SUNY at Albany
June 19-23, 2001
Human Cell Death and Aging: Denatured Single-Stranded DNA
Three forms of cell death, necrosis, apoptosis and terminal differentiation (denucleation) are recognized (1,2). There is no field in cell biology that is more confusing than the area of apoptosis versus necrosis and terminal differentiation. Some researchers view apoptosis and necrosis as being two distinct forms of cell death with major morphological and biochemical differences (3,4, 5). Apoptosis or programmed cell death is a form of regulated destruction characterized by specific morphological changes. These changes include membrane blebbing, cell shrinkage, chromatin condensation and cell fragmentation. The activation of an endogenous endonuclease produces fragmentation of cellular double-stranded (ds-) DNA into oligosomal length fragments (6,7). Terminal differentiation is a form of cell death, which has been less studied than necrosis and apoptosis.
Figure 1: Anti-Single-Stranded (ss-)-DNA Monoclonal Antibody (MAb) (F7-26) Probe Reactivity Towards Different Histological Skin Regions. Selective binding of anti-ss-DNA MAbs to apoptotic cells reflects the decreased stability of apoptotic DNA to terminal denaturation. Decreased DNA stability in apoptotic cells may reflect the effect of activated proteases on histones. For optimal results using the F7-26 MAb, we suggest Carnoy's solution as a primary fixative. The stratum corneum represents the outer most region of the skin.
We have employed two anti-single-stranded (ss-) DNA antibodies in order to immunohistochemically analyze cell death in fixed human tissue sections. The first is a polyclonal antibody (B14) (PAb) which can detect terminally differentiating cells (2), and the second is a monoclonal antibody (MAb) probe (F7-26) which can localize apoptotic cells. This second anti-ss-DNA MAb (F7-26) provides a marker specific for apoptotic cell death that is independent of internucleosomal DNA fragmentation. Additionally, this MAb does not detect necrotic cells (8).
Figure 2: Quantification of Anti-Single-Stranded-DNA Monoclonal Antibody (F7-26) Probe Towards Corneal Cells. Results reveal early and late apoptotic events and precondensation stage of apoptosis in cells. The limbus represents the peripheral (outer boarder) region and the central cornea is located within the apex of the cornea.
Our group has immunohistochemically (2) examined cell death in three different tissues, consisting of samples from younger (<42 years old) and older (>80 years old) humans: normal skin (38, and 91 years old) (Figure 1), normal ocular cornea (41, and 84 years old) (Figure 2), and normal crystalline lens (39, and 81 years old) (Figure 3). Both paraffin (2.5 microns) and plastic embedded (0.5 microns) tissue sections were used. Data were analyzed using a computerized image analysis system (2).
Figure 3: Anti-Single-Stranded -DNA Polyclonal Antibody (B14) Probe Binding Towards Terminally Differentiating (Denucleating) Lens Fiber Cells. Superficial fibers (SF) (0-100 microns), Phase Transition Zone (PTZ) (~ 100 microns), Middle Fibers (MF) (101-700 microns), Deep Fibers (DF) (701-1299 microns), Anucleated Fibers (AF) (1,300 microns), and Primary Anucleated Fiber Cells (PAF).
Within the younger normal tissues (38, 41, and 39 years old, respectively), we observed the standard physiological processes associated with the various forms of cell death (1,2,3). Concerning cell death within the younger human skin tissues (38 years old), we observed an increase in the amount of ss-DNA (F7-26) within the layers of the skins outer epidermal regions (Figure 1). As skin cells are displaced from the inner regions to the outer areas, ready to be shed, we quantified a gradual increase in ss-DNA content. Our group also quantified apoptosis (F7-26) within the four different corneal regions (Figure 2) and observed that cells die (apoptosis) by accumulating larger amount of ss-DNA as they progress from the peripheral regions (limbus: outer boarders), towards the surface and center (apex) region of the central cornea. Terminal differentiation (B11) in the adult lens (Figure 3) also shows an increase in ss-DNA content within fiber cells undergoing death as they are displaced from the outer cortex to the inner nucleus region.
Concerning cell death in the older human tissues (91, 84, 81 years old, respectively), we detected an increase (~ 18%) in ss-DNA content. None of these older tissues developed any pathological conditions, such as skin cancer or cataracts. We believe that each antibody probe recognizes five or more bases of DNA (9). As tissues age, the total amount of denatured ss-DNA increases, this may in turn eventually cause apoptotic and terminally differentiating cells to malfunction, therefore producing disease and/or general manifestations of aging. We conclude that ss-DNA content increases in older nondiseased tissues undergoing cell death. However, in studies involving older diseased tissues, such as human skin (90 years old), cornea (83 years old) and cataracts (82 years old), we calculated a 45 to 57% increase in ss-DNA content. This data indicates the possibility that too much formation of ss-DNA will produce pathological conditions. The chemical nature of DNA lesions may significantly alter subsequent cellular responses. Apoptosis and terminal differentiation suggest that cell death, in younger and older tissues, is a specific goal of a physiological process. However, if right-handed ds-B-DNA undergoes excessive denaturation (ss-DNA), then the gene expression of the programmed cell death is negatively affected. Eventually the DNA degradation will affect higher-order chromatin organization
Claude E. Gagna(1,2,*), Wojciech Golebiewski(2), and W. Clark Lambert(1)
Department of Pathology and Laboratory Medicine(1), University of Medicine and Dentistry of New Jersey-Medical School, Newark, NJ 07103-2714 USA
School of Allied Health and Life Sciences(2), New York Institute of Technology, New York College of Osteopathic Medicine #2, Rm #362, Old Westbury, NY 11568-8000 USA