Laura Mariani

The Proto-Oncogene LRF Is under Post-Transcriptional Control of MiR-20a: Implications for Senescence

MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3′UTR is demonstrated. To validate the interaction miR-20a/3′UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism, alternative to apoptosis; from this point of view the discovery of new physiological “senescence inducer” appears to be promising as this molecule could be used as anticancer drug.

Ribozyme and free alkylated base: a dual approach for sensitizing Mex + cells to the alkylating antineoplastic drug

N-alkyl-nitrosoureas and alkyl-triazenes are alkylating antineoplastic drugs, the efficacy of which is strongly affected by the level of expression of the DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). In tumors, MGMT activity reduces the chemotherapeutic potential of alkylating drugs; therefore, efforts have been made to down-regulate the protein. A partial sensitization of Mex+ cells to alkylating drugs has been obtained using either free alkylated bases or oligonucleotides targeted against MGMT mRNA. In the present work, O6-methylguanine and a chemically modified ribozyme, without a cationic liposome as a carrier, were coadministered to CHO47 cells, which express a high level of human MGMT protein. The reduction of MGMT mRNA and protein enhanced the genotoxicity of the alkylating drug mitozolomide. Furthermore, the sensitivity of CHO47 cells is the same as that of CHO5 cells, which lack MGMT protein. These data indicate that a strategy in which both mRNA and protein are degradation targets can be successfully applied to down-regulate the MGMT gene.

Analysis of electroporation-induced genetic damages in V79/AP4 Chinese hamster cells

Electroporation is a recent technique used to introduce exogenous DNA into eukaryotic cells. It is important to establish that the gene of interest is transferred into a functional, non-mutated recipient cell. V79/AP4 Chinese hamster cells were exposed to high-voltage pulsed electric fields and some biological and genetic effects were measured. The results showed that cytotoxicity was related in a dose-dependent manner to the number of applied pulses. Thioguanine-resistant colony-forming cells as well as chromosomal aberrations were also induced whereas ouabain resistants and sister-chromatid exchanges were not or slightly induced. Spontaneous and electroporation-induced clones that were phenotypically TGR/HATS were used to investigate the hprt locus. Molecular screening of the locus showed that the number of deleted exons was significantly higher in induced than in spontaneous TG-resistant clones, suggesting that the genetic damages induced by electroporation concern the loss of regions well over the size of the hprt locus.

DNA repair systems in early and persistent hepatocyte nodules in the rat

SummaryThe repair of three DNA lesions, namelyO6-methylguanine, 7-methylguanine, and 3-methyladenine, was investigated within early and persistent hepatocyte nodules generated in Fischer 344 rats by a modified Solt-Farber procedure (diethylnitrosamine initiation followed by a 2-acetylaminofluorene/CCl4 cycle). TheO6-methylguanine-DNA methyltransferase concentration within both hepatocyte nodule types was always higher than that found in age-matched controls (normal, initiated-only and promoted-only livers). As far as 3-methyladenine and 7-methylguanine-DNA glycosylases are concerned, the early hepatocyte nodules showed far higher activities for both enzymes than were found in the controls, whereas in the persistent ones they underwent a significant decrease. In conclusion hepatocyte nodules are endowed with a high DNA repair activity, which is partly adaptive, partly constitutive; along with others, such a defence mechanism could allow transformed cells to resist many cytotoxic drugs.

The sensitization of cells treated with O6-methylguanine to alkylation damage is affected by the number of O6-methylguanine-DNA methyltransferase molecules escaped from inactivation

O6-Methylguanine (MeG) can bind to the active site of O6-methylguanine-DNA methyltransferase (MGMT) as a free base. The subsequent methyl transfer reaction inactivates the repair protein. Hence, MeG is used to deplete the active MGMT pools in Chinese hamster cell lines (CHO) transfected to express varying amounts of human MGMT. After treatment with the free base, a residual population of active protein molecules remains localized mostly in the cytoplasm. Depleted cells are then challenged with the alkylating drug mitozolomide. Genotoxicity of this agent varied among the cell lines, and the compound sensitivity seemed to be regulated by a steady state equilibrium of residual MGMT molecules between nucleus and cytoplasm.

Chinese hamster ovary cells deficient or proficient in O6-alkylguanine-DNA alkyltransferase activity are equally sensitive to X-rays

In mammalian cells, under aerobic conditions, ionizing radiations and radiomimetic chemical agents can induce an enzymatic activity involved in DNA repair, O6-alkylguanine-DNA alkyltransferase (O6-AT). This catalytic protein is active against alkyl-radical-induced DNA damages. This induction was proposed to be linked to the formation of hydroxyl radicals. The possible involvement of O6-AT in the defense mechanism of the cell against aerobic radiation damage was investigated by comparing the X-ray sensitivity of two Chinese hamster ovary (CHO) cell lines, the first deficient (CHO mex-) and the second proficient by transfection of O6-AT (CHO mex+). The colony-forming ability after X-irradiation was appreciably reduced in CHO mex- in comparison to CHO mex+ cells. Nevertheless, pretreatment of proficient cells with O6-methylguanine, a specific inhibitor of O6-AT, reduced the DNA repair activity but did not modify the degree of sensitivity to X-rays of the CHO mex+ cells. Since the glutathione concentrations as well as the DNA damage amounts induced by X-irradiation were comparable in the variously treated cell lines, these results suggest that the observed induction of O6-AT by ionizing radiation in aerobic conditions could be a generalized rather than a specific response to damage by radicals.

The Sensitivity of MCF10A Breast Epithelial Cells to Alkylating Drugs is Enhanced by the Inhibition of O6-Methylguanine-DNA Methyltransferase Transcription with a Synthetic Double Strand DNA Oligonucleotide

Cytoxicity of alkylating chemotherapeutic drugs is affected by the cellular content of the enzyme O6-methylguanine-DNA methyl transferase (MGMT). Since high levels of the enzyme confer the efficient repair of DNA alkylation, the chemotherapeutic potential of alkylating chemicals can be maintained either increasing drug dosage or reducing the amount of endogenous MGMT. This study strives to the latter end by competing away a transcriptional activator of the MGMT gene from its native enhancer sequence using a synthetic double strand DNA oligonucleotide (MEBP-ODN). MEBP-ODN was administered in culture medium to MCF10A human breast ep-ithelial cells expressing high level of MGMT. Reverse transcription-polymerase chain reaction and western blotting analyses showed decrease in both MGMT mRNA and protein content. Concomitantly, MEBP-ODN exposed cells were more sensitive to the alkylating drug mitozolomide than their controls, which were not exposed to MEBP- ODN. These results indicate that the cis-acting MEBP-ODN can efficiently deplete MGMT protein by working as decoy binding site for the transcriptional activator MEBP. This approach represents a successful strategy to counteract the protective role of MGMT repair enzyme during an alkylating drug based chemotherapeutic regimen.

Post-transcriptional Modulation of Sphingosine-1-Phosphate Receptor 1 by miR-19a Affects Cardiovascular Development in Zebrafish

Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.