Relationship between nucleic acid structures and sequences on the expression of terminal differentiation (i.e., denucleation): alternative cell death pathway
Authors: Gagna Claude E. , Gupta Megha R. , Haidery Ali E. , Mughal Umay R. , Beague Thomas E. , Pillay Adarsh E. , Sattar Abdallah A. , Lambert Peter , Lambert W. Clark 
Published: June 2021

Terminal differentiation (i.e., denucleation) is a type of cell death, similar to apoptosis and necrosis, and is considered an alternative cell death pathway. Denucleating secondary crystalline lens fiber cells, keratinocytes of the epidermis and erythrocytes use this cell death mechanism to maintain their normal cellular physiology. In all three cases, the overall process of organelle removal is somewhat understood morphologically and biochemically; however, the molecular biology is not fully known. Structural transitions of the canonical A‐RNA into alternative structures such as left‐handed Z‐RNA, and the formation of multistranded DNA's (e.g., quadruplex DNA) are important in cells. We are examining how structural polymorphisms play a role in regulating cell death in the three above mentioned tissues, namely, denucleation, and how it differs from apoptosis and necrosis. Immunohistochemistry was performed to study the distribution and specific amounts of single‐stranded (ss−) DNA, A‐RNA, Z‐RNA and quadruplex DNA during denucleation. Immunohistochemical staining of tissue sections were performed with anti‐ss‐DNA, anti‐A‐RNA, anti‐Z‐RNA and anti‐quadruplex DNA antibodies. We observed ss‐DNA, A‐RNA, Z‐RNA and quadruplex DNA immunoreactivity. Both negative, positive and isotype controls were used. ELISA data correlates well with our immunohistochemical results. Immunohistochemical staining of tissues for the demonstration and quantification of ss‐DNA, A‐RNA, Z‐RNA and G4‐quadruplex DNA, reveals different levels of decreasing and increasing immunoreactivity as dying cells progress thru denucleation. Immunohistochemistry shows different levels of gradually decreasing quadruplex DNA as cells progress thru denucleation, with precipitous declines in certain tissue zones at specific times [i.e., this correlates well with decrease in B‐DNA (conrtols)]. A‐RNA and mainly Z‐RNA content decreases during denucleation. A decline in the presence of quadruplex DNA also reveals how denucleation is eradicating different DNA structures. This gradual decline in quadruplex DNA represents chromatin fragmentation. A precipitous decline in quadruplex DNA occurs towards the end of denucleation, depending on tissue type. We believe that specific types of canonical (e.g. B‐DNA), alternative (e.g., Z‐DNA) and multistranded DNAs (e.g., triplex DNA and quadruplex DNA) regulate pro‐ and anti‐cell death factors (e.g., DNase IIβ, CDK1, and cyclins in lens fiber cells, respectively), which control programmed elimination of cellular organelles and DNA. Pro‐cell death factors allows the denucleated cells to remain viable for a certain period of time. Denucleating keratinocytes, secondary crystalline lens fibers and erythrocytes share some common initial characteristics with apoptosis and necrosis, however, the latter part of denucleation is completely different. Based on our research we believe that pro‐apoptotic proteins are involved in the early stages of denucleation, and then other mechanisms (e.g., anti‐apoptotic) take over the remaining processes of terminal differentiation. The gradual decrease in A‐RNA, Z‐RNA and quadruplex DNA during denucleation may play a role in regulating this alternative cell death pathway. Our results suggest that dissimilar nucleic acid structures cause nucleic acid fragmentation in denucleating cells, which differs from apoptosis and necrosis, in the sequence of events and the time course.

Support or Funding Information

Project was supported by several NYIT ISRC and TriBeta Biology Honor Society grants.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.