Scientific Officers: S. M. Zingde, Ph.D. (Head), R.D. Kalraiya, Ph.D., S.N. Dalal, Ph.D., T.R. Teni, Ph.D., N. R. Naik, Ph.D., M. M. Vaidya, Ph.D., R.B. Govekar, Ph.D., U.M. Warawdekar, Ph.D., S.S. Sawant, M.Sc., A.U. Tarphe, M.Sc.

Research Fellows: P.R. Molli, B.V.V. Reddy, S.S. Bharne, N. Rathore, S.T. Kundu, S. Mallick, A.V. Bhagwat.

Investigations in the Biochemistry and Cell Biology group cover various aspects of the hallmarks of cancer, i.e. cell proliferation, cell death, metastasis, role of intermediate filaments in transformation, and functional differentiation. Studies have also been initiated to investigate specific pathways and molecules involved in each of these events, along with a global analysis of gene expression and protein profiles.

Role of 14-3-3s and plakophilin 3 in regulating cellular response to DNA damage (Department of Science and Technology)

One of the genes required to prevent premature mitotic progression in response to DNA damage is 14-3-3s. This project focuses on understanding the mechanisms by which 14-3-3s and its associated protein plakophilin 3 regulates mitotic progression. A deletion mutant of plakophilin 3 that lacks the head domain (ΔNH₂) is unable to form a complex with 14-3-3s. While the wild type protein is mostly cytoplasmic, the ∆NH₂ mutant shows pan-cellular localisation with an equivalent amount of protein in the cytoplasmic and nuclear compartments. Thus 14-3-3s might regulate plakophilin 3 function by regulating its intracellular localisation. Western blotting reveals that a majority of oral tumour samples show loss of plakophilin 3 expression while 14-3-3s expression is elevated. It is possible that lack of plakophilin 3 expression results in increased DNA damage, thus stimulating 14-3-3s expression. In future plans, 14-3-3s mutants and cell lines showing regulated expression of plakophilin will be generated to permit in-depth study of how the plakophilin 3/14-3-3s complex regulates mitotic progression in response to DNA damage.

Role of altered cell surface glycosylation in organ-specific metastasis and invasion (Department of Science and Technology)

Expression of higher antennary N-linked oligosaccharides (HANLO) on the surface glycoproteins of cancer cells is associated with the metastatic phenotype. Study of HANLO on the tumour cell surface molecules β1 integrin and LAMP, and their regulated expression on metastatic and invasive variants of B16 murine melanoma cells reveal their importance in homing to the lung and invasion into the extra-cellular matrix (ECM). Galectin-3 appears to be the potential ligand for these oligosaccharides, and the anchor for melanoma cells on the lung. The invasion process is facilitated by increased adhesion of B16BL6 cells to fibronectin, collagen IV and matrigel - coated dishes, and higher expression of membrane-tethered forms of matrix metalloproteases as compared to B16F10 cells. The level of expression and extent of different terminal substitutions like a2,3 and a2,6 - linked oligosaccharides, terminal fucose and polylacNAc on HANLO on different glycoproteins from B16F1 and B16F10 cells is being compared.

Cellular fibronectin fragments in blood plasma during malignancy

Tumour invasion is achieved by the enzymatic degradation of protein components of the ECM, viz. fibronectin. Proteolysis-resistant domains of this protein are likely to appear in the blood plasma and can be used as markers of malignancy. Fibronectin exists as a soluble, plasma form and an insoluble, cellular form (cFn) which possesses 3 domains: EDA, EDB and IIICS. Peptides corresponding to EDA and IIICS domains have been synthesized and polyclonal antibodies raised against them, and an ELISA has been set up that is able to detect 2-10 ng/ml of EDA/IIICS peptides of cFn in the plasma. Results indicate that while there is some overlap in the range, tumour values are distinctly higher than normal. Western blots reveal several fragments of cFn in the plasma of normal individuals and cancer patients. A 30 kD fragment is distinctly seen in the plasma of patients with cancer of the stomach, gall bladder and colon. This fragment possesses the EDA domain but does not include the cell-binding domain. The presence of the high affinity heparin-binding domain on this fragment and the possible effect of this fragment on cellular processes are to be determined.

Molecular mechanism/s of defects exhibited by polymorphonuclear leukocytes in chronic myeloid leukemia. (Lady Tata Memorial Trust)

Intracellular trafficking of the chemo-attractant receptor C5aR is regulated by various kinases, phosphatases and intracellular microfilament network. Distinct differences have been seen in the expression, distribution and trafficking of C5aR in polymorphonuclear leukocytes (PMNL) from normal subjects versus chronic myeloid leukaemia (CML) patients. Expression of rhoA – a GTPase that regulates polymerisation and localisation of actin, is being studied to understand the basis of the global defect in stimulation of actin polymerisation in CML PMNL. In normal PMNL, rhoA expression drops by 50% within 45 min of chemo-attractant stimulation, while it is unaltered in CML PMNL, suggesting that altered rhoA dynamics may be a factor leading to actin polymerisation. A cell line expressing bcr-abl and the chemo-attractant receptor FPR has been obtained and characterized. This cell line reveals altered phenotypic characteristics and CD15 expression that is often observed in transformed cells. This cell line will be used to study signaling mechanisms stimulated by occupation of the chemotactic receptors.

Diagnostic and therapeutic modalities for CML

Molecular beacons (MB) have tremendous potential as biomolecular recognition probes. Preliminary studies using bcr-abl positive cell lines and fluorescent-tagged MB against b3a2 have shown that this MB is specific and has the potential to be used in diagnostics. Studies carried out to determine the role of kinase inhibitors – genistein and staurosporine in CML therapeutics show that, while genistein is ineffective, staurosporine induces apoptosis in K562 cells. Preliminary studies using bcr-abl anti-sense phosphorothioate oligonucleotides show inhibition of K562 growth. Further studies using modified oligonucleotides have been initiated.

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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