Aminooxy analog of histamine is an efficient inhibitor of mammalian L-histidine decarboxylase: combined in silico and experimental evidence.

Histamine plays highlighted roles in the development of many common, emergent and rare diseases. In mammals, histamine is formed by decarboxylation of L-histidine, which is catalyzed by pyridoxal-5'-phosphate (PLP) dependent histidine decarboxylase (HDC, EC 4.1.1.22). The limited availability and stability of the protein have delayed the characterization of its structure-function relationships. Our previous knowledge on mammalian HDC, derived from both in silico and experimental approaches, indicates that an effective competitive inhibitor should be capable to form an "external aldimine-like structure" and have an imidazole group, or its proper mimetic, which provides additional affinity of PLP-inhibitor adduct to the HDC active center. This is confirmed using HEK-293 cells transfected to express human HDC and the aminooxy analog of histidine, 4(5)-aminooxymethylimidazole (O-IMHA, IC50 ≈ 2 × 10(-7) M) capable to form a PLP-inhibitor complex (oxime) in the enzyme active center. Taking advantage of the availability of the human HDC X-ray structure, we have also determined the potential interactions that could stabilize this oxime in the active site of mammalian HDC.

Exploring polyamine regulation by nascent histamine in a human-transfected cell model.

There are multiple lines of evidence suggesting interplay between histamine and polyamines in several mammalian cell types. However, the complex metabolic context makes it difficult to elucidate the mechanisms involved. Histamine's effects can be elicited after its binding to any of the four subtypes of G-protein coupled histamine membrane receptors. In addition, intracellular histamine can also interfere with polyamine metabolism, since there are several metabolic connections between the synthesis and degradation pathways of both types of amines. In order to dissect the metabolic effects of intracellular histamine on polyamine metabolism, we chose a well-known cell culture line, i.e., the human embryonic kidney 293 cells (HEK-293 cells). Initially, we show that HEK-293 cells lack a polyamine metabolic response to extracellular histamine, even over a wide range of histamine concentrations. HEK-293 cells were transfected with active and inactive versions of human histidine decarboxylase, and changes in many of the overlapping metabolic factors and limiting steps were tested. Overall, the results indicate a regulatory effect of histamine on the post-transcriptional expression of ornithine decarboxylase and suggest that this effect is primarily responsible for the decrease in polyamine synthesis and partial blockade of cell-cycle progression, which should affect cell proliferation rate.

Development of an expression macroarray for amine metabolism-related genes.

Cationic amino acids are the precursors of biogenic amines, histamine from histidine, and putrescine, spermidine and spermine from arginine/ornithine (and methionine), as well as nitric oxide. These amines play important biological roles in inter- and intracellular signaling mechanisms related to inflammation, cell proliferation and neurotransmission. Biochemical and epidemiological relationships between arginine-derived products and histamine have been reported to play important roles in physiopathological problems. In this communication, we describe the construction of an expression macroarray containing more than 30 human probes for most of the key proteins involved in biogenic amines metabolisms, as well as other inflammation- and proliferation-related probes. The array has been validated on human mast HMC-1 cells. On this model, we have got further support for an inverse correlation between polyamine and histamine synthesis previously observed on murine basophilic models. These tools should also be helpful to understand the amine roles in many other inflammatory and neoplastic pathologies.

Effect of nerve growth factor on the expression of cell cycle regulatory proteins in PC12 cells: dissection of the neurotrophic response from the anti-mitogenic response.

PC12 cells treated with nerve growth factor (NGF) undergo a G1 block and differentiate. Expression of selected cell cycle regulatory proteins was studied under culture conditions which permit observation of a differentiation response independently from a mitogenic or anti-mitogenic response. The expression of all cell cycle regulatory proteins studied is modulated by NGF addition to exponentially-growing cultures in the presence of serum. While levels of most of these proteins decrease, accumulation of cyclin D1 and the cyclin-dependent kinase inhibitor p21 Cip1/WAF1 is observed. Cyclin D1 associated kinase activity is inhibited, correlating with an increase in p21 protein. PC12 cells, synchronized by serum starvation, undergo morphological and functional differentiation in the presence of NGF. Neither cyclin D1 nor p21 are present in such cultures, nor is their expression upregulated by NGF, indicating that they are not required for this process. Removal of serum from differentiated PC12 cells results in loss of these proteins, but has no effect on differentiation or the nonproliferative state in presence of NGF. Together, the results indicate that cyclin D1 and p21 are not necessary for differentiation per se, nor are they required for maintenance of the differentiated state in the absence of serum.

Nerve growth factor-induced accumulation of PC12 cells expressing cyclin D1: evidence for a G1 phase block.

The anti-proliferative effect of nerve growth factor (NGF) on the rat pheochromocytoma cell line PC12 has been previously shown to be accompanied by the accumulation of cells in either the G1 phase with a 2c DNA content, or with a 4c DNA content characteristic for G2/M, as evidenced by flow cytometric analysis of DNA distribution using propidium iodide. Herein, these apparently conflicting results are clarified. The present studies indicate that a simple DNA distribution profile obtained by this technique can confound interpretation of the biological effects of NGF on cell-cycle distribution due to the presence of tetraploid cells. Using cyclin D1 and incorporation of bromodeoxyuridine as markers of respectively, G1 and S phase, we show that PC12 cultures can have a considerable amount of tetraploid cells which, when in the G1 phase, have a 4c DNA content and express cyclin D1. During exposure to NGF, this population increases, reflecting the accumulation of cells in the G1 phase of the cell cycle. The data presented, support the possibility that events affecting the expression or action of G1 regulatory proteins may be involved in the molecular mechanism of the anti-mitogenic effect of NGF.

Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

Structure/function relationship studies on the T/S residues 173-177 of rat ODC.

A well-conserved T/S cluster was detected among vertebrate ornithine decarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdiales, J.M. Matés, F. Sánchez-Jiménez, Trends Biochem. Sci. 19 (1994) 318-319). In the present report we studied the role of these residues (173, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activity and stability by in vitro expression, kinetic characterization and in vitro degradation of site-directed mutants. These T/S residues are substituted by a D/E-enriched fragment in other lower eukaryotic ODCs. The substitution of the T/S-enriched fragment (TLKTS) of rat ODC by the negative charged fragment of T. brucei ODC (KVEDC) did not affect protein stability, but increased Km values of the mutant enzyme. The substitution of the T/S residues by alanine also has a similar effect on rat ODC kinetic values. However, results indicate that polarity of the fragment must be an important factor for protein conformation, since the latter mutant, having no T/S or D/E residue in the fragment (ALKAA), showed reduced stability in vitro.

Expression of different mitogen-regulated protein/proliferin mRNAs in Ehrlich carcinoma cells.

Results from in vivo and from serum-free primary cultures of Ehrlich cells suggest that the expression of mitogen-regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL-related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF-1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.

Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing in vivo.

The antihistaminic (+/-)-chlorpheniramine significantly reduced the progression of Ehrlich carcinoma when it was administered at 0.5 mg/mouse/day from the third day on, after tumour inoculation. The ODC activity of tumour cells was diminished by 70% on day 7 after tumour transplantation, when maximum ODC activity is detected in non-treated tumour growing 'in vivo'. Northern blot analyses indicated that the inhibitory effect of this 1,4-diamine takes place at a post-transcriptional level. Results obtained from serum-free cultured cells indicated that chlorpheniramine inhibits the ODC synthesis rate.

Antiproliferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates.

The biological effects of dehydrodidemnin B(DDB), a novel depsipeptide isolated from Aplidium albicans, were studied on Ehrlich carcinoma growing in vivo and in primary cultures, and compared with those reported for Didemnin B (DB). Daily administration of DB or DDB (2.5 micrograms/mouse) almost duplicated the animal life-span and total number of tumour cells decreased by 70-90%. Results suggest a major effect of DDB when administered in the lag phase of growth. DDB behaved as a very potent inhibitor of protein synthesis; consequently, ornithine decarboxylase activity (ODC, EC 4.1.1.17) is drastically reduced by DDB-treatment.

Early systemic effects on the hepatic mitochondria of tumour bearing mice.

The activities of citrate synthase and cytochrome c oxidase, mitochondrial marker enzymes, were evaluated in the liver of Ehrlich ascites carcinoma-bearing mice during the life span of the inoculated animals. After 10 days of tumour transplantation, when cell proliferation has ceased, a 30-40% decrease of these activities was detected in both liver and kidney. Simultaneously, an increase in the total acidic proteinase activity (50-60%) was observed. The gel filtration profiles of liver proteinase activities from inoculated animal extracts displayed different patterns to those of the controls; low molecular weight proteinase activities appear to be enhanced in the livers of tumour-bearing animals.

The induction of ornithine decarboxylase by ornithine takes place at post-transcriptional level in perifused Ehrlich carcinoma cells.

The increase in ODC activity during perifusion of Ehrlich carcinoma cells with 0.5 mM ornithine correlates with an increase in 'de novo' synthetized ODC protein. ODC synthesis was followed by immunoprecipitation of equal quantities of 35S-labelled proteins after 10, 20 and 30 min of labelling. In addition, the rate of 'de novo' protein synthesis is very much elevated in cells perifused with saline buffer supplemented with 0.5 mM ornithine than in cells perifused with the saline buffer only. In spite of the higher specific ODC activity observed in cells perifused with saline buffer plus 0.5 mM ornithine respect to cells perifused with only saline buffer for 3.5 h, no elevation in ODC mRNA was observed when the cells were perifused in the presence of 0.5 mM ornithine.

Effects of phorbol ester and dexamethasone treatment on histidine decarboxylase and ornithine decarboxylase in basophilic cells.

Both histamine and polyamines are important for maintaining basophilic cell function and viability. The synthesis of these biogenic amines is regulated by histidine decarboxylase and ornithine decarboxylase, respectively. In other mammalian tissues, an interplay between histamine and polyamine metabolisms has been suspected. In this report, the interplay between histamine and ornithine-derived polyamines was studied in a non-transformed mouse mast cell line (C57.1) treated with phorbol ester and dexamethasone, a treatment previously used to increase histidine decarboxylase expression in mastocytoma and basophilic leukemia. Treatment with phorbol ester and dexamethasone increased histidine decarboxylase expression and intracellular histamine levels in C57.1 mast cells to a greater extent than those found for other transformed basophilic models. The treatment also induced a reduction in ornithine decarboxylase expression, intracellular polyamine contents, and cell proliferation. These results indicate that the treatment induces a co-ordinate response of polyamine metabolism and proliferation in mast cells and other immune-related cells. The decrease in the proliferative capacity of mast cells caused by phorbol ester and dexamethasone was simultaneous to an increase in histamine production. Our results, together with those reported by other groups working with polyamine-treated mast cells, indicate an antagonism between histamine and polyamines in basophilic cells.

Polyamine metabolism revisited.

The natural polyamines putrescine, spermidine and spermine play an essential role in cell growth and differentiation. Cellular polyamine depletion results in inhibition of growth, whereas its accumulation appears to be toxic. Intracellular levels of polyamines are regulated by a multitude of mechanisms affecting their synthesis, degradation, uptake and excretion. The three key enzymes in the regulation of polyamine metabolism have short half-lives and are inducible. Ornithine and S-adenosylmethionine decarboxylases regulate polyamine biosynthesis whereas spermidine/spermine acetyltransferase regulates polyamine interconvertion and degradation.

Pharmacological potential of biogenic amine-polyamine interactions beyond neurotransmission.

Histamine, serotonin and dopamine are biogenic amines involved in intercellular communication with multiple effects on human pathophysiology. They are products of two highly homologous enzymes, histidine decarboxylase and l-aromatic amino acid decarboxylase, and transmit their signals through different receptors and signal transduction mechanisms. Polyamines derived from ornithine (putrescine, spermidine and spermine) are mainly involved in intracellular effects related to cell proliferation and death mechanisms. This review summarizes structural and functional evidence for interactions between components of all these amine metabolic and signalling networks (decarboxylases, transporters, oxidases, receptors etc.) at cellular and tissue levels, distinct from nervous and neuroendocrine systems, where the crosstalk among these amine-related components can also have important pathophysiological consequences. The discussion highlights aspects that could help to predict and discuss the effects of intervention strategies.

(-)-Epigallocatechin-3-gallate interferes with mast cell adhesiveness, migration and its potential to recruit monocytes.

Mast cells are multipotent effector cells of the immune system. They are able to induce and enhance angiogenesis via multiple pathways. (-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea and a putative chemopreventive agent, was reported to inhibit tumor invasion and angiogenesis, processes that are essential for tumor growth and metastasis. Using the human mast cell line HMC-1 and commercial cDNA macroarrays, we evaluated the effect of EGCG on the expression of angiogenesis-related genes. Our data show that among other effects, EGCG treatment reduces expression of two integrins (alpha5 and beta3) and a chemokine (MCP1), resulting in a lower adhesion of mast cells associated with a decreased potential to produce signals eliciting monocyte recruitment. These effects on gene expression levels are functionally validated by showing inhibitory effects in adhesion, aggregation, migration and recruitment assays.

The usefulness of post-genomics tools for characterization of the amine cross-talk in mammalian cells.

Evidence is growing in favour of a relationship between cancer and chronic inflammation, and particularly of the role of a polyamine and histamine metabolic interplay involved in these physiopathological problems, which are indeed highly complex biological systems. Decodification of the complex inter- and intra-cellular signalling mechanisms that control these effects is not an easy task, which must be helped by systems biology technologies, including new tools for location and integration of database-stored information and predictive mathematical models, as well as functional genomics and other experimental molecular approaches necessary for hypothesis validation. We review the state of the art and present our latest efforts in this area, focused on the amine metabolism field.

Roles of homocysteine in cell metabolism: old and new functions.

Mild hyperhomocysteinemia has been suggested as a new, independent risk factor for cardiovascular disease. This fact has produced a new, increased interest in the study of homocysteine metabolism and its relation to pathogenesis. This emergent area of biomedical research is reviewed here, stressing the biochemical and metabolic basis of the pathogenicity of increased levels of homocysteine.