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Full Abstracts – CELL CYCLE AND CELL DEATH
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How Do Endocycling Cells Block Apoptosis?
Bingqing Zhang, Christiane Hassel, Suozhi Qi, Brian R. Calvi. Department of Biology, Indiana University,
Bloomington, IN.
Eukaryotic cells employ multiple checkpoints to preserve the integrity of the genome. Excessive DNA damage, however, can trigger a programmed cell
death called apoptosis, which is a major barrier to genome instability and cancer. An important remaining question is how cell cycle programs and
checkpoints differ among cells in development. We have been using
Drosophila melanogaster
as a model system to address this question. We have found
that cells that enter an endocycle, which is a variant cell cycle that consists of only G and S phases, do not apoptose in response to DNA damage. Also unlike
mitotic cycling cells, endocycling cells do not engage apoptosis after over-expression of p53, but do apoptose after over-expression of the pro-apoptotic p53
target genes at the H99 locus. This suggests that apoptosis is repressed because p53 cannot induce transcription of its targets, a hypothesis that we have
confirmed using reporters and qPCR. Chromatin immunoprecipitation (ChIP) from larval brain-disc (mitotic) and salivary gland (endocycle) using
antibodies against modified histones suggests that the H99 locus is epigenetically silenced in endocycling cells. ChIP against Myc tagged p53 suggests that
this epigenetic silencing may partially block binding of p53 to H99 promoters, but also prevent it from activating the promoter once bound. To further
explore mechanism and identify regulators of apoptosis in endocycling cells, we are performing a novel genetic screen using GFP-labeled salivary glands.
Initial results indicate that knockdown of several genes that encode epigenetic silencing proteins sensitize salivary gland endocycling cells to p53 over-
expression. We will also present data that remodeling of the cell cycle is sufficient to repress apoptosis in some cell types of Drosophila, and our efforts to
determine if the repression of apoptosis is conserved in human polyploid cancer cells. This study will provide general insights into the developmental
regulation of the cellular response to stress and the decision to activate the apoptotic pathway.
61
Lack of E2F activity protects cells from irradiation-induced cell death.
Aaron M. Ambrus
1
, Abul B.M.M.K. Islam
2
, Mary Truscott
1
, Núria López-Bigas
2
,
Maxim V. Frolov
1
. 1) Department of Biochemistry & Molecular Genetics, University Illinois at Chicago, Chicago, IL; 2) Research Unit on Biomedical
Informatics, Department of Experimental Health and Sciences, PRBB, Universitat Pompeu Fabra, Barcelona, Spain.
Both overexpression of dE2f1, and the deregulation of endogenous dE2f1 activity have been shown to promote cell death. Moreover, dE2f1 induces a pro-
apoptotic gene expression program. Here we utilized the
Drosophila
eye imaginal disc to examine irradiation-induced apoptosis in cells lacking dE2f
activity (
dDP
mutants). dDP is the dimerization partner for both dE2f proteins and is needed for them to bind DNA and regulate gene expression. It has been
previously reported that following irradiation, cells in
dDP
mutant eye discs fail to undergo apoptosis, despite having full activation of
hid
and
reaper
, two
genes essential for irradiation-induced apoptosis (Moon et al. 2008. PLoS Gen.). By microarray analysis, we confirmed that the normal transcription
program induced in response to irradiation in wild-type animals was also induced in
dDP
mutant animals. Furthermore, irradiated
dDP
mutants showed a
strong enrichment for down-regulated oxidative metabolism related genes. Interestingly, a general block in energy metabolism was sufficient to protect eye
disc cells from irradiation-induced death. Consistently,
dDP
mutant animals have an energy deficiency compared to wild-type animals. Moreover, the
promoters of these metabolic genes were directly bound by dDP. Thus, the absence of irradiation-induced apoptosis in
dDP
mutants is not a consequence of
the failure to induce the normal apoptotic response, but rather the result of a separate gene expression program modulating genes involved in oxidative
metabolism, which protects against irradiation-induced cell death. Therefore, we identified a previously unappreciated set of dE2F-regulated metabolic
genes. This new role for E2f in regulating oxidative metabolism extends our understanding of the function of E2f as a switch between regulating cell cycle
progression, and promoting cell death.
62
JNK and Draper regulate the engulfment of nurse cells by follicle cells during starvation induced mid-oogenesis cell death in the Drosophila ovary.
Jon Iker Etchegaray
1
, Allison Timmons
1
, Adam Klein
1
, Tracy Pritchett
2
, Elaine Welch
1
, Kim McCall
1
. 1) Boston University, Boston, MA; 2) Boston
University Medical School, Boston, MA.
Programmed cell death and the subsequent removal of cell corpses is an important process in animal development and tissue homeostasis. Failure to engulf
cell corpses can lead to leakage of cellular contents, secondary necrosis, and ultimately disease caused by inflammation. The Drosophila ovary provides an
excellent system to study engulfment because the germline can be induced to die by starvation and their remnants are subsequently engulfed by the
surrounding epithelial follicle cells. To address what are the components of engulfment in this system, we have conducted genetic analysis on two genes:
drpr (drpr) and JNK (basket). We have found that drpr, a known effector of engulfment, is required in the follicle cells for proper phagocytosis of the dying
germline. drpr mutants show impaired cell clearance, which is evident by lingering nurse cells remnants as well as reduced enlargement and premature death
of the follicle cells. Moreover, drpr expression is upregulated in the follicle cells during the engulfment process. Interestingly, our lab has also found that
JNK activity is induced in engulfing follicle cells. As with drpr, disruption of JNK expression in the follicle cells disrupts engulfment. By conducting
epistasis analysis we found that drpr is required for JNK activity and JNK activity upregulates drpr expression. Furthermore, we found that expressing a
constitutively active form of Hemipterous (HepCA), a kinase that specifically activates JNK, suppresses the engulfment defects seen in drpr null egg
chambers. We also observed that over-expression of drpr or (HepCA) induces the germline to undergo cell death even when the flies are not starved,
suggesting that JNK and drpr, contribute to the death process. Our overall model is that drpr activation by the dying germline leads to JNK activation in the
follicle cells. Once activated, JNK upregulates drpr through a feed-forward mechanism, as well as other genes involved in engulfment.