KR20010052525A - Method for Triggering Apoptosis in Cells - Google Patents

Abstract

The present invention relates to a method of inducing apoptosis, wherein the C1D gene is overexpressed intracellularly, particularly in tumor cells. This occurs, for example, by introducing corresponding expression constructs in the cell or by exogenous stimulation of the expression of the C1D gene of the cell itself.

Description

Methods for Triggering Apoptosis in Cells {Method for Triggering Apoptosis in Cells}

Apoptosis is programmed cell death. It requires precise control and it is possible to induce or inhibit apoptosis.

As is known, apoptosis can be induced into a number of so-called receptors, namely receptors comprising a death domain (DD) such as CD95, TNF-RI, DR3, DR4 or DR5, and after binding these ligands induce apoptosis signaling pathways. do. For example, the CD95 receptor interacts with the adapter protein FADD / MORT1 after binding of the CD95 ligand, leading to the recruitment and activation of the protease FLICE / Caspase-8 in the DISC "death inducing signaling complex". FADD and FLICE each contain a death effector domain (DED). Induction of apoptosis through these apoptotic signaling pathways can be induced from the outside, for example, by administration of cytotoxin (cytotoxic agents), irradiation, removal of viruses, growth factors, or mechanical cellular injury. However, this possibility for inducing apoptosis involves certain problems. For example, the administration of cytotoxins, such as cytostatic agents, or irradiation in the case of tumor cells, also results in increased resistance and damage to normal cells in which apoptosis should not actually be induced.

It is therefore an object of the present invention to provide a method for inducing apoptosis, for example to combat malignant growth and at the same time reduce the side effects described above.

According to the invention, this is achieved by the content defined in the claims.

The present invention relates to a method of inducing apoptosis in cells, in particular tumor cells.

1 shows the DNA and amino acid sequences of human C1D.

2 shows the DNA and amino acid sequences of C1D in mice.

Figure 3 shows the transient course of the apoptosis process caused by overexpression of C1D in Erlich multi-tumor cells (fluorescence microscope; excitation: 480 nm; emission: 520 nm).

4 shows examples of morphological features that occur during the apoptosis process caused by the overexpression of C1D in cells of Erlich plural tumors.

The present invention is based on the inventor's judgment that in animals, preferably mammals, more preferably humans, there is a protein suitable for inducing apoptosis. Such proteins have a size of about 16 kD and have so far been characterized as DNA-binding proteins (Nehls et al., Nucleic Acids Research, 26, pages 1160-1166 (1998)).

The inventors found that proteins suitable for inducing apoptosis (hereinafter referred to as C1D) are also present in all cells, tumor cells, and are expressed by the organism in predetermined amounts. Each time the C1D gene product is overexpressed, apoptosis is induced in overexpressing cells. However, particularly in tumor cells, apoptosis obtained by overexpression is required. In essence, such overexpression can kill tumor cells. Moreover, it can promote apoptosis affected by conventional tumor therapies such as chemotherapy or irradiation. Moreover, apoptosis may be affected in tumor cells that have already increased resistance with conventional treatments. The inventors have discovered that apoptosis can be induced by overexpressing the C1D gene, ie by increasing the concentration of intracellular C1D gene product. This can be done, for example, by transfecting cells with expression constructs expressing the C1D gene or by stimulating overexpression of the endogenous C1D gene.

The C1D gene product comprises the sequence of FIG. 1 or 2, or an amino acid sequence in which one or several amino acids are different from this. The expression “one or several amino acids are different from this” includes any amino acid sequence encoding for a C1D (related) protein, which DNA sequence hybridizes with the DNA of FIG. 1 or 2. Reference is made to the following description for the expression "hybrid".

Nucleic acids encoding for C1D in DNA, especially cDNA form, are particularly suitable for carrying out the method according to the invention. DNA preferably comprises:

(a) the DNA of FIG. 1 or 2, or the DNA of which one or several base pairs are different, the latter of which is hybridized with the DNA of FIG.

(b) DNA associated with the DNA of (a) via the degenerate genetic code.

Sequence data of the C1D cDNA according to FIGS. 1 and 2 are available in the genetic library under the following registration number:

Mouse cDNA: X95591;

Human cDNA: X95592.

The expression “DNA of one or several base pairs” includes any DNA sequence encoding a C1D (related) protein, which hybridizes with the DNA of FIG. 1 or 2. This DNA can be different from the DNA of FIG. 1 or 2 through addition, deletion, substitution and / or inversion of base pairs, or other modifications known in the art such as, for example, alternative splicing. According to the invention, the expression "DNA" also includes such DNA fragments. The expression "fragment" includes the fragment of the original nucleic acid molecule, and the protein encoded by this fragment still contains the apoptosis-inducing properties of C1D. It also includes allelic variants. Those skilled in the art are familiar with methods of making such nucleic acid sequences, and such methods are standard work in molecular biology, such as Sambrook et al., Molecular Cloning: laboratory manual, 2 ^nd ED, Cold spring harbor laboratory press , Cold spring harbor NY (1989).

Those skilled in the art will also appreciate whether such methods have a biological activity that induces apoptosis by a protein encoded by a modified nucleic acid sequence, for example morphology, multicentric chromatain condensation, conventional membrane changes, and endogenous. It can be determined by detecting apoptosis-normal cell death characterized by antagonistic DNA degradation.

The expression “hybridized DNA with” refers to DNA hybridized with the DNA of FIG. 1 or 2 under normal conditions, especially at 20 ° C. below the melting point of the DNA. In this regard, the expression “hybridize” refers to conventional hybridization conditions, using 5 × SSPE, 1% SDS, 1 × Denhaart solution as a solution and a hybrid temperature of 35 ° C. to 70 ° C., preferably 65 ° C. It is preferable to hybridize under the conditions described above. Washing after hybridization is first performed at 2 × SSC, 1% SDS, and then at 0.2 × SSC at a temperature between 35 ° C. and 70 ° C., preferably at 65 ° C. (For definitions of SSPE, SSC and Denha Art solutions, see Sambrook et al. See above. Particular preference is given to stringent hybrid conditions as described, for example, in the aforementioned literature by Sambrook et al.

To prepare a C1D gene product suitable for carrying out the method according to the invention, it is inserted into a vector or expression vector of a DNA encoding C1D, for example pBlueScript, pQE8, pUC or pBr322 derivatives. In a preferred embodiment, the nucleic acid molecule according to the invention is functionally bound in a vector using a regulator which allows its expression in eukaryotic host cells. Such vectors include, in addition to regulatory enzymes, for example, promoters, specific genes and origins of replication that allow for phenotypic selection of transformed host cells. Regulatory factors for expression in eukaryotes include the CMV, SV40, RVS40 promoter, and CMV or SV40 enhancer. Other examples of suitable promoters are metallothionein I and polyhedrin promoters.

In a preferred embodiment for gene-therapeutic purposes, the vector comprising C1D DNA is a virus such as adenovirus, vaccinia virus or adeno-dependent parvovirus (AAV). Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. For the purpose of gene therapy, the nucleic acid molecules according to the invention can also be transported to the cells of interest in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1998), 682).

General methods known in the art can be used for the construction of expression vectors, in particular therapeutic vectors, which comprise the aforementioned nucleic acid molecules and appropriate regulatory sequences. Such methods include, for example, in vitro recombination techniques, synthesis methods and in vivo recombination methods as described in Sambrook et al. Thus, those skilled in the art know how to insert the DNA according to the invention into an expression vector. One skilled in the art can also insert such DNAs in combination with DNAs encoding other proteins or peptides, so that the DNA according to the invention is a fusion protein, for example, the other portion of the green fluorescent protein of Aequorea Victoria (GFP). It is familiar with the fact that it can be expressed in the form of phosphorus fusion proteins.

For expression of the C1D gene, the expression vectors mentioned above are introduced into the host cell. Host cells include animal cells, preferably mammalian cells, both of which include cultured and living organisms. Animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa are preferred. Methods of transforming these host cells, methods of detecting the resulting transformation, and methods of expressing nucleic acid molecules according to the invention using the vectors described above are known in the art.

In addition, those skilled in the art know the conditions under which transformed and transfected cells can be cultured. Those skilled in the art are also familiar with the methods for isolating and purifying proteins or fusion proteins expressed by the DNA according to the invention.

In order to carry out the method according to the invention, C1D DNA is introduced in a preferred form into expression vectors, in particular gene therapy vectors and cells, preferably tumor cells. This is where expression of C1D protein occurs and induces apoptosis in addition to cell-specific proteins. The vector is introduced into the cell under conditions familiar to those skilled in the art. For in vivo gene therapy see, in particular, "KW Culver, Gene Therapy, A Handbook for Physicians, Mary Ann Libert, Inc., New York, 10994" and "PLChang, Sonatic Gene Therapy, CRC Press, London, 1995". .

In another preferred embodiment, the cell-specific C1D gene is stimulated to exhibit increased expression, for example by exogenous stimulation of the endogenous C1D promoter. The 5'-adjacent sequence of a gene is referred to as a promoter and acts as a starting point for RNA polymerase II, which, in cooperation with transcription factors, affects expression of the gene. For many genes and C1Ds, this process can be induced or stimulated by exogenous factors. There are many factors that influence the specific expression of genes, and physical factors (light, heat, low molecular weight inorganic substances (salts, metal ions, etc.) and low molecular weight organic substances (peptides, nucleic acid forming blocks, bio amines, steroids) Cold air, etc.) to high molecular weight substances (serum, growth factor, immune stimulating factor). Genes specific to the C1D gene are reporter genes such as CAT or EGFP, for example, combining 5′-adjacent sequences present on bacterial artificial chromosome (BAC) clones, exogenous factors, and optionally mass processing methods. By testing them for reporter gene expression and / or stimulation thereof.

Thus, the present invention first offers the possibility of inducing apoptosis through overexpression of certain genes rather than through conventional signaling pathways. This may be particularly important for many diseases, especially tumor diseases. The fact that tumor cells respond much more sensitively than normal cells to overexpression of C1D has proved particularly useful. Therefore, no adverse effects exist for normal cells, whereas tumor cells undergo safe cell death.

The present invention will be described with reference to the following examples.

Example 1: Induction of Apoptosis by Expression of C1D Gene

pcDNA 3-C1D expression construct

CDNA cloned in Bluescript vector (KS +, Stratagene Inc.) and encoded for human or mouse was amplified by PCR. In this regard, the following primers were used:

For human cDNA:

Forward primer:

5'-GGGGTACCATGGCAGGTGAAGAAATTAATGAAGACTAT

Reverse primer

5'-GGGTCGACTTAACTTTTACTTTTTCCTTTATTGGCAAC

(Affects the amplification of the nucleotide sequence from base 118 to base 540 according to FIG. 1)

For mouse cDNA:

Forward primer:

5'-GGGGTACCATGGCAGGTGAAGAAATGAATGAAGATTAT

Reverse primer:

5'-GGGTCGACGTGTTTGCTTTTCCCTTTATTAGCCACTTT

(Affects the amplification of the nucleotide sequence from base 78 to base 500 according to FIG. 2)

By these primers, the Kpn restriction site was introduced before the ATG start codon, and the Sa1 I restriction site was introduced before the stop codon (as a result, the stop codon was removed). PCR reaction was carried out by using a kit component in an amount of 50 µl and using a PCR kit manufactured by Clone Tech Co., Ltd. (K1906-1) according to the manufacturer's instructions.

38.8 μl purified water

5 μl 10x buffer

2.2 μl Mg Acetate

1 μl forward primer (1 μM)

1 μl reverse primer (10 μM)

5 μl (500 ng) C1D template

1 μl 50x dNTP

1 μl kit polymerase

Cycler program:

1) Initial denaturation 94 ℃, 1 minute

2) denaturation 94 ℃, 30 seconds

3) Annealing extension 68 ℃, 3 minutes

4) Final extension 68 ° C., 3 minutes

5) cooling 4 ℃

Step 2) / 3) is carried out 35 times.

After restriction cleavage of the amplification batch using Kpn I / Sa1 I, the fragments were initially recloned in the BlueScript vector (Kpn I / Sa1 I—pretreated). After removing fragments from the BlueScript vector using Kpn I / Not I, the sequence could be cloned into the corresponding preprocessed pcDNA 3 vector (InVitrogen, Inc.).

pcDNA 3-C1D-EGFP expression construct

In a continuous reading frame, the fusion between C1D and GFP (green fluorescent protein of Aequorea Victoria) was affected by pBluescript levels. For this purpose, the pBluescript- (Kpn I) -C1D- (Sa1 I) -plasmids described above were opened by cleavage with Sa1 I / Hind III.

Sequences encoding for EGFP (Clontech Co., Ltd .; EGFP stands for "enhanced green fluorescent protein", a mutant produced by Clontech Co., Ltd., having improved properties for excitation / emission) were amplified by PCR. In this PCR, the following primers were used to insert the Sa1 I site at the 5-terminal and the Hind III site at the 3'-end:

Forward primer:

5'-GGGTCGACATGGTGAGCAAGGGCGAGGAGCTGTTC

Reverse primer:

5'-CCAAGCTTTGGAATTCTAGAGTCGCGGCCGCTTTA

PCR was performed similarly as described above.

After cleavage of the PCR amplification products using Sa1 I and Hind III, they were bound to the prepared Bluescript- (Kpn I) -C1D- (Sa1 I) ... (Hind III) plasmids. The fusion cassette (Kpn I) -C1D-EGFP- (Not I) was then cleaved from the BlueScript vector with the corresponding restriction enzyme and recloned into the corresponding pretreated pcDNA 3 vector (Kpn I / Not I).

Transfection of the vector in tumor cells

The obtained expression vector was subjected to electroporation (Potter et al., Proc. Natl. Acad. Sci. USA, 81, pages 7161-7165 (1984)) or lipofection (SuperFect) in Erlich ascites tumor cells. Transfection Reagent Handbook, Quiagen company, Hilden, 02/97, 1997). Transfected cells (living cells) were observed and photographed under a microscope (fluorescence optical system) (FIG. 3).

After about 12 hours of transfection, 20-60% show weak green fluorescence in the cell nucleus (not shown). This refers to early palliative expression of the fusion protein. From a morphological point of view no features are seen. After about 24 hours in transfection of the vector constructs a lump of fusion protein develops in individual cells (left part of FIG. 3). These agglomerates can also be observed in the phase contrast picture (FIG. 4). In other transient processes, these masses become stronger (FIG. 3, left to right), and the phase contrast picture (FIG. 4) corresponds to the general picture of the cells in which apoptosis has occurred.

Not all infected cells simultaneously show excessive expression of the fusion protein in the photograph. Cells such as the one on the left of FIG. 3 can also be observed up to 48-72 hours later, while other cells have already reached their endpoints (FIG. 3, right). This is a "staggered method" in which cultured cells enter the apoptosis process. With sufficiently high transfection rates, all cultured cells, ie untransfected cells, are also killed in the final assay. This example depends on the initial transfection rate. Apoptosis of the transfected cells releases detrimental factors to the nontransfected cells in the culture and ultimately leads to the death of these nontransfected cells (the so-called “bystander effect”).

It should be noted that the green fluorescent protein of Aequorea Victoria (GFP) was used only to distinguish transfected and nontransfected cells or to visualize overexpression. GFP expression itself has no effect on cell morphology or cell viability. GFP fusion proteins basically have the same functional properties (and intracellular distribution) as functional gene products. Therefore, the apoptotic process shown in the figure is based on C1D function. The depicted form (and loss of cell number) is affected in a regulatory test also by constructs comprising only C1D sequences (eg by the pcDNA 3-C1D expression constructs described above).

<110> Deutsches Krebsforschungszentrum

<120> Method for triggering apoptosis in cells

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<150> DE 198 24 811.3

<151> 1998-06-03

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Pro Leu Glu Gln Ala Lys Val Asp Leu Val Ser Ala Tyr Thr Leu Asn

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Ser Met Phe Trp Val Tyr Leu Ala Thr Gln Gly Val Asn Pro Lys Glu

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His Pro Val Lys Gln Glu Leu Glu Arg Ile Arg Val Tyr Met Asn Arg

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Val Lys Glu Ile Thr Asp Lys Lys Lys Ala Gly Lys Leu Asp Arg Gly

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Ala Ala Ser Arg Phe Val Lys Asn Ala Leu Trp Glu Pro Lys Ser Lys

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aat gca tca aaa gtt gcc aat aaa gga aaa agt aaa agt taactttttg 550

Asn Ala Ser Lys Val Ala Asn Lys Gly Lys Ser Lys Ser

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Pro Leu Glu Gln Ala Lys Val Asp Leu Val Ser Ala Tyr Thr Leu Asn

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Val Lys Glu Ile Thr Asp Lys Lys Lys Ala Gly Lys Leu Asp Arg Gly

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Ala Ala Ser Arg Phe Val Lys Asn Ala Leu Trp Glu Pro Lys Ser Lys

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Pro Leu Glu Gln Ala Lys Val Asp Leu Val Ser Ala Tyr Thr Leu Asn

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Ser Met Phe Trp Val Tyr Leu Ala Thr Gln Gly Val Asn Pro Lys Glu

65 70 75 80

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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

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<221> mat_peptide

<222> (78) .. (500)

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cagaagccgt gtcatggcgt catcatcgtg cgacctattt cccggagaca ggcgtccacg 60

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Met Ala Gly Glu Glu Met Asn Glu Asp Tyr Pro

1 5 10

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Val Glu Ile His Glu Ser Leu Thr Ala Leu Glu Ser Ser Leu Gly Ala

15 20 25

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Glu Asp Asp Met Leu Lys Thr Met Met Ala Val Ser Arg Asn Glu Leu

30 35 40

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Leu Gln Lys Leu Asp Pro Leu Glu Gln Ala Lys Val Asp Leu Val Ser

45 50 55

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Ala Tyr Thr Leu Asn Ser Met Phe Trp Val Tyr Leu Ala Thr Gln Gly

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Val Asn Pro Lys Glu His Pro Val Lys Gln Glu Leu Glu Arg Ile Arg

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Glu Pro Lys Arg Lys Ser Thr Pro Lys Val Ala Asn Lys Gly Lys Ser

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Lys his

140

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Ser Met Phe Trp Val Tyr Leu Ala Thr Gln Gly Val Asn Pro Lys Glu

65 70 75 80

His Pro Val Lys Gln Glu Leu Glu Arg Ile Arg Val Tyr Met Asn Arg

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Val Lys Glu Ile Thr Asp Lys Lys Lys Ala Ala Lys Leu Asp Arg Gly

100 105 110

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115 120 125

Ser Thr Pro Lys Val Ala Asn Lys Gly Lys Ser Lys His

130 135 140

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Met Ala Gly Glu Glu Met Asn Glu Asp Tyr Pro Val Glu Ile His Glu

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20 25 30

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35 40 45

Pro Leu Glu Gln Ala Lys Val Asp Leu Val Ser Ala Tyr Thr Leu Asn

50 55 60

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65 70 75 80

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100 105 110

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<220>

<223> Primer forward

<400> 11

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<210> 12

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ccaagctttg gaattctaga gtcgcggccg cttta 35

Claims (6)

A method of inducing apoptosis in cells by overexpression of the C1D gene. The method of claim 1, wherein the cells are tumor cells. The DNA sequence of claim 1 or 2, wherein the C1D gene product comprises the amino acid sequence of FIG. Or hybridize with DNA of 2. The method of claim 1, wherein the cells are: (a) the DNA of FIG. 1 or 2, or the DNA of which one or several base pairs differ from these, the latter DNA of the DNA of FIG. 1 or 2, or (b) DNA related to the DNA of (a) through the degenerate genetic code; Method for transfecting cells with an expression vector comprising a. The method according to any one of claims 1 to 3, wherein the C1D gene endogenously contained in the cell is stimulated. The method of claim 5, wherein the promoter of the endogenous C1D gene is stimulated by exogenous factors.