Cigarette smoking is a major risk factor in the development of non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers.
QUESTION 3:
Cigarette smoking is a major risk factor in the development of non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. NSCLC is characterized by poor prognosis and resistance to chemotherapy. To understand if nicotine affects this resistance, lung cancer cell lines were treated with the chemotherapeutic drugs gemcitabine, cisplatin and taxol in the presence and absence of nicotine. (Dasgupta et al, Proc. Natl. Acad. Sci. USA, 2006)
A. Three different NSCLC cell lines, A549, NCI-H23, and H1299, were examined by the TUNEL assay, which detects cells that are undergoing apoptosis (programmed cell death). The cells were either untreated or treated with one of the three chemotherapeutic drugs in the absence of presence of nicotine. The following data were obtained.
Why are these chemotherapeutic drugs potentially useful for the treatment of lung cancer, and how does nicotine affect their potential usefulness?
B. A549 cells were incubated with the drugs as in part a, and in an additional chemotherapeutic drug, camptothecin, in the presence or absence of nicotine. The cells were lysed, extracts were run on SDS gels, and then the gels were blotted and probed with antibodies against the indicated proteins. PARP is a protein that is cleaved during apoptosis.
What can you deduce from these data about the effects of nicotine?
What is the purpose of assessing the levels of p53, p21 and actin?
C. Survivin and XIAP are both members of the inhibitor of apoptosis (IAP) family, proteins that protect cells against apoptosis. A549 cells
were treated with the chemotherapeutic drugs in the presence of absence of nicotine and the presence or absence of LY294002, an inhibitor of PI-3 kinase. The levels of PARP, XIAP, Survivin and actin were assessed, as shown in
the western blots below. The graph shows the results of a TUNEL assay in which A549 cells have been transfected with the indicated siRNAs. “None” indicates the amount of cell death with no transfected RNA. Control siRNAs 1 and 2 are irrelevant RNAs added to control for nonspecific effects of having an interfering RNA entering the cells.
What do these studies suggest about the mechanism of nicotine action?
D. Can you provide a biological explanation for why the prognosis is poorer for patients who
smoke during chemotherapy compared with those who quit smoking prior to it?
QUESTION 4: Answer parts a and b:
(a) Activation of Fas activates caspase-8 which cleaves the protein Bid (in addition to other targets) to produce an active fragment, tBid (truncated Bid) that binds to the mitochondrial membrane. tBid promotes oligomerisation of Bax and Bak which stimulates the release of cytochrome c into the cytosol to trigger events leading to apoptosis (see Figure 1). To study this pathway in more detail you have generated mouse embryo fibroblasts (MEFs) that are Bax-/- or Bak-/-, or knockouts for both genes, Bak-/-, Bax-/-. You also construct a vector that expresses tBid so that you can study the process independent of Fas.
In untreated MEFs and in MEFs treated with the empty vector, very few cells are observed to undergo apoptosis (Figure 2). By contrast, when MEFs are treated with the vector that expresses tBid, the wild-type cells and the cells individually defective for Bax or Bak show a dramatic increase in apoptotic cells. MEFs that are defective for both Bak and Bax are resistant to tBid expression showing no increase in the number of apoptotic cells. Among the various mutant cells, cytochrome c is retained in the mitochondria only in the double-knockout MEFs treated with tBid. What do these results tell you about the requirements for Bax and Bak in cytochrome c induced apoptosis? Explain your reasoning.
Figure 1
Figure 2
(b) A variety of treatments can cause cells to undergo apoptosis. You wish to know which of these signals are processed through Bid, Bax, and Bak. You generate Bid-/- and Bax-/-Bak-/- mouse embryo fibroblasts (MEFs) and test them for apoptosis in response to several treatments with the results shown in the following table.
1) based on these results, indicate where each of the signals enters the apoptotic pathway relative to Bid, Bax, Bak.
2) How do you suppose that Fas ligand, which binds to Fas, manages to cause apoptosis in these Bid-deficient and Bax- and Bak- deficient cells?
APOPTOSIS
TREATMENT EFFECT Bid -/- Bax-/- Bak-/-
Fas ligand Activates Fas yes yes
Staurosporine Inhibits protein kinases yes no
UV light Damages DNA yes no
etoposide Inhibits topoisomerase II yes no
tunicamycin Blocks N-linked glycosylation yes no
thapsigargin Inhibits Calcium pump in ER yes no
Table 1: Results in Bid-/- and Bax-/-Bak-/- MEFs of various treatments that cause apoptosis in wild-type cells
QUESTION 5:
Many of the proteins that regulate transit through the cell cycle have been characterized. Xnf7, identified in extracts of Xenopus eggs, binds to the anaphase-promoting complex/cyclosome (APC/C). To elucidate the function of this protein, studies have been undertaken in which Xnf7 either has been depleted from extracts using an antibody raised against it or has been augmented in the extracts through addition of extra Xnf7. The consequences on transit through mitosis were then assessed (see J. B. Casaletto et al.,2005, J. Cell Biol. 169:61–71).
a. Xenopus egg extracts, arrested in metaphase, were either depleted of Xnf7 or were mock depleted (subjected to the same treatment as the first sample but without Xnf7 antibody), then released from metaphase arrest by addition of Ca2+. Aliquots of the extract were then sampled at various times after Ca2+ addition and the amounts of mitotic cyclin determined, as shown on the Western blot below. What information do these data provide about a possible function for Xnf7?
b. In additional studies, exogenous Xnf7 was added to Xenopus egg extracts, arrested at metaphase, so that the total amount of this protein in the extracts was higher than normal. The extracts, released from arrest by Ca2+ addition, were then assessed at various times after release for mitotic cyclin ubiquitinylation (cyclin-Ub conjugates). What is the rationale for examining ubiquitinylation? Using the following figure, determine what information these studies add beyond that obtained from part (a).
c. The spindle checkpoint pathway prevents cells with unattached kinetochores from proceeding into anaphase. Thus cells in which this checkpoint has been activated do not enter anaphase and do not degrade mitotic cyclin. Nocodazole, a drug that prevents microtubule assembly, can be used to activate the spindle checkpoint. Cells in nocodazole become arrested in early mitosis because they cannot form a spindle, and so all kinetochores remain unattached. To determine if Xnf7 is required for a functional spindle checkpoint, Xenopus egg extracts, arrested in metaphase, were subjected to various protocols (see the following figure): untreated (no nocodazole) or treated with nocodazole and either mock depleted (preimmune) or immunodepleted of Xnf7 (?-Xnf7). The extracts were then treated with Ca2+to overcome arrest, and aliquots of the extracts were assessed at various times for mitotic cyclin, as shown on the Western blot below. What can you conclude about Xnf7 from these data?
001BPS WORKSHOP MODULE 5
TECHNIQUES USED TO STUDY APOPTOSIS AND CANCER:
TUNEL assay,
Flow cytometry
Annexin V binding,
DAPI staining,
caspase activation, Cooper 5e: http://www.sinauer.com/cooper5e/animation1702.html
Cytochrome C
release: MOVIE: Reference: Goldstein JC et al Cell Death and Differentiation, (2005) 12, 453–462.
doi:10.1038/sj.cdd.4401596
http://www.nature.com/cdd/journal/v12/n5/suppinfo/4401596s1.html?url=/cdd/journal/v12/n5/full/440
1596a.html
Legend to Movie:
This video is of HeLa cells undergoing apoptosis in response to TNF and CHX. Images were
acquired every 4 minutes and appear at 8 frames/second. The cells express cytochrome c-4CYS
stained with ReAsH (red). Initially cytochrome c-4CYS appears in the mitochondria. Following its
release, cells round and expose phosphatidylserine, as seen by AnnexinV binding (Blue). Many of the
cells express histone H2B coupled to GFP (47), and therefore chromatin condensation and nuclear
fragmentation can also be seen. Finally, plasma membrane integrity is lost and propidium iodide stains
the nucleus (magenta).
QUESTION 1:
(a) Examine the Figure below relating to the S.pombe cell cycle:
1) Describe the function of Cdc25 and Wee1 and why deficit of Cdc25 or excess of Wee1 can
cause G2 arrest
2) b) Describe how deficit of Wee1 or excess of Cdc25 may result in abnormally small cells
(b) The activities of Wee1 tyrosine kinase and Cdc25 tyrosine phospatase determine the state of
phosphorylation of tyrosine 15 in the Cdk1 component of mitotic cyclin-Cdk complexes (MPF). When
tyrosine 15 is phosphorylated, M-Cdk is inactive; when tyrosine 15 is not phosphorylated, M-Cdk is
active. Just as the activity of M-Cdk itself is controlled by phosphorylation, so too are the activities of
Wee1 kinase and Cdc25 phosphatase. The regulation of these various activities can be studied in
extracts of frog oocytes. In such extracts Wee1 tyrosine kinase is active and Cdc25 tyrosine
phosphatase is inactive. As a result M-Cdk is inactive because its Cdk1 component is phosphorylated
on tyrosine 15. M-Cdk in these extracts can be rapidly activated by addition of okadaic acid, which is a
potent inhibitor of serine/threonine phosphatases. Using antibodies specific for each component it is
possible to examine their phosphorylation states by changes in mobility upon gel electrophoresis.
(Phosphorylated proteins generally run slower than their non phosphorylated counterparts).
1) Based on the results with okadaic acid, decide whether the active forms of Wee1 and
Cdc25 are phosphorylated or unphosphorylated. In the figure indicate the
phosphorylated forms of Wee 1 and Cdc25 and label the arrows connecting their
active and inactive forms to show which transitions are controlled by protein kinases
and which by protein phosphatases.
2) Are the protein kinases and phosphatases that control Wee1 and Cdc25 specific for
serine/threonine side chains or for tyrosine side chains? How do you know?
3) How does addition of okadaic acid cause an increase in phosphorylation of Wee1 and
Cdc25, but a decrease in phosphorylation of Cdk1?
4) If you assume that Cdc25 and Wee1 are targets for phosphorylation by active M-Cdk,
can you explain how the appearance of a small amount of active M-Cdk would lead to
its rapid and complete activation?
QUESTION 2
In a recent study by Elizabeth Snyderwine and colleagues, cDNA microarray profiling was used to
compare the expression profiles for 6900 genes in normal and malignant breast tissues from rats.
RNA was extracted from the following tissues:
a.
b.
c.
d.
e.
Breast tissue from virgin rats
Breast tissue from pregnant rats
Breast tissue from lactating rats
Breast carcinoma induced by the meat-derived carcinogen PhIP
Breast carcinoma induced by the experimental carcinogen DMBA
After the microarray slides were hybridized with labeled cDNAs derived from these 5 populations of
RNAs, the data were analyzed by several different comparisons.
In comparison 1, tissues a, b, and c were grouped together as “normal” tissue samples, and d and e
were grouped as “carcinoma” samples. Genes that were either induced (more than twofold increase in
expression) or repressed (more than twofold decrease in expression) in both carcinoma samples relative
to all three normal samples were determined. A partial listing of differentially expressed genes is
shown in the accompanying table.
Induced Genes
Cell-Growth and Cell-Cycle-related Genes
Platelet-derived growth factor A chain (PDGF-A)
Cyclin-dependent kinase 4 (Cdk4)
Cyclin D
Signal-Transduction and Transcription-Related Genes
STAT5a
Repressed Genes
Extracellular Matrix Genes
Alpha 1 type V collagen
Fibronectin 1
Desmin
A) What was the purpose of analyzing breast tissue from pregnant and lactating rats?
B) What characteristic of cancer is promoted by the overexpression of PDGF-A, Cdk4, and cyclin
D?
C) What characteristic of cancer might be promoted by the repression of extracellular matrix
genes?
D) STAT5a is a transcription factor that regulates the expression of cyclin D, Bcl-X L, and other
genes. Why is it possible for these carcinomas that no mutation occurs in the cyclin D gene
despite its overexpression?
E) How does overexpression of Bcl-XL lead to cancer?
F) On the basis of this information, what can be generalized about the molecular profile of breast
carcinomas?
G) Would you anticipate greater or fewer differences in gene expression if two distinct types of
cancer (e.g., breast carcinoma vs. B-cell lymphoma) induced by the same carcinogen were
compared by microarray analysis?
QUESTION 3:
Cigarette smoking is a major risk factor in the development of non-small cell lung cancer (NSCLC),
which accounts for 80% of all lung cancers. NSCLC is characterized by poor prognosis and resistance
to chemotherapy. To understand if nicotine affects this resistance, lung cancer cell lines were treated
with the chemotherapeutic drugs gemcitabine, cisplatin and taxol in the presence and absence of
nicotine. (Dasgupta et al, Proc. Natl. Acad. Sci. USA, 2006)
A. Three different NSCLC cell lines, A549,
NCI-H23, and H1299, were examined by the
TUNEL assay, which detects cells that are
undergoing apoptosis (programmed cell death).
The cells were either untreated or treated with
one of the three chemotherapeutic drugs in the
absence of presence of nicotine. The following
data were obtained.
Why are these chemotherapeutic drugs
potentially useful for the treatment of lung
cancer, and how does nicotine affect their
potential usefulness?
B
A549 cells were incubated with the drugs as in
part a, and in an additional chemotherapeutic
drug, camptothecin, in the presence or absence
of nicotine. The cells were lysed, extracts were
run on SDS gels, and then the gels were blotted
and probed with antibodies against the
indicated proteins. PARP is a protein that is
cleaved during apoptosis.
What can you deduce from these data about
the effects of nicotine?
What is the purpose of assessing the levels of
p53, p21 and actin?
C. Survivin and XIAP are both members of the
inhibitor of apoptosis (IAP) family, proteins
that protect cells against apoptosis. A549 cells
were treated with the chemotherapeutic drugs in
the presence of absence of nicotine and the
presence or absence of LY294002, an inhibitor
of PI-3 kinase. The levels of PARP, XIAP,
Survivin and actin were assessed, as shown in
the western blots below. The graph shows the
results of a TUNEL assay in which A549 cells
have been transfected with the indicated
siRNAs. “None” indicates the amount of cell
death with no transfected RNA. Control
siRNAs 1 and 2 are irrelevant RNAs added to
control for nonspecific effects of having an
interfering RNA entering the cells.
What do these studies suggest about the
mechanism of nicotine action?
D.
Can you provide a biological explanation for
why the prognosis is poorer for patients who
smoke during chemotherapy compared with
those who quit smoking prior to it?
QUESTION 4: Answer parts a and b:
(a) Activation of Fas activates caspase-8 which cleaves the protein Bid (in addition to
other targets) to produce an active fragment, tBid (truncated Bid) that binds to the
mitochondrial membrane. tBid promotes oligomerisation of Bax and Bak which
stimulates the release of cytochrome c into the cytosol to trigger events leading to
apoptosis (see Figure 1). To study this pathway in more detail you have generated
mouse embryo fibroblasts (MEFs) that are Bax-/- or Bak-/-, or knockouts for both
genes, Bak-/-, Bax-/-. You also construct a vector that expresses tBid so that you
can study the process independent of Fas.
In untreated MEFs and in MEFs treated with the empty vector, very few cells are
observed to undergo apoptosis (Figure 2). By contrast, when MEFs are treated with
the vector that expresses tBid, the wild-type cells and the cells individually
defective for Bax or Bak show a dramatic increase in apoptotic cells. MEFs that are
defective for both Bak and Bax are resistant to tBid expression showing no increase
in the number of apoptotic cells. Among the various mutant cells, cytochrome c is
retained in the mitochondria only in the double-knockout MEFs treated with tBid.
What do these results tell you about the requirements for Bax and Bak in
cytochrome c induced apoptosis? Explain your reasoning.
Figure 1
Activated Fas
Pro-caspase-8
caspase-8
Bid
Bax
Bak
Mitochondrial
Cytochrome C
tBid
oligomeric Bax
oligomeric Bak
cytosolic
cytochrome C
Cell death
Figure 2
t Bid
Empty Vector
untreated
% apoptosis
20
10
Wild type
Bax-/Bak+/+
Bax+/+
Bak-/-
Bax-/Bak-/-
(b) A variety of treatments can cause cells to undergo apoptosis. You wish to know which of these
signals are processed through Bid, Bax, and Bak. You generate Bid-/- and Bax-/-Bak-/- mouse
embryo fibroblasts (MEFs) and test them for apoptosis in response to several treatments with
the results shown in the following table.
1) based on these results, indicate where each of the signals enters the apoptotic pathway
relative to Bid, Bax, Bak.
2) How do you suppose that Fas ligand, which binds to Fas, manages to cause apoptosis
in these Bid-deficient and Bax- and Bak- deficient cells?
TREATMENT
EFFECT
Fas ligand
Staurosporine
UV light
etoposide
tunicamycin
Activates Fas
Inhibits protein kinases
Damages DNA
Inhibits topoisomerase II
Blocks N-linked
glycosylation
Inhibits Calcium pump in
ER
thapsigargin
APOPTOSIS
Bid -/yes
yes
yes
yes
yes
Bax-/Bak-/yes
no
no
no
no
yes
no
Table 1: Results in Bid-/- and Bax-/-Bak-/- MEFs of various treatments that cause apoptosis in
wild-type cells
QUESTION 5:
Many of the proteins that regulate transit through the cell cycle have been
characterized. Xnf7, identified in extracts of Xenopus eggs, binds to the anaphase-promoting
complex/cyclosome (APC/C). To elucidate the function of this protein, studies have been
undertaken in which Xnf7 either has been depleted from extracts using an antibody raised against it
or has been augmented in the extracts through addition of extra Xnf7. The consequences on transit
through mitosis were then assessed (see J. B. Casaletto et al.,2005, J. Cell Biol. 169:61–71).
a. Xenopus egg extracts, arrested in metaphase, were either depleted of Xnf7 or were mock
depleted (subjected to the same treatment as the first sample but without Xnf7 antibody), then
released from metaphase arrest by addition of Ca 2+. Aliquots of the extract were then sampled at
various times after Ca2+ addition and the amounts of mitotic cyclin determined, as shown on the
Western blot below. What information do these data provide about a possible function for Xnf7?
b. In additional studies, exogenous Xnf7 was added to Xenopus egg extracts, arrested at
metaphase, so that the total amount of this protein in the extracts was higher than normal. The
extracts, released from arrest by Ca2+ addition, were then assessed at various times after release for
mitotic cyclin ubiquitinylation (cyclin-Ub conjugates). What is the rationale for examining
ubiquitinylation? Using the following figure, determine what information these studies add beyond
that obtained from part (a).
c. The spindle checkpoint pathway prevents cells with unattached kinetochores from proceeding
into anaphase. Thus cells in which this checkpoint has been activated do not enter anaphase and do
not degrade mitotic cyclin. Nocodazole, a drug that prevents microtubule assembly, can be used to
activate the spindle checkpoint. Cells in nocodazole become arrested in early mitosis because they
cannot form a spindle, and so all kinetochores remain unattached. To determine if Xnf7 is required
for a functional spindle checkpoint, Xenopus egg extracts, arrested in metaphase, were subjected to
various protocols (see the following figure): untreated (no nocodazole) or treated with nocodazole
and either mock depleted (preimmune) or immunodepleted of Xnf7 (?-Xnf7). The extracts were
then treated with Ca2+to overcome arrest, and aliquots of the extracts were assessed at various times
for mitotic cyclin, as shown on the Western blot below. What can you conclude about Xnf7 from
these data?