Cloning and characterization of human tumor cell interstitial collagenase.
A full-length complementary DNA (cDNA) for interstitial collagenase was isolated from an A2058 melanoma cDNA library using the pCD-X Okayama-Berg vector. The tumor interstitial collagenase cDNA was sequenced and compared to the published sequences for human fibroblast collagenase. The sequence for the tumor collagenase has two DNA base pairs which differ from the sequence of normal fibroblast collagenase. Restriction enzyme digestion of a specific DNA fragment produced by polymerase chain reaction amplification of genomic DNA from human placenta resolves a discrepancy in the previously reported DNA and amino acid sequences for the fibroblast collagenase. A high level of expression of interstitial collagenase message was found in human A2058 melanoma cells by Northern blot analysis, and this level was slightly increased by phorbol ester (phorbol myristate acetate) stimulation. Interstitial collagenase mRNA expression was significantly decreased by treatment with either transforming growth factor-beta 1 or retinoic acid in A2058 melanoma cells. A high level of the collagenase protein secreted into conditioned media was identified by Western blotting. As shown by gelatin zymogram analysis interstitial collagenase was one of at least two metalloproteinases secreted by this same cell line. Thus, human melanoma cells can directly produce interstitial collagenase without a requirement for host cell interaction.
Templeton
N S
NS
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Brown
P D
PD
Levy
A T
AT
Margulies
I M
IM
Liotta
L A
LA
Stetler-Stevenson
W G
WG
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics; D003012:Microbial Collagenase / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured / Q000201:enzymology*
D004273:DNA, Neoplasm; D007527:Isoenzymes; D015345:Oligonucleotide Probes; D003012:Microbial Collagenase