# Patent

## Title
Cancer cell identification marker and cancer cell proliferation inhibitor

## Abstract

Disclosed is an identification marker which can be utilized for detection of various human cancer cells and whose expression closely relates to malignant alteration of cells, and compositions for human cancer treatment which are based on suppression of cancer cell proliferation through inhibition of expression of the identification marker. The marker is human heterochromatin protein 1γ (HP1γ), and the compositions for cancer treatment comprises one or more agents which suppresses the expression of human HP1γ gene, such as siRNAs to human HP1γ.

## Background
This application is a divisional of U.S. patent application Ser. No. 12/441,747, filed Mar. 18, 2009, which is a PCT National Stage entry based on PCT/JP2007/068129, filed Sep. 19, 2007.


TECHNICAL FIELD
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.
BACKGROUND ART
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 Rev Cancer 3, 89-101 (2003)
    [Non-patent Document 4] Gilbert, N. et al. Formation of facultative heterochromatin in the absence of HP1. Embo J 22, 5540-50 (2003)
    [Non-patent Document 5] Olins, D. E. & Olins, A. L. Granulocyte heterochromatin: defining the epigenome. BMC Cell 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)
    [Non-patent Document 9] Bernstein, E. et al., Nature 409: 363-366 (2001)
    [Non-patent Document 10] Elbashir, S. M. et al., EMBO J 20:6877-6888(2001)
    [Non-patent Document 11] Xu, P. et al., Curr Biol 13: 790-795 (2003)
    [Non-patent Document 12] Martinet, J., et al., Cell 110:533-542(2002)


DISCLOSURE OF INVENTION
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 caner 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 Effect of Invention
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.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1(A) is a set of autoradiograms showing the expression profiles of different proteins (HP1α, HP1αβ, HP1γ) in differentiation-induced 3T3-L1 mouse preadipocytes and human preadipocytes in one of the examples of the present invention;
FIG. 1(B) a set of autoradiograms showing the expression of HP1γ protein in 3T3-L1 in another example of the present invention; and
FIG. 1(C) a set of photographs showing 3T3-L1 cells subjected to oil red-staining in still another example of the present invention.
FIG. 2(A) is a set of autoradiograms showing the time courses of histone modifications in 3T3-L1 cells during cell division in still another example of the present invention,
FIG. 2(B) is a set of autoradiograms showing the time courses of the expression of HP1γ protein and of histone modifications, and
FIG. 2(C) a set of autoradiograms showing the levels of histone modifications and HP1γ expression, after treated with siRNA specific to HP1 genes.
FIG. 3 is a set of photographs showing the result of immunostaining of tissues in still another example of the present invention.
FIG. 4-1 is a set of photographs showing the result of immunostaining of malignant tumor cells in still another example of the present invention.
FIG. 4-2 is a set of photographs sowing the result of immunostaining of malignant tumor cells in still another example of the present invention.
FIG. 4-3 is a set of photographs sowing the result of immunostaining of malignant tumor cells in still another example of the present invention.
FIG. 5 is a graph showing the number (×104 cells) of viable cells of various tumors.
FIG. 6 shows the result of western blotting exhibiting the inhibitory effect of siRNA of the present invention on HP1γ expression in a human cancer cell line.
FIG. 7 is a graph illustrating the therapeutic effect of siRNA of the present invention in human cancer-transplanted nude mice.



BEST MODE FOR CARRYING OUT THE INVENTION
<Cancer Cell Identification Marker and Method for Identification>
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.
<Cancer Cell Proliferation Inhibitor Agents>
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.







TABLE 1







Table 1















SEQ






ID



Gene
CDS
Sequence (5′→3′)
NO:







  40

CCCUUCGGAUGUGGCUUGA
 2



 



  41

CCUUCGGAUGUGGCUUGAG
 3



 



  42

CUUCGGAUGUGGCUUGAGC
 4



 



  43

UUCGGAUGUGGCUUGAGCU
 5



 



  44

UCGGAUGUGGCUUGAGCUG
 6



 



  45

CGGAUGUGGCUUGAGCUGU
 7



 



  46

GGAUGUGGCUUGAGCUGUA
 8



 



  47

GAUGUGGCUUGAGCUGUAG
 9



 



  48

AUGUGGCUUGACCUGUAGG
10



 



  49

UGUGGCUUGAGCUGUAGGC
11



 



  50

GUGGCUUGAGCUGUAGGCG
12



 



  51

UGGCUUGAGCUGUAGGCGC
13



 



 106

CAGCUCGGAGGCGGUGAAU
14



 



 107

AGCUCGGAGGCGGUGAAUA
15



 



 108

GCUCGGAGGCGGUGAAUAA
16



 



 109

CUCGGAGGCGGUGAAUAAU
17



 



 123

AUAAUAGCUCUUCAAGUCU
18



 



 124

UAAUAGCUCUUCAAGUCUG
19



 



 125

AAUAGCUCUUCAAGUCUGC
20



 



 126

AUAGCUCUUCAAGUCUGCA
21



 



 127

UAGCUCUUCAAGUCUGCAA
22



 



 128

AGCUCUUCAAGUCUGCAAU
23



 



 129

GCUCUUCAAGUCUGCAAUA
24



 



 130

CUCUUCAAGUCUGCAAUAA
25



 



 131

UCUUCAAGUCUGCAAUAAA
26



 



 148

AAAAAUGGCCUCCAACAAA
27



 



 149

AAAAUGGCCUCCAACAAAA
28



 



 150

AAAUGGCCUCCAACAAAAC
29



 



 151

AAUGGCCUCCAACAAAACU
30



 



 152

AUGGCCUCCAACAAAACUA
31



 



 153

UGGCCUCCAACAAAACUAC
32



 



 154

GGCCUCCAACAAAACUACA
33



 



 155

GCCUCCAACAAAACUACAU
34



 



 156

CCUCCAACAAAACUACAUU
35



 



 157

CUCCAACAAAACUACAUUG
36



 



 158

UCCAACAAAACUACAUUGC
37



 



 159

CCAACAAAACUACAUUGCA
38



 



 160
  9
CAACAAAACUACAUUGCAA
39



 



 191
 40
AAACAGAAUGGAAAGAGUA
40



 



 194
 43
CAGAAUGGAAAGAGUAAAA
41



 



 194
 43
CAGAAUGGAAAGAGUAAAA
42



 



 216
 65
UUGAAGAGGCAGAGCCUGA
43



 



 222
 71
AGGCAGAGCCUGAAGAAUU
44



 



 223
 72
GGCAGAGCCUGAAGAAUUU
45



 



 231
 80
CUGAAGAAUUUGUCGUGGA
46



 



 232
 81
UGAAGAAUUUGUCGUGGAA
47



 



 234
 83
AAGAAUUUGUCGUGGAAAA
48



 



 247
 96
GGAAAAAGUACUAGAUCGA
49



 



 253
102
AGUACUAGAUCGACGUGUA
50



 



 259
108
AGAUCGACGUGUAGUGAAU
51



 



 265
114
ACGUGUAGUGAAUGGGAAA
52



 



 271
120
AGUGAAUGGGAAAGUGGAA
53



 



 273
122
UGAAUGGGAAAGUGGAAUA
54



 



 330
179
CUUGGGAACCUGAAGAAAA
55



 



 360
209
CAGAAUUGAUUGAAGCGUU
56



 



 361
210
AGAAUUGAUUGAAGCGUUU
57



 



 388
237
UCAGAAAGCUGGCAAAGAA
58



 



 389
238
CAGAAAGCUGGCAAAGAAA
59



 



 391
240
GAAAGCUGGCAAAGAAAAA
60



 



 399
248
GCAAAGAAAAAGAUGGUAC
61



 



 400
249
CAAAGAAAAAGAUGGUACA
62



 



 441
290
GUGAAUCUGAUGACAGCAA
63



 



 453
302
ACAGCAAAUCAAAGAAGAA
64



 



 453
302
ACAGCAAAUCAAAGAAGAA
65



 



 461
310
UCAAAGAAGAAAAGAGAUG
66



 



 469
318
GAAAAGAGAUGCUGCUGAC
67



 



 472
321
AAGAGAUGCUGCUGACAAA
68



 



 482
331
GCUGACAAACCAAGAGGAU
69



 



 484
333
UGACAAACCAAGAGGAUUU
 1



 



 541
390
AGACAGCAGUGGAGAAUUG
70



 



 566
415
CUCAUGAAAUGGAAAGAUU
71



 



 594
443
CAGACUUGGUGCUGGCGAA
72



 



 608
457
GCGAAAGAGGCAAAUAUGA
73



 



 609
458
CGAAAGAGGCAAAUAUGAA
74



 



 684
533
CAGAAGAUGAAGCUCAAUA
75



 



 827

UGAAAGUAGCGUUGGAAGA
76



 



 936

CAUUUGAUACCAUGGUAUA
77



 



 996

GGGAAAUGUCCAUAGUCAU
78



 



 997

GGAAAUGUCCAUAGUCAUU
79



 



1020

AGUCAAAACUUGUGUUCUC
80



 



1196

GCCAUUAUUCCAAGCAAAA
81



 



1219

AGAUAAUCCCUUCAAGUUA
82



 



1220

GAUAAUCCCUUCAAGUUAA
83



 



1258

CCAUACAUUUCAAGUGAAA
84



 



1346

GCAAAAUUCCUAAAAGGAA
85



 



1404

GAUGAGGAAACUAGACAAA
86



 



1409

GGAAACUAGACAAAUGCUA
87



 



1417

GAGAAAUGCUAGUGUGUUU
88



 



1487

GGGCCAUUCCUUAGCAAAA
89



 



1576

AAACCUAAUCAGAUGGUUA
90



 



1684

UCAGAUGGUUAGAGGUGUU
91



 



1590

GGUUAGAGGUGUUGGCAGU
92



 



1621

GUCAUAAAUGUGUGAACAA
93



 



1719

CUUUACUGGUUCAGCAAAA
94



 



1730

CAGCAAAAGCCAGGAAGAA
95



 



1732

GCAAAAGCCAGGAAGAACA
96



 



1799

UGUAAAUACUGGUGAACAG
97










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. Tin (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.















TABLE 2A








SEQ










ID
Position

GC
Position




















NO:
gene
CDS
Candidate sequence
%
Score
19/19
18/19
17/19






















 #1
 39
160
  9
CAACAAAACUACAUUGCAA
31%
137
3
(2)
2
(2)





192


UUGCAAUGUAGUUUUGUUG










 


 #2
127
190
 39
AAAACAGAAUGGAAAGAGU
31%
 80
4
(3)
2
(2)
 7
(6)



193


ACUCUUUCCAUUCUGUUUU






 2
(2)


 


 #3
 40
191
 40
AAACAGAAUGGAAAGAGUA
31%
 92
5
(4)
1
(1)
 4
(3)



194


UACUCUUUCCAUUCUGUUU






 4
(3)


 


 #4
128
192
 41
AACAGAAUGGAAAGAGUAA
31%
 77
5
(4)
1
(1)
 2
(2)



195


UUACUCUUUCCAUUCUGUU




1
(1)
 2
(1)


 


 #5
 41
194
 43
CAGAAUGGAAAGAGUAAAA
31%
124
5
(4)


 6
(6)



196


UUUUACUCUUUCCAUUCUG










 


 #6
129
210
 59
AAAAAGUUGAAGAGGCAGA
36%
 95
5
(4)
2
(2)
11
(9)



197


UCUGCCUCUUCAACUUUUU










 


 #7
130
214
 63
AGUUGAAGAGGCAGAGCCU
52%
 63
5
(4)


 7
(6)



198


AGGCUCUGCCUCUUCAACU






 1
(1)


 


 #8
 43
216
 65
UUGAAGAGGCAGAGCCUGA
52%
104
5
(4)
2
(1)
 4
(4)



199


UCAGGCUCUGCCUCUUCAA






 2
(2)


 


 #9
 44
222
 71
AGGCAGAGCCUGAAGAAUU
47%
127
5
(4)
2
(1)
 5
(5)



200


AAUUCUUCAGGCUCUGCCU






 1
(1)


 


#10
 45
223
 72
GGCAGAGCCUGAAGAAUUU
47%
 90
5
(4)
2
(1)
 7
(6)



201


AAAUUCUUCAGGCUCUGCC






 1
(1)


 


#11
 46
231
 80
CUGAAGAAUUUGUCGUGGA
42%
 93
3
(2)
4
(3)
 2
(2)



202


UCCACGACAAAUUCUUCAG










 


#12
 47
232
 81
UGAAGAAUUUGUCGUGGAA
36%
 91
3
(2)
4
(3)
 2
(2)



203


UUCCACGACAAAUUCUUCA










 


#13
 48
234
 83
AAGAAUUUGUCGUGGAAAA
31%
137
3
(2)
2
(2)
 3
(3)



204


UUUUCCACGACAAAUUCUU






 5
(3)


 


#14
131
235
 84
AGAAUUUGUCGUGGAAAAA
31%
 72
3
(2)
2
(2)
 3
(3)



205


UUUUUCCACGACAAAUUCU






 4
(3)


 


#15
132
241
 90
UGUCGUGGAAAAAGUACUA
36%
 57
3
(2)
4
(3)





206


UAGUACUUUUUCCACGACA










 


#16
133
246
 95
UGGAAAAAGUACUAGAUCG
36%
 66
3
(2)
2
(2)





207


CGAUCUAGUACUUUUUCCA










 


#17
 49
247
 96
GGAAAAAGUACUAGAUCGA
36%
138
3
(2)
2
(2)
 1
(1)



208


UCGAUCUAGUACUUUUUCC










 


#18
 50
253
102
AGUACUAGAUCGACGUGUA
42%
 99
3
(2)
2
(2)





209


UACACGUCGAUCUAGUACU
























TABLE 2B








SEQ










ID
Position

GC
Position




















NO:
gene
CDS
Candidate sequence
%
Score
19/19
18/19
17/19






















#19
 51
259
108
AGAUCGACGUGUAGUGAAU
42%
101
3
(2)
2
(2)
 2
(2)



210


AUUCACUACACGUCGAUCU










 


#20
134
264
113
GACGUGUAGUGAAUGGGAA
47%
 65
5
(4)
2
(2)





211


UUCCCAUUCACUACACGUC










 


#21
 52
265
114
ACGUGUAGUGAAUGGGAAA
42%
107
5
(4)
2
(2)
 4
(1)



212


UUUCCCAUUCACUACACGU










 


#22
 53
271
120
AGUGAAUGGGAAAGUGGAA
42%
 85
5
(4)


 8
(7)



213


UUCCACUUUCCCAUUCACU






 1
(1)


 


#23
135
272
121
GUGAAUGGGAAAGUGGAAU
42%
 63
5
(4)
2
(1)
 5
(5)



214


AUUCCACUUUCCCAUUCAC










 


#24
 54
273
122
UGAAUGGGAAAGUGGAAUA
36%
107
5
(4)
2
(1)
 3
(3)



215


UAUUCCACUUUCCCAUUCA










 


#25
136
293
142
UUCCUGAAGUGGAAGGGAU
47%
 64
5
(4)


 6
(6)



216


AUCCCUUCCACUUCAGGAA






 2
(1)


 


#26
137
294
143
UCCUGAAGUGGAAGGGAUU
47% 
 68
5
(4)
3
(3)
 2
(2)



217


AAUCCCUUCCACUUCAGGA










 


#27
138
296
145
CUGAAGUGGAAGGGAUUUA
42%
 75
5
(4)


 4
(3)



218


UAAAUCCCUUCCACUUCAG






 3
(2)


 


#28
139
301
150
GUGGAAGGGAUUUACAGAU
42%
 60
5
(4)
4
(3)
 4
(4)



219


AUCUGUAAAUCCCUUCCAC






 2
(2)


 


#29
140
302
151
UGGAAGGGAUUUACAGAUG
42%
 72
5
(4)
4
(3)
 4
(4)



220


CAUCUGUAAAUCCCUUCCA






 2
(2)


 


#30
141
310
159
AUUUACAGAUGCUGACAAU
31%
 70
5
(4)


 8
(6)



221


AUUGUCAGCAUCUGUAAAU






 3
(2)


 


#31
142
320
169
GCUGACAAUACUUGGGAAC
47%
 77
5
(4)
2
(1)
 5
(4)



222


GUUCCCAAGUAUUGUCAGC










 


#32
143
322
171
UGACAAUACUUGGGAACCU
42%
 68
5
(4)
2
(2)
 5
(3)



223


AGGUUCCCAAGUAUUGUCA










 


#33
 55
330
179
CUUGGGAACCUGAAGAAAA
42%
102
5
(4)
2
(2)
 7
(5)



224


UUUUCUUCAGGUUCCCAAG










 


#34
144
331
180
UUGGGAACCUGAAGAAAAU
36% 
 81
5
(4)
3
(3)
10
(8)



225


AUUUUCUUCAGGUUCCCAA










 


#35
 56
360
209
CAGAAUUGAUUGAAGCGUU
36%
100
3
(2)
2
(2)





226


AACGCUUCAAUCAAUUCUG










 


#36
 57
361
210
AGAAUUGAUUGAAGCGUUU
31%
 99
3
(2)
2
(2)





227


AAACGCUUCAAUCAAUUCU










 


#37
145
366
215
UGAUUGAAGCGUUUCUUAA
31%
 67
3
(2)


 4
(4)



228


UUAAGAAACGCUUCAAUCA
























TABLE 2C








SEQ










ID
Position

GC
Position




















NO:
gene
CDS
Candidate sequence
%
Score
19/19
18/19
17/19






















#38
146
376
225
GUUUCUUAACUCUCAGAAA
31%
 65
3
(2)


 6
 (5)



229


UUUCUGAGAGUUAAGAAAC










 


#39
 58
388
237
UCAGAAAGCUGGCAAAGAA
42%
114
3
(2)
4
(3)
 2
 (2)



230


UUCUUUGCCAGCUUUCUGA






 3
 (3)


 


#40
 59
389
238
CAGAAAGCUGGCAAAGAAA
42%
106
3
(2)
4
(3)
 7
 (5)



231


UUUCUUUGCCAGCUUUCUG






 5
 (5)


 


#41
147
390
239
AGAAAGCUGGCAAAGAAAA
36%
 78
3
(2)
5
(4)
 9
 (7)



232


UUUUCUUUGCCAGCUUUCU






 3
 (3)


 


#42
 60
391
240
GAAAGCUGGCAAAGAAAAA
36%
104
3
(2)
4
(3)
10
 (7)



233


UUUUUCUUUGCCAGCUUUC






 4
 (4)


 


#43
148
397
246
UGGCAAAGAAAAAGAUGGU
36%
 71
3
(2)
4
(3)
 2
 (2)



234


ACCAUCUUUUUCUUUGCCA






 4
 (3)


 


#44
 61
399
248
GCAAAGAAAAAGAUGGUAC
36%
107
3
(2)
2
(1)
 3
 (3)



235


GUACCAUCUUUUUCUUUGC






 2
 (2)


 


#45
 62
400
249
CAAAGAAAAAGAUGGUACA
31%
100
3
(2)
2
(1)
 4
 (4)



236


UGUACCAUCUUUUUCUUUG










 


#46
149
426
275
AAUCUUUAUCUGACAGUGA
31%
 58
7
(6)
2
(1)
 3
 (2)



237


UCACUGUCAGAUAAAGAUU










 


#47
 63
441
290
GUGAAUCUGAUGACAGCAA
42%
 87
7
(5)
4
(3)
 5
 (4)



238


UUGCUGUCAUCAGAUUCAC






 1
 (1)


 


#48
150
443
292
GAAUCUGAUGACAGCAAAU
36%
 66
7
(5)
3
(3)
 3
 (2)



239


AUUUGCUGUCAUCAGAUUC










 


#49
151
449
298
GAUGACAGCAAAUCAAAGA
36%
 63
5
(4)
2
(2)
 8
 (5)



240


UCUUUGAUUUGCUGUCAUC






 4
 (3)


 


#50
152
452
301
GACAGCAAAUCAAAGAAGA
36%
 64
5
(4)
3
(3)
 4
 (3)



241


UCUUCUUUGAUUUGCUGUC






 5
 (2)


 


#51
 64
453
302
ACAGCAAAUCAAAGAAGAA
31%
 89
5
(4)
1
(1)
15
(11)



242


UUCUUCUUUGAUUUGCUGU






 6
 (3)


 


#52
153
454
303
CAGCAAAUCAAAGAAGAAA
31%
 89
5
(4)
1
(1)
18
(13)



243


UUUCUUCUUUGAUUUGCUG






 4
 (2)


 


#53
154
456
305
GCAAAUCAAAGAAGAAAAG
31%
 72
3
(2)
2
(2)
27
(19)



244


CUUUUCUUCUUUGAUUUGC






 1
 (1)


 


#54
 66
461
310
UCAAAGAAGAAAAGAGAUG
31%
 94
3
(2)
2
(2)
 8
 (7)



245


CAUCUCUUUUCUUCUUUGA




2
(1)
 5
 (4)


 


#55
155
466
315
GAAGAAAAGAGAUGCUGCU
42% 
 72
3
(2)
5
(4)
15
 (9)



246


AGCAGCAUCUCUUUUCUUC






 4
 (4)


 


#56
156
468
317
AGAAAAGAGAUGCUGCUGA
42%
 82
5
(3)
2
(2)
 8
 (7)



247


UCAGCAGCAUCUCUUUUCU


1
(1)


 2
 (2)
























TABLE 2D








SEQ










ID
Position

GC
Position




















NO:
gene
CDS
Candidate sequence
%
Score
19/19
18/19
17/19






















#57
 67
469
318
GAAAAGAGAUGCUGCUGAC
47%
 89
5
(3)
2
(2)
 3
(3)



248


GUCAGCAGCAUCUCUUUUC




1
(1)
 2
(2)


 


#58
157
471
320
AAAGAGAUGCUGCUGACAA
42%
 75
5
(3)
2
(2)
 7
(6)



249


UUGUCAGCAGCAUCUCUUU






 1
(1)


 


#59
 68
472
321
AAGAGAUGCUGCUGACAAA
42%
112
5
(3)
4
(4)
 7
(5)



250


UUUGUCAGCAGCAUCUCUU






 5
(4)


 


#60
 69
482
331
GCUGACAAACCAAGAGGAU
47%
 91
5
(4)
2
(1)
 4
(2)



251


AUCCUCUUGGUUUGUCAGC




2
(1)




 


#61
158
483
332
CUGACAAACCAAGAGGAUU
42%
 61
5
(4)
2
(1)
 2
(1)



252


AAUCCUCUUGGUUUGUCAG






 2
(1)


 


#62
  1
484
333
UGACAAACCAAGAGGAUUU
36%
132
5
(4)


 6
(5)



253


AAAUCCUCUUGGUUUGUCA










 


#63
159
490
339
ACCAAGAGGAUUUGCCAGA
47%
 78
5
(4)


 4
(3)



254


UCUGGCAAAUCCUCUUGGU






 1
(1)


 


#64
160
496
345
AGGAUUUGCCAGAGGUCUU
47%
 85
5
(4)


 3
(3)



255


AAGACCUCUGGCAAAUCCU






 1
(1)


 


#65
161
502
351
UGCCAGAGGUCUUGAUCCU
52%
 65
5
(4)
2
(2)
 3
(2)



256


AGGAUCAAGACCUCUGGCA






 6
(2)


 


#66
162
504
353
CCAGAGGUCUUGAUCCUGA
52%
 76
5
(4)
2
(2)
 4
(3)



257


UCAGGAUCAAGACCUCUGG






 1
(1)


 


#67
163
508
357
AGGUCUUGAUCCUGAAAGA
42%
 79
5
(4)


 4
(3)



258


UCUUUCAGGAUCAAGACCU






 1
(1)


 


#68
164
510
359
GUCUUGAUCCUGAAAGAAU
36%
 60
5
(4)


 2
(2)



259


AUUCUUUCAGGAUCAAGAC




1
(1)




 


#69
165
511
360
UCUUGAUCCUGAAAGAAUA
31%
 81
5
(4)


 5
(5)



260


UAUUCUUUCAGGAUCAAGA






 3
(2)


 


#70
166
537
386
CCACAGACAGCAGUGGAGA
57%
 63
7
(6)


 6
(4)



261


UCUCCACUGCUGUCUGUGG






 7
(6)


 


#71
167
538
387
CACAGACAGCAGUGGAGAA
52%
 76
7
(6)


11
(7)



262


UUCUCCACUGCUGUCUGUG






 4
(3)


 


#72
168
539
388
ACAGACAGCAGUGGAGAAU
47%
 63
5
(4)
2
(2)
 3
(2)



263


AUUCUCCACUGCUGUCUGU






 1
(1)


 


#73
 70
541
390
AGACAGCAGUGGAGAAUUG
47%
 86
5
(4)
2
(2)
 1
(1)



264


CAAUUCUCCACUGCUGUCU










 


#74
169
549
398
GUGGAGAAUUGAUGUUUCU
36%
 57
5
(4)


 4
(3)



265


AGAAACAUCAAUUCUCCAC










 


#75
170
552
401
GAGAAUUGAUGUUUCUCAU
31%
 75
5
(4)


 2
(2)



266


AUGAGAAACAUCAAUUCUC
























TABLE 2E








SEQ










ID
Position

GC
Position




















NO:
gene
CDS
Candidate sequence
%
Score
19/19
18/19
17/19






















#76
 171
554
403
GAAUUGAUGUUUCUCAUGA
31%
 60
5
(4)


 2
(2)



267


UCAUGAGAAACAUCAAUUC






 1
(1)


 


#77
71
566
415
CUCAUGAAAUGGAAAGAUU
31%
 83
5
(4)
2
(2)
 3
(2)



268


AAUCUUUCCAUUUCAUGAG










 


#78
 172
568
417
CAUGAAAUGGAAAGAUUCA
31%
74
5
(4)
2
(2)
 1
(1)



269


UGAAUCUUUCCAUUUCAUG










 


#79
 173
570
419
UGAAAUGGAAAGAUUCAGA
31%
 64
5
(4)
2
(2)
 7
(5)



270


UCUGAAUCUUUCCAUUUCA






1
(1)


 


#80
174
576
425
GGAAAGAUUCAGAUGAGGC
47%
 60
5
(4)


 4
(3)



271


GCCUCAUCUGAAUCUUUCC






 2
(1)


 


#81
175  
577
426
GAAAGAUUCAGAUGAGGCA
42%
80
5
(4)


 3
(3)



272


UGCCUCAUCUGAAUCUUUC






2
(1)


 


#82
176
579
428
AAGAUUCAGAUGAGGCAGA
42%
 71
5
(4)
1
(1)
 7
(5)



273


UCUGCCUCAUCUGAAUCUU










 


#83
72
594
443
CAGACUUGGUGCUGGCGAA
57%
 107
3
(2)
5
(3)
 2
(1)



274


UUCGCCAGCACCAAGUCUG










 


#84
177
595
444
AGACUUGGUGCUGGCGAAA
52%
 67
3
(2)
2
(2)
 4
(2)



275


UUUCGCCAGCACCAAGUCU










 


#85
178
597
446
ACUUGGUGCUGGCGAAAGA
52%
 80
3
(2)
2
(2)





276


UCUUUCGCCAGCACCAAGU










 


#86
179
601
450
GGUGCUGGCGAAAGAGGCA
63%
 70
3
(2)


 13
(10)



277


UGCCUCUUUCGCCAGCACC






 1
(1)


 


#87
180
606
455
UGGCGAAAGAGGCAAAUAU
42%
 73
3
(2)
2
(2)
 1
(1)



278


AUAUUUGCCUCUUUCGCCA










 


#88
73
608
457
GCGAAAGAGGCAAAUAUGA
42%
 86
3
(2)
2
(2)





279


UCAUAUUUGCCUCUUUCGC










 


#89
74
609
458
CGAAAGAGGCAAAUAUGAA
36%
 117
3
(2)
2
(2)
 2
(1)



280


UUCAUAUUUGCCUCUUUCG






 2
(2)


 


#90
181
615
464
AGGCAAAUAUGAAGUGUCC
42%
 66
3
(2)
2
(2)
3
(3)



281


GGACACUUCAUAUUUGCCU










 


#91
182
617
466
GCAAAUAUGAAGUGUCCUC
42%
 73
3
(2)
4
(4)
 2
(1)



282


GAGGACACUUCAUAUUUGC










 


#92
 183
620
469
AAUAUGAAGUGUCCUCAAA
31%
 65
3
(2)
4
(4)
 2
(1)



283


UUUGAGGACACUUCAUAUU










 


#93
184
632
481
CCUCAAAUUGUAAUUGCUU
31%
 57
5
(4)
4
(3)
 3
(2)



284


AAGCAAUUACAAUUUGAGG










 


#94
185
658
507
AGAGAGACUAACUUGGCAU
42%
 61
7
(5)
2
(2)
 1
(1)



285


AUGCCAAGUUAGUCUCUCU






1
(1)
























TABLE 2F








SEQ










ID
Position

GC
Position




















NO:
gene
CDS
Candidate sequence
%
Score
19/19
18/19
17/19






















 #95
186
 659
508
GAGAGACUAACUUGGCAUU
42%
 60
7
(5)
2
(2)





286


AAUGCCAAGUUAGUCUCUC






 1
(1)


 


 #96
187
 673
522
GCAUUCUUGUCCAGAAGAU
42%
 61
7
(6)


 3
(2)



287


AUCUUCUGGACAAGAAUGC






 3
(2)


 


 #97
188
 681
530
GUCCAGAAGAUGAAGCUCA
47%
 82
5
(4)
2
(2)
 4
(3)



288


UGAGCUUCAUCUUCUGGAC










 


 #98
189
 682
531
UCCAGAAGAUGAAGCUCAA
42%
 80
5
(4)


10
(7)



289


UUGAGCUUCAUCUUCUGGA










 


 #99
 75
 684
533
CAGAAGAUGAAGCUCAAUA
36%
 91
5
(4)


 4
(4)



290


UAUUGAGCUUCAUCUUCUG










 


#100
190
 685
534
AGAAGAUGAAGCUCAAUAA
31%
 75
5
(4)


 7
(6)



291


UUAUUGAGCUUCAUCUUCU






 1
(1)


 


#101
 83
1220

GAUAAUCCCUUCAAGUUAA
31%
 95
3
(2)


11
(7)



292


UUAACUUGAAGGGAUUAUC










 


#102
 84
1258

CCAUACAUUUCAAGUGAAA
31%
118
3
(2)


 2
(1)



293


UUUCACUUGAAAUGUAUGG










 


#103
 88
1417

GACAAAUGCUAGUGUGUUU
36%
112
3
(2)







294


AAACACACUAGCAUUUGUC










 


#104
 89
1487

GGGCCAUUCCUUAGCAAAA
47%
114
3
(2)







295


UUUUGCUAAGGAAUGGCCC






 1
(1)


 


#105
191
1538

GGUCAUGAUGAAUGGAAUA
36%
124
3
(2)


 1
(1)



296


UAUUCCAUUCAUCAUGACC






 1
(1)


 


#106
 95
1730

CAGCAAAAGCCAGGAAGAA
47%
110
3
(2)







297


UUCUUCCUGGCUUUUGCUG






 2
(2)









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.
















TABLE 3A







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#5
19/19
Sense
NM_016587
41
194
43







XR_015260.1

322
3U
730552
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC730552), mRNA.





XM_938779.2

155
CDS
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

154
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





XR_015356.1

322
3U
728217
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC728217), mRNA.





NM_016587.2

194
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.



18/19

NM_016587
41
194
43



17/19

NM_016587
41
194
43





NR_002723.2

173365
3U
8327
Homo sapiens GA binding protein










transcription factor, alpha subunit










pseudogene (GABPAP) on










chromosome 7.





XR_019241.1

45
3U
651211
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC651211), mRNA.





NM_006694.2

1309
3U
10899
Homo sapiens jumping translocation










breakpoint (JTB), mRNA.





NM_033106.2

905
3U
85569
Homo sapiens galanin-like peptide










precursor (GALP), mRNA.





XR_016921.1

45
3U
644101
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC644101), mRNA.





NM_001018116.1

1832
3U
347273
Homo sapiens similar to RIKEN










cDNA 2310039E09










(LOC347273), mRNA.



19/19
Antisense
NM_016587
196
194
43



18/19

NM_016587
196
194
43



17/19

NM_016587
196
194
43

























TABLE 3B







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#17
19/19
Sense
NM_016587
49
247
96







XM_938779.2

208
CDS
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

207
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

247
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.



18/19

NM_016587
49
247
96





XR_019241.1

97
3U
651211
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC651211), mRNA.





XR_016921.1

97
3U
644101
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC644101), mRNA.



17/19

NM_016587
49
247
96





NM_001040455.1

1369
CDS
51092
Homo sapiens SID1 transmembrane










family, member 2 (SIDT2), mRNA.



19/19
Antisense
NM_016587
208
247
96



18/19

NM_016587
208
247
96



17/19

NM_016587
208
247
96

























TABLE 3C







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#35
19/19
Sense
NM_016587
56
360
209







XM_938779.2

321
CDS
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

320
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

360
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.



18/19

NM_016587
56
360
209





XR_019241.1

210
3U
651211
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC651211), mRNA.





XR_016921.1

210
3U
644101
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC644101), mRNA.



17/19

NM_016587
56
360
209



19/19
Antisense
NM_016587
226
360
209



18/19

NM_016587
226
360
209



17/19

NM_016587
226
360
209

























TABLE 3D







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#62
19/19
Sense
NM_016857
1
484
333







XM_938779.2

445
CDS
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





XR_019241.1

333
3U
651211
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC651211), mRNA.





NM_007276.3

444
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





XR_016921.1

333
3U
644101
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC644101), mRNA.





NM_016587.2

484
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
1
484
333



17/19

NM_016587
1
484
333





NM_014753.2

844
COS
9790
Homo sapiens BMS1 homolog,










ribosome assembly protein (yeast)










(BMS1), mRNA.





XM_001126283.1

264
CDS

PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC642721), mRNA.





XR_015804.1

887
3U
729096
PREDICTED: Homo sapiens










similar to BMS1-like, ribosome










assembly protein (LOC729096),










mRNA.





XM_01128446.1

273
CDS
642721
PREDICTED; Homo sapiens










similar to chromobox homolog 3










(LOC642721), mRNA.





NM_147128.3

1827
3U

Homo sapiens zinc and ring finger 2










(ZNRF2), mRNA,





XM_944657.2

996
CDS
653468
PREDICTED: Homo sapiens










hypothetical protein LOC653468,










transcript variant 3(LOC653468), mRNA.



19/19
Antisense
NM_016587
253
484
333



18/19

NM_016587
253
484
333



17/19

NM_016587
253
484
333

























TABLE 3E







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#89
19/19
Sense
NM_016587

609
458







XM_938779.2

570
CDS
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

569
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

609
CDS
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
74
609
458





XR_015260.1

737
3U
730552
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC730552), mRNA.





XR_015356.1

737
3U
728217
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC728217), mRNA.



17/19

NM_016587
74
609
458





NM_001077186.1

3046
CDS
79784
Homo sapiens myosin, heavy chain










14 (MYH14), transcript variant 1,










mRNA.





NM_024729.3

3022
CDS
79784
Homo sapiens myosin, heavy chain










14 (MYH14),










transcript variant 2, mRNA.



19/19
Antisense
NM_016587
280
609
458



18/19

NM_016587
280
609
458



17/19

NM_016587
280
609
458





NM_016150.3

242
5U
51676
Homo sapiens ankyrin repeat and










SOCS box-containing 2 (ASB2),










mRNA.





NM_199324.1

6202
3U
54726
Homo sapiens OTU domain










containing 4 (OTUD4),










transcript variant 1, mRNA.

























TABLE 4F







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#101
19/19
Sense
NM_016587
83
1220








XM_938779.2

1177
3U
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

1180
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

1220
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
83
1220



17/19

NM_016587
83
1220



19/19
Antisense
NM_016587
292
1220



18/19

NM_016587
292
1220



17/19

NM_016587
292
1220

























TABLE 3G







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#102
19/19
Sense
NM_016587
84
1258








XM_938779.2

1215
3U
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

1218
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

1258
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
84
1258



17/19

NM_016587
84
1258





NM_003187.4

1150
3U

Homo sapiens TAF9 RNA polymerase II,










TATA box binding protein (TBP)-associated










factor, 32 kDa (TAF9), transcript variant 1,










mRNA.





MM_001015892.1

1387
3U
6880
Homo sapiens TAF9 RNA polymerase II,










TATA box binding protein (TBP)-associated










factor, 32 kDa (TAF9), transcript variant 4,










mRNA,



19/19
Antisense
NM_016587
293
1258



18/19

NM_016587
293
1258



17/19

NM_016587
293
1258

























TABLE 3H







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#103
19/19
Sense
NM_016587
88
1417








XM_938779.2

1374
3U
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

1377
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

1417
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 3, mRNA.



18/19

NM_016587
88
1417



17/19

NM_016587
88
1417



19/19
Antisense
NM_016587
294
1417



18/19

NM_016587
294
1417



17/19

NM_016587
294
1417

























TABLE 3I







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#104
19/19
Sense
NM_016587
89
1487








XM_938779.2

1444
3U
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

1447
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

1487
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
89
1487



17/19

NM_016587
89
1487



19/19
Antisense
NM_016587
295
1487



18/19

NM_016587
295
1487



17/19

NM_016587
295
1487





NM_004872.3

1588
3U
9528
Homo sapiens transmembrane protein 59










(TMEM59), mRNA.

























TABLE 3J







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#105
19/19
Sense
NM_016587
191
1538








XM_938779.2

1495
3U
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

1498
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

1538
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
191
1538



17/19

NM_016587
191
1538





NM_153262.2

1733
CDS
255928
Homo sapiens synaptotagmin XIV










(SYT14), mRNA.



19/19
Antisense
NM_016587
296
1538



18/19

NM_016587
296
1538



17/19

NM_016587
296
1538





NM_153810.4

3335
3U
143384
Homo sapiens chromosome 10 open










reading frame 46 (C10orf46),










mRNA.

























TABLE 3K







Match
Sequence
Accession#
SEQ ID NO:
gene
CDS
GeneID#
Definition
























#106
19/19
Sense
NM_016587
95
1730








XM_938779.2

1687
3U
653972
PREDICTED: Homo sapiens










similar to chromobox homolog 3










(LOC653972), mRNA.





NM_007276.3

1690
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 1, mRNA.





NM_016587.2

1730
3U
11335
Homo sapiens chromobox homolog










3 (HP1 gamma homolog,










Drosophila) (CBX3),










transcript variant 2, mRNA.



18/19

NM_016587
95
1730



17/19

NM_016587
95
1730



19/19
Antisense
NM_016587
297
1730



18/19

NM_016587
297
1730



17/19

NM_016587
297
1730





NM_152292.3

3155
3U
93587
Homo sapiens RNA (guanine-9-)










methyltransferase domain










containing 2 (RG9MTD2), mRNA.





XR_018656.1

451
3U
649307
PREDICTED: Homo sapiens similar to










Olfactory receptor 10R2 (LOC649307),










mRNA.









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.







TABLE 4







Table 4











SEQ




IQ



Sequence
NO:







5′ -CTGTTTTTTTCCCAT-3′
 98



 



5′ -TCTGTTTTTTTCCCAT-3′
 99



 



5′ -TTCTGTTTTTTTCCCAT-3′
100



 



5′ -ATTCTGTTTTTTTCCCAT-3′
101



 



5′ -CATTCTGTTTTTTTCCCAT-3′
102



 



5′ -CCATTCTGTTTTTTTCCCAT-3′
103



 



5′ -TCCATTCTGTTTTTTTCCCAT-3′
104



 



5′ -TCCATTCTGTTTTTTTCCCAT-3′
105



 



5′ -TTCCATTCTGTTTTTTTCCCAT-3′
106



 



5′ -TTTCCATTCTGTTTTTTTCCCAT-3′
107



 



5′ -CTTTCCATTCTGTTTTTTTCCCAT-3′
108



 



5′ -TCTTTCCATTCTGTTTTTTTCCCAT-3′
109



 



5′ -CTCTTTCCATTCTGTTTTTTTCCCAT -3′
110



 



5′ -ACTCTTTCCATTCTGTTTTTTTCCCAT-3′
111



 



5′ -TACTCTTTCCATTCTGTTTTTTTCCCAT-3′
112



 



5′ -TTACTCTTTCCATTCTGTTTTTTTCCCAT-3′
113










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.
<Composition for Cancer Treatment>
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.
<Method for Inhibition of Proliferation>
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.
<Method for Treatment>
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.
<Screening Method>
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
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 %”.
<Method for Cell Culture>
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.
<Antibody>
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 (Abcam); and anti-CAPD antibody to GAPDH (Santacruz) as a control.
Example 1
1. Expression of HP1γ Protein in the Process of Cell Differentiation
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 (ImmobilonTM 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 (LAS-3000TM 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 FIG. 1(A). FIG. 1(A) is autoradiograms showing the time profiles of the expression of different proteins (HP1α, HP1β, HP1γ) in 3T3-L1 mouse preadipocytes and human preadipocytes in both of which differentiation had been induced. In the figure, the results in 3T3-L1 cells are on the left, and those in human preadipocytes on the right.
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 adipocytes. 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 FIG. 1(B). FIG. 1(B) is a set of autoradiograms showing the expression of HP1γ protein in 3T3-L1. In the figure, “tr” indicates the results obtained from the mifepristone-inducible HP1γ-expressing 3T3-L1 (plasmid-introduced cells), and “wt” those from 3T3-L1 in which no recombinant plasmid pGHP1γ had been introduced (plasmid-unintroduced cells). “Mifepristone (+) (−)” indicates whether the culture was performed in the presence, or absence, of mifepristone, and “adipose reagents (+) (−)” indicates whether the culture was performed in the presence, or absence, of the differentiation inducers.
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 FIG. 1(C). The figure is a set of photographs showing the results of the oil red staining of 3T3-L1. In the figure, “transfectant” indicates the results of mifepristone-inducible HP1γ expression system 3T3-L1 (plasmid-introduced cells), and “wt” the results of 3T3-L1 into which no recombinant pGHP1γ had been introduced (plasmid-unintroduced cells). And “mifepristone (+) (−)” indicates a culture in the presence, or absence, of mifepristone, and “adipose reagents (+) (−)” a culture in the presence, or absence, of differentiation inducers.
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.
2. Correlation Between HP1 Expression and Histone Modification
(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 FIG. 2(A). The figure is a set of autoradiograms showing the time course of histone modification during differentiation of 3T3-L1. As shown in the figure, the acetylation levels at K9 and K18 of histone H3, and at K2 and K16 of histone H4, were equally lowered along with the decreasing amount of expressed HP1γ protein which occurred as the cell differentiation proceeded. In contrast to this, and contrary to the decrease in HP1γ protein expression occurring along with cell differentiation, methylation at K4, arginine (R9) and K17 of histone H3 increased with time.
(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 FIG. 2(B). The figure is a set of autoradiograms showing the time course of the expression of HP1γ protein and histone modification. As shown in the figure, along with the over-expression of HP1γ protein, the levels of acetylation of K18 of histone H4 and trimethylation of K20 of histone H4 rose, while those of acetylation of K12 of histone H4 and dim ethylation of K4 of histone H3 declined.
(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 HP1β 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 FIG. 2(C). The figure is a set of autoradiograms showing the levels of histone modification and HP1γ expression when treated with an siRNA specific to each HP1 gene. It was found that trimethylation of histone 4HK20 and the expression of HP1γ protein closely correlate as seen in the figure.
3. Tendency in Terminal Differentiated Cells of Each Tissue
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 FIG. 3. The figure is a set of photographs showing the result of immunostaining of the tissues. In the figure, the results are: (a-c) of adipose tissue, (d-f) of esophageal mucosa, (g-i) of dermal tissue, and (j-l) of colonic normal cells. The left column shows the result of H&E staining of the tissues, the center column the result of immunostaining of the tissues which visualizes localization of HP1γ, and the right column the result of immunostaining of the tissues which visualizes localization of Met3H4K20.
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 HP1γ protein 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.
4. Enhanced Expression of HP1γ Protein in Human Malignant Tumors
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 FIG. 4-1, FIG. 4-2, and FIG. 4-3. These figures are photographs showing the results of immunostaining of the malignant tumors. In these figure, “H&E” indicates the results of H&E staining, “HP1γ” the results of staining which reveals the localization of HP1γ protein, and “TriMeH4K20” the results of staining which reveals the localization of TriMeH4K20 (Met3H4K20). As shown in the figures, HP1γ protein was detected in the cell nuclei of all the cell samples of malignant tumors, and the same result was obtained also in the other samples, which are not presented in figures (n=26). Furthermore, trimethylated K20 of histone H4 was detected in 17 cases out of these 26 case samples.
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 FIG. 4, there are many cases in which HP1γ protein was detected (positive) but histone trimethylation of H4K20 was found negative. Thus, it is thought to be characteristic of malignant tumor cells that the tie between the expression of HP1γ protein and trimethylation of histone H4K20 seen in normal cells is dissociated. The results indicate that normal differentiated cells and tumor cells can be distinguished from each other by detecting HP1γ protein.
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.
Example 2
Inhibition of Proliferation of Malignant Tumor Cells by Means of Suppression of HP1γ Expression Using RNA Interference
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 FIG. 5. The figure is a set of graphs showing the number of the viable cells (×104 cells) among each type of tumor cells. In the figure, “UT” means the results in the cells which were not treated with siRNA, “A” the results of introduction of double-stranded siRNA to human HP1γ gene, and “C” the results of introduction of the negative control siRNA. As seen in the figure, by suppressing HP1γ gene through introduction of siRNA corresponding to human HP1γ gene, proliferation of tumor cells was inhibited. Thus, as proliferation of tumor cells is inhibited by suppressing the expression of HP1γ gene (expression of HP1γ protein) in tumor cells, HP1γ gene is very useful as a target gene for cancer treatment.
Example 3
Examination of the Suppressive Effect of siRNA on HP1γ Expression in Human Malignant Tumor Cells
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 FIG. 6, as well as a mixture of the three (Mix), and human malignant tumor cells transfected with them were examined for any suppression of human HP1γ expression. Malignant tumor cells employed were human cancer cell line DLD-1, which were transfected with each of the siRNAs (5 nM) or their mixture (5 nM each) in the same manner as described in Example 2. As a negative control, siRNA (SEQ ID NO:123)(mftd. by B-Bridge) described in “2.(3)” above was used. After transfection, culture was performed as described in Example 2, and the potency of HP1γ expression was examined by western blotting 3 and 5 days after the start of the culture. The results are shown in FIG. 6.
As evident from FIG. 6, it is confirmed that all the human HP1γ-specific g-1 to g-3 siRNAs strongly inhibit the expression of HP1γ to a similar degree to one another, and that their effect becomes all the more notable with the lapse of days. Further, the mixture of these siRNAs (total concentration being triple) exhibited still more notable effect. On the other hand, the negative control, mouse siRNA, did not show any inhibitory effect. These results indicate that the above-mentioned human HP1γ-specific siRNAs do work specifically to human HP1γ gene as expected, and that their inhibitory effects on HP1γ expression are highly potent. In the figure, “UT DLD-1” indicates the untreated control.
Example 4
Examination of the Therapeutic Effect on Cancer in vivo
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 cance 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 FIG. 7.
As seen in FIG. 7, while a rapid growth of the tumor was observed under the skin of the control agent-administered animals, the tumor growth was notably suppressed in either of the test agent-injected group or the test agent externally-applied group. And according to the microscopic examination of HE-stained slices of the tumors 22 days after the start of the observation, tumor cells were found necrosed in the tumors to which the cream was externally applied. In contrast, no necrosis of cells was observed in the control group which was injected with the negative control.
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.
INDUSTRIAL APPLICABILITY
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.

## Claims

1. A method for treatment of cancer in a mammal comprising administering, to the mammal in need thereof, an effective amount of a cancer cell proliferation inhibitor agent which is;
an siRNA which is specific to heterochromatin protein 1γ (HP1γ) gene of the mammal or
an antisense DNA which is specific to heterochromatin protein 1γ (HP1γ) gene of the mammal.

2. The method for treatment according to claim 1, wherein the mammal is a human and the HP1γ is human HP1γ.

3. The method for treatment according to claim 2, wherein the cancer cell proliferation inhibitor is an siRNA which is specific to human HP1γ.

4. The method for treatment according to claim 3, wherein the siRNA comprises at least one double-stranded RNA selected from #1 to #106, each of which comprises a corresponding RNA strand set forth in the 5′ to 3′ direction in the following Tables 2A to 2F:











TABLE 2A








SEQ





ID





NO:
Sequence









 #1
 39
CAACAAAACUACAUUGCAA




192
UUGCAAUGUAGUUUUGUUG



 



 #2
127
AAAACAGAAUGGAAAGAGU




193
ACUCUUUCCAUUCUGUUUU



 



 #3
 40
AAACAGAAUGGAAAGAGUA




194
UACUCUUUCCAUUCUGUUU



 



 #4
128
AACAGAAUGGAAAGAGUAA




195
UUACUCUUUCCAUUCUGUU



 



 #5
 41
CAGAAUGGAAAGAGUAAAA




196
UUUUACUCUUUCCAUUCUG



 



 #6
129
AAAAAGUUGAAGAGGCAGA




197
UCUGCCUCUUCAACUUUUU



 



 #7
130
AGUUGAAGAGGCAGAGCCU




198
AGGCUCUGCCUCUUCAACU



 



 #8
 43
UUGAAGAGGCAGAGCCUGA




199
UCAGGCUCUGCCUCUUCAA



 



 #9
 44
AGGCAGAGCCUGAAGAAUU




200
AAUUCUUCAGGCUCUGCCU



 



#10
 45
GGCAGAGCCUGAAGAAUUU




201
AAAUUCUUCAGGCUCUGCC



 



#11
 46
CUGAAGAAUUUGUCGUGGA




202
UCCACGACAAAUUCUUCAG



 



#12
 47
UGAAGAAUUUGUCGUGGAA




203
UUCCACGACAAAUUCUUCA



 



#13
 48
AAGAAUUUGUCGUGGAAAA




204
UUUUCCACGACAAAUUCUU



 



#14
131
AGAAUUUGUCGUGGAAAAA




205
UUUUUCCACGACAAAUUCU



 



#15
132
UGUCGUGGAAAAAGUACUA




206
UAGUACUUUUUCCACGACA



 



#16
133
UGGAAAAAGUACUAGAUCG




207
CGAUCUAGUACUUUUUCCA



 



#17
 49
GGAAAAAGUACUAGAUCGA




208
UCGAUCUAGUACUUUUUCC



 



#18
 50
AGUACUAGAUCGACGUGUA




209
UACACGUCGAUCUAGUACU





















TABLE 2B








SEQ





ID





NO:
Sequence









#19
 51
AGAUCGACGUGUAGUGAAU




210
AUUCACUACACGUCGAUCU



 



#20
134
GACGUGUAGUGAAUGGGAA




211
UUCCCAUUCACUACACGUC



 



#21
 52
ACGUGUAGUGAAUGGGAAA




212
UUUCCCAUUCACUACACGU



 



#22
 53
AGUGAAUGGGAAAGUGGAA




213
UUCCACUUUCCCAUUCACU



 



#23
135
GUGAAUGGGAAAGUGGAAU




214
AUUCCACUUUCCCAUUCAC



 



#24
 54
UGAAUGGGAAAGUGGAAUA




215
UAUUCCACUUUCCCAUUCA



 



#25
136
UUCCUGAAGUGGAAGGGAU




216
AUCCCUUCCACUUCAGGAA



 



#26
137
UCCUGAAGUGGAAGGGAUU




217
AAUCCCUUCCACUUCAGGA



 



#27
138
CUGAAGUGGAAGGGAUUUA




218
UAAAUCCCUUCCACUUCAG



 



#28
139
GUGGAAGGGAUUUACAGAU




219
AUCUGUAAAUCCCUUCCAC



 



#29
140
UGGAAGGGAUUUACAGAUG




220
CAUCUGUAAAUCCCUUCCA



 



#30
141
AUUUACAGAUGCUGACAAU




221
AUUGUCAGCAUCUGUAAAU



 



#31
142
GCUGACAAUACUUGGGAAC




222
GUUCCCAAGUAUUGUCAGC



 



#32
143
UGACAAUACUUGGGAACCU




223
AGGUUCCCAAGUAUUGUCA



 



#33
 55
CUUGGGAACCUGAAGAAAA




224
UUUUCUUCAGGUUCCCAAG



 



#34
144
UUGGGAACCUGAAGAAAAU




225
AUUUUCUUCAGGUUCCCAA



 



#35
 56
CAGAAUUGAUUGAAGCGUU




226
AACGCUUCAAUCAAUUCUG



 



#36
 57
AGAAUUGAUUGAAGCGUUU




227
AAACGCUUCAAUCAAUUCU



 



#37
145
UGAUUGAAGCGUUUCUUAA




228
UUAAGAAACGCUUCAAUCA





















TABLE 2C








SEQ





ID





NO:
Sequence









#38
146
GUUUCUUAACUCUCAGAAA




229
UUUCUGAGAGUUAAGAAAC



 



#39
 58
UCAGAAAGCUGGCAAAGAA




230
UUCUUUGCCAGCUUUCUGA



 



#40
 59
CAGAAAGCUGGCAAAGAAA




231
UUUCUUUGCCAGCUUUCUG



 



#41
147
AGAAAGCUGGCAAAGAAAA




232
UUUUCUUUGCCAGCUUUCU



 



#42
 60
GAAAGCUGGCAAAGAAAAA




233
UUUUUCUUUGCCAGCUUUC



 



#43
148
UGGCAAAGAAAAAGAUGGU




234
ACCAUCUUUUUCUUUGCCA



 



#44
 61
GCAAAGAAAAAGAUGGUAC




235
GUACCAUCUUUUUCUUUGC



 



#45
 62
CAAAGAAAAAGAUGGUACA




236
UGUACCAUCUUUUUCUUUG



 



#46
149
AAUCUUUAUCUGACAGUGA




237
UCACUGUCAGAUAAAGAUU



 



#47
 63
GUGAAUCUGAUGACAGCAA




238
UUGCUGUCAUCAGAUUCAC



 



#48
150
GAAUCUGAUGACAGCAAAU




239
AUUUGCUGUCAUCAGAUUC



 



#49
151
GAUGACAGCAAAUCAAAGA




240
UCUUUGAUUUGCUGUCAUC



 



#50
152
GACAGCAAAUCAAAGAAGA




241
UCUUCUUUGAUUUGCUGUC



 



#51
 64
ACAGCAAAUCAAAGAAGAA




242
UUCUUCUUUGAUUUGCUGU



 



#52
153
CAGCAAAUCAAAGAAGAAA




243
UUUCUUCUUUGAUUUGCUG



 



#53
154
GCAAAUCAAAGAAGAAAAG




244
CUUUUCUUCUUUGAUUUGC



 



#54
 66
UCAAAGAAGAAAAGAGAUG




245
CAUCUCUUUUCUUCUUUGA



 



#55
155
GAAGAAAAGAGAUGCUGCU




246
AGCAGCAUCUCUUUUCUUC



 



#56
156
AGAAAAGAGAUGCUGCUGA




247
UCAGCAGCAUCUCUUUUCU





















TABLE 2D








SEQ





ID





NO:
Sequence









#57
 67
GAAAAGAGAUGCUGCUGAC




248
GUCAGCAGCAUCUCUUUUC



 



#58
157
AAAGAGAUGCUGCUGACAA




249
UUGUCAGCAGCAUCUCUUU



 



#59
 68
AAGAGAUGCUGCUGACAAA




250
UUUGUCAGCAGCAUCUCUU



 



#60
 69
GCUGACAAACCAAGAGGAU




251
AUCCUCUUGGUUUGUCAGC



 



#61
158
CUGACAAACCAAGAGGAUU




252
AAUCCUCUUGGUUUGUCAG



 



#62
  1
UGACAAACCAAGAGGAUUU




253
AAAUCCUCUUGGUUUGUCA



 



#63
159
ACCAAGAGGAUUUGCCAGA




254
UCUGGCAAAUCCUCUUGGU



 



#64
160
AGGAUUUGCCAGAGGUCUU




255
AAGACCUCUGGCAAAUCCU



 



#65
161
UGCCAGAGGUCUUGAUCCU




256
AGGAUCAAGACCUCUGGCA



 



#66
162
CCAGAGGUCUUGAUCCUGA




257
UCAGGAUCAAGACCUCUGG



 



#67
163
AGGUCUUGAUCCUGAAAGA




258
UCUUUCAGGAUCAAGACCU



 



#68
164
GUCUUGAUCCUGAAAGAAU




259
AUUCUUUCAGGAUCAAGAC



 



#69
165
UCUUGAUCCUGAAAGAAUA




260
UAUUCUUUCAGGAUCAAGA



 



#70
166
CCACAGACAGCAGUGGAGA




261
UCUCCACUGCUGUCUGUGG



 



#71
167
CACAGACAGCAGUGGAGAA




262
UUCUCCACUGCUGUCUGUG



 



#72
168
ACAGACAGCAGUGGAGAAU




263
AUUCUCCACUGCUGUCUGU



 



#73
 70
AGACAGCAGUGGAGAAUUG




264
CAAUUCUCCACUGCUGUCU



 



#74
169
GUGGAGAAUUGAUGUUUCU




265
AGAAACAUCAAUUCUCCAC



 



#75
170
GAGAAUUGAUGUUUCUCAU




266
AUGAGAAACAUCAAUUCUC





















TABLE 2E








SEQ





ID





NO:
Sequence









#76
171
GAAUUGAUGUUUCUCAUGA




267
UCAUGAGAAACAUCAAUUC



 



#77
 71
CUCAUGAAAUGGAAAGAUU




268
AAUCUUUCCAUUUCAUGAG



 



#78
172
CAUGAAAUGGAAAGAUUCA




269
UGAAUCUUUCCAUUUCAUG



 



#79
173
UGAAAUGGAAAGAUUCAGA




270
UCUGAAUCUUUCCAUUUCA



 



#80
174
GGAAAGAUUCAGAUGAGGC




271
GCCUCAUCUGAAUCUUUCC



 



#81
175
GAAAGAUUCAGAUGAGGCA




272
UGCCUCAUCUGAAUCUUUC



 



#82
176
AAGAUUCAGAUGAGGCAGA




273
UCUGCCUCAUCUGAAUCUU



 



#83
 72
CAGACUUGGUGCUGGCGAA




274
UUCGCCAGCACCAAGUCUG



 



#84
177
AGACUUGGUGCUGGCGAAA




275
UUUCGCCAGCACCAAGUCU



 



#85
178
ACUUGGUGCUGGCGAAAGA




276
UCUUUCGCCAGCACCAAGU



 



#86
179
GGUGCUGGCGAAAGAGGCA




277
UGCCUCUUUCGCCAGCACC



 



#87
180
UGGCGAAAGAGGCAAAUAU




278
AUAUUUGCCUCUUUCGCCA



 



#88
 73
GCGAAAGAGGCAAAUAUGA




279
UCAUAUUUGCCUCUUUCGC



 



#89
 74
CGAAAGAGGCAAAUAUGAA




280
UUCAUAUUUGCCUCUUUCG



 



#90
181
AGGCAAAUAUGAAGUGUCC




281
GGACACUUCAUAUUUGCCU



 



#91
182
GCAAAUAUGAAGUGUCCUC




282
GAGGACACUUCAUAUUUGC



 



#92
183
AAUAUGAAGUGUCCUCAAA




283
UUUGAGGACACUUCAUAUU



 



#93
184
CCUCAAAUUGUAAUUGCUU




284
AAGCAAUUACAAUUUGAGG



 



#94
185
AGAGAGACUAACUUGGCAU




285
AUGCCAAGUUAGUCUCUCU




















TABLE 2F







SEQ





ID





NO:
Sequence








 #95
186
GAGAGACUAACUUGGCAUU




286
AAUGCCAAGUUAGUCUCUC


 



 #96
187
GCAUUCUUGUCCAGAAGAU




287
AUCUUCUGGACAAGAAUGC


 



 #97
188
GUCCAGAAGAUGAAGCUCA




288
UGAGCUUCAUCUUCUGGAC


 



 #98
189
UCCAGAAGAUGAAGCUCAA




289
UUGAGCUUCAUCUUCUGGA


 



 #99
 75
CAGAAGAUGAAGCUCAAUA




290
UAUUGAGCUUCAUCUUCUG


 



#100
190
AGAAGAUGAAGCUCAAUAA




291
UUAUUGAGCUUCAUCUUCU


 



#101
 83
GAUAAUCCCUUCAAGUUAA




292
UUAACUUGAAGGGAUUAUC


 



#102
 84
CCAUACAUUUCAAGUGAAA




293
UUUCACUUGAAAUGUAUGG


 



#103
 88
GACAAAUGCUAGUGUGUUU




294
AAACACACUAGCAUUUGUC


 



#104
 89
GGGCCAUUCCUUAGCAAAA




295
UUUUGCUAAGGAAUGGCCC


 



#105
191
GGUCAUGAUGAAUGGAAUA




296
UAUUCCAUUCAUCAUGACC


 



#106
 95
CAGCAAAAGCCAGGAAGAA




297
UUCUUCCUGGCUUUUGCUG.

5. The method for treatment according to claim 4, wherein the double-stranded RNAs are selected from #5, #17, #35, #62, #89, #101, #102, #103, #104, #105 and #106 in Tables 2A to 2F.

6. The method for treatment according to claim 4, wherein the double-stranded RNAs are selected from #17, #62 and #89 in Tables 2A to 2F.

7. The method for treatment according to one of claim 4, wherein the siRNAs each comprise two-base overhangs on both ends thereof.

8. The method for treatment according to claim 7, wherein each of the overhangs is on the 3′ end of each of the strands forming the double-stranded RNAs.