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Title:  Oncogene-induced apoptosis in drug-resistant cells

United States Patent:  6,555,330

Issued:  April 29, 2003

Inventors:  Fearnhead; Howard O. (Huntington Station, NY); McCurrach; Mila Elena (Cold Spring Harbor, NY); Lowe; Scott William (Cold Spring Harbor, NY); Lazebnik; Yuri (East Norwich, CT)

Assignee:  Cold Spring Harbor Laboratory (Cold Spring Harbor, NY)

Appl. No.:  273220

Filed:  March 19, 1999

Abstract

The present invention relates to a method of identifying a compound (agent) which modulates apoptosis in transformed cells. In one embodiment, the invention is a method of identifying a compound which selectively activates apoptosis in transformed cells. In an alternative embodiment, the present invention can be used as a method of identifying a compound which inhibits apoptosis in cells. The invention also relates to a method of selectively killing transformed cells, wherein the transformed cell is contacted with a compound which selectively activates apoptosis in transformed cells, as described herein. The invention also relates to methods of treating diseases associated with defective apoptotic machinery (e.g., cancer, neurodegenerative disease). The methods of the present invention are useful for defining the biochemical mechanisms of apoptosis. In addition, the invention provides an assay to identify compounds which modulate (inhibit, activate) apoptosis, supplying new drugs which target defective apoptotic machinery associated with disease (e.g., cancer, neurodegenerative diseases such as Huntington's Disease and Alzheimers Disease).

DETAILED DESCRIPTION OF THE INVENTION

Described herein is a system that permits modulation of the apoptotic or suicide machinery in vitro. A system that reproduces inhibition of this activation by an agent e.g., protein) that confers drug-resistance to cancer cells has also been developed.

The present invention is a method of identifying a compound which modulates (activates or inhibits) apoptosis. The method is carried out in a cell-free system and the combination of reagents can be further modified if the assay is used to identify an activator of apoptosis.

In one embodiment, the present invention is a method of identifying a compound which modulates apoptosis in transformed cells. In this method, the following reagents are combined to produce a test sample: the compound to be assessed and a cellular extract comprising apoptotic machinery and a signal capable of activating the apoptotic machinery. The combination is maintained under conditions appropriate for the activation of apoptosis. Modulation of apoptosis can be assessed, for example, by measuring ILP activity.

In one embodiment, the present invention is a method of identifying a compound which selectively activates (induces or enhances) apoptosis in transformed cells. In this embodiment, the following reagents are combined to produce a test sample: the compound to be assessed; and apoptotic machinery and a signal which activates the apoptotic machinery (e.g., obtained from cellular extracts of transformed cells). The combination is maintained under conditions appropriate for activation of apoptosis; activation of apoptosis is detected in the presence of the compound to be assessed by detecting or assessing an event indicative of an increase in activity of the apoptotic machinery. In the method in which a compound which selectively activates apoptosis in transformed cells is to be identified, an inhibitor of apoptosis can also be combined to produce the test sample. In this embodiment, the inhibitor inhibits or uncouples the apoptotic machinery of the cellular extract. Recoupling of the apoptotic machinery (partial or complete activation or reactivation of the ability of the signal to activate the apoptotic machinery) indicates that the compound to be assessed selectively activates apoptosis in transformed cells.

In a second embodiment, the present invention is a method of identifying a compound which inhibits apoptosis in cells. In this embodiment, the following reagents are combined to produce a test sample: a compound to be assessed; an apoptotic machinery and a signal capable of activating the apoptotic machinery. The combination is maintained under conditions appropriate for activation of apoptosis; inhibition of apoptosis is detected in the presence of the compound to be assessed by detecting uncoupling of the apoptotic machinery (partial or complete inhibition of the ability of the signal to activate the apoptotic machinery). Uncoupling of the apoptotic machinery indicates that the compound to be assessed inhibits apoptosis in cells.

Any suitable inhibitor of apoptosis can be used in the method of the present invention. For example, as described in the examples, bcl-2 can be used as an inhibitor of apoptosis in the methods of the present invention. Additional inhibitors of apoptosis which can be used include, but are not limited to, vanadate, E1B-19K, mutated p53, phosphotyrosine; ZN2+, Crm A, and p35 (Beidler, D. R., et al., J. Biol. Chem. 270:16526-16528 (1995); Lazebnik, Y. A., et al., J. Cell. Biol., 123:7-22 (1993); Martin, S. J., et al., EMBO J., 14:5191-5200 (1995); Newmeyer, D. D., Cell, 79:353-364 (1994)).

As described herein, the cell-free assay utilizes apoptotic machinery and a signal capable of activating the apoptotic machinery. The apoptotic machinery, as defined herein, includes all of the signalling pathways that regulate apoptosis or portions of the apoptotic machinery which reproduce the suicide machinery of apoptosis when activated by the signal. For example, interleukin-1.beta.-converting enzyme (ICE)-like proteases (ILPs) can be used with the signal capable of activating the ILPs in the methods described herein to identify compounds which activate or inhibit apoptosis. Other examples of a portion of the apoptotic machinery which can be used with a signal capable of activating the machinery for use in the present invention include the endonuclease activity of apoptosis. Identification of other portions of the apoptotic machinery which are activated by a signal capable of activating the machinery for use in the methods of the present invention can be determined using methods described herein and methods known in the art.

The apoptotic machinery and signal capable of activating the machinery can be obtained from any suitable source. For example, as demonstrated herein, cellular extracts of transformed cells can be used as a source for all or a portion of the apoptotic machinery and signal which activates the machinery. Examples of cellular extracts from transformed cells (preferably mammalian) which can be used to obtained the apoptotic machinery and signal include 293 cells, HeLa cells, transformed fibroblasts (e.g., mouse embryo fibroblasts (MEFS), human fibroblasts). In addition, extracts from other cells transformed with pro-apoptotic oncogenes (e.g., E1A, E7, c-myc, c-myb, cdc-25, E25, E2A-PBX1, ras; see (Bissonnette, R. P., et al., Nature, 359:552-556 (1992); (Fanidi, A., et al., Nature, 359:554-556 (1992); (Galaktionov, K., et al., Nature, 382:511-517 (1996); (Dedera, D. A., Cell, 74:833-843 (1993)) can be used in the methods of the present invention. Alternatively, all or a portion of the apoptotic machinery and the signal which activates the machinery can be chemically synthesized or obtained using recombinant techniques.

In the methods of identifying an activator or inhibitor of apoptosis described herein, the resulting combination is maintained under appropriate conditions (e.g., temperature, pH, sufficient time) for activation or inhibition of apoptosis. The present method can be carried out at a temperature from about 25oC. to about 40oC., preferably between about 30oC. to about 35oC., and even more preferably at 30oC. or 37oC. In addition, the treated sample can be incubated from about 10 minutes to about 120 minutes, preferably between 30 minutes to about 120 minutes, and more preferably for 30 minutes or 60 minutes. The method can be carried out as a series of steps or as a single (one-step) method. In the methods of the present invention, the following reagents must be present: a compound to be assessed, apoptotic machinery and a signal capable of activating the apoptotic machinery. Additionally, in the method in which a compound which selectively activates apoptosis in transformed cells is to be identified, an inhibitor of apoptosis can be present.

In addition, a control sample can be used in the methods of the present invention. For example, a control sample can be produced in which the same reagents are combined and processed in the same manner in the absence of the compound being assessed. Other suitable controls for use with the methods of the present invention can be determined using routine experimentation.

Various methods can be used to detect activation or inhibition of apoptosis in the presence of the compound being assessed. For example, the methods of the present invention can further comprise the step of adding cell nuclei wherein the morphological manifestations of apoptosis (e.g., nuclear chromatin condensation, blebbing of the nuclear, fragmentation of residual nuclear structures into discrete membrane-bounded apoptotic bodies, nuclei shrinkage; See Lazebnik, Y. A., et al., J. Cell Biol., 123:7 (1993)) can be visualized in the presence of the compound to be assessed, using, for example, fluorescence microscopy. Cell nuclei for use in the present invention can be obtained from any suitable source using routine methods. For example, cell nuclei can be obtained from 293 cells, HeLa cells, fibroblasts (e.g., MEFs, human fibroblasts), and Jurkat cells. Alternatively, ILP activation, a biochemical hallmark of apoptosis, can be detected using, for example, an ILP affinity probe (e.g., biotin-YVAD-acyloxymethylketone). ILP activation can also be measured on a synthetic fluorogenic substrate (e.g., DEVD-AFC, DEVD-AMC), on a natural substrate (e.g., PARP), or by processing of cpp32, an ILP implicated in apoptosis. Other suitable methods of detection can be determined by those of skill in the art.

The present invention can also be used to selectively killing transformed cells wherein the transformed cell is contacted with a compound which selectively activates apoptosis in transformed cells, as described herein. The invention can further be used to treat cancer wherein an effective amount of a compound which selectively activates apoptosis in transformed cells is administered to the individual. As described herein, "an effective amount" of a compound is an amount such that when administered, it results in prevention, amelioration or elimination of the cancer in the individual, compared to an individual who does not receive the compound. In addition, the amount of the compound will vary depending on the size, age, body weight, general health, sex, and diet of the host, and the time of administration, duration or particular qualities of the cancer. Adjustment and manipulation of established dose ranges are well within the ability of those of skill in the art.

The compound can be administered to the individual in a variety of ways. The routes of administration include intradermal, transdermal (e.g., slow release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural and intranasal routes. Any other convenient route of administration can be used, for example, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings. In addition, the compound can be administered together with other components or biologically active agents, such as pharmaceutically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, diluents and vehicles.

Thus in the method of the present invention a mammalian cell-free apoptosis system that does not require an exogenous agent to activate the apoptotic machinery is used. The present invention is based on the discovery that certain cells (e.g., E1A transformed cells, E7 transformed cells) contain an endogenous signal which is sufficient to induce apoptosis. The signal is uncoupled from the apoptotic machinery (latent) in drug-resistant cells (e.g., transformed cells resistant to chemotherapy). Thus, agents that re-couple the signal to the machinery selectively kill these cells.

Claim 1 of 17 Claims

What is claimed is:

1. A method of identifying a compound which modulates apoptosis in transformed cells, comprising the steps of:

a) forming a combination by combining:

1) a compound to be assessed and

2) a cellular extract obtained from transformed mammalian cells, wherein the cellular extract comprises an apoptotic machinery and a signal capable of activating the apoptotic machinery;

b) maintaining the combination produced in a) under conditions appropriate for activation of the apoptotic machinery of a) 2); and

c) detecting modulation of the apoptotic machinery of a) 2) in the presence of the compound to be assessed by comparing the modulation of the apoptotic machinery to apoptosis in an appropriate control sample,

wherein detecting modulation of the apoptotic machinery in c) compared to the control sample indicates that the compound to be assessed modulates apoptosis in transformed cells.
 


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