Dietary Calorie Restriction from Adulthood Through Old Age in Rats: Improved DNA Polymerase ß and DNA Gap Repair Activity in Cortical Neurons.

It is well established now that dietary calorie restriction (CR) leads to extension of life span in many species, although the exact mechanism of this effect is still eluding. In the present study, we examined the effect of 40 % CR imposed during a prolonged period of life span (from 6 to 30 months) of rats on the activity of DNA polymerase ß (pol ß) in view of its role in short gap base excision DNA repair and template driven primer extension. DNA pol ß activity is very low at this late age. However, cortical neuronal extracts prepared from CR rats of 30 months age showed significantly higher pol ß protein levels and activity when compared to control 30 month old rats. Yet, one-nucleotide gap repair in old control neurons and an improved efficiency in CR neurons could be visualized only after supplementation of the extracts with T4 DNA ligase indicating the lack of CR affect on ligase activity. No impressive primer extension activity is seen either in the CR or old control neurons. These results are taken to convey that extended CR through adult life leads to improved pol ß activity and therefore, pol ß dependent DNA gap repair activity.

Age-related decline in DNA polymerase ß activity in rat brain and tissues.

Fidelity of DNA polymerases is vital for maintaining genomic integrity. Deficient DNA repair leads to age related disorders or cancer. If the age at which the decline in activity of predominant DNA repair enzymes starts is identified, and the deficient proteins supplemented, then the manifestation of these diseases can be delayed promoting healthy aging. DNA polymerase ß (pol ß) is a predominant repair enzyme in brain. DNA pol ß activity declines with age in rat brain/neurons but the exact age during the life time of rat when this decline begins is not known, and comparison of this activity was not made between post mitotic and proliferating tissues therefore the pattern of pol ß with age was studied in rat brain and tissues. The decline in pol ß activity started between 30 and 45 days postnatal in all the tissues. Post mitotic tissues showed pronounced decline than the proliferating tissues.

Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.

AIMS: Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. METHODS: An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. RESULTS: Several specific APE1 inhibitors were isolated by this approach. The IC(50) for APE1 inhibition ranged between 30 nM and 50 µM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. CONCLUSIONS: Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

On the inhibitory affect of some dementia drugs on DNA polymerase Beta activity.

Some drugs are routinely prescribed for dementia that sets in either due to normal ageing or due to neurodegenerative disorders. We have studied the effect of three of these drugs, Donepezil hydrochloride, Rivastigmine tartrate and Nootropyl, on the activity of DNA polymerases beta, a crucial enzyme in the base excision repair pathway, the most important mode of DNA repair in brain. All the three drugs inhibited DNA polymerase beta activity to varying degrees although the affects of Donepezil being the least and inconsistent. The drugs preferentially bind to and inhibit the activities of 8 kDa domain of DNA polymerase beta that is known to possess the dRP lyase activity. The function of 31 kDa domain dealing with template driven addition of nucleotides at 3' end of the primer is not adversely affected. The inhibitory action of most widely used dementia drugs on DNA repair potential signifies that pharma sector needs to consider this aspect especially while designing drugs targeted towards brain.

DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons.

Linearised pUC 19 DNA with cohesive, blunt and non-matching ends, generated by prior treatment with different restriction enzymes was presented as substrate to measure the NHEJ activity to repair DNA double strand breaks in extracts prepared from isolated neurons from neonatal, young adult and old rat cerebral cortex. Highest end joining activity was noticed with the substrates having cohesive 3' overhang (PstI) or 5' overhang (EcoRI) ends and this activity is significantly reduced with age. However, blunt and non-matching ends were very poorly repaired at all ages. Further, the end joining activity in neurons is not faithful and sequence changes occur during the repair process. Also, the end joining activity in old neuronal extracts, but not in young extracts, was found to decline very rapidly with time of cold storage. These findings, the first of their kind, thus demonstrate that neuronal cells have the capacity to repair DNA double strand breaks through error prone NHEJ mode and that the cohesive end joining activity decreases with age of the animal.

Down-regulation of the global regulator SATB1 by statins in COLO205 colon cancer cells.

Special AT-rich sequence binding protein 1 (SATB1) regulates the expression of more than 1,000 genes in tumor cells. SATB1 expression has been implicated in metastasis, and its silencing results in reduced cancer progression and the reversion of metastatic cells to normal appearance. Therefore, any compound causing down-regulation of SATB1 expression or activity may be exploited for its therapeutic potential in terms of cancer regression. Earlier studies showed that the 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statin drugs), which are widely used to treat hypercholesterolemia, possess other pleotropic activities. These are now increasingly gaining attention for their cancer prevention abilities. However, the downstream interplay of the molecular mechanisms of such anti-cancer activities is unclear. Here, we show that SATB1 is down-regulated by statins in a time- and dose-dependent manner in COLO205 cells. This effect was statin-specific as the down-regulation of SATB1 was brought about by hydrophobic statins, such as simvastatin and fluvastatin, but not by hydrophilic pravastatin. Notably, treatment with mevalonate, an intermediate in the cholesterol and isoprenoid biosynthetic pathways, led to the inhibition of SATB1 down-regulation and cytotoxicity mediated by statins. Treatment with the proteasome inhibitors lactacystine and MG-132 inhibited the statin-mediated down-regulation of SATB1, suggesting that regulation occurs at the post-translational level. Thus, our results demonstrate a novel molecular mechanism for the anti-cancer activity of statin drugs in colon cancer cells, without invoking significant cytotoxicity.