Characterisation of bioenergetic pathways and related regulators by multiple assays in human tumour cells.

BACKGROUND: Alterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells. METHODS: To define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-(14)C]-glucose and [1-(14)C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated (13)C atoms after [U-(13)C]-glucose/[2-(13)C]-acetate labelling. Based on [1-(14)C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated. RESULTS: Both [U-(13)C]-glucose and [1-(14)C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain (13)C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, ß-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-(13)C]-acetate-representing a novel functional test in malignant cells-confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1ß, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency. CONCLUSIONS: The applied methods of energy substrate utilisation and other measurements represent simple assay system using (13)C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.

Leptin-based glycopeptide induces weight loss and simultaneously restores fertility in animal models.

AIM: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction. METHODS: The glycopeptide containing two additional non-proteinogenic amino acids was synthesized by standard solid-phase methods. Normal mice were fed with peanuts until their blood laboratory data and liver histology showed typical signs of obesity but not diabetes. The mice were treated with the peptidomimetic at 0.02, 0.1 or 0.5 mg/kg/day intraperitoneally side-by-side with 0.1 mg/kg/day leptin for 11 days. After termination of the assay, the blood cholesterol and glucose amounts were measured, the liver fat content was visualized and quantified and the remaining mice returned to normal diet and were allowed to mate. In parallel experiments normal rats were treated intranasally with the glycopeptide at 0.1 mg/kg/day for 10 days. RESULTS: The 12-residue glycosylated leptin-based peptidomimetic E1/6-amino-hexanoic acid (Aca) was designed to target a principal leptin/ObR-binding interface. E1/Aca induced leptin effects in ObR-positive cell lines at picomolar concentrations and readily crossed the blood-brain barrier (BBB) following intraperitoneal administration. The peptide initiated typical leptin-dependent signal transduction pathways both in the presence and absence of leptin protein. The peptide also reduced weight gain in mice fed with high-fat peanut diet in a dose-dependent manner. Obese mice receiving peptide E1/Aca at a 0.5 mg/kg/day dose lost weight, corresponding to a net 6.5% total body weight loss, while similar mice treated with leptin protein did not. Upon cessation of the weight loss treatment, several obesity-related pathologies (i.e. abnormal metabolic profile and liver histology as well as infertility) normalized in peptide-, but not leptin-treated, mice. Peptide E1/Aca added intranasally to growing normal rats decelerated normal weight gain corresponding to a net 6.8% net total body weight loss with statistical significance. CONCLUSIONS: No resistance induction to peptide E1/Aca or toxicity in either obese or healthy rodents was observed, indicating the potential for widespread utility of the peptidomimetic in the treatment of leptin-deficiency disorders. We provide additional proof for the hypothesis that difficulties in current leptin therapies reside at the BBB penetration stage, and we document that by either glycosylation or intranasal peptide administration we can overcome this limitation.

KIT Mutation Incidence and Pattern of Melanoma in Central Europe.

Data on the KIT mutation rate in melanoma in the central European region is missing. Accordingly, in a cohort of 79 BRAF/NRAS double wild type cutaneous melanoma and 17 mucosal melanoma KIT mutation was assessed by Sanger sequencing of exons 9,11,13,17 and 18. In this cutaneous melanoma cohort KIT mutation frequency was found to be 34/79 (43.04%) with a significantly higher rate in acrolentiginous melanoma (ALM) as compared to UV-induced common variants (20/34, 58.8% versus 14/45, 31.1%, p = 0.014). In the double wild type mucosal melanoma cohort the KIT mutation frequency was found to be comparable (41.2%). The actual frequency of KIT mutation in the original 227 patient cutaneous melanoma cohort was 34/227, 14.9%. Exon 11 was the most frequent mutation site (44.7%) followed by exon 9 (21.1%) equally characterizing UV-induced common histotypes and ALM tumors. In mucosal melanoma exon 9 was the most frequently involved exon followed by exon 13 and 17. KIT mutation hotspots were identified in exon 9 (c482/491/492), in exon 11 (c559,c572, c570), in exon 13 (c642), in exon 17 (c822) and in exon 18 (c853). The relatively high KIT mutation rate in cutaneous melanoma in this central-European cohort justifies regular testing of this molecular target in this entity, not only in mucosal variants.

Biochemistry and enzyme induction in MC-29 virus-induced transplantable avian hepatoma.

For the biochemical characterization of a new transplantable hepatoma derived from the MC-29 virus-induced liver tumor, the macromolecular content and the inducibility of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and aryl hydrocarbon hydroxylase were compared in chicken liver and in this hepatoma. The alteration of the nucleocytoplasmic ratio was deduced from measurements of DNA, RNA, protein, and phospholipid contents of the whole cell homogenate and cell fractions. The increased nuclear and decreased cytoplasmic content of macromolecules suggests a dominancy of the nuclei in the tumor cells. Glucose-6-phosphatase and aryl hydrocarbon hydroxylase activities were lower by 60 and 80%, respectively, in the highly proliferating hepatoma than in the liver. In contrast, glucose-6-phosphate dehydrogenase activity increased in the hepatoma. However, enzyme inducers, such as methylcholanthrene, hydrocortisone, and insulin, were able to enhance the activity of these enzymes in the liver but had no stimulating effect on the hepatoma.

Expression of syndecan-1 in human B cell chronic lymphocytic leukaemia.

Syndecan-1 is considered an important transmembrane proteoglycan in cell-microenvironment interactions, but its exact function in normal or in transformed B cells is still unknown. In this study, RNA was isolated from peripheral cells of chronic lymphocytic leukaemia (B-CLL) and 'normal', non-leukaemic patients, as controls. Reverse PCR showed no or very low syndecan-1 mRNA expression in controls, while in 11/13 B-CLL the circulating leukaemic cells expressed syndecan-1. Similar results were obtained for interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6). Furthermore, syndecan-1 protein was detected in the majority of circulating B-CLL cells by flow cytometry and immunocytochemistry using anti-syndecan-1 MAb. Control cells were practically negative. Further study is required to understand the biological significance of syndecan-1 on B-CLL cells.

Two human melanoma xenografts with different metastatic capacity and glycosaminoglycan pattern.

Two human melanoma xenografts were compared with respect to their in vivo growth and metastatic potentials as well as glycosaminoglycan patterns. The less differentiated HT 168 tumor showed faster growth at primary sites and a more pronounced capacity for metastasis into the liver. Although chondroitin sulfate was the dominant glycosaminoglycan subtype in both tumors, the more invasive xenograft had a higher heparan sulfate/chondroitin sulfate (HS/CS) ratio. We suggest that tumor progression is influenced by this ratio in this human melanoma system.

Expression of decorin, transforming growth factor-beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis.

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

Syndecan-1 - A new piece in B-cell puzzle.

Syndecans are transmembrane proteoglycans, with core proteins mainly decorated with heparan sulfate chains. Syndecan-1 is expressed in a tissue-, cell-and differentiation-specific manner. Its extra-cellular domain can bind via HS chains to matrix elements, to growth factors (especially "heparin-binding" proteins) and to certain biological agents. The ectodomain released by proteolysis can also be functionally active. The cytoplasmic domain can take part in signaling processes as well as in modifying cell shape. In hematopoietic cells syndecan-1 is expressed in normal pre-B-cells and plasma cells, as well as in plasmocytoid and lymphoplasmocytoid malignancies. According to our study syndecan-1 is expressed in B-CLL cells both in tissue environment and in circulation.

Expression of a decorin-like moleculein human melanoma.

Decorin, a member of the family of small leucin-rich proteoglycans, has originally been described as a secreted proteoglycan component of the connective tissues, and has been implicated in the negative regulation of cell proliferation directly or via interactions with TGF-beta. It was reported to be generally absent from tumor cells. Here we show that human melanoma cell lines express a decorin-like molecule. We detected decorin mRNA by RT-PCR in 7 out 7 human melanoma lines characterized by various metastatic potential. Using polyclonal antiserum against the core protein of decorin, the typical 80-120 kD glycanated form as well as a high molecular weight aberrant version (200-210 kD) of decorin were demonstrated by Western blot technique in the culture supernatants as well as in lysates of human melanoma cells. Finally, decorin epitope was also demonstrated immunohistochemically in human melanoma xenografts, as well as in tumor cells of surgically resected melanomas but not in melanocytes of nevi. The expression of this aberrant decorin did not inhibit the in vitroor in vivogrowth of human melanoma cells, and it was independent of their metastatic potential. Human melanoma cell lines expressing aberrant decorin retained sensitivity to the antiproliferative and gelatinase-stimulatory effects of exogenous TGF-beta.

Antisense tumor therapy (a dream under construction).

Cancer, as a genetic disease, is a logical target for gene-oriented therapy--either by replacing the missing/nonfunctioning gene or by depressing the activity of an unwanted gene. The latter is really the inhibition of gene expression using oligonucleotide-based or "antisense" treatment. There are several strategies to achieve this: anti-gene or anti-code with triplex formation; ribozyme with endogenous catalytic RNase activity; antisense with oligonucleotides through steric inhibition or RNaseH activation; and sense strategy to inhibit or trap proteins by nucleic acids. There are two essential partners of the approach: targeted sequence in the unwanted gene/molecule and the complementary antisense oligo (-ribo- or -deoxyribonucleotide). The antisense sequences require chemical modifications (mostly on the phosphodiester backbone, less in the sugar or in the bases) to avoid nucleases, to form complexes for better delivery (in the organism and also in the cell). The activity of the unwanted target should be non-randomly associated with cancer (e.g. abl/ber). Both aspects of antisense treatment require further improvements to get longer lasting and real sequence-specific antitumor effect which could be competitive with the available therapeutic modalities.

Role of the modified (glycosaminoglycan producing) perisinusoidal fibroblasts in the CCl4-induced fibrosis of the rat liver.

Development and regression of liver fibrosis and cirrhosis induced by CCl4 in male F-344 rats were strictly followed during and after an 8-week treatment. The relative amount of collagen was measured by morphometry and the number of glycosaminoglycan (GAG) containing fat storing cells was counted at each time point. The expression of proteoglycan genes (decorin, versican and BPG-5 HSPG) was studied in parallel with the development of cirrhosis. Collagen content of the liver as well as the number of GAG-containing mesenchymal (fat storing) cells increased in parallel until two weeks after the cessation of CCl4 treatment. Later, both the collagen content and the number of GAG-containing cells decreased in parallel and significantly. Proteoglycan gene expression in the nonparenchymal fraction of liver cells indicated an active proteoglycan synthesis in the course of the development of cirrhosis. It is concluded that modified Ito (fat storing) cells synthesize proteoglycans and play an important role in the formation of connective tissue fibers in liver fibrosis.

Epitopes of cartilage core proteins and GAG pattern in human non-Hodgkin lymphoma xenografts.

Glycosaminoglycan and core protein components of proteoglycans (PGs) have been studied in three human non-Hodgkin lymphoma xenografts of B cell origin. Lymphomas showed similar GAG content, but different composition of GAG subtypes. This variability was accompanied by an individual capacity to adhere to extracellular matrix elements. The core proteins identified by monoclonal antibodies raised against human cartilage chondroitin sulfate PG were also distinctly expressed and released. These proteins shared by different cell types may have biological significance.

Detection of high-risk HPV (16, 18, 33) in situ cancer of the cervix by PCR technique.

OBJECTIVE: The purpose of this study was to collect data about the incidence of high-risk HPV (16, 18, 33) types in in situ cervical cancers, and to evaluate the reliability of the morphological signs of HPV infection by comparing the presence of these signs to the PCR-proven HPV virus infection. METHODS: Fifty patients who underwent conisation at the Department of Obstetrics and Gynecology of Semmelweis University, Budapest, Hungary because of in situ cervical cancer were examined retrospectively for the presence of HPV infection by the PCR technique. The direct and indirect morphological signs of HPV infection identified in the histological and cytological samples were compared to the actual results of virus DNA amplification by PCR in the identical histological sections. The evaluation of the cytological smears and the histological sections was accomplished independently by two different pathologists. RESULTS: E6 open reading frame of HPV 16, 18 or 33 was detected by PCR in 56% (28 cases) of the histological sections of the 50 examined patients with in situ cancer. In 92% (26 patients) of the 28 HPV positive patients one HPV type was detected, while in one of the remaining two cases two HPV types (16/33), or all three types could be detected. The direct morphological signs for HPV infection proved to be 75% sensitive and 50% specific when compared to the results of PCR. Their predictive value for HPV infection was 65%. For the indirect HPV signs the sensitivity was 64% and specificity 31%. The predictive value, prognosticating the presence of HPV 16, 18, 33 infection was 54% in the same sections. Using significance analysis no significant relationship (p = 0.7728) could be detected between the positivity of indirect signs and the presence of HPV 16, 18, 33 infection, while in case of direct signs the relationship was almost significant (p = 0.0675). The joint testing of the direct and indirect signs did not improve the results (p = 0.1338). During the review of the cytological smears the specificity of the cytology in predicting true HPV infections was found to be 68% and sensitivity was 20%. The predictive value was only 50%. A significance analysis was not accomplished by this diagnostic method because of the missing data (see text). CONCLUSION: The method of Nawa et al. seems to be a reliable approach for the detection of HPV DNA in paraffin-embedded material. The three main types of HPV (16, 18, 33) are probably represented in lower percentages in CIN III in Hungary, but a larger survey is needed to obtain reliable data. The direct and indirect morphological signs of HPV infection failed to show a significant relationship with the PCR proven presence of HPV 16, 18, 33.

Further studies on the biochemical characterization of the MC-29 virus derived transplantable hepatoma (VTH). I. Binding of 3H-cortisol to cytoplasmic receptor and DNA.

The molecular mechanisms underlying the failure of steroids to stimulate glucose-6-Pase and arylhydrocarbonhydroxylase activities in the MC-29-virus-derived transplantable hepatoma (VTH) were investigated. Following cellular uptake of 3H-Cortisol, its subcellular distribution, binding to a specific cytoplasmic receptor and the interaction between steroid-bound receptor and DNA were compared in VTH and in normal chicken liver. No appreciable difference was observed either in 3H-Cortisol uptake or in binding to cytoplasmic receptors. However, compared with normal liver, only half as much hepatoma steroid receptor was able to interact with DNA at the protein/DNA ratio of 60. This reduced DNA binding of VTH 3H-Cortisol-receptor was irrespective of the source of DNA (VTH or liver). It is concluded that one of the causes for abnormal gene regulation in VTH may lie at the level of DNA-protein interaction.

[Fibrogenesis in the liver--fibrosis, cirrhosis]. / Máj fibrogenesis--fibrosis, cirrhosis.

Liver fibrogenesis is a delicately balanced process, in which mainly the non-parenchymal liver cells are implicated. Either increased synthesis or decreased catabolism of matrix proteins results in the enhancement of ECM. As further consequence the formation of continuous diffusion and filtration barriers along the Disse space will hinder the bidirectional exchange of macromolecules. Normal structure of ECM is necessary to the normal function of hepatocytes. The quantitative and qualitative changes of ECM observed in liver fibrosis are able to inhibit the liver specific functions of hepatocytes. The mechanisms involved in this effect are not yet clearly understood. In animal experiments liver cirrhosis is reversible and theoretically the chance is open for humans, as well if we will be able to influence the specific steps of fibrogenesis.

[Pathology--on a molecular level (determination of nucleic acids from fixed embedded samples)]. / Pathologia--molekuláris szinten (Nukleinsavak kimutatása fixált, beágyazott mintákból).

Molecular biological techniques may open new avenues to pathological archives. Fixed and paraffin embedded blocks are a suitable source of nucleic acids, especially of DNA, for retrospective analysis. The quality of DNA depends mainly on the fixation procedure. High molecular weight DNA allows Southern hybridization, but fragmented DNA also became a target with the appearance of polymerase chain reaction (PCR). PCR has rising applicability and enables amplification of required sequence even from one section. Using these techniques on archieved materials a wide variety of informations, e.g. correlation between morphology, phenotypic expression and gene alteration, will be available.

[Proteoglycans (their structure, function and role in liver diseases)]. / Proteoglikánok (struktúra, funkció, szerepük májbetegségekben).

Proteoglycans are macromolecules containing a core protein to which glycosaminoglycan chains are covalently attached. The family contains several members with different structures and various functions. Some of them are elements of the extracellular matrix, while others are located to the cell surface playing important role in cell-cell and cell-extracellular matrix interactions. Present paper discusses the possible consequences of the alterations of proteoglycans observed in liver cirrhosis and liver tumors. It has to be emphasized however, that they are also involved in the pathomechanism of arteriosclerosis, Alzheimer-disease, immune diseases, arthritis, tumor progression and metastasis formation.

Experimental and human liver fibrogenesis.

In this work, we provide an overview of our results obtained by studying the role of transforming growth factor beta1 and proteoglycans in liver fibrogenesis. It has been found that transforming growth factor beta1 is one of the most important stimulators of extracellular matrix synthesis in the liver. In chronic liver injury, desmin-positive non-parenchymal liver cells expressed transforming growth factor beta1. The extracellular localization of the growth factor correlated well with types I, III and IV procollagen-alpha, which were detected in the fibrous septa of chronically injured livers. A similar distribution pattern was observed in human specimens. To identify the role of transforming growth factor beta1 in liver extracellular matrix protein synthesis, transforming growth factor beta1-positive transgenic mice were generated. Animals expressing the growth factor in their liver showed spontaneous liver fibrosis. Proteoglycans also participate in fibrogenesis. The majority of liver-specific heparan sulfate proteoglycans, such as syndecan-1 and fibroglycan, are produced by hepatocytes. The extracellular matrix proteoglycans decorin and perlecan are synthesized by non-parenchymal liver cells. The amount of the latter is very low in normal liver, but increases dramatically in liver fibrosis. The effect of regulatory factors on liver proteoglycans seems to be cell type-specific. In contrast to previous observations, elevated amounts of decorin did not inhibit the action of transforming growth factor beta1 in the liver.

Human and experimental hepatocarcinogenesis.

Human liver cancer is increasing worldwide, including in Hungary. The detection of liver tumors in premalignant or early malignant states is essential for successful treatment. MC-29 virus-induced chicken hepatoma and rodent, fish and monkey models for chemical hepatocarcinogenesis were studied and compared to humans. Changes in phenotypic enzyme alterations and in the expression of certain oncogens and growth factors characterize the experimentally induced hepatomas, and might also be characteristic of human premalignant and malignant focal liver lesions. Fish hepatocarcinogenesis is useful for studying compounds in environmental pollution. Increased expression of transforming growth factor á can be observed both in experimental and human liver tumors. Increased tumor incidence was detected in transgene mice containing both transforming growth factor alpha and c-myc genes. Animal models of hepatocarcinogenesis help to understand the development of liver tumors. Methods applied in studies using those models are useful in the study of premalignant and malignant human liver lesions.

Microarray profiling reveals that placental transcriptomes of early-onset HELLP syndrome and preeclampsia are similar.

BACKGROUND: The involvement of the placenta in the pathogenesis of preeclampsia and HELLP syndrome is well established, and placental lesions are also similar in these two syndromes. Here we aimed to examine the placental transcriptome and to identify candidate biomarkers in early-onset preeclampsia and HELLP syndrome. METHODS: Placental specimens were obtained at C-sections from women with early-onset preeclampsia and HELLP syndrome, and from controls who delivered preterm or at term. After histopathological examination, fresh-frozen placental specimens were used for microarray profiling and validation by qRT-PCR. Differential expression was analysed using log-linear models while adjusting for gestational age. Gene ontology and pathway analyses were used to interpret gene expression changes. Tissue microarrays were constructed from paraffin-embedded placental specimens and immunostained. RESULTS: Placental gene expression was gestational age-dependent among preterm and term controls. Out of the 350 differentially expressed genes in preeclampsia and 554 genes in HELLP syndrome, 224 genes (including LEP, CGB, LHB, INHA, SIGLEC6, PAPPA2, TREM1, and FLT1) changed in the same direction (elevated or reduced) in both syndromes. Many of these encode proteins that have been implicated as biomarkers for preeclampsia. Enrichment analyses revealed similar biological processes, cellular compartments and biological pathways enriched in early-onset preeclampsia and HELLP syndrome; however, some processes and pathways (e.g., cytokine-cytokine receptor interaction) were over-represented only in HELLP syndrome. CONCLUSION: High-throughput transcriptional and tissue microarray expression profiling revealed that placental transcriptomes of early-onset preeclampsia and HELLP syndrome largely overlap, underlying a potential common cause and pathophysiologic processes in these syndromes. However, gene expression changes may also suggest a more severe placental pathology and pronounced inflammatory response in HELLP syndrome than in preeclampsia.