Significance of Caspases, Protein Synthesis, and Kinases in Signalling of Apoptosis of
Human B Lymphocytes
BIOL 200 Research Essay
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NP = new paragraph
Significance of Caspases, Protein Synthesis, and Kinases in Signalling of Apoptosis of Human B
Lymphocytes
Apoptosis, or programmed cell death is an important process in development and
maintenance of multicellular organisms (Eldering and van Lier 2005). One reason why apoptosis
may occur is that a harmful cell is generated (Scientific American 1999). B lymphocytes, or B cells
play a significant role in the immune response primarily as either effector B cells – which make
antibodies that can bind to and help eliminate foreign molecules known as antigens – or as memory
B cells which are able to proliferate and differentiate into effector B cells at a later time when the
same antigen is detected (Jacobson et al. 1997). Apoptosis of B cells is necessary to eliminate selfreactive B cells which react against antigens that are actually from the organism itself, and also to
control populations of B cell clones when they proliferate as part of the immune response (Jacobson
et al. 1997). Edwin G. Krebs, who was awarded the Nobel Prize for his work in describing how
reversible phosphorylation activates proteins, did a study in 1998 with Jonathan Graves and other
coauthors on the signalling requirements for apoptosis of human B cells (Snyder 2010, Graves et al.
1998a). In particular, the study attempts to determine to what extent caspases, protein synthesis
and kinases are significant in signalling for apoptosis of human B cells induced by the B cell
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Should define protease
receptor (BCR) or the Fas receptor (Fas) (Graves et al. 1998a). A caspase is a protease involved in
apoptosis and a kinase is an enzyme that transfers phosphate groups from high energy molecules to
proteins to modify their activity. When a B cell receptor (BCR), which consists of a transmembrane
antibody protein, binds to self antigens (molecules belonging to the organism) or an anti-IgM
antibody, apoptosis follows (Mackus et al. 2002). Similarly, a B cell will undergo apoptosis if
Define ligand
another cell surface receptor called the Fas receptor (Fas) of the B cell binds with a Fas ligand
(FasL) (Kokkonen and Karttunen 2010). Graves et al. (1998a) hypothesized that as in the regulation
of many cell processes, phosphorylation and kinases are involved in the initiation of apoptosis. In
the study done by Graves et al. (1998a), signalling requirements were compared for B cell receptor-
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induced and Fas-induced apoptosis with respect to caspases, gene and protein synthesis, and
kinases.
To answer their question and test their hypothesis, Graves et al. (1998a) did experiments
This whole paragraph should be simplified
where they first exposed two populations of B cells to either anti-IgM antisera or the Fas ligand
(FasL) under certain conditions. In the control, there were no additional conditions, and in the
other trials, either a protease inhibitor called ZVAD, transcription inhibitor actinomycin D, or
translation inhibitor cycloheximide was added prior to exposure to anti-IgM or FasL. To measure
level of apoptosis, a protein Annexin V attached to a fluorescent compound was added to the B cells
which would bind to cell membranes, specifically the phosphatidylserine that is exposed in excess
in dead B cells (Eeva and Pelkonen 2004). The number of B cells displaying fluorescence (ie. the
dead cells) were then counted using Fluorescence-activated cell sorting (FACS) – a method that
counts the number of fluorescent cells one at a time. FACS was performed several times – once
before adding the anti-IgM or FasL (0 hours) and a few times afterward up to 12 hours. Graves et
al. (1998a) also sought to determine activity levels of p38 mitogen-activated protein kinase (MAPK)
and stress-activated protein kinase (SAPK) at different times during exposure to anti-IgM or FasL
with and without protease inhibitor ZVAD. They did this using kinase assay, first by lysing samples
of the B cells at different times and extracting the kinases using anti-MAPK and anti-SAPK
antibodies attached to beads (immunoprecipitation). Substrate proteins and radiolabeled ATP
were then added to the beads. More phosphorylation of the substrate proteins will occur if there
are more active kinases on the beads. The now radiolabeled phosphorylated substrate proteins are
run on SDS-PAGE gel (gel electrophoresis) and the gel is then examined by autoradiography which
detects radioactive substances (Cherkasova 2006).
The data collected by Graves et al. (1998a) shows that for both B cell populations, the
number of apoptotic cells as counted with FACS did not increase between 0 and 10 hours when
ZVAD was added in addition to anti-IgM or FasL. When ZVAD was not added, the number of
apoptotic cells did increase significantly between 0 and 10 hours, but this rate of increase was
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higher in Fas-induced than in BCR-induced apoptosis. When actinomycin D was added with antiIgM to the B cells, the number of apoptotic cells did not change significantly over time. However,
the number of apoptotic cells did increase over time when actinomycin D was added with FasL.
Very similar results were found for cycloheximide as for actinomycin. For both B cell populations
with either anti-IgM or FasL added, the amount of radiolabeled proteins increased over time
without ZVAD, but no increase over time was observed when ZVAD was present.
The data indicates that the BCR of the B cell binds to anti-IgM antisera when exposed to it
only, inducing apoptosis. Similarly, B cells exposed to only Fas ligand (FasL) underwent apoptosis
in response to the Fas of the B cell binding to FasL. In the presence of ZVAD, both B cell populations
saw no increase in the number of apoptotic cells. Since ZVAD is a protease inhibitor, it must be
inhibiting some sort of caspase that possesses some necessary function for apoptosis (Graves et al.
1998a). When actinomycin D or cycloheximide is added with anti-IgM to B cells, apoptosis was
inhibited, but this did not occur with Fas-induced apoptosis. Also, the rate of Fas-induced apoptosis
was higher than the rate of BCR-induced apoptosis. This shows that unlike Fas-induced apoptosis,
BCR-induced apoptosis is dependent on some transcriptional and translational event – possibly the
synthesis of a new genes and proteins (Graves et al. 1998a). Graves et al. (1998a) also believe that
the synthesis of new genes and proteins is what causes BCR-induced apoptosis to be delayed
relative to Fas-induced apoptosis. When Fas-induced or BCR-induced apoptosis occurs, Graves et
al. (1998a) show that the amount of radiolabeled proteins in the cell increases over time, which
indicates that kinase activity increases as apoptosis in a population of B cells proceeds. However,
since the amount of radiolabeled proteins does not increase over time in the presence of ZVAD,
activation of the kinases must depend on some activity of caspases (Graves et al. 1998a).
Graves et al. (1998a) found that new gene and protein synthesis likely occurs in BCRinduced and not Fas-induced apoptosis as shown by the inhibition of BCR-induced apoptosis with
transcription and translation inhibitors actinomycin D and cycloheximide, respectively. Also, both
Fas and BCR-induced apoptosis require the activity of proteases called caspases, shown by the
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inhibition of apoptosis by protease inhibitor ZVAD (Graves et al. 1998a). Finally, Graves et al.
(1998a) found that kinases SAPK and MAPK activate during apoptosis but only in the presence of
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the caspases. More research must be done to determine exactly what caspases target to activate
kinases in apoptosis (Graves and Krebs 1999). In their study, Graves et al. (1998a) showed the
importance of caspases in both BCR-induced and Fas-induced apoptosis and that they are required
for special kinases to function. This study is important as it allows further insight into the specific
mechanisms and macromolecules that control apoptosis of B cells, which is necessary to
understand not only how self-reactive B cells are eliminated, but also how some self-reactive
immune cells escape elimination, causing autoimmune disorders. Furthermore, we can learn more
about why apoptosis erroneously does not occur, possibly leading to cancers – we can learn if and
how cancer may be prevented (Shan and Li 2002).
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LITERATURE CITED
Cherkasova, V.A. 2006. Measuring MAP kinase activity in immune complex assays. Methods, 40:
234-242.
Eeva, J. and Pelkonen, J. 2004. Mechanisms of B cell receptor induced apoptosis. Apoptosis, 9: 525531.
Eldering, E. and van Lier, R. 2005. B-cell antigen receptor-induced apoptosis: looking for clues.
Immunology Letters, 96: 187-194. (REVIEW).
Graves, J.D., Draves, K.E., Craxton, A., Krebs, E.G., and Clark, E.A. 1998. A comparison of signaling
requirements for apoptosis of human B lymphocytes induced by the B cell receptor and
CD95/Fas. The Journal of Immunology, 161: 168-174. (PRIMARY).
Graves, J.D. and Krebs, E.G. 1999. Protein phosphorylation and signal transduction. Pharmacology
and Therapeutics, 82: 111-121.
Jacobson, M.D., Weil, M., and Raff, M.C. 1997. Programmed Cell Death in Animal Development. Cell,
88: 347-354.
Kokkonen, T.S. and Karttunen, T.J. 2010. Fas/Fas-Ligand-mediated apoptosis in different cell
lineages and functional compartments of human lymph nodes. Journal of Histochemistry
and Cytochemistry, 58 (2): 131-140.
Mackus, W.J.M., Lens, S.M.A., Medema, R.H., Kwakkenbos, M.J., Evers, L.M., van Oers, M.H.J., van Lier,
R.A.W., Eldering, E. 2002. Prevention of B cell antigen receptor-induced apoptosis by
ligation of CD40 occurs downstream of cell cycle regulation. International Immunology, 14
(9): 973-982.
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Scientific American. [Author Unknown]. Why does programmed cell death, or apoptosis, occur?
Does it take place among bacteria and fungi or only in the cells of higher organisms?
[online]. Available from http://www.scientificamerican.com/article.cfm?id=why-does
programmed-cell [cited 30 October 2010]. (POPULAR).
Shan, C. and Li, J. 2002. Study of apoptosis in human liver cancers. World Journal of
Gastroenterology, 8 (2): 247-252.
Snyder, Alison. 2010. Edwin Krebs. The Lancelet, 375: 634.
-Really good clear writing but it is too complicated for a first year
-Many techniques in the paper are not used in BIO 200 so a first year would not know
them so they must be explained
-I would have generalized the procedure and results while skipping the less important
aspects (e.g. M/SAPK) giving you more space to give background/explain procedures
-still, v. good overall
Level - too complicated - 3/5
Organization - excellent - 10/10
Paragraphing - spacing needed - 3/5
In text citations - 10/10
Bibliography - 9/10
Importance - excellent, some basics should be included in intro - 18/20
Experiment - well-described but simplification needed - 17/20
Results - again, should be simplified, good summary - 17/20
Total - 87/100
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