Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells.
BACKGROUND
We have previously shown that down-modulation (i.e., by antisense expression vector) of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a noninvasive, nontumorigenic cell line led to an acquisition of an invasive, tumorigenic, and metastatic ability in these cells.


OBJECTIVE
Our purpose was to examine whether increased levels of murine TIMP-1 can directly suppress the invasive ability of malignant cells.


METHODS
Murine B16-F10 melanoma cells were transfected with an expression vector to overproduce TIMP-1. Among these transfectants, we isolated five clonal cell lines (2-5, 2-8, 2-10, 6-5, and 6-9) that showed upregulation (i.e., overexpression) of TIMP-1.


RESULTS
These cell lines had an increased basal level of TIMP-1 messenger RNA. TIMP-1 expression was under the control of the mouse metallothionein-I promoter, and four of these five clones (2-5, 2-8, 6-5, and 6-9) showed a threefold to 10-fold induction of TIMP-1 message when they were treated with 20 microM cadmium for 4 hours. An increase in TIMP-1 message led to an increase in TIMP-1 protein activity measured in the conditioned medium of clones 2-10 and 6-5. The invasive ability of the TIMP-1-upregulated cells was tested in a matrigel transwell invasion assay. All of the upregulated clones showed a significant reduction in their invasive ability, relative to the invasive ability of parental B16-F10 and the control 1-2 cell line; this reduction correlated with the level of TIMP-1 overexpression. Cadmium induction of TIMP-1 messenger RNA resulted in a further suppression of the invasive ability of the two inducible cell lines tested (i.e., 2-8 and 6-5).


CONCLUSIONS
Our data demonstrate that a specific upregulation of murine TIMP-1 expression in B16-F10 melanoma cells directly suppresses their invasive ability.
Khokha
R
R
Department of Oncology, London Regional Cancer Centre, ON, Canada.
Zimmer
M J
MJ
Graham
C H
CH
Lala
P K
PK
Waterhouse
P
P
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics / Q000502:physiology*; D008546:Melanoma, Experimental / Q000235:genetics / Q000473:pathology*; D051379:Mice; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D009361:Neoplasm Invasiveness; D012333:RNA, Messenger / Q000187:drug effects; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D006023:Glycoproteins; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D002104:Cadmium; D003012:Microbial Collagenase