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Keywords:

  • renal cell carcinoma;
  • clusterin;
  • Ki-67;
  • TUNEL;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

OBJECTIVES

To evaluate the significance of clusterin expression in surgically resected renal cell carcinoma (RCC) specimens.

PATIENTS AND METHODS

Normal kidney and RCC specimens were obtained from 131 patients who had radical surgery. The expression of clusterin protein was analysed by immunohistochemical staining with an antibody recognizing all isoforms of clusterin. Cell proliferative activities and apoptotic features in these specimens were investigated using Ki-67 immunostaining and the terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling assay, respectively. Findings were evaluated in relation to several clinicopathological factors.

RESULTS

There were various levels of clusterin expression in 128 of the 131 RCC specimens, while 37 of 131 normal kidney tissues (28.2%) had no clusterin staining. Clusterin protein was present in the cytoplasm of both normal and cancer cells, but there was no nuclear staining identified in either type of cell. The expression level of clusterin protein in RCC tissues was significantly related to tumour stage and grade, but not to age, gender or histological cell type. Cell proliferative activity in RCC specimens was significantly associated with clusterin expression, while the apoptotic index was inversely related to clusterin expression. Furthermore, recurrence-free survival in patients with strong clusterin expression was significantly lower than that in those with weak expression.

CONCLUSIONS

These findings suggest that the secreted form of clusterin may be involved in the progression of RCC, and that overexpression of clusterin could be a useful prognostic variable after radical surgery in patients with RCC.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

RCC is the most common malignancy of the adult kidney and annual estimates of newly diagnosed cases have been steadily increasing. Because RCC has a highly resistant phenotype to conventional therapeutic methods, including chemotherapy and radiation, surgical resection of localized disease has been regarded as the only curative treatment; accordingly, the prognosis of patients with advanced RCC generally remains poor [1].

Furthermore, the mechanism underlying the resistance of RCC to several treatments has not yet been well clarified, as RCC has different features from other malignancies in its apoptotic activity, i.e. there is no significant impact of the expression of apoptosis-associated molecules, such as p53, Rb, Bcl-2, Bax and c-Myc, on the prognosis of patients with RCC [2–5].

Clusterin, also known as testosterone-repressed prostate message-2, sulphated glycoprotein-2 or apolipoprotein J, is a heterodimeric highly conserved disulphide-linked glycoprotein that is expressed in a wide variety of tissues and secreted in all human fluids [6]. It is implicated in various pathophysiological processes, including lipid transport, sperm maturation, complement inhibition, tissue remodelling, cell differentiation and transformation, and apoptotic cell death [7]. Recently it was shown that there are two different mRNA transcripts for clusterin, derived from an alternative splicing, one coding for the 76–80 kDa secreted form of clusterin and the other for the 50–55 kDa nuclear form with no leader peptide [8]. Furthermore, several investigators reported that the pattern shift of clusterin isoforms results in the promotion of different functions, particularly in the regulation of apoptosis [8–14]. For example, over-expression of the secreted form of clusterin in human cancer cells gave protection against cytotoxic stimuli that induce apoptosis [9,10].

Similarly, the secreted form of clusterin acts as a molecular chaperone, like small heat-shock proteins, by scavenging denatured proteins outside cells after stress-induced injury [14]. On the contrary, the nuclear form of clusterin exists in the cytoplasm, translocates into the nucleus after exposure to apoptotic stimuli, then initiates cell cycle arrest and cell death [8,11,12].

There is increased expression of clusterin in various renal diseases, e.g. renal dysplasia, membranous glomerulonephritis, inherited polycystic renal disease and RCC [15–17]. We also previously reported that over-expression of clusterin could be a useful prognostic predictor in patients with RCC who had radical surgery [18], and that inhibiting clusterin expression, using antisense oligodeoxynucleotide, enhanced chemosensitivity, resulting in a significant delay of tumour growth in a human RCC tumour model [19]. However, these studies focused on the secreted form alone; therefore, the functional significance of clusterin expression in RCC remains unclear. Considering these findings, we re-evaluated the expression of clusterin in human RCC specimens using immunohistochemical staining with an antibody recognizing all the clusterin isoforms, and further analysed these results according to the apoptotic features of RCC.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

In all, 131 patients who had radical or partial nephrectomy for RCC at our institution between April 1991 and March 2001 were included in this study. Informed consent was obtained from all these patients, and the present study was approved by the Research Ethics Committee of the Hyogo Medical Center for Adults. Samples were examined pathologically by one pathologist and evaluated according to the 1997 TNM system.

The mean follow-up of the patients was 65 (13–156) months; generally, all patients were followed by laboratory and radiological evaluations, and a physical examination, every 6 months to monitor recurrence and metastasis. In the absence of a relapse of RCC 5 years after surgery, the interval between re-examinations was increased.

RCC specimens were stained by immunohistochemistry as previously described [19]. Briefly, sections from formaldehyde-fixed, paraffin-embedded tissue from 131 specimens were deparaffinized by xylene and rehydrated in decreasing concentrations of ethanol. After blocking endogenous peroxidase with 3% hydrogen peroxidase in methanol, sections were boiled on 0.01 m citrate buffer for 10 min and incubated with 5% normal blocking serum in Tris-buffered saline for 20 min. The sections were then incubated with the following antibodies for 2 h at room temperature: antihuman Ki-67 mouse monoclonal antibody (Dako, Carpinteria, CA, USA) and antihuman clusterin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were next incubated with biotinylated goat antimouse or rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for 30 min. After incubation in an avidin-biotin peroxidase complex for 30 min, the samples were exposed to diaminobenzidine tetrahydrochloride solution and counterstained with haematoxylin. Staining results of both normal kidney and RCC tissues were interpreted by two independent observers (M.M. and K.Y.) who were unaware of the clinicopathological data of the patients. Staining intensity was scored from 1+ (no staining), 2+ (weak), 3+ (medium) to 4+ (strong), and staining extent was also scored from 1+ (0–25%), 2+ (26–50%), 3+ (51–75%) to 4+ (76–100%). The product of staining intensity and staining extent was defined as the staining score.

A modified terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling technique was used to detect apoptotic cells in RCC specimens with the ApopTagTM in situ apoptosis detection system (Oncor, Gaithersburg, MD, USA) as previously described [19]. Briefly, after deparaffinization and blocking of endogenous peroxidase as described above, sections were treated with 10 µg/mL proteinase K, labelled with biotinylated poly-dU, introduced by terminal deoxy-transferase, and then stained using antidigoxigenin-peroxidase. Peroxidase activity on sections was detected by immersion in freshly mixed diaminobenzidine tetrachloride and the samples were counterstained with haematoxylin. The number of positively stained cells/high-power field, which is defined as the apoptotic index, was counted in five random fields and averaged.

Several clinicopathological factors of the patients were analysed using the chi-square test, unpaired t-test or Mann–Whitney U-test. The biochemical recurrence-free survival rates of patients were determined by the Kaplan–Meier method, and the differences assessed using the log-rank test, with P < 0.05 considered to indicate significance in all tests.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Normal kidney and RCC tissues obtained from the 131 patients who had radical surgery were immunostained for clusterin and the outcomes are summarized in Table 1. There was positive clusterin staining in 128 of 131 RCCs (97.7%), while 37 of 131 normal kidney tissues (28.2%) had no clusterin staining. Both staining intensity and extent of clusterin protein in RCC tissues were significantly greater than those in normal kidney tissues. In addition, positive clusterin staining was detected in the cytoplasm of both normal and cancer cells, while there was no nuclear staining of clusterin in both types of cells.

Table 1.  Results of clusterin immunostaining in normal kidney and RCC tissues from 131 patients
 Normal kidney, n (%)RCC, n (%)
Staining intensity
1+ 37 (28) 3 (2)
2+ 87 (66)30 (23)
3+  7 (5)51 (39)
4+  047 (36)
Staining extent
1+ 67 (51)27 (21)
2+ 37 (28)39 (30)
3+ 25 (19)40 (31)
4+  2 (2)25 (19)
Staining score
≤8126 (96)70 (53)
≥9  5 (4)61 (47)

The association between the staining of clusterin protein in RCC tissues and several clinicopathological factors showed (Table 2) that the expression level of clusterin protein was significantly related to tumour stage and grade, but no significant association between clusterin expression level and other variables, including age, gender and histological cell type. Furthermore, cell proliferative activity determined by immunostaining of RCC tissues with an antibody against Ki-67 was significantly associated with clusterin expression, while the apoptotic index in RCC specimens was inversely related to clusterin expression (Table 2).

Table 2.  Correlation of clusterin expression in RCC tissues with several clinicopathological factors, and with cell proliferative activity and apoptotic index
VariablesNo. of patientsStaining intensityStaining extentStaining score
1–2+3–4+P1–2+3–4+P≤ 8≥ 9P
  • *

    terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling (TUNEL)

Age, years   0.88  0.83  0.40
 ≤60 611546 3031 3526 
 ≤61 701852 3634 3535 
Gender   0.55  0.60  0.88
 Male 942569 4648 5044 
 Female 37 829 2017 2017 
Stage   0.038  0.035  0.0093
 pT1 882761 5038 5236 
 ≥ pT2 43 637 1627 1825 
Grade   0.039  0.029  0.0041
 1 672245 4027 4918 
 2 or 3 64 1153 2638 2143 
Histological cell type   0.77  0.61  0.13
 Clear cell1092881 5653 5554 
 Nonclear cell 22 517 1012 15 7 
Cell activity and apoptosis
% of positive cells with Ki-67   <0.001  0.022  <0.001
 Absent  9 7 2  7 2  7 2 
 <10% 882563 4840 5830 
 10–25% 30 129  1119  525 
 ≥25%  4 0 4  0 4  0 4 
% positive cells with TUNEL* assay   0.002  0.023  0.043
 Absent 47 839 1730 1928 
 <10% 782058 4434 4632 
 ≥10%  6 5 1  5 1  5 1 

During the period of the study, 33 of the 131 patients (25.2%) developed disease recurrence; considering a staining score of >8 to indicate strong expression, 61 patients were regarded as having RCC with strong clusterin expression and the remaining 70 as having RCC with weak expression. By this classification system, 24 of 33 patients (73%) developing disease recurrence had RCCs with strong expression of clusterin. Representative findings of immunohistochemical studies based on this system are shown in Fig. 1. Recurrence-free survival in patients with strong clusterin expression was significantly lower than that in those with weak expression (Fig. 2).

image

Figure 1. Typical outcomes of immunohistochemical staining of RCC with a clusterin antibody, with (a) weak expression and (b) strong expression of clusterin.

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image

Figure 2. Recurrence-free survival of patients with RCC according to clusterin expression status (weak, green open circles; strong, red closed circles) on Kaplan-Meier analysis.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Although it has been widely accepted that clusterin is an apoptosis-associated gene, a precise relationship between clusterin and programmed cell death has not been clarified [6,7]. Indeed, several experimental and clinical studies report conflicting findings on the role of clusterin in apoptotic cell death induced by several kinds of stimuli; however, recent studies have addressed the dichotomous functions of clusterin, suggesting that pro- and anti-apoptotic functions may be related to nuclear and secreted isoforms, respectively [8–14]. For example, Pucci et al.[13] reported a correlation between enhanced cell survival and the disappearance of the nuclear isoform of clusterin in aggressive colon cancers, with a concomitant increased expression of the secreted form.

Similarly, Scaltriti et al.[12] showed that transient transfection of prostate cancer cells with the unsecreted form of clusterin resulted in massive nuclear localization of the protein, leading to G2-M phase blockade followed by caspase-dependent apoptosis. Currently, a link between differences in clusterin function and the change in pattern of its isoform production in tumour tissue seems to reconcile most of findings in this field.

In the kidney, up-regulation of clusterin expression was shown to protect renal tissues from apoptosis induced by a wide variety of stimuli [15,16]. We also previously reported that there was overexpression of clusterin, compared with normal kidney, in most RCCs, and that the expression level of clusterin in RCC could be a useful prognostic predictor [18,19]; however, the significance of cellular location of clusterin in RCC had not been addressed, and thus we re-evaluated the expression of clusterin protein in 131 RCC specimens by immunohistochemical staining using an antibody recognizing all the isoforms of clusterin. Consistent with our previous study using Northern blotting [18], we confirmed that the expression of clusterin protein in RCC tissues was significantly greater than that in normal kidney tissue.

Interestingly, despite the expression of the secreted form of clusterin, we could not detect the nuclear form of clusterin in either normal or malignant kidney tissues. These findings suggest that the role of the nuclear form of clusterin in the tumorigenesis of RCC may be more limited than that of the secreted form of clusterin. To further characterize the significance of different isoforms of clusterin protein in RCC, it would be necessary to use a more sensitive method than immunohistochemistry, e.g. RT-PCR, to detect these isoforms.

There was a close correlation of clusterin expression in RCC tissues with tumour stage and grade. Furthermore, over-expression of clusterin protein in RCC tissues was closely associated with enhanced cell proliferative activity and suppressed induction of apoptotic cell death. This finding suggests the involvement of clusterin in the progression of RCC, although several recent studies indicated that conventional apoptosis-related molecules play limited roles in the progression of RCC [2–5]. Indeed, recurrence-free survival in patients with strong clusterin expression was significantly lower than that in those with weak expression. Hence, the expression level of clusterin protein in RCC could be an important adjunct to traditional variables for predicting the prognosis of patients with RCC.

We recently reported that the secreted form of clusterin may be an optimum therapeutic target for various kinds of malignant tumours, including prostate, bladder and lung cancer, and osteosarcoma [9,10,20,21]. Also in RCC, we previously reported that over-expression of the secreted form of clusterin causes the acquisition of resistance to Fas-mediated and chemotherapy-induced apoptosis [22,23]. In addition, considering the present findings suggesting a potential role of clusterin in the progression of RCC, a strategy targeting the clusterin gene may be an attractive approach for advanced RCC, i.e. if clusterin expression could be inhibited using a novel nucleotide-based technology, e.g. antisense oligodeoxynucleotide and small interfering RNA, the efficacy of conventional treatments may be enhanced.

In conclusion, the secreted form of clusterin may be implicated in the progression of RCC; therefore, evaluating clusterin expression in surgically resected RCC specimens by immunohistochemistry may help to identify patients who are likely to develop disease recurrence.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES