Showing papers in "Experimental Oncology in 2006"

Reporting recommendations for tumor marker prognostic studies (remark).

Abstract: Despite years of research and hundreds of reports on tumor markers in oncology, the number of markers that have emerged as clinically useful is pitifully small. Often, initially reported studies of a marker show great promise, but subsequent studies on the same or related markers yield inconsistent conclusions or stand in direct contradiction to the promising results. It is imperative that we attempt to understand the reasons that multiple studies of the same marker lead to differing conclusions. A variety of methodologic problems have been cited to explain these discrepancies. Unfortunately, many tumor marker studies have not been reported in a rigorous fashion, and published articles often lack sufficient information to allow adequate assessment of the quality of the study or the generalizability of study results. The development of guidelines for the reporting of tumor marker studies was a major recommendation of the National Cancer Institute - European Organisation for Research and Treatment of Cancer (NCI - EORTC) First International Meeting on Cancer Diagnostics in 2000. As for the successful CONSORT initiative for randomized trials and for the STARD statement for diagnostic studies, we suggest guidelines to provide relevant information about the study design, preplanned hypotheses, patient and specimen characteristics, assay methods, and statistical analysis methods. In addition, the guidelines suggest helpful presentations of data and important elements to include in discussions. The goal of these guidelines is to encourage transparent and complete reporting so that the relevant information will be available to others to help them to judge the usefulness of the data and understand the context in which the conclusions apply.

Epigenetic transfer of metastatic activity by uptake of highly metastatic B16 melanoma cell-released exosomes.

Abstract: AIM To investigate potential role of highly metastatic BL6-10 tumor cell-released exosomes (EXO) in transfer of metastatic activity into poorly metastatic tumor cell line F1. METHODS The highly metastatic B16 melanoma cell line (BL6-10) was generated in our laboratory. EXO from this cell line were isolated and amount of exosomal recovered proteins was measured using Bradford assay. For phenotypic analysis BL6-10 and F1 melanoma cells were stained with FITC-conjugated anti-MHC I (H-2K(b)), MHC II (Ia(b)) and Met 72 antibodies and analyzed by flow cytometry. C57BL/6 mice (8 per group) were injected (i. v.) with 0.5 x 10(6) F1, BL6-10 and F1(EXO) melanoma cells. Lungs were removed 4 weeks after tumor cell injection, fixed in 10% neutral buffered formaldehyde and embedded in paraffin for histological analysis. RESULTS Data revealed that BL6-10 cells expressed metastasis marker (Met 72 tumor antigen), while F1 cells did not display this cell surface marker. All mice inoculated with BL6-10 melanoma cells had numerous lung tumor colonies, while mice injected with F1 tumor cells were free of lung metastatic colonies. BL6-10 tumor cells-released EXO also expressed Met 72 tumor antigen as BL6-10 tumor cells, but in less amount. F1 tumor cells can uptake EXO from BL6-10 tumor cells and express acquired exosomal Met 72 tumor antigen. CONCLUSION The metastatic activity of highly metastatic BL6-10 tumor cells can be transferred to poorly metastatic F1 tumor cells by uptake of highly metastatic BL6-10 tumor-released EXO.

Anticarcinogenic and antiplatelet effects of carvacrol.

Abstract: Aim To investigate the effect of carvacrol on chemical carcinogenesis, cancer cell proliferation and platelet aggregation, and to find possible correlation between all these processes and the antioxidant properties of carvacrol. Materials and methods 3,4-benzopyrene-induced carcinogenesis model using Wistar rats was used. Leiomyosarcoma cells from Wistar rats were used to study carvacrol antiproliferative activity in vitro. The carvacrol antiplatelet properties were investigated with platelet aggregation assay and flow cytometry technique. The production of thromboxane B2, final metabolite of platelet aggregation, was evaluated by radioimmunoassay. Results Our study revealed significant anticarcinogenic properties of carvacrol. We observed 30% decrease of 3,4 benzopyrene carcinogenic activity in vivo. Antiproliferative activity of carvacrol (IC(50)) was 90 microM and 67 microM for 24 h and 48 h of incubation of cells, respectively. Carvacrol possessed also mild antiplatelet effect, inducing the decrease of thromboxane A2 production in platelets and as a result - restrictive expression of the GPIIb/IIIa platelet receptor. Conclusion Our data demonstrated that carvacrol possesses anticarcinogenic, antiproliferative and antiplatelet properties.

The antitumor activity of thymoquinone and thymohydroquinone in vitro and in vivo.

Abstract: Aim: The aim of the study was to investigate antitumor activity of thymoquinone (TQ) and thymohydroquinone (THQ) in vitro and in vivo Materials and Methods: In the in vitro experiments, one L929 mouse fibroblasts and two tumor cell lines (squamous cell carcinoma (SCC VII) and fibrosarcoma (FsaR)) were used The cells were cultured with 01 or 001 mg/ml TQ or THQ for 24 h, and cytotoxicity assay was performed with the use of crystal violet staining technique For in vivo antitumor efficiency evaluation of new compounds two murine tumor models (fibrosarcoma (FsaR) and squamous cell carcinoma (SCC VII)) were used The used dose was equal for both substances Antitumor effect of 4 intratumoral injections of TQ and THQ at the dose of 5 mg/kg was evaluated by comparison of tumor growth kinetics between treated and control animals Results: In vitro study have showh that TQ and THQ exhibit statistically significant cytotoxic activity (p < 001) The cytotoxic activity was dose dependent and more expressed against tumor cells than against L929 fibroblasts The result of antitumor activities of TQ and THQ in vivo reached TGI = 52% and it was statistically significant (p < 005) Conclusion: The results indicate that THQ antitumor activity may be improved with further dose increase of the investigated substance Key Words: thymoquinone, thymohydroquinone, antitumor activity, in vitro, in vivo, mice

Ganoderma lucidum extract inhibits proliferation of SW 480 human colorectal cancer cells.

Abstract: Aim: Ganoderma lucidum is a commonly used Chinese herb and an important ingredient in traditional Chinese medicine herbal formulations for immune dysfunction related illnesses. The effects of this medicinal mushroom on human colorectal cancer cells have not yet been evaluated. In this study, we investigated the effects of Ganoderma lucidum extract using SW 480 human colorectal cancer cell line. Materials and Methods: Two different fractions of Ganoderma lucidum extract, i.e., a fraction containing mainly polysaccharides (GLE-1), and a triterpenoid fraction without polysaccharides (GLE-2) were analyzed. Their antiproliferative activity was evaluated by cell proliferation assay and 3 H-thymidine incorporation assay. Scavenging effects of DPPH radical were assessed using ESR-spectroscopy. Results: Our data showed that both GLE-1 and GLE-2 significantly inhibited the proliferation of SW 480 cells. The inhibitory effect of GLE-2 was much stronger than that of GLE-1. GLE-1 inhibited DNA synthesis in the cells and reduced the formation of DPPH radicals. Conclusion:

Experimental bladder carcinogenesis-rodent models.

Abstract: Several rodent models of bladder cancer development have been established. The aim of this review article is to provide a critical assessment of different animal models available for the study of bladder carcinogenesis, its chemoprevention and therapy. All, except for transgenic and knockout animals, require 8-12 months experimental periods in order to generate a high yield of neoplasias. Spontaneous bladder tumor models are extremely rare. The significance of the results from animal experiments is dependent upon the selection of a suitable animal model. There are no rules regarding the choice of a model, it is however very useful to have knowledge of relevant comparative medical aspects concerning this subject. We describe chemical carcinogens most commonly used to induce bladder cancer, pellet implantation and urinary calculi, agents that promote bladder cancer, and irradiation. We also evaluated other tools such as cell cultures, tumor implantation and transgenic models for bladder cancer, that have been developed to study the process. The review considers how several imaging techniques can be applied to study rodent bladder carcinogenesis.

Antitumor activities of the four sesquiterpene lactones from Elephantopus scaber L.

Abstract: Aim To evaluate antitumor activity of sesquiterpene lactones (scabertopin (ES-2), isoscabertopin (ES-3), deoxyelephantopin (ES-4), isodeoxyelephantopin (ES-5)) isolated from Elephantopus scaber L. in vitro and in vivo. Methods SMMC-7721, Caco-2 and HeLa cell lines were treated with ES-2,3,4,5. Cell viability was determined by MTT assay. Agarose gel electrophoresis was used to detect DNA fragmentation. To evaluate in vivo antitumor activity of ES-4, experimental murine tumor model was used. Results It was shown that ES-2, ES-4, ES-5 exhibited significant antitumor effect in vitro in a concentration-dependent manner. However, the effect of ES-3 on the growth of tested cell lines was relatively weak. In HeLa cells exposed to ES-4 for 48 h, morphological changes and DNA ladder pattern evidencing on apoptosis were detected. ES-4 revealed in vivo antitumor activity. Conclusion Antitumor activity of studied sesquiterpene lactones may be due, at least in part, to induction of apoptosis in vitro. ES-4 possesses also antitumor activity in vivo.

The role of cadherin/catenin complex in malignant melanoma.

Abstract: In the present review article the role of cadherin/catenin complex in cases of malignant melanoma is discussed in some detail. Cadherins represent the most important superfamily of adhesion molecules with epithelial E-cadherin being the most studied. Its role in normal state as well as in cancer invasion and metastasis and some other pathologies is crucial. E-cadherin expression is altered in malignant melanomas and its downregulation or absence is associated with melanoma invasion and metastasis potential. A shift from E-cadherin expression to neural N-cadherin expression in melanocytes is also detected in malignant melanomas formation. In addition, a discussion regarding the role of placental P-cadherin and vascular endothelial VE-cadherin as well as the recently identified molecule of dysadherin, is attempted in brief.

Leptin and resistin levels in serum of patients with hematologic malignancies: correlation with clinical characteristics.

Abstract: AIM To evaluate leptin and resistin levels in patients with various hematologic malignancies. METHODS We included 21 patients with lymphoma, 14 with multiple myeloma (MM), 14 with acute leukemia, 13 with chronic lymphocytic leukemia (CLL), and 25 healthy control subjects into our study. The subjects' body mass indexes (BMI) were calculated; hematological and acute phase response parameters, serum lipid were determined; serum leptin and resistin levels were determined by ELISA. RESULTS Serum leptin level was significantly increased in CLL and MM groups when compared to the control group (p less, similar 0.01). Resistin level was significantly higher in lymphoma patients than in CLL, acute leukemia and control groups (p less, similar 0.01). In the control group, leptin level was negatively correlated with hemoglobin level (r = -0.44, p = 0.047); and in all patients with hematologic malignancies, leptin level was correlated with BMI (r = 0.32, p = 0.02). Leptin in lymphoma subjects correlated with hemoglobin level (r = 0.64, p = 0.005), resistin level correlated with the platelet count in patients with hematologic malignancies (r = 0.26, p = 0.044). In addition, leptin level had negative correlations with international prognostic score (IPS) in Hodgkin lymphoma (r = -0.9, p = 0.002) and with international prognostic index (IPI) in non-Hodgkin lymphoma (r = -0.77, p = 0.03). In CLL patients, leptin level had a correlation with the poor prognostic marker - CD38 level (r = 0.68, p = 0.03). CONCLUSION We found higher leptin levels in MM and CLL patients, and higher resistin levels in lymphoma patients: this fact demonstrates that changes in adipose tissue and metabolism occur in these disease states.

beta-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of Bcl-2 family and activation of caspases.

Abstract: Aim To study in vitro the molecular mechanism of apoptosis caused by beta-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae). Materials and methods The study was carried out on human bladder carcinoma T24 cell line. Determination of cell viability was done using trypan blue exclusion method, apoptosis quantitative estimation - by DAPI staining and agarose gel electrophoresis for DNA fragmentation. Flow cytometry analysis, RT-PCR and Western blot analysis, colorimetric assay of caspase activity were applied as well. Results It was found that in micromolar range of concentrations beta-lapachone inhibited the viability of T24 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation. Treatment of T24 cells with beta-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression. beta-lapachone-induced apoptosis was also associated with activation of caspase-3 and caspase-9, inhibition of IAP expression, and degradation of poly (ADP-ribose) polymerase, phospholipase C-gamma1 and beta-catenin proteins. At the same time Fas and FasL levels were inhibited upon treatment with beta-lapachone in a concentration-dependent manner. Conclusion beta-lapachone-induced apoptosis in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway.

The clinical significance of soluble E-cadherin in nonsmall cell lung cancer.

Abstract: AIM Aberrant expression of the epithelial transmembrane adhesion molecule E-cadherin (E-cad) has been associated with many human malignancies. In the present study the clinical significance of serum levels of soluble E-cadherin (sE-cad) in newly diagnosed patients with non small cell lung cancer (NSCLC) was investigated. MATERIAL AND METHODS An enzyme linked immunospecific assay (ELISA) to determine the circulating levels of sE-cad in 20 newly diagnosed patients with NSCLC as well as in 29 healthy volunteers (control group) was used. RESULTS NSCLC patients exerted increased circulating levels of sE-cad compared with individuals of the control group (p < 0.001). An association was also detected between serum sE-cad levels and the development of distant metastases. On the contrary, no statistically significant correlation could be established with histological type, gender and smoking habits. Patients with increased sE-cad levels at diagnosis had worser outcome, although multivariate analysis failed to demonstrate that sE-cad levels represent an independent prognostic factor of survival. CONCLUSION Our data suggest that E-cad plays a role in the pathogenesis of NSCLC. sE-cad levels may be further studied as a potential prognostic biomarker.

Upper extremity DVT in oncological patients: analysis of risk factors. Data from the RIETE registry.

Abstract: AIM The aim of the study is to up date informations on the clinical characteristics and outcome of patients with upper-extremity deep vein thrombosis (DVT) from the Informatised Registry on Venous Thromboembolism (RIETE). METHODS RIETE is an ongoing registry of consecutive patients with symptomatic, objectively confirmed, acute venous thromboembolism. In this analysis the clinical characteristics and 3-month outcome of all cancer patients with upper-extremity DVT were evaluated. RESULTS Up to February 2006, a total of 14,391 patients with symptomatic, objectively confirmed acute venous thromboembolism had been enrolled in RIETE. Of the 2,945 patients with active cancer 196 (6.7%) had arm DVT: 104 had catheter-associated DVT. Most cancer patients with arm DVT were males, younger than 65, and had a low incidence of additional risk factors or underlying diseases. Twenty of them (10%) had symptomatic pulmonary embolism (PE). Most patients were treated with low-molecular-weigh heparin, both initially (94%) and after discharge (75%). During the 3-month follow-up period 12 patients (6.1%) developed VTE recurrences (PE 6, DVT 6), 8 (4.1%) had major bleeding (fatal in 3), 43 (22%) died. CONCLUSIONS Our data from the RIETE registry show that upper limb DVT is a serious complication in patients with cancer, with a high incidence of recurrences and bleeding complications.

Establishment of serum protein pattern for screening colorectal cancer using SELDI-TOF-MS.

Abstract: AIM The purpose of this study is to develop a proteomic pattern for distinguishing individuals with colorectal cancer from healthy controls and monitoring micrometastasis using SELDI-TOF-MS. METHODS A training set consisting of 63 patients with colorectal cancer, 20 patients with benign colorectal diseases and 26 healthy volunteers was used to develop a proteomic model that discriminated colorectal cancer effectively. The sensitivity and specificity of this model was validated by an independent test set. To explore serum proteins changed after operation, the protein profiles of 31 postoperative patients were compared with those of preoperative patients. We also analyzed protein profiles of patients with and without metastasis to monitor micrometastasis. RESULTS Our study yielded a four-peak model (m/z: 3191.5, 3262.9, 3396.3 and 5334.4) that discriminated cancer from non-cancer samples with sensitivity of 90.3% and specificity of 95.7%. This model was validated in the test set with sensitivity of 87.5% and specificity of 93.8% which was significantly better than the combination use of CEA, CA199 and CA242 (sensitivity 62.4%) for early detection of colorectal cancer. Two peaks (m/z: 2753.8 and 4172.4) were found down-regulated in postoperative samples comparing with preoperative samples. We also detected two proteins (m/z: 9184.4 and 9340.9) that can discriminate patients with primary colorectal cancer from metastatic colorectal cancer. CONCLUSIONS The four-peak model and two peaks (m/z: 2753.8 and 4172.4) detected in this study have the potential for assistance in diagnostics and therapeutic strategies in colorectal cancer and the two proteins (m/z: 9184.4 and 9340.9) were effective biomarkers for monitoring micrometastasis.

Serum interleukin-12 and interleukin-18 levels in patients with oesophageal squamous cell carcinoma.

Abstract: UNLABELLED Interleukin-12 (IL-12) and interleukin-18 (IL-18) play an important role as immunomodulatory factors in cancer pathogenesis. THE AIM of the study was analyze changes of serum IL-12 and IL-18 concentrations in oesophageal squamous cell carcinoma patients depending on the progression of cancer. MATERIALS AND METHODS Blood samples were taken from 41 patients with oesophageal cancer: 5 women and 36 men, mean age 59+/-9 years. 23 patients had surgical resection of oesophagus with II and III tumor stage, 18 patients with IV stage of cancer progression were treated by palliative procedures. The control group included 15 healthy blood donors: 4 female and 11 males, mean age 41+/-6 years. The concentrations of IL-12 and IL-18 were determined by ELISA tests. RESULTS Serum IL-12 and IL-18 amounts detected in blood of oesophageal cancer patients were significantly higher in comparison to control group (p<0.001). Serum IL-12 level was higher in patients with IV stage of the disease than in patients with II and III stages. Also serum IL-18 level was significantly higher in patients with IV stage in comparison to patients surgically treated (p<0.05). Statistically significant differences were found in concentrations of IL-18 according to clinicopathological parameters such as: stage of cancer progression, tumor depth, lymph node metastasis (p<0.05). CONCLUSIONS Serum IL-12 and IL-18 levels are significantly higher in oesophageal cancer patients than in the healthy subjects. A relation between IL-18 content and cancer progression has been registered.

Expression profiling of cyclin B1 and D1 in cervical carcinoma.

Abstract: Aim Cyclins are a family of regulatory proteins that play a key role in controlling the cell cycle. Abnormalities of cell cycle regulators, including cyclins and cyclin dependent kinases, have been reported in various malignant tumors. This study was undertaken to quantitatively detect cyclin B1 and D1 in cervical cancer. Methods A quantitative real-time reverse transcription polymerase chain reaction and Western blot assay were used to analyze the expression of cyclin B1/D1 mRNA and proteins, respectively, in fresh invasive cervical cancer (n = 41) and normal cervical tissues (n = 10). Results There was significantly greater cyclin B1 expression in invasive cervical cancer than in normal cervical tissue (P = 0.019). However, cyclin D1 expression was not significantly different. A Western blot assay yielded similar results. Conclusion Our results were consistent with the concept that up-regulation of cyclin B1 expression occurred in cervical cancer and an aberrant expression of cyclin B1 might play an important role in cervical carcinogenesis.

MicroRNAs in normal and cancer cells: a new class of gene expression regulators.

Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at posttranscriptional level. They are involved in cellular development, differentiation, proliferation and apoptosis and play a significant role in cancer. This review describes miRNA biogenesis, their functions in normal cells, and alterations of miRNA sets in cancer and roles of antitumorigenic and oncogenic miRNAs in cancer development.

The role of CD44 adhesion molecule in oral cavity cancer.

Abstract: CD44 is an adhesion molecule, member of the transmembrane glucoprotein family, with large number of isoforms identified in many human tissues, with particularly high expression in proliferating cells and squamous cell epithelium. This review is focused on the role of CD44 in the carcinogenesis of the oral cavity with special emphasis on its potential use for diagnosis and prognosis of cancer of oral cavity.

Relationship between HPV16/18 E6 and 53, 21WAF1, MDM2, Ki67 and cyclin D1 expression in esophageal squamous cell carcinoma: comparative study by using tissue microarray technology.

Abstract: Waf1 , MDM2, Ki67 and cyclin D1 proteins on TMA slides. In situ hybridization (ISH) targeting HPV gene was also used. Results: In ESCC samples, 18.3% (11/60) were revealed HPV16/18 E6 positive by IHC, while 40.0% (24/60) HPV positive by ISH; HPV16/18 E6 expression was significantly higher than that of control samples. In ESCC samples, the expressions of p53, p21 Waf1 , Cyclin D1, MDM2 and Ki67 were recorded in 60.0% (36/60), 40.0% (24/60), 51.7% (31/60), 65.0% (39/60) and 88.3% (53/60) cases respectively, In ESCC samples, p53, MDM2 and Ki67 expression correlated with the HPV16/18 E6 expression (p < 0.01), p21 Waf1 expression — with these of MDM2 and cyclin D1 (p < 0.01) whilst expression of Ki67 — with ESCC grade (p < 0.01). Conclusion: HPV might be one of etiological factor of esophageal carcinoma in Shantou, China. p53, MDM2 proteins may play important roles in the pathogenesis of HPV-associated ESCC.

Effects of radical oxygen species and NO: formation of intracellular hypoxia and activation of matrix metalloproteinases in tumor tissues.

Abstract: AIM To establish the association between the radical oxygen species (ROS) and NO levels in the tumor cells mitochondria, between cell hypoxia development and activation of matrix metalloproteinases-2 and -9. MATERIALS AND METHODS Electron paramagnetic resonance (EPR) at room temperature and at the temperature of liquid nitrogen (77 degrees K), spin traps technology, enzymography in polyacrylamide gel were applied. RESULTS Redox-centers in the respiration cascade of mitochondria have been revealed, multiple oxidative damage of which in breast and liver cancer tissues of experimental animals as well as in tumor tissue from patients with gastric cancer promote the development of cell hypoxia. Involvement of ROS and NO in activation of latent forms of matrix metalloproteinases in gastric tumor tissues has been shown. CONCLUSION We hypothesize that superoxide radical-anions participate in development of cell hypoxia in tumors and surrounding normal tissues inducing activation of latent forms of matrix metalloproteinases.

Analysis of RAD51 polymorphism and BRCA1 mutations in Polish women with breast cancer.

Abstract: Aim Breast cancer is one of the major killers worldwide. The objectives of this study were to determine the frequency of BRCA1 germ-line mutations and the RAD51 G/C polymorphism in patients with breast cancer. Methods 100 breast cancer women provided blood for mutation analysis. Blood samples age matched healthy individuals (n = 106) served as control. The G/C polymorphism and BRCA1 mutations were determined by PCR-RFLP methods. Results The distribution of the genotypes of the G/C polymorphism RAD51 in both control and patients did not differ significantly from those predicted by the Hardy - Weinberg distribution. There were no significant differences in the genotype distributions and allele frequencies between node-positive and node-negative patients. In present study one Ex20insC mutations of BRCA1 gene was identified in women with breast cancer. Conclusion Our study implies that the G/C polymorphism of the RAD51 gene may not be directly involved in the development and=or progression of breast cancer.

Expression of adaptor protein Ruk/CIN85 isoforms in cell lines of various tissue origins and human melanoma.

Abstract: Aim: Development of monoclonal and polyclonal antibodies against recombinant GST-fused proteins including correspondingly N- and C-terminal parts of Ruk/CIN85 adaptor protein. Analysis of Ruk/CIN85 expression patterns in cell lines of various tissue origins and human melanoma. Methods: Recombinant GST-fused fragments of Ruk/CIN85 were expressed in bacterial system and affinity purified. Monoclonal antibodies against SH3A domain of Ruk/CIN85 were produced using hybridoma technique. The specificity of generated antibodies was examined by ELISA. Polyclonal antibodies against C-terminal coiled-coil region of Ruk/CIN85 were affinity purified from serum of immunized rabbit. Expression patterns of Ruk/CIN85 isoforms and their subcellular localization in cell lines of various tissue origins and human melanoma samples were analyzed by immunoblotting, immunoprecipitation and immunofluorescence microcopy. Results: Ruk/CIN85 is ubiquitously expressed SH3-containing adaptor/scaffold protein which plays important roles in signalling processes. N-terminal half of Ruk/CIN85 molecule, including three SH3 domains, and its C-terminal coiled-coil region were used as antigens to produce monoclonal and polyclonal antibodies, respectively. Hybridoma cell lines secreting monoclonal antibodies (mAbs) to SH3 fragment of Ruk/CIN85 were established. One of the mAbs was extensively characterized and designated as MISh-A1. It was shown that this mAb recognizes an epitope, which resides within first SH3A domain. Polyclonal anti-Ruks Abs affinity purified from serum of immunized rabbit specifically recognized main Ruk/CIN85 isoforms, both endogenous and recombinant, in lysates of HEK293 cells. Notably, produced Abs did not cross-react with CD2AP, the member of the same family of adaptor/scaffold proteins. Multiple molecular forms of Ruk/CIN85 with apparent molecular weights of 130, 80–85, 70–75, 50–56, 34–40 and 29 kD were detected in cell lyzates of NIH3T3, Cos1, L1210, HEK293, Ramos, HeLa S3, MDCK, C6, A549 and U937 using anti-Ruk antibodies. Oligomerization between p85 and p50–56 forms of Ruk/CIN85 was revealed in C6 and NIH3T3 cells, but not in HeLa S3 and HEK293 cells by immunoprecipitation using MISh-A1 antibody following anti-Ruk Western-blot analysis. Using immunofluorescent microscopy and anti-Ruk antibodies, endogenous Ruk-variates were found mostly in cytoplasm of C6, NIH3T3, HEK293 cells and at lower level — in nuclei. Conclusion: Patterns of Ruk/CIN85 molecular forms expression are cell-specific and determined by cellular context. Assembly of oligomeric complexes between p85 and p50–56 Ruk/CIN85 isoforms in C6 and NIH3T3 cells but not in HeLa S3 and HEK293 cells may reflect their specific biological roles in different cell lines. High level of full-length Rukl/CIN85 form expression was revealed in extracts of human melanoma samples. Abs described in this paper may prove useful in future studies of Ruk/CIN85 expression and function in normal and transformed cells.

Patterns of hematological malignancies in Chernobyl clean-up workers (1996-2005).

Abstract: Aim: The question as to whether the incidence of leukemias and malignant lymphomas among the Chernobyl clean-up workers increased in 20 years after the catastrophe is still a point of much controversy. Precise diagnosis of the main forms of hematopoietic malignancies according to FAB classification and new WHO classification and comparison of these data with that in the general population will be helpful in estimating the relative contribution of the radiation factor to the overall incidence of such pathologies. Patients and methods: The data on 218 consecutive cases of malignant diseases of hematopoietic and lymphoid tissues in Chernobyl clean-up workers diagnosed in 1996–2005 are given in comparison with the data of 2697 consecutive patients of general population of the same age group. The morphology and cytochemistry of bone marrow and peripheral blood cells were studied. Immunocytochemical techniques (APAAP, LSAB-AP) and the broad panel of monoclonal antibodies to lineage specific and differentiation antigens of leukocytes were employed for immunophenotyping leukemic cells. Results: Various types of oncohematological diseases developing 10–20 years after Chernobyl accident were registered in a group of clean-up workers under study including myelodysplastic syndromes (MDS), acute leukemias (ALL and AML), chronic myelogenous leukemia (CML) and other chronic myeloproliferative diseases, chronic lymphocytic leukemia (B-CLL) and other chronic lymphoproliferative diseases of B and T cell origin. MDS percentage among patients of clean-up workers group tended to exceed MDS percentage in the group of patients representing the general population examined at the same period (4.58 vs. 3.70%). Among 34 AML cases, leukemia was preceded by MDS in seven patients. The relative contribution of CML to the total number of clean-up workers with leukemia was higher than the corresponding percentage value in general population examined at the same period (9.17 vs. 6.59%). B-CLL was a predominant form of hematopoietic malignancies in clean-up workers under study (25.68%). Nevertheless, B-CLL percentage in patients of clean-up workers group did not differ significantly from that in the patients of general population. The multiple myeloma percentage (7.79%) in the group of patients belonging to clean-up workers in our study turned out to be twice as much as in the patients of general population (4.0%). Conclusion: The verified diagnosis of tumors of hematopoietic and lymphoid tissue according to modern classification (EGIL, WHO) could be the prerequisite for further molecular genetic and analytical epidemiology study of leukemias that may be related to Chernobyl NPP accident consequences.

Immunohistochemical studies of protein kinase D (PKD) 2 expression in malignant human lymphomas.

Abstract: AIM To study the PKD2 expression, autophosphorylation and localization in reactive lymph nodes and tumors of lymphoid tissues. MATERIALS AND METHODS Specific antibodies, which recognize PKD1/2 or PKD2 and autophosphorylated PKD1/2, were used for immunohistochemical and biochemical studies of tonsils, reactive lymph nodes, tumor samples of non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). RESULTS Immunohistochemical and biochemical analysis of PKD1 and PKD2 expression showed PKD2 expression in tonsils, reactive lymph nodes and tumor tissues from patients with NHL and HL. Furthermore, we were not able to reveal PKD1 expression in studied lymphoid tissues. In tonsils and reactive lymph nodes the PKD2 expression was detected in T and B cell zones with highest level in germinal centers of lymphoid follicles and the maximum level of autophosphorylation in the light zones of the germinal centers. We found that low level of PKD2 expression and autophosphorylation was characteristic feature for mantle cell lymphomas, Burkitt's lymphomas, and in 50% of CLL/small lymphocytic lymphomas. Lymphoma cells of germinal center origin and with activated B cell phenotype (diffuse large B cell lymphomas, HL) and anaplastic large cells lymphoma demonstrated the high level of PKD2 expression and autophosphorylation. CONCLUSIONS The level of PKD2 expression and autophosphorylation in neoplastic cells corresponds to the expression pattern of this kinase in their normal analogs, and to the level of cell differentiation and activation.

Matrix metalloproteinase-9 in broncho-alveolar lavage fluid of patients with non-small cell lung cancer.

Abstract: AIM To evaluate concentration of MMP-9 in blood plasma and broncho-alveolar lavage fluid (BALF) from patients with non-small cell lung cancer (NSCLC). METHODS Blood plasma from 40 NSCLC patients and 40 healthy donors was collected and concentrations of blood plasma and BALF MMP-9 were measured using ELISA. Correlation between MMP-9 level and gender, histological type of tumor and stage of disease was analyzed. RESULTS Levels of blood plasma MMP-9 were significantly higher in NSCLC patients (p < 0.0001) then in control group, and were especially high in patients with stage IV of disease (stage I vs stage IV - p < 0.005, stage II vs stage IV - p < 0.01, stage III vs stage IV - p < 0.01). Also, stage IV of NSCLC was characterized by the highest level of BALF MMP-9 (stage I vs stage IV - p < 0.002, stage II vs stage IV p < 0.002, and stage III vs stage IV p < 0.007). Correlation between blood plasma and BALF MMP-9 levels and gender or histological type of tumor was insignificant. CONCLUSION Our data revealed significant correlation between tumor stage and BALF and plasma MMP-9 levels in NSCLC patients.

The efficacy of the thyroid peroxidase marker for distinguishing follicular thyroid carcinoma from follicular adenoma.

Abstract: Aim Expression of thyroid peroxidase (TPO) in the thyroid gland tissue is well known as a sensitive marker of the thyroid malignancy. We have evaluated immunohistochemical assay of TPO for distinguishing follicular thyroid carcinoma from follicular adenoma. Materials and methods Sections of formalin-fixed tissues obtained from 92 patients with thyroid tumors (52 follicular carcinomas and 40 follicular adenomas including the Hurthle cell type) were analyzed using a monoclonal antibody (TPO mAb 47) and the avidin-biotin peroxidase complex immunohistochemical technique. Lesions with staining of more than 80% of the follicular cells/specimen were considered benign, while less than 80% were considered malignant. Results TPO immunostaining correlated with the histopathological diagnosis in 24/40 cases of follicular adenomas and 41/52 cases of follicular carcinomas, giving a specificity of 60% and a sensitivity of 79%. Conclusion These results suggest that immunohistochemical assay of TPO expression has limited value for the differential diagnosis of follicular thyroid carcinoma from thyroid follicular adenoma.

Differential effect of selected methylxanthine derivatives on radiosensitization of lung carcinoma cells.

Abstract: AIM Using caffeine as a reference derivative, this study was performed to investigate how other methylxanthine derivatives, theophylline, 3-isobutyl-methylxanthine and 1,3-dipropyl-7-methylxanthine, sensitize cells to radiation by modifying cell cycle checkpoints and inducing the apoptotic response. The effect of the methylxanthine derivatives was studied in response to gamma and ultraviolet radiation in a human large cell lung carcinoma cell line, null for p53, a normal lung epithelial cell line and the large cell lung carcinoma cell line stably transfected with p53. METHODS Effects of theophylline, 3-isobutyl-methylxanthine and 1,3-dipropyl 7-methylxanthine on cell-radiosensitization in comparison to caffeine tested by clonogenic survival assay, MTT assay, ELISA based apoptotic assay, flow cytometry, caspase-3 activity, TUNEL assay, and western blot analysis. RESULTS All the derivatives, except 3-isobutyl-methylxanthine, increased tumor cell sensitization to radiation by inducing apoptosis in the p53-null lung cancer cell line. The pattern of cell cycle progression revealed that these derivatives increased the number of cells in G1 phase by abrogating the G2/M checkpoint, directing the cells to apoptose through a p53-independent mechanism. In contrast, 3-isobutyl-methylxanthine was more potent than the other derivatives in radiosensitization of normal lung epithelial cells and the lung carcinoma cells stably transfected with wild-type p53. IBMX increased p53 protein level more than caffeine in lung carcinoma cells stably transfected with wild-type p53. CONCLUSION Our results suggest that 3-isobutyl-methylxanthine might function through a p53-dependent mechanism.

Expression of clusterin, XIAP and survivin, and their changes by camptothecin (CPT) treatment in CPT-resistant PC-3 and CPT-sensitive LNCaP cells.

Abstract: Aim Clusterin and IAPs (inhibitor of apoptosis proteins), such as survivin and XIAP, are known to be related to chemo-resistance in several cancer cells. In the current study, we investigated their expression levels in human prostate cancer cell lines, LNCaP and PC-3 which are sensitive and resistant to camptothecin (CPT), topoisomerase I inhibitor, respectively. Methods LNCaP and PC-3 cells were cultured in the presence of CPT, cell death was evaluated using Hoechst 33342 and propidium iodide (PI) double staining. The expression of clusterin, XIAP and survivin on mRNA and protein levels was investigated by semi-quantitative RT-PCR and Western blotting, respectively. Results Our data showed that 24 h treatment of LNCaP cells with 0.5 and 3.0 microM CPT resulted in higher number of apoptotic cells, than that in PC-3 cells. Western blot analysis revealed that the clusterin level in PC-3 cells was 5-fold higher than that in LNCaP cells. In contrast, XIAP expression level in PC-3 cells was lower than that in LNCaP cells, and survivin levels were similar in these two cell lines. Treatment with 0.5 and 3.0 microM CPT resulted in the reduced survivin and XIAP expression in both cell lines, while clusterin expression remained unchanged in LNCaP cells, but was increased in PC-3 cells. Conclusion The results suggest that clusterin may take a greater part in CPT-resistance than survivin and XIAP in PC-3 cells.

Application of DNA from mushroom Pleurotus ostreatus for cancer biotherapy: a pilot study.

Abstract: AIM In present work, the attempt to study immunomodulatory activity and biotherapeutical potential of DNA isolated from the fruit body of P. ostreatus was made. METHODS The efficacy of biotherapeutic application of mushroom DNA was evaluated on the BALB/c mice with subcutaneously transplanted Ehrlich carcinoma. The effect of Pleurotus ostreatus DNA on NK activity was studied in vitro using nonspecific cytotoxicity assay. RESULTS Application of mushroom DNA resulted in augmentation of NK cytotoxic activity in vitro and significant increase of the life span of mice with solid Ehrlich carcinoma. CONCLUSION The observed effects of P. ostreatus DNA administration can be probably explained by the presence of immunostimulatory unmethylated CpG motifs in DNA.

Zinc ribbon domain containing 1 protein: modulator of multidrug resistance, tumorigenesis and cell cycle.

Abstract: Zinc ribbon domain containing 1 (ZNRD1) gene encoding a protein consisting of two zinc ribbon domains was recently cloned from the human HLA locus. So far, ZNRD1 has been found implicated in transcription regulation and might play potential roles in mediating several biological processes, including multidrug resistance, tumorigenesis and cell cycle. This article reviewed these recent findings and provided additional information to support the role of ZNRD1 gene as a novel candidate DNA damage repair related gene.

The function of poliamine metabolism in prostate cancer.

Abstract: In many developed countries prostate cancer is the second leading cause of cancer related death in human population. Prostate tissue is characterized by the highest level of polyamines among organs in human body, and it is even higher in prostate carcinomas. These ubiquitous molecules are synthesized by prostate epithelium and are involved in many biochemical processes including cell proliferation, cell cycle regulation and protein synthesis. In this review we made the attempt to discuss the functions of polyamines, their involvement in apoptosis and potential role as molecular biomarker for prostate cancer. Also we present recent data on generation of drugs, in particular, cyclin dependent kinase inhibitor, developed for therapy of prostate cancer.