Requirements of Cellular Survival and Proliferation
Mark A. Carlson
UNMC Dept Surgery Grand Rounds, September 3, 2003
Overview:
• 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
Requirements of Cellular Survival and Proliferation
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
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 averagehuman, and an abundant repertoire of genes regulating allaspects of cell expansion, it is remarkable that cancers arisein 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)
(University of Arizona Biology Project web site)
Mitosis
[go to “Vid1_mitosis.mov”]
Mitosis
(University of North Carolina Chapel Hill Department of Biology web site)
[go to “Vid2_BloodLilyMitosis.mov”]
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
Trophoblast apoptosis
(University of London St. George’s Hospital web site)
[Go to “Vid3_TrophoblastApoptosis.mov”]
Apoptosisvs.
necrosis
(Karolinska Institute web site)
HeLa cell apoptosis
( Morgan et al., J Cell Biol 2002;157:575)
[Go to “Vid4_HeLaApoptosis.mov”
ApoptoticSignalingPathways
p53 activators
Multiplicity of p53 effects
(Manfredi, Mol Cell 2003;11:552)
(Brooks & Gu, Curr Opin Cell Biol 2003;15:164)
Regulation of p53
ApoptoticSignalingPathways
protein kinase B (PKB, or Akt)
central role in survival signaling& cell cycle regulation
(Brazil et al., Cell 2002;111:293)
Akt interactions
Cell survival & proliferation:Basic requirements
• hydration• ionic• O2• pH• temperature• substrate
• growth factors• adhesion (anchorage dependence)• shape
Cell survival & proliferation:Cell-specific requirements
Human umbilical veinendothelial cells grownon 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) cellsgrown in suspension (noanchorage 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)
Chen et al., Science 1997;276:1425
Dependence of cell growth& survival on cell shape
Human pulmonary capillary endothelial cellsgrown on fibronectin-coated beads
Shape
Chen et al., Science 1997;276:1425
Cell shape (cont’d)
Malignant cells
• Anchorage independence(e.g., growth in soft agar)
• Metastatic potential• GF independence• Shape?
good
tissue loss
response
DEATH
healing
regenerationscar withdisability
scar with minimal or no disability
(quality of life scale)poor
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.
– – – –
Wound Healing ResearchResponse to tissue loss
Burn wound contracture
(Longacre, 1972)
Excisional wound model
5 min after excision postwounding day 5
GT
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 organof healing (= granulation tissue)
Granulation tissue
(open abdomen)
Regulation of granulation tissueproliferation and survival
Wound irrigationduring granulationtissue formation
Neutralization of PDGF duringgranulation 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 withimplanted impermeable barrier
Flap-induced granulationtissue 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)
(Cukierman et al., Science 2001;294:1708)
2D vs. 3D adhesions
The anchored fibroblast-populated collagen
matrix: a model of granulation tissue
attached/mechanically ! loaded (isometric tension) ! ! granulation tissue
released/mechanically ! unloaded
! scar
1 day
detach with spatula
1 day
dissipation of mechanical ! load
Wound model: fibroblast-populated
collagen matrix
(Comparison of thewound and FPCM)
Is the FPCM a good model?
Comparison of models (cont’d)
Fibroblast-populated collagen matrix
Nakagawa et al., J Invest Dermatol 1989;93:792
0 10 20 30 40 500
5
10
15
20
released
attached
incubation time (hr)
%apoptotic
Apoptosis in attached vs. released collagen matrices
*
*
(Grinnell et al., Exp Cell Res 1999;248:648)
ECM
!"
FAK
GF
GFR
PI3K
PI3K
Apoptotic pathways
nucleus
Attached
Akt
p53
FAK
GF
GFR
death
Akt
p53
!"
ECM
Apoptotic pathways
Detached
Regulation of fibroblast survival in the collagen matrix
FAK and Akt activity in the FPCM
ECM
!"
FAK
GF
GFR
PI3K
PI3K
Apoptotic pathways
nucleus
Attached
Akt
p53
FAK
GF
GFR
death
Akt
p53
!"
ECM
Apoptotic pathways
Detached
Regulation of fibroblast survival in the collagen matrix
0 3 60
7500
15000
0
1
2
0 3 6
FAKtub
FAK/tubD
unitsFAK/tub
ratio
FAK
tubulin
post transfection day
vehicle only vehicle + siRNA
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 post-transfection, and immunoblotted for total FAK and α-tubulin. Relative FAK expression was calculated from densiometry (D) of the blots (FAK:tubulin band ratio).
RNA interference (RNAi)
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.
day post-transfection
%apop
2 3 60
5
10
15
20–siRNA+siRNA
RNAi of FAKin the FPCM
ECM
!"
FAK
GF
GFR
PI3K
PI3K
Apoptotic pathways
nucleus
Attached
Akt
p53
FAK
GF
GFR
death
Akt
p53
!"
ECM
Apoptotic pathways
Detached
Regulation of fibroblast survival in the collagen matrix
p53, mdm2, p21 activityin monolayer & the FPCM
monolayer
FPCM
p53 FITC PI
att
rel
p53 FITC PI
transfection: vehicle only vehicle + siRNA
RNAi of p53 in attached vs. released FPCM(p53 immunohistochemistry)
ECM
!"
FAK
GF
GFR
PI3K
PI3K
Apoptotic pathways
nucleus
Attached
Akt
p53
FAK
GF
GFR
death
Akt
p53
!"
ECM
Apoptotic pathways
Detached
Regulation of fibroblast survival in the collagen matrix
p53 IHC (% positive) TUNEL (%positive)
transfection attached detached attached detached
non 3.2 ± 1.9 27.3 ± 4.4* 0.5 ± 0.7 10.7 ± 3.6*
vehicle only 7.9 ± 5.0 26.0 ± 7.5* 1.7 ± 1.3 12.4 ± 2.1*
RNA duplex 8.4 ± 2.0** 15.0 ± 1.5*,** 5.0 ± 2.6** 4.7 ± 1.9**
TUNEL PI
att
rel
TUNEL PI
transfection: vehicle only vehicle + siRNA
RNAi of p53 in attached vs. released FPCM(TUNEL assay)