Blood monocytes and tumor-associated macrophages in human cancer: differences in activation levels.

This study was performed to investigate functional properties of mononuclear phagocytes isolated from ascitic fluid in patients with peritoneal carcinomatosis (PC), and potential immunomodulatory effects of soluble factors produced or induced by human metastatic malignant cells. Phagocytic activity and nitric oxide production of peripheral blood monocytes (PBMo) and tumor-associated macrophages (TAM) or peritoneal macrophages (PEM) were synchronously examined in cancer patients and control individuals. Our results showed that contrary to peripheral blood monocytes, where phagocytic activity was not altered, TAM had impaired phagocytic activity. Moreover, dilutions of crude supernatant from short-term cultures of the peritoneal cells obtained from ascitic fluid of patient with PC, cause a significant, dose dependent inhibition of control PBMo and PEM phagocytosis, comparable to those in TAM, indicating that a soluble factor(s) plays a prominent role in this alteration. Next, we investigated the potential of cancer patients mononuclear phagocytes to produce nitric oxide (NO). It was found that TAM produce fourfold lower levels of NO than PEM from control subject, whereas monocytes produce NO at levels comparable to those of corresponding controls. These data support the hypothesis that depressed TAM function may contribute to the mechanisms of tumor escape from immune destruction.

Therapeutic implications of the kinetics of immunomodulation during single or combined treatment of melanoma patients with dacarbazine and interferon-alpha.

The therapy of metastatic melanoma has not given satisfactory results. Single chemo- or immunotherapeutic agents in the adjuvant setting or combined chemoimmunotherapy for metastatic disease have generally been evaluated only in terms of clinical benefit. Considering that dacarbazine (DTIC) and interferon-alpha (IFN-alpha) are among the most frequently used agents in the treatment of melanoma, the aim of this study was to evaluate the kinetics of immunological changes during adjuvant treatment of melanoma patients with DTIC or with IFN-alpha monotherapy, as well as by their combination in metastatic disease. The evaluated immunological parameters showed significant early increase in the activity of NK (natural killer) cells, CD4/CD8 ratio, CD4+ T cell number in patients treated with combined chemoimmunotherapy and an increase in expression of the early activation antigen CD38 on CD8+ cytotoxic T cells, both, in patients treated with combined chemoimmunotherapy and with IFN-alpha alone, while, no significant change in any one parameter was detected in the group of patients receiving DTIC. The kinetics of the observed immunological changes, restricted to combined chemoimmunotherapy, indicate that the engagement of antitumor immune response appears early but is short-lived and that this favorable effect should be augmented and prolonged by the timely introduction of additional immunomodulating agents.

Expression of CD20 on acute lymphoblastic leukemia cells in children.

CD20 determinant expressed on B precursors is associated with regulation of proliferation, apoptosis and maturation of these cells. The acute lymphoblastic leukemia "common" type (cALL) based on expression of CD20 is subdivided in type I and II. However, the clinical significance of CD20 expression on cALL and significance of cALL type I and II discernment are not fully elucidated. The association of CD20 expression with the expression of multidrug resistance molecule (MDR), CD34, atypical immunophenotypes of leukemia cells and response to induction therapy were determined in the group of 147 patients with acute lymphoblastic leukemia (ALL) B progenitor type (ALL-proB -14 patients) and common type (cALL-133 patients). The expression of CD20 on leukemia cells was studied routinely at diagnosis before the therapy. This expression was noted on leukemia cells of 6 ALL-proB patients (42.8%) and 66 cALL patients (49.6%). The expression of CD20 showed no association with the expression of CD34, CD22 and MDR. The reverse association was observed between CD20 expression and the presence of co-expression of myeloid (CD13, CD33, CD65, CD15) and T lymphoid determinants (CD2, CD5, CD7) on leukemia cells. The effect of induction therapy analyzed as time of blast cells cytoreduction in peripheral blood and time of reaching the complete remission showed the slower clearance of peripheral blood from blast cells associated with expression of CD20. There was no association of CD20 expression with the time of reaching the hematological remission. The above results suggested a "protective" role of CD20 against co-expression of other determinants (myeloid and lymphoid) and no association with the results of induction therapy.

Prognostic significance of c-erbB-2 gene expression in pancreatic cancer patients.

Molecular methods tend to belong to the standard armamentarium of modern pathology. In some instances, these methods are able to identify nosological entities with better accuracy than conventional technique. These methods give useful complementary information to choose appropriate therapeutic strategy. C-erbB-2 overexpression in pancreatic cancer vary widely between 17 to 82%. C-erbB-2 gene is perspective target of anticancer therapies. 57 histologically confirmed tumors (51 pancreatic adenocarcinoma, 5 pancreatic neuroendocrine tumors and 1 carcinoma of Vater's ampullae) were analyzed for the presence of c-erbB-2 expression by immunohistochemistry. Correlation with time from initial symptoms until diagnosis, tumor size and TNM stage at diagnosis, tumor grade, type of operation and overall survival were investigated. C-erbB-2 overexpression was detected in 19.6% samples of pancreatic adenocarcinoma and in one case of Vater's ampullae carcinoma. C-erbB-2 overexpression was found in two of four insulinomas. Univariate statistical correlation stage between c-erbB-2 overexpression and time from initial symptoms until diagnosis, tumor size and TNM at diagnosis, tumor grade, type of operation and overall survival did not reach statistical significans in any parameter studied. C-erbB-2 oncogene was not found to be prognostic factor in pancreatic cancer. Its value to predict therapeutical response remains to be determined in prospective clinical trials.

Basic fibroblast growth factor as a growth factor for SRV-2-infected simian retroperitoneal fibromatosis cells, an animal model for AIDS related Kaposi's sarcoma.

Basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) were demonstrated to be important factors sustaining the growth of Kaposi's sarcoma. RF cells were used to provide a model to study the pathogenesis of Kaposi's sarcoma. In this paper, we demonstrated that bFGF is present in the RF cells, cultured media, and tissues from monkey. The biological activities of bFGF on RF cells were also studied in vitro with serum-free media. The bFGF from serum-free-conditioned media is biologically active to stimulate RF cells in certain media condition. The mitogenic effect was abrogated by sheep neutralizing anti-bFGF antibody. Furthermore, the effect of antibody was reversed by the addition of exogenous bFGF. ELISA measurements indicating the growth potency of conditioned media correlated with the amount of bFGF in the conditioned media. The data from flow cytometry demonstrated the co-existence of SRV-2 and bFGF among RF cells and RF tissues. Immunohistochemical staining of RF tissue blocks for bFGF revealed that bFGF was present in the tumor and the presence of bFGF was not caused by the artifact of tissue culture. These results indicate that bFGF is an important growth factor to promote RF cell growth in vitro and RF tumor in vivo. Further studies are required to determine the relationship between the interaction of bFGF, SRV-2, and VEGF. This model also provides an adequate alternative to the model induced by simian immunodeficiency virus (SIV) to study the Kaposi's sarcoma.

Gemcitabine and vincristine: an effective outpatient regimen with low myelotoxicity for stage IV non-small cell lung cancer.

The activity and tolerability of gemcitabine and the non-overlapping toxicity of gemcitabine plus vincristine were the basis for testing this regimen patients with non-small cell lung cancer (NSCLC). Forty patients (25 male/15 female, median age 52 years) with stage IV NSCLC and a Karnofsky Performance Status score > or = 60 entered the trial. Patients received gemcitabine 1000 mg/m2 on days 1, 8 and 15 and vincristine 1.4 mg/m2 on days 1 and 15, every 4 weeks. The overall response rate was 16/40 (40%) (N = 40); with 2 complete and 14 partial responses; additional 14 patients had minor responses or stable disease. Median duration of remission was 4.5 months, and the median survival was 9 months. In two patients with grade 2 generalized vesicular rash and severe malaise, respectively, treatment-related toxicity led to early termination of treatment. Among patients treated for more than two months, vincristine doses were reduced/omitted for 55% of cycles because of grade 1-2 peripheral neuropathy. Myelotoxicity was frequent but rarely clinically significant. Mean platelet counts on day 1 of cycles 2,3 and 4 were significantly higher than the pre-treatment or post-treatment values. We conclude that vincristine plus gemcitabine is an an active and well tolerated regimen. Its interesting "platelet-saving" effect deserves further investigation.

Cladribine combined with mitoxantrone in the treatment of blastic phase of chronic myeloid leukemia.

A phase II clinical study was performed to evaluate the effectiveness and toxicity of cladribine (2-CdA) combined with mitoxantrone (CM regimen) in the treatment of chronic myeloid leukemia in blastic phase (CML BP). A total of 12 adult patients with CML BP were included in this study. 2-CdA was given at a dose 0.12 mg/kg in 2-hour iv infusion on days 1-5 and mitoxantrone 10 mg/m2 i.v. day 1. The cycles were repeated at 4 week intervals in most cases. Complete remission (CR) was defined as the presence of < 5% of blasts in a normo- or hypercellular bone marrow in addition to normal peripheral blood counts and with normal physical examination. A partial response (PR) required normal peripheral blood counts but 5 to 25% marrow blasts. Toxicity was assessed according to WHO criteria. The patients received 21 courses of CM (median 2, range 1-3). Of 12 patients only 2 (17%), achieved PR. Responses were observed in patients with myeloid BP, after 3 and 2 courses, respectively. Myelosuppression was the main toxicity. Four patients (33.3%) had grade 3 or 4 neutropenia and 3 (25%) had grade 3 or 4 thrombocytopenia. Infections occurred in 4 patients (33.3%) and 2 of them died of sepsis shortly after CM treatment. This preliminary results in a small group of patients suggest that CM programme has limited value in pre-treated patients with CML BP. However, this regimen may be used as palliation in the end stage of disease.

Non-steroidal anti-inflammatory agent ibuprofen-induced apoptosis, cell necrosis and cell cycle alterations in human leukemic cells in vitro.

The cytotoxic activity of the non-steroidal anti-inflammatory agent ibuprofen to human promyelocytic leukemia cell line HL-60, its multidrug-resistant subline HL-60/VCR (MDR-1 gene coded P-glycoprotein), as well as myeloma U266 and B-lymphoblastoid ARH-77 cell lines was demonstrated with the aid of flow cytometric analysis. Ibuprofen inhibited proliferation and induced apoptosis (detected as sub-G0 nuclei, fluorescein diacetate staining, Annexin-V binding cells and agarose electrophoretic detection of nucleosomal DNA fragmentation) in promyelocytic cells and, to a lesser extent, in U266 and ARH-77 cells.

Embryotoxicity of TPPS4 and PS 3 photosensitizers in chicken embryo under different light conditions.

Hematoporphyrin derivatives have been recommended for photodynamic therapy of malignant processes. We administered TPPS4, and Photosan 3 (PS 3) in chick embryo in ovo, with or without subsequent blue light (400-550 nm) irradiation. The aim was to analyze and compare the effects of both substances on organogenesis under different light conditions. The embryotoxic effect (embryonic death and malformations) was detected after a single intra-amniotic injection of 5 different doses (0.3 to 300 microg) of TPPS4 or PS 3 at embryonic day 3-5. The beginning of the embryotoxicity range (minimal embryotoxic dose) was determined in non-irradiated embryos to be between 0.3-3.0 microg PS 3 and 3.0-30.0 microg TPPS4. Malformations of surviving embryos were similar after both substances, represented by trunk hyperlordosis combined with incomplete closure of the ventral body wall and protrusion of viscera as consequences of amnion contraction, reduction limb deformities, eye malformations and cleft beak. Ten minutes light irradiation in ovo following two hours after intra-amniotic injection of TPPS4 or PS 3 increased by one order of magnitude their embryotoxic effects. Even dark-ineffective doses became highly embryotoxic. Contraction of the amniotic sac and extraembryonic vessels seemed to be a common mechanism of photosensitizer action.

Clinical and histopathological evaluation of the adrenal incidentaloma.

Clinically silent adrenal masses (incidentaloma) are incidentally discovered lesions, when noninvasive imaging methods (USG, CT, MRI) are performed for reasons other than known or suspected adrenal disease. Most studies report on a prevalence of adrenal incidentaloma range between 1% and 10% in radiological series. Between 1994 and 1999 we observed in our Department 57 patients with incidentalomas of adrenal glands. After endocrinological evaluation silent Cushing's syndrome was found in 2 cases (3.5%). Fifty two patients were qualified for surgery. Adrenocortical adenoma was diagnosed in 73.1%; adrenocortical carcinoma in 7.7%; pheochromocytoma in 7.7% and less frequent adrenal lesions in 11.5%. All adrenal carcinomas and malignant pheochromocytomas (11.5%) were found in tumors with diameter over 4 cm.

The differential inhibitory effects of genistein on the growth of cervical cancer cells in vitro.

The biological effect of genistein on cervical cancer was studied on two cervical cancer cell lines with different cellular characteristics. Here we report that genistein exhibits inhibitory effects on the growth of HeLa and ME-180 cells. The IC50 was 35 microM and 60 microM for HeLa and ME-180 cells, respectively. ME-180 cells showed obvious G2/M arrest with genistein treatment while most of the HeLa cells were accumulated in S phase. The underlying molecular mechanism was further elucidated by apoptosis analysis and expression levels of cell cycle regulatory proteins. Treatment of the cell lines with genistein also resulted in suppression of invasion through a surrogate membrane in a dose-dependent manner, particularly the HeLa cells. While the underlying mechanism needs to be further studied, the higher suppressive effect on invasion of HeLa cells, an adenocarcinoma cell line, are noteworthy. This in vitro observation may have clinical implication to improve the treatment of cervical adenocarcinoma.

Long-term clinical benefits of the low dose rate endobronchial irradiation of malignant airway obstructions.

Brachytherapy allows the delivery of higher radiation doses, possibly leading to improved locoregional tumor control and subsequent prolonged survival. The purpose of our study was to evaluate the long-term clinical survival in patients with malignant airway compromise treated with endobronchial brachytherapy and to estimate possible influence of other factors on survival and to review complications of the therapy. In a retrospective study 55 patients with malignant inoperable tracheobronchial lesions underwent 71 brachytherapy treatments with 137Cesium. Either MicroSelectron (N=56) or Selectron (N=15) were used. All except 4 patients received external radiation, 20 patients received chemotherapy, 37 patients received laser excision. Major symptomatic improvement was noted in 75% of patients. Substantial or complete relief of hemoptysis was achieved in 85%, of dyspnea in 65% and of cough in 68%. Response evaluation showed no complete response, partial response was achieved in 70.9% and the endoscopic finding was not changed, or recurrence of the tumor was found in 29.1%. A relatively small number of complications of the endobronchial brachytherapy occured. Significant bleeding was observed in 1 procedure and an inability to tolerate in 3 cases. In 2 cases, it was not possible to place an applicator due to extreme hypoxia. Bronchomediastinal fistula developed in 1 patient and tracheal stenosis in 1 patient. The overall incidence of complications was 15%. The median survival from establishing the diagnosis was 510 days. The median survival after the first brachytherapy treatment was 200 days. We compared the survival in the subgroups of patients in relation to TNM status, chemotherapy, laser debulking brachytherapy device used. The stage IIIA patients survived longer from diagnosis than IIIB patients but the difference was on the border of significance (p = 0.090). In the evaluation of chemotherapy, more patients survived 12 months from the diagnosis (p = 0.045) when treated by chemotherapy comparing to the patients treated without chemotherapy. However, this difference disappeared during the further development of the disease. In the Nd-YAG laser treatment, the patients treated by brachytherapy with the previous laser debulking survived significantly longer from the time of the first brachytherapy session (p = 0.005). No statistical difference was found in the survival of patients treated by either the Selectron or MicroSelectron device. The LDR endobronchial brachytherapy is a well tolerated, safe and effective technique for palliation of malignant airway occlusions. In our group of patients, the long-term survival was longer in IIIA stage comparing to the IIIB, in the group treated by the previous chemotherapy compared to the patients without chemotherapy and in the group with the Nd-YAG laser therapy, comparing to the group treated by the brachytherapy only. No difference of the brachytherapy device used was found.

Ursolic acid: an anti-tumorigenic and chemopreventive activity. Minireview.

Ursolic acid, UA, as a pentacyclic triterpene is of interest to scientists in the area of oncology because of its cytotoxicity, induction of differentiation, anti-mutagenic, antiviral and anti-invasive activities. UA is capable of inducing apoptosis in tumor cells on one side and to prevent malignant transformation of normal cells on the other side. It also interferes with numerous enzymes, including the ones serving directly to DNA synthesis. The aim of this review is to summarize reports on UA biological properties and to show its main anti-tumor effects and chemopreventive properties in normal cells.

Lung cancer incidence rates by histologic type: an example of trends in Eastern Europe--Slovakia 1978-95.

During the period 1978-1995 43206 cases of lung cancer--37967 in men and 5239 in women--were recorded in Slovakia. Among 26240 microscopically confirmed cases in men squamous cell carcinomas were the most frequent (57.1%) followed by small cell carcinomas (18.3%) and adenocarcinomas (11.7%). In women from 3190 microscopically confirmed cases squamous cell carcinomas and adenocarcinomas had nearly the same frequency (31.9% and 32.2%, respectively), followed by small cell carcinomas (16.1%). In men after a marked increase of overall lung cancer incidence and mortality the rates started to flatten and even decline from the early 1990s. The main histologic types peaked in the late 1980s and declined thereafter but showed increase of percentage change when the rates at the beginning and the end of the studied period were compared; the highest one was marked for adenocarcinomas. The corresponding rates in women were much lower, but their increase was more pronounced than in men. Adenocarcinomas showed almost twofold increase in women during the first time-period of the study but after peaking in 1984-1986 they stabilized, while squamous cell carcinomas continued to increase also in recent years. The analysis of incidence rates by age groups showed that the initial increase and subsequent decrease of all microscopically confirmed cases in men as well as their gradual increase in women was influenced mainly by the trends of squamous cell carcinomas in younger age groups in men and in all age groups in women. Absence of higher increase and proportion of adenocarcinomas in Slovakia in both sexes could probably be explained by delayed introduction of filter tipped and low tar cigarettes.

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.