Claude E. Gagna

Geographic Distribution of Liver and Stomach Cancers in Thailand in Relation to Estimated Dietary Intake of Nitrate, Nitrite, and Nitrosodimethylamine

It is our working hypothesis that the high rate of the liver and gastric cancers in North and Northeast Thailand is associated with increased daily dietary intake of nitrate, nitrite, and nitrosodimethylamine (NDMA). Samples of fresh and preserved Thai foods were systematically collected and analyzed from 1988 to 1996 and from 1998 to 2005. Consumption frequencies of various food items were determined on the basis of a dietary questionnaire given to 467 adults (212 males and 255 females) from 1998 to 2005. Food consumption data for the preceding and current year were collected and intakes (day, week, and month) of nitrate, nitrite, and NDMA were calculated. The trends in liver and stomach cancer age-standardized incidence rates (ASR) in four regions of Thailand were compared with the dietary intake of nitrate, nitrite, and NDMA in those same geographic regions. Mean daily intakes of nitrate of 155.7 mg/kg, of nitrite of 7.1 mg/kg, and of NDMA of 1.08 μ g/kg per day were found. Significant differences in dietary nitrate, nitrite, and NDMA intakes were seen between various Thai regions (P < 0.0001), and these corresponded to the variations in liver and stomach cancer ASR values between the regions. Dietary factors are likely to play key roles in different stages of liver and stomach carcinogenesis in Thailand.

Binding properties of bovine ocular lens zeta-crystallin to right-handed B-DNA, left-handed Z-DNA, and single-stranded DNA.

Bovine zeta‐crystallin has the ability to bind with different DNAs. Initially, this protein was named regulatory factor 36 (Kang et al., 1985), but it has been shown to be an ocular lens zeta‐crystallin (Jörnvall et al., 1993), which is considered an enzyme‐crystallin (Rodakanaki et al., 1989). The enzyme‐linked immunosorbent assay (ELISA) was used to quantitate the binding of bovine zeta‐crystallin to purified high molecular weight double‐stranded (ds‐) and single‐stranded (ss‐) DNA (bovine and synthetic DNA). ELISA quantitation was achieved by the addition of anti‐zeta‐crystallin antibodies to the DNA—zeta‐crystallin complex, using a novel immunochemical avidin—biotin method. Zeta‐crystallin shows much greater binding intensity for ss‐DNA and for ds‐Z‐DNA than for ds‐B‐DNA. It also reacts slightly more with ds‐Z‐DNA than ss‐DNA. Therefore, we speculate that zeta‐crystallin may act as a transcriptional enhancer (outer lens cortex), possibly binding to Z‐DNA regulatory elements within lens crystallin genes. It may also act to protect DNA from endogenous DNase activity and as a DNA unwinding (destabilizing) protein also involved with transcription, occurring in normal adult bovine lens nucleated secondary fiber cells.

Cell biology, chemogenomics and chemoproteomics

The scientific techniques used in molecular biological research and drug discovery have changed dramatically over the past 10 years due to the influence of genomics, proteomics and bioinformatics. Furthermore, genomics and functional genomics are now merging into a new scientific approach called chemogenomics.

Terminal differentiation and left-handed Z-DNA: a review.

Nucleic acids control the expression of genes, and different conformations of DNA structure may regulate cell death. Left‐handed Z‐DNA, which is speculated to function as a transcriptional enhancer, may be directly influenced by the destructive effects of terminal differentiation. The nicking‐denaturation of double‐stranded Z‐DNA could possibly initiate and enhance terminal differentiation within specific tissues.

Novel multistranded, alternative, plasmid and helical transitional DNA and RNA microarrays: implications for therapeutics

Novel multistranded and alternative DNA, RNA and plasmid microarrays (transitional structural nucleic acid microarrays) have been developed that allows for the immobilization of intact, nondenatured, double-stranded DNA, double-stranded RNA, and alternative and multistranded nucleic acids. It also allows for the study of transitional changes that occur in the structure of DNA and RNA. Alternative types of DNA, RNA and multistranded nucleic acids are immobilized by a variety of different surface chemistries (i.e., noncovalent or covalent) onto a novel substrate surface. This technology represents the next generation of microarrays, which will aid in the characterization of nucleic acid structure and function, and accelerate the discovery of drugs that bind to nucleic acids. In addition, we demonstrate four novel techniques that are the first practical applications of the microarray, that is, transitional structural chemogenomics, transitional structural chemoproteomics, transitional structural pharmacogenomics and transitional structural pharmacoproteomics. These novel nucleic acid microarrays, together with pharmacogenomics, can be used to improve the study of DNA and RNA structure, gene expression, drug development and treatment of various diseases.

The halting arrival of left-handed Z-DNA

Forty-nine years ago Watson and Crick proposed a double-stranded (ds-) model for DNA. This double helix has become an icon of molecular biology. Twenty-six years later, Rich accidently discovered Z-DNA, an exotic left-handed nucleic acid. For many years thereafter, this left-handed DNA was thought to be an artifact. DNA is no longer looked upon as a static molecule but rather an extremely dynamic structure in which different conformations are in equilibrium with each other. Many researchers have spent the last two decades characterizing this novel left-handed DNA structure. Now many investigators are beginning to accept the possibility that this novel ds-DNA conformation may play a significant in vivo role within eukaryotic and prokaryotic cells. However, more research needs to be performed before it is absolutely accepted by all in the scientific community.

Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry

Histotechnological processing of DNA can cause damage to and loss of DNA and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds)-B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were best for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave–vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times) and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated and conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. Results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered and undamaged tissue-bound ds-B-DNA. (J Histochem Cytochem 55: 999–1014, 2007)

Localization and quantification of intact, undamaged right-handed double-stranded B-DNA, and denatured single-stranded DNA in normal human epidermis and its effects on apoptosis and terminal differentiation (denucleation).

Quantification of two types of nucleic acids [double-stranded (ds-) and single-stranded (ss-) DNA] was performed to understand the distribution of DNA within the epidermal strata and to examine the effects of DNA structure on gene expression, viz., apoptosis and terminal differentiation. In addition, we examined the precise starting point of cell death within the epidermis (suprabasal layer); examined how DNA structure affects gene expression of melanocytes; and characterized the “transitional cells” located between the stratum granulosum and stratum corneum, viz., epidermal phase transition zone (EPTZ). Ultrasensitive anti-DNA antibody probes (ds-DNA, ss-DNA), the Feulgen reaction, histological stains (morphological characterization) and the terminal deoxyribonucleotidyl transferase (TUNEL) assay (apoptosis) were used to characterize cell death in normal human epidermis. This study characterized, for the first time, the deterioration of right-handed ds-B-DNA and the increase in denatured ss-DNA during epidermal maturation. For the first time, this approach also allowed for the quantitative and qualitative characterization of DNA content and structure in all epidermal strata, using anti-ds-B-DNA and anti-ss-DNA antibodies. In order to improve the retention and quality of DNA, a novel histotechnological processing procedure was used. The results indicate that the largest decline in DNA occurred within the stratum granulosum, followed by the EPTZ, and the stratum spinosum. Not all epidermal nuclei lost DNA, indicating two differentiating keratinocyte pathways, viz., apoptotic and non-apoptotic. Both pathways united in the stratum granulosum. These results suggest that keratinocyte terminal differentiation and apoptosis are distinct cellular events, cell death begins earlier than expected, and molecular epidermal events take place in a gradual and orderly manner within keratinocytes. During maturation, ds-B-DNA decreases as ss-DNA increases. Therefore, during differentiation of keratinocytes, both DNA content and DNA structure are altered.

Use of anti-single-stranded DNA antibodies to localize and quantify denatured DNA during cell death.

The present discussion is a brief overview which examines the most widely utilized methods for the characterization of cell death. We believe that the development of novel molecular biological techniques to identify cell death in tissue sections has not progressed in a rapid manner. Our group believes that the use of experimentally induced anti-single-stranded (ss-) DNA monoclonal antibodies (MAb; Stollar, 1992) is a specific tool which can be used to characterize cell death, with many advantages over the more frequently utilized procedures. Both singleand double-stranded DNA breaks occur under a normal range of non-apoptotic situations. DNA fragmentation does not always accompany apoptosis. Therefore, the use of positive and negative controls is critical to proper evaluation of cell death. The development of denatured single-stranded breaks in the doublestranded (ds-) DNA molecule is an important event in the genesis of cell death. A variety of techniques can be used to characterize denatured ss-DNA, e.g. denatured DNA can be quantified by the OliGreen ss-DNA quantitation kit (Molecular Probes, Inc., Eugene, OR, U.S.A.). Sophisticated methodologies, employ flow cytometry to identify and quantify normal cells or cells undergoing apoptosis and necrosis, based on changes in light scattering, DNA stainability, DNA sensitivity to denaturation or changed plasma membrane transport of Hoechst 33342 (Li et al., 1995). Two other techniques used for examining DNA strand breaks occurring in apoptotic cells can label individually fixed, permeabilized cells or tissue sections by

Abstract 5354: The guilty bystander model for electrochemotherapy of cancer: Verification in vitro

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Electroporation (EPR) is the induction of pores in cellular membranes in cells by application of a short application of a high voltage, low amperage electric current. If the cells are immersed in a solution containing a large molecule which is normally impervious to this membrane, it may be introduced into the cells in this manner. Electrochemotherapy (ECT) is a clinical procedure in which a drug, such as bleomicin or cisplatin, is introduced into the blood of or intratumorally into an animal or human subject by intravenous injection followed by application of a short high dose, low amperage electric current into a neoplasm after a delay enabling the drug to reach the neoplasm through the bloodstream or by other means. ECT is normally used for palliative care in human subjects, however there are now a number of reports, numbering well over 10, of unexpected complete clearance of neoplasms following ECT. Many of these reports have come from university centers in Europe and elsewhere. Although ECT is not yet approved in the USA, we believe that these reports can no longer be ignored. We and others have used EPR in culture to introduce large molecules into cells for experimental purposes for decades. Even under the controlled experimental conditions we have employed, however, the proportion of cells into which these molecules have been introduced has been well below 100 per cent. To account for the clearance of some tumors by ECT, in which the proportion of cells in which drug is introduced would be expected to be lower than for EPR, we have proposed a “Guilty Bystander Model”, in which cells adjacent to neoplastic cells into which drug has been introduced are also cleared. To test this model in vitro, we optimized conditions for EPR but were unable to produce more than 88 per cent introduction of labeled large molecules into lymphoblastoid cells (GM 1989A, derived from a healthy 33 year old Caucasian male, and GM 3999, derived from a healthy 8 year old Caucasian female), even under conditions which reduced cell viability from 99 per cent to 79 per cent. Increasing voltage produced a marked loss of viability. We conclude that EPR does not produce 100 per cent introduction of large molecules into human cells, even under optimized conditions in vitro, and that there must remain neoplastic cells in tumors treated by ECT that are eliminated by means other than introduction of the drug. Citation Format: W Clark Lambert, Gregory J. Tsongalis, Claude E. Gagna, Muriel W. Lambert. The guilty bystander model for electrochemotherapy of cancer: Verification in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5354. doi:10.1158/1538-7445.AM2015-5354