DNA repair and carcinogenesis

The paper is dedicated to the natural phenomenon of cancer, with its possible causes, life me risks, mechanisms and possible outcomes discussed in fine detail. The molecular events resul ng in uncontrolled cell growth and increased capacity to colonise distant topological sites are reviewed with regards to their impact as separate factors as well as their func on as parts of a common mechanism. The basic classifica ons of cell genes coding for products involved directly or indirectly in carcinogenesis (proto-oncogenes, tumour-suppressor genes, mutator genes and gatekeeper/caretaker genes) are given in parallel in order to provide a be er understanding of the func ons of the encoded proteins. The mechanisms commonly used by cancer cells to evade the control of the DNA damage check/DNA repair/apoptosis system and for deac va on and/or elimina on of an cancer drugs are reviewed. The current and future opportuni es for establishing control over carcinogenesis (for common types of cancer as well as for 'cancer' in general) are evaluated in the light of the theory that cancer is a physiological mechanism set in place by Nature so as to minimise the risk of evolu onary stagna on. Cita on: Chakarov S, Petkova R, Russev GCh, Zhelev N. DNA repair and carcinogenesis. Biodiscovery 2014; 12: 1; DOI: 10.7750/BioDiscovery.2014.12.1 Copyright: © 2014 Chakarov et al. This is an open-access ar cle distributed under the terms of the Crea ve Commons A ribu on License, which permits unrestricted use, provided the original authors and source are credited. Received: March 18, 2014; Accepted: June 3, 2014; Available online /Published: June 8, 2014

Systema c and efficient repair of DNA damage, implemented as soon as possible a er the damage has been detected, is the main mechanism of protec on of living cells against poten ally harmful modifica ons in their major informa on carrier molecule.The DNA repair system of the average individual (apart from the rela vely rare excep ons of inherited DNA repair deficiencies), coupled with the mechanism of programmed cell death, func on together well enough so as not to allow the occurrence of too many altera ons of the gene c content per cell, or, at least, that they would not be passed on to the cell's progeny.There are, however, sources of DNA errors that cannot be eliminated or avoided.
As it was already discussed, despite the accuracy of template copying during replica on and DNA repair, the underlying mechanism is essen ally error-prone.The risk of occurrence of a copying error and the associated risk of the error becoming fixed heritable muta on are minute between two successive cellular genera ons, but tend to accumulate over me.As the cell and the organism age, the efficiency of the mechanisms for detec on and repair of DNA damage generally declines.This is associated with accumula on of unrepaired damage in DNA and, respec vely, with increased risk of introduc on of muta ons.The muta ons may affect various genes, but pro-carcinogenic ac on of random mutagenesis is usually most pronounced when directly affec ng genes coding for products that are associated with s mula on of cell prolifera on (e.g.growth factors and their receptors) and/or products that may suppress cell growth in response to damage-associated signalling (e.g.TP53, ATM, BRCA1 and 2, CHK proteins, etc.).Some of the muta ons that occur de novo in soma c cells may produce a net increase in the genome mutability, which may, in turn, result in higher rate of muta on occurrence per cell genera on.Other soma c muta ons may result in inac va on or evasion of one or more pathways and mechanisms for detec on and/or repair of DNA damage, or the mechanisms that dictate that a damaged cell must die.Thus, the minute error/s occurring in every cell division may, with me, mul ply and augment each other.Eventually, this may result in cell transforma on and, later, in overt cancer.Usually this occurs years and decades a er the ini al event/s that supposedly triggered the process.
It would be safe to say that high-fidelity copying of DNA during replica on, together with the concerted ac on of the mechanisms of DNA repair and programmed cell death are normally sufficient to sustain the individual healthy from concep on up to middle adulthood (45-55 years).Beyond that age, the risk of occurrence of soma c muta on/s eventually resul ng in cancer may become significant.Despite what the media may say, cancer is (and always was) predominantly a disease of middle and advanced age.Not all people beyond the age of 60, however, eventually develop cancer, and many of the oldest old (>85) remain cancer-free to their deaths.
The rate of accumula on of muta ons is usually dependent of the number of divisions the cell goes through.Different cell types have different turnover rates.Cell types with naturally rapid turnover rate are, for example, neutrophil granulocytes (replaced about every 4-5 days); cells in the intes nal crypts (about every 7 days), and erythrocytes (about every 120 days).Adipocytes are exemplary slow dividers, being replaced at a rate of 6-8% per year.Some terminally differen ated cells that were ini ally believed to be irreplaceable, such as neurons in the olfactory bulb in mammals, are actually replaced about every 6 weeks [1].
Cardiomyocytes in the adult heart (which were also ini ally believed to be incapable of division) are replaced at a faster rate (about 1%) up to about 25 years of age, then it slows down to less than 0.5% per year.
There are four basic pathways by which a normal cell may be transformed to a cancer cell.
The origins of all may be traced to instances of DNA damage becoming fixed as permanent altera ons in the sequence and/or the structure of DNA.
Abroga ng the restric ons normally placed upon cell division, resul ng in increased prolifera ve capacity of the cell (e.g.cons tu ve ac va on of a proto-oncogene or cons tu ve inhibi on of a gene coding for product suppressing cell growth); Ignoring or bypassing the pro-apopto c signals and/or the checkpoint/s of the cell cycle where the pro-apopto c decisions are normally made (usually, the G1/S checkpoint) (e.g.loss of func onal TP53 gene copies); Occurrence of molecular or genomic event/s that confer genome instability, increasing the risk for occurrence of addi onal muta ons (e.g.muta ons in genes coding for proteins with roles in the maintenance of genome integrity; accelerated telomere a ri on, etc.); Deregula on of differen a on (differen a on blockage), producing cells with precursor-like phenotype (typically characterised by high prolifera on capacity).
The ini al event that triggers cancerous transforma on may belongs to any of these four types.The others may add up later in any order or virtually simultaneously.
In normal cells, the damage detec on and repair machinery is alerted whenever there is a signal for the presence of damage (priority being given to damage in transcribed genomic regions) and every cycle of division is preceded by extensive damage checks.If the cell has sustained too much damage that cannot be managed by repair mechanisms, it would be instructed to enter permanent replica ve senescence and/or routed to the programmed cell death pathway.Each of these safety mechanisms is very efficient, but they may occasionally not recognise an instance of damage or simply miss it.It is believed that a sta s cal number of muta on events (that is, at least between 3 and 6) must occur in the same cell in order to trigger neoplas c transforma on [2].Considering the turnover rate, different cell types would accumulate a sta s cal number of errors in their DNA over a different me period.At any point, the cell machinery for assessment of genome integrity may evaluate the DNA of the cell as irreparably damaged and route the cell towards the apoptosis pathway.Therefore, many cells that have accumulated enough DNA altera ons to trigger cancerous transforma on would be promptly eliminated before it has actually begun.Only a very small minority of cells that have successfully evaded all mechanisms for detec on of damage may eventually become cancer cells.Since the prolifera on poten al of a transformed cell typically increases as the malignant transforma on progresses, however, even a single transformed cell may (at least in theory) suffice to produce cancer.Of course, this will not happen overnight.Each of the discrete events that may contribute to development of cancer is not enough to turn the normal (that is, non-cancerous) cell instantly to a tumour cell.Carcinogenesis does not occur in leaps and bounds, but is a longterm consequence of expanding and mul plying errors in DNA that happened many months or years ago.The process is largely stochas c (random) in nature and depends on endogenous as well as on environmental factors.

Cancer may sometimes be caused by defined (and, in many cases, eliminable) environmental factors
No ma er what Aristotle says with all his Philosophy, there's nothing like tobacco: it is the passion of respectable men; and the man who lives without tobacco is not worthy to live.Jean-Bap ste Poquelin Molière, Don Juan, or The Feast with the Statue (c.1660).Cancer has always been a mystery disease and the isola on and the characterisa on of the leading pathogene c factors of tumour growth s ll present a major challenge to biomedical research.The simple fact that cancer may be caused by exposure to certain exogenous agents has been known for quite some me before the actual pathogene c mechanisms of cancer were discovered.The earliest a empt for a serious study on the link between factors in the environment and the risk of cancer belongs to Percival Po (1714-1788), a Bri sh surgeon (later knighted), who demonstrated that specific types of cancer were associated with specific occupa ons.Namely, he found that cancer of the scrotum was almost exclusively seen in chimney-sweeps and named exposure to soot as the culprit [reviewed in 3].
It has been known for quite a long me before the nature of the causa ve agent was iden fied, that cervical cancer was more common in married women than in unmarried women that prac ced celibacy.In 1842, the Italian physician Domenico Rigoni-Stern published his observa ons on the epidemiology of cervical cancer, sta ng that cervical cancer was rather common in women with mul ple sexual partners (in his studiespros tutes); and very rare in women living a life of celibacy (as the author bluntly stated, 'nuns, virgins and spinsters'), except for nuns that had chosen the monas c life in later age [Rigoni-Stern, 1842; reviewed in 4].Rigoni-Stern also found that breast cancer was more common in nuns (probably because of the then-prac sed tradi on of breast binding in some Catholic female orders and the low-grade protec on from breast cancer conferred by pregnancy and childbirth).Almost 130 years earlier another Italian physician, Bernardino Ramazzini (1633-1714), proposed that breast cancer was more common in nuns than in married women because of the lack of sexual intercourse (which he found to be 'unnatural') and which presumably caused 'breast ssue instability' that later turned to cancer [Ramazzini, De Morbis Ar ficum (1713)].Ramazzini is also believed to have been the first to voice concerns about physical inac vity in healthy individuals and to encourage the ac ve lifestyle.The sta s cal fact that cervical cancer was more common in married than in unmarried women was not demonstrated in wri ng, however, up to 1949, when the Dutch epidemiologist Versluys published the results of his observa ons on the incidence of carcinoma in the Netherlands and the poten al associa on with the occupa on [5].
Other associa ons between environmental factors and common cancers were not elucidated un l the XX century.For some of them, the connec on simply could not have been made earlier.For example, having suntanned complexion, especially for women, has never been considered a rac ve in Europe (as it was usually considered to be a sign of lower class origin) up un l the 20-es of the XX century, when one of the famous French celebri es of the me, Coco Chanel, had an accidental sunburn during an ocean cruise and decided to show off her new tanned looks instead of trying to conceal them.Very soon, deep tan was considered to be a symbol of health and fitness and 'heliotherapy' was proclaimed to be a cure for all diseases.The finding that skin cancers appeared predominantly on sun-exposed areas of skin was first published in the late 40-es of the XX century [6], over 20 years a er the emergence of heliotherapy.Tobacco was first brought into Europe in early XVI century, but the link between tobacco smoking and lung cancer was demonstrated unequivocally only 60 years ago, in 1950 [7].To explain the huge delay in the acknowledgement of the hazardous effects of tobacco one must take into account that tobacco use was greatly popularised only a er the XVII century; the tobacco industry grew to its fullest extent only a er the industrial revolu on (the end of the XVIII century); and the simple fact that the lifespan of people in the XVI-XVIII century was much shorter than the lifespan of people of the XIX and especially the XX century.
The origins of many cancers cannot be unequivocally linked to any environmental factor/s, and 'healthy living' is not a guarantee than one would remain cancer-free un l their old age; neither is 'unhealthy living' directly associated with development of cancer.It has been accepted that accumula on of unrepaired DNA damage (in the process of ageing, or for other reasons -e.g.high levels of oxida ve stress, defects in recogni on and repair of damage, etc.) is a major mechanism for triggering carcinogenesis, with or without the presence of carcinogenic factors of the environment.

Cancer cells are not that alien to normal cells
All things are the same except for the differences, and different except for the similari es.Thomas Sowell, The Vision of the Anointed (1996).

Cancer cells share some common characteris cs with normal cells
Cancer comes in so many types and the proper es of cancer cells may be so dissimilar, that making a unified defini on of a cancer cell is not easy.The basic proper es of cancer cells may be summarised as follows: 1. Cells that are capable of division beyond the Hayflick's limit or may divide indefinitely; 2. Cells with metasta c poten al (capable of invading and colonising new sites that are a long way away from their place of origin); 3. Cell with proper es characteris c of undifferen ated cells or of cells at earlier stages of differen a on, that are incapable of terminal differen a on, unless under special circumstances.
In cells undergoing cancerous transforma on, the increased prolifera on capacity is usually acquired first and the capacity for metastasising adds up later.
The first two proper es (high prolifera ve poten al and metasta c poten al) are not unique to cancer cells.Other types of cells such as embryonic cells and stem cells also have high prolifera ve poten al (some mes virtually unlimited, e.g. in cultured pluripotent cells).Some non-cancerous cells are naturally capable of colonising new habitats.For example, transplanted haematopoie c stem cells eventually colonise the bone marrow of the recipient, but they are not transferred to the bone marrow during the actual transplanta on.Specifically, the haematopoie c cells are transplanted in the myeloablated recipient by means of a simple IV transfusion of a cell suspension.The cells are then transported by the blood flow to the bone marrow, se le there and replenish the haematopoie c cell niche.
The third characteris c listed above, however -signs of incomplete differen a on -is a defining trait of a cancer cell.As the grade of differen a on of a tumour is a very important characteris c, this will be discussed in more detail below.There have been reports about stabilised stem cell lines (e.g.lines from induced pluripotent stem cells) exhibi ng expression and mRNA profiles characteris c of cancer cells [8,9].This is, in fact, one of the major issues with the use of pluripotent stem cells obtained by reprogramming of soma c cells [10,11].

Cancer cells exhibit traits typical of undifferen ated cells or cells at lower differen a on grades
Cancer cells may express proteins or other molecules that are usually part of the expression profile of undifferen ated cells or of par ally differen ated precursor cells.The differen a on grade of the tumour is one of the basic characteris cs assessed in rou ne histopathology examina on.Differen a on grade may vary from low (undifferen ated or poorly differen ated) to high (moderately to well differen ated).The differen a on grade of tumours is directly associated with their prolifera ve and/or metasta c poten al -the lower the differen a on grade, the higher the aggressiveness of the tumour and, in most cases, the poorer is the prognosis for the pa ent.The survival rates between pa ents with poorly differen ated and well differen ated tumours may be dras cally different, even for the same type of tumour.For example, there are forms of leukemia with minimal differen a on that are very aggressive, and there are leukemias with higher grade of differen a on that may develop slowly or even run a chronic course.This is easily understandable, as the further the cell has gone on the path of differen a on, the lower its prolifera ve poten al usually becomes.Terminally differen ated cells usually have a very limited capacity for division, if at all (for more detail, see 'Cancer stem cells' below).Also, higher grade of differen a on usually means less capacity for invasion of distant loca ons and infiltra on of other ssues (metastasis).Some of the proteins expressed by cancer cells and characteris c of the undifferen ated state are posi ve regulators of cell cycle (for example, growth factor receptors, receptor-associated kinases, or other signalling molecules); substances degrading basal laminae and/or s mulators of angiogenesis, facilita ng the colonisa on of distant sites.Some (but not all) cancer cells may be specifically s mulated towards differen a on.This is usually accompanied with dras c reduc on of the prolifera on poten al of the tumour cells (respec vely, the aggressiveness of the tumour).Induced differen a on is some mes used as a therapeu c approach (differen a on therapy), especially in haematological cancer.Agents known to induce differen a on in cancer cells in vitro as well as in vivo are, for example, trans-re noic acid in the treatment of leukemia [12]; analogues of cAMP (e.g.8-Cl-cAMP [13]); sodium butyrate; some an diabe c drugs of the thiazolidinedione group (e.g.troglitazone) [reviewed in 14]; hormones; cor costeroids; and some 'classic' cytosta c drugs such as methotrexate, cytarabine, 5-azacy dine, and others.Trans-re noic acid has been used in the treatment of leukemia for almost 20 years now [15].Some differen a ng agents (e.g.hormones) are usually efficient in certain types of tumours only, as they alone express the relevant receptor.For example, estrogens and androgens are usually used in the treatment of tumours occurring in ssues dependent on the respec ve hormone -e.g. the mammary gland, the prostate gland, the ovaries and the endometrium.
Estrogen was shown to sensi se some types of tumour cells (e.g.breast and cervical cancer cells) to cytotoxic treatments [16].This has been linked to upregula on of proteins of the HMG family, which are capable of suppression of DNA repair [17, reviewed in 18].Lately, it was demonstrated that sex hormones (e.g.estrogen) may some mes regulate the prolifera on of tumour cells that are seemingly independent of the hormone in ques on (for example, in colorectal cancer) by triggering an -inflammatory and an -tumorigenic signalling networks in the cancer cells [19].
Trans-re noic acid and its deriva ves, 5-azacy dine, and other differen a ng agents have a wider spectrum of ac on and may be applicable for induc on of growth arrest in more than one type of tumour.For example, trans-re noic acid is used in the treatment of leukemia, but also in squamous skin cancer.This is likely to be related to the mul ple targets for the ac on of the drug.For example, trans-re noic acid promotes differen a on of leukemia cells but also suppresses the expression of an -apopto c factors of the BCL-2 protein family [20].The theore cal concept behind the 'double-hit' mechanism is that no single event may result in cancer.This is valid even when there is a gene c factor conferring cancerproneness, as Knudson's model was ini ally developed for a heritable cancer -namely, re noblastoma.For example, carrying a muta on in the BRCA1 or BRCA2 gene/s may eventually result in breast and/or ovarian cancer, but not before a second muta on occurring on soma c level had knocked out the intact gene copy as well.Indeed, from purely sta s cal point of view, the risk for soma c inac va on of the second BRCA1 gene copy by random mutagenesis in cells that already have one defec ve gene copy of the BRCA1 gene is much higher than in cells with both copies intact.The second 'hit', however, may or may not occur, as two 'hits' at the same target is an unlikely event.Even among carriers of BRCA1 muta ons, the penetrance of familial breast cancer rarely exceeds 90%, which means that in about 1 in 10 of proven carriers associated cancers do not develop (although part of these 10% may be a ributed to lack of accurate data).Later, Knudson's double-hit model was developed further by Vogelstein & Kinzler, who postulated that a sta s cal number of DNA muta ons (generally no less than 3, usually more -5 or 6) must occur in the same cell in order to transform it to a cancerous cell (mul ple-hit model) [2].The risk for occurrence of muta ons above the sta s cally significant number of 3-6 increases with age.If we use the example with the BRCA1 gene again, there are virtually no very early (e.g.prepubertal) cases of familial breast and ovarian cancer associated with BRCA1 gene muta ons on record, and cases before the age of 25 are quite uncommon.

Basic mechanisms of cancer
A er 25, the prevalence of cancer smoothly rises, to reach its peak in around the age of 40-45.This age-dependent effect is also valid, however, for cancers developing without any gene c predisposi on.A er all, the longer a cell had been exposed to the everyday genotoxic a acks, the higher the likelihood that it has accumulated enough muta ons to embark on the path of carcinogenesis.As the human body of is made of ≈ 10 cells, the risk that any of these cells may become transformed over the several decades of adult life is fairly high.
The mechanism of accumula on of mul ple 'hits' by random mutagenesis is currently believed to be one of the primary mechanism for triggering cancer growth [2,21].In a genome with a large amount of non-coding DNA, as are mammalian genomes, the majority of the 'hits' would come to nothing, as they are less likely to affect important genomic regions.'Hits' producing detectable (and reparable) DNA damage or damage that is severe enough to trigger the apoptosis pathways may not produce any long-term effects.In the majority of cases, the damage would be repaired before it becomes fixed, or the cell would be made incapable of division, or physically removed from the cell pool.There are, therefore, only very rare single 'hits' that, taken together with others, may eventually produce cancer.Cancer development may be modified (prevented, slowed down or s mulated) by many factors, some of which are gene c and others are factors of the environment.The two types of factors may be equally important for the development of cancer.For example, it has been shown that homozygotes by a rela vely common polymorphism -the 83bp inser on allele in the XPC gene, were at increased risk for development of squamous cell carcinoma and adenocarcinoma of the lung [22].This risk, is, however, was only valid for those carriers who were current or past smokers, while for non-smoker carriers of the polymorphism the risk was found to be negligibly low [22,23].The 'random hit' mechanisms of mutagenesis may play a role in other cancer-related processes as well -for example, in development of resistance to an cancer drugs (for more informa on, see "Bases of cancer resistance to drugs" below. It has been proposed that genomic instability in some cancers (specifically bone cancer, but possibly some cases of breast cancer as well) does not occur as a result of randomly occurring muta ons which happen to 'hit' a crucial gene, but in a single catastrophic event, termed 'chromothripsis', affec ng regions on one or several chromosomes 24,25].
According to the authors, the affected region is literally sha ered into fragments, some of which (but not all) are subsequently assembled together again by the cellular machinery for DNA repair.Since it is not possible to determine the 'correct' sequence of the genomic fragments, these are patched together in a more or less random order using the only possible way -the mechanism of NHEJ, which is inherently error-prone [26].Under such circumstances, it is likely that at least one (possibly more than one) pro-carcinogenic molecular events would occur.Cells that have undergone chromothripsis are, therefore, much more likely to acquire capacity for unlimited prolifera on and for ignoring proapopto c signals.Other authors have theorised that complex rearrangements in cancer may result from disordered DNA replica on in specific genomic regions, ini a ng microhomology-mediated template switching, resul ng in localised complex rearrangements [27].The "50:50" rule Generally, the daughter cells resul ng from the division of a cell are very similar (virtually iden cal) with regard to their proper es, the distribu on of the cellular components, and the fate of the individual cells.The la er means that if more than one alterna ve route is available for a newly formed daughter cell, each of the daughter cells has an equal chance of taking any of these routes.In some types of undifferen ated cells (e.g.stem cells, cancer cells), a specific phenomenon may be observed during cell division.Namely, the daughter cells resul ng from division in these types of cells exhibit different proper es and may have very dissimilar fates.The two daughter cells o en contain different amounts of specific cell compounds and may be intended to take different routes once the division is complete.In stem cells this usually means that one of the two daughter cells (or, more accurately said, 50% of the daughter cells in the popula on of dividing stem cells in a ssue) were des ned from the very start of the cell division to retain the characteris cs of the original stem cell; whereas the other 50% were des ned to take the route of differen a on.Thus, with every cell division, the stem cell popula on is replenished, on the one hand, and a differen a ng precursor cell is produced, on the other hand.One division of the original stem cell eventually results in produc on of many specialised cells, as the precursor cell usually undergoes mul ple divisions before it eventually enters the replica ve arrest that is typical of terminally differen ated cells.The daughter cells that would take on the role of stem cell of the ssue would preserve the characteris c high poten al for prolifera on and the hyperplas c state of their chroma n; while those that were des ned to become differen ated cells would typically lose their capacity for division at some point in the course of differen a on and significant propor on of their chroma n would become condensed and transcrip onally inac ve.
Deciding which of the two daughter cells would retain the stemness quali es and which would take the differen a on route is a complex process.It may be directed by exogenous or endogenous factors.Among the former is, for example, the contact with the cell niche.
The daughter cells may be posi oned differently rela ve to the cell niche where the mother cell belonged to (one in direct contact with the niche, the other away from it).An important factor of endogenous origin may be, for example, unequal distribu on of cell components (mRNA, proteins, membrane-limited compartments, even cell organelles, e.g.mitochondria).DNA may also be distributed in an unequal manner between the daughter cells, as it has been demonstrated that in some cell types the replicated DNA molecules are segregated asymmetrically during division, with all those containing the original 'maternal' DNA strand always dispatched to one of the daughter cells, and those that had been synthesised using a copy made during the previous cycle of division -to the other daughter cell (see below).Not all cell types employ the 50:50 rule of cell division, but cells in ssues with rapid cell turnover o en do.Such are, for example, the adult stem cells in the basal (germina ve) layer of mammalian skin.Dead kera nised cells from the upper layers of the skin are regularly sloughed off and must be promptly replaced in order to preserve the skin integrity.
Epithelial stem cells generally produce progeny in compliance to the 50:50 rule, except in specific cases that take priority over it.For example, in deep penetra ng skin injury the stem cells from the basal layer in the regions adjacent to the injury migrate to the injury site and start dividing, so that eventually new skin grows over the injured site.This means that at some point the progeny of an adult epithelial stem cell must have been comprised predominantly of cells retaining the stemness characteris cs, as the epithelial stem cell popula on at the injury site must be re-established in order to ensure normal skin growth.
A er the stem cell niche had been replenished, the produc on of epithelial cells would comply with the 50:50 rule again.
If, for some reason, the cells of the basal layer of the epidermis start cycling faster than usual, this normally would be compensated by accelera on of the process of differen a on of precursor cells.This is exactly the case with some hyperkerato c states of the skin and mucosa, e.g.skin warts and condylomata acuminata caused by infec on with human papillomavirus (usually of the types 6 and 11).In hyperkerato c epithelia, however, the 50:50 rule is s ll observed (one cell retaining the stemness quali es, including the prolifera on capacity; the other becoming differen ated, gradually losing its ability to divide), only the pace of cell cycle is altered by the virus in order to produce quickly as many viral par cles as possible.
Infec on with HPV may some mes cause shi ing of division of epithelial cells away from the 50:50 rule towards predominant produc on of cells with high prolifera ve poten al and hyperplas c chroma n -in other words, cells with phenotype typical of the undifferen ated cells [28][29][30].Usually, infec on with HPV is cleared rapidly by the immune system and the associated epithelial growths eventually disappear even without treatment.Some mes, HPV infec ons (usually, with types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, possibly others, referred to as 'high-risk' (for cancer) types of HPV) may trigger cancerous transforma on in the infected cells.Briefly, this usually occurs via linearisa on of the viral genome (normally in episomal state) and its subsequent integra on into the genome of the host cell.This results in derepression of the transcrip on of two oncoproteins (E6 and E7), which are normally transcrip onally inhibited in episomal viral genomes.E6 and E7 oncoproteins specifically target and inac vate the major tumoursuppressor proteins of the infected cell -chiefly pRB and p53 [28,31,32], but they may also interact, albeit not always directly, with posi ve regulators of cell prolifera on (e.g. the RAStype of proteins), ensuring that the transformed cell would con nue to divide rapidly regardless of the presence of DNA altera ons that would normally induce cell cycle arrest [33,34].E6 and E7 oncoproteins can also override the inhibitory func ons of CDK inhibitors (e.g.p21 and p27); cause overexpression of cyclin E (responsible for the CDK2-dependent progression though the G1/S checkpoint) [35,36] and modulate the expression of various miRNAs [37], eventually resul ng in abroga on of p53-associated apoptosis of damaged and transformed cells.These two proteins may also induce overexpression of chroma n modifier proteins such as HMGA1, producing chroma n hyperplas city and suppressing DNA repair [38].Finally, the onset of replica ve senescence by reaching cri cal telomere length may be irreversibly lost in the transi on from intraepithelial dysplasia to invasive carcinoma, as the telomerase ac vity is re-ac vated in cervical cancer cells [39,40].Thus, the progeny of transformed cells may not comply with the '50:50 rule, as it consists exclusively of cells with 'stem-like' proper es (high prolifera ve poten al, hyperplas c chroma n).
Asymmetric segrega on of daughter DNA molecules during cell division as an an -cancer mechanism DNA is replicated by a semi-conserva ve mechanism, meaning that the double strands in each of the daughter cells are made of one strand of 'parental' origin (serving as a template in replica on) and one newly synthesised strand.This is valid for every chromosome in eukaryo c cells.In most cell types, the physical segrega on of the two sets of chromosomes in the end of mitosis is at random, that is, each of the daughter cells may receive a chromosome of each pair which contains the 'original' ('ancestral') strand of DNA or a strand that has been copied from the complementary strand, a product of copying in the previous cell cycle.
The likelihood that a cell would receive a set of chromosomes containing the 'original' DNA strand purely by chance is very low indeed, in the order of one in about ten million.In some types of cells, however, e.g. in rodent embryonic cells in primary ssue culture, the chromosomes carrying the same 'ancestral' DNA strand have been shown to be always selec vely targeted together to the same cell [41].The same phenomenon is observed also in several types of adult stem cells, such as the stem cells in the intes nal crypts [42]; the satellite cells in the muscle (the stem cells of skeletal muscle) [43,44]; the epithelial stem cells in the mammary gland [45] and the neural stem cells [46].During division, the cell that receives the set of DNA molecules carrying the 'ancestral' DNA is always the cell that retains the stemness characteris cs and remains within the stem cell niche, while the 'copied copies' of DNA are targeted to the cell that is des ned to the differen a on route (Fig. 2).Thus, the same ancestral DNA strand (also called 'immortal' strand) is transmi ed from one stem cell to another from the first division of the zygote (which is, essen ally, the ul mate stem cell).targeted to the daughter cell that is to remain a stem cell [47].
The hypothesis about the "immortal DNA strand" was first formulated in 1975 by the Bri sh physician John Cairns [48].He proposed that it was a mechanism for protec on of living cells against cancer brought on by accumula on of replica on errors.Nowadays, it is believed that asymmetric division is a specific mechanism of stem cells for protec on from accumula on of DNA altera ons.Some mammalian cells, such as neurons or cardiomyocytes, are naturally long-lived, but the long-term func onality of most ssues is usually ensured by constant renewal of the cells that had been lost because of injury, ageing, or programmed cell death.This requires repeated copying of the cell's DNA throughout many replica on cycles.
The mechanisms of DNA replica on are inherently error-prone although the error rate is normally very low.If the DNA of a stem cell becomes altered, the altera ons would be passed to the cell's progeny and would very likely be mul plied further during subsequent division.This may have various harmful consequences.As the DNA of adult stem cells accumulates errors, their poten al for produc on of new precursor cells gradually declines (one of the hallmarks of ageing); or they may become transformed and start producing mutant cell progeny (for more informa on, see "Cancer stem cells" below).By asymmetric segrega on of the 'original' and the 'copied copy' DNA strands between daughter cells, all the muta ons that had previously occurred because of replica on errors would presumably be passed onto the cell that is des ned to take the differen a on route.Indeed, this 'muta on-laden' cell would divide several (or more) mes before it becomes a specialised cell, but the number of divisions would be finite.It is likely that the impending replica ve senescence would induce permanent cell cycle arrest before the cell has accumulated enough muta ons to become transformed.The daughter cell that retains the stemness quali es would inherit the 'original' blueprint from the mother cell and pass it on in the next division cycle to the next daughter cell des ned to remain a stem cell, thus minimising the risk of accumula on of muta ons in the stem cell popula on of the ssue.This makes perfect sense, as most soma c cells in the adult body are replaceable, but the stem cell popula ons in adult ssues are supposed to last a life me.Not all stem cells employ the mechanism of asymmetric segrega on of DNA.For example, the distribu on of chromosomes containing the 'immortal' and the 'copied copy' DNA strands during division of haematopoie c stem cells was found to be almost completely random [49].There is also the fact that the mechanism of asymmetric segrega on is unlikely to work on its own.Several other condi ons must also be fulfilled in order to ensure the protec on of the cell that retains the 'stemness' characteris cs from accumula on of DNA damage.Among these condi ons, the most important are: the asymmetric segrega on of chromosomes carrying different DNA strands must occur in every cell division; the division of the cell components between the daughter cells must also be asymmetric (so that genome-modifying factors such as proteins and RNAs are also distributed unevenly between the daughter cells); and that another mechanism for maintaining genomic stability in ac vely dividing cells -mito c homologous recombina on -must be suppressed so that the rate of exchange of gene c material between sister chroma ds would become negligibly low [50,51].In only two of the types of adult stem cells men oned above (the satellite cells in adult skeletal muscle and the neural stem cells) the cell division was found to be truly asymmetric (that is, the daughter cell that retains the 'stemness' characteris cs and the daughter cell that takes the differen a on route differ not only in the distribu on of DNA molecules, but also in the distribu on of other cellular components) [43,44,46].It has even been speculated that the mechanism of asymmetric segrega on of cell components and chromosomes with different DNA strands may par ally account for the fact that malignant tumours origina ng from the myogenic progenitor cells are actually quite uncommon [52].Indeed, the incidence of rhabdomyosarcoma is very low compared to other types of so ssue cancer -about <0.5 per 100,000.The same no on may be applied (at least theore cally) to the pathogenesis of the cancer of the small intes ne.The small intes ne makes up for 75% of the length and 90% of the surface area of the gastrointes nal tract, and the epithelial cells making up its innermost layer are replaced at a rapid rate, therefore, from purely sta s cal point of view the risk for cancer anywhere along the small intes ne ought to be higher than in other loca ons in the gastrointes nal tract.Yet, its worldwide prevalence is <1 per 100,000 [53], whereas the prevalence of gastric cancer is about 10 per 100,000 and the prevalence of cancer of the lower intes ne is about 50 per 100,000 (according to Cancer Research UK).Therefore, asymmetric segrega on of cellular components and DNA may be employed as an an -cancer mechanism in cells with high prolifera ve poten al, but it is not commonly used.
Disordered regula on of the asymmetric segrega on mechanism, however, may result in uncontrolled cell growth.

Cellular genes coding for products that play a role in tumorigenesis
To you I'm an atheist.
To God I'm the loyal opposi on.

Woody Allen, Stardust Memories (1980)
Capacity for unlimited prolifera on is one of the main dis nguishing traits of cancer cells.
Muta ons affec ng the cellular genes coding for proteins with func ons in the regula on of the cell cycle make up for the majority of the soma c 'hits' that eventually trigger tumorigenesis.Depending on the type of regulatory func on that these proteins may have in the progression in the cell cycle (posi ve -s mula ng cell prolifera on or nega vesuppressing cell division), a carcinogenic muta on may produce cons tu ve and/or ectopic ac va on of a gene product or, alterna vely, suppress the expression and/or reduce or altogether abolish the ac vity of a gene product.Some pro-carcinogenic muta ons do not alter the regula on of the progression through the cell cycle but, rather, increase the overall muta on rate in the genome, crea ng favourable background for occurrence of other muta ons.At present, the most commonly used type of classifica on of genes and gene products, muta ons in which play a role in carcinogenesis, refers the genes and the encoded proteins to one of three major groups.The criterion for differen a on between the three groups is whether they s mulate or suppress cell prolifera on (directly or indirectly) or work by destabilisa on of genome integrity.The structure of the classifica on is presented below, and major representa ves of each class of pro-carcinogenic genes and proteins are listed.

Proto-oncogenes
Wild type proto-oncogenes (c-onc) are, in fact, the normal cellular genes coding for products func oning in posi ve regula on of the cell cycle as signalling or effector molecules.The products of proto-oncogenes may directly s mulate cell prolifera on (for example, growth factors) or may be capable of inducing the expression of numerous downstream genes that are directly involved in the s mula on of the cell prolifera on and/or the progression through the cell cycle (transcrip on factors), or may ensure the uneven ul passing through cell cycle checkpoints (e.g.components of the cyclin-CDK regulatory system).Proto-oncogenes may also inhibit (directly or indirectly) the expression of genes that are responsible for triggering the pathways of cell cycle arrest, DNA repair and/or apoptosis in damaged cells.
Major human proto-oncogenes are commonly systema sed into several sub-groups, depending on their func ons (Table 1).

Table 1.
Major groups of human proto-oncogenes according to the func ons of their protein products.).Most of the cellular proto-oncogenes have viral homologues.A viral proto-oncogene is usually a fragment of a normal cellular proto-oncogene fused with the viral genome, which may become integrated in the genome of the virus-infected cell and/or may be transported around the genome.Virus genomes may integrate into the genome of the host cell, then excise out of the genome and re-integrate elsewhere, some mes by a 'cut and paste' mechanism and some mes leaving a copy of the viral genome behind.The excision of a virus genome copy is not always accurate, and recombina on may occur between regions of homology belonging to different viral copies integrated in different loca ons of the genome.Thus, virus genomes may leave fragments of their own DNA sequence in the host cell's DNA and/or fragments of the genome of the host cells may be picked up during virus L genome excision, becoming an integral part of the nucleic acid of the virus.The genomic sequences that are carried around by viruses may be func onally ac ve (e.g.complete coding sequences of genes or DNA copies of mRNA transcripts), or may be ac vated a er their transloca on -for example, when placed by viral genome integra on into an ac vely transcribed region of the genome of the host cell; in a loca on within the range of ac on of strong promoters or enhancers, etc.If the incorporated genomic sequence contains poten ally func onal fragments of a proto-oncogene, the integra on of the virus genome copy into the genome of the host cell may result in upregula on of cell prolifera on that may, eventually, produce overt cancer.Inser on of viral genome copies in the host cell's DNA may also cause illegi mate ac va on of the cellular gene/s in the vicinity of the integra on site, because of disrup on of the physiological control over their expression.Such modes of pro-carcinogenic ac on are preroga ve of viruses with life cycle that includes integra on in the genome of the infected cell (transforming viruses, oncoviruses).

Func on of the protein product of the proto-oncogene Examples
Commonly, these are viruses with RNA genomes that propagate via DNA intermediates (retroviruses), although DNA-based viruses such as the papillomavirus and the Epstein-Barr virus may also have transforming proper es, as they are capable of integra on into the genome of host cells.
Most viral proto-oncogenic homologues were discovered before the eponymous cellular proto-oncogene, as part of the genome of a virus with known oncogenic proper es.For example, the src gene, responsible for the transforming func on of Rous sarcoma virus (RSV) was differen ated from the gag, pol and env genes of the virus in 1976 [54].The fact that in vitro transla on of RNA of transforma on-competent virions of RSV in a cell-free system yielded two polypep des that could not be iden fied in transla on products of RNA from a transforma on-defec ve mutant Rous sarcoma virus carrying genomic dele ons was reported an year later, in 1977 [55].The c-SRC homologues in man were iden fied several years later, in 1984 [56].Similarly, the structure of Abelson leukemia virus genome was first reported in 1979, while the human homologue, the c-ABL gene was not iden fied un l 1982 [57].
Pro-carcinogenic muta ons in cellular proto-oncogenes are usually of the gain-of-func on type, resul ng in expression of the gene product at abnormally high levels and/or ectopic and/or cons tu ve expression (this is also the principle of ac on of most viral protooncogenes).Carriership of inherited gain-of-func on muta ons in normal cellular protooncogenes is usually incompa ble with life.The majority of gain-of-func on muta ons are, therefore, of soma c origin.Excep ons to this are, for example, the heterozygous muta ons in the c-RET proto-oncogene.c-RET codes for a receptor protein kinase, involved in the development of the embryonic intes ne and kidneys and the enteric nervous system [58].Heterozygous gain-of-func on muta ons in the c-RET proto-oncogene are associated with mul ple endocrine dysplasia II, a cancer-prone phenotype transmi ed in autosomal dominant pa ern.The affected embryos are carried to term and viable babies are born, but they later develop mul ple endocrine tumours, including carcinoma, pheochromocytoma, and parathyroid adenomas, most likely via the double-hit mechanism [59,60].Similarly, heterozygous gain-of-func on muta ons of the human c-KIT gene, coding for a receptor with tyrosine kinase ac vity func oning in haematopoiesis, melanogenesis, and gametogenesis, are associated with familial gastrointes nal stromal cancer [61,62].Loss-of-func on muta ons in proto-oncogenes may some mes be heritable in man.As could be expected, these are usually not associated with cancer-proneness but, rather, with defects in cell migra on and distribu on of precursor cells in their assigned loca ons during early embryonic life.For example, carriership of heterozygous muta ons in the region encoding the kinase domain of the human c-KIT gene, decreasing the response to KIT ligand binding, may be associated with the phenotype of piebaldism [63,64].Carriership of loss-offunc on muta ons (usually, par al or total dele ons) in the c-RET gene are associated with suscep bility to development of Hirschprung's disease type 1 [59,65].Some mes, same type of muta ons in the same proto-oncogene may be associated with different phenotypes.For example, both gain-of-func on and loss-of-func on muta ons in the c-KIT gene may be associated with familial gastrointes nal stromal cancer [61,66].

Tumour-suppressor genes
The products of tumour-suppressor genes play a role in damage-associated signalling and the induc on of cell cycle arrest and/or apoptosis in response to DNA damage or other type of damage.Pro-carcinogenic muta ons in tumour-suppressor genes are usually of the lossof-func on type and may be heritable.Individuals that have inherited one defec ve copy of a tumour-suppressor gene are at risk of developing a tumours in case the intact gene copy is lost or inac vated at soma c level (e.g. via the double-hit mechanism).Inherited muta ons in tumour-suppressor genes contribute to carcinogenesis in different ways.For example, they may increase the risk for introduc on of 'errors' in DNA during cell division (because of inefficient 'checking' mechanism; inefficient DNA repair and/or inefficient damage-associated signalling); or they may cause suppression of the mechanism for the induc on of cell cycle arrest and/or apoptosis in the presence of DNA damage.Some inherited muta ons in tumour-suppressor genes may indirectly cause s mula on of cell growth, e.g. by increasing the binding of pro-prolifera on factors [67,68].For more informa on on hereditary cancer syndromes associated with carriership of muta ons in tumour-suppressor genes (Li-Fraumeni syndrome, familial breast and colon cancer syndromes, re noblastoma, and others).

Mutator genes
The genome of undifferen ated cells (including cancer cells) is changeable in real me (hyperplas c).Some traits may be lost, while other may be newly acquired, and genes and whole gene clusters may be rapidly switched on and off regardless of the requirements of the normal developmental programme.The hyperplas city of the genome of cancer cells is a crucial part of their capacity to adapt to changing environmental condi ons (e.g.colonising new sites and invading ssues different from the ssue of origin of the tumour) and in response to an cancer treatments.Important players in the induc on and maintenance of genome hyperplas city are the products encoded by the so-called 'mutator genes'.The term 'mutator genes' denotes a large group of genes coding for proteins with diverse func ons.A common feature of mutator genes is that muta ons in them result in a net increase in the overall muta on rate in the genome of the transformed cell.Naturally, this result in increased risk for occurrence of muta ons in proto-oncogenes and 'second hits' in loci in which there is already one inac ve gene copy of a tumour-suppressor or a mutator gene.Thus, muta ons in mutator genes are associated with suscep bility for cancer not because of direct interference in the mechanisms of s mula on or suppression of cell prolifera on, but, rather, because they create a favourable environment for occurrence of other pro-carcinogenic muta ons.Muta ons in mutator genes may occur at soma c level or may be inherited, and the associated diseases and condi ons may be transmi ed in an autosomal dominant as well as in autosomal recessive manner.
The mutant protein products of mutator genes may contribute to destabilisa on of the genome via several different mechanisms.One of these mechanisms is, for example, increasing the risk for introduc on of errors during DNA replica on.Such are, for example, human genes coding for proteins ac ng in repair of mismatched bases in DNA -MSH1, MSH6, MLH1, etc. [69,70].Also, muta ons in mutator genes may result in increased rate of genomic rearrangements.Typical examples are the genes coding for the helicases WRN and BLM, ac ng in repair by homologous recombina on.
The genes coding for the RNA component (TERC) or the protein component (TERT) of the telomerase complex are o en denoted as mutator genes as well, as muta ons in them (soma c as well as inherited) may contribute to cancer development.The carcinogenic mechanisms associated with deregula on of telomere maintenance may significantly vary.Abnormally short telomeres and free reac ve chromosome ends may produce chromosome instability -transloca ons, chromosome fusion and breakage, etc. Inherited muta ons in the TERT and TERC genes associated with disordered telomere elonga on may produce dyskeratosis congenita; idiopathic aplas c anaemia or idiopathic pulmonary fibrosis; and, in case of dele on of a larger chromosome region, containing the TERT locus or the whole 5p chromosome arm -the cri-du-chat con guous gene syndrome [71][72][73][74], which is a is a severe congenital condi on, characterised by mul ple physical anomalies and mental retarda on, plus the issues associated with loss of the TERT locus.The condi on is officially termed '5q dele on syndrome', but is be er known by its trivial name, origina ng from the characteris c shrill crying sound, produced by the affected babies.
Ability to re-synthesise telomeric DNA is usually associated with increased capacity for cell prolifera on, postponing the onset of replica ve senescence; or cell immortalisa on.
Usually, soma c cells have virtually non-existent telomerase ac vity and adult stem cells have but a limited capacity for telomere elonga on.Re-ac va on or upregula on of telomerase ac vity in soma c cells is strongly indica ve of cancerous transforma on [75][76][77].For example, the 3q chromosome arm, containing the TERC gene copy may be mul plied in severe cervical intraepithelial dysplasia.This is usually a hallmark of transi on from CIN to overt cervical carcinoma [78].Amplifica on of the TERT locus has been observed in B-cell lymphoma [79].Some cancer cells are capable of telomere elonga on by an alterna ve mechanism, based on recombina on (alterna ve lengthening of telomeres, ALT, also called alterna ve telomere lengthening, ATL).In ALT, it is not the chromosome's own telomeric DNA used as a template for copying during replica on, but telomeric DNA from another chromosome.Specifically, single-stranded DNA end from one telomere invades double-stranded DNA of another telomere and uses it as a template for copying, eventually producing telomeric DNA with greater length than ini al invading telomere end [80,81].The ALT mechanism is dependent on the MRN (MRE11/RAD50/NBS1) complex that binds to free DNA ends generated by double-strand breaks, processes them and holds them together on order to facilitate the end joining in repair by recombina on.Overexpression of the SP100 nuclear protein is associated with sequestra on of the MRN complex, based on physical interac on between SP100 and NBS1 and eventually resul ng in ALT inhibi on [82,83].Deple on of NBS1, either with or without the other factors of the MRN complex results in inhibi on of the ALT mechanism for telomere elonga on [84].Tumour cells using the ALT mechanism are characterised by heterogeneity in telomere length, rapid changes in length of individual telomeres and high rates of exchange of telomeric DNA between sister chroma ds [85,86].
It is currently believed that in about 10-15% of all tumours the telomerase ac vity is lost at some point, beyond which the telomere length is maintained en rely by ALT [87,88].
Other types of mutator genes normally regulate the expression of other genes implicated in the control of cell growth.Deregula on of the func on of the mutator genes of this type promotes cancerous transforma on by s mula on or inhibi on of the transcrip on of their target genes.For example, muta ons in the gene coding for protein kinase C alpha may promote uncontrolled cell growth [89,90].Protein kinase C (PKC) is an ubiquitously expressed phorbol ester receptor with serine/threonine kinase ac vity that plays a role in prolifera on-associated transmembrane signalling by controlling the transcrip on of some of the major proto-oncogenes and genes coding for an -apopto c proteins (c-RAF1, BCL2, and others) and regula ng the ac va on of signalling cascades that s mulate cell prolifera on [91,92].Some of the invasive tumours of the pituitary glands and tumours of the thyroid gland express mutant variants of PKC-alpha [93,94].
The boundaries between the three categories of cancer-associated genes may some mes become blurred.For example, some of the 'classic' tumour-suppressor genes (prime examples are TP53, ATM, BRCA1 and BRCA2 genes) may also be classed as mutator genes, as they are involved in the maintenance of genome integrity as well.ATM, for example, is usually considered a mutator gene, as the loss of two gene copies produces genome instability, but is some mes viewed as a tumour suppressor, as its protein product func ons in induc on of damage-associated cell cycle arrest and apoptosis.Similarly, muta ons in the proto-oncogene c-RET may produce cancer, but it is usually via the double-hit mechanism that is considered typical of tumour-suppressor genes.
Currently, there is yet another classifica on of cancer-associated genes, assigning 'gatekeeper' func ons to tumour-suppressor genes and 'caretaker' func ons to mutator genes [95].The basic characteris cs of these two groups are quite similar to these defined by the previously discussed classifica on system.Gatekeeper genes are involved directly in the nega ve regula on of cell prolifera on -that is, they inhibit the progression in the cell cycle and/or promote cell death in response to damage.Different ssues have their specific gatekeepers and their inac va on is usually directly associated with a specific type of cancer -e.g.inac va on of the RB1 gene causes re noblastoma, inac va on of the APC gene produces colorectal polyps, etc. Caretaker genes usually play a role in the maintenance of the genome integrity and DNA repair universally, in all ssues.Their inac va on does not promote cancerous growth directly but, rather, by increasing the likelihood of occurrence of muta ons in other genes, including caretaker genes.Examples for caretaker genes are BRCA1 and BRCA2, the XP genes coding for proteins ac ng in NER, the MLH and MSH genes, encoding proteins func oning in mismatch repair, etc. Again, ATM is usually classed together with the caretaker genes.

Bases of cancer resistance to genotoxic drugs
Resistance to an cancer drugs may develop due to various reasons.Among the common causes for development of drug resistance may be upregula on of the expression of gene/s coding for product/s that func on in the sequestering of the drug or an ac ve metabolite so that they become unavailable or subthreshold; and/or their rapid clearance, and/or their degrada on.Some drugs used in an cancer therapy are metabolised by one or more specific enzyme systems.Thus, resistance to the drug may be induced by simple upregula on of the expression of the respec ve enzyme/s in the tumour.For example, many an tumour drugs are substrates for the cytochrome 1B1 (CYP1B1) enzyme of the cytochrome Р450 family.Among these are agents with direct genotoxic effect (e.g.inhibitors of topoisomerase II, such as mitoxanthrone); microtubule stabilisers (taxanes); an estrogens (tamoxifen, flutamide), tyrosine kinase inhibitors (ima nib) and others [96].CYP1B1 is normally expressed at low levels, but many primary and metasta c tumours overexpress CYP1B1, which had been found to be associated with resistance to an cancer agents [97,98].
Cancer cells may also physically mul ply (amplify) the ac ve gene copies coding for the enzyme or a key subunit of an enzyme that degrades or inac vates in any other manner the ac ve compounds of an cancer drugs.Normally, the expression of such proteins is strictly controlled.The gene amplifica on ensures that the synthesis of the protein encoded by the amplified gene is constantly kept at a high level.Such is the case, for example, with dihydrofolate reductase (DHFR) gene, encoding an enzyme inac va ng methotrexate and other cytosta c drugs [99].
Cells resistant to an cancer compounds may not rely on detoxifica on of the ac ve substance/s or metabolite/s but, rather, on decreasing their effec ve concentra ons within the cell.This may be implemented via binding of the compound to drug transporter proteins, such as mul drug resistance proteins (MDPs).MDPs are usually transmembrane proteins with high affinity to different chemical agents.They efficiently bind and export a variety of an cancer compounds outside the cell [reviewed in 100].
The genes coding for MDP may also be subjected to copy number amplifica on and/or upregula on of expression.For example, mul drug resistance in human cancer cell lines to colchicine, vinca alkaloids and an cancer an bio cs such as adriamycin may be due to increased expression of the mul drug resistance gene MDR1 as well as amplifica on of the MDR1 gene copies [101].Lung tumours have been shown to develop resistance to paclitaxel via MDR1 locus amplifica on [102].
Pla num-based an cancer regimens (regardless of whether the pla num compound is used as a single agent or combined with other drugs) are used very o en in the treatment of solid tumours because of the high response rates, comparable only to anthracycline-based regimens.Resistance of cancer cells to pla num-based drugs is a specific area, as the mechanisms of detoxifica on of pla num agents are quite different from these of most an cancer drugs.Generally speaking, out of the wide variety of an cancer compounds, it is only pla num agents that are not 'metabolised' or 'biotransformed', due to their unique structure [103].Resistance to pla num-based regimens is largely unrelated to modula on of enzyme-governed pathways but is strongly dependent on mechanisms such as sequestering the ac ve substance, rou ng it out of the cell or making it inac ve or unavailable before it had found its target.
Unlike many drugs administered intravenously, pla num compounds do not rapidly become bound by plasma proteins, but, rather, it is the pla num ions that are bound and transported inside the cell.Cispla n, the first ever pla num-based drug to be used in treatment of human cancer, and other pla num (II) complexes undergo spontaneous hydrolysis of the two chloride ions in aqueous solu ons.The pla num-containing ca on is bound by plasma proteins such as albumin, transferrin, and gamma globulin within 2-3 hours a er IV administra on.The pla num-protein complexes are then slowly cleared, predominantly by renal excre on, over the next several days.
Resistance to pla num regimens may be based on decreased concentra on of cispla n ca on in the cell due to lower uptake or to intensified export out of the cell.Un l recently, it was believed that pla num ions entered living cells mostly by passive diffusion, but later it was shown that ac ve uptake was also possible.For example, some mammalian copper transporter proteins (e.g.SLC32A1) may transport ac ve pla num compounds as well [104].
Transporter proteins may exhibit substrate specificity with regard to different pla num (II) complexes.For example, SLC22A, an organic ca on transporter protein, was reported to be capable of transpor ng cispla n, but not carbopla n [105].Two other copper transporter proteins, ATP7A and ATP7B, have been shown to be implicated in export of organic pla num-containing ca ons out of the cell [106].High levels of ATP7B proteins in pa ents with cancer have been shown to be associated with poorer response to cispla n-based regimens [106,107].
Binding and inac va on (also called 'trapping') of pla num compounds in the cytosol before they have reached the nucleus may also be a mechanism for development of resistance.A number of normal intracellular compounds (e.g.reducing agents such as glutathione and thioredoxin) may bind pla num deriva ves in a complex that may subsequently be exported from the cell [108].
The genotoxic ac on of pla num deriva ves is based on forma on of adducts in DNA (dG-dG and dG-dA), mainly between nucleo des in the same DNA strand, but also between different strands.Pla num agents may also cause DNA-protein crosslinks, albeit with lower efficiency [109].As pla num-based drugs cannot be 'metabolised', the resistance of cancer cells to them is strongly dependent on the capacity for excision of drug-induced DNA adducts.Indeed, most of the in vivo studies of the impact of individual repair capacity on drug resistance of tumour cells were carried out in pa ents treated with pla num deriva ves.The ability of tumour cells to repair the damage inflicted upon their DNA by pla num compounds is dependent on their gene c background, and specifically, on individual differences in the DNA repair capacity.Subtle as these differences may be, they may become significant under severe genotoxic a ack produced by a therapeu c course with one or several genotoxic agents.It is rarely the case that a cancer cell is a priori resistant to a cytotoxic drug (unless, of course, the mechanism of ac on of the drug employs a pathway that was already blocked or shunted in the tumour).Resistance to an cancer drugs usually develops in the course of treatment in cells that were ini ally sensi ve (some mes -very sensi ve) to the drug.There have been experimental proofs that some cancer cells are capable of restoring the ac vity of previously inac ve or weakly ac ve repair proteins by mutagenesis, resul ng in development of resistance to a drug to which the tumour was ini ally sensi ve.For example, experiments with mouse models of breast cancer carrying inac va ng (frameshi ) point muta ons in the BRCA1 gene showed that some tumours that ini ally were sensi ve to an cancer therapy (specifically, cispla n) subsequently re-acquired the expression of almost full-length BRCA1 and, respec vely, became resistant to cispla n [110].This could be expected, as tumour cells with de novo muta ons restoring the func on of BRCA1 would become able to remove adducts from their DNA more efficiently than the non-mutated BRCA1-defec ve tumour cells.To explain this phenomenon, a mechanism based on error-prone DNA synthesis was discussed.Specifically, it was proposed that damaged template was copied with low fidelity, adding or dele ng nucleo des around the muta on site, un l at some point the reading frame was restored, albeit at the price of dele on/s or subs tu on/s of one or more amino acid residues from the protein; or by inser on of non-template nucleo des [111,112].The rate of de novo occurrence of func on-restoring muta ons in the mouse models was es mated at 1:106 tumour cells.A similar process occurring in vivo in human tumours was described earlier with the BRCA2 gene in an individual with Fanconi anemia [113].The pa ent was a compound heterozygote by the 8415G>T (K2729N) and 8732C>A (S2835X) muta ons in the BRCA2 gene.Pa ents with Fanconi anemia are suscep ble to malignancy, especially haematological cancer.At age 2, the pa ent was diagnosed with acute myeloblast leukemia (AML).The inherited 8732C>A nonsense muta on was not found in leukemic cells from the pa ent, but a missense muta on (8731T>G) was iden fied at the muta on site.Apparently, the stop codon resul ng in trunca on of the BRCA2 protein at posi on 2835 was converted to a codon for a glutamate residue, restoring the open reading frame of the gene.Fanconi anemia cells are usually sensi ve to genotoxic agents (e.g.mitomycin C).Non-leukemic cells from the pa ent were found to be mitomycin C-sensi ve, while leukemic cells were significantly less sensi ve.
In mouse models in which the BRCA1 gene was destroyed beyond repair by targeted mutagenesis (e.g. by introducing large dele ons), resistance to cispla n deriva ves never developed.Indeed, the tumour never disappeared completely, but always grew back, only to regress promptly a er treatment with the same agent [110,112].It was proposed that this type of response of cancer cells to an cancer treatments may be seen, albeit rarely, in vivo, in mice or even in human pa ents.Borst et al. proposed that the remainder of breast cancer cells that was never fully eradicated by pla num-based therapy and was capable of restoring the tumour was made of very slowly cycling cells.The la er were supposedly s mulated towards prolifera on a er the rapidly cycling cells making up the bulk of the tumour had been killed [110].The process is quite similar to the cyclic ac va on of adult stem cells responsible for the renewal of normal ssues, but the new cells bear the hallmarks of cancer.For more informa on on cancer stem cells, see 'Cancer stem cells' below.

Cancer stem cells
The existence of cancer stem cells has been suspected for some years before the first conclusive evidence appeared in 1997, in research on histopathology of acute myeloid leukemia [114].The emergence of the idea of a stem cell from which could grow a tumour caused an almost complete reversal of the basic paradigms of the medical oncology.
Basically, it proposes that tumours (at least some of them) have their origins in altered stem cells, which produce offspring carrying a 'differen a on block'.These altered cells originate from the normal adult stem cells that reside in virtually all adult ssues, supplying new cells to the ssue to compensate for those that had died or had been lost for some other reason (e.g. because of injury).The prolifera on of adult stem cells is usually ghtly controlled.
They usually divide only when they receive a s mula ng signal -mediated by growth factors, hormones, other mediators, or simply signalling acknowledging p53-mediated removal of damaged cells [115].Cancer stem cells, however, have at some point lost their capacity for controlling their own prolifera on.The causes for this may be different -for example, because of soma c muta on that had occurred in the stem cell or its immediate progeny, resul ng in cons tu ve ac va on of a cellular proto-oncogene/s; or, if the cancer clone originates from a precursor cell in later phases of differen a on, because of newly acquired capacity for unrestricted growth [116][117][118].The differen a ng cell may also be converted to a cancer stem cell by acquiring muta ons that render it capable of ignoring pro-apopto c signals.Typically, the earlier the blockage occurs in a differen a ng cell, the lower the differen a on grade of the tumour, and, correspondingly, the higher its aggressiveness.As the differen a on of blood cells is very well studied, the correla on between the meline of the occurrence of poten al differen a on blocks and the type and the proper es of the corresponding haematological cancers is well established.Tumours with low grade of differen a on such as acute blast leukemias have high metasta c poten al and are associated with poorer prognosis for the pa ent, whereas high-grade tumours such as mul ple myeloma and some types of chronic leukemia are characterised by lower invasiveness and the pa ents exhibit be er survival.
According to the cancer stem cell concept, the tumour mass is made of precursor-like rapidly dividing cells that are usually sensi ve to DNA damaging agents.The actual source of these cells, however, has stem cell-like proper es; its cells divide slowly and, respec vely, are only mildly affected by genotoxic agents, if at all.Less than a dozen cancer stem cells (between 4 and 10) may be sufficient to completely restore the bulk of the tumour [118,119].This may explain why all an cancer treatments eventually fail in the end -they do not eradicate the cancer stem cells that cons tute the actual source of the tumour, only their immediate progeny.
Existence of cancer stem cells have been definitely proven so far for some cancers only, such as haematological malignancies and some CNS tumours such as gliomas [120,121], though conclusive evidence has been accumula ng for other types of cancers too, such as colon cancer, breast cancer and non-small-cell lung cancer 110,122,123].As most of the experimental results were obtained in in vitro se ngs and in vivo in animal models (usually, mice), the authors admi ed that the results were not likely to be directly applicable to man [110,112].There have not been definite proofs yet about whether all cancers originate from cancer stem cells or not.

The final checkpoint -cancer as an adaptive evolutionary mechanism
As we already saw, DNA repair/programmed cell death regulatory mechanisms usually manage DNA damage very efficiently, repairing minor damage and elimina ng seriously damaged cells.With ageing, however, the capacity for damage repair and self-renewal of cells and ssues declines and the level of unrepaired damage in the cell (which was low un l that moment) begins to rise.Sustaining unrepaired damage in cells that are capable of division increases the risk for introduc on of muta ons that may be inherited by the cell's progeny.If, as a result of introduc on of muta ons, the cell acquires the ability to bypass the checkpoints in the cell cycle where the integrity of the genome is assessed and the decisions whether to proceed with the cell cycle are made, it may actually evade the general direc ve that damaged cells must stop dividing and/or die.As a result of con nuous prolifera on of the cell carrying the altered genotype and the risk of introducing more muta ons, cancer may eventually develop.Certainly, this does not occur overnight, but, rather, as a long-term consequence of expanding and mul plying DNA errors that had occurred a long me ago.The risk of cancer usually rises with age, as a consequence of the decreased capacity for repair and ssue renewal and the longer me during which the organism had been exposed to damaging factors.
It is the simple and inevitable truth that everything that was ever alive must eventually die.This normally occurs a er a period of gradual but irreversible decline that is commonly called ageing.).This holds true for all living things on Earth, from the simplest prokaryotes to plants, animals and man.Indeed, bacteria are capable of numerous successive divisions, steadily producing (almost) iden cal daughter cells.Bacterial daughter cells may be slightly different from the mother cell -because of random mutagenesis; or via exchanges of discrete units of gene c informa on (e.g. via plasmids) with other bacteria.There is also the error-prone mechanism of translesion DNA copying that may allow prokaryo c cells to survive adverse condi ons, even though their DNA is seriously damaged, at the cost of introducing gene c muta ons.Compared to the limited number of divisions that eukaryo c cells could typically carry out before the onset of replica ve senescence, prokaryotes may be considered very long-lived indeed, prac cally immortal.Nevertheless, it is now known that prokaryotes may also experience gradual restric on of capacity for growth, resembling the process of ageing.The typical phenotype of a malignant cell comprises the capacity for sustaining mul ple divisions (prac cally indefinitely) and to mutate readily, so as to adapt rapidly to changing condi ons (in therapy se ngs -first-line an cancer therapy; second-, third-and so forth lines of therapy; adjuvant therapy (immunomodula ng agents, hormones, biological therapies, etc.)).Cancer cells escape death by actually re-inven ng the ancient ways of prokaryo c cells of living and reproducing for a very long me without showing symptoms of ageing.If we may return again to our hypothe cal example of a popula on of complex living beings that do not age (are prac cally immortal) because of preserved capacity for supplying new cells to make up for those that were lost for any reason throughout their lives we already saw that a er long enough me the popula on would dwindle to a limited number of very old, prac cally immortal individuals.These individuals were ini ally very alike in their gene c background but have since accumulated such an enormous muta on burden that they became hardly similar to each other.With a lifespan that long, and given the random nature of spontaneous mutagenesis, each of these individuals would eventually possess its own unique genotype.That would preclude sexual reproduc on, as it has very strict requirements for the gene c similarity of the ma ng individuals.The la er means that they would be unable to sustain the popula on over me, as old individuals would die, albeit very rarely (e.g. because of a very severe injury) and reproduc on would be hardly possible.This is, once again, a dead end, a gene c stagna on, similar to whatever may occur if all errors in DNA were repaired at a 0% error rate, though not because of too li le, but of too much gene c diversity.One could hypothesise that ageing (of cells, of ssues, and, ul mately, of the organism) is a safety mechanism put in place by Nature during evolu on so as to avoid reversion to the ancient mechanisms (nowadays seen in some prokaryotes only) that may sustain the life of the cell in changing environmental condi ons at the expense of introduc on of unwarranted gene c variability.
Even with all the advancements of modern therapy, cancer eventually kills, as cancer cells are capable of rapid invasion and colonisa on of all types of cells and ssues in mul cellular organisms, and cancer cells are typically not capable of performing the specialised func ons of normal cells.Again, one may speculate that this was the Nature's way to ensure that life based on uncontrolled prolifera on and unrestricted mutability is not a viable op on for living creatures -at least, not beyond the prokaryo c stage of evolu on.Thus, cancer may be viewed as a pre-programmed mechanism, a fail-safe that ac vates when all other op ons to prevent immortalisa on of cells become unavailable for any reason.
Ageing/death of old age, and whenever ageing is not an op on, cancer may then be viewed as the large, popula on-scale equivalents of the cell cycle arrest/DNA repair and programmed cell death mechanisms, designed to work synergis cally in order to maintain the con nua on of life by sacrificing individuals -be it cells, or living beings.On a cell-sized scale, programmed cell death is the only way to extract a damaged cell from its habitat without las ng damage to its neighbours, so that the ssue, the organ and the individual would con nue to live.On a larger scale, death (whether of old age or of cancer) is the only way to sustain life on Earth without permi ng the slow, hit-and-miss evolu onary process to accelerate abnormally and/or go astray.One could hypothesise that ageing is the normal 'default' process designed to ensure that (almost) every member of a popula on is allowed a me period in their life cycle in which they would have an (almost) even chance to contribute their own gene variants into the gene c pool of the popula on, then die, making space for their successors.The la er would, in turn, grow, reproduce, and eventually die, so that many different gene c combina ons would be tried and tested in the course of evolu on.Only in the rare cases when a cell manages to successfully escape the many checkpoints and mechanisms that order it to switch off the ageing mechanism beyond a certain point in their meline and eventually die, thus becoming a threat to the homeostasis of the mul cellular organism, may come cancer, which would eliminate the dangerous cell clone by killing the organism that created it in the first place.Cancer may then actually be a preprogrammed mechanism, the ul mate fail-safe placed in all cells of mul cellular beings in order to eliminate the risk of crea ng and propaga ng genotypes that may poten ally threaten the existence of the popula on, the species and life on Earth as a whole [reviewed in 124].
Certainly, it would be rather simplis c to imagine that the establishment of cancer as the final checkpoint (and any checkpoint or mechanism, for that ma er) were premeditated events.Rather, it was a naturally occurring phenomenon that was subsequently selected for during evolu on, similarly to sexual reproduc on or ageing.

Could we really 'cure' cancer?
It is the laws of Nature that we are dealing with.Figh ng these would be quite pointless.Giving up the fight altogether would be humilia ng, and, once again, pointless.The only viable op on before us is to study the laws of Nature thoroughly, so that we could have them working for us, not against us.

Arkadiy and Boris Strugatskie, One Billion Years
Before The End Of The World (Definitely Maybe), 1974.
Modern biomedical science is waging a real war on cancer, but while winning the individual fights, it is actually losing the ba le in the long term.One in six people in modern socie es eventually succumbs to cancer, despite the advances in research and all the achievements of the medicine and the pharmaceu cal industry.In the light of the theory that cancer is a natural mechanism that prevents complex living beings from living forever because of risk of reaching an evolu onary dead end, this outcome is hardly unexpected.Medicine, however, has all the poten al to become capable, in the near future, to slow down the progression of cancer to terminal phases for long enough so that the life expectancy and the quality of life of the individuals affected by cancer could be comparable to the popula on average.Indeed, the life expectancy of people diagnosed with some types of cancer has drama cally changed in the last decades, and many could live near-normal, fulfilling lives, or at least be as comfortable as possible under the circumstances.It is very rarely the case, however, that the cancer is truly cured (that is, it never relapses throughout the life of the pa ent, un l eventually they die of old age).Such may be the case with cancers of purely soma c origin that were diagnosed and exhaus vely treated at very early stage.Most cancers, however, are never completely eradicated, no ma er what treatments are undertaken, but are merely arrested in their progression.An cancer treatments are usually not a long-term cure, as cancer cells eventually manage to become resistant to all currently available treatments.
Why does the united an -cancer front always fail in the end?We already saw that cell prolifera on is ghtly controlled at mul ple levels to ensure that there is at least one (preferably more than one) op on for induc on of cell cycle arrest and/or apoptosis at every level, if DNA damage or other types of damage are present.Cancer cells eventually achieve resistance to all currently known chemotherapeu c agents exactly because of the opportuni es for interference in the progression through the cell cycle at different stages.Indeed, the capacity for regula on at almost every step of normal cell prolifera on means that there are poten al 'control overrides' at any stage.A checkpoint may be evaded, a crucial checkpoint controller may be eliminated (e.g. by introducing inac va ng muta ons or dele on of the gene copies coding for the wild type protein) or made to work in the opposite direc on (e.g.cancer-specific isoforms of various genes coding for proteins directly regula ng the progression through the cell cycle).In most cases, however, the abnormal cell is iden fied quite early in the course of its cancerous transforma on by any of these selfsame mechanisms for checking the status of DNA and genome integrity; and is usually promptly removed by programmed cell death.It is only very rarely indeed that a cell would accumulate enough muta on events so as to fully unleash its tumorigenic poten al.
One of the topics that commonly come up in mass media, usually under the headline of 'sensa onal news', is 'Scien sts invented a miracle drug [or other type of treatment] that would put a stop to cancer'.This rather bold (for a lack of be er word) announcement may be followed by a short passage made up of mangled sentences compiled from research reports published in the interna onal scien fic data banks (typically ones that came up there years ago) and assembled together in a manner that suggests that this is latestminute news and that the researchers are only a split second away from making a groundbreaking discovery that would eradicate cancer forever.Many real and very useful findings in the field of biology and medicine have suffered this fate (if we would only care to remember the widely publicised idea of stem cells being a cure for all diseases), and many pa ents with cancer and their families have learned the hard way that there is no universal remedy for all diseases, except, maybe faith, hope and love.Indeed, it is hardly conceivable from scien fic point of view that one could possibly invent 'a cure for leukemia' or 'a cure for breast cancer'.The different types of cancer affec ng the same ssue and/or organ may be very different, to the point that the only unifying feature between them may be that they happen to occur in the same loca on in the body.The misconcep on that one cure may work for all varie es of the same type of tumour probably stems from the early days of oncology, when the only tool available to the physician for examina on of a living pa ent was observa on, and when tumours were classed according to the organ or bodily part that they affect.O en, different forms of the same type of tumour are very dissimilar to one another in respect of their aggressiveness, expression profile, eligibility for treatment with different agents, response to various therapies, and other important characteris cs.For example, among leukemias there are very aggressive forms (e.g.acute blast leukemias) which follow a rapidly progressive course despite the modern treatment modali es; and there are indolent forms (e.g.some types of chronic lymphocy c leukemia) that may need treatment only at late stages, if at all.The outcomes of treatment for different varie es of the same generic type of tumour may also greatly vary.For example, the survival rate of Hodgkin's lymphoma, when diagnosed and treated at an early stage, may be between 85 and 98%, while for other types of lymphoma (e.g.angioimmunoblas c T-cell lymphoma) the 5-year survival rate is s ll around 30% [125].Similarly, many types of breast tumours are very sensi ve to conven onal an cancer therapy, even those known to have high invasive poten al.These tumours may be fully manageable by a combined approach consis ng of surgery and several courses of chemo-and/or radiotherapy.One specific type of breast cancer, however -the invasive breast cancer -is usually very aggressive, surgery is typically inefficient and even combined chemoradiotherapeu c approaches have but li le success with it.Finally, the same type of cancer may follow very different course in different pa ents, which is dependent on a myriad of factors, endogenous (e.g.general condi on, gene c background, other co-exis ng diseases and condi ons, mo va on, etc.) as well as exogenous (accessibility to different treatments, living condi ons, etc.).Even cancers that are generally considered very aggressive may be associated with differen al survival rates in different pa ents.For example, the survival rates of Burki 's lymphoma may vary between 30 (in adults with mul ple addi onal risk factors) and >90% (in children with 0-1 risk factors) [126].Thus, it is rather naïve to think that even the best an cancer medicine could work on all cancers and in all pa ents.Every type of cancer ought to be viewed as a separate en ty, with its unique origin, proper es and course, and every cancer sufferer must be treated with regard to their own unique nature.A universal 'cure for cancer' as such does not exist at the moment and is unlikely to be ever invented.

4. 1 .
'Double-hit' and 'mul ple-hit' mechanism of tumorigenesis The classical model of Knudson (1971) presents tumorigenesis as a process dependent on gene c (determinis c) factors as well as environmental (stochas c) factors.A basic schema c of the double-hit mechanism is presented on Fig. 1.

4. 2 .
Should I stay or should I go?Deciding the fate of a new cell

Figure 2 .
Figure 2. Asymmetric segrega on of DNA strands in stem cell division.All DNA molecules (chromosomes) that contain the 'ancestral' DNA strand are specifically