Sister chromatid exchanges induced by sarcolysine in human lymphocytes in vitro

The effect of three concentrations of sarcolysine (0.5 micrograms/ml, 1 microgram/ml and 2 micrograms/ml) on the sister chromatid exchanges (SCE) was investigated in human lymphocytes in vitro. A dose related increase in SCEs frequencies was observed after sarcolysine administration and also a delayed development of cell cycle has been induced by the two last concentrations. The variation range of SCEs per cell was dose-dependent and it was considered to represent the acquired genetic instability induced by the drug.     

Caffeine effects on the chromosomal aberrations induced in vivo by sarcolysine and methotrexate

Wistar adult rats bearing Guérin T8 ascite tumours were intravenously inoculated with 3 mg/kg b.w. sarcolysine and respectively 5 mg/kg b.w. methotrexate on the 7th day after ascite cells grafting. Four hours after cytostatic administration, 5 mg/kg b.w. caffeine was intravenously given. Cytogenetic observations concerning the frequency and the type of induced chromosomal aberrations were performed 24 hrs after cytostatic administration, both in the animals treated with only sarcolysine, methotrexate and caffeine and in those double-treated with cytostatics and caffeine. Chromosome examinations were also performed in untreated controls. Both in the sarcolysine- and methotrexate- treated tumors, the induced chromosome lesions were enhanced by caffeine administration, but this effect was very obvious in the methotrexate experiments and rather weak in the sarcolysine treatments (see table). This different effect of caffeine might be due to the different mechanisms by which sarcolysine and methotrexate are interfering in DNA replicating processes.     

Time- and concentration-dependent production of superoxide anion, nitric oxide, DNA damage and cellular death by ricin in the J774A.1 macrophage cells

The time- and concentration-dependent effects of ricin on some biomarkers of cellular toxicity, including production of superoxide anion (O2-), nitric oxide (NO), and DNA single strand breaks (SSB), as well as cellular death, have been examined in the J774A.1 macrophage cell cultures. Various concentrations of ricin have been added to various cell cultures, and the cells were incubated for 12, 24, 36, and 48 hours. Following 12 hour incubation, ricin did not cause significant increases in any of those biomarkers. However, time- and concentration-dependent increases were observed in the induction of all the biomarkers after incubation for 24-48 hours. Approximately twofold increases in the production of O2- were observed after incubation with 1 and 10 ng/mL of ricin for 24 and 36-48 hours, respectively. The concentrations of ricin that caused approximately twofold increases in the rate of DNA-SSB are 10 and 1-10 ng/mL after 24 and 36-48 hours incubation, respectively. Approximately twofold increases in NO production were only observed after incubation of the cultures with 1-10 ng/mL of ricin for 36-48 hours. Fifty percent reductions in cellular viability were also observed with ricin concentrations of 10-100, 10, and 1-10 ng/mL, after incubation for 24, 36, and 48 hours, respectively.     

Preservation of plastic properties of synaptic transmission in long-lasting hippocampal slices under the effects of a peptide analog of piracetam, L-pGlu-D-Ala-NH2

The tetanic stimulation of the Schaffer collaterals (SC) in rat hippocamp slices after 6 hrs in vitro conditions did not produce long-term potentiation (LTP) of the field response amplitude in the CA1 pyramidal cell layer. In contrast, LTP after the late tetanization was well preserved in the slices that were perfused for 20 minutes with 0.5 mkM L-pGlu-D-Ala-NH2 (PGAA) after 4-4.5 hrs in vitro. There were no significant reactivity changes during the perfusion of the slices with this drug concentration. Two other drugs with nootropic activity, piracetam (100 mkM) and gamma-hydroxybutyrate (100 mkM, Na-salt) did not prevent the disappearance of LTP in the late period in vitro, while enhanced the reactivity during perfusion period. The maintenance of the plastic properties of the SC-CA1 synaptic transmission under the influence of PGAA is thought to be the result of some specific interaction of the drug with LTP induction mechanisms. LTP damaged in the late period in vitro might be a new model of memory disturbances and this model can turn out to be useful for the comparative estimation of the effectiveness of the drugs with proposed nootropic activity and for the analysis of the possible mechanisms of their action.     

Characteristics of beta-adrenoreceptors of L-1210 leukemia and its sarcolysine-resistant variant

Beta-adrenoceptors have been discovered on the surface of cells of leukaemia L1210 and its variant resistant to sarcolysine (D, L-melphalan). One type of functionally active receptors with dissociation constant Kd for L-[3H] dihydroalprenolol about 0.02 nM and 360 receptors per cell have been revealed in leukaemia L1210 cells. In the resistant cells two types of functionally inactive receptors with Kd1 approximately 0.02 nM (420 receptors per cell) and Kd2 approximately 2.5 nM (3000 receptors per cell) have been revealed. This property of beta-adrenoceptors may be one of the causes of tumour cell resistance to sarcolysine.     

Antithrombin III inhibition of the reaction to thrombin excitation of the anticoagulation system

It has been established that intravenous administration of alpha-thrombin-antithrombin III preparations (1 mkM) has practically no effect on anticoagulation parameters (thrombin time, additive fibrinolytic activity, nonenzymatic fibrinolysis and nonenzymatic fibrinolytic activity). Administration of 1 mkM of alpha-thrombin caused a statistically significant increase of all the parameters. The experiments on perfusion of the humorally isolated sinocarotid area of the rabbit with alpha-thrombin-antithrombin III preparations (1.25 mkM) showed no changes peculiar to the induction of anticoagulation response with thrombin. It is concluded that antithrombin III blocks the ability of thrombin to activate anticoagulation system function.     

Unscheduled synthesis induced by thiophosphamide in human lymphocytes

An unscheduled DNA synthesis in human nonreplicative lymphocytes is shown to follow cell exposure to thiophosphamide at a dose level of 1-10 microgram/ml. Incorporation of 3H-thymidine in the absence of the mutagen is probably due to spontaneous reparative processes. Significant variations are found of spontaneous and thio-phosphamide-induced levels of a reparative DNA synthesis in normal individuals.     

Combined action of antiblastoma preparations on lysozyme activity in animal organs]

The lysozyme activity in the spleen, kidneys and lungs of the mice treated with neocid, sarcolysine or Tio-Fef in therapeutic doses increased or remained at the control level by the 5th or 10th day after the drug administration. The use of sarcolysine per se or in combination with neocid increased the activity of lysozyme in the spleen, kidneys and lungs during the whole period of the experiment as compared to the control. The values of the lysozyme activity in the spleen and lungs of the animals treated with neocid in combination with sarcolysine were higher for 5 days, and in all organs examined were higher by the 10the day as compared to the animals treated with neocid alone. Increased lysozyme activity in the spleen, kidneys and lungs was observed under the effect of neocid in combination with sarcolysine as compared to the lysozyme activity in mice treated with sarcolysine per se (assay on the 10th day). Decreased lysozyme activity was determined in the spleen, kidneys and lungs by the 5th day and in the kidneys by the 10th day in the mice treated with sarcolysine in combination with Tio-Tel and in the spleen and lungs by the 5th and 10th days in the animals treated with neocid in combination with sarcolysine or Tio-Tef as compared to the animals treated with sarcolysine. The lysozyme activity in the kidneys under the effect of sarcolysine combination with Tio-Tef was lower by the 5th days and higher by the 10th day as compared to that under the effect of Tio-Tef.     

The effect of 3-aminobenzamide on the mitotic cycle of Chinese hamster cells cultured on a medium with 5-bromodeoxyuridine following ionizing radiation action]

A specific inhibitor of poly(ADP-ribose)polymerase-3-aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-K1 cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU cells), and increase their radiosensitivity; 2) induce G2 delay in BDU cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2 delay in X-irradiated BDU cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

Large-scale fragmentation of dna and the death of tumor cells by the action of the binary system ascorbic acid-metallocomplexes of cobalt in vitro

High-molecular-weight DNA fragments are the markers of the early stage of apoptosis induced in eukaryotic cells by cytotoxins of different nature. The dynamics of the development of large-scale DNA fragmentation in K-562 leukemia cells by the action of the antitumor drug, the binary system ascorbic acid--cobalt phthalocyanine within 48 h of incubation, which correspond to two periods of the doubling of cell number in growing control cultures, have been studied. It was shown that, within the first hours of incubation, hydrogen peroxide generated by the system induces the formation of DNA fragments from 2200 to 50 kbp long. Later on the cell death accompanied by a decrease in the content of fragmented DNA is observed. Within 24 h of incubation, part of fragmented DNA remains unrepaired; after 48 h of incubation, a delay or a slowed down proliferation of K-562 cells, which differ from control cells also by a high level of death and a higher content of high-molecular-weight DNA fragments, is observed.     

Effect of UV radiation on the DNA-membrane complex of Bacillus subtilis]

The influence of UV-light on DNA-membrane complex (DMC) of Bacillus subtilis was studied. An increased DNA content in DMC for strains 168 and rec A-, and a degradation of DMC for strain polA- have been registrated. The increase in DNA in DMC of the two former strains is inhibited by caffeine to be correlated with changes in protein content in DMC, determined by a radioactive label, but not with lipid content. Thus, the association of DNA with the membrane is mediated by proteins. DNA increasing capacity seen in DMC after UV-irradiation and after the following incubation of bacteria in the complete medium is correlated with a relative sensitivity of strains. To explain these data, it is supposed that the reparative synthesis is accomplished in cell on their membranes and that for the normal completion of DNA repair the association between DNA and the membrane is necessary.     

Reactive growth of the mesothelium of the parietal leaf of the peritoneum]

In the process of reparative regeneration of the mesothelium caused by suturing of a portion of the jugular vein into the peritoneum defect the inflammatory mesothelial growings are formed. They have different configuration, consist only of the mesotheliumor the connective tissue covered with the mesothelium and are observed from the 4th day till 3 months after operation, being always faced to the abdominal cavity. The capacity of mesothelial cells to high mitotic activity and wedging out from the layer underlies the formation of posttraumatic structures of the mesothelium.     

Inhibition of rat fibroblast cell proliferation at specific cell cycle stages by cocaine

We have analyzed the role of cocaine in the control of the rat fibroblast (EL2) cell proliferation. Our data show a dose-related effect on the inhibition of DNA synthesis and cell growth when cocaine is added with serum or with a pure growth factor [Epidermal Growth Factor (EGF)]. Pretreatment by drug did not appreciably enhance the inhibition of S-phase entry above that obtained when cocaine and mitogen were added simultaneously. On the contrary, exposure of quiescent EL2 cells to cocaine has little or no effect on DNA synthesis, when drug is removed before the mitogenic stimulus. Moreover, even when cocaine is added after EGF, an exposure only within 1–8 hours is required in order to inhibit stimulation of DNA synthesis. Cocaine also suppressed the general increase in protein synthesis that occurs during the first hour after EGF addition. The combined data suggest that cocaine inhibits the traverse of mitogen-stimulated quiescent EL2 cells from Go to S phase by acting on processes that take place during the initial phase of the cell cycle.     

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, −3, −8, and −9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ‘`Replication Complexes’’ during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-α activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/− aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-α and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 α2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a α2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.     

DNA replication in mammalian cells after exposure to factors of a physical, chemical or biological nature. III. 5-Fluorodeoxyuridine induces DNA synthesis in cells not suppressed by gamma irradiation

The influence of preincubation of HeLa and Chinese hamster V79 cells with fluorodeoxyuridine (FUdR, 10(-6) M) on DNA replication and molecular weight of nascent DNA was studied after gamma-irradiation with a dose as much as 10 Gy. The 60Co-radiation inhibits DNA synthesis in both HeLa and V79 cells by 30-40 per cent. The incubation with FUdR before irradiation suppresses the inhibitory effect of irradiation on DNA synthesis. It is suggested that differences in gamma-radiation inhibition of DNA synthesis may result from the FUdR-induced changes in chromatin structure, rather than from synchronization of cell growth. This suggestion is based on the observation that the radioresistant mode of DNA synthesis occurred 18 hours following the short-term (6 hours) incubation with FUdR in cell cultures differing from each other in almost 2-fold their cell longevity.     

Neurotoxicity of the environmental pollutants: tin (Sn2+) effects on currents induced by acetylcholine in the snail neurons

In isolated single neurone or in whole ganglia preparation, SnCl2 caused a decrease in the acetylcholine-induced inward current in a dose-dependent way. Threshold concentration was 0.1 mkM and saturated at 5 mkM SnCl2. After 10-min. pre-treatment with SnCl2 the effect was more obvious (by 20%) than after 3-min pre-treatment. Sn(CH3)2 decreased the amplitude of Ach-induced current similarly to the inorganic Sn. The effect of Sn(CH3)2 was irreversible. The findings support earlier data that the agonist-activated channels are important targets for toxic metals. Direct action upon neuronal membrane seems to be an important event in modulation of synaptic transmission.     

DNA replication in mammalian cells exposed to the action of physical, chemical or biological factors. IV. 5-fluorodeoxyuridine modifies the effect of hyperthermia on DNA synthesis and the survival of HeLa cells

Preliminary incubation of logarithmically growing HeLa cells with FUdR decreases an inhibitory effect of hyperthermia (43 degrees C, 1 hour) on DNA synthesis. The hyperthermia alone inhibits DNA synthesis considerably: the label in acid-precipitable material accounts for 30% of control level. Preliminary incubation of the cells with FUdR (10(-6)) for 24 or 6 hours (plus 18 hours in fresh medium) decreases the effect: the label yields account for 50 or 90% of the respective control levels. A molecular weight of nascent DNA synthetized in the cells after hyperthermia or incubation with FUdR is lower than the control one but it increases rapidly during postincubation. Nucleoid of cells treated with FUdR has a sedimentation velocity which exceeds that of the control cells by more than 25%. Preliminary incubation with FUdR sensitizes the cells to hyperthermia. The effect is not believed to be associated with cells synchronization since the treatment of the cells with FUdR for 2 or 6 hours, when FUdR itself does not exert its toxic effect, brings about sensibilization of cells to hyperthermia. It is suggested that modification of the cell viability and DNA replication are related to some changes of chromatine structure induced by FUdR.     

Molecular mechanisms involved in the antiproliferative action of protein tyrosine phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate

Potassium bisperoxo(1,10-phenanthroline)oxovanadate, bpV(phen), a powerful protein phosphotyrosine phosphatase inhibitor and a potent insulinomimetic, influenced three fundamental cellular processes in HL-60 human leukemic cells: 1) inhibition of proliferation, 2) induction of differentiation and 3) apoptotic cell death. In the presence of micromolar concentrations of bpV(phen) cell number and DNA synthesis decreased progressively with time of incubation. A single treatment with bpV(phen) (3 microM) activated a differentiation program; after 6 days of incubation 82% of cells were differentiated, but differentiation started already within the first 24 h. Concentrations of 5-10 microM bpV(phen) caused the characteristic DNA ladder pattern, starting after 4.5 h. Differentiation in HL-60 cells appear to be associated with activation of extracellular signal-regulated kinase while apoptosis is connected with phosphorylation and activation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in a concentration and time-dependent manner. The antiproliferative and apoptotic action of bpV(phen) could be exploited in combination chemotherapy in leukemia.