Suppression of metastasis of rat prostate cancer by introduction of human chromosome 13

Shigeru Hosoki¹, Sho Ota², Yayoi Ichikawa^1,3, Hiroyoshi Suzuki¹, Takeshi Ueda¹, Yukio Naya¹, Koichiro Akakura¹, Tatsuo Igarashi¹, Mitsuo Oshimura⁴, Naoki Nihei⁵, J. Carl Barrett⁵, Tomohiko Ichikawa^1,3, Haruo Ito¹

¹Department of Urology, ³Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
² Department of Urology, Teikyo University School of Medicine, Ichihara Hospital, Ichihara 299-0111, Japan
⁴ Department of Molecular and Cell Genetics, Tottori University School of Medicine, Yonago 683-8503, Japan
⁵ Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Asian J Androl  2002 Jun; 4:  131-136             

Keywords: BRCA2; chromosome 13; metastasis; metastasis suppressor genes; prostate cancer; RB1

Abstract

Aim: Chromosome 13 is one of the most frequently altered chromosomes in prostate cancer. The present study was undertaken to examine the role of human chromosome 13 in the progression of prostate cancer. Methods: Human chromosome 13 was introduced into highly metastatic rat prostate cancer cells via microcell-mediated chromosome transfer. Results: Microcell hybrid clones containing human chromosome 13 showed suppression of metastasis to the lung without any suppression of tumorigenicity, except for one clone, which contained the smallest sized human chromosome 13 and did not show any suppression on lung metastasis. Expression of two known tumor suppressor genes, BRCA2 and RB1, which map to chromosome 13, was examined by reverse transcription- polymerase chain reaction analysis. BRCA2 was expressed only in the metastasis-suppressed microcell-hybrid clones, whereas RB1 was expressed in all clones. Conclusion: Human chromosome 13 contains metastasis suppressor gene(s) for prostate cancer derived from rat. Furthermore, the RB1 gene is unlikely to be involved in the suppression of metastasis evident in this system.

Prostate cancer is one of the most common malignancies in men in Western countries [1]. The incidence in Japan is about one-tenth of that in Western countries, but has been steadily rising during the past few years [2]. The introduction of prostate specific antigen into clinical practice has had a profound impact on prostate cancer screening, where it may detect and subsequently result in the treatment of clinically insignificant tumors [3]. On the other hand, approximately 30% of clinically localized prostate cancers that are presently being detected without aggressive screening have already established micrometastatic disease at the time of definitive local therapy [4]. At present, no diagnostic methods are available to individually substage patients with histologically detected, localized prostate cancer into those requiring no therapy, versus those requiring definitive local therapy and systemic therapy. Therefore, identification of molecular and cellular markers for the metastatic ability of prostate cancer would be useful in developing diagnostic methods for substaging histologically localized prostate cancer on an individual patient basis. 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 [5]. The Dunning system has proven to be an excellent model for studying the malignant progression of prostate cancer. Metastatic Dunning sublines have the advantages of being well characterized in vitro and in vivo and of producing spontaneous lung metastasis in nude mice. At present, there is no analogous human xenograft system that is sufficiently metastatic to allow quantitative metastasis suppression assaying.

In cytogenetic and molecular analyses of human prostate cancer, allelic losses have been frequently observed in chromosome 13 [6-14]. In the present study, we introduced human chromosome 13 into highly metastatic Dunning rat prostate cancer cells using microcell-mediated chromosome transfer to clarify the role of human chromosome 13 in rat prostate cancer.

2 Materials and methods

2.1 Cell lines and culture

The cells used in the present study were highly metastatic, androgen-independent, and anaplastic rat prostate cancer cells (AT3.1). The development and characteristics of the AT3.1 subline have been described previously [15]. AT3.1 cells were maintained in RPMI 1640 (Gibco, Grand Island, USA) containing 10 % fetal bovine serum (Gibco), 0.4 % penicillin-streptomycin-Fungizone mixture (Whittaker M.A. Bioproducts, Inc., Walkersville, USA), and 250 nmol/L dexamethasone (Sigma, St. Louis, USA) (i.e., standard medium) at 37 with 5 % CO₂.

2.2 Microcell-mediated chromosome transfer

Microcell-mediated chromosome 13 transfer was performed as described previously [16], using A9(13) cells as the donor cells and AT3.1 rat prostate cancer cells as the recipients. The A9(13) fibrosarcoma cell line carries a single copy of human chromosome 13 tagged with a hygromycin B resistance gene. Resultant AT3.1-13 hybrids were selected in standard media supplemented with 1 mg/mL hygromycin B and individually cloned. The AT3.1-s11 cell lines containing a small portion of human chromosome 11 were used as controls, as this portion of human chromosome 11 has neither tumor suppressor genes nor metastasis suppressor genes [17]. AT3.1-s11 cells were maintained in standard media containing 500 mg/mL G418.

2.3 Cytogenetic and fluorescence in situ hybridization (FISH) analyses

Chromosome spreads of each clone were prepared and banded using the trypsin-Giemsa technique as described previously [18]. Fluorescence in situ hybridization (FISH) analysis was performed using biotin-labeled total genomic human placenta DNA as described elsewhere [19].

2.4 Genomic DNA preparation and molecular analysis of cell lines

Genomic DNA was isolated by using the QIAGEN blood and cell culture DNA midi kit (Qiagen Inc., Valencia, USA). Polymerase chain reaction (PCR) analysis was used to identify the portion of human chromosome 13 retained in the various microcell hybrids. PCR primers obtained from Research Genetics (Huntsville, USA) and had been mapped to 13q, were used for this analysis. The reaction mixture contained 100 ng of genomic DNA, 1.5 mmol/L MgCl₂, 200 mmol/L dNTPs, 1 mmol/L primers, and 0.25 U Taq polymerase (QIAGEN Inc., Valencia, USA) at a final volume of 10 mL. The PCR products were amplified for 35 cycles with annealing temperatures ranging from 55 to 57 by a thermal sequencer (DNA Amplifier MIR-D40, SANYO, Osaka, Japan), fractionated on 3 % agarose gels, and visualized after ethidium bromide staining.

2.5 Spontaneous metastasis assay

To evaluate metastatic ability, 510⁵ cells were injected subcutaneously at the right flank of 5-week-old male athymic nude mice (BALB/cA-nu: Clea Laboratory, Kawasaki, Japan). Tumor doubling time was used as the index of tumor growth and was determined as described previously [20]. Tumor-bearing animals were scored for lung metastases when died spontaneously or sacrificed at day 28 postinoculation.

2.6 Reverse transcription-polymerase chain reaction (RT-PCR)

To examine the expression of the known tumor suppressor genes found on human chromosome 13, BRCA2 and RB1 [21, 22] in the microcell hybrid clones, we performed RT-PCR analysis using primers designed for amplification of these two genes (Research Genetics, Huntsville, USA). RNA was extracted from AT3.1 cells and AT3.1-13 clones using the QIAGEN RNeasy Kit (Qiagen Inc., Valencia, USA). Reverse transcription was then performed using the cDNA Cycle Kit (Invitrogen,USA) with oligo dT primers. PCR was performed using the resultant cDNAs and the BRCA2 and RB1 primers.

Highly metastatic AT3.1 cells were used as the recipients for microcell transfer of human chromosome 13 tagged with a hygromysin B resistance gene. Four AT3.1 clones containing human chromosome 13 (AT3.1-13-1, -2, -3, -4) were isolated (Figure 1 and Table 1). Fluorescence in situ hybridization to metaphase chromosomes from these four AT3.1-13 clones using human genomic DNA probes demonstrated that each one had 2 copies of human chromosome 13 (Figure 2). For controls of the AT3.1-13 clones, three AT3.1 clones containing human chromosome region 11p11.2-11cen (s11) (AT3.1-s11 clones) were used, which contained neither metastasis nor tumor suppressor genes for rat prostate cancer cells.

Figure 1. Giemsa-banding karyotype of AT3.1-13- clone (i.e., AT3.1-13-1). Two copies of human chromosome 13 are evident.
Figure 2. Fluorescence in situ hybridization of AT 3.1-13-1 clone using biotin-labeled total genomic human placenta DNA. Two copies of labeled chromosome in the metaphase are evident (arrowheads).

Table 1. In vivo characteristics of AT3.1, AT3.1-s11 control, and AT3.1-13 microcell hybrid clones. ^cP<0.01 vs AT3.1 (parental).

For further clarification of the portion of human chromosome 13 retained in the four AT3.1-13 clones, PCR analysis was performed with human chromosome 13 specific primers (Figures 3 and 4). Essentially, all of the 24 markers examined were retained in the AT3.1-13-1, AT3.1-13-3 and AT3.1-13-4 clones. The regions between D13S175 and BRCA2, at D13S220, D13S291, and D13S273, and between D13S163 and D13S158 had been spontaneously deleted in the AT3.1-13-2 clone.

Figure 3. Polymerase chain reaction analysis of DNA from A9-13 clone, AT3.1 parental, and AT3.1-13- microcell hybrids (i.e., AT3.1-13-1, -2, -3 and -4) using probes that detect a human chromosome 13-specific region (i.e., D13S260 in this figure). DNA derived from human peripheral blood was used for the control.
Figure 4. Summary of retention of human chromosome 13 loci in A9-13 (donor of human chromosome 13) and AT3.1-13- microcell hybrids examined using polymerase chain reaction analysis. O, retention of loci; , loss of loci.

To test the effect of the human chromosomes transferred into microcell hybrids on the behavior in vivo of AT3.1 cells, we injected 5 x 105 cells of parental AT3.1, AT3.1-s11 control and AT3.1-13 clones into the athymic nude mice. No significant differences in tumor doubling time or in metastatic ability between parental AT3.1 and AT3.1-s11 control microcell hybrid cells was observed (Table 1). Tumorigenicity and tumor growth rate were not suppressed in any of the four AT3.1-13 clones. The three AT3.1-13 clones that contained almost intact human chromosome 13 produced only 0 to 2 lung metastases, whereas the other AT3.1-13 clone (i.e. AT3.1-13-2) which contained the smallest sized human chromosome 13 produced almost the same number of lung metastases as the parental AT3.1 clone.

To examine the expression of BRCA2 and RB1 genes, which are located at 13q12-13q13 and 13q14.3, respectively, in the AT3.1-13 clones, RT-PCR analysis was performed (Figure 5). The three AT3.1-13 clones that showed suppression of metastatic ability expressed the BRCA2 gene, whereas the other metastasis-unsuppressed AT3.1-13 clone (i.e. AT3.1-13-2) did not. The RB1 gene was expressed in all four AT3.1-13 clones regardless of their metastatic potential.

Figure 5. Reverse transcription-polymerase chain reaction analysis of RNA from AT3.1 parental, and AT3.1-13- microcell hybrids (i.e., AT3.1-13-1, -2, -3 and -4) using primers for BRCA2 and RB1. The three AT3.1-13 clones that showed suppression of metastatic ability expressed the BRCA2 gene, whereas the other metastasis-unsuppressed AT3.1-13 clone (i.e. AT3.1-13-2) did not.

The present study is a continuation of work designed to construct a molecular map of prostate-specific and general suppressors of metastasis within the human genome. Using this rat prostate cancer model and microcell-mediated human chromosome transfer into rat prostate cancer cells, human chromosomes 2, 7, 8, 10, 11, 12, 16 and 17 have been shown to contain metastasis suppressor genes for rat prostate cancer [17, 23-32]. In the case of human chromosome 11, spontaneous deletion of portions of human chromosome 11 has been observed in some clones. Molecular and cytogenetic analyses of these clones have demonstrated that one or more metastasis suppressor genes are located on human chromosome segment 11p13-11.2 [17]. In a continuation of the human chromosome 11 study, using the rat system, a metastasis suppressor gene for prostate cancer, KAI1, was isolated [33]. In the case of human chromosome 17, MKK4/SEK1 was identified from 17p12 as a candidate metastasis suppressor gene [34]. In the subsequent study, a statistically significant, direct and inverse relationship between Gleason pattern and MKK4/SEK1 was observed in human prostate cancer specimens [35]. These findings demonstrate that the present rat prostate cancer system is potentially useful for identifying metastasis suppressor genes for human prostate cancer.

In the present study, introduction of intact human chromosome 13 into rat prostate cancer cells suppressed metastatic ability without affecting the tumor growth rate of microcell hybrid clones. Spontaneous deletions of portions of human chromosome 13 were observed in some clones. The smallest sized human chromosome 13 fragment, which had spontaneous deletions within the regions 13q11-12.3, 13q14.1, and 13q14.2-q32, and which was present in the AT3.1-13-2 clone did not suppress the metastatic ability of the AT3.1 recipient cells. This demonstrates that metastasis suppressor gene(s) may be located within the 13q11-12.3, 13q14.1 and 13q14.2-q32 regions.

Frequent allelic loss on 13q has been well documented [6-14]. The deleted regions reported in these studies overlapped with those observed in the present rat assay system. Hyytinen et al. [10] reported that allelic loss on chromosome 13 at q14, q21-22 and q33 occurred in a subset of primary tumors and was a frequent event in metastatic lesions of prostate cancers. Ueda et al. [8] identified a 1-cM region of common deletion on 13q14, with the frequency of the loss higher in metastatic tissue than in corresponding primary legions (67 %-70 % vs. 25 %-39 %). Two known tumor suppressor genes on human chromosome 13, BRCA2 and RB1, are located at 13q12.3 and 13q14, respectively. In the present study, we used RT-PCR analysis to clarify whether these genes act as tumor suppressor genes in the metastasis-suppressed AT3.1-13 microcell hybrid clones. Expression of BRCA2 was observed only in the metastasis-suppressed hybrid clones, whereas that of RB1 was observed in all hybrid clones regardless of their metastatic potential. This result demonstrates that BRCA2 may function as a metastasis suppressor gene in the present rat system, whereas RB1 does not. According to allelotype studies of human chromosome 13, neither BRCA2 nor RB1 is the main target of the loss of heterozygosity, and other tumor suppressor genes play a key role in prostate cancer. Therefore, these unknown genes could also play an important role in suppression of metastatic ability in the present study.

5 Conclusion

In conclusion, human chromosome 13 contains metastasis suppressor gene(s) for prostate cancer derived from rat. The RB1 gene is not involved in the suppression of metastasis in this system, whereas BRCA2 gene may be. However, still unknown is whether all the metastasis suppressor genes on human chromosomes detected in this rat system are equivalent to the suppressors on the same chromosomes, which have been identified in human tissue. Further analysis is necessary to confirm this potentially useful advantage in the identification of metastasis suppressor gene(s) for prostate cancer.

Acknowledgements

This study was supported in part by Grants-in-Aid for Scientific Research (A)(11307029 & 14207061) and a Grant-in-Aid for Encouragement of Young Scientists (11770882) from the Japan Society for the Promotion of Science, a Grant-in Aid from The Japan Medical Association (1999), and a Grant-in-Aid from The Japanese Urological Association (2000).

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Correspondence to: Tomohiko Ichikawa, M.D., Ph.D., Department of Molecular Oncology (M7), Graduate School of Medicine, Chiba University, 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 2002-05-08      Accepted 2002-05-14