Model
Digital Document
Publisher
Florida Atlantic University
Description
The proteolytic activities of the ADAM (a disintegrin and metalloproteinase),
ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and
MMP (matrix metalloproteinase) protein families play important roles in normal and
multiple pathological conditions. These metalloproteases have potential implications
in the degradation of the extracellular matrix and in the processing of bioactive
molecules. Under pathological conditions these proteases are involved in many
diverse processes from tumor cell migration to cartilage destruction in rheumatoid
arthritis. In the present study, the gene expression levels of six ADAMs, eight MMPs,
and four ADAMTSs were analyzed by Real Time PCR. RNA was isolated from
multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic
normal tissue and tumor samples. This method allowed for detected changes in mRNA expressiOn of the individual metalloproteainase genes to be compared
between normal and metastatic states, and also between tissue and cultured cells.
Substantial differences have been observed in the level of ADAM and MMP mRNA
expression between tissue and cell lines. In general, the level of expression is several
folds higher in cultured cells compared to the isogenic tissue they are derived from.
Protein microarrays were utilized in order to evaluate the correlations between MMP
and TIMP mRNA copy numbers and protein abundance in cell culture. In several
cases, distinct differences were observed regarding the localization of the proteins
examined. In order to determine if the metalloprotease genes that were elevated at the
level of RNA expression produce functional proteins, the foundations of an in situ
FRET assay have been established. This will greatly aid in a better understanding of
the behavior of metallopeptidases in a cellular context.
ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and
MMP (matrix metalloproteinase) protein families play important roles in normal and
multiple pathological conditions. These metalloproteases have potential implications
in the degradation of the extracellular matrix and in the processing of bioactive
molecules. Under pathological conditions these proteases are involved in many
diverse processes from tumor cell migration to cartilage destruction in rheumatoid
arthritis. In the present study, the gene expression levels of six ADAMs, eight MMPs,
and four ADAMTSs were analyzed by Real Time PCR. RNA was isolated from
multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic
normal tissue and tumor samples. This method allowed for detected changes in mRNA expressiOn of the individual metalloproteainase genes to be compared
between normal and metastatic states, and also between tissue and cultured cells.
Substantial differences have been observed in the level of ADAM and MMP mRNA
expression between tissue and cell lines. In general, the level of expression is several
folds higher in cultured cells compared to the isogenic tissue they are derived from.
Protein microarrays were utilized in order to evaluate the correlations between MMP
and TIMP mRNA copy numbers and protein abundance in cell culture. In several
cases, distinct differences were observed regarding the localization of the proteins
examined. In order to determine if the metalloprotease genes that were elevated at the
level of RNA expression produce functional proteins, the foundations of an in situ
FRET assay have been established. This will greatly aid in a better understanding of
the behavior of metallopeptidases in a cellular context.
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