Mechanisms of metastasis suppression by introduction of human chromosome 10 into rat prostate cancer

Masaaki Hamano¹, Hiroaki Kuramochi¹, Naoki Nihei², Naoto Kamiya¹, Hiroyoshi Suzuki¹, Tatsuo Igarashi¹, J. Carl Barrett², Tomohiko Ichikawa³, Haruo Ito¹

¹Department of Urology, Graduate School of Medicine, Chiba University, Chiba, Japan
²Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
³Department of Molecular Oncology and Department of Urology, Graduate School of Medicine, Chiba University, Chiba, Japan

Asian J Androl  2002 Jun; 4:  123-129             

Keywords: prostate cancer; human chromosome 10; invasion; neoplasm metastasis; suppression of metastasis

Abstract

Aim: The metastatic ability of a Dunning R-3327 rat prostate cancer subline (AT6.3) was suppressed by the introduction of human chromosome 10, when these hybrid cancer cells were injected subcutaneously into nude mice (Nihei et al., Genes Chromosomes Cancer 14:112-119, 1995). The present study was undertaken to clarify which step of metastasis was suppressed in the human chromosome 10-containing microcell hybrids (AT 6.3-10 clones). Methods: Gelatin zymography, an in vitro invasion assay using a Boyden chamber and an intravenous metastasis assay involving the injection of hybrid cells into nude mice were performed. Results: Gelatin zymography revealed that AT6.3-10 microcell hybrid clones expressed the 72 kD type IV collagenase (MMP-2) at an almost equal level as control microcell hybrid clones. Both the invasiveness as measured by the invasion assay and the number of lung metastases as measured by the intravenous metastasis assay of AT6.3-10 hybrid clones were significantly less than those of the AT6.3 parental clone. Conclusion: The human chromosome 10 suppresses both the local invasion and the metastatic process after entry into the blood circulation of rat prostate cancer. This decrease in local-invasive ability does not seem to require a decrease in MMP-2 activity.

A number of genetic changes is responsible for the initiation and progression of prostate cancer. In cytogenetic and molecular analyses of human prostate cancer, frequent chromosomal deletions [1,2] and allelic losses[3-9] have been observed in chromosome 10. These findings suggest that human chromosome 10 contains tumor suppressor gene(s) that affect human prostate cancer.

In a previous paper, we showed the possibility of the presence of metastasis suppressor gene(s) on human chromosome 10 using a highly metastatic Dunning rat prostate cancer cell line (AT6.3) assay system [10]. When human chromosome 10 was introduced into AT6.3 cells using the microcell-mediated chromosome transfer technique, the metastatic ability of the recipient cells following subcutaneous transplantation was suppressed without any suppression of tumor growth rate or tumorigenicity. However, the mechanism of the metastasis suppression by the introduction of human chromosome 10 was not determined.

The development of cancer metastasis is comprised of several sequential steps, namely, the dissociation of individual cells from the primary tumor, the invasion of underlying connective tissue, the entry into the circulation (intravasation), the arrest of circulating cancer cells, the extravasation, and the growth as metastatic nodules [11, 12]. The function of human chromosome 10 that plays a role in the suppression of the metastatic ability of AT6.3 is most likely related to one or more of these steps. Once the metastatic step suppressed in AT6.3-10 microcell hybrids has been identified, the suppressive effect of human chromosome 10 on the metastatic ability of this system may be clarified. In the present study, the gelatin zymography, an in vitro invasion assay, and an intravenous metastasis assay were performed. The in vitro invasion assay, using a Boyden chamber, examined the effect of the local invasion on the underlying connective tissue. The expression of gelatinases was analyzed by gelatin zymography, to test whether a change in invasion ability was associated with the expression level of gelatinases. An intravenous metastasis assay was performed to examine the effect of human chromosome 10 on processes following intravasation, namely, the arrest of circulating cancer cells, extravasation, and the growth of the metastatic nodules. Therefore, the results of all the examinations would elucidate which step(s) of the metastasis of rat prostate cancer is suppressed by the introduction of human chromosome 10.

2 Materials and methods

2.1 Animals

Four-week-old, male athymic nude mice (BALB/cA-nu; Clea Laboratory, Kawasaki, Japan) were used.

2.2 Cells

The AT6.3 clone is a highly metastatic, androgen-independent, anaplastic subline from Dunning R-3327 rat prostate adenocarcinoma [13]. The 6 AT6.3-10 clones used (i.e., AT6.3-10-1, -2, -3, -4, -5, and -6) are the AT6.3 microcell hybrids containing human chromosome 10 from our previous study[10]. All of these clones show metastasis suppression activity compared with their parental AT6.3 clone. The 4 AT6.3-s11 clones used (i.e., AT6.3-s11-1, -2, -3, and -4) are AT6.3 microcell hybrids containing the human chromosome region 11p11.2-11cen. Since our previous study demonstrated that this small portion of human chromosome 11 contained neither metastasis suppressor genes nor tumor suppressor genes for rat prostate cancer [14], the AT6.3-s11 clones were used as the control for microcell hybrids containing human chromosome 10. AT6.3-10 and AT6.3-s11 clones were established by microcell-mediated chromosome transfer as previously described [10]. Each human chromosome contained the integrated neomycin (i.e., G418) resistant gene. AT6.3 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., Walkersville, USA), and 250 nM dexamethasone (Sigma, St. Louis, USA)(i.e., standard medium) at 37oC in 5 % CO₂. AT6.3-10 and AT6.3-s11 microcell hybrids were maintained by culture in standard medium supplemented with 500 mg/ml of G418 (Sigma).

2.3 Gelatin zymography

The gelatinases in the conditioned media were analyzed by gelatin-zymography. A total of 210⁵ cells of each clone were plated onto 35-mm dishes containing 2 mL of the standard medium. Forty-eight hours later, the medium was replaced with 1 ml of serum-free RPMI 1640 medium after washing twice with this medium. The serum-free and conditioned medium was collected after 24 h of incubation and analyzed by zymography. The proteins from the conditioned media were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10 % (w/v) acrylamide gels containing 0.1 % (w/v) gelatin (Sigma) at 20 mA, 4 for 5 h without heat treatment. The gels were rinsed twice in Triton X-100 (2.5 %, v/w) to remove the sodium dodecyl sulfate, and then incubated in 50 mM Tris-HCl, 50 mM CaCl₂ and 1 mM ZnCl₂ (pH 8.0) at 37 for 16 h. Gelatinases were identified by staining the gels with 0.25 % (w/v) Co-omassie brilliant blue and destaining with 10 % (w/v) methanol and 5 % (w/v) acetic acid. Digested areas appeared clear on a blue background, indicating the location of the gelatinases.

2.4 In vitro invasion assay

A Boyden chamber invasion assay was performed essentially as described by Albini et al.[15], and the conditions of the experiment were as previously described [16]. A Transwell chamber (Costar Co., Cambridge, USA) having a filter with a 6.5 mm diameter and 8 mm pores was used for this assay. After water-repellent treatment of a Transwell chamber with paraffin, 10 mg Matri-gel (Collaborative Research Co., Bedford, USA) diluted to 100 mg/mL with distilled water, was applied to the upper surface of the filters and dried overnight at room temperature. Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma [15]. The lower compartment of the Boyden chamber was filled with 900 ml of fibroblast-conditioned medium as the chemoattractant. The conditioned medium was prepared by incubating proliferative cultures of BALB/3T3 cells (Japanese Cancer Research Resources Bank, Tokyo, Japan) for 24 h in a serum-free medium. A total of 110⁵ cells were suspended in RPMI 1640 containing 0.1 % bovine serum albumin (BSA; Sigma), seeded onto the Matrigel-coated filters, and incubated for 12 h at 37 in 5 % CO₂. At the end of each incubation, cells on the upper surface of the filter were completely removed with cotton swabs. The filter was fixed in 70% ethanol and stained with Giemsa, and cells that had migrated to the lower surface of the filter were counted at 200 magnification. To evaluate the invasive ability, the percentage of the invasion rate (%) [i.e., (the number of cells migrated to the lower surface of the filter/110⁵) 100 ] was calculated.

2.5 In vivo growth rate analysis and subcutaneous metastasis assay

A total of 510⁵ cells of each clone suspended in 0.1 mL Hank's balanced salt solution (Gibco) were subcutaneously injected into the flank of nude mice. Five animals were used for each clone. Tumor doubling time was determined as previously described [17], and used as the index for tumor growth rate. Five weeks after injection, the mice were sacrificed in order to determine the number of lung metastases. The metastases was quantified by counting macroscopically the number of metastatic nodules on the surface of the lung.

2.6 Intravenous metastasis assay

A total of 110⁵ cells of each clone suspended in 0.2 mL Hank's balanced salt solution were injected intravenously into the lateral tail vein of nude mice. Five animals were used for each clone. Three and five weeks after the injection, mice were sacrificed in order to determine the number of lung metastases as mentioned above.

2.7 Statistical analysis

Student's t-test was used for all statistical analyses. Statistical significance was established at the P<0.05 level.

3.1 Subcutaneous metastasis assay

Detailed cytogenetic analyses demonstrated that all of the AT6.3-s11 control and AT6.3-10 clones conserved all the chromosomes from their parental AT6.3 cells. All four AT6.3-s11 control clones had 1-3 copies of the small portion of human chromosome 11 (i.e., 11p12-11cen). All six AT6.3-10 clones essentially contained a single copy of cytogenetically normal human chromosome 10 except the AT6.3-10-2 clone, which contained del(10)(q24), and the AT6.3-10-6 clone, which contained an additional copy of human chromosome 10 [10].

Initially, to reconfirm our previous data[10], AT6.3 parental, AT6.3-s11 control, and AT6.3-10 clones were injected subcutaneously into the flank of nude mice, and their tumorigenicity, growth rate and metastatic ability were examined. All the clones showed tumorigenicity with essentially the same growth rate (Table 1) and with similar histological findings (data not shown). No significant differences in the number of lung metastases were observed between the AT6.3 parental and AT6.3-s11 control clones. However, all the AT6.3-10 clones, except the AT6.3-10-1 clone, produced only 5 to 40 lung metastases, which was significantly smaller than those produced by the AT6.3 parental and AT6.3-s11 control clones (Table 1). These data were essentially consistent with our previous report.

Table 1. In vivo metastatic ability and in vitro invasive ability of AT6.3 parental, AT6.3-s11 control, and AT6.3-10 microcell hybrid clones

^acm³ at 5 weeks after injection.
^bCount, 5 weeks after injection.
^cCount, 3 weeks or 5 weeks after injection.
^dN.D., not determined, since all the mice died within 4 weeks after injection. The number of lung metastases of AT6.3-10 clones at the end of the 5th week was compared with that of the AT6.3 clone at the end of the 3rd week.
^eRate of cells which passed through the Matigel-coated filter in 12 hours by the Boyden chamber invasion assay (see Materials and Methods).
^fMean+S.E.
^gNumbers in parentheses, the number of animals or assays / group.
^hP<0.05 versus AT6.3 (parental).
ⁱP<0.01 versus AT6.3 (parental).

3.2 Expression of gelatinase

The activities of gelatinases that were secreted into the conditioned media by cancer cells were estimated by gelatin zymography (Figure 1). All the clones primarily secreted 72 kD type IV collagenase (MMP-2). No significant differences in the level of gelatin digestion were observed among the AT6.3-s11 control and AT6.3-10 clones.

Figure 1. Gelatin-zymography analysis of gelatinase expression of AT6.3 parental, AT6.3-s11 control (i.e., AT6.3-s11-1, -2, -3, and -4), and AT6.3-10 microcell hybrid clones (i.e., AT6.3-10-1, -2, -3, -4, -5, and -6). The expression level of MMP-2 was almost equal for all clones.

3.3 Invasive ability in the Boyden chamber

The ability of AT6.3 parental, AT6.3-s11 control and AT6.3-10 clones to invade the basement membrane was estimated by the Boyden chamber invasion assay. In the AT6.3 parental clone, 6.4 % of the cells invaded the Matri-gel and passed through the microporous filter after 12 hours. The invasion rate of the AT6.3-s11 clones, except AT6.3-s11-4, was similar to that of the AT6.3 parental clone. The average rate of invasion of AT6.3-10 clone cells ranged from 2.0 to 4.3 % (Figure 2, Table 1). The decrease in invasion rate was statistically significant for all AT6.3-10 clones when compared with both AT6.3 parental and AT6.3-s11 control clones.

Figure 2. Microscopic findings of the cells that passed through the Matrigel-coated filter (stained with Giemsa). AT6.3 parental (A) and AT6.3-s11 control cells (AT6.3-s11-1) (B) passed through the filter at a high rate. AT6.3 microcell hybrid cells (AT6.3-10-5) (C) passed through at a lower rate. Small arrowheads show 8 mm pores. Large arrowheads show cells that have passed through the filter. Bar: 100 mm.

3.4 Intravenous metastasis assay

The cells of the AT6.3 parental, AT6.3-s11 control and AT6.3-10 clones were injected into the lateral tail vein of nude mice and the number of lung metastases was counted 21 days later. In the AT6.3 parental and AT6.3-s11 control clones, numerous metastatic nodules were found on the surface of the lung. No significant difference in the number of lung metastases was observed between these clones. However, the number of lung metastases ranged from 0 to 11 in the AT6.3-10 clones, which was significantly lower than those of the AT6.3 parental and AT6.3-s11 control clones (Table 1). Even 5 weeks after the injection, the average number of lung metastases of the AT6.3-10 clones was still between 5 and 55, which was statistically lower than that of the AT6.3 parental clone after 21 days. In the AT6.3 parental and AT6.3-s11 control clones, all the mice died within 4 weeks after injection.

The results presented here indicate that at least two steps are involved in the suppression of metastasis of the rat prostate cancer cells by the introduction of human chromosome 10. The first is the suppression of local invasion, as demonstrated by the Boyden chamber invasion assay. The second is the suppression of the processes occurring after intravasation of cancer cells, as evaluated using the intravenous metastasis assay. These findings suggest that human chromosome 10 possesses metastasis suppressive function(s) to rat prostate cancer.

Cytogenetic and allelotyping studies have also suggested that human chromosome 10 contains multiple tumor suppressor genes. Several studies in human prostate cancer have reported a common deleted region at the long arm of chromosome 10 [1-9]. Bergerheim et al.[4] found in their detailed mapping that chromosome 10 had allelic deletions not only on the long arm but also on the short arm. These losses occurred at a higher rate in clinically advanced disease [4, 6- 9].

Alterations of human chromosome 10 have been reported in human neoplasms other than prostate cancer, including renal cell carcinoma [18], glioblastoma [19], malignant meningioma [20], malignant melanoma [21], and endometrial cancer [22]. Rempel et al. [20] found allelic losses on chromosome 10 more frequently in morphologically and invasively malignant meningioma. In malignant melanomas on the basis of studies with a series of sequential tumor samples from individual patients whose disease was progressive [21], loss of heterozygosity occurred as a late event in tumor progression,.

PTEN/MMAC1, a candidate tumor suppressor gene located at 10q23.3, was recently isolated and found to be deleted or mutated in cancer cell lines derived from a variety of human tissues, including prostate [23,24]. It is now becoming clear that PTEN plays a significant role not only in cell cycle arrest and programming apoptosis, but also in other aspects of cell physiology, including regulation of cell adhesion, migration and differentiation[25]. LOH at 10q23 is a common event in most primary tumors, but the complete loss of PTEN function in early stage tumors occurs only in endometrial and ovarian cancer [26]. In most other cases, including prostate cancers, complete inactivation of PTEN is associated with late-stage, more aggressive, and usually metastatic tumors[27-29]. Recently, Koul et al. have demonstrated that PTEN/MMAC1 reduces the invasive ability of glioma cells, as determined in Matrigel-coated trans well inserts[30]. They suggested that this suppressive activity of PTEN/MMAC1 expression is due in part to regulation of MMP-2 gene transcription and thereby its enzymatic activity.

The steps of invasion to the connective tissue and intravasation can be evaluated using the Boyden chamber invasion assay [15, 31]. In the present study, the invasion rate of AT6.3-10 clones was significantly lower than that of the AT6.3 parental and AT6.3-s11 control clones. This result suggests that human chromosome 10 plays a suppressive role in the local invasion of rat prostate cancer. As shown in glioma cells [30], PTEN/MMAC1 may also be related to this suppressive ability on rat prostate cancer. Several factors have been found to regulate the invasive ability of prostate cancer in the Boyden chamber assay. Those which have been demonstrated as invasion enhancers include nerve growth factor-like protein [32], epidermal growth factor [33], plasminogen activators [34], autocrine motility factor [35] and uteroglobin, which is an invasion suppressor [36]. Dedhar et al.[37] found an increase in a6b4 integrin expression and a decrease in a3b1 integrin expression in prostate cancer that had a high invasive ability. The function of human chromosome 10 might be the regulation of these factors.

Unregulated secretion of matrix metalloproteinases (MMPs) has also been implicated in tumor invasion. Stearns and Wang [38] found increased 72kD type IV collagenase (MMP-2) expression in malignant human prostate cancer tissue compared to benign controls. Luo et al.[39] showed that conditioned media from a highlymetastatic and invasive Dunning rat prostate subline expressed 72-kD type IV collagenase (MMP-2), the expression of which was reduced by E-cadherin trans-fection. As mentioned above [30], PTEN/MMAC1 suppressed MMP-2 expression and invasion in glioma cells. In the present study, gelatin zymography demonstrated that the MMP-2 expression of AT6.3 microcell hybrid clones is not suppressed by the introduction of human chromosome 10, and therefore MMP-2 might have no association with the suppression of the invasive ability of AT6.3-10 clones. However, the role of PTEN/MMAC1 in this rat system should be examined in future studies to further clarify the mechanism of metastasis suppression by human chromosome 10.

The metastatic steps following entry into the blood circulation namely, arrest of cancer cells, extravasation and growth as metastatic nodules, can be evaluated by direct intravenous injection of tumor cells, as in the present study. The number of lung metastases was significantly lower in AT6.3-10 clones than in AT6.3 parental and AT6.3-s11 control clones. This result suggests that the introduction of human chromosome 10 has a significant effect on the metastatic steps following intravasation of tumor cells. Several factors could be related to these steps. In the arrest of cancer cells, carbohydrate antigens sialyl Lewis A and sialyl Lewis X, which are ligands of E-selectin, have been found to mediate the adhesion of cancer cells to the vascular endothelium [40]. However, no relationship between these adhesion molecules and human chromosome 10 has yet been demonstr-ated.

Our previous study demonstrated that the metastatic ability of Dunning rat prostate cancer cells is also suppressed by the introduction of human chromosome 8 [41]. In a subsequent study, Kuramochi et al. [16] showed that this suppression of metastatic ability is due to the suppression of the in vitro invasion ability. However, in contrast with the present human chromosome 10 study, introduction of human chromosome 8 did not reduce the number of lung metastases. This suggests that these two human chromosomes play different roles in the suppression of the metastatic ability of rat prostate cancer, even though both chromosomes suppress the invasive ability in vitro. This finding also suggests that the manner of the suppression of cancer cell invasion by the primary tumor is different from that of an organ with a metastatic tumor for this rat prostate cancer. Further studies including examination of PTEN/MMAC1 are required to clarify the mechanism of suppression of metastatic ability by human chromosome 10.

Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research (A)(11307029) from the Japan Society for the Promotion of Science.

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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 2002-04-02      Accepted 2002-04-18