Basic Research programs of the Cancer Research Institute have seen the initiation of several novel projects by new Principal Investigators, during 2011-12. These include the study of stem cells in skin, solid and haematological malignancies, monitoring of chemo and radiation resistance in vitro and in vivo using complex vector systems, establishment of in vivo imaging techniques for diagnosis and treatment, monitoring of complex intra and intercellular molecular networks using confocal microscopy, monitoring of the distribution of specific labelled molecules using micro PET-SPECT–CT to evaluate their diagnostic and therapeutic potential, and characterization of somatic mutations in cancer using an integrated NextGen sequence approach combined with high-end computing and bioinformatics.
On-going molecular profiling investigations have moved ahead towards defining signatures for risk assessment and prognosis in cancers of the oral cavity, cervix, breast and brain, and acute lymphocytic leukemia. Investigations into cancer biology have provided insights into the role of histone modifications in gene regulation, and that of specific molecules/ molecular networks in regulating the cell cycle, DNA damage and repair, and cell transformation. A study examining the role of glycosylation in cell invasion and metastasis has revealed the intricacies of sugars and their engagement with cell surface molecules. The study of gamma delta T cells has provided insights into immune surveillance mechanisms in cancer. In-depth analysis of the chemopreventive action of curcumin and tea polyphenols has led to the identification of mechanisms that operate in vivo. Systematic biophysical investigations have revealed key structural signatures for proteasomal recognition and degradation, apoptotic regulation, BRCA1/2 interacting partners involved in breast cancer, and the utility of Raman spectroscopy as a non invasive diagnostic tool.
Head and Neck Cancers form the bulk of cancers affecting males reporting at the Tata Memorial Hospital. High-resolution array comparative genomic hybridization analysis of advanced stage oral cancers reveals that chromosomal gain of region 11q22.1-q22.2 and 11q23-q25 is associated with loco-regional recurrence and shorter survival. Driver genes, associated with oral carcinogenesis and shorter survival, have been identified. Variant alleles of GSTM1 and XPD variant alleles, both independently and in combination, appear to serve as important predictors of clinical outcome in radiotherapy-treated OSCC patients.
The role of anti-apoptotic protein Mcl-1L in survival and radiosensitivity of oral cancer cells is being investigated using RNAi strategy, alone or combined with ionizing radiation and/or chemotherapy. Assessment of Mcl-1L expression in various oral cell lines revealed predominant over expression of anti-apoptotic Mcl-1Lvis a vis pro-apoptotic isoforms in AW8507 and SCC25. siRNA mediated down regulation of Mcl-1L in AW8507 cells alone or with irradiation led to significant (p<0.002) apoptosis accompanied by upregulation of pro-apoptotic Bax protein. A synergistic reduction in clonogenic survival of AW8507 cells was noted. Mcl-1 expression thus influences survival and radiosensitivity of oral cancer cells.
Tumor cell resistance to ionizing radiation and chemotherapy are major obstacles in cancer therapy. Identification of genes/ proteins that are differentially expressed in radiosensitive vs radioresistant cancer cells could help predict the clinical effectiveness of radiotherapy. A radioresistant sub line AW13516-70Gy was established. To undersand the mechanism of radioresistance, the proteins differentially expressed in these cell lines were identified by 2DE and mass spectrometry.
Oral cancer tissues show altered expression of survivin and clusterin. Since both proteins are repressed by p53, their regulation was examined in oral cancers where p53 is mutated. Analysis of five survivin splice variants in 13 oral/ cervical cancer cell lines revealed consistent higher expression of wild type survivin in all the cell lines. Levels of survivin 3B were comparatively high in cervical cancer cell lines, while survivin 2a levels were significantly higher in oral cancer cell lines. Secretory clusterin expression was higher in all the cell lines; nuclear clusterin expression was not detected. Amongst the p63 isoforms, ∆Np63 showed higher RNA expression compared to TAp63, but p63 protein was detected in just three cell lines. p73 transcript analysis showed higher TAp73 levels compared to ∆Np73. Expression of various isoforms in normal and tumor tissues will be examined next.
Patients with cancers of the upper aerodigestive tract often present with multiple primary neoplasia (MPN). Correlation was established between the genotype and molecular phenotype following genotoxic exposure in vitro of lymphoblastoid cell lines generated from MPN patients having cancers of the upper aerodigestive tract (UADT). Twenty two SNPs in candidate genes involved in DNA repair, apoptosis, cell cycle regulation and xenobiotic metabolism were analyzed and a genotype score (G score) from the number of variant alleles was created. Data suggests that the extent of DNA repair, percent cell death and cell cycle delay might be able to identify susceptibility to UADT MPN. Also, differentiation of study participants into subsets based on a distinctive polymorphism-based G score signature might have an important bearing on predisposing an individual to UADT MPN and also in designing chemoprevention studies.
Using an immunoproteomics approach, the utility of autoantibody response to tumor antigens as diagnostic and prognostic markers for cancer is being assessed in the sera of patients with gingivo buccal complex cancer, subjects with leukoplakia, and individuals with chewing habits. Antibody response to Hsp70 and a-enolase correlated significantly with disease free survival of patients, indicating its potential as an additional serological prognostic marker for GBC cancer. Recombinant proteins for identified tumor antigens were expressed and purified to generate a multiplex array for further studies.
ACTREC along with NIBMG has developed Indias participation in the most ambitious international cancer collaborative study ever conducted - The International Cancer Genome Consortium (ICGC). The Indian component of the ICGC focuses on Oral Cancer. The ICGC aims to establish a comprehensive catalogue of Genomic & other abnormalities in 500 tumours each of 50 different Cancer types of Clinical & Societal importance across the globe. India, under the direction and funding from the Dept. of Biotechnology has taken up Comprehensive Genomic and associated analyses of the most prevalent Indian Cancer - Gingivo-Buccal Squamus Carcinoma. A third of the planned 500 cases have been accrued, annotated and biobanked at ACTREC with ongoing work of deep resequencing at NIBMG and associated methylation, HPV, IHC and functional analyses at ACTREC.
Centre for Advanced Research for Cancer Genetics and Genomics at ACTREC. It now serves as the Apex National Referral Centre for comprehensive clinical and laboratory genetic services and genetics research for all major cancer predisposition syndromes and a model of Comprehensive Cancer Genetics Unit with dedicated daily Genetics Clinic and a Cancer Genetics Lab. Best international practices in counselling, genetic testing, screening & medical management have been developed and tailored for Psycho-Social-Cultural dynamics unique to Indian families. It is the only unit in the country which conducts and issues clinically relevant reports with advice for clinical management for carriers of germline mutation in a wide range of cancer associated genes (BRCA1, BRCA2, RET, TP53, RB, CHK2, XP, MMR etc). With 1600 such pedigrees, DNA Bank of 2000 cases & EBV cell lines from 500 cases, it is one of the largest collection of clinical & research resources in diverse hereditary cancers outside USA & Europe. Several founder, novel and recurrent germline mutations have been identified in various common and rare hereditary cancer predisposition syndrome with public health relevance.
Cervical cancer is one of the major cancers affecting females. On-going molecular profiling studies aim at identifying genes affecting disease prognosis. Pretreatment samples of stage IIIB cancer were analyzed using DNA microarray with Agilent 4*44K chip. Samples were grouped into those with no evidence of disease after a median follow up of 65 months or disease recurrence during the follow up period. The gene set obtained after analysis could clearly differentiate good and bad prognosis groups, predicting the prognosis of test samples with 87.5% accuracy. These genes are being validated by real time PCR. In parallel, studies were undertaken to identify genetic alterations in locally advanced (stage IIIB) cervical cancer and correlate the data with clinical outcome after radiotherapy or concomitant chemo radiation. Putative somatic variations were identified using appropriate software, and the genes and their functional implications were predicted. Selected variations, that could serve as biomarkers of cervical cancer and help evaluate drug response or identify potential targets for therapy, are being validated by DNA microarray, PCR and Sanger sequencing.
Genetic polymorphisms that affect pro-/anti- inflammatory cytokine levels and disease risk are being studied in Maharashtrian pre menopausal breast cancer patients and age/ community matched healthy controls. Analysis of genotype combinations of TGF beta and its receptor TGFBR1 revealed lower frequency of weak responders amongst Maharashtrians compared to Parsis or Caucasians. A beneficial effect of higher anti-inflammatory cytokine levels was suggested. Sequence based HLA typing of 41 breast cancer patients and >50 healthy Parsi controls highlighted both distinctness and genetic diversity of this community, with 8-10 alleles covering 80% of the population. Certain enriched alleles and haplotypes (with relative linkage disequilibrium = 1) indicate limited genetic pool and endogamy, while the presence of unique haplotypes and classical Indian alleles like A*0211, A*3301, B*4006 and B*35 variants (B*3504, 3508, etc.) suggest genetic admixture. Both allele and haplotype frequencies highlight the distinct identity of Parsis vis a vis Caucasians or Orientals.
The most frequent HLA allele in the Indian population is the ‘HLA-B44 supertype’ family allele, HLA-B*4006. Its amino acid sequence and structural considerations suggest a small, poorly hydrophobic ‘F’ pocket that might adversely affect interaction with the C terminal residue of the antigenic peptide. Analysis of the modeled structures and HLA-peptide complexes strongly support the adverse effect of Trp95 at pocket F and possible role of the third residue of the antigenic peptide (P3) as an auxiliary anchor in HLA-B*4006 peptide complexes. In the light of suggested promiscuous peptide binding pattern of this allele and association with risk for TB/ AIDS, these findings of preliminary in silico analysis have helped define a direction for wet-lab studies.
Gastric carcinoma remains the fourth most common cancer and ranks second vis a vis cancer deaths worldwide. Epigenetics has recently drawn increasing attention in gastric carcinogenesis. Epigenetic alterations, differential histone modifications and variants are being studied in human gastric cancer to define ‘true cut margin’, to correlate etiological and clinical parameters, and to identify patients who would benefit from adjuvant treatment after an apparently curative resection. Preliminary data from 36 samples shows the presence of H. pylori gene urease A in 80% samples, 65%of which are positive for cagA gene confirming infection with pathogenic strains of H. pylori. Site-specific modification pattern of histone H3 and H4 reveals aberrant patterns seemingly associated with the transformed phenotype, which may have implications in histone-related biomarkers and better treatment modalities.
T acute lymphocytic leukemia (T-ALL) is a phenotypically and clinically heterogeneous disease with a high frequency of treatment failure. On-going studies have identified a distinct subgroup of Indian T-ALL patients with clonal TCRgd+ gene rearrangements that exhibit improved disease free survival and overall survival. Using cDNA microarray technology, 1234 differentially expressed genes were identified. Unsupervised clustering of T-ALL samples resulted in clear differences in immune synapse related genes in gd+T-ALL vs. ab+T-ALL. Results suggest that gd TCR+ leukemic blasts may form better immune synapse with effector cells and thus are killed more efficiently in gd+ T-ALL patients. Analysis of molecules involved in immune synapse formation and validation of expression of respective genes is in progress.
Medulloblastomas are highly malignant brain tumors occurring predominantly in children. Of the four molecular subgroups of medulloblastoma, one is associated with the activation of WNT signalling and another with SHH signalling, while the other two novel subgroups have an overlapping gene signature that is not associated with any known oncogenic/ developmental pathway. Molecular sub-grouping of an independent set of 86 paraffin-embedded tumour tissues was done using a select set of protein-coding genes and miRNAs as markers. Several miRNAs were found to be up-regulated in the WNT signaling subgroup – which has lower metastatic potential and better survival. Examination of the role of specific genes and miRNAs whose expression is deregulated in medulloblastoma revealed that miR-193a, an miRNA up-regulated specifically in WNT subgroup medulloblastomas, inhibits proliferation and anchorage-independent growth of medulloblastoma cells belonging to other subgroups. Gliomas account for almost 80% of primary malignant brain tumours. Glioblastomas are grade IV astrocytic gliomas which are highly resistant to apoptosis. Genome-wide expression profiling of glioblastoma tissues is being undertaken to correlate findings with response to temozolomide treatment.
A new program has been undertaken which involves integrated characterization of somatic alterations of cancer specimens by performing genome-wide analysis of copy number changes using SNP arrays, genomic re-sequencing using next generation sequencing platforms, and low throughput loss-of-function pooled RNAi mediated genetic screen using tumor derived cell lines established in-house. Research effort also focuses on experimental evaluation of functional relevance of somatic alterations identified by genomic approaches, using molecular and cellular approaches.
One of the areas of research is on understanding the classical caspase-dependent apoptotic pathways with an aim at targeting them for disease intervention. Recent reports point to novel extrinsic adapter-independent and caspase-independent mechanisms of apoptosis, mediated by proapoptotic proteins - human papillomavirus E2 and serine protease HtrA2 respectively.
Cervical cancer, the second most common cause of cancer related mortality in women, is caused by human papillomaviruses (HPV). HPV-16 E2 protein has recently been found to induce apoptosis in both HPV positive and negative cell lines. Although the exact role of E2-induced apoptosis is not known, its interaction with p53 (in both HPV+ and HPV- cell lines) and viral DNA binding ability (in HPV+ cell line only) have been implicated. Based on this information, a hypothesis has been developed that an E2 mutant incapable of p53 binding but with enhanced DNA-binding ability would be a potent apoptosis inducer only in HPV positive cells. Using docking studies, residues critical for binding of p53 and E2 C-terminal domain have been identified and mutants of E2C designed. Viral DNA binding mutants have also been designed. Experiments to evaluate their action on HPV positive cells are on-going.
The mitochondrial serine protease HtrA2 was primarily identified as a proapoptotic molecule that antagonizes inhibitor of apoptosis proteins (IAPs) by activating caspases. Recent findings indicate that HtrA2 is also capable of inducing apoptosis in a caspase-independent manner via its serine protease activity; the mechanism needs characterization. Recombinant HtrA2 wildtype and IBM site/ PDZ peptide groove mutants as well as IAP substrates (XIAP and cIAP1) were purified. Experiments showed that the IBM site is essential for interaction of IAPs to HtrA2. Global conformational change occurring upon N-terminal binding of IAPs was compared with C-terminal binding, with the active site orientation being monitored in both cases. Thus, intricate conformational changes and proper active site conformation play a role in regulating HtrA2 functions. Intricate dissection of its structure and dynamics and interactions with its binding partners is on, using biophysical, structural, proteomics and molecular biology probes.
Synthesis of proteins, their ability to change from a linear polypeptide chain into a final folded structure, the specific functions that they carry out and finally their degradation are tightly and spatio-temporally controlled processes. Any aberration in these processes can lead to patho-physiological conditions. On-going projects examine the structural, mechanistic and cell biological aspects of protein degradation by a self compartmentalized ubiquitous, ATP-dependent regulatory protease called the proteasome, and the ATP dependent functions of human 14-3-3 zeta a potential protein disaggregating chaperone and, using the prediction program PNSAS developed in-house, aim to predict natural substrates of proteases. On-going investigations focus on the proteasomal subunits gankyrin and PSMD9. Using structural insights and bioinformatic tools, conservation of short sequences within non-homologous proteins has been identified to discover putative interacting partners of two subunits of the proteasome. Coupled with microarray, proteomic studies, available X-ray crystal structures, modeling and docking studies, these predictions are being validated using in vitro systems and cell based assays.
The Breast Cancer susceptibility genes 1 and 2 (BRCA1/2) play a major role in predisposing carriers to early-onset breast and ovarian cancer. Several cellular proteins are reported to bind to BRCA1/2 and regulate transcription. To understand the complex interaction between the binding partners, several functional domains of BRCA1 and its binding partners BARD1, MDC1 and BAP1 have been cloned, proteins of interest have been purified and efforts are on to obtain their crystals. Pathogenicity of mutant proteins identified in Indian breast cancer families and those from the Breast Cancer Information Core (BIC) will be characterized using bioinformatics, biophysics and structure biology tools. Receptor associated protein 80 (RAP80) has several functionally important domains, and interacts with BRCA1 through an intermediate binding partner. Functional domains of RAP80 and the pathogenic mutants associated with them have been cloned and purified. Full length MERIT40 and several of its functional domains have been cloned, expressed and purified. Functional characterization using biophysical approach has revealed that MERIT40 has properly folded domains. Protein-protein interactions have been analyzed using ITC, BIACORE, and crystallization of purified proteins is under way.
The association between inflammation and cancer is well established. In silico analysis of proteolysis of breast epithelial membrane mediated by cathepsin G is under way. Data on the proteome of normal and cancerous breast epithelium was obtained from the Human Protein Atlas database. Searches were performed for membrane proteins of normal and cancerous breast tissue. The specificity matrix (PSSM) of protease cathepsin G was developed using information from the Merops database. Protease specificity (PoP’s) program was used to predict the cleavage sites in the membrane proteins of the breast tissue using the matrix. Proteins predicted as potential substrates for cathepsin G are being validated. In vitro experiments have been initiated to study the effect of neutrophilic proteases on MCF 10A breast cancer cells.
Keratin 8/18 - the markers of simple epithelia, are reported to be aberrantly expressed in cancer of the buccal mucosa. Immunodetection had revealed differences in the K18 expression pattern in transformed buccal epithelium and adjacent normal tissue. Mass spectrometric assessment of this pattern showed that the molecule identified as K18 by immunodetection was actually K13, indicating that the antibody K18 (CY90) cross reacts with the K13 protein. The keratin profile of tumor and normal tissue of buccal mucosa was therefore re-assessed by mass spectrometry. Results revealed down regulation of K13 and consistent appearance of K14, 16 and 17 in tumor samples, which was validated by immunohistochemistry. The study indicates the need to confirm immunodetection data on families of homologous proteins like keratins with a non-antibody based approach such as mass spectrometry.
Aberrant expression of the structural proteins K8/18 has been shown to contribute to the malignant transformation of stratified epithelial cells. K14 is normally expressed by basal proliferating cells of stratified epithelia. To understand their role in transformation, K8 and K14 expression was knocked down in oral cancer cell line AW 13516 and HaCat cells respectively using shRNA. K8 knock down cells showed reduced number and size of colonies in soft agar, slow growth rate and reduction in the size of tumors in nude mice. Down regulation of desmoplakin, fascin and β4-integrin expression, and reduction of filamentous actins like filipodia and microspike formation was also noted. Over expression of fascin in AW13516 cells (OSCC) resulted in alterations in actin cytoskeleton and a more migratory phenotype. Transfection of phosphorylation mutants of K8 in AW 13516 and K8 knocked down cells resulted in increased motility and tumorigenicity. K14 knock down cells showed a significant reduction in cell proliferation, and increased cell differentiation. These clones also showed K5 down regulation and drastic reduction in K5 filaments along with increased involucrin expression and decreased p-Akt levels. Thus results suggest that K14 helps in maintaining proliferating potential of basal cells in stratified epithelia.
To investigate the role of K8 and 18 in differentiation and transformation, K8 was over-expressed in MDA MB 435 cells and down-regulated in MDA MB 468 (less invasive transformed cells) and MCF10 A (non-transformed) cell line. K8 transfected cells did not show any change in the cell growth profile, motility and soft agar colony formation. Significant differences were observed in the tumor volume, in vitro invasion assays and metastasis of K8 transfected cells as compared to vector control cells. K8 knockdown MDA MB 468 clones showed increase in cell growth, soft agar colony formation potential, motility and also invasion. Clones of MCF10A are being assessed for their neoplastic potential upon down-regulation of K8. Results indicate that K8 might play an important role in prevention of invasion and metastasis of mixed epithelia like breast.
A model rodent system exhibiting different stages of lingual carcinogenesis has been developed by feeding 4NQO to rats. The 4 plex iTRAQ LC-MS proteomics approach was used to obtain the protein profile of each stage. Of the 1851 proteins identified from the rat tongue tumors, 347 proteins were found to be differentially expressed as compared to normal tissues. Proteins seen included K5, K14, K17, K10, vimentin, MMP9, transglutaminase 3 (TGM3), periostin etc, that are reported to be differentially expressed in human OSCC. Their expression will be validated, and iTRAQ analysis will be done on human leukoplakia and tongue SCC samples to validate the results.
The 14-3-3 family of proteins are multifunctional and play key roles in cell proliferation, differentiation and metastasis. Aberrations in these proteins lead to transformation. shRNA mediated knockdown technology has been used to determine the contribution of individual 14-3-3 isoforms to checkpoint control and to identify novel ligands for these proteins. A new method for the generation of transgenic animals has been developed and this is being used to address various questions. Indian and US patent applications for the process have been filed and the protocol has been published this year. On-going investigations show that loss of 14-3-3g leads to sterility in male mice due to the absence of cell-cell adhesion between Sertoli cells and also between Sertoli cells and spermatocytes, seemingly due to decreased desmosome formation. Investigations are on to identify 14-3-3g ligands that play a role in spermatocyte differentiation and Sertoli cell adhesion.
On-going experiments also show that loss of 14-3-3g leads to an increase in centrosome number in cells, leading to genomic instability and aneuploidy, which is accompanied by increased soft agar colony formation and tumor formation in the mouse. Studies to identify the underlying molecular mechanism reveal that over expression of cdc25 family members exacerbates the centrosome phenotype while a knockdown of cdc25 family members led to an inhibition of centrosome duplication. These results could also be phenocopied by over expression of the constitutively active cdk1AF mutant that cannot be inhibited by phosphorylation. Lack of 14-3-3g thus leads to an increase in centrosome number resulting in tumor progression.
Experimental evidence also shows that loss of 14-3-3ε causes lymphoproliferative disorder, leading to splenomegaly and lymphocyte infiltration in the lungs, liver and kidney, ultimately resulting in death of the mice. These mice also show hair loss with decreased hair formation and alterations in the organization of the epidermis. These results are now being confirmed.
Plakophilin 3 is a desmosomal plaque protein whose levels are reduced in poorly differentiated tumors of the oropharyngeal cavity and invasive colon carcinomas. The molecular mechanisms underlying the genesis of structures that mediate cell-cell adhesion such as desmosomes are under investigation. Another focus is to examine how inhibition of desmosome function leads to an increase in neoplastic progression and metastasis. The hypothesis being tested is that plakophilin3 is required for desmosome assembly and maintenance, and that an alteration in plakophilin3 levels could alter desmosomal architecture. These changes might affect migration and cell-cell adhesion, and cause changes in signaling that activate the epithelial mesenchymal transition (EMT), leading to invasion and metastasis. Previous work has demonstrated that another ARM repeat containing the protein plakoglobin is required for plakophilin3 recruitment to the cell border and desmosome formation. Multiple mutants of plakoglobin have been generated to identify regions required for border localization and desmosome formation; these mutants are being tested for their ability to induce desmosome formation and neoplastic progression.
Glycosylation is the major post translational modification that regulates structure and functions of proteins. Expression of β1,6 branched N-linked oligosaccharides correlates with the invasive and metastatic properties of several human and murine cancers. Interaction between poly-N-acetyl lactosamine (polylacNAc) substituted β1,6 branched N-oligosaccharides on surface proteins of melanoma cells and galectin-3 - the high affinity receptor for polylacNAc which is expressed in highest amounts on the lungs, play a key role in mouse lung metastasis. β1,6 branched N-oligosaccharides expression promotes invasion by modulating adhesion, chemotaxis and movement, with increased adhesion inducing MMP-9 secretion and thus invasion. The mechanisms by which these oligosaccharides regulate cellular processes critical for invasion and organ homing are being identified by analyzing terminal substitutions and characterizing the surface proteins that carry them. Preliminary mass spectrometry data indicate that integrins, cadherins, MMPs and glycosyl transferases are potential carriers of polylacNAc. An inducible lentiviral shRNA construct that targets polylacNAc synthesizing genes (GalT I and V) has been generated. The signaling pathways activated downstream of galectin-3/ polylacNAc interaction are being elucidated using lentiviral constructs.
The level of galectin-3 on the lungs seems to dictate the metastatic outcome to some extent. Galectin-3 knock-out mice showed metastasis at the same level as wild type mice, and even inhibition of N-oligosaccharides did not inhibit metastasis in them. Galectin-3 regulates the function and maturation of several immune cells. Replacement of the bone marrow of galectin-3 (-/-) mice with that from galectin-3 (+/+) mice had no effect on metastasis, suggesting that chimeric mice still had compromised immune status and that galectin-3 is required even for maturation of immune cells. The overall immune scenario in galectin-3 (+/+), (+/-), and (-/-) mice is being investigated. Generation of conditional knock outs lacking galectin-3 only on the lungs or the vascular endothelium would be attempted.
The functional importance of the recently discovered O-glcNAcylation on nuclear and cytoplasmic proteins is being explored by analyzing its role in regulating keratin 8/18 properties and its impact on cellular behavior and malignant transformation. On-going studies show that triple mutant of K18 (Ser 29, 30 and 48 to Ala) deficient in all the glycosylation sites was more stable than the K18 wild type. Its stable expression resulted in altered filament architecture and accumulation as intracellular aggregates, ultimately causing cell death. K18 glycosylation triple mutants exhibited a two fold increase in phosphorylation at serine 52. O-GlcNAc site specific mutants of K18 have been generated to investigate which specific site/s regulate filament assembly, stability and site specific phosphorylation. Preliminary results indicate that O-GlcNAc at Ser 29 on K18 regulates its stability, while O-GlcNAc at Ser 48 on K18 regulates its phosphorylation at Ser 52. Role of specific sites of O-GlcNAcylation on K18 in regulating cellular processes like cell spreading, migration and invasion are being studied.
Metastasis is a complex process involving the contribution of many genes, one of which is Metastasis Associated 1 (MTA1) gene. MTA1 is over expressed in cancer cells, and its expression is more in highly metastatic cancers as compared to low or non-metastatic cancers. MTA1 regulates SMAD7, a component of TGFb signaling. TGFb signals are transduced to the nucleus by the Smad family of proteins, which includes Smad7 - a negative regulator of TGFb. SMAD7 expression increases on knockdown of MTA1 and upon treating cells with a histone deacetylase inhibitor. MTA1 is recruited to the SMAD7 promoter region. SMAD7 inhibits activation of SMAD2 and SMAD3; the levels of these active SMAD proteins are decreased in cells expressing shRNA against MTA1. Further when MTA1 is knocked down, the expression of downstream targets of SMAD7 decreases. MTA1 thus appears to regulate a key inhibitor of TGFb signalling, SMAD7. MTA1 might assist the process of tumorigenesis and metastasis by regulating molecules like SMAD7.
Jak/STAT and PI3K/Akt are two parallel pathways whose aberrant activation leads to angiogenesis. The effects of a methylxanthine derivative pentoxyfylline on STAT3 mediated regulation of cytokines involved in tumor development and tumor induced angiogenesis in melanoma cells are being investigated.
Epigenetic modifications are central to cell transformation and cancer progression. Histone variants and/or their post-translational modifications participate in diverse nuclear functions including DNA damage and gene regulation. The role of homomorphous/ heteromorphous histone variants in cancer initiation, malignant transformation and progression in vivo remains unclear. On-going studies in a chemically induced hepatocarcinogensis model show that the homomorphous histone H2A variant H2A.1 is over-expressed during malignant transformation of hepatocyte in vivo. H2A.2, the other H2A variant, shows decreased expression at protein and RNA level. RT-PCR analysis has shown that, out of two H2A.1 coding genes, one gene is expressed in liver and it shows increased expression during carcinogenesis. The results indicate that the increase in proliferating cell population during progressive transformation of hepatocytes to malignant state coincides with increase in H2A.1 expression. Altered expression of histone variants H2A.1 and H2A.2 are indicative of the changing phenotype during malignant transformation. It is also hypothesized that the differential incorporation of H2A.1 and H2A.2 may alter chromatin dynamics, thus influencing gene expression and phenotypic behaviour of the cell. The fundamental question of how chromatin ‘switches’ between varying levels of expression and packaging through the concerted action of histone variants is being investigated.
The repair of DNA double-strand breaks occurs within the context of chromatin and serves as natural barrier in DNA-damage recognition, repair and recovery. Time and dose-dependent screening of H3 and H4 site-specific modifications in G1-enriched cells (WRL68 and HepG2) after low-dose and high dose ionizing radiation shows increase of gH2AX, decrease of H3-Ser10P, H3-K9ac, K56ac, H4-K5ac, K12ac, K16ac in damage and repair phase. The decrease in repair phase of H3Ser10P correlates with comet tail moment and radiation dose. Loss of H3Ser10P is noted in multiple human cell lines of different tissue origin and also in response to various DNA-damaging agents. Data suggests that the loss of H3Ser10P is a universal phenomenon in response to DNA damage and phosphorylation is restored in the recovery phase of DNA damage in mammalian cells. H3Ser10P correlates with chromatin relaxation and gene expression in G1-phase of cell cycle. The decrease in H3Ser10P may cooperate to establish a heterochromatin-like state, which may prevent the premature separation of damaged DNA ends and facilitating efficient DNA repair.
Size and shape of the Golgi and nucleus are receiving increasing attention in relation to cancer. Intracellular Intelligence controls the fundamental features (such as size, shape, number polarity or dynamics) of Intracellular objects. Investigations in a yeast mutant showing abnormal Golgi sizes have identified a gene, NMT1 which may be responsible for this phenotype. Results indicate that NMT1 possibly acts in a pathway that control homotypic membrane fusion, and in turn the size control mechanism of Golgi. To understand the regulation of nuclear size assay systems have been developed to monitor the size variation of nucleus using fluorescent fusion of different proteins. In some of the parameters that can influence ER exit site size and numbers such as protein export are being evaluated, with particular attention to the localization of human Sec12, an important protein involved in early secretory pathways which is reported to be predominantly localized in ER exit sites. Experiments reveal that Sec12 interacts with another ER exit protein Sec16.
Acquisition of resistance to the standard platinum and taxol based therapies in the ovarian cancer patients is a considerable challenge for the clinicians. Alterations in apoptotic activities, drug transporter mechanism, DNA repair mechanism and deregulation of cell survival factors play an important role in the acquisition ofchemoresistance in ovarian cancer. On-going investigations focus on early detection of molecular changes associated with acquirement of cisplatin and paclitaxel resistance in ovarian cancer cells and the role of ‘tumor initiating’ or ‘cancer stem cells’ in development of resistance.
Using PA1 ovarian cells stably expressing a PI3KCA sensor (PI3KCA promoter driving fluc2-tdt reporter gene), the effect of cisplatin in in vitro and in mice tumor xenograft model has been monitored by bioluminescence imaging. Results show that cisplatin is able to significantly reduce the promoter activity and simultaneously induce nuclear localization of p53 and total p53 level in PA1 cells. In tumor xenograft model, attenuation of promoter activity was detected after 4th day of completion of cisplatin treatment which further decreased with time. The control mice, however, had increased luminescence and tumor growth with time.
In cisplatin resistant A2780 cells, the PI3KCA promoter activity is not augmented with increased resistance. Interestingly, the attenuation of PI3KCA promoter activity by cisplatin was found to be completely abolished with resistance. Increased phosphoprylation in Akt, and decreased activation of p53 by cisplatin were also observed in this model. Studies are on to investigate the molecular explanation behind this loss of cisplatin mediated reduction in the PI3KCA promoter activity.
A novel caspase sensor MTF is being used to characterize early molecular changes of acquired paclitaxel resistance. This sensor, comprised of three reporter (bioluminescent, fluorescent and PET) genes, shows higher reporter activity after it is cleaved by activated caspase 3. A dynamic model of paclitaxel resistance in OAW42 cells has been developed which shows decreased level of fold induction of luciferase indicating suppression of activated caspase 3 with increased resistance. A NF-kB sensor is being developed to understand the modulation of NF-kB promoter during acquirement of paclitaxel resistance.
A new series of 1-(2’,4’-difluorophenyl)-3-(substituted phenyl)-1,3 propanediones were evaluated for their cytotoxic activity, using a reporter gene mediated caspase-3 sensor in ovarian cancer cells Combinatorial treatment of most of these propanediones along with paclitaxel significantly augmented its cytotoxicity. These novel propanediones thus have dual potential - enhancement of paclitaxel toxicity and anti-inflammatory activity, to act as anti-cancer agents.
A novel RNA aptamer synthesized against the extracellular domain of EpCAM (EpEx) has been used to evaluate EpCam expression in paclitaxel resistant ovarian cancer cells. Specific membrane localization was observed in MCF-7, TD47 and OAW42 cells with the EpCAM-aptamer as compared to the scrambled one. Increased paclitaxel resistance resulted in reduced membrane localization and diffused cytoplasmic expression of the EpCAM-aptamer. Investigations are on to understand the molecular mechanism underlying these observations.
Increasing evidence suggests that cancers contain a small subset of cancer-initiating cells, so-called cancer stem cells (CSCs) that are capable of regenerating a tumor even after chemo/ radiation therapy. Ovarian cancer stem cells (OCSC) have been isolated from OAW42 and A2780 cells (both control and paclitaxel resistant cells) and characterized, and their role in driving chemoresistance is under investigation.
The majority of glioblastoma patients experience treatment failure due to therapeutic resistance. Radiation and chemotherapy work by damaging the DNA of tumor cells. Therefore, understanding the molecular mechanisms that can modulate DNA repair in resistant gliomas would be clinically very valuable. Studies have been initiated to investigate these mechanisms in glioblastoma cells that act as ‘histone marks’ for local recruitment of DNA damage repair factors and activation of differential DNA damage response. In parallel studies are on-going to investigate the epigenetic modifications of histones and the chemoresistance exhibited by leukemic stem cells.
Homing and engraftment of hematopoietic stem cells is another area receiving attention. Ex vivo expansion of human CD34+ cells manipulated with a mixture of cytokines and stromal cell derived factor 1 has been explored. Gene expression profiles of peripheral blood stem cell (PBSC) harvests, CD34 enriched PBSC andex-vivo expanded cells (unstimulated; SCF+TPO+FLT3 (GF)-treated and GF + chemokine treated) were obtained using the Agilent Platform. Validation of homing related genes - CXCR4, CD44, ITGB1BP3 and ITGAL, which were up regulated after GF+chemokine stimulation over only GF-treated cells is underway.
Studies have been initiated to understand the molecular and cellular mechanisms that govern stem cell regulation , and how perturbation in these mechanisms leads to oncogenesis. Signaling pathways such as Wnt / Notch / Sonic-hedgehog, TGF-β and EGFR etc. regulate stem cell renewal and genes affected in these pathways, and are also associated with oncogenesis. Using the mouse skin model, efforts are on to identify genes involved in stem cell regulation and cancer, and how these genes work at the cellular level to develop normal tissue and also repair injured ones to maintain homeostasis.
The growth factor modulator - enhancing factor (EF), is the mouse homologue of human secretory group II phospholipase A2. Keratin-14 promoter has been used to drive the expression of this protein in the mouse skin. K14-EF transgenic mice show abnormal whiskers, defect in hair formation/ growth and a scaly beaded tail. However, its role in hair follicle stem cell regulation and morphogenesis has not been studied. EGF is known to be involved in stem cell regulation. Importantly, EGF interacts with EF suggesting that EF may be involved in stem cell regulation. Results show high keratinization although hair follicle cycling is normal. Further experiments are on-going to determine if the stem cells have the activation signal or have lost the same. This study will unravel the mechanism involved in hair follicle stem cell regulation.
Over 50% of oral tumors are known to recur post surgery. Recent evidence suggests that tumours are generated and sustained by a small subset of cancer cells defined as cancer stem cells (CSC). It is hypothesized that the non-targeted stem cell compartment present in the adjacent areas of the tumour may be responsible for tumour recurrence. If cancer stem cells can be identified and isolated, it should be possible to identify therapies that will selectively target these cells. Spheroid bodies from AW-13516 and SCC-029B cell lines were derived, and characterized for CSC markers. Further, this CSC enriched population was injected in immunocompromised mice. The tumours derived in response to these injections were surgically removed and the animals were re-stitched. These animals were treated with cisplatin at a concentration of 10 mg/ kg body weight. The recurrent tumours obtained were subjected to histopathology and microarray analysis ill be undertaken. Micrometastasis was seen in lung and liver.
Optimal gene delivery technologies are essential for killing cancer cells. An RNAi based strategy has been used to sensitize cancer cells to radio/ chemotherapy by knocking down genes that protect the cell from apoptosis, thus complementing conventional therapy with gene therapy. shRNA to cyclin D1 and ATM have been designed and cloned, and it has been shown that they sensitize cells to conventional therapy. While synthetic nucleic acid delivery systems appear extremely effective in vitro, they have not been too successful in vivo. In an Indo-French collaborative effort, shRNA have been cloned into plasmid vectors and tested in vitro to overcome this difficulty. Various polyethylenimine (PEI) based vectors were synthesized which could efficiently transfect plasmid DNA into HeLa cells, efficiently deliver oligonucleotide in 293 cells and siRNA in other cells. An in vivo ‘virus-like’ delivery system was also evaluated. The results have provided invaluable information for designing and tailoring nucleic acid systems for in vivo administration.
In another investigation, an Indian HIV-2 isolate based lentiviral vector that can efficiently deliver a heterologous gene with sustained protein production as well as a shRNA targeted to down regulate desired protein expression have been developed. From the parental vector, nine different transfer vector platforms have been made which have two novel features - (a) pseudotyping the vector with HHV-6 virus envelope glycoprotein (heavy and light chains gH and gL together), and (b) use a novel minimal PCNA promoter to drive the vector transgene. A clonally derived HEK293 cell line expressing high level of recombinant human erythropoietin (rhEPO) from the human EPO transgene delivered through the LV has been made. A further increase in protein expression by the producer cell line was achieved through lentiviral delivery of shRNA to elastase. The producer clone with down regulated elastase activity yields an amount of rhEPO in the static flask culture not reported so far. This is the first report of rhEPO production from LV platform from a producer cell line metabolically engineered with LV mediated shRNA. An enhanced lentiviral transgene product biodistribution system has been developed by tagging the transgene with a cell penetrating peptide (cpp) coding sequence. Apart from in vitro assessment, the cpp tagged IFN-a system has been validated effectivelyin vivo inamouse model.
The technology for making HPV-16 major capsid protein L1 derived virus like particles (VLP) from yeast has been established at our centre. In collaboration with IIT-B, the VLP was encapsulated in alginate-coated chitosan nanoparticles. This modification provided long term stability of the coated VLP while maintaining its immunogenicity, thereby providing a platform for vaccine development.
In a multicentric collaborative project on delivery of therapeutic/ anti-HCV gene through a lentiviral vector, distinct inhibition of HCV replication by LV delivered shRNA to viral IRES has been shown. Further, the HCV envelope E1-E2 protein has been used to pseudotype the LV to target human liver cell lines.
Aminoglycoside antibiotic-peptide conjugates can be potential drugs as they interfere with the virus life cycle. A single step reporter assay based on the LV system has been developed for screening putative inhibitors of HIV-1 Tat mediated LTR transactivation. A marked dose-dependent inhibition of reporter profile was obtained in the presence of known potent inhibitors. Using a similar approach, a luciferase based anti-HIV-1 Rev protein activity measuring assay system has also been developed.
Human herpes virus-6 (HHV-6) is a ubiquitous latent herpesvirus that gets activated in various immunodeficient conditions. An HHV-6B isolate was obtained from a B-cell line developed from an individual with chromosomally integrated virus. The viral IE-1 protein up-regulates c-fos, c-myc and hsp70 promoter activation, a feature similar to another herpesvirus, HCMV. This is the only known Indian HHV-6 isolate.
Molecular imaging provides real-time visualization and quantitative measurement abilities of cellular processes at the molecular or genetic level. Triple negative breast cancers lack hormone receptors, and therefore lack effective targeted treatment. hNIS gene induced targeted radiotherapy is being explored as an alternative treatment plan for this subgroup of patients. On-going experiments show that hNIS is over expressed in the majority of breast cancers, raising the possibility of using different radioactive isoforms, I-131, I-125 and I-124 that are compatible with radiotherapy, SPECT imaging or PET imaging respectively for imaging and treatment. An automated IHC scoring method based on digital deconvolution technique has been standardised.
High atomic number metals such as gold particles preferentially absorb X-rays compared to soft tissue and promoted local dose enhancement during treatment with ionizing radiation. A HER2 targeted gold nanoparticle (AuNP) is being developed and its efficacy is being evaluated n a preclinical mouse model of breast cancer.
BRET based optical imaging approaches are being developed to provide tools to monitor dynamic protein–protein interactions within cells and small animals in vivo. The assays in turn can accelerate the screening and evaluation of novel drug candidates that modulate MMP-14 activity in the context of whole-body physiological environment.
Screening and early detection are important tools for overall management of cancer and to reduce cancer burden/ morbidity in the population. Thus there is an urgent need to develop sensitive, preferably non invasive method for early diagnosis. Studies are on-going towards development of Raman spectroscopy based methods. Raman spectral markers (fingerprints) have been identified for diagnosis of oral precancerous lesions and cancer, progression of cervical cancer and prediction of radiosensitivity in oropharyngeal cancer. Animal models to assess the diagnostic efficacy of laser Raman spectroscopy vis a vis histopathology has been initiated for studying oral cancer and breast cancer. Fibre optic probes for Raman spectroscopy are also being developed.
A major effort has been directed towards the identification and delineation of the mechanisms of action of chemopreventive agents from Indian foods and beverages, and development of surrogate endpoint biomarkers. Towards this end, the effect of dietary exposure to turmeric/ curcumin on carcinogen–induced tumors and BPDE-DNA adducts was examined in hamsters/ mice. Dietary turmeric post-treatment decreased DMBA-induced tumor volume and multiplicity in the hamster buccal pouch. This correlated well with increase in DMBA-mediated apoptosis and decrease in DMBA-induced cell proliferation, phosphorylated MAPK, cell inflammation markers and levels of NFkB and AP1 transcription factors. A relatively greater reduction in BPDE-DNA adducts was noted in liver/ lungs of mice administered dietary curcumin for 1, 3 and 5 days, as compared to the B(a)P-treated group. This decrease can be attributed to increased cell turn-over and/or enhanced repair and/or dilution of DNA adducts. Dietary curcumin also augmented apoptosis in B(a)P-treated mice at 1, 3 and 5 day time points, suggesting that removal of adduct-containing cells could indirectly confer protection.
Evaluation of the chemopreventive efficacy of polymeric black tea polyphenols and mechanism(s) of their anti-promoting effects are also on-going. Earlier studies have shown that topical pre-treatment with PBP 1-3, EGCG and curcumin (positive control) led to decreased TPA-induced translocation of PKC isozyme (α, ß, γ, ε, η) levels and activity from the cytosol to the membrane, while PBP 4 and 5 were less effective. Levels of PKC δ and ζ in cytosol and membrane were similar in all the groups. Confocal microscopic evaluation showed decrease in TPA-induced green fluorescence intensity (PKC α) in PBP-3 pre-treated membranes, whereas pre-treatment with PBP-5 did not show a similar decrease in both epidermal layer of skin and HaCat cell membrane. Comparative evaluation of TPA-mediated changes in the activity and levels of PI3K/ AKT/ PDK using specific enzyme inhibitors and phospho-specific antibodies showed a decrease in PBP-3 mediated PKC phosphorylation resulting in decreased TPA-induced translocation of PKC. Reported anti-promoting effects of PBPs are thus likely to be due to modulation of PI3K-mediated signal transduction (see Fig.).
The Department of Biotechnology (DBT), Government of India, has supported the establishment of a laboratory at ACTREC for the development and validation of a sensitive HPLC method for the quantification of curcuminoids in human plasma. In this program, a highly sensitive assay that could be employed for clinical pharmacokinetic studies of curcuminoids has been established. After approval of DBT for calibration and validation of this method, the levels of curcumin/ conjugates in plasma from patients of PK/PD studies in DBT funded projects are being evaluated.
Earlier studies have shown that polyphenols from green and black varieties of Indian grapes possess anti-initiating and anti-promoting activities in different model systems, although the exact mechanism/s of their chemopreventive action are not well understood. Effects of pre-treatment with grape polyphenolic extracts (GPE) were studied in appropriate in vivo experimental models. Evaluation of mechanisms of anti-initiating effects of GPE revealed that GPE pre-treatment led to significantly increased activity of phase II metabolic enzymes and decreased expression of B(a)P-derived DNA adducts in liver and lungs of mice. Mechanistic studies on the anti-promoting effects of GPE revealed down regulation of TPA-induced expression of target molecules associated with cell proliferation (PCNA), inflammation (cyclooxygenase 2) and apoptosis in GPE-treated mouse epidermis.
Understanding the immune scenario and reasons for immune dysfunction in cancer patients has been receiving a lot of attention. The focus areas include study of the (i) role of Notch in regulation of gd T cells and regulatory T cells (Tregs), (ii) role of Toll like receptors (TLRs) in anti-tumor immunity mediated by gd T cells, (iii) antibody dependent cellular cytotoxicity mediated gd T cells, (iv) tumor microenvironment and immune dysfunctions, (v) use of plant derived alkylamines to stimulate anti-tumor potential of gd T cells, (vi) characterization of bioactive compounds that regulate osteoclastogenesis, and (vii) role of Th17 and Tregs in gall bladder cancer.
Multi-color flow cytometry revealed an altered expression pattern of TLRs on peripheral blood mononuclear cells (PBMCs) of oral cancer (OC) patients as compared to healthy individuals (HI). The functional responses of lymphocytes from OC patients to TLR stimulus in terms of proliferation, cytotoxicity and cytokine secretion were significantly lowered as compared to HI. TLR3 and TLR9 were dominantly expressed on gd T cells of OC patients and HI. OC gd T cells showed decreased proliferative response and IFN-g production to TLR/ TCR stimulus compared to HI. Stimulation with TLR agonists led to augmentation of tumor directed cytotoxicity of gd T cells from HI but not from OC patients. However, TLR stimulation rescued gd T cells from activation-induced cell death by increasing Bcl2:Bax ratio. HSP60 treated Tregs showed an increased suppressive potential as compared to untreated Tregs. Expansion of gd T cells was inhibited by Tregs, and their suppressive potential was enhanced when Tregs were treated with SP60.
The activation status of gd T lymphocytes and mechanism of antitumor immune response after stimulation of gd T lymphocytes with alkylamines has been studied. The calcium flux in purified gd T lymphocyte stimulated with five alkylamines was found to be comparable to the response observed with anti-CD3 MAb stimulation, indicating T cell receptor mediated stimulation of the purified gd T cells by all the five alkylamines. As compared to medium control, the cytotoxic potential of gd T cells stimulated with ethylamine and isopentylamine against oral cancer cell line AW8507 cells was found to be augmented at 3mM and 7.5 mM concentration respectively. Dual color flow cytometry revealed increased perforin levels in ethylamine and isopentylamine stimulated (i.e. activated) gd T cells.
Examination of the role of Notch signaling pathway in antigen specific responses of gd T cells revealed that Notch1, Notch2 and Dll1 along with NRARP, HES1 and DTX target genes were predominantly expressed in phosphoantigen stimulated gd T cells. Decreased expression of CD107a and diminished lysis of tumor targets in the presence of g-secretase inhibitor (GSI) confirmed that Notch was involved in cytotoxic effector functions of gd T cells. Cytometric bead array revealed that release of TNF-α and IFN-g by phosphoantigen-stimulated gd T cells was inhibited in the presence of GSI. Oral and breast cancer cells showed dominant expression of Jagged1 ligand, which may exert inhibitory signal as a means to evade immune recognition by gd T cells. Confocal microscopic examination of gd T cells from oral cancer patients revealed Notch1 expression. Expression of Notch target gene HES1 was also observed in gd T cells.
Gall Bladder Cancer (GBC) is the third most common malignancy of the gastrointestinal tract in India. The immune scenario is poorly understood in GBC patients. Th17 and regulatory T (Treg) cells play an important role in modulation of antitumor responses, and an imbalance in their ratio may dictate the disease outcome. Multicolour flow cytometric analysis revealed decreased percentages of CD4+, CD8+ and gd T cells, but higher percentages of IL17 producing component within these cells in the peripheral blood of GBC patients, as compared to HI. Frequencies of IL17 producing cells were higher in tumor infiltrating lymphocytes as compared to peripheral blood. Th17 cells in peripheral blood correlated negatively with Tregs in these patients. Cytometric bead array revealed increased levels of IL6, IL10 but decreased levels of IL17, IFNg in the sera of GBC patients. Low lymphocyte proliferative response upon stimulation with anti CD3 MAb was observed in GBC patients indicating an immunosuppressed state.
Vg9Vd2 T cells are a unique subset of lymphocytes, known to exhibit potent cytotoxicity against various tumor cells. On-going studies examine whether MAb B11F12 that reacts with oral tumors can be used in conjunction with Vg9Vd2 T cells isolated from oral cancer patients and healthy individuals (HI) to increase their antitumor cytotoxicity via antibody dependent cellular cytotoxicity (ADCC). Compared to HI, Vg9Vd2 T cells of OC patients exhibited a marked decrease in oral tumor cell killing, which significantly increased in the presence of this MAb. Treatment of oral tumor cells with aminobisphosphonate zoledronate further enhanced tumor directed ADCC mediated by Vg9Vd2 T cells of OC patients. Increased CD107a expression was observed on Vg9Vd2 T cells when they were co-incubated with zoledronate treated oral tumor cells in the presence of this MAb. In vivo studies using SPECT radioimaging of xenografted oral tumor cells in nude mice demonstrated that 125I labelled MAb B11F12 specifically targeted the oral tumors.
Institutional Scientific Resources and Extramural Funding
The on-going research projects are supported by the institute through organised core scientific facilities: Flow cytometry, Imaging, Proteomics and Mass spectrometry, Genomics and gene expression, Structural biology and Small animal imaging facility. The common instrument rooms (CIR) house most of the frequently required equipment such as high end ultra centrifuges, spectrophometers, sonicators, shaker incubators, deep freezers, luminometer, gradient PCR, etc. During the report year, two new ultracentrifuges and two high speed centrifuges were installed in the CIR, along with several heavily used smaller equipment.
Several projects pursued in CRI are multi-institutional and involve collaboration with faculty from major Indian academic and research institutions, corporate research centres, and international academic institutions. During 2011, the faculty at ACTREC successfully generated extramural fundsto the tune of Rs. 6.14 crore for 54 on-going projects from governmental agencies such as DBT, DST, CSIR, ICMR, MOES, LTMT, etc. Of these, 12 were new projects with total sanctioned funds of Rs. 1.69 crore for a three year period. Research at the Centre resulted in 86 indexed publications by ACTREC faculty during 2011 - 60 of these publications were in the field of basic and translational research and 26 were in clinical and medical technology areas.
Scientists in the basic research wing are investigating and exploring the various mechanisms underlying the conversion of a normal cell to a tumor cell. They use multiple strategies to understand the basic biology of normal and tumor cells including biophysical approaches to study protein structure and function, cell biology based approaches to understand molecular pathways, small animal models that mimic human tumor progression in vivo and the study of human tumors to identify genetic variants associated with tumor progression. The goal of these studies is to identify novel targets for therapeutic intervention, to generate assay systems for testing novel compounds for their anti-cancer potential and to improve our current strategies for cancer prevention.