Expression of five cathepsins in murine melanomas of varying metastatic potential and normal tissues.
The relative levels of mRNAs for cathepsins B, D, H, L, and S in eight normal murine tissues and three murine melanoma variants, B16-F1, B16-F10, and B16a, have been analyzed by RNA dot blot and densitometry. A direct correlation was observed between the levels of cathepsin B mRNA and the metastatic potentials of these three melanoma variants. The relative amount of cathepsin B mRNA in B16a, which is the melanoma variant with the highest metastatic potential, was at least 3 times greater than that found in any of the normal murine tissues surveyed. Similar results were obtained in analyses of either solid tumors or of cultures of tumor cells, confirming that the tumor cells themselves were the source for the elevated expression of cathepsin B mRNA. Northern blot analysis revealed the presence of three cathepsin B transcripts of 5.0, 4.0, and 2.2 kilobases in the melanoma variants, while only the 2.2-kilobase transcript was seen in the normal murine tissues. Concurrently with the mRNA analysis, enzyme assays for cathepsin B activity were also performed using synthetic peptide substrates. The assays revealed increased cathepsin B activities in the melanoma variants, corresponding well with the increased cathepsin B mRNA levels, and in addition demonstrated that all three of the melanoma variants secreted a latent form of cathepsin B into conditioned medium, which could be activated by limited proteolysis with pepsin. The levels of the latent enzyme released by the murine melanoma variants correlated well with the levels of cathepsin B mRNA and with the metastatic potentials as determined by spontaneous metastasis form a s.c. site.
Qian
F
F
Department of Biochemistry and Biophysics, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinios 60153.
Bajkowski
A S
AS
Steiner
D F
DF
Chan
S J
SJ
Frankfater
A
A
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D008297:Male; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000032:analysis; D012016:Reference Values; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D002403:Cathepsins; D002401:Cathepsin B