Changes in protein expression during melanoma differentiation determined by computer analysis of 2-D gels

Abstract

Cytodifferentiation in many melanocytic cells is regulated through the adenylate cyclase-cAMP pathway. To analyse the molecular changes associated with this process we have compared the proteins produced by two closely related cell lines which, though derived from a single cell line, respond very differently to modulation of this signalling pathway. The human melanoma cell line DX3 shows little change in in vitro characteristics following treatment with cAMP elevating agents; in contrast the more malignant DX3 LT5.1 variant, derived from the DX3 parental line, shows pronounced dendrification, decreased proliferation and a reduction in metastatic capacity after similar treatment. The two cell lines were treated with phosphodiesterase inhibitors for 5 days and then processed for two-dimensional gel characterization using an immobilized pH gradient for the IEF dimension. Proteins were detected by silver staining the gels and protein intensities were digitized using a laser densitometer. Two-dimensional gel patterns were edited, matched and a melanoma protein database of 637 spots constructed using PDQUEST software on an Orion 1/05 computer. Eleven proteins were lost and four new proteins were detected in both cell lines following treatment. Twenty-two proteins were present in DX3 LT5.1 after treatment but not in untreated lines or treated DX3. These differentially expressed proteins may be associated with the observed changes in differentiation patterns and metastasis. Our results illustrate the resolving power of this technique and suggest potential applications to the study of cellular differentiation.

Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two‐dimensional gel electrophoresis

“…Along with improved staining protocols, an ever-evolving selection of image analysis software has become available for spot detection and quantification. Such software is proving itself to be extremely valuable for discriminating differences in protein abundance in biological studies [19][20][21][22][23][24][25][26][27]. Two recent investigations have been published comparing the capabilities of some of the commercially available software packages [2,28].…”

A systems biology analysis of metastatic melanoma using in‐depth three‐dimensional protein profiling

“…Several prior studies used conventional 2-DE to compare metastatic human melanoma cells with nonmetastatic cells [9][10][11]. For example, Bernard et al [11] compared the proteomes of normal melanocytes with melanoma cell lines and identified eight proteins as differentially expressed in the transformed cells.…”

A systems biology analysis of metastatic melanoma using in‐depth three‐dimensional protein profiling

“…Several prior studies used conventional 2‐DE to compare metastatic human melanoma cells with non‐metastatic cells 9–11. For example, Bernard et al .…”