Arecoline-induced changes of poly-ADP-ribosylation of cellular proteins and its influence on chromatin organization
Introduction
Betel nut (Areca catechu L.) or areca nut in various forms is consumed by over 600 million individuals across the globe [20]. Consequently, a large segment of the human population is constantly exposed to arecoline, a natural alkaloid of betel nut. There are indications of association of betel nut chewing with cancer of the mouth, oropharyngeal cavity, upper parts of the digestive tract and other sites [11], [20]. Arecoline, believed to be the main cause of cellular transformation and carcinogenesis, gives rise in vitro to at least four nitrosamines, two of which are carcinogenic [11], [20]. These nitrosamines have the potential to interact with the genome and other macromolecules to cause different types of damage [20]. Arecoline has been shown to induce DNA strand breaks, induce unscheduled DNA synthesis in the bone marrow cells (BMC) of mice and show a high rate of mutation in the Ames test [4], [21], [23], [24].
Poly-ADP-ribosylation (PADPR) is a post-translational modification of chromosomal proteins primarily. It is strongly elicited in response to DNA strand breaks [2]. The enzyme-catalyzed reaction modifies a large array of cellular proteins – overwhelming target proteins are chromosomal proteins [2], [5], [22]. The process is completely reversible. The unbranched or branched polymer, poly-ADP-ribose, is synthesized onto the target proteins from its endogenous substrate, nicotinamide adenine dinucleotide (NAD+), by enzyme poly-ADP-ribose polymerase (PADPRP). The polymer is degraded by enzyme ADP-ribose glycohydrolase (ADPRG). PADPR has been suggested to be the most drastic of all post-translational modifications of chromosomal proteins affecting both the charge and size of the modified proteins and, therefore, is likely to modulate the structure of chromatin [2], [5], [10]. The main target proteins for modification are histones (heteromodification) and PADPRP (automodification). Thus, processes dependent on chromatin structure, such as DNA replication, DNA damage and repair, transcription and gene expression, are likely to be influenced by the PADPR reaction [3], [15].
Carcinogenic transformation is a complex multi-step process involving multiple genetic alterations in somatic cells [1], [5], [14]. The most crucial and decisive event of carcinogenesis is the interactions of carcinogens with cellular macromolecules, especially the DNA. This event, at initiation, being irreversible commits a cell or tissue to transformation that ultimately culminates in a tumor [1], [7]. This makes the molecular events during initiation very important and its proper understanding may pave the way for reversing the course of carcinogenesis [1]. During carcinogenic transformation, several neogenes are known to be expressed indicating that gene expression patterns are altered during carcinogenesis [1], [17], [20]. Since alteration in gene expression pattern is also affected by the chromatin superstructure, it is likely that the PADPR reaction could influence carcinogenesis [1], [7], [14]. Fully transformed cells do show higher levels of PADPR of proteins. Further, inhibitors of PADPR have been shown to potentiate the cytotoxicity of DNA damaging agents used in cancer chemotherapy [6].
The aim of this study was to elucidate the relationship between PADPR of chromosomal proteins and chromatin organization during the initiation phase of carcinogenesis in an in vivo situation. Arecoline was chosen for its obvious relevance to humans. Swiss albino mice were chronically exposed to arecoline to mimic the situation of the constant exposure of humans who chew betel nuts. PADPR has been assessed using 32P-NAD+ as the substrate and the state of organization of chromatin was monitored by studying its fragmentation by DNase I.
Section snippets
Chemicals
All chemicals were of analytical grade and were used without further purification. All solutions were prepared in double distilled water.
Carcinogen and its administration
Male Swiss albino mice (6–8 weeks old), used for all the experiments, were housed in polycarbonate cages with rice husk bedding and provided with standard mouse pellets and drinking water ad libitum. An aqueous solution of arecoline hydrobromide (Sigma Chemical Co., MO) was administered ad libitum in the drinking water (10 μg/ml) to separate batches of mice.
Level of poly-ADP-ribosylation of the total cellular proteins following arecoline exposure
Fig. 1 shows that the level of PADPR of cellular proteins of SC declined immediately upon exposure to arecoline and essentially remained at the same level up to the end of the treatment. With regard to BMC, however, the level of PADPR increased in week 2 then declined below the control level in weeks 3 and 4 of arecoline treatment.
Quantitative analysis of arecoline-induced PADPR of histone proteins and poly-ADP-ribose polymerase (PADPRP)
The quantitative analyses of PADPR of individual histone proteins (heteromodification) of BMC and SC are shown in Table 1, Table 2, respectively. The data were
Discussion
Study of carcinogenesis using an in vitro system and extrapolation of results to an in vivo situation has inherent problems. In the present investigation, an in vivo Swiss albino mouse model was used to understand the initial events of carcinogenesis by a natural alkaloid, arecoline, to which a large segment of the population is chronically exposed. BMC and SC were chosen in this investigation for monitoring the level of PADPR after arecoline exposure as arecoline has no tissue specificity for
Acknowledgements
JRS thanks the UGC for grant of JRF and SRF for her studies. Part of this work was supported by a DST grant to RNS.
References (24)
Poly(ADP-ribosyl)ation, DNA strand breaks, chromatin and cancer
Toxicol. Lett.
(1993)Determination of DNA concentration with diphenylamine
Methods Enzymol.
(1968)- et al.
Poly(ADP-ribose) polymerase: a molecular nick sensor
Trends Biochem. Sci.
(1994) - et al.
Chromatin Isolation
- et al.
Poly(ADP-ribose) synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei
J. Biol. Chem.
(1981) - et al.
Low dose exposure of diethylnitrosamine affects mice liver thymidine kinase
Life Sci.
(1995) - et al.
Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro
Mutat. Res.
(1992) New targets for cancer chemotherapy – Poly(ADP-ribosylation) processing and polyisoprene metabolism
Biol. Rev.
(1990)- et al.
Poly(ADP-ribose): Structure, properties and quantification
Poly-ADP-ribosylation: a histone shuttle mechanism in DNA repair
J. Cell Sci.
(1992)
Induction of mutations by different extracts of betel nut, and radiation: Their implications in carcinogenesis
Relation between Carcinogenesis, chromatin structure and Poly(ADP ribosylation) (Review)
Anticancer Res.
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Present address: Department of Biochemistry, Regional Research Laboratory, Jorhat 785006, India.