Biological properties of a human compact anti-ErbB2 antibody.

ErbB2 is a prognostic factor and target of therapy for many carcinomas. In contrast with the other ErbB receptors, ErbB2 lacks a soluble direct ligand, but it is the preferred co-receptor for the ErbB family members, forming heterodimers with more potent and prolonged signalling activity than that of homodimers. We recently produced a new anti-ErbB2 antibody, Erb-hcAb, by fusion of Erbicin, a human, anti-ErbB2 scFv, selectively cytotoxic to ErbB2-positive cells, and a human Fc domain. This fully human antitumour antibody represents a compact version of an IgG1, with the cytotoxicity of the scFv moiety on target cells, combined with the ability of the Fc moiety to induce both antibody- and complement-dependent cytotoxicity. Here, we describe the main properties of Erb-hcAb, using as a reference Herceptin, an anti-ErbB2 humanized monoclonal currently employed in clinical immunotherapy. We found that both bivalent Erb-hcAb and Herceptin increase receptor phosphorylation and downregulation, whereas monovalent Erbicin does not. These results correlate with the finding that Erb-hcAb is capable of inducing apoptosis and inhibiting cell cycle progression in ErbB2-positive cells. Its powerful in vitro antitumour action matched that observed in vivo in experiments with human ErbB2-positive tumour xenografts established in athymic mice. Finally, Erb-hcAb displays a glycosylation profile virtually superimposable to that of a human IgG. These findings suggest that Erb-hcAb is a very promising new agent for the immunotherapy of carcinomas that overexpress the ErbB2 receptor

Effects of a human compact anti-ErbB2 antibody on gastric cancer

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Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops

Histone methylation changes and formation of chro- matin loops involving enhancers, promoters and 3′ end regions of genes have been variously associ- ated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A re- ceptor, at the RARE–promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA ex- posure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethy- lase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elic- its an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nu- cleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5′ tran- scription start site and the 3′ end of the genes. The RARE bound-receptor governs the 5′ and 3′ end se- lection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription

Meccanismi d’azione di nuovi potenziali farmaci ad attività antitumorale, utilizzando sistemi in vitro ed in vivo.

L’insorgenza e la progressione dei tumori maligni ha interessato inizialmente l’aumentata proliferazione cellulare, successivamente si è approfondito il rapporto esistente fra proliferazione emorte cellulare. La morte cxellulare è caratterizzata dalla apoptosis o morte cellulare programmata e necrosi. Da alcuni anni quindi sempre maggior attenzione è stata posta allo sviluppo di farmaci antitumorali che agiscano attraverso l’attivazione del fenomeno apoptotico, infatti, la maggior parte delle cellule neoplastiche muoiono in seguito all’attivazione di una serie di segnali che attivano la via apoptotica e che difetti dell’attivazione di tale fenomeno sono coinvolti nella proliferazione neoplastica. Il lkavoro ha avuto come obbiettivo la determinazione dei meccanismi d’azione di nuove sostanze ad attività antitumorale che presentano diversa origine, attraverso sistemi in vitro ed in vivo. In particolare la ricerca ha riguardato 1) Lo studio di una nuova classe di peptidi ciclici analoghi alle astine naturali 2) La caratterizzazione in vitro degli effetti della 2-metil-arachidoil-2’-fluoro- etilamide ( Met-F-AEA) un analogo metabolicamente stabile dell’anandammide. 3) Immunoagenti diretti contro il recettore tirosin-chinasico ErbB2. 4) La messa a punto di un sistema di imaging ottico e radioisotopico per la rivelazione precoce dei tumori e delle metastasi in vivo

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations

Phosphofructokinase Regulation in Some Transplantable Thyroid Tumors

Some kinetic, molecular, and rate-limiting properties of partially purified phosphofructokinase from normal rat thyroid and three transplantable rat thyroid tumors have been studied, and interesting differences have been found. Citrate inhibition of tumor phosphofructokinase and its reversal by cyclic adenosine 3′:5′-monophosphate were lower than those of the normal enzyme. A direct relationship between tumor growth rate and the decrease of citrate inhibition was observed. The diethylaminoethyl cellulose affinity properties of the rat thyroid phosphofructokinase were slightly but consistently different from those of the three tumors; differences observed among the tumor enzymes with regard to these properties were likewise related to their respective growth rates. When the amounts of lactate, pyruvate, and glycerol 3-phosphate produced from each of the substrates of the glycolytic pathway were measured, it was found that, in contrast to results obtained with normal thyroid extracts, no rate-limiting function was associated with phosphofructokinase activity in tumor extracts. It was concluded that the modulation of phosphofructokinase by its allosteric effectors was similar in all three tumors, although different from that of the enzyme from normal rat thyroid; it was proportional to tumor growth rate and probably related to molecular and regulatory modifications

[Subcellular distribution of the protein kinase activity in the normal and neoplastic thyroid tissue of the rat].

Thyroid tissue has been fractionnated by centrifugation (105 000 q) of its homogenate. Protein-kinase activity in presence of histone is distributed in nuclei (11.5%) mitochondira (22.8%), microsomes (9.8%) and soluble fraction (56%); it is activated by cyclic AMP and GMP, mostly in soluble and nuclear fractions. Protein-kinase activity of total homogenate of neoplasic thyroid (strain 1-5G Wollman) in presence of histone is 3 times higher than in normal tissue and more activated by cAMP. In absence of histone, protein-kinase activity is the more important in mitochondrial and microsomal fractions of normal thyroid and in soluble and nuclear fraction of neoplastic tissue

Differences in phosphofructokinase regulation in normal and tumor rat thyroid cells.

The kinetic and molecular properties of a phosphofructokinase derived from a transplantable rat thyroid tumor lacking regulatory control on the glycolytic pathway were studied. The properties of the near-purified enzyme (specific activity 140 units/mg) were compared with those of phosphofructokinase from normal rat thyroid (specific activity 134 units/mg). The electrophoretic mobilities and gel elution behavior of these two enzymes were almost similar. The thyroid tumor phosphofructokinase showed, however, a greater degree of size and/or shape heterogeneity in the presence of ATP than the normal thyroid enzyme, as determined by gel filtration and sucrose density gradient centrifugation. Kinetic studies below pH 7.4 showed a sigmoid response curve for both enzymes when the velocity was determined at 1 mM ATP with varying levels of fructose-6-P. The interaction coefficient, however, was 4.2 and 2.6 for normal and tumor thyroid phosphofructokinase, respectively. Ammonium sulfate decreased the cooperative interactions with the substrate fructose-6-P in both enzymes. The thyroid tumor enzyme, however, was less sensitive to the inhibition by ATP and by citrate. The reversal of citrate inhibition by cyclic 3':5'-adenosine monophosphate was also less effective with the thyroid tumor phosphofructokinase, while the protective effect of fructose-6-P was stronger. The difference in citrate inhibition between tumor and normal thyroid enzyme was not strongly affected by varying the MgCl2 concentration up to 10 mM. It is concluded that the complex allosteric regulation typical of the normal thyroid phosphofructokinase is still present in the enzyme isolated from the thyroid tumor tissue. The latter, however, is more loosely controlled by its physiological effectors, such as ATP, citrate, and cyclic AMP