The Bronze Melanocyte Award
6th Meeting of the ESPCR (Lausanne, Switzerland) Oct. 1995
Comparison of attachment of human ocular melanocytes and melanoma cells to extracellular matrix (ECM) proteins
M. Wagner, I.G. Rennie*, S. MacNeil
Dept of Medicine, Clinical Sciences Centre, Northern General Hospital
*Dept of Ophtalmology and Orthoptics, Royal Hallamshire Hospital,
The aim of our study was to investigate whether the intracellular signalling systems used by the uveal melanoma cell differed significantly from those used by the uveal melanocyte in their attachment to ECM proteins. Any differences identified could be used to select a pharmacological approach to affect neoplastic but not normal cell attachment.
First of all we compared the substrate preference of the normal versus the neoplastic cell and found that the melanocyte expressed a clear preference for fibronectin over the other substrates studied (collagen I, collagen III, collagen IV, laminin and plastic). In contrast to this, the melanoma cells showed an equal preference for collagens I,III,IV and fibronectin. We then compared the timecourse of attachment of both cell types to fibronectin, but found that both normal and transformed cells attached similarly showing rapid attachment within 20 - 30 minutes.
In this study, we considered the post receptor events and examined which intracellular signalling systems which were important in mediating the early stages of cell attachment to matrix proteins. All initial studies were conducted using melanocytes in their mitogen rich medium that has one-tenth the normal physiological calcium concentration. Under these conditions we found that by manipulating the cyclic AMP system using dideoxyadenosine and, the protein kinase C system, using forskolin and phorbol 12-myristate 13-acetate and staurosporine, had little or no effects on both cell types.
Similarly, we found that inhibition of the calmodulin system using either the experimental calmodulin antagonist drug J8 or tamoxifen produced a profound inhibition of attachment. Both melanocyte and melanoma cells did not differ in their sensitivity to either drug.
We were surprised to discover that by manipulating the the intracellular calcium system using ionomycin and TMB8 was initially without effects on the attachment of the ocular melanocyte, while they inhibited the ocular melanoma cell attachment. As it may have offered an approach for selective pharmacological intervention we examined this result further.
In these further studies the melanocytes were transferred to medium containing physiological concentrations of calcium for two days prior to experimentation. These experiments revealed that the lack of response of the melanocytes to ionomycin was dependent upon the calcium concentration of the medium as all other additions to the medium were kept constant.
We also confirmed that melanocyte and melanoma cells responded equally well to an acute addition of ionomycin with a rapid increase in intracellular calcium. However, the action of ionomycin is entirely dependent on the concentration of extracellular calcium and therefore our results lead us to conclude that if uveal melanocytes are grown under concentrations of low extracellular calcium then ionomycin is probably unable to elevate intracellular to a level which will inhibit cell attachment.
To conclude, we found that both normal uveal melanocytes and transformed uveal melanoma cells use the same intracellular signalling systems in their initial stages of attachment to ECM proteins This signalling involves calcium and calmodulin to a large extent, accordingly any drugs which affect calcium or calmodulin activity could reduce the likelihood of the attachment of either cell.
We can also state that the ocular melanocyte and melanoma cells show a requirement for a calcium and calmodulin sensitive intracellular signal within the first few minutes of adhesion suggesting that this is unlikely to be specific to a particular cell or even to any particular adhesion molecule/ECM substrate interaction.
In summary, while we are unable to identify a pharmacological target for intracellular signalling which would allow one to focus on the transformed rather than the normal cell, it is still possible that drugs affecting calcium or inhibiting calmodulin could be used in preventing adhesion of any unattached cells and this could be of value in preventing metastatic spread.