WO2008011008A1

WO2008011008A1 - Bioluminescent imaging of trail induced apoptosis

Info

Publication number: WO2008011008A1
Application number: PCT/US2007/016165
Authority: WO
Inventors: Wafik S. El-Deiry
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2006-07-17
Filing date: 2007-07-17
Publication date: 2008-01-24

Abstract

This invention relates to methods and kits to be used in the imaging apoptosis in vivo. Specifically, the invention relates to the use of a prolumÊnescent reagent, capsase substrate, operably linked to aminoluciferin in the imaging of TRABL-induced apoptosis in nucleated cells.

Description

BIOLUMINESCENT IMAGING OF TRAIL INDUCED APOPTOSIS

FIELD OF INVENTION

[0001] This invention is directed to a method for imaging apoptosis. Specifically, the invention relates to the use of a proluminescent reagent, capsase substrate, operably linked to aminoluciferin in the imaging of TRAEL-induced apoptosis in nucleated cells.

BACKGROUND OF THE INVENTION

[0002] Anti-neoplastic compounds frequently engage a physiologically important process described as programmed cell death, or apoptosis, to eliminate cancer cells. Apoptosis, an energy-dependent process that is characterized by several molecular and morphological changes to the cell, is crucial to physiological processes including embryogenesis, tissue homeostasis and the prevention of transformation of cells damaged beyond repair. Apoptosis constitutes a fundamental component of cell removal because it lacks the severe bystander effects and inflammation that are observed with tissue damage and subsequent passive cell death, i.e. necrosis. A wealth of data on the regulation of apoptosis in the context of chemotherapy has been generated from experiments on tissues, tissue culture and cells from experimental animals. However, studies of the process in human cells are typically restricted to the use of continuously growing human (cancer) cell lines that may or may not accurately replicate the magnitude of cell death in human tumor or normal tissues in vivo.

[0003] Although apoptosis was first described over thirty years ago, there is still a lack of consensus about how to monitor the process in patients receiving chemotherapy. Due to the heterogeneous nature of cancer, the individual variation among patients and hence the need to individually tailor anti-cancer therapies, apoptosis can be a useful pharmacokinetic marker for response to chemotherapy. Thus several efforts have been made to identify reliable biomarkers of apoptosis. Traditionally morphological and biochemical changes have been monitored in preclinical settings, e.g. by staining with DNA binding fluorochromes and subsequent analysis by flow cytometry. A number of soluble surrogate markers for tumor apoptosis present in peripheral blood, more easily utilized in clinical settings, have also been identified.

[00041 There is a need for improved methods, which allow discrete and accurate detection of the initiation of apoptosis in tumor cells as a method of gauging the efficiency of treatment. SUMMARY OF THE INVENTION

[0005] In one embodiment, the invention provides a method of imaging TRAIL-induced apoptosis in a cell within a region of a mammalian subject, comprising: contacting said cell with a proluminescence reagent; incubating said cell with said proluminescence reagent; contacting said nucleated cells with TRAIL; incubating said TRAIL-contacted cells; and measuring luminescence intensities, wherein the higher the measured luminescent intensity, the higher is the programmed cell death level. In one embodiment, the cell is a nucleated cell.

[0006] In another embodiment, the invention provides a method of evaluating the efficiency of a chemotherapeutic agent in a subject, comprising obtaining a tumor cell from said subject, wherein said tumor is the one treated with said chemotherapeutic agent, contacting said tumor cell with a candidate chemotherapeutic agent, incubating said chemotherapy treated tumor cell with a proluminescence reagent; and measuring luminescence intensity, wherein the higher the measured luminescent intensity, the more efficient is the chemotherapeutic agent.

[0007] In one embodiment, the invention provides a kit for imaging TRAIL - induced apoptosis in a nucleated cell within a region of a mammalian subject, comprising: DEVD-aminoluciferin powder and Dulbecco's PBS.

BRIEF DESCRIPTION OF THE DRAWINGS

^" Figure 1 shows a model of the TRAIL-induced apoptosis pathway.

Figure 2 shows Bioluminescent imaging of TRAIL-induced apoptosis at various incubation times. (A) I h, (B) 2h, (C) 3h, (D) 4h, (E) 18h after addition of DEVD-aminoluciferin compound in lysis buffer (Promega) at the 6h-TRAIL timepoint in HCTl 16, p53^{( /"}}, pGL-2 cells.

Figure 3 shows flow cytometry results of live FLICA assay on HCTl 16, p53^{( Λ)}, pGL-2 cells after 7h TRAIL treatment Figure 4 shows Bioluminescent imaging of TRAIL-induced apoptosis for alternate preparations of reagent (DEVD-aminoluciferin reagent in lysis buffer, lysis buffer only, DEVD-aminoluciferin powder dissolved in PBS, PBS only): (A) Immediately after reagent addition, and (B) Ih after reagent addition at 6h TRAIL treatment timepoint of HCTl 16 cells. 5

Figure 5 shows phase contrast microscope images of HCTl 16 cells Ih after reagent addition (addition after 6h TRAIL treatment) for alternate preparations of addition reagent: (A) DEVD-aminoluciferin reagent in lysis buffer (Promega), (B) lysis buffer only, (C) DEVD-aminoluciferin powder dissolved in PBS, (D) PBS only. Fig. 5E: Flow cytometry results of live FLICA assay on HCTl 16 cells after 6h 10 TRAIL treatment. Fig. 5F: Detecting apoptosis via sub-Gl analysis of HCTl 16 cells by Flow cytometry after 6h TRAIL treatment.

Figure 6 shows Bioluminescent imaging of TRAIL-induced apoptosis for alternate preparations of reagent (DEVD-aminoluciferin reagent in lysis buffer, lysis buffer only, DEVD-aminoluciferin powder 15 dissolved in PBS, PBS only): (A) Immediately after reagent addition, and (B) Ih after reagent addition at 6h TRAIL treatment timepoint of HCTl 16, p53^(πΛ), pGL-2 cells.

Figure 7 shows (7A) a comparison of bioluminescent intensity produced by DEVD-aminoluciferin powder (dissolved in PBS) and D-luciferin at lOOμg/mL in HCTl 16, pSS^07'*, pGL-2 cells. Reagents 20 were added 7h after TRAIL treatment (50ng/mL), and images were captured immediately after and Ih after reagent addition. (7B) Next-day phase contrast microscope images (32x) of 7h-TRAIL treated HCTl 16, pS^^"'^"*, pGL-2 cells incubated with DEVD-aminoluciferin powder (dissolved in PBS) and D- luciferin (diluted in PBS) for Ih.

25. Figure 8 shows Bioluminescent imaging of 7h TRAIL-induced HCTl 16, p53^{( / )}, pGL-2 cells with DEVD-aminoluciferin powder and D-luciferin immediately and Ih after reagent addition for different cell densities: (A) 50,000 cells/well, (B) 150,000 cells/well in clear 24-well plate.

Figure 9 shows flow cytometry results of live FLICA assay on HCTl 16, p53^{( / )}, pGL-2 cells after 6h 30 TRAIL + Ih (A) DEVD-aminoluciferin powder or (B) lOOμg/mL D-luciferin incubation.

Figure 10 shows Bioluminescent imaging of 6h TRAIL-induced apoptosis with heat-treated DEVD- aminoluciferin powder. Figure 11 shows the viability of colonic explants as assessed by Pi-staining.

Figure 12 shows cell death as assessed by the FLICA assay following treatment with 5-FU and/or TRAIL.

Figure 13 shows cell death as assessed by Annexin V binding following treatment with 5-FU and TRAIL.

Figure 14 shows FLICA-assay following treatment with CPT-11 and TRAIL.

DETAILED DESCRIPTION OF THE INVENTION

[0008] According to this aspect of the invention and in one embodiment, the invention provides a method of imaging apoptosis in a nucleated cell, comprising: contacting said nucleated cell with a proluminescence reagent; incubating said nucleated cell with said proluminescence reagent; contacting said nucleated cells with an apoptosis inducing agent; and measuring luminescence intensities, wherein the higher the measured luminescent intensity, the higher is the programmed cell death level.

10009] Apoptosis refers in one embodiment, to "programmed cell death" whereby the cell executes a "cell suicide" program. It is thought that the apoptosis program is evolutionarily conserved among virtually all multicellular organisms, as well as among all the cells in a particular organism. Further, it is believed that in many cases, apoptosis may be a "default" program that must be actively inhibited in healthy surviving cells.

[00010] In one embodiment, the term "proluminescence reagent" or "Proluminscent reagent" refers to a compound that in its native state does not induce luminescence when stimulated by an electromagnetic energy source, nor emits photons as the result of a reaction with another compound. In one embodiment, the proluminscent reagents used in the methods of the invention, require a trigger to convert the proluminscent reagent, to a luminescent reagent, such as in another embodiment, through a reaction with a protease, which releases a luminescent moiety operationally coupled to another non-luminescent moiety. In one embodiment, the proluminscent reagent used, is DEVD operationally linked to aminoluciferin. [0001 1] In one embodiment, the term "apoptosis inducing agents", refer to compositions such as genes encoding the tumor necrosis factor related apoptosis inducing ligand termed TRAIL, and the TRAIL polypeptide (U.S. Pat. No. 5,763,223; incorporated herein by reference); the 24 kD apoptosis-associated protease of U.S. Pat. No. 5,605,826 (incorporated herein by reference); Fas-associated factor 1, FAFI (U.S. Pat. No. 5,750,653; incorporated herein by reference). Also contemplated for use in these aspects of the present invention is the provision of interleukin-lβ-converting enzyme and family members, which are also reported to stimulate apoptosis.

[00012] In one embodiment, bioluminescence images are acquired with the charge-coupled device (CCD) camera and luminescence intensity is quantified using the Living Image software (version 2.5) from

Xenogen. In one embodiment, the luminescence intensities measured, are those captured by the CCD camera, translated to arbitrary luminescence units (ALU). As used herein, higher luminescence refers to those captured bioluminescence images, exhibiting greater ALU values than a standard. In one embodiment, the measured bioluminescence of a cell before being contacted with apoptosis-inducing agent serves as bioluminescence standard and is designated an index ALU number.

[00013] In one embodiment, the increase in ALU following exposure to apoptosis-inducing agent reflects the degree of apoptosis or programmed cell death and therefore, the higher the measured luminescent intensity above and beyond the index ALU, the higher is the programmed cell death level.

[00014] In another embodiment, the proluminescence reagent used in the methods and kits of the invention comprises a DEVD-aminoluciferin powder. In another embodiment, the proluminescent agent is incubated in contact with the cells in the kits and methods of the invention, for about 1 to about 6 hours, or in another embodiment, between about 1 and 2 hours, or in another embodiment, between about 2 and 3 hours, or in another embodiment, between about 3 and 4 hours, or in another embodiment, between about 4 and 5 hours, or in another embodiment, between about 5 and 6 hours.

[00015] Caspase-3 and -7 are members of the cysteine aspartic acid-specific protease (caspase) family, which play an effector roles in apoptosis in mammalian cells. In one embodiment, DEVD- aminoluciferin refers to a proluminescenl caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent form optimized for caspase activity, luciferase activity and cell lysis. The results of cell lysis due to programmed cell death in another embodiment, is followed by caspase cleavage of the substrate and generation of a luminescent signal, produced by luciferase. In one embodiment, luminescence is proportional to the amount of caspase activity present and therefore to the extent of programmed cell death.

[00016] In one embodiment, aminoluciferin represents a leaving group. The liberated aminoluciferin can be luminometrically detected even in smallest concentrations, in one embodiment, the liberated aminoluciferin is reacted with the enzyme luciferase of the firefly Photinus pyralis or of the firefly Photinus plathiophthalamus or of the luciferase of other species or chemically or genetically modified luciferases in the presence of ATP+MgCk. In the course of said reactions photons are emitted; i.e. in the course of the reaction with the enzyme of the firefly Photinus pyralis at 605 nm in one embodiment and in the course of the reaction with the enzyme of the firefly Photinus plathiophthalamus at 549 or 570 nm in another embodiment, or wavelength corresponding to the used luciferin/luciferase system, respectively. The emission at 549 nm takes place if the enzyme originates from the dorsal organ of the firefly mentioned whereas the emission at 570 nm takes place if the enzyme originates from the ventral organ.

[00017] A luciferase is an enzyme that catalyzes a reaction to produce light. There are a number of different luciferase enzymes derived or modified from various sources, including firefly luciferase in one embodiment, and Renilla luciferase in another embodiment. "Renilla luciferase" refers to a luciferase enzyme isolated from a member of the genus Renilla or an equivalent molecule obtained from any other source or synthetically.

[00018] In one embodiment, the apoptosis inducing agent is TRAIL, referring to a membrane-bound cytokine molecule that belongs to the family of tumor necrosis factor (TNF). In one embodiment, TRAIL binds with five different receptor molecules, such as DR4, DR5, DcRl , DcR2, and osteoprotegerin (OPG). These receptor molecules, members of the TNF receptor (TNF-R) family, are type I transmembrane polypeptides with 2-5 cysteine-rich domains (CRD) at the extracellular region. DR4 and DR5 containing a cytoplasmic death domain, that is essential for death signaling, are able to transmit apoptosis-inducing activity of TRAIL across the cell membrane.

LOOOl 9] Four homologous, distinct, human TRAIL receptors exist in one embodiment. In another embodiment two TRAIL-Rl TR AIL-R2 having the ability to initiate the apoptosis signaling cascade after ligation and in another embodiment, two others; TRAIL-R3 and TRAIL-R4 lacking the ability to initiate apoptosis signaling cascade after ligation. TRAIL-R3 and TRAIL-R4 have are protective receptors in one embodiment, either by acting as "decoy" receptors or via transduction of an anti- apoptotic signal.

[00020] The participation of TRAIL-R3 and-R4 in regulating TRAIL sensitivity may be greater, in one embodiment, in normal cells/tissues or primary tumors than in established tumor cell lines. In another embodiment TRAIL-R3 is a key regulator of the sensitivity of normal cells to TRAIL-induced death, but the addition of cycloheximide may inhibit the production of some other protein (such as FLIP in one embodiment) critical for TRAIL resistance. In one embodiment TRAIL-R3 and/or -R4 are used with the methods and kits of the invention to obtain more information about the death-inducing agents used.

[00021] In one embodiment, the term "cell death" includes the processes by which mammalian cells die. Such processes include apoptosis (both reversible and irreversible) and processes thought to involve apoptosis (e.g., cell senescence), as well as necrosis. "Cell death" is used in one embodiment to refer to the death or imminent death of nucleated cells (e.g., neurons, myocytes, hepatocytes and the like) as well as to the death or imminent death of anucleate cells (e.g., red blood cells, platelets, and the like). Cell death is typically manifested by the exposure of PS on the outer leaflet of the plasma membrane. Apoptosis refers in one embodiment to "programmed cell death" whereby the cell executes a "cell suicide" program. In another embodiment, the apoptosis program is evolutionarily conserved among virtually all multicellular organisms, as well as among all the cells in a particular organism. Further, it is believed that in many cases, apoptosis may be a "default" program that must be actively inhibited in healthy surviving cells. All apoptosis pathways appear to converge at a common effector pathway leading to proteolysis of key proteins. Caspases are involved in both the effector phase of the signaling pathway and further upstream at its initiation. The upstream caspases involved in initiation events become activated and in turn activate other caspases that are involved in the later phases of apoptosis.

[00022] In order to monitor cancer therapeutic effects, a bioluminescent imaging technique that measures caspase activity in one embodiment, was measured, since activation of the caspase cascade is an integral event in the apoptotic pathway. When in one embodiment, the proluminescent DEVD-aminoluciferin powder is used with lysis buffer (Caspase-Glo 3/7 reagent, Promega) in HCTl 16, p53(-/-), pGL-2 cells (stable firefly luciferase gene), strong luminescence signals were captured from 6h TRATL-treated cells. In another embodiment, cells treated with TRAIL and 20 μM Z-IETD-FMK (caspase 8 inhibitor) show uniformly low luminescence signals that do not vary across different TRAIL concentrations and are comparable to those signals obtained from control cells, because caspase 8 is required for TRAIL- induced apoptosis (Seol et al., 2001). [00023] In one embodiment, alternate preparations of DEVD-aminoluciferin reagent and their effects on cells are used in the methods and kits of the invention, in order to develop a minimally invasive apoptosis detection technique. In one embodiment, dissolving the lyophilized substrate in Dulbecco's PBS ('powder in PBS') produces a reagent that is the least harmful to the cells. In one embodiment, upon addition of the lysis buffer, cells shrunk together rapidly and could not be recovered, while in another embodiment, cells incubated with 'powder in PBS' for Ih remained healthy by morphological examination under phase contrast microscopy and by apoptosis assessment through flow cytometry. Luminescence intensities produced by 'powder in PBS' are significantly lower in one embodiment, than those produced by the regular powder + buffer reagent, but are still 2.5-fold stronger than luminescence signals produced by D-luciferin at a concentration of 100μg/mL.

[00024] In one embodiment, the methods and kits of the invention utilize a DEVD-aminoluciferin powder whereby the powder is heated to above the denaturation temperature of a present luciferase; followed by cooling of the powder to below 37.3 ⁰C prior to the step of incubating said nucleated cell with said proluminescence reagent. This is done in one embodiment to ensure the accurate quantification of the measured luminescent intensity, by ensuring that no luciferase has entered the reaction mix with the proluminescence reagent.

[00025] In one embodiment, the proluminscent reagent used in the kits and methods of the invention is amnoluciferin operably linked to a protease substrate, which in another embodiment is caspase-7. Caspase enzymes recognize a 4 amino acid sequence (on their substrate) which includes an aspartic acid as the fourth amino acid. The cleavage reaction occurs in one embodiment at the carbonyl end of the aspartic acid residue.

[00026] In one embodiment, aminoluciderin is operably linked to DEVD (benzyloxyycarboπyl aspartyl glutamylvalylaspartic acid fluoromethyl ketone, SEQ ID NO. 1), VEHD (benzyloxyycarbonyl valyl glutamyl histidylaspartic acid fluromethyl ketone, SEQ ID NO. 2), LETD (benzyloxycarbonyl leucylglutamylthreonylaspartic acid fluoromethyl ketone, SEQ ID NO. 3), LEHD (benzyloxycarbonyl leucylglutamylhistidylaspartic acid fluoromethyl ketone, SEQ ID NO. 4), IEPD (benzyloxycarbonyl lsoleucylglutamylprolylaspartic acid fluoromethyl ketone, SEQ ID NO. 5), DETD (benzyloxycarbonyl aspartylglutamylthreonylaspartic acid fluoromethyl ketone, SEQ ID NO. 6), WEHD (tryptophyl glutamylhistidylaspartic acid fluromethyl ketone, SEQ ID NO. 7), YVAD (benzyloxycarbonyl tyrosylvalylalanyl aspartic acid fluoromethyl ketone, SEQ ID NO. 8), VEID (benzyloxycarbonyl valylglutamyl isoleucylaspartic acid fluoromethyl ketone, SEQ ID NO. 9). "Operatively linked" refers in one embodiment to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. In one embodiment aminoluciderin is "operably linked" to DEVD, acting as a substrate for caspase-7, which is involved in apoptosis in one embodiment and whose action results in the release of aminoluciferin from DEVD, thereby making it accessible to react with luciferase.

[00027] In one embodiment, the luminescence activator reagent used in the methods and compositions of the invention comprises a luciferase. In another embodiment, the DEVD-aminoluciferin powder is dissolved in Dulbecco's PBS.

[O0O28J In one embodiment, the cells are incubated in the presence of the apoptosis-inducing agent, which in another embodiment is TRAIL, for a period of up to 6 hours, or in another embodiment, between 1 and about 2 hours, or in another embodiment, between 2 and about 3 hours, or in another embodiment, between 3 and about 4 hours, or in another embodiment, between 4 and about 5 hours, or in another embodiment, between 5 and about 6 hours.

[00029] In one embodiment, the cells of the invention, prior to or in conjunction with the methods of the invention, are contacted with a caspase inhibitor, which in another embodiment is F⁷LICA, such as FITC- VAD-FMK.

[00030] In one embodiment, the invention provides a method of evaluating the efficiency of a chemotherapeutic agent in a subject, comprising obtaining a tumor cell from said subject, wherein said tumor is the one treated with said chemotherapeutic agent, contacting said tumor cell with a candidate chemotherapeutic agent, incubating said chemotherapy treated tumor cell with a proluminescence reagent; and measuring luminescence intensity, wherein the higher the measured luminescent intensity, the more efficient is the chemotherapeutic agent.

[0003 IJ The term "subject" refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects. [00032] In one embodiment, the term "Efficiency" of a chemotherapeutic agent refers to the relationship between a minimum effective dose and an extent of toxic side effects. Efficiency of an agent is increased if a therapeutic end point can be achieved by administration of a lower dose or a shorter dosage regimen. If toxicity can be decreased, a therapeutic agent can be administered on a longer dosage regimen or even chronically with greater patient compliance and improved quality of life. Further, decreased toxicity of an agent enables the practitioner to increase the dosage to achieve the therapeutic endpoint sooner, or to achieve a higher therapeutic endpoint.

[00033] In another embodiment, the chemotherapeutic agent whose efficiency is capable of being evaluated according to the methods and kits of the invention is methotrexate, or 5-fIuorouracil, doxorubicin, daunorubicin, mitomycin, actinomycin D, bleomycin, plicomycin, taxol, vincristine, vinbiastine, cisplatin, VPl 6, carmustine, meiphalan, cyclophosphamide, chlorambucil, busulfan, lomustine, carboplatin, procarbazine, mechiorethamine, camptothecin, ifosfamide, nitrosurea, tamoxifen, raloxifene, estrogen receptor binding agents, gemcitabien, navelbine, farnesyl -protein transferase inhibitors, transpiatinum or temazolomide, or a combination thereof in other embodiments.

[00034] In one embodiment, the candidate chemotherapeutic agent is unknown and need to be assessed for its properties as a chemotherapeutic agent. When the chemotherapeutic agent's properties are unknown, the methods and kits of the invention may further include comparison standards, such as a known concentration of TRAIL and following the incubation of the proluminscent reagent in the presence of the unknown chemotherapeutic agent and identical incubation in the presence of TRAIL for the same time, temperature and concentration of reactants, the luminescence intensity of the unknown chemotherapeutic candidate agent is compared with that of TRAIL incubated cells.

[00035] In another embodiment, the unknown chemotherapeutic agent sought to be evaluated is as a candidate for a specific tumor type, or cancer type in another embodiment, or a diffuse tumor in another embodiment, or a solid tumor in another embodiment. In one embodiment the comparison between the unknown chemotherapeutic candidate agent and the known control standard, is with a control standard proven as effective for the specific cancer type.

[00036] In one embodiment, the kits of the invention are used to carry out the embodiments of the methods described herein. [00037] In one embodiment, the invention provides a kit for imaging an agent-induced apoptosis in-vivo within a region of a mammalian subject, comprising: DEVD-aminoluciferin powder and a suspension media. In one embodiment, the suspension media is Dulbecco's PBS, or lysis buffer. In another embodiment, the agent-induced apoptosis is TRAIL induced apoptosis. In one embodiment, the suspension media used in the kits of the invention is Dulbecco's PBS, or lysis buffer in another embodiment.

[00038] In one embodiment, the kits of the invention, which in another embodiment are used to carry out the methods of the invention, further comprise a standard for comparison of the luminescence intensity obtained from the agent induced apoptosis, to that obtained with a known apoptosis inducing agent, such as TRAIL in another embodiment.

[00039] In one embodiment, the results obtained are compared to a standard, which, in another embodiment, may comprise a series of standards, which, in another embodiment is used in the kits of the invention for quantification of differential expression. In one embodiment, the standard may comprise any embodiment listed herein, or in another embodiment, will be suitable for a particular application of the kit

[00040] In one embodiment, the kits of the invention may further comprises a positive or negative standards, wherein the standard can be assayed and compared to the test sample. It is to be understood that the kits of the invention may be modified and marketed for particular use, which in one embodiment are tumor-specific, or cancer specific or apoptosis-induciπg agent specific in other embodiments.

[00041] In one embodiment, the kit of the invention may further comprise a software package contained on a computer storage medium, with a program for correlating values obtained with a standard, for storing and comparing, by date, or in another embodiment for extrapolating results obtained, for the purpose of determining efficacy of the apoptosis-inducing agent in one embodiment, or determining course of action in therapy, making diagnosis, making prognosis, diagnosing or a combination thereof in other embodiments.

[00042] In one embodiment, the cells used for the methods of the invention are obtained from a biological sample given by the subject. The sample to be analyzed may consist in one embodiment of, or comprise blood, sera, urine, mucosa, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample or chorionic villi, and the like. A biological sample may be processed in another embodiment to release or otherwise make available a nucleic acid or a protein for detection or as a reactant as described herein. Such processing may include in one embodiment steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of RNA from the biological sample. Thus, the nucleic acid to be amplified in one embodiment by the methods of the invention may be DNA or RNA.

[00043] Cytotoxic materials which can be analyzed according to the methods and kits of the invention, induce cellular apoptosis in a variety of manners according to type. Since such variously induced apoptosis eventually induces the activation of effector caspase, like caspase-3 in one embodiment, the various types of apoptosis signals are gathered by the activation of one or two caspases. In another embodiment, caspase-3 has a broad range of intracellular protein substrates, or in another embodiment other substrates such as DEVD-aminoluciferin.

[000441 In another embodiment, contacting the cell with the methods and kits of the invention, comprises amplifying the expression of a compound associated with apoptosis or cell-cycle arrest. In another embodiment, the gene amplified is that encoding caspase-3, or in another embodiment caspase-7 or both in another embodiment. In one embodiment, the term "amplification" or "amplify" refers to one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential in one embodiment, or linear in another. In one embodiment, a target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an "amplicon." While the exemplary embodiments described herein relate to amplification using the polymerase chain reaction ("PCR"), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.) and are considered within the scope of the present invention. The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990, pp 13- 20; Wharam et al., Nucleic Acids Res. 2001 June 1 ;29(11):E54-E54; Hafner et al., Biotechniques 2001 Apr;30(4):852-6, 858, 860 passim; Zhong et al., Biotechniques 2001 Apr;30(4):852-6, 858, 860.

100045] In another embodiment, real time PCR is used in the methods and kits of the invention. The term "real time PCR" refers in one embodiment to the process where a signal emitted from the PCR assay is monitored during the reaction as an indicator of amplicon production during each PCR amplification cycle (i.e., in "real time"), as opposed to conventional PCR methods, in which an assay signal is detected at the endpoint of the PCR reaction. Real time PCR is based in one embodiment on the detection and quantitation of a fluorescent reporter. The signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. For a general description of "real time PCR" see Dehe et al. J. Virol. Meth. 102:37-51 (2002); and Aldea et al. J. Clin. Microbiol. 40:1060- 1062 (2002) (referring to the "LightCycler," where real-time, kinetic quantification allows measurements to be made during the log-linear phase of a PCR).

[00046] The cellular pathway leading to apoptosis involves the activation of members of a family of protease, caspases. To date, fourteen members of the caspase family have been identified in vertebrates, and at least eight are known to be involved in apoptotic cell death (Saunders, et al., 2000, Anal.

Biochem., 284, 114-124)^ Among the various apoptosis-related caspases, extensive research has been performed on caspase-3, because caspase-3 has a broad range of intracellular protein substrates (Han, et al., 1997, J. Biol. Chem., 272, 13432-13436). In one embodiment, significant elevation in caspase-3 activity preceded the apparent physiological apoptotic progress induced by sodium butyrate and that the overexpression of Bcl-2 inhibited the NaBu-induced apoptosis by suppressing the activation of caspase-

3 (Kim and Lee, 2000, Biotechnol. Bioeng., 71, 184-193).

f00047] In the methods and kits according to embodiments of the present invention, data relating to values obtained for the parameters for malignant and non-malignant samples analyzed may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file. The database or data file may contain in one embodiment, a patient identifier such as a name or number, the values obtained, patient prognosis, age of onset of symptoms, therapy regimen, and other identifying and relevant characteristics, as will be understood by one skilled in the art. The database may contain, in other embodiments, the change in any of the parameters analyzed, as a function of time, or chemotherapy regimen, or a combination thereof. In one embodiment, the methods and kits of this invention may further comprise accessing a memory, or a means thereto for storing the obtained values for the parameters, and other data as described herein. In another embodiment, the methods of this invention may further comprise generating and graphically displaying the values obtained. In one embodiment, the analysis is executed by a processor or a virtual computer program.

[00048] In one embodiment the software incorporates statistical tools for determining the significance of the findings. Statistical significance is determined, in other embodiments, by conducting pairwise comparisons, and determining a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. In one embodiment, a p value of 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, 0.0001 , or less is indicative of a significant difference.

[00049] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

[00050] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Materials and Methods: Tumor cell line [00051 J HCTl 16 cells (human colon adenocarcinoma) were from American Type Culture Collection (ATCC, Manassas, VA) and grew in McCoy's 5A medium (Invitrogen Corporation, Carlsbad, CA) with 10% FBS and 1 % penicillin-streptomycin in a 37°C, 5%CO₂ incubator. HCTl 16, p53(-/-) cells stably transfected with a Firefly luciferase gene through the pGL-2 construct were grown under the same conditions.

Apoptosis induction

[00052] Apoptosis was induced by incubating cells in fresh medium containing different His-tagged TRAIL concentrations (0, 10, 50, lOOng/mL). For control experiments with caspase-8 inhibitor, Z- IETD-FMK (R&D Systems, Minneapolis, MN) was added to a final concentration of 20μM.

Bioluminescence imaging

[00053] After 6h apoptosis induction, Caspase-Glo 3/7 reagent (Promega Corporation, Madison, WI) or D-luciferin (dissolved in PBS) at a final concentration of lOOμg/mL was added in a 1 :1 volume ratio. Blank controls of reagent and McCoy's 5A medium (with 10%FBS and 1 % P/S) were always included. Immediately after the addition and l-4h after addition, cells were placed into the dark chamber of the Xeπogeπ IVIS imaging system (Xenogen Corporation, Alameda, CA), where bioluminescence images were acquired with the charge-coupled device (CCD) camera. Luminescence intensity was quantified using the Living Image software (version 2.5) from Xenogen. Caspase-Glo 3/7 preparations

[000541 The Caspase-Glo 3/7 (Promega Corporation, Madison, WI) lyophilized substrate (DEVD- aminoluciferin powder) was dissolved in Caspase-Glo 3/7 buffer (lysis buffer) as recommended by Promega, or, alternatively, was dissolved in an equal volume of Dulbecco's PBS. In order to observe the effects of DEVD-aminoluciferin alone and inactivate luciferases saturated in the DEVD-aminoluciferin powder, DEVD-aminoluciferin powder dissolved in PBS was heated at 85°C for 20 min, and then cooled in a 37°C water bath for 20 min before addition to cells (to prevent heat damage to cells). Experiments with the Caspase-Glo 3/7 buffer alone and with PBS alone (control) were also carried out.

Cell density

[00055] For bioluminescent imaging, cells were plated at 20,000 cells/well in 200μl growth medium per well in a black 96-well plate with clear bottom (Corning Incorporated, Corning, NY), or at 50,000- 150,000 cells/well in 2mL medium per well in a clear 24-well plate the day before experimentation. For FLICA assays, cells were plated at 0.8-lxlO⁶ cells/well in 2mL medium per well in a clear 6-well plate the day before experimentation. For sub-Gi analysis, cells were plated at 0.5xl0⁶ cells/well in 2mL medium per well in a clear 6-well plate 2 days before experimentation.

Phase contrast microscopy

[00056] Cells were viewed under phase contrast at 32x magnification of a Zeiss Axiovert 100 microscope (Carl Zeiss, Inc., Thornwood, NY). Images were captured using the Scion Image 1.62 software (Scion Coφoration, Frederick, MD).

Propidium iodide staining

[00057] In separate experiments, TRAIL-treated cells and control cells were stained with propidium iodide for sub-Gi analysis by flow cytometry. Cells harvested at the indicated time points were fixed in 95% cold ethanol, stained with 50μg/mL propidium iodide and RNase A for 30min at room temperature, and then analyzed using an Epics Elite (Beckman Coulter, Miami, FL) flow cytometer.

FLICA (Fluorescent Inhibitor ofCaspase Activity) assay [00058] In separate experiments, the percentage of TRAIL-treated and control cells undergoing apoptosis was determined by using FLICA assays. Cells harvested at the indicated time points were resuspended in lOμM FITC-VAD-FMK (Promega Corporation, Madison, WI), which irreversibly binds to caspases 1 , 3, 4, 5, 7 and 8, for 20 min in a 37°C, 5% CO₂ incubator, and then analyzed by flow cytometry (Beckman Coulter Epics Elite, Miami, FL) without fixation. Isolation of Primary Colonic Explants (PCE's)

[00059] Parts of the large intestine from a 16-year old female APC-patient who underwent prophylactic colectomy was obtained following informed patient consent and approval from the Institutional Review Boards at both the Children's Hospital of Philadelphia and the University of Pennsylvania. The colon was cut open, the mucosa was separated from the serosa and placed in Base Medium consisting of four parts MCDB 201 and one part Ll 5 (Sigma), supplemented with 2 ng/ml human recombinant EGF (Sigma), 5 μg/ml insulin (Sigma), 5 μg/ml transferrin, 50 μg/ml streptomycin and gentamycin and 2% FCS.18 Tissues were further dissected into 3-7 mm slices, cultured in 100 mm dishes containing DMEM-10 consisting of Dulbecco's Minimum Essential Medium (Invitrogen), 10 % FBS and 1% Penicillin/Streptomycin/Fungizone, and incubated in a humidified chamber at 37°C and 5% CO2. The colonic tissue isolated for these studies was from non-polypoid mucosa. The colon had small polyps that were not isolated.

In vitro Treatments

[00060] Healthy colonic explants were treated with 200 μg/ml of 5-Fluorouracil (American

Pharmaceutical Partners Inc. IL), 100 μg/ml of CPT-I l (Camptosar®, Pharmacia Upjohn, MI) or 100 ng/ml of recombinant murine TRAIL (BioMol, CA) or a combination of 5-Fluorouracil or CPT-11 plus TRAIL for 18 - 48 hours. Recombinant murine TRAIL has been shown to kill Jurkat cells effectively.19 Experiments in our laboratory have also shown that recombinant murine TRAIL effectively kills HCTl 16, a human colon cancer cell line that has been shown in several studies to be sensitive to recombinant human TRAIL.

Apoptosis Assays (I) Anncxin V-staining

[00061] PCE's were transferred to Binding Buffer (1OmM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCh, 1 mM MgCh 5 mM KCl) and 1 μl of Annexin V-EGFP (BioVision, CA), and 25 μg/ml (final concentration) of PI was added to each sample. The colonic explants were washed in PBS and transferred to a glass slide, covered with a glass cover slip and analyzed using an Axiovert 100 inverted fluorescent microscope (Zeiss).

(II) Fluorochrome-Labeled Inhibitor of Caspase Activity (FLICA)- assay In Situ

[000621 The PCE's were incubated with lOμM of FITC-VAD-FMK (CaspACE™, Promega, WI) and 25 μg/ml of propidium iodide for 20 minutes at 37°C protected from light. This was followed by washing in PBS and then transfer to glass slides, cover slip placement and analysis using an Axiovert 100 inverted fluorescence microscope (Zeiss).

Example 1: Bioluminescence imaging of TRAIL-induced apoptosis

[00063] In order to image TRAIL-induced apoptosis, activation of caspases 3 and 7 were measured in HCTl 16, p53(-/-), pGL-2 (firefly luciferase gene) cells by adding DEVD-aminoluciferin reagent (dissolved in lysis buffer) to cells treated with 0-lOOng/mL TRAIL for 6h. Luminescence images were acquired with the Xenogen IVIS imaging system after 1-4 hours of incubation with the reagent, and luminescence intensities were measured as photons and plotted as average values (experiments performed in duplicates). Luminescence intensity increased with increasing TRAIL concentrations (Fig. 2), reflecting increasing apoptosis levels. However, luminescence intensity for TRAIL-treated cells incubated with 20μM Z-IETD-FMK (caspase 8 inhibitor) did not vary across the different TRAIL concentrations and was similar to that for control cells, since caspase 8 is required for TRAIL-induced apoptosis (Seol et al., 2001). For comparison, a living-cells FLICA (fluorescent inhibitor of caspase activity) assay was performed on HCTl 16, p53(-/-), pGL-2 cells after 7h TRAIL treatment. The percentage of apoptotic cells was determined by flow cytometry (Fig. 3).

Example 2; Luminescence signals stable for several hours, but weaker dose-dependence over time for higher TRAIL concentrations

[00054] Luminescence intensity, measured in photon units, remained strong and stable over several hours (up to 4h after reagent addition; Fig. 2A-E). However, the signals' dose-dependence decreased over time for cells treated with higher TRAIL concentrations (50 and lOOng/mL). Luminescence intensity for cells treated with TRAIL plus 20 μM Z-IETD-FMK (caspase 8 inhibitor) did not vary across the different TRAIL concentration; the signal strength was significantly lower than that for TRAIL-treated cells (4-10 fold), and was comparable to that for control cells for several hours after addition of DEVD- aminoluciferin (in lysis buffer), the apoptosis imaging reagent (Fig. 2A-D). After a long incubation period with this reagent, the signal strength for cells treated with only TRAIL decreased significantly by about ten-fold (Fig. 2E).

Example 3: Alternate preparations of proluminescent reagent for noninvasive imaging [00065] To test bioluminescence intensity for modified preparations of the DEVD-aminoluciferin reagent, and to test the reagent's effects on cells, the lyophilized substrate ('DEVD-aminoluciferin powder') was dissolved in Dulbecco's PBS instead of the lysis buffer (Promega). Immediately after addition (Fig. 4A), the DEVD-aminoluciferin powder dissolved in PBS only produced a weak, TRAIL- dependent bioluminescence signal in HCTI 16 cells, weaker than signals from DEVD-aminoluciferin dissolved in lysis buffer (Caspase-Glo 3/7 reagent, Promega) and from the buffer alone. After Ih incubation (Fig. 4B), signals from the 'powder in PBS' grew stronger and exceeded those from the buffer alone. Luminescence signals from PBS alone remained stable and were the lowest at both times of imaging, as expected for this control experiment.

[00066] Bioluminescence for all three preparations of the DEVD-aminoluciferin reagent (powder in lysis buffer, buffer alone, powder in PBS) were at least 2.5-fold stronger for TRAIL-treated cells (50 ng/mL) compared to their corresponding control cells (0 ng/mL), reflecting TRAIL-induced apoptosis that has been imaged through the reagents' prolumincsccnt substrates. Morphologically, the 'powder in PBS' was least harmful to cells: Although cells started to round up Ih after addition, they were still attached to the plate and appeared relatively healthy; on the other hand, cells incubated with 'powder + buffer' or 'buffer' alone rapidly shrunk together and could not be recovered after Ih incubation (Fig. 5 A-C). No morphological changes were observed in cells to which only PBS was added (Fig. 5D). For comparison with other apoptosis assays, a living-cells FLICA (fluorescent inhibitor of caspase activity) assay and sub-Gi analysis through propidium iodide staining were performed on HCTl 16 cells after 6h TRAIL treatment. The percentage of apoptotic cells was determined by flow cytometry (Fig. 5E-F).

[00067] Similar bioluminescent imaging results were obtained for the same experiment using HCTl 16, p53(-/-), pGL-2 (firefly luciferase gene) cells plated at half the density (50,000 cells/well with 500μl media/well in a clear 24- well plate) (Fig. 6).

Example 4: Stronger luminescence signal by DEVD-aminoluciferin than with (100ug/mL) D- luciferin

[000681 In separate experiments, HCTl 16, p53(-/-), pGL-2 (firefly luciferase gene) cells were treated with 0 or 50 ng/mL TRAIL for 7h, at which time DEVD-aminoluciferin powder dissolved in PBS was added to the first row of cells, and D-luciferin diluted in PBS was added to the second row at a final concentration of 100 μg/mL. While all signal intensities increased after an hour of incubation with the reagents (Fig. 7A), the largest increase was observed in 'powder in PBS' wells (2.6-fold increase for TRAIL-treated cells, 2.2-fold increase for control cells; for D-luciferin addition, 1.75-fold increase for TRAIL-treated cells, 1.2-fold increase for control cells). The strongest signal was also produced by the 'powder in PBS' reagent in TRAIL-treated cells after Ih incubation (2.5-fold stronger signal than control cells under same conditions, reflecting TRAIL-induced apoptosis), 2.5-fold stronger than the strongest D-luciferin signal.

[00069] As a life-death exclusion assay, addition of 100 μg/mL D-luciferin produced an approximately 2- fold stronger bioluminescence signal for untreated versus TRAIL-treated cells at its highest intensities (after I h incubation). As expected, addition of D-luciferin did not harm cells (Fig. 7B): After the medium was replaced at the 7h+l timepoint, phase contrast images captured the following day showed healthy, confluent control cells for D-luciferin addition. Furthermore, phase contrast images (Fig. 7B) showed that, for a 1 : 1 'powder in PBS' addition (by volume), most control cells were recovered when the medium was replaced Ih after addition, even though the cells started to round up and gradually detach upon reagent addition (also see above under "alternate preparations of proluminescent reagent for noninvasive imaging").

[00070] Similar results were obtained when the cell density was varied moderately. Bioluminescence intensity increased slightly with increasing cell density: 1.2-fold stronger maximum intensity with 'powder in PBS' addition, 2.2-fold stronger maximum intensity with D-luciferin addition for a clear 24- well plate seeded with 150,000 versus 50,000 HCTl 16, p53(-/-), pGL-2 cells/well (maximum intensities observed at 7h+l timepoint for untreated 'powder in PBS' cells and TRAIL-treated D-luciferin cells) (Fig. 8).

Example 4; Minimal invasiveness of DEVD-aminoluciferin powder tested via FLICA assay and heat inactivation

[00071] Flow cytometric analysis through a FLICA (fluorescent inhibitor of caspase activity) assay was performed on HCTl 16, p53(-/-), pGL-2 cells incubated with DEVD-aminoluciferin (powder dissolved in PBS) for Ih to determine the reagent's harmfulness to cells. After 6h incubation with 0 or 50 ng/mL TRAIL, cells were incubated for another hour with DEVD-aminoluciferin powder dissolved in PBS or with D-luciferin diluted in PBS (final concentration: lOOμg/mL); a flow-cytometry based FLICA assay was then performed on those cells. The measured percentage of apoptotic cells after incubation with the proluminescent DEVD-aminoluciferin powder was on average only 1.3-fold higher than that for cells incubated with D-luciferin containing PBS (Fig. 9). [00072] In order to observe the effects of DEVD-aminoluciferin alone and inactivate luciferases saturated in the DEVD-aminoluciferin powder, DEVD-aminoluciferin powder dissolved in PBS was heated at 85°C for 20min, and then cooled in a 37°C water bath for 20 min before addition to cells (to prevent heat damage to cells). The bioluminescence signal captured in TRAIL-treated cells was much stronger in cells transfected with the firefly luciferase gene (pGL-2) than in normal HCTl 16 cells (Fig. 10), suggesting that most of the luciferases provided in the DEVD-aminoluciferin powder were heat- inactivated, and that DEVD-aminoluciferin alone was a good apoptosis imaging reagent in luciferase- expressing cells. Cells did not have to be lysed for extra-cellular luciferases to interact with DEVD- aminoluciferin cleaved by activated caspases 3 and 7, and thus DEVD-aminoluciferin can serve as a sensitive, noninvasive reagent for bioluminescent imaging of TRAIL-induced apoptosis through detection of activated caspases 3 and 7.

Example 5: Viability assessment of colon explants

[00073] To investigate the role of cell death in viable primary colonic tissues following treatment with established chemotherapy (CPT-I l, 5-Fluorouracil) and the cytokine TRAIL, colonic explants were isolated from a surgically resected colon from a juvenile APC-patient and cultured them in vitro.

[00074] Viability of the isolated explants was determined by the amount of PI that was taken up by the explants upon addition to the medium (see figure 11). PI up-take in colonic explants increased over time following isolation of the explants, suggesting that viability decreased with increased culturing time.

Due to low viability, the colonic explants were cultured for no more than 6 days. At early time points (4 hours, see Figure 1 1 ), only parts of the explants stained for PI suggesting that the reminder of the colonic explants, at least partially, contained viable cells. Thus, in order to minimize background death, experiments were limited to the first 3 days following isolation of the explants.

[00075] Figure 1 1 shows PI added to the medium of the colonic explants at different time-points following isolation. Increased staining intensity (red) was observed over time following isolation.

Example 6: Treatment of colon cell lines with 5-Fluorouracil (5-FU) and/or TRAIL

[00076] 5-FU has been shown to kill several human colon cell lines at doses that are significantly lower than the dose used herein, whereas primary human hepatocytes are resistant to doses of 5-FU up to 200 ug/ml. TRAIL has been shown to kill primary human hepatocytes and primary human esophageal cells at doses as low as 10 ng/ml . However, the TRAIL-induced cell death observed in primary hepatocytes could be related to the preparation of the cells, as toxicity has not been replicated by some investigators even in the presence of proteasomal inhibitors.

[00077] The colonic explants were treated for 24 and 48 hours with 200 μg/ml of 5-FU. At 48 hours, a large number of treated cells labeled positive using the FLICA-assay (HTC-VAD- FMK) (Figure 12A). The high levels of cell death in the tissue at this time point was suspected as an event that could overshadow a potential additive effect of 5-FU and TRAIL. At 24 hours following treatment with 5-FU alone, there was a noticeable elevation of FLICA positive cells in the tissue (Figure 12B). TRAIL alone did not result in a significant increase of FLICA -positive cells, and the combination of 5-FU and TRAIL did not give an increase in the numbers of FLICA-positive cells relative to 5-FU treatment alone. PI uptake was increased following treatment with 5-FU alone for 24 hours whereas no increase was observed when the colonic explants were treated with TRAIL alone for 18 hours (Figure 12B). Combination treatment with 5-FU and TRAIL only very modestly increased the PI-positivity in the tissue.

[00078] Although it was possible to document cell death by FLICA/PI, which cell type in the explants is primarily affected by treatment with CPT-11, 5-FU and TRAIL could not be determined. Upon resection of the colon, only the mucosa was isolated from the organ. Thus, considering the frequency of cell death, this event involves primarily the colonic mucosa.

[00079] Cells that undergo apoptosis translocate phosphatidylserine groups from the inner monolayer to the outer monolayer of their plasma membrane. Phosphatidylserine groups can be detected using fluorochrome-conjugated annexin V, a molecule that binds phosphatidylserine groups with high affinity. Adding EGFP-conjugated annexin to colonic explants treated with 5-FU and TRAIL in an identical manner as described above did not convincingly show specific labeling of apoptotic cells (Figure 13). Thus it seems that this method may not be optimal to detect cells undergoing apoptosis in colonic explants following treatment with 5-FU and/or TRAIL. Therefore, 5-FU can trigger cell death and caspase activation within 24-48 hours in primary colonic explants whereas no TRAIL-induced apoptosis could be detected at 18 hours. The combination 5-FU and TRAIL does not enhance 5-FU mediated caspase activation as assessed by FLICA and only modestly enhances PI-positivity. Thus, in combination with 5-FU, TRAIL may trigger cell death independently of caspase activation.

Example 7: Treatment with CPT-Il and TRAIL [00080] Colonic explants were treated with CPT-11, a member of the family of topotecans and an inducer of lethal DNA-strand breaks. Colonic explants were treated for 36 hours with CPT-I l (100 μg/ml) and 30 hours with TRAIL (100 ng/ml) or a combination of CPT-I l and TRAIL, following 6 hours of pretreatment with CPT-I l. The pro-apoptotic response was monitored using the FLICA-assay. An increase in FLICA labeling and Pi-staining was observed following treatment with CPT-11 for 36 hours as compared to the control (Figure 14). Interestingly, increased labeling by FLICA and PI of the colonic explanted tissues was observed following 30 hours of incubation with TRAIL (figure 14), which stands in sharp contrast to the results obtained when the colonic explants were treated with TRAIL for 18 hours (figure 12). Also, the combination of both CPT-11 and TRAIL yields a more frequent labeling by both the FLICA-assay and PI as compared to either TRAIL or CPT-11 alone (figure 14).

[00081] Thus, exposure of primary human colonic epithelia for 36 hours to CPT-11 (100 ng/ml) yield a readily detectable apoptotic response as measured by FLICA and PI assays. This is also the case for 30 hours of TRAIL treatment. An additive effect of TRAIL and CPT- 11 is also observed.

[00082] The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

What is claimed is:

1. A method of imaging agent-induced apoptosis in-vivo in a cell, comprising: contacting said cell S with a proluminescence reagent; incubating said cell with said proluminescence reagent; contacting said cells with an apoptosis inducing agent; and measuring luminescence intensities, wherein an increased luminescent intensity measured is correlative with an increased cell death.

2. The method of claim 1 , wherein said proluminescence reagent comprises a DEVD-0 aminoluciferin powder.

3. The method of claim 1, wherein said apoptosis inducing agent is TRAIL.

4. The method of claim 1 , wherein the apoptosis inducing agent is unknown and is suspected ass being an apoptosis inducing agent.

5. The method of claim 1 , wherein incubating said nucleated cell with said proluminescence reagent is done for about 1 to about 6 hours. 0

6. The method of claim 4, wherein incubating said nucleated cell with said proluminescence reagent is done for about 1 hour.

7. The method of claim 1 , wherein said luminescence activator reagent comprises a luciferase. 5 8. The method of claim 2, wherein said DEVD-aminoluciferin powder is dissolved in Dulbecco's

PBS.

9. The method of claim 2, wherein said DEVD-aminoluciferin powder is dissolved in a lysis buffer. 0

10. The method of claim 1, wherein incubating said TRAIL-contacted nucleated cells, is done for 0 to about 6 hours.

11. The method of claim 2, further comprising the steps of heating said powder to above the deπaturation temperature of a present lucif erase; and cooling said powder to below 37.3 ⁰C prior to the step of incubating said nucleated cell with said proluminescence reagent.

12. A method of evaluating the efficiency of a chemotherapeutic agent in a subject, comprising obtaining a tumor cell from said subject, wherein said tumor is the one treated with said chemotherapeutic agent, contacting said tumor cell with a candidate chemotherapeutic agent, incubating said chemotherapy treated tumor cell with a proluminescence reagent; and measuring luminescence intensity, wherein the higher the measured luminescent intensity, the more efficient is the chemotherapeutic agent.

13. The method of claim 14, wherein said proluminescence reagent comprises a DEVD- aminoluciferin powder, wherein said powder is dissolved in a lysis buffer or a Dulbecco's PBS (phosphate buffer solution).

14. A kit for imaging an agent-induced apoptosis in-vivo within a region of a mammalian subject, comprising: DEVD-aminoluciferin powder and a suspension media.

15. The kit of claim 14, wherein said agent-induced apoptosis is TRAIL induced apoptosis.

16. The kit of claim 14, wherein said suspension media is Dulbecco's PBS, or lysis buffer.

17. The kit of claim 14, further comprising instructions for use in performing said imaging apoptosis.

18. The kit of claim 14, further comprising one or more of: packaging materials, instructions for using the components, one or more containers for holding the components, standards for calibrating any aminoluciferin bioluminescent emissions.

19. The kit of claim 14, further comprising at least one standard, obtained from a subject, or pool of subjects, treated with FLICA (Fluorescent inhibitor of Caspase activity).

20. The kit of claim 14, further comprising means for amplifying the expression of a compound associated with apoptosis or cell-cycle arrest.

21. The kit of claim 14, further comprising a software package, wherein said software package compares a value obtained from the test subject to a standard to determine the extent of said apoptosis

22. The kit of claim 21 , wherein said software package comprises of a library of intensity of emitted spectra form a sample obtained from a subject with quantified TRAIL induced apoptosis.

23. A method of imaging apoptosis as substantially described herein.

24. A kit for apoptosis as substantially described herein.