Transcript Document

Effects of Vitamin A Metabolites on Apoptosis
A.M. Campbell and R. Barra
Dept. of Biological Sciences, University of Mary Washington
Abstract
Results
Introduction
Retinol is a vitamin A compound present in all mammalian cells (1). Dietary sources of
retinol are consumed, processed as carotenoids, and stored as retinyl esters in the liver. In order
to be used by mammalian cells, retinol must be metabolized (1); retinoic acid (RA) is the most
useful metabolite of retinol. Retinol affects mammalian cells in a variety of ways. RA has been
found to have an effect on normal, malignant and premalignant cells in both in vivo and in vitro
studies (4). The concentration of RA is important when examining its effects on cell lines, which
are said to be “dose-responsive” (9). There is an optimum range for administering retinol for
each cell line as both high and low concentrations of RA can have unfavorable effects on
development (2, 5).
Retinol is metabolized to RA by a two-step mechanism; both steps consist of oxidation
reactions. RA is the most active, functional metabolite (7) and it has the ability to alter the
expression of many genes (5). Multiple cis and trans isomers of RA exist. An isomerization
reaction must take place and act on retinol before the first oxidation reaction occurs in order for
the end product to be a cis or trans isomer (6).
The exact mechanism of action of RA is unknown (8). RA is an important regulator of
morphogenesis, differentiation, and proliferation (3) in normal and tumor cells and is believed to
act through the regulation of retinoid-responsive genes (4). Low concentrations of retinol cause
cells to stop growing and die; however, the dead cells still have intact nuclei, so apoptosis is not
the cause of the cell death (1).
Two of the most common metabolites are 13-cis RA (13cRA) and all-trans RA (ATRA).
13cRA is the main compound found in the acne medication, Accutane. There is evidence that
13cRA is effective in re-differentiating therapy (3) and that is has the ability to prevent
precancerous cells from becoming malignant and invasive (4). ATRA completely stopped the
cell cycle during the G1 phase of development in human embryonic carcinoma cells. ATRA
increased the rate of ubiquitination of cyclin D1; attaching ubiquitin to the cyclin D1 protein
deactivates it (10). Decreasing levels of cyclin D1 are closely associated with G1 phase arrest
(11).
CD95 is a death receptor that belongs to the TNF-R Superfamily. Apoptosis in induced
by death inducing signaling complex (DISC) formation. DISC is formed almost immediately
after CD95 is activated by its ligand (CD95L) or by an agonistic antibody (13).
There is evidence from various studies that RA can affect the cell cycle, can induce
apoptosis, or can alter gene expression by DNA methylation, although the pathways involved
have not been fully elucidated. In this study, I examined the effects of two main metabolites of
Vitamin A, 13cRA and all-trans RA (ATRA), on cell cycle regulation, specifically CD95
expression and cytotoxicity on CRL-1790 epithelial cells. This is a normal epithelial cell line
initially isolated from colon tissue. An enzyme-linked immunosorbent assay was used to
determine CD95 expression and the MTT assay was used to determine cell viability.
Experiment Design
References
Discussion
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I would like to thank the University of Mar y Washington, College of Arts and Sciences for the
funding for this opportunity in under gradua te researc h.