Histone deacetylases (HDACs) in XPC gene silencing and bladder cancer.

Bladder cancer is one of the most common malignancies and causes hundreds of thousands of deaths worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens, and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibition study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased binding of both CREB1 and Sp1 transcription factors at the promoter region of the XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08). In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All of these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the severity of bladder tumors.

Induction of endogenous gamma-globin gene expression with decoy oligonucleotide targeting Oct-1 transcription factor consensus sequence.

Human beta-globin disorders are relatively common genetic diseases cause by mutations in the beta-globin gene. Increasing the expression of the gamma-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the gamma-globin gene. The human gamma-globin genes (both Agamma and Ggamma-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing gamma-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the gamma-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, alpha2gamma2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous gamma-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous gamma-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the gamma-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the gamma-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and beta-thalassemia.

The role of Bcl-x(L) protein in nucleotide excision repair-facilitated cell protection against cisplatin-induced apoptosis.

Many anticancer drugs target the genomic DNA of cancer cells by generating DNA damage and inducing apoptosis. DNA repair protects cells against DNA damage-induced apoptosis. Although the mechanisms of DNA repair and apoptosis have been extensively studied, the mechanism by which DNA repair prevents DNA damage-induced apoptosis is not fully understood. We studied the role of the antiapoptotic Bcl-x(L) protein in nucleotide excision repair (NER)-facilitated cell protection against cisplatin-induced apoptosis. Using both normal human fibroblasts (NF) and NER-defective xeroderma pigmentosum group A (XPA) and group G (XPG) fibroblasts, we demonstrated that a functional NER is required for cisplatin-induced transcription of the bcl-x(l) gene. The results obtained from our Western blots revealed that the cisplatin treatment led to an increase in the level of Bcl-x(L) protein in NF cells, but a decrease in the level of Bcl-x(L) protein in both XPA and XPG cells. The results of our immunofluorescence staining indicated that a functional NER pathway was required for cisplatin-induced translocation of NF-kappaB p65 from cytoplasm into nucleus, indicative of NF-kappaB activation. Given the important function of NF-kappaB in regulating transcription of the bcl-x(l) gene and the Bcl-x(L) protein in preventing apoptosis, these results suggest that NER may protect cells against cisplatin-induced apoptosis by activating NF-kappaB, which further induces transcription of the bcl-x(l) gene, resulting in an accumulation of Bcl-x(L) protein and activation of the cell survival pathway that leads to increased cell survival under cisplatin treatment.

The involvement of ataxia-telangiectasia mutated protein activation in nucleotide excision repair-facilitated cell survival with cisplatin treatment.

DNA damage can lead to either DNA repair with cell survival or to apoptotic cell death. Although the biochemical processes underlying DNA repair and apoptosis have been extensively studied, the mechanisms by which cells determine whether the damage will be repaired or the apoptotic pathway will be activated is largely unknown. We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NER-defective xeroderma pigmentosum (XP) XPA and XPG cells. The caspase-3 activation experiment demonstrated a greatly increased casapse-3 activation in the NER-defective cells following cisplatin treatment. The flow cytometry experiment revealed an altered cell cycle arrest pattern of the NER-defective cells following cisplatin treatment. The results obtained from the Western blot experiment showed that NER defects resulted in enhanced CHK1 phosphorylation and p21 induction after cisplatin treatment. The cisplatin treatment-induced ATM phosphorylation, however, was attenuated in NER-defective cells. The results obtained from our immunoprecipitation experiment further demonstrated that the ATM protein interacted with the TFIIH basal transcription factor and the XPG protein of the NER pathway. It also showed that a functional XPC protein was required for the association of the ATM protein to genomic DNA. These results suggest that the NER process may prevent the cisplatin treatment-induced apoptosis by activating the ATM protein, and that the presence of the XPC protein is essential for recruiting the ATM protein to the DNA template.

Peptide nucleic acid (PNA) binding-mediated gene regulation.

Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a single-stranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The D-loops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.