Minoo Askari

The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone stimulates proliferation of immortalized human pancreatic duct epithelia through β-adrenergic transactivation of EGF receptors

Purpose: Pancreatic ductal adenocarcinoma is an aggressive smoking-associated human cancer in both men and women. The nicotine-derived 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is thought to contribute to the development of these neoplasms in smokers through genotoxic effects. However, NNK has been recently identified as an agonist for both β1- and β2-adrenergic receptors. Binding of NNK to these receptors stimulates proliferation of pulmonary and pancreatic adenocarcinomas cells in vitro and in hamster models. The goal of this study was to elucidate the NNK effects on the signal transduction pathways downstream of both β1- and β2-adrenergic receptors in immortalized human pancreatic HPDE6-c7 cells. Methods: The HPDE6-c7 cells are developed from normal pancreatic duct epithelial cells which are the putative cells of origin of pancreatic ductal adenocarcinoma. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cell proliferation assays, Western blot and cyclic AMP assays were employed to demonstrate the effects of NNK and other β1- and β2-adrenergic agonists and antagonist treatments on these cells. Results: MTT cell proliferation assays demonstrated that NNK and the classic β-adrenergic agonist, isoproterenol, increased cell proliferation in HPDE6-c7 cells. Western blot and cyclic AMP assays demonstrated that NNK treatments also resulted in: (1) transactivation of the epidermal growth factor receptor, EGFR, (2) an increase in intracellular cyclic AMP accumulation, and (3) phosphorylation of mitogen-activated protein kinase, Erk1/2. The proliferative response to NNK and isoproterenol were inhibited by the use of beta-blockers (propranolol), and the inhibitors of adenylyl cyclase (SQ 22536), EGFR-specific tyrosine kinase (AG 1478) and Erk (PD 98059). Conclusion: These findings suggest that the NNK -mediated β-adrenergic receptor transactivation of the EGFR and phosphorylation of Erk1/2 in immortalized human pancreatic duct epithelial cells as a novel mechanism might contribute to the development of tobacco-associated pancreatic carcinogenesis.

DDT mimicks estradiol stimulation of breast cancer cells to enter the cell cycle

Estrogens play a critical role in the etiology of found breast cancer. Estradiol promotes the growth of breast cancer cells in vivo and in vitro. Exogenous estrogens in both the environment and in the human diet increase the growth of breast cancer cells in vitro. A role for xenoestrogens in breast cancer etiology has been proposed but remains controversial. We examined the effects of the xenoestrogenic pesticide 1,1,1‐trichloro‐2,2‐bis(chlorophenyl)ethane (DDT) on estrogen‐receptor (ER)‐positive MCF‐7 and T‐47D human breast cancer cells as well as on ER‐negative HS 578Bst breast cancer cells and rat liver cells. Estradiol and DDT were found to increase the growth of MCF‐7 cells in the presence of insulin. The activity of cyclin‐dependent kinase (Cdk)2 increased in growth‐arrested T‐47D and MCF‐7 cells treated with β‐estradiol or DDT. The steroidal antiestrogen ICI 182,780 prevented both growth and Cdk2 activation induced by estradiol or DDT. Increased phosphorylation of Cdk2 and the retinoblastoma protein (pRb1O5) was observed in ER‐positive cells treated with DDT or estradiol. Cdk2 activity was not affected by DDT or estradiol in ER‐negative HS 578Bst breast cancer cells or in rat liver epithelial cells. Cyclin D1 protein synthesis was increased by DDT and estradiol in MCF‐7 cells. DDT and estradiol‐induced ER‐dependent transcriptional activation of estrogen response elements (EREs) in stably transfected MVLN cells, and ERE activation by low doses of DDT was increased by insulin. These findings suggest that DDT can stimulate breast cancer cells to enter into the cell cycle by directly affecting key regulatory elements. The relative potency of DDT in inducing cell‐cycle progression appears to be only 100–300 times less than that of estradiol when measured in the presence of insulin. Therefore, the cancer risks associated with DDT exposure may be greater than first thought, especially when additional mitogenic stimuli are present. Mol. Carcinog. 18:107–114, 1997.

Application of an Antibody Biochip for p53 Detection and Cancer Diagnosis

Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody‐based detection instrument, the biochip, to detect the presence of the anti‐p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8‐nm He‐Ne laser. Using this immuno‐biochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluorescein‐labeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen‐antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody‐based biochip for simple, rapid and early biomedical diagnostics of cancer.

Effectiveness of Purge-and-Trap for Measurement of Volatile Organic Compounds in Aged Soils.

The U.S. EPA-recommended method for measurement of trace levels of volatile organic compounds (VOCs) in soil, purge-and-trap, measures the readily desorbable organic contaminants from soil pore spaces and external soil surfaces. It does not, however, measure contamination that has diffused into internal micropores of soil matrix. Thus, the purge-and-trap method measures only a small fraction of total soil contaminants, especially in long-contaminated soils, where ∼90-99% of contamination may be in the interior of the soil matrix. We compared three methods for determination of VOCs in aged field samples:  purge-and-trap, methanol immersion, and hot solvent extraction. Hot solvent extraction proved to be much more effective than the U.S. EPA-approved purge-and-trap technique. For three long-contaminated soils containing such VOCs as trichloroethene, benzene, toluene, chloroform, methylene chloride, and cis-1,1-dichloroethylene, recovery from purge-and-trap ranged between 1.5 and 41.3% that of hot solvent extraction. Our data show that purge-and-trap may not be the best methodology for measuring soil VOCs concentrations, particularly in aged soils. It is clear from this and previous studies that the best overall choice for soil VOCs measurements is hot solvent extraction. These results also indicate the inefficiency of purge-and-trap as a method for evaluating vapor extraction remediation technology. Our results suggest that the EPA should review the use of the purge-and-trap method for measuring VOCs concentrations in soils.

Simultaneous detection of the tumor suppressor FHIT gene and protein using the multi-functional biochip

The tumor suppressor gene, fragile histidine triad (FHIT), encompasses the most common human chromosomal fragile site, at 3pl4.2. Detection of FHIT gene is important in cancer diagnostics since its alterations have been associated with several human cancers. A unique multi-functional biochip for simultaneous detection of FHIT DNA and FHIT protein on the same platform was applied. The design of the biochip is based on miniaturization of photodiodes, where functioning of multiple optical sensing elements, amplifiers, discriminators, and logic circuitry are integrated on a single IC board. Performance of biochip is based on biomolecular recognition processes using both DNA and protein bioreceptors, Cy5-labeled probes and laser excitation. Application of biochip for concurrent detection of various immobilized target DNA and protein molecules and multiplex of DNA and protein on the same microarray was accomplished. Linearity of biochip for quantitative measurements was demonstrated. Results demonstrated utility of this multi-functional biochip as a useful detection technology with applications in biological and clinical laboratories.

Advanced biochip: principle and applications in medical diagnostics and pathogen detection

An important factor in medical diagnostics is rapid, selective, and sensitive detection of biochemical substances (proteins, metabolites, nucleic acids), biological species or living systems (bacteria, virus or related components) at trace levels in biological samples (e.g., tissues, blood and other bodily fluids). This manuscript provides an overview of the operating principle and application of an integrated multi-functional biochip based on integrated circuit complementary metal oxide semiconductor (CMOS) sensor array for use in medical diagnostics and pathogen detection. The usefulness and potential of the biochip as a rapid diagnostic tool at the physicians office and as a practical, inexpensive screening tool for pathogens under field conditions are discussed.

Synchronous luminescence: a simple technique for the analysis of hydrolysis activity of the fragile histidine triad protein

Human fragile histidine triad (FHIT) protein has dinucleoside 5′,5′′′-P1,Pn-polyphosphates hydrolysis activity, with AMP being one of the reaction products. Application of synchronous luminescence (SL) spectroscopy, in which both excitation and emission wavelengths are scanned simultaneously while a constant wavelength interval is maintained between them, was investigated for detection of the enzymatic activity of the FHIT protein. Ability of SL to identify reaction components, AMP production and its increase as a result of increase in substrate concentration and inhibition of the hydrolysis activity by ZnCl2 are demonstrated.