms arise from acquisition of somatic mutations during initiation, expansion of clones of initiated cells through the action of proliferative signals in promotion, and emergence of increasingly malignant sub-clones to result in disease progression [1,2]
Expression of mRNA for the inflammatory cytokine Tumour Necrosis Factora
Endothelium and SAOS-2 osteosarcoma cells. human gingival fibroblasts (h-GF) were isolated by explant culture from human gingival biopsies which were obtained with written informed consent from the Oral surgery unit, Westmead Centre for Oral Health, Westmead Hospital, under a protocol approved by the Sydney West Area Health Service Human Research Ethics Committee as outlined earlier [9,19]
Summary
Malignant neoplasms arise from acquisition of somatic mutations during initiation, expansion of clones of initiated cells through the action of proliferative signals in promotion, and emergence of increasingly malignant sub-clones to result in disease progression [1,2]. In that study we observed the exchange of separate membrane and cytoplastmic fluorescent markers in the absence of nuclear exchange, between cultured h-GF and malignant cell lines including: SAOS-2 osteosarcoma; melanoma MeIRMu, NM39, WMM175, MM200-B12; and ovarian carcinoma cells PE01, PE04 and COLO316 [9]. Expression of mRNA for the inflammatory cytokine Tumour Necrosis Factora We demonstrated altered cytokine synthesis in response to TNF-a by h-GF permitted cellular sipping, compared with h-GF denied this contact dependent interaction [14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
