JP4456887B2 - Method for determining drug sensitivity of cells - Google Patents

Description

  The present invention relates to a gene polymorphism of MDR1, which is a gene of P-glycoprotein, which is a protein that transports drugs such as anticancer drugs to the outside of cells, and a method for determining drug sensitivity of normal cells and cancer cells by detecting the polymorphism About.

  Anthracyclines such as doxorubicin, daunorubicin, and epirubicin; Vinca alkaloids such as vincristine; Taxanes such as paclitaxel and docetaxel; Camptothecins such as irinotecan hydrochloride; It is widely used because it is expensive, but it is known to cause side effects such as bone marrow suppression and diarrhea. In addition, it is known that some patients may not be effective even in cancers for which these anticancer agents are considered effective.

  The mechanism of acquiring resistance to these anticancer drugs in cancer cells has been well studied, and it is known that P-glycoprotein, one of the ABC transporters, is involved in the resistance of these anticancer drugs. (Non-Patent Document 1). In other words, it was found that in cancers that express P-glycoprotein, P-glycoprotein has the effect of decreasing the intracellular accumulation of anticancer agents by excreting the anticancer agents out of the cells. The gene encoding this P-glycoprotein is the MDR1 gene.

  P-glycoprotein is expressed in adrenal gland, large intestine, small intestine, kidney, vascular endothelial cells, hematopoietic progenitor cells and the like in normal tissues. Hematopoietic tissue and small intestinal epithelial tissue are tissues that are susceptible to toxicity as a side effect of anticancer agents, and the side effects appear as symptoms such as bone marrow suppression and severe diarrhea. In particular, such side effects are a major problem in cancer chemotherapy using anticancer agents. Since P-glycoprotein transports various anticancer drugs to the outside of the cell, if the expression of P-glycoprotein in normal tissues of cancer patients is high, the side effects of anticancer drugs are less likely to occur. Strong side effects are expected when expression is low.

From this point of view, it is considered that side effects and drug resistance of drugs such as anticancer drugs can be controlled by controlling the expression of the MDR1 gene.
Methods in Enzymology, 292: 248-594 (1998)

  An object of the present invention is to elucidate the mechanism of regulation of P-glycoprotein expression in vivo, and to determine the sensitivity to drugs and the expression of side effects.

  As a result of various studies, the present inventor has found that there are individual differences in the expression of P-glycoprotein in normal tissues. Therefore, we focused on single nucleotide polymorphisms that regulate such individual differences. That is, one or several differences in the nucleotide sequence on the MDR1 gene, which is a gene for P-glycoprotein, increased or decreased MDR1 mRNA expression, increased or decreased P-glycoprotein expression, P-glycoprotein Thought to appear as an increase, decrease or loss of function. As a result of examining the structure of the MDR1 gene in human peripheral leukocytes, the present inventor has found that the MDR1 gene has a gene polymorphism, and the polymorphism greatly reduces the ability to acquire drug resistance by P-glycoprotein. As a result, the present invention has been completed.

That is, the present invention provides a method for determining the sensitivity of a test cell to a drug, characterized by identifying the MDR1 gene polymorphism of the test cell.
The present invention also provides a method for determining the drug resistance of a test cell or the occurrence of side effects upon drug administration, characterized by identifying a polymorphism of MDR1 in the test cell.
The present invention also provides a polymorphic polypeptide of P-glycoprotein having the amino acid sequence represented by SEQ ID NO: 1.
The present invention also provides a partial peptide of the MDR1 gene polymorphic polypeptide having the amino acid sequence represented by SEQ ID NO: 1, wherein the MDR1 gene polymorphism is MDR1 (C3583T).
Furthermore, the present invention provides an MDR1 gene polymorphism having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.
Furthermore, the present invention relates to a partial polynucleotide or partial oligonucleotide of MDR1 having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1, wherein the MDR1 gene polymorphism is MDR1 (C3583T), and the portion A partial polynucleotide or partial oligonucleotide complementary to the polynucleotide or partial oligonucleotide is provided.

  If the gene polymorphism of the present invention is identified, it is possible to predict the degree of side effects when a drug such as an anticancer drug is administered to a subject having the test cell, and the degree of drug resistance can also be predicted. It is possible and effective in formulating safer drug treatment guidelines.

  The gene polymorphism of the present invention is one in which one of the bases constituting the wild type MDR1 gene is substituted with another base, for example, a P-glycoprotein (H1195Y) having the amino acid sequence represented by SEQ ID NO: 1 [The wild-type P-glycoprotein has an amino acid sequence in which the 1195th histidine (H) is mutated to tyrosine (Y)]. The base sequence of MDR1 (C3583T) is represented by SEQ ID NO: 2, for example.

  The gene polymorphism of the present invention can be cloned from the cDNA obtained by performing RT-PCR that amplifies the full length of the wild type MDR1 gene using, for example, total RNA derived from human peripheral white blood cells. That the obtained mutation is not a PCR error but a gene polymorphism may be confirmed by amplifying the DNA of the cell from which the polymorphism was obtained and confirming the nucleotide sequence.

  When the gene polymorphism of the present invention thus obtained is expressed using the same promoter of wild-type MDR1, the amount of P-glycoprotein produced decreases because the amount of mRNA to be expressed decreases or The resulting P-glycoprotein is not expressed on the cell surface or the function of the produced P-glycoprotein is reduced, resulting in an increase in cell sensitivity to the drug.

  In the present invention, sensitivity to a drug such as an anticancer drug is determined by identifying a polymorphism of MDR1 using test cells, for example, leukocytes in peripheral blood. By grasping this sensitivity in normal cells, it becomes possible to determine (predict) the degree of occurrence of side effects upon administration of drugs such as anticancer drugs, and as a result, safe drug administration can be achieved. In addition, when cancer cells (tissue) excised from a patient or a biopsy specimen are used as test cells, the sensitivity can be similarly determined, and as a result, drug resistance such as anticancer drug resistance of the cancer cells (tissue) can be determined.

  In the present invention, when a specific gene polymorphism MDR1 (C3583T) is expressed using the same promoter of wild-type MDR1, the amount of P-glycoprotein expressed on the cell surface is very small. It has been found. Cancer patients with this gene polymorphism have low expression levels of cell surface P-glycoprotein in normal tissues of adrenal gland, large intestine, small intestine, kidney, vascular endothelial cells, and hematopoietic progenitor cells, and therefore patients with the gene polymorphism When the anticancer agent is administered to the cerebral organ, it is expected that the normal tissue is greatly damaged, that is, the side effect of the anticancer agent is strong. In addition, if the test cell has this gene polymorphism, the expression level of the P-glycoprotein on the cell surface is small, and thus it is considered that the test cell is highly sensitive to anticancer agents. Therefore, the identification of the MDR1 gene polymorphism of the present invention is effective for safer anticancer drug treatment such as setting of an anticancer drug dosage and administration method.

  The gene polymorphism MDR1 (C3583T) of the present invention has a lower expression level of P-glycoprotein on the cell surface than the wild type, and the expressed P-glycoprotein does not function sufficiently as an anticancer drug efflux pump. The cells are highly sensitive to anticancer agents, and are particularly useful for determining the degree of occurrence of side effects upon administration of the above anticancer agents and for determining resistance to anticancer agents. In addition, since the degree of occurrence of side effects varies depending on whether the gene polymorphism is homozygous or heterozygous, this point can be used as an index for determination.

  In order to detect or measure the polymorphism of the present invention, for example, PCR and invader assay using primers specific to MDR1 (C3583T), the region containing codon 1195 is amplified by PCR, and the nucleotide sequence is directly determined. Direct sequencing method, PCR-SSCP method, which amplifies the region containing codon 1195 by PCR and electrophoresis to examine whether fragments with different base sequences are amplified, P-glycoprotein (H1195Y) specific antibody It can be carried out according to the immunoassay used.

  Here, as a primer specific for MDR1 (C3583T), one of the two primers is a primer containing a sequence complementary to TAT at codon 1195, and the other is a primer for the other part of MDR1. A primer containing the sequence may be mentioned. More specifically, as a primer containing a sequence complementary to TAT at codon 1195, 5′-ATC CAA AAG CAA AAT ATA AG -3 ′ (SEQ ID NO: 3), 5 as a primer containing the sequence of the other part of MDR1 '-GTG GGG CAA GTC AGT TCA TT -3' (SEQ ID NO: 4) can be mentioned.

  Using the above two primers, PCR is performed using cellular genomic DNA as a template. Specifically, after 5 minutes of reaction at 94 ° C, the reaction was repeated 30 times at 94 ° C for 30 seconds, 52 ° C for 30 seconds, 72 ° C for 1 minute, and finally at 94 ° C for 30 seconds, 72 ° C. An amplification product of 638 base pairs can be obtained by performing a single reaction at 15 ° C. for 15 minutes.

In the present invention, the anticancer agent to be subjected to the determination of the degree of side effects and the drug resistance determination is not limited as long as it is an anticancer agent that produces resistance by P-glycoprotein. For example, anthracyclines such as doxorubicin hydrochloride, daunomycin, epirubicin hydrochloride, vincristine And vinca alkaloids, taxanes such as paclitaxel and docetaxel, camptothecins such as irinotecan hydrochloride and topotecin, etoposide, mitomycin and the like, and mitoxantrone.
Other than anticancer drugs, macrolide antibiotics such as erythromycin and clarithromycin, new quinolone antibiotics such as levofloxacin, anti-HIV drugs such as indinavir sulfate and saquinavir, deep and superficial antifungal drugs such as itraconazole, ketoconazole Superficial antifungal drugs such as Ibermectin, nematode treatment drugs such as ivermectin, antimalarial drugs such as mefloquine hydrochloride, immunosuppressive drugs such as cyclosporine and tacrolimus hydrate, phenothiazine antihistamines such as promethazine hydrochloride, spironolactone, etc. K-retaining diuretics, peripheral sympathetic nerve suppressors such as ruserpine, benzothiazepine antihypertensives such as diltiazem hydrochloride, Ca antagonists such as verabamil hydrochloride, Na channel blockers such as quinidine sulfate and propafenone hydrochloride, hydrochloric acid Beta blockers such as amiodarone, digitalis such as digoxin Remedies for heart failure, antihypertensives, anti-dopamines such as domperidone, antipruritics such as loperamide hydrochloride, phenothiazine antipsychotics such as chlorpromazine hydrochloride, antipsychotics such as pimozide, and 5-HT ₃ receptors such as ondansetron hydrochloride Examples include body antagonist antiemetics.

  In addition, the target cancer for determining the degree of side effects and anticancer drug resistance determination at the time of administration of the anticancer drug of the present invention is not particularly limited as long as it is a cancer to which the anticancer drug is applied. Examples of test normal cells include cells derived from normal tissues in which the P-glycoprotein is expressed, peripheral blood cells, and the like.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not restrict | limited at all by this.

Example 1
(1) MDR1 gene
P-glycoprotein was first identified as an ABC transporter involved in anticancer drug resistance. The human full-length cDNA sequence of the MDR1 gene, a gene for P-glycoprotein, has already been reported by a research group in the United States.
The gene named MDR1 is registered in GenBank accession number M14758, Chen, C., J., et al., Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug- resistant human cells. Cell 47: 381-389 (1986).
However, the sequence of this MDR1 cDNA was obtained from colchicine-resistant cancer cells treated with the mutation agent ethylmethane sulfonate. Compared to the most common type of MDR1 gene (so-called wild type) in Japanese, C540T, G554T, A555T , There is a difference of T1236C. In this case, the base sequence of the wild type MDR1 gene is described on the left side of the number. Among these differences in the base sequence, C540T is a gene polymorphism that is in a codon encoding serine 180 and does not cause an amino acid change due to the polymorphism. T1236C is a gene polymorphism that is in the codon encoding the 412th glycine and does not cause an amino acid change due to the polymorphism. In G554T and A555T, the CCA encoding the 185th glycine of the wild-type MDR1 gene becomes GTT and is mutated to valine. This is a mutation that occurred after the cancer cells were treated with a mutagen, and is considered an artificial mutation.

  In the present invention, a gene called human wild-type MDR1 cDNA is isolated from a human adrenal cDNA library. Kioka, N., et al. P-glycoprotein gene (MDR1) cDNA from human adrenal: Normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance. Yes.

(1) Extraction of DNA DNA was extracted from human peripheral blood cells using QIAGEN RNA / DNA Maxi kit (Qiagen). The extraction procedure followed the protocol attached to Kit. In this way, approximately 100-200 μg of DNA was obtained from 14 mL of peripheral blood.

(2) Amplification of MDR1 gene exon27 by PCR
In order to determine the base sequence of each exon of MDR1 gene, DNA was amplified using human peripheral blood DNA as a template. For exon27 amplification of the MDR1 gene, 5'-CTT TAC TTT CAG TTC TAC TTT CA -3 '(SEQ ID NO: 5) and 5'-GAG AAT ACA GCA TTT TTA AGG A -3' (SEQ ID NO: 6) Using two primers, and using Applied Biosystems AmpliTaq Gold, the reaction was carried out once at 95 ° C for 9 minutes, at 5 ° C for 30 seconds, and at 72 ° C for 1 minute, then at 95 ° C for 30 seconds, 55 The reaction at 30 ° C. for 30 seconds and at 72 ° C. for 1 minute was repeated 30 times. Finally, the reaction at 72 ° C. for 15 minutes was performed once to obtain 256 bp of amplified DNA.

(3) Sequence Using the DNA amplified by the above PCR as a template, sequence using 5'-CAG TTC TAC TTT CAT AAC AAC AA-3 '(SEQ ID NO: 7) primer and BigDye Terminator Cycle Sequence Kit from Applied BioSystems. Reaction was performed. The nucleotide sequence was determined using an ABI PRISM377 DNA sequencer from Applied Biosystems. As a result, a gene polymorphism of C3583T of the MDR1 gene was identified (SEQ ID NO: 2). This polymorphism revealed that histidine (CAT) at codon 1195 was changed to tyrosine (TAT) (SEQ ID NO: 1).

(4) Preparation of a vector expressing MDR1 (C3583T) Using wild-type MDR1 cDNA as a template and using primer 5'-AGA CAG CCT TAT ATT TTG CTT-3 '(SEQ ID NO: 8) for Site Directed Mutagenesis MDR1 (C3583T) was prepared using Mutan-Super Express Km (Takara Shuzo) as a kit according to the attached protocol. The base sequence of the amplified cDNA was determined, and it was confirmed that the gene polymorphism was introduced and that there was no mutation caused by PCR. These cDNAs were inserted into pHaL-IRES-DHFR, which is a bicistronic plasmid vector, using T4 DNA ligase using two types of restriction enzymes, Sst II and Xho I. The prepared plasmid was named pHa-MDR (C3583T) -IRES-DHFR. The vector into which the wild type MDR1 cDNA has been introduced is pHa-MDR-IRES-DHFR.

(5) Presence or absence of appearance of vincristine-resistant colonies due to introduction of MDR1 (C3583T) pHa-MDR-IRES-DHFR or pHa-MDR (C3583T) -IRES using the calcium phosphate method on PA317 cells which are amphotropic retrovirus packaging cell lines in mice -Introduced DHFR. When cells after gene transfer were selected with 30 ng / mL vincristine, more than 100 vincristine-resistant colonies were obtained per dish in cells transfected with pHa-MDR-IRES-DHFR, but pHa-MDR In the dish of the cells into which (C3583T) -IRES-DHFR was introduced, all the cells were killed and no vincristine resistant colonies were obtained. This indicates that cells introduced with MDR1 (C3583T) did not acquire high resistance to vincristine.

(6) Preparation of MDR1 (C3583T) -introduced cells In the same manner as described above, PA317 cells, which are mouse amphotropic retrovirus packaging cell lines, were added to pHa-MDR-IRES-DHFR or pHa-MDR (C3583T) -IRES- using the calcium phosphate method. DHFR was introduced. This time, the cells after gene transfer were selected with 120 ng / mL methotrexate. In both the dish of cells introduced with pHa-MDR-IRES-DHFR and the dish of cells introduced with pHa-MDR (C3583T) -IRES-DHFR, 100 or more methotrexate resistant colonies were obtained per dish. The cells were named PA317 / MDR and PA317 / MDR (C3583T).

(7) Cell proliferation test
The sensitivity of PA317 cells, PA317 / MDR cells, and PA317 / MDR (C3583T) cells to vincristine was examined by a cell growth inhibition test. These cells were seeded in a 12-well plate (Iwaki) at 2500 cells / mL / well, and then 1 mL of drug diluted to each concentration with medium was added per well. This plate was cultured in a 5% carbon dioxide incubator at 37 ° C. for 5 days. Five days later, after washing the cells with phosphate buffer buffer, peel off the cells with 0.5 mL trypsin-EDTA, suspend in 1 mL of medium, and put the cell solution in each well into a beaker containing 9 mL of cell pack diluent (Toho Medical Electronics). Were added, and the number of cells was counted with a Sysmex CDA-500 automatic cell counting device (Toago Medical Electronics). As a result, the vincristine concentration (IC50) that reduces the cell number to 50% was determined. The IC50 for vincristine of the parent strain PA317 cells was 2.09 ng / mL, whereas the IC50 for vincristine of PA317 / MDR cells into which wild-type MDR1 cDNA was introduced was 16.61 ng / mL, which was about 8 times more resistant to vincristine. It was. In contrast, the IC50 for vincristine of PA317 / MDR (C3583T) cells was 4.46 ng / mL, indicating only about twice the resistance to vincristine. From the above, PA317 / MDR (C3583T) cells introduced with MDR (C3583T) showed clearly lower resistance than PA317 / MDR cells introduced with wild-type MDR1 cDNA.

(8) Expression of cell surface P-glycoprotein The expression of cell surface P-glycoprotein was examined by flow cytometry using MRK16, an anti-human P-glycoprotein antibody. Cell suspensions of PA317 cells, PA317 / MDR cells, and PA317 / MDR (C3583T) cells were prepared and suspended in Hanks (Nichirei) solution supplemented with 1% bovine serum albumin and 0.1% sodium azide. To each of approximately 500,000 cells, add the F (ab ') ₂ fragment of MRK16 antibody first labeled with biotin so that the final concentration is 0.1 mg / mL. The reaction antibody was washed away with the Hanks solution. Next, phycoerythrin-labeled streptavidin (Nippon Becton) was added and reacted on an ice bath for 30 minutes, and then unreacted phycoerythrin-labeled streptavidin was washed away with the Hanks solution. The fluorescence of the cells was measured with a FACS Calibur manufactured by Nippon Becton.
The results are shown in FIGS. In FIGS. 1 to 3, the thick solid line indicates the case where MRK16 is added, and the thin dotted line indicates the case where MRK16 is not added. In PA317 cells, the thick solid line and the thin dotted line almost coincide with each other, indicating that no cells responded to MRK16, which is an anti-human P-glycoprotein antibody. In PA317 / MDR cells, when the thick solid line with MRK16 added was seen, most of the cells moved to the right side, indicating that the fluorescence intensity was high, that is, it strongly reacted to MRK16, an anti-human P-glycoprotein antibody. It became clear. On the other hand, PA317 / MDR (C3583T) cells showed weak reactivity to MRK16, an anti-human P-glycoprotein antibody, and most of the cells had lower fluorescence intensity than PA317 / MDR cells. That is, it was shown that the H1195Y-type P-glycoprotein encoded by the MDR1 (C3583T) gene has a low ability to make cells resistant to anticancer drugs because of its low expression on the cell surface.

The result of having investigated the expression of P-glycoprotein on the surface of PA317 cell by flow cytometry using an anti-human P-glycoprotein antibody is shown. The result of having investigated the expression of P-glycoprotein on the surface of PA317 / MDR cell by flow cytometry using the anti-human P-glycoprotein antibody is shown. The result of having investigated the expression of P-glycoprotein on the surface of PA317 / MDR (C3583T) cell by the flow cytometry using the anti-human P-glycoprotein antibody is shown.

Claims (8)

  A method for determining the sensitivity of a test cell to a drug, comprising identifying a mutation MDR1 (C3583T) in the MDR1 gene of the test cell.   A method for determining the degree of occurrence of a side effect upon administration of a drug in a test cell, characterized by identifying a mutation MDR1 (C3583T) in the MDR1 gene of the test cell.   A method for determining drug resistance of a test cell, comprising identifying a mutation MDR1 (C3583T) in the MDR1 gene of the test cell.   The method according to claim 1, wherein the drug is an anticancer drug.   A gene-mutated polypeptide of P-glycoprotein having the amino acid sequence represented by SEQ ID NO: 1.   A mutant gene of MDR1 having the base sequence represented by SEQ ID NO: 2.   A partial polynucleotide or partial oligonucleotide comprising a polymorphic (C3583T) site among the mutant MDR1 having the base sequence represented by SEQ ID NO: 2, wherein the MDR1 gene mutation is MDR1 (C3583T).   Among the mutant MDR1 having the base sequence represented by SEQ ID NO: 2, characterized in that the mutation of the MDR1 gene is MDR1 (C3583T), the partial polynucleotide or partial oligonucleotide containing the polymorphic (C3583T) site Complementary partial polynucleotide or partial oligonucleotide.

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