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Mapping
of metastasis suppressor genes for prostate cancer by microcell-mediated
chromosome transfer
Tomohiko
ICHIKAWA1, Shigeru HOSOKI1, Hiroyoshi SUZUKI1,
Koichiro AKAKURA1, Tatsuo IGARASHI1, Yuzo FURUYA2,
Mitsuo OSHIMURA3, Carrie W. RINKER-SCHAEFFER4,
Naoki NIHEI5, J. Carl BARRETT5, John T. ISAACS6,
Haruo ITO1 1Department
of Urology, Chiba University School of Medicine, Chiba 260-8670, Japan
2Department of Urology, Teikyo University School of Medicine,
Ichihara
Hospital, Ichihara 299-0111, Japan
3Department of Molecular and Cell Genetics,
School
of Medicine, Tottori University, Yonago 683-8503, Japan
4Departments of Surgery,
Section of Urology, University of Chicago, Chicago, Illinois 60637, USA
5Laboratory of Molecular Carcinogenesis, National Institute
of Environmental
Health Sciences, National Institute of Health, Research Triangle Park,
North Carolina 27709, USA
6The Johns Hopkins Oncology Center,
Baltimore, Maryland 21231, USA
Asian J Androl 2000 Sep; 2: 167-171
Keywords:
prostate
cancer; metastasis; metastasis suppressor gene; chromosome
Abstract
Aim: To identify the metastasis suppressor genes for prostate cancer. Methods: A copy of human chromosomes was introduced into the highly metastatic Dunning R-3327 rat prostate cancer cells by the use of microcell-mediated chromosome transfer. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained the metastasis suppressor gene(s) for prostate cancer. To determine portions of human chromosomes introduced, G-banding chromosomal analysis, fluorescence in situ hybridization analysis, and polymerase chain reaction analysis were performed. Results: Each of microcell hybrid clones containing human chromosomes 7, 8, 10, 11, 12, or 17 showed decreased ability to metastasize to the lung without any loss of tumorigenicity. This demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in the human chromosome 7, 10, 11, 12, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal arm deletions of human chromosome 8. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes were located on 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13, 12q24-ter, and 17pter-q23. KAI1 and MKK4/SEKI were identified as metastasis suppressor genes from 11p11.2 and 17p12, respectively. Conclusion: This assay system is useful to identify metastasis suppressor gene(s) for prostate cancer.1 Introduction
Acquisition of metastatic ability is a definitive criterion by which to substage histologically localized prostate cancers. Identification of molecular and cellular markers for the metastatic ability of prostate cancer, therefore, should be useful in developing diagnostic methods for substaging histologically localized prostate cancers on an individual patient basis. We are constructing a molecular map of suppressors of prostate cancer metastasis. To demonstrate the chromosomal location of human prostate cancer metastasis suppressor gene(s), the technique of microcell-mediated chromosome transfer has been used to introduce specific human chromosomes into highly metastatic Dunning R-3327 rat prostate cancer cells[1-4]. The Dunning system has proven to be an excellent model for studies on the malignant progression of prostate cancer. Metastatic Dunning sublines have the advantages of being well-characterized in vitro and in vivo and producing spontaneous lung metastasis assays in nude mice. When these cells were injected s.c. in the flank of nude mice, they produced lung metastases 4-5 weeks later. At this time there is no analogous human xenograph system that is sufficiently metastatic to allow such quantitative metastasis suppression assays. In our studies a single copy of human chromosomes 7, 8, 9, 10, 11, 12, and 17 was introduced into Dunning prostate cancer cells to clarify the role of these human chromosomes.2 Materials and methods
Highly metastatic, androgen-independent, anaplastic Dunning R-3327 AT3.1 and AT6 sublines (i.e., AT6.1, AT6.2 and AT6.3 clones) were used as recipient cells[5]. AT6 cells have the karyotype of 44XY,del(3)(q32q36),+4,+12,del(15)(p14). AT3.1 cells have the complicated karyotype of 61 chromosomes with 20 structural abnormalities. Microcell-mediated human chromosome transfer was performed as described previously using mouse A9 cells containing a single copy of human chromosomes as donor cells[6,7]. In the present study a single copy of human chromosomes 7, 8, 9, 10, 11, 12, and 17 was introduced.
To
determine the portions of human chromosomes introduced, G-banding chromosomal
analysis, fluorescence in situ hybridization analysis, and polymerase
chain reaction analysis were performed as described previously[1,2].
3
Results
3.1
Human Chromosome 7
For
the study of the possible biological significance of frequently observed
deletions on chromosome arm 7q in human prostate cancer, human chromosome
7 was introduced into AT6.3 cells[8]. The introduction of human
chromosome 7 resulted in the suppression of metastatic ability of the
microcell hybrids, whereas no suppression
of tumorigenicity was observed (Table 1).
To identify the portion of human chromosome 7 containing the metastasis
suppressive function gene, the derivative chromosome 7 that was generated
with the initial transfer was retransferred into rat prostate cancer cells.
Cytogenetic and molecular analyses of these clones demonstrated
that loss of segments on 7q (i.e., 7q21-22, 7q31.2-32) was related to
the reexpression of the metastatic phenotype (Figure
1). These regions do
not include 7q31.1, at which frequent allelic losses have been detected
in allelotype analyses of various tumors[9-12].
These results suggest that the metastasis suppressor gene(s) in
this region for rat prostate cancer cells differs from a tumor suppressor
gene involved in human neoplasms.
Figure
1. Location of metastasis suppressor genes identified by the rat assay
system. Each vertical line
indicates the region of metastasis suppressor gene(s).
KAI1 and MKK4/SEK1 were identified as metastasis
suppressor genes from 11p112 and 17p12, respectively.
Table
1. Effect of human chromosomes on lung metastasis of microcell hybrid
clones.
|
Portion
of human chromosome transferreda |
Recipient
clone |
Effect
of human chromosome on lung metastasis |
| 7p137q31.3::7q367qter |
AT6.3 |
suppressive |
| 8pterqter |
AT6.2 |
suppressive |
| 8p2112 |
AT6.2 |
suppressive |
| 9pterqter |
AT6.3 |
no |
| 10pterq24 |
AT6.3 |
suppressive |
| 10pterq11::q23q24 |
AT6.3 |
no |
| 11pterqter |
AT3.1 |
suppressive |
| 11p15cen |
AT3.1 |
suppressive |
| 11p13cen |
AT6.1 |
suppressive |
| 11p11.2cen |
AT6.1 |
no |
| 12p13q22::q24.1qter |
AT6.1 |
suppressive |
| 12p13cen |
AT6.1 |
no |
| 17pterq23 |
AT6.1 |
suppressive |
aPortions
of human chromosomes transferred were determined by chromosomal and/or
polymerase chain reaction analysis.
bAT6.1, AT6.2, and AT6.3 clones are derived from AT6 cells.
These clones have the karyotype of 44,XY,del(3)(q32q36),+4,+12,del(15)(p14),44,XY,del(3)(q32q36),+dup(4)(q11q22),+12,del(15)(p14),
and 43,XY,der(3)(?::3cen3q32::3q363qter),+4,del(15)(p14), respectively.
3.2
Human Chromosome 8
Introduction
of human chromosome 8 into AT6.2 cells resulted in suppression of metastatic
ability of the microcell hybrids without suppression of tumorigenesis[2]
(Table 1). In this study,
it was unknown which portion of human chromosome 8 was associated with
suppression of metastatic ability, since the microcell hybrid clones contained
at least a single copy of intact human chromosome 8. To
enrich chromosomal arm deletions of human chromosome 8, the irradiated
microcell-mediated chromosome transfer technique was performed[13].
Microcells were irradiated
with a dose of 500 rads of 200 KeV X ray and fused to the recipient cells.
Molecular and cytogenetic analyses demonstrated that the portion
of human chromosome 8 containing metastasis suppressor gene(s) for rat
prostate cancer was located on 8p21-12 (Figure
1). This includes the
region at which frequent allelic losses have been detected in allelotype
analysis of human prostate cancer[14-20](Figure
2). This suggests that
one of metastasis suppressor gene(s) for rat prostate cancer on human
chromosome 8 determined by using Dunning rat prostate cancer
system may also play an important role in the progression of human prostate
cancer. We are currently
attempting to identify this metastasis suppressor gene.
Figure
2. Location of tumor suppressor genes and metastasis suppressor gene(s)
on human chromosome 8[13-20].
Regions of tumor suppressor genes identified by allelotype analysis
of human prostate cancer are compared with that of metastasis suppressor
gene(s) by the rat prostate cancer assay system.
Each vertical line indicates the location of tumor suppressor genes
or metastasis suppressor gene(s).
3.3
Human chromosome 9
Human
chromosome 9 suppressed neither tumorigenicity nor metastatic ability
of the AT6.3 microcell hybrid clones (Table 1)[8].
This demonstrates that physical existence
of a complete size of human chromosome itself does not reduce the malignant
potential of the recipient rat cells.
3.4
Human chromosome 10
Frequent
losses have also been detected in the long arm of human chromosome 10
in allelotype analysis of human prostate cancer[21-23].
Therefore, the
role of human chromosome 10 in the Dunning system was investigated.
Introduction of human chromosome 10 into AT6.3 cells resulted in
suppression of metastatic ability[4](Table 1).
In this study, metastatic revertant clones were established in
culture from spontaneous lung metastatic tissues.
Cytogenetic and molecular analysis demonstrated that the short
arm of human chromosome 10 was retained in all of the metastatic revertant
clones. This indicates
that at least one of metastasis suppressor genes is located on the long
arm of human chromosome 10 (Figure 1).
This region (i.e., 10cen-q22) did not include the PTEN/MMAC1(10q23.3)
that was identified as a tumor suppressor gene from human neoplasms[24].
3.5 Human chromosome 11
A
single copy of human chromosome 11 was introduced into both AT6.3 and
AT3.1 clones. A single
copy of cytogenetically intact human chromosome 11 could be introduced
into AT3.1 cells, resulting in suppression of lung metastasis without
suppression of tumor growth rate[1] (Table 1).
However, only small portions of
human chromosome 11 could be introduced into AT6.3 clones (Table 1).
One of the
small portions (i.e., 11p13-cen) suppressed metastatic ability of AT6.3
microcell hybrid clones, whereas the smallest fragment (i.e., 11p11.2-cen)
suppressed neither tumor growth rate nor metastatic ability of AT6.1 microcell
hybrid clones. This
demonstrated that the small chromosomal region 11p13-11.2 contained a
metastasis suppressor gene.
In the continuation study, KAI1, a metastasis suppressor gene was
identified from genomic DNA fragments from the 11p13-11.2 region[25](Figure
1). Immunohistochemical
analysis in human materials showed that expression of KAI1 protein is
inversely correlated with progression of prostate cancer[26,27].
This demonstrates that the current rat assay system is useful to
identify metastasis suppressor genes for human prostate cancer.
3.6
Human chromosome 12
Introduction
of human chromosome 12 suppressed metastatic ability without suppression
of the tumor growth (Table 1)[28].
In this study, a -70cM portion of
human chromosome 12 was identified as the region of metastasis suppressor
activity (Figure 1). The
presence of chromosome 12 does not affect tumorigenicity, latency, or
in vivo growth rate of the AT6.1 rat prostate cancer cells.
This is in contrast to the findings of Berube et al[29],
which identified a tumor suppressor activity
encoded by human chromosome region 12pter-q13.
The recipient cell line used in their study was the DU-145 human
prostate cancer.
3.7
Human chromosome 17
Introduction
of human chromosome 17 suppressed metastatic
ability with no effect on tumor growth rate of microcell hybrid clones
(Table 1)[3]. In this
study it was also demonstrated that the metastasis suppressor activity
encoded by the chromosome
17pter-q23 region was p53-independent and not due to enhanced expression
of NM23 protein. In the
continuation study, MKK4/SEK1 was identified from 17p12
as a candidate metastasis suppressor gene[30].
A significance of
this gene in human prostate cancer is being currently investigated.
4
Discussion
5
Acknowledgements
These
studies were supported in part by Grant-in-Aid for Scientific Research
(A) from Japan Society
for the Promotion of Science (11307029), and Grant-in-Aid of
The Japan Medical Association (1999).
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Correspondence to: Dr. Tomohiko Ichikawa, Department of Urology, Chiba
University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba
260-8670, Japan.
Tel: +81-43-226 2134, Fax: +81-43-226 2136
e-mail: ichikawa@urology1.m.chiba-u.ac.jp
Received 2000-08-13 Accepted 2000-08-30
