Lipids and cell cycle Phospholipid metabolism and the cell cycle

1
Lecture 6. October 1, 2008
Lipids and cell cycle Phospholipid
metabolism and the cell cycle Calcium
independent PLA2 (iPLA2) PC-PLC inhibitor
D609 and stroke Cell cycle regulation D609
and cell proliferation
2
Adult neurogenesis in mammals
Pre-1990s, all regions were categorized as
'non-neurogenic' (grey). Late 1990s, only the
dentate gyrus and olfactory bulb (as well as the
subventricular zone (SVZ), which gives rise to
the rostral migratory stream) were categorized as
'neurogenic' (red). Today, the two known
neurogenic regions are shown in red, and areas
for which there is controversial evidence for
low-level adult neurogenesis are shown in pink.
Gould, Nature Rev Neurosci. 8, 481 (2007)
3
Signaling pathways to transcription
4
Lipid second messengers-DAG and ceramide
regulate the cell cycle
Many enzymes and receptors are switched "on" or
"off" by phosphorylation and dephosphorylation.
Enzymes called kinases (phosphorylation) and
phosphatases (dephosphorylation) are involved in
this process.
Formation of second messengers DAG and IP3 from
PIP2 by action of PI-PLC
DAG Phosphocholine
PLC
PC
Phosphatidic acid choline
PLD
PA phosphohydrolase (PAP)
DAG
5
Phosphatidylcholine, Phosphatidylinositol
DAG (180, 204) from PI-PLC is compositionally
different from DAG from PC-PLC (160, 180,
181) in terms of fatty acid composition.
6
The Cell Cycle
G1 Gap 1 6-12 hrs S Synthetic 6-8 hrs G2
Gap 2 3-4 hrs M Mitosis 1 hr G0 cells that
cease division
7
Phospholipid Metabolism and the Cell Cycle

• G1 (gap) phase High phospholipid (PL) turnover
  (degradation and synthesis of PC). Partial RNA
  and protein synthesis
• S (synthesis) phase. PL Degradation stops. Net PL
  accumulation at the G1-S inter-phase as the cell
  replicate the DNA
• G2-M (mitosis) phase. Net PL synthesis shuts
  down. More RNA and protein synthesis continues.
• After cell division the cell cycle repeats.

PL synthesis shutdown
PL turnover RNA protein synthesis
PC synthesis
RNA protein synthesis
CCT
PC turnover
iPLA2/ PC-PLC
PC hydrolysis
Net PL accumulation, PLA2 activity virtually
down. Synthesis continues
G1 checkpoint
PC more important than DNA Eugene
Kennedy Gordon conference 1997
8
Cyclins and Cyclin dependent kinases (CDKs)

• Cyclins
• Family of proteins involved in the progression of
  the cell cycle.
• Regulatory subunits of the heterodimeric protein
  kinases that control cell cycle.
• Cyclins are so named because their concentration
  varies in a cyclical fashion during the cell
  cycle they are produced or degraded as needed in
  the different stages of the cell cycle.

CDK4 CDK6
CDK2
CDK1

• Cyclins complex with cyclin-dependent kinases
  (CDK), activating the protein kinase function.
  CDKs are a group of protein kinases originally
  discovered as being involved in the regulation of
  the cell cycle. CDKs are also involved in the
  regulation of transcription and mRNA processing.
  CDKs are serine/threonine kinases.
• When cyclin concentrations in the cell are low,
  the cyclin detaches from the CDK, inactivating
  the kinase activity.

9
Cell Cycle
10
Cell Cycle Regulation Retinoblastoma protein

• Retinoblastoma protein (pRb) is a tumor
  suppressor protein that is dysfunctional in many
  types of cancer.
• Important function of pRb is to inhibit the cell
  cycle until a cell is ready to divide the
  dysfunctional protein in many cancers fails to do
  this.
• pRb prevents the cell from replicating damaged
  DNA by preventing its progression through G1 to S
  phase.
• pRb binds and inhibits transcription factors of
  the E2F family, which are composed of dimers of
  an E2F protein and a DP protein. When pRb is
  bound to E2F, progression through the cell cycle
  is halted.
• The pRb-E2F/DP complex also attracts a histone
  deacetylase (HDAC) protein to the chromatin,
  further suppressing DNA synthesis.
• As long as E2F-DP is inactivated, the cell
  remains stalled in the G1 phase.
• Phosphorylation of pRb by cyclin/CDK complexes
  causes pRb to dissociate from its complex with
  E2F
• The transcription activating complexes of E2
  promoter-bindingprotein-dimerization partners
  (E2F-DP) can push a cell into S phase.

11
p53, the guardian of the genome
p53 (protein 53 or tumor protein 53), is a
transcription factor that regulates the cell
cycle and thus functions as a tumor suppressor.
p53 also serves as a master watchman by
preventing genome mutations. Though it runs at 53
kDa on SDS-PAGE, its mass is actually only
43.7kDa. This is due to the high number of
proline residues which slow its migration on
SDS-PAGE.

• Normal cells p53 activity is maintained at low
  levels by murine double minute (MDM2) protein
• (B) Cellular insults trigger post-translation
  modifications of p53 such a phosphorylation of
  Ser 15 and stabilizes by disrupting its
  interactions with MDM2

12
p21Cip1/Waf1 and p27Kip1
p21Cip1/Waf1 (cyclin-dependent kinase inhibitor
1A or CDKN1A), directly inhibits the activity of
cyclin-CDK2 and cyclin-CDK4 complexes. p21
functions as a regulator of cell cycle
progression at G1. p21 expression is controlled
by the tumor suppressor protein p53 and is
activated by ceramide, generated from
sphingomyelin by the action of sphigomyelinase(s).
p27Kip1, a CDK inhibitor protein that binds to
cyclin D either alone, or when complexed to its
catalytic subunit CDK4. p27 inhibits the
catalytic activity of CDK4, and prevents CDK4
from phosphorylating retinoblastoma protein.
Increased levels of p27 cause cell cycle arrest
in G1 phase. p27 also binds to other CDK proteins
when complexed to cyclin subunits such as Cyclin
E/CDK2 and Cyclin A/CDK2. p27 is activated by
transforming growth factor ß (TGF ß)
13
Cell Cycle Regulation
1. CDK phosphorylation
2. C degradation
DNA damage
3. C CDK synthesis
4. CDK inhibition
CDK2
CE
pRb
pRb
pRb
E2F
Enzymes for DNA synthesis
14
Cell Cycle Regulation
15
Cell Cycle
Nocodazole An anti-neoplastic agent, interferes
with the polymerization of microtubules.
Nocodazole treatment serves as a control for
G2-M-transition arrest.
Human cell showing microtubules in green, DNA in
blue, and kinetochores in pink (Wikipedia)
Mitotic human cells microtubules in red, DNA in
purple, and kinetochores in green
cmcbride-bio156.blogspot.com/
16
What is flow cytometry?
Flow cytometry is a technique for counting,
examining, and sorting microscopic particles such
as cells suspended in a stream of fluid. It
allows simultaneous multiparametric analysis of
single cells. Flow cytometry can be used to
measure the amount of DNA in cells and thus
identify the proportions of cells in different
parts of the cell cycle. The data generated by a
flow-cytometer can be plotted in a single
dimension, to produce a histogram.
17
What is flow cytometry?
Data from flow cytometry can be plotted in two
dimensional dot plots or even in three
dimensions. The regions on these plots can be
sequentially separated, based on fluorescence
intensity, by creating a series of subset
extractions, termed "gates."
18
Fluorescence-activated cell sorting (FACS)
Fluorescence-activated cell sorting (FACS), a
specialized type of flow cytometry, sorts a
heterogeneous mixture of cells into two or more
containers, one cell at a time, based upon the
specific light scattering and fluorescent
characteristics of each cell. It is a useful
scientific instrument as it provides fast,
objective and quantitative recording of
fluorescent signals from individual cells as well
as physical separation of cells of particular
interest. The acronym FACS is trademarked and
owned by Becton Dickinson.
19
Cell cycle - Bromodeoxyuridine (BrdU) labeling
BrdU (5-bromo-2-deoxyuridine) is a synthetic
nucleoside analog of thymidine Incorporated into
the newly synthesized DNA (S phase) Antibodies
are used to detect the incorporated BrdU Binding
of the antibody requires denaturation of the DNA,
usually by exposing the cells to acid or heat Use
for comparative growth rates, length of cell
cycle, pulse labelling Can be combined with
dual-labeling BrdU replaces thymidine in DNA
replication, can cause mutations, and therefore a
health hazard
BrdU
Gould E. Nat Rev Neurosci 20078481
20
BrdU labeling
Dentate gyrus granule cells born in the adult rat
over the course of one week. DNA in dividing
cells was labeled with BrdU, injected every other
day for one week. GreenBrdU. RedTUC-4, a marker
of immature neurons. Bluenuclear counterstain.
21
Central dogma of molecular biology
22
Translation

• mRNA -gt protein via triplet code
• What happens if mRNA is destroyed or otherwise
  cant be translated?

23
RNA interference (RNAi)/RNA silencing

• Small/short interfering RNA (siRNA or RNA
  silencing) are a class of 20-25 nucleotide-long
  double-stranded (ds) RNA molecules which enter
  into the RNA interference (RNAi) pathway that
  interferes with the expression of a specific
  gene.
• Also known as post-transcriptional gene silencing
• Double-stranded RNAs, complementary to a specific
  segment of mRNA for a gene, are introduced into
  the cell
• Must be highly specific to prevent degradation of
  other (non-target) mRNA
• As RNAi may not totally abolish expression of the
  gene, this is sometimes referred as a
  'knockdown', to distinguish it from 'knockout' in
  which expression of a gene is entirely eliminated
  by removing or destroying its DNA sequence

24
RNA interference (cont..)

• Endogenous double strand RNAs (dsRNAs) are
  cleaved into 21-23 nucleotide segments (small
  interfering RNAs, or siRNAs) by an enzyme called
  Dicer. The resultant products have 2-3
  nucleotide 3 overhangs with 5 phosphate and 3
  hydroxyl groups.

25
RNA interference (cont..)

• One strand of the RNA duplex is cleaved
  (passenger strand cleavage) and the guide RNA is
  incorporated into RNA-induced silencing complex
  (RISC).
• Guided by base complementarity of the siRNA, RISC
  targets mRNA for degradation
• The complex then releases the cleaved RNA and can
  recognize and cleave additional substrate
  molecules

26
RNA interference why?

• Studying gene function
• Temporarily knock out or inhibit a genes normal
  function
• Can the cell/organism survive?
• What compensatory and/or phenotypic changes are
  observed?
• Therapeutic suppression
• e.g. cancer treatment
• siRNA may have potential to lower cholesterol
  (Future Lipidology 3485(2008)

27
Micro RNA (miRNA)

• Gene expression regulation
• Created by similar process to siRNA
• Generally prevents binding of ribosome

28
Micro RNA (miRNA)

• The nascent primary-microRNA (pri-miRNA)
  transcripts are first processed into
  70-nucleotide precursor miRNA (pre-miRNAs) by
  Drosha inside the nucleus.
• Pre-miRNAs are transported to the cytoplasm by
  Exportin 5 and are processed into miRNAmiRNA
  duplexes by Dicer.
• Dicer also processes long dsRNA molecules into
  siRNA duplexes.
• Only one strand of the miRNAmiRNA duplex or the
  siRNA duplex is preferentially assembled into
  RISC, which subsequently acts on its target by
  translational repression or mRNA cleavage,
  depending, at least in part, on the level of
  complementarity between the small RNA and its
  target ORF open reading frame.

29
RNAi problems

• Not well enough targeted for treating patients
  yet.
• Mismatches result in knockdown of other genes.
• siRNA might get mistaken for miRNAs of which
  there are many, and are much less specific.
• Sense strand mistakenly gets incorporated into
  RISC rendering RNAi nonfunctional.
• Stability -- siRNAs must remain intact long
  enough to work.
• Introduction of large dsRNAs (gt30 nucleotides)
  can trigger interferon response that shuts down
  protein production altogether.
• Delivery is still a problem. Uses lipid as
  carrier (similar to liposomes)
• In vivo experiments are still at infancy.

30
Disruption of G1-phase phospholipid turnover by
inhibition of Ca2-independent phospholipase A2
(iPLA2) induces a p53-dependent cell-cycle arrest
in G1 phase
XH Zhang, C Zhao, K Seleznev, K Song, JJ Manfredi
and ZA Ma J. Cell Sci. 119, 1005-1015 (2006)
The increase of cell-membranous
phosphatidylcholines (PC) containing
polyunsaturated fatty (PUFA) acid residues
induces phosphorylation of p53 through activation
of ATR.
Zhang XH, Zhao C and Ma ZA J. Cell. Sci. 120
4134-4143 (2007)
31
iPLA2 in membrane remodelling and Cell Cycle

• Inhibition of iPLA2 causes
• an increase in polyunsaturated/saturated
  hydrocarbon chain in PC
• profoundly alter the membrane fluidity of the
  nuclear envelope and timing of DNA replication.
• In response ATR-p53-p21 signaling pathway is
  activated to prevent cells from inappropriately
  entering the S phase of the cell cycle.

32
Active iPLA2 is a tetramer Inactive (truncated)
iPLA2
Serine in GxSxG motif that confers iPLA2 activity
was mutated to alanine
Ankyrin Repeat Domain (ARD) proteins act as
negative regulators by binding to full length
iPLA2 and disrupting the formation of active
tetramers.
Manguikian AD, Barbour SE. J Biol Chem
200427952881.
33
METHODS

• Insulinoma (INS-1) cell line
• Human HCT116 colon cancer cells
• P53-wild type
• p53- deficient
• p21-deficient
• DNA constructs
• Wild type iPLA2-GFP
• ARD-iPLA2-GFP, a negative regulator of iPLA2
• Mut-iPLA2-GFP expression vector
• p53 depletion by siRNA
• Cell proliferation by BrdU incorporation
  detection by FACS
• Cell cycle analysis by FACS

34
BEL (bromoenol lactone) inhibits iPLA2

• BEL is an irreversible iPLA2 inhibitor
  (covalently modifies and may cause conformational
  change)
• R- (20-30 µM), S- ( 2 µM) and mixed stereo
  isomer IC50 60 nM
• May also affect phosphatidic acid
  phosphohydrolase (PAP)
• Inhibition of PAP (by propranolol) caused massive
  cell death, rather than cell-proliferation
  arrest.
• The effects proposed in this study are NOT due to
  cell death, rather due to cell cycle arrest.

35
Inactivation of iPLA2 inhibits INS-1 cell
proliferation

• BEL inhibits INS-1-cell proliferation. Cells were
  treated with BEL for up to 6 days and counted
  daily.
• Proliferation of BEL-treated cells recovers upon
  BEL withdrawal. INS-1 cells were cultured in the
  presence of BEL (15 µM) for 2 days and then
  continuously cultured with (red) or without
  (green) BEL. Untreated cells were used as
  controls (blue).
• Transient expression of ARD-iPLA2-GFP reduces
  INS-1 cell proliferation. INS-1 cells were
  transfected with ARD-iPLA2-GFP, then counted
  daily, starting 24 hours after transfection. The
  cell lysates were analyzed for expression of
  ARD-iPLA2-GFP over 6 days.
• Expression of Mutated-iPLA2-GFP inhibits the
  proliferation of INS-1 cells. iPLA2-expressing
  INS-1 cells were transfected with Mut-iPLA2-GFP
  or mock transfected DNA. Counting of cells began
  on the second day after transfection. Cell
  lysates were analyzed by Western blotting for
  GFP.
• siRNA against iPLA2 decreases cell proliferation.
  Mock transfected INS-1 cells, and those
  transfected with psiRNA-iPLA2. A scrambled siRNA
  was served as a negative control.

36
Nocodazole treatment serves as a control for
G2-M-transition arrest
Nocodazole An anti-neoplastic agent, interferes
with the polymerization of microtubules.
Microscopy of nocodazole-treated cells shows that
they do enter mitosis but cannot form metaphase
spindles because microtubules (of which the
spindles are made) cannot polymerise.
37
Inactivation of iPLA2 by BEL arrests INS-1 cells
in G1 phase

• Inactivation of iPLA2 inhibits DNA synthesis.
  Cells were cultured for 24 hours with or without
  BEL (12.5/ 15? µM). Cells were stained with
  fluorescein-conjugated anti-BrdU for BrdU and PI
  for DNA. R2 indicates cells in S phase.
• BEL prevents cell progression to G2-M
  transition. INS-1 cells were treated with BEL
  followed by immunofluorescent staining of the
  mitosis marker phosphohistone H3. Nocodazole An
  anti-neoplastic agent, interferes with the
  polymerization of microtubules. Nocodazole
  treatment served as a control for G2-M-transition
  arrest.
• Inhibition of iPLA2 nocodazole increases the
  number of cells in G1 phase. INS-1 cells were
  treated either with nocodazole only or with both
  nocodazole and different concentrations of BEL
  for 20 hours and 30 hours. The cell DNA was
  analyzed by FACS after PI staining.

Ctrl
Noco
Noco BEL 7.5µM
Noco BEL 10
Noco BEL 12.5
Noco BEL 15
38
Inhibition of iPLA2 induces accumulation of p53
and expression of p21cip1

• Expression of ARD-iPLA2-GFP in INS-1 cells
  induces the accumulation of p53 and expression of
  p21. Increased expression of ARD- caused dramatic
  increase in p53.
• (B) Treatment with BEL (15 µM) for 30 hours
  increases p53, p21 and p27 levels and decreases
  cyclin A levels in INS-1 cells.
• (C) p53 accumulates in the nuclei of 15 µM
  BEL-treated INS-1 cells.

39
G1-phase arrest induced by inhibition of iPLA2
requires p53 HCT 116 colon cancer cells (Note
change of cell line)

• BEL treatment for 10 hours increases the number
  of HCTp53/ cells but not of HCTp53-/- cells in
  G1 phase.
• Accumulation of p53 and expression of p21
  increases with increasing BEL. 10 hrs
• Inhibition of iPLA2 with increasing
  concentrations of BEL dramatically inhibits the
  proliferation of HCTp53/ (blue bars) but only
  mildly inhibits the proliferation of HCTp53-/-
  (red bars) cells.
• (D) Accumulation of p53 and expression of p21
  increases with increasing concentrations of BEL
  in HCTp53/. In HCTp53-/- cells, by contrast,
  p21 expression increased only slightly. 28 hrs

40
BEL treatment induces p53-dependent apoptosis in
p21-deficient HCT cells

• HCTp21 -/- cells have a high death rate after
  inactivation of iPLA2 by BEL. HCTp21-/- cells
  were treated with BEL for 28 hours.
• (B) Inactivation of iPLA2 induces apoptosis of
  p21-deficient cells. Both wild-type HCT and
  HCTp21-/- cells were incubated with or without
  BEL for 10 hrs and analyzed for apoptosis by
  Annexin-V-Fluos staining and FACS. R1 living
  cells. R2 cells undergoing early apoptosis, and
  R3 secondary necrotic cells. In the absence of
  p21 p53 induces apoptosis.
• (C) BEL-associated death of HCTp21-/- cells can
  be prevented by p53 depletion. HCTp21-/- cells
  were cultured overnight in the presence of BEL
  with 1 nmol of SMARTpool-p53.

41
ATM (Ataxia Telangiectasia Mutated) and ATR (ATM
and Rad3 Related) related to PIP3 kinase family.
DNA damage is first detected by sensor proteins
that activate transducers in the signal cascade
and mediate arrest of cell cycle or cause
apoptosis. ATM is activated by ds or ss DNA
breaks caused by ionizing radiation or
chemotherapeutic drugs. BEL treatment did not
cause ds or ss DNA damage
42
iPLA2 in membrane remodelling and Cell Cycle

• Inhibition of iPLA2 causes
• an increase in polyunsaturated/saturated
  hydrocarbon chain in PC
• profoundly alter the membrane fluidity of the
  nuclear envelope and timing of DNA replication.
• In response ATR-p53-p21 signaling pathway is
  activated to prevent cells from inappropriately
  entering the S phase of the cell cycle.

43
Conclusions

• Inactivation of iPLA2 by BEL inhibited INS-1 cell
  proliferation in G1 phase
• Enforced expression of ARD- iPLA2 domain induces
  G1 phase arrest
• LPA or LPC cannot overcome inhibition of cell
  proliferation by BEL
• iPLA2 inhibition induced accumulation of p53 and
  expression p21
• G1 phase arrest induced by iPLA2 inhibition
  requires p53
• BEL induces p53 dependent apoptosis in p21
  deficient cells
• BEL did not cause ds or ss DNA damage suggest
  ATR-p53-p21 pathway involvement
• iPLA2 is involved in membrane remodeling
  maintains the nuclear membrane fluidity for
  proper cell cycle regulation. Inhibition of iPLA2
  causes an increase in polyunsaturated/ saturated
  hydrocarbon chain in PC
• G1 phase PL turnover is systematically monitored
• any cell respond to interrupt this turnover will
  turn on protective mechanisms and
• arrest in G1 phase when both p53/p21 are on
• In the absence of p21 follow the apoptotic route
  guided by p53

iPLA2 inhibition arrests cells in G1 phase of the
cell cycle by inducing p53 accumulation and
expression of p21cip1
44
Stem cells produce new differentiated cells

• Adult stem cells
• Make identical copies of themselves (long-term
  self-renewal).
• Give rise to mature cells with specialized
  functions.
• Generate an intermediate precursor or progenitor
  cell before they become fully differentiated.
• Progenitor or precursor cells
• Partly differentiated, divide and give rise to
  differentiated cells.
• Regarded as "committed" to differentiating along
  a particular development pathway
• May not be definitive

45
Limitations in Brain Repair

• Normal adult neural progenitor cells (NPCs)
• neuroblasts ?mature neurons
• glioblasts ? glial cells. Microvasculature (?)
  signals modulate this NPC differentiation.
  Following insult, the neurogenic milieu activates
  microglia/macrophages that secrete cytokines
  (yellow dots),
• leading to altered NPC cell-cycle progression
• impaired neuronal differentiation

• neuronal apoptosis
• blocking of trophic cues from the
  microvasculature.
• NPC recruitment and guidance are further
  abrogated by increased distance from their
  supporting vasculature. This results in a
  decrease in cycling NPCs and an increase in glia
  at the expense of new neurons. Generation of new
  neurons is virtually eliminated.

Snyder Nat. Med. 20028928 Herrup K, Nat Rev
Neurosci 20078368.
46
Cell cycle regulation in the post-mitotic neuron
oxymoron or new biology?
Neurons are specialized cells that normally never
enter a cell cycle once they are
differentiated Aberrant cell cycle entry results
in death, not cell division. The concept of cell
cycle in a cell that simply does not divide
seemed an oxymoron, but, now we must deal with
the underwriters fine print of developmental
biology the devil is in the details
Herrup K, Nat Rev Neurosci 20078368.
47
Hypothesis D609 neuroprotection is by inhibiting
macrophage/microglia proliferation and
preventing mature neurons entering into cell
cycle and dying after stroke
Stroke
D609 (tricyclodecan-9-yl xanthogenate)
D609 attenuated bFGF induced astrocyte
proliferation Riboni et al JBC 2001
48
D609 inhibited cell proliferation

Bright field
Control
100 µM D609

BrdU incorporation
Control
100 µM D609

• D609 inhibited proliferation (755) of
  neurospheres. BrdU incorporation (red) Hoechst
  nuclear stain (blue) 3 d after exposure to 100µM
  D609.
• D609 arrested the proliferation and did not cause
  cell death (metabolic activity).

49
D609 affecting the cell cycle events ?
Effect of D609 on cyclin D1, phospho-ERK (pERK),
and phospho-AKT (pAKT) in cultured rat progenitor
cells
50
D609 (PC-PLC inhibitor) reduced infarction after
stroke
PC-PLC
Ipsi-/D609 150 25
PC
DAG phosphocholine
Saline
D609
D609

• D609 (xanthogenate compound)
• inhibits PC-PLC
• 50 mg/kg i.p at the onset of reperfusion
  attenuated infarction by 35 5 after 1 hr
  MCAO/24 hr reperfusion.
• Prevented bFGF-induced proliferation of
  astrocytes (may be affecting cell cycle events)

51
D609 Effect on signaling and cell cycle
regulatory proteins after rat model of stroke
IC ischemic/ipsi cortex Penum. Penumbra CC
Contra- cortex

• D609 after 1 hr MCAO and 1 d reperfusion
  significantly, in ipsi- vs contra-cortex
• decreased pERK1/2, cyclin D1, ASMase expression
• increased the expression of p21 and p27
• PKC? (atypical PKC does not need Ca2 or DAG)
  and pAKT expression unaltered.
• ceramide affects PKC? phosphorylation. Need to
  check phospho-PKC? levels

52
Effect of D609 on lipids after stroke at 1 day
rep.
D609 attenuated loss of PC
D609 unchanged SM levels
D609 increased ceramide

• Does D609 inhibit PC-PLC as well as SM synthase?
• Is PC-PLC putative SM synthase (Luberto Hannun
  JBC 1998) ?
• Does ceramide increase expression of CDK
  inhibitors?

plt0.05 compared with contra- plt0.05
compared with ipsi/saline
53
D609 increased pCDC2 and p21Cip1 expression after
stroke

• p21Cip1 expression after 1 hr MCAO and 1 d and 3
  d reperfusion. ICipsi-cortex CCcontra-cortex.
• Phospho-Tyr15CDC2 expression appears to be
  increasing at 3 day rep. with D609 treatment. May
  suggest that D609 affects cell cycle at the G1/S
  phase.
• Need to check
• Does ceramide increase at 3 day rep. vs 1 day
  rep?
• Status of p53?
• Is there any relationship between p53, ceramide,
  p21 pathways?

54
Ceramide can increase p21Cip1 expression
Siddiqui RA,. Biochem J 2003371621.
55
Conclusions

• iPLA2, membrane remodeling and silencing causes
  cell cycle arrest. In the absence of p21, p53 may
  direct the cells to apoptotic pathways.
• D609, a PC-PLC inhibitor
• significantly decreased infarction after tMCAO
• May be affecting the cell cycle (at the G1
  phase). Prevented bFGF-induced proliferation of
  astrocytes
• May prevent macrophage/microglia proliferation
  and protect mature neurons entering into cell
  cycle and commit suicide

56
END OF LECTURE
57
END OF LECTURE

• Schematic representation of the response to
  CPT-11 in wt-p53 and mut-p53 HT29 cells.
  CPT-11-induced DNA damage activates a
  p53-dependent response in HT29-A4 cells,
  resulting in p21WAF1/CIP1 induction leading to
  cell-cycle arrest, and eventually triggering
  apoptosis as a result of a sustained blockage. In
  mut-p53 HT29 cells, no p53-dependent response is
  activated after CPT-11-induced DNA damage, and
  the cell-cycle regulatory machinery progresses
  through the S phase p21WAF1/CIP1 is then
  activated to inhibit the accumulated cdk1/cyclin
  B complex, thus preventing cell progression into
  G2/mitosis. (b) Sensitivity to CPT-11 in wt-p53
  and mut-p53 HT29 cells. The activation of p53 in
  response to CPT-11 eventually leads to apoptosis
  as a result of a sustained cell-cycle arrest. In
  mut-p53 cells, the additional incubation with
  CYC-202 exploits the accumulation of cdk1/cyclin
  B complexes to improve the sensitivity to CPT-11,
  by inducing arrested cells into apoptosis
• Abal Oncogen (2004) 231737

58
(No Transcript)
59
Variation in Cell Cycle Cyclins
Cell cycle phases
60
Enforced expression of the iPLA2 ankyrin-repeat
domain induces G1-phase arrest

• GFP-fluorescence (FL1-H) and DNA content (FL2-A).
  R1, R2 and R3 represent the gated cells with
  increasing GFP-fusion protein expression.
• Cells expressing ARD-iPLA2-GFP remain in G1 phase
  even in the presence of nocodazole. INS-1 cells
  were transfected with ARD-iPLA2-GFP or iPLA2-GFP
  constructs (green), stained for the nuclear pore
  protein mAB414 (red), counterstained for DNA with
  DAPI (blue). Arrows in b and f indicate the
  condensed chromosomes arrows in c and g indicate
  the nuclear-envelope-breakdown events arrows in
  d and h indicate merged cells.
• The number of ARD-iPLA2-GFP-expressing cells in
  G1 phase far exceeds that of iPLA2-GFP-expressing
  cells, even in the presence of nocodazole. When
  GFP-intensity levels were equalized (FL1-H) in
  the presence of nocodazole, 61.5 of
  ARD-iPLA2-GFP-expressing cells versus 37 of
  iPLA2-GFP-expressing cells were in G1 phase. By
  contrast, 27.7 of ARD-iPLA2-GFP-expressing cells
  versus 57.4 of iPLA2-GFP-expressing cells were
  in G2-M transition.

61
Lyso-PA or Lyso-PC treatment cant overcome the
inhibition of cellular proliferation by BEL
LPA and LPC downstream products of PLA2, lyso-PLD
actions on PC LPA or Lyso-PC are mitogens that
stimualte cell proliferation. INS-1 cells were
treated with or without BEL, and 20 µM of LPA and
lyso-PC were added to the BEL-treated group. BrdU
incorporation was then measured.
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Stem cells and Progenitor cells

• Adult stem cells
• Make identical copies of themselves (long-term
  self-renewal).
• Give rise to mature cells with specialized
  functions.
• Generate an intermediate precursor or progenitor
  cell before they become fully differentiated.
• Progenitor or precursor cells
• Partly differentiated, divide and give rise to
  differentiated cells.
• Regarded as "committed" to differentiating along
  a particular development pathway
• May not be definitive

NIH Stem Cell Information web site
stemcells.nih.gov/info/scireport/chapter4.asp
NIH Stem Cell Information web site
stemcells.nih.gov/info/scireport/chapter4.asp