A novel synthetic DNA minor groove binder, MS-247: antitumor activity and cytotoxic mechanism.

PURPOSE: MS-247 is a novel synthetic compound possessing a DNA-binding moiety and a DNA-alkylating residue, chlorambucil. In this study, we evaluated the antitumor activity of MS-247 against murine tumor cell lines and its effects on DNA molecules in both cell-free and cellular systems. METHODS: The in vitro cytotoxic activity of MS-247 was evaluated against four murine tumor cell lines, P388, L1210, Colon26 and B16, and its in vivo antitumor activity was also tested in comparison with Adriamycin (ADM), cisplatin (CDDP) and paclitaxel. The ability of MS-247 to associate with the DNA minor groove was assessed by measuring quenching of Hoechst 33342 fluorescence. DNA-DNA interstrand crosslinks (ICL) were detected by an alkaline elution assay for cellular DNA and a band-shift assay using the plasmid pBR322. The effects of MS-247 on macromolecule synthesis (DNA, RNA and proteins) were examined by measuring incorporation of the radiolabeled precursors. RESULTS: MS-247 exhibited in vitro cytotoxicity with IC(50) values ranging 11 to 500 nM, and MS-247 given i.v. showed strong in vivo antitumor activity against i.p.-implanted L1210 leukemia cells and s.c.-implanted Colon26 carcinoma cells, and moderate activity against i.p.-implanted P388 leukemia cells but no apparent activity against s.c.-implanted B16 melanoma cells. MS-247 reversibly displaced Hoechst 33342 bound to DNA within a few minutes, and irreversibly formed ICL within 1-6 h in both the cell-free system and the cellular system. These results suggest that an association of MS-247 with the DNA minor groove occurred more quickly than ICL formation. The inhibition of DNA synthesis was more prominent than the inhibition of RNA and protein synthesis in L1210 cells exposed to MS-247, and a 6-h incubation with MS-247, which formed apparent ICL in the cellular system, strongly inhibited DNA synthesis. This result suggests that impairment of DNA replication preceded the inhibition of RNA and protein synthesis and that ICL formation greatly contributed to the inhibition of macromolecule synthesis. CONCLUSION: The results of this study suggest that MS-247 exerts its cytotoxic effect through impairment of DNA function by getting into the minor groove of DNA and subsequently forming ICL. MS-247 has potent antitumor activity with a different spectrum from the activity of clinically proven antitumor agents such as paclitaxel, ADM and CDDP against several murine tumor cell lines. This result suggests that MS-247 may be useful for the treatment of human cancers.

Potent antitumor activity of MS-247, a novel DNA minor groove binder, evaluated by an in vitro and in vivo human cancer cell line panel.

We synthesized a novel anticancer agent MS-247 (2-[[N-[1-methyl-2-[5-[N-[4-[N,N-bis(2-chloroethyl) amino] phenyl]] carbamoyl]-1H-benzimidazol-2-yl] pyrrol-4-yl] carbamoyl] ethyldimethylsulfonium di-p-toluenesulfonate) that has a netropsin-like moiety and an alkylating residue in the structure. We evaluated antitumor activity of MS-247 using a human cancer cell line panel coupled with a drug sensitivity database and subsequently using human cancer xenografts. The average MS-247 concentration required for 50% growth inhibition against a panel of 39 cell lines was 0.71 microM. The COMPARE analysis revealed that the differential growth inhibition pattern of MS-247 significantly correlated with those of camptothecin analogues and anthracyclins, indicating that MS-247 and the two drug groups might have similar modes of action. MS-247 exhibited remarkable antitumor activity against various xenografts. A single i.v. injection of MS-247 significantly inhibited the growth of all 17 xenografts tested, which included lung, colon, stomach, breast, and ovarian cancers. In many cases, MS-247 was more efficacious than cisplatin, Adriamycin, 5-fluorouracil, cyclophosphamide, VP-16, and vincristine and was almost comparable with paclitaxel and CPT-11; these are the most clinically promising drugs at present. MS-247 was noticeably more effective than paclitaxel (in HCT-15) and CPT-11 (in A549, HBC-4, and SK-OV-3). The toxicity of MS-247, indicated by body weight loss, was reversible within 10 days after administration. The MS-247 mode of action showed DNA binding activity at the site where Hoechst 33342 bound, inhibited topoisomerases I and II (as expected by the COMPARE analysis) blocked the cell cycle at the G2-M phase, and induced apoptosis. These results indicate that MS-247 is a promising new anticancer drug candidate to be developed further toward clinical trials.

Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.

This study was undertaken to determine whether and how advanced glycation end products (AGE), senescent macroproteins accumulated in various tissues under hyperglycemic states, cause angiogenesis, the principal vascular derangement in diabetic microangiopathy. We first prepared AGE-bovine serum albumin (BSA) and anti-AGE antiserum using AGE-RNase A. Then AGE-BSA was administered to human skin microvascular endothelial cells in culture, and their growth was examined. The AGE-BSA, but not nonglycated BSA, was found to induce a statistically significant increase in the number of viable endothelial cells as well as their synthesis of DNA. The increase in DNA synthesis by AGE-BSA was abolished by anti-AGE antibodies. AGE-BSA also stimulated the tube formation of endothelial cells on Matrigel. We obtained the following evidence that it is vascular endothelial growth factor (VEGF) that mainly mediates the angiogenic activities of AGE. (1) Quantitative reverse transcription-polymerase chain reaction analysis of poly(A)+ RNA from microvascular endothelial cells revealed that AGE-BSA up-regulated the levels of mRNAs for the secretory forms of VEGF in time- and dose-dependent manners, while endothelial cell expression of the genes encoding the two VEGF receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase 1, remained unchanged by the AGE treatment. Immunoprecipitation analysis revealed that AGE-BSA did increase de novo synthesis of VEGF. (2) Monoclonal antibody against human VEGF completely neutralized both the AGE-induced DNA synthesis and tube formation of the endothelial cells. The results suggest that AGE can elicit angiogenesis through the induction of autocrine vascular VEGF, thereby playing an active part in the development and progression of diabetic microangiopathies.

[An experimental study on healing processes of microarterial and microvenous transplantation. The possibility of clinical application in oral reconstructive surgery].

Sixty-three Wistar female rats, weighing from 250 to 350g were used in order to investigate patency rate and histological changes of arterio-arterial and veno-venous autografts, using the right femoral artery and vein. The left femoral artery and vein served as control. The grafts were retrieved over a period of time ranging from 4, 7, 14, 28 and 56th day postoperatively. The patency rate was checked by Hayhurst and O'Brien's method. The histological changes of grafts were evaluated by the light and scanning electron microscope. Arterio-arterial graft maintained high patency rate throughout the experiment (90.0%, 27/30). This high patency rate may be explained by revascularization immediately after anastomosis. The average patency rate in veno-venous graft was 75.8% (25/33). Patency rate in 14th and 28th day in veno-venous graft showed 100%, that seemed to be related to the early completion of endothelial cells lining of the graft. Regeneration proceeded from the preserved endothelial cells in graft as well as endothelial cells of the artery and vein. The number of preserved endothelial cells in the arterial graft was fewer than that in the venous one. Therefore, the completion of covering by endothelial cells in arterial graft was slower than that in venous graft. The obstruction in arterio-arterial graft was caused by thrombus in the rupture of vessels formed by the suture procedures. In veno-venous graft, thrombus on the exposed vessels' wall resulted in the obstruction. Subintimal hyperplasia was seen in all arterial graft. No such change was seen in venous graft. These results indicate that revascularization immediately after anastomosis, tight and atraumatic suture procedures are important for the prevention of obstruction in arterio-arterial graft and veno-venous graft.

[Scanning electron microscopic observation on odontogenic keratocysts].

Five odontogenic keratocysts were examined by using a scanning electron microscope (SEM) on the three-dimensional ultrastructure of epithelial lining cells. The fractured surface of the specimens showed a single row of columnar or cuboidal cells perpendicular to the flat basal lamina, a four- to five-cell layer of spinous cells, and a superficial layer of flattened parakeratinized cells. At high magnification of superficial cell surface, microvillis, short discontinuous branched or anastomosing microridges were observed.

[A case of greater auricular nerve autologous nerve grafting for inferior alveolar nerve injury by mandibular fracture].

The results of peripheral nerve repair have been greatly improved in the last few years following the introduction of micro-surgery and increased application of free autologous nerve transplants. In the field of oral surgery, a rich experience has been made in plastic and reconstructive repair. The inferior alveolar nerve is endangered by a series of mandibular fractures, with fracture lines running along the nerve canal. For plastic repair of the inferior alveolar nerve, we interpose an autologous transplant from the greater auricular nerve.

[General pharmacological studies on isepamicin sulfate (HAPA-B)].

General pharmacological properties of isepamicin sulfate (HAPA-B), a new aminoglycoside antibiotic, were studied in animals and the results obtained were summarized below. Intramuscular injections of HAPA-B at doses of 500 mg/kg inhibited the writing response induced by acetic acid, and at doses of 1,000 mg/kg, caused muscle relaxation, respiratory depression, suppression of spontaneous motor activity and prolongation of thiopental anesthesia. Anticonvulsive action and the effect on the rectal temperature were not observed. Intravenous Intravenous HAPA-B showed no significant effect on the general behavior and the function of the central nervous system at doses of 100 mg/kg. Intravenous injections of HAPA-B to anesthetized dogs resulted increases in the femoral arterial blood flow at doses of 12.5 mg/kg, decrease in the blood pressure and increase in the respiratory rate at doses of 25 mg/kg, and increase in the carotid arterial blood flow at doses of 50 mg/kg. Apparent changes were not recognized in the heart rate and electrocardiograms. In conscious rabbits, intravenous HAPA-B produced increases in the heart rate without significant changes of the blood pressure and electrocardiograms at doses of 100 mg/kg. Spontaneous beatings of isolated atria were depressed by HAPA-B in concentrations of 3 X 10(-4) to 10(-3) g/ml. The HAPA-B inhibited the gastric secretion at intramuscular doses of 500 mg/kg or intravenous doses of 100 mg/kg, and depressed charcoal transport through small intestine and the spontaneous movement of isolated ileum at intramuscular doses of 1,000 mg/kg and at concentrations of 3 X 10(-4) to 10(-3) g/ml, respectively. No irritative effect was found on the gastric mucous membrane. Intravenous HAPA-B inhibited the response of nictitating membrane to pre and post ganglionic stimulations of cervical sympathetic nerve at doses of 100 mg/kg. In in vitro test, HAPA-B inhibited nonspecifically the constrictive responses of trachea, aorta, stomach, ileum and vas deferens to various agonists in concentrations of 3 X 10(-4) to 10(-3) g/ml. Spontaneous movements of uteri of estrous or pregnant animals were depressed by HAPA-B at intravenous doses of 50 to 100 mg/kg and in in vitro at concentrations of 10(-4) to 3 X 10(-4) g/ml. Antidiuretic effect was also observed at intramuscular doses of 250 mg/kg. HAPA-B increased the length of the whole blood clotting time and raised the plasma glucose level at intramuscular doses of 1,000 mg/kg and inhibited the platelet aggregation induced by ADP in vitro at concentrations of 10(-3) g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA repair in competent cells of Bacillus subtilis.

A series of isogenic transformable strains of Bacillus subtilis carrying the uvr-19 or rec-43 mutation or both were constructed. Both mutations made competent cells defective in repairing UV-irradiated cellular or transforming DNA, and their effects were additive in a doubly deficient strain, suggesting that two repair processes, requiring uvr-19+ and rec-43+ gene products, are independently functional in competent cells of B. subtilis.

Familial cystinuria in Ioannina District (Greece). Diagnosis and treatment.

The "Urocystin Test" has been used as a screening procedure for the diagnosis of the incidence of cystinuria in Ioannina District (Northwest Greece). From the 210 investigated urine samples, eight were positive and four of them were also L-cystine stone formers. All positive cases belong to two cystinuric families. The pedigree in village "Kato-Lapsista" is a genetic type of a "completely recessive cystinuria" while the other one in "Marmara" is the type of an "incompletely recessive cystinuria". All patients with L-cystine urolithiasis, except one child, were treated with the drug Thiola.

Timing of gastrulation in fused double-embryos formed from eggs with different cleavage schedules in the starfish, Asterina pectinifera.

The aim of this study is to explore the control mechanism which regulates the timing of gastrulation. We wished to know whether the timing is rigidly determined within the constituting cells of the embryo of whether it is changed by intercellular communication systems. Starfish-fused double-embryos were formed by contact between pairs of denuded eggs with different cleavage schedules, one egg in each pair being distinguished with vital stain. Electron microscopy revealed identical ultrastructural characteristics in every cell contact: Septate junctions were found not only between cells in the contact area between the two counterparts constituting a fused embryo but also between cells within a counterpart. In fused embryos formed from stained eggs and unstained eggs with the former having started the first cleavage 30 min earlier than the latter, the stained counterparts began to invaginate about 30 min earlier than the unstained. When fused embryos were produced by putting stained eggs in contact with unstained eggs which had begun the first cleavage 2 hr later than the stained eggs, gastrulation of the stained counterparts took place about 2 hr earlier than that of the unstained counterparts. These results suggest that the timing of gastrulation is fixed within the cells of an embryo and cannot be altered in spite of the cell-to-cell communication that appears to exist.