CS268903B1 - A method for testing substances having an eytostatic effect - Google Patents

Abstract

The essence of the solution is that testing is carried out on growing protoplasts of yeast of the genus Saccharomyces and the biological effects of substances with cytostatic effects are analyzed by cytological methods including evaluation of the size of protoplasts, the number of nuclei per protoplast and the degree of reduction of the microtubular system. From the microscopic evaluation of the preparations, it is possible to directly determine the uniform response of a set of yeast cells and at the same time determine the relationship between the chemical composition of the tested cytostatic and the molecular level of its effect on the cell structure. The method replaces the testing of substances with cytostatic effect carried out using tissue cultures of animal cells and laboratory animals.

Description

The invention relates to a method for testing substances with cytostatic effect.

The procedure is based on the general latest scientific findings, confirming that the molecular mechanism of eukaryotic cell division is phylogenetically very old and fundamentally identical from yeast to man (3.L. Marx, Science 226, 527-528, 19854 P. Nurse, Nature 313, 631-632, 1985). The main component of this mechanism are proto-oncogenes, homologues of oncogenes - genes of tumor viruses - which are present as genes regulating proliferation in the genome of all vertebrates including man (R.A. Weinberg, 3.Cell Biology 97, 166-166 -1662, 1983). Violation of the interplay of the main prollferators. genes is the first step in initiating some types of tumor transformation (H.E. Varnus, Ann.Rev.Genet. 18. 553-612, 1984).

In the last two years, 8 proto-oncogenes have been identified in the yeast genome /A. E. Wheals. Bioessays 3. 108-112. 1985/. These genes are all involved in the regulation of cell division, their function is relatively well known and their expression is directly linked to the microtubule system, which is the first cytoskeleton component identified in yeast /E. Streiblová and J. Hašek. Microbial Sciences 2. 139-143. 1985/. Inhibitors of the polymerization of the microtubule system have experimentally and clinically useful cytostatic effects due to selective toxicity to microtubules present in the nuclei of rapidly dividing tumor cells /M. G. Fitzgerald. Biochem. Pharmac. 25, 1383. 1975/.

The known methods used for serial testing of substances with cytostatic effect are carried out on laboratory animals and in tissue cultures /De Brabander M. et al. Cancer Res. 36, 905-916 /1976//.

Furthermore, there are known works dealing with the specificity of anti-microtubule substances and testing of substances with cytostatic effect on whole yeast cells /Walker G. K., J. G. Microbio., 128, 61-71 /1982/; Quinlan R. A. et al., J. Cell Sci. 46, 341-352 /1980//.

The techniques used are time-, methodologically and economically demanding.

These disadvantages are eliminated by the method of testing substances with cytostatic effect according to the solution, the essence of which is that in a sterile nutrient medium stabilized osmotically with sorbitol with an osmolarity of 0.7 to 1, protoplasts of Saccharomyces ce8 9 -1 revisiae or Saccharomyces uvarum are incubated submerged in an initial concentration of 10 to 10 cells/ml at a temperature of 25 to 30 °C for 20 to 30 minutes, and then a sample of a substance with cytostatic effect is added at a concentration of 10 to 100 µg/ ^ml dissolved in dimethyl sulfoxide and left for 4 to 6 hours, then the sample is centrifuged, the protoplasts are fixed for detection of nuclei and indirect immunofluorescence of the microtubular system.

Substances with cytostatic effect are tested on yeast protoplasts formed by enzymatic non-digestion of intact yeast cells with a mixture of enzymes from the stomach of the garden snail /Helix pomatia/; this procedure removes the permeable barrier on the yeast surface, which could prevent the entry and transport of the tested substances into the cells /Nečas 0., A. Svoboda, J. F. Peberdy and L. Fevenczy Zeds./ Fungal protoplasts, pp. 115-133, Marcel Dekker, New York 1985/. For testing, the yeast species Saccharomyces uvarum with large cells and high growth rates can be used, for example. In addition, this species is harmless to health, has low nutritional requirements and is traditionally used in brewing.

The resulting yeast protoplasts, which continue to grow (synthesis of proteins and macromolecules) and undergo nuclear division when cultivated in osmotically stabilized medium, represent a dynamic in vitro system that is easily accessible by cytostatics.

The advantage is that growing protoplasts are exposed to the influence of the studied cytostatic substances directly in the osmotically stabilized environment in which they grow; biological effect2

The weights of the test substances are analyzed after four and six hours, i.e. approximately after three nuclear divisions that occur in the control protoplasts.

Knowledge of the kinetics of growth processes, nuclear division and cytoskeletal behavior in yeast protoplasts can be used to understand the ways in which substances with a cytotoxic effect enter the regulation of cellular behavior. Testing allows: a/ to assess the overall sensitivity of the cell to the studied substance, b/ to contribute to the clarification of the sensitive site for the action of a substance with a cytostatic effect, c/ to interpret the mechanism of its effect, d/ to express the inhibition of nuclear division in a specified interval at the cellular level.

For cytological analysis of inhibited protoplasts, an optical microscope equipped with a device for fluorescence microscopy in combination with phase contrast is sufficient. The microtubular system is visualized by indirect immunofluorescence /Progress in Cell Cycle Controls, Microbiological Institute of the Czech Academy of Sciences, 1983, pp. 159 to 160/. Nuclear and mitochondrial ONK and the number of nuclei are visualized by direct fluorescence /H. Giloh and J. W. Sedat, Science 217, 1 252 to 1 255, 1 982/. The size of protoplasts is assessed by measuring their diameters in phase contrast using an ocular micrometer, which is inserted into the ocular for this purpose /Mikrobiologické metody, Prague, Academia 1981, pp. 30 to 53/. The size of protoplasts is expressed as the mean size of protoplasts /x/.

_ · ⁿ i * ^x i

X = . , .10 ε · , 1· x average mean size of protoplasts in pm number of protoplasts in 1 observed field average size of protoplasts in 1 observed field in um ζη^ total number of measured protoplasts

Observation of protoplasts is performed using a Jenalumar (Zeiss Jena NOR) fluorescence microscope, which allows for alternating phase contrast with fluorescence observation at different wavelengths on one preparation. An eyepiece with a 6x magnification with an inserted micrometer and an immersion objective with a 100x magnification using non-fluorescent immersion oil are used. The size of the protoplasts is measured in phase contrast, the distribution of DNA and the number of nuclei are monitored in the fluorescence image using a filter for a wavelength of 340 to 380 nm, the state of the microtubule system is monitored in the fluorescence image using a filter for a wavelength of 450 to 490 nm. An average of 20 fields containing 20 to 50 protoplasts are compared with the same number of reference (control) protoplasts.

The proposed test makes monitoring the effects of cytostatic agents much more economical, because the costs of maintaining the strain, nutrient media, lytic enzymes and antibodies are almost negligible compared to breeding and testing on laboratory animals.

The present method of use enables serial testing of substances with cytostatic effect according to the invention.

The examples document the sufficient universality of the method of using yeast for testing substances with cytostatic effect, namely by using nocodazole /methyl-/-2-thienylcarbonyl/-1H-benzimidazol-2-yl carbamate/, carbendazim /MBC; methylbenzimidazol-2-yl carbamate/ and by using the original Czechoslovak cytostatic Benfluron /5-2-/N-N-dimethylamino/ethoxy-?-oxo-7H-benzo c fluorene hydrochlorides/, which

CS 268903 81 3 is the subject of the Czechoslovak Patent No. 212 669 and its properties are further specified in the Czechoslovak Patents No. 235 429 and No. 242 292. The different effects of cytostatics on the microtubular system and nuclear division of protoplasts of the yeast Saccharomyces uvarua can be documented by fluorescence microscope images. From these images it is also possible to determine the relationship between the chemical composition of the given cytostatic and the molecular level of its effect on the functional organization of the cell.

Further details are given in the examples which illustrate the method according to the invention in more detail, but do not limit it in any way.

Design examples

Example 1

The test uses Saccharomyces uvarum strain P 96, which is continuously maintained on solid sterile medium containing 2X glucose, 2X bactopeptone, 1X yeast extract and 2X agar. The strain grown at 25°C for 48 hours is stored at 4 to 8°C for 3 months. After this time, the culture is subcultured onto liquid medium of the same composition for 48 hours and then transferred again to solid slant agar, grown at 25°C for 48 hours and then stored at 4 to 8°C for 3 months.

To prepare protoplasts, cells are cultured submerged in liquid sterile nutrient medium of the same composition in flasks on a rotary shaker at 28 °C for 6 hours. Cells in the logarithmic growth phase are obtained from the culture medium by vacuum filtration through a membrane filter /Synpor - Synthesis, Pardubice, pore size 0.4 /pm/ and transferred to the digestion mixture so that their final concentration in this mixture is 10$ cells. ml \ .

The digestion mixture contains 3 mL of digestive juice from the Helix pomatia, 1.5 mL of 0.1 M KH^PO^ solution at pH 6.5, 1 mM phenylsulfonylfluor1d /hereinafter PMSF/ and 1.1 M sorbitol solution. The current concentrations in the digestion mixture are given. After 30 minutes of slow shaking at a temperature of 25 °C, the protoplast suspension is carefully diluted with 0.1 M KH^PO^ solution over 10 minutes to a final osmolarity of 0.7 sorbitol. The protoplasts thus obtained are centrifuged into a liquid nutrient medium, the composition of which was described in the first paragraph; the nutrient medium is osmotically stabilized with 0.7 M sorbitol. From this medium, the protoplasts are inoculated at an initial concentration of 10 protoplasts per mL and their further cultivation takes place at a temperature of 25 °C with slow shaking on a reciprocal shaker with a swing rate of 100. min\'

The cytostatic nocodazole is prepared as a stock solution in dimethyl sulfoxide (hereinafter DMSO) and applied to the culture medium at a final concentration of 10 ^jg . mL ^-1 ; the concentration of DMSO used in application to protoplasts does not exceed 0.4 X /vol/vol/. The protoplasts are first grown for 30 minutes in the culture medium as mentioned in the previous paragraph, then nocodazole is added to the growing protoplasts and left to act for 4 hours. At the same time, protoplasts are grown without cytostatic under the same conditions for the same time interval. After four hours, both protoplast samples are collected by centrifugation and fixed at 25 °C in the following way.

Protoplasts are fixed for a total of 90 minutes in two stages. The first stage includes an 80-minute fixation with 4 X /v/v/ formaldehyde in a stabilization buffer containing 0.1 M KH^PO^ solution at pH 6.5, 20 mM ethylene glycol-bis^#-aminoethyl ether/-N,N,n)n-tetraacetic acid (hereinafter referred to as EGTK) and 1 mM MgCl ₂ solution. The actual concentrations in the fixation mixture are indicated. In the second stage, protoplasts are further fixed for 10 minutes with 1 X /v/v/ glutaraldehyde. Then the fixed protoplasts are centrifuged from the culture medium, washed with buffered physiological saline at pH 7.3 (hereinafter referred to as PFR) and incubated for 20 minutes in a cold NaBH^ solution at a concentration of 0.5 mg . mL ^-1 in PFR. Finally, the protoplasts are washed three times with PFR solution and spread in a thick suspension in a thin layer on clean, washed microscope slides (coverslips). After drying, the protoplasts are permeabilized by immersing the slides in methanol for 10 minutes and then in acetone for 30 seconds at a temperature of -20 °C.

For indirect immunofluorescence detection of the microtubular system in protoplasts, the mouse monoclonal antibody TU 01 against porcine leg tubulin is used /V. Viklický et al. Cell Blol. Internát. Reports 6, 725-730, 1982/. Coverslips with permeabilized protoplasts are first preincubated with 1 Z /vol/vol/ solution of bovine serum albumin /hereinafter referred to as HSA/ in PRF and then incubated with the antibody Tu 01 obtained according to Czechoslovak author's certificate No. 223 535. The antibody is diluted 1 : 99 with 1 X /vol/ /vol/ HSA in PFR and incubated with the preparations for 60 minutes. Then the samples on the slides are washed three times with warm 1 X solution of HSA in PFR. In the second stage, the protoplasts are post-incubated with swine anti-mouse immunoglobulin conjugated with fluorescein isothiocyanate (SwAM/FITC - Institute of Serum and Vaccines Prague or, for example, Niles Laboratories, Inc., Research Products, Div. Elkhard. Ind. England). Post-incubation takes place in SwAM/FITC labeled antibody diluted tenfold in 1 X /v/v/ PFR for 60 minutes. All of the above procedures are performed at 37 °C. Then the slides are washed three times with PFR solution and mounted on microscope slides in 0.1 X propyl gallate, which prevents fluorescence fading.

For fluorescent detection of nuclei in protoplasts, the method described in the previous paragraph is followed and finally a 50% (vol/vol) aqueous solution of glycerol containing 10 µg mL- ^{1 of} 4,6-diamino-2-phenylindole (OAPI), which specifically binds to nuclear and mitochondrial DNA (Williamson 0. H. et al. Methods in Cell Biology 1_2, 335-351 /1975//). '

The comparison of the biological effects of a substance with a cytostatic effect on protoplasts with a control sample of protoplasts grown in its absence is carried out using the cytological methods mentioned in the description of the present invention.

The average size of protoplasts inhibited by the added nocodazole was 10.2 µm, the average number of nuclei per protoplast was 1.3. The microtubular system of the inhibited protoplasts was completely depolymerized, which clearly demonstrates the primary effect of the cytostatic on this cellular component.

In the control sample of growing protoplasts grown without the presence of cytostatics, the average size of protoplasts was 15.4 µm, the average number of nuclei per protoplast was 1.8. The microtubule system showed no reduction - normal microtubule configurations were found in the nuclei and distinct cytoplasmic microtubules in the cytoplasm reaching up to the cytoplasmic membrane in the physiological state.

Example 2

Instead of Saccharomyces uvarum, the species Saccharomyces cerevisiae was used for the preparation of protoplasts. Protoplasts of the said yeast species were prepared under the conditions and method given in Example 1. Instead of nocodazole, another compound of the carbamate series - carbendazim - was used at a concentration of 100 µg. mL~\ The procedure was then as given in Example 1, including the detection of biological effects based on selected parameters.

The average size of protoplasts inhibited by carbendazim was similar to that of protoplasts inhibited by nocodazole, including the average number of nuclei, as reported in Example 1. However, the microtubule system in the inhibited protoplasts was only partially reduced. The nuclear microtubules were completely depolymerized, while

CS 268903 B1 5 cytoplasmic microtubules remained preserved in a reduced form. The results indicate that the target cellular structure for the action of carbendazim is the microtubular system. However, its effect on nuclear and cytoplasmic microtubules is differentiated.

Example 3

Cultivation of the Saccharomyces uvarum strain and preparation of protoplasts were carried out according to Example 1. Benfluron was used as a substance with cytostatic effect in a basic concentration of 10 µg. mL. The assessment of the size of protoplasts, the number of nuclei and the state of changes in the microtubular system was carried out as in Example 1 using the described evaluation methods.

The average size of protoplasts inhibited by Benfluron did not differ from control protoplasts /15.4 µm/. Defects were found at the level of nuclear and mitochondrial ONK corresponding to the immediate arrest of ONK replication in nuclei and mitochondria. Nuclear and cytoplasmic microtubules were disorganized without depolymerization. The target structure of this cytostatic is nuclear and mitochondrial ONK.

The method of testing substances with cytostatic effect according to the invention can replace the testing of these substances, which has so far been carried out using tissue cultures of animal cells and laboratory animals, which is economically considerably advantageous.

Claims (1)

A method for testing substances with cytostatic effect, characterized in that in a sterile nutrient medium stabilized osmotically with sorbitol with an osmolarity of 0.7 to 1, protoplasts of Saccharomyces cerevisiae or Saccharomyces uvarum are incubated submersively at an initial concentration of 10 ⁸ to 10 ⁹ cells. mL~ ¹ at a temperature of 25 to 30 °C for a period of 20 to 30 minutes and then a sample of a substance with a cytostatic effect is added at a concentration of 10 to -1 100 µg/mL dissolved in dimethyl sulfoxide and left for 4 to 6 hours, then the sample is centrifuged, protoplasts fixed for detection of nuclei and indirect fluorescence of the microtubule system.