Education

Research Interests

Cancer is a complex disease where failure of a number of molecular pathways contributes to the disease progression. Understanding various cell signaling pathways involved in normal cells and dysregulated in disease progression (Cancer). I want to be a part of translational research aiming at improving current anticancer therapeutics. Chromatin remodeling, DNA damage and repair, stem cells & developmental biology are my major areas of interest. Metabolism and cell migration are most important aspects of tumor development and progression. Free radicals are considered as one of the primary cause of genetic mutations that may lead to cellular transformation. With my past experience with free radicals (ROS) and its relation to cancer, I would like to explore further the implication of ROS during cancer cell metastasis. Since various environmental factors like polluted drinking water may induce carcinogenesis, I intend to test various environmental factors (pesticides and other pollutants) which can cause DNA mutation directly or through ROS. I would also like to test the role of various naturally occurring antioxidants as anti-metastatic agents. Taken together my research plan will be concentrating on role of environmental factors in induction of ROS and subsequently tumor development and metastasis. Understanding role of naturally occurring antioxidants as anticancer agents will help us in reduction of cancer development by slight diet modifications. In research, Regulation of gene expression and the dynamic nature of chromatin has always been an interesting subject for me. I would like to work on gene regulation and chromatin modifications during proliferation, differentiation and disease manifestation. This complex regulation always involves various signaling pathways, thus I feel studying cell signaling is inherent part of studying chromatin remodeling. Another facet of molecular biology that interests me is the tools for gene silencing; RNAi and miRNA. These tools help in better understanding of involvement of particular protein in the complex signaling cascade, which ultimately helps us in delineating the molecular mechanisms governing various cellular events like gene regulation. I would like to explore the implication of miRNA in various disease conditions and develop it into potential therapeutic tool.

My current interests are related to the increased cancer incidence in Malwa region of Punjab. Currently I am working on the following aspects of carcinogenesis:

1. Understanding the carcinogenic potential of underground water

2. Understand the effect of antioxidants on cell migration: testing of natural occurring antioxidants.

3. Analysis of genetic variations in cancer patients of Malwa region of Punjab

4. Role of Environmental Factors in regulation of gene and miRNA expression during carcinogenesis

Research & teaching Experience:

1. Currently working as Assistant Professor at Centre for Human Genetics and involved in theory and practical training in advanced animal physiology, Cancer biology, Immunology, advanced genetics, research methodology, cell biology for MSc and MPhil Students.

2. Research Associate at Central University of Punjab, Bathinda. Theory and practical training in molecular biology, animal physiology, advanced genetics etc. subjects to M.Sc., M.Phil. & M.Pharma students. Oct 2011 - Aug 2012.

3. Postdoctoral fellow with Dr. Ya Huei kuo in hematopoietic stem cells and leukemia research, at City of Hope, Duarte, California, USA. The aim of the research was to delineate the molecular mechanisms associated with inversion-16 recombinant protein in Acute Myeloid Leukemia stem cells. March 2010 - Sept 2011

4. PhD in the field of Chromatin-Cancer biology at National Center for Cell Science (NCCS), Pune, India, under the guidance of Dr. Samit Chattopadhyay.
Title: To study SMAR1 interacting partners by yeast two hybrid and its role in cellular differentiation and tumorigenesis. Feb 2005- Feb 2010

Project Handled:

a. Identification of SMAR1 interacting partners using yeast two hybrid assay.

b. Role of SMAR1-AKR1a4 complex in regulation of free radical stress and tumorigenesis

c. Interplay between SMAR1 and p53 for GAD65 promoter regulation upon streptozotocin treatment.

d. Regulation of SMAR1 expression upon NGF induced neuronal differentiation of PC12 cells

5. Worked as Research Staff in PGIMER, Chandigarh in NIH funded project “Marijuana Abuse: cognition, Neuroimaging and Endocrine”. Worked on detection of SNPs as well as serum analysis and their correlation to the drug abuse with Prof. Madhu Khullar. 2004-2005

6. M. Sc. Thesis “Differential regulation of apoptosis in male and female murine peritoneal macrophages” With Prof. Tapas Mukhopadhyay. Analyzed the effect of heat shock on male and female mice peritoneal macrophages and showed that male macrophages are more prone to heat shock induced apoptosis compared to the female mice peritoneal macrophages. 2002-2004

Publications:

1. Singh S, Raina V, Sreenath K, Pavithra L and Chattopadhyay S. Regulation of GAD 65 promoter by SMAR1. BMC Molecular Biology Accepted for publication (Corresponding author).

2. Singh S, Sreenath K, Pavithra L, Roy S, Chattopadhyay S. SMAR1 regulates free radical stress through modulation of AKR1a4 enzyme activity. Int J Biochem Cell Biol. 2010; 42(7):1105-14. PMID: 20097305

3. Kaul-Ghanekar R, Singh S, Mamgain H, Jalota-Badhwar A, Paknikar KM, Chattopadhyay S. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study. BMC Cancer. 2009; 9:350. PMID: 19799771

4. Sreenath K, Pavithra L, Singh S, Sinha S, Dash PK, Siddappa NB, Ranga U, Mitra D, Chattopadhyay S. Nuclear matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling. Virology. 2010 Apr 25;400(1):76-85. PMID: 20153010

5. Pavithra L, Sreenath K, Singh S, Chattopadhyay S. Heat-shock protein 70 binds to a novel sequence in 5' UTR of tumor suppressor SMAR1 and regulates its mRNA stability upon Prostaglandin A2 treatment. FEBS Lett. 2010 Mar 19;584(6):1187-92. PMID: 20153327

6. Pavithra L, Singh S, Sreenath K, Chattopadhyay S. Tumor suppressor SMAR1 downregulates Cytokeratin 8 expression by displacing p53 from its cognate site. Int J Biochem Cell Biol. 2009;41(4):862-71. PMID: 18822384

7. Singh S, Dubash T, Sreenath K and Chattopadhyay S. Regulation of SMAR1 expression upon NGF induced differentiation of PC12 cells. Manuscript submitted (corresponding author).

8. Dhar A, Mallick S, Ghosh P, Ahmed I, Bhattacharyya S, Mandal T, Manna A, Singh S, Wilder P, Markowitz J, Weber D, Ghosh M, Chattopadhyay S, Bandyopadhyay S, Roy S. A helix mimetic peptide against S100B reactivates p53 regulatory circuit in human melanoma cells causing rapid apoptosis. Submitted.

9. Sreenath K, Singh S, Pavithra L, Ranga UK, Mitra D and Chattopadhyay S. SMAR1 inhibits Transactivation activity of HIV Tat through deacetylation and inhibiting its interaction with coactivator TAP. Submitted.

10. Pavithra L, Sreenath K, Singh S and Samit Chattopadhyay. SMAR1, a target of ATM kinase regulates p53 activation in response to DNA Damage. Submitted.

Awards/Merits

1. Qualified joint CSIR-UGC National Eligibility Test (NET) for Junior Research Fellowship, held in June 2004.

2. Invited Oral Speaker in Graduate Student Meet 2007 and 2008 held in ACTREC.

3. Won Best poster presentation award in “International Symposium on Intermediate Filament Proteins: Recent Trends in Keratin Biology” held on September 28-29, 2007 in ACTREC, Navi-Mumbai.

4. Won travel award for poster presentation in ‘9th International Congress of Cell Biology & 20th Annual Conference of the Korean Society for Molecular and Cellular Biology’ eld on 7-10 October 2008 in Seoul, South Korea.

5. Post Doctoral Fellow at City of Hope National Medical Centre, California, USA (March 2010-September 2011).

6. Invited Guest Lecture in workshop on training in molecular biology techniques for faculty organized by, Panjab University, Chandigarh.

7. Invited Guest Speaker at 1st North States Conference on An Anatomy of Ageing held on 25th August 2012 at Desh Bhagat Dental College at Sri Muktsar Sahib.

Meetings and Conferences

1. Participated in Indian Science Congress meeting held in 2004 at Chandigarh.

2. Poster presentation in All India Cell Biology Conference December 2004 held at Chandigarh.

3. Poster presentation in “International Symposium on Intermediate Filament Proteins: Recent Trends in Keratin Biology” held on September 28-29, 2007 in ACTREC, Navi-Mumbai.

4. Poster presentation in XXXI All India Cell Biology Conference & Symposium on ‘Stem cells: Applications and Prospects’ held on December 14-16, 2007 in Department of Zoology, Banaras Hindu University, Varanasi.

5. Oral talk in Graduate student meet 2007 entitled “SMAR1 regulates AKR1a4 enzyme activity”

6. Poster presentation in ‘The 9th International Congress of Cell Biology & The 20th Annual Conference of the Koran Society for Molecular and Cellular Biology’ held on 7-10 October 2008 in Seoul, South Korea.

7. Invited Oral talk in Graduate student meet 2008.

8. American Society of Hematology (ASH 2010) meet held on December 2010 in Orlando, Florida, USA.

Summary (Post doctoral work)

Title: Molecular Mechanisms associated with inversion-16 in Acute Myeloid Leukemia.

Genetic alterations play an important role during Leukemogenesis. As a Post doctoral fellow, my research is focused on understand the various signaling pathways affected by the inversion-16 recombinant protein. Using normal and leukemic mouse models, I am looking into the differences between normal and leukemic stem cells in the bone marrow. Within a short period of time, I am able to delineate various novel interacting partners, transcriptional targets as well as miRNAs associated with inversion-16 recombinant protein. My further research implicate inversion 16 in induction of leukemogenesis through induction of DNA damage associated mutagenesis and repression on anticancer proteins like p53. I have further shown that inversion 16 induces elevated free radical levels which in-turn leads to DNA mutations.

Summary (Ph.D. thesis)

Title: To study SMAR1 interacting partners by yeast two hybrid and its role in cellular differentiation and tumorigenesis.

The matrix attachment region binding proteins (MARBPs) help in tethering as well as functional and structural organization of chromosomal DNA through biding to matrix attachment regions (MARs). This dynamic spatial organization plays an important role in DNA replication, transcription and DNA damage repair. I have been working with Dr. Samit Chattopadhyay at NCCS. My PhD work was to screen for SMAR1 interacting partners and its role in disease manifestation. During my work we developed a novel in vitro DNA pull down assay to look for DNA sequences bound by SMAR1. After screening of more than 150 different sequences a consensus sequence for SMAR1 binding was made using MEME prediction. We also identified that SMAR1 binds to GAD65 promoter but it does not bind the other isoform GAD67 that is located on different chromosome. Further studies using EMSA and ChIP showed that SMAR1 leads to up-regulation of GAD65 promoter upon streptozotocin treatment in rat insuloma cell line. This work for the first time shows role of SMAR1 as a transcriptional regulator of GAD65. In another project during my PhD, using yeast two hybrid assay, several SMAR1 interacting proteins like AKR1a4, SRm160, Hoxd13, Fbxo40 etc were identified. My work showed that SMAR1 interacts with AKR1a4 enzyme in the cytoplasm and inhibits its enzyme activity. DNA damage leads to disruption of this complex due to ATM dependent phosphorylation and nuclear translocation of SMAR1. Upon cellular transformation and in higher grades of breast cancer, SMAR1 expression is lost leading to hyperactivity of AKR1a4 enzyme thus discovering the free radical scavenging properties of this metabolic enzyme. Using erythrocytes as model system, a small peptide inhibitor for AKR1a4 enzyme activity was developed which may be proved useful therapeutic tool for anticancer and other metabolic disorders.
In the third project during my PhD, regulation of SMAR1 expression upon NGF induced neuronal differentiation of PC12 was studied. My work shows that SMAR1 expression is elevated upon NGF treatment through ATM. Further SMAR1 expression is regulated through Trk A receptor and not by p75NTR. I also studied the regulation of SMAR1 expression during various stages of embryogenesis using confocal microscopy.

Relevant scientific techniques and skills acquired

Hematopoietic/ Leukemic stem cell culture and bone marrow transplantation

Protein-protein interaction studies using the Yeast two hybrid screening, pull down, immuno-precipitation,

precipitation assays and Fluorescence anisotropy

Production, purification and standardization of polyclonal antibodies in Rabbits.

Protein extraction and purification by FPLC (Both His and GST tagged systems).

Cell culture maintenance, transfection, transductions and construction of stable cell lines and maintenance of hybridomas.

Isolation and purification of T Cells, monocytes and macrophages from human blood and primary cell culture.

Studies of DNA protein interaction by Gel Shift assays, South-Western blotting, in-vitro and in-vivo Matrix binding assays and chromatin Immunoprecipitation (ChIP) assays.

Purification of histones, HDAC assays and in vitro chromatin assembly studies, kinase and acetylation assays.

Protein identification and post-translational modification using Mass Spectrometry and 2-D gel electrophoresis.

Analysis and evaluation of Southern, Northern and Western blots.

Genomic analysis (cloning, elaboration of restriction maps, gene structures analysis, reconstruction of gDNAs)

Isolation of DNA and RNA, Primer designing, PCR & RT PCR, agarose gel electrophoresis.

Cell viability, proliferation, migration, co-migration, invasion, colonigenecity assays

RNase protection assays, RNA-pull down assays, RNA-IP (RIP).

Purification of Nuclear Matrix, mitochondria and other cellular organelles.

Fluorescence and confocal microscopy and Atomic Force Microscope related techniques, in-situ Nuclear Matrix staining, FISH.

Multicolor FACS analysis and sorting of live cells (up-to 8 colors).

In-vitro DNA pull down assay and CNBr pull down assay.

Analysis of cell surface and DNA-protein complexes using atomic force microscopy