David Weisblat 385 Life Sciences Addition Phone 642-8309 Email: weisblat@berkeley.edu

David Weisblat385 Life Sciences AdditionPhone 642-8309Email: weisblat@berkeley.edu

Office hours by appointment

Advantages:1) cellular environment can be manipulated2) cell type well defined3) large quantities of cells can be obtained4) many cellular functions can be investigated

Three types of cultured cells:1) primary cultures2) cell strains3) cell lines

Cell culture = propagation of cells outside the organism

Caveat: How well does what we see in the dish correspond to what goes on in the animal?

dish of cell colonies

100 mm

single cell- scanning EM

0.01 mm

colony of cells

1 mm

Primary Cultures

• Cells derived directly from tissues

First developed in 1907:

spinal cord explant+

lymphatic fluid

1 day

axons growin culture!

cultured neuronextending processes

Cell Strains

• Derived from primary cultures

tissue explant

dissociate cells

cells

plate cellsmedia

dish

• Grow and divide while adhering to plastic dishes

• Require media for growth

• Cells die after 50-100 divisions

Cell LinesCan be derived from several different sources:

1) from cell strains (“normal cells”)• rare genetic changes generate variant cells thatcan grow indefinitely (immortal)

• example: BSC-1 cell line (derived from African Green Monkey kidney)

• grow until contacting neighboring cells, then exitcell cycle (experience contact inhibition)• will not form tumors when injected into mice

• cell strains• transformed cells• tumor cells

Mouse Cell Line Derived from Cell Strain

Some Cell Lines:

Retain properties of precursor cells:e.g. liver cell linesT-cell lines

Can differentiate in culture:e.g. epithelial cell linesmuscle cell lines

differentiatingmusclecell line =C2C12

Cell lines

2) from “transformed” cells• further genetic changes: can be caused by radiation, chemical carcinogens, tumor virusessee 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 transformed by virus

contact-inhibited pile up, rounded

normal cells

transformed cells

scanning EM

Properties of Cancer Cells

1. Lack normal growth controlsa) self-sufficiency in growth signalsb) insensitivity to anti-growth signalsc) evade programmed cell death (apoptosis)d) unlimited replicative potential

2. Able to invade tissues and metastasizea) loss of dependency on anchorage for growthb) 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

carcinogentumor formation

+

tumor promoter -

carcinogen + tumorpromoter +++

TPA = phorbal myristate acetate = PMA

• 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

Phorbol ester

Two cytoskeletal elements examined:

Examining cell architecture using fluorescence microscopy ?

• can visualize and localize individual proteins within a cell.

Test the effects of different drugs on the cytoskeletonand cell shape

TPA/PMALatrunculin

TaxolNocodazole

Actin Microtubules

Actin structures in a fibroblast cell

Microtubules = greenDNA= 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 - immunofluorescence1o ab: rabbit anti-tubulin; 2o ab: fluorescein anti-rabbit

3) Fluorescence microscopy

excitation emission

fluorescent molecule

Fluorochrome Excitation wavelength Emission wavelengthFluoroscein 490 - blue 520 - greenRhodamine 550 – green 580 - redHoechst (stains DNA) 345 - UV 455 - blue

wavelength

ex em

inte

nsity

Fluorescence microscope