TNF-alpha induced changes in cell membrane antigen expression on K-562 cells associated with increased lactate dehydrogenase (LDH) release.

TNF-alpha is a pleiotropic cytokine, which induces death of sensitive cells, whose effect depend on cell membrane receptor expression, cell cycle phases, as well as on intracellular ratio of pro- apoptotic and anti-apoptotic molecule expression. Since determination of LDH release from cultured cells in vitro, reflects early membrane alterations, we estimated and compared LDH release from cultured cells with changes in cell membrane antigen expression on K-562 cells after TNF-alpha treatment by flow cytometry. The significant increase in LDH release activity and cytotoxicity values was associated with decrease in membrane molecule expression for CD45 and CD30 as well as for low expressed CD45RA and CD38 after TNF-alpha treatment. However, percentage of decrease of all examined molecules is not uniform, and appears to depend on the respective level of pre treatment values expression and molecule type. These results indicated the complexity of events on cell membrane, including association between increasing LDH release and decrease of antigen expression of membrane molecules following TNF-alpha mediated processes.

Modulation of guinea pig peritoneal macrophage migration in vitro: effects of protease inhibitors.

With the purpose of examining the significance of macrophage neutral proteases for the random migration of guinea pig peritoneal macrophages, we tested the influence of the polyvalent protease inhibitor, Trasylol (100-2000 KIU/ml), and the serine protease inhibitor, phenylmethyl sulphonyl fluoride (PMSF; 10(-4)-10(-3) M), on this function. Using the capillary method, the migration of resident and oil-induced cells was examined under different culture conditions: absence of serum; presence of 10% of either intact (IHS) or acid-treated horse serum (AHS). Protease inhibitors only reduced the locomotion of inflammatory macrophages. Trasylol caused a dose-dependent reversible macrophage migration inhibition the degree of which depended upon the conditions in culture (AHS greater than no HS greater than IHS). The irreversible effects of PMSF were better in the presence of serum. Although the migration of control macrophages occurred at the three different quantitative levels (AHS greater than IHS greater than no HS), there were no differences in the migration kinetics under the action of these antiproteases compared to the corresponding control, which, together with the preserved cell viability indicates that the drugs did not act deleteriously on macrophages. Our results suggest that macrophage neutral proteases do not only play an important role in delayed hypersensitivity, as previously demonstrated, but also in the random migration of inflammatory macrophages. Furthermore, it is shown that the clinical application of Trasylol may have an influence on this vitally important macrophage function.

A comparison of the NK cell cytotoxicity with effects of TNF-alpha against K-562 cells, determined by LDH release assay.

Effects of r h TNF-alpha as a single cytotoxic mediator against K-562 cells was examined by LDH release and compared with NK cell cytotoxicity. The mean values of the percentage of LDH release (x = 6.25 +/- 3.68%, for ten individual experiments) from K-562 cells cultured for 2 h with r h TNF-alpha 100 U/ml of culture medium did not give significant difference in comparison with mean values of percentage LDH release (x = 6.43 +/- 2.97%, for 37 individual experiments) from K-562 cells which were cultured without r h TNF-alpha (Student's t-test, P > 0.05). The results also showed, that in the presence of increasing concentrations of r h TNF-alpha there was no significant increase of LDH release through the cell membrane in these short term incubations. However, significant difference in LDH release from K-562 cells was found after 6 h between cultures treated for 30 min with or without r h TNF-alpha (Mann-Whitney test, P < 0.05). Since TNF-alpha alone shows a lower degree of K-562 cell membrane damage than NK effectors, this suggested that TNF-alpha is neither an only nor a major mediator of cell destruction, based on determination of LDH release.

Reactivity of monoclonal antibodies OK-CLL (anti-CD5) with peripheral blood cells of patients with B cell lymphoproliferative disorders.

Reactivity of OK-CLL monoclonal antibodies that can identify CD5 antigen on peripheral blood mononuclear cells, was investigated in 172 patients with B cell chronic lymphocytic leukemia (B-CLL) in clinical stages RAI O-I and RAI II-IV and in patients with non-Hodgkin's lymphomas, classified according to the Working formulation into high and low grade histologic type. The OK-CLL reactivity with B-CLLs in the initial (RAI O-I), as well as in advanced stages of disease (RAI II-IV) was significantly higher than in controls. Peripheral blood cells of lymphoma patients, regardless of histological type, showed a much lower values of the CD5 positive population than chronic lymphocytic leukemia (CLL) patients, and a non significant discrepancy between the number of cells stained with anti-CD5 and those stained with other T cell markers. In spite of the showed considerable decrease of CD5 positive cells in CLL patients during therapy, elevated number of this population compared to normal individuals, after chemotherapy, was found. However, in lymphoma patients of both types of malignancies, CD5 positive population increased concomitantly with therapy. These results may suggest that analysis of CD5 antigen expression on peripheral blood cells of patients with B cell lymphoproliferative malignancies may have diagnostic, or, in correlation with some relevant clinical parameters, a potential predictive value in the treatment of those patients.

Steroid receptors in pleural effusions of advanced breast cancer patients.

The steroid receptor content in breast carcinoma correlates with the responsiveness of malignant cells to endocrine manipulation. Although the steroid receptor status of the primary tumor is mostly used to select systemic therapy, it was suggested that steroid receptor content should be evaluated in metastatic lesions whenever possible. In this study the estrogen and progesterone receptor content was determined biochemically in 38 pleural effusions from advanced breast cancer patients. In 17/38 patients the steroid receptor status was assessed twice during the course of the disease - at diagnosis in the primary tumor/lymph nodes, and subsequently in metastatic pleural effusion fluid. A trend towards lower receptor values in pleural fluids was evident. There was no correlation between pleural steroid receptor content and pleural response to endocrine or chemo/endocrine therapy, indicating that the usefulness of effusional steroid receptors for therapy planning of advanced breast cancer could not be confirmed in this study.

Estrogen and progesterone receptor content in bilateral breast cancer.

Estrogen and progesterone receptor content was determined in 34 patients with synchronous and 23 patients with asynchronous bilateral breast cancer. Steroid receptor content was measured quantitatively by DCC method. It was shown that progesterone receptor content could not be predicted, as well as, that steroid receptor content of the second tumor significantly influenced the development of asynchronous bilateral breast cancer. The high discordance rate concerning histologic type between two tumors within synchronous as well as asynchronous biopsies was observed. The obtained results indicate that both synchronous and asynchronous bilateral breast tumors may be considered as biologically different tumors whose both steroid receptor levels should be determined whenever possible.

The comparison of spontaneous LDH release activity from cultured PBMC with sera LDH activity in non-Hodgkin's lymphoma patients.

Based on the fact that lactate dehydrogenase (LDH) enzyme is a very sensitive indicator of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status, and malignant transformation, in this study we compared values of the spontaneous LDH release from circulating PBMC with sera LDH activity in 53 different subtypes of non-Hodgkin's lymphoma (NHL) patients. Results shows that serum LDH was significantly (p < 0.05) elevated in comparison to the range values only in the advance clinical stage (III and IV) in all investigated subtypes of NHL according to The Working and REAL classification. On the other hand, the spontaneous LDH release from cultures PBMC is significantly (p < 0.01) elevated in early and advanced stage in all investigated forms of NHL in comparison to healthy controls. Based on consideration that an increase in spontaneous LDH release appears before elevated sera LDH activity, we conclude that determination of spontaneous LDH release by microassay from cultured cells together with other findings may help in the diagnosis of NHL patients, especially in patients with early stage of disease.

Stage dependence of NK cell activity and its modulation by interleukin 2 in patients with breast cancer.

NK cell activity was evaluated in breast cancer patients with different clinical stages of disease prior to surgery. In 58 patients with Stages I-III of breast cancer the peripheral blood NK cell activity was significantly reduced as compared to controls, and NK activity of 11 patients with Stage IV and metastases was significantly reduced as compared to both controls and patients with locoregional disease. In vitro treatment of peripheral blood lymphocytes (PBL) in medium with 10% fetal bovine serum (FBS) alone significantly increased NK cell activity in patients with Stages I-III and Stage IV of disease, although the level of NK cell activity of patients with Stage IV remained below that for less advanced disease. In vitro treatment of PBL of some Stages I-III patients (n = 41) with interleukin 2 (IL 2) gave a significant, dose-dependent enhancement of their NK cell activity so that it was significantly higher than basic NK activity of healthy controls. The results showed decrease of NK cell activity in breast cancer patients especially in advanced disease and enhancement with IL 2 indicating the possibility of immunotherapy in this neoplasm.

Amine oxidase-mediated cytotoxicity of spermine and epinephrine to human myelogenous leukemia K562 cells.

The effects of spermine and beta-adrenoceptor agonists (epinephrine, terbutaline and orciprenaline) in the presence and in the absence of fetal bovine serum (FBS) on human myelogenous leukemia K562 cells viability (V) and survival (N/Nc) were examined. Spermine-FBS significantly decreased both V and N/Nc of K562 cells. Aminoguanidine (AG), an amine oxidase inhibitor, and reduced form of glutathione abolished this effect demonstrating that the spermine-FBS action was amine oxidase-mediated. Epinephrine expressed a strong cytotoxicity to K562 cells which was abolished by pargyline, a specific monoamine oxidase (MAO) inhibitor, as well as by reduced form of glutathione. Terbutaline and orciprenaline exerted no cytotoxic activity to K562 cells cultured in FBS-supplemented medium, independently on the presence of spermine. However, terbutaline at concentrations of over 1 mmol strongly inhibited the cytotoxic effect on spermine-FBS. The relationship between cytotoxicity and chemical structure of beta-adrenoceptor agonists was discussed especially with respect to their stability toward oxidation.

Association among an autocrine parameter (EGF-R) and endocrine parameters (ER and PR) in locoregional breast cancer.

Sixty-three locoregional breast cancer biopsies were examined for association among epidermal growth factor receptors (as parameter of autocrine growth control), and estrogen and progesterone receptors (as endocrine parameters of estrogen responsiveness). In the tumor group with similar steroid receptor levels, low (0-50 fmol/mg), medium (50-100 fmol/mg) and high (above 100 fmol/mg), an inverse quantitative correlation between epidermal growth factor receptors and progesterone receptors was obtained (p < 0.05). In the tumor groups where estrogen receptor levels were higher or lower than progesterone receptor levels, epidermal growth factor receptors were correlated neither with estrogen nor with progesterone receptors (p > 0.05). It seemed that inverse correlation between endocrine and autocrine parameters obtained in previous studies might not be a common behaviour of all breast tumors.

To the mechanism of spermine-FBS cytotoxicity toward K562 human myelogenous leukemia cells.

The effects of several compounds acting through adenylate cyclase system and/or influencing prostaglandin biosynthesis on spermine-FBS cytotoxicity to human myelogenous leukemia K562 cells were studied. Salbutamol, a beta 2-adrenoceptor agonist inhibited to a certain extent spermine-FBS cytotoxic action to K562 cells, and propranolol, a beta 2-adrenoceptor antagonist, did not affect this inhibition. Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase, acted suppressing spermine-FBS cytotoxicity to K562 cells. Pretreatment of the cells with dexamethasone did not significantly alter salbutamol-related inhibition of spermine-FBS cytotoxicity. Indomethacin, an inhibitor of cyclooxygenases directly involved in prostaglandin biosynthesis, did not interfere with protective terbutaline effects against spermine-FBS cytotoxicity to K562 cells during the 24-hour period.

In vitro effect of indomethacin on mitogen-induced lymphoproliferative response in lung cancer patients.

There is some evidence that prostaglandin (PGE)-secreting cells may have a role in immunosuppression in cancer patients. In this work we investigated the effects of indomethacin--a PGE synthesis inhibitor, on PHA-induced lymphoproliferative response in vitro. Twenty patients with lung cancer before therapy were included in this study. When compared to controls, the patients had significant decrease of T cell number and proliferative response to PHA (p less than 0.001) and increased number of mononuclear phagocyting cells (p less than 0.001). The degree of depression of lymphocyte response did not correlate with the number of mononuclear phagocytes. The presence of indomethacin in the culture induced significant (p less than 0.01) improvement of the reactivity in high percentage (75%) of patients with diminished lymphoproliferative response to PHA. In the patients with normal lymphocyte response, indomethacin did not change reactivity to PHA. These results indicate that PGE-secreting cells may contribute to the immune depression in lung cancer patients, and that indomethacin may have therapeutical potential in some patients.

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.

Estrogen dependence of primary breast cancer--correlation with histologic type and grade.

The purpose of this study was to investigate whether histologic type and grade of primary breast cancer are related to the estrogen and progesterone receptors. Our results showed that histologic type influenced the estrogen dependence through histologic grade. There was no difference in the estrogen and progesterone receptor content, when corresponding grades of different histologic types and estrogen dependence were confirmed by quantitative non-parametric analysis. It showed a direct relationship between estrogen and progesterone receptor content in all the three histologic grades and further that histologic grade defines three different groups in regard to progesterone receptor content.

Content of epidermal growth factor receptor in metastatic breast cancer: its role in endocrine sensitivity prediction.

Epidermal growth factor receptor (EGF-R) is known as an indicator of endocrine independence of breast cancer. However, a small proportion of EGF-R expressing tumors was found to respond to endocrine treatments. On the other side, a cut-off point of EGF-R positivity is not yet defined. In the aim to find out whether there exists a cut-off value that sharply discriminate the endocrine sensitive and endocrine insensitive breast cancers, the quantitative EGF-R content was analyzed in a group of 42 female patients with metastatic disease, being routinely treated with chemo-, chemo-endocrine, or endocrine therapy alone. Steroid receptors (SR) and EGF-R were determined by biochemical methods in tissue samples of an unselected group of patients. Patients with metastatic disease, either at diagnosis, or developed after the treatment of operable or locally advanced breast cancer, were included in the present analysis. According to the treatments used, and their therapeutic response, all patients were divided in endocrine sensitive or resistant, and chemo-sensitive or resistant. The SR and EGF-R status and content was analyzed in relation to the sensitivity to both systemic treatments. The EGF-R content was significantly lower in responders to endocrine treatments, compared to non-responders, while there was no difference in EGF-R level, in relation to the sensitivity to chemotherapy. In addition, the EGF-R content was significantly higher in chemo-sensitive tumors, than in endocrine sensitive. On the contrary, ER content was significantly higher in endocrine sensitive, than in endocrine resistant, and in chemo sensitive patients, as well. Similar differences were found in PR content, but they were less pronounced. While the individual ER contents in endocrine sensitive and endocrine resistant tumors overlapped, the EGF-R ranges were different: no one endocrine sensitive tumor exceeded the EGF-R content of 26 fmol/mg, thus suggesting the EGF-R cut-off point of endocrine sensitivity. The clinical use of EGF-R, with the cut-off point of 26 fmol/mg, in addition to clinical criteria of endocrine sensitivity and SRs, would significantly improve the correct endocrine sensitivity prediction (from 52 to 78%). In conclusion, in a group of metastatic breast cancer patients, treated routinely by systemic therapies it was found, that the use of higher cut-off point for EGF-R positivity can improve the prediction of endocrine sensitivity. The prognostic relevance of this cut-off value remains to be analyzed.

Expression of epidermal growth factor receptor in breast cancer, from early stages to advanced disease.

Epidermal growth factor receptor was determined in 106 newly diagnosed breast cancer patients, using the biochemical method. The group consisted of 58 patients in stage I-II, and 48 patients in stage III-IV. Although a significant inverse correlation was found between EGF-R status, and ER or PR status, quantitative content of EGF-R did not correlate either with quantitative ER, or PR levels. The ER/PR content was similar in all clinical stages, suggesting their stability during the clinical course of the disease. EGF-R content was significantly higher in stage IV, compared to stage I, while intermediate clinical stages and all substages did not differ according to the EGF-R content. EGF-R was confirmed as a weak prognostic factor within clinical stages. However, in a whole group, the overall survival was significantly better in patients whose tumors EGF-R content was lower than 26 fmol/mg, compared to those with higher ERF-R content. EGF-R content was highly predictive for the response to systemic endocrine treatment, in metastatic breast cancer patients. In locally advanced breast cancer a trend towards higher levels of EGF-R was found in inflammatory breast cancers, compared to non-inflammatory ones. Slightly higher levels were found in responders to local non-endocrine primary treatments (radiotherapy with or without chemotherapy), compared to non-responders, suggesting the possible predictive role of EGF-R for the response to such treatments. Our results emphasized the usefulness of quantitative receptor determination suggesting the relative stability of EGF-R content during the clinical course of breast cancer, its independence from ER, its significant predictive and weak prognostic values, and a possible correlation with the aggressiveness of the disease, and response to non-endocrine treatments.

The importance of the specific Z-DNA structure and polyamines in carcinogenesis: fact or fiction.

In this work some aspects of carcinogenesis are given. The importance of the emergence of Z or H DNA structure in the gene, or in the flanking gene sequences for the gene deletion and unusual gene recombination, is discussed. Some considerations on the role of selective pressure (of polyamines, of Mg2+, of the various levels of topoisomerase II, and of ATP) in the process of oncogene amplification, are given too.

Impaired perforin-dependent NK cell cytotoxicity and proliferative activity of peripheral blood T cells is associated with metastatic melanoma.

AIMS AND BACKGROUND: Patients with metastatic melanoma often have defects in the percentage and function of peripheral blood NK cells, which are involved in the non-specific innate antitumor immune response, and T cells, which participate in the specific acquired antitumor immune response. The aim of this study was to investigate in more detail not only the percentage but also the activation status and function of NK and T cells in patients with metastatic melanoma prior to therapy. METHODS: The percentage of peripheral blood CD56+ NK cells, CD3+ T cells and their CD4+ and CD8+ subsets, as well as the expression of the activation antigens CD69, CD38 and HLA-DR were analyzed by flow cytometry. The functional capacity of NK cells was evaluated by the 51-chromium release cytotoxicity assay, while the proliferative activity of T cells was estimated by the lymphocyte transformation test to mitogen phytohemagglutinin. RESULTS: The results obtained in this study have revealed a new aspect of NK and T cell dysfunction that is not, as commonly reported, associated with a decrease in their percentage. Moreover, a significant number of the investigated patients had a higher percentage of NK cells that did not lead to improved NK cell cytotoxicity as a result of the detected defect in the NK cell perforin-mediated cytotoxic mechanism of tumor cell lysis. The impaired proliferative response of T cells was associated with a decreased expression of the activation antigen HLA-DR. CONCLUSION: The novel finding in this study of melanoma patients with metastatic disease is the impaired perforin-dependent NK cell cytotoxic mechanism, which was recently shown to be primarily responsible for preventing metastasis. Another interesting finding was the generally hyporeactive status of T cells, possibly resulting from persistent antigenic stimulation. The observed dysfunction of NK and T cells in patients with metastatic melanoma prior to therapy point to the need to supplement chemotherapy with appropriate immunotherapeutic agents in order to overcome the immunosuppression associated with advanced malignancy.