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Bladder cancer is a common neoplasm worldwide, consisting mainly of transitional cell carcinomas, while squamous, adenocarcinoma, and sarcomatoid bladder cancers account for the remaining cases. In the present study, multiplex fluorescence in situ hybridization (M-FISH) has been used to characterize chromosome rearrangements in eight transitional and one squamous cell carcinoma cell line, RT112, of UMUC-3, 5637, CAT(wil), FGEN, EJ28, J82, 253J, and SCaBER. Alterations of chromosome 9 are the most frequent cytogenetic and molecular findings in transitional cell carcinomas of all grades and stages, while changes of chromosomes 3, 4, 8, 9, 11, 14, and 17 are also frequently observed. In the present study, alterations previously described, including del(8)(p10), del(9)(p10), del(17)(p10), and overrepresentation of chromosome 20, as well as several novel findings, were observed. These novel findings were a del(15)(q15) and isochromosome 14q, both occurring in three of nine cell lines examined. These abnormalities may reflect changes in bladder tumor biology. M-FISH represents an effective preliminary screening tool for the characterization of complex tumor karyotypes.

Backgound: The major histocompatibility (MHC) class I antigens act as associative molecules for interaction amongst immuno-competent cells. The grooves of class I antigens are normally loaded with self peptides of between 8 and 11 amino acids. However, when the cells transform to malignant state they may carry peptide(s) of non-self origin within these grooves. Using immuno-bead purification followed by high-performance liquid chromatography (HPLC), this study attempted to isolate peptides from class I antigens of various biological specimens Results: The combination of immuno-bead purification (BP) and HPLC was reliable for peptide isolation. Class I antigens and associated peptides could be isolated from normal peripheral blood leukocyte (PBL). Under the same conditions, the PBL yielded almost twice as much peptide as that of tumour cell lines. The HPLC profile of peptides (range of 8–10 amino acid residues) isolated from a bladder and a cervical tumour cell line showed unique features. In the case of the bladder line there were at least 22 peptides. In addition, the class I-associated peptides could also be isolated from kidney tumour fragments of three individuals. In each the isolated peptides showed a unique HPLC peak profile with some similarities as well as differences. Conclusions: These data indicated a variation in the nature of peptides isolated from different specimens. The approach showed the feasibility of preparing peptide(s) from a relatively small number of cells. The data also showed that peptide isolation could also be carried out from tumour tissue biopsies paving the way for the future of peptide vaccination in cancer patients following the identification of putative tumour-specific replicate(s).

Background/Methods: In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high-performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection. Results: The results showed that: (1) Transfection of the class I negative Fen cell line with normal β-microglobulin (β2-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the β2-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700–3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 × 106 of positive Fen cells contained about 200 µg total protein of which about 0.10 µg was class I and about 2 ng was class I-associated peptides. Conclusions: These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients.

Objective To develop specific monoclonal antibodies (mAbs) against human germ cell tumours. Materials and methods A single-cell suspension obtained from tumour tissue fragments (consisting of both tumour and normal compartments) from a patient with seminoma was used as an immunogen. Spleen cells from immunized mice were used to develop mAbs. Tissue specificity, biochemical characteristics and competitive studies were analysed using immunocytochemical staining, dot blots and a Western blot analysis, to identify target antigen(s). Results The immunization protocol led to the development of 107 hybridomas, 90 of which were negative against the original tissue biopsies. The remaining 17 showed positivity against various tissue compartments. One selected mAb (ATC2) showed specific staining on germ cell tumours but not on normal tissues, and positive staining with some human tumour cell lines. The target antigen for ATC2 was confirmed to be placental alkaline phosphatase (PLAP) based on: Western blot analysis compared with commercially available PLAP; comparison of the data with another well-known anti-PLAP mAb (H17E2, although the two mAbs recognized different antigenic epitopes); heat resistance characteristics; high-performance liquid chromatography of the ATC2 target antigen and purified PLAP. Conclusion The selected mAb ATC2 has high specificity for human germ cell tumours, the target antigen for ATC2 being PLAP, although the antigenic epitope(s) differ from those recognized by H17E2. Thus ATC2 may be useful for monitoring serum levels of PLAP in patients with testis cancer and may be relevant for detecting cancer cells in the semen of individuals with suspected testis cancer, particularly in those with equivocal findings on ultrasonography.

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta2-microglobulin (beta2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients.

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

A chemically induced syngeneic hamster tumour model of human oral squamous cell carcinoma (OSCC) was used to investigate the possible influence of locally transplanted growing rumours on the immune system of the recipient. Cell activation and cell cytotoxicity assays were performed in vitro using the colorimetric MTT assay to measure any possible changes. The fast growing nature of the tumour model if grafted locally as a fragment was confirmed but not if injected as a single cell suspension (SCS). Stimulation (Concanavalin A) of spleen cells from normal and from tumour bearing animals showed that there was a minor though statistically significant decrease in the mitogenic response of the latter. Thus, the respective stimulatory indices (SI) were 4.06+/-1.61 and 2.06+/-0.87 (0.02 0.01). No significant difference was observed when spleen cells were stimulated with interleukin-2 (IL-2), although there was a similar trend. Pre-immunisation of animals with irradiated autologous SCS three weeks prior to grafting, resulted in a significant decrease in the tumour growth rate of subsequently grafted tumour. Thus, the mean If: SD (weight of takes in mg) for the successful takes of untreated (n=10) and treated (n=9) groups were 52.0+/-52.2 and 25.7+/-19.4 (0.02 0.05) respectively. The number of cases with no tumour takes were 2 of 10 (20%) and 6 of 9 (66%) respectively. In a separate experiment groups of 5 animals were immunized with an increasing number of cells as irradiated SCS, the results of which demonstrated an inverse correlation between the rate of tumour growth and the number of injected tumour cells. The addition of irradiated SCS to IL-2 activated normal spleen cells (LAK cells) in vitro led to a dose-related decrease in the efficiency of cytotoxicity of latter when tested against an xenogeneic super-sensitive surrogate tumour target cell line (Fen cells). Thus, the percent killing by IL-2-activated normal spleen cells was 56.4%. The corresponding mean values for IL-2 activated normal spleen cells in the presence of tumour SCS at 25/1 and 50/1 ratios were 35.9% (p

A modified tetrazolium reduction assay (MTT) was used to assess the relation between HLA class I antigen expression on tumour cells and their susceptibility as a target for non-MHC restricted LAK/NK cytotoxicity using interleukin-2 activated peripheral blood mononuclear cells (MNC) from normal individuals. At 20/1 effector/target ratio this ranged from no killing to 77%. The efficiency of killing was dependent on duration of effector cell culture with IL-2, peaking at day 10 and declining thereafter. This killing could be enhanced by addition of other cytokines including interferons alpha, beta and gamma. Study of a panel of 15 tumour cell lines using a single effector showed that there was no statistically significant inverse correlation (using Spearman rank test) between the degree of tumour class I expression and LAK/NK killing at 20/1 (r = 0.23 P = 0.39) and 10/1 (r = 0.30, P = 0.27) and at 5/1 E/T ratio r = 0.47, P = 0.08) respectively. Lack of inverse correlation between these two parameters came from study of one bladder tumour line (FEN), whose absent class I antigens had been corrected by transfection with beta 2 microglobulin gene. At high E/T ratio (20/1) there was an increase in the susceptibility of target cells to lysis (36% parent cell, 45% transfected cell), whilst at lower E/T ratios (1/1) there was significantly more killing of the non-transfected cells (10% vs 31%). The addition of anti-class I antibody W6/32 increased killing by 18% but this was non-specific as the same increase occurred with a class II antibody. These data suggest that overall there was not an inverse correlation between class I expression and LAK/NK killing at high E/T ratios, whilst at low (5/1 or lower) E/T ratios this correlation nearly reached statistical significance suggesting that the conflicting literature reports may be due to a threshold levels of effector cells above which the masking effects of MHC antigens disappears.

Tissue sections from 22 seminoma (Se) and 10 teratoma (Te) patients were investigated for correlation between the presence of tumour infiltrating T-lymphocytes (TIL) and the expression of major histocompatibility complex (MHC) antigens using an immunoperoxidase staining technique. Complete absence of both class I and II antigens was observed in all Te and 20 out of 22 Se. The two positive Se showed only weak expression on 2% of tumour cells. Despite the absence of human leucocyte antigens (HLA) there were a large number of TIL scattered throughout the tissues in the case of Se with no predominance of CD4 or CD8 subpopulations in either group. T gamma positive cells were less than 5% of total CD3 positive cells in both Se and Te. The majority of the TIL were found to express activation markers, i.e. HLA class II antigens. Culture of tumour cell suspension with IL-2 produced passageable IL-2-dependent T cells from 10 out of 15 tumours. Studies with testis cell lines showed the complete absence of class I antigens in 2 out of 5 cases and the inability of interferon gamma (IFN gamma) to induce expression. IFN gamma also failed to induce class II antigens in three out of five of these lines. The immunological paradox of the presence of a large number of activated T-cells in testicular tumours despite the complete absence of MHC antigens remains unexplained and needs further investigation. Possible hypotheses are reviewed.

Placental alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7– 8 min (corresponding to 95 kDa molecule) and one at 12–13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.

Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

In this study the impact of epidermal growth factor (EGF) on chemosensitivity and susceptibility to lymphokine-activated killer (LAK) cytolysis on six cell lines (one super expressing EGFr i.e. HN5, three high expressing i.e. Hep2, KB and MCF-7 and two low expressing i.e. Fen and HN15) was investigated using the tetrazolium salt reduction assay (MTT) as measured by optical density (OD). Hep2, KB, MCF-7 and Fen lines showed a dose-related inhibition to cisplatin (from 19% to 80%). Treatment of EGFr positive cell lines, Hep2, KB and MCF-7 but not EGFr negative Fen by EGF prior to exposure to cisplatin inhibited the cells by between 10-15% (p

Using radiobinding, transfection and colorimetric assays, the biological significance of epidermal growth factor (EGF) and its receptor on established human tumour cell lines was investigated. The intensity of class I major histocompatibility antigen (MHC) and EGF receptor (EGFR) expression on 20 tumour cell lines was investigated and showed no direct correlation (coefficient of correlation r = 0.43 and P = 0.06). furthermore, transfection of the beta 2-microglobulin gene into a class I negative bladder tumour cell line, resulting in the re-expression of fully assembled cell surface class I antigens, did not result in alteration of EGFR expression. However, there was an inverse correlation between the intensity of EGFR expression and the stimulatory response of cells to exogenously added EGF. The per cent inhibitions of cell proliferation by EGF at 100 ng/ml for A431 (highest EGFR expressor) and Scaber (lowest EGFR expressor) were 37 and -7%, respectively. The results also showed that cell lines isolated from testis tumours positive for epithelial markers (using pan keratin antibody LP34 as an epithelial marker), expressed significantly lower EGFR levels than cell lines from bladder tumours. The expression of EGFR receptor was not modulated by interferons (IFN-alpha and -gamma and only a minor effect with IFN-beta) or active supernatant containing a mixture of cytokines. Whilst the pretreatment of tumour cells with IFNs resulted in a significant increase in the susceptibility of tumour cells to interleukin-2-activated peripheral blood mononuclear cells, EGF treatment resulted in their protection. Thus, the per cent killing at an effector:target ratio of 20:1 for untreated cells and EGF (100 ng/ml), IFN-alpha (1000 U/ml), -beta (2000 U/ml) and -gamma (100 U/ml) were 53%, 33% (P = 0.004), 64% (P = 0.004), 69% (P = 0.001) and 66% (P = 0.001), respectively. These results indicate the complex interactions between EGF and EGFR and their relevance in modifying tumour cell behaviour. The hypothesis that the resistance to cytolysis of tumour cells induced by EGF stimulation may be a factor in the accelerated tumour growth seen in patients after traumatic tissue damage is discussed.

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-α (1000 U/ml), β (2000 U/ml) and γ (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.

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.

The induction of major histocompatibility complex antigens by interferons (IFN) on 17 established tumour cell lines was investigated by radio binding. One bladder (Fen) and two testis lines (Tera I and Ha) lacked class I antigens and IFN-gamma failed to induce their expression. However, IFN-gamma upregulated these antigens on lines expressing low class I antigens (Tera II and EP2102) with little or no significant effect on high class I expressing lines (T24 and RT112). In one bladder line (Wil) IFN-gamma, whilst failing to alter monomorphic class I, upregulated polymorphic HLA-A2 and A3 antigens. None of the 17 lines expressed class II antigens, but could all be induced by IFN-gamma except T24, TccSup, Tera II and Lan lines. This defect was not due to the absence of IFN-gamma receptor, since under the same conditions intracellular adhesion molecule 1 was upregulated. IFN-alpha, whilst failing to have any effect on class II, induced class I antigens. IFN-beta showed no activity on either class I or II antigens when used alone. However, in combination, it inhibited IFN-gamma induced class II antigens. Thus, it may be possible to study cells from fresh tumours to preselect the minority of patients who might benefit from cytokine therapy.

Analysis of tissue sections from transurethrally resected bladder tumours using anti-CD3 antibody showed the presence of T lymphocytes in intra-epithelial layers in eight of 12 cases investigated. In a larger group of patients, Tumour Infiltrating Lymphocyte (TIL) growth was established from six of 19 cases using Interleukin-2 (IL-2) and conditioned medium (CM) and resulted in the expansion of TILs up to 100-fold. TILs from these individuals were phenotyped with W6/32 (anti-HLA-A,B,C), HB55 (anti-DR) and anti-CD3 antibodies using FAC sorter. The mean +/- s.d. frequency of positive staining with these antibodies were 96.7 +/- 4.0%, 87.5 +/- 10.0% and 82.5 +/- 7.8% respectively, indicating the activated nature of these T cells. The cytotoxic activity of these TILs against Daudi (ie, LAK activity) cell line at 25/1 E/T ratios varied from 26.3 +/- 3.2 to 62.8 +/- 5.2%. In one case where TILs and autologous tumour cell line were established, cytotoxicity studies showed low level of cytotoxicity against the autologous tumour cells (15.8 +/- 1.6%) compared with 62.8 +/- 5.2% against Daudi. Staining of tumour sections from these 19 individuals with W6/32 and BBM.1 revealed positive staining in six of six that developed TILs but only six of 13 (46%) cases, whose tumour failed to grow TILs (P less than 0.02, Fisher exact test). These results are indicative of the presence of IL-2 passageable T cells in bladder cancer biopsy and demonstrate that the successful expansion of these cells correlates with the normal expression of class I antigens on the tumour cells. Images Figure 5