Use of isogenic human cancer cells for high-throughput screening and drug discovery
AssigneeThe Johns Hopkins University
InventorsChristopher Torrance, Bert Vogelstein, Kenneth Kinzler
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Abstract
A strategy for drug-screening is based on cells that are isogenic except for a gene of interest. Each cell can be tranfected with a vector that encodes a different fluorescent protein that can be differentially detected to monitor cell growth. Co-culture of both cells allows facile screening for compounds with selective toxicity towards a gene of interest. The drug screening is broadly applicable for mining therapeutic agents targeted to specific genetic alterations responsible for cancer development.
Claims55
A pair of cells comprising:
- a first cell;
- and
- a second cell,
- wherein the first cell and the second cell are isogenic but for:
- a gene of interest and a gene encoding a fluorescent protein;
- wherein the first cell comprises a gene that encodes a first fluorescent protein having a first absorption spectrum and a first emission spectrum;
- wherein the second cell comprises a gene that encodes a second fluorescent protein having a second absorption spectrum and a second emission spectrum;
- and
- wherein either:
- the first and second absorption spectra are not identical;
- and/or
- the first and second emission spectra are not identical.
A pair of cells comprising:
- a first cell wherein the Ras genotype of the first cell is c-Ki-RasWT/mutant and wherein the first cell comprises a first gene that encodes a first fluorescent protein having a first absorption spectrum and a first emisson spectrum;
- and
- a second cell wherein the Ras genotype of the second cell is c-Ki-RasWT/null and wherein the second cell comprises a second gene that encodes a second fluorescent protein having a second absorption spectrum that is not identical to the first absorption spectrum and a second emission spectrum that is not identical to the first emission spectrum, wherein the first and second cells are isogenic but for the Ras gene and the gene encoding a fluorescent protein.
A method of making a pair of cells, comprising the steps of:
- genetically modifying a first cell to yield a second cell that is isogenic with the first cell but for a single gene of interest;
- transfecting the first cell with a first gene that encodes a first fluorescent protein having a first absorption spectrum and a first emission spectrum;
- and
- transfecting the second cell with a second gene that encodes a second fluorescent protein having a second absorption spectrum and a second emission spectrum, wherein either the first and second absorption spectra are not identical and/or the first and second emission spectra are not identical.
A method of identifying a test compound as selectively affecting a gene of interest or its expression products or downstream genes or proteins in its pathway comprising the steps of:
- culturing a first and second cell, wherein the first and second cells are isogenic but for a gene of interest and a gene encoding a fluorescent protein, wherein the first cell comprises a first gene that encodes a first fluorescent protein having a first absorption spectrum and a first emission spectrum, and wherein the second cell comprises a second gene that encodes a second fluorescent protein having a second absorption spectrum and a second emission spectrum, wherein either the first and second absorption spectra are not identical and/or the first and second emission spectra are not identical;
- contacting the first and second cells with a test compound;
- and
- identifying the test compound as selectively affecting the gene of interest or its expression products or downstream genes or proteins in its pathway if the growth rate of the first cell is altered with respect to the growth rate of the second cell.
A method of identifying a test compound as selectively affecting a Ras gene, Ras protein, or downstream gene or protein in its pathway in cells comprising:
- contacting a test compound with a co-culture of an essentially equal number of a first and a second cell that are isogenic but for their Ras genes and a gene encoding a fluorescent protein, wherein the Ras genotype of the first cell is c-Ki-RasWT/mutant and wherein the first cell comprises a first gene encoding a first fluorescent protein having a first absorption spectrum and a first emisson spectrum and wherein the Ras genotype of the second cell is c-Ki-RasWT/null and wherein the second cell comprises a second gene encoding a second fluorescent protein having a second absorption spectrum that is not identical to the first absorption spectrum and a second emission spectrum that is not identical to the first emission spectrum;
- and
- identifying the test compound as selectively affecting the Ras gene, Ras protein, or downstream gene or protein in the pathway if the growth rate of the first cell is altered with respect to the growth rate of the second cell.
A composition comprising at least 90% of a compound having a formula selected from the group consisting of:
A method of treating cancer comprising:
- administering to a patient in need thereof a therapeutically effective amount of a compound having a formula selected from the group consisting of:
- or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically appropriate carrier.
A pharmaceutical composition comprising a compound with a formula selected from the group consisting of:
- or pharmaceutically acceptable salts, solvates, or prodrugs thereof, and a pharmaceutically appropriate carrier.
A cytotoxic composition comprising a compound having a formula:
- or a pharmaceutically acceptable salt, solvate, or prodrug thereof;
- and a pharmaceutically appropriate carrier.
Dependencies are inferred from the claim text.