Altered expression of urokinase-type plasminogen activator and plasminogen activator inhibitor in high-risk soft tissue sarcomas.

In recent years, classification of soft-tissue sarcomas (STS) has improved with cytogenetic analyses, but their clinical behavior is still not easily predictable. The aim of this study was to detect alterations in the urokinase-type plasminogen system, involved in tumor growth and invasion, by comparing mRNA levels of its components with those of paired normal tissues, and relating them with patient clinical course. Real-time PCR was performed on human STS cell lines and tissues from highly malignant STS, including leiomyosarcomas and malignant fibrous histiocytomas, to evaluate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of gene products was also performed. Median mRNA values of all genes studied were higher in tumors than in paired normal tissues. In agreement with data on STS cell lines, significant up-regulation for uPA and PAI-1 genes compared to reference values was seen. Moreover, different levels of expression were related to histotype and metastatic phenotype. There was accordance between uPA mRNA and protein expression, while immunodetection of PAI-1 product was weak and scattered. Clearly, the controversial role of PAI-1 protein requires further biological analyses, but evident involvement of uPA/PAI-1 gene overexpression in STS malignancy may highlight a molecular defect useful in discriminating STS high-risk patients.

Molecular alterations of monophasic synovial sarcoma: loss of chromosome 3p does not alter RASSF1 and MLH1 transcriptional activity.

Differential diagnosis of monophasic synovial sarcoma requires the detection of specific biological markers. In this study we evaluated the presence of molecular alterations in 15 monophasic synovial sarcomas. Multiple changes affecting chromosome arms were detected by CGH-array in all microdissected cases available, and an association between gain or loss of specific regions harbouring cancer progression-associated genes and aneuploid status was found. The most frequent alteration was loss of 3p including 3p21.3-p23 region that, however, did not involve the promoter regions of the corresponding genes, RASSF1 and MLH1. Using Real-Time PCR, mRNA levels of both resulted moderately high compared to normal tissue; however, the weak to absent protein expression suggests RASSF1 and MLH1 post-transcription deregulation. Moreover, immunohistochemical analysis revealed that both mesenchymal and epithelial antigens were present in diploid tumours. These findings confirm the genetic complexity of monophasic synovial sarcoma and underline the need to integrate different analyses for a better knowledge of this tumour, essential to investigate new diagnostic and prognostic markers.

Amplification of CDK4, MDM2, SAS and GLI genes in leiomyosarcoma, alveolar and embryonal rhabdomyosarcoma.

We evaluated amplification and overrepresentation of CDK4, MDM2, GLI and SAS genes of the 12q13-15 region, in a group of soft tissue sarcomas including leiomyosarcomas (LMS), alveolar rhabdomyosarcomas (ARMS) and embryonal (anaplastic and classic variants) rhabdomyosarcomas (ERMS), to ascertain genomic alterations and possible differences within histologic subtypes of rhabdomyosarcoma (RMS). Quantitative real-time PCR was performed on DNA samples from 29 LMS, 9 ARMS, 7 anaplastic ERMS and 6 classic ERMS. Alteration of one or more of the 12q13-15 genes was revealed in 13/29 LMS (45%) and 12/22 RMS (54%) including 5/9 ARMS (56%), 5/7 anaplastic ERMS (71%) and 2/6 classic ERMS (33%). The potential importance of overproduction of protein products in neoplastic development, led us also to study a possible high expression of cdk4, mdm2 and gli proteins in immunohistochemical staining experiments on paraffin-embedded tissue samples of the same cases. Among LMS and RMS most cases with CDK4, MDM2 and GLI gene alterations also showed a simultaneous high expression of the relative protein. In summary, these results indicate that amplification or overerepresentation of genes at 12q13-15 region involve both LMS and RMS. Moreover these genes alterations reveal predominantly in the alveolar and in the anaplastic variant of the embryonal subtype. These two seem to have a more similar behavior than anaplastic and classic embryonal that are classified in the same subtype.

Proteases and interleukin-6 gene analysis in 92 giant cell tumors of bone.

BACKGROUND: Giant cell tumor of bone (GCT) is a benign tumor with a significant tendency to recur locally and rarely to produce pulmonary metastases. It is characterized by the presence of multinucleated osteoclast-like giant cells together with mononuclear spindle-shaped cells. Few prognostic markers have been reported to predict the clinical outcome of GCT patients, so is very important to find the factor that can be implicated in its potential aggressiveness. PATIENTS AND METHODS: Different groups of GCT patients were selected for this study, including patients without evidence of disease and patients who recurred locally or with lung metastasis. The total of 92 tumor samples also included the specimens of the local recurrences and the lung metastases. By using immunohistochemistry and real-time quantitative polymerase chain reaction techniques, the genetic and proteic analyses were performed on the urokinase-type plasminogen activator (u-PA), its receptor (u-PAR) and its inhibitor (PAI-1), which have been described to be frequently implicated in the process of degradation of the extracellular matrix during the metastatic process. Interleukin-6 (IL-6), a cytokine released by GCT cells, which stimulates resorption of bone, was also analyzed. RESULTS: IL-6, u-PA, u-PAR and PAI 1 genes were found amplified, respectively, in 7%, 5%, 8% and 12% of total cases (92). In particular, the percentages of amplified genes were higher in the GCT cells that gave rise to metastases (12 cases) and in the samples of lung metastases (nine cases) compared with the disease-free group of patients (60 cases). CONCLUSIONS: These results suggest a possible association of these factors with a higher biological aggressiveness of GCT. Morever, it appears that increased expression of the IL-6, u-PA, u-PAR and PAI1 proteins might not depend on mutation of the corresponding genes.

Tissue and serum loss of metalloproteinase inhibitors in high grade soft tissue sarcomas.

The activity of matrix metalloproteinases (MMPs) in degrading extracellular matrix is controlled by activation of pro-enzymes and inhibition of MMP tissue inhibitors (TIMPs). To assess proteolytic cascade imbalance in malignancy progression, the enzymatic activity of MMP2 and MMP9 and the expression and serum level of their inhibitors, TIMP2 and TIMP1 respectively, was evaluated in selected patients with high-risk soft tissue sarcoma (STS). Gelatinase activity and inhibitor expression was evaluated on 69 biopsies by zymography and immunohistochemistry. TIMP1 and TIMP2 serum concentration was tested in 53 STS patients and in 56 controls using a sandwich enzyme immunoassay. Clinical and biological variables were related to clinical outcome of the patients. A significant gelatinolytic activity was seen in a high percentage of STS. TIMP expression was weak or negative in the majority of samples. The difference between disease-free (p=0.001) and overall survival (p=0.007) curves based on TIMP2 immunoreactivity was statistically significant. TIMP plasma concentration of 53 STS revealed significantly lower levels compared to those of 56 controls (p=0.0001). In conclusion, low levels of negative regulators of proteolysis may be related to tumor biological aggressiveness and used to select patients with poor prognosis to improve cure.

Neuroprotection afforded by some hindered phenols and alpha-tocopherol in guinea-pig detrusor strips subjected to anoxia-glucopenia and reperfusion-like conditions.

2-t-butyl-4-methoxyphenol (BHA), 3,5-di-t-butyl-hydroxyanisole (DTBHA), 2,6-diisopropylphenol (propofol), alpha-tocopherol (alpha-TOC) and two newly synthesised analogues of BHA, namely 1-O-(4-hydroxy-3-t-butyl)phenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose (beta-TAG) and 1-O-(4-hydroxy-3-t-butyl)phenyl-beta-D-glucopyranose (beta-GLU), were tested for their capability to protect the intrinsic nerves of guinea-pig urinary bladder from damage due to anoxia-glucopenia and re-exposure to glucose and O2. Guinea-pig detrusor strips were mounted for tension recording in small organ baths, superfused with warmed Krebs solution and the nerves stimulated electrically either under control or ischaemia-like (anoxia-glucopenia) and reperfusion-like conditions (normal medium re-superfusion). The Ca2+ antagonist activity of the compounds was assessed by their effect on the contraction of detrusor strips induced by 60 mM K+ Krebs solution in the presence of either 0.5 mM or 5 mM Ca2+. The antioxidant activity was illustrated by the ability of the compounds to scavenge peroxyl radicals generated by linoleic acid oxidation. All the compounds, except beta-GLU and alpha-TOC, inhibited in a concentration-dependent manner K+-induced contractions of detrusor muscles, the inhibition being inversely related to the Ca2+ concentration of the perfusion solution; moreover, they exhibited a marked antiperoxidant activity with pIC50 values decreasing in the order: DTBHA > alpha-TOC > BHA > beta-TAG > propofol > beta-GLU. alpha-TOC, BHA, DTBHA and beta-TAG improved significantly the response of the strips to electrical field stimulation either during the anoxia-glucopenia phase or thereafter when recovering during reperfusion, as compared to untreated tissues. The neuroprotection afforded by the phenol derivatives as well as by alpha-TOC was positively correlated to their antioxidant activity, but not to their Ca2+ antagonist activity.

On the photoreactivity of 4,5-dithiophen-3-yl-[1,3]dithiol-2-one. The first preparation of a thieno[3,4-c]dithiine.

[reaction: see text]. Irradiation of 4,5-dithiophen-3-yl-[1,3]dithiol-2-one 1 at lambda > 330 nm gave the thieno[3,4-c]dithiine 2, the first example of this heterocyclic system

Synthesis and antiperoxidant activity of new phenolic O-glycosides.

We describe the synthesis of some 3-tert-butyl-4-hydroxyphenyl D-glycopyranosides by reaction of tert-butylhydroquinone with beta-D-pentaacetyl-glucose, beta-D-pentaacetyl-galactose, 2-acetamido- and 3,4,6-tri-O-acetyl-2-butanamido-2-deoxy-beta-D-glucopyranosyl chlorides as well as the formation of anomeric 3-tert-butyl-4-hydroxyphenyl 4,6-di-O-acetyl-2,3-dideoxy-D-erythro-hex-2-eno-pyranosides by reaction between tert-butylhydroquinone and 3,4,6-tri-O-acetyl-D-glucal. All compounds, except 3-tert-butyl-4-hydroxyphenyl alpha- and beta-D-glucopyranosides, inhibited lipid peroxidation with a degree of potency comparable to that of tert-butyl hydroxyanisole.

Some chemical properties and biological role of thiazolidine compounds.

In this study we have investigated some chemical properties and the biological role of thiazolidine compounds, obtained by condensation of aminothiols (L- or D-cysteine, cysteamine) with pyridoxal-5'-phosphate. These products have been tested in presence of rat liver extracts (supernatant and mitochondria); bacterial suspensions and enzymes (L- or D-aminoacid oxidase, xanthine oxidase) with interesting results which gives evidence to a biological role. Their formation in vivo may represent the regulation of intracellular levels of pyridoxal-5'-phosphate and aminothiols. Moreover, we have analysed the two diastereoisomers of the thiazolidine compounds derived from L-cysteine and D-cysteine: we have succeeded to distinguish by NMR analysis the cis and the trans forms, concluding that the interconversion of the free forms is extremely rapid at pH 7: thus, it may be relevant for the protein bound forms.

Biological role of carbamoyl pyridoxal 5'-phosphate.

A new compound, carbamoyl-pyridoxal 5'-phosphate (C-PLP), was synthetized by condensation of pyridoxal 5'-phosphate (PLP) with KCNO. It may be obtained under certain physiological conditions of pH, temperature and concentration of reagents. Formation and degradation of C-PLP are readily reversible chemical reactions, not involving enzymes, at least in rat tissues. However, different considerations suggest that synthesis and breakdown of C-PLP play a biological role in the cell, providing 'protective synthesis' and a 'variable reservoir' of PLP and KCNO, which can be trapped by other proteins, apoenzymes and metabolites, to regulate many cell metabolic functions.

Characterization and differentiation of heterocyclic isomers. tandem mass spectrometry and molecular orbital calculations on 3-methylisoxazolo- and 2-methyloxazolopyridines.

Metastable mass-analyzed ion kinetic energy (MIKE) and collision-induced dissociation MIKE spectrometries have been applied to the study of all members of two classes of heteroaromatic isomers: 3-methylisoxazolo-and 2-methyloxazolopyridines. The study revealed that tandem mass spectrometry can characterize and differentiate the isomeric ion structures produced by these heterocycles. In particular, the MIKE spectra of both the molecular ions and abundant fragments formed by CO and CH3CN losses show characteristic differences that allow distinction among the isomers dependent on the position of the nitrogen atom in the pyridine ring, and distinction of isoxazole derivatives from oxazoles. The results indicate that the isomerization of the isoxazole moiety to oxazole-proposed for other analogous compounds-does not occur in these heterocyclic systems. The experimental work is supported by molecular orbital calculations both on neutral molecules and on molecular and fragment ions.

The inhibition of rat liver threonine dehydratase by carbamoyl-phosphate. The formation of carbamoylpyridoxal 5'-phosphate.

The effects exerted by carbamoyl phosphate (CP) and cyanate (KCNO) on rat liver L-threonine deaminase have been studied. The two compounds showed that same effects, inhibiting through a competitive mechanism both the holoenzyme and the dialyzed enzyme; inhibition was more evident for the latter. Ki values, both for L-threonine and pyridoxal 5'-phosphate (PLP), were lower for the apoenzyme and the inhibitors also affected the Km of the apoenzyme for PLP. The effects of CP and KCNO are mainly due to an interference in the association reaction apoenzyme + PLP in equilibrium holoenzyme This was clearly demonstrated by the fact that, when PLP was incubated with CP or KCNO, it failed to enhance the activity of the holoenzyme nor did it reactivate the resolved apoenzyme. Such interference of CP and KCNO in the L-threonine deaminase activity was mainly due to a specific mechanism, the formation of a new derivative of PLP. The reaction of PLP with either CP or KCNO occurred readily, at low concentrations, under physiological conditions. The new compound was identified as 3,4-dihydro-2H-pyrido[3,4-e]1,3-oxazin-2-one derivative by ultraviolet-visible spectra, elemental analysis, infrared, NMR and MS spectra. In this paper we formulate the hypothesis that this compound is involved in the regulation of the CP and PLP intracellular content and in the control of PLP dependent enzymes.

Synthesis and NMR properties of 2-(4-pyridyl) thiazolidine-4-carboxylic acids.

Various thiazolidine derivatives, obtained through condensation between pyridoxal or pyridoxal-5'-phosphate with several aminothiols, have been obtained and examined for their NMR spectra. For the compounds tested, we demonstrated the presence in solution of the two diasteroisomeric thiazolidines, A and C, which differ for the configuration at C2, and are interconverted via the open chain Schiff's base, with different rates, depending on the pH. The possibility that, when aminothiols react with PLP bound to the enzyme, only one of the two diasteroisomers may be formed, and the biological role of such derivatives is discussed.