Patent Application
20100168209
The present invention relates to apoptosis inducers for cancer cells, which comprise as an active ingredient a compound that suppresses the expression of a gene belonging to the RecQ DNA helicase family, or a compound that suppresses the function of the protein encoded by that gene, and screening methods to select candidate compounds for the apoptosis inducers.
Genes belonging to the RecQ DNA helicase family are widely present in organisms ranging from prokaryotes such as Escherichia coli (E. coli) to higher eukaryotes including humans. Conserved in the evolution process, these genes diversified along with the multicellularization of organisms. The E. coli RecQ gene was the first of the RecQ family genes to be discovered. This gene was identified as a gene participating in zygotic recombination and in the RecF pathway for UV damage repair (see Non-Patent Document 1). E. coli RecQ gene has been revealed to have the function of suppressing incorrect recombinations (see Non-Patent Document 2). The budding yeast SGS1 gene and the fission yeast Rqh1 gene are the only known RecQ homologues in these yeasts. Both of these genes mainly suppress recombination and play important roles in genome stabilization (see Non-Patent Documents 3 and 4). Higher eukaryotes carry a plurality of RecQ homologues. In humans, there are five types of genes known to belong to the RecQ family: the RecQL1 (see Non-Patent Document 6), BLM, WRN, RTS, and RecQL5 genes. Of these five, the RTS gene (see Non-Patent Document 5, and Patent Documents 1 and 2) and the RecQL5 gene (see Non-Patent Document 5, and Patent Document 3) were identified by the present inventors. BLM, WRN, and RTS genes respectively cause Bloom's syndrome (see Non-Patent Document 7), Werner's syndrome (see Non-Patent Document 8), and Rothmund-Thomson syndrome (see Non-Patent Document 9). These genes all play important roles in genome stabilization in cells.
In fibroblast cells and lymphocytic cell lines derived from patients with Werner's syndrome, chromosomal translocation and deletion, which are indexes for genome instability, have been reported to occur with a high frequency (see Non-Patent Document 10). Chromosomal breakage and sister chromatid exchange (SCE) are frequently detected in cells derived from patients with Bloom's syndrome (see Non-Patent Document 11). Trisomies of human chromosome 2 and 8 are frequently found in lymphocytes derived from patients with Rothmund-Thomson syndrome (see Non-Patent Document 12). These findings suggest that WRN helicase, BLM helicase, and RTS helicase encoded by the various causative genes of these three genetic diseases play important roles in genome stabilization in cells.
Telomere length abnormalities are seen in lymphocytic cell lines derived from patients with Werner's syndrome compared to cell lines derived from normal healthy subjects (see Non-Patent Document 13). In addition, cell immortalization was not observed in lymphocytic cell lines derived from patients with Werner's syndrome, although about 15% of cell lines derived from normal healthy subjects were immortalized after passaging (see Non-Patent Document 14). This finding indicates that WRN helicase contributes to telomere structure maintenance, and is thus essential for the immortalization (canceration) of lymphocytic cell lines.
It has been suggested that WRN helicase is associated with homologous recombination-mediated repair, because the helicase forms foci in the nucleus in response to DNA-damaging agents, and these foci are co-localized with the single-stranded DNA-binding protein RPA (which is a WRN-binding protein) and with the recombination repair factor RAD51 (see Non-Patent Document 15). In addition, WRN helicase has been known to bind to the DNA-dependent protein kinase complex (DNA-PK) and to flap endonuclease 1 (FEN-1). By binding to DNA-PK, WRN helicase plays an important role in the processing of terminals generated by DNA double strand breaks, which are repaired in the pathway of non-homologous end joining (see Non-Patent Document 16). WRN helicase is believed to activate FEN-1 by binding to it, and to provide a site for precise reconstruction of the replication fork through homologous recombination by processing Okazaki fragments (see Non-Patent Document 17). The above findings suggest that WRN helicase plays an important role in DNA repair during DNA replication.
BLM helicase is localized in the PML body, a specific structure found in the nucleus, and it binds to topoisomerase III (see Non-Patent Document 18). The helicase has the unwinding activity of the G-quadruplex structure, and thus is considered to contribute to telomere maintenance (see Non-Patent Document 19). Furthermore, the helicase has been reported to unwind the Holliday junction and to interact with the Rad51 protein (see Non-Patent Document 20). These findings suggest that BLM helicase cooperates with other DNA-metabolizing enzymes and plays important roles in recombinational DNA repair and telomere maintenance.
Of the five human proteins belonging to the RecQ DNA helicase family (RecQ1, BLM, WRN, RTS, and RecQ5), RecQ1, BLM, WRN, and RTS are expressed at negligible levels in resting cells, whereas they are expressed at high levels in cells whose proliferation has been enhanced by transformation with viruses (see Non-Patent Document 21). Furthermore, when the carcinogenic promoter TPA is added to resting cells, the expression of RecQ1, BLM, WRN, and RTS is induced along with the induction of cell division (see Non-Patent Document 21). These findings suggest the importance of the RecQ DNA helicase family in cell proliferation.
Taken collectively, these findings suggest that the RecQ DNA helicase family members may be potential target molecules for anti-cancer therapy because the family members participate in genomic repair in cells (BLM, WRN and RTS) and also the maintenance of telomere structure (BLM and WRN), play important roles in the immortalization of certain cells (WRN), and their expression is induced following cell division (RecQ1, BLM, WRN and RTS).
In addition, the expression levels of genes in the RecQ DNA helicase family are markedly higher in tumor cells. Thus compounds that suppress tumor growth can be screened using the suppression of expression of RecQ DNA helicase family genes as an index (see Patent Document 4). It has also been suggested that compounds suppressing RecQ helicase gene expression may suppress cancer cell growth (see Patent Document 4).
However, no one has reported the correlation between the suppression of RecQ
DNA helicase family gene expression and the cancer cell-specific induction of apoptosis, and there have been no findings that suggest such a correlation.
[Patent document 1] Japanese Patent Application No. Hei 9-200387
[Patent document 2] Japanese Patent Application No. Hei 11-11218
[Patent document 3] Japanese Patent Application No. Hei 10-81492
[Patent document 4] Japanese Patent Application Kokai Publication No. (JP-A) 2000-166600 (unexamined, published Japanese patent application)
[Non-Patent Document 1] Nakayama, H., Nakayama, K., Nakayama, R., Irino, N., Nakayama, Y., and Hanawalt, P. C., “Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway.”, Mol. Gen. Genet., 1984, Vol. 195, pp. 474-480.
[Non-Patent Document 2] Hanada, K., Ukita, T., Kohno, Y., Saito, K., Kato, J., and Ikeda, H., “RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli.”, Proc. Natl. Acad. Sci. USA., 1997, Vol. 94, pp. 3860-3865
[Non-Patent Document 3] Myung, K., Datta, A., Chen, C., and Kolodner, R. D., “SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination.”, Nat. Genet., 2001, Vol. 27, pp. 113-116
[Non-Patent Document 4] Doe, C. L., Dixon, J., Osman, F., and Whitby, M. C., “Partial suppression of the fission yeast rqh1(−) phenotype by expression of a bacterial Holliday junction resolvase.”, EMBO J., 2000, Vol. 19, pp. 2751-2762
[Non-Patent Document 5] Kitao, S., Ohsugi, I., Ichikawa, K., Goto, M., Furuichi, Y., and Shimamoto, A., “Cloning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes.”, Genomics., 1998, Vol. 54, pp. 443-452
[Non-Patent Document 6] Seki, M., Miyazawa, H., Tada, S., Yanagisawa, J., Yamaoka, T., Hoshino, S., Ozawa, K., Eki, T., Nogami, M., Okumura, K. et al., “Molecular cloning of cDNA encoding human DNA helicase Q1 which has homology to Escherichia coli Rec Q helicase and localization of the gene at chromosome 12p12.”, Nucleic Acids Res., 1994, Vol. 22, No. 22, pp. 4566-4573
[Non-Patent Document 7] Ellis, N. A., Groden, J., Ye, T. Z., Straughen, J., Lennon, D. J., Ciocci, S., Proytcheva, M., and German, J., “The Bloom's syndrome gene product is homologous to RecQ helicases.”, Cell, 1995, Vol. 83, pp. 655-666
[Non-Patent Document 8] Yu, C. E., Oshima, J., Fu, Y. H., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, S., Nakura, J., Mild, T., Ouais, S., Martin, G. M., Mulligan, J., and Schellenberg, G. D., “Positional cloning of the Werner's syndrome gene.”, Science, 1996, Vol. 272, pp. 258-262
[Non-Patent Document 9] Kitao, S., Shimamoto, A., Goto, M., Miller, R. W., Smithson, W. A., Lindor, N. M., and Furuichi, Y., “Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome.”, Nat. Genet., 1999, Vol. 22, pp. 82-84 [Non-Patent Document 10] Goto, M., “Hierarchical deterioration of body systems in Werner's syndrome: implications for normal ageing.”, Mech. Ageing Dev., 1997, Vol. 98, pp. 239-254
[Non-Patent Document 11] Ellis, N. A. and German, J., “Molecular genetics of Bloom's syndrome.”, Hum. Mol. Genet., 1996, Vol. 5, pp. 1457-1463
[Non-Patent Document 12] Lindor, N. M., Devries, E. M., Michels, V. V., Schad, C. R., Jalal, S. M., Donovan, K. M., Smithson, W. A., Kvols, L. K., Thibodeau, S, N., and Dewald, G. W., “Rothmund-Thomson syndrome in siblings: evidence for acquired in vivo mosaicism.”, Clin. Genet., 1996, Vol. 49, pp. 124-129
[Non-Patent Document 13] Tahara, H., Tokutake, Y., Maeda, S., Kataoka, H., Watanabe, T., Satoh, M., Matsumoto, T., Sugawara, M., Ide, T., Goto, M., Furuichi, Y., and Sugimoto, M., “Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner's syndrome patients transformed by Epstein-Barr virus.”, Oncogene, 1997, Vol. 15, pp. 1911-1920
[Non-Patent Document 14] Sugimoto, M., Furuichi, Y., Ide, T., and Goto, M., “Incorrect us of “immortalization” for B-lymphoblastoid cell lines transformed by Epstein-Barr virus.”, Virol., 1999, Vol. 73, pp. 9690-9691
[Non-Patent Document 15] Sakamoto, S., Nishikawa, K., Heo, S. J., Goto, M., Furuichi, Y., and Shimamoto, A., “Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51.”, Genes Cells., 2001, Vol. 6, pp. 421-430
[Non-Patent Document 16] Yannone, S. M., Roy, S., Chan, D. W., Murphy, M. B., Huang, S., Campisi, J., and Chen, D. J., “Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase.”, J. Biol. Chem., 2001, Vol. 276, pp. 38242-38248
[Non-Patent Document 17] Brosh, R. M. Jr, von Kobbe, C., Sommers, J. A., Karmakar, P., Opresko, P. L., Piotrowski, J., Dianova, I., Dianov, G. L., and Bohr, V. A., “Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity.”, EMBO J., 2001, Vol. 20, pp. 5791-5801
[Non-Patent Document 18] Johnson, F. B., Lombard, D. B., Neff, N. F., Mastrangelo, M. A., Dewolf, W., Ellis, N. A., Marciniak, R. A., Yin, Y., Jaenisch, R., and Guarente, L., “Association of the Bloom syndrome protein with topoisomerase III alpha in somatic and meiotic cells.”, Cancer Res., 2000, Vol. 60, pp. 1162-1167
[Non-Patent Document 19] Mohaghegh, P., Karow, J. K., Brosh, Jr R. M. Jr, Bohr, V. A., and Hickson, I. D., “The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases.”, Nucleic Acids Res., 2001, Vol. 29, pp. 2843-2849
[Non-Patent Document 20] Wu, L., Davies, S. L., Levitt, N. C., and Hickson, I. D., “Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51.”, J. Biol. Chem., 2001, Vol. 276, pp. 19375-19381
[Non-Patent Document 21] Kawabe, T., Tsuyama, N., Kitao, S., Nishikawa, K., Shimamoto, A., Shiratori, M., Matsumoto, T., Anno, K., Sato, T., Mitsui, Y., Seki, M., Enomoto, T., Goto, M., Ellis, N. A., Ide, T., Furuichi, Y., and Sugimoto, M., “Differential regulation of human RecQ family helicases in cell transformation and cell cycle.”, Oncogene., 2000, Vol. 19, No. 41, pp. 4764-4772.
The present invention was achieved under such circumstances. An objective of the present invention is to provide apoptosis inducers for cancer cells, which comprise as an active ingredient a compound that suppresses the expression of a RecQ DNA helicase family gene, or a compound that suppress the function of the protein encoded by that gene. Another objective is to provide screening methods to select candidate compounds for apoptosis inducers for cancer cells.
The present inventors conducted extensive research to achieve the above-described objectives. The expression levels of RecQ DNA helicase family genes are known to be higher in tumor cell lines (for example, cancer cells). Using siRNAs having the activity of suppressing human RecQ helicase family gene expression, the present inventors studied the influence of suppressing the expression of respective RecQ helicase family genes on cancer cell growth. As a result, the present inventors found, for the first time, that apoptosis can be induced in various cancer cells by suppressing the expression of the RecQ helicase family genes WRN, BLM, or RecQ1, and that cancer cell growth can thus be suppressed through this apoptosis. These effects were not observed in a diploid fibroblast cell line derived from normal human fetal lung. In addition, the present inventors found that, without significant side effects, WRN-siRNA and RecQ1-siRNA can inhibit in vivo tumor growth in cancer-bearing nude mice.
Thus, the RecQ helicase family genes WRN, BLM, and RecQ1 can serve as target molecules for superior anticancer agents having few side effects. Specifically, the above-described compounds are able to suppress the expression of RecQ helicase family genes, or the function of RecQ helicase proteins, and may have the activity of inducing apoptosis. Such compounds are expected to serve as anticancer agents having few side effects. For example, compounds based on the mRNA sequences of WRN, BLM, and RecQ1 that suppress the expression or activity of RecQ helicase family proteins such as siRNA, are expected to serve as anticancer agents having few side effects.
Apoptosis inducers can be screened by using the expression of RecQ helicase family genes as an index.
Specifically, the present invention relates to apoptosis inducers for cancer cells which comprise as an active ingredient a compound that suppresses the expression of a RecQ DNA helicase family gene, or a compound that suppresses the function of the protein encoded by that gene, and screening methods to select candidate compounds for apoptosis inducers for cancer cells. More specifically, the present invention provides the following:
[1] an (cancer cell-specific) apoptosis inducer for cancer cells, wherein the apoptosis inducer comprises as an active ingredient a compound that suppresses the expression of a gene belonging to the RecQ DNA helicase family;
[2] the apoptosis inducer according to [1], wherein the compound that suppresses the expression of a gene belonging to the RecQ DNA helicase family is a double-stranded RNA having RNAi activity towards that RecQ DNA helicase family gene;
[3] the apoptosis inducer according to [2], wherein the double-stranded RNA comprises a sense RNA comprising a sequence homologous to an arbitrary 20 to 30 consecutive nucleotides from the mRNA of a gene belonging to the RecQ helicase family, and an antisense RNA, comprising the sequence complementary to the sense RNA;
[4] an apoptosis inducer for cancer cells, wherein the apoptosis inducer comprises as an active ingredient a DNA that can express the double-stranded RNA having RNAi activity towards a RecQ DNA helicase family gene;
[5] the apoptosis inducer according to [1], wherein the compound that suppresses the expression of a RecQ DNA helicase family gene is either:
(a) an antisense nucleic acid targeting a transcript of the gene belonging to the RecQ DNA helicase family, or a portion thereof; or
(b) a nucleic acid having the ribozyme activity of specifically cleaving a transcript of the RecQ DNA helicase family gene;
[6] an apoptosis inducer for cancer cells, which comprises as an active ingredient a compound that suppresses a function of a protein encoded by a RecQ DNA helicase family gene;
[7] the apoptosis inducer according to [6], wherein the compound that suppresses a function of a protein encoded by a gene belonging to the RecQ DNA helicase family is any one of:
(a) a mutant protein belonging to the RecQ DNA helicase family, where the mutant protein has a dominant-negative activity towards the protein encoded by the gene belonging to the RecQ DNA helicase family;
(b) an antibody that binds to the protein encoded by the gene belonging to the RecQ DNA helicase family; or
(c) a low-molecular-weight compound that binds to the protein encoded by the gene belonging to the RecQ DNA helicase family;
[8] the apoptosis inducer according to any one of [1] to [7], wherein the gene belonging to the RecQ DNA helicase is the WRN gene, the BLM gene, or the RecQ1 gene;
[9] the apoptosis inducer according to any one of [1] to [7], wherein the nucleotide sequence of the gene belonging to the RecQ DNA helicase family is the nucleotide sequence of any one of SEQ ID NOs: 6 to 30;
[10] the apoptosis inducer according to any one of [2] to [4], wherein either strand of the double-stranded RNA with RNAi activity comprises the nucleotide sequence of any one of SEQ ID NOs: 1 to 5, 31 to 40, and 43 to 69;
[11] an anticancer agent which comprises the apoptosis inducer according to any one of [1] to [10], as an active ingredient;
[12] a method of screening for a candidate compound for a cancer cell apoptosis inducer (a screening method for an apoptosis inducer for a cancer cell), which comprises the steps of:
(a) contacting a test compound with a protein encoded by a gene belonging to the RecQ DNA helicase family or a partial peptide thereof;
(b) assaying the binding activity between the protein, or the partial peptide thereof, and the test compound; and
(c) selecting a compound that binds to the protein encoded by the gene belonging to the RecQ DNA helicase family, or the partial peptide thereof;
[13] a method of screening for a candidate compound for a cancer cell apoptosis inducer (a screening method for an apoptosis inducer for a cancer cell), which comprises the steps of:
(a) contacting a test compound with cells expressing a gene belonging to the RecQ DNA helicase family or with an extract of those cells;
(b) determining the expression level of the gene belonging to the RecQ DNA helicase family; and
(c) selecting a compound that reduces the expression level by comparing the level with that determined in the absence of the test compound;
[14] a method of screening for a candidate compound for a cancer cell apoptosis inducer (a screening method for an apoptosis inducer for a cancer cell), which comprises the steps of:
(a) contacting a test compound with cells or an extract of those cells, wherein the cells have a DNA comprising a structure in which a reporter gene is operably linked to a transcriptional regulatory region of a gene belonging to the RecQ DNA helicase family;
(b) determining the expression level of the reporter gene; and
(c) selecting a compound that reduces the expression level by comparing the level with that determined in the absence of the test compound;
[15] a method of screening for a candidate compound for a cancer cell apoptosis inducer (a screening method for an apoptosis inducer for a cancer cell), which comprises the steps of:
(a) contacting a test compound with a protein encoded by a gene belonging to the RecQ DNA helicase family, or a cell expressing that protein or an extract of that cell;
(b) determining the activity of the protein; and
(c) selecting a compound that reduces the activity of the protein by comparing the level with that determined in the absence of the test compound;
[16] the method according to any one of [12] to [15], wherein the gene belonging to the RecQ DNA helicase family is the WRN gene, BLM gene, or RecQ1 gene; and
[17] a method for producing the apoptosis inducer according to [1] or [6] as a pharmaceutical composition, which comprises the steps of:
(a) screening for a compound using the method according to any one of [12] to [16]; and
(b) combining the compound with a pharmaceutically acceptable carrier.
Preferably, the above-mentioned genes belonging to the RecQ DNA helicase family are human genes belonging to the RecQ DNA helicase family.
The present inventors revealed, for the first time, that apoptosis can be induced in cancer cells (tumor cells) by suppressing the expression of RecQ DNA helicase family genes. Thus, the present invention provides apoptosis inducers that comprise as an active ingredient a compound that suppresses the expression of a RecQ DNA helicase family gene. The apoptosis inducers of the present invention comprise the activity of selectively inducing apoptosis in cancer cells in particular. Specifically, a preferable embodiment of the present invention provides pharmaceutical agents comprising the activity of selectively (specifically) inducing apoptosis in cancer cells, where the pharmaceutical agents comprise as an active ingredient a compound that suppresses the expression of a RecQ DNA helicase family gene, or the function of the protein encoded by that gene.
The term “apoptosis” generally refers to cell death actively induced by the cell itself due to a physiological condition. Morphological features of apoptosis include, for example, chromosome condensation in the cell nucleus, nuclear fragmentation, loss of microvilli on the cell surface, and cytoplasmic shrinkage. Thus, as used herein, the term “apoptosis-inducing activity” refers to, for example, the activity of inducing in cells any of the above-described morphological features of apoptosis, but is not limited to those described above. One skilled in the art can appropriately assess whether apoptosis induction in cells is taking place or not.
For example, the present invention's apoptosis inducers for cancer cells can be anticancer agents (carcinostatic agents) having apoptosis-inducing activity as their mechanism of action.
More specifically, genes belonging to the RecQ DNA helicase family (hereinafter sometimes simply referred to as “RecQ helicase genes”) include the RecQ1 gene, WRN gene, BLM gene, RTS gene, and RecQ5 gene. Those skilled in the art can readily obtain information on the nucleotide sequences of these genes from public gene databases (for example, GenBank). Exemplary GenBank accession numbers of the genes described above are listed below.
RecQ1 gene: NM—002907 (SEQ ID NO: 6), NM 032941 (SEQ ID NO: 7), BC001052 (SEQ ID NO: 8), D37984 (SEQ ID NO: 9), and L36140 (SEQ ID NO: 10).
WRN gene: NM—000553 (SEQ ID NO: 11), AF091214 (SEQ ID NO: 12), L76937 (SEQ ID NO: 13), and AL833572 (SEQ ID NO: 14).
BLM gene: U39817 (SEQ ID NO: 15), NM—000057 (SEQ ID NO: 16), and BC034480 (SEQ ID NO: 17).
RTS gene: NM—004260 (SEQ ID NO: 18), AB006532 (SEQ ID NO: 19), BC020496 (SEQ ID NO: 20), and BC011602 (SEQ ID NO: 21), and BC013277 (SEQ ID NO: 22).
RecQ5 gene: NM—004259 (SEQ ID NO: 23), AK075084 (SEQ ID NO: 24), AB006533 (SEQ ID NO: 25), AB042825 (SEQ ID NO: 26), AB042824 (SEQ ID NO: 27), AB042823 (SEQ ID NO: 28), AF135183 (SEQ ID NO: 29), and BC016911 (SEQ ID NO: 30).
One skilled in the art can readily obtain amino acid sequences of proteins encoded by the RecQ helicase genes of the present invention by, for example, using the accession numbers as indicated above. As examples of amino acid sequences of proteins encoded by the RecQ helicase genes of the present invention, the Sequence Listing shows the amino acid sequence of the protein encoded by each of the RecQ1 gene, WRN gene, BLM gene, RTS gene, and RecQ5 gene (the amino acid sequences of RecQ1 protein, WRN protein, BLM protein, RTS protein, and RecQ5 protein are shown in SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively).
Depending on the presence of polymorphic variations in the nucleotide sequences, there may be multiple accession numbers for the same RecQ helicase gene. “Polymorphism” is not restricted to single-nucleotide polymorphisms (SNPs), such as mutations due to the substitution, deletion, or insertion of a single nucleotide, but also includes mutations due to the substitution, deletion, or insertion of several continuous nucleotides. Therefore, the RecQ helicase gene nucleotide sequences are not limited to the sequences available under the accession numbers described above. Likewise, amino acid sequences of proteins encoded by the RecQ helicase genes are also not limited to the amino acid sequences shown in SEQ ID NOs: 70 to 74. Specifically, the above-described proteins of the present invention are not limited to the amino acid sequences shown in SEQ ID NOs: 70 to 74; and include proteins functionally equivalent to any one of the proteins as shown in SEQ ID NOs: 70 to 74, and comprising amino acid sequences with one or more amino acid additions, deletions, substitutions, and/or insertions in the amino acid sequences shown in SEQ ID NOs: 70 to 74.
Preferable RecQ helicase genes of the present invention include, for example, the WRN gene, BLM gene, and RecQ1 gene. The RecQ helicase genes of the present invention typically include, but are not limited to, those derived from animals, more preferably those derived from mammals, and most preferably those derived from humans.
Preferable compounds that suppress the expression of a RecQ helicase gene of the present invention include, for example, double-stranded RNA having RNA interference (RNAi) activity towards the RecQ helicase gene. In general, the term “RNAi” refers to a phenomenon where target gene expression is inhibited by inducing disruption of the target gene mRNA. This disruption is caused by introducing into cells a double-stranded RNA that comprises, a) a sense RNA comprising a sequence homologous to the target gene mRNA sequence, and b) an antisense RNA comprising a sequence complementary to the sense RNA. While the precise RNAi mechanism remains unclear, it is thought that an enzyme called DICER (a member of the RNase III nuclease family) contacts double-stranded RNA, decomposing it into small fragments called “small interfering RNA” or “siRNA”. This siRNA is also included in the double-stranded RNA comprising RNAi activity of the present invention. Furthermore, DNAs that allow the expression of the double-stranded RNA of the present invention are also included in the present invention. Specifically, the present invention provides DNAs (vectors) that allow the expression of a double-stranded RNA of the present invention. These DNAs (vectors) that allow the expression of a double-stranded RNA of the present invention are typically DNAs comprising a structure where a DNA encoding one strand of the double-stranded RNA, and a DNA encoding the other strand of the double-stranded RNA, are operably linked to a promoter. Those skilled in the art can readily prepare an above-described DNA of the present invention with routinely-used genetic engineering techniques. More specifically, expression vectors of the present invention can be prepared by appropriately inserting DNA encoding an RNA of the present invention into various known expression vectors.
The RNA used to achieve RNAi need not be completely identical (homologous) to the RecQ helicase gene or a portion of the gene; however such an RNA is preferable.
The double-stranded RNA comprising RNAi activity of the present invention is typically a double-stranded RNA that includes a) a sense RNA comprising a sequence homologous to an arbitrary RNA region of continuous nucleotide residues from the RecQ helicase gene mRNA, and b) an antisense RNA comprising a sequence complementary to the sense RNA. The above-mentioned “arbitrary RNA region of continuous nucleotide residues” is typically 20 to 30 nucleotides in length, and preferably 21 to 23 nucleotides in length. The length of the double-stranded RNA of the present invention is however not limited, because long-chain RNA that has no RNAi activity when intact may decompose to form siRNA comprising RNAi activity in cells. Furthermore, a long-chain double-stranded RNA corresponding to the full-length or near full-length mRNA of the RecQ helicase gene can be, for example, pre-digested with DICER, and the resulting decomposition products can be used as an apoptosis inducer of the present invention. The decomposition products are expected to contain double-stranded RNA molecules (siRNA) comprising RNAi activity. In this method, it is not necessary to select an mRNA region expected to comprise RNAi activity. Specifically, it is not essential to determine the RecQ helicase mRNA region comprising RNAi activity.
Usually, double-stranded RNAs comprising an overhang of several nucleotides at their ends are known to have a high RNAi activity. It is preferable that the double-stranded RNAs of the present invention comprise an overhang of several nucleotides at the ends. There is no limitation as to the length of the overhang nucleotide sequence, but it preferably comprises two nucleotide residues. A double-stranded RNA comprising an overhang of, for example, TT (a thymine doublet), UU (a uracil doublet), or some other nucleotide (most preferably, a molecule comprising a double-stranded RNA of 19 nucleotides and an overhang of two nucleotides (TT)) can be suitably used in the present invention. The double-stranded RNA of the present invention also includes molecules in which the overhanging nucleotides are DNAs.
Those skilled in the art can appropriately prepare the above-described “double-stranded RNA comprising RNAi activity towards a RecQ helicase gene” of the present invention based on the nucleotide sequence of a RecQ helicase gene that is the target of the double-stranded RNA. The nucleotide sequences of RecQ helicase genes can readily be obtained from public gene databases, as described above. For example, a double-stranded RNA of the present invention can be prepared based on the nucleotide sequence of any one of SEQ ID NOs: 6 to 30. Specifically, the selection of an arbitrary consecutive RNA region of an mRNA transcript based on the nucleotide sequence of any one of SEQ ID NOs: 6 to 30, and preparation of a double-stranded RNA corresponding to this region would be an easy task for one skilled in the art.
It is not essential to have information on the full-length nucleotide sequence of a gene (the target gene) from which the double-stranded RNA of the present invention is derived. It is enough that the arbitrary RNA region comprising consecutive nucleotides (for example, 20 to 30 nucleotides) which is to be selected has been identified. Thus, the double-stranded RNA of the present invention can be prepared based on the nucleotide sequence of a fragment of a gene, such as an Expressed Sequence Tag (EST), whose mRNA sequence has been determined partially, but not completely. The accession numbers and names of EST sequences in the GenBank database with a high homology to human genes belonging to the RecQ DNA helicase family are shown below. However, this list includes only a few examples of the many EST sequences. Those skilled in the art can readily obtain sequence information on appropriate EST fragments from public databases.
(a) RecQ1 gene: BQ436743, AU130503, BI756143, BQ962215.1|BQ962215, AU117557, BQ049370, AU129387, BQ882675, BM015357, BG392546, AU131006, BU190089, BE870195, BM786040, BQ182943, AU138156, AW629737, CA489724, BF031494, AA258050, AW149458, BG113470, BG177944, AV718094, BQ952333, BQ647346, AU127897, AA830035, BQ215072, BE708578, BM467595, AL042375, AA298927, BU858680, CB158017, AA298835, BG536173, AU280724, BU194296, BI090858, BG118947, AA070943, BG530720, BG529492, AW505210, AW673131, BE888299, BE794392, CA394258, Z33439, BX093234, AA298951, AA459772, BG542269, BF344325, BQ359629, CA396832, AA670042, BF061797, AI652053, CB306566, CB155969, BU160790, BM263857, AW338719, AI872607, AI560139, CA450066, CA310830, BQ776691, BM980368, BM976615, BF436358, BE502851, AW864567, AWO57629, AI685944, AI671558, AI636512, AI478320, AI474213, AI434812, AI223294, AA969326, AA939175, AA889357, AA804459, AA456585, AA287557, BM151219, AI261327, AW779519, AW867647, T71941, BI559810, AW182531, BE246521, CA450299, CA311010, and AW672788.
(b) WRN gene: AW965099, AL707510, AL709832, BM785803, BG714322, BM552381, BF103840, BQ305613, T39125, BG992948, BQ305407, BU633322, CB156864, AA344201, AA373157, W40393, AA287985, AA193296, AA193286, BM721721, R58879, AW016548, AI203498, H80461, AA835784, BQ354585, N64051, A1025669, AW102683, BU683758, AA905633, BE939929, AI457716, BE787985, AA778924, BG941850, and BQ774611.
(c) BLM gene: AL556823, AL556853, BM451903, BI091601, BG199179, BI091772, BQ230262, BG772975, BG875917, BM542461, BM040993, BE618504, BM041661, BG574669, BI667071, BE538092, BG721596, AW502890, BM804157, CB243435, AL120858, BX106802, BE889560, BQ316432, BE963549, AI394601, BG397477, BG756262, BX283839, AI097184, AW503829, AW404657, BE535950, AA747832, AI590599, AW504704, AI114820, AA904488, BG531593, BE245802, BU588736, BQ359304, AA974756, BE778486, BM151892, AA769336, BG192554, BG187329, AW575595, AA214549, AA480209, AI423875, AW173139, AI630521, CA488994, AA862803, BE940055, BU431321, AA249737, BM150628, AW138812, AA903504, BE245666, BG210806, BG185919, BG182955, H53763, BG217661, AA887818, AA643177
(d) RTS gene: AL561020, BU902971, CA454998, BM557643, BU944576, BQ065027, BQ649577, BG824628, BQ072016, AL582326, BU173357, BU902969, BE560845, BG388102, BG337750, CA489272, BM763376, BQ646647, BU957442, BG757074, BE514063, BU171530, BQ218075, BE513519, BE379488, BE513709, BQ668351, BG338114, BQ883533, BM724503, BM849415, BE295951, BI457058, BG398209, BM679729, BM794167, BM853252, BM461584, BE466-402, BE281293, BE378846, BQ215879, BM847781, BE906701, BX116159, BE349634, BG337918, H16879, BG339379, AW236527, BM854499, AI858255, BG954668, BF513150, BM144460, BM144537, AI886385, AI590776, BM848938, AI765149, CA449577, BM763607, AA768048, AA595239, BU729459, BM702983, AI872334, AA401146, BF909603, BE790022, AA984927, BM796016, BF935882, H16770, AI471262, AA627850, BG991513, BF800269, BX095851, BQ576069, BG059979, BF000493, AI341050, AI206129, BE243589, AW304768, BF115628, BE869080, AI365167, BQ651717, AA620446, BG822953, and AL042193.
(e) RecQ gene: BU187406, BI916256, BQ954275, BQ932656, BQ924931, BG762566, AL525351, BQ316263, BG424888, BG721800, BQ049337, BQ061205, BQ059196, BM449612, BM469366, BX099953, CB270726, BG763254, BU431740, BQ375153, BF982170, AI567000, AI671940, CB155215, AA155882, BI772198, AW368882, BQ322863, AI310229, BF896710, BF896709, BF182856, AL600850, BI198364, BI200066, AA155835, BE314216, BE313572, BE263134, BE261456, AI363275, AI218469, BQ316256, AW605932, and BI116483.
Specifically, a preferred embodiment of the present invention provides an apoptosis inducer that comprises a double-stranded RNA with RNAi activity, which comprises as one strand an RNA region of continuous nucleotides in mRNA corresponding to a gene of any one of the sequences of SEQ ID NOs: 6 to 30 or to any of the ESTs shown above.
As described above, some identical RecQ helicase genes comprise various polymorphic sequences. Those skilled in the art can appropriately design a sequence of RNA expected to have RNAi activity by basing the design on any one of the above-described SEQ ID NOs: 6 to 30 or EST sequences, for example by evaluating information on a RecQ helicase gene obtained from public databases for polymorphisms. An apoptosis inducer comprising such RNA is also included in the present invention. In addition, those skilled in the art can also prepare an apoptosis inducer by appropriately selecting, from a number of double-stranded RNAs of the present invention, an RNA comprising optimal RNAi activity.
The above-described “double-stranded RNA comprising RNAi activity” of the present invention is preferably exemplified by the double-stranded RNAs described in
Another preferred embodiment of the present invention provides apoptosis inducers in which the compound that suppresses gene expression of the RecQ DNA helicase family is the compound of (a) or (b) described below:
(a) an antisense nucleic acid targeting a transcript of a RecQ DNA helicase family gene, or a portion thereof; and
(b) a nucleic acid having the ribozyme activity of specifically cleaving the transcript of a RecQ DNA helicase family gene.
As used herein, the term “nucleic acid” refers to RNA and DNA. Methods well known to those skilled in the art for inhibiting (suppressing) the expression of a specific endogenous gene include those using antisense technology. Multiple factors contribute to the inhibition of a target gene expression by an antisense nucleic acid. These factors include, for example, inhibition of transcription initiation through triplex formation; inhibition of transcription through hybrid formation with a sequence at the site of a local open loop structure made by RNA polymerase; inhibition of transcription through hybrid formation with the RNA being synthesized; inhibition of splicing through hybrid formation with a sequence at an intron-exon junction; inhibition of splicing through hybrid formation with a sequence at the site of spliceosome formation; inhibition of transfer from the nucleus to the cytoplasm through hybrid formation with mRNA; inhibition of splicing through hybrid formation with a sequence at the capping site or poly(A) site; inhibition of translation initiation through hybrid formation with a sequence at the site of binding of the translation initiation factor; inhibition of translation through hybrid formation with a sequence at the ribosome binding site near the initiation codon; inhibition of peptide chain elongation through hybrid formation with a sequence at the site of the translational region or polysome binding site of the mRNA; and inhibition of gene expression through hybrid formation with a sequence at the site of interaction between the protein and the nucleic acid. Thus, an antisense nucleic acid inhibits target gene expression by inhibiting various processes, such as transcription, splicing and translation (Hirashima and Inoue, Shin Seikagaku Jikkenkoza 2 (New Lecture for Experimental Biochemistry 2), Kakusan IV (Nucleic Acid IV), Replication and Expression of Genes; Ed., Japanese Biochemical Society, Tokyo Kagaku Dozin Co., Ltd., pp. 319-347, 1993).
Antisense nucleic acids used in the present invention may inhibit the expression of a RecQ helicase gene through any one of the actions described above. In one embodiment, an antisense sequence is designed to be complementary to the 5′-untranslated region of a RecQ helicase gene mRNA. Thus such an antisense sequence is expected to effectively inhibit translation of that gene. A sequence complementary to the coding region or 3′-untranslated region can also be used for this purpose. Thus, a nucleic acid comprising the antisense sequence corresponding to the sequence of the translated as well as the untranslated regions of the RecQ helicase gene can be included as an antisense nucleic acid used in the present invention. The antisense nucleic acid to be used is ligated downstream of an appropriate promoter and preferably ligated with a sequence comprising a transcription termination signal at the 3′ end. The antisense nucleic acid to be used for clinical applications is typically a synthetic oligomer. Such synthetic oligomers include the widely used S-oligo (phosphorothioate oligo nucleotide) in which S (sulfur) has been substituted for O (oxygen) at the phosphate ester bond, thus reducing sensitivity to nuclease digestion and maintaining antisense nucleic acid activity. S-oligo is currently being tested as an antisense drug in clinical trials where it is administered directly to affected areas. This S-oligo is also suitable for use in the present invention. It is preferable that the antisense nucleic acid sequence is complementary to the target gene sequence or a portion thereof; however perfect complementarity is not necessary as long as the antisense nucleic acid effectively suppresses target gene expression. The transcribed RNA has preferably 90% or higher complementarity, and most preferably 95% or higher complementarity to the target gene transcript. The length of the antisense nucleic acid used to effectively suppress target gene expression is at least 15 nucleotides or longer, preferably 100 nucleotides or longer, and more preferably 500 nucleotides or longer.
The inhibition of RecQ helicase gene expression can also be achieved using a ribozyme or ribozyme-encoding DNA. The term “ribozyme” refers to an RNA molecule comprising catalytic activity. Ribozymes can have a variety of activities, and can be designed to have the activity of cleaving RNA in a site-specific fashion. Ribozymes such as group I intron-type ribozymes and M1 RNA, which are RNase P ribozymes, are 400 nucleotides or more in length. Others such as hammer-head and hairpin ribozymes have active sites comprising about 40 nucleotides (M. Koizumi and E. Otsuka, Tanpakushitsu Kakusan Koso (Protein, Nucleic acid, and Enzyme), 1990, 35, 2191).
For example, the autolytic domain of a hammer-head ribozyme cleaves the 3′ side of C15 in the sequence G13U14C15. Base pairing between U14 and A9 plays an important role in this activity, and A15 or U15 can be cleaved instead of C15 (Koizumi, M. et al., FEBS Lett, 228: 228, 1988). A restriction enzyme-like RNA-cleaving ribozyme that recognizes the target RNA sequences UC, UU or UA can be produced by designing the ribozyme such that the substrate binding site complements the RNA sequence near the target site (Koizumi, M. et al., FEBS Lett, 239: 285, 1988; M. Koizumi and E. Otsuka, Tanpakushitsu Kakusan Koso (Protein, Nucleic acid, and Enzyme), 35:2191, 1990; and Koizumi, M. et al., Nucl. Acids Res., 17: 7059, 1989).
The hairpin ribozyme can also be used for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, J. M., Nature, 323: 349, 1986). A target specific RNA-cleaving ribozyme can also be produced from a hairpin ribozyme (Kikuchi, Y. and Sasaki, N., Nucl. Acids Res., 19: 6751, 1991; Kikuchi, H., Kagaku to Seibutsu (Chemistry and Biology), 30: 112, 1992). Thus, the expression of a RecQ helicase gene of the present invention can be inhibited by specifically digesting the gene transcript using a ribozyme.
Furthermore, the present invention relates to apoptosis inducers comprising as an active ingredient a compound that suppresses the function (activity) of a protein encoded by a RecQ DNA helicase family gene (herein, also abbreviated as “RecQ helicase protein”).
The RecQ helicase proteins of the present invention further include mutant and homologous proteins functionally equivalent to RecQ helicase proteins, but whose amino acid sequences contain one or more amino acid deletions, substitutions or additions compared to the normal RecQ helicase protein sequence. Herein, the term “functionally equivalent protein” refers to a protein having the same activity as a RecQ helicase protein. Alternatively, a protein functionally equivalent to a RecQ helicase protein can be defined as a protein having, for example, 90% or higher homology, preferably 95% or higher homology, and more preferably 99% or higher homology to the amino acid sequence of the RecQ helicase protein.
A preferred embodiment of the present invention provides apoptosis inducers in which the compound suppressing the function (activity) of a protein encoded by a RecQ DNA helicase gene is a compound of any one of (a) to (c) below. These compounds have the activity of inducing apoptosis in cells, cancer cells in particular, through inhibiting the function or activity of a RecQ helicase protein:
(a) a mutant RecQ DNA helicase family protein comprising dominant-negative activity against a protein encoded by a RecQ DNA helicase family gene;
(b) an antibody that binds to a protein encoded by a RecQ DNA helicase family gene; and
(c) a low-molecular-weight compound that binds to a protein encoded by a RecQ DNA helicase family gene.
The phrase “mutant RecQ DNA helicase protein comprising dominant-negative activity against a protein encoded by a RecQ DNA helicase family gene” described above in (a) refers to a mutant RecQ helicase protein comprising the function of eliminating or reducing the function of the endogenous wild-type protein. An example is a mutant RecQ helicase family protein reported to have no ATP hydrolysis activity. This mutant is formed when alanine or methionine is substituted for lysine residues in motif I (ATP-binding motif), one of seven highly conserved helicase motifs in the helicase domain of a RecQ helicase family protein. This mutant functions as a dominant negative mutant protein. Thus, the dominant negative mutant of the present invention is exemplified by a mutant in which the lysine residues in the above-described motif I have been replaced with other amino acid residues.
The antibody that binds to a RecQ helicase protein, described in (b), can be prepared by a method known to those skilled in the art. When the antibody is a polyclonal antibody, it can be prepared, for example, using the following method: Antiserum is obtained by immunizing small animals such as rabbits with a natural or recombinant RecQ helicase protein, or a recombinant RecQ helicase protein expressed as a fusion protein with GST in microorganisms such as E. coli, or a partial peptide thereof. The antibody is purified from the serum using, for example, ammonium sulfate precipitation, a protein A column, a protein G column, DEAE ion-exchange chromatography, an affinity column where a RecQ helicase protein or synthetic peptide thereof has been immobilized, etc. Alternatively, a monoclonal antibody can be produced by, for example, immunizing a small animal such as mouse with the RecQ helicase protein or a partial peptide thereof, removing the spleen, gently grinding the spleen to separate cells, fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol, and then screening the fused cells (hybridomas) to select clones that produce antibodies that bind to the RecQ helicase protein. These hybridomas are then transplanted into a mouse peritoneal cavity and the ascites are collected from the same mouse. The monoclonal antibody thus prepared can be purified using, for example, ammonium sulfate precipitation, a protein A column, a protein G column, DEAE ion-exchange chromatography, an affinity column where a RecQ helicase protein or synthetic peptide thereof has been immobilized, etc.
There is no limitation as to the type of antibodies of the present invention, as long as they can bind to a RecQ helicase protein of the present invention. The antibodies include human antibodies, humanized antibodies prepared using genetic recombination, antibody fragments thereof, and modified antibodies, in addition to the polyclonal antibodies and monoclonal antibodies described above.
The RecQ helicase protein of the present invention to be used as an immunization antigen to obtain antibodies need not derive from the same species as the protein. However, mammalian proteins are preferable, for example, proteins from mice and humans, and proteins from humans are especially preferable.
Proteins to be used as an immunization antigen in the present invention may be intact proteins as well as partial peptides derived from those proteins. Such partial protein peptides include, for example, protein amino (N)-terminal fragments and carboxyl (C)-terminal fragments. As used herein, “antibody” usually refers to an antibody which reacts with a full-length protein or a fragment thereof.
In addition to obtaining the above-described hybridomas by immunizing non-human animals with an antigen, hybridomas producing a desired human antibody comprising binding activity with the protein can also be prepared in vitro by sensitizing human lymphocytes, for example, human lymphocytes infected with EB virus, with the protein, cells expressing the protein, or a lysate of those cells, and fusing these sensitized lymphocytes with immortalized human myeloma cells, for example, U266 cells. When an antibody of the present invention is intended to be administered into human bodies (antibody therapy), a human antibody or humanized antibody is preferable to reduce the immunogenicity.
Compounds known to bind to a protein belonging to the RecQ helicase family include, for example, monoclonal and polyclonal antibodies against RecQ1 helicase, WRN helicase, BLM helicase, RTS helicase, and RecQ5 helicase.
Compounds of the present invention that suppress RecQ helicase gene expression or the function (activity) of a protein encoded by that gene may be natural or synthetic compounds. Typically, the compounds can be produced, obtained or isolated using methods known to those skilled in the art. Such compounds include, for example, compounds comprising a single molecule, such as organic compounds, inorganic compounds, nucleic acids, proteins, peptides, and sugars; and libraries of compounds, expression products of gene libraries, cell extracts, cell culture supernatants, the products of fermenting microorganisms, marine organism extracts, plant extracts, and compounds purified or isolated from such extracts.
Compounds known to inhibit the function (activity) of a RecQ helicase family protein include, for example, distamycin A and netropsin that have the activity of inhibiting WRN helicase and BLM helicase (Brosh, R. M. Jr, Karow, J. K., White, E. J., Shaw, N.D., Hickson, I. D. and Bohr, V. A., “Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs.”, Nucleic Acids Res. 28, 2420-2430, 2000); 3,6,9-trisubstituted acridine that has the activity of inhibiting WRN helicase and BLM helicase (Li, J. L., Harrison, R. J., Reszka, A. P., Brosh, R. M. Jr, Bohr, V. A., Neidle, S, and Hickson, I. D., “Inhibition of the Bloom's and Werner's syndrome helicases by G-quadruplex interacting ligands.”, Biochemistry 40, 15194-15202, 2001); and N-methyl mesoporphyrin IX that has the activity of inhibiting E. coli RecQ helicase (Wu, X. and Maizels, N., “Substrate-specific inhibition of RecQ helicase.”, Nucleic Acids Res. 29, 1765-1771, 2001).
The present invention also provides screening methods to select candidate compounds for apoptosis inducers for cancer cells (screening methods for apoptosis inducers for cancer cells). Compounds selected by these screening methods are expected to have apoptosis-inducing activity.
In one embodiment of these methods, the binding of a test compound to a RecQ helicase protein or partial peptide thereof is used as an index. A compound that binds to a RecQ helicase protein or partial peptide thereof is typically expected to have the activity of inhibiting the function of that RecQ helicase protein.
In the above-described method of the present invention, first, a test compound is contacted with the protein encoded by a RecQ helicase gene or partial peptide thereof. Depending on the type of index used to detect test compound binding, the RecQ helicase protein or partial peptide thereof can be, for example, a purified protein or partial peptide; a protein or partial peptide expressed within or outside of cells; or a protein or partial peptide bound to an affinity column. The test compounds used in this method can be labeled as required. Such labels include, for example, radiolabels and fluorescent labels.
The next step of this method comprises detecting the binding between the test compound and the RecQ helicase gene protein or partial peptide thereof. The binding between the test compound and the RecQ helicase protein or partial peptide thereof can be detected, for example, by using a label attached to the test compound bound to the RecQ helicase protein or partial peptide thereof. Alternatively, binding can be detected by using as an index a change in RecQ helicase protein activity induced by the binding of the test compound to that protein or partial peptide thereof which is expressed within or outside of cells.
The next step of this method comprises selecting test compounds that bind to the RecQ helicase gene protein or partial peptide thereof.
There is no limitation as to the type of test compound used in the present invention. Such compounds include, but are not limited to, for example, single unmixed compounds of organic compounds, inorganic compounds, nucleic acids, proteins, peptides, sugars, natural compounds, and such; or compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, products of fermenting microorganisms, marine organism extracts, and plant extracts; and artificially synthesized compounds.
In an alternative embodiment of the screening method of the present invention, first, a test compound is contacted with cells that express a RecQ DNA helicase family gene, or with a cell extract prepared from such cells. The phrase “cells that express a RecQ DNA helicase family gene” described above include cells expressing an endogenous RecQ helicase gene, and cells into which an exogenous RecQ helicase gene has been introduced and in which that gene is expressed. The cells in which an exogenous RecQ helicase gene is expressed can typically be prepared by introducing into host cells a vector which contains the gene. Those skilled in the art can prepare such an expression vector using routine genetic engineering techniques. In the screening methods of the present invention, cells expressing a RecQ helicase gene preferably include various tumor cells, for example, MCF7 (breast cancer), A549 (lung cancer), U2OS (osteogenic sarcoma), C33A (cervical cancer), HT1080 (fibrosarcoma), PA-1 (ovarian teratocarcinoma), Tera2 (embryonal carcinoma), T24 (bladder cancer), K562 (chronic myelocytic leukemia), Molt4 (acute lymphoblastic leukemia), A172 (glioblastoma), HeLa (cervical cancer), and HepG2 (hepatic cancer), U251 (glioblastoma), UACC62 (melanoma), Caki-1 (renal cancer), KP4 (pancreatic cancer), MKN45 (stomach cancer), and LNCaP (prostatic cancer).
Typically, but without limitation, a test compound is contacted with cells expressing a RecQ helicase gene by adding the test compound to a culture medium of the cells expressing the RecQ helicase gene. When the test compound is a protein, the contact can be achieved by introducing into the cells a DNA vector that allows protein expression.
The next step of this method comprises determining the expression level of the RecQ helicase gene. Herein, the phrase “gene expression” refers to both transcription and translation. The gene expression level can be determined using a method known to those skilled in the art. For example, mRNA can be extracted from cells expressing the RecQ helicase gene according to a conventional method, and by using this mRNA as a template, the transcriptional level of the gene can be determined using Northern hybridization or RT-PCR. Alternatively, the translational level of the gene can be determined by collecting protein fractions from the cells expressing the RecQ helicase family gene, and then detecting the expression of the RecQ helicase protein using an electrophoresis method such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, the translational level of the gene can be determined by detecting the expression of the RecQ helicase protein by Western blotting analysis using an antibody against the protein. There is no limitation as to the type of antibody used for RecQ helicase protein detection, as long as the protein can be detected. Such antibodies include, for example, both monoclonal and polyclonal antibodies. For example, the above-described antibodies of the present invention can also be used.
In this method, a compound is then selected such that it causes a reduction in expression level when compared to the expression level measured in the absence of a test compound (control). The compound selected by the above-described procedure is expected to have the activity of inducing apoptosis in cancer cells. This compound may be used as an anticancer agent whose mode of action is based on apoptosis induction.
In an alternative embodiment of the screening method of the present invention, a compound that reduces the expression level of a RecQ DNA helicase family gene of the present invention is selected using a reporter gene.
In this method, a test compound is first contacted with cells (or an extract of those cells) that comprise a DNA having a structure where a reporter gene is operably linked to a transcriptional regulatory region of a RecQ helicase gene. As used herein, the phrase “operably linked” means that the transcriptional regulatory region of a RecQ helicase gene is linked to a reporter gene in such a way as to induce reporter gene expression when a transcriptional factor binds to the transcriptional regulatory region of the RecQ helicase gene. Thus, even when the reporter gene is connected with another gene and thus forms a fusion protein with that gene product, such a connection can be expressed by the phrase “operably linked”, as long as the expression of the fusion protein is induced when the transcriptional factor binds to the transcriptional regulatory region of the RecQ helicase gene. Using a known method and based on the cDNA nucleotide sequence for a RecQ helicase gene, those skilled in the art can obtain from the genome the transcriptional regulatory region of that RecQ helicase gene.
There is no limitation as to the type of reporter gene used in this method, as long as it can detect the expression of the gene. Such reporter genes include, for example, the CAT gene, lacZ gene, luciferase gene, and GFP gene. The “cells that comprise a DNA having a structure where a reporter gene is operably linked to a transcriptional regulatory region of a RecQ DNA helicase family gene” include, for example, cells into which a vector with the structure where a reporter gene is operably linked to a transcriptional regulatory region of a RecQ DNA helicase family gene has been introduced. Those skilled in the art can prepare the above-described vector using routine genetic engineering techniques. The introduction of such a vector into cells can be achieved using a conventional method, for example, using calcium phosphate precipitation, electroporation, the lipofectamine method, microinjection, etc. “Cells that comprise a DNA having a structure where a reporter gene is operably linked to a transcriptional regulatory region of a RecQ DNA helicase family gene” also includes cells in which that structure has been inserted into the chromosome. The DNA structure can be inserted into the chromosome by using a method routinely used by those skilled in the art, for example, a gene transfer method using homologous recombination.
An extract of those “cells that comprise a DNA having a structure where a reporter gene is operably linked to a transcriptional regulatory region of a RecQ DNA helicase family gene” includes, for example, a mixture prepared by adding a DNA to a cell extract included in a commercially available in vitro transcription translation kit, where that added DNA comprises a structure where a reporter gene is operably linked to a transcriptional regulatory region of a RecQ DNA helicase family gene.
In this method, the “contact” can be achieved by adding a test compound into a culture medium of “cells that comprise a DNA having a structure where a transcriptional regulatory region of a RecQ DNA helicase family gene is operably linked to a reporter gene”, or by adding a test compound into the above-described commercially available cell extract, which contains the DNA. However, the method of contact is not limited to these methods described above. When the test compound is a protein, the contact can also be achieved, for example, by introducing into the cells a DNA vector that directs the expression of the protein.
The next step of this method comprises determining the level of reporter gene expression. The expression level of the reporter gene can be determined by a method that depends on the type of the reporter gene and which is known to those skilled in the art. For example, when the reporter gene is the CAT gene, expression level can be determined by detecting the acetylation of chloramphenicol, mediated by the CAT gene product. When the reporter gene is the lacZ gene, expression level can be determined by detecting color development in the chromogenic compound, mediated by the catalytic action of the lacZ gene expression product. When the reporter gene is the luciferase gene, the level can be determined by detecting the fluorescence of the fluorescent compound, mediated by the catalytic action of the luciferase gene expression product. Alternatively, when the reporter gene is the GFP gene, the level can be determined by detecting the fluorescence of the GFP protein.
The next step of this method comprises selecting compounds that reduce reporter gene expression level as compared to expression level determined in the absence of a test compound. The compounds selected by the above-described procedure can be candidate compounds for apoptosis inducers for cancer cells.
In an alternative embodiment of the method of the present invention, compounds are screened using as an index the activity of a protein encoded by a RecQ DNA helicase family gene of the present invention.
In this method, a test compound is first contacted with a protein encoded by a RecQ helicase gene, or cells expressing that protein, or an extract of such cells. The activity of the protein is then determined. The activity of the protein includes, for example, DNA helicase activity and DNA-dependent ATP hydrolysis activity. Helicase activity typically means the unwinding activity of double-stranded DNA into two single-stranded DNAs. DNA helicase activity can be determined by a method known to those skilled in the art. For example a radio-labeled complementary oligo DNA is annealed with single-stranded circular M13 phage DNA to form a partially double-stranded structure. Using this structure as a substrate, the enzyme is incubated in the presence of ATP and magnesium ions. The products of this reaction are then fractionated into single-stranded and double-stranded DNA using polyacrylamide gel electrophoresis or agarose gel electrophoresis, and then detected using autoradiography. In an alternative method, complementary single-stranded DNAs, labeled with europium and Qcy7 respectively, are annealed and the resulting double-stranded DNA is used as a substrate and incubated with the enzyme in the presence of ATP and magnesium ions, whereupon the fluorescence of the europium-labeled single-stranded DNA released from the double-stranded DNA by helicase activity is determined. Alternative methods include an electrochemiluminescence-based helicase assay, scintillation proximity assay, homogeneous time-resolved fluorescence quenching assay, and DELFIA helicase assay (Zhang, L., Schwartz, G., O'Donnell, M. and Harrison, R. K., “Development of a novel helicase assay using electrochemiluminescence.”, Anal. Biochem. 293, 31-37, 2001).
The activity of the above-mentioned DNA-dependent ATP hydrolysis can be determined using a method known to those skilled in the art. Such methods include, for example, a method that comprises reacting the enzyme with ATP as a substrate in the presence of magnesium ions and plasmid DNA or salmon sperm DNA, and then quantifying color development associated with the release of inorganic phosphoric acid in the conversion of ATP to ADP using malachite green.
A compound is then selected that reduces RecQ helicase protein activity, compared with activity determined in the absence of the test compound. In the above-described method, it is preferable to use a full-length RecQ helicase protein having no mutations. However, a protein whose amino acid sequence contains partial substitutions and/or deletions can also be used, as long as it has an activity equivalent to that of the RecQ helicase protein.
In the above-mentioned screening method, the RecQ DNA helicase family gene is preferably the WRN gene, BLM gene, or RecQ1 gene.
The present invention also provides anticancer agents (pharmaceutical compounds for treating cancers) which comprise as an active ingredient an apoptosis inducer of the present invention that targets cancer cells (cancer cell-specific).
The present invention also provides a method for producing an apoptosis inducer as a pharmaceutical composition. In this method a candidate compound for the apoptosis inducer for cancer cells is first selected using a screening method of the present invention. Then, the selected compound is combined with a pharmaceutically acceptable carrier. Such a pharmaceutically acceptable carrier can include, but is not limited to, for example, detergents, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspensions, isotonizing agents, binders, disintegrating agents, lubricants, fluidizing agents, and correctives. Other conventional carriers can be also used appropriately.
The pharmaceutical agents of the present invention can be formulated by adding the above-indicated carriers as required and according to a conventional method. More specifically, such carriers include: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylacetaldiethylamino acetate, polyvinylpyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, saccharose, carboxymethyl cellulose, cornstarch, and inorganic salts.
The dosage forms for the agents described above include, for example, oral forms, such as tablets, powders, pills, dispersing agents, granules, fine granules, soft and hard capsules, film-coated tablets, pellets, sublingual tablets, and pastes; and parenteral forms, such as injections, suppositories, endermic liniments, ointments, plasters, and liquids for external use. Those skilled in the art can select the optimal dosage form depending on the administration route, subject, and such. Viral vectors such as retrovirus, adenovirus, and Sendai virus vectors, and non-viral vectors such as liposomes, may be used to introduce, into the living body, DNAs expressing proteins encoded by RecQ DNA helicase family genes, or DNAs expressing antisense RNAs, ribozymes, or siRNAs that suppress RecQ DNA helicase family genes. Alternatively, non-viral vectors such as liposomes, polymer micelles, or cationic carriers, may be used to introduce, into the living body, synthetic antisense nucleic acids or synthetic siRNAs that suppress RecQ DNA helicase family genes. The introduction methods include, for example, in-vivo and ex-vivo methods.
The present invention also includes pharmaceutical compositions comprising the above-described apoptosis-inducing activity.
Ultimately, the dose of a pharmaceutical agent or pharmaceutical composition of the present invention can be appropriately determined by a physician considering the dosage form, administration method, patient's age, weight, symptoms, etc.
In
The present invention is illustrated in detail below with reference to Examples, but it is not to be construed as being limited thereto.
The human cells used were HeLa (uterocervical carcinoma cells), A549 (human lung cancer cells), MCF7 (human breast cancer cells), TIG3 (normal diploid fibroblast cells), U2OS (osteosarcoma), HepG2 (hepatic cancer), U251 (glioblastoma), UACC62 (melanoma), Caki-1 (renal cancer), KP4 (pancreatic cancer), MKN45 (stomach cancer), and LNCaP (prostatic cancer). All human cells were cultured under 5% CO2 at 37° C. in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 μg/ml gentamycin.
Five siRNAs targeting each of the five types of human RecQ helicase genes (WRN, BLM, RTS, RecQ1, and RecQ5) were designed by using the method by Elbasher et al. (Elbasher, M. S. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 (2001)) to examine the effect of suppressing the expression of each of the five human RecQ helicases on cancer cell growth. The siRNAs were synthesized by Dharmacon, Inc. and QIAGEN.
The siRNAs were introduced into human uterocervical carcinoma-derived HeLa cells and fetal embryo-derived fibroblast TIG3 cells as normal cells. Twenty-four hours before siRNA transfection, the cells were plated in 24-well plates at a density of 1 to 3×104 cells/well. When these cells were 20 to 50% confluent, the siRNAs were transfected. Twenty-four hours after plating, cells were transfected with the siRNAs using Oligofectamine (Invitrogen) or Lipofectamine 2000 (Invitrogen) respectively, following the supplier's protocols.
Total RNA was extracted from the cells using RNeasy Mini Kits (Qiagen), 42 and 72 hours after siRNA transfection, respectively. Quantitative PCR was carried out using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). RT-PCR primers for the RecQ helicase genes and beta-actin gene, as well as TaqMan probes were purchased from Applied Biosystems. RT-PCR reactions were carried out using TaqMan One-Step RT-PCR Master Mix Reagents Kits (Applied Biosystems) according to the method described in the manual. The mRNA expression levels were compared quantitatively using beta-actin as a standard.
The expression of each mRNA in cells into which a control RNA (NS) had been introduced was taken as 100%, and then compared with the expression of each mRNA in cells into which each siRNA had been introduced. NS-siRNA is a duplex consisting of the following two strands: 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 41/“dTdT” is represented by “TT” in the Sequence Listing) and 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ ID NO: 42/“dTdT” is represented by “TT” in the Sequence Listing).
As a result, in HeLa cells cultured for 72 hours, the siRNAs which reduced mRNA expression to the following values were identified: 2% in the case of WRN; 15% in the case of BLM; 0% in the case of RTS; 0% in the case of RecQ1; and 7% in the case of RecQ5 (
Next, the present inventors also designed siRNAs against three types of human RecQ helicases, RecQ1, WRN, and BLM, by the same procedure as described above, and selected 14, 11, and 7 types of siRNAs for RecQ1, WRN, and BLM, respectively. The selected sequences also include some sequences already shown above. The selected siRNA sequences are shown in
The siRNAs were introduced into HeLa cells. 48 hours after introduction, the expression levels of RecQ1, WRN, and BLM genes were quantitated by semi-quantitative RT-PCR. The gene expression levels after introduction of each siRNA were compared taking the expression levels after treatment with non-silencing siRNA (NS) as 100%.
As a result, all 14 types of siRNAs for RecQ1 and the siRNAs for WRN3, WRN4, WRN6, and WRN12 exhibited 90% or stronger gene-suppression effect, while the siRNAs for BLM2, BLM3, BLM4, BLM5, and BLM7 suppressed the gene expression level by 80% or more (
Western blot analysis was used to determine the expression levels of the proteins WRN, BLM, RTS, RecQ1, and RecQ5 in HeLa cells in which each mRNA expression was suppressed. The cells were harvested 48 and 72 hours, respectively, after transfection with the siRNAs, and then lysed in RIPA solutions (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X 100, and 1% sodium deoxycholate). The proteins extracted were fractionated using SDS-PAGE on a polyacrylamide gel with a concentration gradient of 4% to 20%. The fractionated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. As the primary antibodies, polyclonal antibodies against RecQ1 and BLM, monoclonal antibodies against WRN, RTS, and RecQ5, and β-actin antibody were used. The secondary antibody was an antibody conjugated with peroxidase. The proteins were detected by chemical luminescence using ECL Plus Western Blotting Detection Reagents (Amersham Biosciences).
As a result, each helicase protein was detected by using an antibody specific for the helicase. It was confirmed that, even at the protein level, the expression of these gene products was suppressed when compared to expression in cells into which control RNA (NS) had been introduced (in a 72-hour cell cultivation;
Each of the five types of siRNAs against human RecQ helicases selected in Example 2 was introduced into HeLa cells and TIG3 cells. The viable cell number after 96 hours was determined by a colorimetric assay for cell viability based on the cleavage of the tetrazolium salt WST-8 by mitochondrial dehydrogenase using Cell Count Reagent SF (Nacalai Tesque). The absorbance of formazan dye at 450 nm was determined three hours after addition of the reagent.
The viability of HeLa cells in which control RNA (NS) had been introduced was taken as 100%. The viabilities of HeLa cells in which RTS and RecQ5 siRNAs had been introduced were 96% and 94%, respectively, showing that viability was not largely affected. However, the viabilities of HeLa cells in which WRN, BLM, and RecQ1 siRNAs had been introduced were 10%, 28%, and 5% respectively, indicating a marked reduction in the viability of HeLa cells (
In addition, the present inventors introduced each of the newly designed siRNAs against RecQ1, WRN, and BLM into HeLa cells, and determined the cell viability by MTT assay after 96 hours. The siRNAs used are shown in
The result showed that RecQ1-9, WRN4, and BLM2 reduced the viability to 20%, 5%, and 7%, respectively, when the number of cells treated with non-silencing siRNA (NS), a control, was taken as 100% (
The effects of the three of WRN, BLM, and RecQ1 siRNAs affecting HeLa cell proliferation in EXAMPLE 3, on the proliferation of human lung cancer-derived A549 cells were examined. WRN, BLM, and RecQ1 siRNAs were introduced into A549 cells, respectively, and 72 hours later, WRN, BLM, RecQ1 mRNA expression was quantified using Taqman PCR. The expression level of each mRNA in cells in which control RNA (NS) had been introduced was taken as 100%. Comparison of the respective mRNA expression levels in the cells into which each siRNA had been introduced revealed that the mRNA expression of WRN, BLM and RecQ1 had been suppressed to 8%, 21%, and 4% respectively (
Western blot analysis was used to determine the expression levels of WRN, BLM, and RecQ1 proteins in the A549 cells in which each mRNA expression had been suppressed. As a result, it was confirmed that even at the protein level the expression of these gene products was suppressed when compared to expression in cells into which control RNA (NS) had been introduced (
Each of the siRNAs was introduced into A549 cells, respectively. The viability of the cells after four days was assessed using an MTT assay. When the viability of the cells in which control RNA (NS) had been introduced was taken as 100%, the viabilities of cells into which WRN, BLM, and RecQ1 siRNAs had been introduced were 36%, 60%, and 32% respectively. As a result, the viability of A549 cells was found to be markedly reduced (
Next it was investigated as to whether or not apoptosis caused the above-described reduction in viability of the cells into which the siRNAs had been introduced. WRN, BLM, and RecQ1 siRNAs were introduced into HeLa cells, respectively. After 48 hours, the presence of apoptosis induction was examined by the TUNEL method using in situ cell death detection kit (Roche Diagnostics) following the supplier's protocols.
Apoptosis induction was clearly recognized in HeLa cells where WRN, BLM, or RecQ1 siRNA had been introduced. However, apoptosis induction was not detectable in HeLa cells in which control RNA (NS) had been introduced (
To determine whether the apoptosis-inducing activities of WRN-siRNA, BLM-siRNA, and RecQ1-siRNA in HeLa cells are specific to the tumor cell or may occur in other tumor cell lines as well, the present inventors evaluated the apoptosis-inducing effects of siRNAs by the same procedure as described in Example 6 using eleven types of tumor cell lines derived from various tumor types: HeLa, A549 (lung cancer), MCF7 (breast cancer), HepG2 (hepatic cancer), U2OS (osteosarcoma), U251 (glioblastoma), UACC62 (melanoma), Caki-1 (renal cancer), KP4 (pancreatic cancer), MKN45 (stomach cancer), and LNCaP (prostatic cancer). The results showed that the siRNAs induced apoptosis in several cancer cells but did not induce apoptosis in normal cells.
The results of investigations into the apoptosis-inducing effects of the siRNAs tested in Examples 6 and 7 are summarized in Table 1.
In the Table above, “+++”, “++”, and “+” indicate that apoptosis was induced in 70% or more, 70% to 40%, and 40% to 20% of cells, respectively.
All eleven types of tumor cell lines listed above were sensitive to the siRNAs against the three types of RecQ helicases, although the level of sensitivity varied depending on the siRNA combination and tumor cell line. When considering transfection efficiency, the effect of each siRNA on tumor cells should be more strictly evaluated. However, what deserves emphasis is that WRN-siRNA, BLM-siRNA, and RecQ1-siRNA are effective for a broad range of tumor types and thus can be candidates for excellent anti-tumor agents. In addition, WRN, BLM, and RecQ1 helicases are promising as tools in screenings for anti-tumor agents.
The present inventors tested whether the RecQ helicase siRNA-mediated selective inhibition of tumor cell growth found in vitro was also achieved in vivo in cancer-bearing animal models.
Male BALB/cA nude mice were purchased from CLEA Japan, Inc. A549 cells (5×106 cells/0.1 ml) were subcutaneously injected into the back of the nude mice (six- to seven-week-old). siRNA administration was commenced eight days after subcutaneous injection of the tumor cells.
WRN-siRNA and RecQ1-siRNA were tested for their tumor-suppression effect on the A549 tumor which had been grown in the backs of nude mice (
The result showed that the development of the A549 tumor was markedly suppressed over 32 days after inoculation of RecQ1-siRNA. However, NS-siRNA mixed with polyethylene imine by the same procedure as described above did not have any effect and caused the A549 tumor to increase in volume (
In view of these results, the in vivo efficacy of another siRNA, specifically WRN-siRNA, was evaluated in addition to RecQ1-siRNA using more mice (N=6 for testing each siRNA) by periodically measuring tumor volume for 50 days and plotting the mean tumor volume. WRN-siRNA and RecQ1-siRNA were introduced together with a polyethylene imine carrier into each subject by the same procedure as described in the figure legend for
Furthermore, it was assessed whether or not apoptosis was induced in RecQ1-siRNA- or WRN-siRNA-introduced solid tumor tissues. Tumor tissues were excised 23 days after introduction of RecQ1-siRNA or WRN-siRNA, and fixed with a 4% paraformaldehyde fixative. Then, the tissues were refixed with a neutralized 10% formalin buffer fixative. After a five-day fixation, the tissues were sufficiently washed with water and embedded in paraffin using an automated embedding system. Then, paraffin sections of about 2 μm were prepared, and apoptosis was detected using the ApopTag Peroxidase in situ Apotosis Detection Kit (Serologicals Co.). The result showed that apoptosis-positive cells were found at a significant level in solid tumor tissues to which RecQ1-siRNA or WRN-siRNA had been introduced, as compared to NS-siRNA (
Compounds that inhibit the expression of a RecQ DNA helicase family gene of the present invention or the function of a protein encoded by the gene, have the activity of selectively inducing apoptosis in cancer cells. Pharmaceutical agents comprising such a compound can be used with very few side effects as anticancer agents whose mechanism is based on this apoptosis-inducing activity. The present invention, for the first time, provides anticancer agents which target RecQ helicase using a mechanism based on apoptosis-inducing activity.
Even if certain compounds are found to have apoptosis-inducing activity, it is difficult to use the compounds as pharmaceuticals when their apoptosis-inducing activities in normal cells are unknown. This is because there may be a risk of adverse effects when the compounds have apoptosis-inducing activities in normal cells. In other words, if the compounds have the apoptosis-inducing activities not specific to cancer cells, in general, it is practically difficult to use the compounds as pharmaceuticals. Accordingly, the pharmaceutical agents (the compounds) of the present invention are very practical and effective because their apoptosis-inducing activities are specific to cancer cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-140685 | May 2003 | JP | national |
This application is a continuation of U.S. patent application Ser. No. 10/556,968, filed Jan. 23, 2007, which application is a U.S. national phase of International Application No. PCT/JP04/007145, filed May 19, 2004, which application claims the benefit of Japanese Application No. 2003-140685, filed May 19, 2003, where these applications are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10556968 | Jan 2007 | US |
| Child | 12643938 | US |
