Barbieri R

Inhibition of 'in vitro' tumor cell growth by aromatic polyamidines exhibiting antiproteinase activity.

Aromatic polyamidines containing two, three or four benzamidine residues inhibit proteinase activity and proliferation of different human tumor cell lines, including leukemic (K562, HEL), melanoma (Colo 38) and B-lymphoid (WI-L2) cell lines. In addition, the benzamidine derivatives analysed in the present study inhibit cell growth of the Chinese hamster FHO6T1-1 cell line, obtained after transfection of primary lung cells with the activated human T24-Ha-ras-1 oncogene.After treatment of FHO6T1-1 cells with benzamidine derivatives, a sharp decrease of the content of Ha-ras-1 mRNA was found, but not of transferrin receptor mRNA.We found that inhibition of cell proliferation by tetra-benzamidine derivatives is not restricted to tumor cells, but concerns also non-tumorigenic cell lines as well as normal primary fibroblasts. Therefore, our analysis was extended to di- and tri-benzamidine derivatives, which could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors. The data obtained demonstrate that these latter compounds and their halo-derivatives also exhibit strong antiproliferative effects onin vitro cultured cells.

Human leukemic K562 cells: Suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.

Distamycin inhibits the binding of a nuclear factor to the -278/-256 upstream sequence of the human HLA-DRα gene

In this study we analyse the effects of the anti-tumor compound distamycin on the binding of nuclear factor(s) to a synthetic oligonucleotide (GTATA/IFN-gamma) mimicking a putative regulatory region of the human HLA-DR alpha gene. This region contains the sequence (GTATA), that is required for nuclear protein binding and is likely to interact with distamycin. The present results, by showing that distamycin inhibits the interaction between nuclear factors and the GTATA/IFN-gamma oligonucleotide, suggest that distamycin might alter the binding of transacting factors to cis-elements containing AT/TA sequences. Alterations of nuclear protein binding to specific target sequences could be one of the molecular mechanism(s) by which distamycin exerts its antiproliferative activity on living cells.

Regulation of the expression of class II genes of the human major histocompatibility complex in tumor cells.

The control of expression of human class II MHC genes has been studied in lymphoid and melanoma cells. Specific unmethylation of all restriction sites nearby the promoter regions has been detected in all cell lines and tissues studied, irrespective of their ability to express class II MHC products. The main functional role of DNA methylation appears, on the contrary, to be the regulation of a fraction of the nucleotide polymorphism of class II MHC genes. Constitutive expression of these genes can be modified by recombinant IFN-gamma and by the demethylating agent 5-azacytidine. Both the modifiers differentially regulate the levels of class II MHC and invariant chain products. In melanoma cells IFN-gamma derepresses transcription of a 1.2-Kb HLA-DR alpha mRNA, but does not affect the levels of a 0.8-Kb HLA-DR alpha specific mRNA. These molecular changes are triggered by IFN-gamma through a protein-synthesis-dependent pathway.

Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DRα genes in a human B-lymphoid tumor cell line

In this paper, we report the effects of aromatic tetra amidines (TAPP-H) on cell growth and gene expression of a B-lymphoid human tumor cell line, WI-L2. The results obtained give evidence (a) for inhibition of cell proliferation by TAPP-H; (b) for stronger antiproliferative activity of TAPP-halo derivatives; (c) for TAPP-mediated inhibition of accumulation of c-myc RNA sequences but not of HLA-DR alpha mRNA and DR antigens. These results suggest that this class of antiproliferative compounds exhibit differential effects on cell-cycle specific and differentiation specific genes. In addition, also TAPP-related compounds containing 2 (DAPP) or 3 (TAPB) benzamidine residues retain inhibitory activity on the proliferation of WI-L2 cells. These latter compounds might be proposed as useful substrate in the synthesis of drug-conjugated monoclonal antibodies or growth factors.

Human HLA-DRα gene: a rare oligonucleotide (GTATA) identifies an upstream sequence required for nuclear protein binding

Synthetic oligonucleotides containing putative regulatory sequences are currently employed to identify and isolate genes coding for nuclear binding factors. Upstream DNA sequences of eukaryotic genes required for transcriptional activity and tissue specificity can be identified by means of biochemical techniques as well as computer analysis using homology searching. An alternative approach has been recently proposed by our research group. Scanning DNA sequences 1.8 megabases in length from a Genetic Sequence Data Bank, we have identified rare oligonucleotides 5 base pairs (bp) long, which are localized within or close to regulatory segments in mammalian promoters. In this paper we demonstrate that the rare GTATA sequence identifies an upstream region of the HLA‐DRα gene which operates in conjunction with the sequence AGAAGTCAG, homologous to a box found in many interferon‐inducible genes, in binding nuclear proteins.

Transgenic mice mimic the methylation pattern of the human HLA-DRα gene

Abstract The methylation pattern of the human HLA-DRα gene has been studied in different tissues of transgenic mice. Offspring from two transgenic lines was selected for this analysis, carrying the integrated HLA-DRα gene in either single or multiple (8–10) copies per diploid genome. In transgenic animals two distinct methylation patterns of the HLA-DRα gene are generated, due to a complete methylation of all the GCGC and CCGG sites the former, and to unmethylation restricted to one or both the GCGC sites located in the 5′ portion of the HLA-DRα gene, the latter. Unmethylation restricted to the 5′ portion of the HLA-DRα gene is a highly conserved feature in human tissues and in vitro cultured cell lines; therefore, it is concluded that the methylation pattern of the human HLA-DRα transgene may be faithfully reconstituted in transgenic animals. Northern blotting analysis of the RNA isolated from tissues of the transgenic mouse carrying single-copy HLA-DRα transgene demonstrates its tissue specific expression, suggesting that transgenic mice may represent an “in vivo” experimental system to study the relationship between methylation state and transcriptional activation.

Hypomethylation of the human HLA-DRα gene in breast carcinomas and autologous metastases

The methylation pattern of the human HLA-DRα gene was analyzed in normal breast tissues, breast primary tumors and lymphonodal metastases isolated from patients carrying breast carcinomas. In breast adenomas and also in normal tissues (including breast, muscle, brain, sperm and T- and B-lymphocytes), the HLA-DRα gene is hypermethylated at the CCGG and GCGC sites. In all tissues studied, the only constantly unmethylated region is located in the 5′ portion of the gene, near the promoter sequence. Further, the results indicate that the HLA-DRα gene is hypomethylated in carcinomas and in the relative metastatic lymph nodes. It is suggested that hypomethylation of the human HLA-DRα gene could be proposed as a molecular marker of malignant breast tumors.

CG Dinucleotides of class II MHC genes are mutation hot-spots.

DNA methylation of human class II genes of the Major Histocompatibility Complex (MHC) was correlated with gene expression and methyl-related CG → TG and CG → CA changes. It was found that cytosine methylation of the CG dinucleotides of MHC class II genes should be involved in generating a fraction of nucleotide polymorphism, rather than in controlling transcription.

Molecular Evolution of the Ha-ras-1 Oncogene: Relationship between DNA Methylation, Frequency of CpG Dinucleotides and Binding to the Sp1 Transacting Factor

One of the molecular events involved in the regulation of the expression of cellular oncogenes is the interaction between transcriptional factors and target DNA sequences present in the 5′ genomic portion1.