WO1999066323A9 - Detection of apoptotic cells in living nematodes - Google Patents
Detection of apoptotic cells in living nematodesInfo
- Publication number
- WO1999066323A9 WO1999066323A9 PCT/US1999/013650 US9913650W WO9966323A9 WO 1999066323 A9 WO1999066323 A9 WO 1999066323A9 US 9913650 W US9913650 W US 9913650W WO 9966323 A9 WO9966323 A9 WO 9966323A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nematode
- apoptotic cells
- apoptosis
- apoptotic
- cells
- Prior art date
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Classifications
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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
Definitions
- 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 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.
- certain neurodegenerative diseases, cancer, or diseases caused by viruses involve the apoptotic pathway. Id. Assays that can screen, in vivo, for conditions or agents that modulate apoptosis, would significantly advance the treatment of these diseases.
- 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 in the nematode; and detecting the presence or absence of apoptotic cells stained with the dye.
- a living nematode e.g., Caenorhabditis elegans
- 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 (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 (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 (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 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 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 gene expression effects apoptosis.
- the invention also includes a gene(s) that modulates apoptosis, as determined by methods described herein.
- 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 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.
- the present invention involves methods of identifying a mutated, living organism having cells that undergo an altered apoptotic 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 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.
- kits that comprise at least one nematode (e.g., Caenorhabditis elegans); and a vital dye (e.g., acridine orange or a SYTO dye).
- nematode e.g., Caenorhabditis elegans
- a vital dye e.g., acridine orange or a SYTO dye.
- the nematode can be mutated so that the nematode is sensitive or insensitive to radiation that induces apoptosis.
- Figure 1 is a diagram of an adult hermaphrodite germ line.
- Figure 2 A contains graphical representations of the amount of germ cell apoptosis at 0, 12, 24 and 36 hours after irradiation of wildtype, ced-3, ced-4, rad-5, ced-9(gf) and egl-l(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).
- 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.
- ENU N-nitroso-N-ethylurea
- Figure 3 is a schematic showing that separate signaling pathways feed into 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 egl-l(n3082) animals.
- Figure 4A is a photograph of adult hermaphrodites that were scored for germ cell death by direct observation under differential interference contrast (DIC) optics.
- DIC differential interference contrast
- 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 (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.
- 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 UN 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 ( ⁇ 2) strains after gamma radiation with 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).
- 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.
- DIC differential interference contrast
- AO Acridine Orange
- 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).
- apoptosis e.g., DNA damage induced apoptosis
- 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.
- 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: ⁇ ⁇ 9- ⁇ 22, 119 (1998).
- Apoptosis also occurs when a cell is exposed to a stress, such as radiation, Ultra Violet (UV) light, toxic drugs, etc.
- a stress such as radiation, Ultra Violet (UV) light, toxic drugs, etc.
- UV Ultra Violet
- Cells that undergo apoptotic death shrink and are engulfed by a nearby cell, before the content 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.
- a receptor referred to as the "Fas receptor”
- 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.
- certain genes are expressed or inactivated.
- Caenorhabditis elegans C. elegans
- the 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.
- ced-9 is either partially or fully inactivated.
- a stress that damages the DNA of the cell can induce apoptosis.
- Studying 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.
- assays did not exist which allow one to quickly and efficiently screen for a large number of apoptotic cells.
- 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 reaction from the vital dye used to stain apoptotic cells for the assay.
- 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 fiuorescently. A fluorescent microscope can be used.
- a fluorescent microscope emits wavelengths that enables one to view the fiuorescently stained cells. Additionally, a machine that detects fiuorescently 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 assay.” Since the apoptotic cells are stained, they are easily and quickly identified.
- 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.
- the assay allows one to study apoptotic pathway in an in vivo system efficiently.
- 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 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 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 present in the background because the dye more easily permeates the retractile 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.
- concentration, kinetics of the dye, the amount of buffer, and amount of worms to be stained are factors that influence the length of time the dye should be in contact with the worms.
- AO Acridine Orange
- 500 ⁇ L of a 300 ⁇ g/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 fresh media or buffer.
- 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). 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 retractile, just as the membrane of germ cells.
- 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.
- a light source e.g., a fluorescent microscope or fluorescent plate reader
- the apoptotic cells can be viewed a low magnification (e.g., 10X-50X magnification) to obtain an overall assessment of whether more or less apoptosis is occurring.
- 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 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 are any number of conditions including time, temperature, pressure, and various media. Conditions can also include exposure to 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 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.
- 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 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 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 sensitive or insensitivity can be determined by exposing the mutant to different degrees of radiation.
- 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 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. Similarly, an agent can be tested to determine its effects on apoptosis.
- the low amount of radiation e.g., 1.0 kRads
- the radiation can be increased in an incremental fashion.
- 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 apopto
- 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.
- the nematodes are exposed to the agent to be tested.
- the length of time (e.g., 5 minutes-12 hours) and the concentration of the agent can be determined using methods known in the art. Various lengths of time and concentrations can be tested to determine optimal parameters for the desired effect.
- 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 concentrations, 5mM and ImM, 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.
- a range of exposure times can be tested and assessed using the apoptotic assay.
- 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 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 is an control in which the nematode is exposed to an agent that induces apoptosis.
- the agent to be tested can be compared 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.
- the nematode 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. After the L4 stage, the larvae becomes an adult. Epstein, H.F. et al,” Caenorhabditis 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.
- the present invention pertains to screening for cancer 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 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 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 lower doses (10-25mM) can reduce toxicity.
- a mutant nematode can then undergo the apoptotic assay to assess the effect the mutated gene has on apoptosis.
- the expression of the gene or genes in their non-mutated 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.
- 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 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.
- a mutant referred to as op 152
- the op 152 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, op 152 induces high levels of germ cell apoptosis, as compared to a control. See Example 3. An increase 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.
- the present invention can be used to screen one or more mutants that have cells that undergo an altered apoptotic cell death.
- Cells that 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 indicate that the nematode contains one ore more mutations that modulates apoptosis.
- the screening of a number of mutants can be done quickly and efficiently.
- 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 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.
- 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.
- C. elegans and its powerful genetics allow for significant contributions to the understanding of radiation-induced apoptosis and its 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 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 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.
- the present invention includes mutant nematodes and, in particular, radiation sensitive or insensitive mutants.
- the present invention also encompasses kit which comprise the wildtype nematodes, or mutants made and screened as described herein, and vital dye.
- C. elegans strains were maintained at 20 °C as described by Brenner (Brenner, S., "The genetics o ⁇ 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," N ⁇ twre 569:318-320 (1994). egl-l(n3082) was described by 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).
- Late L4 stage hermaphrodite worms were irradiated with the indicated doses of gamma radiation and scored after 0, 12, 24 and 36 hours. For each time-point, 15 animals were scored for germ cell death in one gonad arm via direct observation using standard ⁇ omaski optics. Data shown are means +/- sem (standard error of the mean). Strains used were ced-3(n717), ced-4(nll62), ced-9(nl950), egl-l(n3082) and rad-5(mnl59).
- induced germ cell death was also analyzed as described in Figure 2B. 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 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 in M9 buffer with the indicated concentrations of ENU, for 4 hours.
- worms were irradiated with 6000-24,000 ⁇ Joule/cm 2 of UN light (254nm) using a Stratalinker (Model 1800) crosslinker.
- Stratalinker Model 1800 crosslinker.
- synchronized animals 24 hours post the L4/adult molt were irradiated with 120 Gray and analyzed in 1 hour intervals. Counting apoptotic cells in the soma. Cell corpses in the head of ced-
- the brood size of rad-5 (mn 159) 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(mnl59) animals raised at 15 °C (AG, SM and MOH). ENU induced apoptosis. ⁇ U induces germ cell apoptosis. Hermaphrodites were synchronized in late L4 and treated in M9 buffer with the indicated concentrations of ⁇ U for 4 hours. Germ cell death was scored.
- D ⁇ A-damage-induced apoptosis is essential to remove genetically compromised cells that might become dangerous for the entire organism.
- damage-induced apoptosis is hampered by the lack of a genetic model system.
- C. elegans germ cells to undergo induced cell death was analyzed.
- C. elegans 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.
- the hermaphrodite germ line was chosen as an experimental system.
- germ cells progress through various stages of differentiation, as shown in Figure 1.
- Figure 1 at the distal end of the U-shaped ovotestis, mitotic stem cells proliferate throughout the worm's life.
- germ cells 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.
- meiotic cells The most abundant population of meiotic cells are arrested in 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 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" appear to serve a homoeostatic function as they occur under normal growth conditions.
- 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 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- induced germ cell death by time lapse video analysis were studied.
- 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.
- 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 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(nll62). ( Figure 2A). Radiation-induced cell death is also absent in the ced-9(nl950) gain of function mutant and strongly reduced the egl-l(n3082) loss of function mutant, Figure 2A-2C. Both ced-9 (nl950) and egl-l(n3082) completely suppress programmed cell death during somatic development but do not have an effect on physiological germ cell death.
- 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.
- 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.
- many of the resulting embryos die in a radiation dose-dependent manner at various stages of embryonic development, presumably as a delayed consequence 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 engulfment, 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 can not be extensive, as adult worms readily survive radiation doses of up to 480 Gray.
- Genotype extra cells in pharynx germ cell corpses average +/- sem. n avera ge +/- sem. n
- Rad-5 (mnl 59) 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.
- 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 thought to be essential for the elimination of damaged cells that might become harmful for the entire organism.
- damage-induced apoptosis which is often compromised in tumor cells, is thought to be essential for the elimination of damaged cells that might become harmful for the entire organism.
- C. elegans 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 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.
- 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, damage sensing induces a transient meiotic cell cycle arrest, whereas in mammalian spermatogenesis, it triggers either apoptosis or cell cycle arrest. In C.
- rad-5(mnl59) 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.
- the protocol used for staining the C. elegans is as follows:
- worms which stained above background were pulled from the plate. Ifthese 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 positives to be removed before a screen of the F3s was carried out.
- 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.
- the germline continues to proliferate throughout life.
- 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.
- dying germ cells are rapidly engulfed, such that normally only a few germ cell deaths can be observed at any given time.
- an assay was developed using the vital dye acridine orange, which detects apoptotic germ cells in living worms. ( Figure 4).
- 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 gamma or UV radiation, or to N-nitroso-N-ethylurea (ENU) ( Figures 5-7).
- opl52 induces high levels of germ cell apoptosis (see Table 4).
- the op 152 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.
- ced-9(n2812) III No +++ apoptosis ced-9(n2812) III; ced-3 (n717) IV Yes NA
- 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 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
- DIC differential interference contrast
- DIC optics 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 animals using DIC optics.
- Marinees are cellularized and more retractile 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, when compared with the number of apoptotic cells detected using DIC optics.
- FIG. 5 shows that Gamma irradiation induces apoptosis in the adult C. elegans hermaphrodite germ line. L4 larvae were transferred to agar plates seeded with E. coli and exposed to a Cs source (Mark I Model 68A Irradiator, JL Shepherd
- Example 4 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 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.
- 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.
- 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 Stasio E, et al, Genetics, 147:597-608 (1997).
- ENU N-nitroso-N-ethylurea
- 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 (Stratagene, CA), and the extent of germ cell death determined by visual inspection using DIC microscopy as described in Example 4.
- UV Stratalinker 1800 Stratagene, CA
- 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 68 A Irradiator, JL Shepherd & 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.
- Another radiation sensitive mutant, rid-2 was found using the AO vital dye 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 in 26+/- 3.6 corpses.
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