Genetically-Engineered Adenovirus That Kills
Cancer Cells!
~~~~~
Introduction
This article is about something really exciting that may offer hope for our
ability to specifically target certain cancer cells, and kill them - without
harm to our normal cells. You have probably read in the newspaper or heard on
various news broadcasts recently, about a virus which has been purposefully
genetically altered for use in treating cancer patients. And, you may have
heard that certain clinical trials have been started to examine this virus for
possible use as a therapy for cancer patients.
UPDATE
As of April 1999, this cancer treatment, use of a
genetically-engineered form of adenovirus (normally causes colds) in a
formulation named ONYX-015, is now (August, 2000) in Phase II Clinical
Trials with success against head and neck cancer. Please see
additional information on how this "drug" may work, dated April,
1999 from The University of California, San Francisco,
Press
Release, UC San Francisco, April, 1999
There are additional trials presently in progress using ONYX-015 against
lung cancer and against ovarian cancer. Please see the following sites:
Lung
Cancer Trial, January, 2000
Ovarian
Cancer Trial, August, 2000
END UPDATE
The present article, written in 1996,is based upon recent research
published in the October 18, 1996 issue of the scientific journal
Science. The Science article is:
Bischoff, J.R., D.H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna,
L. Ng, J. Nye, A. Sampson-Johannes, A. Fattaey, and, F. McCormick. 1996.
An Adenovirus Mutant That Replicates Selectively in p53-Deficient Human Tumor
Cells. Science 274: 373-376.
All of these scientists listed are or were associated with ONYX
Pharmaceuticals, 3031 Research Drive, Richmond, California 94806.
The Science article is pretty technical, so, I thought you might
wish to know what is going on here in a bit less technical language.... why
this particular approach has been taken, and why this approach offers some hope
for future therapeutic approaches. To explain this information, we need to
examine some background, and then we'll talk about specifics. I must tell you
that I am going to leave out certain precise things - and, I will not be
absolutely, scientifically accurate in my explanations. However, I won't take
too much latitude with my explanations. So, I hope the information I
provide may help you to understand this system a little better.
Our Cells and Cell Division
Except for certain skin cells, cells which appear from our bone marrow,
and certain immune system cells, most normal human cells are usually quiescent,
e.g., not dividing. And, even when certain of our cells are specifically
triggered to divide when they are supposed to divide (like our activated immune
system cells which help us to fight infections, etc.,) the number of
cell divisions "allowed" is usually pre-programmed, and limited.
Our cells have thus evolved in such a way that results in a system which
essentially protects the body from any cell which no longer has cell
division under strict regulatory control - the loss of such regulation is of
course the condition we call, cancer. If a single one of the over 65
trillion cells in the human body loses the ability to regulate cell division,
this loss of regulation can of course eventually lead to the formation of a
tumor, or many, many cells which do not necessarily aggregate but disperse
throughout the body.
What is the Cell Division Regulatory System?
Fortunately for us, there is a suppressor system within each of the
over 65 trillion (over 200 different types) of cells within our body. One of
the major components of this division suppression system is the protein known
as p53 (protein of 53,000 molecular weight). A certain amount of
functional p53 must be available within each of these cells to
maintain this condition of cell division suppression. In the presence of
p53, a cell will enter what is known as G0 or will enter what is known as G1
arrest at the appropriate time.
How Else is p53 Involved?
If a cell has undergone significant DNA "damage" - due perhaps to
exposure of cells to uv-light, certain chemicals - or if the cell's DNA is
perturbed by a DNA-type virus - or - if some event causes some kind of
interference with cellular DNA which leads to inappropriate cellular activation
signals - p53 is also involved. The human cell has evolved with a protective
system which will lead to cell suicide if DNA damage cannot be adequately
repaired, or if inappropriate cell activation signals are initiated by
infectious or other events. Of course, damaged cellular DNA as well as
improper cell activation independently have the potential to lead to very
harmful consequences. This protective cell suicide system is known as programmed
cell death - or - apoptosis (the second letter p is silent). The p53
system becomes very active under such conditions.
DNA can be repaired by a number of different enzymes - and - before the cell is
"allowed" to replicate the DNA (as would be required for cell division) -
the damaged DNA must be repaired prior to any DNA replication/cell
division. The p53 protein normally maintains the cell in G0 or in G1 arrest.
If DNA repair systems "try" to repair damage to the cell's chromosome(s) -
and if the repair doesn't occur well, there are interacting cell materials
which signal p53 protein, and in concert these systems will drive the cell to
commit self-destruction - cellular suicide - in a very precisely controlled
manner. This type of cell death is quite different than cell death
which may be caused by a virus, other intra-cellular infectious agent, or
trauma. When apoptosis occurs, the chromosomes are broken into discrete
fragments, and the inside contents of the cell are never released to the
surrounding tissue. This lack of release of cytoplasmic contents serves to
prevent inflammatory responses - which are induced when a virus or some
other agent causes a cell to break apart. Thus, programmed cell death is a
powerful protective response against cancer cells, against cells improperly
activated to divide for whatever reason - and against any "rogue"
cell(s) with lots of damaged and/or mutated DNA in them.
What Happens If There is Something Wrong With p53?
If there is something wrong with p53 within a single cell - either the p53
doesn't function properly, or is completely missing (damage/mutation of the
gene responsible for p53 appearance), there is the potential for
trouble from that cell if the cell is triggered to begin cell division. If
the cell is activated to cell division by normal circumstances; or, if there
is significant DNA damage (uv-light, chemicals or viral interference with
cellular DNA) which may inadvertently activate the cell to divide, not only will
the cell's DNA not be repaired, without a functional p53 system, the cell is
now incapable of regulating cell division, and is also unable to commit suicide
by apoptosis... a functional p53 protein is necessary. Therefore, a cell
may become activatable because of DNA damage, or the cell may be activated
under normal circumstances... but in each case, without a functional p53
protein, the cell will continue to divide - will never stop; and, all of
the progeny cells will not be able to commit suicide - nor regulate their
cell division either. It is estimated that fifty percent (50%) of all
known cancers have a dysfunctional p53 protein - because of a mutation in
the gene which encodes information for the synthesis of this protein.
Are There Examples of Non-Damaged Cells Which Are Regulated By p53?
Yes; there are such examples. And, it is important to take a look at a
couple of examples.
If it is normal for a particular type of cell to undergo activation and
subsequent cell division - the regulation of cell division by p53 will be
temporally controlled (controlled with respect to time) - will appear only
after a certain time - and thereby will allow a certain number of cell division
cycles to occur. One such example involves the cells which are produced every
day from our bone marrow which become the cells of our entire hematopoietic
system (red blood cells, lymphocytes, lots of other kinds of cells. For example,
we make approximately 2 billion neutrophils per day - which arise from
maturation and cell division paths due to certain hormone-like triggers); and,
any time a mature cell - like a lymphocyte - is activated to undergo cell
division under normal circumstances - as in response to an infectious agent -
these same temporal constraints apply.
If a cell undergoes the proper number of divisions and reaches a certain
temporal stage, p53 accumulates and cell division ceases. Sometimes, depending
on the particular cell, the action of p53 and other cellular constituents, the
cell will enter a yet different condition and will undergo apoptosis -
"programmed cell death."
What's So Unique About Adenovirus; and, What is the Involvement of
p53?
Human Adenovirus normally infects human cells which are quiescent (non-dividing).
But, the wild-type virus (the one which is normal in the environment) can
also infect dividing cells (normal or cancer cells). In all instances -
upon introduction of this virus into a human cell (viral infection), the
genetic material belonging to the virus - which happens to be DNA - is
activated. Two particular gene regions in the DNA of this DNA-virus are
activated immediately: e1a and e1b. This activation leads to the
synthesis of two different viral proteins: E1A and E1B.
What Does the E1A Adenoirus Protein Do?
The viral protein, E1A, interacts specifically with certain proteins within the
cell which results in driving the cell (forced entry) into what is called the
S-phase (the DNA Synthesis phase) of the cell cycle - which ultimately leads to
cell division (M-phase.... mitosis)... if a cell is already in this cycle - no
problem - virus substances will _maintain_ the cell in this cycle. In order
for this virus - Adenovirus - to replicate its own DNA genetic material, the
enzymes necessary for this process and other activation and synthesis substances
within the cell are available only if the CELL's DNA activation and
replication systems are turned on within the cell's nucleus. This viral
activation of cellular processes allows the VIRUS to replicate its
genetic material, and to synthesize all of the proteins the VIRUS needs to
assemble new virus and thereby produce many, many progeny viruses.
What Else Does Adenovirus E1A Protein Do?
The action of E1A also happens to stimulate the cell's p53
system...(remember - viral activation to cell division is an improper thing...
p53 acts in response to this improper activation). Theoretically, therefore,
the cell will initiate activity which would normally result in G0, G1 arrest,
or, cell suicide. If any of these protective events occurred, however, the
virus would not be able to produce progeny - its host cell died too quickly,
or, necessary cell systems were no longer active. For the virus - such
events would be lethal to propagation of the virus. So - the virus has
evolved, too.
How Does Adenovirus Circumvent the Cell's p53 System?
The normal, wild-type Adenovirus has evolved a back-up system which circumvents
the activation of the cell's p53 system by its E1A protein. The virus normally
needs E1A to force the infected cell into cell division mode - but - the cell
has a defense in that p53 is activated by the action of virus E1A protein.
Here is where the second important viral gene, e1b is involved.
Activation of this gene leads to the synthesis inside the infected cell of
Adenovirus protein, E1B. The viral protein E1B interacts specifically
with the cell's p53 protein - and in so doing, prevents p53 from regulating
the cell division process (prevents suppression of cell division - "ties
up" p53.
What Normally Happens If a Wild-Type Adenovirus Infects a Cell?
If wild-type Adenovirus infects a p53-positive cell or if it infects
a p53-negative or p53-dysfunctional cell, the virus can replicate, produce many
progeny, and ultimately lyse (open) the cell - will kill the cell - but - now
all of the virus progeny are released, which can infect adjacent cells. The
virus thus produces more of its kind and survives. So, both normal and even
nondivision-controlled cancer cells can be killed by a wild-type Adenovirus.
In the cells with functional p53, the viral E1B protein blocks p53 action.. therefore,
E1A-driven S-phase entry and viral replication proceed unimpeded. In the cells
with dysfunctional or absent p53, E1A has the same effect; but in this instance,
E1B is no longer necessary for the virus to be successful - since the p53 in
these cells does not work or is absent.
What Does all of This Information Mean With Respect to Cancer Treatment?
Investigators Bischoff, and others (et. al.,) of ONYX Pharmaceuticals, Inc.,
reasoned along the following lines:
1. A NORMAL (wild-type) Adenovirus producing NORMAL E1A and E1B viral proteins
can successfully replicate and ultimately lyse (kill) all human cells which
have either a normal p53 or improper p53 protein. Therefore, the wild-type
virus will replicate successfully within both normal and cancer cells,
and thus can ultimately kill both of these kinds of human cells. But - killing
of normal cells is not the object - only killing of cancer cells is what
is desired.
2. A MUTANT Adenovirus - with a specifically designed (genetically
engineered) mutation which results in a dysfunctional E1B virus protein - should
not be able to replicate very well within a NORMAL cell - containing
NORMAL p53 - but MIGHT be able to replicate in an ABNORMAL cell with ABNORMAL
or ABSENT p53. So - the ultimate aim was to genetically-design a mutant
Adenovirus which could successfully replicate within and lyse, cancer cells -
those 50% of known cancer cells with a dysfunctional p53 - or any
cell not yet cancerous, but with a dysfunctional p53. Here is why such
a genetically-altered Adenovirus would theoretically work in this fashion.
Action of The Mutant Adenovirus on a Normal Cell
This virus has had its DNA purposefully altered such that the e1b gene
region is mutated - activation of this gene leads to an incorrect E1B virus
protein structure (dysfunctional E1B). Consequently, when this Adenovirus
mutant infects a cell, this virus CANNOT produce a functional E1B protein. ...
remember - a functional E1B protein will normally interact with and interfere
with functional p53 - which allows viral DNA replication to proceed well,
and which prevents cell suicide initiated by p53.
A normal cell will have a functional p53. So, when viral protein E1A drives
the cell into S-phase there will be a concomitant response of p53 - which will
lead to cell-division arrest or apoptosis eventually if this action of p53
proceeds unimpeded. Since the mutant virus does not have a functional
E1B protein - the p53 system responds unimpeded - which allows repair,
inhibition of viral replication, and/or which can lead the cell to commit
suicide. All of these things mean that a mutant Adenovirus with this
particular e1b gene mutation, theoretically should not replicate efficiently and
therefore should not easily kill, normal cells.
Action of The Mutant Adenovirus on an Abnormal - p53-Deficient - Cell
The replication of this mutant Adenovirus IN CELLS WITHOUT p53 OR WITH A
DYSFUNCTIONAL p53 protein, like many different kinds of cancer cells - can
proceed without interference. The LACK of synthesis of an available, functional
E1B protein by this mutant virus is really of little consequence to the virus....
E1B is not really "needed" for the virus to properly replicate, since the cell
already has a non-functioning p53. It is only that the absence of E1B
prevents efficient replication of the mutant virus within a cell which has a
functional p53 - e.g., a normal cell.
Thus, the whole idea is based upon the ability to select only cancer
cells - to target only cancer cells for killing by the mutant Adenovirus. Therefore,
the absence of E1B - BY DESIGN prevents replication of the virus
in normal cells, and thus essentially targets the virus for replication
in cancer cells only - or potential cancer cells (dysfunctional p53 - but not
yet dividing for whatever reason, without control).
Data Which Showed It Worked!
Tests by these investigators showed that this mutant virus relative to
p53-dysfunctional cells, replicates 100-times less well in normal cells. But,
this same mutant virus killed all cancer cells the investigators tested.. those
cancer cells which were known to have a p53 dysfunction or where p53 was
completely absent. Conversely, when these investigators transfected
(introduction of a gene into an animal cell) a normal p53 gene into one
of these same originally p53-dysfunctional cancer cell lines, these cells
were no longer sensitive to being killed by this mutant virus - the virus
failed to replicate efficiently in these cells. Thus, the lack of ability to
efficiently replicate, clearly depended upon the presence of a functional p53
protein and this viral mutation - together in the same cell.
Human Clinical Trials
Clinical trials (Phase I) are already underway to test the toxicity level of
this mutant virus (certain selected patients with a p53-negative squamous
cell cancer of the head and neck). If all goes well for these first patients,
ensuing phases will be designed to use progressively more detailed experimentation
and treatment. Many, many phases of a human clinical trial are necessary
before approval of a therapy for general use in cancer treatment. Everything
possible is done to prevent the therapy from causing more harm than the disease.
However, in cases where the early data are abundantly clear that the treatment
has no apparent harmful effect, but only helpful effects, the phases may be
shortened, and the trial stopped in order to immediately begin treating people.
This type of decision is made very carefully, however, because the long-term
negative effects of a new treatment might not yet be obvious.
Some Cautionary Statements
This particular therapeutic approach for cancer treatment, e.g., the use
of a mutant Adenovirus to kill cancer cells, has a potential problem - a
problem of which the designers of this system are also aware, and which
was discussed in the Science article referenced above. This problem
is the specific defense system which all humans have against infections -
viral or otherwise - the human immune system. This system is specifically
active against virally-infected cells. Therefore, it is possible that after
one or more mutant viral treatments, the person would generate an immune
response which would kill the mutant virus prior to the virus' successful entry
and replication in the cancer cells of the patient. However, there is also
the possibility that the immune system would now perhaps be more efficient in
eliminating cancer cells specifically - because of viral material generated in
virally-infected cancer cells (is how our immune system recognizes and kills
one of our cells if the cell is infected with a virus).
In any event, this type of genetic approach is of great interest, and
will hopefully provide if not now, at least in the future, renewed hope
for the treatment of cancer. It is important to note that all of
the information which led to this particular potential cancer treatment, has
been generated by a few individuals over the course of history - and usually
funded by the hard-working people of various nations through their tax dollars.
All kinds of different things had to be understood before this approach could
even be attempted - and prior to the gathering of an astonishing amount of
fundamental research knowledge, some of which at the time had no apparent
relationship to anything obviously worthwhile - prior to the time when the
information had finally been gathered - in no instance was there someone who
"out of the blue" said, "I've got it; let's genetically
engineer a mutant Adenovirus with an e1b gene mutation to prevent the
killing of normal human cells by the absence of the ability to interfere
with the p53 cell-division suppression system when cells are forced into S-phase
of the cell-cycle - and allow the killing of p53-deficient cells - a specific
deficiency which I'll bet that 50% of all cancer cells in humans, have."
Additional Information
There is a very well-written, scientifically-detailed article about p53 on
the Internet. Please see an article written by J. Frank Baker:
A Review Of The
Importance Of The p53 Gene In Cancer Research
and, there is an article I wrote about research funding, which may be of
interest to you. Please also see::
What the Heck is a Research Grant?
For some human interest information, one of the above listed authors, Ali
Fattaey, has a local connection. Dr. Fattaey received the Ph.D. from Kansas
State University, Division of Biology, while working in the laboratory of
Richard Consigli. Dr. Consigli received his Ph.D from the University of
Kansas, Department of Microbiology. My thanks to Dave Rintoul, KSU, for this
information.
Reference
1. Bischoff, J.R., D.H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna,
L. Ng, J. Nye, A. Sampson-Johannes, A. Fattaey, and, F. McCormick. 1996.
An Adenovirus Mutant That Replicates Selectively in p53-Deficient Human Tumor
Cells. Science 274: 373-376.
Copyright John C. Brown, October, 2000. This Page Updated, 08/03/00
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