Generation of Reactive Oxygen Species in Peripheral Blood Lymphocytes of Patients with Prostate Cancer.

The study revealed a two-fold elevation of ROS production in peripheral blood lymphocytes of the patients with prostate cancer (from 0.48±0.04 to 0.93±0.12 rel. units) together with significant variability of individual values. Hormone therapy had no effect on the mean and individual ROS levels. In contrast, organometallic cytostatics cisplatin and morfozol 2-fold increased ROS generation in lymphocytes of patients with prostate cancer above the enhanced level of this parameter determined by tumor growth.

[Comparative studies on the genotoxic activity of a new palladium (II) acidocomplex vs cisplatin in human blood lymphocytes in vitro].

A comparative study on the genotoxic activity of cisplatin versus morfozol, the first representative of a new class of cation-anion complexes of palladium [AH]2[PdCl4] (where A-methylmorpholine) has been performed using human lymphocytes in vitro. The results of the DNA-DNA cross-linking activity investigations showed that both compounds studied exhibited biphasic dose-effect relationship: a linear decrease in the DNA percent in the comet tail and the region of the "plateau". However, in the "plateau" region, morfozol reduced the DNA percent in the comet tail up to 6 times while cisplatin caused a 2-fold decrease only. Morfozol, like cisplatin, inducing DNA-protein cross-linking and generating reactive oxygen species, was more effective than cisplatin.

[Effectiveness of DNA repair and expression of MLH1, MSH2 and FASR in lymphocytes of patients with chemotherapy-responsive, disseminated cutaneous melanoma].

Patients with advanced malignant melanoma have poor prognosis as conventional chemotherapy induces complete response in a very small fraction (not more than 20%). One of research strategies aimed at raising its efficiency is the search for markers predicting individual response to chemotherapy. Our study was concerned with evaluation of the potential of DNA damage, repair (BER, MMR), expression of proteins MLH1, MSH2 and FasR as prognosticators of chemotherapy. These parameters were assessed in lymphocytes sampled from the blood of patients with metastatic cutaneous melanoma before and after one cycle of chemotherapy with lomustine, dacarbazine, cisplatin and interferon-gamma (LDCI). Clinical response was evaluated after a full course of therapy. We established that the major DNA damage induced by chemotherapy occurred on the levels of AP sites and single strand (SS) breaks. Despite the individual variations in BER efficacy, complete repair of SS breaks was reported in lymphocytes of all patients 30 days after the first cycle of chemotherapy. As a consequence, this type of damage and relevant BER efficacy did not correlate with clinical response. Conversely, the number of DNA double strand breaks detected in lymphocytes after the first cycle of chemotherapy was in good correlation with positive clinical response (p < 0.001). This parameter does not fully represent MMR function and, if coupled with cytotoxic effect of chemotherapy on lymphocytes, may be used as a predictive marker for clinical response to LDCI chemotherapy regimens for melanoma.

[Cellular markers based on DNA damage and repair (BER, MMR), expression of MLHI, MSH2, FasR, and cell death of lymphocytes as predictive parameters for clinical response to chemotherapy of melanoma].

Melanoma is a highly aggressive neoplastic disease attributed to transformed melanocytes. The efficacy of regimens of cytotoxic chemotherapy for advanced stage patients does not exceed 20%. Search for lymphocyte markers of patients' sensitivity to chemotherapy provides a rational basis for development of cytotoxic chemotherapy. Using blood lymphocytes we evaluated efficacy of BER and MMR, expression of MLH1, MSH2 and FasR, and cell death in melanoma patients relative to clinical response to chemotherapy. We found that LDCI-chemotherapy (lomustine, dacarbazine, cisplatin and interferon gamma), induced AP sites and DNA ss-breaks which repaired trough BER pathway. However, neither initial DNA damage nor the rate of their repair correlated with clinical response. This result prompts us to think that this type of damage is not crucial in cytotoxic effect of LDCI-regimen of chemotherapy. DNA ds-breakes appeared downstream ss-breakes were attributed to repair of 06-methylguanine by MMR mechanism in PHA-stimulated lymphocytes. The number of ds-breakes appeared by 48 correlated with positive clinical response of patients to chemotherapy. The same link was observed between clinical response and the number of dead lymphocytes. However, there was no correlation between clinical response and expression of MLHI + MSH2 and FasR. These results imply possible contribution of crosslink repair through NER pathway to formation of DNA ds-breaks as well as to cytotoxicity of LDCl-therapy. The observed link between high level of secondary ds-breaks and positive response to chemotherapy indicates the potential of these instruments to serve as prognostic end point in clinical trials.

[Genetic markers of melanoma].

Melanoma is among the most aggressive malignancies. Tumors with a thickness of 4 mm can produce metastases, and the mean survival of the patients is 9 months. The review presents modern classification of the melanoma types based on cytological and morphological indices (Clark model). Alterations of genes in melanomas are discussed in detail. These genes include tumor suppressors, proliferative response genes (oncogenes), and transcription factors. Alterations in the Wnt signaling, MAPK cascade, and Fas signaling pathways are considered. Changes in the mismatch repair (MMR) genes are also analyzed. From practical perspective, understanding the genetic alterations provides identification of potential targets for therapeutic exposure and enables prognosis of the tumor response to chemotherapy.

Sensitivity of human lymphocytes to genotoxic effect of n-methyl-n-nitrosourea: possible relation to gynecological cancers.

UNLABELLED: The aim of this work is to study responses of PHA-stimulated and resting lymphocytes to methylating agent N-methyl-N-nitrosourea (MNU) and to compare sensitivity to this agent of healthy donor lymphocytes and lymphocytes from patients with gynecological cancers. METHODS: Cytotoxicity of MNU, apoptotic death of lymphocytes, was evaluated using two common tests--annexin V-FITC detection assay and live/dead double staining assay (nuclear morphological changes). Genotoxic effect of the agent was determined as delayed (secondary) DNA double strand breaks (DSBs) using neutral comet assay both conventional variant and modified for detection of bromodeoxyuridine-labelled comets, produced by proliferating lymphocytes only. RESULTS: Unstimulated lymphocytes were tolerant to geno- and cytotoxic effects of MNU. In contrast to resting cells, proliferating lymphocytes showed significant genotoxicity (p=0.0054) of MNU followed by increased apoptotic death of cells (p<0.05). Average number of secondary DSBs induced by MNU in lymphocytes from patients with gynecological cancers was about 4-fold less than that of lymphocytes of healthy donors. While lymphocytes from cancer patients did not change proliferative index in response to MNU, the agent decreased 2-fold proliferative indices of stimulated lymphocytes from healthy donors. CONCLUSION: There is a reverse association between geno- and cytotoxicity of MNU in stimulated lymphocytes and the presence of tumor. The relationship appears to be based on MMR-insufficiency in lymphocytes of the cancer patients.

[The use of 1H-NMR spectroscopy for the prognosis of overall survival of breast cancer patients].

The pool of low-molecular metabolites in untreated breast cancer patients was investigated by high-resolution 1H-NMR spectrometry. It was found that the delta = 1.75 m.d signal can serve as a prognostically significant negative factor. Beside other markers, its absence in tumor sample extracts may indicate favorable prognosis.

[Effect of chemotherapy on cytosol proteins of ovarian tumors]. / Vliianie khimioterapii na belki tsitozolia opukholei iaichnika.

Cytosol proteins of malignant ovarian tumors were studied with one- and two-dimensional gel electrophoresis, prior to and after effective chemotherapy with cisplatin and taxotere. Effective chemotherapy caused the bands and spots of 29, 36 and 50-55 kDa to fade significantly or disappear completely. It is suggested that protein 36 kDa is the proliferating nuclear cell antigen while one of the proteins 50-55 kDa--betatubulin. These proteins may serve as additional markers of ovarian tumor sensitivity to chemotherapy.

[Comparative study of cytosolic proteins in normal and neoplastic ovaries by polyacrylamide electrophoresis]. / Sravnitel'noe issledovanie belkov tsitozolia normal'nykh i opukholevykh tkanei iaichnikov metodom élektroforeza v poliakrilamidnom gele.

The report discusses the data on cytoplasmic protein occurrence in ovarian tissue (106) obtained by polyacrylamide gel electrophoresis. They were identified in normal ovarian tissue (15), benign tumor (16), primary malignant tumor (55) and tumor tissue after chemotherapy (20). A band was detected in the area of molecular 36 cD in malignant epithelial tumor tissue which was not seen in either normal, benign tumor tissue or after successful chemotherapy.

The use of 1H-NMR spectroscopy for predicting the efficiency of neoadjuvant chemotherapy of breast cancer.

The pool of low-molecular-weight metabolites was studied in patients with breast cancer by high-resolution 1H-NMR spectroscopy. In order to predict the efficiency of treatment, mathematical regression analysis was carried out with consideration for some clinical morphological characteristics of patients, chemotherapy protocols, and the degree of therapeutic pathomorphosis. The efficiency of drug therapy was largely determined by metabolic status of tumors in untreated patients with breast cancer.

[The new antitumor drug cycloplatam: cytotoxicity and DNA interstrand cross-linking]. / Novyi protivoopukholevyi preparat tsikoplatam: tsitotoksichnost' i mezhnitevye sshivki DNK.

Cytotoxicity genetic mechanisms such as induction of SOS-repair, excision repair and interstrand coupling induced by cycloplatam or ammine (cyclopentyl amine)-S(-) malatoplatinum (II), a new antitumor drug, were for the first time studied in comparison to those of the known drug cis-diammine dichloroplatinum (II) (DDP) in a model system of Escherichia coli. In the cells of E. coli the cycloplatam cytotoxicity was much lower than that of DDP. Both the drugs induced SOS-repair in E. coli PQ37. In a concentration of 25 microM DDP was 20 times as active as cycloplatam. In concentrations of 40 to 100 microM the difference leveled. Both the drugs induced interstrand coupling in specimens of pure DNA from calf thymus and E. coli. When the cells of the wild type E. coli AB1157 were incubated in the presence of the drugs only DDP induced the DNA interstrand coupling. No correlation between the DNA interstrand coupling induced by cycloplatam or DDP and cytotoxicity of the drugs was observed.

[Kinetics of development of human melanoma xenografts in immunosuppressed animals]. / Kinetika razvitiia ksenograftov melanom cheloveka na immunosupressirovannykh zhivotnykh.

An experimental model of development of the human tumor xenografts (melanomas HCMB and Bro) in immunosuppressed phenotypically normal animals (CBA/Ca mice) was developed. Optimal conditions were established for immunodeficiency induction in the animals. Kinetics of development of the xenografts in the immunosuppressed animals was studied. The level of 0(6)-alkyl-guanine-DNA-alkyltransferase, an enzyme responsible for repair of DNA damage in the tumor cells induced by chemotherapeutic agents (nitroalkylurea), was determined.

[The biochemical mechanisms of the action of N-alkyl-N-nitrosoureas. The possible reasons for drug resistance to these compounds]. / Biokhimicheskie mekhanizmy deistviia N-alkil-N-nitrozomochevin. Vozmozhnye prichiny lekarstvennoi ustoichivosti k étim soedineniiam.

N-alkyl-N-nitrosoureas exhibit a wide spectrum of antitumor activity. They react as alkylating agents at nucleophilic sites in purine and pyrimidine moieties of DNA. The predominant site of this alkylation is N7 of guanine, which is followed by the site N3 of adenine and 06 of guanine. The formation and persistence of 0(6)-alkylguanine (0(6)-AG) may be of primary importance in cytotoxicity of the nitrosoureas. 0(6)-AG adducts of DNA of the tumor cells are repaired by protein 0(6)-alkylguanine-DNA transferase (0(6)-AGT) which transfers the alkyl group to internal cysteine residue being the acceptor protein for the alkyl group in an irreversible transfer reaction. 0(6)-AGT can protect the tumor cells against 0(6)-AG adducts by the way of inhibiting the formation of the DNA interstrand cross-links 0(6)-AGT plays an important role in the drug resistance because it repairs the DNA alkyl adducts at the 0(6) position of guanine. The 0(6)-AGT activity inversely correlates with the cytotoxic effect of the nitrosoureas. The agents like 0(6)-methylguanosine, 0(6)-methyl-2'-deoxyguanosine, and some 0(6)-benzylated guanine derivatives are effective inactivators of 0(6)-AGT, and thus can be used to enhance the cytotoxicity of N-nitrosoureas. The activation of 0(6)-AGT and other repairing enzymes such as alpha and beta DNA-polymerases as well as an increase in the level of reduced glutathione may be used in developing the resistance to the nitrosoureas.

[Modulation of the antitumor activity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a by O(6)-methyl-2'-deoxyguanosine--a new inhibitor of O(6)-alkylguanine-DNA-alkyltransferase]. / Moduliatsiia protivoopukholevoi aktivnosti 1-(4-amino-2-metil-5-pirimidinil)metil-3-(2-khlorétil)-3-nitrozomochev iny O(6)-metil-2'-dezoksiguanozinom--novym ingibitorom O(6)-alkilguanin-DNK-alkiltransferazy.

O6-Methyl-2'-deoxyguanosine (O6-MedG), a novel inhibitor of O6-alkylguanine-DNA alkyltransferase (O6-AGT), has been synthesized. The ability of O6-MedG to deplete the O6-AGT activity in leukemia L1210 and melanoma B16 cells in vivo has been studied. After intraperitoneal administration of O6-MedG to mice bearing leukemia L1210 or melanoma B16, the activity of O6-AGT in tumour cells decreased by 50%. Pretreatment of leukemia L1210 bearing mice with O6-MedG (200 mg/kg) 24 hours prior to ACNU (15 mg/kg) administration resulted in six out of seven 60-day survivors. Treatment of mice with ACNU (15 mg/kg) alone increased the life span by 200%. Treatment of melanoma B16 bearing mice with O6-MedG and 3 hours thereafter with ACNU resulted in a 50% inhibition of tumour growth, whereas the inhibiting effect of ACNU alone was 16%. There was no difference in leukemia growth when L1210/BCNU bearing mice were treated with O6-MedG followed by ACNU treatment. In vivo ACNU (15 mg/kg) produced a deep and prolonged inhibition of DNA, RNA and protein synthesis in leukemia L1210 cells. The DNA synthesis in leukemia L1210/BCNU cells was shown to recover more rapidly than in L1210 cells. The activities of DNA-polymerases alpha and beta and, especially, of O6-AGT were elevated in ACNU-resistant leukemia cells as compared with ACNU-sensitive cells. The activation of some repairing enzymes, such as O6-AGT, DNA-polymerases alpha and beta as well as increased levels of GSH may play a role in the development of drug resistance to ACNU.

[Biochemical mechanisms of resistance to a new antineoplastic agent amin(cyclopeptidylamin)-S-(-)-malatoplatinum (II) (cycloplatam)]. / Issledovaniia biokhimicheskikh mekhanizmov rezistentnosti k novomu protivoopukholevomu preparatu amin(tsiklopentilamin)-S-(-)-malatoplatine (II) (tsikloplatamu).

The "in vivo" effect of cycloplatam on DNA synthesis in leukemia P388/o (parent strain), P388/c (cycloplatam-resistant strain) and in some organs of tumour-bearing mice, such as spleen, kidney, gastrointestinal mucosa (GI mucosa) and bone marrow, has been studied. Cycloplatam induced a deep and stable inhibition of DNA synthesis in leukemia cells and kidney. DNA synthesis in normal dividing cells (GI mucosa, bone marrow, spleen) was shown to recover more rapidly than in leukemia cells and kidney after cycloplatam treatment. The GSH level was increased tenfold in leukemia P388/c cells in comparison with P388/o. The glutathione peroxidase and glutathione reductase activities were increased twofold in the resistant strain in comparison with the parent strain, while the activity of glutathione-S-transferase showed a 1.5-fold increase. Administration of cycloplatam to tumour-bearing mice caused a marked increase of the GSH level in the both leukemia strains. Alterations in GSH-dependent enzymes following cycloplatam therapy were expressed in a lesser degree. These data indicate that GSH and GSH-dependent enzymes may play an important role in the resistance of P388 leukemia cells to cycloplatam.

[Changes in the replication apparatus and phosphorus-containing metabolite pool in experimental tumors in animals during development]. / Izmeneniia v apparate replikatsii i sostoianii pula fosforsoderzhashchikh metabolitov éksperimental'nykh opukholei zhivotnykh v protsesse razvitiia.

Activity of replicase complex enzymes involving thymidine kinase (TK), ribonucleotide reductase (RR), DNA-polymerases alpha and beta as well as DNA synthesis and single breaks in DNA were studied during growth of P388 ascites tumor. Under these conditions the rate of DNA synthesis was distinctly decreased via salvage pathway and de novo. Single breaks were not detected in the preexistent DNA within various periods after transplantation of P338 leukemic cells. Retardation of DNA synthesis during tumor growth correlated with a decrease in TK, RR and DNA-polymerase alpha activities, while DNA-polymerase beta activity was markedly increased. Growth of melanoma B16 was accompanied by a decrease in content of ATP, ADP, NAD, phosphocreatine and phosphosaccharides as well as by an increase in the level of inorganic phosphates.

[Biochemical mechanisms of resistance to a new antineoplastic drug CRC 680578 from the nitrosourea class]. / Biokhimicheskie mekhanizmy rezistentnosti k novomu protivoopukholevomu preparatu iz klassa nitrozomochevin CRC 680578.

The biochemical mechanisms of resistance to CRC 680578, a new antitumour chloroethylnitrosourea alpha-amino acid derivative, were studied. Alterations in DNA, RNA and protein syntheses, SH-group content, drug efflux, activities of replicative and repair enzymes, such as ribonucleotide reductase, thymidine kinase, O6-alkylguanine-DNA-alkyltransferase and DNA polymerases alpha and beta and damages of the DNA secondary structure were investigated in sensitive and resistant to CRC 680578 leukemia L1210 cells. It was found that the total SH-group number in drug-resistant cells was increased (about 1.3-fold in comparison with sensitive cells) which seems to be due to the mechanisms of drug resistance. CHC 680578 induced less pronounced inhibition and more rapid restoration of DNA and RNA synthesis in resistant cells. No differences between the ribonucleotide reductase and thymidine kinase activities were found either in intact cells of the both strains or after drug administration. The efficiency of repair of DNA chloroethyl adducts by O6-alkylguanine-DNA-alkyltransferase in leukemia cells of various sensitivity was found to be identical. The differences in enzyme activities in intact cells of the both strains were insignificant. It was supposed that factors other than changes in the level of O6-alkylguanine-DNA-alkyltransferase in leukemia cells may be responsible for the resistance to CRC 680578. The increase in the levels of DNA polymerase alpha and, especially, of DNA polymerase beta, in sensitive (but not resistant) mouse leukemia cells 48 hours after drug administration is though to define the mechanism of resistance to the new antitumour agent CHC 680578.