1 Dipartimento di Medicina Sperimentale e Patologia, Università degli Studi di Roma La Sapienza, Rome; 2 Dipartimento di Patologia e Medicina di Laboratorio, Università degli Studi di Parma, Parma, Italy
Received 28 March 2002; revised 27 May 2002. accepted 8 July 2002
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Abstract |
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It has been suggested that progression of superficial bladder cancer may be regulated at the molecular level by a typical pattern of expression of genes involved in apoptosis. Recently LIVIN, belonging to the inhibitors of apoptosis (IAP) family, has been found to be expressed in most solid tumors, where its expression is suggested to have prognostic significance. No data are available concerning the significance of LIVIN in the progression of bladder tumors.
Patients and methods:
In the present paper we used RTPCR to investigate the expression of LIVIN isoforms and ß, SURVIVIN, BCL-X and BCL-2/BAX expression ratio both in normal and tumoral bladder tissues, and correlated their expression with the emergence of early relapses in a follow-up of 4 years. This study shows that only the
isoform of LIVIN, which is not expressed in normal bladder tissue, is expressed in a proportion of tumors with a high risk of relapse.
Results:
LIVIN was found in 7/30 patients (23%), SURVIVIN in 9/30 (30%), BCL-2/BAX ratio >1 in 16/30 (53%), BCL-2/BAX expression ratio <1 in 14/30 (46.6%) and BCL-X, only in isoform BCL-XL, in 11/30 (36.6%). When we evaluated the dependence between each gene expression and relapse free time of patients, we found that LIVIN, high BCL-2/BAX ratio and BCL-XL, but not SURVIVIN, reached statistical significance in order to predict relapses.
Conclusions:
Our findings suggest that LIVIN may be involved in the progression of superficial bladder cancer and used as a marker of early recurrence; while the expression of SURVIVIN cannot be used to identify patients with high risk of relapse.
Key words: BCL-2/BAX, BCL-X, bladder cancer, LIVIN, relapse, SURVIVIN
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Introduction |
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Detection of LIVIN ß in fetal tissues, and in placenta in particular, seems to indicate that it may play a role during fetal development. In adult tissues, high expression of both LIVIN and ß were detected in heart, placenta, spleen, ovary and lung, while low levels of just LIVIN
were found in lymphocytes, brain and skeletal muscle. Furthermore, high levels of both LIVIN
and ß were detected in melanoma, colon cancer and prostate cancer cell lines. In addition, high levels of just isoform
were detected in some melanoma and lymphoma cell lines. Although extensive studies have been performed concerning the expression of LIVIN in most human tissues, no data were available in the literature concerning the expression of LIVIN isoforms in bladder tissues and in bladder cancer.
Within the IAP family SURVIVIN has also been recently described. Despite its limited expression in normal tissues, SURVIVIN seems overexpressed in a variety of human tumors, including breast, colon, pancreas and prostate carcinoma, neuroblastoma, melanoma and non- Hodgkins lymphoma [7]. Studies performed by immunohistochemistry described presence of SURVIVIN in a variable percentage of tumors, ranging from 30% of gastric cancers to 90% of melanomas [8, 9]. Most of these studies found a positive correlation between SURVIVIN expression and prognosis of disease, which is more evident in neuroblastoma and in colorectal cancer, where a multivariate statistical analysis revealed that SURVIVIN expression is an independent prognostic factor for disease progression [10].
Expression of SURVIVIN has been evaluated in bladder cancer by immunohistochemistry and found to be associated with short disease-free interval [11]. Furthermore, SURVIVIN has been detected in the urine of patients with bladder cancer, leading to the hypothesis that it could be used as a marker of early diagnosis [12].
The first goal of this study was to evaluate the expression of LIVIN isoforms in 24 normal bladder tissues as well as in 30 primary superficial bladder cancer specimens. The same tissue samples were also analyzed for the presence of SURVIVIN, BCL-2, BAX and BCL-X mRNA. Furthermore, we evaluated dependence between expression of all these genes and relapse-free time of patients in 4 years of follow-up.
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Patients and methods |
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The post-surgical pathological stage of each tumor was classified according to the revised tumornodemetastasis (TNM) staging system. Tumor extension limited to the mucosa (pTa), or the lamina propria (pT1) of the bladder wall was defined as superficial.
Tumor specimens were immediately frozen in liquid nitrogen after surgery and stored at 80°C until use. Informed consent was obtained from all patients.
All patients were subjected to periodical follow-up studies. Patients at high risk for relapse, with multiple lesions or high tumor grade, were treated by TUR-B followed by mitomycin C or BCG. The schedule of the treatment post-TUR was one instillation/week for 8 weeks followed by one instillation/month.
These patients were followed every 3 months with urinary cytology and pelvic ultrasonography for a period of 1 year. If no recurrence was observed during this period, patients were then followed every 4 months for the second year, and every 6 months thereafter.
The mean follow-up period for the study was 39 months (2751 months). Clinical features of patients are summarized in Table 1.
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RTPCR
Total RNA (1 µg) extracted from frozen tissues was reverse transcribed in a final volume of 20 µl with 100 pmol of random examer and 50 U MuLV reverse transcriptase (Perkin Elmer Cetus, Norwalk, CT, USA), according to the manufacturers guidelines.
Aliquots corresponding to 100 ng RNA were then amplified in PCR buffer containing 25 pmol each primer and 1 U Taq polymerase in a final volume of 50 µl.
Aliquots of the same cDNA were amplified with ß-actin, LIVIN, SURVIVIN, BCL-2, BAX and BCL-X primers. Each amplification, except for LIVIN, was performed for 30 cycles; a cycle profile consisted of denaturation at 94°C for 30 s, annealing at 60°C (62°C for BAX and BCL-X) for 30 s and extension at 72°C for 30 s. A sample without RNA was included in each RTPCR as a negative control; for positive controls, RNA extracted from M14 cell line for SURVIVIN, HeLa cell line for BCL-2 and CaSki cell line for BAX and BCL-X were used. LIVIN primer sequences and amplification conditions were as described [6].
Sequences of the other primers used are as follows: ß-actin upstream, 5'-TTAGCTGTGCTCGCGCTACTCTCTC-3'; ß-actin downstream, 5'-GTCGGATTGATGAAACCCAGACACA-3'; SURVIVIN upstream, 5'-CAGATTTGAATCGCGGGACCC-3'; SURVIVIN downstream, 5'- CCAAGTCTGGCTCGTTCTCAG-3'; BCL-2 upstream, 5'-GTGGAGGAGCTCTTCAGGGA-3'; BCL-2 downstream, 5'-AGGCACCCAGGGTGATGCAA-3'; BAX upstream, 5'-GGCCCACCAGCTCTGAGCAGA-3'; BAX downstream, 5'-GCCACGTGGGCGGTCCCAAAGT-3'; BCL-X upstream, 5'-TTGGACAATGGACTGGTTGA-3'; BCL-X downstream, 5'-GTAGAGTGGATGGTCAGTG-3'. The primers for BCL-X were designed to recognize both isoforms, BCL-XL and BCL-XS.
The size of the amplified products were 168, 206, 304, 479, 780 and 591 bp for ß-actin, SURVIVIN, BCL-2, BAX, BCL-XL and BCL-XS, respectively. The size of LIVIN isoforms and ß were 368 and 314 bp, respectively.
We then performed a semi-quantitative analysis of BCL-2/BAX ratio in each sample, as described [5]. In order to verify the specificity of the amplified products, we performed a hybridization with specific oligonucleotide probes, as previously described [13].
Statistical analysis
Statistical analysis was performed using BMDP statistical software, version 7 [14].
Time to first recurrence was calculated from the date of initial surgery and used as the endpoint of the study. Relapse-free time was estimated using the product-limit method of KaplanMeier; the difference between the curves was evaluated using the log-rank test (MantelCox method). Chi-square test was used in order to assess the association between expression of LIVIN, SURVIVIN, BCL-X and BCL-2/BAX ratio and clinical prognostic variables, such as stage, grade, age and multicentricity of tumors. A value of P <0.05 was considered statistically significant.
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Results |
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In tumor samples the presence of SURVIVIN expression was found in 9/30 (30%) patients, BCL-2/BAX ratio >1 in 16/30 (53%) and BCL-X expression in 11/30 (36%) (Table 1). BCL-X gene was found to be expressed only in the BCL-XL isoform (Figure 2).
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Discussion |
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Tissue distribution of LIVIN has been recently described: elevated levels of both LIVIN isoforms and ß have been detected in heart, placenta, lung, spleen and ovary, while LIVIN ß alone has been detected specifically in fetal tissues. Adult kidney seems to express only isoform ß. LIVIN
alone has been detected in brain, skeletal muscle and peripheral blood lymphocytes.
Since no data were available concerning LIVIN expression in bladder, our study demonstrates for the first time that expression of both LIVIN isoforms and ß are not detectable in normal bladder tissues, as previously described for BCL-2, a gene with antiapoptotic properties.
Furthermore, while LIVIN expression was detected in a variety of cancerous cell lines, no data were available concerning the expression of LIVIN isoforms in tumor tissues. Our study for the first time demonstrates that LIVIN isoform is expressed in a proportion of superficial bladder tumors, all of which had a clear tendency to early relapse, showing an unexpected short relapse-free time. Data from the literature have demonstrated that, whether both LIVIN isoforms are involved in blocking apoptosis induced by anti-CD95 and TNF-
, isoform
is less efficient than isoform ß in blocking apoptosis induced by DNA damaging agents. Nevertheless, it is conceivable that in bladder tumors the expression of LIVIN isoform
, together with an overexpression of BCL-2 upon BAX, may be sufficient to give cells a strong resistance to chemotherapy-induced apoptosis.
In the same group of tumor samples we investigated the presence of SURVIVIN, another member of the IAP family, which other authors described as being absent in normal bladder tissue and expressed in bladder tumors, and other genes known to be involved in the pathogenesis of bladder cancer, specifically BCL-2, BAX and BCL-X. LIVIN expression does not seem to be correlated with known prognostic variables, such as grade, stage and multicentricity; furthermore, no significant association was found between LIVIN and SURVIVIN expression, or between LIVIN and BCL-2 or BCL-X expression. These results may reflect the different transcriptional pathways of these genes.
Follow-up data obtained in the first 4 years after surgery revealed that patients whose tumors showed expression of LIVIN isoform had a very short relapse-free time (3.5 months), similar to that observed in patients whose tumors expressed a high BCL-2/BAX ratio. In all patients who relapsed during the course of treatment we found a high BCL-2/BAX expression ratio. This further stresses the role of high BCL-2 expression as a mechanism by which tumor cells escape chemotherapy-induced apoptosis.
On the contrary, SURVIVIN expression in our samples does not correlate with the emergence of early relapse, although its expression seems to be associated with histological grade of tumors.
The only data available concerning SURVIVIN in bladder tumors seem to indicate that SURVIVIN, but not BCL-2, detected by immunohistochemical methods, is significantly associated with time of recurrence and histological grade [11]. We cannot really explain the discrepancy between the data of Swana et al. [11] and ours, mainly due to the lack of sufficient clinical data in that series of patients (size of tumors, modalities of treatment after surgery). Moreover, it is known that BCL-2 alone in bladder cancer, as well as in other solid malignancies, is not sufficient to predict disease recurrence. Thus, the lack of association between BCL-2 expression and poor prognosis in that series of patients may be due to altered expression levels of other family members, like BAX, whose presence was not investigated by Swana et al. [11].
The emergence of early relapses in patients with high levels of BCL-2 expression may also depend on the known ability of BCL-2 to inhibit chemotherapy-induced apoptosis, as previously described in bladder, lung and breast cancer [1517]. It is thus conceivable that SURVIVIN does not share with BCL-2 the ability to block mitomycin-C-induced apoptosis. In fact, few data are available concerning the efficacy of SURVIVIN in blocking chemotherapy-induced apoptosis. To date, SURVIVIN has been shown to inhibit apoptosis of NIH3T3 transfectants induced by paclitaxel, and apoptosis of 293 cell line induced by etoposide, but it seems ineffective against microtubule depolymerizing agents such as vincristine [18]. Due to the small number of patients whose results were positive for LIVIN, it is still difficult to establish whether LIVIN expression may interfere with chemotherapy-induced apoptosis.
Unlike other solid malignancies, such as breast, gastric and colorectal cancer, the presence of SURVIVIN in our series of bladder tumor specimens does not seem to be associated with high levels of BCL-2. This seems unsurprising, due to the fact that SURVIVIN and BCL-2 do not completely share common mechanisms of transcriptional activation [6]. In addition, similar to what has been observed in other tissues, no correlation was found between LIVIN isoform and SURVIVIN expression in bladder cancer.
The emergence of local relapses in superficial bladder cancer represents one of the major problems in the clinical management of this tumor. In fact, it is widely acknowledged that stage and grade are often unable to predict the local progression of TaT1 low grade bladder tumors [1], and that 50% of patients affected by superficial disease have local recurrence in the first year of follow-up. This is the reason why in 1996 the European Organization for Research and Treatment of Cancer and Medical Research Council defined the utility of prophylactic treatment also in stage TaT1G1 bladder cancer patients, since it gives a clear advantage in terms of duration of disease-free interval [19]. In view of this, the search for a panel of molecular markers could be useful in the identification of patients with a higher risk of relapse. In our series of patients, the combination of a high BCL-2/BAX ratio, high BCL-X expression and LIVIN isoform seems to identify a subset of patients with shorter relapse-free time.
Our findings, which suggest for the first time a role of LIVIN isoform in the progression of superficial bladder cancer, also indicate that the expression of SURVIVIN cannot be used to identify patients with a higher risk of early relapse.
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Acknowledgements |
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Footnotes |
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References |
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