This November, Mr Cruff and the AP Biology class from Shanley High visited MSUM to learn about the biochemistry / physiology of cancer, about research conducted at MSUM and to work on an experiment. (see below for details)

Dr. Provost and Wallert discussed cancer and apoptosis, fluorescent microscopy and their research.

Cancer and Apoptosis Links Cancer Net  National Cancer Institute  Sugen (a drug company) Focus on Cancer  Angiogenesis and Cancer  Fighting Cancer with Angiogenesis Inhibitors  Signaling Pathways - PKC and others  Apoptosis  Apoptosis: Dance of Death  PubMed - free science search library

The experiment was be conducted in three phases. Each group had a few dishes  of cells each with a negative control. The control cells will not be subjected to any treatment. The students used Chinese Hamster Lung Fibroblast cells (CCL39) grown to about 80% confluence, a measure of the cell density.

Group 1 — Investigation of ultraviolet light to induce cell death.

Group 2 — Investigation of hydrogen peroxide to induce cell death

Group 3 — Investigation of lysophosphatidic acid and staurosporine to induce cell death.

For experimental details see below.

Group I — Only the longer exposure indicated less than 1/2 of the cells were beginning to die. Does that mean that UV light isn’t harmfull? Is the only outcome death? What about mutations to the DNA? Do all of these lead to cell death? What would happen if the cells were mutated and rather than die, they lost the negative control for cell growth or the mutations lead to a hormone receptor being activated with or without the hormone? What are the likely consequences of that?

Group II — Nearly all of your cells were in the initial stages of cell death. Can you determine what might have started the cell death? Search the internet and pub med. Hint — tyrosine kinases. What kind of reaction occurs when you add hydrogen peroxide to an open wound? Is that causing apoptosis? Hint- think of the free radicals, peroxides and enzyme that catalyze them. This is kind of an oxidation step. What is causing the oxidation?

Group III — The cells that had staurosporine were almost all dead. Staurosporine is a protein kinase C (PKC) inhibitor (what is protein kinase C?). It acts to compete with ATP for enzymes, but unlike ATP staurospoine can not react to create energy. What might be the results on a cell if ATP can not be properly utilized? The cells that had at least one hour of high concentrations of lysophosphatidic acid (LPA) also had a good number of cells dying. What kind of molecule is LPA? What is interesting here is that this is the same hormone or growth factor that we use to activate our cells. Now, thanks to your work Dr. Wallert and Provost know that this may also kill the cells. We will have a student look more into this after Christmas!

Questions for all. How is apoptosis different than cancer? What other normal cellular processes undergo apoptosis? Why in inducing apoptosis in a cell specific manner an important part of what many pharmaceutical companies do now?

Phase I — Induction of cell death. Each group will treated the cells with the following treatment.

• Group 1 —Four dishes. One is the control and was be left alone. The second dish was subjected to 15 min of UV light at 8:30 am. This group exposed the remaining two dishes one for 5 min and the other for 15 min.
• Group 2 —Four dishes. One is the control and was left alone. The second dish has already had H₂O₂ (hydrogen peroxide) at 100 µM earlier in the morning. The students added 20 µl (100 µM) to the third dish and 100 µl (500 µM) to the fourth dish.
• Group 3 — Three dishes. One is the control and was left alone. The second dish had 50 µM of the protein kinase inhibitor staurosporine added in the morning. They added 6.6 µl (100 µM) of the lipid lysophosphatidic acid to the last dish.

The students removed the media and washed the cells with phosphate buffered saline. Then they added the two probes and incubated the cells for 45 minutes

Phase IV — Visualize cells under fluorescent microscope. - Fun in the dark.
 
 

The structural aspects of cellular systems and the function which accompany different structures can also be examined using fluorescent probes.  A variety of cell-permeable stains that selectively associate with the mitochondria, lysozomes, endoplasmic reticulum and golgi apparatus in lives cells are commercially available.

Additionally, cells respond to their environment through a complex and interdependent series of signal transduction pathways that frequently begin at the cell membrane.  Many cellular receptors are transmembrane proteins with extracellular domains that selectively bind a ligand (hormone).  In response to ligand binding, the receptor’s cytoplasmic domain may change conformation and transmit the signal across the membrane or individual receptors may aggregate and interact with other membrane proteins in order to generate a response.  Transmembrane signals trigger a cascade of events in the cell, including changes in intracellular calcium levels, enzymatic activity and gene expression.  Each of these events can be monitored using selected fluorescent probes.

This experiment is designed to introduce the concept of using fluorescent probes to visualize cellular events, and to determine the various factors that may induce apoptosis.

II. Viability Determination
A. Introduction to viability determination using fluorescent microscopy

The live/dead viability/cytotoxicity assay kit (molecular probes) provides a two-color fluorescence cell viability assay based on the simultaneous determination of live and dead cells with two probes that measure two recognized parameters of cell viability, intracellular esterase activity and membrane integrity.  Lives cells are distinguished by the presence of ubiquitous intracellular esterase enzymes, The esterase enzymes convert a non-fluorescent probe (calcein AM) to a very fluorescent molecule.  The modified probe is well retained within lives cells which produce an intense uniform green fluorescence n lives cells (excitation 495 nm, emission 515 nm).  Dead cells are identified by the ability of a different probe (EhtD-1) to enter cells with damaged membranes.  The EhtD-1 undergoes a 40-fold enhancement of fluorescence in dead cells (excitation 495 nm, emission 635 nm).  The intact membrane of live cells excludes this probe.