CS268903B1 - Method of substances testing with cytostatic effect - Google Patents

Abstract

The essence of the solution is that it is tested performed on growing for toplasts yeast of the genus Saccharomyces and biological effects of substances with cytostatic effects are analyzed by cytotoxic methods including protoplast size assessment number of nuclei per protoplast and degree of reduction microtubular system. From microscopic evaluation of the preparations is possible directly to find out the uniform response of the yeast group cells while determining the relationship between chemical composition of the cytostatic to be tested and the molecular level of its effect on cell structure. The method replaces testing cytostatic effects of animal tissue culture cells and laboratory animals.

Description

CS 268903 B1 1

BACKGROUND OF THE INVENTION The present invention relates to a method for testing substances having a cytostatic effect.

The process is based on the most recent scientific evidence confirming that the molecular mechanism of eukaryotic cell division is very phylogenetically very old and princi- pally identical from yeast to human (3rd L. Marx, Science 226, 527-528, 19854 P. Nurse, Nature 313, 631-632, 1985). The main component of this mechanism are proto-oncogenes, homo-ogylogs of oncogenes - genes of tumor viruses - which are present as genes regulating proliferation in the genome of all vertebrates, including humans (R. A. Weinberg, 3rd Cell Biology 97, 166-166-1662, 1983). Violation of the major proliferator. genes is the first step to trigger some types of tumor transformation (H.E. Varnua, Ann.Rev.Genet. 18, 553-612, 1984). During the last two years, 8 proto-oncogenes have been identified in the yeast genome / A. E. Uheals, Bioessays 3, 108-112, 1985]. These genes are all involved in cell division regulation, their function is relatively well known and their expression is directly related to the microtubular system, which is the first component of the cytoskeleton identified in yeast / E. Streibl and J. Hasek, Hicrobial Sciences 2, 139-143, 1985). Microtubule system polymerization inhibitors have an experimentally and clinically useful cytostatic effect due to selective toxicity to microtubules present in the nuclei of rapidly dividing tumor cells. G. Fitzgerald, Biochem. Pharmac. 25, 1383 (1975). The known methods of serial testing of cytotoxic agents are carried out in laboratory animals and in tissue cultures (De Srabander M. et al.

Cancer Res. 36, 905-916 (1976). Furthermore, there are known papers dealing with the specificity of anti microtubular substances and the screening of cytostatic agents for whole yeast cells (Ualker G. M., J. G. Micro-Bio., 128, 61-71 (1982)); Quinlan R.A. et al., J. Cell Sci. 46, 341-352 (1980).

These techniques are time consuming, methodical and economical.

These disadvantages are eliminated by the method of testing substances with cytotoxic effect, which is that in the sterile osraotically stabilized nutrient broth with an osmolarity of 0.7 to 1, protyclycites of Saccharomyces ce-8 9-1 revisiae or Saccharomyces uvarum are initially incubated at an initial concentration 10 to 10 cells. ml at 25 to 30 ° C for 20 to 30 minutes and then a sample of eyelet-static substance is added at a concentration of 10 to 100 µg / ml ~1 dissolved in dimethyl sulfoxide and left for 4-6 hours, then the sample is centrifuged protoplasts are fixed for detection of nuclei and indirect immunofluorescence of the microtubular system. Cytotoxic agents are tested for toplasts of yeasts produced by enzymatic digestion of intact yeast cells with a mixture of garden snail helix (Helix pomatia); this procedure removes the permeable barrier to the surface yeast that could prevent the entry and transport of test substances into the cells / No-time 0., A. Svoboda, JF Peberdy and L. Fevenczy /eds./ "Fungal orotope asts", p. 133, Marcel Dekker, New York 1985. For testing, for example, the yeast may be a Saccharomyces uvarum species with large cells and high growth rates. In addition, the species is harmless to health, has low nutritional requirements and is traditionally used in brewing.

The resulting yeast protoplasts, which continue to grow in osmotically stabilized soil in the growth / synthesis of proteins and macromolecules (and in nuclear division), represent a dynamic in vitro system readily available. The advantage is that the growing protoplasts are exposed to the studied cytostatic substances directly in the osmotically stabilized environment in which they grow; biological agents of the test substances are analyzed after four and six hours, ie approximately three nuclear divisions that take place in the control protoplasts.

The knowledge of growth process clinkers, nuclear division and cytoskeletal behavior in yeast protoplasts can be used to recognize the ways in which substances enter the regulation of cellular behavior through cytosatic action. Testing allows: a / to assess the cell's overall susceptibility to the substance under study, b / to contribute to the elucidation of the target site for the action of the agent with cytostatic effect e / To interpret the mechanism of its effect to express nuclear attenuation at a specified interval at the cellular level. An optical microscope equipped with a device for fluorescence mlcroscope in combination with phase contrast is sufficient for cytologic analysis of Inoculated Protoplasts. The nebulous-bulbar system is imaged by indirect immunofluorescence1 / Progress 1n Cell Cycle Controls, Institute of Microbiology of the Czechoslovak Academy of Sciences, 1983, pp. 159-160. Nuclear and mitochondrial and nucleus counts are shown by direct fluorescence / H. Siloh and J. V. Sedat, Science 217, 1252-1255, 1,982]. The size of the protoplasts is assessed by measuring their phase contrast averages by means of an eyepiece micrometer inserted into the eyepiece (Microbiological Methods, Prague, Academia 1981, pp. 30-53). The size of the protoplast is expressed as the mean protoplast size (x). £ 10. n,. x, X =. 10 ε · 1 x the average mean protoplast size in μη n ^ the number of protoplasts in the 1 observed field x ^ the mean protoplast size in the 1 observed field in µ µn total protoplast count measured

Protoplast observation is performed using a Jenalumar / Zelss Jena NOR fluorescence microscope, which allows for alternate phase contrast alternation with fluorescence observation at different wavelengths in a single preparation. An ocular magnifier was used 6 times with a micrometer inserted and an immersion lens magnifying 100 times using a non-fluorescent immersion oil. The protoplast size was previously in phase contrast, DNA distribution and number of nuclei monitored in a fluorescence image using a wavelength filter of 340 to 380 nm, the state of the microtubule system being monitored in a fluorescence image using an oro filter with a wavelength of 450 to 490 nm. An average of 20 arrays containing 20 to 50 protoplasts with the same number of reference / control / protoplasts are compared.

By the proposed test, the monitoring of the effects of cytostatic agents is many times less economical, since the cost of maintaining the strain, nutrient broths, lytic enzymes and antibodies is almost negligible compared to rearing and testing in laboratory animals. The present method of use allows for the serial testing of substances with a cytostatic effect according to the invention. In the examples, sufficient versatility of the method is utilized for the use of cytostatic agents to test the substance by using nocodazole / methyl-2-thienyl-phenylcarbonyl / -1H-benzimidazol-2-yl carbamate (carbendazim / MBC); methyl benzimidazol-2-yl carbamate and the use of the original Czechoslovak cytostatic Ben fluron / 5- 2- (N, N-dimethylamino) ethoxy-7-oxo-7H-benzo-fluorene hydrochloride (CS 268903 81 3) is pass · MS. author's certificate No. 212 669 and whose properties are mentioned in the MS. author certificates Nos. 235 429 and 242 292. Various effects of cytostatics on the microtubular system and nuclear division of protoplasts of yeast Saccharomyces uvarun do not document fluorescent microscope images. From these images it is possible to determine the relationship between the chemical composition of a given cytostatic and the molecular level of its effect by the functional cell organization.

More details are given by way of example, which is not to be construed as limiting the invention. EXAMPLES Example 1 In the assay, Saccharomyces uvarum strain P 96 was used, which was maintained on solid sterile soil containing 2 X glucose, 2 X bactopepton, 1 X yeast extract and 2 X agar. Strain grown at 25 ° C for 48 hours is stored / stored at 4 to 8 ° C for 3 months. After this time, the culture was seeded on liquid soil of the same composition for 48 hours and then transferred to the fixed slanted agar, allowed to grow at 25 ° C for 48 hours, and stored at 4 to 8 ° C for 3 hours. months. To prepare the protoplasts, the cells were cultured submersely in liquid sterile nutrients of the same composition in flasks on a rotary shaker at 28 ° C for 6 hours. Cells, which are in the logarithmic phase of growth, are obtained from the culture broth by vacuum filtration through a membrane filter (Synpor - Synthesia, Pardubice, pore size 0.4 / pm) and transferred to the digestion mixture so that their final concentration in the mixture is 10 ^ cells. ml

The digestive mixture contains 3 mL of the Hellx pomatia digestive tract, 1.5 mL of a 0.1 M KH 2 PO 4 solution, pH 6.5, 1 mM phenylsulfonyl fluoride (further PMSF), and 1.1 H sorbol solution. Current concentrations in the digestive mixture are given. After shaking for 30 minutes at 25 ° C, the protoplast suspension is carefully quenched with a 0.1 M KH 2 PO 4 solution over a 10 minute period to a final sorbitol 0.7 osmolarity. The pro-proteases obtained in this way are centrifuged into a liquid nutrient medium, the composition of which has been described in the first embodiment, the nutrient medium is stabilized osmotically with 0.7 M sorbitol. Of this soil, the protoplasts are inoculated at an initial concentration of 10 protoplasts. mL and further culturing them at 25 ° C with slow agitation on a reciprocating shaker apparatus 100. min 1.

Nocodazole is prepared as a stock solution in dimethyl sulfoxide (hereinafter referred to as OMSO) and applied to the nutrient broth at a final concentration of 10 µg. mL-1; the OMSO concentration used in protoplast application does not exceed 0.4 X / v / v / v. The protoplases are first grown for 30 minutes in the broth as described in the preceding paragraph, then nocodazole is added to the growing protoplasts and allowed to act for 4 hours; At the same time, no cytostatic protoplasts are grown under the same conditions in the same time interval. After four hours, the two protoplast samples were collected by centrifugation and fixed at 25 ° C as follows.

Protoplasts are fixed for a total of 90 minutes in two stages. The first step involves an 80 minute fixation of 4 X / v / v / v formdehyde in a stabilizing buffer containing a 0.1 M KH 2 PO 3 solution at pH 6.5, 20 mM and a medium of 1-b1 s. ^ -arai noe thylene te / -Ν, Ν, N ^N-tetraacetic acid (hereinafter referred to as EGTK) and 1 mM MgCl2 solution · Actual concentrations in the fixation mixture are given. In the second step, the protoplasts are further fixed for 10 minutes with 1 X / v / v / glutardehyde. Thereafter, the fixed protoplasts from the broth are centrifuged, washed with buffered saline pH 7.3 (hereinafter referred to as PFR) and incubated for 20 minutes in cold 0.5 mg NaBH 4 solution. mL-1 in PFR. Finally, the plastics are washed three times with PFR solution and applied in a thick slurry to a cleanly washed microscope glass (topcoat). After drying, the peraeablister protoplasts are immersed in slides for 10 minutes followed by acetone for 30 seconds at -20 ° C.

For indirect immunofluorescence detection of the Mrotrotubular System in Protoplasm, a mouse aoclonal antibody TU 01 against porcine brain tubulin is used. Viklický et al. Cell Blol. Dormitory. Reports 6, 725-730 (1982). The cover glasses with peri-ablated protoplasts are first pre-incubated with 1 Z / v / v / v / v bovine serum solution (hereinafter HSA) in PRF and then incubated with TU 01 antibody obtained by MS. No. 223,535. The antibody is diluted 1: 99 with 1X / v / v / HSA in PFR and incubated with the preparations for 60 minutes. For this, slides are washed three times with warm 1X HSA in PFR. In the second step, protoplasts are incubated with porcine antifungal fglobullin conjugated with fluoreseel isothecanate / SwAM / FITC - Institute of Serum and Vaccine Prague or, for example, Hlles Labatorator, Inc., Research Products, Div. Elkhard. Indian. England /. Postlnkubaee is run with SwAM / FITC labeled antibody diluted ten times in 1X / v / v / PFR for 60 min. all procedures are performed at 37 ° C. Then, the slides were washed three times with PFR solution and plated on microscopic slides into 0.1 X propyl gallate, which prevents fluorescence fading. In the fluorescence detection of the protoplast kernels, the procedure described in the previous paragraph is followed, and finally, a 50 µl / v / v / v / v aqueous glycerol containing 10 µg is added. 4,6-dimethylamino-2-phenylindole (DAPI), which specifically binds to nuclear and cytochrome D DNA / Williamson D. H. et al. Methods in Cell Biology 12, 335-351 (1975).

By comparing the biological effects of a cytostatic agent to the protoplasts of a protoplast control sample grown in its absence, it is performed using the cytological methods described herein. the average protoplast size inhibited by the added nocodazole was 10.2 µm, the average number of nuclei per protoplast was 1.3. The microtubular system of inhibited protoplasts has been completely depolymerized, which clearly demonstrates the primary effect of the cytostatic on this cell component. In the control sample of growing protoplasts grown in the absence of cytostatics, the average protoplast size was 15.4 µm, the average number of nuclei relative to one protoplast was 1.8. The microtubular system showed no reduction - normal corotubular configurations in the nuclei and distinct cytoplastic microtubules in the cytoplasm reaching up to the cytoplastic membrane in the physiological state were detected. Example 2 Instead of Saccharomyees uvarum, the species Saccharomyces cerevisiae was used to prepare protoplasts. Protoplasts of the above-mentioned yeast species were prepared under the conditions and in the manner described in Example 1. Instead of nocodazole, a carbamate series compound - carbendazim - was used at a concentration of 100 µg. The procedure described in Example 1 was followed, including the detection of biological effects on the basis of selected parameters.

The average protoplast size inhibited by the presence of carbendazim was consistent with the nocodazole inhibited antiplast size, including the average number of nuclei, as described in Example 1. However, the microtubular system in inhibited protoplasts was only partially reduced. Nuclear microtubules were completely depolymerized while

Claims (1)

EN 268903 B1 5 Eytoplasmatic mlrotrotules have been retained in reduced form. The results indicate that the target cell structure for carbendazole is the mlcrotubular system. However, its effect on nuclear and cytoplasmic mlcrotubules is differentiated. EXAMPLE 3 Cultivation of the Saccharomyces uvarum strain and the preparation of protoplasts was performed as described in Example 1. Benfluron was used as the cytostatic agent at a basic concentration of 10 µg. mL The protoplast size, number of nuclei, and changes in the mlcrotubule system were evaluated as described in Example 1 using the methods described above. The observed mean size of protoplasts inhibited by Benfluron was not different from that of control (15.4 µm). Defects at the nuclear and mitochondrial ONK levels were found corresponding to the immediate arrest of ONK replication in both nuclei and mitochondria. Nuclear and eytoplasmatic mlkrotubules were disorganized without depolymerization. The target structure of this cytostatic is nuclear and mitochondrial ONK. The method of testing compounds having a cytostatic effect according to the invention can be replaced by the testing of these substances, which are still carried out with the aid of animal cell tissue cultures using laboratory animals, which is economically advantageous. OBJECT OF THE INVENTION A method for testing cytostatic agents, characterized in that in the sterile osmotic sorbitol-stabilized broth with an osmolarity of 0.7 to 1, protoplasts of Saccharomyces cerevisiae or Saccharomyces uvarum are initially incubated at an initial concentration of 10 8 to 10 9 cells for 30 minutes and then a sample of cytostatic agent v-100 / Ug / mL dissolved in dimethylsulfoxide is added and left for 4-6 hours, then centrifuged, protoplasts fixed for nucleus detection and indirect immunofluorescenceimicrotubule system. mL ~ 1 at a temperature of 25 to 30 ° C for 20 to a concentration of 10 to a sample