Stable cell line of human ovarian adenocarcinoma skov-kat

FIELD: medicine.

SUBSTANCE: invention can be used for screening of anti-tumour preparations in vivo, in particular for screening synthetic and natural substances with potential anti-tumour activity.

EFFECT: claimed invention makes it possible to create stable cell line of human ovarian adenocarcinoma.

2 dwg, 2 ex

 

The invention relates to biotechnology and experimental Oncology and can be used for screening of anticancer drugs in vivo, in particular for the screening of synthetic and natural substances with potential antitumor activity.

In the selection of synthetic and natural substances with potential antitumor activity widely used test for the lines of tumor cells in vivo.

The use of panels of diverse genetic and phenotypic characteristics of cell lines provide a very effective screening of synthetic compounds, natural substances and physical effects on searching for their biological activity at the cellular and molecular levels. One of the directions in cancer therapy is the development of drugs with mechanisms for targeted delivery to cancer cells. When working with tumor models in vivo, it is extremely important to have objective methods of observation and monitoring of tumor cells. In recent years, was developed region fluorescence tomography. It allows you to observe cancers non-invasive method, which is especially important for a detailed analysis of the effectiveness of cancer drugs.

Known cancer cell lines with overexpression of HER marker/neu (Oncogene, 11; 19(53):6102-14, 2000):

- SKOV-3 cell line of human ovarian adenocarcinoma (J. Natl. Cancer Inst. 59, 221-226, 1977);

In SK-BR-3 cell line breast cancer man (New York: Plenum Publishing Corp, pp.115-145, 1975).

However, these HER2/neu positive line does not have a pronounced fluorescence, which makes them unsuitable for direct observation using fluorescent imaging.

Known cancer cell lines, modified green fluorescent protein GFP:

1) cancer cell lines human breast MF-7, MDA, MW, MDA-MB435;

2) human cell lines of prostate cancer RES, DU145;

3) cell line human glioblastoma 324;

4) cell line human lung cancer Anip-73, N;

5) cancer cell lines human colon l-205, HCT-15, WiDr;

6) cell line gastric cancer man NVGC-4;

7) cell line kidney cancer human SN12C;

8) cancer cell line of human language SCC-25;

9) cell line human melanoma LOX, SK-mel-5;

10) cancer cell line of Chinese hamster ovary Cho-K1;

11) cell line melanoma mouse 16.

(WO/1998/049336,1 998).

Known cancer cell lines, modified red fluorescent proteins:

cell line human pancreatic cancer MIA-Race-2, modified RFP (Clinical and Experimental Metastasis, Volume 21, Number 1 7-12, 2004);

cell line alveolar rhabdomyosarcoma man is ka Rh30, modified DsRed2 (Cell Proliferation, Vol.41 Issue 2 Page 365, 2008);

cell line melanoma B16F0, modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003);

cell line prostate cancer mouse MMT, modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003);

cell line prostate cancer human PC-3, a modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003);

cell line of colon cancer HCT-116, modified RFP (Proceedings of the National Academy of Science, vol. 100, Issue 24, p.14259-14262, 2003).

Known closest to the claimed cancer cell line human breast MDA-MB-231, modified tdTomato (Neoplasia, 8(10): 796-806, 2006).

Known fluorescent lines do not have the overexpression of this gene marker HER2/neu, and therefore they cannot be used for screening synthetic anticancer compounds and natural substances with targeted delivery to HER2/neu or directional mechanism of action for HER2/neu.

The objective of the invention is the creation of a model cell line bearing a tumor marker HER2/neu and fluorescing in the far red region, for advanced analysis and in vivo screening of substances targeted antitumor activity and detailed research results impact on this type of cancer in vivo.

The problem is solved due to tile the offered lines of human ovarian adenocarcinoma SKOV-kat, do overexpress tumor marker HER2/neu and fluorescing in the far red spectral region from 600 to 700 nm, for use in the screening of anticancer drugs in vivo.

For specified cell line gene of the fluorescent protein Katushka contained in the vector plasmid pKatushka-N, injected with the drug Unifectin56(Unifact Groups) in a line of cancer cells SKOV-3 and perform the selection on the antibiotic gentamicin and G418 (Sigma).

It is known that animal tissues are more transparent to long-wave radiation (Neoplasia, 8(10): 796-806, 2006).

Protein Katushka fluorescent in the far red spectrum has a maximum fluorescence at 635 nm and the maximum absorption at 588 nm and is the brightest at the wavelength of 650 nm, other proteins have 40-60% brightness Katushka in this wavelength range (Nature Methods, 4, 741-746, 2007) - this makes it one of the best tools for monitoring grafted tumors in the tissues of a living organism using fluorescence imaging.

After removing the cells from cryopreservation they retain fluorescence in full. Measurement of the fluorescence produced by using a flow cytometer (Beckman-Coulter, USA) (column 1, 2).

Cells received line SKOV-kat consistently reproduce the acquired fluorescence for six months of in vitro cultivation (more than 500 generations).

Using the test system on the basis of the e immunospecific of staining (RF patent No. 2303783, 2007) indicate the presence of a HER2/neu receptor on the surface of transfected cells in number, are identical to lines SKOV-3 (see table).

Cell line SKOV-kat have intense fluorescence in the far red region of the spectrum (600-700 nm) in contrast to the original cell line SKOV-3 (column 1, 2).

Cell line SKOV-kat resistant to the antibiotic G418 in contrast to the cell source line SKOV-3.

Cell line SKOV-kat have the level of expression of a marker HER2/neu on the surface, identical to the level of expression of HER2/neu on the surface of SKOV-3.

Morphological features

Cell line SKOV-kat morphologically similar to cells of the parental line SKOV-3 cell adenocarcinoma of the ovary.

Cultural properties

The culture of the adhesive type. Vials area of 25 cm2sow 1.0-9.0×105cells in 5 ml growth medium. Passedout every three days. Cell lines are not demanding in terms of cultivation and may persist attached to the tablet in the form of a monolayer.

Growth environment: the environment of a needle in a modification of Dulbecco (DMEM), L-glu, 10% serum.

Marker signs following.

- Overexpression of surface antigen HER2/neu.

Intense fluorescence in the far red region of the spectrum (600-700 nm).

Cryopreservation.

Cell line SKOV-kat resuspended in serum-free medium containing

7, 5% is metilsulfate (DMSO), at a concentration of 2-10×106cells/ml, poured into plastic vials and placed in a heat insulating material (polyurethane foam) and transferred into a low-temperature refrigerator at -70°C for 24 hours, after the initial freeze was placed on long-term storage at -135°C. Thawed quickly at 37°C. Cells diluted in 10 ml serum-free medium and precipitated by centrifugation, resuspended in 5 ml of medium containing 10% fetal calf serum, and transferred into a culture flask. Cell viability assessed by adhesion on plastic for 20 to 50 hours.

Thus, the obtained transfusiona genome Katushka line SKOV-kat has a number of qualities which distinguish it from the original line SKOV-3, as well as other cancer cell lines:

- cell line SKOV-kat resistant to the antibiotic G418 in contrast to the cell source line SKOV-3;

- cell line SKOV-kat have significant fluorescence in the far red region of the spectrum (600-700 nm) in contrast to the original cell line SKOV-3 (column 1, 2);

cells lines have the overexpression of tumor marker HER2/neu unlike other fluorescent cell lines.

The invention is illustrated graphic materials

Figure 1 - histogram 1.

Comparative characteristic fluorescence cell lines SKOV-kat and SKOV-3 620 nm.

Data flowing qi is fluorimeter (Beckman-Coulter,USA) is presented in the form of a histogram of the number of registrations (cells) from the logarithm of the luminosity, presented in arbitrary units luminosity (Cup). In parentheses are shown the average value of the fluorescence, normalized to 100 for SKOV-kat, averaging was performed over 10300 registrations.

Figure 2 - histogram 2.

Comparative characteristic fluorescence cell lines SKOV-kat and SKOV-3 675 nm.

Data from the flow cytometer (Beckman-Coulter,US) presents the histogram of the number of registrations (cells) from the logarithm of the luminosity presented in arbitrary units luminosity (Cup). In parentheses are shown the average value of the fluorescence, normalized to 100 for SKOV-kat, averaging was performed over 10300 registrations.

The invention is illustrated by the examples.

Example 1.

Obtaining cell line.

Transfection make as follows: 200 ng of plasmid DNA (gene fluorescent protein Katushka contained in the vector plasmid pKatushka-N) in 10 µl environment Dulbecco''s Modified Eagle's Medium (DMEM) (paneco) without serum mixed with 1 ál Unifectin56(Unifact Groups) 40 µl (medium without serum), and incubated for 15 minutes and add to the cells SKOV-3 (calculation of 60 mm Cup, 70% of the monolayer). One day after transfection the medium was added to the antibiotic G418 (Sigma)

in the amount of 500 μg/ml and continue to add in the same amount every 5 days, together with the change of environment. After 20 days in selective conditions on the cups are formed of fluorescent colonies, which is s selected using a platinum loop in wells with medium and an antibiotic. Then after the formation of the colony re-selection in the wells with medium without antibiotic. Within 50 days see a stable level of fluorescence. Numerical analysis of fluorescence produced using flow cytofluorometry (column 1, 2). To do this, cells are removed with a solution of Versene (Hyclone) and using centrifugation with a frequency of 800 rpm, the cells are transferred into a solution of PBS (KN2RHO41.7 mM; Na2HPO45.2 mm; NaCl 150 mm) for monitoring on the device. Data from the flow cytometer (Beckman-Coulter, USA) treated for 30,000 registrations.

Example 2.

Demonstration of the presence of cell marker.

Cells are placed in a solution of PBS with 0.5 million cells/ml with molecular structures 4D5-barnase-4D5+barstar-EGFP 4 μg/ml and incubated for 45 minutes at +4°C. as a negative control non-specific binding using the cell line HeLa (Cancer Res., 12: 264-265, 1952), and as positive control - the original cell line SKOV-3. For the numerical analysis on flow cytometer cell is removed from the tablet in a solution of PBS (table). Data from the flow cytometer (Beckman-Coulter, USA) treated for 30,000 registrations. The number of HER2/neu on the surface of cells is directly proportional to the incremental signal from the dye minus nonspecific staining.

Cell line SKOV-kat HER2/neu-positive adenocarcinoma of the ovary of man, fluorescent in the far red region of the spectrum (600-700 nm), obtained by transfection of the cell line SKOV-3 gene of the protein Katushka. Stable cell line SKOV-kat fully preserves the phenotype of HER2/neu-positive adenocarcinoma of the ovary and produces intracellular fluorescent protein Katushka. Cells obtained fluorescent lines reproduce the acquired characteristic for at least six months of in vitro cultivation.

The above properties and evaluation results of these properties allow the use of cell line SKOV-kat of human ovarian adenocarcinoma for screening of anticancer drugs in vivo targeted delivery mechanism to a surface marker of cells of HER2/neu.

Stable cell line human ovarian adenocarcinoma SKOV-kat resistant to the antibiotic G418, do overexpress tumor marker HER2/neu and fluorescing in the far red spectral region from 600 to 700 nm, obtained by a process comprising the following stages:
A. providing the plasmid containing the gene of the fluorescent protein Katushka;
b. transfection of cells SKOV-3 plasmid containing the gene of the fluorescent protein Katushka;
C. selection of fluorescent colonies in the environment in the presence of the antibiotic G418;
d. repeated selection of fluorescent colonies in the environment in the absence of the antibiotic G418.



 

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