Patent Application
20050158775
The invention relates to (i) a plasmid DNA comprising a novel reporter gene DNA, (ii) a transformant transformed with the above plasmid DNA and a DNA encoding a known effector protein, (iii) a therapeutic agent for a cancer or an autoimmune disease, comprising both of the above DNAs as active ingredients, and (iv) a method for detecting a ligand of an intracellular receptor, comprising using the above transformant.
More particularly, the invention relates to (i) a plasmid DNA comprising a novel reporter gene, namely a minimum essential promoter and a DNA encoding an amino acid sequence comprising a transmembrane region and a functional region of an Fas antigen for detecting a gene expression effect, in a region downstream of an enhancer element in which a responsive element of Gal4 protein which is a yeast basal transcription factor (hereinafter referred to as “UAS”) is repeated several times, (ii) a transformant transformed with both of the above plasmid DNA and a DNA encoding a fusion protein in which an amino acid sequence comprising a ligand binding region of an intracellular receptor is linked to the C-terminal of an amino acid sequence comprising the DNA binding region of a known effector protein, namely Gal4 protein, (iii) a therapeutic agent for a cancer or an autoimmune disease, comprising both of the above DNAs as active ingredients and (iv) a method for detecting an intracellular receptor ligand, comprising using the above transformant.
Recently, receptors of humoral factors having important physiological activities, typified by cytokine and hormones, have been revealed by gene manipulation, and a large number of receptors have been isolated and identified. The examples include receptors of lipophilic hormones generally referred to as intracellular receptors. As a result of the analysis of amino acid sequences of various intracellular receptors, it was revealed that they are ligand-dependent transcription factors having a common basic structure and forming one of gene family. That is, they belong to a family including a steroid receptor, such as glucocorticoid receptor, progesterone receptor, mineral corticoid receptor, androgen receptor, estrogen receptor and the like; a retinoid receptors, such as retinoid X receptor, retinoic acid receptor and the like; a peroxisome proliferator-activated receptor; a vitamin D3 receptor; and a thyroid hormone receptor. Since it has been revealed that functional regions, such as a ligand binding domain and a domain which recognizes a target DNA sequence and the like, are distinctively separated in these receptors (see Science, 240, 889 (1988)), they have been isolated based on the homology, though there are certain receptors whose ligands are still unknown.
Recently, a nuclear receptor, peroxisome proliferator-activated receptor (hereinafter referred to as a “PPAR receptor”), is drawing attention in the field of studies on transcription factors related to the expression and induction of fat cell differentiation marker genes. Regarding the PPAR receptor, cDNA has been cloned from various animal species, and a plurality of isoform genes have been found including α, δ and γ types in mammals. In addition, it is known that the γ type is expressed in fat tissues, immunocytes, adrenal gland, spleen and small intestine, and the α type in liver and retinas, but the δ type has no tissue specificity and is expressed ubiquitously.
On the other hand, Fas antigen is a membrane protein whose participation in programmed cell death, named apoptosis has been clearly shown. Yonehara et al. have prepared monoclonal antibodies for human cell surface antigens and obtained an anti-Fas antibody showing lethal activity upon various human cells (see J. Exp. Med., 169, 1747 (1989)). cDNA of a cell surface molecule recognizable by the anti-Fas antibody has been isolated, and structure of the human Fas antigen has been determined (see Cell, 66, 233 (1991)). This Fas antigen is composed of 335 amino acid residues, and 16 amino acid residues in the N-terminus is assumed to be its signal peptide. A transmembrane region composed of 17 hydrophobic amino acid residues is present in a central position of the molecule, and it is considered that 157 amino acid residues in the N-terminus exist as its extracelluar region and the C-terminal side sequence of 145 amino acid residues is its cytoplasmic region.
Since the structure of the Fas antigen is resemble in the structure of a receptor for tumor necrosis factor (TNF), it is assumed that the apoptosis of the Fas antigen occurs by a mechanism similar to the action of TNF. Functional regions of the Fas antigen have also been revealed gradually, and it has been found that the region essential for the signal transduction of apoptosis (functional region) is an amino acid sequence of the 175th to 304th positions (see J. Biol. Chem., 268, 10932 (1993)). In addition, the amino acid sequence of mouse Fas antigen has also been revealed (see J. Immunology, 148, 1274 (1992)), and it has a homology of 49.3% to the human Fas antigen as a whole. It is considered also that its functional region is an amino acid sequence of the 166th to 291st positions corresponding to the region of the human Fas antigen.
Receptors have a ligand binding region and a signalling transduction region. When a ligand is linked to the ligand binding region, the stereostructure of the signal transfer region is changed and a signal is transferred to other protein or DNA.
Recently, many studies have been conducted positively on the intercellular signal transduction by a fusion protein in which the ligand binding region of a receptor is linked to the signalling region of a different protein.
For example, it has been revealed that, in the case of cells transformed with a gene encoding a fusion protein of the ligand binding region of an estrogen receptor which is one of steroid receptors family, and the functional region of human cancer gene c-Myc, the cells are cancerated to abnormally proliferate by the stimulation of the ligand, estrogen (see Nature, 340, 66 (1989)).
Similar studies have been carried out on fusion proteins of the ligand binding region of each of the receptors for glucocorticoid, mineral corticoid and estrogen with the functional region of each of the proteins of E1A (adenovirus), c-Fos, v-Myb, C/EBP, v-Re1, GATA-1, 2 and 3, GAL4-VP16, Rev (HIV) and c-Ab1 (see Cell, 54, 1073 (1988), Proc. Natl. Acad. Sci. USA, 88, 5114 (1991), EMBO J., 10, 3713 (1991), Science, 251, 288 (1991), EMBO J., 11, 4641 (1992), Genes Dev. (in press), Proc. Natl. Acad. Sci. USA, 90, 1657 (1993), ibid., 87, 7787 (1990) and EMBO J., 12, 2809 (1993)), confirming that respective signals are transferred by the binding of ligands to their corresponding nuclear receptors.
Kakizuka et al. have reported a method in which the Fas antigen is used as a signal transduction protein for applying the above fusion protein to the treatment of a cancer (see JP-A-7-316200). In this method, an amino acid sequence of the 136th to 305th positions including not only the functional region but also the transmembrane region is fused with the ligand binding region of the intracellular receptor. As a result, a ligand capable of interacting with the amino acid sequence of the nuclear receptor ligand binding region can be detected effectively.
On the other hand, R. M. Evans et al. have reported a reporter assay method in which a reporter using an enzyme capable of converting a substrate into a chemiluminescent or visible dye product or introducing a substituent (e.g., luciferase, β-galactosidase, secretory alkaline phosphatase, or chloramphenicol acetyltransferase) in a region downstream of the Gal4 responsive element and a protein prepared by fusing the Gal4 DNA binding domain and the PPAR receptor ligand binding region are introduced into cells (see PCT International Application International Publication No. 9640128). Another assay method has also been reported in which, either by expressing an exogenous PPAR receptor protein or along with an endogenous PPAR receptor protein, the above reporter, with the proviso that in this case, the reporter is a reporter using the above enzyme in a region downstream of PPAR receptor responsive element (hereinafter referred to as “PPRE”) are introduced (Cell, 83, 803 (1995)). These assay systems are detection systems which can evaluate the function of intracellular receptors as transcription factors.
However, since these methods require a certain period of time for the activity detection, it is difficult to carry out high speed screening of the effect of ligands and compounds to be tested. In addition, since the introduction of DNA into cells is transient, it causes a problem in that the results cannot easily be compared due to insufficient stability among tests.
For carrying out high speed screening of compounds which bind to ligands of intracellular receptors as an object, the inventors of the invention have conducted intensive studies and found, as a result of the efforts, that the object can be attained using a reporter assay in Which an Fas protein is expressed.
That is, an expression vector comprising a reporter using a functional region of an Fas structural protein and a DNA encoding a protein prepared by fusing a DNA binding region of Gal4 and a nuclear receptor (particularly PPAR receptor) ligand binding region was stably introduced into mouse fibroblast L929 cells. These cells were able to grow under such conditions that ligands or compounds did not bind to the amino acid sequence of the nuclear receptor ligand binding region, but cell death occurred due to expression induction of an Fas protein when the ligands or compounds bound to the sequence. Thereafter, we have succeeded in carrying out high speed screening of a compound which interacts with the ligand binding region of an intracellular receptor and can functionally increase or decrease the activity of transcription factor by separating the thus cell death-caused cells from intact cells, staining the intact cells with a dye, and measuring the absorbance of the stained cells.
The high throughput screening method with the high speed of the invention is a novel method which has been unknown completely.
In the method of the invention, the measurement is carried out by generating cell death through expression of an Fas protein using functional region as a reporter of an Fas protein, so that it is completely different from the method of R. M. Evans et al. using an enzyme which generates chemiluminescence (particularly luciferase) or forms a visible dye (particularly β-galactosidase) as the reporter and measuring the amount of expressed the enzyme (enzyme activity).
In comparing by the standard test methods, the method of R. M. Evans et al. requires an experimental period for 4 days at the shortest in which DNA is introduced into cells and serum stimulation is carried out on the first day, re-inoculation is optionally carried out, a compound to be tested is added after 46 to 48 hours (on the third day), and luciferase activity is measured 24 hours thereafter (on the final day). Also, since the introduction of DNA into cells is transient, it causes a problem in terms of stability and comparison between tests. An expression vector comprising a DNA encoding another enzyme may be co-introduced in order to compare each measurements, but the handling becomes complex. In addition, since the measured value greatly varies, the method using luciferase requires a large number of cases (generally requires an n number of 3 to 4). Alternatively, it is necessary to standardize the method with a simultaneously introduced internal standard gene (e.g., β-galactosidase gene etc.) as described above.
On the other hand, the invention is a method of two-day schedule at the shortest, in which, when cells are prepared in advance, a compound to be tested is added on the first day, and the cells can be measured by a dye staining on the latter half of the next day (after 36 hours). Also, since L929 cells (the same clone) are used, comparison between tests can be made easily. In addition, since the variation of values is small when measured by the method of the invention in which stably introduced cells are used, it does not require a large number of cases (generally, sufficient judgment can be made by an n number of 1 to 2).
Thus, according to the method of the invention, it is possible to carry out high speed evaluation of ligands and compounds which can change functions of intracellular receptors, and the handling per se is simple and easy. Also, since variation of measured values is small, data between tests can be easy to compare and are very stable. These advantages exert full abilities particularly in the high speed evaluation of multiple samples. In addition, since an absorbance measuring apparatus is attached to any generally and practically used automatic analyzers (robotic machine) as a standard measure, the method can be regarded as a robot-corresponding compound evaluation system. This is a fact which cannot be expected from the previous art but confirmed for the first time by experiments carried out by the present inventors. In addition, since the method of the invention require no enzyme substrate, sharp reduction of cost can be made.
The invention relates to
The Fas antigen and intracellular receptor for use in the invention are those which are originated from mammals, such as humans, monkeys, dogs, cats, mice, rats and guinea pigs. The Fas antigen and intracellular receptor are preferably originated from the same species, but those from the different species can also be used. It has been confirmed by the invention that the transfer of signals can be effected when those which are originated from different species are used. For example, a combination of human Fas antigen with a human intracellular receptor, a combination of mouse Fas antigen with a human intracellular receptor, a combination of human Fas antigen with a rat intracellular receptor, a combination of mouse Fas antigen with a rat intracellular receptor, a combination of human Fas antigen with a mouse intracellular receptor or a combination of mouse Fas antigen with a mouse intracellular receptor can be used suitably.
As the amino acid sequence comprising the transmembrane region and the function expressing region of an Fas antigen and the amino acid sequence comprising the ligand binding region of an intracellular receptor for use in the invention, the portion corresponding to each region may be used alone, or the portion corresponding to each region may further contain a flanking region (a region which does not take part in the functional region; several tens, preferably 60 or less, of residues as an amino acid sequence) on its both termini (N-terminal and/or C-terminal). If desired, an amino acid sequence comprising a signal peptide region may be linked to the N-terminal of the transmembrane region of the Fas antigen. The amino acid sequence corresponding to each region may be not only a natural amino acid sequence but also its derivative in which at least one amino acid is deleted, substituted, added and/or inserted in a such extent that the original apoptosis function of the Fas antigen or the ligand binding function of the intracellular receptor is not spoiled.
Among the Fas antigens for use in the invention, the amino acid sequence comprising the transmembrane region and the function expressing region of the human Fas antigen is a sequence of the 145th Ser to the 319th Val, and, in the case of the mouse Fas antigen, a sequence of the 136th Ser to the 305th Leu is preferably used. Also, as the amino acid sequence comprising a signal peptide region, a sequence of the −16th to 23rd positions of human Fas antigen or a sequence of the −21st to 14th positions of mouse Fas antigen is preferably used.
Examples of the intracellular receptor in the effector protein for use in the invention include a steroid receptor, such as a glucocorticoid receptor, a progesterone receptor, a mineral corticoid receptor, an androgen receptor, an estrogen receptor and the like; a retinoid receptor, such as retinoid X receptor, retinoic acid receptor and the like; a peroxisome proliferator-activated receptor; a vitamin D3 receptor; and a thyroid hormone receptor. Among these, human or rat estrogen receptor; human or mouse retinoid X receptor α, retinoid X receptor β or retinoid X receptor γ; human or mouse retinoic acid receptor α, retinoic acid receptor β or retinoic acid receptor y; human or mouse PPAR α receptor; human or mouse PPAR δ receptor; and human or mouse PPAR γ receptor are preferably used.
The ligand binding region of each receptor is already known (see Science, 240, 889 (1988); Mol. Endocrinology, 6, 1634 (1992); Biochem. Biophys. Res. Commun., 224, 431 (1996); J. Steroid Biochem. Molec. Biol., 51, 157 (1994); and Proc. Natl. Acad. Sci. USA, 91, 7355 (1994)), such as 528th to 777th positions in the human glucocorticoid receptor, the 680th to 934th positions in the human progesterone receptor, the 734th to 984th positions in the human mineral corticoid receptor, the 676th to 919th positions in the human androgen receptor, the 311th to 551st positions in the human estrogen receptor, the 307th to 557th positions in the rat estrogen receptor, the 225th to 462nd positions in the human retinoid X receptor α, the 297th to 526th positions in the human retinoid X receptor β, the 230th to 467th positions in the mouse retinoid X receptor α, the 171st to 410th positions in the mouse retinoid X receptor γ, the 229th to 463rd positions in the mouse retinoid X receptor γ, the 198th to 462nd positions in the human retinoic acid receptor α, the 191st to 448th positions in the human retinoic acid receptor β, the 200th to 454th positions in the human retinoic acid receptor γ, the 198th to 462nd positions in the mouse retinoic acid receptor α, the 190th to 448th positions in the mouse retinoic acid receptor β, the 200th to 458th positions in the mouse retinoic acid receptor γ, the 192nd to 427th positions in the human vitamin D3 receptor, the 183rd to 410th positions in the human thyroid hormone receptor α, the 232nd to 456th positions in the human thyroid hormone receptor β, the 280th to 478th positions in the human PPAR γ (human PPAR γ1 subtype) receptor, the 278th to 475th positions in the mouse PPAR γ (mouse PPAR γ1 subtype) receptor, the 273rd to 468th positions in human PPAR α receptor, the 273rd to 468th positions in the mouse PPAR α receptor, the 273rd to 468th positions in rat PPAR α receptor, the 246th to 441st positions in the human PPAR δ receptor, and the 245th to 440th positions in the mouse PPAR δ receptor.
Preferred examples include a sequence of the 311th position to the 551st position of the human estrogen receptor, the 307th to 557th positions of the rat estrogen receptor, the 225th to 462nd positions of the human retinoid X receptor α, the 297th to 526th positions of the human retinoid X receptor β, the 230th to 467th positions of the mouse retinoid X receptor α, the 171st to 410th positions of the mouse retinoid X receptor β, the 229th to 463rd positions of the mouse retinoid X receptor γ, the 198th to 462nd positions of the human retinoic acid receptor α, the 191st to 448th positions of the human retinoic acid receptor β, the 200th to 454th positions of the human retinoic acid receptor γ, the 198th to 462nd positions of the mouse retinoic acid receptor α, the 190th to 448th positions of the mouse retinoic acid receptor β, the 200th to 458th positions of the mouse retinoic acid receptor γ, the 176th to 478th positions of the human PPAR γ (human PPAR γ1 subtype) receptor, the 174th to 475th positions of the mouse PPAR γ (mouse PPAR γ1 subtype) receptor, the 167th to 468th positions of the human PPAR α receptor, the 167th to 468th positions of the mouse PPAR α receptor, the 167th to 468th positions of the rat PPAR α receptor, the 139th to 441st positions of the human PPAR δ receptor, and the 138th to 440th positions of the mouse PPAR δ receptor.
More preferred examples include a sequence of the 281st to 595th positions of the human estrogen receptor, the 286th to 600th positions of the rat estrogen receptor, the 176th to 462nd positions of the human retinoic acid receptor α, the 177th to 458th positions of the mouse retinoic acid receptor α, the 166th to the 478th positions of the human PPAR γ (human PPAR γ1 subtype) receptor (corresponding to a sequence of the 194th to 506th positions of the human PPAR γ2 subtype), the 164th to 475th positions of the mouse PPAR γ (mouse PPAR γ1 subtype) receptor (corresponds to a sequence of the 194th to 505th positions of the mouse PPAR γ2 subtype), the 157th to 468th positions of the human PPAR α receptor, the 157th to 468th positions of the mouse PPAR α receptor, the 157th to 468th positions of the rat PPAR α receptor, the 129th to 441st positions of the human PPAR δ receptor, and the 128th to 440th positions of the mouse PPAR δ receptor.
Examples of more preferred effector protein for use in the invention include:
Regarding the UAS which is a Gal4 protein responsive element contained as a repeating structure in the reporter plasmid of the invention, an already known sequence was used (see Cell, 83, 803 (1995) and Cell, 83, 813 (1995)). That is, a sequence in which a sequence of 5′-CGACGGAGTACTGTCCTCCG-3′ is repeated several times, specifically at least 3 times, preferably 4 times, more preferably 5 times.
Examples of the promoter of the invention include SV40, HSV LTR, a metallothionein promoter, and a thymidine kinase (TK) promoter. A TK promoter is preferred. More specifically, it is preferred to use a sequence of the −727th to 56th positions of the mRNA of the TK promoter, when its transcription initiation point is regarded as the 1 st position, as used in plasmid pTKβ (Clontech Laboratories, Cat. No. 6179-1) (corresponding to a sequence of the 165th to 945th positions in the pTKβ map). A sequence of the −105th to 51st positions of the mRNA of the TK promoter is more preferred.
DNAs encoding the reporter plasmid of the invention are also included in the invention. As well known, one amino acid is encoded by 1 to 6 codons (e.g., 1 codon for methionine (Met) and 6 codons for leucine (Leu)). Accordingly, the nucleotide sequence of a DNA can be changed without changing its corresponding amino acid sequence.
Among DNAs encoding the reporter plasmid of the invention, a group of nucleotide sequences of all DNA sequences which result in the same Fas protein polypeptide are included in the invention. Productivity of the fusion protein may sometimes be improved by changing the nucleotide sequence.
The DNA encoding the reporter plasmid of the invention can be prepared in accordance with the following method.
That is, this method is carried out by
In detail, the step (i) is a step in which necessary parts for fusion are amplified by PCR. In the case of the Fas antigen, a nucleotide sequence encoding an amino acid sequence comprising the transmembrane region and the function expressing region is chemically synthesized and used as a primer. Also, in the case of the intracellular receptor, a nucleotide sequence encoding an amino acid sequence comprising the ligand binding region is chemically synthesized and used as a primer.
A specific restriction enzyme site is arranged on the 5′-end of each of the thus obtained primers. Regarding the template for PCR, mRNA isolated and purified from cells or a cell line of a corresponding mammal, such as human, mouse or the like, can be used. The PCR is carried out by a known method. Since automatic PCR apparatuses have come into wide use recently, the apparatuses are suitably used.
In the step (ii), DNAs corresponding to the DNA binding region of Gal4 and the nuclear receptor ligand binding region are fused in an expression vector. As the plasmid vector for use in this step, a large number of vectors capable of functioning in Escherichia coli (e.g., pBR322) or Bacillus subtilis (e.g., pUB110) are known, and any of them can be used suitably. Also, it is possible to insert these fragments directly into an expression vector.
When expressed in mammal cells, the expression vector is prepared by inserting the PCR product of the Gal4 DNA binding region and then that of the intracellular receptor into a region downstream of an appropriate promoter (e.g., SV40 promoter, LTR promoter or metallothionein promoter) in an appropriate vector (e.g., a retrovirus vector, a papilloma virus vector, a vaccinia virus vector or an SV40 vector). Preferably, pCMX (described in Cell, 66, 663 (1991)) or pSV (described in Anal. Biochem., 188, 245 (1990)) is used.
Subsequently, in the step (iii), a reporter plasmid is produced. The plasmid vector for use in this step may be any one of those which function in E. coli or B. subtilis as described above. It can also be inserted directly into the expression vector. A plasmid comprising a DNA in which a repeating structure of Gal4 responsive element, a herpes simplex virus thymidine kinase promoter and a DNA encoding an Fas structural protein are located in a tandem from the 5′ region upstream can have the purpose depending on whether its origin is a vector which can function in E. coli or mammal cells. As described above, it may further contain an ori region, a promoter controlling factor and at least one selective marker gene. When mammal cells are used, test cells which die in an Fas protein expression-dependent manner can be obtained by transforming appropriate mammal cells using the above plasmid capable of functioning in mammal cells and culturing the resulting transformant in an appropriate medium.
When the expression is carried out in mammal cells, a PCR product of a nucleotide sequence encoding the signal peptide region of an Fas antigen can be optionally inserted into a site immediately downstream of the promoter.
Examples of the replication or expression vector comprising the DNA of the invention include a plasmid, a virus and a phage vector comprising an ori region, and optionally a promoter for the expression of the above DNA and a controlling factor of the promoter or the like. The vector may further contain at least one selective marker gene (e.g., neomycin resistant gene, blasticidin S resistant gene).
Furthermore, as the protein expression system of the invention, the objective fusion protein can be produced by transforming appropriate mammal cells (e.g., monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, mouse fibroblasts, human cancer cells and the like) with the above expression vector which functions in mammal cells, and culturing the resulting transformant in an appropriate medium. The thus obtained polypeptide can be isolated and purified by generally used biochemical means.
The host cells transformed with a replication or expression vector comprising the DNA of the invention are included in the invention.
The thus obtained plasmid DNA of the invention can be used as a therapeutic agent for a cancers and an autoimmune disease. That is, an expression vector comprising the plasmid DNA of the invention is topically administered to the focal part of a cancer or autoimmune disease by a targeting method (e.g., by its inclusion in liposomes). This vector penetrates into cancer cells of the focal part and reaches genes. Thereafter, when a ligand corresponding to the intracellular receptor which constitutes a part of the effector protein of the invention (for example, 9-cis-retinoic acid when a retinoid X receptor is used as the receptor, or vitamin A when a retinoic acid receptor is used) is administered, the effector is activated and the Fas protein is expressed. The signal of apoptosis is transduced by the Fas protein produced in the cancer cells, thereby the cells become extinct.
Furthermore, the plasmid DNA of the invention can be used broadly as a screening method of an agonist or an antagonist for an intracellular receptor. That is, a sample to be tested is added to cells into which an expression vector comprising the DNA encoding the effector protein (an expression vector DNA comprising a DNA encoding the ligand binding region of various nuclear receptors in a region downstream of the Gal4 DNA binding region) and the reporter DNA for use in the present invention has been inserted. Death of the cells indicates that the sample to be tested has agonist activity, and survival of the cells in the presence of an agonist reveals that the sample has antagonist activity.
According to the screening method of the invention, high speed evaluation of ligands and compounds capable of changing functions of intracellular receptors can be conducted. Since the effects (results) are observed as cell death, the judgment is easy and the handling per se is also simple and easy. In addition, when a DNA encoding the ligand binding region of various nuclear receptors is arranged in a region downstream of the Gal4 DNA binding region as an effector protein, it becomes a method generally used which can screen compounds that act upon nuclear receptors including orphan receptors whose ligands are unknown. As a matter of course, it is possible to search for physiological ligands of orphan nuclear receptors. Specifically, it can be applied to the identification of ligands or their purification from fractions having ligand activities by adding a physiological ligand candidate to be expected or a partially fractionated sample of serum to the above cells and observing the presence or absence of cell death.
Also, since variation of values measured by the method of the invention is small, data between tests can be easy to compare and are very stable. These advantages exert full abilities particularly in the high speed evaluation of multiple samples. In addition, since an absorbance measuring apparatus is attached to any generally and practically used automatic analyzers (robotic machine) as a standard measure, the method can be regarded as a robot-corresponding compound evaluation system. Moreover, since the screening method of the invention does not require enzyme substrate, sharp reduction of cost can be made.
The invention is described further in detail and specifically by the following examples, though the invention is not restricted thereto as a matter of course.
The following primers were prepared based on the structure of the mouse Fas antigen in accordance with the method reported by Fukunaga et al. (see J. Immunology, 148, 1274 (1992)) or Kakizuka et al. (see JP-A-7-316200).
F1: A sense primer, 5′-CCAAGCTTGGCGACCAGCAATACAAACTGCAGGAAAC-3′ (SEQ ID NO:1), corresponding to the structure of an amino acid sequence of the 131st to 143rd positions, namely Pro Cys Thr Ala Thr Ser Asn Thr Asn Cys Arg Lys Gln, in which a restriction enzyme HindIII site was introduced into a part of the 5′ side, was synthesized.
R1: An antisense primer, 5′-TCAGGATCCAGACATTGTCCTTCATTTTCATT-3′ (SEQ ID NO:2), corresponding to an amino acid sequence of the 298th to 306th positions (C-terminal), namely Asn Glu Asn Gln Gly Gln Cys Leu Glu, and a portion of the 3′ non-translation region, in which a BamHI site was introduced into a part of the 3′ side, was synthesized.
Using mRNAs obtained from a mouse T cell line, RLM-11 (Cell, 68, 1109 (1992)), as a template, and F1 and R1 as PCR primers, RT-PCR was carried out using a thermal sequencer (GeneAmp PCR System 9600, Perkin Elmer) under conditions of 180 seconds×1 at 95° C.→(60 seconds at 95° C., 60 seconds at 55° C., 60 seconds at 72° C.)×15→180 seconds at 72° C. As a result, a fragment of about 520 bp (hereinafter referred to as “MFas”) was obtained and inserted into the multi-cloning site, HindIII-BamHI restriction enzyme site, of pBluescript (trade name, Promega), hereinafter referred to as “pBSMFas”.
The following primers were prepared based on the structure of human PPAR α, γ or δ receptor described by R. Mukherjee et al. (see J. Steroid Biochem. Molec. Biol., 51, 157 (1994)); M. E. Greene et al. (see Gene Expression, 4, 281 (1995)); A. Elbrecht et al. (see Biochem. Biophys. Res. Commun., 224, 431 (1996)); or Schmidt (see Mol. Endocrinol., 6, 1134 (1992).
F2: A sense primer, 5′-AACCAGCACCATCTGGTCGCGATGGT-3′ (SEQ ID NO:3), corresponding to the 5′ non-translation region and N-terminal amino acids Met1-Val2 of the human PPAR α, was synthesized.
R2: An antisense primer, 5′-AGGTGTGGCTGATCTGAAGGAACTCA-3′ (SEQ ID NO:4), corresponding to the 3′ non-translation region of the human PPAR α and a amino acid termination codon, was synthesized.
F3: A sense primer, 5′-AGAAATGACCATGGTTGACACAGAGA-3′ (SEQ ID NO:5), corresponding to the 5′ non-translation region and N-terminal amino acids Met1-Met8 of the human PPAR γ (γ1 subtype), was synthesized.
R3: An antisense primer, 5′-AAATGTTGGCAGTGGCTCAGGACTCT-3′ (SEQ ID NO:6), corresponding to the 3′ non-translation region of the human PPAR γ (γ1 subtype), was synthesized.
F4: A sense primer, 5′-AGATCAGCCATGGAGCAGCCACAGGA-3′ (SEQ ID NO:7), corresponding to the 5′ non-translation region and N-terminal amino acids Met1-Glu6 of the human PPAR δ, was synthesized.
R4: An antisense primer, 5′-ATTGGAGTCTGCAGGGAGGCCTGGGT-3′ (SEQ ID NO:8), corresponding to the 3′ non-translation region of the human PPAR δ, was synthesized.
Using Superscript Human Liver cDNA Library (trade name, Cat. No. 10422-012, manufactured by GIBCO BRL) as cDNA library, and F2 and R2, F3 and R3 or F4 and R4 as PCR primers, PCR was carried out using a thermal sequencer (GeneAmp PCR System 9600, manufactured by Perkin Elmer) under conditions of 120 seconds×1 at 95° C.→(60 seconds at 95° C., 90 seconds at 60° C., 120 seconds at 72° C.)×30→180 seconds at 72° C. As a result, the human PPAR α of 1450 bp, the human PPAR γ of 1469 bp and the human PPAR δ of 1367 bp were obtained, and they were inserted into a T-vector (pT7Blue-T vector, Cat. No. 69836-1) manufactured by Novagen to confirm their complete nucleotide sequences.
A vector capable of expressing Gal4 chimeric receptor protein as a structural gene under control of SV40 promoter was constructed using Pica Gene Basic Vector 2 (trade name, PGBV2, Cat. No. 309-04821, manufactured by Toyo Ink) as a basic vector. That is, this was completed by excising a luciferase structural gene of PGBV2 using restriction enzymes NcoI and XbaI and inserting Gal4 chimeric receptor protein-encoding cDNA prepared in the following manner.
(1) Insertion of DNA Encoding DNA Binding Region of Gal4 Transcription Factor Into Expression Vector
As cDNA of the DNA binding region of a yeast basal transcription factor Gal4 protein, a DNA encoding an amino acid sequence of the 1st to 147th positions of Gal4 was amplified by PCR under the same conditions of Example 2 using pGBT9 (manufactured by Clontech, Cat. No. K1605-A) as the template. The following F5 and R5 were used as primers.
F5: A sense primer, 5′-GCAAGCTTCACCATGAAGCTACTGTCTTCTATCGAAC-3′ (SEQ ID NO:9), corresponding to an N-terminal side amino acid sequence of the 1st to 8th positions of the DNA binding region of Gal4, in which a HindIII site and, in order to obtain effective protein expression in mammal cells, a kozak sequence were added to a part of the 5′ side, was synthesized.
R5: An antisense primer, 5′-AGCCATGGCCGGCGATACAGTCAACGGTCTTTG-3′ (SEQ ID NO:10), corresponding to a C-terminal side amino acid sequence of the 141st to 147th positions of the DNA binding region of Gal4, in which an NcoI site was added to a part of the 5′ side, was synthesized, and the thus synthesized primer was amplified by PCR under the same conditions of Example 2. As a result, an amplified DNA of about 465 bp, after confirmation of its complete nucleotide sequence, was recombined with the HindIII/NcoI site in the PGBV2 vector (hereinafter referred to as “pGVgal”).
(2) Preparation of DNA Encoding Human PPAR γ Ligand Binding Region and Construction of Gal4-Human PPAR γ Chimeric Receptor Protein Expression Vector
Using the complete human PPAR γ-encoding cDNA isolated from a human liver cDNA library in accordance with the procedure of Example 2 as a template and synthesizing the following primers, a DNA encoding a sequence of Ser176 to Tyr478 including the human PPAR γ ligand binding region was amplified by PCR under the same conditions of Example 1.
F6: A sense primer, 5′-GCCATGGCTCCTAAGAAGAAGCGTAAGGTAGGATCCCATAATGCCATCAGGTTTGGGCGGAT-3′ (SEQ ID NO:11), corresponding to a sequence of Ser176 to Met185, in which an NcoI site, an SV40 T antigen-originated nuclear transport signal (Ala Pro Lys Lys Lys Arg Lys Val Gly) and a BamHI site were arranged in this order on the 5′ side, was synthesized.
R6: An antisense primer, 5′-CCTCTAGACTAGCTGGCATAGTCGGGCACGTCGTAGGGGTAGTCGACGTACAAGTCCTTGTAGATCTCC-3′ (SEQ ID NO:12), corresponding to a sequence of Glu472 to Tyr478, in which an SalI site, an influenza hemagglutinin epitope (Tyr Pro Tyr Asp Val Pro Asp Tyr Ala) as an epitope tag sequence for use in the detection of expressed protein, a translation termination codon and an XbaI site were arranged in this order, was synthesized.
The luciferase structural gene in the above pGVgal was excised out both from the XbaI site of the region downstream of luciferase structural gene and the NcoI site at the region downstream of the DNA encoding the Gal4 DNA binding region, and replaced by a DNA encoding the human PPAR γ ligand binding region, thereby obtaining the effector protein expression vector of interest (hereinafter referred to as “pGVgal/γLBD”).
(3) Preparation of DNA Encoding Human PPAR α Ligand Binding Region and Construction of Gal4-Human PPAR α Chimeric Receptor Protein Expression Vector
A PPAR α ligand binding region structural gene was synthesized by synthesizing the following primers comprising the human PPAR α ligand binding region and carrying out PCR amplification under the same conditions of Example 2. The thus obtained PPAR α ligand binding region structural gene was inserted into the BamHI-SalI restriction enzyme site of the pGVgal/γLBD vector synthesized above which had been digested with these restriction enzymes to selectively excise the PPAR γ ligand binding region structural gene to construct a Gal4-human PPAR α chimeric receptor protein expression vector (hereinafter referred to as “pGVgal/αLBD”).
F7: A sense primer, 5′-CACGGATCCCACAACGCGATTCGTTTTGGACGA-3′ (SEQ ID NO:13), corresponding to an amino acid sequence of Ser167 to Arg 175 of the human PPAR α and having a BamHI site in its 5′ side, was synthesized.
R7: An antisense primer, 5′-ATGGTCGACGTACATGTCCCTGTAGATCTCCTG-3′ (SEQ ID NO:14), corresponding to an amino acid sequence of Gin461 to Tyr468 of the human PPAR α and having an SalI site in its 5′ side, was synthesized.
(4) Preparation of DNA Encoding Human PPAR δ Ligand Binding Region and Construction of Gal4-Human PPAR δ Chimeric Receptor Protein Expression Vector
A PPAR δ ligand binding region structural gene was synthesized by synthesizing the following primers comprising the human PPAR δ ligand binding region and carrying out PCR amplification under the same conditions of Example 2. The thus obtained PPAR δ ligand binding region structural gene was inserted into the BamHI-SalI restriction enzyme site of the pGVgal/γLBD vector synthesized above which had been digested with these restriction enzymes to selectively excise the PPAR γ ligand binding region structural gene to construct the Gal4-human PPAR δ chimeric receptor protein expression vector (hereinafter referred to as “pGVgal/δLBD”).
F8: A sense primer, 5′-CACGGATCCCACAACGCTATCCGTTTTGGTCG-3′ (SEQ ID NO:15), corresponding to an amino acid sequence of Ser139 to Arg7 of the human PPAR δ and having a BamHI site in its 5′ side, was synthesized.
R8: An antisense primer, 5′-ATGGTCGACGTACATGTCCTTGTAGATCTCCTG-3′ (SEQ ID NO:16), corresponding to an amino acid sequence of Gln434 to Tyr441 of the human PPAR δ and having an SalI site in its 5′ side, was synthesized.
An expression vector pTKβ (manufactured by Clontech, Cat. No. 6179-1) under control of herpes simplex virus thymidine kinase (TK) promoter was used. NotI sites upstream and downstream of the β-galactosidase structural gene of pTKβ were digested, and both termini are smooth-ended. Next, a DNA encoding the MFas protein, as a DNA to be inserted, was excised from pBSMFas by using restriction enzymes HindIII and BamHI and blunt-ended. Both of the DNAs were ligated to select a clone in which the MFas structural gene was connected to the region downstream of TK promoter in the forward direction. The thus obtained plasmid pTK-MFas of about 4400 bp contains a DNA encoding MFas protein under control of the TK promoter. Next, the SalI site located in the region upstream of the TK promoter of plasmid pTK-MFas was digested and blunt-ended, and then a multi-cloning site was inserted as a linker DNA (SEQ ID NO:17, EcoRI-SalI-KpnI-EcoRV-SacI NotI):
to obtain pTK-MFas-ML1 (5′-EcoRI-SalI-KpnI-EcoRV-SacI-NotI-3′).
An enhancer region was produced by inserting a Gal4 responsive element, 5′-T(CGACGGAGTACTGTCCCCCG)×4 AGCT-3′ (SEQ ID NO:18), having 4 repetition of a basal unit (UAS) and SalI site/SacI site on both ends, into the SalI-SacI site inside the pTK-MFas-ML1 multi-cloning site. As a result, p4×UAS-TK-MFas (about 4600 bp) in which the 4×UAS, TK promoter and MFas structural gene were arranged in this order was constructed.
Each of the thus obtained expression vector pSVgal/αLBD, pSVgal/γLBD and pSVgal/δLBD, the reporter plasmid and pSV2bsr (manufactured by Kaken Pharmaceutical, Cat. No. KK-500) were subjected to linearization by the treatment with a restriction enzyme AatII or NotI or not treated (circular form), respectively, and introduced at a quantitative ratio of 1:1:0.1 into mouse fibroblast L929 using Lipofect AMINE (manufactured by GIBCO, Cat. No. 18324-012). Stably transformed cells were selected in accordance with the method (blasticidin S selection) described in Exp. Cell Res., 197, 229 (1991).
After 1 to 2 weeks of selection, observed colonies were subjected to cell cloning by limiting dilution and grown. For example, each subclone was suspended in Eagle's MEM containing 10% fetal calf serum (FCS) in a density of 2×104 cells/100 μl, dispensed into wells of 96 well microtiter plate and cultured at 37° C. for 4 to 6 hours in an atmosphere of 5% carbon dioxide. Cells which die significantly in the presence of a ligand were selected using CS-045 and BRL-49653 known as PPAR γ ligands (see Cell, 83, 863 (1995), Endocrinology, 137, 4189 (1996) and J. Med. Chem., 39, 665 (1996)), carbacyclin and ETYA known as PPAR α ligands (cf. J. Steroid Biochem. Molec. Biol., 51, 157 (1994), Cell Structure and Function, 18, 267 (1993), Gene & Development, 10, 974 (1996) and Eur. J. Biochem., 233, 242 (1996)), carbacyclin known as PPAR δ ligand (see Gene & Development, 10, 974 (1996)) and a compound disclosed by Merck (see the compound of Example 7 in PCT International Application International Publication No. 9728149). That is, each sample of these compounds having two times higher concentration than their final concentration 10 μM was added to each of the above culture wells, and the cells were again cultured for 40 hours. After discarding the supernatant, the resulting plate was soaked in a Crystal Violet solution (0.75% Crystal Violet-0.25% NaCl-1.75% formaldehyde in 50% ethanol) at room temperature for 20 minutes and then washed twice with phosphate buffered saline (PBS), thereby effecting staining of survived cells (see Cell, 66, 233 (1991)). Absorbance of each well of the plate was directly measured at 570 nm using a microplate reader.
Each of the clones obtained in Example 5 having the highest responsive ability was cultured by the above method, cell death of the thus cultured cells was induced by the above compounds by the above method and then the survived cells were stained to measure the absorbance. The cell death appeared significantly after 24 hours and was completed during 36 to 40 hours by reaching to the plateau stage.
Dose-dependent changes in the number of intact cells after 40 hours of treatment of the PPAR γ ligand-responsive clone with CS-045 and BRL-49653 are shown in
Also, dose-dependent changes in the number of intact cells after 40 hours of treatment of the PPAR α ligand-responsive clone with carbacyclin, ETYA, Wy-14634 and CS-045 are shown in
Also, dose-dependent changes in the number of intact cells after 40 hours of treatment of the PPAR α ligand-responsive clone with carbacyclin, the compound of Merck, ETYA and CS-045 are shown in
Also, the ratio of the number of intact cells when each compound was replaced by a solvent was expressed as 100% in each drawing.
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Based on the results shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| P.HEI 9-171440 | Jun 1997 | JP | national |
