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US 5,631,133 AGranted
Transition in transcriptional activation by intracellular hormone receptors at the tumor stage of dermal fibrosarcoma development
AssigneeThe Regents of the University of California
InventorsDouglas Hanahan, Keith R. Yamamoto, Maria d. M. Vivanco
Life of this patent
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Abstract
Intracellular hormone receptors are discovered to undergo posttranslational regulation. Assays to assess cancer progression and to permit discovery of a new class of biologically active compounds are provided. Related kits are also provided.
Claims20
A method of detecting posttranslational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. transfecting a eukaryotic cell with a recombinant expression cassette which comprises a marker gene operably linked to a promotor with a DNA binding site for the intracellular receptor;
- ii. transfecting an immortal eukaryotic cell with the expression cassette of step (i) , wherein the cell of step (i) and the cell of step (ii) express the intracellular receptor in a non rate limiting amount;
- iii. contacting the eukaryotic cell of step (i) and the immortal eukaryotic cell of step (ii) with the cognate hormone;
- iv. measuring the level of expression of the marker gene in the eukaryotic cell of step (i) and the eukaryotic cell of step (ii);
- and,
- v. comparing the level of expression of the marker gene in the eukaryotic cell of step (i) and the immortal eukaryotic cell of step (ii).
3 more elements
A method of detecting posttranslational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. transfecting a eukaryotic cell with a recombinant expression cassette which comprises a marker gene operably linked to a promotor with a DNA binding site for the intracellular receptor;
- ii. transfecting an immortal eukaryotic cell with the expression cassette of step (i), wherein the cell of step (i) and the cell of step (ii) express the intracellular receptor in a non-rate limiting amount;
- iii. contacting the eukaryotic cell of step (i) and the immortal eukaryotic cell of step (ii) with the cognate hormone;
- iv. measuring the level of expression of the marker gene in the eukaryotic cell of step (i) and the eukaryotic cell of step (ii);
- and,
- v. comparing the level of expression of the marker gene in the eukaryotic cell of step (i) and the immortal eukaryotic cell of step (ii);
- wherein the eukaryotic cell of step (i) and the eukaryotic cell of step (ii) are human.
4 more elements
A method of detecting posttranslational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. transfecting a eukaryotic cell with a recombinant expression cassette which comprises a marker gene operably linked to a promotor with a DNA binding site for the intracellular receptor;
- ii. transfecting an immortal eukaryotic cell with the expression cassette of step (i), wherein the cell of step (i) and the cell of step (ii) express the intracellular receptor in a non-rate limiting amount;
- iii. contacting the eukaryotic cell of step (i) and the immortal eukaryotic cell of step (ii) with the cognate hormone;
- iv. measuring the level of expression of the marker gene in the eukaryotic cell of step (i) and the eukaryotic cell of step (ii);
- and,
- v. comparing the level of expression of the marker gene in the eukaryotic cell of step (i) and the immortal eukaryotic cell of step (ii):
- wherein the eukaryotic cell of step (i) and the eukaryotic cell of step (ii) are human skin cells.
4 more elements
A method of detecting post translational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. co-transfecting a first primary cell with
- a. a first expression cassette having a nucleic acid encoding the intracellular hormone receptor operably linked to a first promoter;
- and,
- b. a second expression cassette having a marker gene operably linked to a second promoter having a response element for the intracellular hormone receptor;
- ii. transfecting a second cell with the second expression cassette;
- iii. separately contacting the first primary cell and the second cell with the cognate hormone;
- iv. measuring the expression of the marker gene from the first primary cell and the second cell;
- and,
- v. comparing the expression of the marker gene from the first primary cell and the second cell.
6 more elements
A method of detecting posttranslational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. co-transfecting a first primary cell with
- a. a first expression cassette having a nucleic acid encoding the intracellular hormone receptor operably linked to a first promoter;
- and,
- b. a second expression cassette having a marker gene operably linked to a second promoter having a response element for the intracellular hormone receptor;
- ii. transfecting a second cell with the second expression cassette;
- iii. separately contacting the first primary cell and the second cell with the cognate hormone;
- iv. measuring the expression of the marker gene from the first primary cell and the second cell;
- and,
- v. comparing the expression of the marker gene from the first primary cell and the second cell;
- wherein the first primary cell is a human skin cell grown from a tissue sample without serial passage in culture.
7 more elements
A method of detecting posttranslational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. co-transfecting a first primary cell with
- a. first expression cassette having a nucleic acid encoding the intracellular hormone receptor operably linked to a first promoter:
- and,
- b. a second expression cassette having a marker gene operably linked to a second promoter having a response element for the intracellular hormone receptor:
- ii. transfecting a second cell with the second expression cassette;
- iii. separately contacting the first primary cell and the second cell with the cognate hormone;
- iv. measuring the expression of the marker gene from the first primary cell and the second cell;
- and,
- v. comparing the expression of the marker gene from the first primary cell and the second cell;
- vi. transfecting an immortal cell line with the first expression cassette and the second expression cassette of step (i);
- vii. measuring the level of expression of the marker gene expressed by the transfected immortal cell line;
- and,
- viii. comparing the level of expression of the marker gene from the first primary cell and the second cell to the immortal cell line;
- wherein the first primary cell and the second primary cell are human skin cells, and the immortalized cell line is an immortalized skin cell line.
11 more elements
A method of detecting posttranslational regulation of an intracellular hormone receptor in the presence of a cognate hormone, comprising:
- i. co-transfecting a first primary cell with
- a. a first expression cassette having a nucleic acid encoding the intracellular hormone receptor operably linked to a first promoter;
- and,
- b. a second expression cassette having a marker gene operably linked to a second promoter having a response element for the intracellular hormone receptor;
- ii. transfecting a second cell with the second expression cassette;
- iii. separately contacting the first primary cell and the second cell with the cognate hormone;
- iv. measuring the expression of the marker gene from the first primary cell and the second cell and,
- v. comparing the expression of the marker gene from the first primary cell and the second cell;
- vi. transfecting an immortal cell line with the first expression cassette and the second expression cassette of step (i);
- vii. measuring the level of expression of the marker gene expressed by the transfected immortal cell line;
- and,
- viii. comparing the level of expression of the marker gene from the first primary cell and the second cell to the immortal cell line.
- wherein the level of expression of marker gene in the second cell relative to the level of expression of the marker gene in the first primary cell and the immortal cell line indicates whether the second cell is a fibrosarcoma.
10 more elements
A method of identifying a compound which differentially modulates posttranslational activity of an intracellular hormone receptor in an immortal cell compared to a primary cell, comprising:
- (i) transfecting the primary cell, which expresses the intracellular hormone in a non rate limiting mount, with an expression cassette comprising a marker gene operably linked to a promoter, wherein the promoter comprises an intracellular hormone response element;
- (ii) transfecting the immortal cell with an expression cassette comprising a marker gene operably linked to a promoter, wherein the promoter comprises an intracellular hormone response element;
- (iii) contacting the primary cell and the immortal cell with a non rate limiting mount of cognate hormone;
- (iv) measuring the level of expression of the marker gene in the primary cell and the immortal cell;
- (v) contacting the primary cell and the immortal cell with the test compound;
- (vi) measuring the level of expression of the marker gene in the primary cell and the immortal cell;
- and,
- (vii) comparing the level of expression of the marker gene in the primary cell and the immortal cell in step (iv) and step (vi).
5 more elements
A method of identifying a compound which differentially modulates activity of an intracellular hormone receptor in an immortal cell compared to a primary cell, comprising:
- (i) transfecting the primary cell, which expresses the intracellular hormone in a non-rate limiting amount, with an expression cassette comprising a marker gene operably linked to a promoter, wherein the promoter comprises an intracellular hormone response element;
- (ii) transfecting the immortal cell with an expression cassette comprising a marker gene operably linked to a promoter, wherein the promoter comprises an intracellular hormone response element:
- (iii) contacting the primary cell and the immortal cell with a non-rate limiting amount of cognate hormone;
- (iv) measuring the level of expression of the marker gene in the primary cell and the immortal cell;
- (v) contacting the primary cell and the immortal cell with the test compound;
- (vi) measuring the level of expression of the marker gene in the primary cell and the immortal cell;
- and,
- (vii) comparing the level of expression of the marker gene in the primary cell and the immortal cell in step (iv) and step (vi);
- wherein the primary cell of step (i) and the immortal cell of step (ii) are human skin cells.
6 more elements
A kit for the detection of posttranslational regulation of intracellular hormone receptor activity, comprising a container and an expression cassette encoding a marker gene operably linked to a promoter, wherein the promoter comprises an intracellular hormone response element, and wherein the promoter alters expression of the marker gene upon binding of the intracellular hormone receptor response element to an intracellular hormone receptor.
Dependencies are inferred from the claim text.