Download Expression of gelatinolytic activity by oesophageal and colon

Biochemical Society Transactions (1995) 23 391S
Expression of gelatinolytic activity by oesophageal and colon
carcinoma cell lines.
Della Porta Paola', Ryan Norma" M., and Collins J . Kevin*
Departments of Biochemistry" and Medicine and Microbiology*,
University College Cork.
Several in LWO and m v i i m studies have shown a correlation between the
ability of tumor cells to invade the extracellular matrix and their
secretory levels of the active form of type IV collagenases (1-3). Two
major forms of type IV collagenase (Pro-MMP-9 and Pro-MMP-2, of
Mr 92 and 72 kDa respectively) have been identified in the media and
in the cellular extract of several tumor cell lines (4). A 100 kDa type IV
collagenase has also been reported. In order to become active, proMMP-2 and pro-MMP-9 must be converted into 62 and 82 kDa forms,
respectively. In vitro, this activation can be catalyzed by p-aminophenylmercuric acetate (APMA). The in vrvo mechanism through which the
activation occurs has not been clarified to date. MMP-9 and MMP-2 can
also degrade heat-denatured type I collagen, which, incorporated into a
SDS polyacrylamide gel, allows the identification of these
metalloproteases on the basis of their molecular weight and their ability
to degrade gelatin ( 5 ) . The pattern of gelatinolytic activity expression in
the cell media of three oesophageal carcinoma cell lines (OCI, OC2,
both derived from a squamous carcinoma. and OC3, from an
adenocarcinoma) and one colon adenocarcinoma cell line (SW620) is
reported here (6). Cell media concentrated with 80% ammonium
sulfate. resuspended and dialyzed against 10 mM Tris-HCI (pH 7.5)
contaming 0.5 M NaCl and 0.01% Brij, was analyzed on 15%
polyacrylamide resolving gels containing I mdml of gelatin. The
proteinases in the gel were renatured in 2.5% Triton X-100 containing
50 mM Tris (pH 7.5) and incubated in 50 mM Tris-HCI, 5 mM CaC12.
0.2 M NaCl and 0.02% Brij at 37OC for 28 hours. Gels were then
stained with 0.5% Coomassie Brillant Blue and destained. All cell lines
in this study showed a complex pattern in the expression of gelatinases
(Fig. I). Concentrated samples of OC3 and SW620 cell lines exhibited
two bands of activity at 149 kDa and 135 kDa. After preincubation with
APMA. the higher Mr band disappears, while there is an increase in the
intensity of the 135 kDa band. Therefore it would appear that the two
bands correspond to an inactive and an active form, respectively, of high
Mr proteases expressed by both OC3 and SW620 cell lines. This has not
been reported previously. The activation process by APMA is also
evident in the OC2 cell line, in correspondence to the 61 kDa band,
whose intensity increases after APMA preincubation. In contrast with
the activation observed for the OC3 and SW620 cell lines, there is no
disappearance of higher MW band observed for the OC2 cell line. It is
possible that the 61 kDa band corresponds to the active form of the 71
kDa gelatinolytic activity. previously reported by other researchers as
Pro-MMP2 (3).
Fig. 2
A 15?6SDS polyacrylamide gel of samples used in this study was also
stained for protein (Fig.3).
Fig. 3
Using SDS polyacrylamide gels. far greater amounts of concentrated
samples were necessary to show only some of the bands which were
easily visualized by zymographic analysis. No difference in the
electrophoretical mobility was noted between the band pattern of heated
and unheated samples prior to analysis (data not shown). To conclude, it
has been demonstrated that zymographic analysis is a sensitive and
specific technique for the detection of the APMA-sensitive
inetalloproteases, which have not been identified previously in these cell
lines.
Aknowledgment
I would like to thank Doctor D. Sheehan and Mr. M. 0' Donoghue for
their help, support and useful advise, and to express my most sincere
gratitude to Doctor T. McCarthy for his strong support, during the
summer, in the realization of this work.
References
1
Fig. I Zyinogram of gelatinolytic acti\ ity of concentrated samples of
cell inedia
;lliquots of concentrated samples. corresponding to the gelatinolytic
activit). expressed by the same number of cells of each cell line, were
aiialysed by zymography. in order to get an approximate estimate of the
relative amount of proteases expressed by the different cell lines (Fig.2).
'The OC2 cell line clearly showed a stronger expression of gelatinolytic
activity than that observed for the OCI. OC3 and SW620 cell lines (data
not shown). A densitometric analysis will allow us to compare the
relative amounts of activity expressed by OCI. OC3 and SW620 cell
lines.
Stetler-Stevenson W ( i . (1990). Cancer and metastasis Reviews
9: 289-303.
2
Nakajima M., Welch D.R.. Belloni P.R. and Nicolson G.L. (1987)
Cancer Research. 47: 4869-4876,
3
Duffy M.J. (1992). Clin. Exp. Metastasis. lo:145-55.
4
Tryggvason K.. Hoyhtya and Pyke C. (1993). Breast Cancer
Research and Treatment 24. 209-2 18.
5
Mackay A.R.. Corbitt R.W. Hartzler
Research ( 1990). 0:5997-600 I
6
Collins K.J.. O'Sullivan G . O'Donoghue %
et al.
I(1992).
Fibrinolysis. 6: 83-88.
and Thorgeirsson Cancer