Metastasis suppressor gene(s) for rat prostate cancer on the long arm of human chromosome 7

Allelotype analyses of human prostate cancer indicate that allelic losses on human chromosome arms 7q, 8p, 10q, 13q, 16q, 17q, and 18q are observed frequently. For the study of the possible biological significance of the frequently observed deletions on chromosome arm 7q in human prostate cancer, human chromosome 7 was introduced into highly metastatic rat prostate cancer cells by use of a microcell‐mediated chromosome transfer technique. The introduction of human chromosome 7 resulted in the suppression of metastatic ability of the microcell hybrids, whereas no suppression of tumorigenicity was observed. To identify the portion of 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. Human chromosome 7‐containing rat prostate cancer cells could be used as the donor cells, because rodent cells produced a sufficient number of microcells with colchicine treatment. Cytogenetic and molecular analyses of these clones demonstrated that loss of segments on 7q was related to the reexpression of the metastatic phenotype. These results show that human 7q contains a metastasis suppressor gene or genes for rat prostate cancer. The findings also suggest that this gene may play an important role in the progression of human prostate cancer. Genes Chromosomes Cancer 24:1–8, 1999. © 1999 Wiley‐Liss, Inc.     

肝癌细胞微细胞介导染色体转移方法学的建立与探讨

目的建立肝癌细胞微细胞介导染色体转移方法，为肝癌转移抑制基因的染色体功能定位建立技术平台。方法人单染色体供体细胞通过微核化、出核、融合步骤将随机标记有耐药neo基因的正常人8号染色体导入到大鼠肝癌高转移细胞系C5F中，对微细胞杂交克隆进行药物筛选和单细胞克隆，并填序列标签位点-PCR和全染色体涂染荧光原位杂交方法验证人染色体转移的结果。结果获得具有G418和HAT双重抗性的微细胞杂交细胞，通过单细胞分离克隆方法获得15个具有双重抗性的微细胞杂交克隆，序列标签位点-PCR结果发现导入染色体的随机片段丢失，全染色体涂染荧光原位杂交结果发现导入的人8号染色体与大鼠染色体发生了稳定的重组。结论成功建立微细胞介导的染色体转移技术，为肝癌转移抑制基因的染色体功能定位奠定了技术基础。     

Irradiation microcell-mediated chromosome transfer (XMMCT): the generation of specific chromosomal arm deletions

The microcell-mediated chromosome transfer technique has been used to introduce whole chromosomes into malignant cells and revert the tumorigenic phenotype. However, in most instances the limited availability of selectable chromosomes has hindered the ability to reduce the region containing the tumor suppressive information. The work presented here describes a new method to enrich for specific chromosomal arm deletions of selectable chromosomes and thereby more finely focus upon the genetic region of interest. The irradiation-microcell mediated chromosome transfer (XMMCT) technique involves the irradiation of microcells containing single human chromosomes followed by fusion to a nonirradiated host and cytogenetic characterization. The XMMCT procedure was performed on a microcell hybrid containing a der(11) as the only human chromosome. The resultant irradiated microcell hybrids were found to have deletions that ranged from simple interstitial deletions to complex deletions/rearrangements involving only the human der(11) chromosome. The XMMCT procedure has broad applications in generating chromosomal reagents for mapping genetic loci and for use in functional analyses such as tumor suppression studies.     

Identification of tumor metastasis suppressor region on the short arm of human chromosome 20

Acquisition of metastatic ability by prostate cancer cells is the hallmark of their lethal trait and outcome. However, the genetic alterations underlying the clinical progression and pathogenesis of prostate cancer are not well understood. Several studies involving loss of heterozygosity (LOH) and comparative genomic hybridization analysis have identified distinctively altered regions on various human chromosomes, and genomic imbalance of chromosome 20 was implicated in progression and recurrence of prostate tumors. To examine the role of chromosome 20 in prostate neoplasms, we introduced this chromosome into highly metastatic rat prostate cancer cells using the microcell-mediated chromosome transfer technique. Introduction of the chromosome resulted in significant suppression of the metastatic ability of the hybrid cells, by as much as 98%, without any interference with the in vivo growth rate or tumorigenicity of primary tumor in SCID mice. Our STS-PCR analysis on 10 hybrid clones indicates that the suppressor activity of chromosome 20 is located in the p11.23-12 region. Further examination of the hybrid clones by experimental metastasis assay and histologic analysis as well as Matrigel invasion assay suggests the involvement of the suppressor region at an early stage of invasion and extravasation. We also investigated the status of the chromosome 20 suppressor region in pathology specimens from human prostate cancer patients and detected the frequent loss of this region in high-grade tumors. These results suggest the presence of a putative suppressor gene on human chromosome 20 that is functionally involved in development of prostate cancer metastases.     

Localization of a novel tumor metastasis suppressor region on the short arm of human chromosome 2

Much of the lethality of malignant neoplasms is attributable directly to their ability to develop secondary growths in organs at a distance from the primary tumor mass, whereas few patients die from their primary neoplasm. Little is known about the molecular mechanism of tumor metastasis, however, which is controlled by a variety of positive and negative factors. In the search for metastasis suppressor genes, we have used the microcell-mediated chromosome transfer method and a rat prostate tumor model in SCID mice. When human chromosome 2 was introduced into the highly metastatic rat prostatic tumor cell, AT6.1, the metastatic ability of this cell was significantly (>99%) decreased in animals. An STS-based PCR analysis for 8 hybrid clones indicates that the suppressor activity is located in the p25-22 region of the chromosome. Furthermore, the AT6.1 cell with human chromosome 2 showed a reduced ability to invade Matrigel, suggesting that the suppressor activity is involved in the step of tumor invasion during the progression of prostate cancer. We have also examined the status of the suppressor region on chromosome 2 in human prostate cancer specimens and found that this region was often lost in high-grade tumors. These results suggest that the putative suppressor gene on chromosome 2 is functionally involved in the progression of human prostate cancer. Genes Chromosomes Cancer 28:285-293, 2000.     

Suppression of endometrial carcinoma cell tumorigenicity by human chromosome 18

Presumptive tumor suppressor genes may be localized to specific chromosomes by the procedure of microcell fusion, whereby individual chromosomes derived from normal human cells are introduced into tumor cells. Allelic loss on chromosome I 8 is commonly seen in endometrial carcinoma, and the DCC gene on chromosome arm 18q is a potential human tumor suppressor gene. In this study, we investigated the hypothesis that a gene on chromosome 18, possibly DCC, is capable of suppressing the tumorigenicity of endometrial carcinoma cells. Microcells from the mouse A9 cell clone containing one human chromosome I8 tagged with the pSV2-neo plasmid were fused with the highly tumorigenic endometrial carcinoma cell lines HHUA and Ishikawa, and G418-resistant microcell hybrids containing an extra copy of chromosome I8 were isolated. Clones isolated from the HHUA cell line were completely suppressed for tumorigenicity in nude mice, and clones from the lshikawa line were suppressed or inhibited for tumorigenicity. In contrast, growth rates in vitro were not significantly affected in clones from either parental cell line. DCC expression was elevated in most of the suppressed hybrids. These results indicate that a gene on human chromosome I 8 is capable of suppressing the tumorigenicity of endometrial carcinoma cells, and that DCC is a candidate for this endometrial carcinoma tumor suppressor gene. © 1995 Wiley-Liss, Inc.     

Chromosome 18 suppresses the tumorigenicity of prostate cancer cells

Microcell-mediated chromosome transfer allows for the introduction of normal chromosomes into tumor cells in an effort to identify putative tumor suppressor genes. We have used this approach to introduce an intact copy of chromosome 18 into the prostate cancer cell line DU145, and independently to introduce human chromosomes 8 and 18 into the prostate cancer cell line TSU-PR1. Introduction of an extra copy of human chromosome 8 had no effect on the growth properties in vitro or the tumorigenicity in vivo of TSU-PR1 cells. However, microcell hybrids containing an introduced copy of human chromosome 18 exhibited a longer population doubling time, retarded growth in soft agar, and slowed tumor growth in athymic nude mice. These experiments provide functional evidence for the presence of one or more tumor suppressor genes on human chromosome 18 that are involved in prostate cancer.     

Localization of metastasis suppressor gene(s) for rat prostatic cancer to the long arm of human chromosome 10

To examine the role of human chromosome 10 in development of prostatic cancer, we introduced human chromosome 10 into highly metastatic rat prostatic cancer cells by microcell-mediated chromosome transfer. Microcell hybrid cells introduced with human chromosome 10 showed suppression of the metastatic ability to the lung to some extent without any suppression of tumorigenicity, although the tumor growth rate decreased slightly. To minimize the region that contains metastasis suppressive activity, the hybrid cells in metastasis foci of lung were established in culture and reanalyzed for portions of human chromosome 10 retained in the metastasis tissues. Cytogenetic and molecular analyses demonstrated that loss of the region between 10cen and D10S215 on human chromosome arm 10q was related to expression of the metastatic phenotype. These results demonstrate that the region between 10cen and D10S215 on human chromosome arm 10q contains at least one of the metastasis suppressor genes for rat prostatic cancer.     

The human Y chromosome suppresses the tumorigenicity of PC-3, a human prostate cancer cell line, in athymic nude mice

The loss of the Y chromosome is a frequent numerical chromosomal abnormality observed in human prostate cancer. In cancer, loss of specific genetic material frequently accompanies simultaneous inactivation of tumor suppressor genes. It is not known whether the Y chromosome harbors such genes. To address the role of genes on the Y chromosome in human prostate cancer, we transferred a tagged Y chromosome into PC-3, a human prostate cancer cell line lacking a Y chromosome. A human Y chromosome was tagged with the hisD gene and transferred to PC-3 by microcell-mediated chromosome transfer. Tumorigenicity of these PC-3 hybrids was tested in vivo and in vitro, and the results were compared with those of the polymerase chain reaction analyses conducted on the PC-3 hybrids using Y chromosome-specific markers. Among 60 mice injected with 12 different PC-3 hybrids (five mice per hybrid), tumor growth was apparent in only one mouse, whereas tumors grew in all mice injected with the parental PC-3 cells. An in vitro assay showed that the Y chromosome did not suppress anchorage-independent growth of PC-3 cells. We found that addition of the Y chromosome suppressed tumor formation by PC-3 in athymic nude mice, and that this block of tumorigenesis was independent of the in vitro growth properties of the cells. This observation suggests the presence of a gene important for prostate tumorigenesis on the Y chromosome.     

High‐density screen of human tumor cell lines for homozygous deletions of loci on chromosome arm 8p

Tumor cell‐specific homozygous deletions coinciding at a particular genetic location may indicate the inactivation of a nearby tumor suppressor gene. Forty‐six human cancer cell lines of prostate, pancreatic, lung, liver, and colon origin were screened for homozygous deletions of 139 expressed sequence tag (EST) and sequence‐tagged site (STS) loci spanning the entire short arm of chromosome 8. Only one Southern blot–verified homozygous deletion was detected in this set of cell lines. The deletion, in pancreatic tumor cell line MIA‐PaCa‐2, encompassed two screening loci, D8S549 and D8S1992, and overlapped another previously described homozygous deletion of band 8p22 in a metastatic prostate cancer specimen. Both deletions entirely removed the candidate tumor suppressor gene N33. These data define a consensus homozygous deletion region in chromosome band 8p22. Genes Chromosomes Cancer 24:42–47, 1999. © 1999 Wiley‐Liss, Inc.     

Human chromosome 5 carries a putative telomerase repressor gene

Telomerase, the ribonucleoprotein enzyme that maintains the telomere, is active in human germ and stem cells and in a majority of tumor tissues and immortalized cell lines. In contrast, telomerase activity is not detected in most somatic cells, suggesting that normal human cells contain a regulatory factor(s) to repress this activity. To identify which human chromosomes carry a gene or genes that function as telomerase repressors, we investigated telomerase activity in hybrids of the B16-F10 cell line, which contain individual human chromosomes transferred previously by microcell fusion and therefore represent a hybrid panel for the entire genome except for the Y chromosome. Microcell hybrids with an introduced normal human chromosome 5 showed inhibition of telomerase activity, but clones at a late passage exhibited reactivation of telomerase activity. Reactivation of telomerase activity was accompanied by deletion and/or rearrangement of the transferred human chromosome 5. The introduction of other human chromosomes did not significantly affect the telomerase activity of B16-F10 cells. The effect of suppression of telomerase activity in microcell hybrids containing chromosome 5 was accompanied by a reduction in the level of mTERT mRNA, which encodes a component of the telomerase complex. The putative telomerase repressor gene was mapped to human chromosome bands 5p11-p13 by a combination of functional analysis using transfer of subchromosomal transferable fragments of chromosome 5 into B16-F10 cells and deletion mapping of revertant clones with reactivated telomerase activity. Thus, these results suggest that loss of a gene(s) on this chromosome was responsible for telomerase reactivation, indicating that human chromosome 5 contains a gene or genes that can regulate the expression of mTERT in B16-F10 cells.     

Chromosome 18 suppresses tumorigenic properties of human prostate cancer cells

Although prostate cancer is still the most diagnosed cancer in men, most genes implicated in its progression are yet to be identified. Chromosome abnormalities have been detected in human prostate tumors, many of them associated with prostate cancer progression. Indeed, alterations (including deletions or amplifications) of more than 15 human chromosomes have been reported in prostate cancer. We hypothesized that transferring normal human chromosomes into human prostate cancer cells would interfere with their tumorigenic and/or metastatic properties. We used microcell-mediated chromosome transfer to introduce human chromosomes 10, 12, 17, and 18 into highly tumorigenic (PC-3M-Pro4) and highly metastatic (PC-3M-LN4) PC-3-derived cell lines. We tested the in vitro and in vivo properties of these hybrids. Introducing chromosome 18 into the PC-3M-LN4 prostate cancer cell line greatly reduced its tumorigenic phenotype. We observed retarded growth in soft agar, decreased invasiveness through Matrigel, and delayed tumor growth into nude mice, both subcutaneously and orthotopically. This phenotype is associated with a marker in the 18q21 region. Combined with the loss of human chromosome 18 regions often seen in patients with advanced prostate cancer, our results show that chromosome 18 encodes one or more tumor-suppressor genes whose inactivation contributes to prostate cancer progression.     

A hamster-human subchromosomal hybrid cell panel for chromosome 2

We have constructed hamster-human hybrid cell lines containing fragments of human chromosome 2 as their only source of human DNA. Microcell-mediated chromosome transfer was used to transfer human chromosome 2 from a monochromosomal mouse-human hybrid line to a radiation-sensitive hamster mutant (XR-V15B) defective in double-strand break rejoining. The human chromosome 2 carried theEcogpt gene and hybrids were selected using this marker. The transferred human chromosome was frequently broken, and the resulting microcell hybrids contained different sized segments of the q arm of chromosome 2. Two microcell hybrids were irradiated and fused to XR-V15B to generate additional hybrids bearing reduced amounts of human DNA. All hybrids were analyzed by PCR using primers specific for 27 human genes located on chromosome 2. From these data we have localized the integratedgpt gene on the human chromosome 2 to the region q36–37 and present a gene order for chromosome 2 markers.     

Monochromosome transfers to syrian hamster BHK cells via microcell fusion provide functional evidence for suppressor genes on human chromosome 9 both for anchorage independence and for tumorigenicity

We previously identified an anchorage independence-suppressor gene, SAII, on rat chromosome (RNO) 5. RNO5 is homologous to human chromosomes (HSA) I and 9. In order to find the human homolog of the SAII gene, we transferred HSAI and HSA9 to an anchorage-independent and tumorigenic Syrian hamster BHK 191-5C cell line by microcell fusion. For HSA9, we used a t(X; 9)-derivative chromosome to force the retention of this chromosome in hybrids by hypoxanthine-aminopterin-thymidine (HAT) selection. To study the possible effect of the X portion of the der(9)t(X; 9), we also transferred a normal X to 191-5C cells. For HSAI, a neo-tagged chromosome was introduced. Following the transfer of der(9)t(X; 9) to 191 -5C cells, the hybrid cells became anchorage dependent and nontumorigenic, and, upon the loss of this chromosome, the cells regained their tumorigenic and anchorage-independent phenotypes. The transfer of HSAX or HSAI, on the other hand, affected neither of these phenotypes. These results provide functional proof of suppressor genes on HSA9 involving both anchorage independence and tumorigenicity. In addition, our data suggest the presence of another gene on HSA9 that causes a negative growth effect and whose phenotypic expression, contrary to the suppressor genes, is dosage dependent. © 1995 Wiley-Liss, Inc.     

Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion

A method for the production of stable mouse-human cell hybrids containing a single human chromosome is described. As a first step in this method, a cloned selectable marker, the E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, was transferred to human cells to generate cell lines each carrying Ecogpt integrated into a different site. Human chromosomes marked with Ecogpt were transferred further into mouse cells by microcell fusion. Monochromosomal hybrids, in which the human chromosome is maintained by selection, have been produced for chromosomes 2, 5, 16, and a rearranged chromosome involving a translocation between chromosomes 1 and 2. In addition to these monochromosomal hybrids, we have also obtained monochromosomal hybrids for human chromosomes 6, 12, and 17 by selection for the loss of marked chromosome from the microcell hybrids each containing two human chromosomes. Although the human chromosome present in these hybrids cannot be maintained by selection, 80–90% of cells retained the transferred chromosome on continuous growth for 15 days. Monochromosomal hybrids would provide biological materials to construct genetic maps of human chromosomes. In addition, chromosomes marked with dominant selectable markers can be transferred further to any cell line of interest in inter- or intra-species combination.     

Suppression of tumorigenicity in the human prostate cancer cell line M12 via microcell-mediated restoration of chromosome 19

Previously we immortalized human, nontransformed prostate epithelial cells with SV40 large T-antigen (SV40TAg) and derived increasingly aggressive sublines from the immortalized line. The progression of the tumorigenic sublines to metastatic capacity was accompanied by the formation of an unbalanced translocation between chromosomes 16 and 19, resulting in loss of 19p and proximal 19q. To test whether the tumorigenic and/or metastatic phenotype was causally related to this genetic alteration, we restored a neo-tagged human chromosome 19 to M12 cells by microcell-mediated transfer and assessed their growth. In vitro, the resultant hybrids grew more slowly in monolayer culture and showed a significant reduction in anchorage-independent growth as compared to M12neo controls. In vivo, all mice (13/13) injected subcutaneously (SC) with control M12neo cells developed tumors after 9-15 days. In contrast, 9/15 mice injected SC with microcell-transferred chromosome 19 hybrid cells failed to form tumors, with 6/15 producing very small tumors after 120 days. Analysis of three of these six tumors showed consistent, new chromosomal changes. Furthermore, in one of the tumors, loss of a chromosome 19 was noted in 40% of the cells. After intraprostatic injections of the hybrid cells, only 2/7 mice developed microscopic tumors, with no metastases. These data suggest the presence of a gene or genes on chromosome 19 that function to suppress growth.     

Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids

Mouse-human somatic cell hybrids have been extensively used in the molecular genetic dissection of human disease-related chromosome rearrangements because of their ability to selectively and randomly eliminate human chromosomes. This technology allows the isolation of structural chromosome abnormalities, which then allows determination of the precise molecular address of chromosome breakpoints associated with deletions and translocations, down to the nucleotides involved. The main confounding problem with the analysis of somatic cell hybrids is determining the exact chromosome complement unequivocally and quickly. Spectral karyotyping can identify each of the individual human chromosomes in a normal metaphase spread, as well as structural chromosome rearrangements-although, because of potential cross-hybridization between the human probe and mouse DNA sequences during the hybridization reaction, it has not been determined whether the same analysis will selectively identify human chromosomes on a mouse background. We show (to our knowledge, for the first time) that, under modified conditions of chromosomal in situ suppression hybridization, the standard spectral karyotyping probe does not cross-react with mouse chromosomes and can be used to identify subtle structurally rearranged chromosomes in hybrid cells. This analysis allows for the rapid and unequivocal identification of the human chromosome complement in these hybrids, as well as structural chromosome rearrangements that occur between mouse and human chromosomes that might otherwise confound the analysis.     

Genetic background of different cancer cell lines influences the gene set involved in chromosome 8 mediated breast tumor suppression

Several lines of evidence suggest that chromosome 8 is likely to harbor tumor-suppressor genes involved in breast cancer. We showed previously that microcell-mediated transfer of human chromosome 8 into breast cancer cell line MDA-MB-231 resulted in reversion of these cells to tumorigenicity and was accompanied by changes in the expression of a breast cancer-relevant gene set. In the present study, we demonstrated that transfer of human chromosome 8 into another breast cancer cell line, CAL51, strongly reduced the tumorigenic potential of these cells. Loss of the transferred chromosome 8 resulted in reappearance of the CAL51 phenotype. Microarray analysis identified 78 probe sets differentially expressed in the hybrids compared with in the CAL51 and the rerevertant cells. This signature was also reflected in a panel of breast tumors, lymph nodes, and distant metastases and was correlated with several prognostic markers including tumor size, grading, metastatic behavior, and estrogen receptor status. The expression patterns of seven genes highly expressed in the hybrids but down-regulated in the tumors and metastases (MYH11, CRYAB, C11ORF8, PDGFRL, PLAGL1, SH3BP5, and KIAA1026) were confirmed by RT-PCR and tissue microarray analyses. Unlike with the corresponding nontumorigenic phenotypes demonstrated for the MDA-MB-231- and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differed. However, the majority of genes in both gene sets could be integrated into a similar spectrum of biological processes and pathways, suggesting that alterations in gene expression are manifested at the level of functions and pathways rather than in individual genes.     

Unchanged frequency of loss of heterozygosity and size of the deleted region at 8p21-23 during metastasis of lung cancer.

The genetic mechanisms involved in lung cancer development and progression are beginning to be understood. Many studies have documented frequent loss of heterozygosity (LOH) at specific chromosomal regions in cancer cells; this implies that tumor suppressor genes (TSG) are usually present in those regions. Recently, it has been reported that LOH or chromosomal deletions at chromosome 8p21-23 represent early events frequently occurring in lung cancer. In addition, the size of these chromosome 8 deletions, as well as their frequency, was also reported to increase during lung cancer progression. To determine the spectrum and frequency of alterations of chromosome 8p21-23 in human lung cancer and whether these increase with progression of the tumors, we performed LOH analysis of chromosome 8p and 3p in the genomic DNA from cells from primary and metastatic sites of lung cancer, as well as from normal lung. We studied 35 subjects with primary lung cancer including 30 tumors with distant metastasis. Detection of allelic deletion utilized a PCR-based approach of microsatellite polymorphism analysis, which was performed using the microsatellite markers D8S1130, D8S1106, D8S511, D8S1827, D8S549, D8S261, LPL, D8S258, D8S136, NEFL, D3S1295, D3S1313, D3S1234, D3S1300, D3S1351, D3S1339, and D3S1340. The overall allelic deletion rates were 10 of 28 (35.7%) at 8p and 13 of 33 (39.4%) at 3p. The allelic deletions in the primary cancer and its metastatic sites were in each case identical in both frequency and size of the deleted regions. In our analysis, 8p21-23 deletions were not always associated with 3p deletions in primary lung cancer. These results therefore suggest that allelic deletion at chromosome 8p21-23 is an early and frequent event in the carcinogenesis and development of lung cancer, independent of chromosome 3p deletion. However, a continuing increase in the frequency of LOH at 8p21-23 and in the size of the deleted region rarely occurs during the process of metastasis.     

Assignment of retinoblastoma susceptibility gene to mouse chromosome 14

A human cDNA probe was used to screen a panel of mouse-Chinese hamster somatic cell hybrids to determine the chromosomal location of the retinoblastoma susceptibility gene (Rb-1)in mouse. The Rb-1gene mapped to mouse chromosome 14. Thus, the retinoblastoma susceptibility gene is syntenic with esterase 10 (the mouse homolog of human esterase D). The chromosomal assignment of the mouse Rb-1gene was further confirmed by using the same probe to study mouse-rat microcell hybrids. Since the human retinoblastoma susceptibility gene (RB1)along with the gene for esterase D is on chromosome 13q14, these data indicate this linkage group is conserved in man and mouse.