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r Malignant tumors are one of the main problems of modern
medicine and oncology because very frequently it is not
possible to achieve complete recovery of patients and
maintain corresponding quality of their lives even in the
case of favourable course of this disease.
r Proposed schemes of cancer cure employ surgical
methods and high dose chemotherapy, which has such
serious drawback as high toxicity to vitally important
organs and systems of the body.
r Solution of the problem of cancer cure requires use of new
approaches, having high selectiveness and effectiveness of
treatment.
r §atural R§ase exhibit cytotoxicity that can be
increased by their modification with cationic groups.
r Recently, various compounds performing effective.R§
cleavage and known as the artificial Rnases (aR§ases),
have attracted much attention.
r Like natural enzymes, these compounds catalyze the
transesterification reaction and cause irreversible R§
degradation under physiological conditions.
r Example: aR§ases based on conjugates of 1, 4
diazabicyclo[2.2.2]octane and imi dazole effectively cleave
R§ under physiological conditions in vitro at
concentration of 10^5²10^4 M . Within 6-12 hours total
cleavage of R§ was observed.
r Thus, it was interesting to elucidate, whether these
compounds are cytotoxic for human cancer cells and
whether mechanism responsible for cell death involves
mR§ cleavage or other reasons account for toxic effect
of these aR§ases.

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The following cell lines have been used in
experiments:
r wuman epidermoid carcinoma KB31 cells, human weLa
cervical epithelial adenocarcinoma cells, human
neuroblastoma SK§MC cells, human embryonic kidney
(wEK293) cells, and also canine kidney epithelial MDCK
cells.
r Cells were cultivated in the IMDM medium containing 10%
embryonic calf serum, antibiotics(100 U/ml penicillin and
0.1 mg/ml streptomycin), and amphotericin (0.25 Ǎg/ml)
in an atmosphere of 5% CO2 at 37°C.
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r The R§ precipitate was separated by centrifugation (12000
g, 4°C, 15 min), washed with 75% ethanol, dried, dissolved in
Milli Q water and kept at 20°C.
r R§ concentration was determined spectrophotometrically
by measuring the absorbance at 260 nm.
r The quality of the isolated R§ was evaluated by
electrophoresis in 1.5% agarose gel.
r Changes in mR§ levels of à
, 18S rRN , 'actin,
ǃmicroglobulin, (ǃ2m) and cmyc genes in KB31 cells were
performed by reverse transcription polymerase chain
reaction (RTPCR).
r Êor synthesis of cD§ we used total R§ isolated from the
KB31 cells as described above. The reac- tion of cD§
synthesis was performed at 42°C for 1h using the reaction
mixture, containing 50 mM Tris wCl, pw 8.3, 3 mM MgCl2, 5
mM DTT, 75 mM KCl, 0.05 Ǎg/ml of total R§ , 5 ǍM primer
d(T)15, 0.5 mM each deoxynucleoside triphosphate, and 0.5
U/Ǎl of MMuLV R§ dependent D§ polymerase.
r PCR was performed in a reaction mixture containing 250 ǍM
deoxynucleoside triphosphates, 0.25 ǍMof forward and
reverse primers, cD§ obtained from50 ng of total cell R§ ,
and 2 U of Taq D§ polymerase in the buffer containing 10
mM TriswCl,pw 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01%
Tween-20.

r mplification products were analyzed by electrophoresis in

1.5% agarose gel under standard conditions. Gel was stained
with ethidium bromide(0.002%) for 15 min and
photographed.
 
  


r The toxic effect of compounds Dp12 and BL3C3

on cell cultures were evaluated by the MTT assay.
r Cells were seeded into 96well plates as described
above and treated with increasing concentrations
of these compounds (from 10²7 to 10²3 M).
r solution of MTT(3(4,5dimethylthiazol2yl)2,
5diphenyl tetrazolium bromide) (5 mg/ml, final
concentration 0.5 mg/ml) was added in phosphate
buffered saline to cells.
r fter incubation for 3 h under the same conditions
the medium was removed.
r Cells were treated with 100 Ǎl of DMSO causing dissolution
of formazan crystals formed in cells during incubation.

r Optical density was registered using a multichannel

spectrophotometer at 570 and 630 nm ( 570 is formazan
absorption and 630 is back ground absorbance of cells).

r Data represent the percentage of survived cells versus

control. The number of control cells incubated in the
absence of aR§ases was defined as 100%.
 

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r Membranotropic activity of compounds Dp12 and

BL4C3 was determined by means of cell staining
with Trypan blue.

r Data represent the percentage of living (Trypan

blue unstained) cells versus cells with damaged
membrane (and stained with the dye).

r The ratio of stained to unstained cells in control

(cells incubated in the absence of aR§ ses and
detergent) defined as 100%.
  
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r Study of ultrastructural changes of KB31 cells induced
by BL3C3 was performed using electron microscopy.
r Cells grown on Millicell plate membranes were treated
with 20 and 50ǍM BL3C3 aR§ase for 30 min, 2.5, 6
and 24 h, and fixed with 4% paraform aldehyde at 4°C.
r fter washing with wanks solution membranes with
cells were fixed with 1% osmic acid and dehydrated by
increasing concentration of ethanol and acetone using
the standard method and then embedded in the
eponaraldite resin.
r Ultrathin sections were prepared in the plane
perpendicular to the surface of the supporting
membrane using aReichert ultratome (Reichert,
ustria), contrasted with uranyl acetate and citrate
lead solutions and analyzed using a witachi w600
electron microscope.
 
 m 

r It was demonstrated that various compounds

imitating active sites of natural R§ases effectively
cleave R§ under physiological conditions (pw 7.0,
37°C).

r BL3C3, a conjugate of 1,4diazabicy

clo[2.2.2]octane and imidazole is one of the most
effective compounds of this type; BL3C3 has an
oligomethylene fragment at quaternary nitrogen
atom and histidine. t the concentrations(1²5)
×10²4 M this compound caused total cleavage of
R§ within 6²12 h at 37°C.
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