The enzyme ornithine decarboxylase is the key regulator of the synthesis of polyamines which are essential for cell proliferation. Expression of this enzyme is transiently increased upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes. To test whether ornithine decarboxylase could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 cells with expression vectors carrying the complementary DNA encoding human ornithine decarboxylase in sense and antisense orientations. The increased expression of the enzyme (50-100-times endogenous levels) induced not only cell transformation, but also anchorage-independent growth in soft agar and increased tyrosine phosphorylation of a protein of M(r) 130K. Expression of ornithine decarboxylase antisense RNA was associated with an epithelioid morphology and reduced cell proliferation. Moreover, blocking the endogenous enzyme using specific inhibitor or synthesizing antisense RNA prevented transformation of rat fibroblasts by temperature-sensitive v-src oncogene. Our results imply that the gene encoding ornithine decarboxylase is a proto-oncogene central for regulation of cell growth and transformation.

Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-beta signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes-all inducible by transforming growth factor-beta. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-beta receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis.

A novel enzyme responsible for the oxidation of spermidine and spermine has been found in rat liver. Spermidine is shown to be degraded to putrescine and 3-aminopropionaldehyde, and spermine to be cleaved to spermidine and 3-aminopropionaldehyde. A single enzyme catalyzing both reactions and designated as polyamine oxidase has been purified 4000-fold to electrophoretic homogeneity. Polyamine oxidase appears to be a flavoprotein, containing flavin adenine dinucleotide (FAD) as a prosthetic group. Hydrogen peroxide is evolved in the reaction and no other electron acceptors except molecular oxygen have been found. The molecular weight of the enzyme was approximately 60 000 and the sedimentation coefficient 4.5 S. The enzyme appears to be a single polypeptide chain since no evidence for structural subunits was obtained. Polyamine oxidase was sensitive to sulfhydryl and carbonyl group reagents. The optimum pH value for the oxidation of polyamines was close to 10. The reaction velocities were enhanced by various aldehydes, especially certain aromatic aldehydes. Polyamine oxidase appears to be localized in peroxisomes of liver cells, although the existence of an isoenzyme in the cytosolic fraction was not definitively ruled out. No marked changes were observed in the activity of polyamine oxidase in rat liver after partial hepatectomy, carbon tetrachloride poisoning, and after treatment with growth hormone or thioacetamide, conditions which are known to alter profoundly the metabolism and accumulation of polyamines.

anzctr.org.au Identifier: ACTRN12611001173987.

No abstract

Malignant melanomas are characterized by their high propensity to invade and metastasize, but the molecular mechanisms of these traits have remained elusive. Our DNA microarray analyses of benign nevi and melanoma tissue specimens revealed that the genes encoding extracellular matrix proteins tenascin-C (TN-C), fibronectin (FN), and procollagen-I (PCOL-I) are highly upregulated in invasive and metastatic melanomas. The expression and distribution of these proteins were further studied by immunohistochemistry in benign nevi, radially and vertically growing melanomas, sentinel node micrometastases, and macrometastases. TN-C was increased in all invasive tumours and metastases, especially at invasion fronts, but not in benign nevi or non-invasive melanomas. Significantly, the intensity of TN-C staining correlated with metastasis to sentinel lymph nodes, better than tumour thickness (Breslow). Moreover, TN-C, FN, and PCOL-I appeared to co-localize in the tumours and form tubular meshworks and channels ensheathing the melanoma cells. Our data suggest that melanoma invasion is associated with the formation of special channel-like structures, providing a new concept, structured tumour cell spreading. Altogether, these data provide potential new prognostic markers and therapeutic targets/strategies for preventing melanoma dissemination.

No abstract

We have studied the effects of c‐Ha‐ras oncogene in mouse NIH 3T3 fibroblasts by DNA transfection and analysis of gene expression at the mRNA and protein level in a heat‐ and heavy metal‐inducible model system. The human c‐Ha‐ras proto‐oncogene and oncogene were cloned under the hsp70 heat‐shock promoter. Clonal lines of cells with negligible basal expression of the hsp‐c‐Ha‐ras oncogene construct were chosen on the basis of the inducibility of p21c‐Ha‐ras protein and several transformation parameters. We demonstrate that the expression of ornithine decarboxylase (ODC) mRNA is enhanced approximately 4‐6 h after the induction of the p21c‐Ha‐ras oncoprotein. This increase was reversible upon cessation of c‐Ha‐ras mRNA and protein synthesis, while constitutively elevated ODC was characteristic for stably c‐Ha‐ras‐transformed cells. The high‐level expression of ODC in ras‐transformed cells was insensitive to tumour promoter stimulation. A similar mRNA induction by c‐Ha‐rasVal‐12 was also observed for two other serum‐ and tumour promoter‐regulated genes associated with the transformed phenotype: transin (stromelysin) and the glucose transporter. This prompted us to examine also potential changes in the expression of the serum‐ and tumour promoter‐induced transcription factor genes junB and c‐jun after induction of the hsp–c‐Ha‐ras construct. The junB mRNA was enhanced approximately 10‐fold and the c‐jun oncogene mRNA to a lesser degree in the hsp–c‐Ha‐ras‐transfected cells after zinc activation of the hsp70 promoter. These effects were not seen in similarly treated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)