Isolation of genes from complex sources of mammalian genomic DNA using exon amplification.

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.

Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.

Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas.

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.

A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription.

Matrix metalloproteinases (MMPs) facilitate cellular invasion by degrading the extracellular matrix, and their regulation is partially dependent on transcription. Binding sites for members of the Ets family of transcription factors are present within MMP promoters and are potent positive regulators. We report a single nucleotide polymorphism at -1607 bp in the MMP-1 promoter, where an additional guanine (G) creates an Ets binding site, 5'-GGA-3'. This polymorphism displays significantly higher transcription in normal fibroblasts and in melanoma cells than the 1 G polymorphism, and it binds substantially more nuclear extract and recombinant ETS-1. Analysis of control DNAs from the Center d'Etude du Polymorphisme Humain pedigrees reveals that this polymorphism is not a mutation, with a frequency of the 2 G polymorphism at 30%. In contrast, in eight tumor cell lines, this frequency increased to 62.5% (P < 0.0001). Thus, this MMP-1 polymorphism contributes to increased transcription, and cells expressing the 2 G polymorphism may provide a mechanism for more aggressive matrix degradation, thereby facilitating cancer progression.

Frequent loss of chromosome 14 in atypical and malignant meningioma: identification of a putative 'tumor progression' locus.

Formation of meningiomas has been associated with the loss of genetic material on chromosome 22. To approach the additional chromosomal events that underlie progression of these tumors to malignancy, we have examined several other chromosomal regions for loss of heterozygosity (LOH) in these tumors. Fifty-eight tumors, comprising 43 benign meningiomas, 11 atypical meningiomas and four malignant meningiomas, were examined. While the loss of chromosome 22 was seen in approximately half of all these tumors, regardless of their malignancy, the most frequent chromosomal losses observed in the malignant and atypical tumors were on the long arm of chromosome 14. Thirty-nine tumors were informative for at least one of the three markers on chromosome 14 that we tested. Of these, 7/14 malignant and atypical tumors showed LOH in contrast to only 1/25 benign tumors. Other loci that showed LOH in malignant tumors, although at a much lower frequency, were on chromosomes 17p and 1p. The high frequency of LOH for loci on chromosome 14q in atypical and malignant tumors suggests the presence of a tumor progression gene at this locus. In one of the malignant meningiomas heterozygosity was lost at D14S13 and D14S16 but retained at the proximal marker D14S43 as well as the more distal marker D14S23. This suggests that an interstitial deletion occurred in this tumor which should be useful for further refining the position of the putative tumor progression locus.

Interstitial collagenases as markers of tumor progression.

Degradation of the extracellular matrix is the sine qua non of tumor invasion and metastasis. Most of this degradation is mediated by matrix metalloproteinases (MMPs), a family of enzymes that, collectively, degrades the extracellular matrix. Although the basement membrane-degrading enzymes, MMP-2 and MMP-9, have been given considerable attention for their roles in invasion and metastasis, the interstitial collagenases, a subfamily of MMPs that cleaves the stromal collagens types I and III, have received relatively little recognition for their part in these processes. This subfamily is comprised of collagenase 1 (MMP-1), collagenase 3 (MMP-13), and the MT-MMPs, membrane-bound MMPs, and numerous reports over the last several years document the expression of these MMPs in a wide variety of advancing tumors. Of particular interest is a single nucleotide polymorphism in the MMP-1 promoter that increases the transcription of this gene and that is associated with melanoma and with ovarian and endometrial cancers. The collagenases can mediate tumor invasion through several mechanisms, which include constitutive production of enzyme by the tumor cells, induction of collagenase production in the neighboring stromal cells, and interactions between tumor/ stromal cells to induce collagenase production by one or both cell types. Thus, evidence indicates that elevated expression of the interstitial collagenases is associated with a poor prognosis in a variety of cancers, and therefore, these MMPs can serve as a marker of tumor progression.

Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis.

Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all-trans retinoic acid and several RAR-specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR alpha, beta, or gamma agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR alpha antagonist suppressed MMP-1 and MMP-2 synthesis in the melanoma cell line, but not in the FaDu or SCC-25 cells. On the other hand, synthesis of MMP-1 and MMP-9 by the FaDu cells was affected hardly at all, while a RAR gamma antagonist reduced the levels of MMP-2. Only all-trans retinoic acid reduced MMP-1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All-trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.

Posttranscriptional regulation of collagenase-1 gene expression in synoviocytes by adenosine receptor stimulation.

OBJECTIVE: To characterize the transcriptional and posttranscriptional regulation of collagenase-1 by adenosine receptor stimulation in interleukin-1 (IL-1)-stimulated fibroblast-like synoviocytes (FLS). METHODS: FLS were stimulated with IL-1 and either the nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) or the adenylate cyclase activator forskolin. Electrophoretic mobility shift assays were performed to determine AP-1 and cAMP-responsive element binding protein (CREB) activation. Transcriptional activation was determined by transfecting HS68 dermal fibroblasts with a collagenase-chloramphenicol acetyltransferase construct. Finally, collagenase messenger RNA (mRNA) half-life was determined by activating cells in the presence of IL-1, IL-1 + NECA, or IL-1 + forskolin and culturing cells in the presence of actinomycin D. RESULTS: NECA and forskolin had no effect on AP-1 activation, c-jun or c-fos gene expression, or CREB phosphorylation. IL-1 markedly increased collagenase promoter activity, and neither NECA nor forskolin blocked this action. Studies of mRNA half-life showed that both NECA and forskolin decreased the half-life of collagenase mRNA in IL-1-stimulated FLS and HS68 cells. CONCLUSION: The findings of this study demonstrate that NECA and forskolin decrease collagenase gene expression in FLS and dermal fibroblasts due to enhanced mRNA degradation.

Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells.

Interleukin-1 beta (IL-1 beta) is a potent cytokine that stimulates interstitial collagenase-1 (matrix metalloproteinase-1; MMP-1). In this study, we compared the mechanism(s) by which IL-1 beta induces collagenase gene expression in two very different cells, normal human foreskin fibroblasts (HFFs) and an aggressive breast cancer cell line, BC-8701 cells. Northern analysis showed that the time course of collagenase induction was distinct in the two cells: although both cells expressed low levels of MMP-1 constitutively, addition of IL-1 beta increased MMP-1 mRNA in HFFs by 1 h and levels remained high over a 24-h period. In contrast, MMP-1 levels in IL-1 beta-treated BC-8701 cells did not increase until 4 h, peaked by 12 h and then declined. To analyze the transcriptional response, we cloned and sequenced more than 4,300 bp of the human MMP-1 promoter, and from this promoter clone, we prepared a series of 5'-deletion constructs linked to the luciferase reporter and transiently transfected these constructs into both cell types to measure both basal and IL-1 beta induced transcription. When both cell types were uninduced, promoter fragments containing less than 2,900 bp gave only a minimal transcriptional response, while larger fragments showed increased transcriptional activity. With IL-1 beta treatment, significant responsiveness (P < 0.001) in HFFs was seen only with the larger fragments, while in the BC-8701 cells, all fragments were significantly induced with IL-1 beta. Finally, we found that IL-1 beta stabilized MMP-1 mRNA in normal fibroblasts, but not in BC-8701 breast cancer cells. We conclude that both the transcriptional and post-transcriptional regulation of MMP-1 gene expression by IL-1 beta is controlled by cell-type specific mechanisms, and we suggest that IL-1 induced MMP-1 expression in tumor cells and in neighboring stromal cells may amplify the invasive ability of tumor cells.

Transcriptional repression of the human collagenase-1 (MMP-1) gene in MDA231 breast cancer cells by all-trans-retinoic acid requires distal regions of the promoter.

In the present study, we investigated the mechanisms controlling constitutive transcription of collagenase-1 and its repression by all-trans-retinoic acid (RA) in the highly invasive metastatic and oestrogen-receptor-negative breast cancer cell line MDA231. A combination of in vivo and in vitro experiments that include DNAase I hypersensitivity assays, transient transfection of collagenase-1 promoter constructs, and electrophoretic mobility shift assays implicate several PEA3 sites, binding sites for Ets-related transcription factors, in the constitutive expression of the human collagenase-1 promoter. Transient transfection of promoter constructs linked to the luciferase reporter, along with gel retardation assays, revealed that repression of collagenase-1 transcription by RA is not dependent on the proximal AP-1 site, but, rather, requires sequences located in distal regions of the promoter. Transcriptional analyses and electrophoretic mobility shift assays suggest that the PEA3 site located at -3108 bp facilitates, at least in part, the transcriptional repression of the human collagenase-1 gene in MDA231 cells. We conclude that collagenase-1 repression in MDA231 cells occurs by a novel regulatory pathway that does not depend on the proximal AP-1 site at -73 bp, but does depend on distal regions in the collagenase-1 promoter.

p53 down-regulates human matrix metalloproteinase-1 (Collagenase-1) gene expression.

Recent studies show that the p53 tumor suppressor protein is overexpressed in rheumatoid arthritis (RA) synovium and that somatic mutations previously identified in human tumors are present in RA synovium (Firestein, G. S., Echeverri, F., Yeo, M., Zvaifler, N. J., and Green, D. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10895-10900; Firestein, G. S., Nguyen, K., Aupperle, K. R., Yeo, M., Boyle, D. L., and Zvaifler, N. J. (1996) Am. J. Pathol. 149, 2143-2151; Reme, T., Travaglio, A., Gueydon, E., Adla, L., Jorgensen, C., and Sany, J. (1998) Clin. Exp. Immunol. 111, 353-3581). We hypothesize that the abnormality of p53 seen in RA synovium may contribute to joint degeneration through the regulation of human matrix metalloproteinase-1 (hMMP-1, collagenase-1) gene expression. Transcription assays were performed with luciferase reporters driven by the promoter of the hMMP-1 gene or by a minimal promoter containing tandem repeats of the consensus binding sequence for activator protein-1, cotransfected with p53-expressing plasmids. The results revealed that (i) wild-type (wt) p53 down-regulated the promoter activity of hMMP-1 in a dose-dependent fashion; (ii) four of six p53 mutants (commonly found in human cancers) lost this repression activity; and (iii) this p53 repression activity was mediated at least in part by the activator protein-1 sites found in the hMMP-1 promoter. These findings were further confirmed by Northern analysis. The down-regulation of hMMP-1 gene expression by endogenous wt-p53 was shown by treatment of U2-OS cells, a wt-p53-containing osteogenic sarcoma line, and Saos-2 cells, a p53-negative osteogenic sarcoma line, with etoposide, a potent inducer of p53 expression. p53, activated by etoposide, appears to block hMMP-1 promoter activity induced by etoposide in U2-OS cells. In summary, we have shown for the first time that the hMMP-1 gene is a p53 target gene, subject to p53 repression. Because MMP-1 is principally responsible for the irreversible destruction of collagen in articular tissue in RA, abnormality of p53 may contribute to joint degeneration through the regulation of MMP-1 expression.

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.

MTS1/CDKN2 gene mutations are rare in primary human astrocytomas with allelic loss of chromosome 9p.

Human astrocytomas frequently have allelic losses of chromosome 9p, suggesting the presence of a 9p astrocytoma tumor suppressor gene. The MTS1 (or CDKN2) gene on chromosome 9p encodes a cell-cycle regulator and is deleted in approximately 80% of astrocytoma cell lines. To determine whether MTS1 is the tumor suppressor gene involved in human astrocytoma formation in vivo, we have analyzed chromosome 9p allelic loss and the MTS1 gene in 30 primary astrocytomas. Deletion mapping demonstrated 15 cases with allelic loss of chromosome 9p, with all losses either flanking or involving the MTS1 gene. Direct analysis of the MTS1 gene, however, revealed only a single missense mutation in a high-grade tumor that had lost the second allele. The low frequency of MTS1 mutations in primary astrocytomas with allelic 9p loss suggests that MTS1 may be more important for in vitro than in vivo astrocytoma growth, and that another 9p tumor suppressor gene may be involved in astrocytoma formation in vivo. Analysis of the MTS1 gene also demonstrated two intragenic polymorphisms, one in exon 2 and one in the 3' untranslated region, that can be used to assay allelic loss directly at MTS1.

Loss of heterozygosity on chromosome 11q22-23 in melanoma is associated with retention of the insertion polymorphism in the matrix metalloproteinase-1 promoter.

Matrix metalloproteinase-1 (MMP-1, collagenase-1), which degrades interstitial collagen, is expressed at high levels by some tumor cells and is thought to enhance their invasiveness and metastatic potential. We recently described a common single nucleotide insertion polymorphism (2G allele) at -1,607 bp in the promoter of the MMP-1 gene that creates a binding site for the ETS family of transcription factors, and that is associated with enhanced transcription of this gene and increased enzyme activity. Allelic loss at the MMP-1 locus on chromosome 11 occurs in many tumors including melanoma, an invasive and aggressive cancer. We hypothesized that although loss of either the 1G or 2G allele from 1G/2G heterozygotes is random, retention of the transcriptionally more active 2G allele would favor tumor invasion and metastasis. As a result, a higher proportion of metastases would contain the 2G genotype than the 1G genotype. We report here the development of quantitative methods for assessing allelic loss at the MMP-1 locus, and demonstrate that 83% of the metastatic melanomas with loss of heterozygosity at this locus retained the 2G allele. This supports the hypothesis that retention of the 2G allele favors tumor invasion and metastasis in melanoma.

A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor.

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by the occurrence of bilateral vestibular schwannomas and other central nervous system tumors including multiple meningiomas. Genetic linkage studies and investigations of both sporadic and familial tumors suggest that NF2 is caused by inactivation of a tumor suppressor gene in chromosome 22q12. We have identified a candidate gene for the NF2 tumor suppressor that has suffered nonoverlapping deletions in DNA from two independent NF2 families and alterations in meningiomas from two unrelated NF2 patients. The candidate gene encodes a 587 amino acid protein with striking similarity to several members of a family of proteins proposed to link cytoskeletal components with proteins in the cell membrane. The NF2 gene may therefore constitute a novel class of tumor suppressor gene.

Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation.

Manganese-superoxide dismutase (Sod2) removes mitochondrially derived superoxide (O(2)) at near-diffusion limiting rates and is the only antioxidant enzyme whose expression is regulated by numerous stimuli. Here it is shown that Sod2 also serves as a source of the intracellular signaling molecule H(2)O(2). Sod2-dependent increases in the steady-state levels of H(2)O(2) led to ERK1/2 activation and subsequent downstream transcriptional increases in matrix metalloproteinase-1 (MMP-1) expression, which were reversed by expression of the H(2)O(2)-detoxifying enzyme, catalase. In addition, a single nucleotide polymorphism has recently been identified (1G/2G) at base pair--1607 that creates an Ets site adjacent to an AP-1 site at base pair --1602 and has been shown to dramatically enhance transcription of the MMP-1 promoter. Luciferase promoter constructs containing either the 1G or 2G variation were 25- or 1000-fold more active when transiently transfected into Sod2-overexpressing cell lines, respectively. The levels of MMP-2, -3, and -7 were also increased in the Sod2-overexpressing cell lines, suggesting that Sod2 may function as a "global" redox regulator of MMP expression. In addition, Sod2(-/+) mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study provides evidence that the modulation of Sod2 activity by a wide array of pathogenic and inflammatory stimuli may be utilized by the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.