Stage-related decorin and versican expression in human laryngeal cancer.

BACKGROUND: The major proteoglycan of normal human larynx is aggrecan. In laryngeal carcinoma, aggrecan is depleted, with versican and decorin appearing in higher amounts. MATERIALS AND METHODS: Proteoglycans in laryngeal carcinoma samples were characterized immunohistochemically and using Western blotting; their expression was examined by RT-PCR. RESULTS: Aggrecan was totally removed in advanced cancer and its RT-PCR product was not identified. Both versican and decorin were overexpressed in cancer, versican much more than decorin. Decorin expression was higher than that of versican in the normal larynx; therefore, their disproportionate overexpression during cancer resulted in about equimolar expression. Both proteoglycans' expression correlated with their stage-related accumulation within the tissue. CONCLUSION: These data add to our previous findings and support the view that the levels of expression and the extent of accumulation and localization in the tumor stroma of structurally modified versican and decorin could be associated with the degree of aggressiveness of laryngeal carcinoma.

Expression of metalloproteinases and their tissue inhibitors in squamous cell laryngeal carcinoma.

The hallmark of cancer invasion is the degradation of extracellular matrix components. Matrix metalloproteinases are the major enzymes participating in this event and their activity is regulated extracellularly by their presence as proenzymes and the concomitant presence of the specific tissue inhibitors. The present study describes the immunohistochemical localization of gelatinases, matrix metalloproteinase (MMP)-2 and -9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 in human laryngeal carcinoma and their expression with respect to tumor classification and compared with the respective healthy subjects. MMP-2 was immunolocalized in the cytoplasm of the epithelial cells and in the loose connective tissue, whereas MMP-9 was also observed in basement membrane and chondrocytes. Both were also found in tumor cells, but staining was decreased with increasing stage of cancer. TIMP-1 was present exclusively in stroma and totally absent from tumor cells and it was overexpressed in normal cells surrounding the tumor. TIMP-2 was identified in the cytoplasm of epithelial cells, in stroma and sometimes in chondrocytes. In addition, it was present in tumor cells of only stage IV samples. The expression level of both gelatinases and TIMPs increased as the stage of cancer increased, suggesting the possible post-transcriptional removal of their mRNA. These observations, performed in a given head and neck site, suggest that the behavior of head and neck tumors seems to depend on the site and additional studies should be performed to obtain a general understanding of the disease and ascertain the role of the constituents examined.

The increased accumulation of structurally modified versican and decorin is related with the progression of laryngeal cancer.

Versican and decorin, two proteoglycans (PGs) with contradictory roles in the pathophysiology of cancer, comprise important stromal components in many tumor types and play a crucial role in the progression of cancer. In this study, we provide direct evidence for a significant and stage-related accumulation of versican and decorin in the tumor-associated stroma of laryngeal squamous cell carcinoma (LSCC) in comparison to normal larynx. Both PGs were found to be co-localized within the peritumorous stroma. In addition, the accumulated versican and decorin were markedly modified on both protein core and glycosaminoglycan (GAG) levels. Decorin, which was present under both glycanated and non-glycanated forms, perceptibly increased with the progression of LSCC, compared to the normal larynx. Tumor-associated glycanated decorin was found to contain significant amounts of dermatan sulfate (DS) sequences. Versican was also found to undergo stage-related structural modifications since a marked heterogeneity of protein cores was observed, being intense in late stage of laryngeal cancer. The increased accumulation of both versican and decorin was associated with a significant stage-related increase of the molar ratio of Delta di-mono4S to Delta di-mono6S up to approximately threefold in LSCC compared to the normal ones. The modified chemical structure of both PGs could be associated with the degree of aggressiveness of laryngeal squamous cell carcinomas.

Hyaluronidase and CD44 hyaluronan receptor expression in squamous cell laryngeal carcinoma.

Squamous cell laryngeal carcinoma undergoes significant structural-related modifications of the extracellular matrix components (ECM), the most characteristics being the presence of degraded collagen, aggrecan and hyaluronan. We examined the presence of hyaluronidase and of the cellular hyaluronan receptor CD44 during the various stages of cancer. ECM components were extracted by using PBS, 4 M GdnHCl and 4 M GdnHCl-0.1% Triton-X 100 sequentially and hyaluronidase and CD44 analyzed by zymography and immunochemistry techniques. Total RNA was also extracted and the mRNA of the various hyaluronidases and of CD44 was analyzed after amplification with RT-PCR. Hyaluronidase was detected as a double band of 45 and 55 kDa molecular mass, only in cancer samples. The analysis of mRNA indicated an aberrant expression of PH-20, the testicular-type hyaluronidase, at late stages of cancer and an overexpression of HYAL1 only at stage IV. In addition, CD44 was identified in two protein bands of 80 and 64 kDa in cancer samples. The analysis of mRNA showed that hyaluronan receptor was expressed in a stage-related order. Thus, it could be suggested that in laryngeal squamous cell carcinoma, cancer cells migrated and proliferated under the influence of small molecular mass hyaluronan, by expressing increased amounts of its receptor.

Diagnostic and classification value of metalloproteinases in squamous human laryngeal carcinoma.

Metalloproteinases (MMPs) are a class of enzymes largely involved in tumour progression and metastasis. At least twenty different enzymes are recognized that are also present under normal state of tissues. Their activity is regulated by their presence as proenzymes and by the concomitant presence of the respective tissue inhibitors (TIMPs). The present study describes the alterations of MMPs observed in human laryngeal carcinoma with respect to tumour classification and compares their activity in normal and cancerous tissues and biopsy specimens. Samples from five patients who underwent laryngectomy, from five biopsies and three from autopsies were used. The MMPs of normal and malignant human laryngeal cartilage and of biopsy specimens were identified immunochemically and by zymography using gelatin or casein as substrates. Healthy cartilage from autopsies was found to contain almost exclusively MMP-1, proMMP-2 and proMMP-9. Normal parts from laryngectomies contained, in addition, significant amounts of active MMP-2. The respective malignant parts contained both MMP-2 and -9 in increased amounts in their latent and active forms. Similar profile of MMPs was also identified in tissues surrounding affected cartilage. These alterations were found to be in good accordance with tumour stage and were also observed in biopsy samples. Thus, analysis of MMPs in biopsies can be used together with the clinicopathological parameters for the classification or early diagnosis of laryngeal tumours.

Alterations in the content and composition of glycosaminoglycans in human laryngeal carcinoma.

Glycosaminoglycans in normal and cancerous human laryngeal cartilage were isolated and characterized by means of enzyme susceptibility and high performance liquid chromatography. The known mammalian glycosaminoglycans were identified in all samples but their content and composition varied between normal and malignant samples. Chondroitin/dermatan sulphate was the major glycosaminoglycan in all cases, but its relative proportion was decreased in malignant samples. Its sulphation pattern showed that in normal samples it was sulphated mainly at the C6 position of galactosamine, whereas in malignant samples it was sulphated mainly at C4. Dermatan sulphate, expressed as a result of the different digestion of samples with chondroitinases, was present in very small amounts in normal samples (2.7% of total sulphated glycosaminoglycans) but increased in proportion up to 27.7% in malignant samples. The content of oversulphated chondroitin/dermatan was increased twofold in malignant samples. The content of heparan sulphate was increased almost fivefold in malignant samples as compared to normal ones. The content of hyaluronan was increased in malignant samples 3.5-fold, amounting to up to 11.4% of total glycosaminoglycans. These dramatic changes in the content and composition of glycosaminoglycans seemed to be characteristic of the tumour and independent of its status.

Keratan sulphate in cerebrum, cerebellum and brainstem of sheep brain.

Keratan sulphate was identified in sheep brain. We describe here the isolation and partial characterization of keratan sulphate from cerebrum, cerebellum and brainstem of young sheep brains. The galactosaminoglycan was isolated by using ion-exchange chromatography and gel filtration after exhaustive digestion with papain of the delipidated tissues, followed by alkaline borohydride degradation and chondroitinase ABC and heparinases I, II and III treatment. The material isolated by ion-exchange chromatography from each tissue was eluted as single but polydispersed peak from Sephadex G-75, with average molecular masses 8.4, 7.9 and 8.8 kDa for cerebrum, cerebellum and brainstem, respectively. Keratanase I and II totally degraded keratan sulphate from cerebrum and brainstem, but only partially that from cerebellum. The content of keratan sulphate was found to be about 215, 173 and 144 microg/g dry delipidated tissue for cerebrum, brainstem and cerebellum, respectively.

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate.

The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs)--chondroitin sulfate (CS) and dermatan sulfate (DS)--were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.

Determination and structural characterisation of dermatan sulfate in the presence of other galactosaminoglycans.

Chondroitin sulfate and dermatan sulfate are galactosaminoglycans that have similar size and charge density thus making difficult their separation and accurate determination from tissue preparations. A procedure was developed, which was based on the specific action of chondroitinase B, that allowed the determination of dermatan sulfate content in a mixture of chondroitin sulfate/dermatan sulfate, its molecular mass (Mr), and iduronic acid content and distribution throughout the chain. According to this procedure, the galactosaminoglycan sample was treated with chondroitinase B and its profile, upon gel chromatography on Sepharose CL-6B, was compared to that of the initial sample. The differences in uronic acid content of the fractions of the gel chromatographies were plotted and a secondary profile was constructed, which corresponded to the elution profile of intact dermatan sulfate in the sample. From this profile, the size distribution of dermatan sulfate was obtained and its Mr was calculated. In addition, the accurate content of dermatan sulfate in the sample was determined. The digest contained oligosaccharides of variable size that were separated on BioGel P-10. From the separated oligosaccharides the distribution of iduronic acid throughout the dermatan sulfate chains was determined. The procedure was applied to the determination and partial characterisation of dermatan sulfate from sheep nasal cartilage, in which it is reported for the first time that it contains a significant proportion of dermatan sulfate chains of low iduronic acid content.

Pancreatic carcinoma is characterized by elevated content of hyaluronan and chondroitin sulfate with altered disaccharide composition.

The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.

Distribution and changes of glycosaminoglycans in neoplasias of rectum.

The content, composition and physicochemical characteristics of glycosaminoglycans in human rectum and rectum carcinoma were investigated by chemical analyses, enzymic treatments, chromatographic and electrophoretic techniques. The overall glycosaminoglycan content was increased about 2 fold in neoplastic tissues compared to nonneoplastic tissues. The absolute amounts of chondroitin sulfate, hyaluronic acid and dermatan sulfate significantly increased but the amounts of heparan sulfate decreased in neoplastic tissues compared with nonneoplastic tissues. In addition an increased fraction of keratan sulfate and undersulfated chondroitin was identified in neoplastic tissues. HPLC analysis of chondroitinase AC and ABC digests showed a marked increase in delta di-6S and delta di-OS disaccharides in tumor chondroitin sulfate, revealing significant alterations on the sulfation pattern. The results indicate that specific glycosaminoglycan alterations occur in human rectum carcinoma and suggest that proteoglycan metabolism is also altered in this carcinoma.

Kinetic studies on activation of peptide bond formation by hyaluronic acid.

1. In this study, a cell-free system derived from Escherichia coli has been used in order to examine in detail the effect of hyaluronic acid on peptide bond formation with the aid of puromycin reaction. 2. This reaction is activated by hyaluronic acid. 3. The degree of activation of peptide bond formation depends on the molecular size of hyaluronic acid. 4. The kinetic analysis revealed that the hyaluronic acid acts as a mixed-type nonessential activator. 5. The presence of hyaluronic acid improves about 9-fold the activity status of ternary complex as it can be calculated by k3/k5 ratio.

Mechanism of action of sparsomycin in protein synthesis.

Before CI isomerizes to C*I, we detect a competitive phase of inhibition (Ki = k5/k4 = 0.05 microM) which eventually, by increasing the concentration of I, becomes linear mixed noncompetitive and involves C*I in place of CI. The equilibration of C and I according to reaction 2 is much slower than the equilibration between C and S in reaction 1 (time-dependent inhibition). The inactivation plots obey reaction 2 and allow us to estimate k6 as equal to 2.2 min-1. The isomerized C*I, free of excess I, can be studied as a mixture with complex C. From the kinetics of the regeneration of C from C*I, in the presence of puromycin, we can estimate k7 to be between 0.22 min-1 and 0.06 min-1. Although the isomerized C*I survives after adsorption on cellulose nitrate filter disks, it does not survive after gel chromatography on a Sepharose CL-4B column but is converted quantitatively to complex C containing D of unchanged reactivity. This result does not support the proposed [Flynn, G. A., & Ash, R. J., (1990) Biochem. Biophys. Res. Commun. 166, 673-680] chemical reaction between D and I toward new products. The isomerized C*I can be obtained not only from the already-made complex C but also de novo from D, R, and M. In the latter case, the reactions which lead to C are represented by the following hypothetical scheme: D + R + M in equilibrium with DRM or C (binding reaction). When C*I is formed de novo, this reaction is coupled to reaction 2 and the ultimate product is a mixture of C and C*I.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glycosaminoglycans on peptide bond formation in bacterial ribosomes.

1. A cell-free system derived from E. coli has been used in this study. The process of peptide bond formation was assessed with the aid of the puromycin reaction, which is catalyzed by peptidyltransferase. 2. This reaction is inhibited by heparin, in contrast, this reaction is activated by hyaluronic acid. 3. The presence of heparin decreases the percentage of formed initiation complex (complex C), but hyaluronic acid, chondroitin sulphate and keratan sulphate have no effect on the formation of complex C. 4. From other types of glycosaminoglycans, only hyaluronic acid increases the stability of active complex C.

Kinetics of inhibition of peptide bond formation on bacterial ribosomes.

A cell-free system derived from Escherichia coli has been used in order to study the kinetics of inhibition of peptide bond formation with the aid of the puromycin reaction in solution. A similar study has been carried out earlier on a solid support matrix with the same inhibitors. We find that the overall pattern of the kinetics of inhibition is the same in the two systems. At low concentrations of inhibitor there is a competitive phase of inhibition, whereas at higher concentrations of inhibitor the type of inhibition becomes mixed noncompetitive. The values of Ki of the competitive phase in the system in solution are: 5.8 microM (amicetin), 0.2 microM (blasticidin S), 0.5 microM (chloramphenicol), and 0.5 microM (tevenel). The inhibitors amicetin, blasticidin S, and tevenel interact with the ribosome in a reaction which is slower than that of the substrate puromycin, showing clear-cut characteristics of slow-onset inhibition in both systems. Chloramphenicol, on the other hand does not easily show such a delay in solution. It interacts with the ribosome relatively faster than the other three antibiotics. Despite this, chloramphenicol too shows characteristics of time-dependent inhibition.

Glycosaminoglycans in the vitreous body of patients with retinal detachment.

The variation and changes of glycosaminoglycans in human vitreous body from patients with retinal detachment were studied. The isolated glycosaminoglycans from normal vitreous were identified as hyaluronate, which is the main component (92%) and chondroitin sulphate (8%). In contrast, in pathologic samples up to 18% of total glycosaminoglycans were identified as chondroitin.sulphate. In addition, in pathologic vitreous two fractions of glycosaminoglycans about 10% were identified as undersulphated chondroitin and heparan sulphate. The hydrodynamic size of hyaluronate differs between normal and pathologic samples. In samples from the patients with detached retinas the hyaluronic acid was of small hydrodynamic size.

Recovery of active ribosomal complexes from cellulose nitrate membranes.

The ternary Ac-[3H]Phe-tRNA-poly(U)-ribosome complex (complex C) [D. L. Kalpaxis, D.A. Theocharis, and C. Coutsogeorgopoulos (1986) Eur. J. Biochem. 154, 267-271] was used in model experiments aiming at the purification of this complex via adsorption on cellulose nitrate membranes and then desorbing the complex back into solution. The desorption was carried out at pH 7.2 in the presence of the nonionic detergent Zwittergent (ZW). The activity status of complex C was assessed with the aid of the puromycin reaction which characterizes ribosomal peptidyltransferase as part of complex C. The optimal conditions for desorbing complex C were 5 degrees C and a buffered solution containing 0.1% ZW. The kinetic constants of peptidyltransferase in the adsorbed state were kcat = 2.0 min-1, Ks = 0.4 mM. In the desorbed state, in solution, kcat = 3.4 min-1 and Ks = 0.3 mM. The method promises to be suitable for the rapid purification of ribosomal complexes containing mRNA and aminoacyl-tRNA.

Aminoacylaminonucleoside inhibitors of protein synthesis. A new approach for evaluating their potency.

In a model system derived from Escherichia coli, Ac[3H]Phe-puromycin is produced in a pseudo-first-order reaction between the preformed Ac[3H]Phe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin [Kalpaxis et al. Eur. J. Biochem. 154, 267, 1986]. Amicetin and gougerotin inhibit this reaction to various degrees depending on whether or not complex C is allowed to interact with the inhibitor (I) prior to the addition of puromycin (S). The kinetic analysis shows a phase where competitive inhibition can be observed provided that S and I are added simultaneously. After preincubating C with I, the inhibition becomes of the mixed non-competitive type. The Ki (the dissociation constant of the CI complex), calculated from the competitive plot, is 20.0 microM for amicetin and 15.0 microM for gougerotin. This inhibition constant (Ki) cannot distinguish amicetin from gougerotin. Its acceptance as a criterion of potency does not explain why after preincubation amicetin proves to be a stronger inhibitor than gougerotin. The determination of the apparent catalytic rate constants of peptidyltransferase at various inhibitor concentrations and the appropriate replotting of these rate constants distinguish amicetin from gougerotin. A new approach for evaluating the potency of these inhibitors is proposed. The familiar Ki is supplemented with an apparent kinetic constant obtained from a replot in which the intercepts of the double-reciprocal plots (1/kobs versus 1/[S]) are plotted versus the inhibitor concentration.

Kinetic studies on ribosomal peptidyltransferase. The behaviour of the inhibitor blasticidin S.

In a cell-free system derived from Escherichia coli, the reaction between Ac[3H]Phe-tRNA and puromycin (S) is inhibited by blasticidin S (I). In this reaction Ac[3H]Phe-tRNA is part of the Ac[3H]Phe-tRNA--poly(U)--ribosome complex (C). After preincubating the complex C with I and then adding S, the degree of inhibition is greater than that observed when C reacts with a mixture of S and I. Without preincubation, the inhibition is competitive giving a Ki of 2 X 10(-7) M. After preincubation the inhibition becomes of the mixed non-competitive type. A first-order kinetic analysis of the reaction between C and excess S, in the presence or in the absence of I, with or without preincubation, suggests that I acts as a modifier decreasing the catalytic rate constant of ribosomal peptidyltransferase (the putative enzyme that catalyzes the reaction between C and S). The effectiveness of I cannot be expressed by an equilibrium constant such as the above-mentioned Ki. A model is proposed which explains the results obtained. In this model, in the presence of I, C is converted to a modified species C, which is still able to react with S but with a lower catalytic rate constant. This is a novel concept, in which the ribosome can be subjected to modulation of its activity by small ligands. It can be useful in studies on translational control of protein synthesis.

Cartilage keratan sulphate: changes in chain length with ageing.

Keratan sulphate from sheep nasal cartilage of five different ages was isolated by a combination of methods. The mean length of the chains progressively increased with ageing, as assessed by the molar ratio of glucosamine to galactosamine or galactosaminitol. The mean length ranges from eight monosaccharides for the younger to seventeen monosaccharides for the older animals. The results suggest that the increase in keratan sulphate content of cartilage may be due to the increase in the length and not in the number of chains.