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Expression of glutathione-S-transferases isoenzymes and p53 in exfoliated human bladder cancer cells
Urologic Oncology: Seminars and Original Investigations 29 (2011) 538 –544
Original article
Expression of glutathione-S-transferases isoenzymes and p53 in exfoliated human bladder cancer cells Serpil Og˘uztüzün, Ph.D.a,*, Yasemin Sezgin, Ph.D.b, Sertaç Yazıcı, M.D.c, Pınar Fırat, M.D.b, Müzeyyen Özhavzalı, M.S.d, Haluk Özen, M.D.c b
a Department of Biology, Kırıkkale University, Kırıkkale, Turkey Department of Pathology, Faculty of Medicine, Hacettepe University, Ankara, Turkey c Department of Urology, Faculty of Medicine, Hacettepe University, Ankara, Turkey d Department of Mathematics, Kırıkkale University, Kırıkkale, Turkey
Received 19 May 2009; received in revised form 29 July 2009; accepted 3 August 2009
Abstract Objectives: This study investigates the usefulness of glutathione-S-transferases (GST) isoenzymes and p53 immunostaining as a marker of malignancy in urinary cytology, and evaluates their potential effect in increasing diagnostic accuracy in a series of urine cytologic samples. They are also correlated with cytopathology diagnosis and histopathologic diagnosis. Materials and methods: In this study, the slides from 124 bladder carcinoma patients prepared by the cytocentrifugation method were observed. The cytomorphologic properties of these cancer cells were determined. Moreover, the immunocytochemical distributions of GST alpha (GSTA), pi (GSTP), mu (GSTM4), theta (GSTT1) isoenzymes and p53 protein were studied for the patients. Results: The urothelial cancer cells had small cytoplasm and rough nuclear membrane. The chromatin granules were heterogeneously distributed in each malignant cell’s nucleus. There was a pleomorphism of the malignant cells’ nuclei. According to immunocytopathologic observations, the urothelial cancer cells had stronger staining intensity than the benign cells had in 48% of cases for GSTA, 46% of cases for GSTP, 38% of cases for GSTM4, and 42% of cases for GSTT1. For all papillary cases, the malignant cells were stained negative, while the benign cells were positive. For 83% of patients, the malignant cells were stained positive for p53. There was a significant difference in GSTA (P ⫽ 0.006), GSTT1 (P ⫽ 0.004), GSTP (P ⫽ 0.000) and p53 (P ⫽ 0.000) expressions for benign cells whereas, a non-statistical difference in the malignant cells for GSTA, GSTT1, GSTP, GSTM4, and p53 expressions (P ⬎ 0.05). Conclusions: GST isoenzymes and p53 immunostaining were not found to be markers of malignancy in urinary cytology. © 2011 Elsevier Inc. All rights reserved. Keywords: Bladder cancer; Cytology; Immunocytochemistry; Glutathione-S-transferase; p53
1. Introduction Cystoscopy and biopsy are the golden standard for detection of urothelial carcinoma of the urinary bladder but it is invasive and causes significant patient discomfort. Urinary cytology is a noninvasive method for the detection of urothelial carcinoma of the urinary bladder. Urinary cytology has high sensitivity in high grade bladder tumors but is less sensitive in grade-1 tumors with a sensitivity of 0% to 50% and still a high specificity of 99% [1,2]. * Corresponding author. Tel.: ⫹00-90-535-464-2445; fax: ⫹00-90318-357-2923. E-mail address: [email protected] (S. Og˘uztüzün). 1078-1439/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2009.08.001
The correct identification of malignant cells in cytologic specimens is the obvious aim of diagnostic cytopathology. However, the change of many tumors and the consequent low rate of morphologic deviation of neoplastic cells make this goal difficult to achieve. In particular, this proves to be true for urinary cytology, which is considered to be of great help at diagnosing bladder neoplasms at early stages, allowing adequate early treatment. Unfortunately, the validity of urinary cytology is limited by a great number of false negative results [3]. In this context, the methods that contribute to improve diagnostic accuracy in tumor detection would be of great value. Urotoxicities of chemical carcinogens and other noxious compounds depend on the disposition and efficient transport
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and removal of those substances. Glutathione-S-transferases (GSTs) can catalyze metabolic conversions of certain potentially urotoxic substances to facilitate their excretion in urine as mercapturates [4 – 6]. Genotoxic effects of common bladder carcinogens, such as trans-stilbene oxide or aminobiphenyl metabolites could be prevented by the action of GST class [7]. Thus, GST subunit composition could influence the metabolism of carcinogens and affect selective responses to carcinogens that induce bladder cancer. Accordingly, GSTs have received considerable attention in relation to susceptibility and risks of bladder cancer in humans [8] with regard to therapeutic drug resistance [9]. Based on their biochemical, immunologic, and structural properties, GSTs have been placed into seven classes of cytosolic proteins, the most prominent of which are the classes ␣ (A), (M), (P), and (T) [10]. GSTs assemble in homo- and heterodimeric combinations within the same class, and their expression is regulated in tissue-, developmental-, and gender-specific manner [11]. Elevated expression of class member GSTP1-1 was confirmed in a number of solid tumors [12]. In some cases, the amount of GSTP1-1 was correlated with the degree of dedifferentiation and malignancy. It is generally accepted that overall GST activity is also up-regulated in transitional cell carcinoma (TCC) of the urinary bladder [13], with GSTP1-1 accounting for the majority of this increase [14,15]. However, information on the expression of other GST isoenzymes is lacking. P53 mutation seems to be one of the most common genetic events in human tumors [16]. Because of the increased half-life of its mutated gene product, p53 mutation can be detected immunohistochemically in the majority of breast, colon, lung, and bladder cancers [17]. In previous studies, p53 mutation was shown to be a predictive marker in the progression of bladder cancer [18,19]. P53 overexpression has shown to be associated with grade and stage [20]. Hypothetically, the mutated p53 in environmentally
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associated cancers, such as bladder cancer, may therefore be influenced by GST isoenzymes. Accordingly, this study was directed to investigate the usefulness of GST isoenzymes and p53 immunostaining as a marker of malignancy in urinary cytology, aiming at evaluating its potential for improving the diagnostic accuracy in a series of urine cytologic samples. They are also correlated with cytopathology diagnosis and histopathologic diagnosis. To the best of our knowledge, this is the first study examining the role of GST isoenzymes in urine cytology smears.
2. Materials and methods 2.1. Specimens All cytology and surgical pathology specimens used in this study were obtained from the Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. Age, gender, pertinent clinical history, and final clinical outcome were noted from the patient records of the hospital. One hundred-twenty-four cytologic specimens from 124 patients with a history of bladder tumors were selected from the routine submissions to our cytology laboratory. Criteria used in the selection process were adequate preservation of urothelial cells, moderate to high cellularity, an absence of obscuring inflammation, and the availability of a bladder biopsy specimen. Cytologic diagnoses were those rendered during routine cytology sign-out by a cytopathologist. Papanicolaou stain was used in all cases. The diagnostic categories consisted of negative, positive, and atypical cells. In addition, 30 controls from healthy people were included in our study. About 50 ml fresh afternoon voided urine specimens were collected from the patients and controls. Urine was centrifuged for 5 minutes with 1670 g in a Hettich centri-
Fig. 1. (A) Negative staining in benign cell but positive staining in malignant cells for GSTA (⫻400); (B) positive staining in malignant cells for GSTT1 (⫻400). (Color version of figure is available online.)
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Fig. 2. (A) Positive cytoplasmic staining in benign but negative staining in papiller malignant cells for GSTM4 (⫻200); (B) positive staining in benign cell but negative staining in malignant cells for GSTP (⫻400). (Color version of figure is available online.)
fuge. The pellet of each urine sample was fixed in Shandon cytospin collection fluid (Shandon Inc., Pittsburgh, PA), containing 39% ethanol, 3% polyoxyethylene, and 2% isopropanol. Following evaluation of the initial diagnostic PAP stained cytospin slide, five cytospin preparations were made on uncoated slides for immunocytochemistry. Of the urine specimens received by our laboratory, a minority (⬍50%) met our criteria for preservation and cellularity. 2.2. Immunocytochemical staining For an indirect immunocytochemical procedure, cytology specimens were treated with 3% H2O2 for 10 minutes, taken to water, and then rinsed in PBS (pH 7.4) for 5 minutes. Nonspecific protein binding was blocked on specimen by incubating with blocking solution for 10 minutes. The primary antibodies, GSTA, GSTP, GSTM4, GSTT1 and p53 were used at 1:100, 1:100, 1: 50, 1:500, and 1:100 dilutions, respectively, and incubated for 1 hour at room temperature. Specimens were washed with PBS buffer (pH 7.4) and incubated in biotinylated secondary antibody solution for 10 minutes. Diaminobenzidine (DAB) served as the chromagen and Mayer’s hematoxylin as the counterstain. In each batch, a known positive slide of TCC of the bladder was kept as the positive control. For the negative control, the primary antibody was omitted in one of the slides of TCC.
creased nuclear/cytoplasmic (N/C) ratio, and when an abundant number of atypical cells or cell groups was present. A specimen was judged “positive” for malignancy if the cells exhibited severely atypical nuclear morphology, a very high N/C ratio, and many atypical cells or cell groups (Fig. 1, Fig. 2, and Fig. 3). The immunocytochemical staining results were reviewed independently and blindly by two of the authors (Y.S. and S.O.) without the knowledge of clinical history and previous diagnosis. Cytoplasmic positivity was taken into account. The staining intensity was quantitated in each case. A score of 0 to 3 positivity was given to each cell. The smears were
2.3. Specimen evaluation The cytopathology diagnosis was performed on PAP stained specimens with criteria described by Frost [21] and Kern [22]. An “atypical” diagnosis was based on the presence of slightly-to-moderately irregular and enlarged nuclei with hyperchromasia and increased chromatin granularity. A “suspicious” diagnosis was made when nuclei exhibiting coarser chromatin granules, marked hyperchromasia, an in-
Fig. 3. Positive nuclear staining in malignant cells for p53 (⫻200). (Color version of figure is available online.)
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Table 1 Relationship between expressions of GSTA, GSTP, GSTT1, and GSTM4 by benign and malignant cells in bladder cancers GSTT1
(–) (1⫹) (2⫹) (3⫹) Total P value
GSTP
Malignant cells
Benign cells
n
n%
n
24 10 2 2 38
64 26 5 5
7 15 3 1 26
GSTA
Malignant cells
Benign cells
n%
n
n%
n
27 57 12 4
5 6 1 1 13
38 46 8 8
9 3 5 3 20
0.004*
GSTM4
Malignant cells
Benign cells
n%
n
n%
n
45 15 25 15
13 12 — 1 26
50 46 — 4
9 8 11 4 32
0.000*
Malignant cells
Benign cells
n%
n
n%
n
n%
28 25 34 13
18 12 — — 30
60 40 — —
8 7 2 1 18
44 39 11 6
0.006*
0.203
* Correlation is significant at the 0.05 level (2-tailed).
then divided into (1) negative (N); (2) weak (1⫹); (3) moderate (2⫹); and (4) strong (3⫹). One way ANOVA and multiple comparison tests were carried out to test the significance of difference of positivity among all the groups.
3. Results We studied the expression of GST isoenzymes and p53 in the cells collected from the urine of 124 patients admitted for resection of recurrent bladder carcinoma (Table 1). The patients were examined by cystoscopy. Visible tumors were resected transurethrally, and suspicious areas in the bladder were biopsied. Thirty cases were included in the study as the control group. Of the 124 patients, malignant cells were observed in 13 cases for GSTP, 16 cases for GSTA, 38 cases for GSTT1, 30 cases for GSTM4, and 40 cases for p53. GSTP was detected in 8 (62%) malignant samples, whereas GSTA, GSTM4, and GSTT1 were present in 13 (50%), 12 (40%), and 14 (37%) malignant samples, respectively (Table 1). In bladder tumors, 8/13 (62%) displayed GSTP immunoreactivity. Five out of 13 cases (38%) did not show any GSTP expression. Table 1 summarizes the distribution of GSTA, GSTP, GSTM4, and GSTT1 immunostaining in bladder tumors. We found the relationships among GSTA, GSTP, and GSTT1 expressions between benign and malignant groups (Table 1) (P ⬍ 0.05). There was, however, no relationship for GSTM4 expression (Table 1) (P ⬎ 0.05). GSTA, GSTM4, GSTP, and GSTT1 expressions were stronger in malignant cells than those in benign cells in 14, Table 2 Mean and standard deviation values of control group according to GST isoenzymes
Mean Standard deviation
GSTP
GSTM4
GSTA
GSTT1
p53
1.3 0.458258
1 0
0.9 0.031623
1.2 0.415799
1 0
6, 5, and 16 bladder cancer cases (Table 1, Fig. 1). Fig. 2A showed that positive stronger cytoplasmic staining was observed in benign rather than in malignant cells in 5, 6, 3, and 8 cases for GSTA, GSTM4, GSTP, and GSTT1, respectively. Moreover, GSTA, GSTM4, GSTP, and GSTT1 had negative staining in benign cells, but positive staining in papillary malignant cells for the same patients in 10, 4, 3, and 14 cases, respectively (Table 1, Fig. 2B). There was negative staining for GSTA, GSTM4, GSTP, GSTT1, and p53 in all 30 control samples. Of the 40 patients, 33 (12%) possessed tumor p53 protein; 27 of 33 bladder cancer patients with p53 positive had G1 tumors while five of these patients had G3 tumors. Overexpression of tumor p53 protein was found in 33 of 40 patients (82%) examined. Overexpression of tumor p53 protein was found in 82% (27/33) of G1 tumors, 3% (1/33) of G2 tumors, and 15% (5/33) of G3 tumors. No p53 immunoreactivity was found in normal (control) samples. P53 displayed nuclear expression in 82% tumor cells (Table 1, Fig. 3). Histologic grade of the tumors was determined on hematoxylin-eosin (HE) stained slides; 22 cases were grade 1 bladder carcinomas while four cases were grade 2, and 19 cases were grade 3. There was no difference in GST isoenzymes and p53 expression in control cells (Table 2) (P ⬎ 0.05). Similarly, there was no statistically significant difference in GSTA, GSTT1, GSTP, and p53 expressions for benign (P ⫽ 0.201) and malignant cells according to one way ANOVA test (P ⫽ 0.076) (P ⬎ 0.05) (Tables 3 and 4).
Table 3 One way ANOVA test results for the comparison of GST isoenzymes and p53 in malignant cell groups Malignant cell
Sum of squares
df
Mean square
F
Sig.
Between groups Within groups Total
4.392 64.811 69.203
4 128 132
1.098 0.506
2.169
0.76
df ⫽ degrees of freedom; F ⫽ F-test.
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Table 4 One way ANOVA test results for the comparison of GST isoenzymes and p53 in benign cell groups Benign cell
Sum of squares
df
Mean square
F
Sig.
Between groups Within groups Total
2.530 52.617 55.147
3 98 101
0.843 0.537
1.571
0.201
df ⫽ degrees of freedom; F ⫽ F-test.
There was a significant difference in GSTA (P ⫽ 0.006), GSTT1 (P ⫽ 0.004), GSTP (P ⫽ 0.000), and p53 (P ⫽ 0.000) expressions for benign cells (P ⬍ 0.05). However, there was no significant difference in GSTM4 expression (P ⫽ 0.203) for benign cells (P ⬎ 0.05) (Tables 5 and 6).
Table 6 Multiple comparisons between GST enzymes and P53 of benign cells and control groups Mean difference
Std. Error
Sig.
95% confidence interval Lower bound
Dunnett t (2-sided)† GSTA control GSTM4 control GSTP control p53 control
Upper bound
0.507*
0.158
0.006
0.11
0.90
0.277
0.147
0.203
⫺0.09
0.64
0.451*
0.135
0.004
0.11
0.79
0.815*
0.201
0.000
0.31
1.32
* The mean difference is significant at the 0.05 level. † Dunnett t-tests treat one group as a control, and compare all other groups against it.
4. Discussion The incidence of bladder cancer is either increasing moderately or remaining stable in most developed countries, despite improvements in diagnostic and therapeutic methods and greater awareness of important risk factors, such as cigarette smoking and occupational chemical exposure [23]. The balance between the activation and detoxification of carcinogens affects the amount of deoxyribonucleic acid (DNA) damage that accumulates in cells [24]. The entire process leading to DNA damage and the subsequent repair of the damage involve a host of enzymes. DNA damage is a consequence of the balance between the activation and detoxification of carcinogens that involves phase I and II metabolic enzymes, many of which are polymorphic [25]. The genetic polymorphisms in a number of metabolic enzymes and other genes have been found as the modulators of bladder cancer risk. The polymorphisms in genes for some important enzymes, especially GSTs, have been extensively studied in connection with bladder cancer risk. An improved understanding of the biology of urothelial malignancies is helping to define more clearly the role of new prognostic indices and multidisciplinary treatment for this disease. Uroepithelium is capable of metabolizing some precarcinogens in their effective genotoxic metabolites and is not regarded as an inactive target of preformed reactive metabolites of the carcinogens in the urine [26]. Several studies
have reported on overall GST activity, including isoenzymes belonging to the classes ␣, , and , toward 1-chloro-2, 4-dinitrobenzene (CDNB) [27,12], and GST protein expression in the uroepithelium and tumors of patients with TCC [13]. It is generally accepted that overall GST activity is up-regulated in TCC, with GSTP accounting for most of this increase [13,28]. As information on GSTT1 activity is also lacking, it would be of great importance to determine whether GSTT1 is expressed in the uroepithelium and tumors of the patients’ TCC. To the best of our knowledge, the present study represents the first attempt for a comprehensive description of the 4 classes of GSTs isoenzymes in urine cytology smears. The slides from 124 bladder carcinoma patients prepared by the cytocentrifugation method were observed. Of the 124 patients, malignant cells were observed in 13 cases for GSTP, 16 cases for GSTA, 38 cases for GSTT1, 30 cases for GSTM4, and 40 cases for p53. According to this, GSTP was detected in 8 (62%) malignant samples, whereas GSTA, GSTM4, and GSTT1 were present in 13 (50%), 12 (40%), and 14 (37%) malignant samples, respectively. In this study, by comparing the benign and malignant groups, no relation between GSTA, GSTP, GSTM4, and GSTT1 expressions could be found. We observed a statistical difference in staining intensity for GSTP, GSTA, GSTT1, and p53 between benign epithelial cells and normal cells. However, there was no difference in staining intensity for GSTM4 between benign and normal epithelium cells
Table 5 Multiple Comparisons of enzymes expressions between benign cells and control groups
Assume equal variances Does not assume equal variances df ⫽ degrees of freedom; t ⫽ t-test.
Contrast
Value of contrast
Std. error
t
df
Sig. (2-tailed)
1 1
⫺2.05 ⫺2.05
0.448 0.384
⫺4.569 ⫺5.340
146 46.231
0.000 0.000
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(Table 1, P ⬍ 0.05). Cancer cells reveal multiple genetic alterations, resulting in morphologic and functional differences from the normal cells. Tumor cells may change some of their functions (e.g., expression of some proteins) in the malignant transformation process. It can be speculated that higher level of GST expression in the benign cells can be the result of this transformation. In a more extended study, Simic et al. [14] reported significantly higher GST enzyme activity levels, GSTP1 and GSTT1 levels in TCC compared with control tissues. Berendsen et al. [13] found that GSTP1 and GSTM1 significantly increased in TCC relative to adjacent mucosa. Giralt et al. [12] have reported increased levels of GST enzyme activity in samples of TCC in comparison with levels in normal mucosa of the same patients (n ⫽ 34). Several investigators measured high levels of GST enzyme activity in bladder cancer cell lines, and GSTP was the most predominant isoenzyme in these cell lines. GSTM and GSTA were either absent or present at very low levels [29,30]. Being the most commonly mutated gene in human cancer, including bladder carcinoma, p53 has attracted a great deal of interest as a prognostic factor, diagnostic tool, and therapeutic target [31]. Recent studies using immunohistochemical staining following antigen retrieval investigated the expression of p53 in bladder carcinomas, and found that from 29% to 78% of the tumors demonstrated overexpression of tumor p53 protein [32,33]. In the present study, overexpression of tumor p53 protein was found in 33 of 40 patients (82%) examined. The present study demonstrates the wide variability in GST enzyme expression in bladder cancer region. Incorporating such an approach in larger trials may help in elucidating the roles of these enzymes in carcinogenesis as well as in identifying potential targets for chemoprevention. We observed that there was a nonsignificant relationship between the expressions of GSTs and p53, and the tumor grade. The number of bladder cancer patients in this analysis can be considered small. Further studies with substantially larger numbers of bladder cancer patients are needed to examine prospectively for the possible relationship between GST expression and prognostic factors. Expression of xenobiotic metabolizing enzymes within tumors has been identified as a potentially important factor in determining anti-tumor drug resistance. Both the amount and the proportion of different enzymes present in tumors play a role in determining anticancer drug resistance. Immunocytochemistry is a useful method for the investigation of expression and cellular localization of GSTs within tumors, and may be useful in identifying those tumors that are potentially resistant to specific anticancer drugs. More data are needed to improve our understanding of the role of these enzymes in neoplasia and in resistance to cytotoxic drug therapy.
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Acknowledgements This work was supported by the Research Fund of the Kirikkale University, project number: 02/03.04.04. References [1] Planz B, Striepecke E, Jakse G, et al. Use of Lewis X antigen and deoxyribonucleic acid image cytometry to increase sensitivity of urinary cytology in transitional cell carcinoma of the bladder. J Urol 1998;159:384 – 8. [2] Lotan Y, Roehrborn CG. Sensitivity and specificity of commonly available bladder tumor markers versus cytology: Results of a comprehensive literature review and meta-analyses. Urology 2003;61: 109 –18. [3] Eagan JW Jr. Urinary tract cytology. In: Hill GS, editor. Uropathology. Edinburgh: Churchill Livingstone, 1989. p. 873–934. [4] Li D, Wang M, Dhingra K, et al. Aromatic DNA adducts in adjacent tissues of breast cancer patients: Clues to breast cancer etiology. Cancer Res 1996;56:287–93. [5] Hayes JD, Flaagan JU, Jowsey IR. Glutathione-S-transferases. Annu Rev Pharmacol Toxicol 2005;45:51– 88. [6] Coles BF, Kadlubar FF. Detoxification of electrophilic compounds by glutathione S-transferase catalysis: Determinants of individual response to chemical carcinogens and chemotherapeutic drugs? Biofactors 2003;17:115–30. [7] Feng Z, Hu W, Rom WN, et al. Aminobiphenyl is a major etiological agent of human bladder cancer: Evidence from its DNA binding spectrum in human p53 gene. Carcinogenesis 2002;23:1721–7. [8] Aktas D, Ozen H, Atsu N, et al. Glutathione S-transferase M1 gene polymorphism in bladder cancer patients: A marker for invasive bladder cancer? Cancer Letters 2001;125:1– 4. [9] Hung RJ, Boffetta P, Brennan P, et al. GST, NAT, SULT1A1, CYP1B1 genetic polymorphisms interactions with environmental exposures and bladder cancer risk in a high-risk population. Int J Cancer 2004;110:598 – 604. [10] Hayes JD, Strange RC. Glutathione-S-transferase polymorphisms and their biological consequences. Pharmacology 2000;61:154 – 66. [11] Tsuchida S, Sato K. Glutathione transferases and cancer. Crit Rev Biochem Mol Biol 1992;27:337– 84. [12] Giralt M, Lafuente A, Pujol F, et al. Enhanced glutathione S-transferase activity and glutathione content in human bladder cancer. Follow-up study: Influence of smoking. J Urol 1993;149:1452– 4. [13] Berendsen CL, Peters WHM, Scheffer PG, et al. Glutathione-Stransferase activity and subunit composition in transitional cell cancer and mucosa of the human bladder. Urology 1997;49:644 –51. [14] Simic T, Mimic-Oka J, Savic-Radojevic A, et al. Glutathione-Stransferase T1-1 activity up-regulated in transitional cell carcinoma of urinary bladder. Urology 2005;65:1035– 40. [15] Savic-Radojevic A, Mimic-Oka J, Pljesa- Ercegovac M, et al. Glutathione S-transferase-P1 expression correlates with increased antioxidant capacity in transitional cell carcinoma of the urinary bladder. Eur Urol 2007;52:470 –7. [16] Hainaut P, Hollstein M. p53 and human cancer: The first ten thousand mutations. Adv Cancer Res 2000;77:81–137. [17] Hainaut P, Hernandez T, Robinson A, et al. IARC database of p53 gene mutations in human tumors and cell lines: Updated compilation, revised formats, and new visualization tools. Nucleic Acids Res 1998;26:205–13. [18] Brockmöller J, Kaiser R, Kerb R, et al. Polymorphic enzymes of xenobiotic metabolism as modulators of acquired P53 mutations in bladder cancer. Pharmacogenetics 1996;6:535– 45. [19] Spruck CH III, Rideout WH III, Olumi AF, et al. Distinct pattern of p53 mutations in bladder cancer. Cancer research 1993;53: 1162– 6.
544
S. Og˘uztüzün et al. / Urologic Oncology: Seminars and Original Investigations 29 (2011) 538 –544
[20] Fujimoto K, Yamaha Y, Okajs¸ma E, et al. Frequent association of p53 gene mutations in bladder cancers and urine samples. Science 1991; 252:706 –9. [21] Frost J. The cell in health and disease. An evaluation of cellular morphologic expression of biologic behavior. In: Wied G, von Hamm E, Koss L, et al, editors. Monographs in Clinical Cytology, 2nd ed. Basel: Karger, 1986. [22] Kern W. Urinary tract. In: Keebler C, Reagan J, editors. A Manual of Cytotechnology, 6th ed. Chicago: ASCP Press, 1983. [23] Stewart BW, Kleihus P. World cancer report. Lyon, France: IARC Press 2003. [24] Stern MC, Johnson LR, Bell DA, et al. XPD colon 751 polymorphism, metabolism genes, smoking, and bladder cancer risk. Cancer Epidemiol Biomarkers Prev 2002;11:1004 –11. [25] Sanyal S, Festa F, Sakano S, et al. Polymorphisms in DNA repair and metabolic genes in bladder cancer. Carcinogenesis 2004;25: 729 –34. [26] Their R, Golka K, Brüning T, et al. Genetic susceptibility to environmental toxicants: The interface between human and experimental studies in the development of new toxicological concepts. Toxicol Lett 2002;127:321–7.
[27] Lafuente A, Giarlt M, Cervello I, et al. Glutathione S-transferase activity in human superficial transitional cell carcinoma of the bladder. Comparison with healthy controls. Cancer 1990;665: 2064 – 8. [28] Singh SV, Xu BH, Tkalevic GT, et al. Glutathione-linked detoxification pathway in normal and malignant human bladder tissue. Cancer Lett 1994;77:15–24. [29] Singh SV, Ahmad H, Krishan A. Expression of glutathione related enzymes in human bladder cancer cell lines. Biochem Pharmacol 1990;39:1817–20. [30] Ahn H, Lee E, Kim K, et al. Effect of glutathione and its related enzymes on chemosensitivity of renal cell carcinoma and bladder carcinoma cell lines. J Urol 1994;151:263–7. [31] Assimakopoulos D, Kolettas E, Zagorianakou N, et al. Prognostic significance of p53 in the cancer of the larynx. Anticancer Res 2000;20:3555– 64. [32] Lipponen PK. Overexpression of p53 nuclear oncoprotein in transitional cell bladder cancer and its prognostic value. Int J Cancer 1993;53:365–70. [33] Esrig D, Elmajian D, Groshen S, et al. Accumulation of nuclear p53 and tumor progression in bladder cancer. N Engl J Med 1994;331: 1259 – 64.
Original article
Expression of glutathione-S-transferases isoenzymes and p53 in exfoliated human bladder cancer cells Serpil Og˘uztüzün, Ph.D.a,*, Yasemin Sezgin, Ph.D.b, Sertaç Yazıcı, M.D.c, Pınar Fırat, M.D.b, Müzeyyen Özhavzalı, M.S.d, Haluk Özen, M.D.c b
a Department of Biology, Kırıkkale University, Kırıkkale, Turkey Department of Pathology, Faculty of Medicine, Hacettepe University, Ankara, Turkey c Department of Urology, Faculty of Medicine, Hacettepe University, Ankara, Turkey d Department of Mathematics, Kırıkkale University, Kırıkkale, Turkey
Received 19 May 2009; received in revised form 29 July 2009; accepted 3 August 2009
Abstract Objectives: This study investigates the usefulness of glutathione-S-transferases (GST) isoenzymes and p53 immunostaining as a marker of malignancy in urinary cytology, and evaluates their potential effect in increasing diagnostic accuracy in a series of urine cytologic samples. They are also correlated with cytopathology diagnosis and histopathologic diagnosis. Materials and methods: In this study, the slides from 124 bladder carcinoma patients prepared by the cytocentrifugation method were observed. The cytomorphologic properties of these cancer cells were determined. Moreover, the immunocytochemical distributions of GST alpha (GSTA), pi (GSTP), mu (GSTM4), theta (GSTT1) isoenzymes and p53 protein were studied for the patients. Results: The urothelial cancer cells had small cytoplasm and rough nuclear membrane. The chromatin granules were heterogeneously distributed in each malignant cell’s nucleus. There was a pleomorphism of the malignant cells’ nuclei. According to immunocytopathologic observations, the urothelial cancer cells had stronger staining intensity than the benign cells had in 48% of cases for GSTA, 46% of cases for GSTP, 38% of cases for GSTM4, and 42% of cases for GSTT1. For all papillary cases, the malignant cells were stained negative, while the benign cells were positive. For 83% of patients, the malignant cells were stained positive for p53. There was a significant difference in GSTA (P ⫽ 0.006), GSTT1 (P ⫽ 0.004), GSTP (P ⫽ 0.000) and p53 (P ⫽ 0.000) expressions for benign cells whereas, a non-statistical difference in the malignant cells for GSTA, GSTT1, GSTP, GSTM4, and p53 expressions (P ⬎ 0.05). Conclusions: GST isoenzymes and p53 immunostaining were not found to be markers of malignancy in urinary cytology. © 2011 Elsevier Inc. All rights reserved. Keywords: Bladder cancer; Cytology; Immunocytochemistry; Glutathione-S-transferase; p53
1. Introduction Cystoscopy and biopsy are the golden standard for detection of urothelial carcinoma of the urinary bladder but it is invasive and causes significant patient discomfort. Urinary cytology is a noninvasive method for the detection of urothelial carcinoma of the urinary bladder. Urinary cytology has high sensitivity in high grade bladder tumors but is less sensitive in grade-1 tumors with a sensitivity of 0% to 50% and still a high specificity of 99% [1,2]. * Corresponding author. Tel.: ⫹00-90-535-464-2445; fax: ⫹00-90318-357-2923. E-mail address: [email protected] (S. Og˘uztüzün). 1078-1439/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2009.08.001
The correct identification of malignant cells in cytologic specimens is the obvious aim of diagnostic cytopathology. However, the change of many tumors and the consequent low rate of morphologic deviation of neoplastic cells make this goal difficult to achieve. In particular, this proves to be true for urinary cytology, which is considered to be of great help at diagnosing bladder neoplasms at early stages, allowing adequate early treatment. Unfortunately, the validity of urinary cytology is limited by a great number of false negative results [3]. In this context, the methods that contribute to improve diagnostic accuracy in tumor detection would be of great value. Urotoxicities of chemical carcinogens and other noxious compounds depend on the disposition and efficient transport
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and removal of those substances. Glutathione-S-transferases (GSTs) can catalyze metabolic conversions of certain potentially urotoxic substances to facilitate their excretion in urine as mercapturates [4 – 6]. Genotoxic effects of common bladder carcinogens, such as trans-stilbene oxide or aminobiphenyl metabolites could be prevented by the action of GST class [7]. Thus, GST subunit composition could influence the metabolism of carcinogens and affect selective responses to carcinogens that induce bladder cancer. Accordingly, GSTs have received considerable attention in relation to susceptibility and risks of bladder cancer in humans [8] with regard to therapeutic drug resistance [9]. Based on their biochemical, immunologic, and structural properties, GSTs have been placed into seven classes of cytosolic proteins, the most prominent of which are the classes ␣ (A), (M), (P), and (T) [10]. GSTs assemble in homo- and heterodimeric combinations within the same class, and their expression is regulated in tissue-, developmental-, and gender-specific manner [11]. Elevated expression of class member GSTP1-1 was confirmed in a number of solid tumors [12]. In some cases, the amount of GSTP1-1 was correlated with the degree of dedifferentiation and malignancy. It is generally accepted that overall GST activity is also up-regulated in transitional cell carcinoma (TCC) of the urinary bladder [13], with GSTP1-1 accounting for the majority of this increase [14,15]. However, information on the expression of other GST isoenzymes is lacking. P53 mutation seems to be one of the most common genetic events in human tumors [16]. Because of the increased half-life of its mutated gene product, p53 mutation can be detected immunohistochemically in the majority of breast, colon, lung, and bladder cancers [17]. In previous studies, p53 mutation was shown to be a predictive marker in the progression of bladder cancer [18,19]. P53 overexpression has shown to be associated with grade and stage [20]. Hypothetically, the mutated p53 in environmentally
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associated cancers, such as bladder cancer, may therefore be influenced by GST isoenzymes. Accordingly, this study was directed to investigate the usefulness of GST isoenzymes and p53 immunostaining as a marker of malignancy in urinary cytology, aiming at evaluating its potential for improving the diagnostic accuracy in a series of urine cytologic samples. They are also correlated with cytopathology diagnosis and histopathologic diagnosis. To the best of our knowledge, this is the first study examining the role of GST isoenzymes in urine cytology smears.
2. Materials and methods 2.1. Specimens All cytology and surgical pathology specimens used in this study were obtained from the Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. Age, gender, pertinent clinical history, and final clinical outcome were noted from the patient records of the hospital. One hundred-twenty-four cytologic specimens from 124 patients with a history of bladder tumors were selected from the routine submissions to our cytology laboratory. Criteria used in the selection process were adequate preservation of urothelial cells, moderate to high cellularity, an absence of obscuring inflammation, and the availability of a bladder biopsy specimen. Cytologic diagnoses were those rendered during routine cytology sign-out by a cytopathologist. Papanicolaou stain was used in all cases. The diagnostic categories consisted of negative, positive, and atypical cells. In addition, 30 controls from healthy people were included in our study. About 50 ml fresh afternoon voided urine specimens were collected from the patients and controls. Urine was centrifuged for 5 minutes with 1670 g in a Hettich centri-
Fig. 1. (A) Negative staining in benign cell but positive staining in malignant cells for GSTA (⫻400); (B) positive staining in malignant cells for GSTT1 (⫻400). (Color version of figure is available online.)
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Fig. 2. (A) Positive cytoplasmic staining in benign but negative staining in papiller malignant cells for GSTM4 (⫻200); (B) positive staining in benign cell but negative staining in malignant cells for GSTP (⫻400). (Color version of figure is available online.)
fuge. The pellet of each urine sample was fixed in Shandon cytospin collection fluid (Shandon Inc., Pittsburgh, PA), containing 39% ethanol, 3% polyoxyethylene, and 2% isopropanol. Following evaluation of the initial diagnostic PAP stained cytospin slide, five cytospin preparations were made on uncoated slides for immunocytochemistry. Of the urine specimens received by our laboratory, a minority (⬍50%) met our criteria for preservation and cellularity. 2.2. Immunocytochemical staining For an indirect immunocytochemical procedure, cytology specimens were treated with 3% H2O2 for 10 minutes, taken to water, and then rinsed in PBS (pH 7.4) for 5 minutes. Nonspecific protein binding was blocked on specimen by incubating with blocking solution for 10 minutes. The primary antibodies, GSTA, GSTP, GSTM4, GSTT1 and p53 were used at 1:100, 1:100, 1: 50, 1:500, and 1:100 dilutions, respectively, and incubated for 1 hour at room temperature. Specimens were washed with PBS buffer (pH 7.4) and incubated in biotinylated secondary antibody solution for 10 minutes. Diaminobenzidine (DAB) served as the chromagen and Mayer’s hematoxylin as the counterstain. In each batch, a known positive slide of TCC of the bladder was kept as the positive control. For the negative control, the primary antibody was omitted in one of the slides of TCC.
creased nuclear/cytoplasmic (N/C) ratio, and when an abundant number of atypical cells or cell groups was present. A specimen was judged “positive” for malignancy if the cells exhibited severely atypical nuclear morphology, a very high N/C ratio, and many atypical cells or cell groups (Fig. 1, Fig. 2, and Fig. 3). The immunocytochemical staining results were reviewed independently and blindly by two of the authors (Y.S. and S.O.) without the knowledge of clinical history and previous diagnosis. Cytoplasmic positivity was taken into account. The staining intensity was quantitated in each case. A score of 0 to 3 positivity was given to each cell. The smears were
2.3. Specimen evaluation The cytopathology diagnosis was performed on PAP stained specimens with criteria described by Frost [21] and Kern [22]. An “atypical” diagnosis was based on the presence of slightly-to-moderately irregular and enlarged nuclei with hyperchromasia and increased chromatin granularity. A “suspicious” diagnosis was made when nuclei exhibiting coarser chromatin granules, marked hyperchromasia, an in-
Fig. 3. Positive nuclear staining in malignant cells for p53 (⫻200). (Color version of figure is available online.)
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Table 1 Relationship between expressions of GSTA, GSTP, GSTT1, and GSTM4 by benign and malignant cells in bladder cancers GSTT1
(–) (1⫹) (2⫹) (3⫹) Total P value
GSTP
Malignant cells
Benign cells
n
n%
n
24 10 2 2 38
64 26 5 5
7 15 3 1 26
GSTA
Malignant cells
Benign cells
n%
n
n%
n
27 57 12 4
5 6 1 1 13
38 46 8 8
9 3 5 3 20
0.004*
GSTM4
Malignant cells
Benign cells
n%
n
n%
n
45 15 25 15
13 12 — 1 26
50 46 — 4
9 8 11 4 32
0.000*
Malignant cells
Benign cells
n%
n
n%
n
n%
28 25 34 13
18 12 — — 30
60 40 — —
8 7 2 1 18
44 39 11 6
0.006*
0.203
* Correlation is significant at the 0.05 level (2-tailed).
then divided into (1) negative (N); (2) weak (1⫹); (3) moderate (2⫹); and (4) strong (3⫹). One way ANOVA and multiple comparison tests were carried out to test the significance of difference of positivity among all the groups.
3. Results We studied the expression of GST isoenzymes and p53 in the cells collected from the urine of 124 patients admitted for resection of recurrent bladder carcinoma (Table 1). The patients were examined by cystoscopy. Visible tumors were resected transurethrally, and suspicious areas in the bladder were biopsied. Thirty cases were included in the study as the control group. Of the 124 patients, malignant cells were observed in 13 cases for GSTP, 16 cases for GSTA, 38 cases for GSTT1, 30 cases for GSTM4, and 40 cases for p53. GSTP was detected in 8 (62%) malignant samples, whereas GSTA, GSTM4, and GSTT1 were present in 13 (50%), 12 (40%), and 14 (37%) malignant samples, respectively (Table 1). In bladder tumors, 8/13 (62%) displayed GSTP immunoreactivity. Five out of 13 cases (38%) did not show any GSTP expression. Table 1 summarizes the distribution of GSTA, GSTP, GSTM4, and GSTT1 immunostaining in bladder tumors. We found the relationships among GSTA, GSTP, and GSTT1 expressions between benign and malignant groups (Table 1) (P ⬍ 0.05). There was, however, no relationship for GSTM4 expression (Table 1) (P ⬎ 0.05). GSTA, GSTM4, GSTP, and GSTT1 expressions were stronger in malignant cells than those in benign cells in 14, Table 2 Mean and standard deviation values of control group according to GST isoenzymes
Mean Standard deviation
GSTP
GSTM4
GSTA
GSTT1
p53
1.3 0.458258
1 0
0.9 0.031623
1.2 0.415799
1 0
6, 5, and 16 bladder cancer cases (Table 1, Fig. 1). Fig. 2A showed that positive stronger cytoplasmic staining was observed in benign rather than in malignant cells in 5, 6, 3, and 8 cases for GSTA, GSTM4, GSTP, and GSTT1, respectively. Moreover, GSTA, GSTM4, GSTP, and GSTT1 had negative staining in benign cells, but positive staining in papillary malignant cells for the same patients in 10, 4, 3, and 14 cases, respectively (Table 1, Fig. 2B). There was negative staining for GSTA, GSTM4, GSTP, GSTT1, and p53 in all 30 control samples. Of the 40 patients, 33 (12%) possessed tumor p53 protein; 27 of 33 bladder cancer patients with p53 positive had G1 tumors while five of these patients had G3 tumors. Overexpression of tumor p53 protein was found in 33 of 40 patients (82%) examined. Overexpression of tumor p53 protein was found in 82% (27/33) of G1 tumors, 3% (1/33) of G2 tumors, and 15% (5/33) of G3 tumors. No p53 immunoreactivity was found in normal (control) samples. P53 displayed nuclear expression in 82% tumor cells (Table 1, Fig. 3). Histologic grade of the tumors was determined on hematoxylin-eosin (HE) stained slides; 22 cases were grade 1 bladder carcinomas while four cases were grade 2, and 19 cases were grade 3. There was no difference in GST isoenzymes and p53 expression in control cells (Table 2) (P ⬎ 0.05). Similarly, there was no statistically significant difference in GSTA, GSTT1, GSTP, and p53 expressions for benign (P ⫽ 0.201) and malignant cells according to one way ANOVA test (P ⫽ 0.076) (P ⬎ 0.05) (Tables 3 and 4).
Table 3 One way ANOVA test results for the comparison of GST isoenzymes and p53 in malignant cell groups Malignant cell
Sum of squares
df
Mean square
F
Sig.
Between groups Within groups Total
4.392 64.811 69.203
4 128 132
1.098 0.506
2.169
0.76
df ⫽ degrees of freedom; F ⫽ F-test.
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Table 4 One way ANOVA test results for the comparison of GST isoenzymes and p53 in benign cell groups Benign cell
Sum of squares
df
Mean square
F
Sig.
Between groups Within groups Total
2.530 52.617 55.147
3 98 101
0.843 0.537
1.571
0.201
df ⫽ degrees of freedom; F ⫽ F-test.
There was a significant difference in GSTA (P ⫽ 0.006), GSTT1 (P ⫽ 0.004), GSTP (P ⫽ 0.000), and p53 (P ⫽ 0.000) expressions for benign cells (P ⬍ 0.05). However, there was no significant difference in GSTM4 expression (P ⫽ 0.203) for benign cells (P ⬎ 0.05) (Tables 5 and 6).
Table 6 Multiple comparisons between GST enzymes and P53 of benign cells and control groups Mean difference
Std. Error
Sig.
95% confidence interval Lower bound
Dunnett t (2-sided)† GSTA control GSTM4 control GSTP control p53 control
Upper bound
0.507*
0.158
0.006
0.11
0.90
0.277
0.147
0.203
⫺0.09
0.64
0.451*
0.135
0.004
0.11
0.79
0.815*
0.201
0.000
0.31
1.32
* The mean difference is significant at the 0.05 level. † Dunnett t-tests treat one group as a control, and compare all other groups against it.
4. Discussion The incidence of bladder cancer is either increasing moderately or remaining stable in most developed countries, despite improvements in diagnostic and therapeutic methods and greater awareness of important risk factors, such as cigarette smoking and occupational chemical exposure [23]. The balance between the activation and detoxification of carcinogens affects the amount of deoxyribonucleic acid (DNA) damage that accumulates in cells [24]. The entire process leading to DNA damage and the subsequent repair of the damage involve a host of enzymes. DNA damage is a consequence of the balance between the activation and detoxification of carcinogens that involves phase I and II metabolic enzymes, many of which are polymorphic [25]. The genetic polymorphisms in a number of metabolic enzymes and other genes have been found as the modulators of bladder cancer risk. The polymorphisms in genes for some important enzymes, especially GSTs, have been extensively studied in connection with bladder cancer risk. An improved understanding of the biology of urothelial malignancies is helping to define more clearly the role of new prognostic indices and multidisciplinary treatment for this disease. Uroepithelium is capable of metabolizing some precarcinogens in their effective genotoxic metabolites and is not regarded as an inactive target of preformed reactive metabolites of the carcinogens in the urine [26]. Several studies
have reported on overall GST activity, including isoenzymes belonging to the classes ␣, , and , toward 1-chloro-2, 4-dinitrobenzene (CDNB) [27,12], and GST protein expression in the uroepithelium and tumors of patients with TCC [13]. It is generally accepted that overall GST activity is up-regulated in TCC, with GSTP accounting for most of this increase [13,28]. As information on GSTT1 activity is also lacking, it would be of great importance to determine whether GSTT1 is expressed in the uroepithelium and tumors of the patients’ TCC. To the best of our knowledge, the present study represents the first attempt for a comprehensive description of the 4 classes of GSTs isoenzymes in urine cytology smears. The slides from 124 bladder carcinoma patients prepared by the cytocentrifugation method were observed. Of the 124 patients, malignant cells were observed in 13 cases for GSTP, 16 cases for GSTA, 38 cases for GSTT1, 30 cases for GSTM4, and 40 cases for p53. According to this, GSTP was detected in 8 (62%) malignant samples, whereas GSTA, GSTM4, and GSTT1 were present in 13 (50%), 12 (40%), and 14 (37%) malignant samples, respectively. In this study, by comparing the benign and malignant groups, no relation between GSTA, GSTP, GSTM4, and GSTT1 expressions could be found. We observed a statistical difference in staining intensity for GSTP, GSTA, GSTT1, and p53 between benign epithelial cells and normal cells. However, there was no difference in staining intensity for GSTM4 between benign and normal epithelium cells
Table 5 Multiple Comparisons of enzymes expressions between benign cells and control groups
Assume equal variances Does not assume equal variances df ⫽ degrees of freedom; t ⫽ t-test.
Contrast
Value of contrast
Std. error
t
df
Sig. (2-tailed)
1 1
⫺2.05 ⫺2.05
0.448 0.384
⫺4.569 ⫺5.340
146 46.231
0.000 0.000
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(Table 1, P ⬍ 0.05). Cancer cells reveal multiple genetic alterations, resulting in morphologic and functional differences from the normal cells. Tumor cells may change some of their functions (e.g., expression of some proteins) in the malignant transformation process. It can be speculated that higher level of GST expression in the benign cells can be the result of this transformation. In a more extended study, Simic et al. [14] reported significantly higher GST enzyme activity levels, GSTP1 and GSTT1 levels in TCC compared with control tissues. Berendsen et al. [13] found that GSTP1 and GSTM1 significantly increased in TCC relative to adjacent mucosa. Giralt et al. [12] have reported increased levels of GST enzyme activity in samples of TCC in comparison with levels in normal mucosa of the same patients (n ⫽ 34). Several investigators measured high levels of GST enzyme activity in bladder cancer cell lines, and GSTP was the most predominant isoenzyme in these cell lines. GSTM and GSTA were either absent or present at very low levels [29,30]. Being the most commonly mutated gene in human cancer, including bladder carcinoma, p53 has attracted a great deal of interest as a prognostic factor, diagnostic tool, and therapeutic target [31]. Recent studies using immunohistochemical staining following antigen retrieval investigated the expression of p53 in bladder carcinomas, and found that from 29% to 78% of the tumors demonstrated overexpression of tumor p53 protein [32,33]. In the present study, overexpression of tumor p53 protein was found in 33 of 40 patients (82%) examined. The present study demonstrates the wide variability in GST enzyme expression in bladder cancer region. Incorporating such an approach in larger trials may help in elucidating the roles of these enzymes in carcinogenesis as well as in identifying potential targets for chemoprevention. We observed that there was a nonsignificant relationship between the expressions of GSTs and p53, and the tumor grade. The number of bladder cancer patients in this analysis can be considered small. Further studies with substantially larger numbers of bladder cancer patients are needed to examine prospectively for the possible relationship between GST expression and prognostic factors. Expression of xenobiotic metabolizing enzymes within tumors has been identified as a potentially important factor in determining anti-tumor drug resistance. Both the amount and the proportion of different enzymes present in tumors play a role in determining anticancer drug resistance. Immunocytochemistry is a useful method for the investigation of expression and cellular localization of GSTs within tumors, and may be useful in identifying those tumors that are potentially resistant to specific anticancer drugs. More data are needed to improve our understanding of the role of these enzymes in neoplasia and in resistance to cytotoxic drug therapy.
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Acknowledgements This work was supported by the Research Fund of the Kirikkale University, project number: 02/03.04.04. References [1] Planz B, Striepecke E, Jakse G, et al. Use of Lewis X antigen and deoxyribonucleic acid image cytometry to increase sensitivity of urinary cytology in transitional cell carcinoma of the bladder. J Urol 1998;159:384 – 8. [2] Lotan Y, Roehrborn CG. Sensitivity and specificity of commonly available bladder tumor markers versus cytology: Results of a comprehensive literature review and meta-analyses. Urology 2003;61: 109 –18. [3] Eagan JW Jr. Urinary tract cytology. In: Hill GS, editor. Uropathology. Edinburgh: Churchill Livingstone, 1989. p. 873–934. [4] Li D, Wang M, Dhingra K, et al. Aromatic DNA adducts in adjacent tissues of breast cancer patients: Clues to breast cancer etiology. Cancer Res 1996;56:287–93. [5] Hayes JD, Flaagan JU, Jowsey IR. Glutathione-S-transferases. Annu Rev Pharmacol Toxicol 2005;45:51– 88. [6] Coles BF, Kadlubar FF. Detoxification of electrophilic compounds by glutathione S-transferase catalysis: Determinants of individual response to chemical carcinogens and chemotherapeutic drugs? Biofactors 2003;17:115–30. [7] Feng Z, Hu W, Rom WN, et al. Aminobiphenyl is a major etiological agent of human bladder cancer: Evidence from its DNA binding spectrum in human p53 gene. Carcinogenesis 2002;23:1721–7. [8] Aktas D, Ozen H, Atsu N, et al. Glutathione S-transferase M1 gene polymorphism in bladder cancer patients: A marker for invasive bladder cancer? Cancer Letters 2001;125:1– 4. [9] Hung RJ, Boffetta P, Brennan P, et al. GST, NAT, SULT1A1, CYP1B1 genetic polymorphisms interactions with environmental exposures and bladder cancer risk in a high-risk population. Int J Cancer 2004;110:598 – 604. [10] Hayes JD, Strange RC. Glutathione-S-transferase polymorphisms and their biological consequences. Pharmacology 2000;61:154 – 66. [11] Tsuchida S, Sato K. Glutathione transferases and cancer. Crit Rev Biochem Mol Biol 1992;27:337– 84. [12] Giralt M, Lafuente A, Pujol F, et al. Enhanced glutathione S-transferase activity and glutathione content in human bladder cancer. Follow-up study: Influence of smoking. J Urol 1993;149:1452– 4. [13] Berendsen CL, Peters WHM, Scheffer PG, et al. Glutathione-Stransferase activity and subunit composition in transitional cell cancer and mucosa of the human bladder. Urology 1997;49:644 –51. [14] Simic T, Mimic-Oka J, Savic-Radojevic A, et al. Glutathione-Stransferase T1-1 activity up-regulated in transitional cell carcinoma of urinary bladder. Urology 2005;65:1035– 40. [15] Savic-Radojevic A, Mimic-Oka J, Pljesa- Ercegovac M, et al. Glutathione S-transferase-P1 expression correlates with increased antioxidant capacity in transitional cell carcinoma of the urinary bladder. Eur Urol 2007;52:470 –7. [16] Hainaut P, Hollstein M. p53 and human cancer: The first ten thousand mutations. Adv Cancer Res 2000;77:81–137. [17] Hainaut P, Hernandez T, Robinson A, et al. IARC database of p53 gene mutations in human tumors and cell lines: Updated compilation, revised formats, and new visualization tools. Nucleic Acids Res 1998;26:205–13. [18] Brockmöller J, Kaiser R, Kerb R, et al. Polymorphic enzymes of xenobiotic metabolism as modulators of acquired P53 mutations in bladder cancer. Pharmacogenetics 1996;6:535– 45. [19] Spruck CH III, Rideout WH III, Olumi AF, et al. Distinct pattern of p53 mutations in bladder cancer. Cancer research 1993;53: 1162– 6.
544
S. Og˘uztüzün et al. / Urologic Oncology: Seminars and Original Investigations 29 (2011) 538 –544
[20] Fujimoto K, Yamaha Y, Okajs¸ma E, et al. Frequent association of p53 gene mutations in bladder cancers and urine samples. Science 1991; 252:706 –9. [21] Frost J. The cell in health and disease. An evaluation of cellular morphologic expression of biologic behavior. In: Wied G, von Hamm E, Koss L, et al, editors. Monographs in Clinical Cytology, 2nd ed. Basel: Karger, 1986. [22] Kern W. Urinary tract. In: Keebler C, Reagan J, editors. A Manual of Cytotechnology, 6th ed. Chicago: ASCP Press, 1983. [23] Stewart BW, Kleihus P. World cancer report. Lyon, France: IARC Press 2003. [24] Stern MC, Johnson LR, Bell DA, et al. XPD colon 751 polymorphism, metabolism genes, smoking, and bladder cancer risk. Cancer Epidemiol Biomarkers Prev 2002;11:1004 –11. [25] Sanyal S, Festa F, Sakano S, et al. Polymorphisms in DNA repair and metabolic genes in bladder cancer. Carcinogenesis 2004;25: 729 –34. [26] Their R, Golka K, Brüning T, et al. Genetic susceptibility to environmental toxicants: The interface between human and experimental studies in the development of new toxicological concepts. Toxicol Lett 2002;127:321–7.
[27] Lafuente A, Giarlt M, Cervello I, et al. Glutathione S-transferase activity in human superficial transitional cell carcinoma of the bladder. Comparison with healthy controls. Cancer 1990;665: 2064 – 8. [28] Singh SV, Xu BH, Tkalevic GT, et al. Glutathione-linked detoxification pathway in normal and malignant human bladder tissue. Cancer Lett 1994;77:15–24. [29] Singh SV, Ahmad H, Krishan A. Expression of glutathione related enzymes in human bladder cancer cell lines. Biochem Pharmacol 1990;39:1817–20. [30] Ahn H, Lee E, Kim K, et al. Effect of glutathione and its related enzymes on chemosensitivity of renal cell carcinoma and bladder carcinoma cell lines. J Urol 1994;151:263–7. [31] Assimakopoulos D, Kolettas E, Zagorianakou N, et al. Prognostic significance of p53 in the cancer of the larynx. Anticancer Res 2000;20:3555– 64. [32] Lipponen PK. Overexpression of p53 nuclear oncoprotein in transitional cell bladder cancer and its prognostic value. Int J Cancer 1993;53:365–70. [33] Esrig D, Elmajian D, Groshen S, et al. Accumulation of nuclear p53 and tumor progression in bladder cancer. N Engl J Med 1994;331: 1259 – 64.