In the context of the many associations between a virus and a given malignancy, the distinction between associated versus causative agent frequently arises and may be difficult to make . The major pathogenetic role of HCV infection in hepatocellular carcinoma is well documented [18, 19]. The mechanism of its oncogenesis remains unclear; however, alterations in cell cycle, proto-oncogenes, tumour suppressor genes, apoptotic proteins, telomeres are the key events in determining the biological behavior of bladder cancer [2, 17, 30]. In the present study we examined the expression of the telomerase and tumour suppressor genes (Rb, E2F3, TP53, and CDKN1A; p21), and proto-oncogenes (FGFR3) genes in HCV- and non-HCV infected patient with bladder cancer.
In the present study we found that, the BC cases associated with HCV infection were of TCC type, higher grade, and more invasive while, non-HCV-associated cancers were of SCC type, lower grade, and less invasive tumors. Moreover, the present study showed positive correlation between TCC and HCV infection, suggesting that the HCV infection might be a risk factor for bladder cancer of TCC cell type. On the other hand, Abdulamir et al., reported that schistosomal bladder tumors (SBT) were associated significantly with SCC, high grade, and invasive tumors while non-SBT were associated with TCC, a bit lower grade, and less invasive tumors.
Yoshida and Toge hypothesized that telomerase might be activated in early stages of urological carcinogenesis. Expression of hTERT by real time PCR in the present study showed significant increase in normal urothelium of HCV infected patients as well as in malignant bladder tissues from HCV infected patients. Also, detection of hTERT by immunohistochemistry in tissue samples showed expression in 6% in normal urothelium from non- HCV infected patients and 24% in that from HCV infected patients. In malignant tissues, immunostaining was 48% in samples from non-HCV infected patients with mean labeled cells 31.65± 9.02 and 76% in samples from HCV-infected patients with mean labeled cells 44.36 ± 9.84. We found that hTERT is localized predominantly in the nucleolus and this is in agreement with the few previous reports describing hTERT protein localization [25, 33] as the nucleolus is the site of nucleoprotein complex assembly . These findings suggest that HCV infection enhances the expression of telomerase enzyme in normal and malignant tissues. Also, the activity of telomerase was evaluated by TRAP assay which is the most widely used method for monitoring telomerase activity. In this study, TRAP was positive in 4% of samples of normal urothelium from HCV-infected patients and 20% from HCV infected patients. In malignant tissues, TRAP was positive in 64% of non HCV infected patients and 82% of HCV infected patients. Detection of telomerase activity in normal urothelium of patients with BC suggests that telomerase might be activated in the early stages of BC carcinogenesis. Our findings are in agreement with Yoshida and Toge who reported the activity of telomerase by TRAP in more than 70% of bladder cancer and Abd El Gawad et al.. and Abdel-Salam et al. who reported positivity of TRAP in 73.9% of cases with bilharzial cancer and 87% for non bilharzial BC. The absence of telomerase activity in some tumors may be due to the presence of a telomerase inhibitor . Also, the presence of a positive correlation between HCV infection and TRAP and hTERT expression suggests that HCV infection might have a role in development and progression of BC especially of the TCC type.
Tumour suppressor genes are involved in the process of oncogenesis. We tested in our study retinoblastoma (Rb), and TP53 genes. It appears conceivable that TP53 may negatively regulate the expression of genes through the induction of p21/CDKN1A and the consecutive hypophosphorylation of pRb and its relatives . Retinoblastoma tumor suppressor (Rb) gene encodes a nuclear phosphoprotein (pRb) that functions as a cell cycle regulator . Unphosphorylated pRb negatively regulates E2F, a protein transcription factor, by binding with it. The transcription factors E2f1, E2f2 and E2f3 act as promoters of the G/S phase introduction, E2f4, E2f5, and E2f6 are generally regarded either as weak transcriptional activators or as transcriptional repressors . This protein species, in turn, binds to E2F family transcription factors and converts them from transcriptional activators to transcriptional repressors . When pRb is phosphorylated by the cyclin/CDK complex, the transcription factor E2F-1 is released and switches on genes (e.g. thymidine synthetase) whose products drive cells into the DNA synthesis (S) phase of the cell cycle. Normal cells express the Rb protein, while mutations or gene deletions, which often result in lack of protein expression, may be identified by the lack of Rb expression . Previous studies found that Rb gene mutations are seen in approximately 30% of BC  and reported that co-operation between (pRb) removal and over expression of E2F3 may be required for bladder carcinogenesis . In the present study, immunohistochemical detection of Rb protein in tissue samples of Rb expression showed significant increase in altered expression (either negative or increased homogenous positivity in >50% of cells) in bladder tumors associated with HCV infection. In addition, we found that Rb expression by RT-PCR was decreased in bladder tumors from HCV infected patients. As an inhibitor of cyclin-dependent kinases, p21 is known to prevent the phosphorylation of retinoblastoma (Rb)  family proteins and hence lead to the accumulation of hypophosphorylated pRb .
Also, we recorded overexpression of E2F3 in malignant BC when compared to normal urothelium, and this overexpression is enhanced by HCV infection. Moreover, Rb had a negative correlation with HCV infection, and positive correlation with TCC, while, it has no correlation with the grade and invasiveness of bladder cancer.
Functional inactivation of TP53 is the most common event in human malignancies, occurring in at least half of all tumors . In the present study, immunohistochemical detection of TP53 in tissue samples from normal urothelium of non HCV infected patients revealed negative staining, while positivity was 6% of samples from normal urothelium from HCV-infected patients. Malignant tissues from non-HCV infected patients showed positivity in 42% of samples and those from HCV infected patients showed positivity in 84% of samples. In consistence with immunohistochemical findings, assay of TP53 by RT-PCR showed significant increase in its expression in normal urothelium from HCV-infected patients and in malignant tissues from HCV infected patients. Moreover, TP53 expression showed positive correlation with HCV infection. TP21 (CIP1/WAF1) cyclin kinase inhibitor protein; binds to and inhibits the activity of cyclin-CDK2 or -CDK1 complexes, and thus functions as a regulator of cell cycle progression at G1. The expression of this gene is tightly controlled by the tumor suppressor protein TP53, through which this protein mediates the TP53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. Although, CDKN1A (p21) is a transcriptional target of the tumor suppressor gene TP53, unfortunately, the present study, showed significant loss in CDKN1A expression in bladder tumors from HCV- infected patients. In addition, we found low CDKN1A expression by RT-PCR in bladder tumors from HCV infected patients. Moreover, CDKN1A had a negative correlation with HCV infection. However, we could not explain the cause for this controversy in decreased expression of CDKN1A in HCV infected patients in contrast to TP53. Decreased or loss of CDKN1A expression in BC of HCV infected patients could be a sign for bad prognosis in case of BC. In a previous study of patients with advanced bladder carcinoma undergoing radical surgery showed that patients with tumors that maintained CDKN1A (p21) expression had increased survival relative to patients with loss of CDKN1A expression .
The last tested gene is FGFR3 (proto-oncogene) which is associated with early papillary lesions with low malignant potential [9, 10]. The mutations of FGFR3 are found more frequently in superficial than in invasive urothelial cell carcinoma (UCC) , and it has been reported that such mutations are more frequent in UCCs that do not recur . Real time PCR and immunohistochemical examination of FGR3 in the present study showed high expression of FGFR3 in malignant bladder tissues from HCV infected patients. Moreover, there is a positive correlation between FGFR3 and HCV infection. Finally, an interesting and novel finding in the present study was a negative correlation between HCV infection and overall survival rates and disease free survival.
Although, the present study was the first study, up to the best of our knowledge, demonstrating altered expression of telomerase, Rb, E2F3, TP53, CDKN1A (p21) and FGFR3 in BC patient with HCV infection, it has some limitations. This study did not investigate mutations of these genes in BC of HCV infected patients. Moreover, the effect of HCV on chromosomal stability, and DNA repair genes, hence the behavior of bladder cancer, was not studied. Also, decreased overall survival and disease free survival rates of BC with HCV may be due to other factors such as liver affection, other metabolites, medications, and immune system alterations, and this point needs further exploration to study the impact of these factors on survival of HCV infected patients. This would be a future proposal for further study and correlations.