Agents and a method of chromosome preparation using protein phosphatase inhibitors induced premature chromosome condensation (PCC) technique

Agents and a method for generating chromosomes by premature chromosome condensation (PCC) technique utilizes inhibitors of serine/threonine protein phosphatases. When the cells were treated with these agents, they underwent PCC at any phase of cell cycle within 2 hours. This method enables to obtain not only chromosome of mitotic cells but also those from interphase nuclei much easily, quickly and effectively.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the method of obtaining the chromosomes 
more easily and efficiently than the conventional method using colhitin or 
its derivatives (colcemid), in more particular to the method of inducing 
premature chromosome condensation in interphase cell with inhibitors of 
serine/threonine protein phosphatases, that permit one to obtain not only 
mitotic chromosomes but also prematurely condensed chromosomes of 
interphase nuclei. 
2. Description of the Related Art 
Chromosomal analysis is being widely used for screening or diagnosis of 
genetic diseases, assay of the mutagenecity of physical or chemical 
factors or other various cytogenetical purposes in medical, biological, 
agricultural or other fields. Chromosomes are obtained usually from 
mitotic cells using colhitin or its derivative, colcemid, thereby 
inhibiting the assembly of tubulin to form mitotic spindles during mitotic 
process. Therefore it requires cells to pass through the mitosis. However, 
it is well known by those skilled in the art, that it is often difficult 
to obtain chromosomes where cells do not proliferate well. 
Furthermore, many phenomena occur during interphase; for example, 
chromosomal cleavage by irradiation and subsequent reunion occur through 
the interphase nuclei, resulting in chromosomal aberration. It is, thus, 
necessary to obtain a technique that allows one to obtain chromosomes from 
interphase cells. 
The first approach was done by Johnson and Rao (Johnson and Rao, 1970, 
Nature 226: 717). They fused interphase cells and mitotic cells, thereby 
obtaining premature interphase chromosome condensation. (Premature 
Chromosome Condensation; PCC). However, this method of inducing PCC is 
technically demanding; synchronizing and collecting large amounts of 
mitotic cells followed by fusion with target cells. In addition, mixture 
of chromosomes of interphase and mitotic cell makes it often difficult to 
analyses the chromosomes of interphase cells. 
A similar premature chromosome condensation can be induced by caffeine in 
cells blocked in S-phase (Schlegel and Pardee, 1986, Science: 232, 1264). 
More recently, okadaic acid has also been reported to induce PCC in cells 
blocked in S-phase (Ghosh et al., 1992, Exp. Cell Res.: 201, 535, 
Steinmann et al., 1991, Proc. Natl. Acad. Sci. USA: 88, 6843, Yamashita et 
al., 1990, EMBO J: 9, 4331). However, the requirement of block the cells 
in S-phase has limited the use of PCC technique for the analysis of 
chromosomes. 
SUMMARY OF THE INVENTION 
The present invention has been proposed in view of the above-mentioned 
drawbacks inherent to the prior art, and accordingly, one object of the 
present invention is to provide an improved PCC technique for obtaining 
chromosomes prematurely from the interphase cells at any time of cell 
cycle, with quickly, easily, efficiently and in a highly reproducible 
manner. 
According to the present invention, protein phosphatase inhibitors, okadaic 
acid, okadaic acid ammonium salt, 35-methyl okadaic acid, calyculin A, 
tautomycin, cantharidine, cantharidic acid and endothal can directly 
induce PCC in mammalian somatic cells at any time of cell cycles without 
the block of cells in S-phase. When the cells were treated with these 
agents, the cells underwent PCC within 2 hour after exposure to these 
agents. The frequency of PCC obtained by treatment of these agents 
surpassed that of metaphase chromosomes using colcemid. 
These findings indicate that the method, described here, generates the PCC 
using protein phosphatase inhibitors might take the place of conventional 
way of obtaining chromosomes using colcemid. Furthermore, this method 
might permit one to analyze many of the phenomena occuring in interphase 
nuclei.

As used herein "PCC", refers to an abbreviation of premature chromosome 
condensation. Because, chromosomes usually condense during mitosis except 
under certain circumstances. When the chromosomes condense prior to 
mitosis, this phenomenon is known as premature chromosome condensation. 
Prematurely condensed chromosomes also give the same abbreviation "PCC". 
So said "PCC" means both a phenomenon on chromosomes and a condition of 
those. In many cases, its meanings are interchangeable . Therefore, we use 
the term "PCC" for both meanings for a phenomenon and a condition of 
chromosomes, so far not specified. 
Prematurely condensed chromosomes or metaphase chromosomes in all the 
experiment of the present invention were prepared by the usual method 
which is well known by the skilled in the art. Namely, cells were grown 
exponentially at 37.degree. C. in 5% CO.sub.2 atmosphere under 95% 
humidity. After treatment of cells with individual agent for up to 2 
hours, cells were harvested. Then cells were subjected to hypotonic 
treatment in 75 mM KCl at 37.degree. C. for 20 minutes to swell the cells. 
Cells were then fixed with a fixative (3:1 vol./vol. methanol:acetic acid) 
and then they are spread on a glass slide. 
DESCRIPTION OF PREFERRED EMBODIMENTS 
Referring now to FIG. 1 which illustrates the relationship between cell 
cycle and chromosome condensation. The cells proliferate through the 
G.sub.1 (Gap 1) phase, S (Synthesis) phase and G.sub.2 (Gap 2) phase, and 
divide at M (Mitosis) phase. Said "Gap 1" phase means that this phase 
locates behind said "M" phase and ahead of said "S" phase, and said "Gap 
2" phase means that this phase locates behind said "S" phase and ahead of 
said "M" phase. At mitosis, chromosomes condense, attach to mitotic 
spindles and segregate in each cell. Colhitin or its derivative colcemid 
arrest the cells at mitosis, whereby inhibiting the formation of mitotic 
spindles. Therefore, these agents are widely used for obtaining mitotic 
chromosomes. However, it is well known by those skilled in the art that it 
is often difficult to acquire chromosomes from cells which proliferate 
slowly, because they do not pass through mitosis. In addition, many of 
biological events go on during interphase such as chromosome breakage by 
irradiation. So several attempts have been done to obtain interphase 
chromosomes to overcome these problems. One of these is the technique of 
premature chromosome condensation (PCC). 
Referring to FIG. 2 and 3 which are schematic showing of PCC by cell fusion 
method and the prematurely condensed chromosomes obtained by this 
technique, respectively. When the interphase cell is fused in mitotic 
cell, the interphase nucleus undergoes PCC (FIG. 2). Various kinds of PCC 
are shown in FIG. 3. Numerals 3-a and 3-b are G.sub.1 -PCC; well condensed 
but univalent chromosomes. Numeral 3-c is early S-PCC; decondensed and 
filamentous shape. Numerals 3-d and 3-e are S-PCC; a typical "pulverized" 
appearance. Numeral 3-f is G.sub.2 -PCC; well condensed and bivalent 
chromosomes identical with metaphase chromosomes. Small arrows indicate 
the chromosomes prematurely condensed by cell fusion, and large arrows 
indicate the metaphase chromosomes of inducer cells. Bar indicates 10 
.mu.m. This fusion method is very useful to obtain interphase chromosomes, 
but it is technically demanding: synchronize and arrest the inducer cells 
at mitosis, and fuse with target cells. The efficiency of cell fusion is 
not so high. In addition, the chromosomes obtained by cell fusion are a 
mixture of those of inducer and target cells as shown in FIG. 3. 
Therefore, it makes it difficult to use this technique widely. 
FIG. 4 shows metaphase chromosomes obtained after treatment with colcemid, 
as a control study. 
Referring now to the agents used in the present invention. Okadaic acid, an 
agent extracted from a marine sponge Haricondria Okadai, is a specific 
inhibitor of serine/threonine protein phosphatases. Okadaic acid has a 
molecular weight (M.W.) of 805.2 and its agent structure is C.sub.44 
H.sub.68 O.sub.13. Okadaic acid ammonium salt (C.sub.44 H.sub.67 O.sub.13 
--NH.sub.4, M.W. 822.05) and 35-methyl okadaic acid (C.sub.45 H.sub.70 
O.sub.13, M.W. 819.04) are derivatives of okadaic acid, both show 
inhibitory effect on protein phosphatase as okadaic acid does. Calyculin A 
(C.sub.50 H.sub.81 O.sub.15 P, M.W. 1009.18) is another toxin obtained 
from a marine sponge Discodermia Calyx, which is also an inhibitor of 
protein phosphatases. Tautomycin (C.sub.41 H.sub.66 O.sub.13, M.W. 766.97) 
is an antibiotic extracted from a soil bacteria Streptomyces 
spiroverticillatus. Cantharidine (C.sub.10 H.sub.12 O.sub.4, M.W. 196.2) 
is an agent extracted from an erosive secretion of a blister beetle. 
Cantharidic acid (C.sub.10 H.sub.14 O.sub.5, M.W. 214.2) and Endothal 
(C.sub.8 H.sub.10 O.sub.5, M.W. 186.2) are derivatives of Cantharidine. 
These are also inhibitors of protein phosphatase as okadaic acid. All the 
agents presented here are specific inhibitor of serine/threonine protein 
phosphatases, especially of type 1 and type 2A protein phosphatases. In 
addition, all of these show cell permeability. All the agents except for 
okadaic acid ammonium salt were dissolved in 100% ethanol, methanol or 
dimetyl-sulfoxide (DMSO) to make a stock solution of 1 mM concentration 
(okadaic acid, 35-methyl okadaic acid, calyculin A or tautomycin) or 100 
mM (cantharidine, cantharidic acid or endothal). Okadaic acid ammonium 
salt was dissolved in water to make a stock solution of 1 mM 
concentration. 
As used herein "protein phosphatase" or more specifically "serine/threonine 
protein phosphatase", refers to the enzymes that catalyze 
dephosphorylation of phosphorylated protein, especially of 
dephosphorylation of phosphorylated serine and/or phosphorylated threonine 
amino acid residues of protein. These enzymes are generally classified 
into 4 types, namely, type 1, type 2A, type 2B and type 2C according to 
their subunit structure, their dependencies of metal ion for their 
catalyzing activity, or other factors. 
As used herein "inhibitor of protein phosphatase", refer to the agents that 
inhibit the catalyzation of the protein dephosphorylation by said protein 
phosphatases. 
By using protein phosphatase inhibitors listed above as an inducer of PCC, 
the present invention provides a new way to generate chromosomes condensed 
prematurely from interphase nuclei, easily and more quickly than the 
conventional cell fusion method. 
Referring now to FIG. 5 which shows the prematurly condensed chromosomes 
generated by the present invention using 100 nM okadaic acid (as final 
concentration). Hereafter, the concentration of each agent used in the 
present study indicates the final concentration in the culture medium, so 
far not specified. Herein used "G.sub.1 /S-PCC", refers to the terms 
combined the G.sub.1 -PCC and S-PCC. Although G.sub.1 -PCC and S-PCC are 
different from each other in strict meaning, the frequency of G.sub.1 -PCC 
is much less than that of S-PCC and it is difficult to distinguish them 
practically. So we scored both types of PCC together and termed as 
"G.sub.1 /S-PCC". When the human peripheral blood lymphocytes were 
affected with this agent, the cells underwent PCC at any phase of cell 
cycle. FIG. 5-a shows G.sub.2 -PCC, which shape is the same as that of 
metaphase chromosomes obtained after treatment of colcemid (Bar indicates 
10 .mu.m). FIG. 5-b shows G.sub.1 /S-PCC (indicated by arrows) and G.sub.2 
-PCC (Bar indicates 50 .mu.m). G.sub.1 /S-PCC shows a typical form 
"pulverized appearance" as shown in FIG. 5-c and its magnified picture, 
5-d. G.sub.1 /S-PCC is composed of thick and thin parts of chromatin. It 
is thought that the former corresponds to replicated chromosomes, whereas 
the latter corresponds to unreplicated chromatins. Arrowheads indicate the 
thick portion in G.sub.1 /S-PCC (Bars indicate 10 .mu.m). So the 
phenomenon studied here is actually PCC. Preferred embodiment is that this 
technique requires only addition of agent in the culture medium. It does 
not require any laborious procedure; synchronizing and collecting large 
amounts of mitotic cells followed by fusion with target cells. 
Furthermore, the present invention is thus very simple. 
Table 1 shows the dose response of PCC induced by okadaic acid in various 
types of mammalian cells. As human cells, Phytohemaggulitin-P (PHA-P) 
stimulated human peripheral blood lymphocytes from two healthy donors, 
established cell line AT(L)5KY and AT(L)6KY were used. Whereas as mouse 
cells, Concanavalin-A (Con-A) or Lipo-polysaccharide (LPS) stimulated 
mouse splenocytes and established cell line Ba/F3 were used. As used 
herein "PHA-P", "Con-A" and "LPS" are mitogens usually used for stimulate 
the lymphocytes, that is well known by those skilled in the art. These 
cells were treated with okadaic acid of which concentration varies from 1 
nM to 100 nM. After 2 hours' exposure, cells were harvested and subjected 
to hypotonic treatment. Then cells were fixed with methanol:acetic acid 
(3:1) and chromosome spreads were obtained, which is well known by those 
skilled in the art. G.sub.1 /S-PCC or G.sub.2 -PCC was scored and 
frequencies (putted in parentheses as a percentage) were obtained. As a 
control study, cells were treated with colcemid for 2 hours to generate 
metaphase chromosomes. The same hypotonic treatment and fixation were 
done. In the case of the concentration of okadaic acid is lower than 10 
nM, G.sub.2 -PCC was solely induced. In contrast, 100 nM okadaic acid 
induced both G.sub.1 /S-PCC and G.sub.2 -PCC effectively. Furthermore, the 
frequency of G.sub.1 /S-PCC and G.sub.2 -PCC were much higher than that of 
metaphase chromosomes obtained by the conventional colcemid treatment. 
TABLE 1 
__________________________________________________________________________ 
Intrerphase Cell 
G1/S PCC 
G2/M PCC 
Total 
Sample Name Treatment (%) (%) (%) Number 
__________________________________________________________________________ 
Human 
Human lymphocyte #1 Colcemid 
364 (97.3) 10 (2.6) 
374 
Okadaic acid 
1 nM 389 (97.7) 
0 (0.0) 
9 (2.3) 
398 
Okadaic acid 
10 nM 
500 (97.2) 
1 (0.2) 
13 (2.5) 
514 
Okadaic acid 
100 nM 
123 (56.7) 
54 (24.9) 
40 (18.6) 
217 
Human lymphocytes #2 
Colcemid 
622 (95.8) 27 (4.2) 
649 
Okadaic acid 
1 nM 480 (98.0) 
0 (0.0) 
10 (2.0) 
490 
Okadaic acid 
10 nM 
773 (98.6) 
0 (0.0) 
11 (1.4) 
784 
Okadaic acid 
100 nM 
77 (59.7) 
39 (38.2) 
10 (8.0) 
126 
AT(L)5KY Colcemid 
381 (97.9) 8 (2.1) 
389 
Okadaic acid 
1 nM 493 (97.8) 
0 (0.0) 
11 (2.2) 
504 
Okadaic acid 
10 nM 
347 (95.8) 
0 (0.0) 
15 (4.1) 
362 
Okadaic acid 
100 nM 
89 (50.6) 
66 (37.5) 
21 (11.9) 
176 
AT(L)6KY Colcemid 
282 (98.6) 4 (1.4) 
286 
Okadaic acid 
1 nM 453 (98.6) 
0 (0.0) 
6 (1.4) 
469 
Okadaic acid 
10 nM 
394 (96.8) 
0 (0.0) 
13 (3.2) 
407 
Okadaic acid 
100 nM 
179 (60.1) 
97 (32.6) 
22 (7.4) 
298 
Mouse 
Mouse lymphocyte (Con A) 
Colcemid 
264 (93.3) 19 (6.7) 
283 
Okadaic acid 
1 nM 280 (96.9) 
0 (0.0) 
9 (3.1) 
289 
Okadaic acid 
10 nM 
196 (96.6) 
0 (0.0) 
7 (3.4) 
203 
Okadaic acid 
100 nM 
163 (88.5) 
6 (3.3) 
15 (8.2) 
184 
Mouse lymphocytes (LPS) 
Colcemid 
265 (88.0) 36 (12.0) 
301 
Okadaic acid 
1 nM 248 (94.7) 
0 (0.0) 
14 (5.3) 
262 
Okadaic acid 
10 nM 
148 (93.7) 
0 (0.0) 
10 (6.3) 
158 
Okadaic acid 
100 nM 
116 (78.4) 
17 (11.5) 
15 (10.1) 
148 
Ba/F3 Colcemid 
261 (92.2) 22 (7.8) 
283 
Okadaic acid 
1 nM 254 (92.4) 
0 (0.0) 
21 (7.6) 
275 
Okadaic acid 
10 nM 
303 (92.4) 
0 (0.0) 
25 (7.6) 
328 
Okadaic acid 
100 nM 
138 (88.5) 
2 (1.3) 
16 (10.3) 
156 
__________________________________________________________________________ 
FIG. 6 shows the graphical representation of the result shown in above 
Table 1. The frequencies of PCC or metaphase chromosomes are shown as an 
average number of each species, human and mouse cells. With respect to 
colcemid treatment, G.sub.2 -PCC should be interpreted as metaphase 
chromosomes. As clearly shown in the FIG. 6, 1 nM or 10 nM okadaic acid 
favor to induce G.sub.2 -PCC solely in human and mouse cells. In contrast, 
100 nM okadaic acid can induce both G.sub.1 /S-PCC and G.sub.2 -PCC in 
human and mouse cells. 
Table 2 shows the dose response of PCC induced by calyculin A in the same 
kinds of mammalian cells used in the experiment with okadaic acid. 
TABLE 2 
__________________________________________________________________________ 
Interphase Cell 
G1/S PCC 
G2/M PCC 
Total 
Sample Name 
Treatment (%) (%) (%) Number 
__________________________________________________________________________ 
Human 
Lymphocytes Colcemid 
364 (97.3) 10 (2.6) 
374 
Calc A 
1 nM 424 (98.4) 
0 (0.0) 
7 (1.6) 
431 
Calc A 
10 nM 
393 (94.6) 
11 (2.7) 
11 (2.7) 
415 
Calc A 
100 nM 
89 (61.0) 
30 (20.5) 
27 (18.5) 
146 
AT(L)5KY Colcemid 
381 (97.9) 8 (2.1) 
389 
Calc A 
1 nM 331 (96.2) 
0 (0.0) 
13 (3.8) 
344 
Calc A 
10 nM 
101 (54.6) 
65 (35.1) 
19 (10.3) 
185 
Calc A 
100 nM 
27 (26.2) 
50 (48.5) 
26 (25.2) 
103 
AT(L)6KY Colcemid 
282 (98.6) 4 (1.4) 
286 
Calc A 
1 nM 251 (97.6) 
0 (0.0) 
6 (2.3) 
257 
Calc A 
10 nM 
144 (72.0) 
39 (19.5) 
17 (8.5) 
200 
Calc A 
100 nM 
45 (37.8) 
49 (41.1) 
25 (21.1) 
119 
Mouse 
Lymphocytes (ConA) 
Colcemid 
264 (93.3) 19 (6.7) 
283 
Calc A 
1 nM 376 (95.2) 
1 (0.2) 
18 (4.6) 
395 
Calc A 
10 nM 
9 (10.1) 
48 (57.8) 
26 (31.3) 
83 
Calc A 
100 nM 
-- -- -- -- 
Lymphocytes (LPS) 
Colcemid 
265 (88.0) 36 (12.0) 
301 
Calc A 
1 nM 217 (94.8) 
0 (0.0) 
12 (5.2) 
229 
Calc A 
10 nM 
25 (20.8) 
45 (37.5) 
50 (41.6) 
120 
Calc A 
100 nM 
-- -- -- -- 
Ba/F3 Colcemid 
261 (92.2) 22 (7.8) 
283 
Calc A 
1 nM 197 (89.9) 
7 (3.2) 
15 (6.8) 
219 
Calc A 
10 nM 
0 (0.0) 111 (75.0) 
37 (25.0) 
148 
Calc A 
100 nM 
1 (0.8) 69 (59.4) 
46 (39.7) 
116 
__________________________________________________________________________ 
These cells were treated with calyculin A of which concentration varies 
from 1 nM to 100 mM. After 2 hours' exposure, PCC were obtained as 
described above. Control study was also done using colcemid. The 
appearances of PCC were identical to those induced by okadaic acid. 
Therefore I do not present photographs hereafter. When the concentration 
of calyculin A is 1 nM, G.sub.2 -PCC was -solely induced. In contrast, 
more than 10 nM calyculin A induced both G.sub.3 /S-PCC and G.sub.2 -PCC 
effectively. As same as okadaic acid, frequency of both G.sub.1 /S-PCC and 
G.sub.2 -PCC induced by more than 10 nM calyculin A were much more 
effective than that of metaphase chromosomes obtained by the conventional 
colcemid treatment. As to mouse splenocytes, calyculin A showed much 
higher cell toxicity than okadaic acid, thus resulted in loss of cells 
during culture. Therefore we omitted the experiment using 100 nM calyculin 
A for these cell types. Even at 10 nM concentration, calyculin A exerted 
PCC effectively. 
FIG. 7 shows the graphical representation of the result shown in above 
Table 2. The frequencies of PCC or metaphase chromosomes are shown as an 
average number of each species, human and mouse. In the case of colcemid 
treatment, G.sub.2 -PCC should be interpreted as metaphase chromosomes. As 
clearly shown, 1 nM calyculin A favor to induce G.sub.2 -PCC solely in 
human and mouse cells. In contrast, more than 10 nM calyculin A can induce 
both G.sub.1 /S-PCC and G.sub.2 -PCC in human and mouse cells. 
According to the experiments described above, it was found that both 
okadaic acid and calyculin A could induce PCC in mammalian cells at any 
time of cell cycle. These agents have a common feature as a specific 
inhibitor of protein phosphatases, more specifically type 1 and type 2A of 
serine/threonine protein phosphatases But these agent structures are 
somewhat different from each other. So the PCC induced by these agents is 
thought to be contributed by the inhibitory effect of protein phosphatase. 
Therefore we investigated the PCC inducibility using other inhibitors of 
protein phosphatase; okadaic acid ammonium salt, 35-methyl okadaic acid, 
tautomycin, cantharidine, cantharidic acid and endothal. The profiles of 
these agents were shown above. 
Now referring to Table 3, which shows the result of the frequency of PCC 
obtained after treating of this agent in human peripheral blood 
lymphocytes. Cells were stimulated and cultured as done in the experiment 
using okadaic acid or calyculin A. Cells were treated with each agent for 
2 hours, then PCC were obtained as described above. 
TABLE 3 
__________________________________________________________________________ 
Interphase Cell 
G1/S PCC 
G2/M PCC 
Total 
Sample Name 
Treatment (%) (%) (%) Number 
__________________________________________________________________________ 
Human 
Human Lymphocytes Colcemid 
344 (97.7) 8 (2.3) 
352 
Okadaic acid Ammonium salt 
1 nM 244 (96.8) 
0 (0.0) 
8 (3.2) 
252 
Okadaic acid Ammonium salt 
10 nM 
259 (96.6) 
0 (0.0) 
9 (3.4) 
268 
Okadaic acid Ammonium salt 
100 nM 
156 (57.1) 
83 (30.4) 
34 (12.5) 
273 
Human Lymphocytes Colcemid 
363 (97.0) 11 (3.0) 
374 
35-methyl okadaic acid 
1 nM 277 (96.8) 
0 (0.0) 
9 (3.2) 
286 
35-methyl okadaic acid 
10 nM 
263 (96.5) 
0 (0.0) 
10 (3.5) 
273 
35-methyl okadaic acid 
100 nM 
161 (63.1) 
62 (24.3) 
32 (12.6) 
255 
Human Lymphocytes Colcemid 
364 (97.3) 10 (2.6) 
374 
Cantharidin 500 nM 
266 (97.5) 
0 (0.0) 
7 (2.5) 
273 
Cantharidin 5 uM 213 (92.0) 
12 (5.3) 
7 (2.7) 
232 
Cantharidin 50 uM 
260 (78.8) 
61 (18.5) 
9 (2.7) 
330 
Human Lymphocytes Colcemid 
381 (97.9) 8 (2.1) 
389 
Cantharidic acid 
500 nM 
257 (97.5) 
0 (0.0) 
7 (2.5) 
264 
Cantharidic acid 
5 uM 201 (91.0) 
13 (6.0) 
7 (3.0) 
221 
Cantharidic acid 
50 uM 
221 (85.3) 
29 (11.2) 
9 (3.5) 
259 
Human Lymphocytes Colcemid 
364 (97.3) 10 (2.6) 
374 
Endothal 500 nM 
268 (98.2) 
0 (0.0) 
5 (1.8) 
273 
Endothal 5 uM 262 (97.8) 
0 (0.0) 
8 (2.2) 
268 
Endothal 50 uM 
223 (94.6) 
6 (2.5) 
7 (2.9) 
236 
Human Lymphocytes Colcemid 
282 (98.6) 4 (1.4) 
286 
Tautomycin 100 nM 
236 (97.4) 
0 (0.0) 
7 (2.5) 
243 
Tautomycin 1 uM 270 (95.5) 
3 (1.0) 
10 (3.5) 
283 
Tautomycin 10 uM 
200 (84) 28 (11.8) 
10 (4.2) 
238 
__________________________________________________________________________ 
Okadaic acid ammonium salt or 35-methyl okadaic acid could induce G.sub.1 
/S-PCC and G.sub.2 -PCC at their concentration was 100 nM. At 1 nM or 10 
nM of these agents, G.sub.2 -PCC was solely induced. These findings were 
the same as in the case of okadaic acid. So the effect of these 3 agents 
is thought to be identical. 
Cantharidine or cantharidic acid could induce G.sub.1 /S-PCC and G.sub.2 
-PCC at their concentration higher than 5 .mu.M, and G.sub.2 -PCC only was 
induced at 500 nM these agents. Cantharidine or cantharidic acid were 
required to have the higher concentration to induce PCC than that of 
okadaic acid. Inhibitory dose (IC.sub.50) of cantharidine or cantharidic 
acid for protein phosphatase is higher than that of okadaic acid. 
Probably, this is the one reason for explaining the differences of the 
dose required. 
Endothal also could cause G.sub.1 /S-PCC and G.sub.2 -PCC at 50 .mu.M. 
However, it could induce G.sub.2 -PCC solely at less than 5 .mu.M. 
Furthermore, the frequency of G.sub.1 /S-PCC and G.sub.2 -PCC at 50 .mu.M 
endothal were less than those induced with cantharidine or cantharidic 
acid at same concentration. Endothal can inhibit protein phosphatase 
partially, whereas cantharidine and cantharidic acid can do it almost 
completely. Probably, this makes for the differences between their 
inducibility of PCC. 
Tautomycin could cause G.sub.1 /S-PCC and G.sub.2 -PCC at more than 1 
.mu.M, whereas it could induce G.sub.2 -PCC solely at 100 nM. 
Table 4, which shows the result of the frequency of PCC obtained after the 
same experiment shown in Table 3 but in human established cell line 
AT(L)5KY. 
TABLE 4 
__________________________________________________________________________ 
Interphase Cell 
G1/S PCC 
G2/M PCC 
Total 
Sample Name 
Treatment (%) (%) (%) Number 
__________________________________________________________________________ 
Human 
Human AT(L)5KY Colcemid 
361 (96.5) 13 (3.5) 
374 
Okadaic acid Ammonium salt 
1 nM 244 (96.2)) 
0 (0.0) 
10 (3.8) 
254 
Okadaic acid Ammonium salt 
10 nM 
239 (95.8) 
0 (0.0) 
11 (4.2) 
250 
Okadaic acid Ammonium salt 
100 nM 
126 (47.9) 
94 (35.8) 
43 (16.3) 
263 
Human AT(L)5KY Colcemid 
370 (96.5) 13 (3.5) 
383 
35-methyl okadaic acid 
1 nM 257 (96.0) 
0 (0.0) 
11 (4.0) 
268 
35-methyl okadaic acid 
10 nM 
263 (96.1) 
0 (0.0) 
10 (3.9) 
273 
35-methyl okadaic acid 
100 nM 
119 (48.6) 
89 (36.1) 
38 (15.3) 
246 
Human AT(L)5KY Colcemid 
357 (96.9) 11 (3.1) 
368 
Cantharidin 500 nM 
300 (96.2) 
0 (0.0) 
12 (3.8) 
312 
Cantharidin 5 uM 175 (81.0) 
22 (10.2) 
19 (8.8) 
216 
Cantharidin 50 uM 
30 (18.3) 
111 (67.7) 
23 (14.0) 
164 
Human AT(L)5KY Colcemid 
346 (97.0) 10 (3.0) 
356 
Cantharidic acid 
500 nM 
271 (96.4) 
0 (0.0) 
10 (3.6) 
281 
Cantharidic acid 
5 uM 197 (85.6) 
22 (9.6) 
11 (4.8) 
230 
Cantharidic acid 
50 uM 
39 (26.5) 
88 (59.9) 
20 (13.6) 
147 
Human AT(L)5KY Colcemid 
349 (97.5) 9 (2.5) 
358 
Endothal 500 nM 
289 (97.3) 
0 (0.0) 
8 (2.7) 
297 
Endothal 5 uM 298 (96.8) 
0 (0.0) 
10 (3.2) 
308 
Endothal 50 uM 
179 (92.6) 
7 (3.7) 
7 (3.7) 
193 
Human AT(L)5KY Colcemid 
295 (97.7) 7 (2.3) 
302 
Tautomycin 100 nM 
232 (95.8) 
0 (0.0) 
10 (4.1) 
242 
Tautomycin 1 uM 217 (93.9) 
3 (1.3) 
11 (4.8) 
231 
Tautomycin 10 uM 
142 (66.4) 
56 (26.3) 
16 (7.4) 
214 
__________________________________________________________________________ 
As easily recognized, they showed the same inclination as obtained from 
human peripheral blood lymphocytes. So these phenomena are in common and 
not depend on cell types. To make brevity, graphical representations of 
Table 3 and 4 are not shown. 
In conclusion, PCC could be induced at any phase of cell cycle in mammalian 
cells (human and mouse cells were investigated in the present invention), 
using several kinds of inhibitors of protein phosphatases. All of agents 
used herein could induce PCC in all kinds of cells used here. The 
effective dose to induce PCC were different for each agent, probably due 
to the differences in cell permeability, the degradation in cells, or the 
sensitivity of cells to each agent. All of the agents used herein have 
cell permeability, this allows one to use these agents to induce PCC much 
more simply as accomplished by the present invention. The agent having 
inhibitory effect of protein phosphates but not cell permeability (such as 
microcystin-LR), could not induce PCC at all after addition it into the 
culture medium (data not shown). Therefore, besides the inhibitory effect 
of protein phosphatase, the agents should have cell permeability. 
Otherwise, a laborious technique should be required to inject an agent 
into cells, such as micro-injection or cell portion technique. It is 
noteworthy that the method in the present invention gives the way to 
generate the chromosomes from interphase nuclei, and that it permit one to 
investigate the genetically events occur in the interphase nuclei. 
Furthermore the method in the present invention is much more simple than 
the known cell fusion method, which only requires the addition of agents 
into the culture medium, which leads to induction of PCC quickly, easily 
and with highly reproducibility. Thus the method in the present invention 
will provide a way to develop an assay or a diagnosis kit of analyze the 
chromosomes of many fields including clinical, medical, zoology, 
veterinary medicine, fisheries, botany or agriculture. 
Although the preferred embodiments of the present invention have been 
explained, in detail, herein above, the present invention should not be 
limited to these embodiments alone, but various modifications and changes, 
including an improvement or a discovery of new agents that have inhibitory 
effect of protein phosphatase with cell permeability, can be made thereto 
without departing from the scope of the invention defined in the appended 
claims. 
Thus the present invention provides an agent for inducing premature 
chromosome condensation (PCC) and which acts as an inhibitor of protein 
phosphatases which comprises at least one of okadaic acid, calyculin A, 
okadaic acid ammonium salt, 35-methyl okadaic acid, tautomycin, 
cantharidine, cantharidic acid or endothal dissolved in predetermined 
solvent. 
The present invention also provides an agent for inducing PCC and which 
acts as an inhibitor of protein phosphatases and which comprises at least 
one of okadaic acid, okadaic acid ammonium salt, 35-methyl okadaic acid, 
calyculin A, tautomycin, cantharidine, cantharidic acid and endothal, 
dissolved in a predetermined solvent and for diagnostic use. 
The agent may be effective at any stage of the cell cycle: G1 (Gap 1 
phase), S (DNA synthesis phase), G2 (Gap 2 phase) or M (mitosis phage). 
The agent may comprise okadaic acid or calyculin A. 
The present invention also provides use of at least one of okadaic acid, 
okadaic acid ammonium salt, 35-methyl okadaic acid, calyculin A, 
tautomycin, cantharidine, cantharidic acid or endothal dissolved in a 
predetermined solvent as an agent to inhibit protein phosphatases for the 
purpose of inducing PCC. 
In such use, the agent may comprise okadaic acid or calyculin A. 
The present invention also provides use of at least one of okadaic acid, 
okadaic acid ammonium salt. 35-methyl okadaic acid, calyculin A, 
tautomycin, cantharidine, cantharidic acid and endothal which act as 
inhibitors of protein phosphatases and induce PCC, for the manufacture of 
a diagnostic agent. 
Use may be of okadaic acid or calyculin A. 
The present invention also provides a method for generating chromosomes by 
PCC which comprises treating proliferating cells with an agent which 
inhibits protein phosphatases and induces PCC which agent comprises at 
least one of okadaic acid, calyculin A, okadaic acid ammonium salt, 
35-methyl okadaic acid, tautomycin, cantharidine, cantharidic acid or 
endothal dissolved in predetermined solvent. 
In much a method the agent may comprise okadaic acid or calyculin A. 
In such a method the final concentration of okadaic acid or calyculin A may 
be in the range of 1 to 100 nM. 
In such a method the final concentration of okadaic acid may be either (i) 
higher than 100 nM and such that it can generate G1 phase, S phase or G2 
phase PCC; or (ii) in the range of 1 to 10 nM and such that it can 
generate G2 phase PCC. 
In such a method the final concentration of calyculin A may be either (i) 
higher than 10 nM and such that it can generate G1 phase, S phase or G2 
phase PCC; or (ii) 1 nM and able to generate G2 phase PCC. 
In such a method the agent may comprise either okadaic acid ammonium salt 
or 35-methyl okadaic acid. 
In such a method the final concentration of the okadaic acid ammonium salt 
or 35-methyl okadaic acid may be 1 to 100 nM. 
In such a method the final concentration of the okadaic acid ammonium salt 
or 35-methyl okadaic acid may be either (i) higher than 100 nM and such 
that it can generate G1 phase, S phase or G2 phase PCC; or (ii) in the 
range of 1 to 10 nm and such that it can generate G2 phase PCC. 
In such a method the agent may comprise tautomycin. 
In such a method the final concentration of tautomycin may be 100 nM to 10 
.mu.M. 
In such a method the final concentration of tautomycin may be either (i) 
higher than 1 .mu.M and such that it can generate G1 phase, S phase or G2 
phase PCC; or (ii) 100 nM much that it can generate G2 phase PCC. 
In such a method the agent may comprise either cantharidins, cantharidic 
acid or endothal. 
In such a method the final concentration of cantharidine, cantharidic acid 
or endothal may be 500 nM to 50 .mu.M. 
In such a method the final concentration of cantharidine, cantharidic acid 
or endothal may be either (i) higher then 5 .mu.M and such that it can 
generate G1 phase, a phase or G2 phase PCC; or (it) 500 nM such that it 
can generate G2 phase PCC. 
The agents provided may be for chromosomal analysis of human cells for 
clinical or medical purposes. 
The agents provided may be for chromosomal analysis of non-human cells. 
The uses provided may be for chromosomal analysis of human cells for 
clinical or medical purposes. 
The uses provided may be for chromosomal analysis of non-human cells. 
The methods provided may be for chromosomal analysis of human cells for 
clinical or medical purposes. 
The methods provided may be for chromosomal analysis of non-human cells. 
The present invention also provides assays or diagnostic kits or reagents 
comprising an agent as above.