Anna Lipińska

Differences in glycosylation of intracellular proteins between benign and malignant thyroid neoplasms

Differences in glycosylation of nuclear and cytosolic proteins isolated from benign and malignant human thyroid neoplasms were analyzed by lectin blotting and enzyme linked lectino-solid-phase assay using Erythrina cristagalli and Ricinus communis agglutinins. The results reported in this study have not shown any significant differences in lectin binding by nuclear proteins of benign and malignant tumors, however, quantitative and qualitative differences were observed in the patterns of cytosolic glycoproteins. In the majority of carcinomas samples lectin binding to cytosolic proteins was definitely weaker in comparison with adenomas and non-neoplastic specimens, which suggested alterations in glycosylation of cytosolic proteins in malignant tumors.

Expression, Localization, and Phosphorylation of Akt1 in Benign and Malignant Thyroid Lesions

The serine/threonine protein kinase Akt is a key molecule in the phosphatidyl inositol 3-kinase pathway that is often overactivated in human cancers. Three Akt isoforms (Akt1, Akt2, Akt3) have been identified in human cells and they show different distribution and have non-redundant functions. The aim of this study was to determine whether the expression, phosphorylation, and localization of Akt1 isoform in human thyroid malignant lesions are different from those in benign lesions. Nuclear and cytoplasmic fractions were isolated from tissue samples and Western blot method was used to detect Akt1 presence in both cellular fractions. Akt1 expression was also assessed by ELISA method. To estimate Akt1 phosphorylation, kinase was immunoprecipitated from cell lysates and tested with anti-phospho-Akt antibodies. The Akt1 expression in majority of thyroid cancer samples was significantly higher than in benign lesions (p < 0.05). Akt1 both in differentiated cancers (follicular and papillary) and benign lesions was localized mainly in cytoplasmic fraction. In two of three anaplastic cancer samples Akt1 was predominantly localized in nucleus. The ratio of phosphorylated Akt1 to total Akt1 was lower in cancers than in non-neoplastic lesions and adenomas. Thus, although Akt1 seems to be overexpressed in thyroid neoplasms, its high phosphorylation is not characteristic for thyroid cancers.

The activity of chromatin-bound protease extracted selectively with histone h2b from calf thymus and rat liver

Abstract 1. 1. Chromatin-bound protease of calf thymus and rat liver may be extracted selectively with histone H2B. 2. 2. Proteolytic activity seems to be tightly bound with histone H2B in ionic strength of 0.4 and 0.7 M NaCl. 3. 3. The activity of protease coextracted with histone H2B from calf thymus was twice higher than the same one from rat liver.

A comparative study on the sensitivity of Cyprinus carpio muscle and liver FBPase toward AMP and calcium

The activity of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) isozymes is influenced by AMP, Ca2+ and by reversible interactions with subcellular structures. In contrast to mammalian and avian isozymes, the kinetic properties of FBPases from ectothermal vertebrates are not fully described. To get some insight into mechanism of glycogen resynthesis in ectothermal vertebrates we examined the features of FBPases isolated from Cyprinus carpio skeletal muscle and liver. To investigate the evolutionary origin of the sensitivity of FBPase to effectors, we performed a phylogenetic analysis of known animal amino acids sequences of the enzyme. Based on our findings, we hypothesize that the high, mammalian-like, sensitivity of FBPase to Ca2+ is not essential for controlling the stability of glyconeogenic complex in striated muscles, instead it ensures the precise regulation of mitochondrial metabolism during prolonged Ca2+ elevation in contracting muscle fibers. Comparison of the kinetic properties of vertebrate and insect FBPases suggests that the high sensitivity of muscle isozyme to inhibitors has arisen as an adaptation enabling coordination of energy metabolism in warm-blooded animals.

Nuclear glycoproteins of hamster liver and Kirkman-Robbins hepatoma recognized by concanavalin A.

1. Glycoproteins recognized by Concanavalin A (ConA) have been identified in nuclei and nuclear fractions differing in sensitivity to micrococcal nuclease digestion from hamster liver and Kirkman-Robbins hepatoma. 2. The major ConA binding proteins from hamster liver and Kirkman-Robbins hepatoma nuclei have molecular weights about 27,000 and 57,000, and 38,000 and 49,000, respectively. 3. A distinct distribution of glycoproteins between fractions differing in sensitivity to nuclease digestion has not been observed.

Diversity of non-histone protein fraction NHCP2 from hamster Kirkman-Robbins hepatoma and liver

Non-histone protein fraction NHCP2 eluted from hydroxyapatite with 100mM phosphate buffer (pH6.8) of undigested, nuclease-sensitive and nuclease-resistant nuclei of hamster Kirkman-Robbins hepatoma and liver was studied by two-dimensional gel electrophoresis and microcomplement fixation test in the presence of antibodies elicited against NHCP2 of examined tissues. The NHCP2 of undigested nuclei as well as from two chromatin fractions with different susceptibility to nuclease of both tissues, besides many common components, showed some differences in their non-histone patterns especially within molecular weights of 17 000–24 000, 36 000–44 000 and 60 000–90 000. Immunological analysis confirmed the high specificity of hepatoma non-histone components of the NHCP2 fraction. However, these components appeared not to be exclusively localized either in nuclease-sensitive or nuclease-resistant part of chromatin of neoplastic tissue.

Molecular and functional diversity of non-histone protein fraction NHCP1 from hamster Kirkman-Robbins hepatoma and liver

Non-histone protein fraction NHCP1 of micrococcal nuclease-sensitive and nuclease-resistant chromatin from Kirkman-Robbins hepatoma and hamster liver was studied by two-dimensional electrophoresis followed by Coomassie and silver staining and by microcomplement fixation technique in the presence of antibodies elicited against NHCP1 of both tissues. Apart from many common spots several tissue specific components associated with either nuclease-sensitive or nuclease-resistant chromatin were found. The presence of tissue specific components among NHCP1 from hepatoma and liver was confirmed by immunological analysis. It was stated that these components are exclusively localized in nuclease-resistant part of chromatin from neoplastic and normal tissues thus suggesting their structural function.

Expression of hypoxia inducible factor 1α and 2α and its association with vitamin C level in thyroid lesions

BackgroundCells adapt to hypoxia by transcriptional induction of genes that participate in regulation of angiogenesis, glucose metabolism and cell proliferation. The primary factors mediating cell response to low oxygen tension are hypoxia inducible factors (HIFs), oxygen-dependent transcription activators. The stability and activity of the α subunits of HIFs are controlled by hydroxylation reactions that require ascorbate as a cofactor. Therefore, deficiency of intracellular vitamin C could contribute to HIFs overactivation. In this study, we investigated whether vitamin C content of human thyroid lesions is associated with HIF-1α and HIF-2α protein levels.MethodsExpression of HIF-1α and HIF-2α as well as vitamin C content was analyzed in thyroid lesions and cultured thyroid carcinoma cell lines (FTC-133 and 8305c) treated with hypoxia-mimetic agent (cobalt chloride) and ascorbic acid. The expression of HIFs and hypoxia–induced glucose transporters were determined by Western blots while quantitative real-time PCR (qRT-PCR) was performed to detect HIFs mRNA levels. Ascorbate and dehydroascorbate levels were measured by HPLC method.ResultsWe found an inverse correlation between vitamin C level and HIF-1α but not HIF-2α expression in thyroid lesions. These results agree with our in vitro study showing that vitamin C induced a dose - dependent decrease of HIF-1α but not HIF-2α protein level in thyroid cancer cells FTC-133 and 8305C. The decreased HIF-1α expression was correlated with reduced expression of hypoxia-related glucose transporter 1 (GLUT1) in thyroid cancer cells.ConclusionThe results demonstrate that HIF-1α activation is associated with vitamin C content in thyroid lesions. Our study suggests that high tumor tissue ascorbate level could limit the expression of HIF-1α and its targets in thyroid lesions.

Effect of Glucose on GLUT1-Dependent Intracellular Ascorbate Accumulation and Viability of Thyroid Cancer Cells

Enhanced glucose requirement of cancer cells is associated with an increased glucose transport across plasma membrane that is mediated by a family of facilitated glucose transporter proteins, named GLUTs. GLUT1 is the main transporter in thyroid cancer cells. Glucose is the principal physiological substrate of GLUT1; however, it is also capable of transporting of oxidized form of vitamin C [i.e., dehydroascorbic acid (DHAA) which inside the cells is reduced to ascorbic acid (AA)]. The objective of this study was to determine the effect of normo-, hypo-, and hyperglycemia conditions on GLUT1-dependent intracellular ascorbate accumulation and viability of thyroid cancer cells. GLUT1 seems to be the main DHAA transporter in thyroid cancer cells because its knockdown by RNAi reduced DHAA accumulation by more than 80%. The results showed that in thyroid cancer cells high glucose inhibits both transport of AA and DHAA. Inhibition of vitamin C transport by glucose had a cytotoxic effect on the cells. However, stabilization of vitamin C in one of 2 forms (i.e., AA or DHAA) abolished this effect. These results suggest that cytotoxic effect is rather associated with extracellular accumulation of vitamin C and changes of its oxidation state than with intracellular level of ascorbate.

Age-related patterns of lectin-binding nuclear glycoproteins of hamster liver

We studied the age-related patterns of lectin-binding liver nuclear glycoproteins from hamsters between 5 and 35 weeks of age. The examinations of the carbohydrate structures of liver nuclear glycoproteins in relation to the age of hamsters were carried out after electrophoresis and blotting by a very sensitive immunological detection system with highly specific digoxigenin-labelled lectins. The results reported in the present study do not show any significant changes in the patterns of nuclear glycoproteins with regards to the age of hamsters except the decrease in affinity of wheat germ agglutinin (WGA) to 63 kDa glycoprotein bearing single O-linked N-acetylglucosamine residues.