by Jerin Mathew, Raunak Mukherjee, Lloyd Morgan and David López-Lázaro
Figure 2. An image showing oversized polytene chromosomes22
Can heat shock proteins be the answer to molecular deformation, aging, cancer and spinal cord injuries?
A research paper based on study and work on genetically modified organisms and heat shock proteins
The world of science is constantly evolving, month to month, year to year. New discoveries are being made and new techniques are being discovered. In this research paper we will be discussing the impact of a genetically modified organism (GMO) that we have created and formed as a group called DJLR55. The main aim was to tackle a variety of issues including: injuries (mainly ischemic), ageing, cancer, molecular deformation and spinal cord injuries. Nevertheless, what we found was that heat shock proteins (HSPs) are arguably the most versatile proteins ever discovered in the medical field; it can be used to treat/prevent some of the biggest mysteries in the scientific world. Although there are various ethical dilemmas and obstacles that prevent the imminent approval of a treatment like ours, there is definitely a foundation for future developments to occur.
Immortality is a phenomenon that humans have strived to achieve since 3000 BC. Through designing DJLR55, we believe we are now one step closer to making this dream into a reality. This research paper will be comprised of the applications and adaptations of DJLR55 (Figure 1). The GMO we have created consists of a strong promoter11 called J23100. As a result it can always express the gene no matter what the conditions. It is also made up of HSPs, meaning that in certain cases (as explained throughout this paper) it will be expressed to carry out a function - this is all joined to a backbone called pSB1AC310 which has a high copy number of 100-300 per cell. The ribosome binding site (RBS) is called BBa_B0030 and affects the number of copies of the protein that is produced12. The RBS in DJLR55 has a strength of 91.84% relative to B0034- meaning it produces a relatively large number of copies. Finally the terminator used in DJLR55 is called a Rho-independent terminator13. This is a typical terminator that works by creating a hairpin loop to end the transcription process.
Figure 1. Shows a diagram of the structure of the GMO DJLR55
To begin with, a GMO is described as the adaptation of an organism by changing its genetic structure either by inserting genes from another species, the ‘deletion’ of genes or by the mutating of the genes in order to create an organism that has the favourable qualities the researcher is looking for. This process can occur in nature without the influence of man or can be created in a laboratory environment where a syringe is used to insert the DNA into the nucleus of the host. Another way in which this can be done is where the DNA of one organism is transferred to that of another organism through an electric pulse (also known as electroporation2).
The thought of modifying the DNA inside an organism had occurred well over three decades ago. For instance, the first recombinant DNA molecule (where the DNA would have been separated from two organisms and then pieced back together to form one double helix molecule) was thought up and created by Paul Berg, (who constructed this by combining the DNA of a monkey virus and that of the lambda virus). Nonetheless this discovery was short lived; as little over a year later, Boyer and Cohen created the first ever GMO by creating a bacterium that was able to survive in the presence of Kanamycin (a type of antibiotic). Since the mid 1970’s there have been many advancements in the field of GMO’s- the making of genetically modified plants is a prime example.
As aforementioned, there have been a lot of advancements within the past few decades in this field. However we are now at a stage where these GMO’s have been created and can treat a myriad of problems the human body may face. In fact DJLR55 is specialised to help prevent aging, cancer and the protection of cells from stress and other injuries as well as regulation of the immune response. This is done by producing HSPs which are then used in research studies to help further the scientific knowledge in the topics mentioned above.
According to the works of Tower, (2010) ‘Heat Shock Proteins and Drosophila Aging8’, and Benjamin-McMillan, (1998) ’Stress (Heat Shock) Proteins1’, Drosophila melanogaster is a fruit fly that responds to heat and oxidative stress and one that produces oversized polytene chromosomes, shown in Figure 2, in their salivary glands in these conditions as mentioned above21. It rapidly transcribes HSP 70 to enable enzymes and other proteins to remain intact under stressful conditions20.
Figure 2. An image showing oversized polytene chromosomes22
The mechanism for this rapid transcription arises from many transcription factors (a transcriptional activator) that bind along with RNA polymerase to the promoter DNA sequence to initiate the process of transcription. As humans are homeotherms that keep a constant inner body temperature that is contained within a finite range in normal conditions, small changes in the environmental surroundings may start to affect the thermoregulatory systems within body that allows homeostasis to be maintained9. Critically enzymes may start to become denatured that help catalyse vital chemical reactions in a fraction of the time taken for it to occur in the absence of an enzyme. This is where the heat shock protein gene acts best as it can reform the tertiary structure of enzymes that may have started to form bonds in different places.
The GMO is adapted to this as the plasmid can replicate up to 300 times per cell10, providing a facility for rapid transcription to take place through a greater number of coding sequences. Hyperthermia occurs when the internal body temperature is higher than usual, around 105°F, and can be severely damaging due to the systemic nature of the illness with the brain being very vulnerable to damage as well as heart rate being elevated and it can bring on other symptoms such as nausea and seizures15. We can supply hyperthermia patients that may have suffered heat strokes or fever brought about by infections with a treatment containing the heat shock protein gene that allows it to be rapidly transcribed similar to the Drosophila fly. The fly acts as a good source of the protein but does involve the ethical dilemma of using the lab cultured organism for the sole purpose of human treatment but overall Drosophila is a commonly used species that has been applied in many areas of genetic research already such as the findings of Nüsslein-Volhard, Eric Wieschaus, and Ed Lewis in 1995 that concerned how embryos mature from single undifferentiated cells into mature multicellular organisms17. This precedent suggests that the practice of using the Drosophila melanogaster species is acceptable.
There is also the issue as to whether fevers brought about by simple infections that can be overcome in a short period of time by the immune system require treatment at all and whether the potential cost of developing treatment outweighs the benefits to the patient. According to the works of Kenny et al, (2010) ‘Heat stress in older individuals and patients with common chronic diseases’4, certain groups are at high risk of developing heat-related illnesses such as patients affected by cardiovascular and respiratory diseases as well as those with diabetes mellitus and suffering from obesity. The high risk stems from the reduced ability to maintain a stable metabolic activity due to compromises in activity so this in turn leads to difficulties in being able to maintain homeostasis and thermoregulate the body correctly. The heat shock protein can be implanted into these patients to ensure that the body’s main functions are not affected by sudden changes in environment or moments of stress due to the chaperone nature of the protein. However the welfare of the patient must be considered foremost and if patients are already being affected by a particular disease and are on medication for this, it might not be recommended to provide the additional treatment involving the genetically modified organism as the backbone of the GMO, pSB1AC38, shows resistance to two antibiotics, ampicillin and chloramphenicol, so using the plasmid would severely affect a patient that is for example suffering from a bacterial infection like meningitis.
Previous research in this field has also found that HSPs can reverse the effects of aging. Firstly, aging is primarily caused by oxidative stress and the build-up of misfolded proteins. The key evidence for HSPs helping to stop aging was discovered in fruit fly (Drosophila melanogaster); as the HSPs induced a “puffing pattern of polytene chromosomes in response to heat and oxidative stress.” (Ritossa, 1962, 1996). However HSPs can bind to denatured proteins and can change the structure of proteins as a result of heat and oxidative stress which can cause the protein to denature. Furthermore, there are different types of HSPs- each of which can target specific cells. Hence in terms of real life application they can be used in different parts of the body. After several laboratory experiments it was found only particular families of HSPs could help increase the lifespan of fruit fly6. Nonetheless the findings have been too inconsistent to make strong conclusions from. When lab experiments were conducted on other types of animals eg. mice, HSPs that worked on one mice would not work on another. In that light it is fair to say that although there is evidence that HSPs can help increase lifespan, we are far from reaching a stage where strong conclusions can be made.
As a result, many ethical issues arise from the fact that we could potentially reduce the effect of aging in a population- the first of which being who exactly would benefit from this treatment? To many it may appeal as a way of looking younger and more attractive and whilst it may also be able to prolong people's lives, it doesn't by any means mean that it will be a content one. It's quite possible that due to their lives being lengthened many people will start to feel depressed or lonely; they may have to watch others around them pass away. Furthermore, the introduction of this type of technique will most definitely result in social tension. As the question would arise of who would be able to obtain this method of anti-aging and prolonging of life? It isn't yet a reality as to how expensive this could be however it will be clear that based on past occurrences, a new revolutionary treatment would be very expensive and so it wouldn't be accessible to everyone as not everyone would have the money. This can be seen as morally wrong as you are in a way hinting at the fact that only the rich will be able to access what is essentially the “fountain of youth”. Therefore attempts to exploit the anti-aging ability of HSPs could be hindered in the future as a result of the ethical issues discussed above. On the contrary to what has been mentioned, there would also be many positive outcomes to such a treatment being created. For instance, it could mean that getting older doesn’t necessarily mean the start of the stereotypical wrinkles or aches and pains. In a world where self image seems to be everything, improvements in terms of how we look and for example the prevention of wrinkles would be a revolution as we would appear younger and more attractive than ever. This could also lead to people having a better quality of life as we are now at an age where the number of people going under the knife is almost tripling every year since 2002.
Immunity and Cancer
When looking into immunity and HSPs, the one area which HSPs could have a real benefit is looking at how antigens are presented to the immune system by the HSPs. Also HSPs chaperone antigens, helping them be presented to receptors on white blood cells, such as macrophages7. One particular benefit from this process is the ability use HSPs in vaccines, which have already been done in some cancer cells. HSPs are particularly useful in treating cancer because cancer cells produce more HSPs3, giving researchers a target to induce an immune response against the cancer cells.
Pramod Srivastava Ph.D., has shown that HSP vaccines do work14. He showed that the HSP-peptide complex from tumour cells can interact with the antigen on macrophage cells, by the HSPs binding to a receptor (CD91) on the macrophage, escorting the peptide to the macrophage14. The macrophage then presents the antigens of the tumour cells14. The macrophage then activates an immune response using these antigens, destroying the cancer cells via the necrotic pathway, a mechanism that destroys damaged cells14.
Moving on from this study, one hypothesis could be that more cancer HSPs vaccines could be developed, targeting a range of different cancers. This can have huge implications over oncology. One benefit is reduced radiology and chemotherapy rates due to less patients getting to the stage where they require these treatments, which would reduce the number of patients experiencing side effects associated with these treatments. As well as this, vaccines could prevent cancer going to advanced stages, nipping the cancer cells in the bud as only a few cells being dealt with by the immune system.
However, when looking at the disadvantages of vaccines, the main issue is the practicality. There are many different strains of cancer, with new strains being found regularly. An example of this is acute myeloid leukaemia, where it is found that it is made up of 11 different strains5, each presumably with separate antigens which will respond differently to a HSP vaccine and so will require different research to make sure the vaccine is safe. When scaled up to all the different strains of cancer that is hundreds, if not thousands of strains each with potentially different antigens and so different effects from the HSPs. In order to treat each one, the vaccines will undergo vigorous testing that can take years, with no guarantee that the vaccines will be effective. That is not to say that this research should be discounted, but should be viewed with this in mind. Potentially, some vaccines could be prioritised over others, due to the number of people affected and potential for success.
As well as this, even if the vaccines are developed, the sheer number of them would mean that we could not hope to vaccinate the entire community. This means that we would have to prioritise certain areas of the community for certain vaccinations. Of course, one potential use of the vaccines is in patients that have the specific cancer, in order to prevent recurrence of cancer and to potentially treat the cancer. But as well as this, at risk groups e.g. over 65s, those with history of certain cancers and those genetic conditions that increase their likelihood of cancer18.
Finally, in the age of austerity where funding for the NHS is uncertain and NHS trusts with huge deficits, we have to look at how much these vaccines will cost. If the vaccines costs are high, then we will have to discuss how much are we prepared to fund these vaccines. However, the costs in the vaccines could be saved with the reduced later stage intervention in cancer. In the long run, the prevention will save money24 and so be effective in the long run, as long as the success rate is high.
Spinal Cord Injuries
Another ability of HSPs is being able to help the body recuperate following the event of a spinal cord injury. Normally spinal cord injuries are composed of a primary and secondary injury19, the primary being the actual cause of the injury which may be due to a fall or an impact up on the spine causing the damage or dislocation of the spinal cord23. The secondary impact is where the body attempts to fix itself from the injury that has taken place. However, it isn’t as simple as it appears to be, during the primary injury a variety of things can happen for example a fall may cause the spinal cord to press down upon the nerves “pinching” the nerves19, whilst on the other hand depending on the way the injury has taken place the patient may suffer from lack of blood or circulation reaching and flowing through the spinal cord leading to bleeding within the actual cord due to a build up of the blood in the vessels16. It is in this secondary injury that the heat shock proteins are involved, their aim is to regenerate and repair the damaged proteins that have been affected (this is where their name “protein guardians” originates from as they are a type of guardian that helps repair the damage made)6. They do this as they are expressed into the bloodstream via the stressed endothelial cells6 that then leads to the spinal cord which then act as “inflammatory mediators”6. Furthermore it has been scientifically shown that they contribute to the protection of the motor neurons in the spinal cord and prevent them from being inflamed.
In summary, our GMO has the possibility of being tested in many areas of medicine due to the versatile nature of the HSP gene that is always expressed due to the organism containing the strong promoter. DJLR55 can be applied in several exciting fields and we have dedicated our research to applications in extreme conditions, anti-aging, immunity and preventing spinal cord injuries.
HSPs can potentially be applied in extreme conditions as the chaperone function of the HSP means that during periods when cells and enzymes undergo extreme stress in abnormal conditions such as hyperthermia, stability is maintained by the presence of the HSP. This is particularly important in groups of high risk like those affected by cardiovascular disease. Even though there are various obstacles to the use of DJLR55 and the practicality of using it when there may be a simple infection or even endanger patients using other treatments, the benefits are much greater than the hazards as people with thermoregulatory issues could possibly experience fewer effects from their disorder so remarkable developments like this is surely something that deserves to be explored in further detail.
Despite initial trials on the anti-aging properties of HSPs having been promising- the results are too inconsistent to make strong conclusions from. However, as long as future research in the field is not hindered due to the various ethical issues outlined in this paper and the appropriate funding is provided- then we can advance far more quickly in this field than one would assume.
HSPs also have significant applications in immunity. Through HSP vaccines, there is the potential for vaccinations against cancer that could revolutionise the way we treat cancer. However, there are significant hurdles that we need to overcome both in research and in clinical institutions (NHS) which could seriously threaten the legitimacy of these vaccinations. But, hopefully we could have a new tool to tackle cancer.
The GMO can also be applied in the field of sport injuries, specifically spinal cord injuries that could be severely damaging and lead to paralysis. In these injuries, there is a primary injury caused by a fall or trauma to the body and a secondary injury involving the body trying to repair itself. HSPs can help reform damaged proteins and minimise inflammation so reduces the extent of the symptoms that the patient suffers and thus could prevent life threatening injuries having as significant an impact as previously.
Although there are many barriers to DLJR55 becoming an imminent treatment, there is a concrete basis for even more investigations being conducted into GMO’s being a safe and effective therapy in the coming future. We explored various ethical dilemmas such as cost of treatment vs benefit to patients during our reading and have incorporated this into our discussions along with our ideas as to in what ways DLJR55 can be applied appropriately.
1. Benjamin, IJ; et al. (1998). "Molecular Chaperones in cardiovascular biology and disease." Circulation Research 83: 117-152 http://circres.ahajournals.org/content/83/2/117.full
2. Calderwood, SK; et al. (2012). "Heat Shock Proteins, Autoimmunity, and Cancer Treatment." Autoimmune Diseases. 12,10: 1-8 http://www.hindawi.com/journals/ad/2012/486069/
3. Ciocca, DR; et al. (2005). "Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications". Cell Stress and Chaperones 10,2: 86-103 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176476/
4. Kenny, GP; et al. (2010). "Heat stress in older individuals and patients with common chronic diseases". Canadian Medical Association Journal 182: 1053-1060
5. Papaemmanuil, E; et al (2016) "Genomic Classification and Prognosis in Acute Myeloid Leukaemia". The New England Journal of Medicine 374,23: 2209-2221
6. Redy, SJ; et al. (2008). "The role of heat shock proteins in spinal cord injury". Journal of Neurosurgery. 14: 147-153. http://www.ncbi.nlm.nih.gov/pubmed/19695121
7. Srivastava, PK; et al. (1998). "Heat Shock Proteins Come of Age: Primitive Functions Acquire New Roles in an Adaptive World". Journal of Immunity 8,6: 657-665.
8. Tower, J; (2010). "Heat Shock Proteins and Drosophila Ageing". Experimental Gerontology 46: 355-362
10. "Backbone". HotZoc. 2016. http://hotzoc.com/project/backbone/
11. "Promoters - Strong Promoters". HotZoc. 2016 http://hotzoc.com/project/promoters/
14. "Heat Shock Protein Vaccine: How Does It Work?". Journal of the National Cancer Institute. 2 January 2002
15. "Hyperthermia". Medicine Net. 16th October 2013. http://www.medicinenet.com/hyperthermia_symptoms_and_signs/symptoms.
16. "Definition of ischemic". Merriam-Webster. 29 June 2016 http://www.merriam-webster.com/dictionary/ischemia
17. "The Nobel Prize in Physiology or Medicine 1995". Nobel Prize. 9 October 1995 http://www.nobelprize.org/nobel_prizes/medicine/laureates/1995/press.html
18. "Risk Factors for Cancer". National Cancer Institute. 23 December 2015 http://www.cancer.gov/about-cancer/causes-prevention/risk
21. "Polytene Chromosomes- Sedat Lab at UCSF". University of California San Francisco. 19 August 2003 http://msg.ucsf.edu/sedat/polytene_chrom.html
22. "Figure 2. Enlarged image of polytene chromosome". University of California San Francisco. 19 August 2003 http://msg.ucsf.edu/sedat/polytene_chrom_enlarge.html
23. "Spinal Cord Injury". University Of Southern California. Last updated: Unknown. http://www.uscspine.com/conditions/spinal-cord-injury.cfm