Erkki Hölttä

Regulation of ornithine decarboxylase activity by putrescine and spermidine in rat liver

Abstract The marked enhancement of the activity of ornithine decarboxylase (EC 4.1.1.17) in rat liver at 4 h following partial hepatectomy or the treatment with growth hormone could be almost completely prevented by intraperitoneal administration of putrescine. A single injection of putrescine to partially hepatectomized rats caused a remarkably rapid decline in the activity of liver ornithine decarboxylase with an apparent half-life of only 30 min, which is almost as rapid as the decay of the enzyme activity after the administration of inhibitors of protein synthesis. Under similar conditions putrescine did not have any inhibitory effect on the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) or tyrosine aminotransferase (EC 2.6.1.5). Spermidine given at the time of partial hepatectomy or 2 h later also markedly inhibited ornithine decarboxylase activity at 4 h after the operation and, in addition, also caused a slight inhibition of the activity of adenosylmethionine decarboxylase.

ORNITHINE DECARBOXYLASE- AND RAS-INDUCED CELL TRANSFORMATIONS : REVERSAL BY PROTEIN TYROSINE KINASE INHIBITORS AND ROLE OF PP130CAS

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.

Ornithine decarboxylase activity and the accumulation of putrescine at early stages of liver regeneration

intensive stimulation of ornithine decarboxylase (EC 4.1 .1.17, L-ornithine carboxylase) activity after partial hepatectomy of the rat belongs to the earliest biochemical changes in the regenerating liver remnant [l-4] . Enhanced ornithine decarboxylase activity results in a concomitant accumulation of liver putrescine [ 1,5,6] which in turn is supposed to stimulate the synthesis of spermidine [l] probably by activating the decarboxylation of S-adenosyl-L- methionine (Ado-met), the reaction needed for the synthesis of spermidine [7] . The activity of ornithine decarboxylase is almost maximally stimulated as early as at about 4 hr after partial hepatectomy and remains elevated for several days [2,6]. It has been reported that hypophysectomy, but not adrenalectomy, castration, or thyroidectomy, considerably delays, although does not prevent, the stimulation of ornithine decarboxylase after partial hepatectomy [8] . This latter observation might indicate the involvement of some humoral factor(s) in the regulation of putrescine synthesis in the regenerat- ing rat liver. In the present communication we have studied more closely the synthesis and accumulation of putrescine at early stages of liver regeneration. The results of several series of rats revealed that during the first day of regeneration the stimulation of ornithine decarboxylase appeared to occur in two phases, the first peak of the enzyme activity invariably occurring at 4 hr after the operation independently of the age of the animal. The stimulation of ornithine decarboxyl- ase was closely followed by an accumulation of putrescine. Evidence is also presented indicating that

The cellular response to induction of the p21 c-Ha-ras oncoprotein includes stimulation of jun gene expression.

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)

Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.

Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride

Abstract A single intraperitoneal injection of carbon tetrachloride into rats resulted within 12 hours in a marked accumulation of putrescine in liver with a concomitant decrease in the concentration of spermidine. The accumulation of putrescine apparently was partly due to an immense stimulation of ornithine decarboxylase activity occurring at the same time. However, in addition it was found that during the maximal accumulation of putrescine there was a marked incorporation of radioactivity from labelled spermidine to liver putrescine in vivo . The conversion of spermidine to liver putrescine was hardly detectable in control animals. Besides the treatment with carbon tetrachloride, increased conversion of radioactive spermidine to liver putrescine in vivo also occurred after treatment with growth hormone, after partial hepatectomy and after treatment with thioacetamide, i. e. under circumstances characterized by a stimulation of ornithine decarboxylase activity and an increased accumulation of putrescine.

Caspases and mitochondria in c-Myc-induced apoptosis: identification of ATM as a new target of caspases

The mechanism(s) of c-Myc transcription factor-induced apoptosis is still obscure. The activation of c-Myc has been found to lead into the processing/activation of caspases (caspase-3), but the significance of this for the cell demise is debatable. Here we report that several targets of caspases (PKCδ, MDM2, PARP, replication factor C, 70 kDa U1snRNP, fodrin and lamins) are cleaved during c-Myc-induced apoptosis in Rat-1 MycERTM cells, indicating an important role for caspases in the apoptotic process. We further found that the ATM (ataxia telangiectasia mutated) – protein is a novel key substrate of caspases. In in vitro assays, purified recombinant ATM protein was found to be cleaved by the effector caspases 3 and 7. The functional significance of the ATM cleavage is supported by the finding that ectopic expression of ATM protected in part against apoptosis. We also show that c-Myc-induced apoptosis involves loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria into the cytosol and subsequent processing of caspase-9. The cleavage of caspase-9 is, however, minimal and a much later event than the processing/activation of caspase-3, suggesting that it is not the apical caspase. Evidence is provided that there is, nevertheless, an upstream caspase(s) regulating the functions of caspase-3 and mitochondria. Additionally, it was found that p53 becomes upregulated, together with its transcriptional targets MDM2 and p21, upon c-Myc induction, but this occurs also at a later time than the activation of caspase-3.

Metastatic Outgrowth Encompasses COL-I, FN1, and POSTN Up-Regulation and Assembly to Fibrillar Networks Regulating Cell Adhesion, Migration, and Growth

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.

Involvement of CPP32/Caspase-3 in c-Myc-induced apoptosis

c-Myc is a transcriptional activator implicated in the control of cell proliferation, differentiation and transformation, but is also involved in the regulation of programmed cell death, apoptosis. Despite intensive research, the molecular mechanisms by which c-Myc triggers and executes cell death remain still elusive. Here, we made use of Rat 1A MycER cells expressing a conditionally active c-Myc protein and tested first the hypothesis that ornithine decarboxylase (ODC), which is a transcriptional target of c-Myc, were a mediator of c-Myc-induced apoptosis. However, our results show that the activity of ODC is not required for the c-Myc-mediated apoptosis to occur in these cells. We also found that the expression of p53, p21waf1/cip1, Bcl-2, Bax, Bcl-xL, Bad and cyclins D1, E, A and B did not show any significant changes following c-Myc induction. But, our studies revealed that the c-Myc induced apoptosis is associated with a specific cleavage of poly(ADPribose) polymerase (PARP), suggesting that a cysteine protease of the ICE/CED-3 family is involved. Moreover, we found that the cysteine protease CPP32/Caspase-3, which is known to cleave PARP, is processed from its inactive form to an active protease composed of 17 and 12 kDa subunits; whilst Ich-1/Caspase-2 belonging to another subset of this protease family was not processed/activated following c-Myc activation. The activation of CPP32 and apoptotic cell death were inhibited by addition of Z-VAD-fmk, a universal inhibitor of ICE-like proteases. Further, a selective inhibitor of CPP32-like proteases (Z-DEVD-fmk) partly inhibited apoptosis. These results provide evidence that the ICE/CED3-family proteases, CPP32 and likely others, play a critical role in the execution of a nuclear proto-oncogene, c-Myc-induced apoptosis.

Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion

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. Copyright