Divalent Cations Control Cell-Substrate Adhesion and Laminin Expression in Normal and Malignant Human Melanocytes in Early and Late Stages of Cellular Differentiation

Gordon E. Searles, Walter T. Dixon, Panakkezhum D. Thomas, Kowichi Jimbow
1995 Journal of Investigative Dermatology  
Integrins are a class of adhesion molecules that depends on divalent cations for proper function. This study exantined whether human norntal melanocytes and malignant (metastatic) melanocytes with early and late stages of cellular differentiation (G361 and SK-MEL-23, respectively) would differ in integrinmediated adhesion to fibronectin, laminin, as well as collagens type I and type IV, and whether divalent cations could influence the strength of adhesion ability. Integrin subunit expression
more » ... bunit expression was determined by flow cytometry using integrin subunit-specific antibodies as probes. Integrin-specific adhesion was determined using soluble glycine-arginine-glycine-asparagine-serine peptide and integrin subunit-specific antibodies as functional blocking agents. This study shows that both normal and malignant melanocytes adhere to extracellular matrices in a divalent cation-C ell ad h esion to extracellular matrix molecules involves a fam ily of cell-surface molecules call ed integrins, which are a -{3 heterodimers that in tegrate the extracellular matrix with the cell cytoskeleton 1 1in the {3 subunit [1]. The a-subuni t appears to direct ligand specificity by usin g divalent cation-binding domains similar to the "EF-hand " stru c tural motif found in the divalent cation binding regions on the Ca ++ -binding prote in calmodulin (2]. These domains when bound to metal ions ca use conformational changes in the molecule, which may activate th e receptor by uncovering active epitopes [3] . Normal hum an m e lanocytes bind with greater strength to extracellular matrix substrates in the presence of Mn and Mg than with Ca, presumably through conform atio nal changes in the integrin molecule induced by occupation of the metal-ion-binding site [4] . However, it is not kn own how cell s utilize these conformationa l chan ges to selectively bind to M.anuscript received N ovember 2, 1994; final revision Abbreviations: AJJTS, 2,2' -azin obis(3-ethylbenzothi azolin c sul fonic acid); GRGDS, glyci ne-arginin e-glyci ne-glycir lc-asparagin e-scri nc; MTT, 3-[ 4,5-dimcthylthiazol-2-yl]-2,5-diphenyltetrazo lium bromide: SIU3, sulforhodaminc B. dependent manner, and adhesion strength varies with the cation species. Integrins can be rapidly activated by small alterations in cation concentration, manganese being the most potent. There were marked differences in substrate adhesion between normal melanocytes and metastatic malignant ntelanoma cells, but these differences were not related to the stage of cellular differentiation. All the three cell types, however, expressed the same integrin subunits at approximately the same levels. This suggests that substrate adhesion of melanocytes and melanoma cells lllight involve some integrin-independent mechanisnts as well. Manganese, in particular, appears to cause adhesion by activating both integrindependent and -independent mechanisms. Key JVord: mela11oma. J l1west Dermato/105:301-308, 1995 substrates or whether tlus ability can vary with the state of cell difFerentiation. Human maJjgnant melanom:1 may provide a mod el for studying cell differentiation and cell-substrate adhesion i11 vir' o and in "itro, and the latter can be best illustrated by compar.ing w ith nonmetastatic normal melanocytes [5] . Normal melanocytes and their malignant metastatic counterparts differ in th eir mobility and adhesion to extracellular matt-ix (ECM), presumably due to differences in adhes io n molecule expression and in tegratio n with the cytoskeleton. Normal and malignant melanocytes express several members of the integrin family [6] . Furthermore, different integrin subunits h ave been ide ntified during malignant progression (7, 8] . However, it is not known to what extent integrin eJ..']Jressio n changes according to the cellul ar differentiation state, including metastatic potentials, and h ow su ch alte rations in in tegrin eJ..']Jression affect adhesion to substrates. This study compares integrin expression and adhesion to extracellular matrices between nonnal and malignant human melanocytes. Specifically, w e were interes ted in determi1ung whether divalent cations cou ld influence adhesive abili ty to · extracellu lar matri.x su bstrates. lntegrin (protein) expression was measured by flow cytometry using monoclonal antibodies, and cell-substrate ad hesion was assayed by a modified version of the centrifugation detachment assay [9-10) . [n addition to normal h uman m elanocytes, a nonadh e rent small cell lung ca •·cinoma cell line and two 0022-202X/95/S09.50 • SSDI0022-202X(95)00252-G • Copyri ght
doi:10.1111/1523-1747.ep12318988 pmid:7636317 fatcat:6yis4f44off2rax3ogdhmdcuoe