Cancer Initiation

Friday, January 29, 2016

The danger of "Dimension Reduction" in science and education

   Science education exists at the cusp of the epistemology known as science, and the cognitive ability recognized as "education". The historical foundation of science has built around finding the proper function, as determined by various types of statistical analysis, such as correlation. Beyond correlation, scientists use various logical methods to "prove" causation between two variables.
    This foundation has worked very well for physics and chemistry.  We can easily define an ion's concentration as a function of pH, or a particle's position as a function of time and force. As such, we are tempted by conditioning to apply the same principles to biomedical science, such as physiology.
  The focus of this note is to demonstrate that we are wrong when we try to force biological systems, systems of multiple dimensions, into the single dimension systems  that give us such great comfort in physical and chemical systems.
  Homeostasis is a fundamental concept in physiology. In simple terms, components of complex systems feed back into each other to keep vital variables at physiological levels. Each component of these systems has both positive and negative response elements.  An M.D. intuitively looks at vital variables not only to see if they are at best levels, but to see if the components of a system are responding properly to perturbations of the system of interest.
  All of this seems very rational, even obvious. The aforementioned discussion seems to be relatively intuitive for a century at least. So how can top scientists still reduce multidimensional functionality in a system down to a single largely non-relevant value? This seems to happen in the discussion of telomerase (hTERT), a gene system responsible for maintenance of the ends of chromosomal DNA. As I have discussed in a previous post detailing a system I call the Rattle Snake Hypothesis expression of telomerase is a double edge sword. On one hand, cells known as stem cells need to express telomerase as a component of the stem cells ability to continue producing a relatively infinite number of new cells. A good example is bone marrow where blood cells are produced. On the other hand, differentiated cells, or somatic cells ( of the body ) always seem to have telomerase repressed by being blocked epigenetically.
    It has long been observed these somatic cells can only divide a limited number of times and then become senescent. This observation has been attributed to Hayflick , and as a result is known as the Hayflick Limit.  The Hayflick limit is generally regarded as a cells first line of defense against cancer.
  In cancer cells, the Hayflick Limit has always been defeated either by expression of telomerase or acitvity of the ALT pathway which accomplishes the same purpose.
   The Hayflick limit is by no means the last line of defense against cancer. The immune system is an important next level of defense against cancer.  As a conceptual model, we have introduced this level of defense as the Rattlesnake  Hypothesis.
  So what then is the best expression level for telomerase? The answer depends upon the physiological function of the particular cell in question. None the less, the question continually seams to come up, not only for telomerase, but for all genes, each of which has an appropriate expression level depending upon the physiological function of the particular cell.
  The "it depends" answer never seems to be sufficient as an answer in science. In todays assignment, Tom Cech describes the best gene expression level for telomerase. Hey says at 1:01:46  :
It's on a knife edge, you don't want two times too much or two times to little, it has to be like Goldy Locks, it has to be just right. And so that could be, that is an issue with the people with too little.
So my point is that on a large discussion of a cutting edge issue on a critical topic related to cancer, regulation of telomerase, it always has to be reduced at some point to a single dimension. This seems to be a compulsory  requirement of people, not physical or biological systems.
   With  that point made, I would like to thank Tom Cech for placing this video online for the science education community, and for people interested in cancer research at the molecular level. Since Mr. Cech already has a Nobel Prize and a successful laboratory, I believe he will survive the accusation of "dimension reduction" by a blogger. Hopefully in the end, students will be encouraged to think in more dynamic terms about dynamic systems in equilibrium.

Thursday, January 28, 2016

Checkpoint Loss and Paul Nurse's "Wee Mutants"

In previous posts we have referred to "Checkpoint Loss" as a presumed underlying cause of loss of control of the cell cycle. Here, we discuss further the historical science behind the concept of checkpoints and their loss. The first "popular" discussion of checkpoints comes as part of Paul Nurse's Nobel Lecture of 2001.        In it, he refers to the "wee mutants" as those members of his fission yeast culture which divided before they reached full size. He reasoned that that there must be some cellular mechanism that kept cells from progressing through the cell cycle before their "Synthesis" or "S" phase was complete. As such, cells which had a defective mechanism, or checkpoint, would appear smaller under the microscope. These cells could be isolated and geno-typed to find the specific genetic, or DNA based mutation that was responsible.
  Based upon isolating the "Wee Mutants" and development of a genetic toolkit for working with fission yeast, Paul Nurse and his associates decoded the mechanism of cell cycle regulation in Eukaryots.
   Possibly Foremost in this system are the "Cyclins" or those protein products that accumulate as each stage of the cell cycle progresses. When the level of  a specific cyclin in the cell reaches a threshold level, it activates an enzyme known as a cyclin dependent kinase ( CDK ) .  A kinase is an enzyme which phosphorylates, or adds a phosphate group, and these CDKs act to phosphorylate a substrate known as E2F. When E2F has been phosphorylated, it can no longer bind to retinoblastoma (RB, pRB ), and as such it is free to translate to the nucleus where it is a transcription factor, and acts to move the cell through the cell cycle.
  We already have a great cancer research flag flown up here.  The gene retinoblastoma ( pRB ) has already been predicted and described as associated with cancer. The prefix "retinoblast" is a type of precurser cell in the retina of the eye.  A particular type  of cancer then has been characterized as " cancer arisen from the precursor cells of the retina" or "retinoblastoma". Susceptibility to retinoblastoma had been studied as a genetic condition, and lead to the formation of the "two hit" hypothesis of Alfred Knudson.
  The "two hit" hypothesis would have worked like this. When a patient suffers a mutation in the second allele of rb, it fails to function. As such, it can no longer bind to E2F and prevent progression through a checkpoint to the next stage of the cell cycle. This was the foundation for cancer causation for most of modern medical history.
  Nevertheless, with the development of more sophisticated medical technology, it was discovered that in fact, more often than not, the gene sequence of rb is in good shape. What has happened is that its expression has been suppressed by promoter methylation. This promoter methylation is a foundation of what is now called  epigenetics.
   A missing link in the logic here is that fission yeast are single cell biota. As such, they do not suffer cancer. Cancer is a disease of organisms which have some degree of differentiation. This differentiation is increasingly being as a function of DNA methylation. As such, we have proposed a methylation maintenance model to describe the molecular/genetic organization of higher animals.
  As such, our cancer model is currently more sophisticated than Nurses "wee mutant" mode. When a checkpoint is lost, dividing cells fail to complete the duplication of DNA methylation patterns upon division, or mitosis.  This leads to a clinically observable condition known as global hypomethylation which is observed on the molecular level as a particular cancer progresses. As such, not only does DNA sequence mutate as cancer progresses, regulation of expression disintegrates as well.
  On an cellular level, this happens quite often. There are multiple levels of defense. The first is epigenetic suppression of telomerase. A somatic cell can divide a certain number of times until its telomeres expire and all descendants become senescent. 
  The next level of defense involves triggering the immune system. We refer to this as the rattlesnake hypothesis.  In brief, this states that a cell contains a number of "danger flags" known as wingspans antigens. When these epigenetically suppressed genes become expressed, their sole purpose is to trigger the immune system to attack the cell. In the presence of a healthy immune system, this mechanism is sufficient to protect against cancer.
  In summary, although much of what has been known as cancer research has been swept to the side by the advance in molecular analytics, the original concept of the checkpoint, and associated "wee mutant" seems to be one that will stay as a foundation of future cancer models.

Tuesday, January 12, 2016

What is meaning of the word "epigenetics"

 The word epigenetics has become the term of the decade in cancer research, and in some degree, almost all degenerative diseases. Epigenetics appears as though it will fill in missing gaps in developmental diseases as well. It appears now that there is a diverging opinion of that this word actually means.
   At to core of the word, is the well understood base known as genetics. In general terms, genetics refers to the heritable information which is passed from one cell generation to the next cell generation. This heritable information was almost always presumed to be the DNA sequence.
  As the tools of molecular biology have been refined, automated and personalized, it has become apparent the DNA sequence does not account for all of the information passed from each cell generation. In particular, methyl groups can be added to specific sequences in the cells DNA known as CpG islands, where C and G represent nucleotides and p in the connecting phosphate.
   The regulatory region of a cells DNA known as a promoter is typically rich in CpG islands. When these become methylated, a protein known as Methyl CpG Binding protein 2 binds to the promoter, and "epigenetically" blocks expression of that gene.
   A key to the concept of epigenetics is that these methylation patterns are copied upon cell division, the same as DNA sequence. That is, modifications to promoter status are passed from one generation to the next. The prefix "epi" means "on top of" in medical and scientific terminology. Thus DNA methylation is a type of genetics which "piggy backs" on DNA sequence genetics.
  In a recent article in The scientist reviewing RNA methylation, the authors refer to this as "RNA epigenetics".   
   In fact I feel that "RNA modification" or "RNA decoration" would be a better term. There does not seem to be any evidence presented that these modified RNAs can be duplicated and perpetuated from generation to generation. As such, it is not quite in the same class as DNA modifications which are duplicated by a "DNA methyltransferase"


 By Dan Dominissini, Chuan He and Gidi RechaviRNA Epigenetics, The Scientist | January 1, 2016


Monday, November 2, 2015

The foundatons of the emerging field of epigenetics provides the long sought link between development and cancer

In 1996 Pierre Beland released the book "Beluga - A farewell to whales" as a next step in the emerging field of environmental toxicology, or ecotoxicology.  The concept of transfer of biological toxins through the web of the food chain had already been introduced by Rachel Carsons "Silent Spring", but Beland added the next critical realization to our popular understanding by adding the observation that mammals, including us, have an extra level in our food chain which makes eco-toxins particularly harmful and insidious.
  It had previously been known that some agents which I am going to call eco-toxins, bio-accumulate as they rise through the food chain. Beland extends the bio-accumulation model to mammals, in this case, the whales of the St. Lawrence Seaway, and shows that these toxins achieve another level of bio-concentration in the milk of female mammals.  Thus, Beland's story is one of cancer, developmental failure and death, but the bio-chemical link between cancer development has remained somewhat of a mystery.
   Molecular biology and eco-toxicology was missing an important keyword - epigenetics.  As organisms and tissues develop, they accumulate methylation "marks" on the promoters of genes. Toxins that inhibit or interfere with the process methylating the CpG islands associated with promoters, likewise inhibit proper development and differentiation.
  Once an organism has reached an adult development stage, cells continue to divide in a "maintenance" stage. There are many genes in an adult somatic ( of the body ) cell that are suppressed in order to keep the cell out of the cell cycle. If the DNA methylation duplication system fails on an oncogene, or gene that causes a cell to enter the cell cycle, a cancer, or run away cell division can occur.
   In most cases, internal controls, or the immune system will catch these runaway cells.
 The immune system itself is dependent upon the mechanisims of differentiation. If the toxins has thus suppressed the immune system, then other cancerous cells can continue to divide and grow without natural inhibition.
  Thus, there is another chapter in the eco-toxicology model which is yet to be written. That is, using new molecular based tools to visualize and quantify how  individual "epigentic toxins" accumuate and lead to cancer and development failure.

Saturday, April 25, 2015

The principles of epigenetics imply a lack of equivalence between mutagenicity and carcinogenesis

   Inherent in the current system of carcinogenic risk analysis is the  precondition that the mutagenicity of a compound is equivalent to its carcinogenic potential. This equivalence dates back to the work of Bruce Ames during the 1980's, and his refinement of the "Ames Test".  In brief, the Ames test is a "reversion test", in which a small mutation is placed in a model organism ( salmonella ), and the ability of a test compound to perform random mutations that reverse the effects of the inserted mutation is quantified.
   This quantified assessment of a compounds mutagenicity, or ability to interfere with the proper replication of DNA is a priory assumed to be equivalent to its carcinogenic risk.  This causal-equivalence presumably arises from early work in genetics, primarily in fruit flies ( drosophila ) .
In fruit fly work, the eggs or larvae are exposed to radiation or known mutagenic chemicals, and resulting mutations (genes) are named after the development phenotype of the affected organism.
  So far so good. But, as I have mentioned, this particular model has since broken down as a model of carcinogenesis. Time after time, in studies of actual tumors in patients, the gene sequence of the presumed causative gene is normal, but the gene has been suppressed at the transcription level. The details of the particular suppression are part of a field of study known as "epigenetics".
    A PubMed search on "cancer epigenetics" returns thousands of hits. This has become the modern direction of cancer research. On the other hand, the "Ames test" remains the standard of risk determination, even though it only quantifies an effect that is no longer uniquely associated with carcinogenic risk.
    As a "literature experiment", I first placed the term "mutagenicity carcinogenisis ames" in the PubMed search bar. The resulting list ( April 2015 ) returned  13 pages of 247 articles.
  I then added the term "epigenetics" to the search and the result was "no items found"

Figure 1. Two searches, showing no review of the findings of "epigenetics" on the validity of the Ames test.
 Let me further clarify that carcinogensisis as a result of "genomic" mutation is a completely different process thatn that of "methylomic" mutation.  Genomic fidelity is associated with proteins known as DNA polymerases. On the other hand, the methylome is associated with the fidelity of the "maintenance DNA methyl transferase"  (DNMT).
   The continuing lack of differentiation between these two processes in the research and science education community is one of the primary reasons for proposing that a new foundation of biology be developed around maintenance of the methylome.

Saturday, April 18, 2015

The 9/11 Anthrax Investigation accused of being a psychological operation without actual evidence by its one time leader

An article published today details the charges filed by Richard Lambert.  According to the complaint:

 Mr. Lambert holds a Doctor of Jurisprudence degree, and three
Master’s degrees in Political Science, Public Administration, and Business Administration.  As a result of Defendants’ grossly negligent and erroneous legal opinion, Mr. Lambert was fired from  his DOE-UT-Battelle position on June 10, 2013 and his contract with the University of Tennessee has not been renewed.  Due to the notoriety and stigma surrounding Defendants’ erroneous legal opinion and its plain implication that he is a federal felon, Mr. Lambert is currently unemployed and unemployable. bert, former head of the governments investigation into the 9/11 anthrax mailing.
  Well, it appears that Mr. Lambert is not your typical flat foot. After a 24 year career with the FBI, including heading the anthrax investigation, the FBI retaliated for a whistle blower suit he filed related to the psy-op that was perpetrated in coordination with the Ivins case. The original whistle blower case never seems to have made widely published news sources.

   Mr. Lambert retired from the FBI in 2012, approximately 11 years after 9/11. One curious aspect of this case is that if the FBI had not continued harassment of Mr. Lambert, the public would have never known that there was dissension within FBI regarding the Ivins case. This case should be an interesting one to watch.
  As the Washingtons Blog post details, the scientific community was never able to sign on to the FBI's theories about the Ivins case.

Saturday, March 14, 2015

The Methylome Maintenance Model, a medically relevant organizational paradigm for biological sciences based upon DNA promoter methylation status

 This article introduces the concept of the "Methylome Maintenance Model".  It is proposed as an alternative to the current "central dogma of molecular biology" as proposed by Francis Crick.  Although the central dogma has provided an organizational framework for cancer research since the inception of molecular cancer research, it has ultimately proven to offer nothing in terms of insight or organizational characterization of clinical molecular observations.

We start our discussion with a very brief review of the history, failure  and future of biological organizational frameworks, or paradigms, as I prefer to call them here. First was taxonomy, or classification based upon physical characteristics, followed by evolution, or the notion that taxonomy originates as a function of evolution.   As such Charles Darwin was recognized as the founder of modern biological science for his exhaustive, yet empirical argument that evolution was based upon small incremental changes that result from random mutations, and preferential adaptation to the environment.
  Although Mendel was a contemporary of Darwin, his work was temporarily lost, only to be rediscovered decades after his passing.  Upon rediscovery, Mendel's concept of a genetic factor seemed to reinforce Darwin's work. Mendel's description of the workings of the factors that came to be called genes led to a race for the identification of the genetic material. The finish line for the description of the genetic material seemed to be crossed when Watson and Crick elucidated the structure and function of DNA,  The case seemed closed as far as a search for organizational paradigms.
  Francis Crick stated the so called "central dogma" of molecular biology in 1956. It is summarized in the Wikipedia as follows:
 The central dogma has also been described as "DNA makes RNA and RNA makes protein,"[3] a positive statement which was originally termed the sequence hypothesis by Crick. However, this simplification does not make it clear that the central dogma as stated by Crick does not preclude the reverse flow of information from RNA to DNA, only ruling out the flow from protein to RNA or DNA. Crick's use of the word dogma was unconventional, and has been controversial.
Since the word "dogma" usually refers to a concept that is repeated automatically and religiously,  even when incorrect or not relevant, Crick's choice of the word was correct, and in fact prophetic. Today, the "central dogma" remains the operative paradigm for what molecular biology laboratories do.
 With respect to cancer, a landmark paper was delivered in 1971 by Alfred Knudson.[1] which described cancer ( retinoblastoma ) as a genetic event operating in adherence to the central dogma and Mendelian genetics. He proposed, that A first hit could be inherited, and a second hit ( to DNA sequence ) could case  cancer.  At least in terms of cancer, this was the first major event linking medical  (epidemiological)  observations to the genetic / central dogma paradigm.
  There was a gap of quite a few decades between the Knudson's proposal and the ability of clinical researchers to definitively evaluate the DNA sequence of pRB ( protein retinoblastoma, the predicted, and discovered  ) gene responsible for the observed cancer.
   Surprisingly,  ( and very quietly I might add)  in many, if not most cases, the DNA sequence of  pRB is intact. By strict interpretation of Crick's central dogma, this is impossible.
   Lets make clear that pRB is the right gene ( at least in the cases we are presently discussing ). What has happened is that although the DNA sequence is intact,  the promoter associated with pRB has become methylated, and consequently bound by methyl CpG binding protein 2 (MeCPB2 ), so the gene is not functional,  and is blocked from being expressed.
    Obviously, billions of dollars of research spent on potential problems with pRB function became dubious. What is even more breathtaking is that billions of dollars of research money continue to be spent on similar studies almost a decade after many of the particulars of pRB's function have became quaint, at least in clinical terms.
   For a description of the political inertia that continues, please browse Mastrangelo [2][3][4]. It is unfortunate that practitioners have to doubt that clinical evidence is even relevant, or that the research community has any interest in all at obtaining actual medical data. Consider the following from Mastrangelo[4]

The presumed genetic origin of Rb and its relationship with the Rb1 gene represent a clear example of how an entire body of prominent researchers may fail to question a flawed pathogenetic hypothesis (i.e., the “two-hit” theory), for the sake of personal, academic, or other interests. It was not by chance that we had to approach many different scientific journals to have access to the medical community about the role of aneuploidy and genomic instability in the genesis of Rb [4, 5, 39]. We are still optimistic, however, because our alternative pathogenetic explanation has finally appeared in recent ophthalmologic literature [40].
Case Made
   It is time to start from scratch in terms of an organizing paradigm. The new paradigm is based on the concept of cellular differentiation. An organism begins its existence as a clump of similar, undifferentiated stem cells.
    These stem cells have a few important characteristics.
  • The first of these cells are totipotent, they can differentiate to any somatic tissue.
  • The earliest of these cells are immortal, either telomerase ( hTERT) or other mechanisms exist to maintain telomere ends. relatively speaking, the promoters of respective genes are unmethylated, so genes can respond to any transcription factor which will interact with its promoter
From the "stem cell" stage, the process of development is directed by two major factors:
  •  Signals received from outside the cell in the form of chemical or electrical signals.
  • DNA methylation patterns inherited from the cells predecessor(s)
Yes, the amount of signals that a cell can receive is immense, but since we are talking about organizational paradigms, here, we can choose to simplify here, and detail another day. That is the function of an organizational paradigm.

The major advance of the MMM is that we have long known that all cells have the same DNA. Each cell works from a complete genome, and yet, each cell has an accounting system which integrates its current signaling environment with its inherited cell type. In more common terms different tissues may have unique response to common signals.
   The currency of this cellular accounting system is DNA methylation.  Methylation of the promoters of  the DNA of a cells genome can give the cell a unique response to signals.
   In stead of speaking of a cells genome, which is static, and the same for all cells of an organism, we speak now of a genome, a transcriptome, and a methylome. As such, these cumbersome terms are sometimes referred to as "omics".
    Due to advances in technology, a cells DNA can be sequenced relatively rapidly. Likewise for those genes that are currently being transcribed, messenger RNA can be recovered, and rapidly processed with "gene chips" to give relatively automatic printout of those genes that are currently being transcribed. The "transcriptome", unlike the genome is unique for each cell, or more practically, tissue. The methylome refers to those genes that may be behind responsive promoters for the particular cellular environment, but for which transcription is blocked by methylation, ( and subsequent binding by MeCPB2. Increasingly, it is the methylome that is of interest in cancer research.
   In fact our ability to collect automatic data about cancer progression has outstripped our ability to process effectively.
  Perhaps as an aid to automatic data analysis ( informatics ) of automatically generated data (omics) we really need to advance our organizational paradigm. Neither genomics or the central dogma address the present problem.
  At the core of historic cancer research are the concepts of "cell fate" and apoptosis, or programmed cell death. Failure of apoptosis is seen as a common clinical feature in cancerous clones of cells. Likewise, according to the cell fate model, a cell differentiates into its terminal somatic tissue type, and then functions in support of metabolism until it is senescent and goes through the previously mentioned programmed cell death.
  What I am going to do here is no less than re-define metabolism. Metabolism in the maintenance of fidelity of the methylome. When the cell detects a failure, which questions this fidelity, the first response should be apoptosis. Failing that, we go into additional defenses, which as I have mentioned are telomere length related, or immunologic. These additional defenses are what we commonly call cancer.

   All models need a name. I feel "methylome maintenance model" is as descriptive as we can get for our exercise of redefining metabolism as methylome fidelity maintenance.    In the MMM, it is the goal, not only of every cell, but of the organism as a system to maintain it's methylome in a fully differentiated state.
  We need to recognize that this  maintenance system operates both at the level of the individual cell, and at the level of the organism. At the cellular we have many ( hundreds at least ) biochemical pathways that lead to apoptosis. Failing that, we have what I have previously defined as the Rattlesnake Hypothesis in which loss of methylation fidelity causes  Wingspan's antigens to become expressed, and signal an immune response against the offending ( cancer ) cell.

[1] Alfred G. Knudson, Jr.
Mutation and Cancer: Statistical Study of Retinoblastoma
Proc Natl Acad Sci U S A. 1971 Apr; 68(4): 820–823. [PubMed]

[2] Mastrangelo D1, De Francesco S, Di Leonardo A, Lentini L, Hadjistilianou T.
Retinoblastoma epidemiology: does the evidence matter?
Eur J Cancer. 2007 Jul;43(10):1596-603. Epub 2007 May 31. [PubMed]

[3]  Mastrangelo D, De Francesco S, Di Leonardo A, Lentini L, Hadjistilianou T
Does the evidence matter in medicine? The retinoblastoma paradigm.
Int J Cancer. 2007 Dec 1;121(11):2501-5.[PubMed]

[4] Mastrangelo D1, Hadjistilianou T, De Francesco S, LorĂ© C   Retinoblastoma and the genetic theory of cancer: an old paradigm trying to survive to the evidence. J Cancer Epidemiol. 2009;2009:301973. [PubMed Central]