Cancer Initiation: 2014

Thursday, May 29, 2014

An emerging role for cancer/testis antigens in cancer progression and innate defences

The family of cancer/testis antigens ( CTA ) represents a class of several families of surface antigens that are expressed on the surface of cancer cells, testis cells, and only in very limited quantities, some somatic tissues. Here we propose a developmental and cancer defense function of these mostly epigenetically suppressed antigens. Cancer/testis antigens are a significant component of the Rattlesnake hypothesis ( alternatively, Wingspans rattlesnake ) model for cancer detection and immunological defense by the host organism. The emergence of CT antigens on the surface carcinogenic clones of cells is regarded by the host immune system as a warning ( rattle ) of a progression of global hypomethylation , in turn a sign that a cell cycle checkpoint has been lost and the cell (or colony)  has lost control of it's division process and is carcinogenic.
   CT antigens are currently a portion of the  "stem cell model" for carcinogenisis.[1]. It is summarized as follows( also from Costa):

Cancer stem cells were first identified in acute myeloid leukemia when surface markers were used to distinguish the stem cell population from the remaining cells with limited proliferative potential [5]. In solid tumors, cancer stem cells have been identified in breast cancer [6, 7], glioblastomas [8], lung cancer [9], ovarian cancer [4], prostate cancer [3], and epithelial gastric cancer [10]. Based on these observations, a cancer stem cell model has been proposed, and it is based on the concept that the great majority of the tumor cells have a limited proliferative potential, but a small cell population—the cancer stem cells—are able to self-renew and proliferate, maintaining the tumor cell mass. In this model, cancer is a disease of deregulated self-renewal of normal stem cells. Thus, in the cancer stem cell model, tumor recurrences and even metastases may occur due to residual cells—probably chemotherapy-resistant—that are able to expand to form secondary tumors [11]      ( Costa et. al. [1] )
The cancer stem cell model as it stands ( stood in 2006, and remains without significant change ) does not tie together loss of a checkpoint with immune system cancer defences. Costa does recognize the potential importance of CT antigens.
  To date, nearly 40 distinct CTAs have been identified based on immunogenic properties [41], expression profiles [42], and by bioinformatic methods [43]. However, little is known about their specific functions, and their functional connection with stem cell biology and cancer is widely unexplored. In this regard, it was recently reported that some CTAs such as N-RAGE, NY-ESO, MAGE-1, and SSX are expressed in human mesenchymal stem cells of the bone marrow, suggesting that CTA expression may not only be a hallmark of gametogenesis but also a stem cell marker [28] (Fig. 1).
We see the statement " little is known about their specific functions, ...", where as in fact under the  Rattlesnake hypothesis, their specific function is to tell the immune system that global hypomethylation is under way in a cell, or clone of cells, and to activate the immune system.  CTAs are a critical component of the hosts defense against cancer. They are a "dead mans switch" in case that a sells methylation "fidelity" has been lost and the cell is careening out of control.

It is also recognized that there is a significant difference between mesenchymal stem cells ( MSC) in that they do not stimulate an immune response where as cancer cells do.  It is evident then that there is some type of "fidelity" involved in normal mesenchymal cells that is lost in cancer cells.  The  following is also from Costa and describes experimental differences betwen MSCs and cancer cells with respect to interaction with cytolytic T  lymphocytes ( CTLs).
It is also possible that MSCs differ from cancer cells and escape recognition by therapeutically infused CTA-specific CTLs. This is supported by the fact that MSCs are not immunostimulatory in vitro when cultured with allogeneic lymphocytes [53, 54]. Furthermore, MSCs can escape lysis by allogeneic cytotoxic CD8+ T cells (CTLs). After transplantation of fully HLA-mismatched MSCs into an immunocompetent fetus, the cells persisted for a long term [55]. The transplanted MSCs did not induce any immune response in the child, again indicating that MSCs have immunoevasive properties

CTAs and the Germ Line Cells
  It has long been known that the cells of the testis generate an immune reaction, not only in foreign hosts, but in the hosts own immunologically environment. As such, the testes must be
compartmentalized in an immunologically privileged area. If the containment of the testicular area is broken, an immune reaction occurs, presumably mediated by cytolytic T cells. The concept of immunologic privilege is one that has been consistently overlooked, particularly given the prevalence of testicular and prostate cancer incidence. The prostate is an organ that must function in contact with cells of the testes, and as such, we might guess ( hypothesize ) that the prostate exists within the immunologically privileged area associated with the testes.

Research history
  C/T antigens were first identified by Van Der Bruggen [3]  ( Boon corresponding author ) and their first characterization noted that when expressed, they attracted the defensive and destructive attention of cytotoxic T lymphocytes( CTLs );
  The behaviorial observations are as follows in  Van Der Bruggen [3]:
 For human tumors, autologous mixed cultures of tumor cells and lymphocytes can generate CTLs that lyse tumor cells(4). These anti-tumor CTLs do not lyse targets of natural killer cells and autologous control cells such as fibroblasts or EBV-transformed B lymphocytes. However, it is difficult to evaluate to what extent the antigens recognized on human tumors by autologous CTLs are relevant for tumor rejection.
So we see that even though the property of tumor attack by CTLs , before Van Der Bruggen,  ( their reference 4 ), and families of specific signaling antigens ( CT antigens ) were identified from 1991 on, researchers have still not described their significance and roll in the larger picture of cancer defenses. That is, they have not connected the central components of the Rattlesnake Hypothesis, that is:
  •  telomerase expression status, ( repression is defense 1 - Hayflick )
  •  loss of a component of a checkpoint, and resulting
  •  progressive global hypomethylation,
  •  emergence of CT antigens as the result of progressive loss of suppression on their promoters, and promoters that are responsive to multiple ubiquitous transcription factors such as sp1
  • initiation of tumor specific immune defenses triggered by recognition of CT antigens by cytotoxic T lymphocytes
Implications for cancer therapy
  Cancer therapies that seek to stimulate and take advantage of the immune system are now, as always relegated to "alternative" therapies, or second line, prospects. We can see from the implications of Van Der Bruggen and Boon [3] [4] [5] [6] there was never really any basis for this in research. As such there is much new interest in designing therapies that stimulate and take advantage of the immune systems natural targeting ability for cancer.

The hTERT ( telomerase ) promoter
  The responsiveness of the telomerase promoter has been characterized by Zhao et. al. [7]. Like CT antigens, the expression of hTERT is suppressed in normal somatic cells by promoter hypermethylation. The promoter itself has been shown to be responsive to ( the ubiquitous ) transcription factor sp1 as described by Zhao:
We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner.[7]
So we see that the ubiquitous promoter sp1 activates both telomerase and CT antigens, so that in both cases, expression is completely dependent upon the promoter methyation state. Thus, in the pathological case of global hypomethyation, both genes will become, at some point expressed, and presumably, the CT antigens are "warnings" or "rattles" that a clone of cells has defeated it's Hayflick limit defense, and presumably needs help from the immune system.

Therapeutic implications and applications
  Although the role of CT antigens has yet to be integrated into a "big picture" of cancer theory , their potential in relation to cancer detection ( biomarkers ), as well as implications for immune system based therapies has been recognized.[8] [11] [12].  A  logical framework for cancer defenses that actively included Cancer/Testis Antigens would therefore be of value in the therapeutic  and clinical  end of the cancer research spectrum because a logical and evidence based foundation for decision making is even more important in clinical practice than it is in education and basic research.

  There is a gene expression symmetry which exists between telomerase and CT antigens that is dictated by the evidence that both genes exist behind promoters that are responsive to ubiquitous transcription factors, and as such, they must be suppressed epigenetically by promoter hypermethylation in somatic cells.  Complementing this parallel expression profile, is the observation that one ( telomerase, hTERT ) is a significant danger to the host as a result of an immortalized clone of cells ( cancer ) and the other is a group of warning signals to the immune system, specifically cytolytic T cells. Underlying both these complementary functions is what has been called "global hypomethylation", or the observed progressive loss of promoter methylation occurs in a colony of cancer cells, presumably as a result of the loss of a cell cycle check point, and incomplete duplication of methylation patterns on the daughter strand of the duplicated DNA.

See Also:

Description of the "Rattlesnake Hypothesis" in developmental biology and cancer defense


[1] Costa F1, Le Blanc K, Brodin B. Concise review: cancer/testis antigens, stem cells, and cancer. Stem Cells. 2007 Mar;25(3):707-11. [PubMed] [Full Text]

[2]Yang F, Zhou X, Miao X, Zhang T, Hang X, Tie R, Liu N, Tian F, Wang F, Yuan J.
MAGEC2, an epithelial-mesenchymal transition inducer, is associated with breast cancer metastasis. Breast Cancer Res Treat. 2014 May;145(1):23-32. [PubMed Central]

[3] van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A,
 Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 1991;254(5038):1643–1647. [Abstract] [Full Text]

[4] Gaugler B, Van den Eynde B, van der Bruggen P, Romero P, Gaforio JJ, De Plaen E, Lethé B, Brasseur F, Boon T. Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med. 1994 Mar 1;179(3):921-30. [PubMed Central]

[5]Van den Eynde B, Peeters O, De Backer O, Gaugler B, Lucas S, Boon T. A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma.
J Exp Med. 1995 Sep 1;182(3):689-98.[PubMed Central]

[6] Boon T, van der Bruggen P. Human tumor antigens recognized by T lymphocytes. J Exp Med. 1996 Mar 1;183(3):725-9.[PubMed Central]

[7] Zhao JQ1, Glasspool RM, Hoare SF, Bilsland A, Szatmari I, Keith WN. Activation of telomerase 8na gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3. Neoplasia. 2000 Nov-Dec;2(6):531-9.[PubMed Central]

[8]Caroline J. Voskens,Duane Sewell, MD, Ronna Hertzano,Jennifer DeSanto, Sandra Rollins,  Myounghee Lee,  Rodney Taylor,  Jeffrey Wolf,  Mohan Suntharalingam, Brian Gastman,  John C. Papadimitriou,  Changwan Lu,  Ming Tan,  Robert Morales,  Kevin Cullen, Esteban Celis,  Dean Mann, and Scott E. Strome,  Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma Head Neck. Dec 2012; 34(12): 1734–1746. [PubMed Central]

[9] Lim JH1, Kim SP, Gabrielson E, Park YB, Park JW, Kwon TK. Activation of human cancer/testis antigen gene, XAGE-1, in tumor cells is correlated with CpG island hypomethylation. Int J Cancer. 2005 Aug 20;116(2):200-6.[PubMed] [Full Text]

[10] James SR, Cedeno CD, Sharma A, Zhang W, Mohler JL, Odunsi K, Wilson EM, Karpf AR.
DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11.  Epigenetics. 2013 Aug;8(8):849-63. [PubMed Central]

[11] Stacey S Willard and Shahriar Koochekpour  Regulators of gene expression as biomarkers for prostate cancer Am J Cancer Res. 2012; 2(6): 620–657. [PubMed Central]

[12]  Comber JD1, Philip R2. MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.  Ther Adv Vaccines. 2014 May;2(3):77-89. [PubMed Central]

Tuesday, May 20, 2014

The paradox of global hypomethylation and local hypermethylation in terms of the sea-saw molecular logic of epigenetics

The observation of global DNA hypo-methylation is relatively easy to understand given our simplistic model of loss of a checkpoint in the cell cycle coupled incomplete duplication of methylation patterns during the DNA "maintenance" operations of the synthesis and growth phase phase of the cell cycle. The actual duplication if the cells methylation imprint is mediated by DNA Methyl - Transferase 1, (DNMT1) the catalytic portion of a replication complex. For a review and brief description of the DNA Methyl Transferases, we provide a link to Kim[1]. From Kim we have the following description of DNMT1:

 In proliferating cells, DNMT1 is found to be associated with replication foci (Leonhardt et al., 1992), ensuring methylation of the daughter strand during DNA replication. Knockout of Dnmt1 in the mouse genome resulted in global demethylation and embryonic lethality (Li et al., 1992).    (Kim[1] )
  Here, demethylation refers to complete loss of DNA CpG Island methylation, where as the word hypo-methylation refers to under, or incomplete methylation.
As further evidence that the loss of complete methylation is sufficient to induce loss of control of the cell cycle, Pacaud et. al. [3] disrupted the DNMT1 DNA maintenance complex ( DNMT1/PCNA/UHRF1) induces tumorigenesis.
 But  loss of methylation is only half the story, the other half is that tumor suppressors, such as our now familiar mediators of checkpoints ( pRB ) and apoptosis "guardian" genes, ( p53, BRCA1/2 ) become hyper- methylated, and as a result are suppressed.
 Lets look at an actual example related to Non Hodgkins Lymphoma. In Yim[4] We are introduced to our familiar sea/saw regulation of cancer related genes by micro-RNA's:

MicroRNAs (miRNAs) are short, non-coding RNA sequences of 18–25 nucleotides, which can repress the translational of multiple protein-coding mRNAs by sequence-specific binding to the 3′untranslated region. Depending on the genes targeted, miRNA can be tumor suppressive if an oncogene is repressed, or it can be oncogenic when a tumor suppressive gene is repressed. Recently, aberrant methylation of tumor suppressive miRNAs has been reported in different types of cancers including lymphomas. (Yim[4])
So we see, that if a micro-RNA that should be suppressed by promoter methylation is in fact expressed due to the progression of "global hypomethylation", then it may become expressed. If the molecular target gene ( transcript ) of the micro-RNA  is a "tumor supressor", then that target gene will become promoter hyper-methylated and its expression will be blocked.. Yim[4] also provides a slightly more technical description of the expression of a micro RNA, and the details of how it blocks a target.
These intermediates pre-miRNAs are exported via Ran-GTP-dependent exportin-5 (XPO-5) into the cytoplasm, where these pre-miRNA stem-loops are further processed into mature miRNA duplex (Yi et al., 2003). Eventually a single-stranded mature miRNA is produced, ready to function when it is loaded onto the DICER1-TAR RNA-binding protein-containing RNA-induced silencing complex (RISC; Liu et al., 2004). The biosynthesis and processing of miRNA is summarized in Figure Figure11. (Yim[4])
In general terms, the micro-RNA is processed by Dicer into a short single stranded piece of RNA that is incorporated into a silencing complex ( RISC ), which will mediate the hyper-methylation of the promoter of the target gene. By this process, we complete the pathway from loss of checkpoint, global hypomethylation and hyper-methylation of tumor suppressors, thus describing the "paradox".

 In conclusion, we note that micro- RNAs are relatively new addition to the molecular biology soup. Here we see that they provide an important logical component in describing a mechanism behind the often observed phenomenon of tumor suppressor hyper-methylation, while in fact, the majority of the cells DNA is undergoing hypo-methylation as a result of the loss of checkpoints  and/or DNMT1 integrity.


[1]  Gun-Do Kim, Jingwei Ni, Nicole Kelesoglu, Richard J. Roberts, and Sriharsa Pradhan Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases EMBO J. Aug 1, 2002; 21(15): 4183–4195. [PubMed Central]

[2] Sceusi EL1, Loose DS, Wray CJ.
Clinical implications of DNA methylation in hepatocellular carcinoma. HPB (Oxford). 2011 Jun;13(6):369-76.  [PubMed Central]

[3] Pacaud R, Brocard E, Lalier L, Hervouet E, Vallette FM, Cartron PF.
The DNMT1/PCNA/UHRF1 disruption induces tumorigenesis characterized by similar genetic and epigenetic signatures. Sci Rep. 2014 Mar 18;4:4230.  [PubMed Central]

[4] Yim RL1, Kwong YL, Wong KY, Chim CS.  DNA Methylation of Tumor Suppressive miRNAs in Non-Hodgkin's Lymphomas.   Front Genet. 2012 Nov 8;3:233.[PubMed Central]

Sunday, May 11, 2014

Description of the "Rattlesnake Hypothesis" in developmental biology and cancer defence


The Rattlesnake Hypothesis  (RsH)  is an organizing principle which describes organization of gene expression in the developmental biology of higher organisms, and presents an underlying principle which describes natural cancer defenses.
   First must make some notes about the word "hypothesis" in the title and in the concept Rattlesnake hypothesis. In the epistemology of science, hypothesis means something close to "testable conjecture", where testable refers to a controlled laboratory study.  This is not quite what the rattlesnake hypothesis is. In science, it would be more closely related to the word theory which translates to explanation. such as in Darwins's theory of natural selection as a foundation of evolution.
 By way of note, this is not a blog that adheres to the epistemology of science. This is a blog that hopes to contribute to science education. Education has it's own epistemology which is a branch of cognitive science. In the field of cognitive science, the Rattlesnake Hypothesis is more akin to what might be called a  graphic organizer. As is often the case, this description rather awkwardly straddles the epistemologies of science and education, which is often the case in the field of science education.
  The role of the RsH in cancer defense
  We have previously described the suppression of telomerase and the concept of the Hayflick Limit in a previous post. The epigenetic suppression of telomerase can be thought of as a laboratory observable definition of the difference between what might be classified as "germ line" and "stem" cells and what might be described as "somatic" cells, where somatic means "of the body". In broad terms, germ line cells  and stem cells must be "immortal", where as somatic cells are terminally differentiated to their physiologically relative function, and have exited the cell cycle. The fate of somatic cells is presumed to be apoptosis, or programmed cell death at the end of their functional life.
 So, in general terms, two things can go wrong, stem cells can loose control of their cell cycle, and somatic cells can loose suppression of telomerase. In this sense, telomerase is one end of the "Rattlesnake", that is, it is the fangs. If a cell does not have "stem" cell markers and is expressing telomerase, it is a threat to the whole biological system. There must be a marker for the system to throw up warning signs to activate the immune system to destroy the threat. (cancer)
  The role of progressive hypomethylation in cancer progression.
Once checkpoints in the regulation of the cell cycle have been defeated, the process of DNA synthesis becomes compromised. As such, the cell enters into mitosis before the machinery of the S ( synthesis ) phase has completed duplicating DNA and copying promoter methylation markers. Incomplete promoter methylation during duplication can now be thought of as an underlying explanation of the commonly observed phenomenon of "global hypomethylation" associated with cancer stage progression. As a result of global hypomethylation, epigenetically suppressed antigens called cancer testis antigens, (C/T antigens) become expressed. C/T antigens get their name because they are (were originally ) found in two places, cancer and testes. That poses the question "What do cancer and testis have in common?" The RsH proposes an answer to the C/T question, which is that both of these undifferentiated immortal cells pose a threat to the organism since they can be carcinogenic if not properly defended.
  As a note, it has long been noted that cells of the testes (sperm) initiate an autoimmune reaction if their protective encapsulation within the body is breached. The conspicuous location of the testes may be a result of their potential danger in the case of loss of cell cycle control. Also, it is worth while to note that testicular cancer is one of the more common forms of neoplasm.
   The Rattle of the RsH
We have described telomerase as the "fangs" of the rattlesnake, now we are going to go ahead and denote C/T antigens as the "rattle" of the rattlesnake. The purpose of C/T antigens has not been previously proposed, but here we are going to go ahead and say that C/T antigens form a second line of defense against cancer after the Hayflick limit. In other words, we are going to say that if a clone of mitotic cells have breached the Hayflick limit due to loss of suppression of telomerase, global hypomethylation will begin to occur. As it progresses, somaticly suppressed C/T antigens will loose their epigenetic suppression. Since the promoters for C/T antigens are responsive to ubiquitous transcription factors, cells will express these C/T antigens, and as such, such cells will be targeted by the immune system.
 Cells that are potentially cancerous must be, in some way immortal. In about 90% of fully progressed cancers, telomerase is expressed. Likewise the immune system has always been known to target cancer cells. Here we propose that there is an "dead mans switch" in each cell such that if cancer/testes antigens are not continually suppressed at each mitotic generation, an immunological attack will be mounted against the cell. In most cases, potential neoplasms are destroyed by the immune system. In some cases, cancerous cells develop ways to suppress immunological attack, or merely overwhelm the immune system by brute force. In any case, the rattlesnake hypothesis provides an organizing principle by which each form of cancer can be further described in terms of it's mitotic and immunologic state.
  That is each clone of cells can be described, and possibly quantified in terms of it's immortality potential  ( telomerase, fangs ) , and it's immunological potential ( Cancer testis antigens, rattle ). Together they describe both  ends of the cancer rattlesnake.

Tuesday, April 15, 2014

The epidemiology of epigenetic pathologies

The title of this article contains two words that appear similar, but are not closely related. The first of these two words is epidemiology. Epidemiology is the social and laboratory science of the study of disease occurrence.  For a pattern of disease to be classified as an "epidemic", there must by some pattern in its occurrence which statistically implies that there is a cause related to each case of occurrence. Epidemiology is an important input to the medical science of etiology, or the study of causation of disease or pathology. In practice, epidemiological studies provide one input to determine the etiology of a particular set of symptomatically related pathologies. Of most interest in the field of health is the epidemiology and etiology of pathologies related to bacterial infections, viruses, and environmental or workplace toxins.
   An example of an epidemic and etiology is the occurrence of hepatitis C infection. Hepatitis C is known to be caused by an RNA virus. For quite a few reasons, RNA is difficult to deal with in the laboratory compared to DNA and proteins which are more environmentally stable, and as such, are easier to isolate from tissue and blood samples. As such, if Hepatitis C Virus (HCV) is suspected as a causation of a pathology, the first step is to test for antibodies to proteins produced by the patient to viral proteins.  Immunological diagnostics are relatively inexpensive. If a preliminary study implies that HCV infection is probable, then RNA can be isolated from patient samples, and then reverse transcribed into DNA. The now double stranded DNA sample can then be expanded, or amplified with the use of a common laboratory procedure called polymerase chain reaction ( PCR ). PCR is a fairly standard and sensitive procedure, but requires a piece of specialized equipment called a thermo-cycler, which makes the test expensive relative to immunology based preliminary tests. By careful choice of DNA primers in PCR, specific genes of interest, or specific viral DNA can be amplified, and this in turn can be an input into a DNA sequencer which will determine the exact sequence, and the exact strain of the pathogen.
  This is an example of a traditional study, but not what we are here to talk about today. The other big word in the title is epigenetic.  The etiology and diagnosis of epigenetic pathologies is a relatively recent phenomenon which relates to the ability of laboratories to determine the promoter state of particular genes as well as to isolate particular micro RNAs.

An example in breast cancer
  As an example of the type of etiological input promoter state provide lets look directly at a recent article related to some of the most trendy terms in breast cancer pathology. In Stefansson et. al. [1], the first three words of the title, CpG island hypermethylation, refers to the previously mentioned promoter state. In this case the related gene is the well known cancer prediction gene breast cancer 1 (BRCA1). A promoter is a region on DNA upstream of a particular genes start of translation, which is the site of the assembly of the genes transcription engine, RNA polymerase. In the case of genes that can be "switched off", the promoter region of the DNA contains numerous copies of the sequence CG. Since these DNA bases are connected by a phosphate ( phosphodiester bond ), and because they occur in bunches, they are referred to in literature as CpG islands. As the result of an gene expression blocking process I will touch on briefly later, a methyl group can be added to these sequences in the DNA. Where as the prefix hyper means "over" or "too much",  the word hypermethylation in the title of [1], means that the these CpG sequences in the promoter have been methylated to the point where they attract the binding of methyl CpG binding protein 2 . ( MeCP2 ). When MeCP2 is bound to a promoter, expression of the gene is blocked. When placed in the context described in Stefansson[1], the expression of the tumor suppressor BRCA1 is blocked, and as a result, the tissue has a greater propensity, or probability of becoming tumorogenic.
  Moving through the title of Stefansson, the next term is "loss of pRB as co-occuring events". The gene pRB refers to protein Retinoblastoma, and was one of the first genes studied in relation to the long time "two hit" theory of cancer developed by Knudson. It has long been known that pRB is a critical component of cell cycle checkpoint regulation machinery, and as such when its expression has been suppressed, its role in checkpoint management is eliminated, more specifically, the transcription factor E2F is not bound and inhibited from entry into the next stage in the cell cycle. I've done a whole discussion of that topic here:
  When two or more loss of expression events occur simultaneously, they first thing we presume, or suspect, is that a micro RNA has been aberrantly expressed, and that that particular micro RNA has targeted numerous genes.  In the case of BRCA1, one such candidate micro RNA is miR-9,  which according to Sun [2] regulates BRCA1 epigenetically as follows:
Reverse miR library screening revealed that miR-9 reduced the normalized luciferase activity to 60.3% (95% confidence interval [CI] = 52.0% to 68.5%; P < .001). miR-9 bound directly to the 3'-UTR of BRCA1 and downregulated BRCA1 expression in ovarian cancer cells.
What this short passage says is that the investigators created an assay in which a light emitting protein, luciferase, was used as a reporter to detect activity of micro RNA of micro RNAs in regulating targets, and miR-9 was found to bind to the 3' untranslated region of the messenger RNA of BRCA1. As such, translational activity was blocked, and the presumptive initiation of a silencing complex ( RISC) was initiated for BRCA1. A silencing complex, ( RISC), is the active tool that leads to promoter hypomethylation.
  In terms of the concept of cancer etiology, the implications are huge here. BRCA1 is commonly considered as one of the primary actors, or "causes" in terms of etiology, but in fact, a perfectly normal BRCA1 may be lost from cellular activity is it has been silenced by a micro RNA (miR-9). In this case, loss of BRCA1 decreases the cells ability to repair DNA, and increases the cells sensitivity to chemotherapy ( cisplatin ).
  Far from being unique in the role of cancer causation, epigenetic changes to cancer suppressors seems to be the rule, not the exception. That includes the "bedrock" gene of cancer theory itself, retinoblastoma.  Where as pRB became the foundation of the "two hit", primary structure ( DNA sequence) theory of cancer initiation, it is now known that the overwhelming evidence is that pRB itself is the subject of epigenetic changes. The summary from the abstract of Mastrangelo[3] is as follows:
Through the analysis of the specialised literature and a set of original epidemiological and biological data concerning retinoblastoma, the authors illustrate the evidences arguing against the 'two hit' hypothesis and propose that epigenetic factors and aneuploidy play central roles in the disease.

  Most of the cases of autism are classified as "idiopathic" or of unknown causes. An exception to that rule is Rett syndrome which is classified as an autism spectrum disorder, and is known to arise from mutations to the gene MeCP2 [4]. As we have mentioned previously, methyl-CpG binding protein 2 ( MeCP2 ) plays an important role in the course of development as it is in charge of actuating promoter blockage once a promoter has been hyper-methylated. Presumable mutations is MeCP2 could corrupt the entire epigenetic aspect of development. As such, Rett syndrome is only found in girls since the MeCP2 gene is found on the X chromosome, and patients only survive in the heterozygous case. Since males have one X chromosome, if they carry the mutation, they are not viable. Although, as the reference shows, Rett is the subject of considerable study,  the only take away point that I want to make here is that autism type symptoms result from processes that interfere with the maintenance of epigenetic promoter markers on genes.
   The implication with respect to epidemiology of the previous point is that is the case of idiopathic autism, on logical place to start looking is processes  and factors that relate to maintenance of epigenetic markers ( methylation patterns ) on DNA. THere are two ways DNA can become methylated, de-novo methylation as a result if cellular processes, likely involving micro RNAs and RNA Induced Silencing Complexes ( RISC), and inherited methylation patterns which result from duplication of methylation patterns over cell division.
  In terms of inherited mechanisms, these are to be considered the most important where the pathology occurs in an area of  high cellular proliferation. Areas of cellular proliferation are breast tissue, testicular tissue, hippocampus ( short term memory ) portion of the brain, skin and intestinal lining.
  The loss of methylation pattern fidelity over mitosis can logically be attributed to one of two general ( pathological ) processes, where it is not the result of random error ( small, and correctable ).  The first potentially pathological process is the introduction of toxins that chemically interfere with the duplication process,  The second potentially pathological process is the incomplete duplication which may occur as a result of the loss of a checkpoint in the regulation of the cell cycle.
   These to mechanisms are not mutually exclusive, but rather potentially synergistic. For example, a toxin from the environment may interfere with the correct methylation pattern duplication of a gene responsible for regulation of the cell cycle. As a result, the cell may loose an important checkpoint. From that point, additional epigenetic alterations may "snowball" as a result of the loss of the checkpoint and incomplete methylation pattern duplication. This "snowball" effect is commonly observed in tumors, and commonly characterized in cancer research literature as "global hypomethylation". ( Note here the prefix "hypo" or under, as opposed to "hyper", or over used before) .
   Just quick note here, for one of the main points of this article, related to epidemiology, heavy metal toxins such as mercury, arsenic, and other environmental heavy metals are known to interfere with this exact epigenetic process, and are also associated with symptoms that parallel those, or are indistinguishable from autism.
Parkinsons Disease
   The epigenetic revolution in molecular biology has extended to Parkinson Disease.  It has recently been shown that not only can epigenetic changes be detected in tissue recovered from deceased patients, by those changes
  As a background lets review a short introductory passage from Masliah[5] to place it into the context of our discussion.
Epigenetic processes control several neurobiological and cognitive functions, from early brain development and neurogenesis7 to memory formation, learning and synaptic plasticity.8 Altered epigenetic mechanisms have also been associated with neurological disorders, including Rett syndrome, autism, schizophrenia and Alzheimer, Huntington and Parkinson diseases.9 We recently reported a decay on DNA methylation in the brain of PD subjects, associated with the interaction of α-syn with DNA methyltransferase 1 (DNMT1) that results in sequestration of DNMT1 in the cytoplasm.
This is a good time to bring up the topic of DNMT1, or DNA methyltransferase 1.  We discussed previously two methods by which DNA methylation patterns could be disrupted over cell division, ( the duplication process is mediated by DNMT1 ) Here, the job of methylation maintenance is interrupted because the mediator, is "sequestered by α-syn in the cytoplasm", and as a result cannot accomplish its purpose in the nucleus of the cell, where of course, the DNA is.

  It has recently been shown by Mastroeni et. al. [6] that progression in alzheimers disease is associated with the failure of the ability of affected neurons to maintain methylation patterns. The following is a short passage from the abstract[6]
We evaluated immunoreactivity for two markers of DNA methylation and eight methylation maintenance factors in entorhinal cortex layer II, a region exhibiting substantial Alzheimer's disease (AD) pathology in which expression changes have been reported for a wide variety of genes. We show, for the first time, neuronal immunoreactivity for all 10 of the epigenetic markers and factors, with highly significant decrements in AD cases.
The markers that show decrements are members of the maintenance complex that binds to  CpG islands in the promoters of affected genes. As mentioned, a complex binds to these regions that can orchestrate a large number of related gene expression changes. As described in the introduction in [6]:
MeCP1 is not bound directly to methylated DNA, but rather to a single methyl-CpG-binding domain protein, MBD2. The resulting MeCP1/MBD2 complex is composed of 10 known proteins that include the complete nucleosome remodeling and histone deacetylase (NuRD) core, as well as MBD2. This group of proteins, in conjunction with CDK2AP1 (Doc1), make up a complex capable of nucleosome remodeling and histone deacetylation (Feng and Zhang, 2001, Feng and Zhang, 2003).
This is a pretty important passage, at least as far as understanding the primal nature of methylation patterns in understanding epigenetics as it relates not only to Alzheimers, but to various diseases. The term "epigenetics" brings back many responses when put into a medical search engine such as PubMed. Many of them are related to "nucleosome remodeling and histone remodeling". This passage is describing, and giving a reference, to a system where the "MeCP1/MBD2 complex" is the dominant machinery controling other nucleosome remodeling and histone deacetylation. It is the methylation patterns that are copied carried over cell division, and subservient modifications, nucleosome remodeling and histone deacetylation, result from the reassembly of an associated complex on the duplicate DNA strand.

An example of epigenetic pathology in the Kidney
  As I have mentioned previously here,  polycystic kidney disease (ADPKD) has also been shown to arise from loss of expression of genes required to maintain cellular polarity in the collecting ducts of the kidneys of affected patients. Of note here, is this model, on a molecular basis, fits into our understanding of a wide array of pathologies that are similar on a molecular level, but only differ in the symptoms that result from these often repeated pathologies.


  Here we have discussed how common epigenetic mechanisms underly the etiology of cancer, autism,  polycystic kidney disease, Parkinson disease and Alzheimer's disease. The implications going forward is that any factor that impacts maintenance proper DNA methylation can lead to a broad spectrum of seemingly unrelated pathologies. These pathologies are unrelated only in the sense that a different medical specialist is required for each one. They are similar in that the medical specialist that one would seek attention from would likely have no knowledge of "epigenetic" factors as they relate to their organ. what is needed is a broad reeducation in the public health system concerning environmental and dietary toxins that impact DNA methylation maintenance.

[1] Stefansson OA, Jonasson JG, Olafsdottir K, Hilmarsdottir H, Olafsdottir G, Esteller M, Johannsson OT, Eyfjord JE. CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer. Epigenetics. 2011 May;6(5):638-49. [PubMed Central]

[2]Sun C1, Li N, Yang Z, Zhou B, He Y, Weng D, Fang Y, Wu P, Chen P, Yang X, Ma D, Zhou J, Chen G.  miR-9 regulation of BRCA1 and ovarian cancer sensitivity to cisplatin and PARP inhibition. J Natl Cancer Inst. 2013 Nov 20;105(22):1750-8. [PubMed]

[3] 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.[Abstract]

[4]LaSalle JM1, Yasui DH. Evolving role of MeCP2 in Rett syndrome and autism. Epigenomics. 2009 Oct;1(1):119-30. [PubMed Central]

[5] Masliah E, Dumaop W, Galasko D, Desplats P. Distinctive patterns of DNA methylation associated with Parkinson disease: identification of concordant epigenetic changes in brain and peripheral blood leukocytes.  Epigenetics. 2013 Oct 1;8(10):1030-8. [PubMed Central]

[6] Mastroeni D, Grover A, Delvaux E, Whiteside C, Coleman PD, Rogers J. Epigenetic changes in Alzheimer's disease: decrements in DNA methylation. Neurobiol Aging. 2010 Dec;31(12):2025-37. [PubMed Central]

Sunday, March 2, 2014

The cells first line of defence against loss of control of the cell cycle depends on epigenetic repression of telomerase, a "molecular fossil"

 As mentioned previously, the event most closely associated with cancer initiation is loss of control of the cell cycle. This very often is associated with loss of a "checkpoint" at one of the key cell cycle transitions. What natural defenses does the cell have against what may be a common occurrence? There are apparently multiple levels of defenses, but the first level seems to be a limitation on the number of times a cell can divide as a function of the length of the telomeres.
  The DNA of the cell is organized into units called chromosomes. Each chromosome is a single molecule.  On each end of the chromosome is a highly repetitive structure called the telomere. During mitosis, ( the M phase of the cell cycle), the chromosomal DNA is duplicated. The enzymes that mediate this process are called DNA polymerases. The function of  DNA polymerase is such that each time a duplication cycle takes place, a small amount of DNA on the telomere is lost.  Thus in somatic cells, or cells of the body, there is a limited number of replications that can take place. This number of generations is called the Hayflick limit after the scientist who determined that cell lines made from somatic cells could only divide a limited number of times.
   If there were not exceptions to the "Hayflick Limit" rule, then life would not be immortal, in the sense of perpetually maintaining a genome. The exceptions to the Hayflick Limit rule are cells we call "germ line" cells and "stem cells". These cells have an enyme called telomerase, whose function is to elongate telomeres that have been shortened by replication. The active subunit of the telomerase enzyme is called human telomere reverse transcriptase ( hTERT ). A reverse transcriptase is an enzyme that transcribes ( copies ) RNA to DNA, where the forward direction for a transcriptase (polymerase) is to copy DNA to RNA.
  Enzymes that are ribo-proteins, or consist of a portion of the enzyme that is a protein and a portion that is an RNA, are thought to be "molecular fossils" in the terms of White[1]. Typically, the RNA is the enzymatically active portion of the enzyme. We call these catalytically active nucleotides prosthetic groups, in that they help the function of the core protein. The molecular fossils hypothesis, or alternatively RNA World hypothesis holds that there was an earlier state in the evolution before the emergence of DNA and proteins, where RNA molecules performed both information storage and catalytic functions. A genome of these early progenitors is thought to consist of an RNA coding for an ensemble of early RNA enzymes. From these early progenitors, the catalytic machinery for proteins emerged, including ribosomal RNA. The subsequent appearance of proteins created a class of riboproteins in a manor described by White as follows[1].

The appearance of coded proteins would provide new opportunities for a metabolic system composed of nucleic acid enzymes. Proteins would bind to the nucleic acid enzymes and provide specific substrate binding sites not previously available. In time, proteins would completely displace nucleic acid enzymes in all but the catalytic core. Gene duplication and independent evolution would create families of homologous enzymes.[1]
  Thus, White, in 1976 described an archaic evolutionary system  which seems to accurately describe the emergence of telomerase while at the time, observing  different systems, nucleotides (co-enzymes) associated with the citric acid cycle and generation of ATP.
  One might understandably ask why we would take such a diversion into admittedly fringe topics when discussing regulation and deregulation of the cell cycle in cancer. The answer is that it always seems to be a good idea to keep the unending antics of RNA in mind. In particular, micro RNAs and their interactions with cellular components are now of primary interest in the study of cancer initiation. In taking the broad view, we hope to avoid overlooking significant clues.
   In other words, the reason to take a circumspect view is because there never seem to be any hard and fast laws in biology in the same sense as in physics or chemistry. Even the previously described mechanism for limiting the number of cell divisions seems to have exceptions. It seems to have been shown that even if the telomere lengthening catalytic activity is eliminated by introducing a mutation, ectopic (scientist mediated)  reintroduction if the disabled gene back into the cell is enough to transform some cell lines into a  cancer causing phenotype. First, we must recognize that there seems to be some other telomere maintenance system in addition to telomerase/hTERT. It is referred to as ALT by Stewart et. al. as follows: [2]
Telomerase activation is not the only mechanism by which cells can stabilize their telomeres. As many as 10% of human tumor-derived cell lines are telomerase-negative and rely on an alternative mechanism of telomere maintenance termed ALT [2]

Telomerase Shown to have function outside of telomere extension
   The Stewart group designed a disabled hTERT designated HA, and combined it with fluorescent marker protein GFP. When the cell cycle was stimulated with oncogene Ras,  the presumed  Hayflick limit was defeated  with the ectopic addition of the HA, or catalytically deficient hTERT. It was described as follows:[2]
 As expected, the hTERTHA-GFP expressing cells did not form tumors. In contrast, the hTERTHA-Ras expressing cells formed tumors at the same efficiency as the hTERT-Ras-expressing cells (Table 2 ), indicating that telomere elongation was not critical for tumor formation.
So Stewarts 2002 paper is something that many scientists would be tempted to overlook because it does not fit into some ideological "dogma" associated with the most commonly interpreted function of hTERT.  In fact, Stewart may have set the stage so to speak for later observations about the role of telomerase in tumor transformation.
  Just as the process of differentiation can be regarded as the transformation of cells from germ line cells to mesenchymal cells, or stem like cells, to fully differentiated cells ( epithelial cells in the case of organs ), cancer is the reverse of that process. Progression of cancer through its stages consists of an epithelial to mesenchymal ( EMT ) transition.

More about the epithelial mesenchyme transition
   The role of telomerase in the  EMT is well known [3], and has been observed in multiple types of cancer. Below is a quote from a paper related to gastric cancer as opposed to breast cancer. It is apparent that the role of telomerase in forcing a cell back to a "stem cell" like phenotype ( mesenchymal tissue like ) is a generally observed progression of neoplasms ( cancer ) .
    Because epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are key factors in cancer metastasis and relapse, and hTERT has been shown to exhibit multiple biological activities independently of its telomere-lengthening function, we address a potential role of hTERT in EMT and CSCs using gastric cancer (GC) as a model.[3]

   We additionally  learn that the mechanism related to hTERTS function in elongation and maintenance of telomeres, is related that JAK2/STAT3 pathway.[4]
  Here we demonstrate that STAT3 physically interacts with CD44 and NF-kB and activates the catalytic subunit of telomerase (hTERT) in human breast cancer stem cells. STAT3 plays a role as a signal transducing molecule between CD44 and NF-kB. In addition to functioning as a catalytic subunit of telomerase, hTERT has been reported to function as a transcription co-factor which drives EMT and cancer stem cell phenotype in human cancer.[4]
   As a matter of reference, and for better understanding of the histology, Singh and Settleman [6] provide a nice reference figure if the EMT transition.  The EMT is a natural occurring process in the case of wound repair, where de-differentiation and re-differentiation is a naturally regulated process, that seems to get "hi-jacked" in the case of cancerous cell transformation.

EMT cell surface markers
   Two of the most potentially lethal properties of the acquired mesenchymal phenotype ( often referred to grade ) are that that they tend to dis-associate as a function of loss of cell adhesion, and they tend to "home" for bone. It seems that these two lethal results  of the EMT transition can be attributed to markers stimulated by activation of hTERT.
  As mentioned in the above quite hTERT is a key factor in the transition to the more aggressive "mesenchymal" cell type, or phenotype. The markers for this more "stem like" grade of cancer are cd44+ and cd24-  where ( of course ) cd stands for cluster of determination, and usually means cell surface marker by which cells can be labelled immunologically or sorted in a cell sorter ( flow cytometry).

 EMT and loss of cell-cell adhesion 
   If this section  seems overly general, it is because all the details of loss of cell adhesion have not yet been decoded, and loss of cell adhesion in the EMT is still somewhat of a "paradox". [5] In general, normal (mammary) glandular tissue consists of differentiated epithelial cells that enclose lumenal caviities guareded by tight junctions between cells. In addition, the basal portion of these glandular cells recognize and bind to extra-cellular signalling molecules in the basement membrane. When an EMT is initiated by a cancer causing event, these cell surface proteins and associated cellular infrastructure disintegrate. The resulting sells loose their (histologically) visible structure, and become free to migrate through the blood stream to other parts of the body. Thus, the most dangerous phase of breast cancer has been enabled.

Homing to Bone
  The other major result of the EMT transition is that resulting disassociated "stem like" cells begin to "home for bone". This is one of the (again) most lethal aspects of breast cancer. Once cancer cells have metastasized to bone, they become impervious to immune defenses and surgery. The tendency to home to bone is now presumed to be due to the inappropriate expression of cd44 / HCELL. [7][8]. Sackstein provides the best description of the roll of the HCELL "glycoform" of cd44:

The molecular effectors that mediate cancer metastasis are best known for their role(s) in directing normal leukocyte trafficking, such as to lymphoid organs and to sites of inflammation and tissue injury. The braking adhesive interactions of flowing cells onto the vascular endothelium consist of shear-resistant tethering and rolling of circulating cells onto the endothelial surface. This primary step is mediated principally by the selectin class of adhesion molecules. The selectins comprise a family of three lectins that bind sialofucosylated glycans on their respective ligands. One of these selectin ligands, known as HCELL (Hematopoietic Cell E/L-selectin Ligand), is a specialized sialofucosylated glycoform of CD44 that is characteristically expressed on human hematopoietic stem cells.[8]
In essence, what Sackstein is saying is that HCELL is a normal marker and binding surface protein that has a normal role in the trafficking of white blood cells ( leukocytes). When it is mis-expressed on free circulating cancer cells, it causes them to leave the circulation system in a manor similar to leukocytes, and take residence in bone marrow. The result is new colonies of cancerous cells in bone marrow. They recognize bone marrow by "binding" or tethering to the suface of the blood vessel. The blood vessel signals that initiate rolling are carbohydrate decorations on proteins. Proteins that bind to carbohydrates are called "lectins".  The HCELL marker is a member of a special subset of lectins called selectins.

   Epigenetic repression of telomerase/hTERT in somatic cells provides a first line of defense against loss of control of the cell cycle. When telomerase is expressed, it aids in transforming the cell to a more "stem like" phenotype. It contributes in many ways, some unknown, to what is called the epithelial mesenchymal transition. As a result of this transition, cells change bio-markers to become cd24- cd44+ . This transition creates a new population of cells which loses its tendency to form regular cuboidal/columnar epithelium as a result of loss of cell to cell adhesion markers. The loss of cell adhesion leads to cell disassociation. Likewise, the cd44 marker when expressed, causes the disassociated "stem like" cells to home for bone. Those cells which defeat immune defenses and lodge in bone marrow stroma are likely to be protected from the defenses of the immune system by mechanisms that have not been completely described. It is these bone resident cells which begin new untreatable colonies ( metastasis ), and initiate the final stage of the cancer.


 [1] White, H.B. Coenzymes as fossils of an earlier metabolic state J. Mol. Bio. 7 101-104 (1976)

 [2]Sheila A. Stewart, William C. Hahn, Benjamin F. O'Connor, Elisa N. Banner, Ante S. Lundberg,Poonam Modha, Hana Mizuno, Mary W. Brooks, Mark Fleming, Drazen B. Zimonjic, Nicholas C. Popescu, and Robert A. Weinberg Telomerase contributes to tumorigenesis by a telomere length-independent mechanism  Proc Natl Acad Sci U S A. 2002 October 1; 99(20): [PubMed Central]

[3]Liu Z, Li Q, Li K, Chen L, Li W, Hou M, Liu T, Yang J, Lindvall C, Björkholm M, Jia J, Xu D. Telomerase reverse transcriptase promotes epithelial-mesenchymal transition and stem cell-like traits in cancer cells.Oncogene. 2013 Sep 5;32(36):4203-13. [PubMed]
[4] Noga Bloushtain-Qimron, et. al. The JAK2/STAT3 signaling pathway is required for growth of CD44+CD24– stem cell–like breast cancer cells in human tumors J Clin Invest. 2011 July 1; 121(7): 2723–2735.[PubMed Central]

[5] Mei Chung Moh and Shali Shen  The roles of cell adhesion molecules in tumor suppression and cell migration A new paradox Cell Adh Migr. 2009 Oct-Dec; 3(4): 334–336. [PubMed Central]

[6] Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010 Aug 26;29(34):4741-51. [PubMed Central]

[7] Chung SS1, Aroh C1, Vadgama JV2. Constitutive Activation of STAT3 Signaling Regulates hTERT and Promotes Stem Cell-Like Traits in Human Breast Cancer Cells. PLoS One. 2013 Dec 30;8(12) [PubMed Central]

[8] Sackstein R, Merzaban JS, Cain DW, Dagia NM, Spencer JA,
Lin CP, Wohlgemuth R (February 2008). Ex vivo glycan engineering of CD44 programs
human multipotent mesenchymal stromal cell trafficking to bone.
Nat. Med. 14 (2): 181–7. [PubMed]

[9] Jacobs PP, Sackstein R.  CD44 and HCELL: preventing hematogenous metastasis at step 1. FEBS Lett. 2011 Oct 20;585(20):3148-58 [PubMed]

Thursday, January 30, 2014

The danger of misrepresentation of scientific studies is demonstrated by "The Scientist" article on vitamin deficiency

 The translation and interpretation of scientific research for educational use is fundamental to our education system, and in fact researchers often seek to have their research disseminated beyond the scientific community. None the less, care must be taken by those who attempt to translate research  to insure that no unintentional misrepresentation is made. My feeling is that a severe, yet unintentional misrepresentation has been made in a recent article by The Scientist.
  In the field of science education, scientific terminology is a required foundation. Likewise, in medicine and related health science field, medical terminology must be mastered and used correctly. Most often, when used correctly, there is no misunderstanding when using correct scientific terminology. In the case of this article, there seems to be a major problem involving terminology.
   The immune system is divided into the innate division and the adaptive division. When we study the acquired immunity that we receive from environmental exposure, as well a immunization from vaccines, we are speaking of the adaptive immune system.
  The title chosen by The Scientist was as follows:

Vitamin Deficit Can Boost Innate Immunity

By enlarging the text of the title, versus the subtitle and the article text, it seems to imply to readers without specific immunology backgrounds, that vitamin deficits may be beneficial. In the context of the title, the word innate does not seem to imply one specific subset of the immune system, which in this case is probably trying to compensate for a deficiency in another, likely more important part of the immune system. None the less, the article text quickly notes that no major reversal of nutritional theory is necessary.

 Vitamin A deficiency is associated with several health problems including night blindness and increased asthma risk. And as with other nutritional deficiencies, it is also known to compromise adaptive immunity mediated by the specialized T cells of the immune system. So it came as a surprise when researchers found that vitamin A deficiency could also activate the immune system and help protect mice against worm infections.[1] ( bold face mine )
Although the text of the article is largely correct, the word innate in the title seems intended to misrepresent the overall findings of the study. The "social context" of the word innate is that of unsupplemented by antibiotics and medication.
  The medical interpretation of this particular type of study is also a matter of debate. While mice are valid model systems for many types of studies, particularly cell cycle or cancer studies, the same is not necessarily true in the field of immunology.  Although the most basic constructs of the mouse immune system are similar, there are enough differences between the species such that immunological comparisons between murine ( mouse ) studies and humans are not immediately definitive.
  The defense of an organism against foreign threats is an active process that requires cell division ( mitosis ) and extensive translation ( generation of new proteins ). These processes are dependent not only on vitamin A, but all vitamins.
   In addition to the complex job of manufacturing and selecting new antibodies to arm the bodies defenses against foreign threats, the immune system must be constantly on guard for rogue "self" cells. When a cell looses control of its cell cycle, it begins to signal to the immune system that it needs help to extinguish a potential neoplasm. (cancer) .
   The process of cell division is a complicated process in which not only all of the DNA needs to be correctly copied, all of the epigenetic markers such as promoter methylation patterns ( CpG islands ) need to be correctly.  If a gene such as an oncogene ( gene which causes cancer ) needs to be suppressed in a particular tissue, it is imperative that the gene not only be copied correctly, but that it is suppressed by CpG island methylation of its promoter.  Accordingly, epigenetics is the direction that cancer research has now turned, and its importance to cancer causation research cannot be understated.
  Non the less, even under the best conditions, it seems to be a relatively common for the process of creating a high fidelity duplication to fail, and a mutant cell to arise. In this case, it often becomes the job of the immune system to assist in extinguishing and disposing of the offending cell.

  In conclusion, in science words matter. Wherever possible it is best to try to use the most correct scientific terminology, particularly if a particular term ( such as innate ) may have a different meaning in common usage.

[1]  Laasya Samhita    Vitamin Deficit Can Boost Innate Immunity
Researchers show that vitamin A deficiency can help protect mice against parasitic worm infections.
    The Scientist Jan. 23, 2014  [ Article ]

Thursday, January 23, 2014

Micro RNAs that regulate cell cycle check point components are implicated in loss of control of the cell cycle in glioblastoma

 When a cell is fully differentiated into a functional somatic cell of a particular tissue, it is thought to have permanently exited the cell cycle. The presumed normal fate of a cell that has differentiated and exited the cell, is to serve its physiologically relative functional life, and then die through a process of programmed cell death called apoptosis. When a cell has been transformed to a cancerous cell, it re-enters the cell cycle and begins to divide out of control.
   So, precisely where is the mechanism that fails and sends the cell back into the cell cycle?  This is a foundation of cancer research. There are multiple pathways by which cells exit and enter the cell cycle. These pathways most often must work in conjunction with molecular regulatory mechanisms called checkpoints .  When we use the term checkpoint, we most correctly are referring to these molecular mechanisms that work at the G1 to S transition and G2 to M transition, where (of course ) the four phases of the cell cycle are G1, S, G4 and Mitosis (M) .

Checkpoint implementation
  As a bit more review of cell cycle regulation, we must say that as the cell progresses through each phase of the cell cycle it produces proteins called cyclins, which are a bit like sands in an hourglass. When enough cyclins of a specific type have been produced, the cell reaches a threshold, or critical point where it is capable of moving thorough a checkpoint to the next phase.
   When this happens, it is because enough cyclins have been produced to activate a member of a family of proteins called cyclin dependent kinases. (CDK)   A kinase is an enzyme that phosphorylates ( adds a phosphate group ) to a substrate. In the case of cell cycle checkpoint implementation, the phosphorylation substrate of cyclin dependent kinase is retinoblastoma, or pRB when referring to the protein associated with the retinoblastoma ( rb ) gene.
  Retinoblastoma itself gets its name from a type of cancer with which it was originally associated. A retinoblast is a particular type of developmental cell in the retina of the eye, and cancer arising from these retinoblasts had been designated retinoblastoma before the discovery of molecular mechanisms underlying cancer. If fact retinoblastoma was predicted from genetics of afflicted patient families before the gene was discovered.  Retinoblastoma ( pRB) can be a cell cycle regulation component in all cells, not just retina cells.
   When a checkpoint is in the "blocked" state, pRB is bound to a protein called E2F. After pRB has been phosphorylated by a cyclin dependent kinase, it becomes unable to bind to E2F, and thus E2F is free to provide it's own signalling function.
  E2F itself is a transcription factor. When it is in its activated, it translocates ( moves ) to the nucleus of the cell and binds to the promoter region of appropriate genes on the cells DNA. In the case where the promoter has not been methylated,  and thus blocked, this is the initial step of gene expression for the genes that represent the next step in the cell cycle.
   In practice, specific ( numbered or lettered ) Cyclins, CDKs and E2Fs are associated with each checkpoint.  Below is a table:

Checkpoint Cyclin Cyclin Dependent Kinase
G1-S Cyclin A CDK4/6
G2-M Cyclin B CDK 1

Epigenetic factors controlling checkpoints

   More recently, epigenetic regulation of checkpoint genes has come under scrutiny of researchers. When we speak of epigenetics, we are referring to  micro RNAs that bind to and regulate specific messenger RNAs, promoter methylation, and reversible changes to the histones that package genes in the nucleus.
  Promoter methylation plays a large role in determining whether an activated gene ( transcription factor available ) actually becomes an expressed gene ( promoter unblocked by methylation and binding of MeCP2 ). Methylation patterns themselves are caused by an RNA/RNA interaction between the expressed gene and an associated micro RNA ( mi-RNA ). As such, most recent research has begun to focus on genes and their micro RNA regulators.
    Now we will transfer our general theory to actually observed cancer research. Much or most of the previously mentioned principles of cyclin/checkpoint regulation was actually done by Nobel Laureate Paul Nurse in fission yeast. The following quite is from the abstract of a recent paper [1] on the aggressive brain brain cancer glioblastoma multiforme (GBM).
Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138.  
After observing that gene expression of micro RNA 138 had been lost in lines of glioblastoma cells, expression was artificially induced (ectopic expression ) using a cloned version of the gene and a vector (  replication deficient viral transporter ). As a result of reinserting active miRNA-138 into cell lines from glioblastoma multoforme, the G1/S checkpoint was restored, and the cells exited their abnormal growth program.

In addition to the checkpoint proteins we previously discussed, We see an unfamiliar one called EZH2. EZH2 itself has been found to be implicated in many cancers, but its precise role was inknown. Its normal function relates to epigenetics, in the role of histone modifier as described in the following from Qui[1]:
 EZH2 possesses the histone methyltransferase activity and can lead to gene silence through methylating histones [15] and [16]. EZH2 is over-abundant in a broad range of human malignancies including GBM, and contributes to tumor proliferation and cell cycle control [12], [16] and [17]. The Cyclin-dependent kinase 4 and 6 (CDK4/6) are critical regulators of cell cycle G1/S transition and are specialized to phosphorylate and inactivate the cell cycle controller Retinoblastoma protein (Rb) [18].
 The new hypothesis is that EZH2 itself is a target of E2F, and as such a feedback loop can develop. What they go on to show is that model cell lines can be forced out of the cell cycle by introducing a micro rna which down-regulates members of the checkpoint pathway.
 Additionally, it is exhibited that the transcription factor E2F1 is able to drive EZH2 transcription [21]. Therefore, we come to the hypothesis that an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1 is involved in gliomagenesis, and this signal loop could be suppressed by the candidate miRNA.

   The addition of another, epigenetic level of understanding to neo-genic ( cancer causing ) pathways multiples the number of candidate therapies. As a result of new information, scientists can work on drug candidates that are more specific for a certain type of cancer. In this case a microRNA that is shown to inhibit EZH2 activity/expression is shown to rescue cancer cells, and force them out of the cell cycle by breaking a feedback loop associated with a checkpoint at the G1-S checkpoint.
   As a more general observation about the introduction of epigenetic models and hypothesis generation into cancer research, computational expertise, relating to RNA/RNA interaction is playing a greater role in research planning and interpretation. We are moving toward an era in which discovers will come from unique computational methods only.

[1 ] Qiu S, Huang D, Yin D, Li F, Li X, Kung HF, Peng Y.
Suppression of tumorigenicity by microRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme.
Biochim Biophys Acta. 2013 Oct;1832(10):1697-707. [Full Text]

[2] Chase A, Cross NC. Aberrations of EZH2 in cancer.Clin Cancer Res. 2011 May 1;17(9):2613-8. [Abstract]

[3] DeGregori J, Johnson DG. Distinct and Overlapping Roles for E2F Family Members in Transcription, Proliferation and Apoptosis. Curr Mol Med. 2006 Nov;6(7):739-48.  [PubMed]