Detection of Hypermethylated Genes in Women With and Without Cervical Neoplasia

Qinghua Feng1, Akhila Balasubramanian1*, Stephen E. Hawes1, Pap Toure2, Pap Salif Sow2, Ahmadou Dem2, Birama Dembele2, Cathy W. Critchlow1, Longfu Xi1, Hiep Lu1, Martin W. McIntosh3, Alicia M. Young3, Nancy B. Kiviat1

1University of Washington, Seattle, USA; 2University of Dakar, Dakar, Senegal; 3Fred Hutchinson Cancer Research Center, Seattle, USA. *

Introduction: DNA methylation changes are an early event in carcinogenesis and are often present in the precursor lesions of various cancers. Objectives: To examine whether DNA methylation changes might be used as markers of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). Methods: We used methylation-specific polymerase chain reaction (PCR) to analyze promoter hypermethylation of 20 genes in exfoliated cell samples and matched tissue biopsy specimens from 3 19 Senegalese women (histology negative/atypical squamous cells of undetermined significance= 142, CIN-1=39, CIN-2=23, CIN-3/carcinoma in situ [CIS]=23, ICC=92). Logic regression was used to determine the best set of candidate genes to use as disease markers. Results: Similar promoter methylation patterns were seen in genes from exfoliated cell samples and corresponding biopsy specimens. The best panel of hypermethylated genes included DAPK1, RARB, or TWIST1. At least one of the three genes was hypermethylated in 57% of CIN-3/CIS and in 74% of ICC but in only 5% of samples with


Avenues for Potential Therapeutic Use of Anticancer Properties of Vinca rosea, Withania somnifera and Ocimum sanctum

A Nath*, Priyanka Sinliu and Preety Sinha

Cell Biology & Toxicology Lab, Department of Zoology, A N College, Patna University, Patna. *

One of the most dreadful diseases threatening the existence of mankind in the 21th century is cancer. Various kinds of cancer are reported due to pesticides which are very liberally used to increase the yield of crops, leading to their entry into the ecosystem, ultimately causing mutation and genotoxicity in human being. Malathion, parathion, rogor, carbamate, endosultan, DDT, Aldrin α, β & γ HCH etc are commonly used as pesticides. In the present investigation LM, EM, enzyme and hormone assessment in Swiss albino mice show that endosulfan, malathion and parathion cause cytotoxic effects in various organs but are not carcinogenic. Rogor produces carcinogenic effects on liver and testes of test animals. In the fifth and sixth successive generation of rogor treated mice tumors appear on back and near the neck in male. Herbal extracts tend to lower pesticide induced carcinogenicity in liver and testes. Vinca rosea and Withania somnifera were chosen as probable antidotes against such carcinogenicity. Vinca rosea extract has vinblastin, which acts as cell-cycle inhibitor and thus can be a potent anticancer device. W. Somniferti extracts contain GABA a and GABA b, which activate neuroendocrine system ultimately leading to hyperactivity of endomembrane system giving smooth biosynthetic pathways to various biomolecules. Root extracts of W. somnifera was used to lower subcellular toxicity caused by pesticides in liver and testes. Ocimum sanctum leaf extract with its antioxidant effects was also found to be beneficial. Administration of sublathal doses of endosulfan in male mice results in complete arrest of spermatogenesis and spermiogenesis, blebbing on the sperm head and deformities in testicular cells leading to reduced sperm count and testosterone level. Administration of V. rosea and W. somnifera extracts shows remarkable recovery in the testes. In rogor treated mice several abnormalities like fenestrations, blebs, ruffles, labopodia and deformities occur on surface of hepatocytes, white their nuclei become irregular in shape. In testes there is high incidence of changing of Sertoli cells to macrophage like structure. TEM of liver cells show vacuolizations increase in RER, SER and simultaneous increase in ribosomes as well as vast increase in mitochondria number revealing high protein and energy requirement. Serum SGPT is highly raised raised along with total bilirubin content. The supplementary feed mixed with medicinal plant extracts when fed to the test animals for 2-3 months show remarkable regeneration of organelles, which were in damaged state earlier due to pesticide toxicity. It tends to normalize the cellular and subcellular damages like diffusion of plasma membrane increased lysosomal activity, dilation of mitochondrial cristae disrupted secretory pathways and major carcinogenic effects caused due to pesticide toxicity. Vinca rosea along with Ocimum sanctum leaf extracts acts syneraistically and provide ameliorating effects against carcinogenicity in mice to some extent. It may be administered intraperitoneally or applied as ointment as a potent antidote against cervical cancer as vinblastin and vincristin in Vinca rosea might check the proliferation of cancer cells.


N-myristoyltransferase: A Novel Therapeutic Target for Cancer

Anuraag Shrivastav1*, Jonathan R Dimmock2, Nand K. Singh3 and Rajendra K Sharma1.

1Department of Pathology, College of Medicine, University of Saskatchewan and Health Research Division, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, S7N 4H4, Canada, 2College of Pharmacy and Nutrition. University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5C9, Canada, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221 005, India. *

Protein N-myristoylation is a lipidic modification which involves the covalent attachment of myristate, a 14 carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral and fungal proteins. N-myristoylation ensures the proper function and intracellular trafficking of proteins. Many proteins involved in a wide variety of signaling, including cellular transformation and oncogenesis, are myristoylated. The myristoylation of proteins is catalyzed by N-myristoyltransferase (NMT). Earlier, we have reported that NMT activity is higher in colonie epithelial neoplasms than in normal appearing colonie tissue and that the increase in NMT activity appears at an early stage in colonie carcinogenesis. Furthermore, we observed that NMT expression is elevated in colorectal and gallbladder carcinoma. Results from our laboratory have established NMT as a novel therapeutic target for cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. A large number of compounds of diverse structures and proteins are capable of inhibiting NMT. On the basis of their effects on enzyme reaction, inhibitors may be broadly classified into two general categories namely: proteins and synthetic organic/inorganic compounds. We have discovered recently that enolase potently inhibited NMT activity in a dose dependent manner with the half maximal inhibition at 4.5 0.35 nM. Synthetic compounds such as mannich bases of 2-arylidenecyclohexanones and transition metal complexes of thiohydrazides showed inhibition of NMT activity. Further characterization of inhibitor protein and compounds in terms of their mode of action will serve as a new set of treatment protocol for cancer.


Dynamic Spectral Imaging for in vivo Diagnostics, Screening and Guided Therapeutics of Cervical Neoplasia.

Costas Balds*1,2, Cliristos Simos2, Yiannis Sciadas2

1 Department of Electronic and Computer Engineering, Technical University of Crete, 73100 Chania, Crete, Greece. 2 FORTH-Photonics, 19-21 Theophonotis str, 1 1523. Athens. Greece. *

Introduction: The visual assessment of the acetowhitening characteristics is not effective, since it is qualitative and subjective and depends on the examiner’s visual acuity and training. Objectivrs: To develop an alternative digital imaging technology for diagnosis and screening of cervical neoplasia, relying on the measurement, processing and mapping of the acetowhitening characteristics. Methods: The DySIS technology, developed at FORTH-Photonics, measures the acetic acid-induced temporal alterations in the optical properties of the cervix. From the measured data, quantitative parameters expressing the acetowhitening characteristics are calculated and displayed, for every image pixel. Different (pseoudo-) colours are used to represent different parameter values, enabling the direct visualization of the various acetowhitening degrees, over the entire tissue surface. The obtained pseoudocolor kinetic map can be displayed and stored together with the actual tissue image, facilitating lesion’s localization, mapping and follow-up. Results: The unique advantage DySIS technology over visual examination is that it enables the accurate and standardized assessment of the acetowhitening effect. The provided quantitative data improve both sensitivity and specificity, since the discrimination between CIN and non-CIN lesions as well as between different grades of CIN is facilitated. Moreover, the measured acetowhitening kinetic map provides valuable information for the location, size and grade of the lesion, thus improving both biopsy sampling and treatment accuracy. Furthermore by enabling the direct comparison of kinetic maps, obtained during subsequent examinations, possible recurrences can be detected. Conclusion: DySIS technology has the potential to become a stand-alone, cost-effective tool for screening, diagnosis and guided biopsy sampling and treatment of cervical neoplasia in developing countries.


Identification of a Spliced Variant of Human Sin3b in Adult Lungs and Placenta

Harish Batra1, Mushook Ali1, Rakesli Singh1, Vineet Kumar1, Shambhavi Mishra2, Bahijl Prakasli2, Daman Saluja*1

1Medical Biotechnology laboratory, Dr B.R. Ambedkar Center for Biomedical Research. University of Delhi, India. 2Department of Biological Sciences & Bioengineering, Indian Institute of Technology, Kanpur, India. *

Introduction: Sin3 family of proteins contains highly conserved, multifunctional, nuclear oncoproteins that have been implicated as corepressors utilized by several transcriptional repressers regulating diverse cellular functions. Although ySin3 is not essential for viability, null mutations of Sin3 are lethal in Drosophila. In yeast and several other organism a single isoform of Sin3 has been identified while mouse and human contains atleast two distinct genes for Sin3 namely Sin3A and Sin3B. The two isoforms are close paralogs. Primarily Sin3 proteins have been shown to act as transcriptional repressers by working as a scaffold for assembly of specific HDAC and Swi/SNF components. In mammals, several AML family members fusion oncoproteins were found in a ternary complex with AML-I and Sin3A, suggesting that they may acts by recruiting transcriptional corepressors and histone deacetylases. hSin3 A associates with N-CoR or SMRT and ETO independently. Other DNA binding transcription factors including Max, Unieo. and nuclear hormone-receptors can also recruit hSin3/HDAC/N-CoR complex. Recently, Mxilmediated inhibition of Myc has been shown to require interaction with mSin3A proteins through its PAH2 domain. Objective: To identify the structure and function of human Sin3B. Methodology: Using silico’ analysis primers were designed to amplify hSin3B cDNA and clone in bacterial and mammalian vector. Complete sequencing of hSIN3BcDNA was deciphered by designing internal primers. The exon intron boundaries were predicted using Gene Mark program. Various motifs for the Sin3B protein were searched and aligned at their respective positions using PROSITE and CDART. Structural modeling was done using Jpred, 3D-PSSM and MODELLER6.v2 The homology models thus obtained were examined for significant interactions with MADl in Insight. The Connolly surfaces of the homology models were generated using INSIGHTII software package. PAHl interacting protein region of NCoR was modeled using MADl interacting helix as a template. Tissue specific expression was checked using human multiple tissue panels (clonetech). Results: We here report the characterization and sequence analysis of two isoforms of hSin3B. The gene for hSin3B is localized on chromosome 19p and spans approximately 50 Kb. An alternate spliced form is differentially expressed in adult lungs and placental tissue. The long isoform contains 20 exons and the short isoform lacks exon X. The exon X codes for 32 amino acids and lies downstream of PAH3 domain and this may hold importance in differential gene regulation. In silico structure analysis of the conserved domains predict an alternate protein-protein interaction domain present in hSin3B. Functional interaction studies of hSin3B with other proteins will be presented.


Rx of Cancer: Silencing of a Gene using Novel Liposome Entrapped siRNA

Imran Ahmad*

NeoPharm Incorporation, Lake Forest, Illinois, USA. *


Interleukin-8 Expression in Urine of Patients with Superficial Bladder Cancer after Bacillus Calmette-Guerin in Instillation: A Potential Prognostic Marker

Kishore M. Amin*, 1Hemant B. Tongoankar, 1Yuvaraja T.B., Sujata S. Hake, 2Asha S. Raste., Shubhuda V. Chiplunkar

Immunology, Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC). Kharghar, Navi Mumbai, 1Genito-urinary and Gynecological Oncology Service, 2Biochemistry Department, Tata Memorial Hospital, Parel, Mumbai, India. *

Adjuvant intravesical Bacilli Calmette-Guerin (BCG) is an effective method of treating superficial bladder tumors. The mechanism(s) by which BCG inhibits tumor growth and release of cytokine/s are not known. In the present investigation we propose to study the efficacy of BCG instillation by evaluating urinary cytokine response before and after intravesical BCG instillation in Superficial Bladder Cancer (SBC) patients and investigate if Toll Like Receptors are involved. Urinary cytokines IL-2. IL-8, TNF-α and IFN-γ were quantitated in patients (n=45) with SBC using sandwich ELISA kits. Reverse Transcriptase polymerase chain reaction (RT-PCR) was used to study mRNA expression of these cytokines in transurethral resection (TURBT) biopsies and peripheral blood lymphocytes (PBL). Results demonstrate that baseline levels of IL-2 and IL-8 were increased in urine of SBC patients when compared to healthy controls. At four weeks interval and after four hours of BCG instillation a marked increase in urinary IL-8 levels were observed in these patients. TNF-oc and IFN-γ levels did not show any significant increase after 4 hr of BCG instillation. Tumor biopsies of SBC patients showed an increase level of IL-8 mRNA expression but the other three cytokines (IL-2, TNF-oc and IFN-γ) were poorly expressed. Results demonstrate that elevated levels of IL-8 in urine of SBC patients after BCG instillation serves as a good prognostic marker and further establishes that BCG mediates its action via TLR-2 and TLR-4. Studies are in progress to investigate if ectopic IL-8 released by tumor cells correlates with tumor progression and metastasis.


Gene Therapy: Trials and Tribunals

Nurendra Chirmule*

Vaccine and biology Research, Merck and Co. Inc., Wayne. PA, USA. *

Monumental advances have occurred in molecular biology over the past quarter of a century. The discovery of DNA ligase, reverse transcriptase and restriction endonuclcases has laid the ground work for the construction of recombinant DNA molecules. These advances have had a profound impact of understanding gene structure, function and control. The mapping of the human genome is completed and those for various other species and micro-organisms have also been defined. An understanding of genetic basis of various human diseases has also accelerated. In this respect, advances in identifying single and multi-gene defects have pinpointed etiology of diseases. On this landscape of important progress, an entirely new paradigm for the prevention, cure, and treatment of human disease – gene therapy – has evolved. “Human Gene Therapy may be defined as the insertion of a normal or modified gene into somatic cells of patients to correct genetic or acquired diseases, through in vivo synthesis of missing, defective or insufficient gene products”. Since treatment of the first human experiment on September 14, 1990, a new era of treatment has begun. Extensive studies in animal models have demonstrated proof-of-concept, and efficacy of utilization of this technology in treatment of genetic disorders. Several clinical trials are in progress for therapeutic application of genetic diseases, such as hemophilia, cystic fibrosis, ornithine transcarbamylase deficiencies. In addition, novel approaches utilizing genetic tools have been utilized for treatment of various types of cancers; these include inducers of apoptosis, immune stimulation, targeting susceptibility to drugs. More recently a clinical trial serious adverse events, namely, a death of a patient and another trial with integration of the vector/transgene has underscored the importance of innate immune responses and molecular analyses in gene therapy. This lecture will cover the “holy grails” of gene therapy which include: long term gene expression, readministration, targeting, regulation, site-specific integration and gene replacement.


Molecular Diagnostic Modalities for CML: Molecular Beacons

Nishigandha R Naik1* and PKR Kumar2

1Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer, TMC, Kharghar, Navi Mumbai – 410208. :National Institute of Bioscience and Human Technology, Tsukuba, Japan. *nnaik(6i)

Molecular diagnostics, that is, the use of diagnostic testing to understand the molecular mechanisms of an individual patient’s disease, will be pivotal in the delivery of safe and effective therapy of many diseases in the future. Ideally, techniques used in molecular diagnostics, especially in MRD detection should provide some quantification of the target and be rapid, inexpensive, readily standardized and disease specific. Molecular beacons (MB) are short, hairpin shaped, synthetic oligonucleotides with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid. Therefore, they may be useful in molecular diagnostics, especially in situations where it is either not possible or desirable to isolate the probetarget hybrids from an excess of hybridization probe. Use of MB in chronic myeloid leukemia (CML) diagnostics was evaluated. MB were designed against the most common bcr-abl chimera – b3a2 that is consistently associated with CML. After checking specificity in vitro by gel shift assay, intracellular specificity of fluorescent label tagged b3a2 MB was checked flow cytometrically and by laser confocal microscopy, using bcr-abl positive cell lines – K.562 and 32DCbcr/abl. Data analysis of b3a2 MB uptake by these cells showed that specific signals are obtained within few minutes and are maintained till few hours. These studies show that MB have tremendous potential to be used as biomolecular recognition probes for CML.


Combinatorial Chemoprevention of Hereditary Colon Cancer: Models and Biomarkers

Nitin Telang*, Meena Katdare

Strang Cancer Prevention Center and Department of Surgery, Weill Medical College of Cornell University, New York/USA. *

Mutations in tumor suppressor and DNA-mismatch repair genes represent primary genetic defects in the human hereditary nonpolyposis colon cancer (HNPCC) syndrome. Current 5-Fluorouracil based combination therapy produces modest efficacy and substantial systemic toxicity. Alternate approach using low dose combination of Coxibs and DNA inhibitors may enhance efficacy and minimize toxicity. Unlike the human HNPCC syndrome, preclinical animal models exhibit accelerated small intestinal carcinogenesis, and therefore, limit clinically relevant translation. Reliable preclinical models with relevant genetic defects in appropriate target organ site may reduce the need for clinical extrapolation. Subculturable epithelial cell lines established from histologically normal colon of Ape [+/-] / DNA MMR [+/-] mice represents a novel preclinical model. Status of cell proliferative kinetics, cell cycle progression and anchorage-independent colony formation represent the mechanistic endpoint biomarkers. Selected mechanistically distinct, clinically relevant pharmacological agents represent the test compounds for combinatorial efficacy. Relative to the wild type Ape [+/+] / DNA-MMR [+/+] C57 COL cells, the mutant cells exhibit a 41-56% decrease in the population doubling time, and a 95-217% increase in the saturation density. The mutant cells also exhibit enhanced risk for carcinogenesis as evidenced by a 33-100% increase in anchorage-independent colony formation. Treatment of mutant cells with low dose combinations of mechanistically distinct sulindac (SUL) + Difluoromethyl ornithine (DFMO) or Celecoxib (CLX) + 5-Fluorouracil (5-FU) produce at least a 2.5 fold and 7.2 fold increase respectively, in efficacy for growth inhibition relative to that obtained by individual test compounds. The enhanced efficacy is predominantly due to arrest of the cells in the S and/or G2/M phase of the cell cycle. These results provide phenomenological leads for future studies on relevant molecular mechanisms for enhanced efficacy of these combinations. The present data therefore, validates a cell culture model for a rapid mechanism-based screening and rational prioritization of combinations of new synthetic or naturally occurring chemopreventive agents for subsequent clinical trials. [Support: The Irving Weinstein Foundation and NCI MAO # CN 75029-63].


Ablation of Peripheral Dopaminergic Nerves Stimulates Malignant Tumor Growth by Inducing Vascular Permeability Factor/Vascualr Endothelial Growth Factor Mediated Angiogenesis

Sujit Basa1,2, Chandrani Sarkar3, Debanjan Chakroborty3, Janice Nagy4, Rita Basil Mitra5, Partha Sarathi Dassupta3*, Debabrata Mukhopadhya1

1Mayo Clinic Cancer center, Rochester, USA. 2Dept of Medical Oncology, CNCI, Calcutta. 4Signal Transduction &Biogenic Amines Lab, CNCI, Calcutta. 4Dept of Pathology, BIDMC & Harvard Medical School, Boston, USA. 5Dept of Pathology, IPGMR, Calcutta. *

Initiation of angiogenic process has been shown to be an essential early step in the progression of malignant tumors. We report here that the ablation of peripheral dopaminergic nerves markedly increased Angiogenesis. microvessel density , microvascular permeability and growth of malignant tumors in mice. Endogenous peripheral dopamine acted throughD2 receptors as significantly more Angiogenesis and tumor growth was observed in D2 dopamine receptor knock out mice in comparison with controls. The vascular endothelial growth factor receptor 2 phosphorylation which is critical for promoting Angiogenesis, was also significantly more in tumor endothelial cells collected from the dopamine depleted and dopamine D2 receptor knock out mice. These results reveal that peripheral endogenous neurotransmitter dopamine might be an important physiological regulator of vascular endothelial growth factor mediated tumor angiogenesis and growth and suggest a novel link between endogenous dopamine angiogenesis and tumor growth


L1 Variants of HPV Types 16, 33, 45 and 58 in Indian Patients with Cervical Neoplasia

Priya Abraham1*, Narayanan Sathish1, Abraham Peedicayil2, Gopalan Sridliaran1, Subhashini John3, George C handy4

Departments of Clinical Virology1, Obstetrics and Gynaecology2. Radiation Oncology3, Clinical Gastroenterology and Hepatology4, Christian Medical College, Vellore, India. *

Background: Studies in south India reveal the presence of high-risk oncogenic HPV types 3 1, 33, 35, 45, 51, 52, 56, and 58 in addition to 16 and 18. Only few reports of intratypic variations in the L1 gene of these types exist. Objectives: To look for nucleotide variations in the L1 gene of these high-risk HPV types among Indian women with cervical neoplasia. Methodology: In a cross-sectional study, 118 women with cervical neoplasia were recruited comprising 16 with cervical intraepithelial neoplasia and 102 with invasive carcinoma. HPV genotypes were identified by PCR followed by restriction fragment length polymorphism (RFLP). Untypeable isolates were analyzed by nucleotide sequencing. Results: Of 111 women positive for HPV DNA, 96 women (86.5%) were typed by RFLP while 15 women (13.5%) were untypeable. These 15 samples were resolved by nucleotide sequencing revealing: HPV 16 (n=6), HPV 33 (n=2), HPV 45 (n=3) and HPV 58 (n=4). Untypeable isolates of HPV types 33, 45 and 58 and 5 of 6 isolates of HPV 16 had consistent RFLP digestion patterns with identical nucleotide variations in the Ll region. Some of these Ll nucleotide variations produced a change of the encoded amino acid. Conclusions: A proportion of HPV types 16, 33, 45 and 58 exhibited nucleotide variations in the Ll gene. Studying intratypic variants could shed more light on the evolutionary and taxonomic status of HPV types in a given geographical region. Whether Ll variations amounting to amino acid change could have a bearing on future vaccine formulations needs to be further evaluated.


Cervical Cytology or HPV Testing or Both What is Practicable in India?

R Nijliawun*and A Rajwansh

Department of Cytology & Gynae Pathology, Post Graduate Institute for Medical Research, Chandigarh, India. *

Introduction: The West has witnessed a dramatic fall in the rate of cervical cancer, thanks to the well-organized screening programmes. Current approaches vary from cervical cytology alone, to cervical cytology and HPV testing, to HPV testing alone. In India, approximately 10 crore women fall in the age groups (30-60 years) most susceptible to cervical cancer. Current screening programmes are grossly unsatisfactory. Mass HPV testing is a distant dream. Even cervical cytology cannot be offered to all who need it. However, cytology being the cheaper of the two, it is worthwhile to study if there are any cytomorphological features in addition to koilocytosis which may indicate an HPV infection. Aims and Objectives: 1) To study cervical smears with koilocytes for additional morphological features which might suggest an HPV infection. 2) To study cervical smears for special morphological features in women who show the presence of high-risk HPV DNA. Materials and Methods: 1) 30 consecutive cervical smears which showed evidence of koilocytosis were scanned for additional morphological features. 2) 21 cervical smears from women who tested positive for high-risk HPV DNA by HC2 system were screened for special morphological features. Results: 1 ) Of the 30 smears with koilocytosis, four were classified as inflammatory, one as AGUS, 11 as ASUS, five as LSIL, and nine as HSIL. 19 smears showed dyskeratosis, and 17 showed cytoploasmic clearing. 14 displayed both. Five of the ASUS smears revealed occasional atypical parabasal cells, and two of the LSIL smears revealed such cells. 2) Of the 21 smears from women who tested positive for high-risk HPV DNA, 13 showed koilocytosis, and 11 revealed dyskeratosis. Eight smears showed both. Of the 21 (HPV DNA positive) smears, 10 fell in the ASCUS/LSIL group. Of these six revealed occasional atypical parabasal cells. Conclusion: In addition to koilocytosis, features like dyskeratosis and cytoplasmic clearing hold promise as additional morphological indicators of cervical HPV infection. Much larger studies are required to confirm the same.


Effect of Rapamycin on Akt Signaling in Hepatoma HepG2 and HepG2 Cells Over-expressing Constitutively Active Akt/ PKB

Shailly Varma, Jenny Oberg, Dhananjay Gupta and Ramji L Khundelwal*

Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, S7N 5E5, Canada. *

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase, which is known to play an important role in the regulation of cell growth. It activates ribosomal p70^sup S6k^ and inhibits elongation factor eIF4E inhibitor (4E-BP). Rapamycin inhibits mTOR and its downstream signalling. The mechanism of action of rapamycin is that it forms a complex with immunophilin FK506 binding protein 12 (FKBP 12) which in turns complexes with mTOR thereby inhibiting its activity. mTOR phosphorylation levels are controlled by the activation of Akt /PKB. Therefore, the effects of rapamycin on Akt /PKB signal transduction pathway both in the absence and presence of insulin were examined in parental HepG2 and HepG2 cells constitutively over-expressing Akt /PKB (HepG2-CA Akt /PKB). Cells were treated with insulin (1-100 nM) in the presence and absence of rapamycin (20 nM). The phosphorylation status of mTOR (Ser 2448) and p70S6K (Thr 389) were investigated. In HepG2 cells, the phosphorylation of mTOR was stimulated 4- fold by insulin and it was decreased to 2-fold in the presence of rapamycin. Since, HepG2-CA Akt/PKB already have high levels of Akt /PKB activity, phosphorylation status of mTOR was high and did not change in the presence of insulin ± rapamycin. The phosphorylation of p70^sup S6K^ was completely abolished by rapamycin in both types of cells. The reason for differential effects of rapamycin on the phosphorylation of mTOR and p70^sup S6K^ in HepG2 and HepG2-CA Akt/PKB cells is not clear and requires further investigation. It can be concluded, however, that rapamycin can regulate p70^sup S6K^ (and protein synthesis) both in parental hepatoma and cells overexpressing Akt /PKB.


Altered Expression of Anti and Proapoptotic Proteins during Chemoprevention of Hamster Buccal Pouch Carcinogenesis by Tomato and Garlic Combination

V. Bhuvaneswari(a), K. S. Raoh and S. Naaini(a*)

aDepartment of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India. bCentre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500 007, India. *r_kumar24@red

Effective combinations of dietary agents are promising candidates for cancer chemoprevention because of their safety and the fact that they are not perceived as medicine. The present study was designed to investigate the apoptosis-inducing effect of combined administration of tomato and garlic during 7, 12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Hamsters were divided into four groups. The right buccal pouches of animals in group 1 were painted with 0.5% DMBA three times a week. Animals in group 2 painted with DMBA as in group 1, received in addition, intragastric administration of a combined dose of tomato and garlic on days alternate to DMBA application. Group 3 animals were given chemopreventive agents alone. Animals in group 4 served as control. All the animals were killed after an experimental period of 14 weeks. DNA fragmentation and the apoptosis-associated proteinsBcl-2, Bax, Bim, p53 as well as caspases 8 and 3 were used as markers of apoptosis. Topical application of DMBA for 14 weeks resulted in well-developed squamous cell carcinomas (SCCs) associated with increased expression of Bcl-2 and decreased expression of Bax, Bim, P53 and caspases 8 and 3. Combined administration of tomato and garlic significantly inhibited the development of HBP carcinomas and induced apoptosis. This was evidenced by downregulation of Bcl-2 and upregulation of Bax, Bim, p53 and caspases 8 and 3. These observations suggest that induction of apoptosis may be one of the mechanisms through which functional foods such as tomato and garlic exert their anticancer properties.


Role of Vascular Endothelial Growth Factor-A (VEGF-A) in 2 Methoxyestradiol-Induced Tumour Cell Proliferation Inhibition

Samarendra Nath Banerjee1*, Snigdha Banerjee and Susltunia K. Banerjee

Cancer Research Unit, University of Kansas Medical Centre and Division of Hematology and Oncology, Department of Medicine. University of Kansas Medical Centre, Kansas, U.S.A. 1 Present Address: Department of Zoology. Rammohan College, 102/1 Raja Rammohan Sarani. Kolkata-700009, India. *

Angiogenesis, the sprouting of new blood vessels, is essential for progressive solid tumour growth and thus constitutes a very promising therapeutic target. Tumor angiogenesis process is regulated by enhanced secretion of growth factors. Among several tumour angiogenic factors, vascular endothelial growth factor (VEGF) has been recognized as a prime regulator of tumour angiogenic process. It stimulates the proliferation of endothelial cells for the formation of new blood vessels as well as increasing their permeability, which may also be important for the provision of nutrient to tumours. Recent developments in understanding of the molecular mechanisms involved in tumor blood vessel formation provide a rational basis for anti-angiogenic drug development. 2-Methoxyestradiol (2-ME^sub 2^), once considered an inactive end metabolite of estradiol, has recently emerged as a very promising agent for cancer treatment. It is synthesized by sequential hydroxylation of the parent compounds followed by methylation in the liver. 2ME^sub 2^ was reported to elicit both stimulation and inhibition of tumor angiogenesis and growth depending on the dosage used. However, the mechanism(s) of the biphasic action of 2ME^sub 2^ has been elusive. The present study was undertaken to determine the dose dependent effect of 2ME^sub 2^ on VEGF Mrna and protein levels in MCF-7 human breast cells, MIA-PaCa-2 pancreatic cancer cells and GH3 rat pituitary tumor cells, and to correlate these with cell proliferation. Mrna and protein levels were determined using Northern blot and Western blot analyses. Cell proliferation and death rates were measured using 3H-thymidine incorporation into untreated and 2ME^sub 2^ treated cells. A dose-dependent biphasic effect of 2ME^sub 2^ on VEGF-A Mrna and protein expressions and cell proliferation was found in MCF-7 and GH3 cells, while this biphasic effect was undetected in MIA-PaCA-2 cells. One µm 2ME^sub 2^ significantly increased VEGF Mrna and protein as well as ^sup 3^H-thymidine incorporation in both MCF-7 and GH3 tumor cells as compared to untreated cells. In contrast, a trend of decreasing expression of VEGF-A Mrna and rate of cell proliferation was noted with higher (5-10 µM) 2ME^sub 2^ concentrations. A low dose of 2-ME^sub 2^ also increased the VEGFA Mrna expression in ER-cc-transfected normal breast epithelial cells. Moreover, the enhanced expression could be blocked by an antiestrogen ICI 182,780 and suggest that 2-ME,-induced VEGF-A expression is mediated through estrogen receptor. Furthermore, the studies demonstrate that induced VEGF protein is functionally active because it potentiates proliferation of adjacent endothelial cells. Taken together, the results of these studies indicate that 2ME^sub 2^ is a novel therapeutic drug for breast cancer.


Identification of the Signal Transduction Pathways Associated with the Enhanced Survival Ability of Fibroblasts after Exposure to Repetitive Low-grade Stress: Reversal of the Anti-apoptotic Effects by Tea Polyphenols and Resveratrol

Sunghamitra Raha*

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata-700064. India. *

V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 µM) H^sub 2^O^sub 2^ in culture for 4 weeks. p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Transcriptional activity driven by Nuclear Factor kappa B (NF-κB) was significantly (4 fold) enhanced by repetitive oxidative stress and was completely blocked by inhibition of p38MAPK activation. Catalase activity, protein levels and mRNA levels rose markedly (5-6 fold) during this time. Furthermore, Akt/Protein kinase B (PKB) became gradually phosphorylated at Serine^sup 473^ and Threonine^sup 308^ during this period of repetitive stress. The repetitively stressed cells demonstrated a significant resistance to apoptosis by subsequent acute stress in the form of ultraviolet radiation (UVR) at 5J/m^sup 2^ or H^sub 2^O^sub 2^ (7.5 mM). Inhibition of p38MAPK activation by repetitive stress either through exposure to pharmacological inhibition or siRNA-induced silencing of p38MAPK resulted in inhibition of down-stream activation of some anti-apoptotic signaling pathways. Pharmacological blockage of Akt activation also resulted in partial inhibition of the survival effects. The opposition to apoptosis conferred by repetitive stress was drastically reduced by constant exposure to tea polyphenols or resveratrol during the stress period through their restraining effects on the signaling pathways associated with cellular survival. An overview of the multiple signaling events triggered by repetitive low-grade stress and inhibition of these pathways by natural compounds will be presented.


Role of 5’Untranslated Regions of Human Cathcpsin L mRNA Species in Determining Their Stabilities and Translational Efficiencies

Shyam S. Chauhun*

Department of Biochemistry, All India Institute of Medical Sciences. Ansari Nagar, New Delhi-1 10029, India. *

Cathepsin L, is over expressed in a variety of human tumors. A majority of the procathepsin L synthesized by transformed cells in culture is secreted into the medium for which it requires an intact carboxy terminus. It has been established that the switch from nonmetastatic to highly mctastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L. Blocking the secretion of this protease by anti cathepsin L SC-FV could abolish the tumorigenic and metastatic potential of melanoma cells in nude mice. Others and we have established that human cathepsin L is encoded by at least five mRNA species namely hCATL A, AI, All. AIII and hCATL B. All these mRNA species contain identical open reading frames but differ in their 5’untranslated regions. These mRNA species are transcribed from two different promoters which are differentially regulated. HCATL A, AI, All and AIII are produced by the alternate splicing of the same primary transcript transcribed from proximal promoter while hCATL B is transcribed from the alternate promoter. My laboratory identified hCATL AIII for the first time, and established that this most abundant splice variant of cathepsin L is also most efficiently translated. It also exhibit higher stability as compared to other splice variants. We further demonstrated that the 5′ UTR of hCATL AIII is enough to confer translational advantage to a hetrologous mRNA. Thus the 5′ untranslated regions of human cathepsin L mRNA species is sufficient for determining their translational efficiencies and stabilities.


Investigation on the Role of Thymic Peptides in Tumor-Induced Immunosuppression

Pratima Shrivastava, Sukli Mahendra Singh*

School of Biotechnology, Banaras Hindu University, Varanasi, India. *

Introduction: Progressive growth of a spontaneous T cell lymphoma, designated as Dalton’s lymphoma (DL), causes suppression of immune responses and thymic atrophy. It was hypothesized that the decline in the production of thymic peptides could be one of the causes of the inhibited immune responses of DL-bearing host. Objective: The effect of thymic peptide: thymosin 1 (thyal) on the functions of tissue macrophages, tumor-associated macrophages (TAM), TAM-derived dendritic cells (DC) and tumor-induced apoptosis of thymocytes was studied. Methods: Effect of in vivo administration of thyal on the activation of various types of macrophages, DC, bone marrow hematopoiesis of myeloid cells and tumor-induced apoptosis of thymocytes was investigated using cell culture based techniques and bioassays. Results: In vivo administration of thyal or thymus extract resulted in augmentation of cytotoxic and accessory functions of TAM and DC. Macrophages displayed heterogeneity in their response to thyal. Thyal was also found to prime the macrophages to show an augmented responsiveness to activation signal of endotoxin. Thyal administration and adoptive transfer of thyα1 treated macrophages or DC to tumor-bearing mice prolonged their survival along with inhibition of tumor progression. Thyal suppressed myeloid differentiation of bone marrow progenitor cells. Thyal antagonized tumor-associated apoptosis of thymocytes by altering the expression of apoptosis related genes. Conclusion: Phis study shows that thyal can be used to overcome the iminunosuppressed state of macrophages and DC in a tumor-bearing host. Thus these findings may have long lasting impact on the development of tumor immunotherapy using thymic peptides.


Exposure Estimates of Indian Chronieplaters: A Prefatory Study

Roli Budhwar, Mohan Das, Vipin Behari and Sushil Kumar*

Industrial Toxicology Research Centre, Lucknow. *

Introduction: Chromate compounds are occupational carcinogens. Exposure to carcinogenic Cr results in a variety of adverse health effects ranging from dermatitis or skin ulcer to nasal septum perforation, inflammation & carcinoma of respiratory tract. A substantial population of chromeplating subjects in India gets exposure to carcinogen occupationally. However there is a paucity of information on their exposure estimates and health risk assessments in India. Objectives: A preliminary study of chromeplaters (n=24) and suitable control (n=35) with a cross-section study design was, therefore, undertaken. Methods: A structured interview recorded the prevalent adverse health effects. Analyses of Cr and DPC content in biological specimens (blood/urine) provided internal Cr exposure and related biological effect estimates. The metal was quantified by direct dilution method using AAS & graphite furnace. The DNA protein crosslink (effect biomarker) was investigated bio-chemically in peripheral lymphocytes. Result: Clinical symptoms were present in 1/3rd population of chromeplaters (n=8). These included redness in conjunctiva with prominent bulbar vessels, congestion of nasal mucosa and dermatological ailments. No clinical symptoms were present in non-chromeplating subjects. The urinary Cr levels were found to be greater in chromeplaters (n=18). Blood Cr levels (n=24) also showed an increasing trend. DPC content was greater significantly in chromeplaters (n=17). An investigation of the DPC coefficient (a quotient of DPC and total DNA) also revealed a similar result. Conclusion: This exploratory study validated the Cr monitoring biomarkers. It has revealed the importance and need for similar endeavors, albeit with a large sample size. More studies are needed to explore (a) the prevalent morbidity pattern, (b) its association with (both systemic as well as topical) exposure and (c) intervention strategies in Indian chromeplating subjects.


Transdifferentiated Monocytes as Markers for Endothelial Differentiation, Tumor Angiogcncsis and Growth

Vimlarani Chopra1*, Edward V Hannigan1 and Cherylyn A Savary2

The University of Texas 1 Medical Branch, Galveston, TX and 2 M.D. Anderson Cancer Center, Houston, Texas, USA. *

We have detected the presence of CD1a^sup +^ Langerhans/Dendritic cells (DCs) in monocultures of tumor-derived epithelial cells and their cocultures with mononuclear cells from patients with cervical cancers (>CIN II) maintained as three-dimensional (3-D) culture in bioreactors. These cells were also found in circulation of cervical cancer patients. In the 3-D culture environment, the generation of CD1a^sup +^cells was accompanied by changes in expression of pSTAT3, IL-8, IL-10, IL-12, TNF-α, and NO radicals. We are using the bioreactors (mimics the in vivo host microenvironment) to gain insight into the effects of tumor environment on DC differentiation, maturation and functions. Phase contrast microscopy confirmed the formation of very large, nonadherent, and loose 3-D aggregates of developing CD1a+cells (in presence of GM-CSF+IL-4) which consisted of LinHLA-DR’, LiivB7-2+ (CD86+) and Lin- CD40+ cells. These plastic adherent cells cultured in the presence of conditioned medium from 3-D grown HeLa were CD34+ (stains endothelial precursors and mature endothelial cells), and showed evidence oftube formation and formation of network-like structures was observed by adherent cells after iinmunofluorescence staining. These cells were also PECAM-1^sup +^(CD31+, constitutive endothelial marker), and VCAM-1 (CD106+) and ICAM-1 (CD54+, inducibIe endothelial markers). This transdifferentiation appeared to be due at least in part to the presence of VEGF, since no CD31+ cells were detected in the presence of neutralizing antibodies to VEGF. Hence under local angiogenic conditions myeloid cells, including DC progenitors might be directed towards differentiation into endotheliallike cells and can serve as early markers of tumor growth, angiogenesis and metastasis.


Trends in Incidence of Breast Cancer-Indian Scenario

NS Murtliy*1, S Saxcna2, K Chaudhry1 and A Pandey3

1 Indian Council of Medical Research, 2 Institute of Pathology (ICMR), 3 Institute for Research in Medical Statistics (ICMR), New Delhi. *

Globally, Breast cancer is the most frequent female malignancy. It is the most frequent cancer among women in Delhi, Mumbai, Ahmedabad, Calcutta and Trivandrum. In other registries, it is listed as the second leading site among women. Reports of increasing rates of breast cancer in several Indian registries prompted this analysis of trends in the incidence rate of breast cancer by age, period and cohort. Incidence data for the various Indian registries for 5-year periods from 1968-72 to 1993-97 were obtained from the different volumes of Cancer Incidence in Five Continents or from the publications made by the individual registries. Annual percentage changes (APC) in incidence rates were computed using relative difference between the two time periods for crude rate (CR), age adjusted rate (AAR) and age-specific incidence rates (ASIR). In 1993-1997, AAR of breast cancer were highest in Mumbai (28.9/100,000 woman-years) and lowest in the rural registry at Barshi (8.1 per 100,000 woman-years) The rise in ASIR of breast cancer was seen up to ages of 59 years in most of the registries followed by a slight drop and a slower increase in older age groups. The APC by age revealed different patterns of changes in different registries. Trend analysis by period revealed that breast eancer ineidenee rates rose among women in all the registries with an exception of Ahmedabad registry. The highest annual percentage increase in AAR was noted in Mumbai (2.0%) and lowest for Pune (0.5%). Risk was found to be increasing in successive birth cohorts. Efforts should be made to detect breast cancer at an early stage by educating the population about the risk factors and through periodic screening either by physical self-examination or by self-breast examination. Mammography will be difficult to implement in Indian for the prevention and control of breast cancer.


Detection of DNA Damage (Strand Breaks) in Marine Mollusks – An Early Warning Signal of Carcinogenesis

A. Sarkar*, D. Gaitonde and D. Ray

Marine Pollution Assessment and Ecotoxicology Group, National Institute of Oceanography, Dona Paula, Goa -403004, India. *

DNA strand breaks are regarded as one of the primary causes of cancer. It can occur due to various factors. DNA strand breaks in marine organisms can occur due to interaction with various genotoxic substances such as PCBs. PAIIs, PCDDs, PCDFs, TBT, methylmercury, lead etc. which are found to be prevalent in the marine environment. Cytochrome P450 enzymes generally metabolize PCBs to mono and dihydroxy-PCBs. It is evide’nt that dihydroxy PCBs can potentially be oxidized to corresponding quinones which could probably produce reactive oxygen species leading DNA- strand breaks. In case of PAHs, Benzo (a) pyrene is generally converted to chemically reactive diol epoxide (BaPDE) at cellular level and subsequently interact with DNA to form stable adduct resulting into single strand breaks. In order to protect human health from carcinogen it is indeed of prime importance to identify the sources of geotaxis contaminants along the coastal region. In this context, detection of DNA damage (strand breaks) in terms of DNA integrity in marine mollusks (snails, oysters and clams) acts as early warning signal of carcinogen sis. DNA strand breaks in marine mollusks were determined in terms of DNA integrity (expressed as F) following the technique of partial alkaline unwinding assay in which three different parameters were measured – percentage of double strandedness, single strandedness and alkaline unwinding under a defined condition of pH and temperature. The results show significant variation in the integrity of DNA in marine mollusks at different locations. The DNA integrity in oysters (Saccostrea cuculata) was found to be quite low at Murmugao harbour (F, 0.177) and Chicalim (F, 0.266) while in clams (Grafrurium divaricatum) it was considerably higher at Dona Paula (F, 0.473) and Chicalim (F, 0.3 13). In the case of snails (Plunaxis sulcutas) it was found to be in the higher side. (F 0.33 and F 0.553). The lower the integrity of DNA higher will be the strand breaks. Thus it is clearly shows presence of genotoxic substances in the marine environment, which could be transmitted to human tissues through the food chain leading to carcinogenesis.

Copyright Indian Council of Medical Research Feb 2005

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