MRC National Institute for Medical Research, London

Nietzsche once said, ‘That which does not kill us makes us stronger’. Nowadays, people would vehemently disagree with this philosophy, cite horror-story articles about fatal cat diseases and contaminated foods, while squirting hand-sanitizer by the bucketful. However, this was the thinking of previous generations, where a little dirt never did any harm, children played with mercury like playdough (ignorant of its poisonous properties) and if there was a little mould on top of the jam, you scraped it off. It’s certainly the philosophy behind the hygiene hypothesis.

The hypothesis postulates that the industrialized age’s increased use of vaccines, antibiotics and general hygiene standards prevents the body’s natural development of the immune system, causing an immunological imbalance [1]. The body then reacts inappropriately to harmless antigens. The main suggested mechanism behind this hinges on T-helper (Th) cells.

Th cells differentiate into two subtypes, known as Th1 and Th2 cells, which produce different cytokines (cell-signalling proteins). Th1 cytokines are produced in response to intra-cellular antigens, such as bacterial and viral antigens, as well as causing certain organ-specific autoimmune diseases such as Type-I diabetes and inflammatory bowel disease (IBD). Th2 are produced in response to extra-cellular antigens and are responsible for allergic reactions, including asthma. When an allergen (with foreign, but not necessarily harmful antigens) enters the body, Th2 cells stimulate production of B-cells that differentiate into plasma cells (humoral response). They then produce IgE, an antibody that binds to receptor molecules on mast cells (in body tissues) and basophils (in blood) that trigger the release of histamines and leukotrienes, that cause symptoms of allergy, such as airway constriction in asthma. An excessive Th2 response causes Type-I hypersensitivity, leading to atopy, a predisposition to allergic reactions. Therefore, the hygiene hypothesis postulates that a lack of exposure to bacteria and viruses early in life (i.e. under-stimulation of the Th1 response) polarises the immune system towards a more dominant Th2 response later in life (i.e. a tendency towards an antibody-driven response).

The hygiene hypothesis has been controversial. When first proposed by David P. Strachan in 1989 [1], it was met with scepticism: childhood infections were known to contribute to the risk and severity of asthma, so could they protect against it? But with allergies and other auto-immune diseases, particularly asthma, becoming more common [2], more evidence for the hypothesis has emerged. The inverse relationship between hay-fever prevalence and family size was originally noticed by Strachan [3]. A 2011 study reported that the whey protein fraction in milk might be responsible for the ‘protective’ effect, with children who drink raw milk (in comparison to those that drink supermarket milk) having a 41% lower risk of developing asthma [4].

There has also been a recent study demonstrating a potential mechanism for the hygiene hypothesis at work in mice, published recently in Science [4]. One group of mice was kept in a perfectly sterile environment and given antibiotics, while the other was left to normal microbial contact. The ‘sterile’ mice developed inflammation in the lungs and bowel, the rodent equivalent of human asthma and IBD (irritable bowel syndrome). The inflammation was caused by increased levels of iNKT (invariant natural Killer cells); a specialized white blood cell and, crucially, iNKT levels and inflammation did not decrease when the mice were exposed to microbes later in life. It is clear from this, that the higher numbers of iNKT could be similar to overactive Th2 immune responses. This marked an exciting step forward in immunology, but also raised two problems. Does the same mechanism apply to humans? And if so, what specific microbes have a ‘protective’ effect?

Firstly, humans and mice are very different; humans have much greater genetic diversity. Extrinsic conditions can also be easily controlled in a lab: not so for humans. Overall it is highly unlikely that the level of ‘hygiene’ imposed experimentally on mice could ever be replicated for humans and therefore the proposed mechanism must be taken with a pinch of salt.

Secondly, there is much evidence to support the view that allergies are not caused by particular microbes, but that the immune response is shaped by the sum total of its immunological experience [6]. It is the cumulative pressure of constant exposure to pathogens, in diet, in the air, from runny-nosed siblings in the general humdrum of life during the formative years that develops the immune system. Indeed, efforts to try and pin down a particular childhood infection that protects against allergies have failed, despite accounting for family size and older siblings [6].

Importantly, there is epidemiological evidence against the hygiene hypothesis itself, with Th1-mediated auto-immune diseases such as Type-I diabetes, multiple sclerosis and IBD rising at the same time as Type-2 mediated allergies. The common factor between them is the lack of immuno-regulation: it is not an imbalance of Th1 and Th2 immune responses, but the over-activity of both types that causes chronic inflammatory diseases.

Enter the ‘Old Friends’ hypothesis [7], a variant of the hygiene hypothesis, which brings evolution centre stage: by suggesting some microbes have evolved that provide a ‘background hum’ that help us produce an effective immuno-regulatory network. These ‘old friends’ that stimulate the activity of T regulator cells were once universal, but significant changes in domestic life, the lack of farm animals, better healthcare and vaccines, we have been deprived of their benefits. The hypothesis has proven so successful, there are multiple studies and trials using helminths, parasitic worms that work as ‘old friends’, as a form of treatment for the auto-immune diseases mentioned previously [8,9].

If these clinical trials validate the ‘old friends’ hypothesis, then we may indeed be too clean for our own good. Medicine would then need to find ways to selectively reintroduce these benign microbes. Or, as Douglas Adams so presciently put it in the ‘The Hitchhiker’s Guide to the Galaxy’, ‘So doctors will be back in business, recreating the diseases they had abolished, in popular easy-to-use forms’.

References

1. Strachan DP (August 2000). Family size, infection and atopy: the first decade of the "hygiene hypothesis".
2. Kuehni C, Davis A, Brooke A, Silverman M (2001) Are all wheezing disorders in very young (preschool) children increasing in prevalence? Lancet 357:1821–1825.
3. http://download.journals.elsevierhealth.com/pdfs/journals/0091-6749/PIIS0091674911012346.pdf
4. Microbial Exposure During Early Life Has Persistent Effects on Natural Killer T Cell Function http://www.sciencemag.org/content/early/2012/03/21/science.1219328.
5. De Swert LF (1999). Risk factors for allergy. Eur. J. Pediatr. 158 (2): 89–94
6. Holt P. Mucosal immunity in relation to the development of oral tolerance/sensitization.
7. http://evmedreview.com/?p=103
8. Hunter MM, McKay DM (2004). Review article: helminths as therapeutic agents for inflammatory bowel disease.
9. Croese J, O’Neil J, Masson J, Cooke S, Melrose W, Pritchard D, Speare R. (2006). A proof of concept study establishing Necator americanus in Crohn's patients and reservoir donor.

Most normal cells are sociable; they respond constructively to the world around them; monitored by myriads of mechanisms that co-operate with neighbouring cells. When the ‘neighbourhood’ is too crowded, mitosis is inhibited, in a process known as contact inhibition; if their DNA or organelles are damaged, they repair themselves, or self-destruct through apoptosis. However, cancer cells continue to proliferate under these circumstances, ignoring normal signals and making new copies of cells containing a variety of genetic changes. Their emergence has even been likened to ‘natural selection’ within the body, with cancer cells competing for space and nutrients.

In fact, normal cells will not divide unless they receive outside signals causing them to enter into the cell cycle [1]. These signals are detected on the cell membrane and sent into the cell. They come from three different sources: growth factors, cell-cell adhesion molecules, and extracellular matrix components [2]. Normally these signals and other factors control the growth of cells and prevent them dividing uncontrollably. Mutations allow cancer cells to develop the ability to grow in the absence of these external factors and no longer obey the normal regulation of cell division. They do this by producing their own growth factors, over-express or change growth receptors, and even prompting surrounding cells to produce growth signals, such as “epidermal growth factor”, whose normal and important role is to regulate cell growth [3]. In cancer cells, genetic mutations can put receptors into a permanently activated state that results in uncontrolled cell division.

Cancer cells therefore do not rely on their surroundings and have an independence accounted for by their lack of anchorage and freedom to roam. They can invade the lamina, the non-cellular barrier that lines the surfaces of internal cavities, and organs (epithelial tissue). As a result, they travel through the body via the circulatory and lymphatic systems to other sites destroying tissue - a process known as metastasis that starts when a cancer becomes clinically malignant. To accomplish this, individual cells must move away from the primary tumour, which occurs through a change in adhesion molecules present on the cell surface. In normal cells, adhesion molecules ensure that cells remain closely linked to each other and the basement membrane. However, in some tumour cells these molecules are lost allowing the cell to move away from the tumour and enter the blood stream. Normally, loss of adhesion molecules results in cell death, a protective mechanism that helps prevent metastasis. However, metastatic cells usually have developed mutations that allow them to disable this mechanism and survive in the absence of adhesion.

These tumours need a blood supply for food and oxygen. Cancer cells secrete various growth factors such as VEGF, that make nearby blood vessels produce branches that grow into cancerous tissue [4] - a process known as ‘angiogenesis’. In 2007, a mutation was discovered in one kind of cancerous cells that stops cells producing the anti-VEGF enzyme PKG (protein kinase G). In normal cells PKG limits the activity of beta-catenin, which normally drives angiogenesis. Angiogenesis is required for the metastatic spread of a tumour. Single cancer cells can break away from an established solid tumour, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumour.

Normal human cells have a finite life-span of approximately fifty two divisions which is known as the Hayflick Limit [5]. This is determined by repetitive nucleotide sequences that protect the end of chromosomes known as telomeres. With each replication, these shorten, until they reach the Hayflick Limit when the cell stops dividing or undergo apoptosis. Cancer cells also have telomeres, but they do not control cell division as in normal cells. The reason they continue to divide indefinitely compared to normal cells is that cancer cells up-regulate telomerase. This is a special enzyme complex that elongates telomeres, enabling them to ignore the Hayflick Limit.

Another way that cancer cells avoid cell-suicide is through anaerobic respiration. Mitochondria, the cell’s power-house, play a crucial role in apoptosis of normal cells but in cancer cells this organelle is dysfunctional, as the cancer cells do not rely on aerobic respiration. . Instead, they thrive by the non-oxidative breakdown of glucose or ‘glycolysis’ occurring in the cytoplasm. As a result the mitochondria go into shut-down mode, and cancer cells become immortal.

Normal cells start off undifferentiated cells but become specialised and capable of specific functions. For example, muscle cells are specialized for contraction; nerve cells (neurons) are specialized for conducting signals, etc. Differentiated cells divide relatively little; most cell division involves stem cells or undifferentiated cells. These divide into daughter cells that are identical, because during mitosis, the centrosomes will pull identical sets of chromosomes into each new cell, resulting in two genetically identical copies. Cancer cells can have three or four centrosomes so that the DNA may separate unevenly, creating genetic chaos.

The immune system can recognize foreign cells and invaders (bacteria, viruses, etc.) because they have proteins and other structures that are different from the body’s "self" markers. Cancer cells may have mutated "self" markers and are often destroyed by the immune system.

Evidently, cancer cells differ from normal cells in numerous ways. It is through understanding the differences between cancer cells and normal cells that the nature of the disease can be truly understood and to pave the way for new treatments.

References

1. PubMed-Cancer cells
2. http://en.wikipedia.org/wiki/Angiogenesis
3. http://en.wikipedia.org/wiki/Cancer_cell
4. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/C.html
5. Wikipedia: Telomeres

When I asked my mother about what makes cancer cells different to normal cells, she replied that cancer cells were "the red spiky ones with evil faces". Unfortunately, cancer cells are not quite so different from other cells. In this case, I am considering "other" cells to be all non-cancerous cells in a healthy human, though looked at collectively, they pose a slight challenge because of their diversity, which means there are occasional exceptions to generalisations. On top of that cancer cells, though easily recognisable, are quite hard to define because of their variation. Cancer cells are caused by a series of mutations, which differentiate them from regular cells by three main factors: an ability to move around the host, unlimited reproductive potential and immunity to messages that trigger apoptosis (programmed cell death) or signals that would stop uncontrolled proliferation (cell division).

The underlying differences between cancerous and regular cells are caused by changes in the DNA — mutations, duplications and rearrangements of genetic material [1]. During cell division, the DNA is replicated by an enzyme called DNA polymerase, but errors are frequently made during copying — understandably, given that DNA replication must be a like copying-out the Encyclopaedia Britannica — and although there are in-built mechanisms to prevent mistakes (the equivalent of spell-check), some errors evade detection. These mutations can be point mutations, (substitution of one base for another), insertions, and deletions. For a cancerous cell to emerge, more than one mutation must occur, which means that it takes time for sufficient mutations to accumulate. There are numerous causes of alterations in DNA structure, but I find the most interesting to be the changes caused by specific viruses that incorporate bits of their genetic material into infected cells that code for proteins that interfere with mechanisms for regulating cell proliferation and make cells cancerous [2]. Some of these mutations cause over-expression of oncogenes (genes for proteins that can cause cancer) or may damage tumour suppressor genes. Oncogenes mutate from proto-oncogenes and cause an increase in cell growth and division. The genes that normally prevent this are the tumour suppressor genes, but the mutations that affect them are recessive so that both must be mutated to make a cell cancerous.

Mutations of proto-oncogenes and tumour suppressor genes drive one of the primary characteristics of cancer cells — unregulated cell division. Mutations in the proto-oncogenes are often genetically dominant, as only one is needed to cause over-expression. In contrast tumour suppressor gene mutations are normally recessive so that both copies must mutate to contribute to malignancy. Most cells from adults are not dividing and are arrested in the G₀ phase. On the other hand, cancer cells are constantly engaged in the cell-cycle. The human body has genes to police the processes that regulate cell numbers and ensure that new cells receive correctly replicated DNA [3]. When these genes are mutated, DNA copying becomes less strictly regulated, resulting in even more mutations and unregulated cell division. An example of a tumour suppressor gene is the p53 gene; the protein it codes for normally can stop the cell cycle or can initiate apoptosis when necessary. Cell cycle arrest allows time for damaged DNA to be repaired, and apoptosis eliminates potentially dangerous cells forever. The roles of the p53 gene are important for the prevention of cancer and mutations in this gene are likely to make cells cancerous and indeed, damaged p53 genes are common characteristics of cancerous cells.

Another important feature of cancer cells is their potential immortality. Each human chromosome ends in sections called telomeres composed of the repeated sequence TTAGGG that are thought to keep the chromosome stable. Each time a normal cell divides they lose sub-units in a process that contributes to the ageing process and which puts a limit on the number of cell divisions a cell can make (the Hayflick limit) [4]. In contrast, the telomeres of cancer cells do not shorten but are maintained through many cell divisions. They have no Hayflick limit and can theoretically divide indefinitely.

Uncontrolled cell division causes tumours, but tumours are not always cancerous. More mutations must occur on top of those that cause rapid cell proliferation. The main difference between a benign and malignant tumour is that cells of the latter have the ability to move around the body and grow in new sites where they may form a secondary tumour in a process known as metastasis. These migratory cancerous cells move through the surrounding tissue and through the protein layer surrounding the organ, called the basement membrane. The cancer cells secrete proteases that digest these membranes and then break into the bloodstream (or lymphatic system), and can then re-enter a different organ — a process called metastatic colonisation. The tumour may also undergo angiogenesis (forming blood vessels) to provide nutrients and oxygen that allow the tumour to develop [3].

The differences between healthy human cells and cancer cells arise from just a few mutations. It is perhaps disturbing that our own bodies can malfunction in such a critical way through the fault of just a handful of changes to the DNA. The mutations cause unregulated proliferation, immortality, and the ability of metastasise, and the mutated cells have the potential to fatally damage the remaining healthy cells. The difficulty is that cancer cells are not so different from our own — they don’t even have evil faces on them — but they severely upset the fine balance of the way we function and they are an extremely unwelcome addition to the human body.

References

1. Bray, D. Wetware. 2009
2. Skloot, R. The Immortal Life of Henrietta Lacks. 2010
3. Pecorino, L. Molecular Biology of Cancer. 2008
4. Ball, P. Molecules: A Very Short Introduction. 2003