We all know that our immune system protects us from bacteria and viruses.  But it also protects us from cancer.  The immune system is designed to recognize molecules (proteins, DNA, RNA etc.) that are not from us as individuals.  Not self.  Cancer cells are genetically unstable and while in general cells with genetic instability die, cancer cells have acquired the ability to survive this instability. With genes rearranging and mutating new proteins are made by cancer cells and protective proteins are lost.  New proteins are recognized by our immune system as not self and proteins that are lost open the cancer cell to attack by our immune cells.  This was the basis for a novel theory published over 50 years ago called “Immune Surveillance”.  According to this theory cancer cells would be recognized by the immune system and attacked just like other pathogenic invaders. The argument further posited that if not for our own immunity cancer would afflict all of us. 

NK cell attached to a target cell.  Activation will release cell killing chemicals. Image from Online Biology Notes, source: Biocenter.com

In the decades of research since this theory scientists have argued passionately about its validity. For quite a while most cancer biologists felt that the immune system could not perform this function.  Then they realized that indeed it might, in so far as a key part of our system comprising what are called T-cells (as in derived from the thymus) that are responsible for cell-based immunity could attack tumor cells and kill them.  However, a consequence of immune surveillance should be that experimental animals lacking an immune system should have more cancers than those with an intact immune system.  When this was tested in special mice called nudemice (because they lack hair) that lacked a robust immune system there was no difference in cancer incidence between these two types of animals.  Later it was learned that nude mice are not completely immunodeficient and have an important part of the immune system functioning.  The so-called NK (for natural killer cells) system functions in these mice. NK cells are a special type of lymphcyte that do not require prior exposure to a cell or protein in order to react to it but that does not kill ones own cells (self). When the activity of these cells was removed from mice tumor incidence increased. Moreover,  pathologists and tumor biologists noted that many cancers had immune lymphocytes through their tumor mass.  These were named tumor infiltrating lymphocytes or TIL.  Importantly it was discovered that patients whose cancers displayed TIL did better than those that did not.   Immunodeficient humans and patients with HIV AIDS (with a compromised immune system) have many more cancers than people with intact immune systems.

NK cells are one type of lymphocyte (white blood cells) that can attach to and destroy cancer cells and cells infected with viruses. Our own cells have surface MHC (major histocompatibility complex) molecules that bind to NK cell receptors and inhibit them.  Target cells activate the NK cell to release chemicals that kill the target cell. http://www.stream.wum.edu.pl/en/knowledge-base/96-nk-cells-applications-in-immuno-oncology

So, if our T-cells infiltrate tumors and kill the cancer cells why do we get cancer?  It turns out that cancer cells can defeat the cytotoxic T-cells by binding a receptor on the T-cell surface that induces the T-cell to die.  It’s called PD-1 for programmed death-1 and when that receptor is activated it induces a series of changes in the T-cell that leads the cell’s death, a process called apoptosis.  Tumor cells can produce a surface protein, PD-L1, known as a ligand, that binds to the PD-1 receptor on T-cells and thereby thwart the T-cell based tumor cell killing. Current immunotherapy for cancer is largely aimed at preventing this activation by interrupting the cancer cell’s ability to bind PD-1.  This done in a number of ways.  By treating the cancer patient with antibodies against the PD-1 T-cell receptor the tumor produced ligand PD-L1 can no longer bind to it and the T-cell remains active and able to participate in tumor cell killing. Similarly, antibodies against PD-L1 (on the tumor cell) can be administered and that likewise inhibits the binding to PD-1 (on the T-cell) and so allows T-cell mediated tumor cell killing. Small molecule drugs have been  developed to interfere with PD-1/PD-L1 interaction as well.  The antibody treatments are in widespread clinical trials at the moment with very encouraging preliminary results for some types of cancers.  

Cytotoxic T-cells can bind surface molecules on a “foreign cell” such as a cancer cell.  This should trigger a cancer cell killing process but if the cancer cell has a PD-L1 surface molecule that binds to the PD-1 receptor the cancer cell triggers the death of the T-cell instead.  This reaction can be blocked by giving the cancer patient antibodies preventing the PD-1 interaction with PD-L1.  Similarly small molecule drugs can prevent the interaction that otherwise would kill the T-cell leaving the cancer cell alive and able to proliferate. Image prepared with BioRender.

Additionally, it is possible to attack the problem by reducing the PD-1 receptors on the T-cells.  This has been achieved by using small molecule inhibitors of an enzyme (GSK) that is involved in a network of proteins that regulate the expression of PD-1.  Inhibiting GSK with these chemicals reduces the expression of PD-1 and clears cancer cells in animals.  In fact, it is as effective as the anti-PD-1 antibodies. This is a very promising approach to harnessing the body’s immune response to cure cancer.

Another approach that is much in the news is CAR-T (chimeric antigen receptor on T-cells) cell therapy.  This consists of removing bone marrow stem cells from the patient and then genetically modifying them so that their surface has a receptor protein that both reacts with the tumor cell and activates the T-cell to participate in killing the tumor cells. 

CAR-T (Chimeric Antigen Receptor T-cell) therapy for cancer modifies the patients T-cells to express a surface molecule that reacts with the cancer cell activating the T-cell to produce chemicals that kill the cancer cell. The modified T-cells are re-infused in the patient where they circulate and attack the cancer cells.   Image prepared with BioRender.

Chemicals called cytokines are released which amplify the effect. It is a complicated therapy involving manipulating isolated bone marrow cells in the lab, genetic modification and extreme immune suppression of the patient including removal of most of the patients unmodified lymphocytes and T-cells so the genetically modified T-cells have a growth advantage when returned to the patient.  Numerous clinical trials are underway to establish if this approach will work in extending the cancer free life of patients. Currently results show persistent responses in some cancers offering hope that this approach will add important weapons in the fight against cancer. Obtaining the NK cells, T-cells and other lymphocytes to use in this fight is a difficult limiting step in these therapies.  Recently, though, it has become possible to obtain essentially unlimited numbers of these cells from the patients themselves by reprogramming some of their adult cells into stem cells and then differentiating them to the necessary lymphocytes.  ,

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Spark of life! Image from Mel Brookes Young Frankenstein 1974

The engineers say that you do not truly understand a system until you can build it from scratch. Previously we have explored the cell, the building blocks of our tissues that in turn make up our organs.  They are a sort of basic unit of human structure and function.  Clearly it is an amazingly complex system with many interlocking parts.  Also, many mysteries.  Thus, we might conclude that there is no way to make one synthetically from simple chemicals.  Certainly, that was the prevailing attitude.  Schrödinger a brilliant physicist famously said that for the most part biologic life was far too obscure to generate first principals.  Introducing the subject matter of his book What is Life he argued that he would avoid mathematical deduction “not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

But daunting complexity has not deterred engineers in the past and that can-do spirit continues undiminished.  Many years ago in order to understand the origins of life, attempts were made to recreate the primordial conditions that led to the generation of life. Reasoning that primitive life was a culmination of favored chemistry scientists tried to create a closed system that contained the bare minimum necessary to produce the chemicals that formed the basis of life.

Known as the Miller-Urey experiment special vessels were employed to combine methane, ammonia and hydrogen in a chamber with water vapor. The water vapor came from heating water in a separate chamber of the sealed vessel. Continuous electrical sparks were discharged through water vapor mixed with the chemicals.  At the end of the reaction time the mixture was cooled and contents collected and analyzed.  The idea was to simulate the early earth’s atmosphere, in part a product of volcanic activity and lightening.  The result was striking and resonates even today as the experiment has been recently reanalyzed and has been repeated many times in many different ways.  The reaction produced the building blocks of life…amino acids that when combined produce proteins and peptide chains.  In fact, although they didn’t know it at the time, reanalysis has shown that many more amino acids were produced than originally thought, including some that do not appear naturally in extant life.

Synthetic pieces can build a cell Image from Pixabay

Proteins alone though cannot make a cell, they do not reproduce robustly and do not transmit genetic information.  That is the domain of RNA and DNA.  The conundrum though is that producing RNA and DNA as we know it now requires biosynthesis in living cells.  So how did they come about before there was life.  Just like for the amino acids that make up our proteins the basic chemical bits that are needed to synthesize RNA and DNA could plausibly have been generated by conditions and materials on early Earth before life began.  

Recently scientists have taken on the challenge of creating artificial genetic information and creating cells where all of the genetic information is wholly synthesized in the lab and then added to an existing cell with its genetic material removed to create a new cell, in this case a kind of bacteria. The methods to produce the synthetic DNA genome in these bacteria utilized the same chemical bits that were likely generated on early Earth.  That is, chemically defined synthesis without materials from living cells.  In fact, the entire genetic code of the bacterium was digitized and stored in a computer! Modifications to the code were made to show that the synthesized DNA that was put into cells were the man-made molecules. Initially scientists replaced the DNA and thus the genes of the bacterium Mycoplasma with DNA synthesized in the lab, containing 1 million base pairs, the basic letters of the genetic code. Recently they have synthesized a much larger DNA complement of 4 million base pairs and used it to rebuild the bacterium E. coli having edited out redundancy in the genetic code.  This to try and understand what are the minimum requirements for life!

Bacteria with wholly synthetic DNA, genes built in the lab Image by Derinck and Ellisman, UCSD, Sci 329, 35, 2010, Gibson et al.

So, since the synthesized DNA was put into existing cells it could be argued that the other components of the cells, proteins, lipids etc. were not man-made.  However, it could be shown that after several dozens of divisions there was no natural cellular material left, it having been diluted at each cell division. The startling fact is that the engineered bacteria could divide, metabolize and remain viable solely on the basis of the synthesized genetic code. The authors of the work said, ”the DNA software builds its own hardware”!

In fact, in a startling advance on the approach other scientists have created artificial genetic codes that utilize 2 base pairs (the letters of the code) that are not seen in nature!  These alien base pairs were taken up into the DNA and faithfully copied when the cells divided.  This leads directly to the possibility of using new, synthetic genetic information to make novel proteins, structures or therapeutics.  It is an amazing affirmation of the notion that the genetic codes found in our natural world are not the only possibilities for renewable and heritable traits. There are great implications both in practical and ethical terms for this, including how extraterrestrial life might form.

Gene expression (blue) in artificial cells Image from Dekker Lab, Delft, part of the BaSyC consortium

Of course, a cell needs to be packaged in a stable format that allows for existence in an environment, for the movements of nutrients and signals into and out of the cells and yet remains self-renewable and scalable upon division of the cells.  Here as well the engineers of cellular life have made progress with synthetic materials.  Scientists working in a new field of engineering called microfluidics have taken major steps toward accomplishing this. Tiny jets of chemicals can be controlled to produce hollow droplets like miniscule bubbles that are the size of cells. 

Tiny droplets flow in microfluidics to produce synthetic cells, then components can be added (bottom) the bar = 0.0001 meters Image from Nature Communications 2018, https://phys.org/news/2018-06-path-artificial-cell.html

Advanced engineering has provided the means to add proteins to these synthetic cell-like structures that perform specific cellular functions, importantly including generation of energy. Not surprisingly these scientists have teamed up with the groups synthesizing genetic material, who as discussed have determined that some few hundred genes are all that are required to sustain synthetic life. 

Artificial cells made from plastic can send and receive signals. Purple cells signal grey cells and change gene expression to make green cells.  Bar = 0.000025 meters Image by Henrike Niederholtmeyer from M. Leslie doi:10.1126/science.aaw1173

A similar approach used a plastic to form an outer envelope (membrane) and these structures also had “nuclei”.  Genetic material containing instructions for a green glowing protein was added to the “nucleus” and it functioned.  The scientists also demonstrated that the completely artificial “cells” could signal each other turning on the production of this green protein thus mimicking an important characteristic of natural cells.

While these advances hardly herald the zombie apocalypse the work raises fascinating and important ethical questions that not surprisingly are more or less left on the table with no particular approach to resolution as the engineering proceeds like a train rushing through the night!

Building organs for repair or replacement. Image by Barbara Gilhooly

Regenerative Medicine.  What does that phrase mean?  We know that regeneration seems like a good thing…. invoking regenerative power, or regenerating tissue, that is, a renewal of something lost.  In high school biology we learned that salamanders (including newts and axolotls)  could lose a tail or a leg and it will grow back.  We are all pretty certain we don’t have the same ability. 

Reproduction of the Gaudi salamander Parc Guell de Barcelona

Even while scientists are trying to find why we can’t regrow lost limbs in the hope that we can learn how to actually make it happen, the phrase regenerative medicine has taken on a larger meaning.  In fact humans do have the ability to regenerate lost tissue but that ability is very limited. In disease our organs lose function often because they lose the cells that comprise them.  But organs also have special cells, cells of renewal, known as stem cells.   When disease kills cells in some organs the body tries to compensate by inducing these resident stem cells to repopulate the organ and reestablish function.  It is a very inefficient process for us humans.  Regenerative medicine then is the attempt to understand how to make the process of renewal of organ function more efficient.  

 Newt!  Image by Holger Krisp, Ulm, Germany

Stages of salamander limb regeneration.  At the 3 o’clock position is a picture of a normal limb before amputation.  Proceeding clockwise are pictures of the limb growing back, taking place over 30 days involving wound healing and dedifferentiation (return to embryonic state), then redevelopment of a normal limb.  Many genes are involved many of which are known and understood (genes identified by new gene expression technologies shown in the center). Image from Biology Open, Oct. 15, 2012, Monaghan et al. (doi: 10.1242/bio.20121594)

We know how to take any adult cell and by manipulating a few genes turn back the genetic clock of the cell so that it thinks its fate is not yet determined, that it can become (differentiate into) any type of cell and participate in the function of any organ. In other words become a stem cell. The use of these cells in bioengineering new tissue is a bold new science that aims for nothing less than the replacement of diseased organs with a patient’s own cells.

We have learned a great deal about how to do this in the more than 15 years since Japanese scientists first described how to turn an adult cell into a stem cell.  Biologists have many ways to study this process and its ramifications but one method particularly useful to regenerative medicine is with structures grown in a dish called organoids.  This is important as the cells used to create stem cells are initially obtained from adults, no embryos involved.  The cells can be manipulated in the lab without creating moral dilemmas and without having to conduct the research on human patients.  By manipulating the stem cells while they are growing they can create structures remarkably like the organs we would like to induce to repair. Organoids are small, three dimensional structures that recapitulate the structure and function of organs.  They are simple, relatively inexpensive test beds for development, physiology and drug testing that allow the development of approaches in advance of animal modeling and allow investigators to cycle through many more iterations of approaches than would be practical with intact animals or with humans.

A striking example of this is the eye. By placing stem cells in a dish and providing them with environmental and genetic cues the cells develop a structure that recapitulates the embryonic eye cup and begin to turn into retinal cells and self assemble in the dish into morphologically recognizable retina. The structures develop photoreceptors that can respond to light with electrical signals showing they are electrophysiologically functional.  Currently human trials are starting to determine if they can cure or ameliorate serious retinal diseases.

Eye structure developed from stem cells in a dish!  Closely resembles a normal embryonic eye.  © 2011 Yoshiki Sasai

When eyes grow in a dish from stem cells the retina organizes (top picture) with similar anatomy and gene expression as normal retinal development (bottom picture, red is Pax6).  Images from Manuela Völkner, et al., Stem Cell Reports. 2016 Apr 12; 6(4): 525–538; 10.1016/j.stemcr.2016.03.001

Similar remarkable results have been obtained for the intestine, prostate and the kidney.  But most remarkable of all is the ability to create little mini-brains in a dish that reveal many normal morphological and patterning similarities to developing brain.  These organoids allow formal investigations of physiology, function, toxicity, and even to test therapeutic approaches to diseases.

Dish with cerebral organoids comprising neural cells differentiated from stem cells, i.e. mini-brains!  Insert: microscopic view of histological section through a cerebral organoid showing gene expression, cortical patterning, and ventricle-like vesicles all reminiscent of normal embryonic brain development. © 2017 Magdalena Renner, Madeline A Lancaster, Shan Bian,Heejin Choi,Taeyun Ku,Angela Peer, 1 Kwanghun Chung,  and  Juergen A Knoblich; EMBO J. 2017 May 15; 36(10): 1316–1329.

As if this weren’t enough very recently scientists have managed to combine two types of stem cells (derived from the two types of cells in very early embryos) in a dish with a matrix that supports 3-D growth and to develop a normal looking and behaving mouse embryo!  This embryo, at what is known as the blastocyst stage, expresses normal genes in the normal place and at the correct time when compared with natural mouse embryos.  This is stunning confirmation of the remarkable ability of stem cells to self organize and direct normal development. This was most recently followed by demonstration that by adding another type of stem cell into the mix synthetic embryos could go through gastrulation, a critical early step of embryonic development, showing the astounding ability of these cells to communicate with each other and self organize into a developing embryo.

Synthetic mouse embryo with embryonic (pink) and extraembryonic (blue) cells. These structures are remarkably like the normal mouse embryo at the time of implantation.  They proceed to mature with normal patterning steps and gene expression in time and place. Zernicka-Goetz lab, University of Cambridge

The ability to create models of the brain and intact embryos in a dish from individual cells and potentially from humans, as in the case of mini-brains should provoke profound ethical concerns.  The dilemmas presented by these capabilities are obvious but have attracted relatively little attention.  Given what is at stake this is remarkable for it would be far better to thoroughly consider ramifications of this technology now.

On the other hand, as scientists learn more about these amazing abilities the cells and the conditions under which they are grown will provide an incredible future for the field of regenerative medicine. We will have in our grasp the tools needed to repair damaged organs with the patient’s own cells!

image by MorgueFile

What do we mean by saying we want to cure a disease?  What does it mean to cure a disease?  Alleviate pain, restore function, improve the quality of life of the patient? All of these things? Someone close to me once said to cure a disease means that you go on to die of something else.  So what does it mean to seek a molecular cure or to use genetics to cure a disease?

Is it even possible? 

We know the answer to that question is definitely yes.  We have discussed some examples before; Gleevec stops some leukemias in many patients and Tarceva takes the debilitation of one type of lung cancer away.  But in many patients the cancer returns and treatment ultimately fails.  Before that happens though patients lead what can be considered a pretty normal life. These drugs were developed based on very fundamental scientific discoveries about the way those cancers arise.

Indeed if proteins are responsible for how our cells function and what makes our organs work then a failure of proteins to form normally or an alteration that results in loss of normal function is the root cause of disease.  Understanding the specific failure and how it comes about in a given disease provides the keys to its cure.

We are entering a new phase of molecular understanding of diseases of nerves and muscle cells.  Previously I described muscular dystrophy and how genetics gave us a way to think about treatment.  We are now on the brink of curing one of the most heartbreaking of muscle disorders, spinal muscular atrophy or SMA.

SMA is a terrible disease of progressive weakness due to the death of muscle cells. As the cells die the body tries to regenerate them.  At its worst babies with this disease cannot sit, their muscles progressively weaken and they die within a year or two unable to breathe.  SMA is the leading genetic cause of death in infants and 1 in 50 Americans have a mutation that makes them genetic carriers of the disease.   The muscle cells die because of a failure of the nerves connected to them.  Those nerves fail and die because they lack an important protein critical to their survival.  Indeed the protein is called survival motor neuron (SMN).  This protein has 2 forms coded by 2 different genes, SMN1 and SMN2. The difference between these two genes is a single base pair alteration that arose in humans (it doesn’t exist in monkeys or chimps).

Neuromuscular junctions where nerves (dark purple) connect to muscle cells (pink).   (image by Thomas Caceci https://doctorc.net/Labs/Lab10/lab10.htm)

Remember that our genes are DNA, comprising 3 billion individual letters (or base pairs).  The DNA acts as a template for RNA, which leaves the cell’s nucleus to direct production of proteins. The result of the single base pair change is a profound alteration in the way RNA is processed and spliced (a necessary step for RNA to direct protein formation). The result is that SMN2 lacks an important part of the protein that renders it nearly non-functional.  A very small amount of functional protein can be made from the SMN2 gene.  If the SMN1 gene is mutated the absence of normal SMN protein causes this terrible disease. If patients have many copies of the SMN2 gene their disease is less severe in proportion to the amount of functional SMN made. At the Stanley Manne Children’s Research Institute Christine DiDonato and YongChao Ma have been pioneers in helping to understand and battle this tragic disease.

Horton Web Design

Our genes are DNA, which is used as a blueprint to make RNA and the RNA, is a template to produce protein. The DNA sequences of a gene include parts that are the blueprint for proteins and parts that help to regulate gene function but do not contribute to the protein.  When RNA is produced from the gene (DNA) it must be processed so that only the parts needed to make the protein are in the final template. This is done by removing the other regulatory parts and then stitching together the bits that instruct the cell how to make the protein.  This process is called RNA splicing.

By Ministry of Information Photo Division Photographer [Public domain]

“Here”...the parts of the gene that make the protein called exons. “Absent”...regulatory parts that don’t leave the nucleus called introns.  The exons are stiched together or spliced to make a mature messenger RNA. From biocomicals.blogspot

An elaborate machinery resides within each of our cells to take the RNA produced from the DNA of our genes and splice together the protein coding parts.  There are many parts to this machine and by studying patients with SMA scientists have learned much about how the machine works.  They have learned how to make very small RNAs called antisense oligonucleotides that can block the effect of inhibitors of the machine and promote different processing of the SMN2 RNA such that it makes a functional protein.  This has worked spectacularly well and by allowing splicing to proceed in the SMN2 gene more and more SMN can be made by cells particularly the nerve cells that are in the spinal cord.  These antisense oligonucleotides are injected into the spinal canal by spinal tap. More SMN is produced and the nerve cells that promote muscle development and make our muscles move, survive and function.

These drugs have been in clinical trial for several years, including here at Lurie Children’s Hospital. Scientists and drug companies working with neurologist Nancy Kuntz have been testing the treatment and the results are amazing.  Children that could barely move are now standing and, in some cases, walking. The results were so positive that the FDA has ruled that the treatment should be made more widely available and within the last month approved its general use in SMA patients. Unfortunately, Biogen has priced this treatment at such a high cost it is effectively unaffordable and so far insurance companies have not agreed to pay for it.

So here we are on the brink of the cure of a horrible disease (if we can figure out how to pay for it!) affecting thousands of children, the most common genetic cause of death in children under 4, because of our understanding of the most fundamental aspects of the biology involved. 

Even with that so many questions remain.  These questions will open new avenues of research that will impact other childhood diseases and as our knowledge increases the intersection of genetics, disease biology and clinical care will lead us to a new era of treatment and, yes, cures based on fundamental science!

July 29th, 2016:

Stem cells can give rise to many types of cells that are necessary for a normal organ to function properly. These cells can be grown in a dish essentially indefinitely. This gives rise to the hope that scientists specializing in regenerative medicine can learn how to repair or replace damaged organs like kidneys or bladders in children or adults. Stem cells were discovered and isolated from very early embryos but required the destruction of the embryo. In humans this was ethically unacceptable for many.

By Lars Frantzen (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

More than ten years ago Japanese scientists discovered a way to take any adult cell, like a skin cell or a fibroblast and turn it into a stem cell. NO embryo NO ethical dilemma! The process required inserting genes with special properties into the adult cell. Because these genes are inserted into the cell from outside they are called transgenes. The good news is that the process is robust and can be used from a patient’s own cells. The bad news is that some of the genes inserted can cause cancer. But, the transgenes are turned off as part of the process of turning the stem cells into the desired cell type. They are said to be silenced.

Replay! from Galat et al. 2016. Cells with re-activated transgene appear red or green.

Transgene silencing in these induced stem cells (they are called induced pluripotent stem cells or iPSC for short) is generally accepted as a fact. Scientists at the SMCRI recently discovered that this article of faith might not be completely justified. In a surprising report just published they demonstrate that the silenced transgenes can turn back on. The result is that cells that were well on their way to becoming useful differentiated cells (cells of a desired type and with desired functions) revert to stem cell status and once again behave as pluripotent (capable of making many different cell types).

These findings are surprising and while the phenomenon may be unusual it provides a cautionary tale for scientists working with these cells. They may need to rethink results of some experiments and the re-emergence of stem cell properties may pose a risk of cancer when the cells are used. Newer methods are being developed that result in the elimination of the transgene right at the beginning of the process. In principal this should prevent the transgenes from getting turned on again because they are not there. However, it may not be that simple and this needs to verified in light of this new discovery.

We have discussed the way genes regulate our earliest development and described the way that master switches turning genes on and off are functioning at critical junctions in decision points about what a given cell will become.  Remember that while all of our cells have the same DNA and the same genes not all of the genes are active or turned on and the active genes are different from cell type to cell type.  We now know that the type of cell is determined by which of these genes are active and which are inactive.  An active gene is one that is “expressed”, that is a gene that produces RNA.  The RNA (except for specialized RNAs that have their own regulatory functions) is sent out of the nucleus and used to pattern a protein.  The proteins define the cell type and function.  We talked about a kind of decision tree in which an active gene sends a cell down a given pathway and prepares it for the next decision branch until the final state or “fate” of the cell is determined.

But what makes the gene active?  How does the cell decide what genes will be turned on and what genes will be turned off? Previously we described a key element of this process, proteins known as transcription factors.  These proteins have the remarkable ability to recognize certain sequences of the four “letters” of the genetic code in our DNA. These recognizable sequences occur near the gene in the DNA.  The transcription factor sticks to the recognition sequences in a protein –DNA interaction.  This sets in motion a cascade of interactions between proteins that result in the assembly of the machinery that will produce the RNA that defines the protein.

Is that all there is to it? Of course not.  Remember that we discussed how while the gene is a linear sequence of A.T, C, and Gs that are the basic letters of the DNA code the DNA is itself enmeshed in proteins in a highly ordered and specific way that helps pack 3 billion “letters” that make a chain of DNA meters long fit into a nucleus that is about .0001 inches in diameter!   We now know that the proteins that accomplish this also have the remarkable ability to regulate whether or not a gene is turned on or expressed. The proteins and DNA are interacting to make this happen and are collectively referred to as chromatin. The state of the chromatin can be conducive to the active expression (RNA production) of a gene or inhibitory of expression in a so-called repressive state.  There is an amazing degree of specificity to this regulation that defines what genes in which cells at what time are ready and able to be expressed. For the most part the proteins that accomplish this are the same through the entire DNA system of genetic code (known as the “genome”) but the combinations of these proteins confer an amazing degree of specificity to the process.

 The 3 meters of DNA in our cells is coiled tightly to pack into a chromosome or into the nucleus.  Part of the coiling is from winding around “nucleosomes”, the small blue balls above somewhat reminiscent of “spoolies” the rage hair curlers of the 1960′s.  The chromatin image from http://study.com/academy/lesson/nucleosome-definition-structure-quiz.html

The proteins that make up the chromatin include histones that create small barrel shaped structures that DNA winds itself around.  The histone and DNA are known as nucleosomes and they are reminiscent of the 1960’s rage, spoolies!  There are four histones in each spoolie and very remarkably the histones have protein “tails” that literally stick out from the structure.  Those tails in turn have the ability to alter their chemical composition at the hands of protein enzymes whose job it is to make the alterations. These chemical modifications in turn alter the ability of other proteins that actually create the RNA to come together at the beginning of the gene to start making RNA.  The proteins that make these modifications can be “writers”, “erasers” or “readers” and function much as their names imply.  The combination of these proteins and the modification to the tails define the chromatin state.  The chromatin state is what is modified as genes activate to expression or deactivate to repression.  Normal development is a delicate balance between cell division and cell differentiation, which is established through the time of the pregnancy to successfully complete the decision tree of gene expression.  This four dimensional problem is solved by creating astronomically large numbers of combinations of proteins.

 A gene switch! Notice the spoolie has little tails with chemicals that connect it to other proteins that operate the switch.  How cool!  From Petell, et al., 2016

The protein enzymes that alter chromatin are said to “remodel” the chromatin and without that process we could not develop normally.  Indeed just a few examples of what happens when this remodeling process is messed up should convince us of the central role chromatin plays in our health. 

We have talked about how simple organisms that are not like us still teach us very relevant lessons about our lives.  Yeast is a great example, many basic processes have been discovered using them.  A gene called switch was discovered in yeast and then an equivalent gene in humans was subsequently discovered.   This gene codes for a protein that makes modifications to the chromatin. In fact there is a family of these proteins that function in the same way in different contexts. When these genes are broken in human beings the result is cancer.  A wide range of terrible human malignancies result from mistakes in the genes. In the case of mistakes in a gene called SMARCA5 patients may develop Ewing’s sarcoma. This is a terrible tumor of the bone that primarily affects children; modern treatment thankfully can save half of these kids. 

The proteins of the gene switches are assembled into “transcription factories” in the cell’s nucleus. Machines like Top2 below are in there and together they remodel the chromatin landscape and recruit other machines to make the RNA.

A representation of the chemical structure of Top2 (a topoisomerase).  The golden spiral at the top represents DNA winding through the protein.  It is almost like a nutcracker! It modifies chromatin and regulates the expression of genes. Image from Wikipedia.

DNA is wound up pretty tightly.  This makes moving important protein machinery needed to create RNA into the gene difficult. Proteins called topoisomerases work to change that winding during regulation of gene expression.  One, known as Top2B, is very important in modifying the chromatin landscape or the “topology” of the gene during regulation of gene expression.  When mutations occur in the gene that codes for Top2B the result can be colon cancer or leukemia.

One final example of the importance of chromatin remodeling in regulation of gene expression is a protein called RBBP4.  This protein is very important in changing the chemical tails of histones that we discussed above.  In fact it operates both as a histone writer and a histone eraser and results gene repression.  When mistakes occur in the gene coding for this protein the result is a devastating childhood cancer of the eye called retinoblastoma.

As we develop starting as a single cell, the fertilized egg, we become trillions of cells with thousands of functions arrayed into more than 200 tissues.  Each of the cells mostly has the same DNA and the process of turning genes on and off is what makes a cell achieve its ultimate fate, what make cells different from each other, in other words what drives differentiation.  Key to that process is the landscape or topology of proteins that the DNA lies in and a constant process of modification of that landscape creates an amazingly dynamic and robust control mechanism for coordinating gene action that results in all of the cells that make us up.  So remodeling, important in so many aspects of our lives is also critical to giving us life!

This work is licensed under a

Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License

By New York Zoological Society

April 1st, 2016:

Previously we discussed some ways that genes can be changed on purpose. Now there is a new gene editing technique that allows scientists to change the DNA code at an unprecedented level of exactitude and at high efficiency. Called CRISPR, an acronym for the approach (clustered regularly-interspaced short palindromic repeats), scientists can create an RNA that homes in on a specific location in DNA that is of interest. The so-called guide RNA (gRNA) is combined with a gene for an enzyme from bacteria that will cause a break in both DNA strands at this precise spot. In an attempt to repair the break, the cell’s own machinery will create a mutation at this spot. The process takes advantage of the adaptive immune system bacteria use to fight off viral infections!

CRISPR genome editing video [4:12]

Scientists can also use this process to place a piece of DNA that has specific genes in it, or a correction for a specific gene at the spot where the cell’s DNA strands were broken. This occurs frequently enough that the procedure is completely accessible in the lab. It has been shown to work in a wide variety of cells and animals and has been used successfully by many many labs.

An immediate question might be: “Can we use this process to correct a faulty disease causing gene?”

The answer appears to be yes, ushering in a truly exciting and also challenging new era of genetic medicine. In the early 1980’s the isolation of disease causing genes in humans with muscular dystrophy was achieved by the only method available at the time, brute force. These methods including using a microscope to see structural abnormalities in chromosomes that were associated with the disease and indicated where the faulty gene was in the chromosome. But these structural markers were a crude signpost at best. Not surprisingly then the first human disease genes isolated in this manner were very big genes.

One of these large broken genes is a cause of the childhood muscle disease Duchenne Muscular Dystrophy (DMD, which affects 1/5000 births in male children). The DMD gene carries the information necessary to produce the protein dystrophin (critical to the function, integrity and longevity of muscle fibers). Dystrophin stabilizes muscle fibers by connecting key structural elements of the muscle cell. When altered by mutations, the protein cannot perform these functions and the muscle fibers are weak and die early leading to regeneration, inflammation and more destruction of muscle. The muscles of the extremities, the heart and in blood vessels are particularly affected. The defect is present at birth and eventually the children die from heart failure or respiratory failure as the muscles of the heart or for breathing fail.

Heart muscle cells: the protein dystrophin is in red  (University of Michigan Health System)

Normal muscle cells and muscle cells from patients with a mild form of muscular dystrophy (BMD) and a severe form (DMD).  Dystrophin is shown in red.  There is some dystrophin in the milder form of the disease and that encouraged scientists to believe that patient’s lives could be improved even if only some normal protein was restored. ( D.J. Blake, et al. Physiological Reviews (2002) Vol. 82 no.  2,  291-329 DOI: 10.1152/physrev.00028.2001)

In other genetic diseases, remediation has been achieved by just replacing the deficient protein. Unfortunately, in the case of muscular dystrophy the effected proteins are those of structural importance. Just having properly formed protein around won’t help, it needs to be properly integrated into the existing cellular structures. It would be like trying to repair a faulty skyscraper that has malformed girders by dropping a load of new steel in the lobby. It was clear that correction of the defect in DMD would require correction of the gene so that the normal regulation and placement of the protein could resume.

Because the gene for dystrophin is very large, it is susceptible to many types of mutations (genetic mistakes) that arise spontaneously as accidents either during replication of the DNA, repair of DNA, or during cell division. These genetic mistakes have a variety of effects on the resulting dystrophin protein translated from the gene. One type of mistake places the stop codon in the wrong place in the gene. A stop codon is a 3-basepair nucleotide code that stops protein synthesis and resides in an exon, which are the parts of the gene that exit the nucleus of the cell as messenger RNA to be translated into protein at the ribosomes. If that stop codon is inserted too early, the dystrophin protein stops it’s synthesis too early, which results in a protein that is shortened significantly and unstable to the point it can’t function.

Recently, in exciting scientific publications, three groups independently have shown that by using a CRISPR system, the misplaced stop codon can be excised from the aberrant gene in a mouse model of DMD. The scientists removed the exon that contained the stop codon allowing a nearly full-length dystrophin protein to be made. This protein was shown to be functional and reversed the disease in the mouse! When the essential elements of dystrophin are present the muscle cell can function normally, so removal of the small part of it with the bad stop codon resulted in nearly normal muscle cells.  While a misplaced stop codon accounts for a small proportion of the cases scientists have already applied this approach to other mutations in the dystrophin gene that cause DMD.

Getting the components of the CRISPR system inside the diseased muscle cell could have been a major hurdle, but it turns out that there is a friendly virus that particularly likes to attach to muscle cells and does not cause disease on its own. Many normal healthy people have this virus and when isolated in the lab provides an excellent vehicle to deliver the CRISPR gene editing materials right where they are needed.

The 3 billion a,t,c, and g base pairs in the human DNA stretched out would be much longer than this ticker tape!

The future of using these technologies to fix devastating diseases like Duchenne Muscular Dystrophy is very exciting and we now face a myriad of questions in this disease and others about how this therapy might work and how we can prove it in humans. For example, it has been shown possible in mice to use the editing system to alter the DNA code in embryos of mice and this results in alteration of the mutant gene that persists in future generations.

As is so often the case, the incredible promise of technological advance brings with it concerns over how the innovations can be safely and ethically applied in society. Most scientists are very concerned about gene editing approaches in human embryos and there is a call to prevent this type of research from going forward because editing genes in the embryo would change the DNA in all of that individual’s children and their children. Indeed such gene editing has already been reported in human embryos that were not developed beyond the earliest embryonic stages. The researchers stopped the work over fears that unintended consequences not foreseen by them or others posed a great potential risk to individuals and society.  The promise of gene editing to children with terrible diseases like Duchenne Muscular Dystrophy though relies on methods that do not alter future generations. Still like for any new therapeutic approach careful study of both safety and effectiveness will be required before it can be used widely in humans.

January 11th, 2016:

Previously we saw that when tissues in an organ were analyzed with a tracer or marker that allowed visualization of the ways cells assort themselves during development that patterns of assortment were the same in a given organ from animal to animal and from species to species, but that these patterns were different in different organs. This suggested that something was regulating the forces (cell movement, cell division and cell death) that created them.  

Presuming the position of cells within the organ is important there must be some way for the positional information to be stored and utilized to affect the needed distribution and spatial arrangement of the cells. With trillions of cells in hundreds of tissues but only 30,000 genes it seems unlikely that our DNA contains the spatial information for correct placement of all cells.

But what if there was some code that represented a kind of short hand instruction set for where cells should go?

In fact it looks like such a short hand exists. Computer simulations have shown that repetitive application of simple cell division rules can create pattern that correlates with the real world. Repetitive applications of rules creating a new state that becomes the input for the next round of application (or iteration) are known as cellular automata and they have the remarkable ability to generate complexity. The patterns then represent an emergent property that occur naturally by self-organization.  The Koch snowflake is a classic example. Take a triangle, on each side replace the middle third with another triangle, and repeat this for every side over and over.  Viola! a snowflake.  It is called a Koch snowflake after the mathematician that invented it in the early 1900s.

The Koch snowflake belongs to a curious class of geometric objects called fractals. A fractal object is one that is self-similar (little bits of it are the same geometrically as the whole of it) and has detail nested with in detail. Imagine the coast line of England; as one looks at it from an airplane one sees bays and harbors; nooks and crannies. As one gets closer, one sees very similar bays and harbors and nooks and crannies just on a smaller scale, and as one crawls along the same again is seen as pebbles and rocks. Indeed if one were to measure the length of the coastline at these various scales of observation the length obtained would increase much more that predicted simply because of the new scale of measurement. This property is the manifestation of the fact that the object has a higher dimensionality than that of the measurement.

A straight line line has a dimensionality of 1, a flat plane 2, and a regular volume 3. For complex structures when you calculate the dimensionality you obtain non-integer values. This is where fractals get their name:  from their fractional dimension. The more complex the structure the larger the fractional portion of its dimension.

Generating fractals is surprisingly simple given their convoluted geometry. By repeating a process of removing or adding to an existing structure often leads to a fractal after many iterations. As we’ve already seen with the Koch snowflake we only needed to add a triangle in the middle of every edge and after a few iterations its dimensionality is fractal.

This set-up is very similar to how organs begin to form in biology. You start with a small set of cells which begin to grow and divide and add repetitively to the structure.

So, if growth in tissues are iterative and repetitively adding as they develop shouldn’t they have fractal patterning?

In short:  YES! By tracking the lineage of cells and watching the tissues develop we do see fractal patterns. The complex patterns in the tissue is the result of repetitive application of cell division rules like when the cell divides, what direction the daughter cell is placed, and how and to shuffle existing cells out of the way. Changing these rules drastically changes the patterning in the tissue and its resulting fractal dimension. This allows us to measure the complexity of the patches that are created in mosaic mice or rats (chimeras where distinct parental lineages can be easily visualized) by measuring their fractal dimensions.

The iteration of rules over and over is a common theme in nature. From shorelines to mountain ranges, nature is filled with fractals. Indeed since fractal images are so easily generated they are often employed to make computer generated images (CGI) look more natural in the movies.

But back to the biology at hand:  Does the fractal dimension have practical implications in human health?  Can the alteration in fractal dimension tell us anything about individual patients?

The answer to both of those questions is yes.  Cancer is an important example, looking at the morphology of the edge of a tumor, or at the ramifications of blood vessels within it can be used to rapidly aid and augment diagnosis in many cancers including lung cancer.  

Another interesting example is the analysis of frequencies in voice pattern to help diagnosis early Alzheimer’s disease (AD).  Analysis of spectrograms (these are the spiky line graphs that are often depicted on TV or in the movies as the voice signature of a person), shows they are characteristic for a given individual. The spectra are self-similar and methods exist to establish their fractal dimension. An important aspect of AD is the progressive loss of language and it turns out that is reflected in changes in the fractal dimension of the spectrogram.

Retinal damage in young diabetics can also be characterized with fractal dimension as one considers the increasing number and complexity of retinal blood vessels in diabetic patients.

Understanding the the biology behind patterns that surround us will lead to a deeper understanding of diseases in which pattern is disrupted. From snowflakes to rivers to diagnosis!

December 23rd, 2015

Previously we discussed the ways in which GMO (genetically modified organisms) have been used in medical research. We talked about transgenic animals that have genes from other organisms and how significant that has been to establishing the mechanisms and pathogenesis of serious human disease. We described the use of animals with four parents, called chimeras, that had identifiable cell lineages and how that system was used to establish that tumors were clonal, that they originated as single abnormal cells.

In the course of studying tumor formation in chimeras examination of normal tissues yielded insight into the way cells were positioned during development. Normal tissue displayed characteristic patterns that while different from organ to organ, were the same in a given organ from animal to animal and even from species to species. Indeed this means that the patterns were both regulated, and conserved. Actually there is no way for the animal to know that a given pattern is present or not so it is actually the processes that control or produce the pattern that are regulated or conserved.

Those processes logically would include cell division, cell movement and cell death. As an organ grows the cells that comprise it divide many many times beginning with the first cell or cells that are committed to make the organ. The pattern of cell lineages observed will depend on which cells divide when and where the daughter cell of the division is placed. The patterns can be modulated by where and how cells move and further refined by what cells die during organ formation and where they are.

Making chimeras! A and B show early rat embryos that are a mixture of green and white cells made by sticking 1 green embryo to 1 white embryo and letting the cells mix.  In C we see that when the embryos develop fully into an animal aggregates of green cells (patches) are present in tissues like skin.

In general the cell division is thought to occur in stem cells responsible for making the cells of an organ. One place that doesn’t work is with the epithelial layer of the cornea. Here chimeras show a fascinating spiral pattern with characteristic shape. We have shown that the stem cells that produce the corneal epithelium do not divide fast enough to explain the speed with which the pattern arises. Something else must be making it appear. We used computer modeling again to point out that shear stress strains might cause the cells of the epithelium to slide past one another in sheets and that force might result in a spiral pattern with the correct characteristics.

Spirals have fascinated us forever and the patterns are really pretty but does it matter?

The ability to find the forces that help shape an organ is inherently valuable as it feeds or strongest intellectual desire – curiosity! But beyond that many diseases of the cornea that can compromise vision, one of the most ancient of senses, are associated with alterations in that pattern. Understanding the relation between the pattern and organ development can offer clues about the disease.

The cornea is the outer layer of the eye and is very important to normal sight.  In these pictures we see that the epithelium of the cornea assorts itself into spirals. What does this pattern tell us about how the cornea is formed?  How does it come about?

Genes and the Environment

In an op-ed I wrote some years ago, I described waiting to speak with my representative in Washington, DC to advocate for research support for environmental health and for the National Institute of Environmental Health Science.  A staffer asked “So what is the next big problem?” I said “Air!” Then thinking for a moment added “Water, air and water!”

The former Director of the National Institute of Environmental Sciences (NIEHS), Ken Olden, famously said, “The genes load the gun; the environment pulls the trigger.”  For many years the mantra of the NIEHS was “The environment is your health.”

Many of us believe the work with water is complete; after all, the fisheries in the Great Lakes are back, Lake Erie no longer catches fire, and indeed tremendous strides have been made. On the other hand we think of lead as being successfully removed from the environment and there is in fact a much-reduced lead burden with respect to continuing contamination. But lead is far from a solved problem. Bruce Lanpher from the Simon Fraser University in British Columbia Canada reminds us that low levels of lead in the blood can have adverse health effects in children including ADHD. Recent troubling news from Flint Michigan reminds us that  lead contamination in water supply is a continuing threat and is often not tested for or tested inappropriately.

There are many pesticide threats to our water supply from farming activity. For example, Atrazine is used to kill broad-leaf plants (weeds) that interfere with corn or other crops. It is very effective against weeds but has contaminated water supplies in both Europe and the US. Currently Atrazine is banned in Europe because it is an endocrine disruptor, mimicking powerful hormones and disrupting normal developmental circuits, especially for gonadal development. Exposure in humans is associated with miscarriage, birth defects, low birth weight, infertility and prostate cancer. A class action lawsuit against the manufacturer Syngenta lead to a settlement in which Syngenta agreed to pay for filtration systems to remove Atrazine from the drinking water of  more than 1000 municipalities. As an expert witness in the case I had the opportunity to describe the mechanisms by which compounds like Atrazine exert their effects. Atrazine increases the activity of enzymes that metabolize it but also participate in steroid and estrogen metabolism. Because these hormones regulate the actions of genes important in development, the environmental exposure directly alters gene expression thereby exerting adverse health effects.

Bellerophon slaying the Chimera with the help of the winged horse Pegasus. Terracotta by Boreads (Greek) 570-565 BCE.

October 16th, 2015:

The cellular origins of cancer long intrigued and perplexed biologists. Did the cancer arise from a generally disabled tissue with all of its cells participating together to create the dreaded tumor or did some rare event trigger chaos in a single cell that then went on a rampage of proliferation and destruction? Another type of genetically modified organism (GMO) provided key evidence to establish that tumors come from single cells starting form the very beginning of the process.

One of the earliest description of a GMO is in Homer’s Iliad. Bellerophon, at the behest of King Iobates of Lycia, was tasked to kill a monster, the Chimera, in the hope that the fire-breathing beast would destroy Bellerophon instead. In this myth Bellerophon, riding astride Pegasus, the winged horse, won. The creature was lion in front, snake behind and goat in the middle.

A representation of the Chimera. Bronze Etruscan circa 400 BCE.

But can such a fanciful mixture of animals occur in the real world?

Well, yes and they have been made for many years to do specific medical research. In the laboratory the combination is achieved by mixing the cells or tissues of very early embryos from genetically distinguishable strains or species to form a combined embryo. In the simplest case this embryo has four parents! If there is a way to see which cells cell came from which parents (called lineage analysis) then the cancer question can be answered.

Two methods for making experimental chimeras. The result is the same in both cases.  Two distinguishable lineages of cells are present derived from four parents.

The constellation Cancer (zastavki.com)

How GMOs Help Us Understand Human Disease

In the last post we discussed how animals could be produced with foreign genes for use in medical research. We will look at a few examples of how this technology has revolutionized many aspects of research into human disease.  

We mentioned that a very early example of this technology began 50 years ago when it was discovered that by inducing two cells to fuse or join together their chromosomes and hence their genes were also combined.  It was then learned that if the cells came from different species the chromosomes from one of the species would be preferentially but randomly lost as the cells grew and divided. That is to say fewer of one type of chromosome and hence its genes would eventually be present in the fused cells but any of the chromosomes from that species might be lost.  This was exploited to do somatic cell genetics, the study of genes in isolated cells without breeding.  When human cancer cells were fused with normal mouse cells, following the fusion the cells no longer behaved as cancer cells. After a time in culture, as the chromosomes of one of the types of fused cells were lost, the cancer-like behavior returned.  In the case of human cancer cells fused to mouse cells it was the human chromosomes that were lost.  

A red cell and a green cell fuse to form one cell with both nuclei, all the chromosomes and all the genes.  Over time chromosomes from one of the original cells can be lost. (iskweb.co.jp)

The explanation is that there are genes on human chromosomes that suppress cancer, and if they are broken or absent the cells behave like cancer.  When the chromosome containing those genes is added back following fusion with a normal cell, the fused cells behave as if they were not cancer.  Once the chromosome containing the special genes is lost from the fused cells they once again behave as cancer.  Thus these genes are called tumor suppressor genes. But is this true of cancer in intact animals?  Do the genes behave in the same way with cancer in an animal or just with cancer ‘in a dish’?  Answering this question was accomplished by putting DNA coding for mutated tumor suppressor genes into mice.  In one example, transgenic animals were produced by inserting a tumor suppressor gene (a gene called p53) that was mutated or not functional.  The transgenic mice were more susceptible to forming lung cancer after treatment with cancer causing chemicals than animals that did not have the mutated form. This worked even though the normal suppressor genes were still present in the transgenic animal.  The normal tumor suppressor was overwhelmed by the mutated p53 and the result was cancer.   So in this case the p53 tumor suppressor is like a brake stopping cancer and when the brake is broken and installed in the cell it develops cancer even though the normal, cancer suppressing gene of p53 is still there. This turns out to be important in the terrible childhood brain cancer medulloblastma. 

Following work with cell fusion the investigation of how viruses can cause cancers in animals led to the discovery of a class of genes, known as oncogenes that cause cancer under special circumstances.  Originally it was thought that the virus carried these genes as their own and inserted them into the animal’s genome.  It is now known that the virus, when it invades a cell and takes over the cell’s genetic machinery, picks up genes that are normal in the cell and then in another cycle of infection inserts them into the animal’s genome in the wrong place where the genes escape normal controls. Without normal controls they can act as oncogenes, cancer causing genes.  Genes that are normally not active in adults can become active either because a mutation prevents their regulation or because a piece of chromosome carrying the oncogene moves, or translocates to some other chromosomal location that keeps the gene active.  It can then cause cancer.

The proof of the pudding for a cancer-causing gene is to insert it into an animal and determine if it results in cancer formation.  For medulloblastoma the oncogene N-myc (MYCN) is a causative oncogene and by using GMOs we now know that N-myc can induce mutations in p53.  So N-myc is the gas pedal and p53 is the brake.  When the gas pedal  (for cell division) is stuck down and the brake  (tumor suppression) is broken the result is brain cancer.  Our lab showed (with Dr. David Walterhouse) that in medullblasotma of children another important oncogenic pathway was interacting with p53, the Sonic Hedgehog/PTCH/GLI pathway discussed in an earlier post.  We now know that GLI, a molecular switch that turns other genes on and off, competes with p53 for a rate-limiting co-factor (another part of these switches).  When GLI wins the result is cancer.  

So childhood cancers are a complex series of overlapping and interacting networks that can be worked out using GMOs.  Once they are worked out therapeutic targets emerge and the pharmaceutical industry can develop drugs to attack those targets.  Early testing of the utility of the drugs also occurs in the transgenic GMO models developed to identify the mechanism of cancer formation.  They have contributed greatly to our understanding of cancer and to our ability to treat it.

There has been a great deal written about genetically modified organisms (GMO) with particular focus on foods that we buy. Altering the genes of crops has been used to increase crop tolerance to pesticides allowing their use to increase yield, to increase resistance to insect pests and to increase nutrient value of the plants.

The main argument against GMOs is that making big leaps in modification of a plant or animal using genetic trickery may make unhealthy food or has unknown consequences although this seems not likely. But there is a side to GMOs that gets very little attention: the use of genetically modified organisms in medical research. It is hard to overstate the importance of such organisms to the discovery of life process or mechanisms of human disease.

The movement of genes from one organism to another at its most fundamental level is an important aspect of reproduction. This process ensures genetic diversity within a population and creates strength but also provides the template for adaptation. The fact that parental genes re-assort themselves during reproduction assures robustness of the offspring. It also allows for selection of desirable traits in plants and animals and from time immemorial man has exploited breeding to improve the agricultural aspects of domestic animals.

The genetic analysis of cancer gained a big boost in the 1960s and 1970s when it was learned how to swap genes between cells directly. This was done by forcing the cells to fuse and creating cells with an amalgam of chromosomes (and their genes) from the fusion partners. The behavior of the cells changed in a way that allowed the discovery of genes that promote cancer and other genes that inhibit cancer.

The age of molecular biology ushered in a period more than 40 years ago that allowed genes to be isolated directly from cells grown in a dish. Scientists then developed methods to put  many many copies of small stretches of DNA into other cells. These altered cells could be used for a wide variety of studies. The DNA managed to fit into existing chromosomes in the recipient cell. This process of incorporation of DNA into intact chromosomes is called integration. You will recall that in earlier posts we discussed the way that genes are organized and regulated in chromosomes and the newly acquired cells get the DNA in a way that allows them to function by producing RNA and ultimately proteins. This allowed the study of the function of individual genes in a convenient experimental setting.

Soon after scientists were encouraged to try and put foreign genes into animals. Remarkably this worked! The first attempts to do this were presented to the scientific community in the early 1980s. The approach was to use a teeny tiny needle to inject DNA containing the coding sequence of a gene, without any of regulatory machinery or proteins that normally package DNA, into the nuclear structure of a fertilized egg (where the chromosome are).

Recall from an earlier post that following fertilization the sperm enters the egg and develops into one of two pronuclei, which are small nuclear structures that will join together in order to combine the chromosomes, and thus the genes of the mother and the father in the offspring. Injecting DNA into a pronucleus is quite a feat since the target structure is about 20 microns (20 millionths of a meter or 0.0008 inches; 50 of them would fit on the head of a pin!). When injected, the foreign DNA integrates into the DNA of the egg. Once this occurs the regulatory machinery and normal proteins of the chromosomes surrounding the injected DNA take over or adopt the foreign gene which then begins to function in its new home.

The injected egg is transplanted into the uterus of a mouse and allowed to develop into a mouse that now has the foreign gene. This gene transcribes RNA and ultimately the protein it codes for is produced. Such animals are called transgenic and they have allowed the study of thousands and thousands of proteins and biological processes in impactful studies from diabetes to stroke to cancer and psychiatric disorders.

The insertion of a gene from jellyfish that makes a fluorescent protein, for example, has allowed many many studies of how cells move about during development, how proteins interact and how genes effect behavior. Transgenic animals are made from many species for specific purposes including zebrafish, rats and pigs. Transgenic pigs with green fluorescent protein are a critically important model of transplantation studies and may eventually lead to successful use of pig organs in humans!

There are other methods for moving foreign genes into organisms including the use of embryonic stem cells, viruses or even specialized bacteria used to make transgenic plants.

In the next few posts we will explore how transgenic animals have impacted our understanding of human disease and how to treat it along with brief discussion of alternate methods of their production.

Did you ever wonder why humans live for around 80 years, elephants for 70 years, mice for 2 years or round worms for 3 weeks? Or why some people live to more than 100 years of age but others die very young?  For a long time a rough correlation between metabolism and lifespan has been talked about or relatedly between size and life span.  A resurgence of interest in this and in longevity in general has been recently sparked by again our lowly worm friend.

Animals with with lower heart rates general have slower metabolism and live longer

Last time we saw that the round worm C.elegans goes through four larval stages before becoming an adult. At each stage the developing worm sheds its epidermis (its skin) a process called molting.  Then it grows a new larger “skin”. In the worm this “skin” also serves as a skeleton!  The structural integrity it gives to the animal is in part due to hydrostatic pressure that builds in the tissue. In between the first and second larval stages or between second and third, a remarkable event can occur in round worms. Occasionally the development stops as the worm enters a paused state called dauer (from the German word for endurance or duration).

This event is triggered by environmental factors like a lack of food or other adverse conditions. Then the developmental program switches away from reproduction and into a kind of stasis. This state can persist for more than 4 months and then the animal can reenter normal life. This is a remarkable feat considering that the average lifespan of C. elegans is only 3 weeks!

Can this state teach us something about aging and longevity?  You bet!  

One of the genes essential to establishing the dauer state is called DAF2 (Decay Accelerating Factor 2). Scientists have found and engineered mutations in DAF2 that result in a most remarkable outcome. The little worm will live for more than twice the normal span! More importantly, this longevity is not associated with aging events normally seen in the worm. Thus it is longevity of youth, and it has sparked the imagination of scientists and non-scientist alike. DAF2 codes for a protein called Insulin like growth factor 1 (or IGF1), a critical part of metabolic pathways in worms and humans alike.

Changes in the Insulin like growth factor levels activate a cell surface receptor that begins a cascade of events in the cell that results in the activation of another gene, DAF16, which is a transcription factor (which we know act like molecular switches) that turns on more than 100 other genes so that they make RNA and ultimately proteins that protect the worm from harsh environmental conditions.  Longevity effects of mutations in DAF2 require a functioning DAF16. The route to longevity involves other networks of genes and these are under intense scrutiny. There are mutations (in a gene called EAT!) that affect the pumping apparatus of the pharynx and produce a partial starvation and dietary restriction that increases the life span of the worm by up to 50%. Another gene called AGE1 functions in the same pathway as DAF2 and mutations in this gene also increase the life span of the worm by more than 50%.

The intimate relationship of insulin, IGF1, and metabolism make us recall another set of fascinating experiments that were largely ignored for decades. In the 1930’s scientists at Cornell thought that our life span was critically related to our diet. So they restricted the caloric intake of rats by changing their diets. In those animals that had restricted diets life was extended by almost 50%. Those few who did pay attention to these experiments objected to the conclusions arguing that many factors unknown and related to dietary differences might be responsible or even that the results might not be true.

As scientists once again became interested in longevity, reexamination of this work has shown it to be reproducible and compelling. Then came the results from C. elegans that showed a possible genetic explanation and gave the same results. Wow!  It now known that longevity can be altered by dietary restriction in many species, rat, mouse, fruit fly, monkeys and of course our little worm.

Original report of dietary restriction’s effect on lifespan

Aging beyond longevity appears in other fascinating and devastating diseases of  children. Progeria is one of the more tragic diseases of childhood.  It is due to a rare mutation in a protein that helps hold the nucleus together and make platforms for the machinery that makes genes work. The mutation is not passed on by inheritance because the affected children die before they can reproduce.   They essentially die of old age while teenagers. All the usual effects of ageing and diseases (such as coronary heart disease, atherosclerosis, and heart attacks) appear in these children at a very young chronological age. For an inside perspective on this disease, Sam Berns gave a TEDx talk about growing up with progeria  back in 2013 shortly before his early demise.

A 15 year old child with progeria poses for a photograph from 1911.

On the other side of the coin another rare condition results in delayed aging referred to as disorganized development where in one case a girl of 16 years had morphological body features of an 11 month old.  The condition is known as Syndrome X and does not have a known cause. You can read more about Syndrome X in this paper from the journal Aging.

As the genetic networks that regulate aging are dissected, their relationship to childhood conditions that disrupt development offer us great promise of meaningfully extending quality life spans.

Food is great challenge to our individual health and to the health of society.  The challenges of climate change and population growth will make this increasingly so and could become an existential threat to some.  Not just nutritional aspects but availability will be problems, more so than today.  The World Bank projects population growth of 35% by 2050 and a whopping 75% increase in food demand.  Increasing nutrition and efficiency of food production means that understanding diseases of plants is as important as understanding human disease, indeed the two are intimately intertwined.  Paramount in this paradigm is the lowly worm nematode.

Vegetable roots infected by worms.

Nematodes make very effective plant parasites.  There are more than 4000 species of nematodes that live on plant tissues. The worms prefer high moisture content in soil, either freshwater or saltwater. They can survive in soil water or by carrying moisture about in their cuticles or outer layer. They may be found everywhere to a depth of 20 feet. They can remain quiescent for decades. They are very little plant parasites and requiring a microscope to be seen. Nematodes are an effective vector for virus transmission to plants.  In tropical and sub-tropical countries up to 15% of production crops are estimated to be lost to nematodes.  Losses in the UK are estimated at more that 70 million dollars a year. Ironically these little creatures are also enormously beneficial to soil adding nitrogen and other much needed nutrients.

The history of the study of these little creatures (called nematology) goes way back. Many “sightings” are sought and found in ancient writing but these are largely fanciful as knowing whether or not observers saw nematodes required them to have been defined in someway, a process that developed over time very slowly.

In 1743 Needham discovered Anguina tritici in wheat gall by crushing the seeds and describing worm like creatures. This began a rich tradition of looking for the parasite as an explanation of disease in economically relevant plants.  In the US in 1913 Nathan Cobb published the first paper on nematodes in the US. His work resulted in US Department of Agriculture opening at least nine new fields of study.

Nathan Cobb at his microscope

Cycle of Life

Organisms have life cycles, stages through which they pass, the cycle of birth to death and on to the next generation.  In a real sense the life cycle is the underlying motivation for curiosity about and research on developmental biology.  It is said that the most fundamental driver in our nature is that we understand that our time is finite and that we must die at some point.  What would be more natural then to want to understand how we get to that point.  A motivation that is expressed in a deep commitment to experimentation with organisms that can help us understand.  Approaching a model system to study developmental process begins with a description of that organism and then a description of the life cycle.  In that way we can begin to approach the mechanisms that are responsible for life transitions and for some of try to make useful extrapolations to the human condition, both of normal development and of disease states.

For the roundworm there is an egg,  four “larval” stages  and an immature adult. These stages are not true larval stages since no metamorphosis occurs as in insects. The egg resides in the female reproductive system. They are laid singly in the soil before the embryo divides. Depending on the species though they may be laid in a cluster, some in gelatinous material.

Rapid cell division ensues and creates a basketball like Blastula stage that involutes creating multiple cell layers and the Gastrula stages. Cell division continues and the embryo elongates and begins to fold but is still inside the egg.  At this point the animal is distinctly worm-like in appearance.  Then motion begins.

Life Cycle of the nematode C. elegans from www.wormatlas.org

In the 1st stage after “hatching” a new cuticle forms over the surface of the larvae as the old one is shed to accommodate growth.

In the 2nd stage the animal lengthens. At the 2nd stage batch feeding occurs and is followed by the 2nd molt (shedding the cuticle allowing more growth of the animal).  Indeed in some species the nematode goes through these stages in the tissues of an insect host.  The nematode parasite releases bacteria to its insect host, which then proliferate, they poison the insect, but are harmless to the nematode, and they provide the food supply for the growing nematode.

In the 3rd stage the “larva” continue to feed and then molt again, in the 4th stage, after the 4th molt, the animal is an adult.

Most nematodes have distinguishable sexes. Male sperm is produced and passed on by copulation. Sperm-egg fusion occurs in the hermaphrodite and an embryo is formed. When there are no males the animals reproduce hermaphroditically or parthenogenetically, for example in C. elegans reproduction can be by copulation or by self-fertilization of eggs.

C. elegans has two sexes determined by the ratio of sex chromosomes to autosomal chromosomes. If that ratio is 1:1 then a true hermaphrodite results with individuals cytologically XX that have sperm and egg. A ratio of 1:2 results in mating males that are cytologically XO are have sperm only.  The sperm from an XX animal can fertilize an egg in the same animal.

Previously we learned of the tragic range of serious childhood diseases caused by species of nematode. Understanding the biology of round worms though is important for many reasons beyond learning how to treat the diseases they cause in us.  Our ever more precious food is at risk from worm based pestilence and we will see next time that the worm has taught us a great deal about our own life span.

Howard Florey who developed penicillin as a drug often said that one should always focus research on the practical. Study what you know can do some good. In the world of modern research this means pursuing what is called translational research, as in translating what happens at a research bench into something that can be used to treat a disease or otherwise improve the health of people. This is an obviously important aspect of medical research.

Less obvious is the indirect, circuitous, frustrating, expensive and time consuming process of making the discoveries that lead to translation in the first place. There is no way to predict what line of investigation or series of experiments will ultimately lead to important approaches to treating the health of patients. While the penicillin story is familiar to us and in modern research rational drug design does provide a clear pathway from biology to treatment we cannot discount the importance of unencumbered curiosity on the development of transformative discoveries. My favorite example of this doesn’t begin in a chemistry or biology lab at all.

We are familiar with the Hindu/Arabic numbering system that features 10 numbers, 1-9 and 0. We all grew up using it and most likely because we mostly have 10 fingers and toes we are comfortable with it. This system has placeholders or significant digits. For example, take the number 147:  there are 7 ones, 4 tens and 1 hundreds. But this system is not the only one. In the 1500s mathematicians began to lay the groundwork for using other numbering systems. Notably logarithms were developed. The place holding significant digits were in fact exponential and derived from the base of the numbering system. Thus in our familiar base 10 decimal system there are 10^0=1 (the ones), 10^1=10 (the tens), 10^2=100 (the hundreds), 10^3=1000 (the thousands), and so forth. So the number 147 can be rewritten as the sum of these place holding digits:  147 = 1x10^2 + 4x10^1 + 7x10^0.

When 1 + 1 = 10:

What if we use another number as the base? In the 1600s Leibniz described a base 2 number system where there were only 2 digits, 0 and 1, and the place holding digits were then 2^0=1, 2^2=4, 2^3=8 and so on. Let’s look at an example number in this base 2 system: 10110. It can be read as having no 1s, one 2, one 4, no 8s and one 16. In our more familiar base 10 system we can add this up as 1x2^1 + 1x2^2 + 1x2^4, or 2 + 4 + 16 = 22. So the base 2 number 10110 is equivalent to the base ten number 22. Our previous example of the number 147 can be translated into base 2 (also called binary from the Latin bi- for “two” and -nary for “belonging to”) as 10010011.

This system of numbers had been described by several cultures in the distant past but not explicitly as a mathematical tool. Most historians credit Gottfried Leibniz with formally describing the binary number system. Leibniz allowed that such a system would make calculations and geometry easier than in base 10 (what we mostly use) but also described its importance in metaphysical religious terms. He believed that 0 represented the void and that 1 was God, that these two states described how the universe came into being ex nihilo (from nothing). He also derived meaning from the ancient Chinese tablet writing of FuXi credited with beginning Chinese language by lining symbols from his early writings up with a table of binary numbers.

First depiction of binary numbers originally from I Ching, a classic canonical text said to be from FuXi ca. 2600 BCE. This version from the 1700s was given to Leibniz who then interpreted the dashes as 0 and the lines as 1.

From Leibniz’s original description of binary numbers with a table converting FuXi’s pictograms to numbers

So in the absence of a compelling use for these number systems those who pursued them were considered effete purists.  Leibniz achieved fame as a mathematician and inventor (including mechanical calculators!), but even he could not know that a time would come when this number system (along with Boole’s logical operators: AND, OR, and NOT) would be an intimate requirement of all digital computation or that virtually nothing in these undreamed of future societies could be done without it. From dishwashing machines to cars to planes to supercomputers, they function today based on the work done in arcane areas of research in the 15th and 16th century. The passionate pursuit of curiosity informed by the scientific method and hard work is like planting seeds. We may not know which seeds will bear fruit, but we do know that if the seeds are not planted there will be no harvest. We must be vigilant in our protection of the most basic of research no matter that practical outcomes are not obvious if we dare to hope that new unimagined solutions to our most intractable problems are to be realized and new undreamed of human endeavor and enterprise are to be found.

March 30th, 2015

Terrestrial worms are not the only ones we can learn from. As man learned to explore the deep reaches of our oceans (arguably less explored than planetary space) a remarkable observation was made. Robotic exploration at amazing depths that should not be compatible with life, so deep no light is present and pressures are hard to imagine revealed a truth we are more and more aware of:  Where there is liquid water there is life.

Near the vents of volcanic activity along the rifts in massive broken sections of the earths crust under water in a soup of toxic chemicals life thrives. The most amazing of these life forms are worm-like and live in tubes they produce for themselves form minerals in their environment.

They move in and out of these tubes, they metabolize hydrogen sulfide, they reproduce and thrive miles under the surface of the ocean (Jules Verne couldn’t make these animals up)! They also represent members of a new classification of life: the extremophiles.  Extremophiles live in hot temperatures, extreme pressures, low oxygen, acid baths or volcanic gases - and thrive there. They are a challenge to delivered notions of what conditions we associate with being compatible for life.

There are several known types but they do not survive decompression and so detailed studies have been difficult. One species the giant tubeworm, Riftia pachyptila, lives by oxidizing hydrogen sulfide, which is present in abundance in and around the thermal vents they live in and near called black smokers. Riftia has a bright red plume from the very special hemoglobin they make.

Unlike the hemoglobin we produce to help red blood cells carry oxygen, the Riftia’s hemaglobin it is not poisoned by the very high hydrogen sulfide concentrations around the black smokers. They metabolize the sulfide with the aid of bacteria that live in symbiotic relationship with the worm and may comprise more than half of the animal’s body weight. The bacteria produce ammonium and amino acids that the worm uses. In turn the worm concentrates nitrates for the bacteria to use.

Another species called the Pompeii worm lives by a very similar mechanism. Pompeii worms were among the first extremophiles identified and live on the edge of survivable zones. In fact Pompeii worms are the most heat resistant animals currently known. The protruding end of the worm is in a comfortable 72˚ F but the other end is often in excess of 170˚ F! This remarkable attribute may also be due to bacteria that live on the worm and provide special proteins that are thought to protect the worm from damaging temperatures.

Imagine the possible uses of materials we might develop from knowledge of how this works.These and other strange life forms that have adapted to extreme environments have already provided us with materials that have opened whole new approaches to problems and indeed new industries. Temperature resistant enzymes have been routinely used for many years to diagnose diseases, identify new drug treatments and develop new genetic approaches to treatment. What new medical advances are yet to be discovered from the strange world that surrounds us?