Download DAW Lecture 1 PPT

David Weisblat
385 Life Sciences Addition
Phone 642-8309
Email: [email protected]
Office hours by appointment
Cell culture
= propagation of cells outside the organism
Advantages:
1) cellular environment can be manipulated
2) cell type well defined
3) large quantities of cells can be obtained
4) many cellular functions can be investigated
Caveat: How well does what we see in the dish
correspond to what goes on in the animal?
Three types of cultured cells:
1) primary cultures
2) cell strains
3) cell lines
single cell- scanning EM
colony of cells
0.01 mm
1 mm
dish of cell colonies
100 mm
Primary Cultures
• Cells derived directly from tissues
First developed in 1907:
1 day
spinal cord explant
+
lymphatic fluid
axons grow
in culture!
cultured neuron
extending processes
Cell Strains
• Derived from primary cultures
dissociate
cells
tissue explant
media
plate cells
cells
dish
• Grow and divide while adhering to plastic dishes
• Require media for growth
• Cells die after 50-100 divisions
Cell Lines
Can be derived from several different sources:
• cell strains
• transformed cells
• tumor cells
1) from cell strains (“normal cells”)
• rare genetic changes generate variant cells that
can grow indefinitely (immortal)
• example: BSC-1 cell line (derived from African
Green Monkey kidney)
• grow until contacting neighboring cells, then exit
cell cycle (experience contact inhibition)
• will not form tumors when injected into mice
Mouse Cell Line Derived from Cell Strain
Some Cell Lines:
Retain properties of precursor cells:
e.g. liver cell lines
T-cell lines
Can differentiate in culture:
e.g. epithelial cell lines
muscle cell lines
differentiating
muscle
cell line =
C2C12
Cell lines
2) from “transformed” cells
• further genetic changes:
can be caused by radiation, chemical carcinogens, tumor viruses
see changed morphology, loss of growth control, loss of
contact inhibition
• example: 3T3-21F
• will form tumors in mice
3) from tumor cells
• similar to transformed cells
• example: HeLA - from cervical carcinoma
(Henrietta Lacks, 1951)
normal
contact-inhibited
transformed by virus
pile up, rounded
normal cells
scanning
EM
transformed cells
Properties of Cancer Cells
1. Lack normal growth controls
a) self-sufficiency in growth signals
b) insensitivity to anti-growth signals
c) evade programmed cell death (apoptosis)
d) unlimited replicative potential
2. Able to invade tissues and metastasize
a) loss of dependency on anchorage for growth
b) loss of contact inhibition
3. Able to develop vasculature - blood supply
“angiogenesis”
Tumor Promoters
• Enhance tumor formation when combined with
carcinogens, but are not themselves carcinogenic
carcinogen
tumor promoter
carcinogen + tumor
promoter
tumor formation
+
+++
TPA = phorbal myristate acetate = PMA
Phorbol ester
• mimics 1, 2 diacylglycerol (DAG)
• DAG + Ca++ activates protein kinase C (PKC)
• causes phosphorylation of PKC substrates
• changes in cell growth, cell shape and the cytoskeleton
Examining cell architecture
using fluorescence microscopy ?
• can visualize and localize individual proteins
within a cell.
Two cytoskeletal elements examined:
Actin
Microtubules
Test the effects of different drugs on the cytoskeleton
and cell shape
TPA/PMA
Latrunculin
Taxol
Nocodazole
Actin structures in a fibroblast cell
Microtubules
= green
DNA
= blue
interphase
mitosis
Visualizing the cytoskeleton
using fluorescence microscopy
1) Prepare samples:
Fixation - kills and immobilizes cells
A. aldehydes - cross-link amino groups in proteins
(formaldehyde, glutaraldehyde)
B. alcohols - denature proteins, precipitate in place
(methanol)
Permeabilization - detergents make proteins
accessible to staining reagents (Triton X100)
2) Staining
Actin - phalloidin covalently linked to rhodamine
(red) - binds to filamentous actin only
Microtubules - immunofluorescence
1o ab: rabbit anti-tubulin; 2o ab: fluorescein anti-rabbit
3) Fluorescence microscopy
excitation
ex
em
intensity
emission
fluorescent molecule
Fluorochrome
Fluoroscein
Rhodamine
Hoechst (stains DNA)
Excitation wavelength
490 - blue
550 – green
345 - UV
wavelength
Emission wavelength
520 - green
580 - red
455 - blue
Fluorescence microscope