JPH09299077A - Control of apoptosis and composition therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To control cell apoptosis through U1-70-contg. tumor necrosis factor receptor route by transducing nucleic acid molecules having CrmA biological activity into cells to proliferate the cells under such conditions that the nucleic acid is transferred and translated in the cells.

SOLUTION: The purpose of this method is to control cell function subject to regulation through U1-70-contg. tumor necrosis factor receptor(TNF-R) route in appropriate cells. This cell function is regulated by cell-damaging T- lymphocytes(CTL) and the CTL-mediating route is apoptosis. The cell apoptosis is controlled as follows nucleic acid molecules having CrmA biological activity are transduced into the cells to proliferate them under such appropriate conditions that the nucleic acid is transferred and translated in the cells to produce a gene product having CrmA biologic at activity to control the activation of the U1-70 route.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION
No. 08 / 389,812 and No. 08 / 457,731, filed on February 13, 1995 and June 1, 1995, the content of which is hereby incorporated by reference. Incorporated.

This invention is partially supported by the US Government under respective grant numbers CA64803 and CA61348 from the National Institutes of Health. Accordingly, the US Government has certain rights in this invention.

The present invention relates to proteases, which are effector components of the mammalian cell death pathway. More particularly, the invention relates to nucleic acid molecules encoding proteases, recombinantly produced proteases, purified proteases, and uses of the proteases. Further, the invention relates to methods of regulating apoptosis in a cell population.

[0004]

BACKGROUND OF THE INVENTION Apoptosis, or programmed cell death (PCD), is involved in biological processes including embryogenesis, maintenance of tissue homeostasis, normal cell development of multicellular organs, elimination of virus-infected cells, and development of the immune system. Fundamentally important (Ellis et al., (1991) Ann. Rev. Cell Bio
l. 7: 663-698). This is a type of cell death that is fundamentally distinct from degenerative or necrosis, in that it is an active process of gene-directed cell self-destruction, which plays a biologically important homeostasis function, for example. Is. On the contrary, necrosis is cell death that occurs as a result of severe wound changes in the environment of infected cells. For a review of apoptosis, see Tomei, LD and Cope, FO Apoptosis: The Mol.
ecular Basis of Cell Deth (1991) Cold Spring Harbo
r Press, NY; Tomie, LD and Cope, FO Apoptos
is II: The Molecular Basis of Apoptosis in Disease
(1994) Cold Spring Harbor Press, NY; and Duval
See l and Wyllie (1986) Immun. Today 7 (4): 115-119.

Morphologically, apoptosis is characterized by rapid condensation of cells with membrane retention. Concomitant with chromatin compaction, several biological changes occur intracellularly. Nuclear DNA is cleaved at the internucleoside linker region, yielding a fragment that is readily demonstrated by agarose gel electrophoresis in which a characteristic ladder is developed.

A number of apoptotic triggers that induce cell death have been identified. Fas antigen (CD95 / APO-1)
Is a member of the tumor necrosis factor (TNF) receptor superfamily. Fas antigen is a transmembrane protein with a wide tissue distribution. Upon activation of binding to a ligand or an agonistic anti-Fas antibody, Fas triggers apoptosis. Cytotoxic T lymphocytes activate Fas on target cells to induce cytolysis. TNF is also known to induce apoptosis when cross-linked with its ligand or agonist antibody.

In addition to being linked to many of the biological processes identified above, apoptosis also induces CD
Human immunodeficiency virus (HIV) of 4 ⁺ T lymphocytes (T cells)
It occurs as a result of infection. In fact, one of the main features of AIDS is the progressive reduction of CD4 ⁺ T lymphocytes during disease development.
Several mechanisms, including apoptosis, have been suggested to be responsible for CD4 ⁺ depletion. The decrease of CD4 ⁺ T cells is
It leads to aggravation of the cellular immune response. Inappropriate activation-induced T cell PCD is guided to substantially disrupt the patient's immune system, can cause functional and numerical abnormalities of T _H cells from HIV-infected patients, it has been proposed. The mechanism by which the Fas antigen initiates the apoptotic cascade, and its various components, remain poorly understood. However, the present invention has identified several components of the apoptotic cascade and methods of regulating apoptosis.

[0008]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above problems, and an object of the present invention is to provide a tumor necrosis factor receptor (TNF-R) containing U1-70 in appropriate cells. ) Methods of modulating cellular functions regulated by the pathway, methods of preventing and inhibiting apoptosis in the appropriate cells by inhibiting activation of the U1-70 pathway, identifying agents involved in the apoptotic pathway in the appropriate cells The present invention provides a method for screening a candidate drug having a biological function in the apoptotic pathway, and a drug identified by the above method.

[0009]

The method of the present invention is directed to tumor necrosis factor receptors (T1-T) containing U1-70 in appropriate cells.
NF-R) pathway is a method of modulating cellular function, the step of introducing into the cell a nucleic acid molecule having CrmA biological activity, such that the nucleic acid is transcribed and translated in the cell Growing the cells under suitable conditions.

According to a preferred embodiment, the cell function includes a cell function regulated by cytotoxic T lymphocytes (CTL).

In a preferred embodiment, the CTL-mediated pathway is CTL-mediated apoptosis.

In a preferred embodiment, the CTL mediated pathway is calcium (Ca ²⁺ ) dependent CTL mediated killing.

The method of the present invention is a method for preventing and inhibiting apoptosis in an appropriate cell by inhibiting activation of the U1-70 pathway, which encodes a gene product having CrmA biological activity in the cell. The step of introducing an effective amount of a nucleic acid molecule of the invention under suitable conditions such that the cleavage of U1-70 is prevented or inhibited.

The method of the present invention is a method for identifying an agent involved in an apoptotic pathway in a suitable cell, comprising the steps of: a) providing a cell culture or tissue culture having a cell surface receptor that mediates apoptosis. B) exposing the cell or tissue culture to preliminary conditions required for apoptosis; c) dividing the cell or tissue culture into test and control samples; d) contacting the agent to be tested with the cell or tissue culture of the test sample; e) a nucleic acid molecule or CrmA encoding a gene product having CrmA biological activity relative to the control sample. Contacting the gene product with the cell or tissue culture of the control sample under conditions that inhibit apoptosis; f ) Contacting the test sample with a nucleic acid molecule encoding a gene product having a CrmA biological activity for the test sample or a CrmA gene product under conditions that inhibit apoptosis of the cells; g) step e ) And f) culturing the sample under conditions that inhibit apoptosis; and h) assaying the sample in step g) for apoptosis by assaying for cleavage of the U1-70kDA protein. .

Agents involved in the apoptotic pathway in suitable cells of the invention are identified by the methods described above.

The method of the present invention is a method for screening a candidate drug having a biological function in the apoptotic pathway, which comprises the following steps: a) U1-70 in an effective amount with an activating drug and U1-70 at 40 kDa
Incubating under conditions suitable for cleaving into molds; and b) assaying to determine if cleavage of U1-70 has occurred.

A candidate drug having a biological function in the apoptotic pathway of the present invention is identified by the above method.

[0018]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides non-natural and isolated natural nucleic acids encoding proteins designated Yama, Pro Yama, activated Yama, p20 Yama, p11 Yama, and mutant Yama. Provide a molecule (polynucleotide).

The present invention also provides a recombinant nucleic acid molecule having the nucleic acid molecule sequence shown in FIG. Furthermore, the present invention provides fragments of the above nucleic acid molecules. In one embodiment, the fragment has at least 8 nucleobases for use as a probe or primer. Specific examples of these fragments are nucleic acid molecules that encode the polypeptides referred to herein as p20 Yama and p11 Yama. Also provided are DNA and RNA polynucleotides complementary to these nucleic acid molecules.

The present invention also provides a recombinant nucleic acid molecule encoding a polypeptide having the amino acid sequence shown in FIG. 2 or a fragment of this amino acid sequence or polypeptide. The invention further provides non-natural nucleic acid molecules encoding a variant CrmA protein and a dominant negative Yama. Further provided are vectors and host cells containing these nucleic acid molecules.

Professional Yama, Activated Yama, p20 Yama, p11 Ya
Also provided herein are purified and recombinantly produced proteins and polypeptides designated ma, mutant CrmA, and mutant Yama.

The present invention also provides a non-natural nucleic acid molecule encoding a mutant CrmA protein, and nucleic acid molecules complementary to this molecule (RNA and DNA) and polypeptides encoded thereby.

These nucleic acid molecules can be inserted into a vector, and such a vector is further provided by the present invention. Also provided herein are host cells, ie, prokaryotic and eukaryotic cells, containing the inserted nucleic acid molecules.

[0024] In one embodiment, the Yama protein of the present invention promotes apoptosis upon activation of SDS-PA.
It is a substantially purified natural zymogen polypeptide with an apparent molecular weight of about 32 kDa as measured by GE. In another embodiment, the Yama protein is a non-natural zymogen polypeptide with an apparent molecular weight of about 32 kDa as measured by SDS-PAGE that promotes apoptosis upon activation. In a further embodiment, the Yama protein is a non-naturally occurring polypeptide having an amino acid sequence that includes the sequence shown in Figure 2. In a further embodiment, the Yama protein is a non-naturally occurring polypeptide comprising fragments having apparent molecular weights of about 20 kDa and 11 kDa as determined by SDS-PAGE. The actual molecular weights of these polypeptides are 17 kDa and 12 kDa, respectively.

The present invention also provides a complex comprising a CrmA polypeptide and a protein called Yama.

A process for producing a Yama protein or polypeptide is also provided. This process is carried out as described above for a host cell, wherein the host cell contains a nucleic acid molecule encoding a Yama protein, and wherein the nucleic acid molecule is operably linked to a promoter of RNA transcription. A step is required to grow the host cell under suitable conditions such that the nucleic acid is transcribed and translated into Yama protein or polypeptide protein.
Another process of the invention is to chemically synthesize a Yama protein or polypeptide by chemically linking amino acids according to a directed amino acid sequence under conditions suitable to produce the Yama protein or polypeptide. The process of doing is provided. Further provided is a method for chemically replicating a nucleic acid molecule encoding a Yama protein or polypeptide by chemically linking nucleotides according to an oriented nucleic acid sequence under conditions suitable to produce the nucleic acid. To be done.

Also provided by the invention is a method of modulating cellular function that is regulated by the Fas receptor pathway in appropriate cells. In one embodiment, the method comprises introducing a Fas regulatory nucleic acid molecule into the cell and growing the cell under suitable conditions such that the nucleic acid is transcribed and translated into the Fas regulatory protein in the cell. It requires a process to Two examples of these proteins are Yama nucleic acids and nucleic acids with CrmA biological activity. Introduction into cells can be effected in vitro, in vivo, or ex vivo.

[0028] Also herein, an effective amount of a nucleic acid molecule encoding a gene product having CrmA biological activity, and under appropriate conditions so that apoptosis is prevented or inhibited, is applied to cells. By introducing
Methods for preventing or inhibiting apoptosis in suitable cells are also provided. An example of this nucleic acid molecule is
Cowpox virus CrmA DNA or a biologically active fragment thereof. Alternatively, the method uses a dominant negative Ya
It may be carried out using a nucleic acid molecule encoding ma.

Further, a method of maintaining T cell viability in a subject infected with human immunodeficiency virus is described in which an effective amount of a gene product having CrmA biological activity in the subject, and T cells survived. It is provided herein by administration under suitable conditions as such. This method can also be performed using a nucleic acid molecule that comprises a polynucleotide encoding a dominant negative Yama.

Antibodies that specifically recognize and bind the Yama protein or fragments thereof are also provided herein. The antibody can be polyclonal or monoclonal. The invention further provides fragments of these antibodies, and recombinant or chimeric molecules comprising the antibodies or antibody fragments. Anti-idiotypic antibodies to these antibodies, and hybridoma cell lines that produce the claimed monoclonal antibodies, are also provided herein. Compositions containing these antibodies are also provided.

Provided herein are methods of identifying agents associated with the apoptotic pathway in suitable cells. The method comprises the steps of (a) providing a cell or tissue culture having cell surface receptors that mediate apoptosis; (b) pre-conditioning the cell or tissue culture with apoptosis. Exposure to; (c) dividing this cell or tissue culture into a test sample and a control sample; (d) contacting the agent to be tested with the cell or tissue culture of the test sample And (e) contacting a control sample with a gene product having CrmA biological activity or a nucleic acid molecule encoding the CrmA gene product under conditions that inhibit apoptosis in cell culture or tissue culture of the control sample. The step of: (f) the test sample,
Contacting a test sample with a gene product having CrmA biological activity or a nucleic acid molecule encoding a CrmA gene product under conditions that inhibit cell apoptosis; (g)
Culturing the sample of steps (e) and (f) under conditions that inhibit apoptosis; and (h) assaying the sample of step (g) for apoptosis. One embodiment of this method includes the additional step of identifying factors that increase apoptosis and are inhibited by the CrmA gene product by the absence of apoptotic morphological changes or inhibition of cell death in the test sample. To be done. The assay for cleavage of the PARP or U1-70kDA protein is one means for testing for apoptotic activity and whether the agent modifies the apoptotic cascade.

The present invention also provides a screening method for a candidate drug having a biological function in the apoptotic pathway. This method comprises the steps of: (a) incubating an effective amount of pro-Yama with an activating agent under appropriate conditions to activate Yama; (b) partitioning activated Yama into at least two reaction solutions. (C) contacting activated Yama with an effective amount of the agent to be tested; (d) each activated Yama under appropriate conditions such that Yama is inhibited. , Contacting an effective amount of CrmA; (e) contacting an effective amount of PARP with the solution of steps (c) and (d); and (f) analyzing the solution of step (e) , A step of determining whether the agent inhibits cleavage of PARP to its 85kDa form (the absence of the 85kDa form indicates that the drug is a candidate with a biological function in the apoptotic pathway), Need. This method can also be performed by incubating with the U1-70 kDa protein described herein and assaying to determine if the drug inhibits cleavage of the protein into the 40 kDa form. Drugs identified by this method are within the scope of the invention.

The present invention further introduces into a suitable cell a nucleic acid molecule encoding a gene product or a gene product having a CrmA biological activity such that induced apoptosis is prevented or inhibited. Thus, compositions and methods for preventing or inhibiting this suitable intracellular apoptosis are provided. Nucleic acid molecules suitable for this method include, but are not limited to, a dominant negative Yama or CrmA gene product.

By subjecting a subject infected or susceptible to human immunodeficiency virus with a nucleic acid molecule encoding a gene product having apoptotic biological activity or an effective amount of this gene product, the Compositions and methods for maintaining T cell viability in a subject include:
Further provided by the present invention. These are, for example:
It may be a CrmA nucleic acid, a dominant negative Yama nucleic acid, or the gene product of these nucleic acid molecules.

Throughout this application, various publications, patents, and published patent applications are referred to by specific citations. The disclosures of these publications, patents, and published patent applications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention belongs.

As is known to those skilled in the art, apoptosis is
It is an active process of gene-controlled cell self-destruction. The present invention provides compositions and methods for preventing or inhibiting apoptosis in appropriate cells or appropriate cell populations. In one embodiment, the method comprises CrmA.
Introducing an effective amount of a nucleic acid molecule encoding a biologically active gene product into the cell (s). The method can also be performed using the gene product itself. Apoptosis inducers (eg anti-TCR, tumor necrosis factor (TNF), HIV, SIV cytotoxic T lymphocytes (CT
It is important to note that the method of the invention inhibits apoptosis even in the presence of L), or receptor ligands such as anti-Fas antibodies). Thus, this method provides an improvement over prior art methods, in which apoptosis interferes with the induction pathway at the ligand-induced level (eg, interferes with ligand binding to its cell surface receptor). Antibodies or anti-ligand antibodies). However, the present invention may be combined with the use of such prior art methods to inhibit apoptosis. The present invention has also been shown to inhibit apoptosis
bcl-2 type factor, nucleic acid, etc. (Lacronique, V. et al. (1996) Na
ture Medicine 2 (1): 80-86).

The present invention also provides a purified or recombinantly produced protease called Yama, which comprises:
Involved in the mammalian cell death pathway and nucleic acid molecules encoding this protease. Upon activation, purified Yama constructs poly (ADP-ribose) polymerase (PARP) with the structure (signa
ture) has been shown to be a zymogen responsible for the proteolytic reaction type, which cleaves into an 85 kDa apoptotic fragment. PARP was identified as a death substrate that is specifically cleaved during apoptosis. Kaufmann et al. (1989) Cance
r Res. 49: 5870-5878 and (1993) 53: 3976-3985, 11
Reported that the 6-kDa nuclear protein is specifically cleaved to produce an 85-kDa fragment in many types of PCD, including chemotherapeutic-induced PCD in cell lines and dexamethasone-induced PCD in thymocytes. To do. Later, Lazebnik, Nature (1994) 37
1: 346-347 ensures that cleavage occurs at the C-terminus of Asp in a cell-free system, and that proteases are similar to ICE in their sensitivity to chemical inhibitors, and purified ICE does not cleave PARP and therefore ICE It was reported to be different. Yama is further characterized in that its proteolytic and apoptotic activities are inhibited by purified CrmA, but not by the equivalent amount of inactive point mutants of CrmA. As described in detail in the Examples section below, CrmA blocked proteolytic cleavage of PARP in cells in which progression of apoptosis was induced.

Definitions The terms "protein", "peptide", and "polypeptide" are used interchangeably and are purified and recombinantly produced to contain amino acids linearly linked through peptide bonds. It is intended to include such molecules.
The amino acids of the invention can be in the L or D form, so long as the biological activity of the polypeptide is maintained. For example, the protein can be modified to be secreted from the cell for recombinant production or purification. The proteins of the invention also include proteins that are post-translationally modified by reactions that include glycosylation, acetylation, and phosphorylation. Such polypeptides may also contain analogs, alleles, and amino acid derivatives or non-amino acid moieties that do not affect the biological or functional activity of the protein as compared to the wild-type or naturally occurring protein. Includes allelic variants. The term “amino acid” refers to naturally occurring amino acids, as well as Ty.
Refers to rMe and their derivatives such as PheCl, as well as other moieties characterized by the presence of both available carboxyl and amine groups. Non-amino acid moieties that can be contained in such polypeptides include, for example, amino acid mimetic structures. A mimetic structure is one that exhibits substantially the same spatial arrangement of reactive groups as an amino acid, but does not necessarily have both the α-amino and α-carboxyl groups characteristic of amino acids.

A "mutein" is, for example, a protein or polypeptide with a small modification in the amino acid sequence caused by site-directed mutagenesis or other manipulations; errors in transcription or translation; or synthetically prepared by rational design. A protein or polypeptide having a small modification in the amino acid sequence. These small modifications result in amino acid sequences in which the biological activity of the protein or polypeptide is modified compared to the wild-type or naturally occurring polypeptide or protein. Examples of muteins include the CrmA mutants and Yama mutants described herein.

[0040] As used herein, the term "peptide bond" or "peptide linkage" refers to an amide linkage between the carboxyl group of one amino acid and the α-amino group of another amino acid.

As used herein, the term "hydrophobic" is intended to include amino acids, amino acid derivatives, amino acid mimetics, and chemical moieties that are non-polar. Hydrophobic amino acids include Phe, Val, Trp, Ile, and Le.
Including u. As used herein, the term "positively charged amino acid" refers to positively charged amino acids, amino acid derivatives, amino acid mimetics, and chemical moieties. Amino acids having a positive charge are, for example, Lys, A
rg, and His are included.

“Purified” when referring to a protein or polypeptide is distinguishable from a naturally occurring or naturally occurring protein or polypeptide as they exist in the purified state. These “purified” proteins or polypeptides, or any intended variants described herein, are, in the narrow sense, the compounds or molecules with which they are ordinarily associated in their natural or natural environment. It is to be substantially free of impurities. The terms "substantially pure" and "isolated" refer to
It is not intended to exclude mixtures of polynucleotides or polypeptides with substances that are not naturally associated with the polynucleotides or polypeptides.

A "native" polypeptide, protein, or nucleic acid molecule refers to a polypeptide, protein, or nucleic acid molecule that is removed from a source that exists in a natural or "wild type."

“Composition” is intended to mean the combination of an active agent with an inactive (eg, detection agent or label) or other active compound or composition (eg, adjuvant).

"Pharmaceutical composition" is intended to include the combination of an active agent with an inert or active carrier, whereby it can be used for diagnostic or in vitro, in vivo, or ex vivo purposes. Make a composition suitable for use.

As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier such as phosphate buffered saline solution, water, and oil / water. Or water / oil emulsions), as well as various types of wetting agents. The composition may also include stabilizers and preservatives. Examples of carriers, stabilizers, and adjuvants are Martin, Remi
ngton's Pharm. Sci. , 15th Edition (Mack Publ. Co., Easton
(1975)).

The term "nucleic acid" refers to single-stranded and double-stranded DNs.
By A, cDNA, genomic DNA, and RNA, and the positive and negative strands of nucleic acids that are complementary to each other, including antisense RNA. "Nucleic acid molecule" means "polynucleotide"
The term is used interchangeably with and each refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. It also includes known types of modifications (eg, labels known in the art (eg, Sa
Mbrook et al. (1989)), methylation, "cap"), 1
Substitutions with analogs of one or more naturally occurring nucleotides, internucleotide modifications (eg, those with uncharged linkages (eg, methyl carbamate, etc.), those with pendant moieties (eg, proteins (eg, nucleases, toxins, (Including antibodies, signal peptides, etc.)), those having an intercalator (eg, acridine, psoralen, etc.), those containing a chelator (eg, metal, radioactive metal, boron, metal oxide, etc.), containing an alkylator Those having a modified linkage (for example, α-anomeric nucleic acid) and unmodified polynucleotides. The polynucleotide may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases. Nucleotides are mRNAs that encode polypeptides
Can be complementary to. These complementary nucleotides are
Includes, but is not limited to, nucleotides that can form triple helices and antisense nucleotides. Recombinant polynucleotides having other non-naturally occurring sequences are also provided by the invention. This includes modifications of the wild-type polynucleotide sequence, including modifications by deletion, insertion, and substitution of one or more nucleotides, or modifications by fusion with other polynucleotide sequences. Not limited.

A polynucleotide "encodes" a polynucleotide if it is capable of being transcribed and / or translated to produce a polypeptide or mature protein when engineered by its natural state or by methods well known to those of skill in the art. It will be done. " Thus, the term "polynucleotide" should include the coding sequences as well as processing sequences and other sequences that do not encode amino acids of the mature protein. The antisense strand of such a polynucleotide is also said to encode the sequence.

The term “recombinant” polynucleotide or DN
A is achieved by artificial manipulation of a segment of DNA isolated by genetic engineering or chemical synthesis 2
A polynucleotide made by the combination of two, otherwise separated segments of sequence.
By doing so, the DNA segments of the desired function may be linked together to create the desired combination of functions.

"Analogs" of DNA, RNA, or polynucleotides occur naturally in form and / or function, particularly the ability to ensure sequence-specific hydrogen bonding to the base pairs of complementary polynucleotide sequences. A macromolecule that is similar to a polynucleotide, but differs from DNA or RNA, for example, by having unusual or unnatural bases or a modified backbone. For example, Uhlm
See ann et al. (1990) Chemical Reviews 90: 543-584.

"Isolated," when referring to a nucleic acid molecule, means separated from other cellular components normally associated with natural or wild type DNA or RNA within the cell.

"Hybridization" refers to hybridization reactions that can be performed under conditions of different "stringency". Conditions that increase the stringency of a hybridization reaction are well known and published in the art: see, eg, Sambrook et al., Supra. Examples of suitable conditions (to increase stringency) include: 25 ° C, 37 ° C, 50
Incubation temperature of ℃, and 68 ℃; 10 × SSC, 6
× SSC, 1 × SSC, 0.1 × SSC (where SSC is 0.15M NaCl
And 15 mM citrate buffer) and their equivalents using other buffer systems; 0%, 25%,
Formamide concentrations of 50%, and 75%; incubation times of 5 minutes to 24 hours, and increased duration, increased number of times, or reduced buffer concentration washes.

"T _m " means that, by Watson-Crick base pairing, 50% of a polynucleotide duplex consisting of complementary strands hydrogen-bonded in antiparallel directions is dissociated into single strands under experimental conditions. The temperature is in degrees Celsius. T _m can be predicted according to standard formulas: for example, T _m = 81.5 + 16.6log [Na ⁺ ] ⁺ 0.41 (% G / C) -0.61 (% F)-
600 / L where Na ⁺ is the cation (usually sodium ion) concentration of mol / L; (% G / C) is the G and C residue as a percentage of all residues in the duplex. Is the number of groups; (% F) is
Percent formamide in solution (wt / vol); and L is the number of nucleotides in each strand of the duplex.

A "stable duplex" of a polynucleotide, or a "stable complex" formed between any two or more components in a biochemical reaction, is the formation of a duplex or complex and subsequent formation. A duplex or complex that is sufficiently long lasting to persist between the detection of Duplexes or complexes are those that are the effect of the assay or reaction being performed, whatever the conditions are, or what conditions are introduced between the time of formation and the time of detection. It must be able to withstand the conditions. Can exist at will, and 2
Interfering conditions that can remove heavy chains or complexes include washing, heating, addition of additional solutes or solvents to the reaction mixture (eg, denaturants), and competition with additional reactive species. A stable duplex or complex can be irreversible or reversible, but must meet the other requirements of this definition. Thus, transient complexes can form in the reaction mixture, but are stable if they dissociate spontaneously or as a result of newly imposed conditions or manipulations introduced prior to detection. Does not form a complex.

A strand is essentially "complementary" when a stable duplex is formed in an antiparallel configuration between two single-stranded polynucleotides, particularly under conditions of high stringency. . A double-stranded polynucleotide may be "complementary" to another polynucleotide when a stable double strand can be formed between one strand of the first polynucleotide and the second polynucleotide strand. Complementary sequences deduced from single-stranded polynucleotide sequences are standard nucleotide optimal sequences predicted to form hydrogen bonds with single-stranded polynucleotides according to generally accepted base pairing rules. Is.

"Sense" and "antisense" strands, when used in the same context, refer to single-stranded polynucleotides that are complementary to each other. They can be the opposite strands of a double-stranded polynucleotide, or one strand can be predicted from the other according to the generally accepted rules of base pairing. Unless specified or implied, assigning one or the other strand as "sense" or "antisense" is optional.

A linear sequence of nucleotides is "identical" to another linear sequence if the sequence of nucleotides in each sequence is the same and there is no substitution, deletion or substantial substitution. Purine and pyridine nitrogenous bases with similar structures can be functionally equivalent with respect to Watson-Crick base pairing; and mutual substitution or methylation of similar nitrogenous bases (especially uracil and thymidine). Modification of the nitrogenous base as such does not constitute a substantial substitution. RNA and DNA polynucleotides refer to sequences for RNA that reflect the order of nitrogenous bases in polyribonucleotides, sequences for DNA that reflect the order of nitrogenous bases in polydeoxyribonucleotides, and When an array meets the other requirement of this definition, it has an identical array. When at least one sequence is a degenerate oligonucleotide having ambiguous residues, the two sequences are such that at least one alternative form of the degenerate oligonucleotide is identical to the sequence being compared. , Are the same. For example, AYAAA is A
It is identical to ATAAA when YAAA is a mixture of ATAAA and ACAAA.

Complementary strands are readily created when a comparison is made between polynucleotides and a sense or antisense strand is chosen that maximizes the degree of identity between the compared polynucleotides, or What is expected is implicitly understood. For example, if one or both of the polynucleotides being compared is double-stranded, when one strand of the first polynucleotide is identical to one strand of the second polynucleotide, then their sequences are identical. . Similarly, when a polynucleotide probe is described as being identical to its target, it is understood that the polynucleotide probe is the complementary strand of the target involved in the hybridization reaction between the probe and the target. It

A linear sequence of nucleotides is "essentially identical" to another linear sequence when both sequences are capable of forming a duplex with the same complementary polynucleotide by hybridization, or "Equivalent". Applicants have not always explicitly stated to refer to a particular nucleic acid molecule, but it should be understood that equivalents thereof are also contemplated. More preferred are sequences that hybridize under conditions of higher stringency. It is understood that hybridization reactions can accommodate insertions, deletions, and substitutions in nucleic acid sequences. Thus, a linear sequence of nucleotides can be essentially identical, even if some of the nucleotide residues do not exactly correspond or align. Sequences that more closely correspond to, or align with, the invention disclosed herein are equally more preferred. Generally, a polynucleotide region of about 25 residues is referred to when its sequences are at least 80% identical, more preferably when they are at least 90% identical; more preferably they are at least 95% identical. When they are identical; even more preferably when they are 100% identical,
It is essentially the same as the other areas. A polynucleotide region of 40 residues or more, after alignment of the homologous portions, when the sequences are at least about 75% identical; more preferably when they are about 80% identical; more preferably at least about 85% identical; more preferably they are at least about 90% identical; even more preferably, they are essentially identical to other regions when their sequences are 100% identical.

In determining whether the polynucleotide sequences are essentially identical, sequences that retain the functionality of the compared polynucleotides are particularly preferred. Functionality can be determined by different parameters. For example, when a polynucleotide is used in a reaction involving hybridization with other polynucleotides, the preferred sequences are those that hybridize to the same target under similar conditions. In general, the T _m of a DNA double strand decreases by about 10 ° C. for every 1% decrease in sequence identity for double strands of 200 residues or more, depending on the position of the mismatch residue.
For duplexes less than residues, there is a reduction of about 50 ° C (eg,
See Meinkoth et al.). Essentially identical or equivalent sequences of about 100 residues form stable duplexes with individual complementary sequences to each other at about 20 ° C. below the T _m ;
Preferably, they form stable duplexes below about 15 ° C; more preferably they form stable duplexes below about 10 ° C; more preferably about 2 ° C below 2 ° C. Form duplexes; even more preferably, they form stable duplexes at about T _m . In another example, when the polypeptide encoded by the polynucleotide is an important part of its functionality, the preferred sequence is the sequence encoding the same or essentially the same polypeptide. Therefore, nucleotide differences that cause conservative amino acid substitutions are preferred over differences that cause non-conservative substitutions, and nucleotide differences that do not alter the amino acid sequence are more preferred, while identical nucleotides are more preferred. Nucleotide insertions or deletions that result in insertions or deletions in the polypeptide are preferred over those that result in out-of-phase downstream coding regions; polynucleotide sequences without insertions or deletions are more preferred. The hybridization characteristics and the relative importance of the polynucleotide encoded polypeptide sequences depend on the application of the invention.

Polynucleotides have the same characteristics, or are equivalent to other polynucleotides, when both are capable of forming stable duplexes with a particular third polynucleotide under similar conditions of maximum stringency. It is a thing. Preferably, in addition to similar hybridization properties, the polynucleotides also encode essentially the same polypeptide.

A "conserved" residue in a polynucleotide sequence is a residue that is present unchanged at the same position in two or more related sequences being compared. Residues that are relatively conserved are those that are more conserved between related sequences than are residues that occur elsewhere in the sequence.

A "related" polynucleotide is a polynucleotide that shares a major portion of the same residues.

A "degenerate" oligonucleotide sequence, as used herein, is a designed sequence derived from at least two related source polynucleotide sequences: Residues that are conserved in the degenerate sequence but not in the original sequence may be provided as some alternatives in the degenerate sequence. For example, the degenerate sequence AYASA can be designed from the origin sequences ATACA and ACAGA. Where Y is C or T and S is C or G. Y and S are examples of "ambiguous" residues. A degenerate segment is a segment of polynucleotide having a degenerate sequence.

It is understood that synthetic oligonucleotides containing degenerate sequences are virtually a mixture of closely related oligonucleotides sharing the same sequence, except at ambiguous positions. Such oligonucleotides are usually synthesized as a mixture of all possible combinations of nucleotides at ambiguous positions. Each oligonucleotide in the mixture is called an "alternative."

As used herein, a polynucleotide "fragment" or "insert" generally refers to a subsection of full length form, although full length polynucleotides may also be included.

Different polynucleotides "correspond" to each other when one is essentially due to the other. For example, messenger RNA corresponds to the gene for which it is transcribed. A cDNA corresponds to the RNA for which it is produced, for example, by the reverse transcription reaction or by chemical synthesis of DNA based on knowledge of the RNA sequence. cDNA also corresponds to the gene encoding RNA. Polynucleotides also "correspond" to each other in the different species, strains, or mutants with which they are compared, when they serve a similar function (eg, encode a related polynucleotide).

A "probe" when used in the context of polynucleotide manipulation is an oligo that is provided as a reagent for detecting a target that may be present in a sample of interest by hybridizing with the target. Refers to nucleotides. Usually the probe is a label or
It contains means by which the label can be attached either before or after the hybridization reaction. Suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins including enzymes.

A "primer" is a general primer that binds to a target that may be present in a sample of interest by hybridizing with the target and then promotes the polymerization of a polynucleotide complementary to the target. In addition, it is an oligonucleotide having a free 3'-OH group.

The process of producing duplicate copies of the same polynucleotide (eg, PCR or gene cloning) is collectively referred to herein as "amplification" or "replication." For example, single-stranded DNA or double-stranded DNA can be replicated to form other DNA having the same sequence. R
NA can be replicated, for example, by RNA-specific RNA polymerase or by reverse transcribing DNA and performing PCR. In the latter case, the amplified copy of RNA is DNA having the same sequence.

"Polymerase chain reaction" PCR "is the process whereby a replicative copy is derived from a target polynucleotide using one or more primers and a polymerization catalyst such as reverse transcriptase or DNA polymerase and especially a heat stable polymerase enzyme. It is a reaction. In general, PCR involves three iterative steps: "annealing", in which the oligonucleotide primers can form a duplex with the polynucleotide to be amplified.
The temperature is adjusted; "extension", in which the temperature is such that the double-stranded oligonucleotides are extended by a DNA polymerase using the double-stranded polynucleotides as templates. Is adjusted; and “melting”, in which the temperature is adjusted so that the polynucleotide and the extended oligonucleotide dissociate. This cycle is then repeated until the desired amount of amplified polynucleotide is obtained. The PCR method is taught in US Pat. Nos. 4,683,195 (Mullis) and 4,683,202 (Mullis et al.).

The elements in the gene include, but are not limited to, a promoter region, an enhancer region, a repressor binding region, a transcription initiation site, a ribosome binding site,
Included are translation initiation sites, protein coding regions, introns and exons, and transcriptional and translational stop sites. An "antisense" copy of a particular polynucleotide refers to complementary sequences that can hydrogen bond with the polynucleotide and thus regulate the expression of the polynucleotide. These are DNA, RNA, or analogs thereof, including analogs with altered degenerate backbones, as described above. The polynucleotide to which the antisense copy binds can be single-stranded or double-stranded.

As used herein, the term "operably linked" refers to a DNA molecule that requires the necessary regulatory sequences (eg,
(Promoter or enhancer) means that the promoter is arranged so that it is directed to RNA transcription from DNA in a stable or transient manner.

“Vector” means a self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. This term refers to vectors that primarily function to insert nucleic acid molecules into cells, replication vectors that primarily function to replicate nucleic acids, and DNA or RNA.
It is intended to include expression vectors that function for transcription and / or translation of the. Vectors that provide one or more of the above functions are also contemplated.

"Host cell" is meant to encompass a vector or individual cell or cell culture that can be or is a receptor for uptake of exogenous nucleic acid molecules and / or proteins. Intent. It is also intended to encompass the progeny of a single cell, which progeny are, due to natural, accidental, or deliberate mutations, perfectly matched to the original parental cell (morphology or (In genomic or total DNA complementarity) may not necessarily be required.

An “antibody” is an immunoglobulin molecule capable of binding an antigen. As used herein, the term
It includes complete immunoglobulin molecules as well as anti-idiotypic antibodies, variants, fragments, fusion proteins, humanized proteins, and variants of immunoglobulins having antigen recognition sites of the desired specificity.

An "antibody complex" is a combination of an antibody (as defined above) and its binding partner or ligand.

“Suitable cells” for the purposes of the present invention include, but are not limited to, cells expressing the Fas receptor, such as bone marrow cells, endothelial cells, breast cancer cells, fibroblasts, epithelial cells, epithelial tumor cells (Spriggs, DR et al. (1988) J. Clin. I
nves. 81: 455-460), T cells (TCR ⁺ , CD
8 ⁺ or CD4 ⁺ T cells), peripheral blood lymphocytes, leukocytes, and mixed leukocyte cultures (MLC), B-lymphoma cells (ATC
C, A202J), colon cells, small intestine cells, ovary cells, testis cells, prostate cells, thymocytes, spleen cells, kidney cells, hepatocytes,
Includes lung cells, brain cells, and monocytes. Fas (APO-1 / C
D95) Since the cell surface receptor is a member of the nerve growth factor (NGF) / tumor necrosis factor (TNF) receptor superfamily, any cell bearing this family of receptors is included within the scope of the present invention. Is intended. Fas and TNF receptor expression has also been identified in a number of tissues: see, for example, Watanabe-Fukunaga et al.
92) J. Immun. 148: 1274-1279 and Owen-Schaub, LB.
Et al . (1994) Cancer Res. 54: 1580-1586; Dhein et al. (1995) N.
ature 373: 438-441; Brunner et al. (1995) Nature 373: 441-
444; and Ju et al. (1995) Nature 373: 444-448. Assays to identify additional "suitable" cells that are susceptible to induction or activation (eg, TCR-TNF-Fas-related apoptosis) are well known to those of skill in the art (eg, Opipari et al ., J. Biol. Chem. (1992) 267: 12424-124
27; Yonehara et al., J. Exp. Med. (1989) 169: 1747-1756;
Dhein et al. (1995) supra; Brunner et al. (1995) supra; and Ju.
(1995) supra).

Suitable cells or "target cells" for practicing the present methods also include PCs by endogenous factors such as, but not limited to, HIV, anti-TCR antibodies, TNF and anti-Fas antibodies.
Includes cells that are induced to D. In one embodiment, the cells are cytokine Tumor Necrosis Factor (TN).
F) or the cell death transduction receptor Fas or TCR, both constitutively and inducibly, which were activated by their respective ligands. Recently, three separate groups have reported that Fas-induced apoptosis is associated with T cell death. In particular, one group is that Fas receptors, which, when linked, may transduce potential apoptotic signals are
It was shown to be rapidly expressed after being activated on cell hybridomas. This suggested that Fas receptor-ligand interaction induces cell death in a cell-autonomous manner. Dhein et al. (1995) Nature 373: 438-441; Brunne
r et al. (1995) Nature 373: 441-444; and Ju et al. (1995) Natu.
See re 373: 444-448.

The cells can be mammalian or animal cells such as guinea pig cells, rabbit cells, monkey cells, mouse cells, rat cells, chicken cells or human cells. They can be continuously cultured or isolated from animals or humans. In another embodiment of the invention neurological cells are specifically excluded.

A "bioequivalent" of a nucleic acid molecule is defined herein as having substantial identity with the reference nucleic acid molecule. A fragment of a reference nucleic acid molecule is an example of a bioequivalent.

A “bioequivalent” of a polypeptide or protein is one that contains an active pentapeptide, QACRG, whose cysteine residues are catalytic and retain the same characteristics as the reference protein or polypeptide.
It also contains a fragment of the reference protein or polypeptide.

A "dominant inhibitory" protein or polypeptide is one in which a cysteine residue has been altered such that the catalytic activity is abolished but the protein or polypeptide still binds to its intracellular target. For example, substitution of alanine or methionine for its catalytic cysteine abolishes its catalytic activity but still retains its binding properties.

"CrmA biological activity" means having the ability to inhibit Fas-induced apoptosis in a manner as described herein. The predominant inhibitory Yama protein or polypeptide (and the nucleic acid encoding this protein or polypeptide) is an example of a factor having CrmA biological activity.

When applied to apoptosis, the term "block" or "inhibit" refers to a reduction in cell death number, or 1.
It is intended to mean prolonging the survival of one cell or multiple cells. They are also intended to mean a reduction or delay in the appearance of morphological and / or biochemical changes normally associated with apoptosis. Thus, an "increased" apoptotic cell death is an increase in the total number of cell deaths,
Or it means a decrease in the survival time of the cells. "Increased" also means a decrease in the time to the appearance of morphological and / or biochemical changes normally associated with apoptosis after contacting the cell with an apoptotic factor. Apoptosis
It is considered programmed cell death (PCD) and can be detected and monitored by a number of morphological and biochemical changes. Methods useful for monitoring and detecting these changes include light microscopy, measurement between doubling times of latent and actual tumors, radiolabeling.
Includes loss of DNA precursors, measurement of DNA fragmentation, or measurement by FCM. These methods are described by Vermes and Haanen.
"Apoptosis and Programmed Cell Death in Health and Disease.
Health and Disease) '' Adv. In Clin. Chem. (1994) 3
1: 177-246, and the references cited herein. Light microscopy and potential tumor doubling time versus actual tumor volume doubling time are most applicable in mammalian pathology. "Inhibition" when used in this context refers to apoptosis or
It means a decrease in the number of cells undergoing PCD, or an increase in the survival time or proliferation rate of the treated cell population as compared to the cell or control population. "Increase" means an increase in the number of cells undergoing apoptosis or PCD, or a decrease in the survival time or proliferation rate of the treated cell population as compared to the cell or control population. A "treated cell" is a cell or population of cells that has been exposed to a protein or antibody, or having a nucleic acid molecule of the invention inserted therein by any number of methods.

As used herein, pro-Yama protein, activated Yama, p20 Yama and p11 Yama are wild-type or naturally occurring proteins as well as mutants (eg dominant inhibitor Yama), analogs, organisms. It is intended to include biological equivalents, and fragments thereof. In some embodiments, the term is also anti-Yama.
Antibodies and anti-idiotypic antibodies, as well as chimeras containing antibodies or antibody fragments are included.

Proteins and Polypeptides The present invention relates to pro Yama, p20 Yama, p11 Yama, activated Yam purified from a natural environment or obtained recombinantly.
a, and a protein or polypeptide called variant Yama, and their bioequivalents. Bioequivalents are proteins or polypeptides that have the same or essentially similar biological activity, as determined by the assays described herein. Unless otherwise identified, the term Yama protein or polypeptide should include all forms and bioequivalents described herein. Proteins and polypeptides can be purified from animal sources such as rat, chicken, human, or mouse. Recombinant forms can be obtained from a number of prokaryotic and eukaryotic recombination systems or can be chemically synthesized. The present invention further provides mutant CrmA proteins that do not inhibit apoptosis.

Pro-Yama is a zymogen that upon "activation" cleaves PARP to form the 85 kDa form. In one embodiment, pro-Yama has an apparent molecular weight of about 32 kDa as measured by PAGE. In another embodiment, it has the 277 amino acid sequence shown in Figure 2. Activated Yama consists of two subunits shown herein as consisting of p20 Yama and p11 Yama. The actual molecular weight of this subunit is
As shown in FIGS. 38 and 42, 17 and 12 kDa
It is. Pro-Yama is cleaved by ICE after aspartic acid to form activated Yama consisting of the p20 and p11 subunits. Accordingly, the present invention also provides p20 Yama and p11 Yama that are purified from their natural environment or are recombinantly obtained. Activated Yama (p20 Yama and
p11 Yama) is a heterodimeric polypeptide in combination characterized by having a biological or functional ability to modulate cellular functions associated with Fas receptor pathways such as Fas-related apoptosis. Especially p20
Yama and p11 Yama heterodimers promote apoptosis in appropriate cells, whose activity is inhibited by CrmA,
It is not inhibited by the mutant CrmA. They also form inhibitory complexes with CrmA but not mutant CrmA.

In one embodiment of the invention activation
Overexpression of the DNA encoding the Yama protein promotes apoptosis. Examples of such proteins include, but are not limited to, p20 Yama and p11 Yama. In another embodiment, the biological activity of the p20 Yama or p11 Yama protein or its equivalent can be inhibited by CrmA as described herein, but not by mutant CrmA. The CrmA gene or nucleic acid is described in Pickup et al. (198
6) It can be isolated from natural or original sources as described in PNAS 83: 7698-7702. Those skilled in the art are familiar with Tewari et al.
(1995) J. Biol. Chem. 270: 3255-3260, or using the methods described below to determine when and whether the biological activity of a protein is inhibited by CrmA. You can decide.

The Yama and Yama subunits were epitope-expressed (293) highly expressed in 293T (ATCC) cells using the method disclosed in Chiang and Roeder (1993) Peptide Research 6 (2): 62-64. It can be isolated from the appropriate cell lysate by using epitope tagged versions).

According to the invention, further purified or recombinantly produced pro Yama, activated Yama, p20 Yama, or p1.
1 Yama, or a polypeptide fragment of a protein having the amino acid sequence shown in Figure 2 is provided.
These peptides are characterized by either being activated into a pro-apoptotic form (activated Yama) or capable of promoting apoptosis in activated cells. One such fragment is the pentapeptide
QACRG, where cysteine is catalytic.

It is understood that the functional equivalents of the Yama proteins defined above are also within the scope of the invention. For example purposes only, a functional equivalent is Ya
ma fusion protein 6 × His-Yama or those having a chemical structure other than amino acids, those functionally mimicking the biological activity of any purified pro Yama, allelic variants thereof, activated Yama, purified p20 Yama, Purified p11 Yama, a recombinant homolog thereof, or a protein having the amino acid sequence shown in FIG. 2 (“analog”), which retains the biological activity of the corresponding purified or recombinant protein or polypeptide. . Further examples of analogs are proteins or polypeptides containing another protein or polypeptide (eg GST fusion protein) linked to Yama or a fragment thereof,
It is an equivalent that alters the primary sequence of the protein of the present invention derived from the amino acid sequence shown in FIG. However, 1 of the present invention
In one embodiment, CPP32β, Mch2, Ced-3, Nedd-
The proteins called 2, TX / ICH2, ICE rel-III, and ICE-rel II are specifically excluded (Fernandes-Alnemri
(1994) J. Biol. Chem. 269: 30761-30764. ) C for activated Yama or p20 and / or p11 Yama
Also provided by the present invention are agents characterized by having the ability to inhibit binding to rmA. Such factors include, but are not limited to, anti-CrmA antibody, or any
The dominant inhibitory fragment of CrmA, or p20, p11 or pro-Yama. The "dominant inhibitory fragment" of the invention is intended to include, but is not limited to, muteins that irreversibly bind to intracellular CrmA or p20 and p11 Yama heterodimer complexes, respectively. Ya
An example of a ma dominantinhibitory mutein is one in which the functionally important cysteine sequence QA C RG in p20 and proYama is mutated to alanine or methionine. These mutants, although lacking their apoptotic potential, still bind their substrates, thereby inhibiting endogenous native Yama and its biological activity or function. These Yama muteins can be made by the amino acid sequence shown in Figure 2 and variations of the methods provided herein and the method of Higuchi et al. (1988) supra.

The proteins and polypeptides of the present invention can be obtained by a number of processes well known to those of skill in the art. It includes purification, chemical synthesis, and recombinant methods. Full-length Pro Yama, Activated Yama, p20 Yama, and p1
1 Yama proteins can be derived from Fas ⁺ cell or tissue lysates by methods such as immunoprecipitation with appropriate antibodies, and standard techniques (eg gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography). Chromatography). For such a methodology, see Deutscher et al., Guide to Protei.
n Purification: Methods in Enzymology (1990) No. 182
See Volume, Academic Press. Thus, the invention also provides processes for obtaining the proteins and polypeptides of the invention, as well as the products obtainable and obtainable by this process.

Proteins and polypeptides may also be commercially available from automated peptide synthesizers (eg, Applie
Model 430A or 431A manufactured by Biosystems, Inc., Foster City, CA), and by chemical synthesis using the amino acid sequences shown in FIGS. 2 and 22. Synthesized proteins and polypeptides can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Thus, the present invention also includes protein sequences (eg, FIG. 2 for Yama and FIG. 5 for mutant CrmA).
And FIG. 22), and methods for chemically synthesizing the proteins of the invention by providing reagents (eg, amino acids and enzymes) and supplying the amino acids in the proper orientation and linear sequence. .

Alternatively, proteins and polypeptides can be prepared as described, for example, in Sambrook et al., Molecular Cloning: A Labor.
atory Manual Volume 2 (Cold Spring Harbor Laboratory
(1989) by well-known recombinant methods, for example using the host cell and vector systems exemplified below and below. The present invention further provides for the growth of pro-Yama, activated Yama, p20 or p20 by proliferating host cells containing a nucleic acid molecule encoding the desired protein, which nucleic acid is operably linked to a promoter of RNA transcription. Provide methods for producing p11 Yama proteins, analogs, muteins or fragments thereof. Host cells are grown under suitable conditions so that the nucleic acid is transcribed and translated into protein.

The present invention also provides the proteins described herein conjugated to a detectable agent for use in a diagnostic method. For example, p20 and p11
Detectable labeled proteins and polypeptides containing the heterodimeric Yama can be bound to a column and Cr
It can be used for detection and purification of mA. They are also useful as immunogens for antibody production as described below. The proteins and fragments of the invention are for screening for agents or drugs that inhibit or increase Fas-related functions such as apoptosis, and
Abnormalities associated with this pathway (eg, lps, immunosuppression, CD4
⁺ T cell depletion, and carcinogenicity), is useful in an in vitro assay system.

More specifically, the in vitro cell method is based on TC
Providing a cell or tissue culture having any cell surface receptor that mediates apoptosis such as R, TNF receptor, or Fas receptor. The cells are cultured under conditions (temperature, growth or culture medium, and gas (CO ₂ )) and for a suitable time to reach exponential growth without concentration-dependent inhibition. The cells are then exposed to the preliminary conditions required for apoptosis. For example, an effective amount of an inducing agent (eg, TCR ligand, HIV, CTL, SIV, TNF, or anti-Fas
Fas ligands such as antibodies) are added to the culture. Anti-F
As antibodies and mitogens (ConA) are well known to those of skill in the art (Itoh, N. et al. (1991) Cell 66: 233-243 and Yoneha.
ra et al. (1989) J. Exp. Med. (1989) 169: 1747-1756). These cells are then “induced” to apoptosis. The cells are cultivated again under the conditions of appropriate temperature and time. In one embodiment, HIV or S
IV is added to the culture. In other embodiments, the drug or agent to be tested is added in varying concentrations at the same time as, before, or after the inducer.

When cells express constitutively Yama,
No addition of Yama is required. In other embodiments, the pro-Yama nucleic acid molecule or protein is provided under an effective amount and under conditions in which the cells internalize the nucleic acid or protein,
Add to culture. In some embodiments, IC
An effective amount of E or ICE nucleic acid is added to activate pro Yama. The cells are cultured under conditions of appropriate temperature and time to induce apoptosis. The cells are split into two samples. The first set may have CrmA nucleic acid or protein added prior to, concurrently with, or after the agent to be tested. The cells are assayed for apoptotic activity using methods well known to those of skill in the art and those described herein. It will be apparent to those skilled in the art that at least two cell cultures must be treated and maintained as the test population. One is maintained without the addition of inducer to measure background release, the second is not added the drug to be tested. The second cell population acts as a control.

In vitro use of the compositions and methods provides a powerful bioassay for screening for drugs that are agonists or antagonists of CrmA and Yama function in these cells. Therefore,
CrmA to prevent or inhibit apoptosis
Can be screened for drugs that have similar or enhanced potency, or that have the same ability as Yama to induce apoptosis. One of ordinary skill in the art
It can be measured by noting the absence of apoptotic morphological changes, or, more simply, when successfully performed by the absence of cell death. The in vitro method further provides an assay for determining whether the method of the invention is useful in treating a pathological condition or disease in a subject associated with apoptotic cell death in an individual.

For example, a T cell hybridoma cell line line such as Jurkat was constructed using a CrmA expression construct, Pro Yama, activated Ya.
Expression vectors containing ma, CrmA or mutant Yama, or vector alone, can be stably transfected and clonal cell lines can be induced. Transfection of Jurkat cells by electroporation is described by Laherty et al., J. Biol. Chem. , (1993) 263: 50.
It may be carried out as described in 32-5039. Cells were ⁵¹ Cr-labeled and untreated or anti-CD3 (cell line 145-2C11 (AT
(Available from CC)) treated tissue culture plastic plates (5 × 10 ⁵ / ml). Cells cultured on uncoated cells are used to measure background release. Percentage of cell death is measured according to the formula at various times after incubation: Cpm released from experimental group minus background released cpm released by 0.5% Triton X-100. (Completely dissolved) Divide by the value obtained by subtracting the background emission cpm from cpm. The drug is then added to the culture and the effect on apoptosis is measured with or without exogenously added CrmA, Yama, mutant Yama nucleic acids or proteins. Using the methods described above, various agents can be tested for their ability to inhibit, prevent, or increase apoptosis.

In another embodiment, a T cell line called CEM (ATCC) is obtained and used. Because it was shown to go through PCD upon HIV infection. CEM cells are transfected by electroporation with the CrmA expression construct and the vector alone as a control. Clonal strains are induced and infected with HIV at various multiplicity of infection rates. Cytotoxic effect is assayed by microscopic observation,
Apoptosis is quantified after propidium iodide staining.
Using the methods described above, various agents can be tested for their ability to inhibit or prevent apoptosis.

The proteins of the present invention can also be combined with a variety of liquid phase carriers such as sterile solutions or solutions, pharmaceutically acceptable carriers, suspensions, and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies, the carrier can also include an adjuvant, which is useful for nonspecifically increasing the specific immune response. Those of ordinary skill in the art will readily determine whether an adjuvant is required and will select one. However, for exemplary purposes only, suitable adjuvants include, but are not limited to, Freund's complete and incomplete adjuvants, mineral salts, and polynucleotides.

The present invention also includes any protein, analog, mutein, polypeptide fragment, antibody, antibody fragment, or anti-idiotype antibody of the invention, alone or in combination with each or other agents, and Provided are pharmaceutical compositions containing a combination with an acceptable carrier. These compositions are useful in a variety of diagnostic and therapeutic methods.

Nucleic acid pro-Yama, activated Yama, Yama muteins, CrmA mutein analogs, nucleic acids and isolated nucleic acid molecules encoding amino acid sequences corresponding to p20 or p11 Yama polypeptides, antibodies, anti-idiotype antibodies, and antibody fragments,
As well as complements of these sequences are also provided by the present invention. In addition to the sequences shown in FIGS. 1, 5 and 22, the present invention also provides antisense polinkreotide strands (eg, antisense RNA). Antisense RNAs include, for example, the sequences shown in FIG.
It can be obtained using the methodology described in ander Krol et al. (1988) Bio Techniques 6: 958. Unless otherwise identified, the term “Yama” nucleic acid refers to all Ya's described herein.
should include the ma nucleic acid molecule.

In one aspect of the invention, the nucleic acid molecule encoding the Yama protein or polypeptide is defined to be the entire sequence shown in Figure 1 or a portion thereof. Further provided are nucleic acid molecules having the pentapeptide QACRG, and oligonucleotides encoding the predominant inhibitory polypeptides and proteins described herein. DNA and RNA complements of these nucleic acid molecules are also included within the scope of the invention.

The present invention also includes nucleic acid molecules which differ from the nucleic acid molecules described above, but which confer the same phenotypic effect, such as an allele. These altered but phenotypically equivalent nucleic acid molecules are referred to as "equivalent nucleic acids." The invention also includes nucleic acid molecules characterized by changes in the non-coding regions that do not alter the phenotype of the polypeptide produced therefrom when compared to the nucleic acid molecule herein. The invention further includes nucleic acid molecules that hybridize to the nucleic acid molecules of the invention under stringent conditions.
Nucleic acid molecules having a sequence different from that shown in FIG. 1, but producing a protein with enhanced or decreased biological activity are also within the scope of the invention.

In one embodiment, CPP32β, Mch
2, Ced-3, Nedd-2, TX / ICH2, ICE-relIII, and ICE-r
A nucleic acid molecule that encodes a protein called el II is
Especially excluded.

The nucleic acid molecule may be linked to a detectable marker (eg, an enzyme label or radioisotope) to detect nucleic acid and / or expression of the Yama-encoding gene in the cell. Briefly, the present invention further encodes an amino acid sequence that is at least part of the Yama protein under conditions that allow hybridization of complementary single-stranded nucleic acid molecules, preferably stringent hybridization conditions. A single-strand encoding an amino acid sequence that is at least part of Yama by contacting the single-stranded nucleic acid molecule with a labeled single-stranded nucleic acid molecule (probe or primer) that is complementary to the single-stranded nucleic acid molecule A method for detecting a nucleic acid molecule is provided.
The hybridized nucleic acid molecule is separated from the single-stranded nucleic acid molecule. Hybridized molecules are detected by methods well known to those of skill in the art and, for example, those described in Sambrook (1989), supra.

Nucleic acid molecules of the invention can be isolated by the methods described in the Examples section and replicated using PCR (Perkin-Elmer). For example, sequences can be chemically replicated using PCR, which, in combination with oligonucleotide synthesis, allows for easy reconstruction of DNA sequences. PCR technology
U.S. Pat.Nos. 4,683,195, 4,800,159 and 4,754,
065, and the subject of 4,683,202, PCR: The
Polymerase Chain Reaction Mullis et al., Birkhauser
Press, Boston (1994) and references cited herein. Alternatively, one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate DNA. Accordingly, the present invention also chemically replicates or ligates polynucleotides, nucleotides, suitable primer molecules, chemicals such as enzymes, and their replication and nucleotides in the proper orientation to obtain a polynucleotide. By providing instructions for providing a process for obtaining a polynucleotide of the invention. In another embodiment, these polynucleotides are also isolated. In addition, one of skill in the art can insert the nucleic acid into a suitable replication vector for replication and amplification, and the vector into a suitable host cell (human B cells or BJAB or 293 T cells). Amplified DNA can be isolated from cells by methods well known to those of skill in the art. The process by which the nucleic acid molecule is obtained by this method is also provided herein, as well as the nucleic acid molecule.

RNA can be obtained by using isolated DNA, operably linking it to control regions suitable for the host cell, and inserting into the host cell. Suitable cells for this purpose include, but are not limited to, human B cells, BJAB, or 293 cells.
Examples include T cells. The DNA may be inserted by any suitable method, such as the use of a suitable insertion vector or electroporation. The cells replicate, and
When the DNA is transcribed into RNA, the RNA can then be isolated by methods well known to those of skill in the art, such as those described in Sambrook et al. (1989) supra.

The present invention further provides an RNA transcription promoter,
And nucleic acid molecules operably linked to other regulatory sequences for DNA and RNA replication and / or transient or stable expression. As used herein, the term "operably linked" refers to when the promoter is DNA
It means that they are arranged in such a way as to direct RNA transcription from the molecule. Examples of such promoters are SP6, T
4, and T7. In a particular embodiment, cell-specific promoters are used for cell-specific expression of the inserted nucleic acid molecule. Vectors containing promoters or promoters / enhancers, along with stop codons and selectable marker sequences, and cloning sites at which an insert of DNA may be operably linked to the promoter, are well known in the art and are commercially available. . For general methodologies and cloning strategies, see Gene Expression Technology , Goeddel, Academi.
c Press, Inc., (1991) and references cited therein, Vectors: Essential Data Series Gacesa and Ra.
See mji ed., John Wiley & Sons, NY (1994).
This includes references to maps, functional characteristics, vendors, and GenEMBL accession numbers for various suitable vectors. Preferably, these vectors allow RNA transcription in vitro or in vivo.

Fragments of the polynucleotide shown in FIG. 1 are also included within the scope of this invention and preferably have at least 8 nucleotides, more preferably at least 10 or 18 nucleotides. These are useful as hybridization probes and PCR primers.

In one embodiment, these fragments are activated Yama, or p20 Yama and p11 Yama.
Is a nucleic acid molecule that encodes the protein called and the pentapeptide QACRG. The nucleic acid molecule encodes a polypeptide that heterodimerizes in activated cells, binds CrmA, and induces apoptosis. This, and further fragments of the invention, are useful for coding for proteins having diagnostic and therapeutic utility as described herein, as well as proteins that may or may not be present. It is useful as a probe for identifying the transcript of Escherichia coli. These polynucleotide fragments can be prepared, for example, by restriction enzyme digestion of the nucleic acid molecule of Figure 1, and then labeled with a detectable marker, such as a radioisotope, using well known methods. Alternatively, random fragments are created using nick translation of the molecule. Methodologies for the preparation and labeling of such fragments are described in Samb.
rook et al. Molecular Cloning: A Laboratory Manual Cold
Spring Harbor Press, Cold Spring Harbor, NY (198
See 9). Polynucleotide fragments are also useful for making new peptides. next,
These peptides are useful as immunogens for making polyclonal and monoclonal antibodies.

As mentioned above, the nucleic acid molecule of the present invention comprises RNA
It may be operably linked to a transcription promoter (inducible or non-inducible promoter). These nucleic acid molecules are
It is useful for recombinant production of Yama proteins and polypeptides, or as a vector for gene therapy. Therefore, the present invention also includes a vector (insertion, replication, or expression vector) having the above-described nucleic acid molecule to be inserted, for example, a viral vector (bacteriophage, baculovirus, and retrovirus), or a cosmid, a plasmid, a YACS, Yeast, and other recombinant vectors are provided. The nucleic acid molecule is inserted into the vector genome by methods well known in the art. For example, both the insert and the vector DNA are exposed to a restriction enzyme to create complementary ends that base pair with each other in both molecules and then ligated with a ligase. Alternatively, a synthetic nucleic acid linker can be ligated into the insert DNA corresponding to the restriction sites in the vector DNA and then digested with a restriction enzyme that recognizes the specific nucleotide sequence. In addition, oligonucleotides containing stop codons and suitable restriction sites can be ligated for insertion into a vector containing, for example, some or all of the following: Selection for stable or transient transfection in mammalian cells. A selectable marker gene, such as the neomycin gene, for: a human cytomegalovirus (CMV) immediate early gene-derived enhancer / promoter sequence for high-level transcription;
SV40-Derived Transcription Termination and RNA Processing Signals for mRNA Stability; Origin of SV40 Polyoma Replication and ColE1 for Proper Episomal Replication; Multipurpose Multiple Cloning Sites; and Sense and Antisense RNA
And SP6 RNA promoters for in vitro transcription of.

Further examples of vector constructs of the invention are:
A promoter, such as the lac promoter, and the Shine-Dalgarno sequence for initiation of transcription and the start codon AUG
Is a bacterial expression vector containing (Sambrook et al. A (1989)
Supra). Similarly, eukaryotic expression vectors are a heterologous or homologous promoter of RNA polymerase II, a downstream polyadenylation signal, a start codon AUG, and a stop codon for elimination from the ribosome. Such vectors can be either commercially obtained or assembled using the sequences described herein. In one embodiment of the invention, the expression vector should be specifically targeted to T cells. For these methods, it is contemplated that the DNA will be operably linked to a promoter that is highly active in T cells. Such promoters include, but are not limited to, IFN-α; IL
-2; IL-3; IL-4; IL-5; IL-9; IL-10; TNF-β; GM-CS
F; CD4, CD8 and IL-2 promoters.

Expression vectors containing these nucleic acids are
It is useful to obtain host vector systems for producing proteins such as the Yama proteins and polypeptides described herein, as well as mutant CrmA. Of course, these expression vectors must be replicated in the host organism either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include viral vectors including adenovirus, adeno-associated virus, retroviruses, cosmids and the like. Adenovirus vectors are particularly useful for introducing genes into tissues in vivo for their high level expression and efficient cell transformation both in vitro and in vivo. The nucleic acid is inserted into a suitable host cell (eg, a prokaryotic or eukaryotic cell),
The protein may be produced recombinantly as the host cell replicates. The appropriate host cell depends on the vector,
It can include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells constructed using well known methods. See Sambrook et al. (1989), supra. In addition to the use of viral vectors for insertion of exogenous nucleic acid into cells, the nucleic acid is transformed into bacterial cells; transfection with mammalian phosphate precipitates into mammalian cells; or DEAE-dextran; electroporation: Alternatively, by a method well known in the art such as microinjection,
It can be inserted into a host cell. For this methodology,
See Sambrook et al. (1989). Accordingly, the present invention also provides a host cell, for example, a nucleic acid molecule encoding a Yama protein or polypeptide, or a mammalian cell, animal cell (rat or mouse), human cell containing a Yama protein or polypeptide or antibody, or Providing bacterial cells.

Recombination using the host vector system described above
A process for producing and / or obtaining a Yama, analog, mutein, or anti-Yama antibody, or an active fragment or variant CrmA thereof, can be performed using the host cells described herein as a Yama or anti-Yama protein, polypeptide. , Or by growing under suitable conditions such that the nucleic acid encoding the antibody is expressed. Appropriate conditions can be determined using methods well known to those of ordinary skill in the art. See, eg, Sambrook et al. (1989), supra. The recombinant product is then purified from the cell extract. Accordingly, the invention further provides for recombinant production of host cells having exogenously added nucleic acid molecules of the invention, as well as proteins, polypeptides and antibodies of the invention by carrying out the steps described above. And the product so produced.

Yama, Activated Yama, Anti-Yama Protein, Ya
Vectors containing nucleic acids encoding ma antisense RNA, nucleic acid molecules encoding Yama antisense RNA or antibodies are also used to modulate or regulate cellular functions such as apoptosis and immune disorders mediated by the Fas pathway. It is useful for gene therapy. The term "Fas cell function"
By cell binding affected by its binding to its extracellular ligand, ie alone or in combination with each other. In some cases, it is desirable to increase Fas apoptotic function and induce apoptosis, eg, in neoplasms or cancer cells. This is a professional
Yama and ICE or p20 and p11 heterodimer Yama
It can be achieved by introducing into cells cells activators such as proteins, or nucleic acid molecules encoding polypeptides and proteins having biological activity. In other cases, down-regulating Fas cell function is desired. This is activated Yama or p20 or p11
Antibody fragment that is the dominant inhibitor of Yama, Yama antisense RNA (or its encoding DNA) or Cr
It can be achieved by introducing into the cell a nucleic acid molecule encoding the mA protein or these agents. In addition, antisense Yama RNA can be used to inhibit the production of pro Yama proteins. This treatment inhibits or regulates intracellular Fas signaling and is therefore useful in treatments that should avoid apoptotic cell death, such as in HIV infected T cells.

When used for in vivo or ex vivo gene therapy, pharmaceutically acceptable vectors are preferred, eg replication incompetent retroviral vectors. The pharmaceutically acceptable vector containing the nucleic acid of the present invention contains, for transient or stable expression of the inserted nucleic acid molecule,
It can be further modified. The term "pharmaceutically acceptable vector" as used herein includes, but is not limited to, a vector or delivery vehicle that has the ability to selectively target and introduce nucleic acids into dividing cells. An example of such a vector is a "non-replicable" vector that is incapable of producing viral proteins in infected host cells and is defined by disabling the spread of the vector. An example of a replication incompetent retroviral vector is LNL6 (Miller, AD et al. (1989) Bio Techniques 7: 980-99.
0). The methodology for using replication incompetent retroviruses for retrovirus-mediated gene transfer of genetic markers is as follows:
Well established (Correll et al. (1989) PNAS USA 86:
8912; Bordignon (1989), PNASUSA 86: 6748-52; Culve
r, K. (1990), PNAS USA 88: 3155; and Rill. DR.
(1991) Blood 79 (10): 2694-700). Clinical studies have shown that there are no or few side effects associated with viral vectors. For example, Anderson (1992) Scie
See nce 256: 808-13.

A composition containing the nucleic acid molecule of the present invention is also provided in an isolated form or in a vector or host cell. When these compositions are used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.

Antibodies The present invention also provides antibodies capable of specifically forming complexes with the proteins, polypeptides and nucleic acid molecules of the present invention. These are Pro Yama, Activated Yama, QA
CRG, dominant inhibitory polypeptides and proteins, Yama
Fragments of (for example, p20 Yama and p11 Yama),
As well as, but not limited to, nucleic acid molecules that encode antibodies. Vectors and host cells containing these nucleic acid molecules are also encompassed by the present invention. The term "antibody" includes polyclonal and monoclonal antibodies. Antibodies include mouse antibodies, rat antibodies,
Includes, but is not limited to, rabbit or human antibodies.

As used herein, an "antibody" or "polyclonal antibody" is produced in response to immunization with an antigen or receptor and has specificity and affinity effective for its intended purpose. It means a protein that reacts with an antigen by sex. The term "monoclonal antibody"
Means immunoglobulin derived from a single clonal cell. All of the monoclonal antibodies from this clone are chemically and structurally identical and specific for a single antigenic determinant. Hybridoma cell lines that produce monoclonal antibodies are also within the scope of the invention.

Laboratory methods for producing polyclonal and monoclonal antibodies, as well as deducing their corresponding nucleic acid sequences, are known in the art. Harlow and Lane, Antibodies ； A La
boratory Manual , Cold Spring Harbor Laboratory, Ne
w York (1988), US Pat. No. 5,411,749 and Sambrook
(1989) supra. The monoclonal antibody of the present invention can be produced biologically by introducing the Yama protein or a fragment thereof into an animal (eg, mouse or rabbit). Antibody producing cells in the animal are isolated and fused with myeloma cells or heteromyeloma cells to produce hybrid cells or hybridomas. Accordingly, hybridoma cells producing the monoclonal antibodies of the invention are also provided.

Thus, using the Yama protein or fragments thereof, and well known methods, one of skill in the art would appreciate that
The hybridoma cells and antibodies of the invention can be made and screened for antibodies that have the ability to bind to.

If the monoclonal antibody tested binds to the Yama protein or polypeptide, the antibody tested and the antibody provided by the hybridoma of the invention are equivalent. The antibody to be tested is similar to the monoclonal antibody of the invention by determining whether it prevents the monoclonal antibody of the invention from binding to Yama, with which the monoclonal antibody of the invention normally reacts. Moreover, it is also possible to determine, without undue experimentation, whether the antibodies have the same specificity. As shown by the reduced binding by the monoclonal antibodies of the invention,
When the antibody being tested competes with the monoclonal antibody of the invention, the two antibodies appear to bind to the same or closely related epitopes. Alternatively, the monoclonal antibody of the invention may be pre-incubated with the Yama protein to which it normally reacts and it may be determined whether the monoclonal antibody being tested is inhibited in its ability to bind antigen. If the monoclonal antibody being tested is inhibited, then, in all likelihood, it has the same or a closely related epitopic specificity as the monoclonal antibody of the invention.

The term “antibody” is also intended to include antibodies of all isotypes. Specific isotypes of monoclonal antibodies can be prepared directly by selection from the first fusion, or by Steplewski et al. (1985).
Proc. Natl. Acad. Sci. 82: 8653 or Spira et al. (1984).
Secreting different isotypes of monoclonal antibodies by using the sibselection technique to isolate class switch variants using the procedure described in J. Immunol. Methods 74: 307 . Can be prepared indirectly from the parental hybridoma.

The present invention also provides biologically active fragments of the polyclonal and monoclonal antibodies described above. These "antibody fragments" retain some ability to selectively bind with its antigen or immunogen. Such antibody fragments include, but are not limited to, (1) antibody molecules produced by digestion with the enzyme papain to obtain Fab, an intact light chain and one heavy chain in part. (2) Fab ', a fragment of an antibody molecule obtained by treatment with pepsin to obtain an intact light chain and a partial heavy chain, and subsequent reduction; an antibody; 2 per molecule
Two Fab 'fragments are obtained; (3) F (ab') ₂ fragments of the antibody obtained by treatment with the enzyme pepsin without subsequent reduction; F (ab ')
₂ is a dimer of two Fab 'fragments linked by at least one disulfide bond: (4) Fv, a genetically engineered containing light chain variable region and heavy chain variable region expressed as two chains. (5) SCA, as a genetically fused single chain molecule, genetically engineered to contain the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker. Is defined as a molecule

Particular examples of "biologically active antibody fragments" include the CDR and VH regions of antibodies.
Methods of making these fragments are known in the art. See, for example, Harlow and Lane, (1988) supra and Davies et al. (1995) Bio / Technology 13 (5): 475-479.

The antibodies of the invention can also be modified to create chimeric and humanized antibodies (Oi et al. (1986) B
io Techniques 4 (3): 214). A chimeric antibody is a DNA derived from one or more of the various domains of the antibody heavy and light chains.
Is an antibody encoded by.

Isolation of other hybridomas secreting a monoclonal antibody having the specificity of the monoclonal antibody of the invention can also be accomplished by those skilled in the art by producing anti-idiotypic antibodies (Herlyn et al., (1986 )
Science 232: 100). Anti-idiotypic antibodies are antibodies that recognize the unique determinants present on the monoclonal antibody produced by the antibody of interest. These determinants are located within the hypervariable region of antibodies. It is this region that binds to a given epitope, and thus this region is responsible for the specificity of the antibody. Anti-idiotypic antibodies can be prepared by immunizing animals with the monoclonal antibody of interest. The immunized animal recognizes and responds to the idiotypic determinants of the immunizing antibody by producing antibodies to these idiotypic determinants. For immunization by using an anti-idiotypic antibody of a second animal, which is specific for the monoclonal antibody produced by the single hybridoma used to immunize the second animal. It is now possible to identify other clones with similar idiotypes to the hybridoma antibodies used in.

The idiotypic identity between the monoclonal antibodies of the two hybridomas indicates that the two monoclonal antibodies are identical with respect to their recognition of the same epitopic determinant. Thus, by using an antibody against an epitopic determinant on a monoclonal antibody, it is possible to identify other hybridomas expressing the same epitope-specific monoclonal antibody.

It is also possible to use anti-idiotype technology to produce monoclonal antibodies that mimic an epitope. For example, an anti-idiotypic monoclonal antibody raised against the first monoclonal antibody has a binding domain within the hypervariable region that is a mirror image of the epitope bound by the first monoclonal antibody. Therefore, in this example, anti-idiotype monoclonal antibodies could be used for immunization for the production of these antibodies.

The term “epitope” as used in the present invention
Is meant to include any determinant having a specific affinity for a monoclonal antibody of the invention. Epitopic determinants usually consist of chemically active surface groupings of molecules (eg, amino acid or sugar side chains) and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

A recombinantly produced, biochemically synthesized, chemically synthesized, or chemically modified protein or polypeptide that corresponds to a natural polyclonal or monoclonal antibody. , Pro Ya
Proteins or polypeptides that retain the ability to bind ma, p20 Yama, or p11 Yama, or fragments thereof, are also encompassed by the present invention.

The antibodies of the present invention can be linked to a detectable agent or hapten. This complex was isolated from the Fas in the sample using standard immunochemistry techniques, such as the immunohistochemistry described by Harlow and Lane (1988) supra.
It is useful for detecting receptors or Yama proteins or fragments, or for detecting agents that interfere with Yama-Fas receptor binding. Examples of types of immunoassays that can utilize the monoclonal antibodies of the invention are competitive and noncompetitive immunoassays, either in direct or indirect format. Examples of such immunoassays are enzyme linked immunoassays (ELISA), radioimmunoassays (RIA) and sandwich (immunoassay) assays. Detection of the monoclonal antibodies of the invention involves a forward forma involving immunohistochemical assays on physiological samples.
t), reverse format or simultaneous format (sim
It can be performed by utilizing an immunoassay performed in any of the ultaneous modes). Other immunoassay formats are known or can be readily appreciated by those skilled in the art without undue experimentation.

Another technique which may also give more sensitive results consists of coupling the antibody to a low molecular weight hapten. These haptens can then be specifically detected by the second reaction. For example, biotin, which reacts with avidin, or dinitrophenyl (d
initropherryl), pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies
The use of haptens such as is common. Harlow and La
See ne (1988) supra.

The monoclonal antibodies of the invention can be attached to many different carriers. Accordingly, the present invention also provides
Compositions comprising antibodies and other substances (active or inactive) are provided. Examples of well-known carriers are glass, polystyrene, polypropylene, polyethylene, dextran,
Includes nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. One of ordinary skill in the art would know of other suitable carriers for binding monoclonal antibodies, or could use routine experimentation to ascertain such.

There are many different labels and labeling methods known to those of skill in the art. Examples of this type of label that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. The person skilled in the art is aware of other suitable labels for attaching monoclonal antibodies,
Alternatively, routine experimentation can be used to confirm such things. In addition, conjugation of these labels to the monoclonal antibodies of the invention can be done using standard techniques common to those of ordinary skill in the art.

For the purposes of the present invention, Yama, when present in biological fluids and tissues, can be detected by the monoclonal antibodies of the present invention. Detectable amount of Yama
Any sample of cell or tissue lysate containing can be used.

Compositions comprising an antibody, fragment thereof or cell line producing the antibody are encompassed by the present invention. When these compositions are used pharmaceutically, the compositions are combined with a pharmaceutically acceptable carrier.

Compositions The present invention also provides compositions comprising any of the above proteins, muteins, polypeptides, nucleic acid molecules, vectors, host cells, antibodies and fragments thereof, and an acceptable solid or liquid carrier. When the composition is used pharmaceutically, it is combined with a "pharmaceutically acceptable carrier" for diagnostic and therapeutic use. These compositions can also be used to prepare a medicament for the diagnosis and treatment of pathologies associated with the Fas receptor and apoptotic pathways.

Industrial Applicability The compositions described above provide the components for an assay for screening for drugs or pharmaceutical compounds that are agonists or antagonists of Fas-related apoptosis in appropriate cells. Suitable cells are those containing Fas / CD95 or TNF receptors, or endogenous factors such as HIV, CTL, anti-TCR antibodies, Fas agonists,
Or PCD by TNF, or anti-Fas antibody)
Is a cell that induces. In one embodiment, the cells are the cytokine tumor necrosis factor (TNF).
Alternatively, it expresses either or both of the cell death transduction receptor, Fas or TCR, constitutively or inducibly. And it is activated by the respective ligands. Recently, three different groups of researchers have reported that Fas-induced apoptosis is involved in T cell death. In particular, one group showed that, when bound, the Fas receptor could transduce potential apoptotic signals, but the Fas receptor was rapidly expressed after activation on T cell hybridomas. It was suggested that Fas receptor-ligand interaction induces cell death in a cell-autonomous manner. Dhein et al. (199
5) Nature 373: 438-441; Brunner et al. (1995) Nature 37.
3: 441-444; and Ju et al. (1995) Nature 373: 444-448.

For purposes of illustration only, examples of suitable cells are T lymphocytes (T cells such as TCR ⁺ , CD4 ⁺ and CD4 ^+.
8 ⁺ T cells), leukocytes and mixed leukocyte cultures (MLC), B
Lymphoma cells (eg, A202J (ATCC)), peripheral blood lymphocytes, colon cells, small intestine cells, ovary cells, testis cells, prostate cells, thymocytes, spleen cells, kidney cells, liver cells, neoplastic cells, carcinoma cells, Includes lung cells or brain cells. These are cells from each mammalian species (eg mouse, rat, monkey or human).

As described in further detail below, proteins and fragments thereof are used to screen for agents and pharmaceutical compounds that either inhibit or enhance the Fas receptor pathway and apoptosis, and this pathway. Disorders (eg, lps, immunosuppression, CD4 ⁺ T cell depletion and carcinogenesis)
Are useful in cell-free and cellular in vitro assay systems for testing possible treatments for. Embryonic development can also be regulated.

The cell-free screen is performed essentially as shown below. For example, an effective amount of professional Yama
Is activated by incubation with ICE in reaction buffer at 37 ° C. After activation, the reaction is split into two parts. To one part is added an effective amount of the drug to be tested. On the other hand, add an effective amount of CrmA. Each reaction mixture is incubated for about 30 minutes. An effective amount of PARP is added to each mixture and the solution is further incubated at 37 ° C. for an effective amount of time or about 2 hours. After this incubation, samples from each reaction mixture were loaded with anti-PARP monoclonal antibody (eg, C-2-1
0) to analyze by immunoblotting or by gel electrophoresis to see if this agent inhibits the cleavage of PARP to its characteristic 85 kDa form. this
The presence of the 85kDa form is an indication that the drug is not an inhibitory drug. The absence of this 85 kDa type is an indicator that the drug is a candidate for inhibiting Fas-related functions (eg, apoptosis). The method also includes
It can also be carried out by replacing PARP with an effective amount of U1-70 kDa protein. Cleavage to the 40 kDa form, which can be detected by the use of antibodies, indicates that the drug is not a candidate to inhibit apoptosis.

According to the present invention, the agents detected by these methods, the nucleic acid molecules encoding them, and the use of these agents and nucleic acid molecules in the therapeutic methods described herein are also included. As will be apparent to one of skill in the art, the above compositions can be combined with instructions for use to provide commercially available screening kits.

The methods described above also allow screening for drugs that have comparable or greater ability to prevent or inhibit apoptosis, eg, as compared to CrmA.

In the in vitro method of cells, a suitable cell or tissue culture is provided. These cells were subjected to conditions (temperature, growth or culture medium and gas (C
O ₂ )) and an appropriate amount of time for incubation, density-dependent constraint
Obtain exponential growth without (density dependent constraints). The cells are then subjected to preliminary conditions necessary for apoptosis, for example, an effective amount of inducer (e.g.,
A TCR ligand, HIV, SIV, TNF, CTL, or Fas ligand (eg, anti-Fas antibody) is added to the culture and exposed. Anti-Fas antibodies and mitogens (ConA) are well known to those of skill in the art (Itoh, N. et al. (1991) Cell 66: 233-243).
And Yonehara et al. (1989) J. Exp. Med. 169: 1747-175 .
6). These cells are now “induced” to apoptosis. Alternatively, cells can be contacted with an inducer after transfection with Yama nucleic acid and drug. The cells are cultivated again under appropriate temperature and time conditions. An effective amount of a drug that would inhibit apoptosis in this system is added to the cell culture. For example, professional Yama,
An effective amount of a nucleic acid molecule encoding p20 Yama or p11 Yama is contacted with a culture of cells or tissue to insert the nucleic acid. Alternatively, an effective amount of polypeptide or protein product is added to the cell culture. The cells are cultivated again for expression of the inserted nucleic acid molecule. An effective amount of the agent to be tested is then added to the cell or tissue culture at various concentrations. In another embodiment where the cells constitutively express Yama, the method can be performed without the addition of cells.

Activated Yama or p20 and p11 Yam
Since the activity of a can be inhibited by CrmA, another culture of cells that can act as a comparison was cultured under the same conditions as above except that CrmA nucleic acid was added to the culture rather than the drug. To do. CrmA nucleic acid or protein is added to the culture in an effective amount, and the cells are cultured under suitable temperature and time conditions that inhibit apoptosis. The CrmA nucleic acid or protein can be added prior to the inducer, simultaneously with the inducer, or after the inducer.

It is also desirable to maintain additional cell cultures; one without the addition of inducer to measure background release and the other.
One is to add no drug to be tested.

Each sample of cells is then assayed for apoptotic activity using the methods well known to those of skill in the art and described herein. An example of this screen is provided below.

The compositions provided herein modulate the Fas receptor pathway and cellular functions associated with this pathway by preventing or inhibiting Fas-regulated apoptosis or cell proliferation and differentiation. Useful for. As used herein, the term "cellular function mediated or regulated by Fas receptor" refers to any cellular response associated with binding of Fas or Fas / TNF receptor complex to its extracellular and / or intracellular ligands. Or features may be included. Apoptotic cell death is one such response.

Provided herein are methods of modulating cell function (eg, apoptotic cell death). These methods can be applied to a subject (eg, animal or human) in an effective amount of pro-Yama, activated Yama, p20 and / or p11 Yam.
a. Administering the nucleic acid, antibody or protein of a. Alternatively, the method can be performed with an inhibitory nucleic acid, antibody or protein. If the cell function is increased apoptotic cell death, an effective amount of the pro-Yama or nucleic acid molecules encoding p20 and p11 Yama or their protein products can be administered to the subject. If the cell function is to inhibit or prevent apoptotic cell death, the subject is administered an effective amount of antisense Yama RNA, anti-Yama antibody fragment, predominant inhibitory Yama, a nucleic acid molecule encoding CrmA, or their protein product. To be done.

When carried out in vivo, the compositions and methods are for modulating or modulating Fas receptor-induced function in a subject or individual suffering from or prone to suffer from receptor-related dysfunction. Or to maintain T cell viability and function in a subject or individual suffering from or prone to abnormal lymphocyte death (eg, CD4 ⁺ T cell depletion associated with HIV infection). Especially useful for. If the method is performed in vivo in a human patient, it is not necessary to provide the inducer. This is because the inducer is provided by the patient's immune system. When the method is performed in vivo, the delivery vector, polypeptide, polypeptide equivalent, or expression vector is added to a pharmaceutically acceptable carrier and the subject, e.g., human patient or animal (e.g., Mouse, guinea pig, monkey, rabbit or rat). Alternatively, it may be directly injected intracellularly by microinjection into the tumor or local administration. A fusion protein comprising a T cell specific ligand for targeting T cells is also
Can be built. Such T cell-specific ligands are
Antibodies to the CD3, anti-CD4, anti-CD28 and anti-IL-1 receptors are included, but are not limited to.

The present invention is also particularly useful for preventing lymphocyte death or immunosuppression in AIDS patients. Preventing or inhibiting apoptosis not only prevents cell death, but also restores functionality (eg, immune proliferative capacity) to the cells and sustains or restores the responsive immune system. Thus, the compositions and methods of the present invention are suitably combined with compositions and methods that prevent or inhibit HIV infectivity and replication.

The method is also described by Lum et al. (1993) Bone Marrow.
Transplantation 12: 565-571 or a modification of the method described in US Pat. No. 5,399,346 may be used ex vivo. In general, a sample of cells (eg, bone marrow cells or MLC) can be removed from a subject or animal using methods well known to those of skill in the art. An effective amount of a CrmA or Yama nucleic acid molecule or expression vector is added to the cells, and the cells are cultured under conditions that favor internalization of the nucleic acid by the cells. The transformed cells are then returned to the same subject or animal (autologous) or one of the same species (allogeneic) in an effective amount and in combination with a suitable pharmaceutical composition and carrier. Or reintroduced.

Alternatively, fresh peripheral blood mononuclear cells (MNC) isolated from mammals or patients are separated from red blood cells and neutrophils by Ficoll-Hypaque density gradient centrifugation. The MNCs were then washed, counted, and at approximately 1 × 10 ⁶ cells / well in 24-well tissue culture plates, 2 mM glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin, 2.5 μg / ml fungizone and 25-1,000. U / ml IL-2
The cells were cultured in AIM-V consisting of AIM-V (GIBCO) having (Cetus). Cells are 5% in a humidified incubator
Cultured in CO ₂ at 37 ° C.

After T cells started to proliferate, CrmA or Ya
The Yama nucleic acid is inserted into proliferating cells by contacting the cells with an appropriate insertion vector containing the ma nucleic acid molecule. It may be necessary to perform multiple transfections of cells. The cells are maintained for an additional 2-7 days with fresh medium and under conditions where the cells restore exponential growth. About 0.1-2.5 × 10 ¹⁰ T cells (or whole culture
80%) are injected into a mammal or patient, and the remaining cells can be cryopreserved for future injections. A sample of cells can also be removed for its expression using the Southern and Northern analyzes of the Yama nucleic acid molecule insert.

As used herein, the term “administering” for in vivo and ex vivo purposes modulates Fas-related cellular function in a subject (eg, prevents or inhibits apoptosis of target cells). Or increasing) is provided to provide an effective amount of the nucleic acid molecule or polypeptide. Methods of administering pharmaceutical compositions are well known to those of skill in the art, and microinjection,
Including but not limited to intravenous or parenteral administration. The composition is intended for topical, oral or local administration, as well as intravenous, transdermal or intramuscular administration. Administration can be continuous or intermittent throughout the course of treatment. Methods of determining the most effective means of administration and dosage are well known to those skilled in the art and are the vector used for therapy, the polypeptide or protein used for therapy, the therapeutic purpose, the treatment. Varies with target cell and subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. For example, the composition can be pre-administered to a subject already suffering from a disease or condition associated with apoptosis. In this context, an effective "therapeutic amount" of the composition is administered to prevent, or at least partially arrest, apoptosis and its associated pathologies, such as immunosuppression in HIV-infected individuals.

However, the composition may be administered to a subject or individual at suspected or at risk of developing an apoptosis-related disease to prevent pathological cell death. In one embodiment, the composition can be administered to a subject suspected of having HIV-associated lymphocyte dysfunction to maintain lymphocyte cell function and viability. In these embodiments, a "therapeutically effective amount" of the composition is administered so as to maintain cell viability and function at pre-infection levels.

The compositions and methods of the invention are for treating, preventing or ameliorating conditions associated with diseases characterized by cell apoptosis by preventing or inhibiting unwanted cell death in a subject or individual. It goes without saying that the method is provided again. Such diseases include AIDS, acute and chronic inflammatory diseases, leukemia, myocardial infarction, stroke, traumatic brain injury, neurodegenerative diseases, muscle degenerative diseases, aging, tumor-induced cachexia and hair loss. Illness, but is not limited thereto.

The present invention also provides pharmaceuticals that can be used to prevent or inhibit apoptosis and maintain the function and viability of cells in appropriate cells, or the treatment of diseases characterized by unwanted death of target cells. Vectors and protein compositions useful in the preparation of a medicament that can be used for.

It is also contemplated to combine the compositions and methods of the present invention with other suitable compositions and treatments, such as the use of CrmA, anti-idio TCR antibodies, antagonists and Fas receptors.

One aspect of the invention is cowpox virus CrmA.
The gene product is a cell surface receptor whose ligand (eg, TCR ligand, CTR, HIV, Fas ligand or TN).
Based on Applicants' discovery that it is a very potent inhibitor of apoptosis induced by binding to F).

The only reported target of the CrmA protein is the cysteine protease interleukin.
-1β convertase (ICE). In one embodiment utilizing the TNF and Fas pathways; CrmA can block the cell death program even at death-stimulating pharmacological doses. CrmA is a cowpox virus gene and serpin
(serpin) family of protease inhibitors. The nucleic acid sequence of CrmA and the corresponding amino acid sequence have been previously reported (Pickup et al . , Proc. Natl. Acad. Sc.
i. (1986) 83 : 7698-7702), and this is shown in FIG. However, Applicants have found that CrmA is a very potent inhibitor of apoptosis. Therefore, a new important function of CrmA is the prevention or inhibition of ligand-induced or cytokine-induced apoptosis. Furthermore, this data suggests that proteases of either ICE or related CrmA inhibitory proteins are components of the Fas- and TNF-induced death pathways. Accordingly, the invention provides compositions and methods for preventing or inhibiting ligand-induced or cytokine-induced apoptosis, assays for the determination of drugs or agents that promote or prevent or inhibit apoptosis, diseases resulting from apoptosis or Assay for drugs to treat or ameliorate symptoms associated with pathological conditions (e.g. AIDS), assay for detection of Fas- and TNF-induced cell death pathway proteases, and methods Providing the protease discovered using.

In one embodiment of the invention, the expression vector is specifically targeted to T cells. In these methods, CrmA DNA is intended to be operably linked to a promoter that is highly active in T cells. Such promoters include IFN-α, IL-2, IL-3, IL
-4, IL-5, IL-9, IL-10, TNF-β, GM-CSF, CD4, CD8 and IL-2 promoters, but are not limited thereto.

Although these methods are preferably carried out with the CrmA gene, it will be apparent to those skilled in the art that the polypeptide product of the CrmA gene and its bioequivalents will be useful in the methods of the invention. Should be. CrmA
The gene product is a 38 kDa protein and is known to be a specific inhibitor of IL-1β. This protein can be purified from natural sources as described in Ray, CA (1992) Cell 69: 597-604, or Pickup et al.
986) (supra), Ray et al. (1992) (supra), and Moss, B. (199).
0) Virology , pages 2079-2112, Raven Press, NY, can be recombinantly produced using the expression vectors described above in a host-vector system. The protein is used in a substantially pure form. "Substantially pure" means
It means that the protein is substantially free of other biochemical moieties with which it is normally associated in nature. Proteins can also be produced using the sequences provided in Figure 9 and methods well known to those of skill in the art.

Accordingly, the present invention also provides CrmA polypeptides, proteins, their bioequivalents and fusion proteins containing them for use in the methods described herein. For targeted delivery to T cells, these polypeptides or proteins are
It can be bound to a targeting antibody (eg anti-CD3 or anti-CD4).

This method may be performed in vitro, ex vivo or in vivo. When this method is performed in vitro, the expression vector, protein, or polypeptide can be added to the cultured cells or to a pharmaceutically acceptable carrier as defined below. further,
The expression vector or CrmA DNA can be inserted into the target cells using well known techniques such as transfection, electroporation, or microinjection.

More specifically, the in vitro method comprises:
Providing a cell or tissue culture having any cell surface receptor that mediates apoptosis such as TCR, TNF receptor, or Fas receptor. These cells are cultured under conditions (temperature, growth or culture medium, and gas (CO ₂ )) for an appropriate time to obtain exponential growth without density-dependent constraints. These cells are then exposed to the preliminary conditions required for apoptosis. For example, TCR ligand, HIV, SIV, TNF, or Fas
An effective amount of inducer such as a ligand (eg anti-Fas antibody) is added to the culture. Anti-Fas antibody and mitogen (Con
A) is well known to those skilled in the art (Itoh, N et al. (1991) Cell 66: 233-2).
43 and Yonehara et al. (1989) J. Exp. Med. (1989) 169: 1.
747-1756). Thus, these cells are "induced" to apoptosis. The cells are again cultured under appropriate conditions (temperature and time). In one embodiment, HIV or SIV is added to the culture. In other embodiments, the drug or agent to be tested is added at various concentrations concurrently with, before, or after the inducer.

CrmA nucleic acid or protein is then added to the culture in an effective amount, and the cells are cultured under conditions (temperature and time) appropriate to inhibit apoptosis. CrmA
The nucleic acid or protein may be added before the inducer, simultaneously with the inducer, or after it. Cells are assayed for apoptotic activity using methods well known to those of skill in the art and those described herein. It will be apparent to those skilled in the art that two separate cultures of cells should be treated and maintained as a test population. One is maintained without giving inducer,
Background emission is measured. The other does not give the drug to be tested. The latter cell population is used as a control.

The use of the compositions and methods in vitro provides a powerful bioassay for screening for drugs that are agonists or antagonists of CrmA function in these cells. Therefore, one may screen for drugs that have similar or enhanced ability to prevent or inhibit apoptosis. It is also useful to assay for drugs that have the ability to inhibit HIV infection and replication. This is because CD4 ⁺ cells do not die by concurrent viral infection. By noting that morphological changes in apoptosis do not occur, or more simply by not causing cell death, one of skill in the art can determine when the method has been successfully completed. In vitro methods further provide assays to determine whether the methods of the invention are useful for treating a non-experimental pathological condition or disease associated with apoptotic cell death in an individual.

In another embodiment, a T cell line called CEM (ATCC) is obtained and used. Because this cell line
It has been shown to undergo PCD when infected with HIV. CEM cells are transfected by electroporation using the CrmA expression construct and vector alone as a control. Clonal strains are obtained and infected with HIV at various rates of multiplicity of infection. Cytopathological effects are assayed by microscopic observation and quantification of apoptosis after staining with prodium iodide. A variety of agents may be tested for their ability to inhibit or prevent apoptosis using the methods described above.

[0174]

【Example】

Example 1 Assay for Assessing Apoptosis Apoptosis was assessed by several methods described below.

Fluorescent DNA staining dye-A fluorescent DNA staining dye was used to reveal the morphology of the nucleus. A transmission electron microscope was also used. 6 for prodium iodide staining
22mm placed in a 35mm well of a well culture dish (Costar)
MCF7 cells were grown on No. ² glass coverslips (Corning). After treatment with TNF, anti-Fas cycloheximide (CHX) or untreated, the medium is removed and the wells are rinsed twice with phosphate buffered saline (PBS) and in -20 ° C in 100% methanol. Fixed for 10 minutes, washed 3 times with PBS, and stained in 100 μg / ml solution (propidium iodide (Sigma) prepared in PBS) for 10 minutes at room temperature. The coverslips were then washed 3 times with PBS, blotted dry, and mounted with Vectashield mounting medium for fluorescence.
ia) (Vector Laboratries) was used to load glass slides. 30 μl cell suspension in wet mount (approximately 3 x 10 ⁵
Cell / ml density) by mixing with 5 μl of acridine orange solution prepared in PBS at 100 μg / ml
BJAB cells were stained with acridine orange (Sigma). Nuclei of both MCF7 stained with propidium iodide and BJAB stained with acridine orange were analyzed by fluorescence microscopy on a FITC range barrier filter cube (range bar).
It was visualized using a carrier filter cube). Bio-Rad MRC60
Laser scanning confocal microscopy was performed using 0 confocal microscopy and digitized images obtained artificially with color.

For electron microscopy, cells were fixed and processed by standard electron microscopy analysis procedures.

Quantitative Apoptosis Assay—MCF7 cells or the resulting transfectants were treated with 2.5 × 10 ⁵ cells /
Plated on coverslips at the concentration of wells. After 2 days, cells attached and expanded, then TNF or anti-Fas
+ CHK was added. TNF at a final concentration of 20 ng / ml, anti-Fas 25
CHX (Sigma) was added at a final concentration of ng / ml and 10 μg / ml. After 22 hours for TNF-treated samples or 18 hours for anti-Fas + CHX-treated samples, cells were fixed, stained with propidium iodide and loaded as above. Apoptotic and non-apoptotic cells were quantified and the percentage of non-apoptotic cells was calculated based on nuclear morphology using fluorescence microscopy analysis. A minimum of 100 cells were counted for each sample, and each experiment was performed in at least duplicate. Spontaneous apoptosis in untreated and CHX alone treated samples was also quantified, as a small fraction of cells undergo apoptosis in any normally growing cell culture.
The percentage of non-apoptotic cells in TNF-treated or anti-Fas + CHX-treated samples was then normalized by correcting for the frequency of spontaneous apoptosis in untreated or CHX-only treated samples, respectively.

BJAB cells were grown to 3 × 10 ⁵ cells / ml,
Treated with anti-Fas antibody at a concentration of 250 ng / ml (unless otherwise indicated) for 18 hours, then aliquots were stained with acridine orange as described above. Apoptotic and non-apoptotic cells were quantified and normalized to untreated samples. Assays were performed in at least duplicate.

MTT Conversion Assay-The secondary assay for cell death is the MTT conversion assay (Opipari, AW et al., J. Biol. Chem.
(1992) 267: 12424-12427) and crystal violet staining (Tartaglia, LA et al. (1993) Cell 74:
845-853) was used.

DNA Fragmentation Assay-The assay of CTL-induced target cell DNA fragmentation was performed with modifications as previously described by Duke (1992) (below). Human PBMC were prepared as described above. 1 × 10 ⁷ BJAB cells were incubated with [methyl- ³ H] thymidine pool. The cells were then plated as above. After 4 hours, 100μ of 95% ethanol was added to each well, the contents were mixed and the plates were incubated for an additional hour. The addition of ethanol caused the release of fragmented DNA, but the high molecular weight chromatin remained intracellular. The plates were then centrifuged, collected and analyzed as above.

Cell Culture-MCF7 cells, BJAB cells, and the resulting vector, and a stable transfectant of CrmA, together with a stable strain transfected with a CrmA mutant, were heat-inactivated 10% fetal calf serum ( Hyclone), L-glutamine, penicillin / streptomycin, and non-essential amino acids and supplemented with 500 μg / ml for MCF7 transfectants and 3 mg / ml for BJAB transfectants (Life Technologies, Inc. ) Was maintained in RPMI 1640 medium supplemented further.

Isolation of Human Peripheral Blood Mononuclear Cells (PBMC) -20 ml
~ 50 ml heparinized human whole blood was obtained from healthy donors and depleted of red blood cells with a Ficoll-Hypaque gradient. The obtained PBMC was used as RPMI supplemented with 10% fetal bovine serum.
After stimulation with 10 μg / ml PHA-P in for 3 days at 37 ° C., it was used for cell lysis and DNA fragmentation assay. Approximately 40% of the small lymphocyte pool in stimulated PBMC was CD4 + lymphocytes, measured by flow cytometry
0% were CD8 + lymphocytes. Subset depletion experiments confirmed that CTL activity at both 4 and 24 hours was almost completely mediated by CD8 + T lymphocytes. For isolation of natural killer (NK) cell activity, freshly isolated human PBMC were used without further stimulation.

Selection of plasmids, transfections, and stably transfected strains--The CrmA gene and its mutant forms shown in FIG. 22 were isolated from pcDNA3 (I
nvtrogen) was cloned into a mammalian expression vector. C
The rmA gene was obtained from Dr. David Pickup (Duke University) and used as a template in a PCR reaction to amplify the entire coding sequence. This PCR reaction uses custom oligonucleotide primers with built-in restriction enzyme sites. The sequences of the primers are: CrmA / 5 '/ R1 5'CAC CGG AAT TCC ACC ATG GAT ATC TTC AGG GAA ATC G CrmA / 3' / XbaI 5'GCT CTA GAC TCG AGT TAA TTA GTT GTT GGA GAG CAA TAT C.

This PCR fragment was digested with EcoRI and Xba1 restriction enzymes and subcloned into similarly digested pcDNA3 vector. After transformation into XL-1 Blue host E. coli competent cells (Strategene), individual colonies were grown, plasmids were extracted, and the presence of the CrmA gene was confirmed by both restriction mapping analysis and DNA sequence analysis. .

The resulting expression construct or pcDNA3 itself
(As a control) was introduced into both MCF7 and BJAB cells by electroporation. MCF7 cells to 0.
Electroporated in a 4 cm cuvette (Biorad) at 330 V, 960 TF, plated in 100 mM culture dishes at various dilutions and selected with G418 sulphate (Gibco-BRL) at a concentration of 500 μg / ml. After 3 weeks of selection, pooled populations from each transfection were prepared by trypsinizing culture dishes containing hundreds of colonies. In addition, clonal cell lines were obtained by picking individual colonies from selective culture dishes. BJAB cells in 0.4 cm cuvette (Bio-Rad)
Electroporated at 220V, 960TF, and 3mg / ml G
Selected in 418 sulfuric acid. One day after transfection, a portion of the cell population was diluted to a concentration of 2500 cells / well in a 96-well culture dish from which the clone strain was obtained after G418 selection. The remaining cells were retained as a pooled population.

Cell Lines, TNF and Anti-Fas Antibody-MCF Cell Lines are TNF sensitive subclones and were obtained from Dr. David R. Spriggs (University of Wisconsin). MCF7 is a breast cancer epithelial cell line that expresses the TNF receptor and
Sensitive to killing. BJAB cell line is Fred
It was a gift from Dr. Wang (Harvard). Recombinant TNF (specific activity 6.27 × 10 ⁷ U / mg) was produced by Genentech (South San Francisc
o, CA). Anti-Fas monoclonal antibody (Clone CH
-11, IgM) was obtained from Pan Vera (Madison, WI). The anti-PARP monoclonal antibody was clone C-2-10, which was previously described (Lamarre et al. (1988) Biochem. Biophy.
s. Acta. 950: 147-160), recognizing an epitope near the N-terminus of PARP (located between amino acids 216 and 375). The affinity purified UI-70kDa reactive antiserum was derived from human autoimmune serum, which was a gift from Dr. Antony Rosen (John Hopkins University, Baltimore, MD).

Treatment with anti-Fas antibody or TNF and preparation of cell lysates—MCF7 cells or the resulting transfectants were plated in 100 mm culture dishes at a concentration of 2 × 10 ⁶ cells / culture dish. On day 2, cells were treated with 40 ng / ml TNF for the indicated times. After rinsing with PBS, by scraping the cells into 15 ml PBS + protease inhibitor (1 mM phenylmethylsulfonylfluoride, 0.5 mg / ml aprotinin, 0.5 mg / ml antipain, and 0.5 mg / ml pepstein) Collect, collect by centrifugation, and
2.5 ml sample buffer (50 mM Tris-HCl (pH 6.8), 6 M urea, 6% 2-mercaptoethanol, 3% SDS, and 0.
Dissolved in 003% Bromophenol Blue. If non-adherent cells were present in the culture medium (eg at later time points), floating cells were also harvested by centrifugation and combined with the adherent cell pellet prior to lysis in sample buffer. BJAB cells or the resulting transfectants were aliquoted into 4 wells of each well in 6 well culture dishes at a concentration of 5 × 10 ⁵ / ml. Next day, cells were anti-Fas antibody (250ng / ml)
Treated for the time indicated, collected by centrifugation, washed once with PBS + protease inhibitor and lysed in 2 ml sample buffer.

Western blotting Western blotting-Immunoblotting to assess the status of UI-70kDa was performed using Casciola-Rosen, LA.
(1994) J. Biol. Chem. 269: 30757-30760. Briefly, cell lysate (2
0 μl) was separated by SDS-polyacrylamide gel electrophoresis and transferred by electroblotting to a nitrocellulose membrane (Schleicher and Schuell). Blocking buffer (10 mM Tris (pH 7.6), 150 mM NaC
Blots were blocked for 1 hour at room temperature with 0.5% Nonidet P-40, 3% bovine serum albumin). UI-70k
Da affinity purified antiserum was diluted 1: 5000 in blocking buffer and used, and incubated at room temperature for 1 hour. Second reagent, biotinylated goat anti-human IgG
Antibody (Southern Biotech) in blocking buffer 1:67
It was diluted to 00, used, and incubated at room temperature for 50 minutes.
A third reagent, streptavidin horseradish peroxidase conjugate (Southern Biotech) was used diluted 1: 10,000 in blocking buffer and incubated for 30 minutes at room temperature. Signal visualization was performed using electrochemiluminescence (Amersham Corp.).

Cytotoxicity Assay Cytotoxicity Assay—PHA-stimulated allegeneic CTL using human PBMC stimulated with PHA.
Cellular Assays by Grabstein, K. (1980) Selected Methods
in Cellular Immun. (Mishell and Shiigi) 125-13
The description was made on page 7, WH Freeman and Co., NY, NY with some modifications. Briefly, 2 × 10 ⁷ BJAB target cells were stimulated with 200 μCi in a total volume of 400 μl Hanks balanced salt solution, 0.2% bovine serum albumin after PBMC stimulation.
Incubated with (Na) ₂ ⁵¹ CrO ₄ (ICN) at 37 ° C. for 2 hours.
Target cells were then washed and plated at 10,000 cells / well in round bottom 96 well plates. Human PBMC
Plated with killer to target ratios ranging from 0.25: 1 to 32: 1, and PHA-P was added to a final concentration of 10 μg / ml and a total volume of 200 μl. In some experiments, EGTA and MgCl ₂ were added at 10 mM and 4 mM, respectively, to clamp intracellular calcium levels. Centrifuge plates at 4 and 24 hours,
Then, a 30 μl aliquot was collected and dropped on a glass fiber filter. Samples were analyzed using a β scintillation counter. Specific cytotoxicity (%),
((Sample cpm-Spontaneous release cpm) / (Total release cpm-Spontaneous release
It was calculated as cpm)) × 100. Background emissions of chromium are typically 5-15% at 4 hours, and
It was 20 to 30% at the 24th hour. Addition of EGTA / MgCl ₂ did not itself affect cell viability as measured at both time points by both background chromium release and morphological examination of cells. There was no significant difference in background ⁵¹ Cr release between vector and CrmA transfected strains. The NK cytotoxicity assay was performed as described above, except that it did not stimulate cells and PHA was not present during the assay.

RNA Isolation and Northern Analysis RNA Isolation and Northern Analysis of CrmA—RNA isolation and Northern analysis was performed according to Dixit et al. (1990) J. Biol. Chem. 265: 297 .
Performed as described in 3-2978. As above, PCR (Perkin
-Elmer) was used to generate a probe spanning the coding region of the CrmA gene. Clont β-actin cDNA probe
ech (Palo Alto, CA), and hybridization signals were obtained from Molecular Dynamics Phosphorima.
Visualized as digitized image on ger.

1 and 2 show the open reading frame and deduced amino acid sequence of the protein referred to herein as pro-Yama. In the nucleotide sequence shown in Figure 1, the starting methionine begins at 224.

FIGS. 3 and 4 show that pro-Yama is a protease zymogen which upon activation cleaves PARP into an 85 kDa apoptotic fragment in vitro.

In the upper panel of FIG. 3, the 6 × His tagged Yama was expressed and used [ ³⁵ S] -Met in an in vitro transcription / translation reaction as described in the Examples section below. Shown to be labeled and purified by affinity chromatography on continuous DEAE Sepharose and Nickel chelate columns. 0.586 μg purified PARP
Were incubated with either buffer alone (lane 1), ICE (lane 2), purified pro-Yama (lane 3), or purified pro-Yama after activation with ICE (lane 4) for 2 hours at 37 ° C. An in vitro reaction was set up.
After incubation, 1/5 of each reaction was added to SDS-PA
Analyzed by immunoblotting with monoclonal antibody C-2-10 against GE and PARP. Whole cell lysates from BJAB cells that underwent anti-Fas-induced apoptosis (lane 5) or whole cell lysates from untreated BJAB cells (lane 6) were run side by side with in vitro reaction samples. Equal amounts of the in vitro reactions shown in lanes 1 to 4 of Figure 3 were separated by SDS-PAGE and the dried gel
The results obtained by subjecting to Phosphorimager analysis to evaluate the status of the radiolabeled Yama protein are shown in the panel at the bottom. The black arrow indicates the mobility of purified Pro Yama, which is referred to as the full-length p32 form. The white arrow shows the professional Yama by ICE
The two major proteolytic fragments observed after activation are shown and are believed to correspond to the predicted p20 and p11 subunits predicted from cleavage at the Asp residue in proYama.

FIG. 4 shows that cleavage of PARP into the 85 kDa fragment is a characteristic feature of Fas- and TNF-induced apoptosis. In the upper panel,
BJAB cells were left untreated (UnRx) or treated with agonist anti-Fas antibody (250 ng / ml) for the indicated times, and as described in the Examples section below, and Cell lysate was prepared and anti-PARP monoclonal antibody C-
Analyzed by immunoblotting with 2-10.
In the lower panel, MCF7 cells are left untreated (UnRx) or for the indicated times recombinant TNF (40 ng /
ml). Cell lysates were similarly analyzed.

5 to 7 show that a point mutation in the reactive site loop (RSL) of CrmA inactivates its ability to inhibit ICE.

In FIG. 5 (top), the reactive site loop sequences of CrmA and CrmA variants are compared. Amino acid 291 of wild-type CrmA was changed from Thr to Arg by site-directed mutagenesis. The lower panel shows protein expression and purification in E. coli as a 6xHis fusion as described in the Examples section below. Briefly, a 44 ng aliquot of ICE was titrated with the indicated amounts of purified CrmA protein (open squares) or CrmA mutant protein (filled circles). Residual IC
E activity (expressed as the ratio of inhibition rate (V _i ) to non-inhibition rate (V ₀ )) was measured using a chromogenic ICE substrate,
And it plotted against the amount of CrmA. ICE activity is 300ng
, But no inhibition with CrmA muteins was detected using 30 μg in a 500-fold molar excess over the enzyme.

FIG. 6 shows that the CrmA mutant does not complex with ICE. [ ³⁵ S] -Met-labeled CrmA or CrmA mutant protein is coupled to each gene by transcription / transcription.
Produced by translation. The indicated amount of ICE was added directly to the diluted lysate as described in the Examples section below and incubated, after which the samples were separated by non-denaturing PAGE and radioactive signals were detected using a Phosphorimager. Was detected. Mutant CrmA did not form a complex with ICE, in fact ICE did not appear to have an effect on this protein. Compared to this,
Some of the wild-type CrmA formed a complex, while the rest were ICE
And was cleaved in a manner that characterizes the interaction of CrmA with (Ko
See miyama et al. (1994) J. Biol. Chem. 269: 19331-19337).

FIG. 7 shows transverse urea gradient polyacryla gel electrophoresis.
Mide gel electrophoresis) (TUG-PAGE) shows that the tertiary structure of the CrmA protein and the CrmA mutant protein cannot be distinguished. [ ³⁵ S] -Met labeled CrmA or CrmA muteins were generated by coupled transcription / translation and analyzed by TUG-PAGE as described in the Examples section below. The gel was dried and analyzed using a Phosphorimager to detect the unfolded signature of each protein. Construction
No difference in (signature) was observed, indicating that the point mutation in the CrmA mutant did not disrupt the tertiary structure of the protein.

FIG. 8 shows that PARP cleavage by activated Yama in vitro can be inhibited by CrmA, but not by equivalent amounts of CrmA mutants. The upper panel shows the results of [ ³⁵ S] -Met labeled Yama produced and purified and activated by ICE as described in the Examples section below. Next, the purified and activated Yama as described below was added to buffer (lane 1), 270 pmol of CrmA.
0.586 μg in the presence of either protein (lane 2) or 270 pmol of CrmA mutein (lane 3)
Of purified PARP at 37 ° C. for 2 hours. 1 of each reaction after incubation with PARP
/ 5 was analyzed by immunoblotting with anti-PARP monoclonal antibody C-2-10. In the lower panel, the equivalent amount of each of the above reaction products was subjected to SDS-PAGE,
The dried gel was analyzed using a Phosphorimager to assess the status of labeled Yama protein. White arrows indicate the positions of the two major products found in the activated Yama preparation, and these are the predicted p20 predicted from the amino acid sequence of Yama.
And may be thought to correspond to the p11 subunit.

FIG. 9A and FIG. 9B show that CrmA activates Ya.
It is a figure which shows that it directly interacts with ma but does not interact with professional Yama.

FIG. 9A is a Phosphorimager scan of the reaction sample prior to immunoprecipitation analysis. Radiolabeled Pro Ya
ma (lanes 1 and 3) or radiolabel activated Yama
80 μl reactions were assembled in which (lanes 2 and 4) were mixed with 358 pmol of CrmA (lanes 1 and 2) or 358 pmol of CrmA mutant (lanes 3 and 4) recombinant protein. SDS-PAGE 10 microliters of each reaction
And pro-Yama or activated Yama by phosphorimaging analysis.
Detected by Black arrows indicate mobility of pro-Yama (p32), while white arrows indicate putative p20 and p11 of activated Yama.
Indicates a subunit.

FIG. 9B shows a polyclonal sample of the reaction sample.
Immunoprecipitation with CrmA antiserum. Each of the above reactants
35 μl of rabbit polyclonal C as described below
Subjected to immunoprecipitation using rmA antiserum. Precipitate SDS-P
Radiolabeled proteins separated by AGE are labeled with Phosphorima
It was detected using ger. White arrow indicates p20 of activated Yama
And the p11 subunit are shown.

10 to 12, in FIG. 10, CrmA or CrmA mutants are expressed in stably transfected clones. The left panel shows vector control (BJAB V1, BJAB V4), CrmA (BJAB CrmA2, BJ).
AB CrmA3), or CrmA mutant (BJAB CrmA mutant # 12,
BJAB CrmA variant # 17) shows a cloned BJAB cell line stably transfected with either of the expression constructs and analyzed by Western blotting with anti-CrmA antiserum. In the right panel, the vector control (MCF7 V
4), CrmA (MCF7 CrmA2, MCF7 CrmA3, MCF7 CrmA4),
Alternatively, cloned MCF7 cell lines stably transfected with either CrmA mutant (CrmA mutant # 1, CrmA mutant # 2) expression constructs were similarly analyzed.

FIG. 11 shows 8 during Fas-induced apoptosis.
We show that PARP cleavage into the 5 kDa fragment is inhibited by CrmA but not by CrmA mutants.
Cloned BJAB trans that does not express CrmA (BJAB V1, BJAB V4), expresses CrmA (BJAB CrmA2, BJAB CrmA3), or expresses CrmA mutant (BJAB CrmA mutant # 12, BJAB CrmA mutant # 17) Fectants were treated with agonist anti-Fas (250 ng / ml) antibody for the indicated times and lysates prepared and analyzed by Western blot with anti-PARP monoclonal antibody C-2-10.

FIG. 12 shows 8 during TNF-induced apoptosis.
We show that PARP cleavage into the 5 kDa fragment is inhibited by CrmA but not by CrmA mutants.
Does not express CrmA (MCF7 V4, MCF7 CrmA2), expresses CrmA (MCF7 CrmA3, MCF7 CrmA4), or expresses CrmA mutant (MCF7 CrmA mutant # 1, MCF7 CrmA mutant # 2)
The cloned MCF7 transfectants were treated with TNF (40 ng / ml) for the indicated times and lysates prepared,
It was analyzed by Western blot with anti-PARP monoclonal antibody C-2-10.

FIG. 13 shows that CrmA, but not CrmA mutant
It is shown to block s-induced cell death and TNF-induced cell death. In the upper panel, MCF7 stable transfected clones were either untreated (UnRx) or TNF (40 ng / m2).
1) for 18 hours, after which the cells were fixed and stained with propidium iodide and examined for nuclear morphology by confocal microscopy. CrmA conferred significant protection against TNF-induced apoptosis, while both vector-transfected expression lines and CrmA mutant expression lines were susceptible to TNF-induced apoptosis.
In the lower left panel, the BJAB stable transfectant clones shown were transfected with Fas-induced P using the acridine orange-based apoptosis assay as described below.
Their sensitivity to CD was evaluated quantitatively. CrmA mutant expressing cell lines were uniformly sensitive, but CrmA expression conferred significant protection. In the lower right panel, the MCF7 stable transfected clones shown are
Their susceptibility to TNF-induced cell death was assessed quantitatively as described in the Examples section below.

FIG. 14 shows that TNF and anti-Fas + CHX showed MCF7.
It is shown to induce apoptosis in cells. MCF7
Cells were treated with TNF or anti-Fas + CHX as described in the Examples section below. Top row: Nuclei of untreated, TNF-treated, or anti-Fas + CHX-treated MCF7 cells were stained with propidium iodide and visualized by laser scanning confocal microscopy. Arrows indicate examples of apoptotic nuclei. Bottom row: Transmission electron microscopy of untreated, TNF-treated, or anti-Fas + CHX-treated MCF7 cells. FIG. 15 shows that anti-Fas antibody induces apoptosis in BJAB cells. BJAB cells were treated with anti-Fas antibody as described below. The figure shows fluorescence microscopy of untreated or anti-Fas treated acridine orange stained BJAB cells. Arrows indicate examples of apoptotic nuclei. Inset: Laser scanning confocal microscopy of untreated or anti-Fas treated acridine orange stained BJAB cells.

Figure 16 shows inhibition of apoptosis by CrmA in pooled transfectant populations. Pooled populations of the indicated vector or CrmA transfected cells were analyzed for susceptibility to TNF- or Fas-mediated apoptosis.

FIGS. 17, 18 and 19 show inhibition of TNF and anti-Fas + CHX-induced apoptosis of MCF7 cells by CrmA. Upper row: Susceptibility of MCF7 vector-transfected clones (V1-V5) or CrmA-transfected clones (CrmA1 to CrmA5) to TNF and anti-Fas + CHX-induced cell death was assessed by propidium iodide apoptosis assay. Inset: Sensitivity of selected clones to TNF and anti-Fas + CHX-induced cell death by MTT conversion assay. Middle row: Northern analysis of the corresponding cell line to detect CrmA transcripts. Bottom: Northern analysis to detect β-actin transcript load of RNA. In the figure, the left bar indicates TNF and the right bar indicates anti-Fas + CHX. Figure 18: Crm of apoptosis induced by increasing doses of anti-Fas antibody
Shows A-mediated inhibition. FIG. 19 shows that CrmA inhibits apoptosis induced by increasing doses of anti-Fas antibody. Vector-transfected clones (BJAB V
1) or CrmA-transfected clone (BJAB CrmA
2, BJAB CrmA3) was analyzed for susceptibility to apoptosis induced by the indicated doses of anti-Fas antibody.

FIG. 20 shows inhibition of anti-Fas-induced apoptosis in BJAB cells by CrmA. Top: Analysis of susceptibility of BJAB vector-transfected clones (V1-V6) or CrmA-transfected clones (CrmA1-CrmA10) to anti-Fas-induced apoptosis. Cell death was assessed by the acridine orange apoptosis assay. Inset: Sensitivity of selected clones to anti-Fas-induced cell death assessed by MTT conversion assay. Middle row: Northern analysis of corresponding cell lines for detection of CrmA transcript expression. Bottom: Northern analysis to detect β-actin transcripts to assess loaded RNA. Figure 21 shows CrmA
Inhibits apoptosis by increasing doses of anti-Fas antibody. Vector-transfected clones (BJ
AB V1) or CrmA transfected clones (BJAB Cr
The sensitivity of mA2, BJAB CrmA3) to apoptosis induced by the indicated doses of anti-Fas antibody was analyzed.
In the figure, the left bar indicates BJABV1, the center bar indicates BJAB CrmA2, and the right bar indicates BJAB CrmA3.

FIG. 22 shows the nucleic acid sequence and the corresponding amino acid sequence of the cowpox virus CrmA gene and gene product.

FIG. 23 and FIG. 24 show that wild-type CrmA is CTL.
We show that it inhibits mediated apoptosis but not mutant CrmA. In FIG. 23, BJAB cells stably transfected with either vector controls (clones V1 (filled squares) and V4 (filled circles)) or CrmA expression constructs (clones CrmA2 (filled triangles) and CrmA3 (filled circles)) are shown. The clonal strain was used as target cells in a 24-hour PHA-stimulated allogeneic CTL-mediated cytolytic assay based on ⁵¹ Cr release, as described herein. In FIG.
Vector control (clone V1 (black square)), CrmA
(Clone CrmA2 (closed circle)) or mutant CrmA (clonal mutant CrmA12 (closed triangle) and mutant CrmA17 (open circle)) expression constructs stably transfected
A clonal line of BJAB cells was similarly loaded with ⁵¹ Cr and analyzed in a 24-hour CTL-mediated cytolytic assay. Each data point shown in both FIG. 23 and FIG. 24 represents the average of samples run in triplicate, and the standard deviation was always less than 5% of the average. Each experiment was repeated at least 3 times independently and had similar results. The absolute value of chromium release cannot be directly compared between experiments due to changes due to differences in blood donors, but the change in the degree of protection by CrmA compared to vector or mutant CrmA was less than 5%. It was 100% chromium release corresponded to values between 4000 and 12,000 cpm depending on the individual experiment.

FIGS. 25 and 26 show that CrmA completely blocks the Ca ²⁺ -independent component of CTL-mediated killing. In FIG. 25, the vector (clone V1 (black square) and
V4 (black circle) or CrmA (clone CrmA2 (white triangle) and C
rJA3 (open circles)) expression constructs were stably transfected with BJAB cells as described herein.
It was analyzed in a 24-hour CTL-mediated cytolytic assay in the absence of EGTA. In FIG. 26, the same cell line was used in the presence of 10 mM EGTA and 4 mM Mg ²⁺ as described herein.
Were analyzed in a 24-hour CTL-mediated cytolytic assay supplemented with. Each data point shown in both FIG. 25 and FIG. 26 represents the mean of samples run in triplicate, and the standard deviation was always less than 5% of the mean. Each experiment was repeated at least 3 times independently and had similar results. The absolute values of chromium release cannot be directly compared between experiments due to changes due to differences in blood donors, but the change in the degree of protection by CrmA compared to vector was less than 5%. 100% chromium release corresponded to values in the range between 4000 and 12,000 cpm depending on the individual experiment.

27 and 28 show that CrmA does not block the Ca ²⁺ -dependent component of CTL-mediated cytolysis.
Figure 27: Vector (clones V1 and V4) or CrmA
BJAB cells stably transfected with either of the (clone CrmA2 and CrmA3) expression constructs are shown.
In the figure, the white square is V1, the white triangle is V4, and the plus is CrmA.
2 # 2, and white circles indicate CrmA2 # 3. These were analyzed in a 4-hour CTL-mediated cytolytic assay in the absence of EGTA as described herein. In FIG. 28, the same cell line was used for 4 hours CTL as described herein except in the presence of 10 mM EGTA and supplemented with 4 mM Mg ^2+.
It was analyzed in a mediated cytolysis assay. In the figure, white squares indicate V1, white triangles indicate V4, plus indicates CrmA2 # 2, and white circles indicate CrmA2 # 3. Each data point shown in both FIG. 27 and FIG. 28 represents the mean of samples run in triplicate and the standard deviation was always less than 5% of the mean. Each experiment was repeated at least 3 times independently,
It had similar results. The absolute values of chromium release cannot be directly compared between experiments due to changes due to differences in blood donors, but the change in the degree of protection by CrmA compared to vector was less than 5%. 100% chromium release corresponded to values in the range between 4000 and 12,000 cpm depending on the individual experiment.

Figure 29 shows that CrmA blocks CTL mediated DNA fragmentation. Vector (Clone V1 (black square)
And V4 (black circle) or CrmA (clone CrmA2 (white triangle))
And CrmA3 (white squares)) stably transfected BJAB cells with either the expression construct, as described herein below, [methyl - ³ H] labeled thymidine, 4 and CTL DNA fragmentation was induced by time incubation. Each data point represents the mean of samples run in triplicate and the standard deviation was always less than 5% of the mean. Two independent experiments gave similar results, and the degree of protection by CrmA compared to vector varied by less than 5% between experiments. 100% DNA fragmentation corresponded to absolute values in the range of 400 cpm and 1,000 cpm depending on the individual experiment.

FIGS. 30 and 31 show that activation of either Fas or TNF receptors induces U1-70 kDa cleavage. In FIG. 30, BJAB cells are left untreated (UnRx) or treated with agonist anti-Fas monoclonal antibody (250 ng / ml) for the indicated times and cell lysates prepared and Analyzed by Western blotting with U1-70kDa reactive antiserum as described in the specification. The arrow labeled "b" indicates a background protein that cross-reacts with antiserum but is not cleaved during apoptosis. In FIG. 31, MCF7 cells were either left untreated (UnRx) or treated with TNF (40 ng / ml) for the times indicated and cell lysates were similarly analyzed. did. "B"
The arrow indicated by indicates a background protein that cross-reacts with antiserum but is not cleaved during apoptosis.

32 to 34 show that CrmA is F1-70 kDa F
It shows that as-induced cleavage is blocked, but mutant CrmA does not. In FIG. 32, cloned BJAB cell lines that do not express CrmA (V4, V1, CrmA5) are left untreated (UnRx) or for the duration of the indicated agonist anti-A.
Either treated with Fas antibody (250 ng / ml), and lysates were prepared and analyzed by Western blotting with U1-70 kDa reactive antisera as described herein. The arrow labeled "b" indicates a background protein that cross-reacts with antiserum but is not cleaved during apoptosis. FIG.
Leaves cloned BJAB transfectants expressing CrmA (CrmA2, CrmA3) untreated (UnRx)
Or the agonist anti-Fas antibody (250
ng / ml) and analyzed in the same manner. The arrow labeled "b" indicates a background protein that cross-reacts with antiserum but is not cleaved during apoptosis. In FIG. 34, the mutant CrmA
(Mutant CrmA / Clone # 12, Mutant CrmA / Clone # 1
Cloned BJAB transfectants expressing 7) were either left untreated (UnRx) or treated with anti-Fas antibody (250 ng / ml) for the times indicated, and similar Analyzed. The arrow indicated by "b" is
It represents a background protein that cross-reacts with antisera but is not cleaved during apoptosis.

35 to 37 show that CrmA is a T of U1-70 kDa.
We show that it blocks NF-R-induced cleavage, but mutant CrmA does not. In Figure 35, cloned MCF7 transfectants that do not express CrmA (V4, CrmA2).
Left untreated or during the indicated time, TN
Either treated with F (40 ng / ml) and lysates prepared and used as described herein, U1-70
Analyzed by Western blotting with a kDa-reactive antiserum. The arrow labeled "b" indicates a background protein that cross-reacts with antiserum but is not cleaved during apoptosis. In FIG. 36, Cr
A cloned MCF7 transfectant (Cr
mA3, CrmA4) were either left untreated (UnRx) or treated with TNF (40 ng / ml) for the times indicated and analyzed in the same way. The arrow labeled "b" indicates a background protein that cross-reacts with antiserum but is not cleaved during apoptosis. In Figure 37, a cloned MCF7 transfectant expressing the mutant CrmA (clone # 1, clone
# 2) was either left untreated (UnRx) or treated with TNF (40 ng / ml) for the times indicated and analyzed similarly. The arrow indicated by "b" is
It represents a background protein that cross-reacts with antisera but is not cleaved during apoptosis.

FIG. 38 shows that a mammal, Yama, stimulates apoptosis (eg, staurosporine).
Is activated by. Jurkat cells are left untreated or treated with 2 μM staurosporine or 200 ng / ml anti-APO-1 antibody for 3 hours. 10 x 10 ⁶ Ju
Cytosolic extracts from rkat cells were analyzed by SDS-PAGE followed by immunoblots with antibodies to the 17kDa and 12kDa subunits of Yama. Similar results were obtained using BJAB or CEM cells. It was observed that the intermediate form of Yama's 17kDa subunit was also thought to contain the prodomain (specific only to antibodies to the 17kDa subunit).

39 to 42 show bcl-2 and bcl-xL.
Shows that it works upstream of Yama. Figure 39: Jurkat
Expression of bcl-2 and bcl-xL in cell lines. Jurka
t cells are control vectors, pEBs-bcl-xL, or
Transfected with pEBs-bcl-2 and selected with hygromycin, SDS-PAGE and immunoblot were performed as described herein. Figure 40 shows bcl-2 and bcl
It is shown that -xL prevents staurosporine induced cell death but not Fas / APO-1 induced cell death. Jur
Kat cells (2 × 10 ⁶ ) were left untreated, treated with staurosporine (2 μM) for 18 hours, or treated with anti-APO-1 antibody (200 ng / ml) for 6 hours. The cells were then analyzed for DNA fragmentation as described herein. In addition, nuclear morphology assessed by acridine orange staining gave similar results. Figure 41 shows the death matrix
staurosporine-induced cleavage of poly (ADP-ribose) polymerase, which is the (death substrate), is associated with bcl-2 and bcl.
Indicates blocked by -xL. Jurkat cells were left untreated, treated with 2 μM staurosporine, or treated with 200 ng / ml of anti-APO-1 antibody for 6 hours, and treated with PARP as previously described herein. The cleavage was analyzed. Figure 42 shows that bcl-2 and bcl-xL
We show that it blocks staurosporine-induced Yama activation. Jurkat cells were left untreated, treated with 2 μM staurosporine for 3 hours, or 200 ng / ml anti-APO-
Treatment with 1 antibody for 1.5 hours was performed and analyzed as in FIG.

43 to 46 show that the target of CrmA is upstream of Yama. In Figure 43, Jurkat cells were transfected with control vector or pZEM-CrmA and selected with neomycin. 7 anti-APO-1
Resistant clones were identified and pooled. SDS-PA
GE and immunoblotting was performed as described in the specification. In FIG. 44, CrmA prevents Fas / APO-1 induced cell death but not staurosporine induced cell death. Jurkat cells (2 × 10 ⁶ ) were left untreated, treated with staurosporine (2 μM) for 18 hours, or treated with anti-APO-1 antibody (200 ng / ml) for 6 hours. . The cells are then treated as described herein.
DNA fragmentation was analyzed. Furthermore, nuclear morphology assessed by acridine orange staining gave similar results. In Figure 45, Fas / APO-1 induced PARP cleavage is CrmA
Blocked by staurosporine-induced PARP
The cut indicates that it is not blocked. Jurkat cells were left untreated, treated with 2 μM staurosporine, or treated with 200 ng / ml of anti-APO-1 antibody for 6 hours, and PARP cleaved as described herein. Was analyzed. In Figure 46, CrmA blocks Fas / APO-1 induced Yama. Leave Jurkat cells untreated or 2
Treatment with μM staurosporine for 3 hours or 200ng / ml
A 1.5 hour treatment with anti-APO-1 antibody was performed and analyzed.

Experiment 1 Induction of Apoptosis by TNF and Anti-Fas-Subclones of the MCF7 breast cancer epithelial cell line, which express the TNF receptor and are sensitive to TNF killing, were selected for these studies. This cell line is Spri
ggs et al. (1988) J. Clinc. Invest. , 81: 455-460. Further analysis demonstrated that Fas was also expressed on these cells and that cross-linking with anti-Fas monoclonal antibody in the presence of the protein synthesis inhibitor cycloheximide induced cell death.

Cycloheximide treatment over the duration of the assay did not induce cell death other than the spontaneously occurring apoptosis with the infrequent frequency found in any untreated cell culture. Anti-Fas alone was not cytotoxic. But this is not surprising. Because Fas
This is because the induction of cell death of non-lymphoid cells by activation has been reported to require the coexistence of either a transcription inhibitor or a translation inhibitor. Itoh et al. (19
91) Cell 66: 233-243.

The B cell lymphoma cell line (BJAB) was also examined. It expresses high levels of Fas. It was also killed by the addition of anti-Fas antibody in the absence of protein synthesis inhibitors.

Cell death can occur by two biochemically and morphologically distinct processes: apoptosis and necrosis. In these studies, cell death was first confirmed to be the result of TNF or anti-Fas inducing apoptosis rather than necrosis. Although various apoptotic markers have been reported, this phenomenon is preferably defined at the morphological level, and chromatin condensation and edging along the inner nuclear membrane, cytoplasmic condensation, and membrane fluid that does not disrupt cell membranes. Is characterized as blebbing. See Duvall et al. (1986) Immunol. Today 7: 115-119.
Conversely, necrosis is defined as cytosolic swelling and lysis of cell membranes and, importantly, does not show the chromatin edging that is characteristic of apoptosis. DNA laddering, which represents cleavage in situ between nucleosomes, is found in some, but not all, types of apoptosis. This further emphasizes the importance of morphological features in defining apoptosis. Barres et al. (1992) Cell 70: 31-46. T
The nuclear morphology of cells staining in response to NF or anti-Fas antibodies was examined after staining with the DNA-binding dyes propidian iodine (MCF7 cells) and acridine orange (BJAB cells). Fluorescence microscope Laser scanning confocal microscope
microscopy laser scanning confocal microscopy)
MC in response to either TNF or anti-Fas-CHX
There was a marked change in the nuclear morphology of BJAB cells in F7 cells and in response to anti-Fas. Chromatin condensation was clearly visualized by immunofluorescence microscopy of both lines, and formed the basis for the apoptosis assay of the later transfected cell lines. Confocal microscopy confirmed edging along the inner nuclear membrane. These morphological features of apoptotic cell death
Further confirmation by transmission electron microscopy. MCF7 cells are
In response to either TNF or anti-Fas + CH, clearly showed chromatin condensation and edging along the inner nuclear membrane, as well as increased cytoplasmic condensation and membrane vacuolization. Anti
BJAB cells treated with Fas antibody showed chromatin rimming and cell atrophy typical of lymphoid cell apoptosis. Therefore, both TNF and Fas induced intrinsic apoptotic cell death in these cell lines.

Experiment 2 Generation of CrmA Mutant Plasmids for Eukaryotic, Bacterial, and In Vitro Expression-4
PCR-based method using two primers (Higuchi et al.
(1988) Nucleic Acids Res. 16: 7351-7367) was used to convert the codon 291 of the CrmA gene from Thr to Arg. Wild type cDNA and protein sequences are shown in FIGS. 14 and 15. First, two independent PCR reactions were performed on the plasmid pcDNA3 / CrmA (Tewari and Dixit (1995) J. B.
iol. Chem. 270: 3255-3260) and above) was used as template. The first reaction consists of an upstream primer (primer A) corresponding to nucleotides 682 to 711 of the sequence encoding CrmA (the first nucleotide of the methionine start codon is designated as nucleotide 1), and a downstream mutation complementary to nucleotides 853 to 896. Consisting of a primer (primer M2). Primer M2 is a G to A transition that removes the Pst 1 site and A instead of Thr.
It contains a base change that changes codon 291 to encode rg, and also introduced a diagnostic Nru1 site. The second PCR reaction consisted of an upstream sense mutation primer complementary to primer M2 (primer M1) and a downstream primer complementary to the last 26 nucleotides of the CrmA coding region with the conventional Xba 1 and Xho 1 sites (primer M1). B) was used. PCR products were gel purified, ligated,
It was denatured by heating and cooled slowly to room temperature for annealing. After an extension reaction for 10 minutes, flanking primers A and B were used for PCR. The amplification product was cleaved with Cla 1 (cut at nucleotide 692 of the CrmA coding sequence) and Xba 1 , and similarly cut pcDNA.
It was cloned into 3 / CrmA. The mutation was confirmed by DNA sequencing, as was all sections obtained by PCR amplification. This recombinant plasmid was designated as pcDNA3 / CrmA mutant.

The sequences of the oligonucleotide primers were as follows: Primer A: 5'GCT ATG TTT ATC GAT GTG CAC ATT CC
C AAG; Primer M2: 5'GCA CAA GTT GCT GCG GCT GCT TCG C
GA TAC TCT TCA TTG ACATC; Primer B: 5'GCT CTA GAC TCG AGT TAA TTA GTT G
TT GGA GAG CAA TAT C; Primer M1: 5'GAT GTC AAT GAA GAG TAT CGC GAA G
CA GCC GCA GCA ACT TGT GC.

The natural CrmA gene and its mutants were cloned into Nco 1 and X for transcription and translation in vitro.
p that encodes a Met-His ₆ tag at the N-terminus in-frame to cleave at ho 1 and facilitate purification.
Ligated to a plasmid based on TM1 (Moss et al. (1990) Nature 348: 91-92). The coding sequence begins with the initiation factor methionine, followed by 6 histidine residues, 1 serine residue,
Then follows the complete coding region for CrmA or a variant thereof.

[0229] For expression in E. coli, cleaves native and mutated CrmA genes in pTM1 with Nco 1 and Xho 1, and isopropyl-1-thio containing similar His ₆ fusion tag
-J-D-galactopyranoside (IPTG) -derived plasmid pFLAG
It was linked to a derivative of (IBI). In addition, pcDNA3 / CrmA (Tewar
i and Dixit (1995) supra) the CrmA gene from producing chimeric glutathione -S- transferase (GST) -CrmA open reading frame Nco 1 / Xho 1 cut pGSTa
g Bacterial expression vector (provided by Dr. Holly Dressler, a Massachusetts General Hospital, and Ron
And in Dressler (1992) BioTechniques 13: 866-869).

Preparation of GST-CrmA Fusion Protein and Generation of Rabbit Polyclonal Antiserum-Antibodies were raised against recombinant CrmA fusion protein. A priming was performed with 6xHis- tagged CrmA recombinant protein and a boost was previously described (Hu et al. (1994) J. Biol. Chem. 269: 30069-30072).
The GST-CrmA fusion protein prepared as described above was used. Briefly, the BL21pLyS E. coli strain was transformed with the pGSTag-CrmA plasmid and IPTG was added up to 50 μM to induce production of the fusion protein in culture. 1.5 at 25 ° C
After incubation for a period of time, the cells were harvested by centrifugation and lysed with a lysis buffer (20 mM Tris (pH8.0), 0.5 M NaCl,
10% glycerol, 1 mM PMSF, 1 mg / ml leupeptin, 1 m
g / ml aprotinin, 10 mg / ml soybean trypsin inhibitor, 1 mg / ml pepstatin, and 0.1% Triton X-100)
Resuspended, sonicated, debris removed by centrifugation, and glutathione-agarose beads (Sigma).
Was adsorbed. Soluble GST-CrmA fusion protein was eluted by incubating with 5 mM free glutathione (Sigma). The typical yield of fusion protein was 1 mg per liter of bacterial culture. Rabbit immunity and antisera screening have been previously described (O'Rou
rke et al. (1992) J. Biol. Chem. 267: 24921-24924).

Expression and Purification of Recombinant 6xHis Tagged CrmA Protein from E. coli-6xHisCrmA or 6xHis
E. coli transformed with either of the CrmA mutant constructs
Strain TG1 was induced with IPTG for 3 hours, harvested, and cells lysed by sonication were pelleted by centrifugation. The supernatant containing soluble CrmA was passed through a 0.22 μm filter, loaded onto a 2 ml Ni-NTA column (Qiagen), and
It was washed with 50 mM Tris (pH 8.0) containing 0.5 M NaCl. Crm
A was eluted with 50 mM Tris, 0.1 mM NaCl, 50 mM imidazole (pH 8.0). This material was diluted with 9 volumes of 20 mM Hepes (pH 7.4) containing 2 mM dithiothreitol and developed on a 2 ml DEAE Sepharose column. When this column was developed with a linear gradient of 0 to 1 M NaCl in 20 mM Hepes buffer (pH 7.4),
mA elutes at approximately 0.4 M NaCl, 4 mg from 6 liter culture
The yield of protein was obtained. The material was estimated to be> 95% pure by Coomassie blue staining and stored at -70 ° C until use. In all experiments using this material, the CrmA was treated with 2 mM DTT for 5 minutes immediately before use. This resulted in the highest inhibitory activity for CrmA.

In Vitro Assay of ICE Inhibition by Recombinant CrmA or CrmA Mutant Protein—To assay for inhibition, 44 ng of purified ICE was added to 10 mM at room temperature for 5 minutes.
Activated with DTT and then in a total volume of 95 μl reaction buffer (100
20 mM containing mM NaCl, 0.5% NP40, and 10 mM DTT
Different amounts of purified CrmA or Cr in Hepes buffer (pH 7.4)
Incubated with mA mutein at 37 ° C. Fifteen
Minutes later, 5 μl of a 10 mM stock of Boc-Ala-Ala-Ala-Pro-Asp-p-nitroanilide in DMSO was added, using a Molecular Devices V _max plate reader operating in kinetic mode,
Remaining by observing p-nitroaniline release at 410 nm I
CE activity was determined. The data were expressed as the kinetics in the presence of the inhibitor (V _i ) divided by the kinetics in the absence of the inhibitor (V ₀ ). This is a residual IC
Represents E activity. The data represent the mean + standard deviation of the values obtained from two independent experiments. Purified recombinant human IC
E was provided by Nancy Thornberry (Merck).

In Vitro Transcription / Translation of CrmA and CrmA Variants-Transcription / translation in pairs was performed using the TNT® kit (Promega) according to the manufacturer's recommendations.
Briefly, 0.5 μg of plasmid DNA was incubated for 1 hour at 31 ° C. in a total volume of 50 μl containing kit reagents and 20 μCi of translation grade [ ³⁵ S] Met. Once translated, the reaction mixture was either used immediately or stored at -20 ° C until needed.

Gel Shift Assay for Detection of Complex Formation between ICE and CrmA or CrmA Variants-Neoserpin Reaction with Target Protease Generated by Purified Proteinase and In Vitro Translation
It can be analyzed by gel shift analysis using [ ³⁵ S] Met labeled neoserpin. (For gel shift analysis, see Komiyama et al. (1994) “ Techniques in Protein Chemistry ” Acad. P.
ress, San Diego, CA, pp. 305-312 and Komiyama et al. (19
94) J. Biol. Chem . 269: 19331-19337. ) A CrmA or CrmA variant transcribed and translated in vitro,
Equal volume of 100 mM NaCl, 10% sucrose, and 0.1%
Diluted with 50 mM Hepes buffer (pH 7.4) containing CHAPS.
10 μl of diluted lysate was incubated with 10 μl of serial 3-fold diluted solution of ICE in the same buffer containing 10 mM DTT for 30 min.
Incubated at 7 ° C. Samples were then analyzed by native gel electrophoresis and Molecular Devices
Visualization was performed using Phosphor imager.

Transversal Urea Gradient PAGE
-In vitro translated CrmA or CrmA muteins were electrophoresed on a transverse urea gradient (TUG) polyacrylamide gel (0 to 8M) (Goldenberg (19
89) `` Protein Structure: A Practical Approach '' IRL P
ress, NY pages 225-250 and Mast et al. (1991) Biochem .
30: 1723-1730). The gel was dried and analyzed using a Molecular Devices Phosphorimager.

Stable transfection of BJAB and MCF7 cells-MCF7 or BJAB cells were electroporated with pcDNA3 / CrmA mutant plasmids and stable clonal cell lines were previously described (Tewari and Dixit (199
5) Generated as in the above).

MCF7 cells, BJAB cells, as well as inducible vectors and CrmA stable transfectants, along with the CrmA mutant transfected stable strains generated in this study, were heat-inactivated in 10% fetal calf serum (Hyclone), L-. Supplemented with glutamine, penicillin / streptomycin, non-essential amino acids, and supplemented with G418 sulfate (Gibco-
BRL / Life Technologies, Inc.) were maintained in RPMI-1640 medium supplemented with 500 μg / ml for MCF7 transfectants and 3 mg / ml for BJAB transfectants.

CrmA induces TNF-induced apoptosis and anti-Fas
Block induced apoptosis-To determine if CrmA can function to inhibit cytokine-induced apoptosis, the MCF7 and BJAB cell lines were treated with the expression vector pcDNA3 alone or as an expression vector as a CrmA expression construct. Transfected with pcDNA3. CrmA
Gene expression was confirmed by Northern analysis. Stable transfectants were generated by neomycin selection and pooled neomycin resistant populations were assayed for CrmA expression. These pool populations were analyzed for susceptibility to TNT-induced and anti-Fas-induced apoptosis by directly counting apoptotic cells based on nuclear morphology after staining with a DNA-binding dye and visualization by fluorescence microscopy. Dramatic resistance to either TNF or Fas was found in both cell lines. Considering that in the pool population of neomycin resistant cells transfected with CrmA, a significant proportion of the cells are believed not to express CrmA, especially due to the non-productive integration of the expression construct into the genomic DNA. This was amazing.

In addition to its pool, clonal strains were derived from both MCF7 and BJAB transfectants, and TN
Challenged by activation of the F and Fas death pathways. In the MCF-7 cell line, the vector clones are uniformly sensitive to apoptosis induced by either TNF or anti-Fas + CHX, whereas among the transfected clones, all clones that detectably detect CrmA are It was resistant to apoptosis (Fig. 5). In fact, the strains expressing the highest levels of CrmA were totally resistant to apoptosis (Figure 5) and showed no morphological cytopathic effect (Figure 6). This indicates that CrmA can completely block the TNF-mediated and Fas-mediated death pathways. Similarly, among the transfected BJAB clones, the CrmA expressing strain was strikingly resistant to anti-Fas-induced apoptosis, whereas vector clones were universally sensitive (FIG. 8A). The important thing is that MCF7
Those clones that expressed low or undetectable levels of CrmA in both BJAB and BJAB transfectants were most susceptible, whereas high levels of CrmA
That is, those clones that expressed .LAMBDA. Were most resistant (FIGS. 5 and 8A). Although direct counting of apoptotic nuclei is the preferred method of measuring apoptosis, cell death assay based on MTT conversion (Fig. 5 insert and Fig. 8A insert) or crystal violet staining (Fig. 7) was used to assess cell viability. At times, comparable results were obtained.

The dose of cell death stimulant was increased to determine if the protection against cytokine-induced apoptosis conferred by CrmA could be attenuated. Notably, CrmA has a relatively high level of protection from anti-Fas-induced apoptosis in response to antibody doses 250-fold higher than that required to kill more than 95% of vector-transfected cells. Was provided (FIG. 8B). Similar results were obtained when the TNF dose was similarly varied for MCF7 transfectants. This means that CrmA functions as a highly effective cell death inhibitor at a probably important step in the cell death pathway.

These results describe an important new function of CrmA. This function is associated with TNF-mediated apoptosis and F
Inhibition of as-mediated apoptosis. Given the importance of both TNF and Fas in the host antiviral response, this function of CrmA is thought to be important for productive viral infection in vivo. CrmA represents yet another example of viral economy, in which one protein has two important functions: inhibition of IL-1β production and prevention of apoptosis.

Furthermore, this data generally means the unification of cell death pathways. First, the fact that CrmA blocks both TNF-mediated and Fas-mediated apoptosis, especially in MCF7 cells that harbor both receptors, is due to the fact that they block cell death via biochemically universal pathways. It suggests that it becomes a signal. This hypothesis is supported by the finding that the cytoplasmic regions of both of these receptors are defined by mutational analysis as being "cell death domains" and probably contain regions of homology that interact with a common set of signaling molecules. . In addition, CrmA has two very different stimuli: recovery of growth factors and activation of cytokine receptors in nerve cultures.
It is not clear that it blocks cell death induced by. It is important to suggest that apoptosis in these two systems occurs via biochemically distinct pathways, the former system's apoptosis is dependent on de novo protein synthesis, and cell death by cycloheximide. Although blocked, in contrast, TNF and Fas-mediated cytotoxicity is independent of de novo protein synthesis and, in fact, is enhanced by cycloheximide. Thus, at some point, the cell death pathways of both systems converge at CrmA inhibitory steps such as activation of proteases. Applicants have identified this protease as pro-Yama, which is activated by PARP.

(Experiment 3) To determine whether CrmA could function to inhibit the lethal cascade induced by the interaction of its target with CTL, BJAB cells (Fas expressing human B cell line) as a target cell for a 24-hour PHA-stimulated allogeneic CTL-mediated cytolytic assay based on ⁵¹ Cr release from target cells, stably transduced with either a vector control, CrmA, or an inactive point mutant CrmA expression construct. It was perfect. ⁵¹ Cr release was examined after 24 hours.
Both Ca ²⁺ -dependent and Ca ²⁺ -independent cytotoxicity were expected to occur at this point. Two vector-transfected clones (V1 and V4), two CrmA-transfected clones (CrmA2 and CrmA) previously described and characterized above.
3), and two mutant CrmA expressing clones (mutant CrmA12
And mutant CrmA17) were selected. (Vaux, D. et al. (1994)
Cell 76: 777-779; Gagliardini, V. et al. (1994) Scienc.
e , 263: 826-828; Enari, M. et al., (1995) Nature , 375: 78.
-81; Los, M. et al. (1995) Nature , 375: 81-83; Quan, L.
T. et al., (1995) J. Biol. Chem. , 270: 10377-10379; Grab
Stein et al. (1980), supra; and Duke, RC and Cohe.
n, JJ (1992) Current Protocols in Immun. (Coliga
n, JE et al.) Volume 1, 3.17.11-13.17.16, John Wile
See yand Sons, NY, NY). Mutant CrmA has codon 291
Thr → A at (the important site in the reaction site loop of CrmA)
Mediates point mutations in rg. This mutation destroys the ability to inhibit proteases without significantly altering the tertiary structure as determined by transverse urea gradient gel electrophoresis as described herein. As shown herein, this mutation has also been shown to disrupt CrmA's ability to inhibit tumor necrosis factor- and Fas-induced cell death.

MCF7 cells, BJAB cells, and inducible vectors and CrmA stable transfectants were maintained as described above, along with CrmA mutant transfected stable strains. The following experiments were used to determine if cell killing and apoptosis had occurred.

PHA-stimulated allogeneic CTL using PHA-stimulated human PBMC
Assays described previously (Grabstein, K. (1980) supra).
This method was carried out with a slight modification. This assay is
As described above.

NK cell cytotoxicity assays were performed as described above, except that cells were not stimulated and PHA was not present during the assay.

When examined for sensitivity to CTL-mediated cytolysis, the parental BJAB cell line was similar to vector-transfected cells (FIG. 23, clones V1 and V4) in a dose-dependent over a range of killing factor: target cell ratios. By style, was effectively killed. However, strains expressing CrmA were significantly protected from CTL-mediated killing (Figure 23, clone CrmA2.
And CrmA3). There were no significant differences between vector and CrmA strains when assessing the spontaneous background of ⁵¹ C release from BJAB strains incubated for 24 hours without the addition of CTL. Therefore, the difference in susceptibility to killing between the vector and the CrmA expressing strain in the CTL-mediated cytolytic assay is not simply due to the diminished characteristic nature of the CrmA strain that undergoes spontaneous lysis.

To determine whether the ability of CrmA to inhibit CTL-mediated cytolysis requires its ability to inhibit proteases, one can compare CrmA point mutants that lack protease inhibitory activity as described above. Cell lines expressing levels were tested. Wild-type CrmA was clearly protective when tested in a similar cytotoxicity assay, whereas both strains expressing mutant CrmA were similar to vector transfectants in CTL-mediated cytolysis. Was sensitive to (FIG. 24).

Since killing by CTLs is the result of activation of both granzyme and Fas pathways, it was interesting to determine whether one or both of these were regulated by CrmA (in previous studies, CrmA has been shown to potentially inhibit both pathways). To study this, using Ca ²⁺ -independent Ca ²⁺ dependent and Fas pathway of granzyme-mediated killing. The calcium chelator, EGTA, can be used to block granule exocytosis. This is because calcium is required for granzyme release from CTL. Therefore, Ca ²⁺ -independent killing can be selectively assessed. In the absence of EGTA,
CrmA was protective compared to the vector control, but there was some residual cytotoxicity (Figure 25).
However, in the presence of EGTA, CrmA was totally protective (Figure 26). It shows that CrmA can completely block the Ca ²⁺ -independent component of CTL killing.

Studies of granzyme B knockout mice suggest that ⁵¹ Cr release in the first 4 hours after CTL target interaction appears to involve only granzyme-based pathways. The experiments reported in the cytolysis assay above were performed in place of the 24-hour assay. There were no significant differences in CTL sensitivity between vector-transfected and CrmA-transfected target cells in this 4-hour assay (Figure 27). Furthermore, EGTA abolished all cytotoxicity in the 4-hour assay (Figure 28). Cell lysis at this point was confirmed to be entirely due to a Ca ²⁺ -dependent mechanism and, therefore, CrmA
Was shown to not ^confer protection from the Ca ²⁺ -dependent pathway. CrmA also did not confer any protection from death induced by NIK cells. This result is consistent with the hypothesis that CrmA does not block the Ca ²⁺ -dependent cytotoxic component, as NK-mediated cytolysis appears to be entirely responsible for the granule elimination pathway.

The 4-hour ⁵¹ Cr release assay measured only Ca ²⁺ -dependent cell lysis, whereas the DNA fragmentation assessment was a more sensitive measure of total apoptosis at this early time point. This is because fragmentation precedes membrane lysis in the case of Fas-mediated apoptosis. When both the vector control transfected and CrmA transfected cell lines were examined for induction of DNA fragmentation by CTLS, the CrmA expressing strain showed significantly less DNA.
Fragmentation was shown (Figure 29). This is consistent with protection from early DNA fragmentation induced by Fas ligation.

(Experiment 4) Cloning of cDNA encoding Yama-cDNA clone corresponding to EST T10341 (b4HB3MA-
COT8-HAP-Ft280 5 '), M. Bento Soares (Columbia Univ
, and were used to screen a random primed cDNA library constructed from human umbilical vein endothelial cells treated with TNF and cycloheximide. Double-stranded DNA sequencing is based on Ya of 277 amino acids.
Clarified an open reading frame called ma.

Expression and Purification of 6 × His-Tagged Yama-The 2.3 kb NcoI / BamHi fragment from the Yama cDNA described above was ligated into a vector containing an N-terminal His ₆ fusion (pTM1) to facilitate purification. did. This construct contained 1800 bp of 3'untranslated DNA plus the complete coding region of Yama after the 6xHis tag. TNT for paired transcription / translation
Modifications were performed using the (registered trademark) kit (Promega) according to the manufacturer's recommendations. Briefly, 4 μg of plasmid DNA was incubated for 1 hour at 31 ° C. in a total volume of 400 μl containing the kit reaction mixture and 160 μCi of translation grade [ ³⁵ S] Met. 20mM Hep of the translation reaction product
Diluted 1:20 with es buffer (pH 7.4) and equilibrated to 500
Load on a μl DEAE Sepharose (Pharmacia) column,
It was then washed with 8 ml Hepes buffer. 5 ml column
Elution was performed using 20 mM Hepes and 0.5 M NaCl. This eluate was loaded onto a 300 μl Ni-NTA column (Qiagen) and then 5 ml of reaction buffer (50 mM Hepes (pH 7.4), 0.1 M NaCl,
Washed with 0.1% CHAPS, and 10% sucrose). The protein was eluted with 5 × 400 μl fractions of reaction buffer with 50 mM imidazole.

Yama activation and in vitro reconstitution experiments-Purified Yama (20 μl) was incubated with 1.5 pmol ICE in reaction buffer supplemented with DTT (10 mM) in a total volume of 25 μl for 4 hours at 37 ° C. After activation, 30 μl reaction buffer is added and the reaction is incubated at 37 ° C.
Incubated for another 15 minutes. After activation, control reaction buffer or 270 pmol of recombinant 6xHisCr
30 μl of either reaction buffer containing mA or 270 pmol recombinant 6 × HisCr mA − mutant was added and incubated at 37 ° C. for 30 minutes. Recombinant protein and control buffer used DT to preactivate CrmA.
Preincubated with T (2 mM). After incubation for 30 minutes, 2 μl (0.586 μg) of purified P
ARP was added and the DTT concentration was raised to 10 mM, after which the reaction was allowed to proceed for 2 hours at 37 ° C. PARP-only control reactions were performed under the same conditions except that no Yama, ICE, or CrmA protein was added during the procedure. The ICE + PARP reaction was similarly performed under identical conditions except that no Yama or CrmA protein was added during the procedure.

After incubation with PARP, one fifth of each reaction sample was analyzed by immunoblotting with anti-PARP monoclonal antibody C-2-10 as described below. In addition, equal amounts of each sample were separated by SDS-PAGE and analyzed using a Molecular Devices Phosphorimager to assess the status of radiolabelled Yama present in the reaction. Purified ICE is a gift of Nancy Thornberry (Merck). PARP to Zahradka and Ebisuzaki (198
4) Purified as described in Eur. J. Biochem. 142: 503-509.

CrmA to detect complex formation with Yama
Immunoprecipitation-reactions were treated with 20 μl of [ ³⁵ S] -Met-labeled pro Yama.
Or ICE activated Yama and 358 pmol of natural CrmA or 358 pmo
Collected by combining with any of the 1 mutant CrmA proteins. Each reaction was diluted in reaction buffer to a final volume of 80 μl. Complex formation was allowed to occur for 30 minutes at 37 ° C. 10 μl of each reaction was separated by SDS-PAGE,
It was then subjected to phosphorimaging to visualize radiolabelled pro Yama or activated Yama. 35 μl of each reaction was added to PBS-TDS (O'Rourke et al. (1992)
J. Biol. Chem. 267: 24921-24924) and immunoprecipitated with 25 μl of rabbit polyclonal CrmA antiserum (described later in this section). Immunoprecipitations were performed as described by O'Rourke et al. (1992) supra,
The precipitate was then separated by SDS-PAGE and subjected to phosphorimaging analysis to detect the presence of radiolabeled pro Yama or activated Yama. Coomassie blue staining of gel shows that equal amounts of native 69, CrmA and mutant CrmA are CrmA
It was revealed that it was precipitated by the antiserum.

Treatment with anti-Fas or TNF for PARP analysis and preparation of cell lysates-MCF7 cells or induced transfectants in 100 mM culture dishes at a concentration of 2 x 10 ⁶ cells per culture dish. Plated with. On day 2, cells were treated with 40 ng / ml TNF for the times indicated. After rinsing with PBS, cells were harvested by scraping in 15 ml PBS + protease inhibitors (1 mM PMSF, 0.5 mg / ml aprotinin, 0.5 mg / ml antipain, and 0.5 mg / ml pepstatin) and harvesting by centrifugation, And 2.5 ml
Sample buffer (50 mM Tris-HCl (pH 6.8), 6M urea, 6
% 2-Mercaptoethanol, 3% SDS, and 0.003%
Bromophenol blue). If non-adherent cells were present in the culture medium (eg at later time points), floating cells were also harvested by centrifugation and combined with the adherent cell pellet before lysing in sample buffer.

BJAB cells or induced transfectants were aliquoted into 6-well culture dishes at a concentration of 5 × 10 ⁵ / ml, 4 ml per well. The next day, the cells were treated with anti-Fas antibody (250
ng / ml) for the indicated times, harvested by centrifugation and 1 with PBS + protease inhibitor.
Washed twice and dissolved in 2 ml of sample buffer.

For detection of CrmA, whole cell lysates (2 × 10 ⁵ cells per lane) were separated by SDS-PAGE, transferred to nitrocellulose and processed as previously described herein. did. Anti-GST-CrmA rabbit antiserum 1: 1,
Used at a dilution of 000 and horseradish peroxidase conjugated donkey anti-rabbit secondary antibody (Amersham Life Sciences) at a dilution of 1: 15,000. Signal visualization is performed by ECL (Am
ersham).

Immunoblotting of lysates for PARP was performed by Desnoyers et al. (1994) Anal. Biochem. 218: 470- .
Performed as described in 473. Anti-PARP mouse monoclonal antibody was used at a 1: 10,000 dilution, and secondary antibody, horseradish peroxidase-labeled anti-mouse Ig, was used at a 1: 1,000 dilution. Signal visualization is also ECL
According to

A human umbilical vein endothelial cell library was screened. This cloned the cDNA encoding the 277 amino acid open reading frame called Yama. Yama is homologous to the Ced-3 / ICE family of proteins. Yama was then assayed for protease activity and to determine if it met the putative requirements for dead proteases. The amino acid sequence suggested that it is an Asp-specific cysteine protease because the residues that are thought to be important for Asp specificity in ICE are conserved in Yama. Yama was expressed in vitro as a fusion to a 6xHis purification tag at the N-terminus, and PARP
Was isolated by ion exchange and nickel chelate affinity chromatography to determine whether it had proteolytic activity capable of cleaving the. Full length
Purified Yama in the p32 form has no proteolytic activity on PARP (Fig. 3, top, lane 3), and therefore it was
It was called Yama. Purified ICE cleaved pro-Yama to yield two major proteins (Figure 3, bottom, lane 4; putative p20 and p).
11 subunits are indicated by white arrows) and, more importantly, Yama activated in this manner.
Acquired proteolytic activity, and PARP 85 kDa
(Fig. 3, top, lane 4).
Purified ICE does not cleave PARP (Figure 3, top, lane 2), L
Confirmed previous results of azebnik et al. (1994) Nature 371: 346-347 and eliminated the possibility that PARP cleavage was mediated by added ICE. The next investigation was on whether PARP cleavage occurred in these cell death systems and whether the cleavage products were similar to those observed in in vitro experiments. Fas (in BJAB lymphoma cells) or T
Activation of either of the NF receptors (in MCF7 breast cancer cells) induced PARP cleavage to the characteristic 85 kDa form (Fig. 4), and this product was produced by purified Yama PA.
It co-migrated with the RP cleaved fragment (FIG. 3, top, lanes 4 and 5). Therefore, Yama has 85kD which is characteristic of PARP.
a A protease that cleaves into apoptotic fragments.

Since mammalian cell death proteases are expected to be sensitive to inhibition by CrmA, then Yama
It was investigated whether it was CrmA inhibitory. To finally address this question, the PARP cleavage reaction was reconstituted in vitro with purified protein so that purified recombinant CrmA protein could be added. A CrmA point mutant was constructed to serve as a control. The CrmA variant has a single amino acid substitution of Arg for Thr at amino acid 291 (Figure 5). CrmA proteins and CrmA muteins were expressed in E. coli as 6xHis tag fusion proteins and purified by nickel chelate affinity chromatography. In vitro characterization of CrmA muteins shows that under conditions where CrmA binds and inhibits ICE, the muteins do not bind to ICE (Fig. 6) nor inhibit its proteolytic activity (Fig. 5). Revealed. However, the tertiary structure of the CrmA mutant is a transferase urea gradient PAGE (Goldenberg (1989) supra; Mast, whose conformational feature is a method used to study the tertiary structure of neoserpin).
Et al. (1991) supra; and Komiyama et al. (1994) supra) (FIG. 7), which was indistinguishable from that of wild-type CrmA and was not significantly altered by the point mutation.

Using these purified recombinant proteins, CrmA significantly inhibited the cleavage of PARP by Yama in vitro (FIG. 8). No inhibition of PARP cleavage was observed when CrmA was replaced by an equivalent amount of CrmA mutein (Figure 8). This indicates that the effect of CrmA is a function of its ability to act specifically as a protease inhibitor.

To confirm that CrmA interacts directly with Yama, either pro Yama or activated Yama
The ability to form complexes with either native CrmA or mutant CrmA was examined. Pro Yama or Activated Yama (both
[ ³⁵ S] -Met labeled) was incubated with either native CrmA or equal amounts of mutant CrmA recombinant protein, respectively. Each reaction was subjected to immunoprecipitation analysis with a polyclonal CrmA antiserum, and immunoprecipitates were separated by SDS-PAGE and visualized by phosphorimaging. CrmA associates with the activated 2 subunit form of Yama, but not with pro Yama (Figure 9, panel B). The CrmA mutant did not bind to activated Yama or pro Yama (FIG. 9, panel B). It was determined that CrmA interacts directly with activated Yama to form an inhibitory complex.

Since CrmA inhibited the cleavage of PARP by Yama in vitro, it was deduced that CrmA must inhibit PARP cleavage in vivo if Yama was actually responsible for PARP cleavage during apoptosis. . To investigate this, the MCF7 breast cancer cell line and BJAB lymphoma cell line stably transfected with either vector (CrmA or CrmA mutant expression construct) was utilized. A clonal cell line expressing the indicated proteins was selected and protein expression was confirmed by immunoblotting with anti-CrmA polyclonal antisera (Figure 10). Consistent with our in vitro findings, CrmA expression was associated with Fas receptor (B
Inhibits the proteolytic cleavage of PARP into a characteristic 85kDa fragment normally produced during apoptosis induced by either JAB cells) or TNF receptors (MCF7 cells), whereas the CrmA mutant vector In, the cleavage of PARP proceeded unabated (FIGS. 11 and 12).

To investigate whether in vivo blocking of PARP cleavage by CrmA and its lack by CrmA mutants correlated with the ability of these proteins to inhibit apoptosis, BJAB and MCF7 transfectants were examined. Examined. CrmA, as expected, conferred protection from TNF-induced apoptosis, whereas the CrmA mutant expressing strain showed no protection and was as sensitive as the vector-transfected strain (Fig. 13, bottom). ). The protection conferred by CrmA and its lack by the CrmA mutant were readily apparent in examination of the nuclear morphology of propidium iodide stained cells (Figure 13, top). When BJAB transfectants were tested for susceptibility to Fas-induced PCD, CrmA was protective, while the CrmA mutant expressing strain remained as sensitive as the vector control strain (Figure 13, bottom). Therefore, CrmA blocks PARP cleavage
It was concluded that the different abilities of the and CrmA variants correlate with the ability of these proteins to block cell death. The finding that CrmA inhibits the proteolytic activity of Yama (FIG. 8) is important, which evidences the well-proven ability of CrmA to inhibit apoptosis, now explained by its inhibition of Yama. Show that you get.
Also, the finding that CrmA inhibits PARP cleavage in vivo is consistent with CrmA inhibiting Yama.

(Experiment 5) The cleavage of U1-70kDa was caused by Fas or T
To determine if any of the events unique to NF-R-induced apoptosis was constructed the following.

Treatment with anti-Fas antibody or TNF and preparation of cell lysates—MCF7 cells or induced transfectants were plated in 100 mM culture dishes at a concentration of 2 × 10 ⁶ cells / culture dish. . On day 2, cells were treated with 40 ng / ml TNF for the times indicated. After rinsing with PBS, 1 cell
5 ml PBS + protease inhibitor (1 mM phenylmethylsulfonyl fluoride, 0.5 mg / ml aprotinin,
0.5mg / ml antipain, and 0.5mg / ml pepstatin)
Collected by scraping and collecting by centrifugation.
Then 2.5 ml of sample buffer (50 mM Tris-HCl (pH 6.
8), 6 mM urea, 6% 2-mercaptoethanol, 3% SD
S, and 0.003% bromophenol blue). When non-adherent cells are present in the culture medium (for example,
At later time points), floating cells were also harvested by centrifugation and combined with the adherent cell pellet before lysis in sample buffer. BJAB cells or induced transfectants were aliquoted into 6-well culture dishes at a concentration of 5 × 10 ⁵ / ml, 4 ml per well. The next day, the cells were anti-Fa
s antibody (250 ng / ml) for the indicated times, harvested by centrifugation, washed once with PBS + protease inhibitor and lysed in 2 ml of sample buffer (Figure 30 and Figure). 31).

Western Blotting-Immunoblotting to assess U1-70 kDa status was performed by Cascio.
lla-Rosen, LA, et al. (1994) J. Biol. Chem., 269: 30757.
-30760. Briefly, cell lysates (20 μl) were separated by SDS-polyacrylamide gel electrophoresis and electroblotted to a nitrocellulose membrane (Schleicher and Schnell).
Transferred to Blot the blocking buffer (10 mM T
ris (pH7.6), 150 mM NaCl, 0.5% Nonidet P-40, 3%
Bovine serum albumin) was used for blocking at room temperature for 1 hour. U1-70 kD affinity purified antiserum was used at 1: 5000 dilution in blocking buffer and incubated for 1 hour at room temperature. The secondary reagent, biotinylated goat anti-human IgG antibody (Southern Biotech) was used at a 1: 6700 dilution in blocking buffer and incubated for 50 minutes at room temperature. Streptavidin-the third reagent-
Horseradish peroxidase conjugate (Southern Biotec
h) was used in blocking buffer at a 1: 10,000 dilution,
Then, it was incubated at room temperature for 30 minutes. Signal visualization is based on electrochemiluminesence
(Amersham Corp.).

(Experiment 6) Yama is activated by various apoptosis stimulating factors. Jurkat T cells express 32kDa pro-Yama and contain staurosporine or anti-APO-
Treatment with 1 antibody produced active 17 kDa and 12 kDa subunits referred to herein as p20 and p11. (FIG. 38). An intermediate form of the larger 17 kDa subunit was also observed and appears to contain the smaller Yama prodomain.

(Experiment 7) Bcl-2 and Bcl-x upstream of Yama
The L-function-bcl-2 family of proteins has been shown to prevent cell death induced by various signals and in many cell types. But bcl-2
Little is known about the functioning of ss against apoptotic proteases. Jurkat overexpresses bcl-2 or bcl-xL to address this key issue
Cells were constructed with the episomal expression construct. Expression of bcl-2 and bcl-xL in the pooled population was confirmed by immunoblotting (Figure 39). Staurosporine has been shown to reliably induce bcl-2 inhibitory cell death in various cell lines. As expected, Jurkat cells expressing bcl-2 and bcl-XL were resistant to staurosporine-induced apoptosis as assessed by DNA fragmentation (Figure 40) and nuclear morphology. An apoptosis stimulator that is not consistently blocked by bcl-2 is Fas / APO-1 activation. This Jurka
In the t cell line, expression of bcl-2 and bcl-XL did not inhibit anti-APO-1-induced cell death or PARP cleavage (Figure 40 and Figure 41). Overexpression of bcl-2 and bcl-XL eliminated staurosporine-induced production of activated Yama (Fig. 4).
2). Similar results were obtained with another bcl-2 / bcl-XL inhibitory death stimulator, thapsigargin. Thapsigagulin is a specific inhibitor of endoplasmic reticulum Ca ²⁺ -ATPase. The inhibitory effects of bcl-2 and bcl-XL can be “bypassed” by treatment with anti-APO-1 antibody, which results in activation of apoptotic proteases (FIG. 42). This result is also well characterized bc
It was observed using the l-2 expressing CEM cell line (Martin (1995) Ce
ll Death and Diff. , 2: 253-257).

Evidence for a proteolytic cascade in the Fas / APO-1 cell death pathway-unlike staurosporine and thapsigargin treatment, Fas / APO-1 induced Yama activation and the resulting apoptosis were found in this Jurkat cell line. Not blocked by bcl-2 or bcl-XL. CrmA-expressing Jurkat cells were resistant to anti-APO-1 induced cell death, but not to staurosporine induced apoptosis (FIG. 44). PARP integrity is also monitored, and CrmA is also Fas / APO-
Blocked PARP cleavage induced by 1 but not staurosporine. To determine whether CrmA directly inhibited Yama in vivo, the activation of apoptotic proteases was evaluated directly. Consistent with DNA fragmentation and PARP analysis (Figure 44 and Figure 45), staurosporine-induced Yama activation was
It was not blocked by CrmA (Figure 46).

Although the present invention has been described in connection with the above embodiments, it is understood that the foregoing description and examples are intended to be illustrative and not limiting the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention include
It will be apparent to those skilled in the art to which the present invention pertains.

[0274]

INDUSTRIAL APPLICABILITY The present invention provides a non-natural nucleic acid molecule encoding a protein called Yama and an isolated natural nucleic acid molecule. The invention also provides polypeptides and proteins encoded by these nucleic acids, as well as purified naturally occurring polypeptides or proteins. The invention also provides a non-natural nucleic acid molecule encoding a mutant CrmA protein and a dominant negative Yama. Vectors and host cells containing these nucleic acid molecules are also provided. Also provided herein are methods for modulating cell function that is regulated by the Fas receptor pathway in cells. In one aspect, the method involves introducing into a cell a nucleic acid molecule encoding a CrmA biologically active gene product (eg, a dominant negative Yama) or a CrmA gene product. Ya
ma nucleic acid molecules and proteins can also be introduced into cells to regulate cellular functions regulated by the Fas receptor.

[Brief description of drawings]

1 shows the nucleotide sequence of the open reading frame of Pro Yama.

FIG. 2 shows the deduced amino acid sequence of pro Yama.

FIG. 3 is an electrophoretogram showing that pro-Yama is a protease zymogen that cleaves PARP into an 85 kDa apoptotic fragment in vitro upon activation.

FIG. 4 is an electrophoretogram showing that cleavage of PARP into an 85 kDa fragment is a characteristic identification of Fas- and TNF-induced apoptosis.

FIG. 5 shows that a point mutation in the reactive site loop (RSL) of CrmA inactivates its ability to inhibit ICE.

FIG. 6 is an electrophoretogram showing that a point mutation in the reaction site loop (RSL) of CrmA inactivates its ability to inhibit ICE.

FIG. 7 is an electrophoretogram showing that a point mutation in the reaction site loop (RSL) of CrmA inactivates its ability to inhibit ICE.

FIG. 8 is an electrophoretogram showing that PARP cleavage by activated Yama in vitro can be inhibited by CrmA, but not by equivalent amounts of CrmA mutants.

FIG. 9 shows that CrmA interacts directly with activated Yama but not with pro Yama.

FIG. 10 is an electrophoretogram showing that CrmA or CrmA mutants are expressed in stably transfected clones.

FIG. 11 is an electrophoretogram showing that PARP cleavage into the 85kDA fragment during Fas-induced apoptosis is inhibited by CrmA but not by CrmA mutants.

FIG. 12 is an electrophoretogram showing that PARP cleavage into the 85kDA fragment during TNF-induced apoptosis is inhibited by CrmA but not by CrmA mutants.

FIG. 13. UnRx or TNF induced transfectant vector, CrmA3, and CrmA variant #
2 micrographs and CrmA, but not CrmA variant,
It is a figure which shows inhibiting Fas induced cell death and TNF induced cell death.

FIG. 14 is a micrograph showing that TNF and anti-Fas + CHX induce apoptosis in MCF7 cells.

FIG. 15 is a micrograph showing that TNF and anti-Fas + CHX induce apoptosis in MCF7 cells.

FIG. 16 shows inhibition of apoptosis by CrmA in pooled transfectant populations.

FIG. 17: TNF and anti-Fas + CHX of MCF7 cells by CrmA
It is a figure which shows the inhibition of induced apoptosis.

FIG. 18 is a micrograph showing CrmA-mediated inhibition of apoptosis induced by increasing doses of anti-Fas antibody.

FIG. 19 is a morphological photograph of an organism showing that CrmA inhibits the apoptosis induced by increasing doses of anti-Fas antibody.

FIG. 20: C of anti-Fas-induced apoptosis in BJAB cells
It is the figure and the electrophoretic photograph which show the inhibition by rmA.

FIG. 21: C of anti-Fas-induced apoptosis in BJAB cells
It is the figure and the electrophoretic photograph which show the inhibition by rmA.

FIG. 22 shows the nucleic acid sequences of cowpox virus CrmA gene and gene products and the corresponding amino acid sequences.

FIG. 23 shows that wild-type CrmA inhibits CTL-mediated apoptosis but mutant CrmA does not.

FIG. 24 shows that wild-type CrmA inhibits CTL-mediated apoptosis but mutant CrmA does not.

FIG. 25 shows that CrmA completely blocked the Ca ²⁺ -independent component of CTL-mediated killing.

FIG. 26 shows that CrmA completely blocked the Ca ²⁺ -independent component of CTL-mediated killing.

FIG. 27. CrmA did not block the Ca ²⁺ -dependent component of CTL-mediated cytolysis.

FIG. 28. CrmA did not block the Ca ²⁺ -dependent component of CTL-mediated cytolysis.

FIG. 29. CrmA blocks CTL-mediated DNA fragmentation.

FIG. 30: Activation of Fas or TNF receptor is shown by U1-70
3 is an electrophoretic photograph showing that it induces cleavage of kDa.

FIG. 31. Activation of Fas or TNF receptor is shown by U1-70
3 is an electrophoretic photograph showing that it induces cleavage of kDa.

FIG. 32 is an electrophoretogram showing that CrmA blocks Fas-induced cleavage of U1-70 kDa, but mutant CrmA does not.

FIG. 33 is an electrophoretogram showing that CrmA blocks Fas-induced cleavage of U1-70 kDa, but mutant CrmA does not.

FIG. 34 is an electrophoretogram showing that CrmA blocks Fas-induced cleavage of U1-70 kDa, but mutant CrmA does not.

FIG. 35 is an electrophoretogram showing that CrmA blocks TNF-R-induced cleavage of U1-70 kDa, but mutant CrmA does not.

FIG. 36 is an electrophoretogram showing that CrmA blocks TNF-R-induced cleavage of U1-70 kDa, but mutant CrmA does not.

FIG. 37 is an electrophoretogram showing that CrmA blocks TNF-R-induced cleavage of U1-70 kDa, but mutant CrmA does not.

FIG. 38 is an electrophoretic photograph showing that mammalian Yama is activated by an apoptotic stimulus.

FIG. 39 is an electrophoretic photograph showing that bcl-2 and bcl-xL function upstream of Yama.

FIG. 40 is an electrophoretic photograph showing that bcl-2 and bcl-xL function upstream of Yama.

FIG. 41 is an electrophoretic photograph showing that bcl-2 and bcl-xL function upstream of Yama.

FIG. 42 is an electrophoretic photograph showing that bcl-2 and bcl-xL function upstream of Yama.

FIG. 43 is an electrophoretic photograph showing that the target of CrmA is upstream of Yama.

FIG. 44 is an electrophoretic photograph showing that the target of CrmA is upstream of Yama.

FIG. 45 is an electrophoretic photograph showing that the target of CrmA is upstream of Yama.

FIG. 46 is an electrophoretic photograph showing that the target of CrmA is upstream of Yama.

 ─────────────────────────────────────────────────── ─── Continuation of front page (71) Applicant 597015922 Wolverine Tower, Room 2071, 3003 S.M. State Street, Ann Arbor, MI 48109-1280, U.S.S.N. S. A. (72) Inventor Bishbaem. Dixto USA Michigan 48105, Ann Arbor, Pepper Pike (No house number)

Claims (9)

[Claims] 1. A method of modulating cell function regulated by tumor necrosis factor receptor (TNF-R) pathway, including U1-70, in a suitable cell, the nucleic acid having CrmA biological activity in the cell. Introducing a molecule, and growing the cell under suitable conditions such that the nucleic acid is transcribed and translated in the cell. 2. The method of claim 1, wherein the cellular function comprises cytotoxic T lymphocyte (CTL) regulated cellular function. 3. The method of claim 2, wherein the CTL mediated pathway is CTL mediated apoptosis. 4. The CTL-mediated pathway is calcium (Ca ²⁺ ).
The method of claim 3, wherein the method is dependent CTL mediated killing. 5. A method of preventing and inhibiting apoptosis in a suitable cell by inhibiting activation of the U1-70 pathway, the nucleic acid molecule encoding a gene product having CrmA biological activity in the cell. A method comprising the step of introducing an effective amount of C. under a suitable condition such that cleavage of U1-70 is prevented or inhibited. 6. A method of identifying an agent involved in an apoptotic pathway in a suitable cell, comprising the steps of: a) providing a cell or tissue culture having cell surface receptors that mediate apoptosis; b) exposing the cell or tissue culture to preliminary conditions required for apoptosis; c) dividing the cell or tissue culture into test and control samples; d) testing Contacting said agent to said cell culture or tissue culture of said test sample; e) a nucleic acid molecule or a CrmA gene product encoding a gene product having CrmA biological activity relative to said control sample. Contacting the control sample with the cell or tissue culture under conditions that inhibit apoptosis; f) the test sample. Contacting a nucleic acid molecule encoding a gene product having CrmA biological activity or a CrmA gene product to the test sample under conditions that inhibit apoptosis of the cells; g) steps e) and f). Culturing the sample of claim 1 under conditions that inhibit apoptosis; and h) assaying the sample of step g) for apoptosis by assaying for cleavage of the U1-70kDA protein. 7. A drug identified by the method of claim 6. 8. A method for screening a candidate drug having a biological function in the apoptotic pathway, comprising:
The following steps: a) Effective dose of U1-70 with activating agent, U1-70 at 40 kDa
Incubating under conditions suitable for cleaving into molds; and b) assaying to determine if cleavage of U1-70 has occurred. 9. A drug identified by the method of claim 8.