Localization of gelatinolytic activity can be detected in breast cancer tissues by film in situ zymography.
BACKGROUND
Extracellular matrix-degrading proteinases secreted by malignant tumor cells have been thought to play an essential role in the processes of invasion and metastasis. However, existence and localization of gelatinase activity in breast cancer tissues have not been clarified.


METHODS
We developed a novel film for highly reproducible detection and the localization of gelatinolytic activity. This film has a gelatin layer with a constant thickness 7 microm, and adequate crosslinking to control the speed of degradation by proteases. Cryosections of several breast cancer tissues were put on this gelatin film and incubated for 16 hrs at 37 degrees C. After staining with ponceau 3R dye, the digested area was evaluated under light microscopy.


RESULTS
Digestion of gelatin was detected in more than 90%of breast cancer specimens, although it varied in degree and area for each case. In most cases, the gelatinolytic activity was located within cancer nests, and was not detected in stromal cells surrounding cancer cells. The gelatinolytic activity was inhibited by 1,10-phenanthroline, an inhibitor of matrix metalloproteinases (MMPs).


CONCLUSIONS
In this study, the localization of net MMP activity was confirmed in breast cancer nest using film in situ zymography. Detailed analysis on the relationship between the strength or distribution of MMP activity and malignancy are anticipated in the future.
Iwata
H
H
Department of Breast Surgery, Aichi Cancer Center hospital, 1-1 kanokoden, chikusa-ku, Nagoya 464-8681, Japan.
Yamamoto
M
M
Nemori
R
R
Mizutani
M
M
Iwase
T
T
Miura
S
S
Obata
Y
Y
Hara
Y
Y
Omoto
Y
Y
Toyama
T
T
Yamashita
H
H
Iwase
H
H
Kobayashi
S
S
D001391:Azo Compounds / Q000378:metabolism; D001943:Breast Neoplasms / Q000737:chemistry / Q000201:enzymology*; D002467:Cell Nucleus / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005780:Gelatin / Q000737:chemistry / Q000378:metabolism*; D018093:Gelatinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006416:Hematoxylin / Q000378:metabolism; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
D001391:Azo Compounds; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D010618:Phenanthrolines; C015233:gelatin film; D005780:Gelatin; D018093:Gelatinases; D020782:Matrix Metalloproteinases; C020478:ponceau 3R; C025205:1,10-phenanthroline; D006416:Hematoxylin