AU4573199A - Creación de Inventos y Patentes con Inteligencia Artificial

AU4573199A – Detection of apoptotic cells in living nematodes
– Google Patents

AU4573199A – Detection of apoptotic cells in living nematodes
– Google Patents
Detection of apoptotic cells in living nematodes

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AU4573199A

AU4573199A
AU45731/99A
AU4573199A
AU4573199A
AU 4573199 A
AU4573199 A
AU 4573199A
AU 45731/99 A
AU45731/99 A
AU 45731/99A
AU 4573199 A
AU4573199 A
AU 4573199A
AU 4573199 A
AU4573199 A
AU 4573199A
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nematode
apoptotic cells
apoptosis
apoptotic
cells
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1998-06-19
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AU746694B2
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Anton Gartner
Michael O. Hengartner
Stuart Milstein
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Cold Spring Harbor Laboratory

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Cold Spring Harbor Laboratory
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1999-06-18
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G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 – G01N31/00

G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Description

WO 99/66323 PCT/US99/13650 DETECTION OF APOPTOTIC CELLS IN LIVING NEMATODES RELATED APPLICATION(S) This application claims the benefit of Provisional Application, No. 60/090,057, filed on June 19, 1998, the entire teachings of which are incorporated 5 herein by reference. BACKGROUND OF THE INVENTION Apoptosis refers to a cellular process involving the programmed death of a cell. Cell death is an important part of animal development. For example, programmed cell death helps sculpt parts of the body, carving out cavities or 10 separating digits. Raff, Martin, Nature, 396:119-122, 119 (1998). Apoptosis also occurs in various disease states, for example, in cells affected by heart attack or stroke. Acute damage results in necrotic cell death, but less damaged cells can die by apoptosis. Similarly, certain neurodegenerative diseases, cancer, or diseases caused by viruses involve the apoptotic pathway. Id. 15 Assays that can screen, in vivo, for conditions or agents that modulate apoptosis, would significantly advance the treatment of these diseases. Studying the effect of an agent or condition in a living organism would provide a more accurate picture of how the agent or condition can be utilized. Accordingly, a need exists for an assay that can quickly and efficiently screen apoptotic modulating agents or 20 conditions in a living organism. SUMMARY OF THE INVENTION The present invention pertains to methods for determining the presence or absence of one or more apoptotic cells in a living nematode, comprising contacting a living nematode (e.g., Caenorhabditis elegans) with vital dye to stain apoptotic cells 25 in the nematode; and detecting the presence or absence of apoptotic cells stained with the dye. The nematode is preferably an adult. The vital dye used in the present WO 99/66323 PCT/US99/13650 -2 methods can be any vital dye, including acridine orange or a SYTO dye (e.g., SYTO12). The apoptotic cells can be visually detected. Another embodiment of the present invention relates to methods of determining the effect of an agent or condition on apoptosis in a living nematode 5 (e.g., Caenorhabditis elegans), comprising exposing the living nematode to an agent or condition to be tested; contacting the nematode with vital dye to stain apoptotic cells in the nematode; and detecting the presence or absence of apoptotic cells stained with the dye. The nematode is preferably an adult. The vital dye used in the present methods can be any vital dye, including acridine orange or a SYTO dye 10 (e.g., SYTO12). The apoptotic cells can be visually detected. The method can further comprise determining the amount of apoptotic cells and comparing the amount to a control. An increase in the amount of apoptotic cells, as compared to a control, indicates an agent or condition that induces apoptosis. A decrease in the amount of apoptotic cells, as compared to a control, indicates an agent or condition 15 that inhibits or reduces apoptosis. The invention also includes the agent that modulates apoptosis, as determined by methods described herein. The present invention also encompasses methods for determining the apoptotic effect of the expression of at least one gene to be tested in a living nematode (e.g., Caenorhabditis elegans), wherein a mutation has been made to at 20 least one region of the gene, comprising contacting the living nematode with vital dye to stain apoptotic cells in the nematode; and detecting the presence or absence of apoptotic cells stained with the dye. The method further comprises determining the amount of apoptotic cells and comparing the amount to a control. An increase or decrease in the amount of apoptotic cells, as compared to a control, indicates that the 25 gene expression effects apoptosis. An increase in the amount of apoptotic cells indicates that the expression of the gene enhances apoptosis. A decrease in the amount of apoptotic cells indicates that the expression of the gene inhibits or reduces apoptosis. The invention also includes a gene(s) that modulates apoptosis, as determined by methods described herein.
WO 99/66323 PCT/US99/13650 -3 The present invention pertains to methods for determining the presence or absence of apoptotic cells in a live nematode, that comprise providing a nematode that has been subjected to a mutagen that alters the nematode’s sensitivity to radiation; contacting the live nematode with vital dye to stain apoptotic cells in the 5 nematode; and detecting the presence or absence of apoptotic cells stained with the dye in the live nematode. The method includes exposing the nematode to an agent or condition to be tested. In another embodiment, the present invention involves methods of identifying a mutated, living organism having cells that undergo an altered apoptotic 10 cell death, that comprise contacting an organism with an agent or condition that modulates apoptosis, thereby creating a mutated, living organism; contacting the mutated organism with vital dye to stain the apoptotic cells in the organism; detecting the presence or absence of apoptotic cells stained with the dye in the living, mutated organism; and selecting the mutant organism having cells that 15 undergo an altered apoptotic cell death, as compared to a control. The mutant can be more or less sensitive to radiation, as compared to a control. The invention includes the mutated nematode, as identified according to this method. The invention also pertains to kits that comprise at least one nematode (e.g., Caenorhabditis elegans); and a vital dye (e.g., acridine orange or a SYTO dye). The 20 nematode can be mutated so that the nematode is sensitive or insensitive to radiation that induces apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of an adult hermaphrodite germ line. Figure 2A contains graphical representations of the amount of germ cell 25 apoptosis at 0, 12, 24 and 36 hours after irradiation of wildtype, ced-3, ced-4, rad-5, ced-9(gf) and egl-1(lf) nematodes. Figure 2B is a graphical representation of germ cell corpses 1-8 hours after irradiation (0 Gy and 120 Gy) of rad-5 mutants and the wildtype (WT).
WO 99/66323 PCT/US99/13650 -4 Figure 2C is a graphical representation of germ cell corpses after exposure to 0-5 mM of N-nitroso-N-ethylurea (ENU) to wildtype nematode, rad-5 mutant, ced 3 mutant and ced-4 mutant. Figure 3 is a schematic showing that separate signaling pathways feed into 5 the core apoptotic machinery to mediate somatic cell death, physiological germ cell death and radiation-induced germ cell death in C. elegans. Arrows indicate activation; T-bars indicate repression. The dashed arrow indicates the partial block of radiation-induced cell death in egi-1(n3082) animals. Figure 4A is a photograph of adult hermaphrodites that were scored for germ 10 cell death by direct observation under differential interference contrast (DIC) optics. Figure 4B is a photograph of adult hermaphrodites that were scored for germ cell death by staining with the vital dye Acridine Orange. Figure 5 is a three dimensional graph that shows the number of worms exhibiting an amount of apoptotic germ cell corpses for ced-3-n717 and wildtype 15 (N2) strains after gamma radiation with 6, 12 or 24 kRad, 28 hours after irradiation. Figure 6 is a three dimensional graph that shows the number of worms exhibiting an amount of apoptotic germ cell corpses for ced-3-n717 and wildtype (N2) strains after 5 mM and 1 mM of N-nitroso-N-ethylurea (ENU) exposure, 28 hours after treatment. 20 Figure 7 is a three dimensional graph that shows the number of worms exhibiting an amount of apoptotic germ cell corpses for ced-3-n717 and wildtype (N2) strains after UV irradiation with 6, 12 or 24 kJ, 24 hours after irradiation. Figure 8 is a three dimensional graph that shows the average number of apoptotic germ cell corpses for a wildtype (N2) strains after gamma radiation with 25 12 or 24 kRad, 0, 1, 2, 3, 4, 5, 6, 12 and 24 hours after irradiation. Figure 9 is a photograph of apoptotic germ cells in a wildtype (N2) strain 24 hours after 12 kRad of gamma irradiation, as identify by differential interference contrast (DIC) optics and Acridine Orange (AO).
WO 99/66323 PCT/US99/13650 -5 Figure 10 is a three dimensional graph that shows the number of worms exhibiting an amount of apoptotic germ cell corpses for rad-5 and wildtype (N2) strains after gamma radiation with 12 kRad, 24 or 48 hours after irradiation. DETAILED DESCRIPTION OF THE INVENTION 5 The present invention relates to an assay that screens for agents, conditions or genes that effect (e.g., enhance or inhibit) apoptosis (e.g., DNA damage induced apoptosis). In particular, the invention pertains to methods for detecting apoptotic cells in a living organism that has been subjected to the agent, condition or gene mutation being tested. 10 Apoptosis is a cellular process of programmed cell death. Cells can be programmed to die for various reasons. Programed cell death occurs to form parts of the body, for example, organs or separating digits. Programed cell death also occurs to eliminate structures that once served a function, but is no longer needed, such as the tail of a tadpole that is now a frog. Raff, Martin, Nature, 396:119-122, 15 119(1998). Apoptosis also occurs when a cell is exposed to a stress, such as radiation, Ultra Violet (UV) light, toxic drugs, etc. Such stresses can cause damage to the DNA of the cell, which leads to a cascade of events that cause apoptosis. Cells that undergo apoptotic death shrink and are engulfed by a nearby cell, before the content 20 of the cell spills out. A cell which is exposed to acute stresses can undergo a necrotic, unplanned death that results in swelling and bursting of the cell. The bursting causes the contents of the cell to spill out to their neighboring cells, thereby eliciting a damaging inflammatory response. Id. The apoptotic pathway involves several events that lead to a cell’s death. In 25 one pathway, a receptor, referred to as the «Fas receptor», is expressed on the cell that is programmed to die. Binding of a Fas ligand to the Fas receptor causes the receptors to aggregate. Proteins, referred to as «procaspases,» cleave and activate each other to begin the suicide sequence. Id. As a result, certain genes are expressed or inactivated. In the worm, Caenorhabditis elegans (C. elegans), the WO 99/66323 PCT/US99/13650 -6 genes, ced-3 and ced-4, are expressed, and activate apoptosis. Expression of another gene, ced-9, prevents ced-3 and ced-4 from activating apoptosis. See Figure 3. Thus, when apoptosis is triggered, ced-9, is either partially or fully inactivated. A stress that damages the DNA of the cell can induce apoptosis. Studying 5 the DNA damage induced apoptotic pathway provides insight into the progression and/or treatment of several diseases such as proliferative diseases, neurodegenerative diseases, cardiac or cerebral conditions that are associated with cell death. Until now, assays did not exist which allow one to quickly and efficiently screen for a large number of apoptotic cells. Also until now, there were no apoptotic assays that 10 allow one to study the DNA damage induced apoptotic process in a living organism (in vivo). The present invention allows one to readily screen a large number of apoptotic cells in a live organism. An organism is any living plant or animal including nematodes (e.g., C. elegans) that would not suffer a toxic, deadly effect or 15 reaction from the vital dye used to stain apoptotic cells for the assay. In particular, the assay involves contacting a live nematode with a vital dye. The vital dye should be contacted with the live nematode for a period of time sufficient for the organism to ingest or absorb the dye and to stain the apoptotic cells. The apoptotic cells can be detected, for example, visually or fluorescently. A fluorescent microscope can be 20 used. A fluorescent microscope emits wavelengths that enables one to view the fluorescently stained cells. Additionally, a machine that detects fluorescently stained cells (e.g., an fluorescent plate reader) can also be used. The steps of contacting the live nematode with the vital dye and then detecting the apoptotic cells is referred to herein as the «apoptotic assay» or the «DNA damage induced apoptotic 25 assay.» Since the apoptotic cells are stained, they are easily and quickly identified. In particular, C. elegans is a transparent worm, and stained apoptotic cells can be viewed under a microscope while the worm is alive. The worm does not need to be dissected. Accordingly, the assay allows one to study apoptotic pathway in an in vivo system efficiently.
WO 99/66323 PCT/US99/13650 -7 The organism utilized in this assay is a nematode, and in particular, C. elegans. The assay is performed on a living (e.g., alive or not dead) nematode, preferable an adult nematode. Performing the assay on a living organism provides a significant advantage because the assay provides a more accurate picture of 5 apoptosis, and agents, conditions or genes that effect apoptosis. The dye that is utilized in the assay is a vital dye or a viability stain. A vital dye is one that stains living cells. Surprisingly, the dye also stains DNA damage induced apoptotic cells after the dye is ingested or absorbed by a nematode. Vital dyes that are now available or later developed can be used in the assay. Examples of 10 vital dyes are acridine orange (Molecular Probes, Eugene, OR), Trypan blue, and SYTO dyes such as SYTO12 (Molecular Probes, Eugene, OR). Dyes that can be used in the present invention are dyes that can be ingested or absorbed by the nematode, without being toxic to the nematode, and can stain apoptotic cells. The vital dye stains the apoptotic cells with more intensity than live cells or the dye 15 present in the background because the dye more easily permeates the refractile apoptotic cell membrane. The present invention involves contacting the nematode with a vital dye for a sufficient amount of time to stain the apoptotic cells that are present. The concentration, kinetics of the dye, the amount of buffer, and amount of worms to be 20 stained are factors that influence the length of time the dye should be in contact with the worms. For example, to stain cells with Acridine Orange (AO), 500,UL of a 300pg/ml solution of AO in M9 buffer is spread evenly over a plate containing 150 200 worm non-starved worms. These worms (nematodes) and the stain are left to incubate for about 2 hours in the dark. The worms can be washed and placed in 25 fresh media or buffer. These worms can then be mounted and detected visually under a microscope. The length of time to expose the dye to the worms is determined by methods known to those of skill in the art. Sulston, J.E. et al., «Methods In: The nematode Caenorhabditis elegans,» Cold Spring Harbor: CSHL Press, pp 587-606 (1988).
WO 99/66323 PCT/US99/13650 -8 Cells that often undergo DNA damage induced apoptosis are germ cells. Hence, the apoptotic assay is preferably performed on germ cells. However, apoptotic somatic cells should also stain with the vital dye because the membrane of apoptotic cells become refractile, just as the membrane of germ cells. 5 After the vital dye step, the stained, apoptotic cells are detected. They can also be counted or scored by visually detecting the cells through a light source (e.g., a fluorescent microscope or fluorescent plate reader) at the appropriate magnification. When performing a screen of several worms at once (e.g., a preliminary screen of the effects of an agent), the apoptotic cells can be viewed a 10 low magnification (e.g., 1OX-50X magnification) to obtain an overall assessment of whether more or less apoptosis is occurring. When counting or scoring the specific apoptotic cells, a higher magnification can be used, for example, a magnification of 430X-1000X. Cells in the worms naturally undergo apoptosis, and can be studied with the 15 methods of the present invention. Additionally, various agents, conditions or genes can be tested to determine whether they induce apoptosis, and assessed with the methods of the present invention. Conditions, that can be tested, are any number of conditions including time, temperature, pressure, and various media. Conditions can also include exposure to 20 various types of light or radiation, such as UV light, infrared light and gamma radiation. The worms can be exposed to one or more of these conditions, contacted with the vital dye, as described herein, and then the apoptotic cells can be detected. One or more conditions can be tested and compared to a suitable control. A control can be a nematode not exposed to the one or more conditions, but contacted or 25 exposed to the vital dye. The amount of apoptotic cells in the worm not exposed to the condition, can be compared to the amount of apoptotic cells found in the worm that is exposed to the one ore more conditions. An increase in the number or amount of apoptotic cells, as compared to the control, indicate that the condition(s) induce apoptosis, whereas a decrease in the number or amount of apoptotic cells indicate 30 that the condition(s) inhibit or reduce apoptosis.
WO 99/66323 PCT/US99/13650 -9 In particular, gamma radiation is a condition that can be assessed for its apoptotic effects. This condition was tested on wildtype and mutated nematodes, as described herein. A range of radiation amounts (e.g., between about 1 and 30 kRads) and times can be tested. In particular, radiation sensitive or insensitive 5 mutants can be screened and tested. Once the mutants are made, as described herein, they can be tested for radiation sensitivity. The mutants can be exposed to the radiation at various amounts, and compared against a control, such as a wildtype nematode that has been exposed to a similar amount of radiation. Both the mutant and the control nematodes are then contacted with the vital dye, so that the dye 10 stains the apoptotic cells. The apoptotic cells can then be detected, for example, under a microscope. An increase in the amount of apoptotic cells in the mutant, as compared to the control, indicates that the mutant is sensitive or more sensitive to radiation. A decrease in the amount of apoptotic cells, as compared to the control, indicates that the mutant is insensitive or less sensitive to radiation. The degree of 15 sensitive or insensitivity can be determined by exposing the mutant to different degrees of radiation. For example, to determine the sensitivity of a radiation sensitive mutant, the mutant can be exposed to low amounts of radiation to assess whether the low amount of radiation (e.g., 1.0 kRads) can induce DNA damage induced apoptosis. If the amount of radiation does not induce apoptosis, then the 20 radiation can be increased in an incremental fashion. Similarly, a radiation insensitive mutant can be exposed to large amounts of radiation (e.g., 30 kRads) to determine whether the high amount of radiation will not induce DNA damaged induced apoptosis. If the amount of radiation induces apoptosis, then the radiation tested can be decrease in increments. 25 Similarly, an agent can be tested to determine its effects on apoptosis. In particular, a type of agent that can be tested is a genotoxic agent. A genotoxic agent is an agent that alters the nucleic acid of the a cell in a nematode. To assess or identify an agent that modulates (reduces or enhances) apoptosis, the nematodes are exposed to the agent to be tested. The length of time (e.g., 5 minutes-12 hours) and 30 the concentration of the agent can be determined using methods known in the art.
WO 99/66323 PCT/US99/13650 -10 Various lengths of time and concentrations can be tested to determine optimal parameters for the desired effect. For example, an agent, referred to as N-nitroso-N ethylurea (ENU) is an agent that was tested and found to induced DNA-damage apoptosis. See Example 5, Figure 6. This agent was tested at two different 5 concentrations, 5mM and 1mM, and was in contact with the nematodes for 4 hours. Another factor that can be considered is the kinetics of the agent being tested, which may or may not be known. For example, the agent to be tested could either be absorbed into the worm, or ingested by the worm. The kinetics of the drug to be tested can impact the length of exposure to the worm. In the situation where the 10 kinetics are unknown, a range of exposure times can be tested and assessed using the apoptotic assay. Once the worm is exposed to the agent, then the vital dye and detection steps can be performed, as described and shown herein. The amount of apoptotic cells are determined and compared to a control. One type of control, referred to as a negative control, is the assessment of apoptotic cells in a nematode 15 that has not been exposed to the agent, but has been through the other steps of the assay (e.g., the vital dye and detection steps). The negative control can be maintained in conditions that allow apoptosis to occur (e.g., naturally or induced). Another control, called a positive control, is an control in which the nematode is exposed to an agent that induces apoptosis. The agent to be tested can be compared 20 to a negative control as well as a positive control to determine the extent the agent induces or inhibits apoptosis. An agent that increases the amount of apoptotic cells, as compared to a negative control, indicates an agent that induces apoptosis, whereas a decrease in the amount of apoptotic cells, as compared to the negative control indicates an agent that reduces or inhibits apoptosis. 25 When testing an agent, the nematode is preferably exposed to or subjected to the agent to be tested when the nematode is in a «pre-adult» stage. There are several stages of larvae. For example, the entire life cycle for a C. elegans nematodes is about 3 days at 25 C, the Li stage is reached at about 12 hours, L2 at about 7 hours after Li, L3 at about 7 hours after L3, and L4 is reached at about 9 hours after L3. 30 After the L4 stage, the larvae becomes an adult. Epstein, H.F. et al.,»Caenorhabditis WO 99/66323 PCT/US99/13650 -11 elegans,» Modern Biological Analysis of an Organism, vol. 48, pp. 4-6 (1995). The vital dye staining step is preferably performed when the nematode reaches the adult stage. In particular, the present invention pertains to screening for cancer 5 modulating agents. A cancer cell is deadly because the cancer cell cannot undergo apoptosis. Cancer cells often have a defect in their apoptotic pathway. Agents can be tested to determine their effect on the apoptotic pathway and used in developing treatments for cancer. Agents that are chemo-sensitive and chemo-resistant can be screened using the methods described therein. The present invention also includes 10 the chemo-sensitive and chemo-resistant agents that are identified using the methods of the invention. Another application of the present invention is a method for identifying one or more genes, or one or more regions of a gene that modulate apoptosis. Nematodes can be mutated in vivo. Mutations that occur in the germ lines are 15 inheritable. The most effective development stage for mutagenesis is the late L4 lava or young adult. Mutations can be performed chemically or by radiation. A popular and widely used chemical mutagen is Ethyl Methane sulfonate (EMS). EMS performs G/C to A/T transition at a frequency of about 7 x 10-6 per mutagenized G/C base pair. EMS concentrations of 50 mM are typically used, but 20 lower doses (10-25mM) can reduce toxicity. The protocols for performing mutations using EMS and other methods are described in Epstein, H.F. et al., «Caenorhabditis elegans,» Modern Biological Analysis of an Organism, 48:2, pp 31 58, 37 (1995). To reduce the number of unwanted or extraneous mutations, several solutions exist. The newly isolated mutants can be thoroughly outcrossed to 25 unmutagenized genetic backgrounds. Mendelian segregation and crossing over will separate most extraneous mutations from the selected mutations. Id. at p36. Other methods of removing unwanted mutations are known in the art. Id. The mutated gene can be isolated cloned also by methods known in the art. Sambrook, et al., «Molecular Cloning, A Laboratory Manuel, 2nd Ed. Cold Spring Harbor Press, 30 1989.
WO 99/66323 PCT/US99/13650 -12 A mutant nematode can then undergo the apoptotic assay to assess the effect the mutated gene has on apoptosis. An increase in the amount of apoptotic cells, as compared to a control, indicates a mutant that has an enhanced ability to undergo apoptosis. In this case, the expression of the gene or genes in their non-mutated 5 form inhibit or reduce apoptosis. A decrease in the amount of apoptotic cells, as compared to a control, indicates that the mutant had a reduced ability to undergo apoptosis. In this case, the expression of the gene or genes in their non-mutated form enhance apoptosis. A control can be a nematode that does not contain the mutation being tested (e.g., a wildtype), and subjected to the same conditions as the 10 mutants, except those that induce the mutation. A control can be a wildtype nematode that is contacted with a vital dye so that the apoptotic cells are stained, and then the apoptotic cells are detected. The number of apoptotic cells of the control can be compared to those stained in the mutant. For example, a mutant, referred to as op152, was made and assessed using 15 the methods of the present invention. The op152 mutant was exposed to the acridine orange vital dye and the apoptotic cells were stained. The apoptotic cells were detected and scored using a microscope. The mutants were maintained in conditions that induce apoptosis by exposing the mutants to radiation. op152 induces high levels of germ cell apoptosis, as compared to a control. See Example 3. An increase 20 in the number of apoptotic cells was observed. Thus, the mutant induces DNA damage induced apoptosis. Accordingly, the gene can be isolated and cloned using known methods. Additionally, the present invention can be used to screen one or more mutants that have cells that undergo an altered apoptotic cell death. Cells that 25 undergo an altered apoptotic cell death means that there is an increase or a decrease (e.g., a modulation) in apoptotic cells, as compared to a control. The mutants are maintained in conditions that induce apoptosis (e.g., radiation) and then they are contacted with the vital dye. The stained apoptotic cells are detected and scored. An increase or decrease in the number of apoptotic cells, as compared to a control, 30 indicate that the nematode contains one ore more mutations that modulates WO 99/66323 PCT/US99/13650 -13 apoptosis. The screening of a number of mutants can be done quickly and efficiently. These mutants can also be used to screen for agents that modulate apoptosis. For example, several mutants were made and screened. A mutant that was isolated, referred to as rid-2, exhibits a supersensitivity to radiation induced 5 DNA damage apoptosis. See Example 6. The rid-2 mutant was made in accordance with the methods described herein and in Epstein, H.F. et al., «Caenorhabditis elegans,» Modern Biological Analysis of an Organism, vol. 48, pp. 4-6 (1995). Upon radiation with 1.6 k Rad of a wildtype C. elegans nematode, 6.6 +/- 0.6 corpses were obtained, whereas the same amount of radiation of rid-2 resulted in 10 26+/- 3.6 corpses, or 3-4 times the amount of apoptosis. This mutant can be used to screen for agents that inhibit apoptosis, as described herein. A radiation insensitive mutant can be used to screen for agents that increase or enhance apoptosis. In summary, the simplicity of C. elegans and its powerful genetics allow for significant contributions to the understanding of radiation-induced apoptosis and its 15 role in preventing genetic imbalance in cancer cells. The present invention can be used to identify, through the apoptotic assay, genes that mediate damage-induced apoptosis in C. elegans, as described herein. Homologues of worm genes involved in important signal transduction pathways will likely perform similar functions in humans because the such pathways remain conserved through evolution. Other 20 applications discussed herein are: to genetically screen for mutations that prevent or enhance apoptosis, and therefore damage-induced cell death; to screen for compounds that prevent or cause radiation-induced or DNA damage induced apoptosis; and to monitor or assay for toxicity of compounds. This apoptotic assay can be done either on pure compounds or samples from the environment. While this 25 work on damage-induced apoptosis was done in the germ line, there is nothing that would prevent the apoptotic assay from applying to damage-induced apoptosis in the somatic tissues. Accordingly, the present invention includes mutant nematodes and, in particular, radiation sensitive or insensitive mutants. The present invention also WO 99/66323 PCTIUS99/13650 -14 encompasses kit which comprise the wildtype nematodes, or mutants made and screened as described herein, and vital dye. The invention will be further illustrated by the following examples which are not intended to be limiting in any way. The teachings of all the references cited in 5 this document are incorporated herein by reference in their entirety. EXAMPLES Example 1: Genetic Control of Radiation Induced Apoptosis MATERIALS and METHODS: Strains. C. elegans strains were maintained at 20’C as described by Brenner 10 (Brenner, S., «The genetics of Caenorhabditis elegans,» Genetics 77:71-94 (1974)). All strains used are described by Jonathan Hodgkin unless otherwise stated. ced 3(n2438) was described by Hengartner, M.O. and Horvitz, H.R., «Activation of C. elegans cell death protein CED-9 by an amino-acid substitution in a domain conserved in Bcl-2,» Nature 369:318-320 (1994). egl-1(n3082) was described by 15 Conradt, B. and Horvitz, R.R., «The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9,» Cell 93:519-29 (1998). Radiation of Worms: Late L4 stage hermaphrodite worms were irradiated with the indicated doses of gamma radiation and scored after 0, 12, 24 and 36 hours. 20 For each time-point, 15 animals were scored for germ cell death in one gonad arm via direct observation using standard Nomaski optics. Data shown are means +/ sem (standard error of the mean). Strains used were ced-3(n717), ced-4(n1162), ced-9(n.1950), egl-1(n3082) and rad-5(mn159). To confirm that ced-9(n1950) completely-, and egl-1(3082) partially block radiation induced cell death, induced 25 germ cell death was also analyzed as described in Figure 2B.
WO 99/66323 PCT/US99/13650 -15 Rapid induction of germ cell death following radiation. Synchronized animals (24 hours post the L4/ adult molt) were irradiated with 120 Gray and analyzed 1 hours intervals after irradiation. Germ cell death was scored. Counting radiation-induced germ cell corpses. For corpse counting 5 synchronized animals were mounted under standard conditions (Epstein, H.F. and Shakes, D.C., Methods in Cell Biology (Academic Press, 1995)) and corpses were identified using Nomaski optics. For irradiation, a Cs source (JL Shepherd and Associates, model 69 A irradiator; 3.984 Gray per minute) was used. To induce germ cell death with ENU hermaphrodites were synchronized in late L4 and treated 10 in M9 buffer with the indicated concentrations of ENU, for 4 hours. To induce germ cell death with UV, worms were irradiated with 6000-24,000 pJoule/cm 2 of UV light (254nm) using a Stratalinker (Model 1800) crosslinker. To determine how fast germ cell death is induced following radiation, synchronized animals (24 hours post the L4/adult molt) were irradiated with 120 Gray and analyzed in 1 hour intervals. 15 Counting apoptotic cells in the soma. Cell corpses in the head of ced 1 (e1 735) engulfiment defective animals at the first larval stage were counted 3 hours after irradiation with 60 Gray. Video time lapse analysis. Animals were mounted under standard conditions in M9 buffer containing 2 mM levamisole (levamisole prevents animals from 20 moving but does not affect the germline or the gonad). For video analysis, a Cohu 4910 series camera attached to a Zeiss (Akioskope) microscope was used. Images were processed using the NIH image program. rad-5(mn159). rad-5(mn159) is likely to be a hypomorphic allele. However, at 20’C the only significant phenotype of rad-5(mn159) is a defect in radiation 25 induced death. The brood size of rad-5(mn159) animals is approximately 50% of wild-type at 20’C but rapidly drops at higher temperatures due to embryonic lethality at various stages of development (AG, SM and MOH). Radiation induced apoptosis is still blocked in rad-5(inn159) animals raised at 15 C (AG, SM and
MOH).
WO 99/66323 PCT/US99/13650 -16 ENU induced apoptosis. ENU induces germ cell apoptosis. Hermaphrodites were synchronized in late L4 and treated in M9 buffer with the indicated concentrations of ENU for 4 hours. Germ cell death was scored. Results: 5 DNA-damage-induced apoptosis is essential to remove genetically compromised cells that might become dangerous for the entire organism. However, it is believed that damage-induced apoptosis is hampered by the lack of a genetic model system. Towards the development of such a model system, the potential of C. elegans germ cells to undergo induced cell death was analyzed. C. elegans 10 meiotic germ cells, in contrast to somatic cells, can readily be induced to undergo cell death following genotoxic stress. Radiation-induced germ cell death is morphologically identical to developmentally-regulated apoptosis in the soma and to physiological apoptosis in the germ line. Consistent with the apoptotic nature of radiation induced death it utilizes the core apoptotic machinery. However, 15 mutations in the cell death regulators egl-1 and ced-9 have different effects on somatic-, physiological germ cell-, and radiation induced deaths. Furthermore, the radiation-sensitive mutant rad-5(mn159), which has previously been shown to have a mutator phenotype, is only defective in damage-induced apoptosis. Thus, separate signaling pathways feed into the core apoptotic machinery to mediate physiological 20 and radiation-induced apoptosis in C. elegans. To determine the effects of genotoxic stress in C. elegans, the hermaphrodite germ line was chosen as an experimental system. Within the adult C. elegans hermaphrodite ovotestis, germ cells progress through various stages of differentiation, as shown in Figure 1. In Figure 1, at the distal end of the U-shaped 25 ovotestis, mitotic stem cells proliferate throughout the worm’s life. Upon passage through the transition zone, germ cells cease dividing and initiate meiosis. Germ cells are only partially enclosed by the cell membrane and thus share a common cytoplasm. In accordance with the literature these partially separated nuclei will be referred to as cells. The most abundant population of meiotic cells are arrested in WO 99/66323 PCTIUS99/13650 -17 pachytene of meiosis I and reside between the transition zone and the bend of the gonad. Upon exit from pachytene, germ cells progress into meiotic diplotene, cellularize, undergo the final stages of oogenesis, and finish meiosis after fertilization upon passage through the spermatheca. Under normal growth 5 conditions, approximately 50 percent of female germ cells are fated to die by programmed cell death. Apoptotic germ cells arise from a population of syncytical cell nuclei arrested in pachytene of meiosis I and are eliminated rapidly by the engulfment machinery resulting in a steady state level of 0 to 4 apoptotic germ cells throughout adulthood. These deaths, termed, «physiological germ cell deaths» 10 appear to serve a homoeostatic function as they occur under normal growth conditions. To determine whether genotoxic stress can induce apoptosis in C. elegans, worms at the fourth (final) larval stage (L4) were exposed to increasing levels of gamma radiation and analyzed the germ line of the matured adult worms after 15 irradiation. Radiation causes a dramatic increase in the number of apoptotic germ cells, as irradiated worms contained up to 45 dead cells in the pachytene region of the germ line (Figure 1 and Figure 2A). To confirm that the increased level of germ cell death is due to an enhanced level of apoptosis rather than an inhibition of engulfment of dead cells, worms were irradiated and the kinetics of radiation 20 induced germ cell death by time lapse video analysis were studied. Time lapse video microscopy of dying cells was performed. Eight representative pictures derived over a 1.5 h time course were done. Various morphological stages of programmed cell death of 5 cells were studied. After «pinching off’ from the surrounding syncytium of meiotic nuclei, dying germ cells undergo morphological 25 changes similar to those seen during physiological germ cell death and during somatic apoptosis. Within approximately 20-minutes the cytoplasm of dying cells becomes increasingly refractile and finally meld with the nucleus to form a uniform highly refractile corpse. Following this change, the corpse persists between 20 and 60 min before it loses its refractile character and dissolves within less than 1 minute.
WO 99/66323 PCT/US99/13650 -18 To determine the genetic requirements for the radiation-induced cell death, the studies were repeated with animals mutant for key components of the C. elegans cell death machinery. Briefly, apoptotic death in C. elegans requires the activity of the caspase homologue CED-3 and the Apaf- 1 homologue CED-4, which promotes 5 CED-3 activation. CED-3 and CED-4 killing activity is antagonized by the Bcl-2 family member CED-9, which has been proposed to sequester CED-4 and CED-3 in an inactive ternary complex. In cells fated to die, the pro-survival activity of CED-9 is inactivated by the BH3 domain-containing protein EGL-1, likely via a direct protein-protein interaction. Figure 2A shows that while germ cell death can readily 10 be induced in a dose dependent manner by 30 to 120 Gray of gamma radiation in wild-type (N2) animals, no cell death is induced in the germ line of animals homozygous for the loss of function alleles ced-3(n717) or ced-4(n1162). (Figure 2A). Radiation-induced cell death is also absent in the ced-9(n]950) gain of function mutant and strongly reduced the egl-](n3082) loss of function mutant, 15 Figure 2A-2C. Both ced-9 (n1950) and egl-1(n3082) completely suppress programmed cell death during somatic development but do not have an effect on physiological germ cell death. Thus, while developmental somatic death, physiological germ cell death and damage-induced death use the same execution machinery, the differential behavior of ced-9(gf) and egl-1(Wf mutations suggests 20 that the various types of programmed cell death in C. elegans are likely to use different mechanisms to activate this machinery (Figure 3). To analyze how quickly apoptosis can be induced, a synchronous population of wild-type adult worms was irradiated 24 hours after the L4/Adult molt and counted dead cells in 1 hour intervals. Apoptosis increases 2 hours after irradiation, 25 peaks 3 to 4 hours after irradiation and then stabilizes at a slightly lower level for the remainder of the time course (Figure 2B). This rapid induction, which is similar to the kinetics of apoptosis observed in irradiated mammalian thymocytes, suggests that induced apoptosis is likely to be a direct response to radiation-induced damage (Figure 2B).
WO 99/66323 PCTIUS99/13650 -19 To confirm that radiation-induced apoptosis is due to DNA damage, the ability of other DNA modifying agents to induce apoptosis in the germ line was tested. The drug N-nitroso-N-ethylurea (ENU) strongly induces apoptosis (Figure 2C). Ultraviolet (UV) radiation also induces apoptosis, although the effect is weaker 5 and difficult to score owing to the high lethality caused by the UV treatment (AG, SM and MOH). In contrast to the meiotic region of the hermaphrodite germ line, any radiation-induced programmed cell deaths in the germ line of young hermaphrodites undergoing spermatogenesis or in the germ line of males could not be identified. 10 Within the hermaphrodite germ line, cell deaths invariably occur only in the pachytene region and never in the mitotic region. Germ cells that are not eliminated by programmed cell death complete oogenesis and are successfully fertilized. However, many of the resulting embryos die in a radiation dose-dependent manner at various stages of embryonic development, presumably as a delayed consequence 15 of unrepaired DNA damage. Somatic cells also appear to be resistant to DNA damage-induced apoptosis, as the number of apoptotic cell corpses present in the head of young ced-1 larvae, which are deficient in corpse engulfinent, was not affected by radiation. (0 Gray, 18+/-0.4 corpses; 60 Gray, 17.6 +/- 0.4 corpses; See the Materials and Methods in this Example). If apoptosis occurs at all in the soma, it 20 can not be extensive, as adult worms readily survive radiation doses of up to 480 Gray. Given the above results, genes might exist that sense DNA damage and transmit this information to the apoptotic machinery. Previous genetic screens in C. elegans had led to the identification of nine radiation-sensitive (Rad) mutants. Upon 25 screening through those, it was found that rad-5(ins]59) is defective in mediating radiation-induced apoptosis (Figure 2A and Figure 2B). To show that rad-5(mn159) specifically affects DNA damage induced germ cell apoptosis rather than all programmed cell deaths, the ability of rad-5 and two ced-3 alleles to prevent radiation-induced germ cell apoptosis was compared to their ability to prevent 30 developmental cell deaths in the pharynx. As expected, ced-3 mutations prevent WO 99/66323 PCT/US99/13650 -20 both types of cell death to approximately the same extent. In contrast, rad-5(mn159) strongly prevents radiation-induced apoptosis but has only a marginal effect on developmental cell death (see Table 1). TABLE 1 5 Genotype extra cells in pharynx germ cell corpses average +/- sem. n average +/- sem. n Wild Type 0 15 22.9 +/-3.2 15 rad-5(mn159) 0.8 +/- 0.3 19 0.3 +/-0.2 15 ced-3(n717) 12 +/-2.6 11 0.1 +/-0.1 15 ced-3(n2438) 1.7 +/- 1.2 12 15.4 +/-2.4 15 10 Table 1) Rad-5(mn159) specifically suppresses DNA-damage-induced apoptosis. Extra cells in the pharynx (resulting from the inhibition of programmed cell death) were quantified as described previously. Germ cell corpses were determined 36 h after irradiation of late L4s. Results shown are mean ± sem.; sem is the standard deviation of the mean; n is number of animals counted. 15 Data presented herein are consistent with the existence of a genetic pathway that senses DNA damage and signals to the apoptotic core machinery to effect programmed cell death. Such a pathway is likely to be evolutionary conserved, as apoptosis can be readily induced in various mammalian systems by DNA damaging agents. Damage-induced apoptosis, which is often compromised in tumor cells, is 20 thought to be essential for the elimination of damaged cells that might become harmful for the entire organism. As in mammalian systems, only a subset of cell types are competent to respond to the DNA damage triggered pro-apoptotic signal. This restriction of the apoptotic potential is particularly evident in C. elegans, as only meiotic germ cells that produce oocytes within the hermaphrodite are 25 competent to undergo radiation-induced apoptosis. Mitotic cells, and meiotic germ cells undergoing spermatogenesis (in the hermaphrodite and male germ line) are resistant to death.
WO 99/66323 PCT/US99/13650 -21 Radiation-induced apoptosis of pachytene cells might be part of a rad-5 dependent meiotic checkpoint that normally senses unresolved recombination intermediates. Indeed, such meiotic checkpoints have been described to operate during budding yeast sporulation and during mammalian spermatogenesis. In yeast, 5 damage sensing induces a transient meiotic cell cycle arrest, whereas in mammalian spermatogenesis, it triggers either apoptosis or cell cycle arrest. In C. elegans, the evidence for a meiotic checkpoint leading to apoptosis is supported by the finding that many of him mutants, which cause meiotic defects ultimately leading to an elevated level of chromosomal non-disjunction, have an increased level of germ cell 10 apoptosis (Table 2). Consistent with the role of rad-5 in sensing DNA alternations, rad-5(mn159) also suppresses the extra cell death phenotype caused by him 8(e1489) (Table 2). In addition to its ability to relay a DNA damage-triggered cell death signal to the apoptotic core machinery, rad-5 might also function as a more general sensor of DNA damage. This additional role is revealed by the mutator 15 phenotype of rad-5(mn159), which results in the accumulation of mutations even in the absence of mutagenesis.
WO 99/66323 PCTIUS99/13650 -22 TABLE 2 Genotype Germ cell corpses Wildtype 0.4± 0.1 him-5 (e 1467) 1.4± 0.3 5 him-5 (e 1490) 4.4± 1.6 him-8 (mn243) 1.4 ± 0.4 him-8 (e 1489) 3.1 ± 0.5 him-8 (e 1489), rad5 (mn 159) 0.4 ± 0.1 rad-5(mn159) 0.3 ± 0.1 10 (Table 2) rad-5(mn159) suppresses the extra germ cell apoptosis observed in strong non-disjunction mutants. Non-disjunction mutants occur when homologous chromosomes do not separate. Mutations causing strong chromosomal non disjunction also lead to extra germ cell death which is suppressible by rad-5. Germ cell corpses were determined 24 h after the L4 stage. Results shown are mean 15 sem., n=15 (n=12 for him-8(mn243) and him-5(e1490)).
WO 99/66323 PCT/US99/13650 -23 In summary, it has been shown herein that DNA damage-induced signaling leads to the induction of rad-5 dependent programmed cell death in the germ line of C. elegans. The differential behavior of rad-5(mn159), egl-](f and ced-9(gf) mutants suggests that separate signaling pathways feed into the core apoptotic 5 machinery to mediate somatic cell death, physiological germ cell death and radiation-induced germ cell death in C. elegans (Figure 3). Genetic analysis of radiation-induced apoptosis in the worm might help to elucidate the molecular mechanisms leading to DNA-damage-induced apoptosis. Example 2: Protocols for Performing the Apoptotic Assay 10 The protocol used for staining the C. elegans is as follows: – Acridine Orange (AO) at 30-50 ug/ml in M-9 (Stock is l0mg/ml so use 3-5ul of stock per ml of M-9) – Added 0.5 ml of solution to small plate with 150-200 nonstarved worms. 15 – Rotated plates to make sure that AO solution is distributed evenly. – Placed in the dark at 200 C for 2 hr (1.5-2.5 hr is fine) – Washed worms off plate with 1.5 ml M-9, and transfer to amber eppendorf tube using a pasture pipette. – Spin for a couple of seconds, and aspirate off M-9. 20 – Washed twice more with M-9. * Replated worms onto small plate with a pasture pipette in no more then 100-150 ul M-9 e Destained at 200 C in the dark for 2 hrs (1.5-6 is acceptable, depending on the intensity of the stain) WO 99/66323 PCT/US99/13650 -24 The assay: Worms were screened in the F2 generation as follows: Large plates with many L4 N2s were mutagenized with EMS using a standard protocol. Worms were washed off the plate in 3ml of M-9, which was 5 transferred to a 15ml tube with 20ul of EMS dissolved in 1 ml of M-9. This was rotated for 4 hours at room temp, washed 3 times in a total of 12 ml of M-9, then replated onto a large plate for 1-2hrs. When the worms recovered, L4s were picked to new plates. Several strategies were used in order to optimize this screen with respect to 10 time spent/genome screened, as well as materials used. The basic plan was to pick L4s, as PO, allow them to lay embryos on a dish for about 24hrs, then remove these POs to new dishes for two more days. The F Is from these plates were then removed to new plates in groups of 5 to 10 for 4-16 hours, after which time they removed, leaving the F2 embryos. When the F2s were adults, they were stained with AO, and 15 observed under a dissecting microscope which allowed for visualization of the dye. Staining was done when adults were older to insure that if mutations occurred causing a developmental delay, the worms would have had sufficient time to grow. In addition to this, in many cases the F2s had been laid over 16 hours, giving a relatively large range of adults on the plate. 20 Worms were viewed directly on the plate where they were destained. If they looked good, they were removed, in order to be rescreened. If the candidate was not sterile, the F3 progeny were rescreened, to ensure homozygosity. If the candidate was sterile, siblings (sib) were picked, in order to clone the strain in a heterozygous form. 25 In order to make sure the screen would be useful in the above assay, control strains were examined under the dissecting scope, after AO staining. Although these examinations were not quantitative, it was observed that engulfment mutants do not stain visibly under the dissecting scope, while other mutants such as ced-9, ced-3 and N2 do in an amount which would be predicted based on their genotypes. Table 30 3 shows the number of nematodes that were screened and observed.
WO 99/66323 PCT/US99/13650 -25 TABLE 3 In total about 6500 genomes were screened in 5 separate trials. Mutagenesis # F Is/plate F1 time on plate F2/plate 1 10 4hr 50-100 5 2 5 -6hr 100-150 3 10 -6hr 150-200 4 10 ~16hr 150-200 5 5 ~l6hr 100-150 In mutagenesis 1-3, worms which stained above background were pulled 10 from the plate. If these worms were not sterile F3s were screened. If they were sterile, sibs were picked and F3s were screened, in order to clone the strain. In the later screens, worms were picked more stringently. They were first pulled out under the dissecting scope, then looked at under Normarski and higher powered fluorescence. This second method was much more efficient, allowing most false 15 positives to be removed before a screen of the F3s was carried out. Example 3: Development of a Fluorescent Assay for Germ Cell Apoptosis in C. elegans The C. elegans germ line was developed as a powerful genetic system to study how cells control activation of the apoptotic program. See Example 1. In 20 contrast to the somatic tissues of the worm, the germline continues to proliferate throughout life. In addition to giving rise to differentiated gametes, a high proportion of germ cells are eliminated by apoptosis. Germ cell death requires the same apoptotic core machinery as is needed for somatic cell death. As in the soma, dying germ cells are rapidly engulfed, such that normally only a few germ cell 25 deaths can be observed at any given time. To simplify the detection of germ line corpses, an assay was developed using the vital dye acridine orange, which detects apoptotic germ cells in living worms. (Figure 4).
WO 99/66323 PCTIUS99/13650 -26 To determine whether germ line cell death can be used as a model system to study the mechanism(s) underlying DNA damage-induced apoptosis, the effects of genotoxic agents on germ cell death were analyzed. Unlike somatic cells, germ cells readily undergo apoptosis in a dose-dependent manner following exposure to 5 gamma or UV radiation, or to N-nitroso-N-ethylurea (ENU) (Figures 5-7). Radiation-induced germ cell death requires ced-3 and ced-4 activity, confirming that it is apoptotic in nature (Figures 5-7). Induction of death is apparent within 3 hours (Figure 8), and can be detected both by differential interference contrast (DIC) optics and Acridine Orange. (Figure 9). 10 Given the above results, genes exist that sense DNA damage and transmit this information to the apoptotic pathway. Upon screening through a panel of existing candidate mutations, radiation-induced cell death is almost completely eliminated in rad-5 mutants, whereas the basal level of radiation-independent germ cell deaths is not affected (Figure 10). Since rad-5 has a mutator phenotype and has 15 no strong effect on apoptosis during normal development, rad-5 acts as a sensor of DNA damage, rather than as an general effector of programmed cell death. See Example 1. To determine whether C. elegans germ cells can also undergo apoptosis when exposed to pathological conditions other than DNA damage, a mutation, 20 op152, was isolated. op152 induces high levels of germ cell apoptosis (see Table 4). The op152 mutation was isolated in a screen for mutations with increased germ cell apoptosis. The L4 wild-type hermaphrodites were mutagenized with EMS using a standard protocol (Anderson, P. Mutagenesis. Methods Cell Biol 48:31-58 (1995)), transferred to fresh seeded plates, and replated every 24 hours for three days. Adult 25 F1 animals were transferred to new plates (5-10 worms / plate) and left to lay eggs for 4 – 16 hours creating synchronized, semi-clonal populations of F2 animals. F2 animals were assayed for germ cell apoptosis 36 hr. after reaching adulthood, using the vital dye acridine orange staining procedure described in Figure 4. Interestingly, when apoptosis is suppressed in op152 worms (through the 30 generation of an op152; ced-3 double mutant), no apoptotic germ cells are observed; WO 99/66323 PCTIUS99/13650 -27 however, the worms are still sterile, and now many necrotic deaths can be observed (Table 4). Thus, in an op152 background, germ cells become sick, and rather than dying by necrosis, which is a «messy death» (because it damages surrounding tissues and, in mammals, causes inflammation), they activate the apoptotic pathway and 5 undergo a «clean death» (without damage or inflammation). Germ cell apoptosis can also be induced by stimuli other than DNA damage, and that it can thus be used as an assay for a wide range of toxic agents. TABLE 4 Mutations in ced-3 prevent op]52-induced apoptosis, but not germ cell death. 10 Genotype Fertility ? Germ cell death Extent Type wild type (N2) Yes + apoptosis ced-3(n717) IV Yes NA ced-9(n2812) III No + apoptosis ced-9(n2812) III; ced-3(n717) IV Yes NA 15 op152 III No +++ apoptosis op 152 III; ced-3(n717) IV No +++ necrosis The extent of germ cell death was determined both by direct observation under DIC optics and by Acridine Orange staining. Only apoptotic germ cells stained efficiently with Acridine Orange. Fertility was determined by scoring at 20 least 6 animals for brood size. Fertile animals gave at least 50 viable progeny. Sterile animals gave fewer than 2 viable progeny. NA: not applicable. Example 4: Acridine Orange Vital Dye Stains Apoptotic Germ Cells in C. elegans To stain apoptotic germ cells with the vital dye Acridine Orange (AO, Molecular Probes, Eugene, OR), 500 Fl of a 30 Fg/ml solution of AO in M9 buffer 25 is evenly spread over a 60 mm plate containing 150-200 non-starved worms. The worms are left to stain for 2 hr. in the dark. Worms are subsequently washed off the WO 99/66323 PCT/US99/13650 -28 plate with 1.5 ml of M9, and transferred to an amber microfuge tube using a Pasteur pipette. After a brief spin in a microfuge to pellet the worms, the supernatant is removed, and fresh M9 is added to the worm pellet. After two washes, the worms are replated onto a 60 mm agar plate seeded with E. coli, and left to recover and 5 destain for 2 hr. in the dark. Worms are then ready for mounting and observation. Apoptotic cells were assayed by using the differential interference contrast (DIC) optics, and compared to vital dye stained apoptotic cells. The appearance and number of cell corpses in the germline of nematodes were studied by mounting animals in a drop of M9 salt solution containing 30 mM NaN 3 and observing the 10 animals using DIC optics. Corpses are cellularized and more refractile than syncytial nuclei or oocytes, and can be identified under high magnification. The apoptotic cells stained with the AO enabled quick and efficient detection of the apoptotic cells. Detection using DIC optics is more time consuming. Approximately, 80-90% of the apoptotic cells were stained with AO, and detected, 15 when compared with the number of apoptotic cells detected using DIC optics. Example 5: Gamma Irradiation, Agents, and UV Radiation Induced Apoptosis, and Radiation Sensitive Mutants Can Be Detected Figure 5 shows that Gamma irradiation induces apoptosis in the adult C. elegans hermaphrodite germ line. L4 larvae were transferred to agar plates seeded 20 with E. coli and exposed to a Cs source (Mark I Model 68A Irradiator, JL Shepherd & Associates, San Fernando, CA) and the extent of germ cell death determined by visual inspection using DIC microscopy as described in Example 4. Figure 8 shows that kinetics of apoptosis in the adult C. elegans hermaphrodite germ line following gamma ray irradiation. Staged adult 25 hermaphrodites were transferred to agar plates seeded with E. coli and exposed to gamma radiation as described above. The extent of germ cell death at various time points following irradiation was determined by visual inspection using DIC microscopy as described in Example 4.
WO 99/66323 PCT/US99/13650 -29 Figure 9 shows that Gamma irradiation-induced germ cell apoptosis can be detected by Acridine Orange staining. L4 larvae or adult hermaphrodites were transferred to agar plates seeded with E. coli and exposed to a Cs source (Mark I Model 68A Irradiator, JL Shepherd & Associates, San Fernando, CA), and the 5 extent of germ cell death determined by visual inspection using DIC microscopy and by Acridine Orange staining, as described in Example 4. Figure 6 shows that the chemical mutagen N-ethyl-N-nitrosourea induces apoptosis in the adult C. elegans hermaphrodite germ line. L4 larvae were mutagenized with N-nitroso-N-ethylurea (ENU) using an established protocol, De 10 Stasio E, et al., Genetics, 147:597-608 (1997). The extent of germ cell death determined by visual inspection using DIC microscopy as described in Example 4. Figure 7 shows that UV irradiation induces apoptosis in the adult C. elegans hermaphrodite germ line. L4 larvae or adult hermaphrodites were transferred to agar plates seeded with E. coli and exposed to UV radiation using a UV Stratalinker 1800 15 (Stratagene, CA), and the extent of germ cell death determined by visual inspection using DIC microscopy as described in Example 4. Figure 10 shows that rad-5 mutants are resistant to gamma ray-induced apoptosis. L4 hermaphrodite larvae were transferred to agar plates seeded with E. coli and exposed to a Cs source (Mark I Model 68A Irradiator, JL Shepherd & 20 Associates, San Fernando, CA), and the extent of germ cell death determined in adults by visual inspection using DIC microscopy, as described in Example 4. Example 6: Screening for Radiation Induced Death Mutants Using the Acridine Orange Vital Dye Assay Another radiation sensitive mutant, rid-2, was found using the AO vital dye 25 to stain the apoptotic cells that resulted from the radiation. The mutant was made using the methods described herein and in Epstein, H.F. et al., «Caenorhabditis elegans,» Modern Biological Analysis of an Organism, vol. 48, pp. 4-6 (1995). RID stands for radiation induced death. Radiation with 1.6 KRad of N2 (wildtype) results in 6.6 +/- 0.8 corpses, whereas radiation of rid-2 with the same does resulted WO 99/66323 PCT/US99/13650 -30 in 26+/- 3.6 corpses. Worms at the L4 larval stage were irradiated with a does of 1.6 KRad and corpses were counted 24 hours later by observation under Normaski optics (n=10). This mutant is supersensitive to radiation, and can be used in the apoptotic assay to screen for agents that reduce the apoptosis in the rid-2 mutant. 5 While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (33)

1. A method for determining the presence or absence of one or more apoptotic cells in a living nematode, comprising: 5 (a) contacting a living nematode with vital dye to stain apoptotic cells in the nematode; and (b) detecting the presence or absence of apoptotic cells stained with the dye.

2. The method of claim 1, wherein the nematode is Caenorhabditis elegans. 10

3. The method of claim 2, wherein the C. elegans is an adult.

4. The method of claim 3, wherein the vital dye is acridine orange or a SYTO dye.

5. The method of claim 1, wherein the apoptotic cells are DNA damage induced apoptotic cells. 15

6. The method of claim 4, wherein step (b) is carried out by visually detecting the apoptotic cells.

7. The method of claim 1, wherein the nematode is exposed to an agent or condition to be tested prior to step (a).

8. The method of claim 1, wherein the nematode contains a mutation that alters 20 apoptosis. WO 99/66323 PCT/US99/13650 -32

9. A method of determining the effect of an agent or condition on apoptosis in a living nematode, comprising: (a) exposing the living nematode to an agent or condition to be tested; (b) contacting the nematode with vital dye to stain apoptotic cells in the 5 nematode; and (c) detecting the presence or absence of apoptotic cells stained with the dye.

10. The method of claim 9, further comprising determining the amount of apoptotic cells and comparing the amount to a control. 10

11. The method of claim 10, wherein an increase in the amount of apoptotic cells, as compared to a control, indicates an agent or condition that induces apoptosis.

12. The method of claim 10, wherein a decrease in the amount of apoptotic cells, as compared to a control, indicates an agent or condition that inhibits or 15 reduces apoptosis.

13. An agent that modulates apoptosis, as determined by the method of claim 9.

14. A method for determining the apoptotic effect of the expression of at least one gene to be tested in a living nematode, wherein a mutation has been made to at least one region of the gene, comprising: 20 (a) contacting the living nematode with vital dye to stain apoptotic cells in the nematode; and (b) detecting the presence or absence of apoptotic cells stained with the dye. WO 99/66323 PCT/US99/13650 -33

15. The method of claim 14, further comprising determining the amount of apoptotic cells and comparing the amount to a control.

16. The method of claim 15, wherein an increase or decrease in the amount of apoptotic cells, as compared to a control, indicates that the gene expression 5 affects apoptosis.

17. The method of claim 16, wherein an increase in the amount of apoptotic cells indicates that the expression of the gene enhances apoptosis.

18. The method of claim 16, wherein a decrease in the amount of apoptotic cells indicates that the expression of the gene inhibits or reduces apoptosis. 10

19. A gene that modulates apoptosis, as determined by the method of claim 14.

20. A method of determining the presence or absence of one or more DNA damage induced apoptotic cells in a living Caenorhabditis elegans nematode, comprising: (a) contacting the living nematode with acridine orange to stain apoptotic 15 cells in the nematode; and (b) visually detecting apoptotic cells stained with acridine orange in the live nematode, thereby determining the presence or absence of apoptotic cells.

21. A method of determining the presence or absence of apoptotic cells in a live 20 nematode, comprising: (a) providing a nematode that has an altered sensitivity to radiation; (b) contacting the live nematode with vital dye to stain apoptotic cells in the nematode; and WO 99/66323 PCT/US99/13650 -34 (c) detecting the presence or absence of apoptotic cells stained with the dye in the live nematode.

22. The method of claim 21, wherein the nematode is exposed to an agent or condition to be tested. 5

23. The method of claim 22, wherein the nematode is sensitive to radiation, as compared to a control.

24. The method of claim 22, wherein the nematode is insensitive to radiation, as compared to a control.

25. The method of claim 22, comprising determining the amount of apoptotic 10 cells present and comparing the amount to a control.

26. A method for determining the presence or absence of one or more apoptotic cells in a living nematode, comprising: (a) maintaining a living nematode under conditions that modulate apoptosis; 15 (b) contacting the nematode with vital dye to stain apoptotic cells in the nematode; and (c) detecting the presence or absence of apoptotic cells stained with the dye in the living nematode.

27. A method of identifying a mutated, living organism having cells that undergo 20 an altered apoptotic cell death, comprising: (a) contacting an organism with an agent or condition that modulates apoptosis, thereby creating a mutated, living organism; (b) contacting the mutated organism with vital dye to stain the apoptotic cells in the organism; WO 99/66323 PCTIUS99/13650 -35 (c) detecting the presence or absence of apoptotic cells stained with the dye in the living, mutated organism; and (d) selecting the mutant organism having cells that undergo an altered apoptotic cell death, as compared to a control. 5

28. The method of claim 27, wherein the mutant is more sensitive to radiation that induces apoptotic cell death, as compared to a control.

29. The method of claim 27, wherein the mutant is less sensitive to radiation that induces apoptotic cell death, as compared to a control.

30. A mutated nematode, as identified according to claim 27. 10

31. A kit comprising: (a) at least one Caenorhabditis elegans nematode; and (b) a vital dye.

32. The kit of claim 31, wherein the vital dye is acridine orange or a SYTO dye.

33. The kit of claim 31, wherein the nematode is mutated so that the nematode is 15 sensitive or insensitive to radiation that induces apoptosis.

AU45731/99A
1998-06-19
1999-06-18
Detection of apoptotic cells in living nematodes

Ceased

AU746694B2
(en)

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US9005798P

1998-06-19
1998-06-19

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1998-06-19

PCT/US1999/013650

WO1999066323A1
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1998-06-19
1999-06-18
Detection of apoptotic cells in living nematodes

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2000-01-05

AU746694B2

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2002-05-02

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AU746694B2
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1998-06-19
1999-06-18
Detection of apoptotic cells in living nematodes

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AU746694B2
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CA
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CA2335549A1
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HU0103709A2
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GB9924983D0
(en)

1999-10-22
1999-12-22
Biocolor Limited
Apoptosis assay

US7083947B2
(en)

2000-05-19
2006-08-01
Devgen Nv
Assay techniques using nematode worms

CN101149366B
(en)

2007-10-16
2015-03-04
李红玉
Method for quickly quantifying eelworm death rate

US20110124523A1
(en)

2009-09-11
2011-05-26
Institute Of Life Sciences
High Throughput Method for Detecting Apoptosis of Embryonic Stages of Metazoan Helminthic Parasites

CN102213672B
(en)

2010-04-03
2015-12-02
李红玉
A kind of method of fast quantification nematode survival rate and kit

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US5593879A
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1993-01-15
1997-01-14
Massachusetts Institute Of Technology
Cell death genes of Drosophila melanogaster and vertebrate analogs

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1999-06-18
PL
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1999-06-18
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1999-06-18
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1999-06-18
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1999-06-18
AU
AU45731/99A
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CN1305588A
(en)

2001-07-25

HU0103709A2
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2002-01-28

AU746694B2
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2002-05-02

JP2002518674A
(en)

2002-06-25

WO1999066323A9
(en)

2000-04-06

PL345518A1
(en)

2001-12-17

CA2335549A1
(en)

1999-12-23

WO1999066323A1
(en)

1999-12-23

EP1088226A1
(en)

2001-04-04

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