Effect of hydralazine upon G9A histone methyltransferase.

<p>A. Quantitative RT-PCR of <i>G9A</i> shows that resistant CaLoGR cells had an increase as compared to basal and that hydralazine reduces its expression (basal vs resistant, p<0.05). B. At protein level, there was an increase in G9A in the resistant cells that decreased after hydralazine treatment. C. H3K9 methyltransferase <i>in vitro</i> inhibition assay. Hydralazine at 2 µM and 10 µM strongly reduced H3K9 methylation (untreated vs hydralazine, p<0.05).</p

GO processes affected by hydralazine.

<p>MetaDrug™ program predicted that hydralazine may affect not only DNA methylation in cytosine but also can be involved in negative regulation of H3K9 methylation.</p

Gemcitabine sensitivity, gene expression and effects of hydralazine in CaLo cells.

<p>A. After acquiring gemcitabine resistance, the IC₅₀ in CaLoGR cells was 927 µM (280-fold concentration). B. Expression of <i>hENT1</i>, <i>dCK</i> and <i>CDA</i> were reduced almost by half, RRM1 and RRM2 had minor changes. C. The reduction of <i>hENT1</i> and <i>dCK</i> genes occurred progressively as resistance developed. D. Treatment of CaLoGR cells with hydralazine at 2 µM for 10 days, 10 µM and 30 µM for 5 days reverted gemcitabine resistance. The corresponding IC₅₀s for these treatments were 1.7 µM, 2.7 µM and 6.6 µM respectively.</p

HDAC activity and promoter acetylation in CaLo cells.

<p>A. Total histone deacetylase activity showed no major changes between basal and resistant cells, actually a small decrease was observed in the resistant cells as evaluated with a HDAC activity kit. B. Total acetylation of H3 and H4 as evaluated by ChIP showed a decrease in H3 and H4 acetylation at the <i>hENT1</i> promoter, a mild increase in acetylation of H3 and not change in H4 at <i>dCK</i> promoter.</p

iRNA assay for G9A and gemcitabine resistance.

<p>A. Inhibitory concentration of gemcitabine in CaLo cells not reached. B. Depletion of G9A restored gemcitabine sensitivity (IC₅₀ 10.3 µM). C. No further changes in IC₅₀ were seen when hydralazine was added to the G9A depleted cells. D. qPCR of G9A confirming the partial depletion of <i>G9A</i> messenger.</p

H3K9 and H3K4 methylation.

<p>For <i>hENT1</i> gene, no changes were observed in H3K4m3 methylation but H3K9me2 mildly increased in the resistant cells and decreased after hydralazine treatment. In <i>dCK</i> gene there was a slight reduction in H3K4me3 in the resistant cells which was not modified by hydralazine. The methylation at H3K9me2 mildly increased in resistant cells and reduced after hydralazine. The changes in band intensity were normalized against their respective inputs.</p

Gemcitabine sensitivity in cervical cancer cells and basal gene expression.

<p>A. The IC₅₀ of gemcitabine for HeLa, CaLo, SiHa and C33A cells were 3.3 µM, 0.3 µM, >1000 µM and 0.1 µM respectively as evaluated with the crystal violet assay. B. Basal expression of <i>hENT1</i>, <i>dCK</i>, <i>RRM1</i>, <i>RRM2</i>, <i>CDA</i> genes as evaluated by RT-PCR. There was no correlation between IC₅₀ and the intrinsic sensitivity/resistance status. Expression was adjusted to the expression in normal cervix.</p

Histone deacetylase activity.

<p>The five patients assayed showed a reduction in enzymatic activity. In Y axis are the absolute values in optical density (OD) units. Higher and lower decreases were 0.3845 and 0.0175, for a mean decrease of 0.1624. This difference was statistically significant (p = 0.042).</p

Hierarchical Cluster Analysis.

<p>The cluster shown represents 3,117 genes. Each row represents a gene, whereas each column corresponds to a tissue sample. The relative abundance of the gene in the tissue correlates with color intensity (red, induced; green, repressed; black, no change). On the dendogram, post-treated clinical samples clustered together.</p

Gene expression by RT-PCR.

<p>RT-PCR of NDUFA13 and DAPPER genes in biopsies from the primary tumor showing that these genes were reactivated after treatment. This corresponds to one of the three patients whose biopsy (only post-treatment) was analyzed by microarray and showed over-expression of these genes.</p