Is the ABCB1 gene associated with the increased risk of gastric cancer development?--preliminary research.

One of the most common malignant diseases, both worldwide and in Poland, is gastric cancer. The pathogenesis of gastric cancer development is not entirely clear. Next to the environmental risk factors, such as Helicobacter pylori infection or dietary habits, the host genetic factors as predispositions to gastric cancer development are discussed. A transmembrane protein that could be associated with predisposition to cancer development is P-glycoprotein (P-gp). Physiologically, P-gp is present in normal tissue of the gastrointestinal tract, where it plays a protective role by transporting xenobiotics from a cell into extracellular environment. P-gp is encoded by the highly polymorphic ABCB1 gene. The most frequent polymorphisms at positions 1236, 2677, and 3435 may affect both the function and amount of protein, thereby leading to a loss of its physiological function, which could increase the predisposition to development of many diseases, including cancer. In this study, the potential significance of the ABCB1 gene in the development and progression of gastric cancer was evaluated. In 19 tissue samples collected from patients with gastric cancer, the ABCB1 gene polymorphisms were identified at positions 1236 and 2677 by automated sequencing and SNP 3435 by the RFLP method. The relative level of ABCB1 expression was measured in 10 samples of gastric cancer and morphologically normal tissues by real-time PCR. For SNPs at positions 1236, 2677, and 3435, no statistically significant differences in genotype frequencies between gastric cancer patients and healthy individuals were found. However, genotype TT for all studied polymorphisms occurred more frequently in the group of gastric cancer patients (31.6, 26.3, 42.1%, respectively) than in the group of healthy individuals (14.6, 13.5, 21.9%, respectively). The lowest relative expression levels of ABCB1 mRNA were observed for genotypes CC of SNP 1236, CC of SNP 3435, and GG of SNP 2677 (median: 0.215, 0.160, 0.160, respectively). There was a tendency that mutant homozygote TT for SNPs at positions 1236, 2677, and 3435 occurred more frequently in the subgroup of patients with Tis or stage I of TNM classification (SNP 1236 p = 0.0760; SNP 2677 p = 0.0813; SNP 3435 p = 0.0760) than in the subgroup of patients with stage II or III. Also the expression levels were lowest (median 0.740) in the group of patients with the less advanced clinical stage of cancer (Tis or I). Preliminary research showed that the ABCB1 gene polymorphisms at positions 1236, 2677, and 3435 were not related to an increased susceptibility of gastric cancer development. However, they may be associated with the inhibition of gastric cancer progression.

Maspin and Nm23-H1 expression in colorectal cancer.

The aim of the study was to analyze the expression of Nm23-H1 and maspin proteins in a series of colorectal adenocarcinoma and to assess their applicability as prognostic factors in this type of cancer. 102 specimens of colorectal carcinoma were analyzed by immunohistochemistry with the use of anti-Nm23-H1 and anti-maspin monoclonal antibodies. Cytoplasmic expression of Nm23-H1 and maspin was found in 90 of all investigated cases. In 60 cases maspin protein was found also in nucleus. Medium/high Nm23-H1 cytoplasmic expression level was associated with tubular type of adenocarcinoma with deeper invasion of cancer into intestinal wall (T3, T4) and presence of vascular invasion. Medium/high expression level of maspin was connected uniformly with bad prognostic features: low differentiation of tumors (G3), deeper invasion of cancer (T3, T4) presence of nodular and distant metastases, higher Astler-Coller stage (C1, C2, D) and presence of vascular invasion. No statistically significant associations between presence of nuclear maspin expression and any clinicopatological and biological features were stated. Cytoplasmic medium/high expression level of maspin but no Nm23-H1 and no presence of maspin nuclear expression was found as independent bad prognostic factor in the investigated group of patients. Measurement of level and cellular pattern of maspin expression could be valuable for predicting disease course in patients suffering from colorectal cancer.

Quantitative analysis of the p65 gene expression in patients with coloroctal cancer.

P65 gene expression level was determined in colon cancer cases by means of real-time PCR. 51 cases of colorectal carcinomas showing positive RT-PCR signals for P65 gene expression selected from 109 frozen samples were further investigated by quantitative real-time PCR. P65 levels were higher in cancer with metastases to lymph nodes and distant metastases. Higher levels were observed in more advanced cases classified as III and IV according to pTNM classification. In two groups of patients with vessel invasion and absence of lymphocytes in tumour tissue, the presence of P65 expression correlated with shorter survival time. Quantitative results confirmed that P65 gene expression in colon cancer is engaged in the process of metastasis formation and could be correlated with worse prognosis for the patients.

Expression of the P65 gene in gastric cancer and in tissues with or without Helicobacter pylori infection.

A 65-kDa tumor-associated protein (P65) is a potential non- specific tumor marker expressed by many types of tumor cells. Our recent studies indicate that P65 gene expression is connected with poor prognosis for the patients with colorectal cancer. In the present study P65 gene expression was determined by means of RT-PCR in the group of 22 gastric cancer and adjacent normal gastric mucosa. Its presence was correlated with some parameters of clinical staging. P65 gene expression was also determined in 102 tissue antral gastric endoscopic biopsy specimens from the patients suspected of H. pylori infection. The presence of H. pylori infection was determined by urease test. We found that in the group of gastric cancers, similarly to colorectal cancer, P65 gene expression was connected with poor clinicopathological parameters as T3, lymph nodes and distant metastases. There was no dependence between P65 gene expression and H. pylori infection. However, more often P65 gene expression was detected in the group of infected men than women. There was also a statistically significant dependence between age and P65 gene expression in the group of people above 60 years old. It could be then postulated that P65 gene expression is connected with poor prognosis for the patients suffering from gastric cancer and that this expression does not depend on H. pylori infection.

Cyclin D1 protein and CCND1 gene expression in colorectal cancer.

AIMS: To report the expression of cyclin D1 protein and its gene in a series of colorectal adenocarcinoma. METHODS: One hundred and eleven specimens of colorectal carcinomas and adjacent normal colorectal mucosa were investigated by staining with a monoclonal antibody against cyclin D1 and by RT-PCR. RESULTS: Expression of CCND1 gene was found in 54 out of 111 cases of colorectal cancers, while in normal mucosa the expression of this gene was not observed. Cyclin D1 protein expression was checked in the same group of adenocarcinoma cases. Presence of this protein was observed in 69 cases and for 43 of them also expression of its gene was found. Dependence between the presence of protein and the gene expression was statistically significant (p=0.0002). In the group of cases where CCND1 gene expression was detected, high level of its protein expression was found in 20 cases. The CCND1 gene expression was associated with metastases to lymph nodes (p=0.0181) and also with distant metastasis (p=0.0204). CONCLUSIONS: The combined measurement of both the gene and its protein product, is an important contribution to the study of molecular markers in histological material.

Expression of p65 gene in experimental colon cancer under the influence of 5-fluorouracil given alone and in combination with hormonal modulation.

The effect of tamoxifen (TAM), lanreotide (LAN) and 5-fluorouracil (5-FU), given separately or together, on p65 gene expression in murine Colon 38 cancer was investigated by RT-PCR method. The findings were compared with cell proliferation determined by bromodeoxyuridine (BrdU) labeling index, apoptosis visualized by TUNEL method and tumor mass. It was found that in the control group (mice bearing colon cancer without treatment) the expression of p65 gene was present in 57% of investigated samples. In the groups treated with TAM or LAN p65 gene expression was detected in 87.5% and 83.3% of analyzed cases, respectively. Both these substances increased apoptotic index in Colon 38 cancer and LAN also decreased the proliferation index. After a combined treatment with TAM and LAN a percentage of p65 positive cases was similar to that of the control group and equaled approximately 60%. This treatment did not increase proapoptotic effects of these drugs, and even reduced the antiproliferogenic effect of LAN. In the group treated with 5-FU and LAN p65 gene expression was also close to the control value (about 66%). Similarly in this group the combined treatment with these two drugs did not cause any favorable effect on proliferation and apoptosis. Moreover, in this group even reduced antiproliferogenic effect of LAN was observed. In the group with 5-FU alone the expression of p65 was present in about 80% of samples. The treatment with 5-FU increased apoptotic index and did not change proliferation. In the group treated with a combination of TAM and 5-FU all analyzed cases showed the presence of p65 gene expression. Previously, we observed in this group the most pronounced and synergistic effect of these substances on the inhibition of cell proliferation and tumor mass reduction. Based on these findings we conclude that p65 gene expression in murine Colon 38 cancer tissues can be modulated via chemotherapy (5-FU) and also via hormonal modulation (TAM and LAN).

Usefulness of polyclonal antibodies raised against P65 oncofetal protein in immunohistochemical diagnosis of ductal breast cancer.

Paraffin-embedded infiltrating ductal breast cancer tissue slides (135) were analyzed by immunohistochemistry with the use of rabbit polyclonal anti-P65 oncofetal protein and mouse monoclonal anti-estrogen/progesterone receptor (ER, PR) antibodies. Analysis with anti-P65 antibody revealed the positive cytoplasmic reaction in 83 cases, 98 showed the nucleic reaction and 3 were immunologically negative. Among the analyzed cases 49 revealed both cytoplasmic and nucleic reactions. For the whole group of cancers the correlation was found between ER or PR level and P65 cytoplasmic reaction (r = 0.77 and 0.66, respectively) and low inverse correlation with nucleic localization of P65 protein. The percentage of positive cells with cytoplasmic expression of P65 was significantly higher in more histologically differentiated cancers (grade I and II according to Bloom and Richardson) than in grade III. Opposite tendency was observed for the nucleic expression of P65 protein. The percentage of immunopositive nuclei grew with the advance of the disease and was the highest in poorly-differentiated (grade III) tumors. The tumors with P65 cytoplasmic reaction were mainly small (T1, T2), without metastases to lymph nodes (N0) and distant metastases (M0). The dependence between P65 protein localization and clinical stage of disease (TNM classification) was evaluated statistically. The straight dependence existed between P65 nucleic reaction and tumor size (p = 0.0002), metastases to lymph nodes (p = 0.0032) and distant metastases (p = 0.0006). The obtained results suggest that the transfer of P65 protein from cytoplasm to nuclei of the breast cancer cells is connected with more clinically advanced stages and worse prognosis for the patients.

HMGI(Y) gene expression as a potential marker of thyroid follicular carcinoma.

BACKGROUND: We assessed HMGI(Y) gene expression in thyroid tumors, control thyroid tissue and in the blood of patients diagnosed with papillary and follicular thyroid cancers to try to differentiate between malignant and benign disease. METHODS: HMGI(Y) gene expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 cases of thyroid tumors. Among this number 11 were diagnosed as papillary carcinoma, 37 as follicular carcinoma, and 12 as follicular adenoma. All carcinoma cases selected for this study were classified according to the tumor, lymph node metastases, distant metastases (TNM) classification. RESULTS: HMGI(Y) gene expression was detected only in follicular carcinomas, whereas in papillary carcinomas, follicular adenomas and control tissues there was no positive reaction. In follicular carcinomas the percentage of positive cases (number of samples with presence of HMGI(Y) gene transcript) was the highest and reached approximately 84. There was no statistical dependence between the presence of HMGI(Y) gene expression and tumor size or the presence of lymph node and distant metastases. HMGI(Y) gene expression was also analyzed in whole blood taken from a selected group of patients diagnosed with papillary or follicular carcinomas. Among follicular carcinomas there were 83% of positive cases, whereas among papillary carcinomas there were only 6%. CONCLUSIONS: On the basis of our study, we conclude that HMGI(Y) gene expression analysis could be helpful in differentiation between follicular carcinoma and adenoma.

Uptake of radiolabeled morphiceptin and its analogs by experimental mammary adenocarcinoma: in vitro and in vivo studies.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and its analogs modified at position 3: [D-Phe(3)]morphiceptin, [D-ClPhe(3)]morphiceptin and [D-Cl(2)Phe(3)]morphiceptin were synthesized and labeled with [(125)I] or [(131)I]. Their binding to membranes isolated from experimental adenocarcinoma was examined in vitro with the use of a cross-linking assay followed by the Western blot technique. The radioactive complex had molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor polyclonal antibody. Expression of the mu-opioid receptor in mouse mammary adenocarcinoma was confirmed by reverse transcriptase-polymerase chain reaction. The binding studies showed the highest affinity and capacity for [D-Phe(3)]morphiceptin (K(d) 0.39 and B(max) 1112) and [D-ClPhe(3)]morphiceptin (K(d) 1.8 and B(max) 220). Morphiceptin and its D-Cl(2)Phe analog had significantly lower B(max) values (131 and 83, respectively). Biodistribution experiments in tumor-bearing C3H/Bi mice with the use of the (131)I-labeled peptides confirmed the results of our in vitro studies. The highest accumulation of radioactive peptides in the tumor tissue was also found for peptides with D-Phe and D-ClPhe.

Expression of the p65 gene in patients with colorectal cancer: comparison with some histological typing, grading and clinical staging.

AIMS: P65 protein/gene is a potential, non-specific tumour marker expressed by different types of neoplasms. In this study p65 gene expression was estimated in a group of colorectal cancers and compared with some histological features, grading and clinical staging of the neoplasms to assess its role as a prognostic marker for colorectal cancer. METHODS: 109 pairs of frozen samples of colorectal carcinomas and adjacent normal colorectal mucosa and four samples of tissue from patients without neoplastic diseases were analysed by means of RT-PCR. RESULTS: Expression of p65 gene was found in 51 out of 109 cases of colorectal cancers. For 19 of them expression of examined gene was observed both in cancer and corresponding healthy mucosa. p65 Gene expression was associated with more advanced tumours (T3, T4; p=0.0003) with metastases to lymph nodes (N1, N2; p=0.0003) and distant metastases (p=0.0005). We did not find association between the age, gender, tumour localization, histological type and p65 gene expression. CONCLUSIONS: p65 Gene expression in primary tumour tissue is associated with poor prognosis for the patients with colorectal cancer in this series.

HMGI(Y) gene expression in colorectal cancer: comparison with some histological typing, grading, and clinical staging.

We investigated HMGI(Y) gene expression in 81 pairs of frozen samples obtained from colorectal carcinomas and adjacent normal colorectal mucosas and in four samples from colorectal mucosa from patients without neoplastic diseases. In this group, HMGI(Y)-positive/-negative expression was compared with some histological features, grading, and clinical staging of neoplasms investigated to assess its potential role as a prognostic marker for colorectal cancer. Expression of HMGI(Y) gene was found in 51 of 81 cases of colorectal cancers, while, in normal mucosa, expression of this gene was not observed. HMGI(Y) gene expression was associated with more advanced tumors (T3, T4) and metastases to lymph nodes (N1, N2). The most interesting finding was that expression of this gene correlated with distant metastases. HMGI(Y) gene expression was detected in all cases classified as M1 (n = 19, p = 0.0008). We did not find any association between age, gender, tumor localization, histological type and this gene expression.

p65 and c-erbB2 genes expression in breast tumors: comparison with some histological typing, grading and clinical staging.

Using PCR technique we have analyzed p65 and c-erbB2 genes expression in 47 frozen tissue slides taken from patients diagnosed as ductal and lobular breast cancer, classified as G3, and in a limited panel of proliferative breast disease cases. Expression of p65 was generally connected with small tumor size and with absence of metastases in regional lymph nodes. We have found interdependence between p65 gene expression and negative states of lymph nodes. On the contrary, c-erbB2 expression was observed in patients with large tumors and with metastases to the regional lymph nodes. Between both genes (p65 and c-erbB2) opposite interdependence was found. No statistical dependence between estrogen/progesterone receptor levels and p65 or c-erbB2 expression were noticed. The presence of p65 expression appeared in the group of proliferating breast disease cases which were connected with higher risk of breast cancer. Lack of p65 expression accompanied cases which were classified as fibroadenoma.

Expression of C-ERBB2 and P65 genes and their protein products in follicular neoplasms of thyroid gland.

C-ERBB2 and P65 gene expression was investigated by reverse transcriptase polymerase chain reaction method (RT-PCR) in thirty follicular thyroid cancers and twenty follicular adenomas. Additionally, the cancers and adenomas were stained by immunohistochemistry for the expression of their protein products. We did not observe P65 gene expression in any of the analyzed follicular cancers (n=30) but it was observed in 13 out of 20 (65%) follicular adenomas. The presence of C-ERBB2 gene expression was found in 18 (90%) follicular adenomas but not in cancers. There were 10 (50%) adenomas and 11 (36.7%) cancers with positive staining for C-ERBB2 protein and 15 (75%) adenomas and 2 (6.7%) cancers with positive staining for P65 protein. We conclude that expression of C-ERBB2 and P65 genes is associated with follicular adenoma but not with cancer of thyroid gland.

Expression of p65, DD3 and c-erbB2 genes in prostate cancer.

The expression of p65, DD3 and c-erbB2 genes was analyzed in 39 histologically verified human prostate cancers. The expression of p65 and DD3 genes was observed in significant percentage in well- and moderately-differentiated tumors. Both genes expression was lower in poorly differentiated tumors. On the contrary, c-erbB2 gene expression increased with advanced histological grading and reached the highest percentage in poorly-differentiated cancers. In the all investigated groups straight dependence between p65 and DD3 genes expression occurred. Opposite dependence was noticed in expression of p65/DD3 and c-erbB2 genes.

Expression of gene encoding P65 oncofetal protein in acute and chronic leukemias.

In this study we have established conditions for p65 gene expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR). On the basis of this technique we analyzed p65 gene expression in various types of leukemia: acute myeloblastic leukemia (AML) (n=26); acute lymphoblastic leukemia (ALL) (n=26) and chronic lymphocytic leukemia (CLL) (n=40). The highest frequency of p65 gene expression was found in the patients with CLL (66%). No relationship between the expression of p65 gene and clinical stage of leukemia was observed. The lower percentage of positivity (presence of gene transcript) was seen in patients with ALL (42%) and AML (46%).

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia.

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.