Anticancer activity of HMGA1 promoter targeting triplex forming oligonucleotide in HeLa cell line

High mobility group protein A1 (HMGA1) acts as an architectural transcription factor and regulates transcription of various genes. Upregulation of HMGA1 has been described in a large number of human malignancies and serves as a ‘tumor marker’. Due to its role in neoplastic transformation and tumor progression, hmga1 is considered as a promising therapeutic target. In the present study, we investigated the interaction of triplex forming oligonucleotide (TFO) of 18 bps targeted to hmga1 promoter (-1917 to -1940) and its influence on the expression of HMGA1 in HeLa cells. Stability of DNA triplex was characterized using various biophysical and thermodynamic methods and was confirmed by gel retardation assay using γ-32P [ATP]. Treatment of HeLa cells with hmga1 specific TFO significantly downregulated HMGA1 expression at mRNA, protein levels (~48%) and inhibited cell proliferation as investigated by RT-PCR, Western blot and Flow cytometry. The findings of the study suggest that TFO-mediated inhibition of hmga1 expression can be a promising strategy for modulation of gene expression and for inhibition of cancer cell proliferation. Moreover, DNA triplex-based therapeutic approaches hold promise in combating cancers associated with HMGA1 overexpression.

DNA triplex structures in neurodegenerative disorder, Friedreich’s ataxia.

It is now established that a small fraction of genomic DNA does adopt the non-canonical B-DNA structure or ‘unusual’ DNA structure. The unusual DNA structures like DNA-hairpin, cruciform, Z-DNA, triplex and tetraplex are represented as hotspots of chromosomal breaks, homologous recombination and gross chromosomal rearrangements since they are prone to the structural alterations. Friedreich’s ataxia (FRDA), the autosomal recessive degenerative disorder of nervous and muscles tissue, is caused by the massive expansion of (GAA) repeats that occur in the first intron of Frataxin gene X25 on chromosome 9q13-q21.1. The purine strand of the DNA in the expanded (GAA) repeat region folds back to form the (R∙R*Y) type of triplex, which further inhibits the frataxin gene expression, and this clearly suggests that the shape of DNA is the determining factor in the cellular function. FRDA is the only disease known so far to be associated with DNA triplex. Structural characterization of GAA-containing DNA triplexes using some simple biophysical methods like UV melting, UV absorption, circular dichroic spectroscopy and electrophoretic mobility shift assay are discussed. Further, the clinical aspects and genetic analysis of FRDA patients who carry (GAA) repeat expansions are presented. The potential of some small molecules that do not favour the DNA triplex formation as therapeutics for FRDA are also briefly discussed.

Analysis of penicillin-binding proteins (PBPs) in carbapenem resistant Acinetobacter baumannii.

Background & objectives : Acinetobacter baumannii is a Gram-negative, cocco-bacillus aerobic pathogen responsible for nosocomial infections in hospitals. In the recent past A. baumannii 0had developed resistance against β-lactams, even against carbapenems. Penicillin-binding proteins (PBPs) are crucial for the cell wall biosynthesis during cell proliferation and these are the target for β-lactams. Therefore, the present study was carried out to identify the PBPs in three (low, intermediate and high MICs) groups of carbapenem resistant isolates strains of A. baumannii. Methods: ATCC 19606 and 20 β-lactam resistant isolates of A. baumannii were obtained. Selective identification of the PBPs was done using Bocillin FL, a non-radioactive fluorescent derivative of penicillin. Results: The fluorescence emission from Bocillin-tag in SDS-PAGE gel of native strain identified eight PBPs, with apparent molecular weight of 94, 65, 49, 40, 30, 24, 22 and 17 kDa, however, these PBPs revealed alteration in carbapenem-resistant isolates. Interpretation & conclusions: A comparative analysis of PBPs in the resistant isolates with those of ATCC revealed a decreased expression of all PBPs except that of 65 and 17 kDa PBPs which were marginally downregulated, and simultaneous appearance of new 28 kDa PBP (in low and intermediate resistant isolates) and 36 kDa in high meropenem resistant group of A. baumannii. The present study indicated an association between alteration in PBPs and β-lactam resistance in A. baumannii.

Interaction of nalidixic acid and ciprofloxacin with wild type and mutated quinolone-resistance-determining region of DNA gyrase A.

The quinolones exert their anti-bacterial activity by binding to DNA gyrase A (GyrA), an essential enzyme in maintenance of DNA topology within bacterial cell. The mutations conferring resistance to quinolones arise within the quinolone-resistance-determining region (QRDR) of GyrA. Therefore, quinolones interaction with wild and mutated GyrA can provide the molecular explanation for resistance. Resistant strains of Salmonella enterica of our hospital have shown mutations in the QRDR of GyrA of serine 83 (to phenylalanine or tyrosine) or aspartic acid 87 (to glycine or tyrosine). In order to understand the association between observed resistance and structural alterations of GyrA with respect to quinolone binding, we have studied the interaction of mutated QRDR of GyrA with nalidixic acid and ciprofloxacin by molecular modeling using GLIDE v4. Analysis of interaction parameters like G-score has revealed reduced interaction between nalidixic acid/ciprofloxacin with QRDR of GyrA in all four mutated cases of resistant strains. The mutation of Ser83 to Phe or Tyr shows least binding for nalidixic acid, while Asp87 to Gly or Tyr exhibits minimal binding for ciprofloxacin. The study also highlights the important role of arginines at 21, 91 and His at 45, which form strong hydrogen bonds (at < 3 Å) with quinolones. The hydrophilic OH group of Serine 83, which is in close proximity to the quinolone binding site is replaced by aromatic moieties of Tyr or Phe in mutated GyrA. This replacement leads to steric hindrance for quinolone binding. Therefore, quinolone resistance developed by Salmonella appears to be due to the decreased selectivity and affinity of nalidixic acid/ciprofloxacin to QRDR of GyrA.