An inverse relationship between KAI1 expression, invasive ability, and MMP-2 expression and activity in bladder cancer cell lines.
OBJECTIVE
To investigate the relationship between the expression of the cancer metastasis suppressor gene KAI1 and MMP-2 and MMP-9 in human bladder cancer cell lines that express variable levels of KAI1.


METHODS
Five bladder cancer cell lines (BL-28/0, BL-13/0, BL-17/0/×1, B10, and D2) were grown in standard culture conditions. Gelatinase activities in serum-free conditioned medium were assessed using gelatin zymography. Whole cell lysates were prepared and Western blotting used to detect the protein expression of MMP-9, MMP-2, TIMP-1, TIMP-2, and KAI1. Semiquantitative RT-PCR was performed to analyze the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, and KAI1.


RESULTS
Western blotting analysis confirmed that KAI1 was expressed in BL-28/0 and Bl-13/0 but not in D2, B10 and BL-17/0/×1 cell lines. This was consistent with in vitro invasion assays reported previously which showed that cell lines lacking KAI1 expression were 2× to 10× more invasive than cell lines that expressed KAI1. MMP-2 protein was detected in BL-28/0, BL-13/0. and BL-17/0/×1 only. Very low levels of MMP-9 were present in BL-28/0, BL-13/0, B10, and BL-17/0/×1 but not D2, whilst very low levels of TIMP-1 were present in all cell lines. No TIMP-2 was detected. Gelatin zymography showed detectable MMP-2 expression in conditioned medium from BL-28/0 and BL-13/0. Very weak MMP-9 was detected in BL-28/0 conditioned medium only. mRNA expression of MMP-2 was only detectable in BL-28/0 and BL-13/0 cell lines. MMP-9 mRNA levels were extremely low in all lines and not detectable in D2 cells. TIMP-1 and TIMP-2 mRNA were detected in all lines.


CONCLUSIONS
We found that KAI1 expression in bladder cancer cell lines is related to a poor invasive potential and expression of latent MMP-2 but not MMP-9. These results are unexpected given other studies showing high levels of MMP-2 and MMP-9 protein expression in patients with invasive bladder cancer. This may reflect differences in the regulation and secretion of MMP-2 and MMP-9 in vitro compared with the in vivo situation, where tumor cells interact with the surrounding environment.
You
JingJing
J
Oncology Research Centre, Prince of Wales Hospital, Randwick, Australia. youjingjing@yahoo.com
Madigan
Michele C
MC
Rowe
Alexandra
A
Sajinovic
Mila
M
Russell
Pamela J
PJ
Jackson
Paul
P
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051861:Kangai-1 Protein / Q000235:genetics* / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
C497688:CD82 protein, human; D051861:Kangai-1 Protein; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9