Requirements of Cellular Survival and Proliferation
Mark A. Carlson
UNMC Dept Surgery Grand Rounds, September 3, 2003
Overview:
Requirements of Cellular Survival and Proliferation
• proliferation (cell cycle)
• cell death
• basic requirements
• cell anchorage
• growth factor dependence
• cell shape
• granulation tissue survival and proliferation
• fibroblast survival in a 3-D matrix
Interesting facts & figures
Approximate number of cells in the human body:
50 trillion (50,000,000,000,000)
Cells undergoing apoptosis per day (and replaced):
10 billion (< 0.1%)
Interesting facts & figures (cont’d)
“With an estimated mutation rate of some 1 in 2 × 107
per gene cell division, some 1014 target cells in the average
human, and an abundant repertoire of genes regulating all
aspects of cell expansion, it is remarkable that cancers arise
in only 1 in 3 lifetimes.”
(Evan & Vousden, Nature 2001;411:342)
Proliferation control: the cell cycle
(Milde-Langosch and Riethdorf, J Cell Physiol 2003;196:224)
Cell cycle:
G1 checkpoint
(Stewart et al., Trends Pharmacol Sci 2003;24:139)
Cell cycle: G2 checkpoint
(Stewart et al., Trends Pharmacol Sci 2003;24:139)
Mitosis
[go to “Vid1_mitosis.mov”]
(University of Arizona Biology Project web site)
Mitosis
[go to “Vid2_BloodLilyMitosis.mov”]
(University of North Carolina Chapel Hill Department of Biology web site)
Types of cell death
• necrosis
• apoptosis
(anoikis)
• nonapoptotic, nonnecrotic cell death (?)
• fixation
Necrosis (“cell murder”)
• sudden disruption of homeostasis
• swelling, cell lysis
• inflammatory infiltrate
Acute myocardial infarction
(University of Illinois College of Medicine Urbana-Champaign web site)
Necrosis (viral hepatitis)
(Curran, Color Atlas of Histopathology, 1985)
Apoptosis (“cell suicide”)
• cell shrinkage
• blebbing/phagocytosis
• no inflammation
[Go to “Vid3_TrophoblastApoptosis.mov”]
Trophoblast apoptosis
(University of London St. George’s Hospital web site)
Apoptosis
vs.
necrosis
(Karolinska Institute web site)
HeLa cell apoptosis
[Go to “Vid4_HeLaApoptosis.mov”
( Morgan et al., J Cell Biol 2002;157:575)
Apoptotic
Signaling
Pathways
p53 activators
Multiplicity of p53 effects
(Manfredi, Mol Cell 2003;11:552)
Regulation of p53
(Brooks & Gu, Curr Opin Cell Biol 2003;15:164)
Apoptotic
Signaling
Pathways
protein kinase B (PKB, or Akt)
central role in survival signaling
& cell cycle regulation
Akt interactions
(Brazil et al., Cell 2002;111:293)
Cell survival & proliferation:
Basic requirements
•
•
•
•
•
•
hydration
ionic
O2
pH
temperature
substrate
Cell survival & proliferation:
Cell-specific requirements
• growth factors
• adhesion (anchorage dependence)
• shape
Human umbilical vein
endothelial cells grown
on a culture dish
Fujio & Walsh, J Biol Chem 1999;274:16349
Growth factor signaling
(receptor tyrosine kinase)
(Garrett & Grisham, Biochemistry, 2nd Ed.)
Anchorage-dependent survival & proliferation
Anoikis (detachment-induced apoptosis)
Renal epithelial (MDCK) cells
grown in suspension (no
anchorage to a substratum)
Khwaja et al., EMBO J 1997:16:2783
Most primary cells require anchorage for survival
Exceptions:
• leukocytes
• lymphocytes
• monocytes
(i.e., hematogenous cells)
• gamete cells (spermazoa, ova)
Shape
Dependence of cell growth
& survival on cell shape
Human pulmonary capillary endothelial cells
grown on fibronectin-coated beads
Chen et al., Science 1997;276:1425
Cell shape (cont’d)
Chen et al., Science 1997;276:1425
Malignant cells
• Anchorage independence
(e.g., growth in soft agar)
• Metastatic potential
• GF independence
• Shape?
Wound Healing Research
Response to tissue loss
tissue loss
response
healing
scar with scar with minimal regeneration
disability
or no disability
DEATH
poor – – – –
good
(quality of life scale)
Figure 1: overview of response to tissue loss.
A mammal will respond to nonfatal tissue loss with
healing or with (in select tissues) regeneration.
Healing results in scar formation which, depending
on the size and location of the tissue loss, can
produce disability, poor quality of life, and
premature death.
Burn wound contracture
(Longacre, 1972)
Excisional wound model
GT
5 min after excision
postwounding day 5
Interesting facts & figures in healing
Number of wound cells immediately post-excision: zero
Number of wound cells on PWD 5: ~200 million
Number of cells in scar (PWD 30): < 1 million
My research:
A focus on the intermediate organ
of healing (= granulation tissue)
Granulation tissue
(open abdomen)
Regulation of granulation tissue
proliferation and survival
Wound irrigation
during granulation
tissue formation
Neutralization of PDGF during
granulation tissue formation
Wound splinting model
Effect of wound splinting on granulation tissue proliferation
effect of wound anchorage on granulation tissue survival
Wound flap model
Chronic inhibition by a flap
Moderation of flap effect with
implanted impermeable barrier
Flap-induced granulation
tissue regression
Fibroblast survival & proliferation
in the collagen matrix
Three-dimensional vs.
two-dimensional culture systems
• 2-D: monolayer
• 3-D: fibroblast-populated collagen
matrix (FPCM)
• in vivo systems (animal models)
2D vs. 3D adhesions
(Cukierman et al., Science 2001;294:1708)
The anchored fibroblast-populated collagen
matrix: a model of granulation tissue
1 day
detach with
spatula
attached/mechanically
! loaded (isometric tension)
! !
granulation tissue
dissipation of mechanical
! load
1 day
released/mechanically
! unloaded
! scar
Wound model:
fibroblast-populated
collagen matrix
Is the FPCM a
good model?
(Comparison of the
wound and FPCM)
Comparison of models (cont’d)
Fibroblast-populated collagen matrix
Nakagawa et al., J Invest Dermatol 1989;93:792
Apoptosis in attached vs. released collagen matrices
20
*
15
%
apoptotic
released
*
10
5
attached
0
0
10
20
30
40
50
incubation time (hr)
(Grinnell et al., Exp Cell Res 1999;248:648)
Regulation of fibroblast survival in the collagen matrix
Attached
GF
GFR
PI3K
nucleus
ECM
FAK
PI3K
Akt
!"
Apoptotic pathways
p53
GF
GFR
Detached
Akt
ECM
FAK
!"
p53
Apoptotic pathways
death
FAK and Akt activity in the FPCM
Regulation of fibroblast survival in the collagen matrix
Attached
GF
GFR
PI3K
nucleus
ECM
FAK
PI3K
Akt
!"
Apoptotic pathways
p53
GF
GFR
Detached
Akt
ECM
FAK
!"
p53
Apoptotic pathways
death
RNA interference (RNAi)
vehicle only
15000
vehicle + siRNA
FAK
tub
D
7500
units
2
FAK/tub
1
0
FAK/tub
ratio
0
0
3
6 0
post transfection day
3
6
FAK
tubulin
Figure A-1: RNA interference of FAK in foreskin fibroblasts. Cells (24-well
plates; 10,000 cells per well) were treated with 200 nM of FAK-specific siRNA
duplex in 0.5% lipid vehicle for 4 hr. Lysates were made on days 1-6 posttransfection, and immunoblotted for total FAK and α-tubulin. Relative FAK
expression was calculated from densiometry (D) of the blots (FAK:tubulin band
ratio).
20
15
–siRNA
+siRNA
%
apop 10
5
RNAi of FAK
in the FPCM
0
2
3
6
day post-transfection
Figure A2: RNAi of FAK in the FPCM
induces cell death. Matrices were incubated
in the attached state for 72 hr prior to
transfection with 67 nM of FAK-specific siRNA
duplex in 0.67% of lipid transfection vehicle for
4 hr. The cell were retrieved after enzymatic
degradation of the matrix, and TUNEL with PI
counterstain was performed on cytospin
preparations. Each bar represents the average
of 4 fields. The experiment was performed
twice with similar results.
Regulation of fibroblast survival in the collagen matrix
Attached
GF
GFR
PI3K
nucleus
ECM
FAK
PI3K
Akt
!"
Apoptotic pathways
p53
GF
GFR
Detached
Akt
ECM
FAK
!"
p53
Apoptotic pathways
death
monolayer
FPCM
p53, mdm2, p21 activity
in monolayer & the FPCM
RNAi of p53 in attached vs. released FPCM
(p53 immunohistochemistry)
transfection:
vehicle only
p53 FITC
att
rel
vehicle + siRNA
PI
p53 FITC
PI
Regulation of fibroblast survival in the collagen matrix
Attached
GF
GFR
PI3K
nucleus
ECM
FAK
PI3K
Akt
!"
Apoptotic pathways
p53
GF
GFR
Detached
Akt
ECM
FAK
!"
p53
Apoptotic pathways
death
RNAi of p53 in attached vs. released FPCM
(TUNEL assay)
transfection:
vehicle only
TUNEL
vehicle + siRNA
PI
TUNEL
PI
att
rel
transfection
non
vehicle only
RNA duplex
p53 IHC (% positive)
attached
detached
3.2 ± 1.9
27.3 ± 4.4*
7.9 ± 5.0
26.0 ± 7.5*
8.4 ± 2.0**
15.0 ± 1.5*,**
TUNEL (%positive)
attached
detached
0.5 ± 0.7
10.7 ± 3.6*
1.7 ± 1.3
12.4 ± 2.1*
5.0 ± 2.6**
4.7 ± 1.9**