Expression of p21 and MDM-2 proteins on tumor cells in responding and non-responding patients with Hodgkin's disease.

Since the prevention of apoptosis can produce resistance to chemotherapy, abnormal expression of oncoproteins engaged in the regulation of this phenomenon in tumor cells may give some prognostic information in patients with neoplasma. To assess this problem in Hodgkin's disease (HD), the cellular expression of two p53 downstreams proteins p21 and MDM-2 was evaluated in the lymph nodes specimens obtained from 68 patients at the time of diagnosis, and compared with some clinical and pathological data. Positive immunoreaction for p21 and MDM-2 on Reed-Sternberg/Hodgkin's (R-S/H) cells was found in a majority of cases (58.8 and 63.2%, respectively). High expression of p21 protein correlated with poor response to the first-line treatment and disease free survival in both univariate and multivariate regression analysis, whereas expression of MDM-2 did not give prognostic information in patients with HD. Expression of p21 and MDM-2 was also compared with p53 protein immunoreactivity of R-S/H cells. The p21+/p53+ immunophenotype occurred to give particularly negative prognostic information in patients with HD. Diversity of p21/MDM-2/p53 patterns of R-S/H cells observed in particular patients may reflect heterogeneity of apoptosis regulatory mechanisms, as well as differences in p53 gene status.

Changes in apoptotic and mitotic activity in rectal carcinoma after short-term cytostatic therapy as possible predictive factors.

Fifteen patients with rectal adenocarcinoma were endoscopically biopsied and given short-term [5 fluorouracil (5FU) (600 mg/m2) and Ca-Folinate (60 mg/m2) for two days] cytostatic therapy. Seven days later the tumor was resected or a second biopsy was performed. Apoptotic and mitotic indices were determined in the tumor tissue before and after the short-term chemotherapy. The patients were treated thereafter with long-term, intermittent 5 FU administration and followed up clinically for 4-13 months. Three patients showed progression of the disease, twelve improved or showed no tumor progression. An increase of the apoptotic index and decrease of the mitotic index after the short-term cytostatic treatment were seen in the tumor tissue of responder cases. Non-responders showed increase or no change in mitotic activity, and decrease or no change in apoptotic activity. These findings suggest that apoptotic and mitotic response to short-term cytostatic therapy may be additional predictive factor in rectal adenocarcinoma.

Lyophilized whole human melanoma cells stimulate human PBMC proliferation and enhance suppressive action of PBMC toward survival of the same malignant cell line in vitro.

The goal of this work was to determine: a) do lyophilized human melanoma BG or Fem-X cells affect the proliferative capacity of normal human peripheral blood mononuclear cells (PBMC) and b) does the PBMC six-days preincubation in nutrient medium with FBS with, or without lyophilized human melanoma BG or Fem-x cells, affect their suppressive action on the survival of the same malignant cell line in vitro. In the aim to avoid any stimulating effect of FBS, other group of experiments were done in nutrient medium with human AB serum in order to determine: c) does the PBMC six-day-preincubation with lyophilized human melanoma BG or Fem-x cells affect their antiproliferative action on the corresponding malignant cell line in vitro and d) does the PBMC six-day preincubation with lyophilized normal PBMC, obtained from healthy volunteer (as a source of allogenous, but not of tumor antigens), affect their suppressive action on the survival of both melanoma BG and Fern-x cell lines in vitro. Results obtained in the presence of FBS in nutrient medium, showed that lyophilized BG cells induced a proliferation of the healthy PBMC, depending on the number of stimulating lyophilized cells. Lyophilized Fem-x cells induced healthy PBMC proliferation in lesser degree than lyophilized BG cells. This stimulation was almost constant, not dependent on the number of stimulating lyophilized Fem-x cells. Six-day stimulation in vitro by both lyophilized melanoma cells enhanced the suppressive action of PBMC on the survival of the corresponding malignant cell line. Experiments done in nutrient medium with normal human AB serum showed that six-day stimulation with lyophilized melanoma cells enhanced, again, the suppressive action of PBMC on the survival of the corresponding malignant cell line. Contrary, six day preincubation of normal PBMC with the lyophilized healthy PBMC (obtained from other healthy person) inhibited their suppressive action on the survival of both malignant cell lines in vitro.

Separation of structurally related flavonoids by GC/MS technique and determination of their polarographic parameters and potential carcinogenicity.

The present study deals with the investigation of the naturally occurring derivatives of the benzo[b]pyran-4-one - flavonoids - chrysin, tectochrysin and galangin, and with the effect of minor changes in their chemical structure on their separation using GC/MS. In the relation to their close chemical structure, their basic polarographic parameters were also investigated. Their potential carcinogenicity index tg alpha was determined by DC polarography experiments in the presence of alpha-lipoic acid. The tg alpha values for chrysin, tectochrysin and galangin were all under 0.180. This indicates a very minor carcinogenic potential that does not prevent the use of the investigated flavonoids in human.

Combined effect of lipoic acid and doxorubicin in murine leukemia.

Our experiments indicate that administration of a toxic drug with high rate of free-radical formation (doxorubicin, DOX) combined with an antioxidant (alpha-lipoic acid, LA) may lead to a decrease in drug-toxicity. However, the effects of antioxidant may be concentration-dependent and it is therefore crucial to choose its appropriate dosage. LA at a low concentration (1 micromol/l) acts as a growth factor and at a higher concentration (100 micromol/l) acts as an antiproliferation agent. Both concentrations of LA in combination with DOX were examined in cytotoxic and antitumor effects in L1210 mouse leukemia cells employing a MTT chemosensitivity assay. In most concentration combinations, DOX and LA effect were antagonistic and synergistic action was only found at the higher concentration of both agents (DOX 2.5 micromol/l and LA 100 micromol/l). Use of LA in doxorubicin therapy lead to an increase (though marginally significant) in survival of animals. Combined single-dose administration of DOX (5 mg/kg) and LA (16 mg/kg) lead to super-additive effect of the combination on survival of leukemic mice.

Evaluation of 2-(methylaminosulfonyl)-1-(arylsulfonyl)hydrazines as anticancer agents.

Seven new 2-(methylaminosulfonyl)- 1-(arylsulfonyl)hydrazines were prepared and evaluated as potential antitumor agents in vivo against murine Ehrlich ascites carcinoma (EAC). Borderline in vivo activity in EAC was exhibited by two compounds. All of them were screened in vitro against a battery of human tumor cell lines at the National Cancer Institute (NCI), USA. One of them, namely compound 2f(NSC No. 649 752) displayed highly significant specificity in two different cell lines as non-small cell lung cancer line HOP-18 and in CNS cancer line SNB-19. The compounds assessed in vitro for anti-HIV activity also at the NCI, however, have not reached the criteria of significant activity. The alkylating activity of the compounds was determined by measuring the absorbance of the alkylated product of 4-(4-nitrobenzyl)pyridine. It was found that they are capable of acting as chemical alkylating agents.

Pentavalent Tc-99m dimercaptosuccinic acid imaging of hepatocellular carcinoma.

Tc-99m (V)-dimercaptosuccinic acid (DMSA) has been used to image various kinds of tumors. However, from a review of the literature, it has never been applied to hepatocellular carcinoma (HCC). Low uptake of Tc-99m (V)-DMSA has been demonstrated in normal liver tissue, thus, Tc-99m (V)-DMSA may be useful for the detection of HCC. Nine male patients with focal nodular HCC were studied with sequential X-ray CT, Tc-99m (V)-DMSA and Tc-99m phytate liver scan. Our data showed that eight patients had increased uptake of Tc-99m (V) DMSA in HCC. Four cases demonstrated higher Tc-99m (V) DMSA uptake in HCC than in adjacent liver, and four cases demonstrated HCC uptake equal to liver uptake. One case showed no uptake of Tc-99m (V) DMSA in HCC. The detection sensitivity of Tc-99m (V)-DMSA was 88.9%. From our early results, Tc-99m (V)-DMSA is a readily available tumor imaging agent that appears to accumulate in HCC.

Doppler flowmetry in the differentiation of biological characteristics of ovarian cancer.

On basis of ultrasonographic structural criteria and Doppler flowmetry in a group of 76 patients with ovarian cancer, benign lesions were discovered in 59 patients and malignant lesions in 17 patients (77.6% and 22.4%, respectively). Surgical intervention was indicated according to the clinical findings and following of the trend of organ-specific oncomarkers in 32 out of 76 patients (42.1%). The stratification involved 17 patients with sonomorphologically/Doppler-flowmetry - diagnosed malignancy as well as 15 patients with sonographically diagnosed benign ovarian tumor. The clinical findings were histologically verified in all 32 patients. Histology confirmed malignant tumor in 16 patients, 1 histologically borderline malignancy and 15 benign tumors. The Doppler-flowmetry showed 94.1% sensitivity, 93.3% specificity, 93.3% positive predictive value, and 93.75% accuracy.

Relevance of the Doppler flowmetry in the diagnosis of pathological alterations in the post-menopausal endometrium.

The sonopathomorphology of the menopausal endometrium was investigated by measuring the endometrium thickness using the Doppler flowmetry technique. The endometrium thickness extending five millimeters has been considered critical, indicating possible neoplastic disorders. Such clinical status has been discovered in 21 out of 58 post-menopausal patients inspected (36.2%). Furthermore, the Doppler blood-flow analysis, based on the evaluation of the resistance indices (RI) in the ascendent branches of uterine arteries bilaterally, and - in case of the vascularization analysis - in the edometrial and perimetrial regions, has been performed. These investigations brought evidence indicating full capacity of the method in detecting blood flow through the uterine arteries, while significant differences in the endometrial flow-through parameters were detected only in 14 (66.7%) patients. In all 21 patients with endometrium thickness extending 5 millimeters, bioptic examination followed by histology confirmed endometrial cancer in 9 patients, epidermoid cancer in 3 patients, and cystic hyperplasia in 3 patients, respectively. The remaining 6 patients showed either secretory or quiet endometrium (5 cases), or necrotic endometrial regions (1 case). In all 12 cases of malignant endometrial cancer and in 2 out of 3 cases of histologically verified benign hyperplasia, intra- and periendometrial vascularization has been confirmed. It is to note that the mean RI values measured in the intra- and periendometrial vessels in case of endometrial cancer were significantly lower than in patients with benign cystic hyperplasia of the endometrium. Our results brought evidence indicating that the estimation of differences in the RI values in patients with detectable intra- and periendometrial vascularization has a significant relevance in the distinction of endometrial cancer from nonmalignant endometrial lesions, predominantly the benign atypical hyperplasia. The reliability of the test was 100%, however - in case of malignant disorders - a significant decrease in the RI values has been seen in the intratumoral vs. peritumoral vascularization.

Tumor necrosis factor-alpha: molecular-biological aspects minireview.

Tumor necrosis factor-alpha (TNF-alpha) a proinflammatory cytokine with multiple actions was first identified for its anticancer activity. However, TNF-alpha has a beneficial function in activation of host defense, its uncontrolled production can lead to pathological consequences. At the cellular level, it is able to exert obviously opposing effects: apoptosis and activation. It modulates survival and activates genes through various intermediates, including protein kinases, protein phosphatases, reactive oxygen intermediates, phospholipases, proteases, sphingomyelinases and transcription factors. In this review, the INF-alpha is characterized at the molecular and cellular level (TNF-alpha mediated signal transduction is discussed in the first part, regulation of its expression in the second one), as well as methods of its determination in biological materials, giving special emphasis to the molecular-biological approach. The full understanding of the molecular mechanism of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.

Construction and testing of gene therapy retroviral vector expressing bacterial cytosine deaminase gene.

A retroviral vector containing gene for bacterial enzyme cytosine deaminase (CD) under the control of viral LTR sequences was constructed and transfected into packaging cell line GP+envAm12. High virus titer producing single cell clone (1 x 10(7) cfu/ml, determined on NIH 3T3 cells) was isolated and used to transfer CD gene into human mammary carcinoma cell lines in vitro. Transduced cells exhibited high sensitivity to the antifungal drug 5-fluorocytosine (5-FC), whereas parental cells did not. Cocultivation of CD-positive and CD-negative parental cells showed bystander effect, dependent on the ratio of CD-positive cells. No enhancement of 5-FC cytotoxicity by leucovorin was observed in cells expressing cytosine deaminase.

Aberrant markers expression in T- and B-lymphoid and myeloid leukemia cells of different differentiation stages.

The aim of the study was to ascertain if in T acute lymphoblastic leukemia (T-ALL), B acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML) of different differentiation stages the coexistence of aberrant markers correlate with the degree of leukemic blasts maturation. We evaluated the results of surface and intracellular markers in 42 T-ALL, 86 B-ALL and 71 AML cases. A large panel of monoclonal antibodies (MoAbs) against T-cell, B-cell, myeloid cell and non-lineage specific structures has been used. Patients had dual-color flow cytometric immunophenotyping performed by FACStar flow cytometer. The correct immunological diagnosis of followed new cases before any treatment has been performed and simultaneously the presence of atypical/aberrant phenotypes has been studied and correlated with leukemia cells differentiation stage. A great deal of T-ALL and AML, in opposite to B-ALL cases, revealed a high proportion of atypical phenotypes (55, 75 and 36%, respectively), which are absent in nonleukemic cells. We found out that these atypical phenotypes were present in T-ALL, AML (not clearly in B-ALL) through all differentiation stages and so we obtained an evidence that they might represent an abnormal/atypical rather than an immature phenotype, as it was postulated till now by several authors.

Angiogenesis inhibitor TNP-470: cytotoxic effects on human neoplastic cell lines.

Angiostatic substance TNP-470 displayed moderate cytotoxicity towards human leukemia HL-60, HL-60/ADR, HL-60/VCR and myeloma ARH77 cell lines with IC50 in the range 5-10 microM of concentrations and slightly higher IC50 for myeloma cell line U266. IC50 for ovarian CH-1, A2780 and A2780/ADR cell lines was in the range 10-15 microM with the exception of platinum-resistant SKOV3 cell line (more than 40 microM ). The IC50 values for MDA-MB-231 and MCF-7 breast carcinoma cell lines were 15 and 25 microM, respectively. In human hemopoietic neoplastic cell lines examined, TNP-470 induced the appearance of subpopulation with sub-G0 DNA content, suggesting the apoptosis-inducing potential of TNP-470 in these cells. No TNP-470-induced drug uptake modulation in drug-resistant leukemia cell line HL-60/VCR was observed. TNP-470 induced accumulation of cells in G0/G1 phase of cell cycle. There was no TNP-470-induced inhibition of MMP collagenase activity or MMP (MMP2 and MMP9) production in the human fibrosarcoma cells HT 1080 in vitro.

Characterization of APC exon 15 germ-line mutation in FAP family with severe phenotype showing extracolonic symptoms.

The adenomatous polyposis coli (APC) gene plays a crucial role in colorectal carcinogenesis. Germ-line mutations of APC gene give rise to familial adenomatous polyposis coli (FAP) - autosomal dominant syndrome manifesting hundreds to thousands of colorectal polyps, if untreated with malignant progression. We have used the techniques of heteroduplex analysis (HDA), protein truncation test (PTT), single strand conformation polymorphism (SSCP) and DNA sequencing for the identification and detailed positional analysis of mutations in IFAP family with the expressive phenotype characterized by polyposis and extracolonic lesions. Detailed analysis revealed a 5bp deletion in a mutation cluster region (MCR) in exon 15 of APC gene in codon 1308. Two screened members of the FAP family exhibited this novel mutation.

A note on nucleoli in granulocytic precursors of the granulopoietic proliferating compartment in patients suffering from the chronic phase of chronic myeloid leukemia treated with two different drugs with different mode of action.

The incidence of main nucleolar types in granulocytic precursors was studied in the granulopoietic proliferating compartment (GPC) of patients suffering from chronic phase of the chronic myeloid leukemia (CML) who were treated by the widely used therapy with two different drugs with different mode of action - hydroxyurea (HU) and interferon alpha (IFN-alpha). In comparison with IFN early stages of GPC, i.e. myeloblasts and promyelocytes in patients treated with HU possessed more frequently micronucleoli which are known to reflect the direct as well as indirect inhibition of the nucleolar biosynthetic activities. On the other hand, the incidence of micronucleoli in these cells of a small percentage of patients treated with IFN also reached the average values of these nucleoli which were noted in patients treated with HU.

Expression of CD26 and DPP IV in T-acute lymphoblastic leukemia: comparison of immunocytochemistry with enzyme cytochemistry.

Tile possible identity ofdipeptidyl peptidase IV (DPP IV) enzymatic activity and CD26 antigen expression in phenotypically defined T-acute lymphoblastic leukemia cells (T-ALL) was examined. For comparative studies, the combination of immunocytochemistry and enzyme cytochemistry methods was used. T he strong correlation between the CD26 antigen expression and DPP IV positivity in the majority of T-lymphoblasts in T-ALL patients was evident. No CD26 antigen was expressed on DPP IV negative T-cells. The variable CD4 and/or CD8 antigen expression, frequent CD5 and CD7 positivity and absence of surface membrane CD3 antigen were the characteristic immunophenotypic features of CD26/DPP IV positive T-lymphoblasts. Moreover, the clear CD71 and CD26/DPP IV coexpression suggested the association of CD26/DPP IV positive cells with proliferation. The immunophenotype of CD26/DPP IV positive T-lymphoblasts seems to be characteristic for the relative immature cell population. In addition, noteworthy was the slight disassociation between the very high CD26 antigen expression and moderate DPP IV activity in cells of some T-ALL patients. The possible existence of enzymatically inactive structures of CD26 antigen or inactive precursors of DPP IV detectable only by immunocytochemistry was discussed. Our study indicates that CD26 antigen expression is tended to identify cells with DPP IV enzymatic activity in T-ALL patients. The results provide some more information of CD26 antigen involvement in the pathology of leukemic cells via its DPP IV enzyme activity.

Comparison of antileukemic immunity against U937 cells in atopic asthmatics versus healthy controls.

To assess the antitumor effects in atopic asthmatics versus healthy adults, we designed this study using in vitro mononuclear cells (MNC) culture as an immunity model with human leukemic U937 cells as the target. MNCs were collected from asthmatic subjects and healthy controls. Conditioned media from the MNC cultures (MNC-CM) were collected after stimulation with various concentrations of phytohemagglutinin (PHA). We treated U937 cells with these MNC-CMs, then assayed their proliferation and differentiation after 5 days of culture. At lower PHA doses (1.25 microg/ml), as well as in absence of PHA, the asthmatic MNC-CMs inhibited U937 cells growth to a slightly greater extent than did the MNC-CMs from controls. In contrast, when higher doses of PHA were used (5, 10 microg/ml), this growth-inhibiting effect was dramatically reversed. The dual effect of MNC-CM in these two groups was also shown in U937 cell differentiation assay, assessed as follows: morphological change by Liu's staining, functional change by NBT reduction test and CD 14 expression by flow cytometric detection. We suggest that the antileukemic effects of MNCs from asthmatic patients result from a slightly immunopotentiated status. This immunity may be dramatically reversed, however, after marked activation of MNCs.

Metastasis as the first sign of thyroid cancer.

The aim of this paper is to review our experience with patients who presented with a metastatic tumor in the lymph nodes or other organs as the first sign of thyroid cancer. In 1974-1998, 18 602 patients were operated on due to goitre. There were 975 (5.2%) patients with thyroid malignant neoplasms. The group comprised 449 (46.1%) patients with papillary carcinoma, 309 (31.7%) with follicular carcinoma, 54 (5.5%) with medullary carcinoma, 106 (10.9%) with anaplastic carcinoma, and 57 (5.8%) with other types of thyroid malignant neoplasms. Out of these 975 patients, thyroid cancer was diagnosed on the basis of the detection of a metastatic tumor in 26 (2.7%) patients. In 16 (61.5%) of these patients the metastatic tumor was located in the regional lymph nodes. In 10 (38.5%) patients distant metastasis beyond the regional lymph nodes was the first sign of thyroid cancer. In (50%) patients metastasis was located in the bones, in 2 (20%) in the lung, in 1 (10%) in the heart, in 1 (10%) in the buttock, and in 1 (10%) in a central neck cyst. Metastasis was the initial manifestation of thyroid cancer in 18 (4%) of 449 papillary carcinoma patients, in 6 of 309 (1.9%) follicular carcinoma patients, and in 2 (3.7%) of 54 medullary carcinoma patients. Lymph node metastasis was the first sign of thyroid cancer in 13 (2.9%) patients with papillary carcinoma, 1 (0.3%) patients with follicular carcinoma and in 2 (3.7%) medullary carcinoma patients, and distant metastasis in 5 (1.1%) patients with papillary carcinoma and in 5 (1.6%) patients with follicular carcinoma. After the detection of the primary focus of thyroid cancer total thyroidectomy and modified neck dissection were performed in all patients. Differentiated thyroid carcinoma patients were treated complementarily with 131I and TSH suppressive doses of L-thyroxine, and medullary cancer patients with teleradiotherapy and substitutive doses of L-thyroxine.

Osteoblasts and osteoclasts in bone marrow smears of cancer patients.

Osteoblasts and osteoclasts are the unique cells occurring in bone marrow smears in situations with high bone metabolic turnover (children, trauma, rachitis, Paget disease or tumors). The collection of 2706 sternal or iliac crest aspirates from patients with hematologic malignancies and solid tumors are presented. We demonstrated significantly higher positivity for osteoblasts-osteoclasts presentation in bone marrow smears for hematological malignancies (p < 0.05), solid tumors (p < 0.01), and especially breast cancer (p < 0.001). We found a significant association between osteoblast-osteoclast positivity and dissemination of breast cancer (p < 0.05). None of the breast cancer patients without signs of dissemination (X-ray, sonography or scintigraphy) had positivity for osteoblasts or osteoclasts. We suppose that the osteoblast-osteoclast positivity in bone marrow smears can serve as a cheap marker for breast cancer dissemination.

Clodronate in the management of bone metastases: a clinical study of 91 patients.

The aim of our study was to evaluate the efficacy of oral clodronate supplementing systemic therapy and/or palliative irradiation in 91 patients with painful bone metastases. Clodronate was administered at a daily dose of 1600-3200 mg for a median of 11 months (range 3--36 months). Partial or complete pain relief was achieved in 61 of 88 evaluable patients (69%). Response rate to clodronate in patients who additionally received palliative bone radiation was similar to that in patients who did not receive irradiation (68 and 71%, respectively). Eleven out of 12 bed-ridden patients with metastatic bone pain regained the ability of walking after the treatment with clodronate. Bone pain relief lasted from 1.5 to 36 months (mean 9.3 months). Clodronate was well tolerated in all but three cases (3%) in whom the treatment was discontinued due to intensive adverse gastrointestinal effects. In conclusion, we observed satisfactory symptomatic effect and low rate of adverse reactions in patients with metastatic bone lesions treated with oral clodronate. Further large controlled studies with thorough patient monitoring are warranted to evaluate the real benefit of clodronate, and to define its optimal scheduling.

Quantitative analysis of modes of invasion and lymph node metastases in oral squamous cell carcinoma.

The mode of tumor invasion has been suggested to have a relationship to the occurrence of cervical metastasis and to prognosis in oral squamous cell carcinoma (OSCC). However, a tumor usually does not have a single mode of invasion, and the importance, if any, of the relative proportions of different modes for metastatic potential has not been studied. Forty two cases of OSCC resected with cervical lymph nodes were selected, 20 of which had nodal metastases and 22 which had not. The mode of invasion in the tumor-host interface was classified as: I - pushing borders, II - bands, III - thin cords, IV - single cells and analyzed in 20 consecutive medium power fields. Also studied were other morphological parameters: perineural and angiolymphatic invasion, tissue eosinophilia, mitosis and intensity of inflammatory infiltrate at the tumor-host interface. The majority of the cases (95.2%) showed two or more modes of invasion. Modes I, II and III occurred with similar frequency in cases with and without metastases. Mode II was the commonest and most extensive in both groups. No mode of invasion was significantly associated with metastases, independent of its extension. The other morphological parameters were neither significantly associated with cervical metastasis. In conclusion, OSCC usually shows two or more modes of tumor invasion if a large extension of tumor-host interface is analyzed. However, the relative proportions of the modes have no correlation with the metastatic potential.

Cells producing recombinant retrovirus with thymidine kinase gene from Herpes simplex virus suitable for human cancer gene therapy.

Therapeutic cells producing amphotropic retrovirus, which are able to transduce in vivo thymidine kinase gene of Herpes simplex virus were prepared. Single-cell clone cells with high virus productivity (PA-3 17JH5c113) were obtained by cell cloning. The cells were found free of replication competent retrovirus, they were non-tumorigenic in xenogeneic host and highly sensitive to ganciclovir treatment in vitro and in vivo. The therapeutic efficacy of PA-317JH5c113 cells was tested in rat brain tumor model. Increase in survival in the group of treated versus untreated rats was observed. Therefore, these cells are suitable for application in human clinical trial.

Reversal of carboplatin resistance in human laryngeal carcinoma cells.

The effectiveness of carboplatin in the treatment of patients with tumors is limited by drug resistance. Because of that, there is a great interest to find a way to revert the resistance and improve the success of cancer treatment. The aim of the present study was to examine five potential modulators of carboplatin resistance with different mode of action: buthionine sulfoximine, ethacrinic acid, amphotericine B, cyclosporine A and aphidicoline. The effect of these compounds on the sensitivity of human laryngeal parental (HEp2) and carboplatin-resistant (HEp7T) cells to carboplatin was examined by MTT spectrophotometric assay. The results have shown that buthionin sulfoximine and, to a lesser extent, ethacrinic acid reduced the resistance of HEp7T cells to carboplatin. Aphidicolin increased the sensitivity of both HEp2 and HEp7T cells to carboplatin, but this effect was more expressed in parental HEp2 cells. Our data suggest that human laryngeal carcinoma cells treated with clinically relevant doses of carboplatin became resistant to this drug due to multifactorial molecular mechanisms. Accordingly, the resistance to carboplatin could be reduced by different modulators.

Lack of correlation between repair of DNA interstrand cross-links and differential sensitivity of G0 and proliferating CD4+ lymphocytes towards cisplatin.

G0 cells in a tumor are insensitive to the chemotherapeutical agents. The nature of this resistance is not completely understood. One of the factors modulating sensitivity of cells may be DNA repair of drug induced DNA damage. In this study we have compared gene-specific formation and repair of cisplatin-induced interstrand cross-links (ICL) in human G0 and proliferating CD4+ lymphocytes. Cisplatin killing of G0 CD4+ lymphocytes is inefficient, and these cells resemble those in a tumor. After exposure to cisplatin under similar conditions, the frequency of ICL introduced is twice as high in the proliferating compared to the resting lymphocytes. Repair of ICL was measured in the housekeeping gene, dihydrofolate reductase (DHFR), in the proliferation inducible c-myc gene, and in the inactive delta-globin gene. We observed similar relative rates and extent of ICL repair in all three genes studied, in G0 or proliferating CD4+ lymphocytes. The mechanisms responsible for the resistance of G0 CD4+ lymphocytes towards cisplatin are discussed.

Flow cytometric analysis of some activation/proliferation markers on human thymocytes and their correlation with cell proliferation.

We immunophenotyped cells from ten human thymuses with emphasis on expression of the CD38 and CD71 antigens. These antigens play role in activation cells and increased expression of them was observed in some leukemia. Simultaneously, certain attention has also been devoted to some further activation markers, e.g. CD25, CD26 and HLA-DR. The classification of leukemia is based on comparison of normal and pathological cells. The study of expression of CD38, CD71 and other markers on thymocytes simultaneously with DNA analysis can be useful for answer if expression of CD38 and CD71 on pathologic cells is a sign of their proliferative ability, a part of immature phenotype in some leukemia, or it is a case of aberrant immunophenotype. In our study, 94% thymocytes were CD38+ and only 16% were CD71+. From our immunophenotypic results including MESF (molecules of equivalent soluble fluorochrome) values and analysis of the cell cycle, the conclusion could be drawn that antigen CD71 can participate in regulation of thymocyte development and presence of both -CD38 and CD71 on pathologic cells will be in all probability the case of aberrant phenotype. We observed a clear correlation of the percentage and MESF values of CD71-positive cells with the cell proliferation only after in vitro thymocytes stimulation with PHA and IL-2. In summary, a strong parallelism was observed regarding the positive relationship between the proliferative rate (assessed by the number of S-phase cells) of stimulated thymocytes and the quantitative (% and MESF) values of some markers - CD71, CD25, CD26 and HLA-DR and negative one with CD38 marker values.

Reduction of genotoxic effects of MNNG by butylated hydroxyanisole.

Butylated hydroxyanisole (BHA) is a food preservative with markedly contradictory effects. On one side many studies showed its antimutagenic and anticarcinogenic effects but on the other side dietary levels of BHA were reported to cause gastrointestinal hyperplasia in rodents. We studied the influence of BHA on cytotoxicity, mutagenicity, and DNA-damaging activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster V79 cells cultured in vitro. Our results showed that BHA significantly reduced the frequency of 6-thioguanine resistant (6-TGr) mutations and micronuclei induced in V79 cells by MNNG. These antimutagenic effects of BHA were, however, accompanied by a very marked increase of MNNG toxicity and also slightly increased level of MNNG-induced DNA damage. For evaluation of toxicity we used three methods: (i) trypane blue exclusion; (ii) plating efficiency; and (iii) intensity of cellular macromolecule synthesis. The level of DNA damage was measured by the comet assay. On the basis of obtained results we suggest that BHA, which induces phase II detoxifying enzymes, probably doesn't reduce the level of DNA damage induced in time of MNNG-treatment but it reduces the level of DNA damage created during a long-term period needed for expression of 6-TGr mutations and micronuclei.

7-ethoxyresorufin O-deethylase (EROD) activity is not capable of reflecting the overall malignant potential of breast cancer tissue.

7-Ethoxylesorufin O-deethylase (EROD) (mainly catalyzed by cytochrome P450 (CYP) 1A1 and used as a marker for CYP 1A1) activity was measured in the breast tumor and surrounding tumor free (normal) tissues of 37 female breast cancer patients with infiltrating ductal carcinoma. About 11% of the tumor and normal breast tissue samples lacked the enzyme activity. Large interindividual variations in the activities of EROD were found in both tumor and normal tissues ranging from 0 to 283 and 0 to 801 fmol/mg/min, respectively. However, no significant difference was noted between the mean EROD activities of tumor and normal breast tissues. This tendency did not change with the stage and grade of the malignancy and menopausal status. No significant correlation was observed between the EROD activity and stage or grade of malignancy (p > 0.05). Thus, it appears that EROD activity is not capable of reflecting the overall malignant potential of breast cancer tissue.

Flow cytometry of p53 protein expression in some hematological malignancies.

p53 is a tumor suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of the p53 protein expression in peripheral blood (PB) and/or bone marrow (BM) cells of patients with some hematological malignancies. A recently developed fixation/permeabilization method was modified for flow cytometric assessment of p53 protein expression using two anti-p53 monoclonal antibodies. p53 quantitation expressed as molecules of equivalent soluble fluorochrome per cell (MESF) providing valuable data contributing to a more precise definition of leukemic cells, was also applied. Our findings showed higher percentage of p53 expression in cells of AML patients at the time of diagnosis opposite to the controls. These data, in association with immunophenotype of cells, accompanied diagnosis of relapse or definition of remission after allogeneic BM transplantation. We observed also elevated levels of p53 protein at initial diagnosis of early B-ALL. According to our results quantitation of p53 protein allows better characterization of selected population of BM cells and should be used for the monitoring of blast persistence during and after therapy and might also be one of the methods to indicate early relapse. Percentage of p53 protein positivity varied in our group of B-CLL patients tested in connection with progression of disease. We documented also one case of Burkitt's lymphoma with high percentage of p53 positivity. Measurement of p53 protein expression by flow cytometry may be of clinical importance by indicating levels of positivity. Our results suggest, that p53 alteration is frequently involved at initial diagnosis of AML, in some T-cell disorders and on the contrary more frequently during early B-ALL relapse, in advanced stages of B-CLL and in Burkitt's lymphoma. p53 protein quantitation is of value to ascertain malignancy and provides additional parameter suitable for the evaluation of residual disease and for the monitoring of therapy.

Incidence of micronuclei in cytokinesis-blocked lymphocytes of medical personnel occupationally exposed to ultrasound.

In order to investigate possible DNA damaging effects of ultrasound, the micronucleus assay on cytokinesis blocked human lymphocytes was performed. Preparations were stained by conventional Giemsa staining technique combined with additional staining techniques using fluorescent dye DAPI and silver nitrate. Blood samples were taken from medical personnel employed on ultrasonic scanning in medical diagnosis and unexposed control subjects from general population. Lymphocytes were cultivated in vitro at 37 degrees C. Cytochalasin-B in final concentration of 6 microg/ml was added 44 h after mitogen stimulation and cultures were harvested 28 h thereafter. Staining with both additional techniques can be used to distinguish micronuclei originating from breakage or mitotic loss of certain human chromosomes bearing DAPI-positively stained or silver-positively stained regions. The results obtained indicate statistically significant increases in total number of micronuclei and changes in their distribution in exposed subjects compared to control. Based on different intensity of DAPI staining signal-positive and signal-negative "type" of micronuclei are distinguished, while silver staining has revealed Ag-NOR+ and Ag-NOR- micronuclei. In exposed subjects a prevalence in number of Ag-NOR+ micronuclei over Ag-NOR-micronuclei compared to control was observed, indicating greater susceptibility of chromosomes from D and G groups to damage caused by continuous occupationally exposure to ultrasound. In spite of their limitations, our results indicate that combination of conventional Giemsa staining of micronuclei with fluorescent dye DAPI and silver nitrate staining techniques can be valuable complement to the standard micronucleus assay.

p53 status in breast carcinomas revealed by FASAY correlates well with p53 protein accumulation determined by immunohistochemistry.

The prognostic and predictive value of p53 mutation in breast cancer is still conflicting. The choice of the p53 status detection method may account for some discrepancies. In this pilot study we compared two differently-based methods for detection of p53 alteration in 32 breast carcinoma samples: the immunohistochemical method using Bp53, DO1 and DO11 monoclonal antibodies for analysis of the p53 protein accumulation in cell nuclei and the functional method FASAY. FASAY - functional analysis of the separated alleles in yeast - tests the capability of the human p53 to transactivate a reporter with a p53 binding site RGC driving the ADE2 gene in yeast. In our group the percentage of breast cancers with accumulated p53 protein was 50%, as well as percentage of mutant p53 scored by FASAY was 50%. Although the agreement of both methods, when comparing the results of individual patients was high (94%), our results show that immunohistochemistry does not reflect the p53 status quite exactly.