Irma Colombo

The primary structure of UK 114 tumor antigen

UK114 is a tumor antigen expressed by various malignant neoplasms. The complete amino acid sequence of UK114 purified from goat liver has been determined by automated Edman degradation of CNBr and endoproteinase Lys‐C peptides. The protein contains 137 amino acid residues, which corresponds to a molecular mass of 14 229 Da. MALDITOF analysis resulted in a molecular weight of 14 290, suggesting that the N‐terminal Met residue is acetylated. Sequence comparison shows that UK114 from goat liver (1) has 77% identity with a previously described 23 kDa protein from rat liver (Levy‐Favatier et al. (1993) Eur. J. Biochem. 212, 665–673), (2) shares a very high degree of similarity with a family of prokaryotic and eukaryotic hypothetic proteins whose function have not yet been characterized, and (3) exhibits a significant similarity to a group of tumor‐associated antigens which belongs to a superfamily of heat shock proteins, acting as possible targets for the hosts antitumor immunity.

Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells

We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine‐kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2–GM3 colocalization in wild‐type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epidermal growth factor receptor (EGFR) and Shc‐p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by (+/–)‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol hydrochloride ([D]‐PDMP) induced a drastic cell‐surface redistribution of ErbB2, EGFR and Shc‐p66, within the Triton‐soluble fractions, as revealed by linear sucrose‐gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside‐depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal‐transduction‐correlated proteins in lipid rafts.

Purification, characterization and partial amino acid sequences of carnitine palmitoyl-transferase from human liver

Carnitine palmitoyl‐transferase has been extracted with 0.5% Tween‐20 from human liver homogenatc and purified to homogeneity. The purified enzyme has a native M r of 274 kDa. The subunit M r is of 66 kDa, as shown by SDS‐PAGE and immunoblots obtained with antibodies raised against human CPT. Purified CPT shows high affinity for palmitoyl‐CoA and palmitoyl‐carnitine and is not inhibited by malonyl‐CoA, Seven tryptic peptides and the N‐terminal of purified human CPT have been sequenced, and found homologous to rat CPT sequence. Both antibodies and peptide sequences are important tools for the investigation of the molecular basis of CPT deficiency in man.

INCREASED TUMORIGENICITY AND INVASIVENESS OF C6 RAT GLIOMA CELLS TRANSFECTED WITH THE HUMAN ALPHA -2,8 SIALYLTRANSFERASE CDNA

Gangliosides are thought to be involved in tumor cell proliferation, migration and invasiveness as so far demonstrated by the addition of exogenous gangliosides to the culture medium. To better understand the direct influence that alterations in ganglioside synthesis can exert on these functional aspects of cell biology, in the present study, we investigated the behaviour of C6 rat glioma cells after stable transfection with the human CMP-NeuAc:NeuAcα2-3Galβ1-4GlcCer α2,8-sialyltransferase (SAT-II, EC 2.4.99.8) gene. The enzyme synthesizes ganglioside GD3 by adding a sialic acid residue to ganglioside GM3. Stable transfection of the constructs into C6 cells and expression of the human SAT-II gene were evaluated using PCR and RT-PCR amplification, respectively. Qualitative and quantitative analysis of the ganglioside profile was performed by conventional HP-TLC and identity of de novo synthesized species was assessed by TLC immunostaining. Results show that whereas C6 parental cells and C6 cells transfected with the empty expression vector synthesize, almost exclusively, ganglioside GM3, de novo synthesis of GD3 is clearly observed in clones expressing the α2,8-sialyltransferase. Subcutaneous grafting in athymic nude mice of cells expressing high levels of GD3 induces tumors growing faster and more aggressively than controls. In in vitro assays, the same cells demonstrate increased proliferation rate, motility and invasiveness. Chemotaxis and chemoinvasion were assayed using the modified Boyden chamber. Data obtained suggest that endogenously neosynthesized GD3 is able to modify proliferation rate, motility and invasion of C6 rat glioma cells, enhancing the features of malignancy of this tumor cell line.

cDNA cloning and Escherichia coli expression of UK114 tumor antigen

Experimental evidence indicates that the antineoplastic effects of UK101, a goat liver perchloric acid extract, is likely due to one of its constituent proteins: the 14 kDa protein named UK114. The cDNA encoding UK114, obtained by PCR methodologies, contains an open reading frame coding for a protein of 137 amino acids with a theoretical molecular mass of 14298 Da. It shows high sequence homology with a 14 kDa protein identified in human, rat and Mus musculus tissues which is likely involved in the inhibition of cell-free protein synthesis. Northern blot analysis indicated that the transcript is present in variable amounts in a wide range of human tissues. Genomic Southern blots revealed that the UK114 mRNA in goat as well as in human is encoded by a single gene, as is the case in rat. The expression system for UK114 was constructed under the control of the PL promoter from bacteriophage lambda and the cDNA coding region has been highly expressed in Escherichia coli as a thioredoxin fusion protein. The recombinant UK114, purified to homogeneity, is immunoreactive to rabbit antisera prepared against UK101 or native UK114, as well as to sera of UK101-treated cancer patients. It inhibits cell-free protein synthesis at 8 microM concentration.

Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation.

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM(3) to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/-)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and beta-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM(3) have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM(3) added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, beta-casein gene expression appeared strongly down-regulated, and beta-casein mRNA levels were partially restored by exogenous GM(3) treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM(3) as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

Two active and differently N-glycosylated isoforms of human ST3Gal-V are produced from the placental mRNA variant by a leaky scanning mechanism

Previously, we identified a human ST3Gal‐V mRNA variant peculiarly characterized by the presence of a translational start codon localized up‐stream and in‐frame with the one that is usually considered as unique translation initiation site in the human gene. In this study we demonstrate, by cDNA transfection experiments, mutational analyses, enzyme activity assays, and endoglycosidase‐H treatments, that the in vivo expression of this transcript gives rise to two human ST3Gal‐V isoforms with distinct characteristics. Produced by a leaky scanning mechanism, they carry different N‐glycan structures and exhibit differences in their GM3 synthase activity that might be relevant for the modulation of GM3 cellular content.

Stable transfection of human β-1,4N-acetylgalactosaminyltransferase and α-2,8-sialyltransferase cDNAs in C6 rat glioma cells induces modifications in ganglioside metabolism

Ganglioside distribution in cells undergoes deep modifications during physiological and pathological events, possibly depending on the activity of glycosyltransferases involved in their biosynthesis. To understand how the ganglioside pattern can be altered by the selective expression of specific glycosyltransferases, C6 rat glioma cell line was stably transfected with two human glycosyltransferase cDNAs: β-1,4N-acetylgalactosaminyltransferase (GaINAcT) and a-2,8-sialyltransferase (ST-II). GaINAcT and ST-II are key enzymes in ganglioside biosynthesis; whereas ST-II synthesizes GD 3 , precursor of the b pathway, GaINAcT produces GM 2 , GD 2 and asialo-GM 2 and it is, therefore, involved in a, b and asialo pathways. C6 cells were subjected to three independent transfections: one with a construct containing GaINAcT cDNA, one with a construct containing the ST-II cDNA, and one with both constructs simultaneously. Whereas control cells present mainly N-acetyl- and N-glycolyl-GM 3 , selected transfected clones show more complex ganglioside profiles: GaINAcT-expressing cells are enriched in the a series gangliosides, ST-II-expressing cells synthesize the b series species, cells expressing contemporarily the two glycosyltransferases produce gangliosides of both series. Furthermore, among the selected clones, expression of GaINAcT and ST-II correlates with changes in the ST-I and ST-IV activities, indicating that the switching on of the biosynthetic enzymes we investigated influences the activity of endogenous glycosyltransferases, possibly through the modification of the amount of their substrates or products.

Omega-3 Long Chain Polyunsaturated Fatty Acids as Sensitizing Agents and Multidrug Resistance Revertants in Cancer Therapy

The efficacy of chemotherapy depends on sensitivity and intrinsic or acquired drug 14 resistance of cancer cells. The n-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs) are 15 considered chemosensitizing agents and revertants of multidrug resistance by pleiotropic 16 mechanisms. The specific mechanisms are not fully understood, but nowadays, it is widely accepted 17 that there are a complex network of mechanisms, including alteration in gene expression, 18 modulation of cellular proliferation and differentiation, induction of apoptosis, generation of 19 reactive oxygen species and lipid peroxidation. A crucial mechanism in the control of cell drug 20 uptake and efflux is related to n-3 LCPUFA influence on membrane lipid composition. The 21 incorporation of docosahexaenoic acid in the lipid rafts produces significant changes in their 22 physical-chemical properties affecting content and functions of transmembrane proteins, such as 23 growth factors, receptors and ATP-binding cassette transporters. Of note, n-3 LCPUFAs often 24 impact on the lipid compositions more in chemoresistant cells than in chemosensitive cells, 25 suggesting their adjuvant role in cancer treatment. 26