Comparative assessment of MHC antigen expression in bladder and testis tumour biopsies and established urological tumour cell lines: the relevance of cytokine and gene transfection for correction of defective MHC antigens.

The aim of this study using radio-binding (RB), peroxidase-anti-peroxidase (PAP) and immunoprecipitation (IP) techniques was to investigate the pattern of major histocompatibility complex (MHC) antigen expression in urological malignancies and to compare the results with those seen in established urological human tumour cell lines. The results showed that using PAP technique, the percent bladder cases showing complete loss or cases with greater than 90% of tumour cells negative with W6/32 (detects all class I antigens), HC10 (detects free heavy chain) and BMM.1 (detects beta2-mirogobulin) monoclonal antibodies (Mab) were 16%, 44% and 2% respectively. In a subgroup of 37 cases, the intensity of MHC class II antigen expression for strong, weak and negative cases were 9 (24%), 8 (22%) and 20 (54%) respectively. The expression for class I antigens on testis tumours was mainly negative and when positive, it was present in a small percent of tumour cells. This was also observed for class II antigens where only 8% of cases showed some degree of positivity. Using RB technique, 10 of 12 (85%) of tumour lines expressed class I antigens spontaneously and following interferon gamma (IFNgamma) stimulation, the 2 negative lines one testis (Tera I) and one bladder (Fen) remained negative and 2 lines (both testis lines Tera II and Ep2102) showed a significant class I up-regulation. None of the lines expressed class II antigens spontaneously and following IFNgamma stimulation, 8 of 12 (66%) were induced. The absence of class I and II antigens in the negative lines was confirmed using IP technique. In the case of one class I negative bladder cell line i.e. Fen, the biochemical analysis showed the absence of beta2-m gene product which could not be restored by IFNgamma stimulation. However, transfection of the cells with beta2-m gene resulted in the expression of fully assembled class I antigens, indicating that the loss of antigens was due to the absence of functional beta2-m gene. These results indicated the similarity between the pattern of expression of MHC antigens on tumour biopsies and established tumour cell lines. They also demonstrated that both cytokine stimulation and gene transfection could be used to correct defective class I antigens in tumour cell lines. These approaches might have important implications for pre-selection of bladder cancer patients for cytokine or gene therapies.

Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC-restricted cytotoxicity.

Using colorimetric MTT assay the susceptibility of a newly established bladder epithelial cell line, Fen cells was compared with conventional target cells, i.e., K562 and Daudi and other epithelial lines for investigation of non-specific killing activity (NK/LAK) of effector cells previously activated with interleukin-2 (IL-2). The results showed that Fen cell line was more sensitive than K562 and Daudi targets and this was seen whether the effector cells were IL-2-activated or not. The percent killing of effector cells from nine normal donors against Fen, K562 and Daudi targets at effector/target (E/T) ratio of 10/1 after IL-2 activation were 63.4 +/- 7.3, 42.6 +/- 4.3 (p = 0.0001) and 38.6 +/- 5.1 (p = 0.0001) respectively. The corresponding values for inactivated effector cells at 50/1, E/T ratio were 44.8 +/- 9.0, 25.1 +/- 8.3 (p = 0.0001) and 24.4 +/- 9.4 (p = 0.0001) indicating exquisite sensitivity of Fen cells to NK/LAK killing. The susceptibility of Fen cells was found to increase by pre-treatment of target cells with interferons (IFN). Thus the percent killing of untreated, IFN-alpha (1000 U/ml), beta (2000 U/ml) and gamma (100 U/ml) treated cells were 52%, 64% (p = 0.005), 70% (p = 0.001) and 67% (p = 0.001) respectively. These results indicated that Fen cells were more susceptible to NK/LAK killing than the conventional K562 and Daudi target cells. These results and the epithelial origin of Fen cells indicate that this cell line might prove to be a more realistic system to replace the conventional approach for assessment of NK/LAK activity in patients with cancer of epithelial origin.

Accurate and rapid assessment of MHC antigen upregulation following cytokine stimulation.

Three human tumour cell lines, A431 (cervical), Scaber (bladder) and Fen (bladder), were studied using immunohistochemical staining (IC), radiobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major histocompatibility antigen (MHC) induction in response to interferon-gamma (IFN-gamma). Induction of class II antigens by IFN-gamma was observed on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive for intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta 2 microglobulin (beta 2-m) (Mab BBM.1), while Fen cells were positive only with HC10. The IP technique demonstrated the presence of a 45 kDa band on precipitation of the Fen lysate with HC10 Mab, whereas no such band was observed when W6/32 was used. The DB technique confirmed the negative reaction with W6/32 and BBM.1 Mabs, while HC10 showed positive staining which was upregulated by IFN-gamma. Transfection of the Fen cells with the beta 2-m gene resulted in the surface expression of fully assembled class I molecules. The DB technique showed upregulation of class I antigens following IFN-gamma stimulation, while RB detected no significant increase in cell surface expression (t test; p = 0.104). The binding values for transfected Fen cells before and after IFN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectively. These results demonstrate that the DB technique facilitates an accurate assessment of cytokine induced antigens, corrected against a background of total cellular protein synthesis. The ease of execution, simplicity, non-radioactive nature and economy make it the method of choice for routine screening prior to the selection of suitable patients for cytokine therapy.