Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.

Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.

A single ataxia telangiectasia gene with a product similar to PI-3 kinase.

A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

Metabolic engineering of astaxanthin production in tobacco flowers.

Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value. To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding beta-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids. Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3'S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red. This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin. The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance.

Heparanase expression in primary and metastatic pancreatic cancer.

The human endoglycosidase heparanase (hpa) degrades heparan-sulfate proteoglycans, which constitute prominent components of basement membranes and extracellular matrix. Due to the critical function of hpa in cancer cell invasion and metastasis, we have analyzed the expression of hpa in human primary and metastatic pancreatic cancer as well as in the normal pancreas and in chronic pancreatic inflammation. By real-time quantitative PCR, there was a 7.9- and 30.2-fold increase of hpa mRNA in chronic pancreatitis and pancreatic cancer tissue samples, respectively, in comparison with normal pancreatic tissues. There was a significant correlation between enhanced hpa mRNA expression and shorter postoperative patient survival. hpa mRNA and protein localized in the cancer cells of primary and metastatic pancreatic cancer, with a preferentially higher expression at the primary tumor site. Cultured pancreatic cancer cells transfected with a full-length hpa construct displayed enhanced invasiveness in an invasion chamber assay. These results suggest that hpa overexpression in human pancreatic cancers facilitates cancer cell invasion, thereby enhancing the metastatic potential of the tumors.

Recombinant ATM protein complements the cellular A-T phenotype.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability and radiation sensitivity. The cellular phenotype of A-T points to defects in signal transduction pathways involved in activation of cell cycle checkpoints by free radical damage, and other pathways that mediate the transmission of specific mitogenic stimuli. The product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycle progression in various organisms. A recombinant vector that stably expresses a full-length ATM protein is a valuable tool for its functional analysis. We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that overcame an inherent instability of this sequence. Recombinant ATM was stably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vector. An amino-terminal FLAG epitope added to the protein allowed highly specific detection of the recombinant molecule by immunoblotting, immunoprecipitation and immunostaining, and its isolation using immunoaffinity. Similar to endogenous ATM, the recombinant protein is located mainly in the nucleus, with low levels in the cytoplasm. Ectopic expression of ATM in A-T cells restored normal sensitivity to ionizing radiation and the radiomimetic drug neocarzinostatin, and a normal pattern of post-irradiation DNA synthesis, which represents an S-phase checkpoint. These observations indicate that the recombinant, epitope-tagged protein is functional. Introduction into this molecule of a known A-T missense mutation, Glu2904Gly, resulted in apparent instability of the protein and inability to complement the A-T phenotype. These findings indicate that the physiological defects characteristic of A-T cells result from the absence of the ATM protein, and that this deficiency can be corrected by ectopic expression of this protein.

Mammalian heparanase as mediator of tumor metastasis and angiogenesis.

Expression of heparan sulfate-degrading endoglycosidases, commonly referred to as heparanases, correlates with the metastatic potential of tumor cell lines, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. We purified a 50 kDa heparanase from human hepatoma and placenta and cloned a cDNA and gene encoding a protein of 543 amino acids. Only one heparanase sequence was identified, suggesting that this enzyme is the dominant endoglucuronidase in mammalian tissues. Expression of the cloned cDNA in insect and mammalian cells yielded 65 kDa and 50 kDa recombinant proteins. The 50 kDa enzyme represents an N-terminal processed enzyme that is at least 200-fold more active than the full-length 65 kDa form. Processing was demonstrated following incubation of the full-length recombinant enzyme with intact tumor cells. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and in specimens of human melanomas and carcinomas. In the colon, both the heparanase mRNA and protein are expressed already at the stage of tubulovillous adenoma, but not in the adjacent 'normal-looking' colon epithelium. Non-metastatic murine T lymphoma and melanoma cells transfected with the heparanase gene acquired a highly metastatic phenotype in vivo. Apart from its involvement in the egress of cells from the vasculature, heparanase is tightly involved in angiogenesis, both directly--by promoting invasion of endothelial cells (vascular sprouting), and indirectly--by releasing heparan sulfate-bound basic fibroblast growth factor, and generating HS degradation fragments that promote bFGF activity. The angiogenic potential of heparanase was demonstrated in vivo (Matrigel plug assay) by showing a three to fourfold increase in neovascularization induced by Eb T lymphoma cells following their transfection with the heparanase gene. The ability of heparanase to promote both tumor angiogenesis and metastasis makes it a promising target for cancer therapy.

Cloning a gene coding for norflurazon resistance in cyanobacteria.

The herbicide norflurazon inhibits carotene biosynthesis in photosynthetic organisms by blocking the enzyme phytoene dehydrogenase (= phytoene desaturase). We have isolated norflurazon-resistant mutants of the cyanobacterium Synechococcus PCC7942. The herbicide-resistance gene from the mutant NFZ4 has been cloned by genetic complementation of the resistance trait in wild type cells. The experiment described here illustrates the usefulness of employing cyanobacteria to clone herbicide-resistance genes in a quick and simple way.

Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes.

Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three zeta-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as intermediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and beta-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic beta-zeacarotene and its monohydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehydrolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of zeta-carotene and only small amounts of trans-zeta-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.

The molecular basis of resistance to the herbicide norflurazon.

We have cloned and sequenced a gene, pds, from the cyanobacterium Synechococcus PCC7942 that is responsible for resistance to the bleaching herbicide norflurazon. A point mutation in that gene, leading to an amino acid substitution from valine to glycine in its polypeptide product, was found to confer this resistance. Previous studies with herbicide-resistant mutants have indicated that this gene encodes phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids. A short amino acid sequence that is homologous to conserved motifs in the binding sites for NAD(H) and NADP(H) was identified in PDS, suggesting the involvement of these dinucleotides as cofactors in phytoene desaturation.

Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum).

The gene Pds encodes phytoene desaturase, a key enzyme in carotenoid biosynthesis that converts phytoene to zeta-carotene. We have cloned and analyzed the genomic DNA sequence of Pds from tomato. In tomato Pds is comprised of 15 exons that, together with the introns occupy over 8 kb. A putative promoter sequence has been identified by comparison with the cDNA sequence of Pds. A consensus nucleotide sequence around intron splicing sites in tomato genes was determined by compiling data on 137 introns in 34 genes. This consensus sequence generally agrees with the consensus sequence of other higher plants with only minor differences that are unique to tomato.

Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening.

The cDNA which encodes lycopene cyclase, CrtL, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN) and functionally expressed in Escherichia coli. This enzyme converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8') position. Inhibition in E. coli indicated that lycopene cyclase is the target site for the inhibitor MPTA, 2-(4-methylphenoxy)tri-ethylamine hydrochloride. The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but different from that of the non-photosynthetic bacteria Erwinia. mRNA of CrtL and Pds, which encodes phytoene desaturase, was measured in leaves, flowers and ripening fruits of tomato. In contrast to genes which encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the 'breaker' stage of fruit ripening, the level of CrtL mRNA decreases at this stage. Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis.

Expression of heparanase in normal, dysplastic, and neoplastic human colonic mucosa and stroma. Evidence for its role in colonic tumorigenesis.

The human heparanase gene, an endo-beta-glucuronidase that cleaves heparan sulfate at specific intrachain sites, has recently been cloned and shown to function in tumor progression and metastatic spread. Antisense digoxigenin-labeled heparanase RNA probe and monoclonal anti-human heparanase antibodies were used to examine the expression of the heparanase gene and protein in normal, dysplastic, and neoplastic human colonic mucosa. To our knowledge, this is the first systematic study of heparanase expression in human colon cancer. Both the heparanase gene and protein were expressed at early stages of neoplasia, already at the stage of adenoma, but were practically not detected in the adjacent normal-looking colon epithelium. Gradually increasing expression of heparanase was evident as the cells progressed from severe dysplasia through well-differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells showed the highest levels of the heparanase mRNA and protein associated with expression of both the gene and enzyme by adjacent desmoplastic stromal fibroblasts. A high expression was also found in colon carcinoma metastases to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts. Moreover, extracts derived from tumor tissue expressed much higher levels of the heparanase protein and activity as compared to the normal colon tissue. In all specimens, the heparanase gene and protein exhibited the same pattern of expression. These results suggest a role of heparanase in colon cancer progression and may have both prognostic and therapeutic applications.

A single polypeptide catalyzing the conversion of phytoene to zeta-carotene is transcriptionally regulated during tomato fruit ripening.

The cDNA of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined. Cells of Escherichia coli that expressed the tomato pds gene could convert phytoene to zeta-carotene. This result suggests that one polypeptide, the product of the pds gene, can carry out phytoene desaturation in the carotenoid biosynthetic pathway. Transcripts of the pds gene accumulate in orange tomato fruit, indicating transcriptional control of pds expression during fruit ripening. The deduced amino acid sequence of phytoene desaturase indicates that this enzyme in tomato contains 583 amino acids that are highly conserved with respect to the homologous enzymes in cyanobacteria and algae. The deduced amino acid sequences of the phytoene desaturases from other microorganisms (purple bacteria and fungi) appear to be evolutionarily unrelated to those from green photosynthetic organisms.

Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.

Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.

Expression pattern and secretion of human and chicken heparanase are determined by their signal peptide sequence.

Cleavage of heparan sulfate (HS) proteoglycans affects the integrity and function of tissues and thereby fundamental phenomena, involving cell migration and response to changes in the extracellular microenvironment. The role of HS-degrading enzymes, commonly referred to as heparanases, in normal development has not been identified. The present study focuses on cloning, expression, and properties of a chicken heparanase and its distribution in the developing chicken embryo. We have identified a chicken EST, homologous to the recently cloned human heparanase, to clone and express a functional chicken heparanase, 60% homologous to the human enzyme. The full-length chicken heparanase cDNA encodes a 60-kDa proenzyme that is processed at the N terminus into a 45-kDa highly active enzyme. The most prominent difference between the chicken and human enzymes resides in the predicted signal peptide sequence, apparently accounting for the chicken heparanase being readily secreted and localized in close proximity to the cell surface. In contrast, the human enzyme is mostly intracellular, localized in perinuclear granules. Cells transfected with a chimeric construct composed of the chicken signal peptide preceding the human heparanase exhibited cell surface localization and secretion of heparanase, similar to cells transfected with the full-length chicken enzyme. We examined the distribution pattern of the heparanase enzyme in the developing chicken embryo. Both the chicken heparanase mRNA and protein were expressed, as early as 12 h post fertilization, in cells migrating from the epiblast and forming the hypoblast layer. Later on (72 h), the enzyme is preferentially expressed in cells of the developing vascular and nervous systems. Cloning and characterization of heparanase, the first and single functional vertebrate HS-degrading enzyme, may lead to identification of other glycosaminoglycan degrading enzymes, toward elucidation of their significance in normal and pathological processes.

Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.

Tumor spread involves degradation of various components of the extracellular matrix and blood vessel wall. Among these is heparan sulfate proteoglycan, which plays a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Expression of an endoglycosidase (heparanase) which degrades heparan sulfate correlates with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin-binding angiogenic proteins are stored as a complex with heparan sulfate in the microenvironment of tumors. These proteins are released and can induce new capillary growth when heparan sulfate is degraded by heparanase. Here, we describe the molecular properties, expression and involvement in tumor progression of a human heparanase. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic human cell lines and in tumor biopsy specimens, including breast carcinoma. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also released ECM-resident bFGF in vitro, and its overexpression elicited an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, two critical steps in tumor progression. Mammary glands of transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts and alveolar development, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland.

Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene.

Atm, the mouse homolog of the human ATM gene defective in ataxia-telangiectasia (A-T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescence in situ hybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22-q23.