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“Wolf’s liver taken in thin wine, the lard of a sow that has been fed upon grass, or the flesh of a she-ass taken in broth.” –Pliny the Elder

Ring of Inhibition

We’ve come a long way in some ways, at least since “flesh of a she-ass” was considered a specific remedy from disease.  Though, did you know that in early Greece moldy bread was a remedy against infection of wounds?  The mold was presumably a kind of penicillium that produced chemicals inhibiting bacterial growth.  On that fine day in 1928, Alexander Fleming had no idea he would discover something that would change medicine forever.  After coming back from vacation and sifting through the piles of clutter in his office he noticed a culture of staphylococcus in which a blue mold was growing.  The mold had pushed back the staph in a characteristic ring of inhibition.  As the story goes, he hypothesized that the mold must be producing something that retarded bacterial growth.  Penicillium chrysogenum produces the chemical backbone used to produce penicillin, an antibiotic which caused a great leap forward in western medicine.  The effect has been so great that the old killers of yesteryear, the terrible diseases that for ages struck fear in the hearts of man are no longer thought of in the same grave tone and are considered more of a nuisance to public health, a mere trifle in consideration of the far greater concerns of the general populace.  Perhaps the greatest triumph is the reaction of surprise that “people still get that?!”

The back bone of penicillin antibiotics, 6-aminopenicillanic acid, was originally isolated from cultures of Fleming’s Penicillium

Various functional groups can be added to the amino side chain to produce different drugs.

notatum.  It is a secondary metabolic product, released only when the mold is under environmental stress.  Early manufacturing methods were hampered, because finding just the right conditions for the mold to release the chemical proved difficult, thus production was initially low.  When the first human trial in the U.S. began in 1942, doctors used half of the nation’s total production of penicillin to cure him. The medical value placed upon it began a race to massively increase its production. Researchers from both Britan and the US worked tirelessly to not only elucidate the shape of the molecule, but develop better processes for producing it.  In 1943 a nationwide search was held to find better strains from which the B-lactam skeleton could be obtained.  The lucky winner was provided by a moldy cantaloupe from Illinois, which produced many times the amount of penicillin than Flemming’s original.  This strain exceeded expectations and was quickly put to work manufacturing the raw penicillin components.  At the time, it was so prized that during World War II doctors would collect their patient’s urine to isolate and reuse the excreted portion of penicillin.

Today genetically engineered strains manufacture it in large tanks where conditions such as the pH, temperature and diet are all automatically controlled to elicit maximum output (S).  The growth medium is key to the entire process, where the goal is to put them under nutritional strain.  Growth mediums are usually proprietary information, but they typically contain a source of carbon and nitrogen, which are chosen to make them work harder to gain the nutrition they need to survive.  Penicillium notatum prefers the simple 6 carbon sugar glucose, but can also digest a similar sugar called lactose.  This change in sugars forces them to work harder for the same amount energy.  Looking for an edge to outpace their competitors, they produce chemicals like 6-aminopenicillanic acid that impede bacterial growth.  The incubating contents are constantly aerated to allow for maximum growth throughout the tank.  When the growth phase has completed the contents are filtered and the raw product is extracted, concentrated and purified.  The liquid can then be altered chemically and turned into a number of antibiotics by adding functional groups to the base skeleton.

Upon discovery of the antibiotic nature of this compound, researchers put the skeleton through many thousands of reactions in order to find compounds with similar properties.  There are many kinds of penicillin, which differ by the addition of unique functional group.  For example, adding phenyl acetyl chloride to the purified preparation produces benzyl penicillin, or penicillin G, as it is more commonly known.  These new additions can change the properties of the original compound, endowing it with activity against a wider range of bacteria or a specific subset of them.  Penicillin G, for example, is strongest against Gram positive organisms, like staphylococci and streptococci, and gram negative cocci, like N. gonorrhoeae, but has low activity against bacteria that produce enzymes that inactivate the compound, called B-lactamases.  With N. gonorrhoeae, however, there has been increasing resistance of this kind reported and penicillin G is no longer considered the drug of choice to treat them.  According to William Strohl, “Over twenty percent of current isolates of N. gonorrhoeae are resistant to penicillin, tetracycline, cefoxitin, and/or spectinomycin.” (S)

B Lactams usually block the enzyme that ties these pieces together.

With such a wide range of activities against so many organisms one might wonder how they work.  Like all poisons, penicillins inhibit critical life processes in the species attacked. In this case, they inhibit bacterial growth by stopping the repair and replacement of their cell walls.  In affected strains, the cell walls are composed of long chains of six carbon sugars with amino acid side chains that weave the chains together like a net.  The long chains are made by polymerizing N-acetylglucosamine and N-acetlymuramic acid sugars into alternating units.  The side chains are made of repeating amino acid units, which differ per species, but for susceptible species they end with the combination D-alanyl-D-alanine amino acids.  B-Lactam antibiotics are natural analogues of these amino acids, which is to say they can fit into some of the same places as the natural bacterial amino acid piece can.  The antibiotics attach themselves to a special protein (the aptly named ‘penicillin binding protein’) that transports and tethers the D-alanyl-D-alanine amino acids to other chains in the cell wall, blocking its activity(S).  With these proteins blocked the cell wall can’t be replaced or repaired. Lysis results when the cell wall is unable to withstand the osmotic pressure upon it.

It is important to point out that these antibiotics are most active when bacteria are growing and synthesizing cell walls, which is fine for fast growing colonies, like the streptococci.  However mycobacteria, which are famous for causing tuberculosis and leprosy, have a dormant states where they don’t actively synthesize new cell wall components as well as impregnate them with certain lipids that impede the entry of penicillins.  In this context, an attack of this kind would be useless or at the very most a stop gap measure.  Since the colony isn’t completely killed off during treatment, this situation is favorable for the development of antibiotic resistances and the selection of hardy strains of the bacteria.  In a clinical setting these factors complicate issues, with treatments for tuberculosis taking upwards of a year or more to complete.

In a colony of 10^6 bacteria, the probability is that at least one will have resistance to a given antibiotic.  A typical mycobacterial colony will have something around 10^8 members, making the selection of strong, antibiotic resistant strains almost a certainty.  This problem is further compounded by a form of bacterial communication called horizontal gene transfer or plasmid exchange.  This is where a circular strand of DNA, typically encoding a gene producing a resistance, is absorbed by other bacteria allowing the technical means of resistance to spread. This is how bacteria such as the dreaded MRSA have developed and why infections from them are most common in hospitals. In the case of tuberculosis, multi-drug therapies utilizing non-B-lactam antibiotic combinations, like isonaizid-rifampin, can cure 95-98% of cases (S).  The probability that one will have a resistance against both antibiotics is very low and using two will usually kill the bacteria that have a selected immunity to one antibiotic alone.  The other 2-3% typically require specialized treatment and top of the line antibiotics, like moxifloxacin or rifabutin.  However, moxifloxacin resistant strains have been recently identified as a cause for concern (S).

Bacterial resistances usually present via four different routes: Reduced membrane permeability to the drug, production of an enzyme that deactivates the antibiotic (a B-lactamase, for example), a mutation in the PBP site where the antibiotic binds and finally, the creation of a means for drug removal (S).  With penicillins, B-Lactamase production is the most common form of resistance, some of which are highly specific to only one kind of drug (S).  B-lactamases attack the core structure of the penicillin molecule, breaking a critical carbon-nitrogen bond.  Mutations in penicillin binding proteins alter the shape of the binding site, making it harder for the B-lactam structure to fit.  In theory mutations could be great enough to block the penicillin from binding completely.  In practice, however, mutations that cause a PBP to be blocked entirely are often less useful functionally, because these will also block the D-alanyl-D-alanine amino acids they were originally intended for.  Small mutations are more useful to inhibit rather than block the penicillin and keep them from reaching a concentration necessary to inhibit bacterial growth. (S)  Gram negative bacteria usually develop resistances to antibiotic entry by changing the shape of receptors that transport the antibiotic across its membrane.  If the drug can’t pass through the membrane, it can’t attack the cell wall manufacturing machinery.  Another means that changes the membrane permeability is the creation of what is called an efflux pump.  These work to transport the antibiotics back across the membrane after entry.  These are of varied efficiencies, but always work to increase the amount of antibiotic required for efficacy.  Both mutation and changes in permeability are often paired with a B-Lactamase enzyme, making an effective, multi-front strategy for resistance.

Even though we have produced effective weapons against pathogenic bacteria, they have begun to fight back.  They have been largely successful in this regard, calling into question how long the usefulness of these drugs can be maintained.  This has spurred the demand for newer drugs to combat the potential ‘super-bugs’ on the horizon and limited use policies to maintain drug effectiveness in the future.  All in all, penicillin is generally considered non-toxic.  The worst adverse reactions are due to hypersensitivities.  Allergic reactions occur, however, this is a rarity occurring in less than 1% of people and is usually not life threatening (a rash, fever, etc).  Even less have a serious reaction, like anaphylactic shock (0.05% of patients).  In a worst-case scenario, though, desensitization to the drug can be used to effectively treat a given disease.

One thing is for sure is that we will continue to look in awe at the complexity life has to offer.  We hope that new attempts to cultivate and extract new antibiotics will be successful in the future.

{sources available upon request}

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As I was running yesterday a thought occurred to me as the steady, percussive hum of wings buzzed by me several times: one giant Asian hornet (an osuzumebachi) is startling, but several dozen is pretty scary. Apparently, I had run too close to a nest and had to hot foot it out; I was lucky.

These things are pretty amazing creatures, with an average body length of 2 inches and a wing span of 3, you will stand up and take notice when they fly past. As with many bee and hornet stings single stings can be deadly as a result of anaphylactic shock, but this is fortunately a rare occurrence. Nonetheless, the hornets are nothing to take lightly as the venom contains an interesting mix of nastiness, as well as a barbless stinger, which it can use repeatedly unlike regular honey bees. The venom contains neurotransmitters like serotonin and acetylcholine, which is responsible for pain transmission, a neurotoxin called mandaratoxin, tissue dissolving enzymes such as phospholipase-B, a mast cell degranulating peptide called mastoparan and a pheromone that instigates other hornets to swarm and attack. Aside from pain, the stings usually leave a dime-sized scar not unlike those of certain poisonous spiders in the southwestern US.

The hornets terrorize smaller, weaker prey like praying mantii and Japanese honey bees. The bee’s stingers are too small to penetrate the hornet’s tough, armored exoskeleton and thus have little defense from a full on attack. They reputably work with ruthless efficiency, a single hornet able to kill as many as 40 bees per minute and a few hornets only a few hours to slaughter a 30,000 bee colony. Interestingly, the bees have a small defense against them in their greater tolerance for heat. Typically, the bees will entice a scout inside the hive, where they will attempt to smother him in a bull rush. Once he is covered, the bees will vibrate their flight muscles increasing the heat in the area to around 47C. The hornets can’t survive past 45C, preventing the scout from calling reinforcements.

Link: http://www.youtube.com/watch?v=JDSf3Kshq1M
Link: http://www.youtube.com/watch?v=hpcHH1EpTZM

Even though, these may seem like kings of the forest, human initiative has once again found ways to make use of nature. The Japanese love energy drinks, one of the more famous products, VAAM is of particular note. As advertising shows it has been the favorite of many a marathon runner and endurance athlete. Yet a little less advertised fact is that it’s a chemical copy of the stomach contents of these hornets. More specifically, it is a copy of the secretions of the colony’s larvae, which the adults feed upon as they lack the ability to digest raw protein themselves. How’s that for wild? This mixture of predigested amino acids and sugars supposedly allows them to fly distances of 60 miles per day at rates of 25-40 miles per hour.

Sounds perfect for runners, now lets have a toast!

As gross as that sounds many professional athletes here swear by its supposed benefits and from personal experience it tastes pretty good. The question of the day, though, is one of efficacy: Does it work? Dr. Takashi Abe of the Institute of Physical and Chemical Research in Japan seems to think so. “VAAM works by helping the body burn the energy it stores more efficiently”…”[It] expedites the metabolism of fat and promotes better hydration,” said Dr. Abe. Animal studies conducted by Abe concluded that mice that were fed VAAM could swim twice as long as those that were fed only water and 25% longer than those fed casein, a protein found in milk. Their blood also contained fewer fatty acids than those of the other groups, an indication of how efficiently the mice were burning their fat reserves. Details are sketchy as to how it might actually accomplish these feats. One possible route may be through the branched chain amino acids in it, which increase the effect of insulin in the body. The product contains a mixture of leucine, isoleucine and valine, of which leucine is considered the most effective at this. Leucine reportedly “increases insulin output by 221%,” if taken with carbohydrates after exercise.

If you’re thinking: “At last the perfect supplement to my admittedly weak dietary habits, that will propel me to epic heights of fame and fortune,” then you’re most likely going to be disappointed (mostly in yourself). I’ll save you the trouble and clue you in to something important: the supplement industry is mostly a joke played on the lazy for the fun of a few bored chemists. Though there might be a slight benefit to some supplementation, there is no substitute for hard work, but we delight in trying to make you think there is. (Next year’s big craze will be whale sperm marketed under the name SPEaRM—->.) On the other hand, if you’re looking to try something that tastes good to replenish your vital fluids during or after a workout, then this might be up your alley.

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With over half a billion deaths attributed annually, Malaria is one of the world’s worst infectious diseases. The WHO, spurred on by previous success with small pox, has long had the dream of eradicating this disease, but has had to deal with limited success as the mosquitoes have developed resistances to the insecticides traditionally used against this primary vector. Currently, the Global Malaria Action Plan (GMAP) has had ambitious plans “to spray 172 million homes and distribute 730 million insecticide impregnated nets” by 2010, but there is a hidden danger to the plan: natural selection. More and more we are finding that the traditional insecticides (DDT, Pyrethrins, etc) are becoming less able to control populations of Anopheles mosquitoes, because the selective pressure on populations is already powerful even without intervention.

The long term benefits of using ‘quick kill’ insecticides are limited, because they add to the selective pressure already existent in the normal breeding cycle, thus quickly creating populations that are resistant to these traditional methods. The problem is one of diminishing returns. Consider the impracticality of perpetually designing an endless chain of active, weakly human toxic insecticides ad infinitum. Insecticides are toxic chemicals by their nature and very few have been found to date that have the necessarily low human toxicity and insect killing capacity. This paper suggests that using new or old pesticides in new ways might limit or all together remove this selective pressure, while at the same time accomplish the stated objective of controlling malaria.

The Big Idea: How They Work:

Malaria is caused by a eukaryotic protest called Plasmodium falciparum, which is transmitted by the bites of Anopheles mosquitoes. Their life cycle is an important part of the problem. After eating, the females produce and deposit a batch of eggs in water, which in total takes 2-4 days to complete. Even without insecticides exacerbating the problem, the egg mortality rate is around 20-40% per batch. With such a turnover rate, breeding selection will always be a strong factor in mosquito populations. At some point, the mosquitoes encounter and become infected with the malarial parasites. These generally go through an incubation period of 10-14 days before becoming active in mosquito salivary glands. This is ironic considering that most mosquitoes will not live long enough to become infectious and spread the disease. Since they only spread the disease at late stages, there is a window of opportunity to curb not only the selective pressure that encourages resistances, but also dramatically control the malaria transmitting mosquitoes.

Late Life Acting insecticides (LLA) have been proposed that disproportionately kill only the older mosquitoes. A major benefit being that selective resistances would spread much slower, than as with the use of current insecticides, while at the same time cull the infective mosquitoes. Conventional insecticides reduce the mosquito reproductive success by about 85% at first, but the LLA’s, which would allow for a normal reproductive cycle would target only older mosquitoes. Selectively targeting only the older mosquitoes relieves this selective pressure. If only the older insects are killed, it allows the younger ones to proliferate and spread their genes giving little selective benefit for having a resistance to the insecticides. Furthermore, the authors point out that:

The strength of selection declines with age. Beneficial genes that act late in life can fail to spread if they are associated with fitness costs earlier in life.

Though LLA insecticides would leave a large population of mosquitoes, the key point is this is meant to be disease control, rather than insect control.

Several methods have been proposed. First, cumulative exposure to ordinarily sub-lethal doses of pesticides. The idea is that the low doses build up over time killing only the older mosquitoes before they transmit the disease. Second, micro-encapsulation techniques designed to release over a long period of time. (Think about your Imodium 24 hour extended release, except with horrible poison!!) Third, chemicals that negatively affect detoxification pathways in mosquitoes. This exploits the fact that as mosquitoes age they become less able to detoxify chemicals. Fourth, compounds or dosages of pesticides that take advantage of an infected mosquitoes weakened state (malarial parasites negatively affect them as well). By using dosages, which are lower than recommended (especially those which are non-lethal for most healthy mosquitoes), it may be possible to target only those older mosquitoes with a weakened composition. Fifth, fungal bio-pesticides, which are active against mosquitoes, killing them 7-14 days after contact. The final possibility mentioned is using Wolbachia bacteria or a densovirus to control the older populations. Wolbachia are inherited bacteria that infect many kinds of insects, in particular affecting the reproduction system of their hosts and in some species causing parthogenesis (reproducing only one sex). Densovirii are virii that are thought to only infect insects. Both of these could be applied in much the same manner as the above methods, either building up over time or causing a metabolic pitfall as they age, shortening their life spans to die before the malarial parasites become active.

Source:

Evolution Proof Insecticides for Malaria Control

Penelope A. Lynch, Andrew F. Read and Matthew B. Thomas

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First and foremost, cancer is a disease of broken genes. For as long as medicine has existed, cancer has defied our attempts to cure it. It is estimated that one in 3 people will experience cancer in their life time(1). More so, “greater than 60 years ago there were no treatments for children with leukemia that could cause remission of the disease, but today this is possible in about 96% of cases!”(2) Though we have made leaps in our understanding and treatment of cancer, the golden fleece: freedom from this oldest plague of man has long eluded us. Perhaps some day we will achieve this ultimate prize, but it seems for the time being we must be satisfied with our current progress. Here’s a few reasons why the Tigers still stalk us in the halls when Sally Kimball is away:

1. Biological Immortality and unlimited growth:

To keep cells fit and in good working order, ordinary cells are limited by how many times they can divide. This is known as the Hayflick limit. A cell’s DNA has a non-coding region at the end of the strand called the telomere, which can be likened to a docking station for the DNA replication machinery. Every time the strands are copied this machinery takes a small piece of the telomere leaving behind a smaller and smaller piece. Once the telomere runs out, the cell enters senescence, stops cell division and dies. Somatic cells have about 50 divisions in them before this happens.

In cancer cells, an enzyme called telomerase actively replaces these lost pieces of telomeres, allowing them to divide beyond the Hayflick limit and evade the natural cell death program. Normally, this limitation keeps a cell line’s genetic package healthy and mutations from accumulating. However, for cancer cells this is not a problem, as the mutations sometimes serve to provide a survival advantage over normal cells. The tumor suppressing genes Rb1 and TP53, for example, are known to have roles in controlling cell growth, but once they are damaged or turned off they allow a cell to break free of the systemic growth of normal cells.

Retinoblastoma protein (Rb), the protein produced by the Rb gene, does its work by prohibiting a cell with damaged DNA from replicating. When this situation occurs, it binds and blocks the transcriptional machinery in cells, keeping it from copying the genome and consequently, proceeding into the next stage of cell division. Many cancer cells have mutations to this gene. In fact, the protein produced by this gene was discovered in a kind of eye cancer called retinoblastoma. This should not be misconstrued as only affecting the eyes, however, as it is a significant regulatory protein in almost every cell in the body.

P-53’s main job is to protect the genome from mutation. It acts as a watchdog right before cell division, making sure that the DNA has been faithfully reproduced. If there is DNA damage, then P-53 can stop the cell cycle and activate DNA repair proteins that can hopefully fix the damage. If the damage is too extensive to repair, then it can also signal apoptosis, the cell’s death program. Without this protein functioning properly, the risk of developing cancer is increased greatly. More so, “greater than 50% of all human cancers contain a deletion or mutation of the TP53 gene” (3).

Mutations of these genes generally allow a cell unrestrained, unlimited growth and are a cause of the massive growth of cells, known as a tumor. In this situation, cancerous cells have an advantage over normal cells, in that they have easier access to nutrient resources from the surrounding tissues.  They will grow in size until they are limited only by the availability of resources. An interesting observation is that the inner portion of many tumors is lower in oxygen (hypoxic), than the outer portions. This is a direct result of Fick’s law of diffusion, which describes the flow of nutrients and oxygen from blood vessels into tissues. It shows that as a tumor grows increasingly larger, it becomes harder to provide cells further away from blood vessels nutrition. This is a natural limitation on cell growth and body structure, but tumor cells have a way to bend this rule, which is described in the next section.

2. Decreased dependence on natural growth factors:

Cancer cells are like ‘rebels without a cause’, the James Deans of human cells. Normally, the signals for growth are usually sent by the endocrine/paracrine system. Platelet derived growth factor, epidermal growth factor and insulin-like growth factor are just a few regulatory chemicals that trigger cell division and cell proliferation. The collective mass of these signals make up a system that tells cells when and under what circumstances to begin division. To have a body and the organizational benefits that it brings, individual cells must perform the tasks they’ve been assigned in the developmental process. Cancer cells, however, produce their own growth factors, often self signal and/or develop a means to receive more signaling. Since they require no outside signals for growth, they are not limited by this systematic control. They effectively work outside the system to effect their own self centered proliferation, at the expense of the body supporting them.

One example of this is an activity called angiogenesis, the production of new capillaries and blood vessels from main branches. Normally, fetal tissue relies on the function of vascular endothelial growth factor (VEGF) to produce new capillaries in its rapidly growing tissues. More capillaries means that a greater volume of nutrients and oxygen can diffuse to important tissues. In adults this process is usually indicative of cancerous activity, with wound healing and the female reproductive cycle being the major exceptions. Tumors produce VEGF to get around the limitations posed by Fick’s law of diffusion. Tumor growth is limited to the short distance nutrients and oxygen can diffuse through tissues. Without the production of new blood vessels the mass of tumor cells would slowly starve.

VEGF has been found in almost every kind of human tumor and is at peak concentration around newly formed blood vessels and the hypoxic, inner regions of the tumors. Research has shown that by blocking VEGF by binding it to monoclonal antibodies, one can suppress tumor growth in mice(4). Other efforts are currently targeting VEGF receptors that are specific to kinds of tumors. Inhibition of a VEGF receptor called FLK1 has been similarly shown to reduce growth of tumors of certain cancer cell lines such as fibrosarcoma cell line HT-1080(5).

Another method cancer cells use to circumvent normal regulation involves the over expression of epidermal growth factor receptors (6). Normally, epidermal growth factor signals limited cell proliferation and its receptor is usually expressed in epithelial cells. However, cancer cells over-express it allowing for greater than normal signaling and as a result, increased proliferation. This over-expression is usually indicative of a poor prognosis, a situation occurring in many advanced stage cancers. In certain types of breast cancer, which comprise a little more than 20% of the total number of cases, the EGF receptor uses a protein called ‘HER’ to pass on the growth signal inside the cell. The bio-engineering giant Genentech recently made headlines for passing its drug herceptin through clinical trials. This is a monoclonal antibody that specifically targets HER proteins. Since cancer cells that over express EGF receptors have a greater preponderance of this protein, they will be targeted for destruction by the immune system specifically over ordinary cells.

Recent drug advances have allowed for the selective targeting of these receptors, effectively blocking this excessive, cancerous signaling. Specifically, several new tactics have been devised to target these receptors and block their activation. Recent successes with monoclonal antibodies, selective toxins and small molecules that block receptor signaling activity have made it into general therapy. Monoclonal antibodies are specifically targeted to these receptors to bind and block them from being activated. Toxins can take a variety of roles to block receptor activity, whether as allosteric inhibitors or by covalently binding to the receptor’s active site. Essentially, the end result is the same with these two kinds of weapons. With small molecule drugs, the tactic is usually to inhibit signal transduction from the receptor. The drug erlotnib is an example of this, in which it blocks a critical signaling component so signals can’t be sent from the receptor. Erlotnib is from a class of drugs called tyrosine kinase inhibitors, that have found great usefulness as selective inhibitors of these kind of over-expressed tumor receptors.

3. Loss of anchorage in tissues and cell adhesion:

Normally, cells will undergo apoptosis once they become separated from their host tissue. It’s tricky to grow normal cells outside of their home tissues, because they communicate with neighboring cells and require paracrine interaction for normal function. However, cancerous cells don’t require these regulatory signals and can be grown in solution or on agar medium. Normal cells are usually held in place by a matrix of proteins and structural elements, as well as being linked directly to neighboring cells by physical junctions. These structural components often give important regulatory information to a cell. The point where this breaks down is the beginning of what is called metastasis, the ability of a cancer cell to travel to other tissues, invade them and start a tumor there.

Tumor cells express a set of metal containing enzymes called matrix metalloproteinases (MMPs). These are responsible for the matrix degradation seen in metastatic cancers. They chop away at the extracellular matrix holding them in place, eventually freeing the cells to do damage elsewhere. The expression of these proteins is considered indicative of progression to metastasis, because ordinary adult cells do not express them. High levels of matrilysin (MMP-7), for example, is a known indicator of prostate cancer. Additionally, MMPs are known for attacking cell to cell adhesion proteins like E-cadherins, β-catenin and α-catenin. MMP-3, also known as stromelysin, is known for cutting apart E-cadherin cell junctions. The loss of their function is a known cause of tumorigenicity and cancer cell invasiveness.

Once a tumor cell escapes it has two routes it can metastasize through: blood or lymphatic. The tumors often have patterns of invasion that are indicative of their tissue origin. For example, tumors of the head and neck are often spread through regional lymph nodes (7). Tumor cells manufacture special tools they use to attach to new host tissues called invadopodia. These are similar in a way to bacterial pilli in that they contain various proteases and adhesive proteins that aid in attachment to the new cells and help the cell cross barriers.

The invadopodia bind to membrane components such as laminin, fibronectin, type IV collagen, and proteoglycans. Normally, these interact with receptor regulatory proteins called integrins that send regulatory signals to the cell. Cancer cells often change the binding preferences of their integrin receptor subunits to match those of degraded extracellular matrix proteins. The new types are thought to match the pieces damaged by MMP degradation and are often associated with invasive, metastatic cells (8). Binding to these pieces functions as a discovery signal that the cell has found a place that is susceptible to invasion. The fact that cancer cell integrin expression has changed in a distinct way, might also reveal a way to specifically target these cancerous cells. This could lead to therapeutic advances targeting the most aggressive, late stage types of cancers.

4. Loss of sensitivity to apoptotic stimuli:

Apoptosis is a form of cell death that is essential for tissue remodeling during embryogenesis and maintaining the number of cells in adult life. It functions to cull damaged, nonviable and potentially dangerous cells by acting as a failsafe that kills a cell when it has acquired too many mutations or unusual activities. It functions through cellular signaling pathways to block cell mitochondrial function, which eventually leads to the degradation of critical cell components and then ultimately, death.

The apoptotic program can be divided into three phases: initiation phase, decision/effector phase and the degradation/execution phase. The initiation phase typically is a response phase to outside stimuli, like death receptor ligands, or to inside stimuli like DNA damage. The decision/effector phase works to clarify the signal and open the door for action. Changes that occur in mitochondrial membrane permeability signal the end: the release of an key respiratory protein called cytochrome C into the cytoplasm. The degradation/ execution phase sees the activation of proteases and nucleases, which degrade proteins and nucleic acids, respectively. The key target is the mitochondrion, as well as other important cell machinery. As this is the main power source for the cell, this is typically fatal for it.

Apoptosis acts through signal transduction pathways controlled by receptors of the Fas cluster (CD95), tumor necrosis factor receptor 1 (TNRF1) and death receptors 3, 4 and 5 (DR 3, 4, 5). Each of these kind of receptors contains a special amino acid sequence called the “death domain” that functions as a specific binding site for special death signaling proteins. These proteins pass the baton on to other proteins in the chain, much like a relay. Eventually they activate the main downstream effectors, the actual ‘doers’ of the hard work, the caspases. Caspases are a family of cysteine proteases that are responsible for much of the cellular degradation mentioned earlier.

The point of no return in this deadly relay is the release of cytochrome C, which is controlled by a set of proteins in the mitochondrial membrane that regulate its release. The first of these was identified in a cancer called B-cell lymphoma and was aptly named the B-cell lymphoma-2 (Bcl-2) protein. This particular protein is a negative regulator of apoptotic signals. This group of regulatory proteins contains both positive (Bax, Bak, Bik, Bid) and negative regulators (Bcl-2, Bcl-x) of these signals. Positive regulators encourage apoptosis, whereas negative regulators discourage it. The key factor determining cytochrome C release is the relative ratio of positive and negative signals.

Cancer cells evade apoptosis by overexpression of Bcl-2. The Bcl-2 gene is usually moved to a different, more active location in the chromosome (typically the IgH promoter, a highly active portion). With more of this protein floating around, the pro-apoptotic signals are drowned out and sensitivity to them is significantly reduced. Lastly, cancer cells suppress apoptotic receptors by mutations that affect binding and proper function of the pathway. Slight mutations to the receptors or relay proteins can drastically affect the ability of the apoptotic signal to reach the critical stage.

5. Genetic instability:

Genetic instability is considered a major causative problem in cancers and one that we have few effective weapons against. Our battle strategy is reactive and revolves around destroying the cells that display these characteristics when they become a noticeable problem, which is often the time with the least efficacy in treatment. A truly proactive strategy will require advances in technology and bioengineering that will enable us to manipulate our genetic code, to edit out or silence problematic parts and correct mutations when they occur. The promise of gene therapy holds the hopes of many to pick up and wear that mantle, but these experiments are a ways off from useable therapies. Today though, like any good general, we make do with the tools we have on hand.

Cancer cells are often distinguished from normal cells by the loss or gain of a specific chromosome, or even the accumulation of an entire extra set of chromosomes. Extra chromosomes can provide cancer cells with extra copies of growth promoting genes. They can utilize these extra copies to amplify ordinary levels of signaling, causing the increased growth response and proliferation seen in tumors. Mutation is common in human tumors where changes at the sequence level can affect growth controlling genes, DNA repair or decreased fidelity during replication. Furthermore, it has long been known that misreplicated DNA can provide a causative explanation for some inherited cancer prone syndromes.

Translocations of genetic material from different chromosomes can lead to the abnormal gene expression seen in cancer. Chromosomal rearrangements, for example, are known to cause cancers like chronic myelogenous leukemia (CML) and Burkitt’s lymphoma. CML is caused by an abnormal chromosome, called the Philadelphia chromosome (named after two scientists from Philly). In CML, two unrelated genes from chromosomes 9 and 22 switch places and parts of the genes are spliced together. This causes a new protein called “BCR/abl” to be produced that remains constantly active, driving cell division by activating cell cycle control proteins continuously and inhibiting DNA repair responses.

Fortunately, since CML is caused by a single protein, it represents a good target for drug based therapeutics. New tyrosine kinase inhibitors, like imatinib, are used to block BCR/abl’s activity and has become the standard treatment for the disease over previous less specific antimetabolite chemotherapies and bone marrow transplants. This drug represents a different level of complexity in the treatment of the disease. An intimate molecular understanding of the disease, lead to the search for drugs that could effectively inhibit the protein’s activity. Drug designers searched protein libraries, through thousands of possible candidates to find the best ones, eventually coming up with imatinib.

Imatinib is representative of a new kind of drug design, where a drug is designed from known specifications of the biological machinery. Specific understanding of the processes involved will ideally improve drug activity and binding specificity and hopefully open the door to more effective therapies. Even though it can not cure this disease, it makes management of the disease possible and improves the lives of patients over that of previous chemotherapy based treatments.

6. The Loss of Cell Cycle Control: Until next time…

Some sources:

(1)http://en.wikipedia.org/wiki/Cancer (In epidemiology section.)

(2) Gale encyclopedia of cancer, pg. 11 intro, Helen A. Pass, M.D., F.A.C.S.

(3) http://en.wikipedia.org/wiki/P53

(4) ask me later, lost it…

(5)B. Millauer, M. P. Longhi, K. H. Plate, L. K. Shawver, W. Risau, A. Ullrich, and L. M. Strawn, Cancer Res., 56, 1615-1620 (1996).

(6) 60. N. Ferrara, and W. J. Henzel, Biochem. Biophys. Res. Commun., 161,851-858 (1989).

(7) Burger’s Medicinal chemistry and Drug Discovery 6th ed., vol. 5 Chemotherapeutic agents. Wiley 2003. Pg12

(8) Burger’s Medicinal chemistry and Drug Discovery 6th ed., vol. 5 Chemotherpeutic agents. Wiley 2003. Pg13

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This time our tale begins in the 1970’s with the supposed existence of “slow viruses” as proposed by Bjorn Sigurdsson in 1954 to explain the long incubation period of certain neuro-degenerative diseases called transmissable spongiform encephalopathies. This group includes diseases like scrapie in sheep, BSE in cows (“mad cow disease”) and Creutzfeldt-Jakob disease in humans. Noting the similarities between the above diseases, the hunt was on for the pathogen that caused them. Most at the time believed in the existence of a virus with an extremely long latency as the cause, hence the name ‘slow virus’. At that point, research had failed to find any virus or similar pathogen complicit in the disease. Two British researchers, John S. Griffith and Tikvah Alper noticed that TSE cultures stayed infectious even after being blasted with doses of radiation that would destroy normal DNA/RNA containing samples. The bonds in DNA or RNA are easily broken by this method, whereas the bonds in proteins are generally stronger and more resistant to high levels of radiation. This led them to suggest a new, radical hypothesis: that the disease-causing agent was made of proteins rather than of nucleic acids. This was controversial at the time, because it was widely believed that only DNA or RNA could transfer hereditary information like diseases, a belief typified in Francis Crick’s seminal “Central Dogma”. As such it was ignored and the search went on for a virus or a more acceptable culprit.

In certain parts of Papua New Guinea the disease Kuru reached epidemic proportions and the pathology of it in the Fore people was similar to the others the TSE group of diseases. It shared the same long latency period and caused similar neural damage manifesting as a sponge-like appearance of the brain as neurons died. Daniel Carleton Gajdusek spent several years studying the disease’s progression and performed autopsies on the victims. He developed the theory that the contagion was transferred by blood through open wounds and by eating the cadaver during the Fore’s cannibalistic funeral rites. They would eat the dead of their tribe so their collected spirit would manifest and protect the tribe. The disease was especially prevalent among children and women, supposedly because they got the less prized cuts of ‘meat’ like the brains and organs. This was particularly unfortunate as these portions were the ones most heavily infected with the later discovered pathogen. He transplanted infected tissue into chimpanzees causing the same symptoms over an extended period of time, showing that the diseased tissue could infect healthy individuals. Clearly, this was not an issue of a genetic malady, but a transmissible pathogen. His efforts won him the Nobel prize in Medicine in 1976, even though he was unable to determine the specific entity that caused the disease. This task was left to another scientist a few years later.

Enter the Prusiner

Stanley B. Prusiner had long been involved with scrapie research and developed a strong interest in Griffith and Alper’s previous research. By the late 70’s scientists had still not identified a virus or similar nucleic acid containing culprit. They were at a loss to describe the cause and the obvious similarities in this class of diseases.  The general consensus was that the virus had special qualities that allowed it to evade every kind of detection available. For example, the virus was thought to have such a high resistance to UV radiation, because it encased itself inside a strong protein shell. Suspiciously, it even resisted techniques to detect this and for such a large entity, it evaded electron microscopy as well. Another theory proposed was the exact opposite: that it was extremely small, but this came with its own problems as well. Prusiner grew skeptical of the idea of a mystery virus and his first paper on the subject is truly brilliant. It takes the viral based position and systematically attacks it, clearing away possibilities like weeds in an unkempt yard. He establishes that a protein must be required for infection and describes the qualities it must have to fit the known evidence.

Prusiner’s big leap came when they tried to purify the infectious substance based upon known qualities of the agent in hamster and mice brains, in particular the LD50’s of homogeneous preparations. Using information gleaned from its sedimentation profile they found that the agent in question would have to fall within a small range of sizes and weights. Given these constraints, it was shown that even if the supposed virus was contained inside a protein shell, its nucleic acid content must be quite small and “too small to code for a protein.” Later, he used Sarkosyl aragose gel electrophoresis to separate the proteins from the nucleic acids in the purified samples. The only samples that maintained their toxicity were those that contained protein. This was useful information and fueled further searches and the narrowing of possibilities.

Solubility is a central concept in chemistry, as such Prusiner invested a lot of time to discover what it was soluble in leading to even more clues to its identity. He used a variety of compounds that typically dissolve nucleic acids, but achieved mixed results. He found many of the compounds used were ionic or suggested that a negatively charged compound was at the root of the issue. The amino acids in proteins have negatively charged end groups, so this seemed to suggest, albeit inconclusively, that it was a protein. Curiously, he found that nucleases had no effect on the agent as would be expected of a virus containing them. In light of this, he tried to digest the preparation with protease K, an enzyme that inactivates proteins, finding a positive result. He repeated the same experiment with trypsin, another protease, with the same results. This was strong evidence that the compound in question was, in fact, a protein. Similarly, he tried to find what chemicals would deactivate its toxic properties. He found that chemicals like phenol and urea, both very strong protein denaturants, destroyed the infectivity of the purified preparations. He also tried to reverse the reactions to attempt to restore the toxicity without success, showing that what happened to the solution wasn’t a predictable A + B = C type reaction, but the product of a reaction the destroyed the toxic essence of the chemical in question.

The information above shows that a protein must be necessary for infection. After establishing these findings, he promoted a new idea: the cause of these diseases is not a virus or other similar entity, but rather a new infectious agent. “The molecular properties of the scrapie agent differ from those of viruses, viroids and plasmids. Its resistance to procedures that attack nucleic acids, its resistance to inactivation by heat, and its apparent small size suggest the scrapie agent is a novel infectious agent.” This novel agent later came to be known as a prion. “Prions are small proteinaceous infectious particles which are resistant to most procedures that modify nucleic acids.”

Not only did Prusiner establish a protein-based mechanism for the disease, but two years later he also purified the protein involved. Toxic prions were discovered to be an evil doppelganger of a naturally occurring protein, which purpose is unknown, but thought to have a basis in the maintenance of memory and certain stem cells. The toxic protein is misfolded and by nature of its poor shape induces similar proteins to bind and form insoluble clumps of protein called amyloid-beta plaques in neural tissue. This could explain its apparent ability to reproduce without a set of nucleic acid instructions and its long latency period.

Recent research has connected these plaques with a wide range of diseases, including the big one: Alzheimer’s disease. Researchers Adriano Aguzzi and Stephen Strittmatter have published a study of the damaging effects of smaller, semi-soluble plaques on neurons in mice. They showed that the prion proteins are necessary for the damaging effects of the semi-soluble plaques. “Researchers removed the prion protein middleman from mice and examined brain slices. When the team washed A-beta oligomers* over the brain slices, the oligomers no longer had an effect on cell activity in the hippocampus.” In a similar study, other researchers got the same results using an antibody primed to block the section of prion protein that binds to the A-beta oligiomer. Without the prion proteins the damaging effects of the disease weren’t seen. “Blocking prion protein binding may be a new therapeutic target for Alzheimer’s disease. Get rid of the prion protein middleman, or its ability to bind A-beta oligomers, and get rid of the disease”, said Strittmatter.

Exciting times these are, perhaps….

1.http://www.sciencenews.org/view/gene…80%99s_disease
Pirons may be complicit in Alzheimer’s disease article

2. http://www.pnas.org/content/95/23/13363.full  
P’s 1991 reiteration

3.http://www.idm.pitt.edu/IDM2004LecturePPT/Prusiner.pdf.
Prusiner’s first prion paper

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Back in the early days of photosynthesis, the oxygen released into the atmosphere created an oxidative environment. This converted more soluble forms of iron into the familiar orange-red, insoluble Fe+3 we see on a daily basis as rust. Iron is needed by microbes to support all sorts of biological processes, like respiration, but in the Fe+3 state it is hard to acquire and even harder to manipulate in a useful manner. Its use in cytochrome proteins of the electron transports system should underline its importance in this role. Further, in mammals the element is tightly bound to proteins such as the famous hemoglobin in red blood cells and transferrin, a mammal iron scavenger. This sequestration of iron creates a bactericidal environment inside the body and forces the invading bacteria to compete for sources of iron to fuel their biological processes.

Since it is critical to the growth and function of bacteria, especially pathogenic bacteria, it is not surprising that they have developed a variety of means to acquire this element. Some of their negative effects on a host organism directly result from this scavenging activity. Some produce proteins called hemolysins to puncture cells and release their contents, and/or produce compounds called siderophores, which are iron scavenging compounds used to leach iron and iron containing proteins from the environment.

These compounds are ubiquitous throughout the microbiological world and are usually produced under conditions of low iron concentration, like those of sterile sites in the body. While finding bacteria in cultures from skin and naso-pharyngeal swabs is not necessarily an indication of disease, finding bacteria in cultures taken from cerebral spinal fluid and blood (etc) is considered an important point in diagnosis. The skin functions as a barrier to infectious microbes, but it can be breached by pathogenic critters. Essentially, for entry into the body a microbe (or chemical) will have to cross a tissue called an epithelium. Hemolysins and cellular matrix degrading proteins are used to damage this layer and gain entry into the body and access to essential nutrients like iron. Siderophores are used to scavenge the iron from the contents of dead cells.

Siderophores vary widely in structure per species, but one thing they have in common is their preference for Fe+3. Fe+3 ions are unlike the typical organic ‘binds 4 times’ carbon atoms. Iron has a variety of oxidation states available to it and the Fe +3 state has six coordination sites arranged in the shape of an octahedron. The siderophore binding site usually accommodates this by being shaped similarly and employing atoms that also have a strong affinity for iron, specifically oxygen. Often, they employ functional groups such as catecholates and hydroxamates that can bind strongly to ions in general. These are usually arranged in such a way as to virtually guarantee that Fe+3 will be the only ion taken in by the protein. Enterobactin is a prime catecholate example of this set up, with is produced generally by enteric bacteria like Escherichia coli. Ferrichrome is another well known example of a hydroxamate compound. The binding affinity for these kind of +3 ions is very high (Ga+3 binding is also very strong), often selectively much more than +2 ions like Al+2. The fact that there is a big difference between the binding efficiency of these two oxidation states affords an efficient means of releasing the iron once it has been captured. Once the bacteria have the iron, they can reduce it to a more soluble and useful Fe+2 state.

Most bacteria have specialized receptors to accommodate these proteins, but this fact also leaves them open to attack. Bacteriophages, bacteriocins and antibiotics have been known to utilize the ferrichome transport receptors for entry into the cell. Bacteriocins are a large class of bacterially produced antibiotics used to compete with other similar strains of bacteria. Some of these have found use in medicine and agriculture to inhibit some pathogenic strains of bacteria in humans and livestock. More to the point, a new thought in antibiotic production is using these siderophore functional groups to cause their uptake by bacterial iron transport systems. The high specificity for these groups and the difficulty in changing the receptor’s structure by mutation (mutation may cause less specificity and inhibit the uptake of a vital resource) make this an interesting tactic.

By attaching a sidereophore moiety to the skeleton of an ordinary antibiotic, its potency can be increased and the effective dose lowered. In the case of attachments to the cephalosporin class of antibiotics, this effect has been studied and proven since the late 1980’s. Studies have shown that experimental catecholate cephalosporins have lower minimum inhibitory concentrations, than comparable antibiotics (frontline ceftriaxone and/or ceftazidime) making the effective dose lower than ordinary antibiotics.

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A molecular understanding of the human body and its processes has enabled us to produce relatively effective treatments for diseases that 100 years ago were death sentences for those who had the misfortune of getting them. One of the biggest discoveries to come out of this in depth study in the last 30 years was the knowledge of G-proteins and their regulation of receptor signaling. This led directly to new drug treatments that paved the way to making diseases like cancer survivable and manageable to a certain extent. A direct understanding of the processes led to treatments that were focused on blocking specific targets and for the most part had less severe side effects than their earlier chemotherapeutic counterparts. Recently, there has been a flurry of research on the new discovery of micro RNAs. As a direct importance to genetic therapy these hold great promise to produce further advances and more effective treatment of diseases like cancer and developmental disorders. Micro-RNAs are involved heavily in the regulation of messenger RNA transcripts and the repression and silencing of genes. They also may hold the key to specifically targeting cancer cells and thus may allow for more effective diagnosis and treatment.

Any discussion on this topic must begin with the process of gene transcription. This is the process of converting the information in our DNA into the instructions RNA ribosomes can use to produce the proteins at the center of system control. Our genome is written in DNA with the familiar sequence of nucleotides thymine (T), guanine (G), cytosine (C) and adenine (A). However, before this can be turned into proteins, it must first be converted into RNA. The major physical difference between DNA and RNA is that the thymine (T) nucleotide is replaced with uracil (U). During the process the DNA strand is unzipped and an enzyme called RNA polymerase reads and converts it into an RNA copy of the information called mRNA, or messenger RNA. mRNA is used by ribosomes to manufacture proteins from the raw amino acids in a process called translation. Here is where micro RNAs come in. MicroRNAs are small (~20 nucleotide long) sequences that match portions of the mRNA products of certain genes. These bind to the mRNA and can either promote translation or stop it from happening and cause degradation of the mRNA. In this way, miRNAs have the ability to regulate gene expression and other important cell processes.

miRNAs were first discovered in a species of flatworm commonly used in genetic experiments called C. elegans(1). Scientists were looking to sequence their genome and found that these small pieces of RNA seemed to have regulatory functions in some of their gene expression. At the time it was thought to be an idiosyncrasy of the species, but the same finding began to pop up in other animals as well. Eventually, they were discovered in the human genome and it was shown that these are highly conserved in most animals. To date, we have discovered about 550 of these in humans and it is probable that there are many more. They are thought to correspond with approximately 60% of human genes and have been found to be involved with the regulation of developmental timing, cell differentiation, cell fate, aging, metabolism and the cell cycle just to name a few critically important processes. It is an understatement to note that when these systems receive aberrant signaling caused by a mutation of some kind the result is usually disease.

Recent studies have shown that all cancers have alterations in their micro RNA expression and in general miRNA genes map to known areas of cancer causing mutations, especially those of tumor suppressor and oncogenes(2). This finding is big news, because at the very least this could hold a path to more efficient diagnosis, quicker testing and more effective therapeutic intervention. One of the major problems with cancer therapy is diagnosis is often late stage, and at the time when our current collection of drugs is the least effective. Too little, too late. The ability to selectively differentiate between cancer and normal cells has been long sought after for this reason and it’s possible that they may have found a good method with miRNAs. Numerous studies have shown connections between irregular miRNA expression and specific kinds of cancer. For example, Calin et’al published results in the NEJM showing mutations in miR15a and miR16-1 are associated with CCL and breast cancer(3). They later noted 13 different miRNAs were linked to specific kinds of cancers and individual expression patterns correlated with CLL prognosis. Furthermore, Yanaihara et al showed that lung cancer prognosis is identifiable by its miRNA signature(4). They found that mutations involving hsa-miR-15 had almost a 3.5 fold increase in pathology and mutations to hsa-let-7a-2 had greater than 2 fold increase compared with other mutations.

It’s good to be able to identify who has cancer, but the whole point is to help those stricken by it. Aside from better diagnostics, this knowledge of miRNAs could also lead to better therapeutic drugs. In cancer cells miRNAs follow two patterns of expression: over-expression and down regulation. Over-expression patterns usually function to promote a stem cell-like state where uncontrolled proliferation is established outside of the normal structured growth. Y. Hayashita in 2005, showed that over-expression of miR-17-92 enhanced lung cancer cell proliferation (5). By using drugs that inhibit this activity, it may be possible to pull a cancerous cell back in line with normal function. The hopeful result in this case is apoptosis of the rouge cells and reduction of the tumor. Drugs that bind to and block the function of over-expressed miRNAs can be specifically designed to match the miRNA sequence, leading to therapies that favor the cancer cells over normal cells. H. Matsubara in 2007 published a study using antisense oligonucleotides to attack over-expressed miR-17-5p and miR-20a in human lung cancer cell lines (6). Antisense means that a complimentary copy of the mRNA had been made that matches the opposite ‘sense’ coding piece of the miR-17-5p. When a ‘sense’ copy interacts with an ‘antisense’ copy it blocks translation of the mRNA script stopping it from being copied at the ribosome. Without the extra miRNAs suppressing the cell’s normal function, the chemical induced apoptosis in the cells, destroying the tumor.

The opposite route with miRNA down regulation also has a variety of possible therapies. Because of the small size of miRNAs (only ~ 22 nucleotides long) it may be possible to use viral vectors to reintroduce the missing or blocked components into cancer cells (7). This tactic has recently been shown successful in proof of concept studies for the treatment of Duchenne muscular dystrophy. In the study, using knowledge gleaned from mutations of the dystrophin gene (the gene producing the critical disfunctional protein in the disease), they trimmed down and produced a smaller version of the gene that was just able to fit inside an adenovirus capsid. The gene had enough functionality to replace the activity of the broken protein in affected muscle cells. In mouse studies it had enough effect to reduce symptoms to a less serious variant of the disease: Becker’s muscular dystrophy. In this way, miRNAs could be used as the payload in a viral capsid or other similar vector and reintroduced into rouge cells. Reintroduction may replace the regulatory function and protective action of the miRNAs in question and hopefully cause target specific apoptosis of damaged, tumorous cells.

The ability to discern the fundamental difference between cancer cells and normal cells has long been seen as the holy grail of cancer research. It was thought that this could lead to quicker diagnosis and targeted, more effective therapies. It is very possible that the patterns of miRNA expression found in cancer cells could hold the key to these kinds of therapies, but in any case more research needs to be done before this yields benefits in clinical medicine. The is a long road ahead to prove that these techniques will not only be safe, but also controllable in a clinical situation.  Time will tell if miRNAs will live up to their promise, but at the very least a small piece of the puzzle has been discovered.

1. Lee RC, Feinbaum RL, Ambros V (December 1993). “The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14”. Cell 75 (5): 843–54. doi:10.1016/0092-8674(93)90529-Y. PMID 8252621.
2. A large list of papers: Calin et al, PNAS 2002; Lu et al, Nature, 2005; Volinia & Calin et al, PNAS 2006; Landgraf et al, Cell 2007, MicroRNA genes map to cancer loci. Calin, G.A., et al.,2004. PNAS101:2999-3004 (specifically)
3. Calin et al, N Engl J Med, 2005; Raveche et al, Blood 2007 Germline abnormalities in miR15a/miR16-1 transcripts are associated with CLL and breast cancer aggregation.
4. Yanaihara et al, Cancer Cell, 2006 A unique miRNA signature is associated with lung cancer prognosis
5. Hayashita Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res. 2005 65(21):9628-32.
6. Matsubara H, et al. Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR-17-92. Oncogene. 2007 Mar 26;
7. Wang CL, et al. Activation of an oncogenic microRNA cistron by provirus integration. Proc Natl Acad Sci U S A. 2006 103(49):18680-4.

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