Analysis of the molecular species generated by MDM2 gene amplification in liposarcomas.

MDM2 gene is overexpressed in several tumors and its product may be processed into different isoforms, some of which have been demonstrated to possess transforming activity. In a panel of liposarcomas characterized by displaying 4 different combinations of mdm2/p53 immunoreactivity, molecular analysis of amplified MDM2 gene revealed a coexistence of mutated full-length MDM2 messenger RNAs, an out-of-frame splicing mRNA and finally aberrant spliced forms. Two of the latter are reported here for the first time. The molecular differences in this heterogeneous mRNA population seem to mirror distinct functional aspects of the altered encoded mdm2 proteins. In fact, besides the deleted transcripts defective in their ability to bind p53 and known to possess a transforming activity, here we describe both mutated full-length forms and deleted transcripts that still maintain the ability to bind p53 but, based on their mdm2+/p53+ immunophenotype, probably fail to signal its degradation. These aberrant forms, which are responsible for the accumulation and inactivation of p53, can contribute, together with the p53 independent transforming forms, to liposarcoma transforming pathway.

Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus species.

The gene adh encoding a NAD-dependent alcohol dehydrogenase from the novel strain RC3 of Sulfolobus sp. was cloned and sequenced. Both the adh gene from Sulfolobus sp. strain RC3 and the alcohol dehydrogenase gene from Sulfolobus solfataricus (DSM 1617) were expressed at a high level in Escherichia coli, and the recombinant enzymes were purified, characterized, and compared. Only a few amino acid replacements were responsible for the different kinetic and physicochemical features investigated.

Suramin modulates cellular levels of hepatocyte growth factor receptor by inducing shedding of a soluble form.

Several growth factor receptors undergo shedding from the cell surface as a result of limited proteolysis via mechanisms that are at present poorly understood. By Western blotting of the conditioned media and cell lysates of several cell lines expressing the hepatocyte growth factor receptor, we found that suramin, a pharmacological agent that inhibits the activity of many growth factors, was able to induce shedding of this receptor. Increased levels of soluble hepatocyte growth factor receptor were observed in the conditioned media of GTL-16, a cell line over-expressing the receptor, as early as ten minutes after initial exposure to the agent, and incubation of this line with 300 microM suramin caused a 50% reduction in cell-associated levels of receptor after 6 hours. Although protein kinase C activation by treatment of cells with phorbol esters has previously been found to stimulate shedding of the hepatocyte growth factor receptor, this hitherto undescribed activity of suramin was not affected by protein kinase C inhibitors. Since shedding represents a possible means of down-modulation of receptor activity, suramin may inhibit the hepatocyte growth factor ligand/receptor system, not only by abrogation of hepatocyte growth factor binding to intact receptor, but also by induction of receptor shedding.

Synthesis and biological activity of five-coordinate platinum(II) complexes including organotin fragments.

The synthesis and characterization of two five-coordinate platinum(II) complexes of general formula [PtCl(SnMe(n)Cl3-n)(2,9-Me2-1,10-phenanthroline) (diethylfumarate)] (n = 1, 2) are described. The bimetallic species were tested on the neuroblastoma cell line SK-N-BE and a considerable inhibition of cellular proliferation was detected; the bimetallic species were also able to induce neuronal differentiation, as did retinoic acid, a well-known neuroblastoma differentiating agent.

Antiproliferative effects and DNA hypomethylation by 5-aza-2'-deoxycytidine in human neuroblastoma cell lines.

5-Aza-2'-deoxycytidine (5-AZA-CdR) is an inhibitor of DNA methylation, and its antileukemic activity has been shown in preclinical and clinical studies. This paper describes the ability of 5-AZA-CdR to inhibit DNA methylation, DNA synthesis and cell growth in several human neuroblastoma cell lines. The stability of cell growth inhibition was ascertained, as well as the ability of the metabolite thymidine to enhance the antiproliferative effect of 5-AZA-CdR. The activity of phosphorylating enzyme deoxycytidine kinase (dCK) was correlated to different levels of sensitivity in several cell lines. The results obtained indicate that 5-AZA-CdR may be an agent for the chemotherapy of neuroblastoma.

Effect of cytidine analogs on cell growth and differentiation on a human neuroblastoma line.

The cytidine analog 5-AZA-2'-deoxycytidine (5-AZA-CdR) has been demonstrated to induce cellular differentiation; on the other hand, induction of differentiation has been suggested as a possible form of therapy for leukemic cells. We have evaluated the possibility that the neuroblastoma malignant tumor growth could be controlled by treatment that promotes the differentiation of immature tumor cells. We have previously reported on differentiation of murine neuroblastoma cells (41A3) treated with 5-AZA-CdR. In this paper, we describe the effect of 5-AZA-CdR on human neuroblastoma cell line CHP-100. The drug-treated cells show some degree of differentiation, demonstrated by morphological and biochemical markers. A significant DNA hypomethylation and partial inhibition of DNA synthesis and cell proliferation is also observed. This effect is more stable than that caused by another cytidine analog, Cytosine-beta-D-Arabinofuranoside (ARA-C).