Up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression: an important role for SAPK signalling in prostatic neoplasia.

Identification of the signalling cascades that are differentially activated during prostatic tumourigenesis is a crucial step in the search for future molecular targets in this disease. The stress-activated protein kinase (SAPK) signalling cascade culminates in the phosphorylation of the JNK and p38 mitogen-activated protein kinases (MAPKs). Recently, the upstream activators of these proteins, the MAPK kinases (MKKs), have been implicated as inhibitors of tumour progression in a variety of clinical and experimental tumour models. This study evaluates MKK4, MKK6 and MKK7 expression during prostate cancer progression in humans and in the transgenic adenocarcinoma of a mouse prostate (TRAMP) model of prostate tumourigenesis. Benign prostate, prostatic intraepithelial neoplasia (PIN) lesions and tumour tissues were collected from 37 TRAMP mice. Additionally, six tissue microarrays were constructed with tumours from a matched group of 102 men who underwent radical prostatectomy. Tissues from 20 patients with extensive high-grade prostatic intraepithelial neoplasia (HGPIN) were also analysed. For all samples, immunohistochemical staining for MKK4, MKK6 and MKK7 was scored in normal and neoplastic glands. Staining intensities of MKK4, MKK6 and MKK7 were significantly increased in HGPIN and prostate cancer compared to surrounding normal glands in both the TRAMP and human samples (p < 0.0001 for all markers). Increased levels of MKK4 or MKK7 correlated with higher pathological stage at prostatectomy (p = 0.01 and p = 0.04). Using multivariate analysis, there was no association between protein levels and time to biochemical recurrence in the human samples. The up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression in both TRAMP and human tissues highlights an important role for the SAPK signalling cascade in prostatic neoplasia. The finding that higher MKK4 and MKK7 expression is associated with higher-stage prostatic tumours underscores the dynamic regulation of these proteins during prostatic tumourigenesis.

Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers.

We have shown recently (B. A. Yoshida et al., Cancer Res., 59: 5483-5487) that mitogen-activated protein kinase kinase 4 (MKK4) can suppress AT6.1 rat prostate cancer metastases in vivo. Evaluation of the expression of components of the MKK4 signaling cascade showed a loss or down-regulation of expression of MKK4 or c-Jun, a downstream mediator of MKK4, in six of eight human prostate cancer cell lines. Given these findings, we next assessed whether MKK4 dysregulation occurs during the development of clinical prostate cancer. Immunohistochemical studies showed high levels of MKK4 expression in the epithelial but not the stromal compartment of normal prostatic tissues. In neoplastic tissues, a statistically significant, direct, inverse relationship between Gleason pattern and MKK4 was established. These results demonstrate that MKK4 protein is consistently down-regulated during prostate cancer progression and support a role for dysregulation of its signaling cascade in clinical disease. To test the possibility that down-regulation of MKK4 protein is the result of allelic loss, metastatic prostate cancer lesions were examined for loss of heterozygosity (LOH) within the MKK4 locus (D17S969). These studies showed a 31% (5 of 16) LOH of MKK4 that is not associated with coding region mutations, which suggests that the nucleotide sequence of the gene in the remaining allele is infrequently mutated.

Metastasis suppression in prostate cancer.

Due to a lack of effective treatments, the development of metastases remains the most lethal aspect of prostate cancer. In order to help overcome this problem there has been an ongoing effort to develop strategies for early intervention. This includes the development of strategies that allow histologic lesions and disseminated cells that are highly likely to cause metastatic disease to be distinguished from those that are not, as well as therapeutic approaches to specifically target bone metastases. Such approaches will be expedited by the identification of genes and signaling cascades that regulate metastatic growth. Genes that specifically suppress metastasis are strong candidates for these studies. This review will focus on metastasis-suppressor genes that have been identified functionally, particularly those found to play a role in prostate cancer, and discuss how the identification and study of these genes has influenced our overall understanding of the metastatic process.

Northern and southern blotting and the polymerase chain reaction to detect gene expression.

For cancer cells to form a metastasis, cells from the primary tumor must overcome the local adhesive forces, migrate and invade the microcirculation, arrest at a secondary site, and then finally proliferate (1). As implied by its mult]step nature, cancer metastasis is a complex and dynamic process that is likely to be regulated by a series of genes at each step (2). A variety of approaches have been used to discern the molecular events that regulate this process. It is likely that the ability of a cancer cell to form clinically detectable metastases is influenced by a variety of factors, including alt]rations in the pattern of gene expression within the cancer cell. Such changes could be the result]of genetic or epigenetic modifications (3). Alt]ough there has been a growing emphasis on array-based techniques for high-throughput screening of gene expression patterns, there are several well established protocols that can be used to identify such molecular changes. This chapter describes two of these techniques: Northern and Southern blotting.E. M. Southern first described a method for immobilizing size-fractionated DNA fragments on a nitrocellulose membrane in 1975. Since then, a number of different variations of this blotting method have been developed, as well as a variety of ways by which scientists can generate and hybridize probes to detect specifically the sequences thus immobilized. Southern blotting is now a general term for a number of different methods by which DNA is transferred from a gel to a membrane, and because nitrocellulose is relatively fragile, improved membranes have been developed that are more durable and that have been optimized for allowing binding of nucleic acids.

Angiogenesis inhibitors.

Angiogenesis inhibitors target the neovascular development that is hypothesized to underlie tumor growth. The inhibitors that are undergoing the clinical testing phase can be divided into five categories based on their target activity: 1) drugs that block matrix breakdown; 2) drugs that inhibit endothelial cells directly; 3) drugs that block angiogenesis activators; 4) drugs that inhibit endothelial cell integrins or survival signaling; and 5) drugs with a currently unknown mechanism of action. The properties of these drugs and some specific agents in each class are reviewed in this article. Because growth inhibition rather than tumor shrinkage is expected to be the clinical effect of angiogenesis inhibitors, some of the challenges and potential solutions for clinical trial design are also discussed.

Metastasis-suppressor genes: a review and perspective on an emerging field.

Metastasis is the most lethal attribute of a cancer. There is a critical need for markers that will distinguish accurately those histologic lesions and disseminated cells with a high probability of causing clinically important metastatic disease from those that will remain indolent. While the development of new diagnostic markers of metastasis was the initial motivation for many studies, the biologic approach used to identify metastasis-suppressor genes has provided surprising insights into the in vivo mechanisms regulating the formation of metastases. This review and perspective describes the evolving view of the mechanisms that regulate metastasis and the importance of metastasis-suppressor genes in this process. The known metastasis-suppressor proteins or genes and the microcell-mediated chromosomal transfer strategy used to identify many of them are reviewed. New evidence for the role of these metastasis-suppressor proteins or genes in regulating the growth of disseminated cancer cells at the secondary site, the potential for the identification of novel therapeutic targets, and the multidisciplinary approach needed to translate this information into clinical tools for the treatment of metastatic disease are discussed.

Defining the biologic role of genes that regulate prostate cancer metastasis.

Metastasis is the most lethal attribute of a cancer. There is a critical need for markers that will accurately distinguish those histologic lesions and disseminated cells that have a high probability of causing clinically important metastatic disease from those cells that will remain indolent. Despite the explosion in new information regarding the genetics of cancer, only six human genes have thus far been shown to functionally suppress metastasis. The present review and perspective describes the evolving view of the mechanisms that regulate metastasis, and the importance of metastasis-suppressor genes in this process. Specifically, the clinical problem of metastatic prostate cancer, the identification of metastatic colonization as a therapeutic target, and the identification and functional characterization of prostate cancer metastasis-suppressor genes are discussed.

Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer.

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.

Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis.

The present is an overview of recent data that describes the genetic underpinnings of the suppression of cancer metastasis. Despite the explosion of new information about the genetics of cancer, only six human genes have thus far been shown to suppress metastasis functionally. Not all have been shown to be functional in breast carcinoma. Several additional genes inhibit various steps of the metastatic cascade, but do not necessarily block metastasis when tested using in vivo assays. The implications of this are discussed. Two recently discovered metastasis suppressor genes block proliferation of tumor cells at a secondary site, offering a new target for therapeutic intervention.

Mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1), a prostate cancer metastasis suppressor gene encoded by human chromosome 17.

The introduction of a discontinuous approximately 70-cM portion of human chromosome 17 significantly suppresses the metastatic ability of AT6.1 rat prostate cancer cells without affecting tumorigenicity (M. A. Chekmareva et al., Prostate, 33: 271-280, 1997). We have recently demonstrated that AT6.1 cells containing the approximately 70-cM region (AT6.1-17-4 cells) escape from the primary tumor and arrest in the lung but are growth-inhibited unless the metastasis suppressor region is lost (M. A. Chekmareva et al., Cancer Res., 58: 4963-4969, 1998). A series of in vivo studies indicated that the observed growth inhibition was due to the effect of a gene(s) at the metastatic site (M. A. Chekmareva et al., Cancer Res., 58: 4963-4969, 1998). We have now identified the mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1) gene as a candidate metastasis suppressor gene encoded by the approximately 70-cM region. AT6.1 cells were transfected with a MKK4/SEK1 expression construct, and the cells were tested in standard spontaneous metastasis assays. Whereas the metastatic ability of the AT6.1-MKK4/SEK1 cells was significantly reduced as compared with that of transfection controls, the growth rate of the primary tumors was not affected; the average tumor volume at day 29 after injection was approximately 2 cm. Furthermore, histological examination of the lungs of AT6.1-MKK4/SEK1 tumor-bearing animals revealed that the suppression by MKK4/SEK1 is due to an effect at the metastatic site, consistent with the phenotype conferred by the original approximately 70-cM chromosomal region. These studies implicate MKK4/SEK1 as a metastasis suppressor gene encoded by human chromosome 17.

In vitro evaluation of calphostin C as a novel agent for photodynamic therapy of bladder cancer.

OBJECTIVES: Calphostin C, a highly specific protein kinase C inhibitor, induces apoptosis in the presence of visible light. We report the photoactivatable cytotoxicity of calphostin C in a series of well-characterized human bladder cancer cell lines: RT4, UM-UC-3, and 5637. METHODS: The human bladder cancer cell lines RT4, UM-UC-3, and 5637 were chosen on the basis of their p53, pRb and 9p21 deletion status. Using standard tissue culture techniques, the cytotoxicity of 10 to 100 nM calphostin C in combination with increasing exposures of visible light was examined. Controls consisted of cells treated with calphostin C without visible light and cells exposed to visible light without calphostin C treatment. Cell viability was determined by MTT assay. The induction of apoptosis by activated calphostin C was determined by 4,6-diamidino-2-phenylindole (DAPI) staining/fluorescence microscopy of nuclei. RESULTS: In the absence of light, calphostin C did not demonstrate a cytotoxic effect on any of the cell lines tested. Increasing the duration of light exposure resulted in a concomitant decrease in cell viability. Significant cell death was seen with calphostin C concentrations as low as 10 nM. These studies also demonstrated that calphostin C induced apoptosis by a mechanism independent of p53 and pRb status and the presence or absence of 9p21 deletions. CONCLUSIONS: We demonstrated the ability of activated calphostin C to induce apoptosis in a light-dependent and concentration-dependent fashion in a bladder cancer model system. Activated calphostin C cytotoxicity is independent of tumor genetic background and the status of p53 and pRb. Further development of calphostin C as a photosensitizer for photodynamic therapy of superficial bladder cancer may be warranted.

Chromosome 17-mediated dormancy of AT6.1 prostate cancer micrometastases.

To improve the diagnosis and treatment of cancer, an increased understanding of the molecular and cellular changes that regulate metastatic ability is required. We have recently demonstrated a prostate cancer metastasis-suppressor activity encoded by a discontinuous approximately 70-cM region of human chromosome. The presence of this region suppresses the spontaneous metastatic ability of AT6.1 rat prostatic cancer cells by greater than 30-fold (M. A. Chekmareva et al., Prostate, 33: 271-280, 1997). Interestingly, a number of potentially important genes which have been mapped to human chromosome 17, including TP53, NM23, and BRCA1, are not retained (M. A. Chekmareva et al., cited above) or are not expressed in these microcell hybrids (B. A. Yoshida et al., In Vivo, in press), which suggests the presence of a novel metastasis-suppressor gene(s) or novel function of a known gene(s) encoded by this region(s). We hypothesize that identification of the "step" in the metastatic cascade that is inhibited by the presence of the approximately 70-cM metastasis-suppressor region will facilitate the identification of candidate metastasis-suppressor genes. For a cancer cell to metastasize, it must escape from the primary tumor, enter the circulation, arrest in the microcirculation, extravasate into a tissue compartment, and grow. This suppression of spontaneous macroscopic lung metastases could be due to the inhibition of a number of steps within this cascade. Results of the current study demonstrate that AT6.1 cells containing the approximately 70-cM region (AT6.1-17-4 cells) escape from the primary tumor and arrest in the lung but are growth-inhibited unless the metastasis-suppressor region is lost. This growth inhibition seems to result from an effect of one or more genes at the metastatic site and not from a circulating angiogenesis inhibitor. Our findings suggest that the approximately 70-cM region of human chromosome 17 may encode a gene(s) that regulates the "dormancy" of AT6.1-17-4 micrometastases.

Human chromosome 16 suppresses metastasis but not tumorigenesis in rat prostatic tumor cells.

Genomic aberrations at the chromosome 16q arm are one of the most consistent abnormalities observed by loss of heterozygosity and comparative genomic hybridization analyses in human prostate cancer, suggesting that there are tumor suppressor or metastasis suppressor genes encoded by this chromosomal region. To functionally identify such suppressor genes, we have conducted microcell-mediated chromosome transfer to introduce human chromosome 16 into the highly metastatic Dunning rat prostatic cancer cell line, AT6.1. The metastatic ability of the resultant microcell hybrid clones was then tested in a standard spontaneous metastasis assay using SCID mice. When the microcell-mediated chromosome transfer hybrid cells containing whole human chromosome 16 were injected, the number of metastatic lesions in the lung was significantly reduced as much as 99% on average. Therefore, chromosome 16 has a strong activity to suppress the metastatic ability of AT6.1 cells while it did not affect the tumorigenesis and tumor growth rate. A PCR analysis of various microcell hybrid clones with sequence-tagged site markers indicates that the metastasis suppressor activity is located in the q24.2 region of chromosome 16. Our results are consistent with the previous finding that the region of human chromosome 16q has frequent loss of heterozygosity in prostate cancer patients and suggest that there is a metastasis suppressor gene in this region that may play an important role in the progression of prostate cancer.

Activated calphostin C cytotoxicity is independent of p53 status and in vivo metastatic potential.

The development of novel therapeutic agents to modulate programmed cell death independent of genetic background or malignant potential is a primary goal of modern cancer therapy. In this report, the light activation- and concentration-dependent cytotoxicity of calphostin C, a photoactivatable perylenequinone, is carefully evaluated using a series of nine well-characterized human and rodent prostate cancer cell lines representing the spectrum of disease progression (e.g., variations in metastatic ability, ploidy, and tumor suppressor gene status). Treatment of these cancer cell lines with nanomolar concentrations of calphostin C in combination with increasing amounts of light exposure established a relationship between light and dose dependence of calphostin C cytotoxicity. The induction of apoptosis is rapid, as evidenced by the fact that immediately after treatment, cells exposed to calphostin C with light activation exhibit both morphological and biochemical changes consistent with apoptosis (cellular and nuclear shrinkage and chromatin condensation). For example, 78% of cells treated with 100 nM calphostin C in combination with 2 h of light activation underwent apoptosis within 24 h of treatment. DNA ladder formation could be detected within 12 h of treatment. In the absence of light activation, treatment with calphostin C at all concentrations tested had no acute or durable cytotoxic effects in any of the cell lines. Our findings demonstrate that calphostin C cytotoxicity is strictly light dependent. Furthermore, its efficacy is independent of the genetic background, p53 status, or in vivo malignant potential of a cell, making it a suitable candidate for the treatment of heterogeneous tumor cell populations.

Identification of a novel metastasis-suppressor region on human chromosome 12.

There is a critical need for markers that can be used to predict accurately the malignant potential of histological prostate cancers (J. T. Isaacs. Am. J. Pathol., 150: 1511-1521, 1997). Metastasis-suppressor genes are attractive candidates for marker development because, by definition, their loss should be associated with the acquisition of metastatic ability. In an effort to identify such genes, a single copy of human chromosome 12, tagged with the neomycin resistance gene, was introduced into highly metastatic Dunning AT6.1 prostate cancer cells by microcell-mediated chromosomal transfer. Thirty-two AT6.1-12 clonal cell lines were established and the region(s) of chromosome 12 retained was determined by sequence tagged site-based PCR analysis. Representative AT6.1-12 clones containing overlapping regions of chromosome 12 were characterized cytogenetically and were shown to have a normal complement of parental AT6.1 rat chromosomes. Fluorescence in situ hybridization, performed on representative AT6.1-12 hybrids, demonstrated a single human chromosome 12-specific signal. The metastatic ability of six representative clones was tested in immunodeficient mice. All of the AT6.1-12 clones showed the same in vivo growth rates as the control AT6.1-neo cells. Clonal cell lines that contained a conserved approximately 70-cM portion of chromosome 12 (e.g., AT6.1-12-8, -8-1, and -8-3), showed a >30-fold suppression in the number of macroscopic surface lung metastases. Mice that received injections of these cells developed a mean number 4 lung metastases whereas mice that received injections of other AT6.1-12 hybrids (lacking the approximately 70-cM region) or AT6.1-neo control cells, developed a mean number of 140 metastases. Interestingly, histological examination of the lungs of the mice that received injections of AT6.1-12-8 cells showed essentially no microscopic metastases. These findings suggest that a gene(s) encoded by the approximately 70-cM portion of human chromosome 12 suppresses an early step in the metastatic cascade.

Prostate cancer metastasis-suppressor genes: a current perspective.

Prostate cancers account for 43% of all cancers diagnosed in American men. It is estimated that in 1996, 317,000 new cases of prostate cancer were diagnosed and 41,000 men died of the disease. The challenge of treating prostate cancer lies in accurately distinguishing those histologically-localized cancers which will complete metastatic progression from those that will remain indolent. At this time, we lack appropriate histological markers to make such distinctions, therefore, it is often difficult to accurately predict the clinical course of an individual patient's disease. There is growing evidence that a critical event in the progression of a tumor cell from a non-metastatic to metastatic phenotype is the loss of function of metastasis-suppressor genes. These genes specifically suppress the ability of a cell to metastasize. Work from several groups has demonstrated that human chromosomes 8, 10, 11 and 17 encode prostate cancer metastasis suppressor activities. As a result of these efforts the first prostate cancer metastasis-suppressor gene, KAI1, was identified and mapped to the p11-2 region of chromosome 11. In subsequent studies, an additional gene encoded by the same region, CD44 was also determined to have metastasis-suppressor activity. Recent studies have shown a correlation between decreased expression of KAI1 and CD44 and an increased malignant potential of prostate cancers. It is anticipated that the identification of other metastasis suppressor genes may allow for the development of diagnostic markers useful in the clinical substaging of individual tumors. This manuscript is intended to present our perspective on the importance of these genes in the understanding of prostate cancer progression. More importantly, we present new findings from our laboratory's effort to identify the metastasis-suppressor genes encoded by human chromosome 17. Specifically we report the strategy currently being used to evaluate a series of candidate genes and the approach being utilized to pinpoint the metastasis-suppressor region on human chromosome 17.

Effects of polyamine analogues on prostatic adenocarcinoma cells in vitro and in vivo.

PURPOSE: The overall purpose of this study was to determine the potential usefulness of 1,19-di-(ethylamino)-5,10,15-triazononadecane (BE-4-4-4-4) in the treatment of prostate cancer using in vitro and in vivo models. More specifically the objectives were: (1) to determine the in vitro and in vivo sensitivity of human and rat prostate cancer cells to two polyamine analogues N1,N11-di(ethyl)norspermine (DENSPM) and BE-4-4-4-4; (2) to determine whether the mechanism of cell kill occurred through an apoptotic pathway; and (3) to determine the toxicity associated with therapeutic doses of BE-4-4-4-4 using an animal model. METHODS: In order to determine the ability of these drugs to cause in vitro cytotoxicity, colony-forming assays were performed utilizing the well-characterized Dunning rat prostate cancer cell lines AT3.1, AT6.1 and AT6.3, and the androgen-insensitive human prostate cancer cell lines DU145, DuPro-1 and TSU-Pr1. Apoptotic cell death was determined using DNA laddering and DAPI staining of nuclei. The antitumor activity of BE-4-4-4-4 was evaluated by treatment of DuPro- and PC-3 xenograft tumors in nude mice. RESULTS: BE-4-4-4-4 was shown to be approximately 4 to 86 times more cytotoxic in clonogenic assays than DENSPM in both rat and human prostate carcinoma cell lines. Cells treated with cytotoxic doses of DENSPM or BE-4-4-4-4 showed no signs of apoptosis using either DNA laddering or DAPI staining of nuclei. There was a significant inhibition of DuPro-1 tumors for animals treated with BE-4-4-4-4 compared with control animals. Equitoxic doses of BE-4-4-4-4 resulted in greater tumor inhibition than DENSPM, although the difference was not significant. After treatment with therapeutic doses of BE-4-4-4-4, histopathologic evaluation indicated minimal to mild necrosis and inflammation in the kidneys on days 15 and 22 following treatment. On day 35, there was no necrosis or regeneration present in the kidney, indicating that the toxicity was transient and that regeneration of epithelial cells was complete with apparent return to normalcy. CONCLUSIONS: These initial studies demonstrate that BE-4-4-4-4 is cytotoxic against rat and human prostate cancer cells in culture and effective against DuPro-1 xenografts in nude mice. Polyamine analogues, such as DENSPM or BE-4-4-4-4, should be considered for clinical use in the treatment of prostate adenocarcinomas.

Localization of prostate cancer metastasis-suppressor activity on human chromosome 17.

BACKGROUND: Prostate cancer is the most commonly diagnosed malignancy in American men. Currently, it is difficult to accurately predict the clinical course of histologically localized prostatic cancer in the individual patient. Identification of markers for metastatic potential of prostate cancer may improve the diagnosis and treatment of this disease. We have previously demonstrated that human chromosome 17 (17pter-q23) suppresses the metastatic ability of AT6.1 rat prostatic cancer cells. In this study we report on the further localization of the metastasis suppressor activity encoded by human chromosome 17. METHODS: A series of AT6.1-17 microcell hybrids was constructed using microcell-mediated chromosomal transfer of human chromosome 17 into highly metastatic AT6.1 cells. Hybrids which had spontaneously deleted regions of chromosome 17 were analyzed by PCR for the presence of 32 sequence-tagged sites (STS) markers as well as the prostate cancer tumor-suppressor loci reported on 17q. In addition, we examined a number of candidate genes and markers that previously have been mapped to chromosome 17. The in vivo metastatic potential of these AT6.1-17 deletion hybrids was determined. RESULTS: We have localized metastasis-suppressor activity to a approximately 70-centiMorgan (cM) portion of chromosome 17, consisting of three distinct regions of 30 cM (D17S952-->D17S805), 6 cM (D17S930-->D17S797), and 34 cM (D17S944-->D17S784). Three of the four markers on 17p13, including HIC1 and TP53, and 12 of the 13 markers in 17q21-23, including BRCA1 (D17S855) and NM23 (NME1), were not retained in the conserved approximately 70-cM metastasis-suppressor region. CONCLUSIONS: These results support a role for a novel metastasis-suppressor gene(s) or a novel metastasis-suppressor function on chromosome 17. Complementary candidate gene and positional cloning approaches are being used to identify the gene(s) within the approximately 70-cM conserved region responsible for metastasis suppression.