WO2001032933A1 - Assay method for determining product r's effect on induction of ifn-gamma in cultured cells - Google Patents

Assay method for determining product r's effect on induction of ifn-gamma in cultured cells Download PDF

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WO2001032933A1
WO2001032933A1 PCT/US2000/041856 US0041856W WO0132933A1 WO 2001032933 A1 WO2001032933 A1 WO 2001032933A1 US 0041856 W US0041856 W US 0041856W WO 0132933 A1 WO0132933 A1 WO 0132933A1
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gamma
product
cultured
cultured cells
ifn
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PCT/US2000/041856
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French (fr)
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WO2001032933A9 (en
WO2001032933B1 (en
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Chaoyuan Chen
Shalom Z. Hirschman
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Advanced Viral Research
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Publication of WO2001032933B1 publication Critical patent/WO2001032933B1/en
Publication of WO2001032933A9 publication Critical patent/WO2001032933A9/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a biological assay method. Particularly, it pertains to a method for determining the effects of Product R on the production of interferon-gamma (hereinafter "IFN-gamma”) in cultured cells.
  • IFN-gamma interferon-gamma
  • Product R is an antiviral agent useful for treating a wide range of viral infections, such as infections of human immunodeficiency virus (HIV) , herpes simplex virus, adenovirus. It has become known that Product R is effective in stimulating the production of chemokines, including interferon-gamma, interleukin- ⁇ and interleukin-1. Product R is described in great detail in U.S. Patent Application Serial No. 09/344,095, which is incorporated herein by reference in its entirety. However, the mechanism of Product R in treating human viral infections is yet to be fully understood. Improved methods for detecting and measuring Product R's existing or potential biological activities are thus desired. To applicant's knowledge, no one has heretofore taught or suggested an assay directed to measuring Product R's effect on the production of IFN- gamma in cultured cells.
  • HIV human immunodeficiency virus
  • the present invention provides a method for determining the effects of Product R on the production of IFN-gamma in H9 cells.
  • the method including three main steps. First, Product R is introduced into the H9 cells through a process of electroporation. Second, electroporated cells are cultured under predetermined conditions and total RNA of the cells is extracted with any conventional methods. Third, the comparative amount of the IFN-gamma mRNA (or other RNAs) in the total RNA sample is determined by a coupled reverse transcription-polymerase chain reaction (RT-PCR) procedure.
  • RT-PCR coupled reverse transcription-polymerase chain reaction
  • Fig. 1 depicts the experimental data obtained in an assay conducted according to the present invention, showing the effects of Product R on the expression of various cytokines in H9 cells.
  • H9 T lymphoma cells (ATCC, Rockville, MD) are cultured in RPMI1640 medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 20% fetal bovine serum (HyClone, Logan, UT) , L-glutamine at 2 M and penicillin/streptomycin (GIBCO-BRL) at 100 U/ml at 37°C with 5% C0 2 in the atmosphere.
  • the initial culture is screened for mycoplasma using a commercial kit (ATCC) and for HIV-1 by RT-PCR, as process described below.
  • step (1) To introduce Product R (Advanced Viral Research Corporation, Yonkers, NY) into H9 cells, The cells cultured in step (1) are harvested at the exponential phase by centrifugation at 1200 rpm at 4° C for 10 mm. After removing the supernatant, the cell pellet ( ⁇ 4 x 10 6 cells) is collected and resuspended in 20 ml of serum-free RPMI 1640 medium. Centrifuge the suspension and discard the supernatant. The resulting cell pellet is ready for electroporation. Prepare a series of containers so that they contain varying concentrations of Product R in cold serum-free RPMI 1640 medium.
  • Electroporation was performed by using a BTX electroporator (ECM 395, BTX, San Diego, CA) at 150V.
  • ECM 395, BTX, San Diego, CA BTX electroporator
  • the electroporation condition is selected because it does not the cell's viability and expression of IL-2 as determined in the control cells undergone electroporation under the same condition. Other conditions may also be satisfactorily used as long as they do not affect the cell's viability and cytokines expression, which can be ascertained by running a control test.
  • the electroporated H9 cells following the completion of step (2) is harvested by centrifugation at 1200 rpm at 4° C for 10 mm and discard the supernatant.
  • the cells are used for preparation of total RNA, which is conducted by using Ultraspec-LI RNA kit (Biotecx, Houston, TX) according to manufacturer's instruction.
  • the amount of RNA in each individual sample is determined from the O.D. reading at 260 nm and the quality of the RNA preparation is evaluated by the ratio of O.D.260 nm to O.D. 280 mn.
  • the RNA samples with a ratio between 1.6 and 1.8 are used for synthesis of 1 st strand cDNAs in step (4) .
  • RT- PCR Reverse transcription-polymerase chain reaction
  • 1 st strand cDNAs are synthesized using a modified manufacturer's protocol (Promega, Madison, WI). Specifically, the total RNA (800 ng per sample) from step (3) is heated in the presence of oligo(dT) primers (0.5 g) at 65 °C for 10 mm and then at room temperature for 2 mm. The RNA sample is mixed with AMV reverse transcriptase buffer, dNTP at 1 mM each, MgS04 at 5 mM and AMV reverse transcriptase (20 U) with a total reaction volume of 20 ⁇ l. The reverse transcription is performed at 42° C for 60 min to produce 1 st cDNA. Two microliters of the resulting 1 st cDNA solution are taken for the PCR procedure.
  • PCR is performed according to manufacturer's instruction (Promega). Optimal concentrations of Mg 2+ for individual oligonucleotide primers are determined in preliminary experiments.
  • Primers specific for the mRNAs of IL-2 (1), IL-6 (2), IFNgamma (3), TNF-alpha (1), GAPDH (4) or HIV-1 (5) are synthesized by Genosys, Woodland, TX and used in PCR at 1 ⁇ M each. The reaction is performed at 95° C for 1 min 55° C for 2 min and 72° C for 2 min for 35 cycles with an extension at 72° C for 10 min at the end of the reaction.
  • PCR products of 458 bp, 628 bp, 462 bp, 305 bp and 195 bp are corresponding to IL2, IL-6, IFN-gamma, TNF- alpha and GAPDH, respectively.

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  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

An assay method for determining the effect of Product R on expression of IFN-gamma in cultured cells. The method comprising the following step: 1) introducing Product R into cultured cells; 2) culturing the Product R-containing cultured cells for a period of time; 3) preparing total RNA from the cultured cells following the culturing; and 4) determining the amount of the INF-gamma mRNA in the total RNA preparation, which reflects the level of IFN-gamma expressing in cultured cells.

Description

Assay Method for Determining Product R's Effect on Induction of IFN-Gamma in Cultured Cells
BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to a biological assay method. Particularly, it pertains to a method for determining the effects of Product R on the production of interferon-gamma (hereinafter "IFN-gamma") in cultured cells.
2. Description of the Related Art
Product R is an antiviral agent useful for treating a wide range of viral infections, such as infections of human immunodeficiency virus (HIV) , herpes simplex virus, adenovirus. It has become known that Product R is effective in stimulating the production of chemokines, including interferon-gamma, interleukin-β and interleukin-1. Product R is described in great detail in U.S. Patent Application Serial No. 09/344,095, which is incorporated herein by reference in its entirety. However, the mechanism of Product R in treating human viral infections is yet to be fully understood. Improved methods for detecting and measuring Product R's existing or potential biological activities are thus desired. To applicant's knowledge, no one has heretofore taught or suggested an assay directed to measuring Product R's effect on the production of IFN- gamma in cultured cells.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a method for determining the effects of Product R on the production of IFN-gamma in H9 cells. The method including three main steps. First, Product R is introduced into the H9 cells through a process of electroporation. Second, electroporated cells are cultured under predetermined conditions and total RNA of the cells is extracted with any conventional methods. Third, the comparative amount of the IFN-gamma mRNA (or other RNAs) in the total RNA sample is determined by a coupled reverse transcription-polymerase chain reaction (RT-PCR) procedure. Using this method, applicants have determined that Product R can increase the expression of IFN-gamma in H9 cells in a dose-dependent fashion while having no significant effect on IL-2 and GAPDH's expression under the same conditions. Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the description and drawings are provided solely for purposes of illustration and not as a definition of the limits of the invention, for which reference should be made to the claims .
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings, like reference characters denote similar elements throughout the several views:
Fig. 1 depicts the experimental data obtained in an assay conducted according to the present invention, showing the effects of Product R on the expression of various cytokines in H9 cells.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS The physical and chemical properties of product R and the process of producing it are fully described in U.S. Patent Application Serial No. 09/344,095, which is incorporated herein in its entirety by reference. A specific embodiment of the present invention is described herein in detail, which comprising the following steps:
(1) Cell Culture:
H9 T lymphoma cells (ATCC, Rockville, MD) are cultured in RPMI1640 medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 20% fetal bovine serum (HyClone, Logan, UT) , L-glutamine at 2 M and penicillin/streptomycin (GIBCO-BRL) at 100 U/ml at 37°C with 5% C02 in the atmosphere. The initial culture is screened for mycoplasma using a commercial kit (ATCC) and for HIV-1 by RT-PCR, as process described below.
(2) Ξlectroporation :
To introduce Product R (Advanced Viral Research Corporation, Yonkers, NY) into H9 cells, The cells cultured in step (1) are harvested at the exponential phase by centrifugation at 1200 rpm at 4° C for 10 mm. After removing the supernatant, the cell pellet (~ 4 x 106 cells) is collected and resuspended in 20 ml of serum-free RPMI 1640 medium. Centrifuge the suspension and discard the supernatant. The resulting cell pellet is ready for electroporation. Prepare a series of containers so that they contain varying concentrations of Product R in cold serum-free RPMI 1640 medium. 400 μl medium from each of container is then transferred into a electroporation cuvettes (4 nun gap, BTX) for electroporation. Electroporation was performed by using a BTX electroporator (ECM 395, BTX, San Diego, CA) at 150V. The electroporation condition is selected because it does not the cell's viability and expression of IL-2 as determined in the control cells undergone electroporation under the same condition. Other conditions may also be satisfactorily used as long as they do not affect the cell's viability and cytokines expression, which can be ascertained by running a control test. After electroporation, the cells are immediately transferred into individual culture flasks, each of which contains 15 ml complete medium, and is cultured for 14 to 18 hrs . at 37 °C and 5% C02. (3) Preparation of total RNA:
The electroporated H9 cells following the completion of step (2) is harvested by centrifugation at 1200 rpm at 4° C for 10 mm and discard the supernatant. The cells are used for preparation of total RNA, which is conducted by using Ultraspec-LI RNA kit (Biotecx, Houston, TX) according to manufacturer's instruction. The amount of RNA in each individual sample is determined from the O.D. reading at 260 nm and the quality of the RNA preparation is evaluated by the ratio of O.D.260 nm to O.D. 280 mn. The RNA samples with a ratio between 1.6 and 1.8 are used for synthesis of 1st strand cDNAs in step (4) . (4) Reverse transcription-polymerase chain reaction (RT- PCR) :
1st strand cDNAs are synthesized using a modified manufacturer's protocol (Promega, Madison, WI). Specifically, the total RNA (800 ng per sample) from step (3) is heated in the presence of oligo(dT) primers (0.5 g) at 65 °C for 10 mm and then at room temperature for 2 mm. The RNA sample is mixed with AMV reverse transcriptase buffer, dNTP at 1 mM each, MgS04 at 5 mM and AMV reverse transcriptase (20 U) with a total reaction volume of 20 μl. The reverse transcription is performed at 42° C for 60 min to produce 1st cDNA. Two microliters of the resulting 1st cDNA solution are taken for the PCR procedure. PCR is performed according to manufacturer's instruction (Promega). Optimal concentrations of Mg2+ for individual oligonucleotide primers are determined in preliminary experiments. Primers specific for the mRNAs of IL-2 (1), IL-6 (2), IFNgamma (3), TNF-alpha (1), GAPDH (4) or HIV-1 (5) are synthesized by Genosys, Woodland, TX and used in PCR at 1 μM each. The reaction is performed at 95° C for 1 min 55° C for 2 min and 72° C for 2 min for 35 cycles with an extension at 72° C for 10 min at the end of the reaction. The PCR products of 458 bp, 628 bp, 462 bp, 305 bp and 195 bp are corresponding to IL2, IL-6, IFN-gamma, TNF- alpha and GAPDH, respectively.
(5) Evaluation of PCR products:
Ten microliters of each PCR product are electrophorized in 1.5% agarose gel with ethidium bromide at 0.2 μg/ml both in the gel and in the running buffer (TAR, pH 7.5). A 100-bp ladder standard (Promega) is used as reference for molecular weights. The amplicons of the PCR are visualized under UV light and photographed . With reference to FIG. 1, in a particular experiment embodying the present invention, a dose response in terms of expression of IFN-ganima has been observed in H9 cells electroporated with 75% and 100% of Product R compared to the cells in medium alone or lower concentrations of Product R, indicating a potentiated production of IFNgamma. In contrast, no significant changes are found in the expression of IL-2 and GAPDH genes, suggesting equivalent loading of starting RNA materials for reverse transcription and specific action of Product R on the LEN-gamma gene. Furthermore, no specific IL-6 gene product is detected in H9 cells electroporated with or without Product R (data not shown). In two independent experiments, however, inconsistent expression of TNF-alpha in Product R- treated H9 cells is observed.
While there have been shown, described and pointed out the features of the invention as applied to a preferred embodiment thereof, it will be understood that various omissions and substitutions and changes in the form and details of the devices illustrated, and in their operation, may be made by those skilled in the art without departing from the spirit of the invention. For example, the invention is not limited to the specific materials and equipment described in the foregoing embodiment. Similar materials from different sources or equivalent assay equipment may be satisfactorily used. It is expressly intended that all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the invention.
The following references are incorporated herein by reference in their entirety. References
1. Than, S., R. Hu, N. Oyaizu, J. Romano, X.-P. Wang, S. Sheikh, and S. Pahwa . 1997. Cytokine pattern in relation to disease progression in human immunodeficiency virus-infected children. J. Infect. Dis. 175:47 - 56.
2. Ito, N., S. Kawata, S. Tamura, S. Kiso, H. Tsushima, Y. Maeda, E. Yamasaki, T. Igura, and Y. Matsuzawa. 1996. Induction of interleukin-6 by interferon Alfa and its abrogation by a serine protease inhibitor in patients with chronic hepatitis C. Hepatology 23:669 - 675.
3. Kenney, R. T., D. L. Sacks, A. A. Gam, H. W. Murray, and S. Sundar. 1998. Splenic cytokine responses in Indian Kala-Azar before and after treatment. J. Infect. Dis. 177:8 15 -819.
4. Kumar, A., M. Commane, T. W. Flickinger, C. M. Horvath, and G. R. Stark. 1997. Defective TNF-induced apoptosis in STATl-null cells due to low constitutive levels of caspases. Science 278:1630 - 1632.
5. Zhou, P., 5. Goldstein, K. Devadas, D. Tewari, and A. L. Notkins. 1997. Human CD4+ cells transfected with IL-16 cDNA are resistant to HTV-1 infection: Inhibition of mRNA expression. Nature Medicine 3:659 - 664.

Claims

CLAIMSWhat is claimed is:
1. A method of measuring production of interferon-gamma (INF-gamma) in cultured cells, comprising the steps of
(a) introducing Product R into a cultured cell,
(b) culturing said cell in a cell culture medium, and (c) determining the amount of INF-gamma mRNA in said cultured cell.
2. The method of claim 1, wherein Product R is introduced into said cultured cell by electroporation.
3. The method of claim 1, wherein said cultured cell is cultured for about 14-18 hours.
4. The method of claim 1, wherein said cultured cell is H9 cell.
5. The method of claim 1, wherein said the amount of said INF-gamma mRNA is determined by reverse transcription-polymerase chain reaction (PCR).
6. The method of claim 5, wherein said INF- gamma mRNA determined by PCR has a length of 462 bases.
7. The method of claim 6, wherein Product R is introduced into said cultured cell by electroporation.
8. The method of claim 7, wherein said cultured cell is cultured for about 14-18 hours.
9. The method of claim 7, wherein said cultured cell is H9 cell.
PCT/US2000/041856 1999-11-04 2000-11-03 Assay method for determining product r's effect on induction of ifn-gamma in cultured cells WO2001032933A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8084239B2 (en) 1996-10-22 2011-12-27 Ohr Pharmaceuticals, Inc Preparation of a therapeutic composition

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5807840A (en) * 1997-11-04 1998-09-15 Advanced Viral Research Corp. Method for treating canine distemper
WO1998046240A1 (en) * 1997-04-15 1998-10-22 Advanced Viral Research Corp. Topical treatment of skin diseases and eye afflictions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046240A1 (en) * 1997-04-15 1998-10-22 Advanced Viral Research Corp. Topical treatment of skin diseases and eye afflictions
US5807840A (en) * 1997-11-04 1998-09-15 Advanced Viral Research Corp. Method for treating canine distemper

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIRSCHMAN ET AL.: "Peptide nucleic acids stimulate gamma interferon and inhibit the replication of the human immunodeficiency virus", J. INVEST. MED., vol. 44, no. 6, August 1996 (1996-08-01), pages 347 - 351, XP002938645 *
LAZZARINO ET AL.: "CXCR4 and CCR5 expression by H9 T-cells is downregulated by a peptide-nucleic acid immunomodulator", IMMUN. LETT., vol. 74, 2000, pages 189 - 195, XP002938644 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8084239B2 (en) 1996-10-22 2011-12-27 Ohr Pharmaceuticals, Inc Preparation of a therapeutic composition

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