Expression of glutathione S-transferase
class isoenzymes in human colorectal and gastric cancers
W.C.C. de Bruin,
M.J.M. Wagenmans,
P.G. Board1 and
W.H.M. Peters2
Department of Gastroenterology, St Radboud University Hospital, Nijmegen, The Netherlands and
1 Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra, Australia
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Abstract
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Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes, which play an important role in the protection of tissues against potentially harmful compounds. In humans, two
class isoenzymes, GSTT1-1 and GSTT2-2, have been described so far. Both enzymes were claimed to have an important role in human carcinogenesis. In colorectal and gastric tissues, the expression of the other isoenzymes changes after malignant transformation. No data on the expression levels of the
isoenzymes in these tissues are available. The aim of this study was to determine the protein levels of the two
class isoenzymes in human colorectal and gastric cancers and paired normal tissue. Cytosolic fractions of normal and matched tumor tissue samples from 20 patients with colorectal or gastric adenocarcinomas were analyzed on immunoblots using specific antibodies against GSTT1-1 and GSTT2-2, respectively. In addition paraffin-embedded sections of these tissues were examined immunohistochemically for GSTT1-1 expression. In both types of tissue,
class isoenzymes were highly expressed. Expression of GSTT1-1 was higher in gastric than in colorectal tissues. The GSTT2-2 levels were comparable in both tissues. A great interindividual difference in expression was demonstrated. In colon, there was no change in the
class isoenzyme levels after malignant transformation. Gastric tumors had significantly lower expression of both
class isoenzymes compared with the normal mucosa. In colon, GSTT1-1 was expressed in the enterocytes and goblet cells. In gastric tissues, staining was seen in upper and deeper mucous cells, chief cells and, to a lesser extent, in parietal cells. In both types of tumors, staining was seen in adenomatous cells. In conclusion, in both normal human colonic and gastric mucosa, GSTT1-1 and GSTT2-2 are present at high levels, whereas after malignant degeneration, expression is not influenced or is even downregulated.
Abbreviations: GST, glutathione S-transferase
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Introduction
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The glutathione S-transferases (GSTs; EC. 2.5.1.18) represent a multigene family of enzymes that play an important role in protection of tissues against potential toxic and carcinogenic compounds. Based on their homology, GST isoenzymes are divided into at least five classes
, µ,
,
and
(1,2).
In humans, two
class izoenzymes GSTT1-1 and GSTT2-2 have been described so far (37). GSTT1-1 is differentially expressed in a wide variety of human tissues (810), but presence of this
class isoenzyme may be less abundant compared with the
and
class GSTs (7). The human
class GSTs display activity against a broad range of compounds, including methyl halides and sulfate esters, and recently GSTT1-1 was shown to be a potent bio-activator of dihalomethanes (11). In addition, they also have distinct lipid-peroxidase activity (3,12,13).
In rats, induction of the expression of the
class isoenzymes by a variety of chemopreventive agents has been demonstrated (11,14,15), indicating that the
GST genes might be regulated in a similar way to the other class GSTs, via xenobiotic and antioxidant responsive elements (1,11).
For GSTT1-1, a genetic polymorphism has been described. Homozygous carriers of the GSTT1 gene deletion do not have detectable enzyme activity and are considered to have a higher risk for developing certain types of malignancies (1,7). There are indications that polymorphisms in the GSTT2 gene might exist (16).
The characteristics of the
class enzymes make it reasonable to assume that GSTT1-1 and GSTT2-2 might have a role in carcinogenesis and in the sensitivity of tumors against anticancer drugs.
For colorectal and gastric tumors, which in general are highly refractory to chemotherapy, changes in GST
,
and µ levels as compared with the surrounding normal tissue have been described (1722). No data on the levels of the
class GSTs in these tissues are available. In this paper, expression of GSTT1-1 and GSTT2-2 in gastric and colorectal tumors and matched normal mucosa was examined on immunoblot. Moreover, the immunohistochemical localization of GSTT1-1 in these tissues was studied.
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Materials and methods
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Tissue
Twenty patients (15 males and five females, median age 60 years, range 4381 years) with gastric adenocarcinoma, and 20 patients (11 males and nine females, median age 69 years, range 4587 years) with colorectal adenocarcinoma were operated on at the Department of Surgery of the St Radboud University Hospital (Nijmegen, The Netherlands). Representative specimens of the tumor as well as the adjacent macroscopically normal tissue were washed in ice-cold phosphate-buffered saline, frozen in liquid nitrogen and stored at 80°C within 30 min of resection. The local ethical committee on human experimentation approved this study.
Tissue preparation
The normal and matched colorectal or gastric tumor specimens were thawed in ~2 vol of ice-cold homogenizing buffer (250 mM sucrose, 20 mM TrisHCl, 1 mM dithiotreitol, pH 7.4) and homogenized using glassglass tissue grinders. The homogenates were subsequently centrifuged at 150 000 g at 4°C for 1 h. The supernatants were frozen in small aliquots at 80°C prior to use. Protein levels were determined according to the method of Lowry et al. (24) using bovine serum albumin as the standard.
Western blot analysis
GSTT1-1 and GSTT2-2 were quantified by laser densitometric analyses of western blots treated with antibodies directed against the
class GSTs. For detection of GSTT1-1, a monoclonal antibody (9,10) was used, whereas for GSTT2-2, a polyclonal antibody raised against purified recombinant protein was used (3). For detection of GSTT1-1 and GSTT2-2, 25 or 50 µg of cytosolic protein were subjected to SDSPAGE (11% acrylamide, w/v) and transferred to nitrocellulose (Schleicher & Schüell, Germany) using a semi-dry blotting system (Novablot; Pharmacia, Uppsala, Sweden). The western blots were subsequently incubated with the
class antibodies according to standard procedures (18). Blots were stained with 3,3'-diamino benzidine and hydrogen peroxide (Sigma, St Louis, MO). Staining intensity was determined using a laser densitometer (Ultrascan XL; LKB, Bromma, Sweden). Known quantities of purified recombinant
class isoenzymes were run in parallel and used for calculation of the absolute amounts of the isoenzymes in the samples. The concentrations of isoenzymes were calculated as ng/mg total cytosolic protein. The detection limit of this assay is ~40 ng/mg protein. The Wilxocon rank sum test was used for statistical analysis of the experimental data. All data are expressed as means ± SEM.
Immunohistochemical analysis of human
GSTs
Tissue sections were formalin fixed and paraffin embedded using standard procedures. Immunohistochemical staining for GSTT1-1 and GSTT2-2 was performed essentially as described previously (22), using the same antibodies as for the immunoblots detection. The anti-GSTT2-2 antiserum, however, showed too much background staining and therefore no immunohistochemical data on the GSTT2-2 are provided.
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Results
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The GSTT1-1 and GSTT2-2 isoenzyme levels of normal mucosa and matched carcinoma tissue of patients with gastric or colorectal adenocarcinoma were quantified by densitometric analyses of western blots after immunodetection with specific antibodies against these human
class isoenzymes. Results are shown in Table I
.
The levels of GSTT1-1 in normal gastric mucosa were significantly higher than in normal colon tissues (mean values 1575 ± 876 versus 640 ± 550 ng/mg cytosolic protein; P < 0.01). In both normal gastrointestinal tissues GSTT2-2 levels were similar (1159 ± 520 or 1027 ± 672). Gender and age had no influence on the levels of the
class isoenzymes in both types of tissue.
Of the patients with gastric or colorectal cancer, 38 and 30% did not express GSTT1-1 and probably were homozygous for the T1 gene deletion. In the tissue samples of one patient with colorectal cancer (no. 32), no detectable amounts of GSTT2-2 were present.
In normal gastric mucosa GSTT1-1 and GSTT2-2 levels were comparable (P = 0.15). Gastric tumors had significantly lower GSTT1-1 and GSTT2-2 levels than corresponding normal tissues (479 ± 450 versus 1575 ± 876 ng/mg cytosolic protein for GSTT1-1, P = 0.0009; and 815 ± 625 versus 1159 ± 520 ng/mg cytosolic protein for GSTT2-2, P = 0.0005). The decrease in GSTT1-1 was more profound than the change in GSTT2-2 expression (ratio tumor:normal 0.46 ± 0.45 for GSTT1-1 versus 0.70 ± 0.25 for GSTT2-2).
In colon, GSTT1-1 isoenzyme levels were lower than GSTT2-2 levels in normal mucosa (mean values: 640 ± 550 ng/mg cytosolic protein versus 1027 ± 672 ng/mg). In contrast to gastric tissues, no change in levels of
class isoenzymes was observed between colorectal normal mucosa and paired tumors (P = 0.09 for GSTT1-1 and P = 0.25 for GSTT2-2).
In Figure 1
, representative results of immunohistochemical staining for GSTT1-1 in gastric and colorectal normal mucosa and matched tumors are shown. In normal gastric tissues, staining was seen in upper and deeper localized mucous cells, in chief cells and to a lesser extent in parietal cells. Connective tissue components and nuclei were consequently negative. In tumor samples, the characteristic loss of tissue organization resulted in a heterogeneous staining of the adenomatous tumor cells. The overall staining intensity in the tumor was lower as compared with the matched normal tissue.

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Fig. 1. Sections of human gastric (ac) and colorectal (fh) normal tissue and their corresponding adenocarcinomas (d and e, and i and j) stained with the anti-GSTT1-1 monoclonal antibody (b, c and e, and g, h and j, respectively) or hematoxylineosin (a and d, and f and i, respectively). Bars represent 25 (b), 50 (g) or 100 µm (j). In (b), a high magnification gastric section is shown where parietal cells (P), chief cells (Ch) and mucous cells (M) are indicated. In (g), a normal colon tissue section is shown, where enterocytes (E) and goblet cells (G) are outlined.
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In normal colorectal tissues, a positive reaction was seen in enterocytes and goblet cells throughout the crypts, predominantly at the basal side of the cell. Connective tissues and nuclei did not show reactivity. In the matched tumor tissues, adenomatous tumor cells stained positive with comparable intensity to that found in the epithelial cells in normal colon tissue sections.
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Discussion
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In many studies, it was found that in both gastric and colorectal cancers, GST isoenzyme expression changed after malignant transformation. In colorectal tumor tissues, GST
levels most often were elevated, whereas the GST
and µ levels were unaffected (17,19,23). In gastric tumors, an increase in GST
expression was also seen, whereas GST µ and
isoenzyme levels decreased in this tissue type (22,23). This elevation of GST
, which is the main GST isoenzyme in both gastric and colonic tissues, has been claimed to contribute to the multidrug resistance of these tumors (1,19). For the recently identified
class GSTs, no data on expression levels in normal and malignant gastric and colon tissues were available. As seen for the other GSTs, the expression levels of the
class isoenzymes display great interindividual variation; GSTT1-1 levels range from 326 to 3249 ng/mg cytosolic protein in normal stomach and from 0 to 1829 in normal colon. The GSTT2-2 levels range from 558 to 1693 ng/mg cytosolic protein in stomach and from 0 to 2686 ng/mg cytosolic protein in colon tissue samples. Previously, the protein contents of the
, µ and
isoenzymes in stomach and colon were measured (18,19), and now it can be concluded that in human stomach, the
class isoenzymes are expressed to the same extent as the
class GSTs. In colon, the
and
levels are lower than in the stomach and the amount of the
class GSTs is between these two enzyme levels. These results indicate that the occurrence of the
class GSTs in human tissues is more profound than assumed earlier by Pemble et al. (7). This might have implications for their contribution to carcinogenesis.
In colon, similar to the
and µ GSTs, no difference in
class isoenzyme levels between normal and tumor tissues was observed. In gastric tumors, the levels of both GSTT1-1 and GSTT2-2 were decreased by 59 and 27%, respectively. Moreover, the
and µ class GST levels also declined in the tumor. The behaviour of the
class GSTs in colorectal and gastric cancers therefore seems to be similar to that of the
and µ GST class enzymes and different from that of the GST
regulation. This might indicate that the expression of the GST
enzymes is coordinately regulated in gastric and colorectal cancers.
In the tissue samples of one patient (no. 32), no GSTT2-2 expression was detectable. Coggan et al. (16) recently described the existence of a GSTT2 gene deletion, and it will be interesting to investigate whether this GSTT2 gene polymorphism is present in the DNA of this patient.
Immunohistochemical analysis of GSTT1-1 shows that the expression of this isoenzyme is cell-type specific. In normal stomach, mucous cells, chief cells and parietal cells are stained, whereas in normal colon mucosa, the enterocytes and goblet cells are reactive. In the tumor specimens, especially those of the stomach, the expression is very heterogeneous. In general, a tendency for lower expression of GSTT1-1 in the gastric carcinomas was noticed, whereas in the colorectal tumor sections, no such change could be observed, and in this respect the immunohistochemical data do confirm the biochemical observations.
In conclusion, the present study has shown that both normal gastric and colonic mucosa contain high levels of GST
enzymes. Only in gastric tumors was expression of the
class enzymes reduced.
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Acknowledgments
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We thank Dr M.Pruszczynski (Department of Pathology, St Radboud University Hospital) for his help with the interpretation of the immunohistochemical data. This study was supported by the Dutch Cancer Society (grant 95-913).
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Notes
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2 To whom correspondence should be addressed Email: w.peters{at}gastro.azn.nl 
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Received February 25, 1999;
revised April 23, 1999;
accepted April 27, 1999.