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Introduction

The isolation and characteristics of circulating immune complexes (CIC) present in the sera of cancer patients have shown both good, and poor correlation with the disease progress [1,2,3,4,5,6,7,8]. As far as prognosis is concerned many authors have observed a variety of findings, ranging from partial to very good correlation between CIC levels and prognosis in the disease [2,4,9,10,11,12,13]. For example, melanoma tumour-associated antigen (TAA) containing CIC were detected in 75% of patients with recurrence of the disease, as compared to their absence in 71% of the patients, who remained disease-free 14. Mavligit and Stuckey [15] have found the presence of carcino-embryonic antigen (CEA) in CIC isolated from the serum of colorectal cancer patients. It has been demonstrated that detection of antigen-specific CIC in the sera of cancer patients may provide an approach for more precise defining of the nature of tumour antigens that is important for immune prognosis [16,17,18,19]. The detailed characterisation of CIC should enable both detection, as well as identification of their antigenic components [9,16,17,20].

Objective

The aim of this study was to separate and determine the antigenic components of CIC isolated from the sera of 76 patients with urinary bladder cancer to find atypical proteins within CIC in bladder cancer progression.

Materials and methods

Patients

We assayed the serum samples originated from 21 patients with bladder cancer in differentiation grade G-1, 32 patients with grade G-2 and 23 patients with grade of bladder cancer G-3. Mean age of cancer patients was 64.2 +/- 13.4 years (range of age 21-87 years). Control group consisted of the serum samples taken from 45 healthy men aged between 25 and 69 (41.1 +/- 10.8 years).

CIC assays by PEG-test

Serum samples (2ml) were diluted in 2 ml of 7% PEG-6000 solution in borate buffer (0.1M, pH 8.4). The samples were incubated at 4oC for 18 h and centrifuged at 15.000g for 30 min at the same temperature. The supernatant was decanted and the precipitate was washed with the 3.5% PEG-6000 solution in borate buffer, suspended in 2ml of 0.1M NaOH and incubated at 25oC. After 30 min, the optical density was read on a LKB spectrophotometer at 280nm (0.1 optical density unit was read as 0.07 g/l of CIC protein) [21].. The results were considered as positive when optical density value was higher than 0.112 (mean value +/- 0.018 observed in 45 men).

CIC isolation

The quantity of 1 ml of patient serum was incubated with 1 ml of 7% PEG in borate buffer (0.1M, pH 8.4) for 18 h at 4oC [22,23]. The precipitate was then washed twice with 3.5% PEG in borate buffer, centrifuged at 15.000 rpm for 30 min at 4oC and subsequently resuspended in a volume of 1 ml PBS. The CIC suspension in PBS was then diluted in the buffer sample and used for polyacrylamide gel electrophoresis.

Gel electrophoresis and silver staining

The nature of the protein present in CIC was studied by SDS-PAGE gel electrophoresis, according to the method of Laemmli [24]. The CIC sample in PBS was diluted 1: 1 in the Tris-HCl buffer of pH 6.8, (1% glycerol, 6% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue), then heated at 100oC for 3 min. and subsequently applied to the plate composed of a stacking gel (3% acrylamide) and resolving gel (11% acrylamide). Approximate molecular weights were estimated by comparison with standard protein markers (MW-SDS-200, Sigma, USA). At the end of the electrophoretic run, the proteins were stained with Coomassie brilliant blue R250 and silver [25,26].

Data analysis

Statistical analysis of data was done by Student´s t-test.

Results

PEG test from cancer and normal serum samples

The CIC level was studied in the sera of 76 patients and 45 healthy men by means of PEG test; CIC-protein concentration are presented on Fig. 1. In a normal serum mean optical density as measured at 280nm was 0.112 +/-0.018 (0.0784+/-0.0126 g/l CIC protein). The concentration of CIC in-patients with bladder cancer was significantly higher. The analysis of 76 cancer serum samples divided according to cancer differentiation grade showed the following mean concentration values: 0.097+/-0.047 g/l (P<0.05) in stage G-1; 0.1112+/-0.039 g/l (P<0.01) in stage G-2 (P<0.01); and 0.1092 +/-0.041 g/l (P<0.01) in stage G-3 (Fig. 1). The percentage of CIC positive patients in the G-1, G-2, and G-3 groups was as follows: 38.1% in G-1 (elevated in 8 of 21 patients), 56.2% in G-2 (elevated in 18 of 32 patients), and 60.8% in G-3 (elevated in 14 of 23 patients). In general, an increase in the level of CIC was observed in 40 out of 76 patients with bladder cancer (52.6%).

SDS-PAGE analysis of CIC originating from control and cancer serum

The distribution and frequency of appearance of the protein fractions in the CIC of control sera showed 20 bands of molecular weight between 22 and 198 kDa (Table 1). This mode of the bands distribution was highly reproducible. In contrast, the protein fractions of CIC obtained from sera of tumour patients showed up to 30 bands. The results, showing the distribution of atypical proteins in relation to molecular weight are displayed in the Fig. 2. Their frequency is shown in the Table 2. Generally sera of patients show the presence of 14 protein additional, atypical fractions with molecular weight 20, 31, 33, 39, 41, 46, 50, 61, 84, 92, 114, 124, 213 and 240 kDa.

Discussion

Results presented above indicate that 52.6% of urinary bladder cancer patients under study had an increase in the level of CIC in the serum. The values described here are higher than those reported by other researchers [2,6,9]. Babaian et al. [9] have found considerably lower percentage (25%) of tumor patients with increased level of circulating immune complexes. Anyway, quoted researchers examined smaller groups of patients which makes difficult to compare our and their results. Babaian et al. [9] indicated the dependence among the level of CIC, the size of tumor, its invasive propensity and the level of its malignancy. The results of this work are consistent with the quoted authors observations concerning the increase of immune complexes level in bladder cancers and their differentiation depending on advancement stage of the cancer.

The origin of the antigens consisting immune complexes in sera of examined cancer patients is unknown. Perhaps the antigens in CIC are related to the level of the cancer progress. Wiederkehr et al. [20] carried out an electrophoresis of immune complexes, isolated from sera of healthy persons and persons with cancer. It turned out that the protein profiles in electrophoretic sections of control group were similar, but there were some small quantitative differences. In the available literature we did not found any work concerning the electrophoretic analysis of CIC present in the sera of urinary bladder cancer patients, so it is hard to compare our results with the results of others. We have found so far only one paper dealing with an analysis of the protein fractions present in the CIC originated from cancer patients. Bartoloni et al. [16] examined CIC isolated from the sera of patients with colon, pancreas, stomach cancers and melanoma. The analysis of purified CIC proteins in most cases indicated existence of a few common fractions: of the albumin (64-72 kDa), of the heavy immunoglobulin chains (52-61 kDa) and of the light immunoglobulins chains (24-28 kDa). The results of this work are consistent with the results previously described by others. Bartoloni et al. [16] indicated the presence of atypical protein fractions with molecular weight ranging from 29-33 kDa to 78-88 kDa found in the serum of colon, pancreas and stomach cancer patients. A protein fraction of molecular mass 99 kDa was defined only in the sera of colon cancer patients. In the CIC originating from the sera of melanoma patients the atypical protein fractions with molecular weight of 30-49 kDa and 71-76 kDa were found. By comparison, our results dealing with bladder cancers indicated the presence of 30 protein fractions of molecular weight ranging from 20 to 240 kDa. So the molecular weight of protein fractions obtained in our study is bigger than the one in the results of Bartoloni´s work.

This may depend on much more sensitive technique (silver method) used by us for the detection of proteins present in CIC (sensitivity 1-10 ng). This technique is much better than the classical method of Coomassie Brilliant Blue R-250, with sensitivity ranging from 1-10 ug [25,26].

The electrophoretic analysis of isolated CIC from sera of bladder cancer patients indicated the presence of 14 atypical protein fractions. Molecular masses of these fractions were as follows: 20, 31, 33, 39, 41, 46, 50, 61, 84, 92, 114, 124, 213 and 240 kDa. The elevated levels of CIC are present in the sera of cancer patients. The level of CIC strictly depends on the histological grade of malignancy.

In CIC isolated from sera of slow progress patients and the bladder cancer there were defined proteins which were absent in immune complexes isolated from healthy persons. Analysis and characterisation of CIC can be both helpful in the diagnostic procedure and in studies on proteins specific for pathomechanism of this group of diseases.

Conclusions

For this reason, the determination and characterization of CIC levels can be helpful in both the diagnostic procedure and the analysis of atypical proteins forms generated by the neoplastic process. There is a need for further research to define the value of CIC atypical proteins analysis more precisely.

Source of support: This work was supported in part by a research grant from the The Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Torun, Poland (BW 127/97).

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Ryszard Gołda
Department of Immunology
The Ludwik Rydygier Collegium Medicum in Bydgoszcz
Nicolaus Copernicus University in Toruń
Maria Skłodowska-Curie Street 9, 85-095 Bydgoszcz, Poland
tel. (0... 52) 585 35 78
goldar@cm.umk.pl