Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-08
2002-11-12
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091100, C435S091200, C435S325000, C435S344000, C536S024300, C536S024330, C536S023100
Reexamination Certificate
active
06479241
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a high-throughput method for screening potentially active compounds which affect the expression of one or more mRNAs. More particularly, the invention relates to an in vitro mRNA expression method employing cell lines.
Most effective, specific anti-cancer drugs affect the expression of one or more mRNAs, and therefore proteins, in the cells that are the targets of these drugs. Analysis of the altered expression patterns of cell lines treated with a novel compound allows researchers to more accurately predict the effect a novel compound will have in vivo as well as determine which other drugs currently in use or development may be synergistic with the compound being analyzed. Furthermore, the analysis of a broad spectrum of mRNAs from cancer cells during exposure to anti-cancer agents allows scientists to more fully understand the molecular mechanism of action of novel anti-cancer agents. This understanding contributes to rational, molecular based drug design.
To date, the analysis of the altered expression pattern of even a single MRNA species over a time course and at different drug concentrations is a labor intensive and costly proposition. There has been a need for the development of a rapid methodology for analyzing altered MRNA expression of a large number of genes in a large number of cell lines in response to treatment of these cell lines with various concentrations of anti-cancer agents over an extended time course. This technology will allow quick and efficient analysis of the effect a novel agent has on expression of numerous mRNAs associated with apoptosis, signal transduction, cell proliferation, cellular differentiation and other cell functions associated with the ultimate effectiveness of an anticancer agent. This information will prove invaluable in predicting the efficacy of novel anticancer agents using in vivo models and in patients.
Although structure-function relationships and rational drug design have been successfully employed in research, a major component of such research still involves high-volume screening of large numbers of compounds. Accordingly, screening methods should be robust, sensitive, fast and capable of accommodating large numbers of samples simultaneously. In addition, experimental drugs often are available only in small quantities. A successful screening method therefore advantageously uses small cell culture volumes and sensitive detection methods. Currently, the standard 96-well microtiter plate format is preferred for these types of procedures.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method for simultaneously screening a plurality of compounds for antitumor activity against a cell line involves the steps of:
(1) cultivating cells, which are capable of serving as targets for anticancer agents in culture medium in wells of a microtiter plate, each well containing a culture volume of from about 50 &mgr;L to about 200 &mgr;L;
(2) adding each compound being tested to separate cell-containing wells at a concentration at which the drug activity is to be tested;
(3) culturing the cells for a time sufficient for the cells to express target genes;
(4) subjecting a sample of cell lysate to quantitative nucleic acid amplification of a sequence from at least one target gene to determine the extent of expression for the target gene in each well; and
(5) correlating the extent of gene expression, as determined by quantitative nucleic acid amplification, to the concentration of each compound to determine the effectiveness of the compounds in altering cellular gene expression.
It has been found that quantitative nucleic acid amplification provides a sensitive and reliable method for determining the extent of gene expression. The sensitivity of this detection method permits the use of very small cell numbers and culture volumes, in contrast to the relatively large culture volumes and conditions described in the prior art. The small culture volumes, in turn, permit the screening assay to be performed in a microtiter plate format and permit the use of very small amounts of test compounds. By using the microtiter plate format, numerous compounds can be tested simultaneously to provide a high-throughput screening method.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a high-throughput method for screening potentially active compounds which affect the expression of one or more mRNAs. The invention is a combination of robotics, standard molecular biology techniques (e.g., cell lysis, RNA isolation, reverse transcription), and real time PCR. The combination of these techniques yields a high throughput analysis that is unique. Preferably the methods are performed robotically in a clean environment using sterile disposables.
More particularly, the invention relates to an in vitro mRNA expression method employing cell lines. A method is described for screening compounds for antitumor activity by cultivating cells in small volumes in microtiter plates in the presence of test compounds and performing quantitative nucleic acid amplification of target genes to determine the effect of the test compound upon the expression of the genes. The advantage of this approach over current methodologies is that real time PCR allows accurate quantitation of the messages expressed in a cell population undergoing treatment with novel agents. It is generally accepted that anti-cancer agents may cause upregulation or downregulation in mRNA expression that may effect the ultimate outcome of treatment. Most genes are not turned completely on or completely off. It is this quantitatively altered expression pattern that is of interest in many cases. The use of this new technology will allow researchers to rapidly and accurately assess the changes in mRNA expression induced by novel anti-cancer compounds. Earlier technologies for performing such studies were manual methods of molecular biology. The advantage of the method disclosed here is the high speed capacity. This high speed capacity allows large scale molecular analysis which would not be feasible with manual methods.
It will be appreciated by those skilled in the art that the screening method described herein may be applied to a wide variety of genes. Such genes include, but are not limited to, the genes listed in Table I. Any cell line which can be cultivated in vitro can be employed in this method. Advantageously, the cells employed in the screening method express the genes that are associated with the condition being studied, e.g., a specific cancer gene, and which is a target for therapeutic interference. In addition to being used for screening for anticancer agents, other uses for the described method include analyzing cell lines for induction of apoptosis, analyzing cell lines for tubulin polymerization, analyzing cell lines for cell surface protein expression, analyzing cell lines for DNA damage such as loss of heterozygosity (LOH) and specific mutations via PCR, analyzing cell lines for cytotoxicity, and other similar uses as will be recognized by those of skill in the art.
TABLE I
Functional Categories of Genes
1) Cell growth and regulation
AIBC1
bax
bcl-2
Beta Catenin
BRCA1
CDIC-4
Cyclin D1
Cyclin E
c-abl
c-flk
c-fos
c-jun
c-kit
c-mpl
c-myc
E cadherin + gp78
EGFr
erb B2
FAA
fas
fas-ligand
gadd45
gadd153
GST
h-ras
Ki67
MDR1
MRP
NKX3
p21
p53
p110
PTEN
Telomerase
hTRT
VEGF
waf-1
ZAB C1
2) Apoptosis
Annexin V
Apaf-1
Bcl-2
Bcl-X
Bik
Mcl-1
Bak
Bad
Bax
PARP (Poly-ADP-Ribose-Polymerase)
Caspases-1
Caspases-2
Caspases-3
Caspases-4
Caspases-5
Caspases-6
Caspases-7
Caspases-8
Caspases-9
Caspases-10
Caspases-13
Caspases-14
Cytochrome C
c-IAP-1
d4-GD1
Daxx
DFF
DNase I
FADD
FAF1
Fas
1-FLICE
ICE
IkB
MEKK
Nc1-x1
NFkB
Rb
RIP
SGP-2
TNF-Rp55
TRADD
TRAF-3
TRAF-4
TRAIL
3) Oncogenes
4) Cell surface receptors/ligands
5) Adhesion molecules
6) Signal transduction molecules
7) Nuclear transcriptional regulators
8) DNA binding molecules
9) Glycolysis/gluconeogenesis molecules
10) DNA repair enzymes
The cells may be cultured by tec
Benzion Gary
Rothwell Figg Ernst & Manbeck
Southern Research Institute
Wilder Cynthia
LandOfFree
High throughput screening of the effects of anti-cancer... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with High throughput screening of the effects of anti-cancer..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and High throughput screening of the effects of anti-cancer... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2991904