Journal of Histochemistry and Cytochemistry, Vol. 49, 929-930, July 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

Comparison of Apoptosis and Terminal Differentiation: The Mammalian Aging Process

C.E. Gagnaa,b, H.-R. Kuob, E. Floreaa, W. Shamia, R. Taorminaa, N. Vaswania, M. Guptaa, R. Vijha, and W.C. Lambertb
a School of Allied Health & Life Sciences, New York Institute of Technology, Old Westbury, New York
b Department of Pathology and Laboratory Medicine, University of Medicine & Dentistry of New Jersey-Medical School, Newark, New Jersey

Correspondence to: C.E. Gagna, School of Allied Health & Life Sciences, NY Institute of Technology, NYCOM 2, Rm. 362, Old Westbury, NY 11568. E-mail: dr.c.gagna@att.net


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Apoptosis is the ordered chain of events that lead to cell destruction. Terminal differentiation (denucleation) is the process in which cells lose their nuclei but remain functional. Our group examined cell death in three tissues using two different fixatives and a postfixation procedure, involving young (5 months) and old (2 years) guinea pigs. The data reveal that B-DNA and Z-DNA content decreases, whereas single-stranded (ss-) DNA increases, in older tissues undergoing apoptosis (skin and cornea) and terminal differentiation (ocular lens). We speculate that some of the factors that contribute to the aging process might also be responsible for the enhanced amount of damaged DNA in older tissues undergoing cell death. (J Histochem 49:929–930, 2001)

Key Words: apoptosis, terminal differentiation, B-DNA, Z-DNA, denatured DNA


  Introduction
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Summary
Introduction
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APOPTOSIS is the organized form of cell death in which cells self-destruct by cutting themselves into membrane-packaged pieces (Arends et al. 1990 ). Another type of cell death is necrosis (Williams et al. 1992 ). Apoptosis regulates adult physiology by contributing turnover of cells in the cornea and skin. Concerning the adult ocular lens, a unique type of cell death plays a role in maintaining its supramolecular order and metabolism: terminal differentiation (Gagna et al. 1997 ). Terminal differentiation is considered a specialized type of apoptosis (Kokileva 1994 ). Terminally differentiating adult lens fibers undergo denucleation but remain viable anucleated fiber cells. If too little cell death occurs, humans can develop cancer, and if there is too much apoptosis we may detect Alzheimer's disease (Hickman 1992 ).

The objective of this study was to investigate a possible relationship between the increase in denatured ss-DNA in guinea pig tissues, each undergoing cell death. This study also examined the effects of aging on nucleic acid structures, involving different types of tissues, two fixatives, and a postfixation method. We wanted to compare apoptosis and terminal differentiation in terms of ss-DNA formation. We wanted to determine if ss-DNA content was altered during cell death in older tissues.

In this study we used a variety of anti-nucleic acid IgG polyclonal antibody (PAb) (Gagna et al. 1997 ) and anti-nuclear matrix PAb (Rickwood and Hames 1982 ; Miller et al. 1993 ) probes to characterize two types of cell death. Normal tissues were examined from 5-month and 2-year-old guinea pigs. Half of the tissues were fixed in 10% neutral buffered formalin (10% NBF: protein fixative), and the remaining tissues processed in Carnoy's solution (nuclear fixative). All tissues were fixed for 24 hr and processed identically (3 µm) (Gagna et al. 1997 ). Paraffin-embedded tissue sections were immunohistochemically stained using the biotin–avidin method (Gagna et al. 1997 ). Antibodies consisted of anti-ss-DNA PAb, anti-Z-DNA PAb, anti-double-stranded (ds-) B-DNA PAb, and anti-nuclear matrix PAb probes. Both positive and negative controls were used. Immunohistochemistry was quantified using a computerized image analysis system (Gagna et al. 1997 ). H&E-stained tissue sections allowed proper orientation of cell components. Guinea pigs were sacrificed by lethal injection conforming to the guidelines of the Institutional Animal Use Committee. Agarose gel electrophoresis (SYBR Green I) (Rickwood and Hames 1982 ) was also employed to analyze DNA from apoptotic and terminal differentiated tissues.

Our results show that, in older tissues, the amount of terminal differentiation (lens) and apoptosis (cornea and skin) increases compared to the younger tissues. Control tissues (older), which do not undergo cell death, did not reveal as much ss-DNA. As these guinea pig tissues age ss-DNA increases, whereas ds-B-DNA and Z-DNA content decreases. Our electrophoresis data suggest that during cell death (5 months), change occurs in chromatin structure, which is associated with fragmentation of DNA into oligonucleosomal fragments of about 180 bp. In the older tissues we observed much greater fragmentation. Our results also indicate that ds-B-DNA and Z-DNA are gradually reduced, with a significant increase in denatured ss-DNA content (older tissues). Finally, our data reveal that as tissues grow older there is an increase in the destruction of the nuclear matrix.

We present two fixation methods for paraffin-embedded sections. The choice of fixatives and postfixation procedures (45% acetic acid, 15 min: antigen retrieval) are critical for properly characterizing cell death. Carnoy's proved to be superior for the detection of DNA structures, and the 45% acetic acid postfixation significantly enhanced the staining signal. NBF 10% produced superior results for quantification of the nuclear matrix. Antigen retrieval methods had no effect on nuclear matrix immunohistochemistry.

We speculate that the initial cleavage (nicking) of DNA occurs with the production of ds-DNA fragments. Towards the end of this process, denatured ss-DNA content increases as the ds-DNA fragments are exposed to more nicks. The ss-nicks (denatured DNA) may be very common in the internucleosomal regions but may also occur in the core particle-associated DNA, especially in older tissue. The chromatin-induced multistep sequence of DNA destruction can be viewed as shrinkage. We speculate that apoptosis and terminal differentiation are clearly controlled by a gene-directed pathway started by physiological or environmental stimuli. This pathway may also regulate the destruction of the nuclear matrix. Alternatively, proteases could be required to promote proteolytic activation. The cleavage of DNA-binding proteins and histones would also be required to increase DNA accessibility to the endonucleases. We speculate that cell death in older tissues may begin to malfunction and exaggerate the production of ss-DNA.

One possible explanation for the decrease in left-handed Z-DNA immunoreactivity within the older tissue sections is that as denatured ss-DNA content increases in right-handed B-DNA, the potential for B-DNA to Z-DNA transitions is decreased. The production of ss-DNA within the mainly ds-B-DNA prevents the development of negative supercoiling, a process that is required for the B- to Z-DNA transition (Gagna et al. 1997 ). The Carnoy's fixative removes chromosomal and non-chromosomal proteins from DNA, allowing torsional strain to be released into the DNA, and this acts as a driving force for the conversion of B-DNA to Z-DNA.

Great progress has been made in understanding the signals, conditions, and molecular sequences that regulate apoptosis and necrosis. Much less is known about terminal differentiation. More research is required to examine the structure and expression of the genes that regulate cell death. We need to know how right-handed B-DNA to left-handed Z-DNA transitions are effected by endogenous endonuclease cleavage. Identifying and characterizing genes that regulate cell death is of paramount importance. Understanding the involvement of B-DNA and Z-DNA binding proteins on in vivo transcription of these genes is critical.

An understanding of the mechanisms involved in apoptosis and terminal differentiation will have a major impact on the therapy of skin cancer, cataracts, and corneal diseases. It may be possible to manipulate cell death in order to therapeutically intervene in diseases such as cancer or AIDS, in which a dysregulation of programmed cell death is suspected. The molecular biological phenomenon of cell death encompasses great potential which may be employed to develop novel approaches for diagnosis, prevention, and treatment of human diseases.


  Acknowledgments

Supported by an NYIT-AAUP grant.

Received for publication December 3, 2000; accepted February 16, 2001.


  Literature Cited
Top
Summary
Introduction
Literature Cited

Arends MJ, Morris RG, Willy AH (1990) Apoptosis: the role of the endonuclease. Am J Pathol 136:593-608[Abstract]

Gagna CE, Lambert WC, Kuo H-R, Farnsworth P (1997) Localization of B-DNA and Z-DNA in terminally differentiating fiber cells in the adult lens. J Histochem Cytochem 45:1511-1521[Abstract/Free Full Text]

Hickman JA (1992) Apoptosis induced by anti-cancer drugs. Cancer Metast Rev 11:121-139[Medline]

Kokileva L (1994) Multi-step chromatin degradation in apoptosis: DNA breakdown in apoptosis. Int Arch Allergy Immunol 105:339-343[Medline]

Miller T, Beausang LA, Meneghini M, Lidgard G (1993) Death-induced changes to the nuclear matrix: the use of anti-nuclear matrix antibodies to study agents of apoptosis. BioTechniques 15:1042-1047[Medline]

Rickwood D, Hames BD (1982) Gel Electrophoresis of Nucleic Acids: A Practical Approach. 2nd ed Oxford, UK, IRL Press

Williams GT, Smith CA, McCarthy NJ, Grimes EA (1992) Apoptosis: final control point in cell biology. Trends Cell Biol 2:263-267[Medline]





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