Twenty years ago, a protein was discovered that had the ability to shrink tumors, and hence was called TUMOR NECROSIS FACTOR (TNF). This stimulated new research in the field. However, to their dismay, it was seen that TNF cannot kill all types of cancer cells and left the question of its possible function as a treatment for cancer, unexplored....until recently.
It has been shown that TNF alpha has a dual nature. It triggers an internal pathway that results in eventual cell death. However, it also activates a molecule that can block this very pathway, which thus sets up a "delicate life-death balance" in the cell (1). This finding raised many eyebrows and brought with it increased hope again for TNF alpha as a possible treatment against cancer.
Several years ago it was seen that drugs like actinomycin D that inhibit
RNA polymerase made cells more susceptible to TNF alpha (2). This is due
to inhibition of gene expression, which indicates that genes are most likely
being turned on in these cells, thus protecting them from cell death. Researchers
suspected that these genes may be getting turned on by NF-kappa B, which
- in turn - become involved in the body's response to inflammation and infection.
NF-kappa B is a transcription factor that consists of 2 subunits: a 50 kilodalton subunit (p50) and a 65 kilodalton subunit (p65, also known as RelA) (1). Therefore, if this heterodimer is defective, it would not be expected to function correctly. Thus, cells without RelA should be unable to transcriptionally activate genes with NF-kappa B binding sites (TNF alpha-inducible genes). This fact was experimentally supported. However, p50 mutant embryonic fibroblasts showed normal transcriptional activation by TNF alpha. Thus, RelA plays a more integral role in transcriptional activation than does the p50 subunit.
It was further interesting to determine the effects of long-term treatments of TNF alpha on cells. It was seen that when embryonic fibroblasts that were RelA-/- were treated with TNF alpha, a dramatic decrease in viability occurred with increasing time of treatments. Past a threshold, TNF alpha treatment showed no additional cell death. Upon applying the same treatment on RelA+/+ or p50-/- embryonic fibroblasts, no cell viability was lost. This substantiates the fact that RelA regulates a cellular protective mechanism against the cytotoxic effects of TNF alpha. This experimental procedure was repeated on tumorous (3T3) embryonic fibroblasts, (1). Again, the results showed a similar effect, and as was the case in every experiment, some RelA-/- cells survived TNF alpha treatment. This creates a problem in that the cells that lived must either have had an inborn ability to subsist that did not depend on RelA or that TNF alpha cannot supply a concentrated enough signal to cause cell death.
The two receptors that have been shown to bind TNF alpha are TNFR1 and TNFR2, of which only TNFR1 contains what has been termed a "death domain". A death domain is the region within the cell that has been shown to be connected to cell death. Because human TNF alpha only interacts with mouse TNFR1, this can be used to test whether TNF alpha's cytotoxicity functions mainly through TNFR1. When experimental embryonic fibroblasts RelA-/- (3T3) were treated with hTNF alpha, a significant decrease in viability occurred, indicating the importance of mediation through TNFR1 (1).
Macrophages are a type of leukocyte (4). It is known that macrophages
can both secrete the TNF alpha cytokine as well as be affected by its
presence. Thus, by testing these theories of RelA on another cell lineage,
it would lend support to its functional role in TNF alpha induction. Macrophages
that were RelA-/-, RelA+/+, and RelA+/- were grown using hematopoietic precursors
from liver cells (1). Through experimentation, it was shown that these macrophages
decreased in viability from RelA+/+ to RelA-/- when subjected to TNF alpha.
This indicated that both fibroblasts and macrophages require RelA for survival
in the presence of TNF alpha .
However, the big question was: Is RelA actively protecting these
cells, or does being RelA-/- developmentally cause the cells to die
in the presence of TNF alpha. This was tested by placing the RelA gene in
a plasmid (vector), along with another gene Lac Z, which was used as a marker
to determine if transfection occurred correctly. The vector was then transfected
into RelA-/- 3T3 cells, and after 36 hours TNF alpha was added. Some cells
survived and were inspected for ß-Gal and shown to be correctly transfected.
Increasing the dosage of RelA in these cells resulted in a dose-dependant
increase in cell viability (1). Thus RelA-/- cells are not predisposed to
die by this cytokine; the lack of RelA causes them to die. RelA somehow
is providing an active protection from TNF alpha .
As described above, TNF alpha seems to relay two signals, one of which induces cell death, the other opposing cell death. This hypothesis has been supported through experimentation. A20 has been found to be an anti-apoptotic gene that gets transcribed upon induction of RelA+/+ 3T3 cells by TNF alpha. In RelA-/- 3T3 cells, no A20 can be detected upon treatment with TNF alpha. However, introduction of A20 into RelA-/- 3T3 cells did not protect them from the cytotoxicity of TNF alpha (1). This simply means that A20 is not the only gene involved in preventing cell death; there can be several genes involved in this protective process.
TNF alpha is a cytokine. There are several types of cytokines in the
human body, and each has a specific 3D structure and receptor. TNF alpha as
of today is known to have two membrane receptors termed Tumor Necrosis Factor
Receptor (TNFR) 1 and 2, with TNFR1 having the intracellular 'death domain'.
Cytokines are glycoproteins that are secreted by individual cells and act
as humoral regulators to modulate the functional activities of individual
cells (5). Thus, during an immune response, specific leukocytes called monocytes
are attracted to a region and begin phagocytosing foreign cells and in doing
so they become macrophages. These then secrete TNF alpha, which can act
on themselves as well as other cells with the appropriate receptors (4).
Upon receipt of a signal by TNF alpha, a cytoplasmic retention protein known
as I B is degraded in the cytoplasm of these cells. I B is attached to another
protein known as NF kappa B and functions to anchor it in the cytoplasm.
Upon its degradation, it releases NF kappa B, which is now free to enter
into the nucleus. NF kappa B is a transcription factor and thus must not
only have a site on the DNA to bind to, but also a specific method of binding.
It has been determined that NF kappa B utilizes the zinc finger to bind
DNA (2). NF kappa B as a transcription factor has the ability to activate
genes involved in inflammatory responses such as those encoding hematopoietic
growth factors, chemokines and leukocyte adhesion molecules, which has made
it an appealing target for controlling inflammatory diseases such as rheumatoid
arthritis. It is further understood that TNF alpha is not only involved
in inflammatory response, but it has cytotoxic activity against tumors.
RHEUMATOID ARTHRITIS is a disease that causes severe pains around
joints. Arthritis causes joint inflammation, and in this disease the immune
system (which normally protects the body from disease) seems to have gone
awry. Instead of fighting disease, the immune system turns against parts
of the body, especially joints, in a process known as the autoimmune response.
This results in severe inflammation. As of now, there is no known cure for
this disease and much speculation has surfaced. One possibility for treatment
involves controlling NF kappa B, which would then potentially prevent these
inflammatory responses in joints. Anti-inflammatory drugs exist (i.e. glucocorticoids)
that are inhibitors of NF kappa B, but it is uncertain if the inflammatory
cells simply die or if the inhibitors have an alternative effect (3).
CANCEROUS CELLS. Further experiments have been performed in which
a mutant I B has been constructed, which is known as "super-repressor."
It permanently binds NF kappa B in the cytoplasm and thus these cells, when
activated by TNF alpha, are unable to protect themselves and subsequently
die. It has been seen that some breast cancer cells have continuous NF kappa
B free from I B and thus will not be killed and can proliferate into a tumor.
Normal breast cells have no NF kappa B and thus can be induced to die by
TNF alpha and consequently will not form tumors. The results of these experiments
have brought about some exciting possibilities to treat proliferative diseases.
If NF kappa B expression can be controlled in cancer cells, TNF alpha can
more effective in killing cancer cells. This would make radiation treatment
more effective in killing the proliferating cells involved in cancer (6).
1. Beg, A.A. and Baltimore, D. 1996. Science. 274: 784-786.
2. Friefelder, D.M. 1987. Molecular Biology. Jones and Bartlett Publishers, Boston, MA.
3. Ellis, J.W. et al. 1990. Medical & Health Guide. Publications International Ltd., Lincolnwood, Illinois.
4. Levine, J.S. et al. 1994. BIOLOGY - discovering life. 2nd edition. D.C. Heath & Company. Lexington, MA. pp.877-881.
5. Habenicht, A. et al. 1990. Growth factors, differentiation factors, and cytokines, Springer, Berlin.
6. Barinaga, M. 1996. Science. 274: 724-725.
Copyright © 1996 Anish Acharya, Farzana Sayani, Kevin Teague
and Bryce Weber.This material may be reproduced for educational purposes
only provided credit is given to the original source.
December 11, 1996
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