Appearance of aberrant mitosis in murine leukemia cells upon combined treatment with low concentrations of cisplatin and teniposide.

The combination of cis-diamminedichloroplatinum (II) (DDP, cisplatin) and topoisomerase II inhibitor teniposide (VM-26) has been shown to exert a synergistic effect in the clinical treatment of cancer. In this study, the combined effect of DDP and VM-26 on the growth and induction of apoptosis in synchronized murine erythroleukemia (MEL) cells, treated at the beginning or in the middle of S-phase of cell cycle, was examined. MEL cells, clone F4 N, were synchronized by a double thymidine block leading to accumulation of 70% of cells at the G1/S boundary. The growth-inhibitory effect of DDP and VM-26 applied alone were stronger in the middle of the S-phase than at the beginning. Morphological analysis showed that the majority of the cells revealed typical signs of apoptosis: nuclei fragmentation and appearance of apoptotic bodies. The combination of both agents at low concentrations had a synergistic effect on cytotoxicity. At higher concentrations the effect was additive. The remainder of the cells were characterized by unbalanced growth, aberrant mitosis and appearance of multinucleated cells. These processes led to delayed cell death. The appearance of aberrant mitosis was more expressed after treatment in the middle of the S-phase. It is likely that as a result of the combined action of cisplatin and VM-26, cells become supersensitive to the ability of topoisomerase II inhibitor to influence mitosis, and this increased sensitivity may contribute to the observed synergism.

Synthesis, cytotoxicity and antitumor activity of platinum(II) complexes of cyclopentanecarboxylic acid hydrazide.

New platinum(II) complexes of cyclopentanecarboxylic acid hydrazide (cpcah) were prepared, characterized by elemental analysis, IR and 1H NMR spectra, and evaluated for in vitro cytotoxicity in Friend leukemia (FL) and A2780 ovarian tumor cells, induction of apoptosis in FL cells, as well as for in vivo antitumor activity toward murine L1210 leukemia and Lewis lung carcinoma. The spectral analyses indicated a cis-square planar structure of the complexes with hydrazide ligand coordinated via the NH2 group. The compounds exerted significantly lower in vitro and in vivo toxicities as compared with those of cisplatin (cis-diamminedichloroplatinum(II), DDP). On the other hand, the complex [Pt(NH3)(cpcah)Cl2] exhibited antitumor activity against L1210 leukemia in mice comparable to that of cisplatin, resulting at a dose of 42 mg/kg (administered 3 times) in a T/C (mean survival time) of 280%. This compound displayed an in vitro macromolecular synthesis inhibition pattern similar to that of DDP. At concentrations close to the cytostatic ones (10-20 microM) this complex, as well as DDP, was able to induce apoptosis in FL cells as shown by neutral comet assay and morphological analysis. We concluded that there is a correlation between the ability of platinum complexes to induce apoptosis and their antitumor activity.

Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency.

The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression.

Inhibition of DNA synthesis in different tissues of L1210-leukemia-bearing mice by new platinum (II) complexes.

The inhibition of [14C]thymidine incorporation into DNA of tumor and normal tissues of L1210-leukemia-bearing mice by single doses of cis-diamminedichloroplatinum (II) (cisplatin, cis-DDP) and two newly synthesized platinum (II) complexes containing as ligands dimethyl aminomethylphosphine oxide (complex I) and methyl bis(aminomethyl)phosphine oxide (complex II) was studied and used as an indication of drug toxicity. All three complexes caused selective inhibition of precursor incorporation in L1210 cells as compared to host tissue cells. cis-DDP caused a complete block of incorporated thymidine in tumor cells during more than 48 h, whereas in intestinal mucosa and bone marrow reverse inhibition was observed. In spleen, liver and kidney the inhibition was about 50% and endured up to 96 h without reversal. Complex I treatment of L1210 cells resulted in an earlier recovery of thymidine incorporation into DNA in comparison with cis-DDP. Towards all other normal tissues compound I was less toxic than cis-DDP. Unlike cis-DDP and complex I, complex II was less active against L1210 cells and most toxic against bone marrow and kidney.

[The action of amino acid di[2-haloethyl]hydrazides on DNA synthesis in normal and tumor cells and on the cell cycle kinetics in mice with melanoma B-16]. / Deistvie di-[2-galoétil]gidrazidov aminokislot na sintez DNK v normal'nykh i opukholevykh kletkakh i kinetiku kletochnogo tsikla u myshei s melanomoi B-16.

A comparative study was carried out with two alkylating agents IMB-MM and IMB-97 which are di-(2-halogenoethyl) hydrazides of amino acid derivatives. They have been found to exert a high activity towards wide spectrum of experimental tumours. Both agents caused inhibition of incorporation of 3H-thymidine into DNA of melanoma B16, marrow, intestinal mucosa, spleen and liver cells of mice with tumours. A maximal inhibition of DNA synthesis in all tissues was observed 24 h after the single doses of drugs. However 96 h later this effect was removed excluding the tumour cells. The cytofluorimetric study have shown that IMB-MM, like sarcolysine, caused an accumulation of tumour cells in G2/M phase of cell cycle, while IMB-97 increased accumulation of S-phase cells. The difference in phase sensitivity of tumour cells towards IMB-MM and IMB-97 is due to the differences in aminoacid carriers of di-(2-halogenethyl) hydrazide groups.

Lack of genotoxic and cytotoxic effects of the herbicide lenacil on mouse tumor cells and on some Salmonella typhimurium strains.

The effects of 3-cyclohexyl-6,7-dihydro-1H-cyclopentapyrimidine-2,4(3H,5H)-dione (lenacil) on macromolecular synthesis, thymidilate synthetase activity, viability and cell cycle progression were studied using Friend leukemia (FL). P388 and Ehrlich ascites tumor cells in suspension, and its cytogenetic effects were studied in a Salmonella/mammalian microsome assay using both frameshift and base-substitution tester strains. At a concentration of 0.5 mmol/l lenacil inhibited 45 to 70% thymidine incorporation into DNA fraction, while incorporations of uridine into RNA and leucine into protein were less affected. Thymidilate synthetase activity in P388 cells as assayed by the release of tritiated water from 5-3H-deoxyuridine was inhibited by the compound to about 20%. Lenacil neither showed an in vivo inhibitory action on thymidine incorporation into acid-insoluble material in P388 cells, nor on thymidilate synthetase activity after a 24 or 48 h treatment. The compound did not change the melting temperature of isolated DNA. Studies of lenacil's effect on cell cycle kinetics of FL cells demonstrated that 48 h treatment increased the percentage of S-phase cells. Lenacil exerted a weak cytotoxic effect on FL cells. At concentrations above 0.1 mmol/l it inhibited cell growth the effect being nonlethal. Cytogenetic studies of lenacil revealed no indication of its mutagenicity against Salmonella typhimurium TA97, TA98, TA100 and TA102.

Cyanopyrazoles as analogs of purine precursors--II. Effects on macromolecular synthesis and cell cycle progression.

The cytotoxic and cytokinetic effects, and in vitro inhibition of macromolecular synthesis by cyanopyrazoles were studied using Friend leukemia and Ehrlich ascites tumor cells. At concentrations in the range of 2.5 mM to 50 microM analog 3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (I) was highly cytotoxic and completely inhibited thymidine, uridine and leucine incorporation into macromolecular material. 24 hr incubation of FL cells with cytostatic concentrations of compound I (in the range of 2 to 0.5 microM) resulted in an accumulation of cells in the G2 + M phase. Analogs N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (II) and 3(5)-amino-4-cyanopyrazole (III) were not cytotoxic at concentrations up to 5 mM and did not substantially inhibit precursor incorporation into macromolecules but exhibited a cytostatic activity. These compounds caused a decrease of FL cells in the G2 + M phase and an accumulation in the S phase. Analogs I and II displayed a similar in vivo inhibitory effect on thymidine incorporation into DNA in EAT cells. The results indicate that the cytotoxicity of cyanopyrazoles correlates with their ability to inhibit precursor incorporation into macromolecular material. On the other hand, the cytostatic action of compound I is not coupled to a block of nucleic acid synthesis.

[Osmotic activity and ionic permeability of membrane vesicles from Streptococcus faecalis and Micrococcus lysodeikticus cells]. / Osmoticheskaia aktivnost' i ionnaia pronitsaemost' membrannykh puzyr'kov iz kletok Streptococcus faecalis i Micrococcus lysodeikticus.

Membrane fractions containing osmotically active vesicles with sufficiently low membrane permeability for K+, Na+ and Cl- ions typical for the intact cell membrane were isolated from the cells of the glycolyzing bacterium Streptococcus faecalis. In their osmotic properties and ionic permeability the membrane fractions of S. faecalis were found similar to those of the respiring bacterium Micrococcus lysodeikticus, which are capable of the energy-dependent potassium transport. It may be thus assumed that the S. faecalis fractions obtained may be used to study ionic transport. The removal of proton-dependent ATPase of the S. faecalis membrane preparations did not affect the permeability of membranes for K+ ions which is indicative of different mechanisms of proton and potassium translocation.

[Selective permeability of bacterial membranes for monovalent thallium ions]. / Izbiratel'naia pronitsaemost' bakterial'nykh membran dlia ionov odnovalentnogo talliia.

Transport of monovalent thallium ions in bacterial cells was studied. An energy-dependent transport of T1+ against electrochemical gradient into the cells of S. faecalis and Micrococcus lysodeikticus, according to the Michaelis-Menten kinetics was observed. T1+, being a K+ analog, is involved into active K+ transport. Unlike K+, T1+ readily penetrates bacterial membranes, reaching the level of stationary distribution between the cells and the medium. This permits to use T1+ as a penetrating cation to study the mechanism of potassium transport in bacteria without the use of ionophores, which can destroy the integrity of cell membranes.

[Energization of membrane vesicles from the cells of glycolyzing bacterium Streptococcus faecalis]. / Energizatsiia membrannykh pyzyr'kov iz kletok glikoliziruiushchei bakterii Streptococcus faecalis.

Membrane fractions were isolated from Streptococcus faecalis cells of a glycolyzing microorganism, devoid of the respiratory chain, using the methods of osmotic shock of the protoplasts, ultrasonic treatment of the cells and ultrasonic treatment of the protoplasts. All fractions possessed the ATPase activity, the highest activity being observed in the fraction isolated by ultrasonication of the protoplasts. All preparations were estimated with respect to the presence of vesicles, formed by the "inside-out" and "inside-in" membranes, using ATPase as a marker of the membrane orientation. In the membrane fractions obtained by ultrasonication of the protoplasts, the "inside-out" vesicles were prevalent. ATP-dependent energization of the membranes, sensitive to the action of dicyclohexylcarbodiimide and tetrachlorotrifluoromethyl benzimidazole, was demonstrated by measuring the transport of the lipophylic anion of phenyldicarbaundecaborane and aniline naphthalene sulfonate fluorescence.

Effect of valinomycin on ion transport in bacterial cells and on bacterial growth.

The antimicrobial action of valinomycin relative to the K+ and Na+ contents of the medium has been investigated in several species of bacteria, particularly in Streptococcus faecalis, which effects energy-linked transport exclusively via degradation of glycolytic ATP, Micrococcus lysodeikticus, effecting active ion transport by respiration and Staphylococcus aureus, the energy-dependent ion transport of which is due to both glycolytic ATP degradation and respiration. It was demonstrated that valinomycin does not act on K+ transport in the glycolysing cells in the same manner as it does on respiring cells under similar conditions. Addition of valinomycin to respiring cells leads to an increase in K+ influx against the concentrational gradient in both growing and resting cells. In contrast to this, antibiotic-treated glycolysing cells experience passive K+ outflow down the concentrational gradient. It was thus concluded that the electrical potential cannot be the driving force for the energy-linked K+ transport in glycolysing cells.