Sailaja V. Elchuri

Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM +ve/let-7bdown-regulated), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM −ve/let-7bup-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM +ve/let-7bup-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

Synthesis and characterization of surface-enhanced Raman-scattered gold nanoparticles.

In this paper, we report a simple, rapid, and robust method to synthesize surface-enhanced Raman-scattered gold nanoparticles (GNPs) based on green chemistry. Vitis vinifera L. extract was used to synthesize noncytotoxic Raman-active GNPs. These GNPs were characterized by ultraviolet-visible spectroscopy, dynamic light-scattering, Fourier-transform infrared (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Raman spectroscopy. The characteristic surface plasmon-resonance band at ~ 528 nm is indicative of spherical particles, and this was confirmed by TEM. The N–H and C–O stretches in FTIR spectroscopy indicated the presence of protein molecules. The predominant XRD plane at (111) and (200) indicated the crystalline nature and purity of GNPs. GNPs were stable in the buffers used for biological studies, and exhibited no cytotoxicity in noncancerous MIO-M1 (Müller glial) and MDA-MB-453 (breast cancer) cell lines. The GNPs exhibited Raman spectral peaks at 570, 788, and 1,102 cm−1. These new GNPs have potential applications in cancer diagnosis, therapy, and ultrasensitive biomarker detection.

Proteomic profiling of retinoblastoma by high resolution mass spectrometry

BackgroundRetinoblastoma is an ocular neoplastic cancer caused primarily due to the mutation/deletion of RB1 gene. Due to the rarity of the disease very limited information is available on molecular changes in primary retinoblastoma. High throughput analysis of retinoblastoma transcriptome is available however the proteomic landscape of retinoblastoma remains unexplored. In the present study we used high resolution mass spectrometry-based quantitative proteomics to identify proteins associated with pathogenesis of retinoblastoma.MethodsWe used five pooled normal retina and five pooled retinoblastoma tissues to prepare tissue lysates. Equivalent amount of proteins from each group was trypsin digested and labeled with iTRAQ tags. The samples were analyzed on Orbitrap Velos mass spectrometer. We further validated few of the differentially expressed proteins by immunohistochemistry on primary tumors.ResultsWe identified and quantified a total of 3587 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 899 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 402 proteins were upregulated and 497 were down regulated. Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1), chromogranin A, fetuin A (ASHG), Rac GTPase-activating protein 1 and midkine that were found to be overexpressed in retinoblastoma were further confirmed by immunohistochemistry by staining 15 independent retinoblastoma tissue sections. We further verified the effect of IGF2BP1 on cell proliferation and migration capability of a retinoblastoma cell line using knockdown studies.ConclusionsIn the present study mass spectrometry-based quantitative proteomic approach was applied to identify proteins differentially expressed in retinoblastoma tumor. This study identified the mitochondrial dysfunction and lipid metabolism pathways as the major pathways to be deregulated in retinoblastoma. Further knockdown studies of IGF2BP1 in retinoblastoma cell lines revealed it as a prospective therapeutic target for retinoblastoma.

Identification of effective substrates for the direct analysis of lipids from cell lines using desorption electrospray ionization mass spectrometry.

RATIONALE Various disease conditions, particularly tumours, can be understood easily by studying changes in the lipid profile of cells. While lipid profiles of tissues have been recorded by desorption electrospray ionization mass spectrometric (DESI-MS) imaging, there is paucity in standardized protocols for sample preparation involving cell cultures to generate reliable results. In this study, we report a method for the direct analysis of lipids from cultured cells by incorporating them onto Whatman 42 filter paper as a substrate for reliable DESI-MS analysis. METHODS The WERI-RB1 cell line was spotted on commonly used substrates for DESI-MS analysis, such as glass slides, Teflon coated glass slides, thin layer chromatography (TLC) plates, and Whatman 42 filter paper. A comparison of mass spectrometric images with two different lipids was made to understand the behaviour of different surfaces when the same sample was spotted on them. Relative intensities of different lipid peaks in the WERI-RB1 cell line were compared and relative lipid abundances were also compared across two different human retinoblastoma cell lines; WERI-RB1 and Y79. RESULTS The study demonstrates that good lipid signals can be obtained by DESI-MS when the cells are spotted on Whatman 42 filter paper. Tandem mass spectrometry was performed to identify the lipids as glycerophosphocholines (PC). Better lipid images from assembly of cells were obtained with distinct boundary when they were spotted on Whatman 42 filter paper than other surfaces. CONCLUSIONS We demonstrate the use of a simple substrate for reliable DESI-MS analysis of cultured cells. This method has the potential to understand various interactions of cells with other external agents. The current method would help in the application of DESI-MS for biology in general and medical sciences in particular.

Membrane Proteome of Invasive Retinoblastoma: Differential Proteins and Biomarkers

Purpose: Retinoblastoma (RB) is a pediatric ocular cancer which is caused due to the aberrations in the RB1 gene. The changes in the membrane proteomics would help in understanding the development of the retinoblastoma and could identify candidates for biomarkers and therapy.

Phosphoproteomics of Retinoblastoma: A Pilot Study Identifies Aberrant Kinases

Retinoblastoma is a malignant tumour of the retina which most often occurs in children. Earlier studies on retinoblastoma have concentrated on the identification of key players in the disease and have not provided information on activated/inhibited signalling pathways. The dysregulation of protein phosphorylation in cancer provides clues about the affected signalling cascades in cancer. Phosphoproteomics is an ideal tool for the study of phosphorylation changes in proteins. Hence, global phosphoproteomics of retinoblastoma (RB) was carried out to identify signalling events associated with this cancer. Over 350 proteins showed differential phosphorylation in RB compared to control retina. Our study identified stress response proteins to be hyperphosphorylated in RB which included H2A histone family member X (H2AFX) and sirtuin 1. In particular, Ser140 of H2AFX also known as gamma-H2AX was found to be hyperphosphorylated in retinoblastoma, which indicated the activation of DNA damage response pathways. We also observed the activation of anti-apoptosis in retinoblastoma compared to control. These observations showed the activation of survival pathways in retinoblastoma. The identification of hyperphosphorylated protein kinases including Bromodomain containing 4 (BRD4), Lysine deficient protein kinase 1 (WNK1), and Cyclin-dependent kinase 1 (CDK1) in RB opens new avenues for the treatment of RB. These kinases can be considered as probable therapeutic targets for RB, as small-molecule inhibitors for some of these kinases are already in clinical trials for the treatment other cancers.

RNA-sequencing of primary Retinoblastoma tumors provides new insights and challenges into tumor development

Retinoblastoma is rare tumor of the retina caused by the homozygous loss of the Retinoblastoma 1 tumor suppressor gene (RB1). Loss of the RB1 protein, pRB, results in de-regulated activity of the E2F transcription factors, chromatin changes and developmental defects leading to tumor development. Extensive microarray profiles of these tumors have enabled the identification of genes sensitive to pRB disruption, however, this technology has a number of limitations in the RNA profiles that they generate. The advent of RNA-sequencing has enabled the global profiling of all of the RNA within the cell including both coding and non-coding features and the detection of aberrant RNA processing events. In this perspective, we focus on discussing how RNA-sequencing of rare Retinoblastoma tumors will build on existing data and open up new area’s to improve our understanding of the biology of these tumors. In particular, we discuss how the RB-research field may be to use this data to determine how RB1 loss results in the expression of; non-coding RNAs, causes aberrant RNA processing events and how a deeper analysis of metabolic RNA changes can be utilized to model tumor specific shifts in metabolism. Each section discusses new opportunities and challenges associated with these types of analyses and aims to provide an honest assessment of how understanding these different processes may contribute to the treatment of Retinoblastoma.

Characterization and Molecular Mechanism of Peptide-Conjugated Gold Nanoparticle Inhibiting p53-HDM2 Interaction in Retinoblastoma

Inhibition of the interaction between p53 and HDM2 is an effective therapeutic strategy in cancers that harbor a wild-type p53 protein such as retinoblastoma (RB). Nanoparticle-based delivery of therapeutic molecules has been shown to be advantageous in localized delivery, including to the eye, by overcoming ocular barriers. In this study, we utilized biocompatible gold nanoparticles (GNPs) to deliver anti-HDM2 peptide to RB cells. Characterization studies suggested that GNP-HDM2 was stable in biologically relevant solvents and had optimal cellular internalization capability, the primary requirement of any therapeutic molecule. GNP-HDM2 treatment in RB cells in vitro suggested that they function by arresting RB cells at the G2M phase of the cell cycle and initiating apoptosis. Analysis of molecular changes in GNP-HDM2-treated cells by qRT-PCR and western blotting revealed that the p53 protein was upregulated; however, transactivation of its downstream targets was minimal, except for the PUMA-BCl2 and Bax axis. Global gene expression and in silico bioinformatic analysis of GNP-HDM2-treated cells suggested that upregulation of p53 might presumptively mediate apoptosis through the induction of p53-inducible miRNAs.

Vallabhaneni Sita Rama Das, 1933–2010: teacher and mentor

Abstract We present here the life and research of V. S. Rama Das, a distinguished Indian botanist who specialized in photosynthesis. He was the first to purify chloroplasts that were free of mitochondrial contamination. He then studied C4, C3–C4 intermediate and CAM pathways, as well as their taxonomic distribution in tropical climates. His most valuable legacy is that he, as a philosopher, inspired and guided many students to pursue their research career in India. Also see Narayana and Pullaiah (Eminent Indian Botanists: Past and present: Biographies and contributions, pp 394–401, 2010) and Raghavendra and Reddy (Curr Sci 101:798–799, 2011) for further information on Rama Das.