Moganty R. Rajeswari

Circulating (cell-free) nucleic acids--a promising, non-invasive tool for early detection of several human diseases.

Circulating nucleic acids (CNA) are present in small amounts in the plasma of healthy individuals. However, increased levels of plasma CNA have been reported in a number of clinical disorders like cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy‐associated complications. CNA has received special attention because of its potential application as a non‐invasive, rapid and sensitive tool for molecular diagnosis and monitoring of acute pathologies and the prenatal diagnosis of fetal genetic diseases. This review throws light on the current status of blood CNA as a diagnostic marker and its potential as a powerful tool in the future.

Overexpression of high mobility group (HMG) B1 and B2 proteins directly correlates with the progression of squamous cell carcinoma in skin.

High mobility group B (HMGB) chromosomal proteins, which plays important role in cancer and inflammation, were followed at various stages of the squamous cell carcinoma of skin. Present results were analyzed by histopathology, BrdU assay, immunohistochemistry, western blot and RT-PCR, which indicate that at early stages of tumorigenesis, expression of HMGB (B1, B2), raised only by about 20%. However, the advanced (⩾12 weeks) tumors showed significant (⩾80%) increase in HMG levels. Using skin cancer model, we demonstrated that high levels of HMGB directly correlate with the extent of neoplastic changes, and it appears that HMGB is an effective stimulus for cell differentiation, tumor progression, and metastatic invasion.

Formation and thermodynamic stability of intermolecular (R*R.Y) DNA triplex in GAA/TTC repeats associated with Freidreich's ataxia

Abstract It is well established that GAA/TTC base triplet expansion is the cause of degenerative disorder in Freidreichs Ataxia. It is also known that these repeat sequences fold back to form the unusual intramolecular triple helix structures in DNA of the type Pyrimidine *Purine •Pyrimidine or Purine *Purine •Pyrimidine. In this paper we report on the stability of Purine *Purine•Pyrimidine intermolecular triple helix DNA containing GAA/TTC repeats under physiological conditions. Using the oligonucleotides 5′ TCGC GAA GAA GAA GAA GAA CGCT3′, 5′-AGCG TTC TTC TTC TTC TTC GCGA-3′for duplex and 5′- AAG AAG AAG AAG AAG −3′ as triplex forming strand (TFO), we have established the formation of triplex by UV-melting temperature (Tm), stoichiometry of mixing and circular dichroic spectra. This was further confirmed by gel-retardation assay. The thermodynamic parameters ΔG, ΔH and ΔS for both duplex and triplex formation were determined at different salt concentrations. The results suggest the formation of a stable intermolecular, anti—parallel triplex in GAA/TTC repeat sequences where each repeat unit contains two A*A•T and one G*G•C triplet. The therapeutic agents and TFOs, which competitively inhibit the in-vivo intra-molecular triplex by formation of a more stable inter-molecular triplex, could be used to reverse the transcription deficit in GAA/TTC expansions in Frataxin gene.

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.

Quantification of Circulating Plasma DNA in Friedreich's Ataxia and Spinocerebellar Ataxia Types 2 and 12

DNA triplet repeat expansion-associated ataxias, Friedreichs ataxia, and different types of spinocerebellar ataxias (SCAs) are progressive multisystem neurodegenerative disorders. The diagnosis of this wide group of inherited ataxias is essentially based on clinical findings. Cell-free circulating DNA in plasma has been considered as a powerful tool in clinical diagnosis and prognosis of several human diseases. In the present study, clinically suspected patients were assessed on the International Co-operative Ataxia Rating Scale and further confirmed by molecular analysis of DNA triplet repeats. Quantification of plasma DNA using a highly sensitive and DNA-specific PicoGreen fluorescent assay was done. We found significantly high levels (p < 0.001) of plasma DNA of 167 ± 43 ng/mL in Friedreichs ataxia patients (n = 15), 148 ± 29 ng/mL in SCA2 patients (n = 10), and 137 ± 29 ng/mL in SCA12 patients (n = 25), whereas those of healthy controls (n = 20) was only 59 ± 15 ng/mL. Therefore, we were able to distinguish between ataxia patients and healthy controls using plasma DNA. Although the precise mechanism by which plasma DNA enters into circulation is not known, significantly higher concentrations of plasma DNA appears to be due to neuronal and muscular degeneration in these patients. Identification of genes in plasma DNA, which are overexpressed or novel, can be a promising tool for the prognosis of these diseases.

Purification and characterization of a major 40 kDa outer membrane protein of Acinetobacter baumannii

Acinetobacter baumannii, an opportunistic pathogen, is well known to cause a wide spectrum of nosocomial infections particularly in intensive care units. The major outer membrane (OM) protein, OmpAb, of 40 kDa from A. baumannii has been identified and purified to homogeneity from cultures grown at 30°C and 100 mM NaCl. The synthesis of OM proteins of A. baumannii is thermoregulated and osmoregulated. The pore forming ability of the purified OmpAb and the diffusion of uncharged solutes in proteoliposomes has been demonstrated by following the liposomal swelling assay. The trimeric OmpAb is characterized as a porin with a pore size of 1.3 nm and is found to be similar to the OmpF of Escherichia coli and can possibly be classified as a general diffusion pore. It appears that OmpAb plays an important role in the diffusion properties of the outer membrane of A. baumannii.

Involvement of high mobility group B proteins in cisplatin-induced cytotoxicity in squamous cell carcinoma of skin.

Cis-diamminedichloroplatinum (II) (cisplatin) is a well-known anticancer drug with high potency and efficacy against various types of human cancers. Although it is widely accepted that the mechanism of cisplatin action is via apoptosis, there is enough evidence to support that cisplatin-induced cell death also occurs by other nonapoptotic pathways. Nonhistone, high mobility group (HMG) proteins are known to bind cisplatin-damaged DNA, and we studied their expression during cisplatin-induced cell death using immunohistochemistry, Western blot, and RT-PCR. Results show that the cell death is primarily apoptotic during initial stages of cisplatin treatment of skin tumors, and there is only marginal increase in high mobility group B (HMGB) levels, indicating that HMGB are still bound to nucleus. However, extended treatment of skin tumors with cisplatin caused necrosis and showed significantly increased levels of HMGB, which suggests that HMGB thus released from nuclei act as cytokine and trigger inflammatory response leading to necrosis. Present results clearly indicate a strong association between HMGB proteins and cisplatin-induced cell death that is dominantly apoptotic or necrotic depending on the duration of cisplatin exposure. Because of their important implication in the outcome of cancer chemotherapy, HMGB proteins can be interesting therapeutic targets.

A structural insight into major groove directed binding of nitrosourea derivative nimustine with DNA: a spectroscopic study.

Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas. The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) has been used to determine the binding sites of nimustine on DNA. Circular dichroism (CD) spectroscopy has been used to confirm conformational variations in DNA molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases guanine (C6 = O6) and thymine (C4 = O4) in DNA major groove. CD spectra of nimustine-DNA complexes point toward the perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction. Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about the drug’s action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea therapeutics with better efficacy and fewer side effects.

Evaluation of Skin Tumors by Magnetic Resonance Imaging

In vivo magnetic resonance imaging (MRI) is a powerful noninvasive technique in medical diagnosis; however, its application to analyze skin disorders is still at initial stages. To check whether MRI can be used as a noninvasive tool to analyze skin tumors, we carried out MRI of mice after treatment with benzo[a]pyrene (BP), a well known carcinogen. MRI was done on whole mice and was particularly focused on various layers and regions of interest of the skin: dermis, epidermis, and tumor. Initial MRIs of mice bearing skin tumors of 4, 8, 12, and 16 weeks after inducing BP clearly revealed the appearance of tumor. The MRIs of tumor-bearing mice with 20-week-old tumor development showed invasion to adjacent internal anatomic structures. The MRI data were in good agreement with the extent of cellular atypia and neoplastic changes that are typical of squamous cell carcinoma as noticed from the histopathologic findings. Therefore, MRI seems to have the potential to evaluate the tumor invasions equally well as that of histopathology or other clinical findings.

Interaction of actinomycin D with promoter element of c-met and its inhibitory effect on the expression of c-Met.

Abstract c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at −142 to −119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4–5 × 105 M−1 with ΔΔG of −47 ± 1.5 Kcalmol−1; (ii) marked increase by + 10 °C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Spl binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC50) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.