ONCOLOGY REPORTS 14: 909-913, 2005 Expression of HIF-la and Glut-1 in human bladder cancer V. PALIT 1 ' 2 , R.M.PHILLIPS 1 , R. PURI 2 , T. SHAH 2 and M.C. BIBBY 1 Cancer Research Unit, University of Bradford, BD7 1DP; and 2 Bradford Royal Infirmary, Bradford, BD9 6RJ, UK Received March 29, 2005; Accepted June 9, 2005 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ Abstract. HIF-1 is a heterodimer consisting of the HIF-la and HIF-1|3 subunits, and HIF-la is the unique oxygen regulated subunit that determines HIF-1 activity. HIF-la upgrades many gene products which include the glucose transporter protein 1 (Glut-1). Immunohistochemical studies using a monoclonal antibody specific for HIF-la indicate that the overexpression of HIF-la occurs in the most common forms of human cancer, including bladder cancer. The expression of Glut-1 in human bladder cancer is associated with poor prognosis and a low survival rate. To our knowledge this is the first study to compare the expression of both HIF-la and Glut-1 with clinico-pathological characteristics in superficial and invasive human bladder cancer (all invasive bladder cancer patients received radical radiotherapy). The Kaplan-Meier survival analysis curve shows a significant association of HIF-la expression with recurrence and survival in superficial bladder cancer and shows a significant association of Glut-1 with survival in invasive bladder cancer [chi 2 (4)= 10.52; Pr >chi 2 =0.0012]. Introduction Like many other solid tumours, hypoxia is prevalent in bladder cancer. The transcriptional activator, Hypoxia Inducible Factor 1, consists of the HIF-la and HIF-18 subunits (1,2). HIF-la, the unique oxygen regulated subunit that determines HIF-1 activity, is expressed in response to cellular hypoxia, and mediates multiple cellular and systemic homeostatic responses to hypoxia. HIF-1 activates a number of factors which include the glucose transporter proteins, of which the glucose transporter protein 1 (Glut-1) is an important member. In a study by JoneszyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al, all bladder cancer cell lines showed hypoxic up-regulation of HJJF-la (3). In the same study, an increased expression of HIF-la protein was observed in human bladder cancer compared with normal bladder tissue. HIF-la protein was expressed to a greater extent in invasive tumours than in superficial, although there was a significant Correspondence to: Dr Victor Palit, 11 Mildred Avenue, Royton, Oldham, OL2 6AD, Lancashire, UK E-mail: victorpalit@hotmail.com Key words: HIF-la, Glut-1, bladder cancer inter-tumour variation, with the highest expression in one of the superficial tumours (3). Younes et al first showed the expression of Glut-1 in transitional cell carcinoma of the bladder and correlated its association with poor patient survival (4,5). This study explored the correlation of HIF-la and Glut-1 expression with clinico-pathological characteristics in superficial and invasive bladder cancer respectively. Materials and methods Tissue. A total of 37 superficial bladder cancer (PTA, PT1), 26 invasive bladder cancer (PT2), and 10 benign bladder cancer patients were included in this study. All superficial bladder cancer patients were treated by TURBT (Transurethral resection of bladder tumour) and intravesical chemotherapy with mitomycin C. Grades of these superficial tumours (Gl, G2, and G3) were recorded, and the time until recurrence of the bladder tumour was noted in this group. Overall survival was also recorded. The 26 invasive bladder cancer patients were all T2, and all were treated with radiotherapy following their initial TURBT and one intravesical dose of mitomycin post- operatively. Different grades (Gl, G2, and G3) of these T2 tumours and overall survival was noted in this group. Formalin-fixed paraffin embedded wax blocks from the above bladder cancer patients were obtained for this study. Hematoxylin and eosin (H&E) staining was performed on the sections to confirm the presence of transitional epithelium which might be denuded during sectioning. The appropriate wax blocks were identified by looking at these H&E slides and were marked for antibody staining. Immunohistochemical staining for HIF-la. All immuno- histochemistry was performed with anti-HIFla antibody in a dilution of 1:50 (This is a monoclonal antibody obtained from Calbiochem, and the immunogen used was a GST- fusion protein containing amino acid residues 432-528 of the HIF-la protein in a concentration of 3 mg/ml). This antibody detects several bands at -120 kDa, representing post- translationally modified forms of HIF-la, and has been used previously (6). Sections were deparaffinized in xylene, rehydrated through decreasing concentrations of alcohol ending in PBS, incubated for 30 min in 0.6% H 2 0 2 and microwaved in antigen unmasking solution for 30 min. Following this, sections were incubated in blocking serum (horse) for 20 min, they were then incubated overnight at 4°C