AU754686B2 - Method for triggering apoptosis in cells - Google Patents

Abstract

The invention relates to a method for triggering apoptosis, whereby the C1D gene in cells, more particularly, in tumor cells, is overexpressed. This occurs, for instance, by introducing corresponding expression constructs in the cells or by exogenous stimulation of the expression of the cell's own C1D gene.

Description

K 2686 Method for Triggering Apoptosis in Cells The present invention relates to a method of triggering apoptosis in cells, in particular in tumor cells.

Apoptosis is the programmed cell death. It is subject to accurate control, it being possible to induce or inhibit apoptosis.

As is known, apoptosis can be induced by a number of what is called death receptors, i.e. receptors containing a death domain such as CD95, TNF-RI, DR3, DR4 or DR5, which after binding their ligands induce apoptosis signal paths.

For example, the CD95 receptor interacts with the adapter protein FADD/MORT1 after the binding of the CD95 ligand so as to induce the recruitment and the activation of the protease FLICE/Caspase-8 at the DISC "death inducing signalling complex". FADD and FLICE each contain death effector domains (DED). The induction of apoptosis via these apoptosis signal paths is possible from outside by e.g. the administration of cytotoxins (cytotoxic substances), irradiation, viruses, removal of growth factors or mechanical cell injuries. However, these possibilities of apoptosis induction are accompanied by certain drawbacks.

For example, the administration of cytotoxins, such as cytostatic agents, or the irradiation in the case of cancer cells results in the development of a resistance and in addition in a damage of normal cells in which apoptosis should actually not be induced.

Hence it would be desirable to provide a method serving for inducing apoptosis, e.g. to combat malignant growth, and, simultaneously reduce the above described side-effects.

According to the invention there is provided a method of inducing apoptosis in cells by overexpression of the C1D gene.

The present invention is based on the inventors' insights that in animals, in particular in mammals, more particularly in human beings, there is a protein which is suitable to "induce apoptosis. Such a protein has a size of about 16 kD and has been characterized as a DNA-binding protein thus far (Nehls et al., Nucleic Acids Research, 26, pages 1160-1166 (1998).

The inventors recognized that the protein suitable to induce apoptosis (hereinafter referred to as CID) is present in every cells, also in tumor cells, where it is expressed in an amount predetermined by the organism. Whenever the C1D gene product is overexpressed, apoptosis is induced in the overexpressing cells. However, especially in tumor cells Sapoptosis obtained by overexpression is desired. This overexpression per se can kill the tumor cells. Furthermore, it could enhance the apoptosis effected by common tumor treatment, such as a chemotherapy or irradiation. Moreover, apoptosis could be effected in tumor cells in which a resistance has already developed by common therapeutic methods. The inventors now found that apoptosis can be induced by overexpressing the CID gene, i.e. increasing the concentration of the cellular CID gene product. This can be done e.g. by transfecting the cells with expression constructs which express the CID gene or by stimulating Soverexpression of the endogenous CID gene.

The CiD gene product comprises the sequence of fig. 1 or 2 or an amino acid sequence differing therefrom -by one or several amino acids. The expression "an amino acid sequence differing therefrom by one or several amino acids" comprises any amino acid sequence coding for a CID (related) protein, whose DNA sequence hybridizes with the DNA of fig. 1 or fig.

2. As to the expression "hybridizes" reference is made to the below explanations.

A nucleic acid coding for CID in the form of a DNA, in particular a cDNA, is particularly suitable to carry out the method according to the invention. A DNA is preferred which comprises: the DNA of fig. 1 or 2 or a DNA differing therefrom by one or several base pairs, the latter DNA hybridizing with the DNA of fig. 1 or 2, or a DNA related to the DNA from via the degenerated genetic code.

The sequence data of the ClD cDNAs according to fig. 1 and 2 are available in the gene library under the following accession numbers: Mouse cDNA: X95591; Human cDNA: X95592.

The expression "a DNA differing by one or several base pairs" comprises any DNA sequence coding for a ClD (related) protein, which hybridizes with the DNA of fig. 1 or 2. The DNA may differ from the DNA of fig. 1 or 2 by additions, -deletions, substitutions and/or inversions of one or several base pairs or other modifications known in the art, e.g.

alternative splicing. According to the invention the expression "DNA" also comprises fragments of this DNA. The expression "fragment" shall comprise a segment of the original nucleic acid molecule, the protein encoded by this fragment still comprising the apoptosis-inducing properties of CID. This also comprises allele variants. A person skilled in the art is familiar with methods of producing the above modifications in the nucleic acid sequence, and such methods are described in standard works of molecular biology, e.g. in Sambrook et al., Molecular Cloning: A laboratory manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).

The person skilled in the art can also determine whether a protein encoded by a nucleic acid sequence modified in this way still has the biological activity of inducing apoptosis, e.g. by the detection of apoptosis-typical cell death characterized by e.g. morphology, multicentric chromatin condensation, typical membrane changes and endogenous DNA degradation.

The expression "DNA hybridized with refers to a DNA which hybridizes with a DNA of fig. 1 or 2 under common conditions, in particular 20 0 C below the melting point of the DNA. In this connection, the expression "hybridizes" refers to conventional hybridizing conditions, preferably to hybridizing conditions under which 5xSSPE, 1 SDS, lx Denhardt's solution is used as a solution and the hybridization temperatures are between 35°C and 70 0

C,

preferably at 65 0 C. Following the hybridization washing is carried out first with 2xSSC, 1 SDS and then with 0.2xSSC at temperatures between 35 0 C and 70 0 C, preferably at 65 0

C

(for the definition of SSPE, SSC and Denhardt's solution see S T* 4 Sambrook et al., supra). Stringent hybridization conditions, as described e.g. in Sambrook et al., supra, are particularly preferred.

In brder to produce the CID gene product which is suitable for carrying out the method according to the invention, the DNA coding for CID is inserted in an vector or expression vector, e.g. pBlueScript, pQE8, pUC or pBr322 derivatives.

In a preferred embodiment, the nucleic acid molecule according to the invention is functionally linked in the vector with regulatory elements which permit its expression in eukaryotic host cells. Such vectors contain, in addition to the regulatory elements, e.g. a promoter, typically a replication origin and specific genes which permit the phenotypic selection of a transformed host cell. The regulatory elements for the expression in eukaryotes include the CMV, SV40, RVS40 promoter, and the CMV or SV40 enhancer.

Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter.

In an embodiment preferred for gene-therapeutic purposes, the vector including the ClD DNA is a virus, e.g. an adenovirus, vaccinia virus or adeno-dependent parvovirus (AAV). Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. For the purposes of gene therapy the nucleic acid molecules according to the invention can also be transported to the target cells in the form of colloidal dispersions.

They comprise e.g. liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682).

General methods known in the art can be used for the construction of expression vectors, and in particular gene therapy vectors, which contain the above-mentioned nucleic acid molecules and suitable control sequences. These methods comprise e.g. in vitro recombination techniques, synthetic methods and in vivo recombination methods as described e.g.

in Sambrook et al., supra. Thus, the person skilled in the art-knows how to insert a DNA according to the invention in an expression vector. He is also familiar with the fact that this DNA can be inserted in combination with a DNA coding for another protein or peptide, so that the DNA according to the invention can be expressed in the form of a fusion protein, e.g. in the form of a fusion protein in which the other part is GFP (the green fluorescent protein of Aequorea Victoria).

For the expression of the CID gene the above-mentioned expression vectors are introduced into host cells. The host cells comprise animal cells, preferably mammalian cells, in both culture and the living organism. The animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa are preferred. Methods of transforming these host cells, detecting the transformation as taken place, and expressing the nucleic acid molecules according to the invention by using the above-described vectors are known in the art.

In addition, the person skilled in the art knows conditions under which transformed and transfected cells can be cultured. He is also familiar with methods of isolating and purifying the protein or fusion protein expressed by the DNA according to the invention.

In order to carry out the method according to the invention the C1D DNA is introduced in a preferred embodiment in an expression vector, in particular a gene therapy vector, and in cells, preferably tumor cells. This is where the expression of the C1D protein occurs, which in addition to i the cell-inherent protein results in the induction of apoptosis. The vectors are introduced into the cells under conditions with which the person skilled in the art is familiar. As to the in vivo gene therapy reference is made in particular to Culver, Gene Therapy, A Handbook for Physicians, Mary Ann Libert, Inc., New York, 1994" and "P.L.

Chang, Sonatic Gene Therapy, CRC Press, London, 1995".

In another preferred embodiment the cell-inherent CID gene is stimulated to an increased expression, e.g. by exogenous stimulation of the endogenous CID promoter. sequences of a gene are referred to as a promoter, which serve as starting points of RNA polymerase II which in cooperation with the transcription factors effect the expression of the gene. In many genes, and also in CID, this process can be induced or stimulated by exogenous factors.

Factors which effect the specific expression of a gene are very numerous and range from physical factors (such as light, heat, cold) via low-molecular inorganic substances (such as salts, metal ions) and low-molecular organic substances (peptides, nucleic acid building blocks, biogenic amines, steroids) to polymolecular substances (serum, growth factors, immunostimulators). The stimulators specific of the CID gene are recognized by combining sequences, present on e.g. the BAC (bacterial artificial chromosome) clones, with a reporter gene, e.g. CAT or EGFP, and examining them for the reporter gene expression and/or its stimulation by exogenous factors, optionally by means of a high-throughput method.

Thus, the present application provides for the first time the possibility of triggering apoptosis not via the common signal paths but through overexpression of a certain gene.

,This can be of special significance for many diseases, in 8 particular for tumoral diseases. The fact that tumor cells respond to an overexpression of CID with much more sensitivity than normal cells proved to be especially advantageous. Therefore, no side-effects exist for normal cells, whereas tumor cells undergo safe cell death.

Brief description of the figures: Fig. 1 shows the DNA and amino acid sequence of CID from a human, Fig. 2 shows the DNA and amino acid sequence of CID from a mouse, fig. 3 shows the temporal course of an apoptosis process triggered by overexpression of CID in cells of the Ehrlich ascites tumor (fluorescence microscopy; excitation: 480 nm; emission: 520 nm) fig. 4 shows examples of morphological characteristics occurring in the course of an apoptosis process triggered by overexpression of CID in cells of the Ehrlich ascites tumor (phase contrast pictures).

The present invention is explained by the below examples.

Example 1: Induction of apoptosis by expression of the C1D gene pcDNA 3 CID expression constructs The cDNA cloned in the Bluescript vector company of Stratagene) and coding for human or murine CID was amplified by PCR. In this connection, the following primers were used: For human cDNA: Primer forward: primer reverse: (effects the amplification of the nucleotide sequence from base 118 to base 540 according to figure 1) For mouse cDNA: Primer forward: primer reverse: (effects the amplification of the nucleotide sequence from base 78 to base 500 according to figure 2) By means of these primers a Kpn restriction site was introduced before the ATG start codon and a Sal I restriction site was introduced before the stop codon (so that the stop codon was omitted). The PCR reaction was carried out by means of the PCR kit from the Clontech company (K1906-1) according to the manufacturer's instructions using the kit components in 50 pl volumes: Water buffer Mg acetate Primer forward Primer reverse C1D template 38.8 pl 5 pl 2.2 pl 1 p1 (1 pM) 1 p1 (10 pM) 5 pl (500 ng) i dNTP 1 p1 kit polymerase 1 p 1 cycler program: 1) initial denaturation 94 0 C, 1 min 2) denaturation 94 0 C, 30 sec 3) annealing extension 68 0 3 min 4) final extension 68 0 C, 3 min cooling 4 0

C

steps are carried out 35 times.

Following the restriction digestion of the amplification batch using Kpn I/Sal I, the fragments were initially recloned in the Bluescript vector (Kpn I/Sal I pretreated). After excising the fragments from the Bluescript vector using Kpn I/Not I, the sequences could be cloned into the correspondingly pretreated pcDNA 3 vector (InVitrogen company).

pcDNA 3-C1D-EGFP expression constructs The fusion between CID and GFP (green fluorescent protein of Aequorea Victoria) with continuous reading frame was effected on the pBluescript level. For this purpose, the above described pBluescript-(Kpn I)-C1D-(Sal I)-plasmids were opened by digestion with Sal I/Hind III.

The sequence coding for EGFP (company of Clontech; EGFP means "enhanced green fluorescent protein" and is a mutant produced by the Clontech company, which has properties improved as regards the excitation/emission) was amplified by PCR. In this PCR, the following primers were used: Primer forward: m primer reverse: to insert a Sal I site at the 5-end and a Hind III site at the 3'-end (here after the stop codon). The PCR was carried out analogously as described above.

After the digestion of the PCR amplification products using Sal I and Hind III they could be ligated into the prepared Bluescript-(Kpn I)-ClD-(Sal I) (Hind III) plasmids.

Thereafter, the fusion cassette (Kpn I)-ClD-EGFP-(NotI) was excised by corresponding digestion out of the Bluescript vector and recloned into the correspondingly pretreated pcDNA 3 vector (Kpn I/Not I).

Transfection of the vectors in tumor cells The above-obtained expression vectors were separated from one another by means of electroporation (Potter et al., Proc. Natl. Acad. Sci. USA, 81, pages 7161-7165 (1984) or lipofection (SuperFect Transfection Reagent Handbook, Quiagen company, Hilden, 02/97, 1997) in cells of the Ehrlich ascites tumor. Transfected (living cells) were observed in the microscope (fluorescence optical system) and photographed (fig. 3).

About 12 hours after the transfection, 20 60 have a weak green fluorescence in the cell nucleus (not shown). This refers to the initially moderate expression of the fusion protein. From a morphological point of view no characteristics can be seen. Approximately 24 hours after the transfection of the vector construct agglomerations of the fusion protein occur in individual cells (fig. 3 left side). These agglomerations can also be observed in the phase contrast picture (fig. In the further temporal course, these agglomerations become stronger (fig. 3, from left to right) and the phase contrast picture (fig. 4) corresponds to the typical picture of a cell undergoing apoptosis.

Not all of the cells which were simultaneously transfected show the excess expression rate of the fusion protein in the picture at the same time. Cells such as on the left side in fig. 3 are also observed even after 48 72 hours, whereas other cells have already reached their end point (fig. 3, right side). This shows that the cells of a culture enter the apoptosis process "in staggered manner". With a sufficiently high transfection rate all cells of a culture, i.e. also those which were not transfected, are killed in the final analysis. This instant depends on the initial transfection rate. By the apoptosis of the transfected cells factors are released which are harmful for non-transfected cells in the culture and finally result in the killing of these non-transfected cells as well (what is called "bystander effect").

It is to be remarked that GFP (green fluorescent protein of Aequorea Victoria) was only used to make a distinction between transfected and non-transfected cells or to make visible the overexpression. GFP expression alone has no effect on the cell morphology or the ability of survival of cells. GFP fusion proteins have basically the functional properties (and also the intracellular distributions) equal to the functional gene product. The apoptotic processes shown in the figures are therefore based on a ClD function.

The morphology shown (and the loss of cell number) was ,effected in control experiments also by constructs which 13 only included the CID sequence by the above described pcDNA 3-C1D expression constructs).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

*o o, EDITORIAL NOTE NO: 51528/99 Sequence listing pages 1-8 are part of the description.

The claims are to follow.

SEQUENCE PROTOCOL GENERAL INDICATIONS:

APPLICANT:

NAME: Deutsches Krebsforschungszentrum STREET: Im Neuenheimer Feld 280 TOWN: Heidelberg COUNTRY: Germany POSTAl CODE: 69120 (ii) TITLE OF THE INVENTION: Method for Triggering Apoptosis in Cells (iii) NUMBER OF SEQUENCES: (iv) COMPUTER-READABLE VERSION: DATA CARRIER: floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SORTWARE: PatentIn Release version #1.30

(EPO)

DATA OF THE CURRENT APPLICATION: not yet known (vi) DATA OF THE PRIOR APPLICATION: APPLICATION NUMBER: DE 198 24 811.3 FILING DATE: June 3, 1998 INDICATIONS AS TO ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1156 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: cDNA (iii) HYPTHETICAL: no (iv) ANTISENSE: no (ix) CHARACTERISTIC: NAME/KEY: CDS POSITION: 118..540 (ix) CHARACTERISTIC: NAME/KEY: mat_peptide Position: 118..540 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTTTCCGGGA GACTGGAGTC GAAGGCCGTG AGTATTTTCT TGTGAGGCAA GAGTACCTAT AGAACCCGGA GGAGGGTGAG AAGCCAGTGT TTAGAGAGTA GAGCAGAGCT GGCCATA ATG -CA Met Ala 1 GGT GAA GAA ATT AAT GAA GAC TAT CCA GTA GAA ATT CAC GAG Gly Giu Glu 5 Ile Asn Glu Asp Tyr 10

GGT

Gly Pro Vai Gu Ile His Giu ATG CTG Met Leu 117 165 213 261

TAT

Tyr

AAG

Ly's TTG TCA GCG Leu Ser Ala ACC ATG ATG Thr Met Met TTT GAG AAT TCC Phe Giu Asn Ser

ATT

Ile 25

AAT

Asn GOT GTG GAT Ala Val Asp

GAG

Giu TCT GTT TCT Ser Vai Ser

AGA

Arg 40 GAG TTG TTG Glu Leu Leu

CAG

Gin AAG TTG GAT Lys Leu Asp OCA OTT Pro Leu TCA ATG Ser Met GAA CAA GCA AAA Glu Gin Ala Lys

GTG

Vai 55

TTG

Leu GAT TTG GTT TOT Asp Leu Val Ser

GCA

Ala

GTT

Val TAO ACA TTA AAT Tyr Thr Leu Asn TTT TGG GTT Phe Trp, Val

TAT

Tyr 70 GCA ACC CAA Ala Thr Gin AAT OCT AAG Asn Pro Lys

GAA

Giu

CAT

His CCA GTA AAA Pro Val Lys

CAG

Gin

ACA

Thr GAA TTG GAA AGA Giu Leu Glu Arg

ATC

le 90 AGA GTA TAT ATG Arg Val Tyr Met AAC AGA Asn Arg GTC AAG GAA Val Lys Giu GCA GCT TCA Ala Ala Ser 115 AAT GCA TOA Asn Ala Ser 130

ATA

Ile 100

AGA

Arg GAO AAG AAA Asp Lys Lys

AAG

Lys 105 GOT GGC AAG OTG Ala Giy Lys Leu TTT GTA AAA Phe Val Lys

AAT

Asn 120

AAA

Lys GOC OTC TGG GAA Ala Leu Trp Giu

OCA

Pro 125 GAO AGA GGT Asp Arg Gly 110 AAA TO!G AAA Lys Ser Lys

TAACTTTTTG

405 453 501 AAA GTT GCO Lys Val Ala

AAT

Asn 135 GGA AAA AGT Gly Lys Ser AAA AGT Lys Ser 140

GTTTTGATGT

TAAATACTTO

GTGATATATT

GGTOAGTCTT

TAAACATTTG

AAATTATAGG

TTCATTT'FrT ACACATATTC AAAAAGTACA TITAATATGTA

CTCTOOAAAG

ATATATTTAT

GCAAGTACOA

TAOTATTTTA

TACATCTGTT

TCTGTATAGA

ATOATTATCT

AATTTACCAT

TTTTATAAG4C

AATGAATAAT

TTOAOTATAT

TACTTTATCA

TTATTGATTA

CTCTTGATGA

AGCTGTGAAA

GACCTTATGA

GATATTAAGA

TGTTTTCATG

ATOACAGTAA

GOACTGAGGA

GACTOTTATT

TTTAAGTGAA

AGTATGOTAT

AAGO!GTGAAT

ATTTTAGGAA

TATGTAAAGC

TTTTAAOATT

TOTTTATATA

ATGTTCTTTG

CTGTAGGOTG

GACTTAAATG

TTACTGCTTT

610 670 730 790 850 910 970 3 GTTGATATTC AAAGTGTGAA ACTAAAAGTT TATGGTTGTA CTTTAATTCT TGGCATGTTG 1030 CCTCTATGTC CCATTTAAAA TAAAATACAT TCTCATTAAC TTTAGATGGG AAATAAGGTT 1090 GTATGTTGAT GGATGAATTT TGGCATGATG ACTGTACTCT CAATAAAGGC TGAAAATGTT 1150 GTAAAA 1156 INDICATIONS AS TO ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 141 amino acids KIND: amino acid TOPOLOGY: linear (ii) KIND OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Met Ala Gly Glu Glu Ile Asn Glu Asp Tyr Pro Val Glu Ile His Glu 1 5 10 Tyr Leu Ser Ala Phe Glu Asn Ser Ile Gly Ala Val Asp Glu Met Leu 25 Lys Thr Met Met Ser Val Ser Arg Asn Glu Leu Leu Gln Lys Leu Asp 40 Pro Leu Glu Gln Ala Lys Val Asp Leu Val Ser Ala Tyr Thr Leu Asn 55 Ser Met Phe Trp Val Tyr Leu Ala Thr Gln Gly Val Asn Pro Lys Glu 70 75 His Pro Val Lys Gln Glu Leu Glu Arg Ile Arg Val Tyr Met Asn Arg 90 Val Lys Glu Ile Thr Asp Lys Lys Lys Ala Gly Lys Leu Asp Arg Gly 100 105 110 Ala Ala Ser Arg Phe Val Lys Asn Ala Leu Trp Glu Pro Lys Ser Lys 115 120 125 Asn Ala Ser Lys Val Ala Asn Lys Gly Lys Ser Lys Ser 130 135 140 INDICATIONS AS TO ID NO: 3: 4 SEQUENCE CHARACTERISTICS: LENGTH: 1040 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: cDNA (iii) HYPTHETICAL: no (iv) ANTISENSE: no (ix) CHARACTERISTIC: NAME/KEY: CDS POSITION: 78..500 (ix) CHARACTERISTIC:

NAME/KEY:

Position: mat_peptide 78..500 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CAGAAGCCGT GTCATGGCGT CATCATCGTG CGACCTATTT CCCGGAGACA GGCGTCCACG GTATTGAGTT GGTCACA ATG GCA GGT GAA GAA ATG AAT GAA GAT TAT CCC Met Ala Gly Glu Glu Met Asn Glu Asp Tyr Pro 1 5 GTA GAA ATT Val Glu Ile GTG GAC GAC Val Asp Asp

CAC

His GAG TCT TTA ACA Glu Ser Leu Thr CTG GAG AGC TCC Leu Glu Ser Ser CTG GGT GCT Leu Gly Ala AAC GAG TTG Asn Glu Leu ATG CTG AAG ACC Met Leu Lys Thr

ATG

Met 35 ATG GCT GTT TCT Met Ala Val Ser

AGA

Arg 110 158 206 254 302 TTG CAG Leu Gin AAG TTG GAC CCA Lys Leu Asp Pro

TTG

Leu 50 GAA CAA GCA AAG GTG Glu Gln Ala Lys Val GAT TTA GTT TCT Asp Leu Val Ser

GCA

Ala TAC ACC TTA AAT Tyr Thr Leu Asn

TCA

Ser 65 ATG TTT TGG GTT Met Phe Trp Val

TAT

Tyr 70 TTG GCA ACT CAA Leu Ala Thr Gin

GGA

Gly GTT AAT CCC AAA Val Asn Pro Lys

GAG

Glu CAT CCA GTG AAG His Pro Val Lys

CAG

Gln 85 GAA CTG GAA AGA Glu Leu Glu Arg ATC AGA Ile Arg GTC TAC ATG Val Tyr Met

AAC

Asn AGA GTT AAA GAA Arg Val Lys Glu

ATA

Ile 100 ACA GAC AAG AAG Thr Asp Lys Lys AAG GCT GCC 398 Lys Ala Ala 105 AAG CTG GAC AGA GGT GCT GCT TCG AGA TTT GTC AAG AAG GCA CTC TGG Lys Leu Asp Arg Gly Ala Ala Ser Arg Phe Val Lys Lys Ala Leu Trp 110 115 120 GAA CCC AAA CGA AAA AGC ACA CCA AAA GTG OCT AAT AAA GGG AAA AGC Glu Pro Lys Arg Lys Ser Thr Pro Lys Val Ala Asn Lys Gly Lys Ser 712 5 130 135 AAA CAC TAATCTTTTG GTTTTGATGT ACATGTTTTC AAAAAGTACA TCCTTTTTAA Lys His 140

TCAGTTTACA

TGTATAAATT

GTTGTCTTTG

ATAAGCAGTT

TGATATTATG

TGATGAGAAA.

TTCGTGATTT

CCTTAAGGCT

TGATGGAAAA

ATGTAGTTAT

TACACATTAC

TTGATTTTCA

GTGAAATCCA

AAGTGTGCTA

TACAGTGACT

TTTTTTTTTT

GTACTTTAAT

GTGACCATGT

ATTTGTGATA

TATAAAGCAT

AATGTTCTCT

TCTGTAGACC

TAAATACCCA

TTGAGTAATT

TCTTCATGTT

GGTGTTTAAA

CTGAATCTTT

CATGATGTGT

GTAAACATTT

TCGAGGTGTA

CTCTGTTTCT

CTGTCTTGAT

CCATTTAAAA

TGGATTCCTT

TTTTTGCTGA

TTAATATTGT

GTAGTGTTTG

AGGACATTTG

GTTCAGTTAG

ATTCAAAGTC

TAAAATGTTC

TTGGAATTCA

GAAAGATTAA

AAGATATTCT

AAATGAACAA

TTTTCAGTAA

TTCAACATGT

AAAATTGAAA

TCATTAACTC

494 550 610 670 730 790 850 910 970 1030 1040 INDICATIONS AS TO ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 141 amino acids KIND: amino acid TOPOLOGY: linear (ii) (xi) Met Ala 1 KIND OF MOLECULE: protein SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gly Glu Glu Met Asn Glu Asp Tyr Pro Val Glu Ile 10 Gly His Glu Met Leu Ser Leu Thr Lys Thr Met Pro Leu Glu Ala Met Leu Glu Ser Ser Leu 25 Ala Val Asp Asp Ala Val Ser Arg 40 Asn Glu Leu Leu Gin Lys Leu Asp Gin Ala Lys Ser Met Val 55 Asp Leu Val Ser Ala Val Tyr Thr Leu Asn Asn Pro Lys Glu Phe Trp Val Tyr Leu Ala Thr Gin Gly His Pro Val Lys Gin Glu Leu Glu Arg Ile 90 Arg Val Tyr Val Lys Glu Ala Ala Ser 115 Ile 100 Thr Asp Lys Lys Lys 105 Ala Ala Lys Leu Met Asn Arg 95 Asp Arg Gly 110 Lys Arg Lys Arg Phe Val Lys Lys 120 Ala Leu Trp Glu Pro 125 Ser Thr Pro Lys Val Ala Asn Lys Gly Lys Ser Lys His 130 135 140 2) INDICATIONS AS TO ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPTHETICAL: no (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGGGTACCAT GGCAGGTGAA GAAATTAATG AAGACTAT INDICATIONS AS TO ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPTHETICAL: no 7 (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGGTCGACTT AACTTTTACT TTTTCCTTTA TTGGCAAC 38 INDICATIONS AS TO ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPTHETICAL: no (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGGGTACCAT GGCAGGTGAA GAAATGAATG AAGATTAT 38 INDICATIONS AS TO ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPTHETICAL: no (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGGTCGACGT GTTTGCTTTT CCCTTTATTA GCCACTTT 38 8 INDICATIONS AS TO ID NO: 9: SEQUENCE CHARACTERISTICS LENGTH: 35 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: other nucleic acid DESCRIPTION: /desc "primer" (iii.) HYPTHETICAL: no (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GGGTCGACAT GGTGAGCAAG GGCGAGGAGC TGTTC INDICATIONS AS TO ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs KIND: nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) KIND OF MOLECULE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPTHETICAL: no (iv) ANTISENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCAAGCTTTG GAATTCTAtA GTCGCGGCCG CTTTA

Claims (7)

1. A method of inducing apoptosis in cells by overexpression of the CID gene.

2. The method according to claim 1, wherein the cells are tumor cells.

3. The method according to claim 1 or 2, wherein the C1D gene product comprises the amino acid sequence of fig. 1 or 2 and/or an amino acid sequence differing therefrom by one or several amino acids, the DNA sequence of the latter amino acid sequence hybridizing with the DNA of fig. 1 or 2.

4. The method according to anyone of claims 1 to 3,, wherein the cells are transfected with an expressionl vector comprising the DNA of fig. 1 or 2 or a DNA differing therefrom by one or several base pairs, the latter DNA hybridizing with the DNA of fig. 1 or 2, or a DNA related to the DNA from via the degenerated genetic code.

The method according to anyone of claims 1 to 3, wherein the ClD gene which is included endogenously in; the cells is stimulated.

6. The method according to claim 5, wherein the promoter of the endogenous CID gene is stimulated by extracellular factors. P:\PDOCS\CRNSPECI755962.sp.dom- 14A

7. Methods of inducing apoptosis in cells by overexpression of the ClD gene, substantially as hereinbefore described with reference to the Examples. DATED this 30th day of August, 2002 DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNG DES OFFENLICHEN RECHTS AND PETER NEHLS By their Patent Attorneys DAVIES COLLISON CAVE