Scientific Officers: R. Mulherkar, Ph.D. (Head), R. Rai, Ph.D.

Research Fellows: P.J. Koppikar, A.L. Kotnis, A.V. Ambade.

Squamous cell carcinomas are a major challenge as these cancers do not respond to conventional treatment and hence are difficult to treat. The goals of the Genetic Engineering laboratory include: (i) understanding the genetic and environmental interactions in the process of tumour formation at the molecular level, using the transgenic mouse model for squamous cell carcinomas, and (ii) identifying and using critical pathways that can be targeted by gene therapy.

Genesis of multiple primary neoplasms with at least one primary in the head and neck

Patients with primary head and neck cancer frequently (~20%) develop second malignancies. The present study proposes to investigate genetic factors that predispose individuals to multiple primary neoplasms (MPN). Mutagen sensitivity assay using bleomycin has been carried out on peripheral blood lymphocytes (PBL) from MPN patients and controls. Their metaphase plates have also been scored. Genomic DNA isolated from PBL of MPN patients has been used to analyse point mutations in the p53 gene, at exons 4 - 8. The only mutation that has been detected is in p53 exon 8 in a Li-Fraumeni syndrome patient. Polymorphism at codon 72 in exon 4 has been noted in p53 gene in some patients. Polymorphisms in the genes encoding xenobiotic detoxifying enzymes - GSTM1 and GSTT1, have also been studied. Future plans include screening for p53 mutations and protein expression, aberrations in DNA repair genes, and prevalence of human papilloma viruses (HPV 16 / HPV 18) in tumour tissues of MPN patients.

Novel and prevalent HPV types in Indian head and neck squamous cell carcinoma (HNSCC) patients

Recent evidence suggest causal association of certain high risk HPV types with a subset of head and neck cancers. In this study, DNA from tumour and adjacent clinically normal appearing mucosa of 109 HNSCC patients was used to amplify a conserved sequence in the L1 region of the HPV genome, cloned and sequenced to determine the HPV type. In all, HPV DNA was detected in 35 patients (32%), in either the tumour or normal tissue. HPV-16 and HPV-18 were present in 6 patients each (8%) while cutaneous HPV 8 was identified in 14% of the patients. A non-invasive method for extraction of high molecular weight DNA was used to isolate DNA from each of 57 age, sex and habit matched normal individuals serving as controls in the study. Five percent of these individuals were found to harbour HPV DNA. High HPV positivity in HNSCC leads to speculation about how early and important HPV infection is for head and neck carcinogenesis. It will be interesting to see if low risk HPV, in conjunction with tobacco carcinogens, can push normal epithelial cells to dysplasia and eventually convert them into neoplastic cells.

Characterization of a transgenic mouse model for chemically induced squamous cell tumours

Transgenic mice serve as good models to study cancer and other human diseases. Transgenic mice, generated earlier in this laboratory, carry enhancing factor (EF) cDNA under the control of human keratin-14 (K14) promoter, which can achieve targeted expression of the transgene in squamous epithelial cells. The aberrant hair follicles and hyperproliferative epidermis of these mice led to the suggestion that they may be sensitive to skin tumorigenesis. Therefore, TgK14-EF mice are being used as a model to study the development of chemically-induced squamous cell carcinomas using the two-stage skin carcinogenesis protocol. A statistically significant increase in tumour occurrence and reduction in time taken for tumour formation is noted in TgK14-EF mice, as compared to normal littermates. These mice also show increased papilloma incidence and a 2-fold increase in the rate of conversion of papilloma to carcinoma upon application of 4NQO in the progression phase. Since expression of EF and hyperproliferation is seen not just in the epidermis but also in the buccal mucosa, tongue epithelium and oesophagus, it is felt that TgK14-EF mice may serve as good models for chemically-induced squamous cell carcinomas of the skin as well as upper aero-digestive tract (UADT). Experiments have therefore been initiated to study whether the UADT of these mice is susceptible to chemical carcinogenesis using the carcinogen – DEN, and to identify the genetic events in pre-cancerous and cancerous lesions in these mice.

Production of clinical grade vector producing cells for gene therapy of oral cancer

HNSCC is the fifth most frequent cancer in the world, and the dominant cancer among Indian males. Conventional treatment cannot be offered to the large number of HNSCC patients who present at a very advanced stage. It is therefore proposed to investigate the ‘pro-drug activation’ strategy for gene therapy of oral cancer - a strategy that has shown success in pre-clinical studies on a xenograft nude mouse model for head and neck cancer established in this laboratory. The aim of this project is to produce clinical grade virus producing cells (VPC) under good manufacturing practice (GMP) conditions and to establish a Master Cell Bank for use in phase I clinical trials. Towards this end, a retroviral vector pLTKSN, carrying herpes simplex virus thymidine kinase (HSV-tk) gene from the plasmid pTK26, has been reconstructed. This construct has been used for large scale DNA preparation and, following restriction mapping using several endonucleases to confirm HSV-tk orientation, LTKSN DNA has been purified. Future studies involve transfection of this purified DNA into 293 cells along with an ecotrophic packaging construct and selection of transfected cells on G418. After checking the supernatant for the presence of infective viral particles with the ability to transfer HSV-tk to the target cells, the ecotrophic VPC will be used to infect PA317 cells to obtain amphotropic VPC. The highest VPC will be used to set up the Master Cell Bank.

Expression of mouse enhancing factor gene in Drosophila

Enhancing factor (EF), a secretory phospholipase A2 isolated earlier in this lab, increases the binding of epidermal growth factor (EGF) to A431 cells. It was proposed that EF might be binding to a 100 kD receptor on the cell surface, trapping EGF and making it available to the cells, thus giving them a growth advantage. Expressing a gene in cells in which it is not normally expressed is a powerful way of determining its function. In order to understand the interaction of EF with EGF and the down-stream events involved, it is proposed to express EF in Drosophila. A number of mutants in the EGF pathway are available in this model and the GAL4 system allows selective expression of any cloned gene in a wide variety of cells in tissue-specific patterns in Drosophila. The mouse enhancing factor cDNA is cloned into the pUAST vector for introduction into the Drosophila germ line. The pUAST-EF clone has been characterized by restriction digestion, EF-specific PCR amplification and sequencing. In order to create transgenic lines, pUAST-EF is micro-injected into embryos of strain yw; +/+. Different GAL4-dependent phenotype is examined in the progeny of the cross. Ectopic expression is monitored by RT-PCR and antibody against EF or by in situ hybridisation to the target gene transcript.

Molecular mechanism of cancer chemopreventive action of curcumin

While curcumin has entered clinical trials as a cancer chemopreventive agent, the mechanism of its action is unknown. Data reveal that it inhibits proliferation of HeLa cells (IC₅₀ = 34 ± 4.2 mM) and RINm5F cells (IC₅₀ = 32 ± 6.5 mM). At the cellular level, curcumin disorganizes the microtubule (MT) network and induces MT depolymerisation, probably by binding to tubulin at a site that is close to or is the same as the colchicine-binding site. Further studies will be targeted towards understanding the cross talk between the cytoskeleton, rho-GTPases and cytoskeleton modulators, using various cell lines.

Protein kinase C isoforms in erythrocytes from CML patients (Council of Scientific and Industrial Research)

Erythrocytes from CML patients are fragile and exhibit molecular alterations, which can be attributed partly to altered protein kinase C activity. CML erythrocytes specifically express PKC d isoform. CML erythrocytes show higher levels of PKC a and x and lower levels of PKC i, as compared to control. Higher PKC activity in CML erythrocytes is due to atypical isoforms (i, x and m) and also due to PKC d. However, activity attributable to PKC a has not been detected in CML erythrocytes, in spite of high protein levels. Whether PKC d is expressed in erythrocytes of patients with other haemopoietic malignancies (AML and ALL) and solid tumours (oral cancer) is now being investigated.

Apoptosis-related genes - FHIT and ERK 3, in human oral cancers and oral lesions

This project evaluates the involvement of apoptosis in oral tumorigenesis. While apoptotic index increases significantly during progression of normal oral mucosa to pre-malignant oral lesions, in oral cancers it is decreased. Proliferative index, on the other hand, increases significantly from normal tissue to oral cancer. Thus, transformation of oral lesions to cancer seems to be accompanied by inhibition of cell death and enhancement of proliferation. LOH - indicative of allelic inactivation of the apoptosis-related Rb gene is seen in 2% of oral tumours, while 24% tumours show loss of Rb protein expression, suggesting the involvement of Rb in a small proportion of oral cancers. Analysis of the transcriptional status of FHIT and ERK3 (cloned earlier in this lab) genes reveals the presence of aberrant FHIT gene transcripts in 10% of oral cancers. Future plans include analysis of the global expression of apoptosis-related genes vis a vis sensitivity/resistance to chemotherapeutic agents, in oral cancer tissues and cell lines.

Regulation of keratin gene expression in human oral cancer and pre-cancer

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