Smoking as risk factor for cervical cancer.

In a matched case-control study which comprised 33 cases with cervical carcinoma in situ, 67 cases with invasive cervical cancer, and 100 hospital controls, ever-smoking was found to be significantly related to cervical cancer (Odds ratio = 5.9, 95% confidence interval = 1.2-29.3) after adjustment for a variety of confounding variables.

Mortality trends of lung cancer in Austria and the Czech Republic.

This paper concerns itself with possible reasons for differences in lung cancer (ICD9-162) mortality rates in Austria and the Czech Republic. Lung cancer mortality in Austrian men reached its peak in 1973 and decreased gradually after a plateau by 23% since then, while the Czech mortality rate in men was constantly increasing till 1986 and then started to decline by 21% till 1998. As far as women in both countries are concerned, the risk of dying from lung cancer has risen dramatically for the last 20 years. In Czech men the mortality rate between 1970-1998 was significantly higher than in Austrian men while in women the death rates were closely similar. Differences cannot be explained by different smoking habits. In the past occupational exposure to cancerogenic agents in the Czech Republic was certainly one of the futile factors for the different lung cancer mortality rates. However, nowadays, Austria and the Czech Republic have to cope with similar problems particularly with an increasing number of children and adolescents (especially females) starting smoking very early. Activities to prevent children and adolescents from starting or stopping to smoke will be the only way to control lung cancer epidemic in the 21st century.

Diet and the risk of lung cancer among women. A hospital-based case-control study.

Variation in diet has been suspected to be one of cofactors related to geographic variation in lung cancer risk, namely for women, or other population groups with a low exposure to cigarette smoking. The study has been designed to obtain more insight into possible associations between diet and lung cancer risk among women in a country with a Central European socioeconomic background. In a hospital-based case-control study personal interviews of 282 female lung cancer cases and 1120 female controls were done using a structured standard questionnaire. Cigarette smoking was the most important factor associated with excess risk for lung cancer among women. Significantly increased risk was found both among current smokers (OR = 9.22), and ex-smokers (OR = 7.11). Positive dose-response gradients (p < 0.001) were observed between lung cancer risk and the daily number of cigarettes, duration of smoking, and number of pack-years. For squamous-, small- and large-cell cancers combined, significant associations of lung cancer risk with the consumption of red meat and poultry (OR = 2.33, and OR = 8.67, respectively), and an inverse association with the consumption of vegetables (OR = 0.55) were found. No such variations in risk were observed for adenocarcinoma, including the bronchioalveolar cancer type. For all lung cancer types combined, coffee drinking showed a significant inverse association with lung cancer risk risk (OR = 0.66). While smoking is the major risk for lung cancer, diet may have a contributory role. Variations in the intake of some components of diet, namely red meat, poultry, vegetables, and coffee may contribute to understanding variations in the risk of lung cancer among Czech women.

Quantitative determination of telomerase activity in breast cancer and benign breast diseases.

Telomerase plays an important role in maintaining the stability of chromosomes. This ribonucleoprotein prevents chromosome ends (telomeres) from gradual loss with each cell division. It enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Telomerase activity has been detected in the majority of tumor and germ cells and in immortalized cell lines. Quantitative telomerase PCR-ELISA (TeloTAGGG Telomerase PCR ELISA(PLUS)) was evaluated for distinguishing benign and malignant breast tissue. Activity of telomerase was determined in 27 samples of fibrocystic and dysplastic tissues, 28 fibroadenomas and phylloid tumors, and 154 breast cancer tissues; 59 specimens were analyzed retrospectively. Analytical precision and linearity of the assay was tested using breast carcinoma cell line ZR-75-1 and breast tumor tissue extracts. About 4% of tumor samples were excluded from analysis due to interferences in the PCR reaction. Relative telomerase activity differed significantly in the groups of dysplastic tissues, fibroadenomas and carcinomas. The highest activity was found in breast cancer tissue. This method can identify breast cancer tissue with 73% clinical sensitivity and 93% specificity as compared to benign breast tumors. We did not find a correlation between telomerase activity and the tissue levels of estrogen and progesterone receptors, HER-2/neu oncoprotein concentration, tumor size, and lymph node positivity. Probability of disease-free survival was significantly lower for patients with telomerase activity higher than median value. As the assay for telomerase activity has very high analytical sensitivity and high specificity for cancer cells, this routinely used method may prove useful for distinguishing malignant phenotype of breast tissues.

DNA ploidy correlates with grade, proliferation and clinical outcome but not with presence of human oncogenic HPVs or expression of Bcl-2 in preneoplastic and neoplastic lesions of the uterine cervix.

The aim of this study was to analyse whether DNA ploidy correlates with proliferative activity as measured by PCNA expression, presence of oncogenic human papillomaviruses (HPVs), histological grade, expression of antiapoptotic protein Bcl-2 and clinical outcome in a cohort of 57 preneoplastic and neoplastic lesions of the uterine cervix. The samples were analysed using computer image cytomorphometry of Feulgen stained sections, standard indirect immunohistochemistry and hybridisation of HPV DNA in situ. The ploidy data were found to be significantly different between low/high grade preneoplasia and invasive carcinoma. Significant positive relationships were also found between DNA content and proliferation of lesions, and DNA content and clinical behavior of the lesions. Nevertheless, no relationship between DNA ploidy and HPV/Bcl-2 status was established.

Incidence of chromosomal aberrations and micronuclei in cave tour guides.

An analysis of structural chromosomal aberrations (SCA) and micronucleus tests (MN) were performed in 38 subjects, cave tour guides and in appropriate control group. The dominant type of chromosomal aberrations in tourist guides were chromosomal breaks (0.013 per cell) and acentric fragments (0.011 per cell). In the control group, these aberrations were present up to 0.008 on cells. Considering the analysed cells of the guides in total (33,556), the incidence of dicentric and rings range is below 0.0008 on cells, even though three dicentric and ring chromosoms were found already in the first 1000 in vitro metaphases of some guides. Only 0.0003 dicentrics and neither other translocations were found in control group (ambiental exposure). The incidence of micronuclei in cytokinesis blocked lymphocytes ranged from 12-32 per 500 CB cells in the cave tour guides and from 4-11 per 500 CB cells in control group. Measurements of radon and its daughters were performed at different locations in the cave. Annual doses from 40-60 mSv were estimated per 2000 work hours for cave guides. The changes found in the genome of somatic cells may be related to the exposure doses of radon and its daughters, although smoking should not be ignored.

Estimation of changes in vimentin filaments induced by etoposide and doxorubicin in human leukemia cell line K-562 by using immunofluorescence microscopy.

This study was undertaken to examine the influence of etoposide and doxorubicin on the distribution of vimentin in cells of human leukemia cell line K-562 by using immunofluorescence microscopy. The cells were cultured with 5 different doses of etoposide: 0.02, 0.2, 2, 20, 200 microM/l and three doses of doxorubicin: 0.5, 5, 10 microM/l. Changes in vimentin filaments were dependent on concentration of drugs compared to untreated control cells. Cells treated with 20 microM/l, especially with 200 microM/l etoposide were much bigger from other cells exposed to lower doses of etoposide and control cells, and their number decreased. In most control cells vimentin was seen as a ring with the increased concentration on one pole of the cells. In 20 microM/l and 200 microM/l etoposide the cells showed rather a diffuse cytoplasmic staining pattern. Vimentin filaments were organized as a dense network in cytoplasm of these cells. Immunofluorescence studies on K-562 cells treated with doxorubicin showed that cells incubated with 5 microM/l doxorubicin have much diffuse staining pattern of vimentin with delicate reticular structure and with intense staining near one pool of the cells. Addition of 10 microM/l doxorubicin to cells resulted in increasing of fluorescence staining, which appeared in the cells as enough dense network with intense staining rather in the centre of the cell.

P53 protein expression in human leukemia and lymphoma cells.

The purpose of this study was to determine the value of p53 protein overexpression in human leukemia and lymphoma cells. We examined PB and/or BM samples on a series of 111 patients with immunophenotypically defined hematological malignancies at diagnosis, in remission and in relapsed disease comparing to 20 control samples of healthy individuals. p53 protein has been studied by flow cytometry using three monoclonal antibodies specific for epitopes on N-terminus (Bp53-12, DO-1) and central region (DO-11) of p53 protein. Our findigs showed, that p53 expression may contribute to phenotype of leukemic cells and that overexpression of this protein is often associated with progression of disease. All samples of early B-ALL patients and samples of patients with immunophenotypically defined T- cell disorders examined at diagnosis of disease were p53 positive. Eleven of 19 patient samples from AML at diagnosis showed also increased expression of p53 protein. The cells of all patients who responded to therapy with complete immunophenotypically defined remission were p53 negative. Relapsed T-, B- ALL and AML develop p53 alteration. We reported positive p53 expression in cells of patients with advanced stages of CLL in comparison to them with initial stage of disease at examination. As well as in the group of B- cell lymphomas only samples of patients with generalized FCC lymphoma at diagnosis were p53 positive. We detected p53 positive cells in immunologically defined myeloid blast crisis of CML opposite to p53 negativity in chronic phase of disease. The finding of p53 positive BM cells without immunophenotypic blast markers in two of followed cases documented the contributing value of p53 detection in their characterization. On the basis of above findings we conclude, that cytofluorometric determination of p53 expression may contribute to the better definition of leukemic phenotype. Loss of the normal p53 function may be important in the genesis of some leukemias. Elucidation of the mechanisms of p53 inactivation needs some more study.

bcl2, p53 and bax in thyroid tumors and their relation to apoptosis.

Twenty five thyroid tumors were investigated by immunohistochemistry for the presence of apoptosis, expression of p53, bax and bcl2. In nine adenomas (average age 40) the rate of p53 positive cells was 5.7%, while it was 13.3% for bax and 41.7% for bcl2 in the tumor cells. In six follicular carcinomas (average age 56) p53 positivity was obtained for 71.1%, while bax and bcl2 positivity constituted 3.4% and 2.5%, respectively. In ten papillary adenocarcinomas (average age 42) p53 was positive in 39.6%, but bax and bcl2 were positive in 3.4% and 3% of the tumor cells, respectively. Almost no signs of spontaneous apoptosis were found in any of the cases. Determination of p53, bax and bcl2 ratio in thyroid tumors may contribute to the differentiation between follicular adenomas and carcinomas.

Hematopoietic stem cell transplantation in children with hematological malignancies across HLA barriers--reasonable alternative?

The aim of this study was to evaluate the efficiency and risks of T-cell depletion in prevention of graft versus host disease (GVHD) using HLA haploidentical family donors as an alternative source of hematopoietic stem cells (HSC) in children with hematological malignancies without suitable matched donor. Ten children, median age 12 years (range, 3-17), were transplanted from haploidentical family donors for acute lymphoblastic leukemia (n = 4), acute myelogenous leukemia (n=2), chronic myelogenous leukemia (n = 2), non-Hodgkin lymphoma (n = 1) and myelodysplastic syndrome (n = 1). Parents were donors for nine, sibling for one patient. T-cell depletion of HSC was performed using CellPro followed by antiCD2/CD3 depletion in 7, and CliniMacs magnetic sorting in 3 grafts. Primary engraftment was achieved in nine patients. Patient with graft failure was successfully re-grafted. Primary acute GVHD was diagnosed in one patient who got higher amount of T-cells in the graft. Secondary GVHD was induced by add-backs of lymphocytes in four patients. Three patients developed chronic GVHD. Four patients died due to transplant related mortality (40%), one from veno-occlusive disease, two due to CMV pneumonia and one of aspergillosis with extensive chronic GVHD. Four patients relapsed with leukemia within 35-98 days post transplant, three without previous signs of GVHD, and all died. Two patients are alive and well 26 and 42 months after transplant. Haploidentical family donors appear to be a reasonable alternative option for patients with urgent indications for allogeneic transplant and/or without a matched donor.

Effects of diallyl disulfide and other donors of sulfane sulfur on the proliferation of human hepatoma cell line (HepG2).

Effects of potential sulfane sulfur precursors (diallyl disulfide, cystamine, 2-mercaptoethanol disulfide, thiosulfate, immunothiole and pyridoxal phosphate jointly with cystine) on [3H]-thymidine incorporation in human hepatoma (HepG2) cells were studied. Of the tested compounds, diallyl disulfide, cystamine and 2-mercaptoethanol disulfide were found to cause significant inhibition of HepG2 cells proliferation. Moreover, pyridoxal phosphate jointly with cystine suppressed [3H]-thymidine incorporation, but the differences between that system and control cells were insignificant. In the case of thiosulfate, no significant difference was observed. The present study shows that diallyl disulfide, found in garlic, is effective in inhibiting [3H]-thymidine incorporation in human hepatoma HepG2 cell cultures. Similar antiproliferative effects on HepG2 cells are shown by such systems being a source of sulfane sulfur as cystamine or 2-mercaptoethanol disulfide. Thus, it may be concluded that these donors of reactive sulfane sulfur may be responsible for inhibition of the proliferation of HepG2 cells. It is suggested that the observed antiproliferative properties of the investigated compounds are connected with the presence of the highly reactive sulfane sulfur.

Preventive effects of raloxifene and melatonin in N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats.

The aim of this study was to evaluate preventive effects of raloxifene (RAL), melatonin (MEL) and their combination in N-methyl-N-nitrosourea (NMU)-induced rat mammary carcinogenesis. MEL-treatment began 12 days and RAL treatment began 10 days prior to carcinogen administration and continued till the end of experiment (24 weeks after first carcinogen administration). RAL was administered subcutaneously twice a week in the dose of 5 mg/kg b.w. MEL was administered diluted in drinking water in a concentration 4 microg/ml daily from 3 p.m. to 8 a.m. At the end of experiment, tumor incidence, frequency, latency period and tumor volume as parameters of mammary carcinogenesis were evaluated. Moreover, the effect of chemopreventives on body and uterine weight, food and water intake were recorded. In RAL-treated group, tumor incidence was decreased by 67% (p < 0.001), tumor frequency per group was reduced by 90% (p < 0.0002) and latency period lengthened by 27 days in comparison with control group. After MEL-treatment tumor incidence was decreased by 19%, tumor frequency per group was decreased by 50% (p < 0.05) when compared to control animals. The effect of RAL+MEL-treatment was very similar to that of RAL-treatment. In groups with RAL administration, significant decrease (p < 0.0001) in body weight gain and relative uterine weight was recorded. As to food intake no significant differences in comparison with control group were found. Consequently, groups were pooled and in RAL-treated groups (RAL, RAL+MEL) a decrease in food intake, when compared to groups without RAL administration (control group, MEL) was recorded (p<0.04). The water intake was markedly decreased in RAL-treated groups (P < 0.0001). RAL and RAL+MEL proved to be very effective in prevention of experimental mammary carcinogenesis in female rats, isolated MEL appeared to be of lower oncostatic activity.

Postoperative radiotherapy of childhood medulloblastomas.

The purpose of this work is to review the result of radiotherapy in the treatment of medulloblastoma in pediatric patients. Between 1986 and 1998, 66 children (45 boys and 21 girls) received postoperative irradiation in our institute. Their mean age was 8.29 years. Irradiation was performed by linear accelerator, 36 Gy were applied in the high risk group (partial tumor resection, tumor cell positivity in the liquor, metastases within the central nervous system) and 30 Gy in the low risk group (total tumor resection, negative liquor cytology, no metastases within the central nervous system) on the entire cerebrum and spinal cord. This was followed in both groups by the application of 20-20 Gy boost irradiation on the posterior scala. Studying the survival it has been found that the surgical radicality did not significantly influence the survival chances of patients, however, with the increase in the tumor size the survival chance significantly decreases (p = 0.03). When predicting life expectancy, however, the stage of tumor, the age of patients, the risk group and the M stage yielded essential information. At the age of 8 years and less, the rate of survivors is 67.6%, for those over 8 years is 75.9% (p = 0.21), however the younger age was not significant. The appearance of metastases considerably deteriorates the chances of survival (from 81.5% to 66.7%, p = 0.02). In the low risk group of patients the 5-year survival is 80%, while in the high risk group it is significantly lower, 67.4% (p = 0.04).

Cancers connected with mutations in RET proto-oncogene.

Germline mutations of RET proto-oncogene are connected with inherited cancer syndrome multiple endocrine neoplasia type 2. The syndrome is characterized by incidence of medullary thyroid carcinoma frequently associated with pheochromocytoma and hyperparathyroidism. Genetic testing of family members at risk significantly contributed to diagnosis and management of MEN 2. Early genetic screening for RET mutations allow to detect people who have inherited the MEN2 specific RET mutation with subsequent possibility to of prophylactic thyroidectomy. On the other hand those family members at risk of MEN 2 who had not inherited the mutation do not require further testing. The involvement of RET proto-oncogene in tumorigenesis is reviewed.

Cysteine proteinases in tumor cell growth and apoptosis.

During their evolution tumor cells acquire and mobilize various mechanisms that crucially affect their capability of proliferation, invasiveness and metastasis. Recent findings provide evidence that tumor cell associated cysteine proteinases such as some lysosomal cathepsins and apoptotic caspases are fundamentally involved in specific developmental traits of tumor cell populations. Tumor cell exterior-associated cysteine cathepsins B and L promote tumor growth, invasion and metastasis through degradation of extracellular connective matrices and through endothelial cell growth-directed activities. On the other hand, caspases -3, -7 and -6, generated in tumor cell cytoplasm via a robust activation of their zymogens, suppress tumor cell growth, invasion and metastasis through proteolytic devitalizing and remodeling of tumor cells into readily phagocytable apoptotic corpses. Tumor cell variants that are deficient in expression of effector caspase zymogens or are capable to suppress the extrinsic and intrinsic activation mechanisms of effector caspase zymogens and the activity of effector caspases have a significant survival advantage in environments of various death stimuli. Advancements in pharmacological targeting of tumor associated pathogenic lysosomal cysteine cathepsins and in apoptotic caspases-oriented conditioning of tumor cells may substantially contribute to therapeutic control of tumor diseases.

Flow cytometry of peripheral blood and bone marrow cells from patients with hairy cell leukemia: phenotype of hairy cells, lymphocyte subsets and detection of minimal residual disease after treatment.

By flow cytometry (FC) and an extensive panel of markers we characterized leukemia cells from the peripheral blood (PB) and bone marrow (BM) of 13 symptomatic patients with hairy cell leukemia (HCL). Hairy cells (HCs) identified in the large cell gate always expressed B-cell markers - CD19, CD20, CD22, HLA-DR, and 'HCL-restricted' markers - CD22+CD11c, CD25 and CD103. Other markers, not followed regularly, were occasionally expressed, such as CD34, CD38, CD71, CD15, CD10 and kappa/lambda light chains. Furthermore, in one patient with suspect but not proved HCL in PB or BM, neither morphologically nor immunologically, we confirmed the diagnosis of HCL. Only the immunophenotyping of splenic cells after splenectomy confirmed HCL diagnosis. Flow cytometry was repeated at 3-5 month intervals, after treatment with 2-Chlorodeoxyadenosine (CdA) or less frequently alpha-interferon (IFN). We investigated serially lymphocyte subsets after treatment and we found profound and persistent CD4+ lymphopenia in majority of studied patients after CdA treatment. Simultaneously we investigated the value of FC to detect minimal residual disease (MRD) and to establish, whether MRD+ could predict relapse. Detection of MRD in our series predicted hematological relapse only in one case with persistent MRD+, in majority of cases with occasionally found MRD+ phenotype, did not. Using quantitative immunophenotyping we observed significantly higher values of molecule numbers of hairy cell B-cell markers, comparing to B-cells in nonleukemic gate of the same sample. Our study showed 1) the diagnostic value of FC in management of HCL patients, 2) long-lasting response in the majority of patients after CdA, 3) a profound and persistent CD4+ lymphopenia in CdA treated patients, 4) some correlation between persistent MRD staining and hematological relapse, and 5) further, till now not described activated feature of HCs, given by the increased values of molecular numbers (molecules of equivalent soluble fluoresceine - MESF) in B-cell antigens of HCL.

Potentiated antitumor effects of interleukin 12 and interferon alpha against B16F10 melanoma in mice.

Systemic administration of cytokines has not found broad application in cancer immunotherapy due to its toxicity and lack of effectiveness in a broad spectrum of tumors. Among the most promising cytokines used often in pre-clinical and clinical trials are interferon alpha and interleukin 12. We have shown in our study that combining IL-12 with IFN-alpha in a dose which alone does not show antitumor activity results in potentiated antitumor effects without inducing toxicity.

Comparison of methods used to study cell death in an adherent tumoral cell line with moderate clonogenic radiosensitivity.

Our objective was to compare different methods for studying programmed cell death in adherent H460 non-small lung cancer cells of moderate clonogenic radiosensitivity. The major effect of gamma-radiation was found to be the release of cells from the substratum. The different methods gave complementary and unexpected information: a) with the TUNEL method, a few non-apoptotic cells were found in the culture medium; b) with the flow cytometry after propidium iodide labeling, some hypodiploid cells which remained attached to the substratum were apoptotic, as demonstrated by the effect of a caspase inhibitor; c) with the annexin V labeling, the detached cells were demonstrated either necrotic or very late apoptotic; d) the mitochondria transmembrane potential (deltapsim), measurements demonstrated that the mitochondria were implicated in cell death induced by gamma-radiation. These data illustrate the need to use several complementary methods in the study of apoptosis in adherent cells exposed to gamma-radiation.

AgNOR pattern and PCNA analysis in fine needle biopsies of liver cell carcinoma.

The aim of this study was to evaluate the prognostic and diagnostic value of AgNOR and PCNA staining mainly in fine needle biopsies of 34 liver cell carcinomas using an image analyzing system. The AgNOR number per nucleus and the relative AgNOR area, but not the PCNA index, showed a significant correlation with the histological tumor grade according to the classical and the modified Edmondson-Steiner's classification. Regarding univariate survival, only the grade of the classical Edmondson-Steiner's classification was of prognostic significance. The parameters sex, age, maximum tumor diameter, mean AgNOR number, total nuclear area and relative AgNOR area, nuclear area, PCNA index or the grade of the modified Edmondson-Steiner's classification did not reach statistical significance regarding survival. By a factor analysis, two factors could be created, which could explain together 72% of the variance of all parameters included in the study. In a linear discriminant analysis, the AgNOR variables could separate between normal liver cells and cells from high grade or low grade carcinomas in 83.7% of the cases. Therefore, we think that the AgNOR technique can be usefully applied in needle biopsies of liver carcinomas for the differential diagnosis and tumor grading.

Comparison of two different methods for CD34+ selection and T cell depletion in peripheral blood stem cell grafts--our experiences with CellPro, E rosetting and CliniMACS technique.

The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.

Conditioned medium from HeLa cells enhances motility of human monocyte-derived dendritic cells but abrogates their maturation and endocytic activity.

Progressive tumor proliferation may be associated with suppression of the immune response. Several different mechanisms can contribute to immune evasion. It is generally proposed that inhibition of dendritic cell functions would be a key mechanism by which tumors could escape immune surveillance. Therefore, the purpose of this study was to evaluate the capacity of HeLa cells conditioned medium (HeLa-CM) to modulate phenotypic and functional parameters of human peripheral blood monocyte-derived dendritic cells (DCs). Two types of reference DCs population were generated in vitro, the first cultured in the presence of IL-4 and GM-CSF which represented immature DCs (iDCs) and the second, representing mature DCs (mDCs), was raised from the iDCs by additional stimulation with a maturation cocktail - TNF-alpha, IL-1beta, IL-6, PGE2. In parallel, the iDCs were treated with HeLa-CM collected from the tumor cells. The analysis of DC populations demonstrated that the HeLa-CM prevented maturation of these cells and also impaired their capacity to uptake an antigen and stimulate proliferation of allogeneic T cells. In contrast, HeLa-CM modulated DCs exhibited a 3-fold increase mobility over iDCs. The latter functional capacity did not correlate with the levels of matrix metalloproteinase expression in the analysed cells. Altogether, our results provide evidence that HeLa cells produce soluble factors that might dramatically alter basic phenotypic and functional characteristics of DCs.

Leukemic transformation of polycythemia vera after treatment with hydroxyurea with abnormalities of chromosome 17.

The leukemogenic risk attributed to therapy of polycythemia vera with radiophosphorus and alkylating drugs has led, over the last 20 years, to the increased use of myelosupressive nonmutagenic drugs, especially hydroxyurea. But there exist reports, which showed the development of polycythemia vera into acute leukemia not only in patients treated with alkylating agents and radiophosphorus but also with single hydroxyurea. In this article we present two cases of polycythemia vera, in which the development to acute myeloblastic leukemia occurred after long-term treatment with hydroxyurea. Significant is the fact, that in both presented cases cytogenetic and FISH analysis showed abnormalities of chromosome 17, in the one of case fullfilled criteria for "17p-syndrome". Due to the possibility of leukemogenic potential in the time of hydroxyurea treatment, it is necessary to be careful especially in young patients. The dynamic follow up of cytogenetic analysis is necessary, especially, in those, where long-term hydroxyurea therapy is supposed.

P-glycoprotein expression in adult acute myeloid leukemia: correlation with induction treatment outcome.

Drug resistance has become a major cause of the treatment failure in patients with acute leukemia. P-glycoprotein (P-gp), which is associated with multidrug resistance (MDR) phenotype, has been reported to be an important predictor of the treatment outcome. The aim of this study was to analyze the value of P-gp expression in bone marrow cells as a predictor of the response to remission induction chemotherapy, as well as duration of remission in adult patients with newly diagnosed acute myeloid leukemia (AML). We examined the expression of P-gp in 31 patients using the monoclonal antibody UIC2. Direct immunofluorescent labeling was performed and samples were analyzed by flow cytomery. Kolmogorov-Smirnov test (D-value) was used to estimate UIC2 staining. A D > or = 0.3 for labeling of gated leukaemic blasts as compared to that of the isotypic control was defined positive (+) and compared to clinical data. P-gp expression was found in 14/31 (45.6%) patients, 17/31 (54.8%) of the samples were found P-gp negative(-). No correlation was found regarding age, sex and FAB subtype, altough 6/14 (43%) cases with more than 50% of cells having P-gp expression, were CD34+/CD7+. Complete remission rates were significantly lower in UIC2+ patients than in UIC2- cases (70% vs 35%, p < 0.01). Complete remission duration was also shorter in UIC2+ patients (6 vs 12.4 months). Our data indicate, that P-gp expression is a reliable marker of resistance to induction treatment in patients with de novo AML and can help to identify patients who may require alternative regimens designed to overcome therapy resistance.

Human glioma cells expressing herpes simplex virus thymidine kinase gene treated with acyclovir, ganciclovir and bromovinyldeoxyuridine. Evaluation of their activity in vitro and in nude mice.

Human glioma cell lines 8-MG-BA and 42-MG-BA were infected with retrovirus vector containing the herpes simplex virus thymidine kinase (HSVtk) gene. The effect of acyclovir (ACV), ganciclovir (GCV), and bromovinyldeoxyuridine (BVDU) on both, parental and HSVtk expressing glioma cells was studied in vitro. BVDU displayed the most potent cytotoxic properties in HSVtk-containing cells, however bystander killing of nontransduced parental cells in a mixture with HSVtk-containing cells was less potent, than observed for ACV or GCV. Taking into account the cytotoxic effect of different prodrugs used, as well as their ability to kill nontransduced bystander cells, ganciclovir was shown to be the most effective. Therefore the effect of GCV treatment on 8-MG-BA xenografts inoculated with PA-317JH5cl13 virus producer cells was further studied on nude mice.

Polymorphism of the p53 gene within the codon 72 in lung cancer patients.

We tested the codon 72 single nucleotide polymorphism (SNP) of the tumor suppressor gene p53 for association with lung cancer. In our hospital-based case-control study, 168 lung cancer patients (134 males and 34 females) and 148 controls without malignant diseases were recruited. The genotype characteristics were determined by PCR-based RFLP method using DNA extracted from peripheral blood. Only in lung cancer patients but not in the controls we found both significant decrease of A1 allele of the p53 codon 72 (p=0.024, OR 0.56, 95% CI 0.43-0.72) and A1/A1 homozygous genotype (p=0.006, OR 0.27,95% CI 0.15-0.51). The results of this study suggest a protective effect of A1 allele against lung cancer.

Inhibition of RNA synthesis in vitro and cell growth by anthracycline antibiotics.

New derivatives of doxorubicin and daunorubicin with amidine group bonded to daunosamine at C-3' atom and bearing the morpholine ring attached to the amidine group have been recently synthesized. Their cytotoxic activities and effects on RNA synthesis in vitro were assayed. The drug concentrations inhibiting mouse leukaemia L1210 cell growth to 50% were about two- and three fold higher for the derivatives compared to doxorubicin and daunorubicin respectively. Inhibition of phage T7 RNA polymerase by the non-covalently interacting derivatives was also slightly lower than that by the parent compounds. As doxorubicin and daunorubicin, their amidine derivatives in the presence of dithiothreitol and Fe(III) ions are activated and covalently bind to DNA. The adducts formed affect RNA polymerase activity. Several bands corresponding to prematurely terminated RNA chains are observed by means of polyacrylamide gel electrophoresis. The patterns of bands are virtually identical for all the anthracyclines studied here and are similar to the terminations induced by actinomycin D. This observation is consistent with a notion that the adducts are formed at guanine in GpC sequences which are also binding sites of actinomycin D. A substantial difference between daunorubicin and its amidine derivative is shown by means of high performance liquid chromatography. The derivative undergoes rapid rearrangements in the presence of dithiothreitol and Fe(III) ions, while daunorubicin is stable for several hours under these conditions. The results presented here indicate that the amidine derivatives despite bulky morpholine substitution exhibit biological activity in the systems used here.

Androgen sensitivity related proteins in hormone-sensitive and hormone-insensitive prostate cancer cell lines treated by androgen antagonist bicalutamide.

Members of the bcl-2 gene family and endogenous inhibitors of cyclin-dependent kinases participate in the regulation of apoptosis and cell cycle in a diverse range of cell types and are implicated in the development of hormone refractory prostate cancer and resistance to anti-cancer therapy. The expression of several of these genes can be regulated by steroid hormones and related agents via their nuclear receptors. However, insufficient information considering the protein expression after the treatment by hormone antagonists is available. The aim of this study was to evaluate the expression of anti- and pro-apoptotic proteins, (Bcl-2, Bax), and to correlate this with the appearance of some nuclear receptors and cell cycle related proteins in androgen sensitive and androgen insensitive prostate cancer cell lines, LNCaP and DU-145, after the treatment by androgen antagonist bicalutamide. Our results revealed that androgen receptor (AR) expression in LNCaP cells decreased, however in DU-145 cells AR slightly increased following anti-androgen treatment. The same agent stimulated expression of p21Waf1/Cip5 and p27Kip1 in LNCaP, as well as in DU-145 cell lines. Bcl-2 level increased slightly in LNCaP cells and, in DU-145 cells was almost undetectable. Bax expression was not changed in LNCaP but significantly decreased in DU-145 cells. Similarly, retinoid X receptor beta (RXRbeta) level was significantly down regulated after 24 hours in DU-145 and also in LNCaP cells after 72 hours. These results confirm that androgen withdrawal therapy employing anti-androgens may elicit different signalling pathways in various types of prostate cancer that may be dependent on AR status and AR sensitivity.

Contemporary trends in in vivo and in vitro testing of chemical carcinogens.

In the sixties of the last century it was realized that many human cancers are caused by environmental carcinogens and that the best way how to reduce cancer is first to identify in environment chemical carcinogens and second to prevent people from being exposed to such carcinogens. Epidemiological studies are probably the only way to confirm human carcinogenesis, however, this approach is so retrospective that carcinogens can be identified only after many victims have appeared. Carcinogenicity testing in long-term, medium-term, and short-term studies is therefore the only way for the prospective identification of possible human carcinogens. End-points of interest in a carcinogenicity study are primarily preneoplastic and neoplastic changes, but also include degree of malignancy, time to tumor appearance, multiplicity of (pre)neoplasia, and occurence of metastases. Long-term bioassays are designed and conducted to detect all of these end-points. Medium-term bioassays are mainly based on the detection of putative preneoplastic lesions and short-term tests can provide very important information concerning genotoxic effects of studied compounds.

Flavonoids as chemoprotective agents in civilization diseases.

Flavonoids, a class of polyphenolic compounds widely distributed in the plant kingdom, are capable of protecting against several chronic and degenerative diseases, among them cancer, cardiovascular diseases, stroke, cataracts and brain and immune dysfunctional states. These substances are common dietary components. They exhibit many biological properties through various mechanism of activity. Early studies of flavonoids investigated their significant antioxidant, antitumor, anti-inflammatory, antiallergenic and hepatoprotective effect. Additionally, their ability to modulate the activity of various enzymes affects normal as well as malignant systems. This review summarizes data on the beneficial effects of flavonoids in humans.

Radiation-induced meningiomas.

High dose radiation-induced meningiomas are a rare, severe and late complication of craniospinal radiotherapy for brain tumors. Radiation-induced meningiomas are, according to the literature, several times more frequent than radiogenic gliomas and sarcomas. It is suggested that every new case of radiogenic meningioma has to be reported to elucidate this particular pathologic entity with its many grey areas. In addition to high dose radiation-induced meningiomas, intracranial meningiomas were observed in patients who underwent low-dose radiation for tinea capitis in childhood, applied en mass to immigrants coming to Israel from the North Africa and the Middle East during the 1950. Authors summarize the data on radiogenic meningiomas from the literature and, as the previous radiotherapy may confer a low, but life-long risk for meningioma occurrence, they suggest that surveillance MRI after high dose cerebrospinal radiotherapy should be extended to several (3-5) decades after radiotherapy.