Scientific Officers: K.V.K. Rao, Ph.D. (Head), S. Sarkar, Ph.D., S. Gupta, Ph.D., H.B. Bodke, M.Sc., M.G. Kamble, M.Sc.

Research Fellows: S. A. Banerjee, B.B. Bose, H.S. Ashra, P.R. Parekh.

The major thrust areas of the Chemical Carcinogenesis group continue to be: investigation of cell cycle checkpoints, cell signaling pathways, transcriptional regulators, identification of markers of neoplastic transformation, transplacental carcinogenesis as well as chemoprevention of cancer using in vivo animal models and in vitro systems.

Investigation of cell cycle checkpoints during liver tumor promotion (Indian Council of Medical Research)

The cellular and molecular mechanisms associated with abrogation of checkpoint control during tumour promotion are being investigated using malachite green (MG) and metanil yellow (MY), earlier identified as tumour promoters in the rat liver model. The inter-relationship of cyclin D1 over-expression during cell growth with Rb phosphorylation, MAP kinase and PKB/Akt signaling proteins during rat hepatocarcinogenesis is now being studied. Up-regulation of cyclin D1, cyclin B1 and CDK4 suggests that the mRNA over-expression during tumour promotion with MY and MG (noted earlier) is due to an increase in the transcriptional activity of these genes. Levels of total ERK1 and ERK2 proteins increase with progression of liver tumour. Over-expression of cell cycle - related genes, during tumour promotion with MG and MY, is thus due to an increase in transcriptional activity. Role of cell survival signaling mechanisms and their contribution to abrogation of checkpoint controls will be investigated using the hepatocarcinogenesis model.

Role of MAP kinases in initiation of G2/M checkpoint signal transduction during transformation of mammalian cells in culture

Exposure of cells to genotoxic agents triggers a wide range of cellular responses including altered gene expression, delay in cell cycle progression, initiation of cell cycle arrest, etc. This project examines the involvement of the MAPK signaling pathway - responsible for altering several downstream events, and the mechanism/s involved in its induction, using MG–treated Syrian hamster embryo (SHE) fibroblasts in primary culture. In completely transformed SHE cells, persistent activation of p38 molecule is responsible for maintenance of transformation. While basal intracellular levels of total ERK, JNK and p38k remain unchanged, increased expression of activated JNK and p38 is noted during the 1st and 2nd week of transformation. Nuclear localisation of activated JNK and p38 molecules is also noted at this time point. Activation of particular isoforms of MAP kinase is not linked to a specific cell cycle phase. Late passage MG-transformed SHE cells show increased expression and persistent nuclear localisation of activated p38, as well as increase in S-phase cells. Downstream events of p38 pathway will be studied in-depth during sequential transformation of SHE cells and in the transformed SHE cell line.

Mechanisms of G2/M checkpoint abrogation in SHE cells (Council of Scientific and Industrial Research)

Eukaryotic cells utilise cell cycle checkpoints to maintain genomic integrity and minimise tumorigenesis. The objective of this study is to determine the possible role of G2/M checkpoint machinery during transformation of SHE cells brought about by MG - which is known to damage DNA by generating reactive free oxidative radicals. Data reveal that MG affects the expression profile of Chk1, 14-3-3, Cdc25C, Cdc2 and cyclin B1 proteins, all of which are crucial to the G2/M phase transition. Untransformed SHE cells follow a normal pattern of G2/M arrest while transformed cells show abrogation of the G2/M checkpoint. MG arrests normal SHE cells at the G2/M phase whereas transformed SHE cells show a reduction in G2/M population. Increased phosphorylation of Chk1 and Chk2 is seen in transformed cells. Thus, while transformed cells show a decrease in percentage of cells arresting at G2/M and a reduction in total protein levels, high levels of the phospho form of most of the kinases is noted. The relationship between expression and levels of G2/M checkpoint proteins and the cell cycle profile will be studied in-depth in normal and transformed SHE cells.

Mechanism of masheri - induced transplacental carcinogenesis

Recent evidence suggests an association between smokeless tobacco use during pregnancy and disturbance of reproductive outcome and development of cancer in the offspring. Since the nature and disposition of PAH metabolites in the foetus depend on the metabolic activity of the placenta, the pathophysiology of the placenta following transplacental masheri (a smokeless form of tobacco) exposure is being examined using an animal model simulating the human condition. At term, the placenta weight is significantly higher in the masheri-treated group than in controls. Masheri-treated group shows histopathological changes such as thickening of sinusoidal capillaries due to fibrin deposition, erythropenia and intermittent necrotic areas, and ultrastructural changes such as diffuse desmosomes and accumulation of lipid droplets in placenta cells. A significant increase in lipid peroxides, superoxide dismutase, glutathione reductase, glutathione peroxidase and glutathione S-transferase and concomitant down-regulation of catalase and reduced glutathione levels are seen in placental tissue of the treated group. At term, maternal hepatic lipid peroxidation and cytochrome P450 level increases while the activity of hepatic antioxidant enzymes is unchanged. Thus, masheri exposure adversely affects placental development - both morphologically and functionally, more than maternal hepatic tissue, and this could be the first step towards aberrant reproductive outcome.

Chemical analyses of masheri, a smokeless tobacco product

Masheri is reported to contain several potential mutagenic, clastogenic and carcinogenic compounds. In view of the toxic effect of masheri on the placenta and foetus, chemical analysis of raw tobacco / masheri samples and masheri extract used in transplacental carcinogenesis studies is now being undertaken. In contrast to earlier reports, a high concentration of benzo(a)pyrene (B(a)P, 530 ng/gm) has been detected in masheri extract, while its concentration in masheri and raw tobacco is 25.15±3.95 and 9.92±2.90 ng/gm, respectively. Nicotine content of masheri extract is 164.65±0.73 mg/gm, while that of masheri and raw tobacco is 19.14±0.00 and 25.88±0.09 mg/gm respectively. Both B(a)P and nicotine have low molecular weight and can easily cross the transplacental barrier to induce foetal toxaemia, and retard growth and development. The increase in placental weight noted in this study might be the result of compensatory hypertrophy to overcome the reduced oxygen supply due to nicotine-induced vasoconstriction. Future studies will aim at detecting the presence of other toxic/carcinogenic substances such as hydrogen cyanide, phenols, trace elements, etc in these products.

Molecular mechanisms of cyclin D1 gene expression during DEN-induced sequential hepatocarcinogenesis

Cyclin D1, a G1/S cyclin also known to be a proto-oncogene, is dysregulated in various cancers. While expression of cyclin D1 is regulated - at least partly, at the transcriptional level by a p21ras-dependent pathway, it is not clear which cis-acting elements in the cyclin D1 promoter are involved in cyclin D1 over-expression during the development of N-nitrosodiethylamine (DEN)-induced hepatocellular carcinoma (HCC). The findings of this study indicate that increased expression of cyclin D1 correlates with up-regulation of its transcription rate during sequential hepatocarcinogenesis. Sensitivity of the cyclin D1 promoter region to DNaseI increases with progression of the liver lesions to HCC. Levels of AP1 and cyclic AMP-responsive element binding proteins increase in neoplastic nodules and HCC. Thus, increased transcription of cyclin D1 seems to be partially mediated through changes in chromatin structure - specifically by decondensation of cyclin D1 chromatin. DNA-protein interaction studies will be undertaken to examine the role of trans-acting proteins that bind to 5’ regulatory sequences of cyclin D1 gene during sequential hepatocarcinogenesis.

Molecular mechanisms of chromatin remodeling-mediated gene regulation during liver cancer progression

This project focuses on determining the role of histone modifications in regulating chromatin structure and how deregulation of these modifications may contribute to tumorigenesis using a DEN-induced rat liver tumour model. Data reveals increased historne H1 and H3 phosphorylation as a function of sequential development of liver tumours. H3 and H4 histones show increase in Tyr/Thr phosphorylation with tumour progression without affecting histone protein levels. Acetylated histones are undetectable in control liver but H1, H3 and H4 show increased acetylation at the lysine residue with tumour progression. Levels and expression of histone deacetylase HDAC1 and histone acetyltransferase PCAF, both of which control acetylation state, are altered in neoplastic nodules and HCC. Post-translational modification(s) of histones, a focal point for both positive and negative transcriptional control, may be regarded as an epigenetic counterpart of genomic instability and linked to the onset and progression of liver cancer. Future studies will focus on the significance of histone modification and chromatin structure on progression through the cell cycle as well as development of HCC.

Chemopreventive effect of orange oil on DEN-induced hepatic pre-neoplasia in rat

Chemoprevention of solid tumours by orange oil has been reported earlier. However, the mechanism of orange oil-induced tumour regression has not been studied at the cellular level. Therefore, this project examines the effect of orange oil at different stages of DEN-induced rat hepatocarcinogenesis, at the ultrastructural level. Administration of orange oil leads to a 75% inhibition of DEN-induced rat hepatic tumours. Ultrastructural changes associated with the chemopreventive effect of orange oil include: restoration of damaged cell membranes, re-induction of bile canaliculi (which were lost in DEN-treated liver) and up-regulation of gap junctional complexes. These changes are probably responsible for the reversal of neoplastic changes to the normal phenotype. The mechanism of suppression of liver tumours by orange oil will now be studied in an in vitro model.

Cellular and molecular markers of early and late stages of lung cancer development in an animal model