Patent Application
20100144830
The present invention relates to a cancer cell identification marker, a method for identification of cancer cells, cancer cell proliferation inhibitor agents, a composition for cancer treatment which contains one or more of the agents, a method for inhibition of cancer cell proliferation using the composition, a method for treatment of cancer in a human and other mammalian animals, and a method of screening for cancer cell proliferation inhibitor agents.
In recent years, so-called life-style related diseases such as cancer, heart diseases, cerebrovascular diseases, etc., have come to account for a major part of mortality in Japan, instead of infectious diseases like pneumonia and tuberculoses, which were dominant up to the early years of the Showa Era. Among others, lethality rate associated to cancer continuously showed a rapid increase, and has been ranking the top cause of death since around 1980 up to the present. Though some difference are found between sex and age, cancer has been found to develop in any organ or tissue of the body, including the brain, skin, blood, bronchi, lungs, stomach, liver, colon, uterus, breasts, pancreas, prostate gland, etc.
To diagnose cancer in such a variety of organs and tissues, while various measures have been employed, such as X-ray CT, MRI, ultrasonography, etc., identification markers for cancer cells also have come to be in use in clinical laboratory tests in recent years. Cancer cell identification markers are, for example, proteins produced by corresponding types of cancer cells, and they can be used to determine whether cancer cells are present in a sample, and also to know the type of the cancer cells which occur. In performing diagnosis of cancer based on a cancer identification marker, it is enough just to sample some cells from an organ or tissue to be diagnosed and then to examine the cells for any presence of that marker, by one of any convenient means, like antibody. It thus contributes to the convenience and reliability of the test, and also eases the burden on the patient as well. However, though some cancer identification markers can be used for detection of several different types of cells, the other, majority of markers are exclusively for a certain specific type of cancer cells. Thus it is in general necessary to use different identification markers in accordance with different organs or tissues to be examined. This increases the costs in preparing detection reagents corresponding to various identification markers, and, moreover, requires a change reagents and modification of the procedure during a detection process, thereby resulting as a whole in somewhat greater burden on the person who are engaged in diagnosis.
That there are a number of different types of cancers has also been posing a problem in cancer treatment by administration of drugs (chemotherapy). Namely, as its mechanism of development generally is intrinsic to each type of cancer, it is required in conducting chemotherapy to select the most suitable drugs for the type of the cancer about to be treated. Therefore, a wide range of drugs must be in stock to cope with a various types of cancers, and this poses a substantial burden due to cost increases and to the workload required in preparing drug formulations and giving treatment (see Non-patent Documents 1-8). Furthermore, there is a more serious problem in chemotherapy, that is, acquisition of resistance by cancer cells to the drugs that have been employed, and consequent reduction in efficacy of the treatment with them. In the etiology of cancer, a defect in cell differentiation is considered to be the major factor (see Non-patent Document 3). Thus, there would be possibilities of blocking cancer cell proliferation if a defect in cell differentiation (dedifferentiation) could be prevented.
On the other hand, a phenomenon called RNA interference (RNAi) has now been found in living organisms in common, from plants, insects, protozoa to mammalian animals, etc. RNAi is the phenomenon that a double-stranded RNA (dsRNA) which consists of a short sequence homologous to the mRNA produced by a target gene and a sequence complementary to the former, induces decomposition of that mRNA in the cell, thereby inhibiting the expression of the target gene.
It is assumed that regulation of the expression of a gene is done based on the formation of siRNA (small interfering RNA) and miRNA (microRNA, single-stranded RNA consisting of 21-23 bases) by the action of an enzyme “dicer”, an endoribonuclease (see Non-patent Document 9). It is thought that in animals siRNAs take part in the cleavage of their respective target mRNAs (see Non-patent Document 10), and that miRNAs prevent the translation of their respective target mRNAs (see Non-patent Document 11). It has been found that either an siRNA or miRNA forms a complex with common proteins to convert them into an active form, which then was identified as RNA-induced silencing complex (RISC) containing as components a plurality of such proteins (see Non-patent document 12). So far, hundreds of miRNAs have been isolated and identified from animals and plants, and the physiological functions of at least four animal-derived miRNAs have been elucidated.
In 2001, it was reported that a 21-base, short double-stranded RNA induced RNAi effects more efficiently than others in mammalian animals. RNAi thus has been expected to be useful as a therapeutic means for intractable disorders such as cancer, viral diseases and neovascularization. In particular, siRNA has been found to be capable of efficiently decomposing and eliminating a certain mRNA at a very low concentration (1 nM), suggesting the presence of some enzymatic amplification.
Long double-stranded RNAs induce interferon synthesis and non-specific mRNA decomposition (interferon response). On the other hand, short dsRNAs inhibit also the expression of other genes than the one whose expression is intended to be suppressed in the case where their sequences are the same as or highly homologous to the mRNA of those genes (off-target effect).
Considering these, it is desirable that no such mRNA should exist that a given siRNA strongly binds to, among the mRNAs derived from other genes than the one whose expression is intended to be suppressed, or that, even if such a mRNA exists, it is the mRNA derived from a gene having a function similar to the very gene suppression of whose expression is intended. It is because, in such cases, it can be prevented that the siRNA should exhibit a wide-ranging non-specific suppressive effect on other functions than is intended.
[Non-patent Document 1] Zhang, C. L., McKinsey, T. A. & Olson, E. N. Association of class II histonedeacetylases with heterochromatin protein 1: potential role for histonemethylation in control of muscle differentiation. Mol Cell Biol 22, 7302-12 (2002)
[Non-patent Document 2] Cammas, F., Herzog, M., Lerouge, T., Chambon, P. & Losson, R. Association of the transcriptional corepressor TIF1beta with heterochromatin protein 1 (HP1): an essential role for progression through differentiation. Genes Dev 18, 2147-60 (2004)
[Non-patent Document 3] Tenen, D. G. Disruption of differentiation in human cancer: AML shows the way. Nat RevCancer 3, 89-101 (2003)
[Non-patent Document 4] Gilbert, N. et al. Formation of facultative heterochromatin in the absence of HP1. EmboJ 22, 5540-50 (2003)
[Non-patent Document 5] Olins, D. E. & Olins, A. L. Granulocyte heterochromatin: defining the epigenome. BMCCell Biol 6, 39 (2005)
[Non-patent Document 6] Popova, E. Y., Claxton, D. F., Lukasova, E., Bird, P. I. & Grigoryev, S. A. Epigeneticheterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood. Exp Hematol 34, 453-62 (2006)
[Non-patent Document 7] Arney, K. L. & Fisher, A. G. Epigenetic aspects of differentiation. J Cell Sci 117, 4355-63 (2004)
[Non-patent Document 8] Fraga, M. F. et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histoneH4 is a common hallmark of human cancer. Nat Genet 37, 391-400 (2005)
Against the above-mentioned background, an objective of the present invention is to provide a means which is commonly applicable for detection of cancer cells of a variety of origins.
Another objective of the present invention is to provide cancer cell proliferation inhibitor agents and compositions for cancer treatment both of which are commonly applicable to a wide variety of cancers.
Still another objective of the present invention is to provide a novel method of screening for proliferation inhibitor agents of a wide variety of cancers.
In the study in search of a method for identification of cancer cells and for treatment of cancer, the present inventors focused on the heterochromatin protein 1 (HP1) family, which are proteins involved in chromatin packaging and gene silencing. There are three homologues (α, β, γ) of the HP1 family in mammals, of which HP1α and β are present in heterochromatin, while HP1γ is present both in heterochromatin and euchromatin. Therefore, though it is thought to differ functionally from HP1α and HP1β, the function of HP1γ has not been known yet (Non-patent Document 1, Non-patent Document 2). The present inventors performed a study focusing on the relation between HP1γ and cell differentiation.
As a result, it was discovered that HP1γ protein, which is detectable in undifferentiated normal cells, gradually decreases in its expression levels as the cells differentiate, and becomes no longer detectable in fully differentiated normal cells; and further that HP1γ protein has not been lost but is expressed in various cancer cells, i.e., undifferentiated abnormal cells. On the basis of this finding, the present inventors have come to find that cancer cells can be distinguished from normal cells by utilizing HP1γ protein as an identification marker, i.e., by using its expression as an index. Furthermore, the present inventors surprisingly found that though its mechanism is yet unknown, proliferation of cancer cells can be potently inhibited by inhibiting the expression of HP1γ gene in those cells. The present invention was completed on the basis of these discoveries.
Thus, the present invention provides what follows.
1. A method for identifying a cell presented for examination as either being a cancer cell or a non-cancer cell, comprising the steps of
detecting expression of HP1γ in the cell presented for examination, and
identifying the cell as being a cancer cell if expression of HP1γ is detected in the cell, and as being a non-cancer cell if no expression of HP1γ is detected in the cell.
2. The method according to 1 above, wherein the cancer cell is an epithelial cancer cell and/or a non-epithelial cancer cell of a mammalian animal including a human, and wherein the HP1γ is HP1γ of the mammalian animal.
3. The method according to 1 or 2 above, wherein the cell presented for examination is a human cell and the HP1γ is human HP1γ.
4. A cancer cell proliferation inhibitor agent consisting of an inhibitor compound of HP1γ gene expression.
5. The cancer cell proliferation inhibitor agent according to 4 above, wherein the cancer cell is an epithelial cancer cell and/or a non-epithelial cancer cell of a mammalian animal including a human, and wherein the HP1γ is HP1γ of the mammalian animal.
6. The cancer cell proliferation inhibitor agent according to 4 or 5 above, wherein the inhibitor compound of HP1γ gene expression is an siRNA specific to HP1γ gene or an antisense DNA specific to HP1γ gene.
7. The cancer cell proliferation inhibitor agent according to one of 4 to 6 above, wherein the cancer cell is a human cancer cell, and wherein the HP1γ gene is human HP1γ gene.
8. The cancer cell proliferation inhibitor agent according to 7 above, wherein the inhibitor compound of HP1γ gene expression is an siRNA comprising at least one of the double-stranded RNAs each of which comprises a corresponding RNA strand set forth in the 5′ to 3′ direction in the following Tables 2A to 1F;
9. The cancer cell proliferation inhibitor agent according to 8 above, wherein the double-stranded RNAs are selected from the group consisting of the double-stranded RNAs set forth as #5, #17, #35, #62, #89, #101, #102, #103, #104, #105 and #106 in Tables 2A to 1F.
10. The cancer cell proliferation inhibitor agent according to 8 above, wherein the double-stranded RNAs are selected from the group consisting of the double-stranded RNAs set forth as #17, #62 and #89 in Tables 2A to 1F.
11. The cancer cell proliferation inhibitor agent according to one of 8 to 10 above, wherein the siRNA has two-base overhangs on its both ends.
12. The cancer cell proliferation inhibitor agent according to one of 8 to 11 above, wherein each of the overhangs is on the 3′ end of each of the strands forming the double-stranded RNA.
13. A composition for the treatment of cancer in a mammal including a human comprising, in a pharmaceutically acceptable carrier, one or more of the cancer cell proliferation inhibitor agents according to one of 6 to 12 above.
14. The composition for the treatment of cancer according to 13 above, wherein the cancer is cancer in a human, the HP1γ gene is human HP1γ gene, and the inhibitor compound of HP1γ gene expression is an siRNA specific to human HP1γ gene.
15. The composition for the treatment of cancer in a human comprising, in a pharmaceutically acceptable carrier, one or more of the cancer cell proliferation inhibitor agents according to one of 8 to 12 above.
16. A method for the treatment of cancer in a mammal including a human comprising administering an effective amount of one or more of the cancer cell proliferation inhibitor agents according to one of 6 to 12 above, in a pharmaceutically acceptable carrier, to the mammal including a human in need thereof.
17. A method for the treatment of cancer in a human comprising administering an effective amount of one or more of the cancer cell proliferation inhibitor agents according to one of 8 to 12 above, in a pharmaceutically acceptable carrier, to the human in need thereof.
18. Use of one of the cancer cell proliferation inhibitor agents according to 8 to 12 above, for the production of a composition for the treatment of human cancer.
19. A method of screening for cancer cell proliferation inhibitor agents comprising the steps of;
bringing part of cancer cells into contact with candidate compounds,
separately detecting expression of HP1γ gene in those cancer cells which were brought into contact with the candidate compounds and in those cancer cells which were not brought into contact with a candidate compound,
determining whether or not HP1γ gene expression was inhibited in the cancer cells which were brought into contact with the candidate compounds by comparing the amount of expression of HP1γ gene in the cancer cells which were brought into contact with the candidate compounds with that in the cancer cells which were not brought into contact a candidate compound,
selecting, as cancer cell proliferation inhibitor agents, those candidate compounds which were brought into contact with those cancer cells in which inhibition of the expression was found.
The present invention as identified above can be used in pathological examination and clinical diagnosis to distinguish not only some particular types cancers but also a wide variety of cancer cells from normal cells. Further, the present invention can be used to inhibit proliferation of not only some particular cancers but also of a wide variety of cancer cells, and therefore to treat cancers in a mammal including a human, in particular human cancers. Furthermore, the present invention enables screening for compounds which inhibit not only some particular cancers but also a wide variety of cancer cells.
The cancer cell identification marker according to the present invention is characterized in that it comprises HP1γ protein. And the method of identification of cancer cells according to the present invention is characterized in that it detects HP1γ protein in the cells. The nucleotide sequence for HP1γ gene is registered with GenBank accession NM—016587 (SEQ ID NO:125), within which the sequence consisting of 152-703 is the coding sequence (CDS) for HP1γ protein, and the sequence for HP1γ protein is registered with GenBank accession NP—057671.2 (SEQ ID NO:126).
According to the present invention, it is possible to detect HP1γ protein contained in the cell and thereby identify the cells in which HP1γ protein occurs as being cancer cells, thereby allowing to distinguish between cancer cells normal cells (normally differentiated cells).
There is no particular limitation regarding the types of cancer cells which can be identified by the method according to the present invention, and they include epithelial cancer cells, non-epithelial cancer cells, as well as those of solid and non-solid cancers. Cancers consisting of epithelial cancer cells include, for example, lung cancer, breast cancer, gastric cancer, colorectal cancer, uterine cervical cancer, uterine cancer (e.g., laryngeal cancer, pharyngeal cancer, lingual cancer, etc.), colon cancer, squamous cell carcinoma, adenocarcinoma and the like; cancers consisting of aforementioned non-epithelial cancer cells (sarcoma) include, for example, liposarcoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, angiosarcoma, and the like. Cells of other cancers also can be identified by the present invention, including, for example, basalioma, Merkel cell carcinoma, myxoma, non-small cell tumor, oat cell tumor, papilloma, bronchiolar tumor, bronchial tumor; leukemia such as B cell tumor, mixed cell tumor, null cell tumor, T cell tumor; HTLV-II related tumors such as lymphocyte acute leukemia, lymphocytic chronic tumor, mastocytoma, and myeloma; histiocytic malignant tumors such as Hodgkin's tumor, non-Hodgkin's lymphoma, malignant melanoma, mesothelioma, Ewing sarcoma, periosteoma, adenofibroma, adenolymphoma, craniopharyngioma, dysgerminoma, mesenchymoma, mesonephroma, ameloblastoma, cementoma, odontoma, thymoma, adenocarcinoma, cholangioma, cholesteatoma, cylindroma, cystic adenoma, cystic tumor, granulosa cell tumor, ovarian tumor, hepatic cancer, syringocarcinoma, islet cell tumor, Leydig cell tumor, Sertoli cell tumor, theca cell tumor, leiomyoma, myoblastoma, ependymoma, neural myoma, glioma, medulloblastoma, periosteoma, neurilemma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, nonchromaffin paraganglioma, angiokeratoma, hematolymphangioma, sclerosing hemangioma, glomus tumor, angioendothelioma, lymphangioma, lymphangiomyoma, lymphagiosarcoma, pineocytoma, carcinosarcoma, colorectal sarcoma, neurofibroma and the like.
It is not limited how to detect HP1γ protein in the cells. Conventional western blotting may be employed, for example. More specifically, there is a method, for example, in which proteins are extracted from the cells to be examined which have been isolated from the living body, then are allowed to react with an HP1γ-specific antibody, and the antigen-antibody complex thus formed is detected. Examples of other methods for analysis include ELISA, immunohistochemical staining, flowcytometory, etc.
The proliferation inhibitors according to the present invention, as aforementioned, are cancer cell proliferation inhibitors which are characterized in that they contain one or more HP1γ gene expression inhibitors. As will be mentioned later, since lack of expression of HP1γ protein is closely correlated with differentiation of cells, and HP1γ protein thus is considered to work as a lock that blocks differentiation of cells, the proliferation inhibitors according to the present invention is expected to be useful for inducing cancer cells to differentiate.
In the present invention, there is no other particular limitation in the selection of aforementioned expression inhibitor compounds as far as they target the process of HP1γ gene expression and inhibit it. For example, it may inhibit any step of the process, which includes the steps of transcription of DNA into RNA, splicing of pre-mRNA (hnRNA) to form mRNA, translation of mRNA into HP1γ protein, and the like. Typical examples of such expression inhibitor compounds include siRNAs and antisenses, of which particularly preferred is siRNAs.
“SiRNA” (small interfering RNA) is a short double-stranded RNA that mediates RNA interference and generally a low molecular-weight double-stranded RNA which is 21 to 27 base long including overhangs consisting of some 2 bases (2 mer) on the both ends.
The aforementioned siRNAs that inhibit HP1γ expression are those siRNAs which contain sequences complementary to the transcript of HP1γ presented as SEQ ID NO:125, and preferably double-stranded siRNAs consisting of 21-base strands each of which consists of 19 bases that forms the double-stranded portion of the RNA, and a 2-base (2 mer) overhang on one end of each strand. In general, the overhangs are preferably 3′-end overhangs.
The term “3′-end overhang” means a nucleotide portion that projects on each 3′-end of a double-stranded RNA which is formed of two RNA strands comprising complementary sequences and paired with each other. Examples of 2-base sequences which form the aforementioned 3′-end overhangs include, but are not limited to, TT (-thymine-thymine), AU (-adenine-uracil), AG (-adenine-guanine), etc. The overhang portions of the sense strand of siRNA (having the same nucleotide sequence as that of the target transcript) and the antisense strand of it (having the complementary sequence to that of the target transcript) may be of the same or different sequences with each other. It may be, for example, that the overhang of the sense strand is AG, while the overhang of the antisense strand is AU, or that the overhang of the sense strand is AU, while the overhang of the antisense strand is AG.
Further, the 2-base sequences that may form 3′-end overhangs are not limited to the above-mentioned sequences, but they may be any one of naturally occurring nucleotide bases (adenine, guanine, thymine, cytosine, and uracil) or any other naturally occurring or artificial modified bases known in the art, as far as they do not substantially affect the RNAi effect. The nucleotides forming the 3′-end overhang may generally be, but are not limited to, ribonucleotides, but deoxyribonucleotides, modified ribonucleotides, or other nucleotide analogues, as far as they do not substantially affect the RNAi effect. Still further, in the present invention, the aforementioned double stranded siRNAs, when needed, may have 5′-end overhangs instead of 3′-end ones.
Thought not limited to them, candidate sequences that may be employed as the aforementioned 19-base pair portion may be the sequences presented in Table 1 (it should be noted that only the sense sequences are presented). In the table, the column “Gene” indicates the position of the target base (corresponding to the 5′-end of the sense strand of siRNA) in the transcript (mRNA) of HP1γ gene set forth as SEQ ID NO:125 presented in Table 1 (the headmost base is assigned number 1), and the column “CDS” indicates the position number of the target base of the siRNA as counted from the headmost base of the coding sequences for the amino acids of HP1γ protein. In the table, the sequences up to the sequence presented at Gene 684 are in the coding region, and the sequences following the sequence presented at Gene 827 are in the non-coding region.
Within the nucleotide sequence of the transcript (mRNA) of HP1γ including the nucleotide sequences listed in Table 1, every 19-base fragment sequence starting from each position was examined as to whether the short RNA of 19-base pairs having the same sequence as the fragment sequence is (a) expected to have desired potent RNAi effects on HP1γ mRNA, and whether (b), in order for avoiding the problem of off-target effects, the short RNA is highly specific to HP1γ mRNA, i.e., has only a little probability of binding to the nucleotide sequences of other genes than HP1γ. Those sequences that met the both purposes were picked out.
In the above, analysis of RNA interference with each of the sequences was done based on their Tm value, GC content, and the distribution of particular bases. Tm (melting temperature) is the temperature at which 50% of a double-stranded nucleotide will be dissociated to single-stranded nucleotides, and can be calculated according to a well known method (see e.g., Breslauer K J et al. (1986) Proc. Natl. Acad. Sci. USA. 83: 3746-3750., Rychlik W et al. (1990) Nucleic Acids Res. 18, 6409-6412. or Owczarzy R et al. (1997), Biopolymers 44: 217-239). In consideration of the distribution of particular bases, the disclosure in WO 2006/060454 (Title of invention: “Methods of Designing Small Interfering RNAs, Antisense Polynucleotides and other Hybridizing Polynucleotides”) was followed.
Selection of sequences which are highly specific to HP1γ mRNA was conducted by searching, within the sequences of the genes that are registered in GenBank (either the genes actually identified or hypothetical genes which were only predicted based on computer analysis), for those containing a sequence which is either fully identical to (19/19), differs only in a single base from (18/19), or differs only in two bases (17/19) from the sense or the antisense strand of each siRNA that has a double-stranded portion made of any one of the sequences presented above as a sense strand, and an antisense strand having a sequence complementary to it. Heaviest regard was given to the fact that no other gene than HP1γ is found that contains a sequence fully identical (19/19) to it, and less heavy regard was given to the fact that a sequence exists which differs only in one base (18/19) or in two bases (17/19), in the order. This is because as the number of the bases increases at which two sequences do not match, the probability of their forming a pair reduces rapidly, therefore making any off-target effect weaker or negligible. Again, the less heavy regard was given to such genes that are not HP1γ gene but were registered as being similar to HP1γ, for they were considered to be performing similar functions to that of HP1γ. Furthermore, the less heavy regard was given to hypothetical genes whose existence was predicted merely on computer, since their real existence had not been verified.
The following Tables 2A to 2F shows (the double-stranded portions) of 106 siRNA candidates selected through the process described above. In each double-stranded RNA portion in the Tables, the upper one is the sense sequence and the lower one the antisense sequence, both of which are presented with their 5′-end placed at the top. Further, in the Tables, the numbers in brackets indicate the number of registrations of a gene which, though being one and same gene, is registered with different accession numbers.
On both ends of each double-stranded RNA shown in Tables 2A to 2F may be attached desired overhang sequences (e.g., TT, UU and the like), and an siRNA thus obtained exhibits a strong RNA inhibitory effect, with minimized off-target effect.
Furthermore, among the 19-base RNA listed above in Tables 2A to 2F, those which are particularly preferred both from the viewpoint of their high RNAi effect and high HP1γ specificity are the following eleven siRNAs: #5 (sense sequence No. 41, antisense sequence No. 196), #17 (sense sequence No. 49, antisense sequence No. 208), #35 (sense sequence No. 56, antisense sequence No. 226), #62 (sense sequence No, 1, antisense sequence No. 253), #89 (sense sequence No. 74, antisense sequence No. 280), #101 (sense sequence No. 83, antisense sequence No. 292), #102 (sense sequence No. 84, antisense sequence No. 293), #103 (sense sequence No. 88, antisense sequence No. 294), #104 (sense sequence No, 89, antisense sequence No. 295), #105 (sense sequence No. 191, antisense sequence No. 296), and #106 (sense sequence No. 95, antisense sequence No. 297).
Namely, siRNAs based on the eleven double-stranded RNA portions presented above are highly specific to HP1γ mRNA, as is seen in the following Tables 3A to 3K, which presents the result of a search of genes which include sequences that are either fully identical to (19/19), differ only in one base (18/19) from, or differ in two bases (17/19) from them, and they therefore are particularly unlikely to cause any off-target effect. One may use either any one of these siRNA alone or two or more of them concomitantly (e.g., in a mixture, or through simultaneous administration of them). Among these eleven, those having particularly great specificity to HP1γ mRNA are those based on #17 (sense sequence No. 49, antisense sequence No. 208), #62 (sense sequence No. 1, antisense sequence 253), or #89 (sense sequence No. 74, anti sense sequence No. 280), which are most preferred.
In Tables 3A to 3K, “Accession#” indicates Accession numbers of the sequences in GenBank, and under “NM—016587”, which is the accession number of human HP1γ, are listed accession numbers of matching sequences that were hit in the search. “GeneID#” indicates identification numbers assigned to the genes, and “11335” in the Tables indicates human HP1γ gene itself. “Predicted” means that the indicated gene is hypothetical one which is predicted on computer. Further, “CBX3” and “chromosome homolog 3 (HP1 gamma homolog, Drosophila)” are the official code and the official name, respectively, assigned to HP1γ by NCBI.
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens GA binding protein
Homo sapiens jumping translocation
Homo sapiens galanin-like peptide
Homo sapiens similar to RIKEN
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens SID1 transmembrane
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens BMS1 homolog,
Homo sapiens zinc and ring finger 2
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens myosin, heavy chain
Homo sapiens myosin, heavy chain
Homo sapiens ankyrin repeat and
Homo sapiens OTU domain
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens TAF9 RNA polymerase II,
Homo sapiens TAF9 RNA polymerase II,
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens transmembrane protein 59
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens synaptotagmin XIV
Homo sapiens chromosome 10 open
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens chromobox homolog
Drosophila) (CBX3),
Homo sapiens RNA (guanine-9-)
There is no particular limitation as to how the above siRNAs are synthesized, and they thus may be synthesized either in vitro, chemically or enzymatically, or in vivo as well. It is preferred, however, that they are chemically synthesized by a method known in the art. Synthesized siRNAs are advantageous, because they, for example, allow easy adjustment of their concentration. They are also advantageous in respect of safety for they allow easy prevention of their contamination. For preparing an siRNA having overhangs and comprising the double-stranded sequences set forth as SEQ ID NO:1, for example, an RNA strand comprising the sequence set forth as SEQ ID NO:1 and having a 2-base overhang at the 3′-end thereof, and an RNA strand comprising the sequence that is complementary to the sequence set forth as SEQ ID NO:1 and having a 2-base overhang at the 3′-end thereof, are separately synthesized. Then the two RNA strands are allowed to form a pair to give a double-stranded siRNA with overhangs. Before use, as needed, they are preferably purified as desired by a method known in the art.
In the case where an antisense (single-stranded DNA) is used according to the present invention to suppress the expression of HP1γ gene, there is no particular limitation in choosing an antisense except than it has a nucleotide sequence which is complementary to the nucleotide sequence of HP1γ gene, and inhibits the expression of HP1γ gene. Such an antisense, as aforementioned, may inhibit either splicing into mRNA or translation into the HP1γ protein. There is no particular limitation as to the length of such antisenses, but it is preferably, for example, 10-40 mer, more preferably 17-30 mer, and still more preferably 20-30 mer in length. Examples of antisenses that may be employed according to the present invention are listed below.
Since the cancer cell proliferation inhibitors, especially the siRNAs according to the present invention act to potently inhibit occurrence of “loss of differentiation”, which is common to a variety of cancer cells, the types of cancers which are to be treated with them are not limited, but a wide variety of cancers, as aforementioned, may be treated, regardless of whether they are epithelial or non-epithelial cancers, or whether they are solid or non-solid cancers. Further, the cancer cell proliferation inhibitors according to the present invention can be used for the treatment of cancers in mammalian animals including human, and, among others, of human cancers. Cancer cells whose proliferation can be inhibited are not limited but are the same as the aforementioned cancer cells that can be identified with the method according to the present invention.
The compositions for cancer treatment according to the present invention are medicinal compositions for the treatment of cancer, and comprises one of the aforementioned cancer cell proliferation inhibitors according to the present invention, inter alia, siRNAs specific to HP1γ in a pharmaceutically acceptable carrier well known in the art. Such a medicinal composition administered to a patient can inhibit proliferation of cancer cells and thereby potently suppress the progression of cancer. There is no specific limitation as to the types of cancers which may be treated, but they are the same as the aforementioned cancer cells that can be identified by the method according to the present invention. Furthermore, because the cancer cell proliferation inhibitors according to the present invention, inter alia siRNAs specific to HP1γ, are highly specific to cancer cells compared with other conventional agents which so far have been available, they enable either to minimize or eliminate the probability of affecting normal cells (non-cancerous cells), thereby remarkably reduce any risks of side effects.
The medicinal composition according to the present invention may further contain one or more cell differentiation-inducing agents. Addition of differentiation inducing agents enables, for example, to further promote the differentiation of the cancer cells and to effectively induce their apoptosis while suppressing proliferation of cancer cells with the aforementioned proliferation inhibitor agents. As cell differentiation-inducing agents mentioned above, those which are known in the art may be employed without particular limitation, and examples of them include, for example, adipose differentiation inducers such as thiazolidine derivatives (PPARγ-ligands) and the like. In addition, one or more anticancer drugs known in the art may also be contained, for they can further suppress proliferation of cancer cells and promote cell death. They include, for example, taxol, cisplatin, herceptin, 5-FU, glivec, rituxan, iressa, etc.
The medicinal composition according to the present invention may further contain a pharmaceutically acceptable carrier. Examples of such pharmaceutical carriers include, but not limited to, those carriers which can enhance the efficiency of penetration of the aforementioned expression inhibitors into target sites, tissues or cells (such as liposome, cation liposome, etc.). Examples of pharmaceutical forms of the medicinal composition according to the present invention include, but not limited to, an injection, cream, ointment, tablet, suspension, or the like. Examples of the way of administration include, but not limited to, injection, and oral, topical, intranasal and intrarectal administration, etc.
The invention provides a method for inhibition of cancer cell proliferation comprising bringing cancer cells into contact with a proliferation inhibitor according to the present invention. It may also be the medicinal composition according to the present invention that are brought into contact with cancer cells.
There is no particular limitation as to the amount of the proliferation inhibitors according to the present invention to be applied to cancer cells, but it may be determined as desired in accordance with the types and amount of the expression inhibitor compounds contained therein. In the case where the expression inhibitor compound is an siRNA, it is applied preferably in an amount of 1-100 nmole per 1×104 cells, more preferably 5-50 nmole, and most preferably 5-10 nmole. In the case where the expression inhibitor compound in an antisense, it is applied preferably in an amount of 1-100 μmole per 1×104 cells, more preferably 5-50 μmole, and most preferably 5-20 μmol.
In practicing the method for inhibition of proliferation according to the present invention, it is sufficient to bring one of the aforementioned cancer cell proliferation inhibitor agents into contact with cancer cells. There is no particular limitation as to how this is done, which, for example, may be determined in accordance with the type of the expression inhibitor compound contained in the proliferation inhibitor agent. In the case where the proliferation inhibitor agent is an siRNA, it may be brought into contact with the cancer cells together with a transfection reagent known in the art to introduce the siRNA into the cells. In the case where an antisense is employed, the same procedure may be followed. The medicinal compositions according to the present invention may also be used in the same manner.
The proliferation inhibitor agents and the medicinal compositions according to the present invention can not only be applied to cancer cells or tissues in vitro, but also be used to treat cancer patients. Namely, the proliferation inhibitor agents or the medicinal compositions according to the present invention may be administered to cancer patients to bring the cancer cells into contact with the expression inhibitor compounds. According to this method, reduction of side effects is expected, for example, on normal cells, the cells that have been fully differentiated. The reason for this is as follows. As will be shown later in Examples, HP1γ protein cannot be detected in normal, differentiated cells. Namely, even if administered with a proliferation inhibitor agent according to the present invention, normal, differentiated cells already lacks expression of HP1γ protein. Therefore, normal, differentiated cells, even if brought into contact with a proliferation inhibitor agent according to the present invention, are hardly thought to be affected by any suppression of expression of HP1γ protein.
Examples of patients include humans, mammalian animals other than humans, and other animals. There is no particular limitation as to the way in which the proliferation inhibitors and medicinal compositions according to the present invention are administered, and in accordance with the site to be treated, such a way of administration may be chosen as injection, topical application, or surgical treatment to implant the inhibitors in the affected site or under the skin. A delivery system known in the art may also be employed in accordance with the site where administration is to be made. Further, as needed, it is also possible to construct an siRNA expression vector which will express an aforementioned siRNA, and thus to utilize a delivery system based on the technique of gene therapy.
The screening method according to the present invention is a method of screening for cancer cell proliferation inhibitor agents, which comprises the steps of; bringing HP1γ gene into contact with candidate compounds, detecting expression of HP1γ gene, and selecting, as cancer cell proliferation inhibitor agents, those candidate compounds which were found to inhibit the expression of HP1γ gene. Employing this method, it becomes possible to construct novel agents which inhibit expression of HP1γ gene. There is no further limitation as to such compounds insofar as they inhibit expression of HP1γ gene (expression of HP1γ protein), and they may be polynucleotides (oligonucleotides), proteins, or low molecular-weight compounds.
Examples of the present invention then will be described below together with Comparative Examples. The present invention, however, is not limited by the Examples and Comparative Examples. Unless otherwise noted, “%” means “w/v %”.
3T3-L1 mouse preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% bovine serum (BS). Colon cancer cells DLD-1, HCT116 and HT-29; lung cancer cells NCI-H23; and gastric cancer cells MKN1 and MKN28 were cultured in RPMI1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS); uterine cervical cancer cell HeLa and SiHa in DMEM medium supplemented with 10% FBS. To any of the media used in Examples were added 1% penicillin and 1% streptomycin. The condition of the culture was set at 37° C. in 5% CO2 atmosphere.
Antibodies employed were; mouse monoclonal antibodies to HP1α, HP1β and HP1γ, respectively (Chemicon); rabbit polyclonal antibody to Met3H4K20 (Upstate); rabbit polyclonal antibodies to AceH3K18, Met2H3R17, Met2H3K4, AceH4K12, AcetH4K16, AceH3K9, Met2H3K9 and AceH4K8, respectively (Abeam); and anti-GAPD antibody to GAPDH (Santacruz) as a control.
The relation between cell differentiation and expression of HP1γ protein was examined.
(1) Expression of HP1γ Protein Concurrent with Cell Differentiation
3T3-L1 mouse preadipocytes were cultured to confluence in DMEM medium supplemented with 10% BS, and their differentiation into adipocytes were induced by culturing them under the following condition. First, the preadipocytes were transferred to a first differentiation-inducing medium (10% FCS, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone), and cultured for two days. The cells then were transferred to a second differentiation-inducing medium (DMEM medium contain 10% FBS and 10 μg/mL insulin), in which they were cultured for two days, and then to a third differentiation-inducing medium (DMEM medium containing 10% FBS), in which culture was performed for a predetermined length of time (for; 0 day, 3 days, 6 days, 9 days, or 14 days) for induction of differentiation. 3-Isobutyl-1-methylxanthine, dexamethasone, and insulin are differentiation inducers (adipose reagents), and, by being cultured in the presence of these differentiation inducers, 3T3-L1 cells generally will change their shape from a one that is characteristic of a fibroblast-like phenotype into a round shape, and form lipid droplets, which will accumulate in the cells. On the other hand, human preadipocytes were cultured in a preadipocyte medium (DMEM/Ham's F12 (1:1) Gibco BRL, 10% FBS) to confluence, then cultured in a forth differentiation-inducing medium (3% FBS, 1 nM dexamethasone, 100 nM human insulin, 0.25 mM 3-isobutyl-1-methylxanthine, 10 μM PPARγ agonist) for two days, and then transferred to a fifth differentiation-inducing medium (3% FBS, 1 nM dexamethasone, 100 nM human insulin), in which they were cultured for a predetermined length of time (treatment for differentiation induction for; 0 day, 3 days, 6 days, 9 days, or 14 days). Regarding the term during which the treatment for differentiation induction was performed in the above, day 0 was assigned to the point of time in the case of 3T3-L1 mouse preadipocytes, when the first differentiation-inducing medium was added, and, in the case of human preadipocytes, to the point of time when the forth differentiation-inducing medium was added, respectively.
The cultured cells thus prepared then were examined for the expression of HP1 proteins (α, β, γ) by western blotting.
First, proteins were extracted from the cultured cells by a method known in the art, and suspended in an SDS-PAGE buffer. The suspension thus prepared was heat-treated to denature the proteins and then applied, in an amount containing 10 μg proteins, to 15% SDS-PAGE for electrophoresis. The proteins thus electrophoresed were transferred to a membrane (Immobilon™ membrane; mftd. by Millipore) by semi-dry method, and antibodies to the proteins to be detected (HP1α, HP1β, HP1γ) were applied to the membrane for letting the proteins immobilized on the membrane undergo an antigen-antibody reaction with the antibodies. Thus obtained antigen-antibody complexes were detected on a detector (LAS3000™ mini: mftd. by Fuji Film) following exposure of an autoradiography film after causing chemical luminescence using a kit containing an enzyme-labeled secondary antibody and a fluorescent reagent (product name: ECL plus: mftd. by Amersham). As a control, the expression of GAPDH protein was examined in the same manner.
The results are shown in
As shown in the above figure, of HP1 proteins, HP1α and HP1β proteins were found expressed at all the stages (from day 0 to day 14) of differentiation into adipoeytes. In contrast, the expression of HP1γ protein was found reduced on day 9 of treatment for differentiation induction, and no longer detectable on day 14. The fact that the amount of expressed HP1γ protein thus reduced as differentiation proceeded and became hardly detectable in differentiated cells suggests that the decline of HP1γ protein expression is playing some role in cell differentiation. Although it has been reported that the all three HP1 homologues have reduced in their amount in fully differentiated cells (e.g., blood cells) (Non-patent documents 4, 5, and 6), HP1γ alone, of the HP1 homologues, as mentioned above, is thought to play a role in differentiation. This was first revealed by the present inventors.
(2) Effect of Ectopic Expression of HP1γ Protein on Cell Differentiation
Examination was performed to find out whether ectopic expression of HP1γ protein has some effect on cell differentiation.
(2-1) Establishment of an HP1γ-Expressing Cell Line
As follows, starting with 3T3-L1, a cell line was established in which the expression of HP1γ protein is constantly induced with mifepristone.
Using total RNA form HCT116 cells, which were colon cancer cells, as a template, reverse transcription PCR was carried out to amplify HP1γ cDNA. HP1γ cDNA thus amplified was directly subcloned into the pCR2.1 vector (mftd. by Invitrogen) to prepare a recombinant vector pCRHP1γ. The pCRHP1γ was cleaved with EcoRI, and the EcoRI fragment containing HP1γ cDNA was subcloned into the EcoRI site of the pGene-V5 (mftd. by Invitrogen) to prepare pGHP1γ. Then, to 3T3-L1 placed in a cell culture dish of 100 mm in diameter were introduced 3 μg of a regulating plasmid (pSwitch vector) and 7 μg of pGHP1γ were introduced. As a control, the empty vector pGene-V5 was introduced instead of pGHP1γ. Their introduction was performed using DoFect GT1 transfection reagent (mftd. by Dojin) according to the product's manual. Selection of the cells that harbored these introduced plasmids was done using 400 μg/mL of Zeocin (registered trademark, mftd. by Invitrogen) and 50 μg/ml, of hygromycin (product name, mftd. by Invitrogen). The cells thus obtained was a mifepristone-inducible HP1γ gene expression cell line (hereinafter referred to as “plasmid-introduced cells”).
(2-2) Confirmation of Ectopic Expression of HP1γ Protein
The cell line thus obtained and plasmid-unintroduced 3T3-L1 (hereinafter referred to as “plasmid-unintroduced cells”) were separately cultured in the presence of 1×10−7 M mifepristone, the inducer, to induce the expression of HP1γ protein. The culture was performed using multiple inducing media as described in “1.(1)” above, and mifepristone was added to every medium. Further, to examine the effect of differentiation induction and that of expression induction, additional culture was also performed in the absence of mifepristone or the inducing agents (adipose reagents). The cells thus cultured were analyzed by western blotting in the same manner as described in “1.(1)” above, and this confirmed that the expression of HP1γ protein had been induced. As a control, the same analysis was made on GAPDH protein.
The results are shown in
As shown in the figure, it was confirmed that in the plasmid-introduced cells, unlike plasmid-unintroduced cells, expression of HP1γ gene was induced in the presence of mifepristone, and this caused an increase in the amount of HP1γ protein.
(3) Effect of HP1γ Expression on Cell Differentiation
The plasmid-introduced cells and plasmid-unintroduced cells, both cultured in the same manner as is described in (2) above, were examined for accumulation of lipid droplets within the cells. As aforementioned, 3T3-L1 cells change their phenotype when differentiation is induced, and they form lipid droplets and accumulate them within the cells. Therefore, assay of cells as to whether they have differentiated or not can be done by detecting lipid accumulation within the cells. Assay of cells differentiation was performed with oil red staining using an analysis kit (product name: Adipogenesis Assay kit; mftd. by Chemicon).
The results are shown in
As shown in the figure, in the plasmid-unintroduced cells cultured in the presence of differentiation inducers (adipose reagents +), positive staining (i.e., accumulation of lipid droplets) was detected regardless of whether the culture had been done in the presence or absence of mifepristone. Thus, it was proved that differentiation is surely induced in the plasmid-unintroduced cells when differentiation inducers are present. In contrast, in 3T3-L1 cells which had been transformed into an HP1γ expression system (plasmid-introduced cells), though positive staining was detected in the presence of differentiation inducers when mifepristone was absent, no positive staining was detectable when mifepristone was present. Thus, in the presence of mifepristone, cell differentiation was not induced either in the presence or absence of differentiation inducers. This result shows that HP1γ protein, which was induced by mifepristone, inhibited cell differentiation, and that inhibition of the expression of HP1γ protein is necessary for cell differentiation to take place.
(1) Time Course of Histone Modification During Cell Differentiation
Modification of histone, which is a component of chromatin, is one of epigenetic mechanisms, and is known to be important for cell differentiation (Non-patent Document 7). Thus, we examined the time course of histone modification in 3T3-L1 during cell differentiation. First, culture of 3T3-L1 mouse preadipocytes was performed in the same manner as described in “1.(1)” above except that the duration of culture in the aforementioned third differentiation-inducing medium was set at certain length of time (0 day, 1 days, 2 days, 3 days, 4 days, 6 days, 8 days, 10 days, or 14 days). And, except that the above-mentioned various antibodies were employed, western blotting was performed in the same manner as described in “1.(2)” above to detect HP1γ, AceH3K9, AceH3K18, Met2H3K4 (DiMetH3K4), Met2H3K9 (DiMetH3K9), Met2H3R17 (DiH3R17), AceH4K8, AceH4K12, AcetH4K16, Met3H4K20 (TriMetH4K20), as well as GAPDH which was a control.
The results are shown in
(2) Relation Between HP1γ Protein Expression and Histone Modification Levels in Differentiation
Further, examination was conducted to find out whether the expression of HP1γ protein directly effects on the levels of the above histone modifications. This was done, using the mifepristone-inducible HP1γ expressing 3T3-L1 (plasmid-introduced cells) prepared in “1.(2)” above, by western blotting of histone modification, at 4-hour intervals for 72 hours after the start of mifepristone treatment. The condition for cell culture was the same as that in “1.(1)” above, and the western blotting was performed in the same manner as described above.
The results are shown in
(3) Relation Between Suppression of HP1γ Gene Expression and Histone Modification Levels
By means of RNA interference using siRNAs which inhibit HP1γ gene expression, examination was conducted to find out a relation between trimethylation levels of histone H4K20 and the expression of HP1γ protein.
Using 12 μl, of HiPerFect reagent (product name, mftd. by Qiagen) per 60-mm culture dish, 3T3-L1 mouse preadipocytes were transfected following the manual attached to the reagent, with 50 nM siTrio (registered trademark) Full Set (mftd. by B-Bridge) containing siRNAs specific to mouse HP1α, HP1β and HP1γ gene, respectively. The siRNA sequences (shown by sense strands only) specific to HP1α were 5′-GGGAGAAAUCAGAAGGAAATT-3′ (SEQ ID NO:114), 5′-GCGAAGAGCUAAAGGAGGATT-3′ (SEQ ID NO:115), and 5′-GGAUACAGUCUGAGAGUUATT-3′ (SEQ ID NO:116); siRNA sequences specific to HP1μ were 5′-GGUACUAGAAGAAGAGGAATT-3′ (SEQ ID NO:117), 5′-GGCGAGUUGUCAAGGGCAATT-3′ (SEQ ID NO:118), and 5′-GAAAACAGCUCAUGAGACATT-3′ (SEQ ID NO:119); siRNA sequences specific to HP1γ were 5′-GGACCGUCGUGUAGUGAAUTT-3′ (SEQ ID NO:120), 5′-CCGACUUGGUGCUGGCAAATT-3′ (SEQ ID NO:121), and 5′-GGAAAAUGGAAUUAGACUATT-3′ (SEQ ID NO:122). In each of these sequences, “TT” on the 3′-end is the overhang. Following the transfection with those siRNAs, the 3T3-L1 cells were cultured for 72 hours, and their whole cell lysates were subjected to western blotting in the same manner as described above. As a negative control, siRNA (SEQ ID NO:123, 5′-AUCCGCGCGAUAGUACGUAdTdT-3′) (mftd. by B-Bridge) was used to transfect 3T3-L1 with.
The results are shown in
Examination was carried out to find out whether HP1γ protein and trimethylated K20 of histone H4 disappeared not only in differentiated adipocytes but also in terminal differentiated cells of various other tissues.
As tissue samples, fat, esophageal mucosa, skin tissues, and colon were employed. These tissues was examined for localization of HP1γ protein and trimethylated histone H4K20 (Met3H4K20) by immunohistochemical analysis. Specifically, slices of formalin-fixed, paraffin-embedded samples were prepared from the above-mentioned tissues according to a conventional method, and then examined on an automatic immunostaining apparatus (product name: Ventana HX System Benchmark, mftd. by Ventana Medical Systems). Antibodies used were anti-human HP1γ monoclonal antibody and anti-Met3H4K20 polyclonal antibody, which were applied after diluted to the ratios of 1:800 and 1:200, respectively (antibody:diluent). Furthermore, hematoxylin and eosin staining (H&E staining) of each tissue was performed for histological examination.
The results are shown in
Occurrence of HP1γ protein was confirmed in premature cells of the tissues (figure not presented). However, as shown in b and c of the figure, neither HP1γprotein nor Met3H4K20 was detected in mature adipocytes (terminal differentiated cells) in the adipose tissue, and, likewise, neither HP1γ protein nor Met3H4K20 was detected in terminal differentiated cells in the esophageal mucosa or the dermal tissue, as shown in e, f, h and i of the figure. Moreover, as shown in k and l of the figure, neither HP1γ protein nor Met3H4K20 was detected on the surface of differentiated mucosa of the colonic tissue. These results indicates that the disappearance of HP1γ protein and Met3H4K20 is related to the differentiation of the cells regardless of which tissue the cells belong to. HP1γ protein is thought to be bound to methylated K20 residues of histone H4 and bind to Suv4-20h1, Sub4-20h2 and/or histone methyl transferase. Thus, the molecular interaction between HP1γ gene and histone H4K20 is thought to be a key mechanism to cell differentiation.
Human various malignant tumors (cancers) were examined for expression of HP1γ protein and trimethylation levels of histone H4K20.
Tissue samples of malignant tumors employed were those of esophageal cancer, uterine cervical cancer, colorectal cancer, breast cancer, lung cancer, and myxoid liposarcoma, which had been surgically excised (n=26). These cells were examined for localization of HP1γ protein and trimethylated histone H4K20 (Met3H4K20) by immunohistochemical staining in the same manner as described in “3.” above.
Typical examples of these results are shown in
As shown in “3.” above, a correlation was found between the expression of HP1γ protein and trimethylation of histone H4K20 in normal differentiated cells. For example, there was a correlation that when HP1γ protein was found positive, trimethylation was also found positive in the undifferentiated cells. On the other hand, in the differentiated cells there was a correlation that where HP1γ was found negative, trimethylation was also found negative. In contrast to this, with malignant tumor cells, as shown in
It has already been reported that the loss of trimethylated histone H4K20 is a notable characteristic of cancer cells (Non-patent document 8). Considering this in combination with the aforementioned results obtained in normal differentiated cells, it must be difficult, by detection of trimethylation of histone H4K20 alone, to distinguish between normal differentiated cells and malignant tumor cells. However, as aforementioned, there is a tendency that HP1γ protein is found negative in normal differentiated cells and positive in malignant tumor cells. Therefore, while a loss of trimethylation of histone H4K20 reported so far alone will not serve to distinguish tumor cells, further detection of the expression of HP1γ will allow one to identify cells as being normal ones if the expression of HP1γ in them has been reduced, and tumor cells if the expression of HP1γ has been increased.
The suppressive effect of siRNAs on proliferation of malignant tumor cells was examined.
Malignant tumor cells employed were: human cell lines DLD-1, HCT116 and HT-29 (colon cancer); MKN1 and MKN28 (gastric cancer); HeLa and SiHa (uterine cervical cancer); NCI-H23 (lung cancer); and 402/91 and 2645/94 (myxoid liposarcoma). These cells were transfected with 5 nM or 50 nM of a double-stranded siRNA oligonucleotide (SEQ ID NO:124: 5′-UGACAAACCAAGAGGAUUUdTdT-3′, mftd. by B-Bridge), which corresponds to human HP1γ gene, with HiPerFect reagent (product name, mftd. by Qiagen) according to the manual attached to the reagent. The siRNA presented as SEQ ID NO:124 is the one indicated as #62 (sense strand of SEQ ID NO:1, antisense strand of SEQ ID NO:253) in Table 2D, on the 3′-end of which is attached an overhang consisting of two thymidine (T) bases. As a negative control, the siRNA mentioned above in “2.(3)” (SEQ ID NO:123: 5′-AUCCGCGCGAUAGUACGUAdTdT-3′) (mftd. by B-Bridge) was used in the same manner to transfect 3T3-L1. After transfection, the cells were cultured for four days as aforementioned in accordance with the type of the cells, and after treated with a cell staining dye (product name: trypan), counted for viable cells on hemocytometer (product name “erythrometer”). Analyses were performed three times for each cancer cell types. As a control, siRNA-unintroduced tumor cells of each cancer type were also counted in the same manner for viable cells.
The results are presented graphically in
Examination was performed of siRNAs which are specific to human HP1γ on their suppressive effect on human HP1γ gene expression. SiRNAs employed were: #17 (sense strand: SEQ ID NO:49, antisense strand: SEQ ID NO:208), #62 (sense strand: SEQ ID NO: 1, antisense strand: SEQ ID NO:253), and #89 (sense strand: SEQ ID NO:74, antisense strand: SEQ ID NO:280) presented in Table 2A-F, each of which had two deoxythymidine nucleotides(dTdT) as the 3′-overhang sequences, and which are shown as g-1 to g-3 siRNAs in
As evident from
Nude mice were transplanted with human malignant tumor cells, and the inhibitory effect of siRNAs of the present invention on the proliferation of them was examined. Namely, nude mice (3 animals per group) were subcutaneously transplanted with 1×106 cultured DLD-1 cells, cells originating from human colorectal cancer. One week after the transplantation, when the cancer had grown to a sufficient size under the skin, (a) the animals of the control group was injected, at the site of tumor, with a mixture solution of one μL of a 10 μM negative control siRNA, 2 μL of Oligofectamine (mftd. by Invitrogen) and 89 μL Opti-MEM (mftd. by Invitrogen), (b) the animals of the test agent-injected group were injected with a mixture solution of one μL of a 10 μM human HP1γ-specific siRNA (SEQ ID NO:124), 2 μl, of Oligofectamine (Invitrogen) and 89 μl, of Opti-MEM I (mftd. by Invitrogen), and (c) the animals of the test agent externally-applied group were externally applied with a preparation which was a cream containing human HP1γ-specific siRNA (SEQ ID NO:124). The cream was prepared by mixing an aqueous solution of the siRNA with a roughly equal weight of neutral fat, and stirring, in the presence of a minute amount of a surfactant and with warming, the mixture to homogeneity. Then, these preparations were administered alike at the interval of three days. Observation of the animals at their cancer-transplanted site was performed for 22 days after the transplantation. The results are shown in
As seen in
The results shown above demonstrate the usefulness of the HP1γ protein as a cancer cell identification marker, and further the usefulness of the human HP1γ-specific siRNAs of the present invention as cancer cell proliferation inhibitor agents to various cancers, and also as an agent for cancer treatment.
By the method for identification of cancer cells according to the present invention, it is possible to distinguish between cancer cells and normal cells by detecting the presence of HP1γ protein, the identification marker, in the cells. Further, the cancer identification marker according to the present invention, unlike conventions ones, makes it possible to determine whether the cells being examined are cancer cells or not, without regard to the cancer cell types. Moreover, of the HP1γ gene expression inhibitors according to the present invention, the human HP1γ-specific siRNAs can be used for the treatment of a wide variety of human cancers because they inhibit growth of cancers in general regardless of their types.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-253258 | Sep 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/068129 | 9/19/2007 | WO | 00 | 1/11/2010 |
