Cancer Letters

Cancer Letters

Volume 139, Issue 1, 3 May 1999, Pages 59-65
Cancer Letters

Arecoline-induced changes of poly-ADP-ribosylation of cellular proteins and its influence on chromatin organization

https://doi.org/10.1016/S0304-3835(99)00008-7Get rights and content

Abstract

Arecoline, the major alkaloid of betel nut (Areca catechu L.) and a suspected carcinogen, has been implicated in human cancers of various sites. A considerable portion of the world's population is constantly exposed to arecoline due to the habit of masticating betel nuts. The present work relates to the study of early molecular events following chronic arecoline exposure at a dose of 10 μg/ml to Swiss albino mice. Poly-ADP-ribosylation of all cellular proteins, histones and poly-ADP-ribose polymerase were studied in bone marrow and spleen cells and correlated with the organizational status of the chromatin. While most proteins showed lowering of their poly-ADP-ribosylation following arecoline treatment, only histone protein H1 in spleen cells and H2B in bone marrow cells exhibited an increase. The chromatin of both the tissues was progressively relaxed upon arecoline exposure. The implications of these changes have been discussed regarding the process of initiation of arecoline-induced carcinogenesis.

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)

  • B. Balachandran et al.

    Induction of mutations by different extracts of betel nut, and radiation: Their implications in carcinogenesis

  • T. Boulikas

    Relation between Carcinogenesis, chromatin structure and Poly(ADP ribosylation) (Review)

    Anticancer Res.

    (1991)

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    Present address: Department of Biochemistry, Regional Research Laboratory, Jorhat 785006, India.

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