Epigenetic transcriptional repression of tumor suppresor genes and its reversion by drugs.

Resum

Genetic alterations and deregulation of the epigenetic mechanisms collaborate in the initiation and progression of cancer. In contrast to the genetic defects, the epigenetic abnormalities are potentially reversible. This fact has driven the search for drugs that induce selectively changes in the epigenetic patterns of the tumor cells and thus lead to differentiation, cell death and/or cell growth arrest. Inhibitors of DNA (cytosine-5)-methyltransferases (DNMTs) and inhibitors of Zn(II)-dependent histone deacetylases (HDACs) have been developed with this purpose. The DNMTs inhibitors allow the reactivation of genes, including tumor-suppressor genes, silenced through hypermethylation of the CpG island at their promoter. The HDACs inhibitors allow the reexpression of genes silenced by ypoacetylation of the histones associated at their promoters. Despite all these chemicals have promising effects on cultured cancer cells, many of them have side-effects that limit their use in anticancer chemotherapy. Because of that, we analyzed the properties of the anesthetic procaine (4-aminobenzoic acid 2-diethylaminoethyl ester) as a new inducer of DNA demethylation and we also compared the effects of seven HDACs inhibitors. In both cases, the breast cancer cell line MCF7 was the model system. 1. Procaine reduces the proportion of 5-methylcytosine into global genomic DNA, achieving its maximum effect within 24-48 h of treatment. Low concentrations of procaine decrease the amount of 5- methylcytosine at RAR¿Â2 promoter, which is hypermethylated in MCF7 cells, and reactivate its expression with only small decrease in global DNA methylation. This fact could be an advantage, since global DNA hypomethylation leads to chromosomal instability. Finally, procaine reduces cell proliferation and arrests cell cycle in mitosis, but does not induce apoptosis in MCF7 cells after treatments 3 days long. 2. The seven inhibitors of Zn(II)-dependent HDACs chosen for comparison were: two carboxylic acids (butanoic and valproic acid); one N-(2¡¯-aminophenyl) benzamide (MS-275); and four hydroxamic acids(trichostatin A, suberoylanilide hydroxamic acid, CX and CY). The results of in vitro HDAC activity assays performed on MCF7 nuclear extracts show the existence of a relationship between the chemical structure of these compounds and their activity: low micromolar concentrations of hydroxamic acids are sufficient for inhibiting almost completely the deacetylase activity, whereas millimolar concentrations of carboxylic acids are required for similar effects. Also the alterations that the drugs cause on cell growth and cell cycle arrest depend on its chemical structure. The IC50 for cell treatments 24 h long is in the range of millimolar concentrations for butanoic and valproic acids and low micromolar for the rest of the chemicals. At the IC50, MS-275 induces cell growth arrest in G1/G0, whereas the hydroxamic acids stop cell cycle mostly at G2/M and the carboxylic acids seems to arrest the cycle at both G1/G0 and G2/M. Despite all these inhibitors induce similar changes in the global acetylation of H4 and H3 when employed at their respective IC50, not all of them are able of reactivate the expression of the same genes. Moreover, it seems that the induced expression levels of CDKN1A and GADD45¿Â determine the alterations induced by the drugs on cell cycle. The changes on histone modifications at the promoters of six genes ( CDKN1A, GADD45¿Â, JunD, IGFBP3, MT1X and MT2A) upon CY treatment were studied. HDACs inhibition induces an increase in histone H4 tetraacetylation and in dimethylation of lysine 4 in H3, as well as a decrease in dimethylation of lysine 9 in H3. Additionally, HDAC2 is released from the promoters upon CY treatment. These changes take place also in the promoters of MT1X and MT2A, the genes whose expression remains unaltered after the treatment with CY. __________________________________________________________________________________________________ RESUMEN Las alteraciones gen¿¿ticas y la desregulaci¿(r)n de los mecanismos epigen¿¿ticos colaboran en la iniciaci¿(r)n y progresi¿(r)n del c¿¢ncer. Los defectos epigen¿¿ticos son potencialmente reversibles, lo que ha suscitado la b¿²squeda de f¿¢rmacos que selectivamente causen cambios en los patrones epigen¿¿ticos de las c¿¿lulas tumorales, con la consiguiente diferenciaci¿(r)n, muerte y/o parada de crecimiento de las mismas. Se han estudiado especialmente inhibidores de metiltransferasas de DNA (DNMTs) e inhibidores de desacetilasas de histonas (HDACs) dependientes de Zn(II). Los inhibidores de DNMTs posibilitan la reactivaci¿(r)n de genes silenciados mediante hipermetilaci¿(r)n de la isla CpG de su promotor y los inhibidores de HDACs, de genes silenciados v¿ªa hipoacetilaci¿(r)n de las histonas asociadas a su promotor. A pesar de sus prometedores efectos en cultivos celulares, muchas de estas sustancias presentan inconvenientes que limitan su aplicaci¿(r)n en quimioterapia. Por ello, en esta tesis: 1. Se ha estudiado por primera vez la capacidad del anest¿¿sico proca¿ªna para reducir la metilaci¿(r)n de DNA gen¿(r)mico y la proliferaci¿(r)n de la l¿ªnea celular MCF7. Proca¿ªna reduce la cantidad de 5-metilcitosina en DNA, reactiva genes silenciados por hipermetilaci¿(r)n ( RAR¿Â2) e induce parada del ciclo celular en mitosis. 2. Se han comparado siete inhibidores de HDACs dependientes de Zn(II): ¿¢cido butanoico, ¿¢cido valproico, MS-275, tricostatina A (TSA), SAHA, CX y CY. Existe una relaci¿(r)n estructura-actividad para los efectos de estas sustancias en ensayos in vitro y en el crecimiento de MCF7. Adem¿¢s, aunque estos inhibidores inducen cambios similares en acetilaci¿(r)n global de histonas, no todos reactivan los mismos genes. Los niveles expresi¿(r)n de CDKN1A y GADD45¿Â parecen determinar los efectos de estos compuestos en el ciclo celular. En los promotores de los seis genes estudiados, la inhibici¿(r)n de HDACs aumenta la acetilaci¿(r)n de H4 y la dimetilaci¿(r)n de la lisina 4 de H3, mientras disminuye la dimetilaci¿(r)n de lisina 9 de H3.