The term cancer describes a larger number of complex diseases affec...

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Cancers are named according to where they originate from in the body, carcinomas are the most common type of cancer and arises from cells that cover external and internal body surfaces. Sarcomas arise from cells originating from the body's supportive tissues, for example, bone, cartilage, fat, connective tissue and muscle. Lymphomas originate from the lymph nodes and tissues of the body's immune system and finally leukaemias are cancers of the immature blood cells created in the bone marrow. Categorising cancers aids in prognosis and optimal management.

It is important to understand how a cancer cell develops. All cancers are the product of a series of micro-evolutionary stages. Tumour development has been described as being analogous to Darwininian evolution whereby a series of genetic changes each conferring a growth advantage lead to the conversion of a normal human cell into a popluation of proliferating invasive cancer cells.

This essay will discuss the genetic basis of cancer. Three main categories of genes have been found to be involved in cancer development; oncogenes, tumour suppressor genes and DNA repair genes. Furthermore, the cell cycle will also be discussed taking into consideration the normal role of each of these genes within the cell cycle and how each of these have been implicated in the development of cancers.

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Cancer as a multi-evolutionary process

There are two types of cancers, sporadic and hereditary. In a sporadic cancer two independent hits are needed before an individual develops cancer. Mutations occur at a relatively low rate in the human genome, approximately 105 to 10-7/gene/generation. The human body is composed of ~1014 cells so that the probability of 6 mutations occurring in any one cell is 1/1014 x (10-6) = 1 in 1022. It is unlikely that at any one time a cell will accumulate so many mutations (Full London Ideas Reference).

Cancer is mainly a genetic disease which is clonal in nature beginning when a single cell gains a series of successive mutations. It is the accumulation of mutations which convert a normal cell into a cancerous cell. A cell evading normal cell growth mechanisms is known as a tumour, and these can be either benign (non invasive) or malignant (invasive). As a malginant tumour grows in size angiogenesis is stimulated. Angiogenesis involves the cell obtaining a new source of blood vessels to provide the tumour cell with oxygen and nutrients. Metastasis follows angiogenesis with the tumour cell invading surrounding tissue to spread to other parts of the body. Six hall marks of cancer proposed by Hanahn and Weinberg define a tumour cell (Hanahn & Weinberg, 2000). These are:

Independant development from external growth signals

Insensitivity to external anti-growth signals

Loss of apoptosis

Indefinate replication

Ability to trigger angiogenesis and vascularisation

Metastasis of other tissues and establishment of secondary tumours

Genomic instability facilitates the micro-evolutionary process involved in transforming a normal somatic cell to a metastatic one. Stability can either be chromosomal causing abnormal karyotypes, for e.g. chromosomal rearrangements creating a fusion gene composed of the BCR and ABL genes located on chromosomes 9 and 22 respectively or via microsatellite instability (MIN). MIN is a DNA instability seen in certain tumours, in particular colon carcinomas. Indeed, the best example understood model for tumour development is colorectal cancer.

Development of a tumour

In approximately 1% of cases people who develop colorectal cancer do so through inheritance of an autosomal dominant disorder known as familial adenomatous polyposis (FAP). Individuals affected with FAP develop many polyps of the large bowel. The earliest indications of normal colonic epithelium developing abnormally is seen under the microscope as aberrant cryptic foci; these progress into polyps, or adenomas. Adenomas are benign epithelial growths which can develop from early (less than 1cm in diameter), intermediate (greater than 1cm but not necessarily carcinomic) to greater than 1cm with carcinomic features when metastasis occurs. The transition from a small adenomatous polyp to an invasive cancer takes between 5 to 10 years (Fearon & Vogelstein, 1990).

A possible five-hit scenario for colorectal cancer, showing the mutational events that correlate with each step in the adenoma-carcinoma sequence.

Figure 1 illustrates that a series of successive mutations is required and not the order in which they appear which is important in the development of a carcinoma. These mutations present in various genes as the size of the tumour grows. In the figure, the loss of the adenomatous polyposis coli (APC) gene on chromosome 5q is the first gene to be mutated, this is followed by activation of the K-ras gene (50% of tumours), followed by loss of chromosome 18q in 70% of tumours where three genes are located these are the DCC gene (deleted in colorectal carcinoma), SMAD4 and SMAD2, members of the a gene family involved in cell signalling involving the transforming growth factor beta pathway. Other genes mutated are the TP53 (guardian of the genome) and Nm23 (suppressor in metastasis).

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Chromosomal instability refers to both structural aberrations caused by DNA replication for e.g. inversions, large deletions, translocations to numerical aberrations arising from segregation defects (Venkitaraman, 2009). The main source of the many numerical abnormalities seen in cancer cells involves a defective spindle checkpoint where sister chromatids separate before being correctly attached to spindle fibres, this is discussed within the context of the cell cycle in more detail in section ???. The second is due to a defective signalling mechanism which fails to recognise damaged DNA. In a normal situation, mutated DNA is repaired before it can enter another round of cell cycle division, or if it is irreparable cells undergo apoptosis. In tumour cells this signalling mechanism is defective and cells either undergo further rounds of division without having repaired their DNA or evade the signals for apoptosis. The third mechanism causing chromosome instability is due to the absence of telomeres,

Telomeres are structures which protect chromosome ends. Under normal circumstances chromosome replication causes telomere length to decline by 50-100bp with each cell generation as DNA polymerase cannot use the extreme 3' end of a DNA strand as a template for replication. The length is then restored by an RNA enzyme called telomerase which is present in the germ line but not in somatic cells. In the absence of telomeres, cells treat the ends as double stranded breaks which are then repaired by either homologous recombination or non homologous end joining. This causes chromosomal translocations or dicentric chromosomes, and during anaphase of mitosis cells fail to divide properly causing further rounds of fusion and breakage. In a tumour cell, indefinite replication results in the absence of telomeres, the chromosome ends are then seen as double strand breaks and are either repaired randomly using the mechanism mentioned or fuse with other chromosomes.

At the gene level three main categories of genes are involved in cancer development, oncogenes, tumour suppressor genes and DNA repair genes.

Oncogene and tumour suppressor genes

Oncogenes

Oncogenes (non mutated versions were initially termed proto-oncogenes) function in cell proliferation. Dominant gain-of-function mutations within tumour cells generate varieties which are either excessively or inappropriately active to cause uncontrolled proliferation. Oncogene activation can be both quantitative and qualitative. Quantitative activation involves the increase of production of a mutant protein whilst qualitative involves the production of a modified product as a result of a mutation. Oncogenes can be activated in 1 of four ways over-amplification of the gene, point mutations, deletions in the coding region, and finally the translocation of an oncogene to a chromosomal region of high transcriptional activation.

Amplification

Examples of oncogenes altered by amplification include ERBB2 also known as Her2 and MYCN, These are involved in breast, ovarian, gastric, non-small-cell lung and colon cancer and neuroblastomas respectively. Mutations in oncogenes result in an increase in the quality of the gene product. Techniques such as fluorescent in situ hybridisation (FISH), antibody detection and comparative genome hybridisation can identify oncogenes having undergone amplifcation. These same techniques can also be used to identify mutant tumour suppressor genes, since this will be indicated by a loss of material.

Point mutations or deletions in the coding region

The RAS oncogenes are a family of genes comprised of HRAS, KRAS and NRAS. Mutations in the RAS genes are found in tumour cells of colon, lung and bladder cancers. The encoded proteins mediate signalling via receptor tyrosine kinases. The RAS proteins typically have GTPase activity. When a ligand binds to the receptor this triggers the binding of GTP to the RAS protein; and the GTP-RAS in turn transmits the signal within the cell. The GTPase activity of the Ras protein converts GTP-Ras to GDP-Ras. In this form it is inactive and the signal is switched off. Mutations in RAS decrease the GTPase activity of the protein, causing a slower inactivation of GTP-Ras resulting in an abnormally excessive cellular response to the signal from the receptor.

Activation by chromosomal rearrangements creating a novel chimeric gene

Chronic myeloid leukaemia (CML) is a cancer of the white blood cells, caused by a reciprocal translocation between chromosome 9 and the acrocentric chromosome 22; the translocation is designated t(9:22). This chromosome is more commonly known as the Philadelphia chromosome and designated Ph1. The breakpoint at chromosome 9 is within an intron of the ABL1 oncogene and joins the 3' translocation part of the ABL1 genomic sequence onto the 5' part of the BCR (breakpoint cluster region) gene on chromosome 22. This creates a fusion gene which is expressed to produce a tyrosone kinase with properties similar to the ABL1 product but with abnormal transforming properties. Ph1 is present in almost 90% of white blood cells in affected patients.

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Other tumour-specific rearrangements that produce chimeric oncogenes have been identified. This mechanism is seen in 15-25% of leukemias, lymphomas, and sarcomas but has been reported in only 1% or less of the common solid epithelial tumours.

Activation by translocation of an oncogene to a region of transcriptionally active chromatin

Burkitt's lymphoma is a rare cancer predominantly affecting young children in central Africa, although it has been reported in other areas. Although the pathogenic mechanism is unknown the form seen endemically in Africa appears to be associated with the Epstein-Barr virus. Burkitt's lymphoma occurs due to the activation of the MYC oncogene. The MYC oncogene located at 8q24 translocates close to an immunoglobulin (IG) locus, which may be IGH at 14q32, IGK at 2p12 or IGL at 22q11. The characteristic t(8;14;q32) translocation is seen in ~ 75-85% of patients with the remainder have t (2;8)(p12;q24). These differ from the previous translocations described in that chimeric genes are not produced. Instead, the translocated oncogene is under the control of regulatory elements that normally produce high expression of the immunoglobulin genes in antibody-producing B cells.

These four mechanisms are illustrated in figure ????

oncogene

Four mechanisms where an oncogene cause cancer. (a) deletion or point mutation in a coding sequence, gene amplification and chromosome rearrangement and (b) insertional mutagenesis where recombination occurs between the retroviral DNA and the oncogene. This has effects similar to those of chromosome rearrangement, bringing the proto-oncogene under the control of a viral enhancer and/or fusing it to a viral gene that is actively transcribed. Insertions of the virus indicated by arrows can activate transcription of the oncogene from distances of more than 10,000 nucleotide pairs away and from either side of the gene. This effect is attributed to a powerful enhancer DNA sequence present in the terminal repeats the viral genome.

Five broad categories of oncogens have been identified and these are summarisd in table ???

IMPLICATION OF ONCOGENES AND TUMOUR IN THE DEVELOPMENT OF CANCERS

Function

Cellular

Proto-oncogenea

Location

Viral oncogenea

Viral disease

SECRETED GROWTH FACTORS

Platelet-derived growth factor B subunit

PDGFB

22q13.1

v-sis

Simian sarcoma

CELL SURFACE RECEPTORS

Epidermal growth factor receptor

EGFR

7p11.2

v-erbb

Chicken erythroleukemia

Macrophage colony stimulating factor receptor

CSF1R

5q32

v-fms

McDonough feline sarcoma

SIGNAL TRANSDUCTION COMPONENTS

Receptor tyrosine kinase

HRAS

11p15.5

v-ras

Harvey rat sarcoma

Protein tyrosine

kinase

ABL1

9q34.1

v-abl

Abelson mouse leukemia

DNA BINDING PROTEINS

AP-1 transcription factor

JUN

1p32.1

v-jun

Avian sarcoma 17

DNA-binding transcription factor

MYC

8q24.21

v-myc

Avian myelocytomatosis

DNA-binding transcription factor

FOS

14q24.3

v-fos

Mouse osteosarcoma

CELL CYCLE REGULATORS

D-type cyclins:

Cyclin D1

Cyclin D2

Cyclin D3

CCND1

11q13

K-cyclin of Kaposi sarcoma-associated herpesvirusb

Kaposi Sarcomab

a The viral oncogenes are designated v-sis, v-myc, etc., their cellular counterparts can be described as c-sis, c-myc, etc. b Kaposi sarcoma-associated herpesivurs encodes a virus-specific D-type cyclin, which is an independant gene, not an activated version of one of the human cyclin D genes. However, oncogenic activated versions of all three human D-type cyclins, the result of somatic mutations, have been identified in certain leukemias.

Tumour Suppressor Genes

The second major class of genes involved in the development of cancer are tumour suppressor genes. In contrast to oncogenes, these genes act to limit normal cell proliferation by preventing inappropriate cell cycle progression or leading mutant cells into apoptosis. Mutations affecting tumour suppressor genes are recessive loss of function, thus both alleles must be inactivated.

Knudson's two hit hypothesis uses the retinoblastoma protein as a paradigm for understanding how tumour suppressor genes are involved in causing hereditary cancer. In hereditary cancer an individual has a greater predisposition to develop cancer. They need only one hit inherited in the germline and a single mutation in a somatic cell for tumorigenesis to occur. Therefore a mutation in both alleles is required to knock out the function of a gene (Knudson, 1971).

Figure ???? Knudson's two-hit hypothesis for tumourigenesis involving a tumour suppressor gene (TSG). A pair of chromosomes is illustrated, with one TSG [the normal gene (grey), the mutated gene (red), and deletion of the gene (absence) are shown]. (a) Normal individuals have two normal copies of the TSG, so two independent 'hits' (mutations) are required in the same cell to initiate a cancer. (b) Individuals with a germline mutation of the TSG already have a first 'hit' in every cell and require only one subsequent 'hit' in a cell to initiate a cancer.

Tumour suppressor genes thus function as recessives at the cellular level although cancer syndromes involving tumour suppressor genes (e.g. FAP and breast cancer) are inherited in a dominant Mendelian fashion. Examples of tumour suppressor genes involved in cancer are given in table ?????

Tumour suppressor gene

SYNDROME

TUMOUR

ASSOCIATED CANCER/TRAITS

CHROMOSOME LOCATION

GENE/PROTEIN PRINCIPAL FUNCTION

RB1

Familial retinoblastoma

Retinoblastoma

Osteosarcoma

13q14

Transcriptional regulation (E2F mediated) and cell cycle regulation

APC

Familial polyposis

Colorectal cancer

Intestinal polyposis, duodenal tumours, desmoids tumours, jaw ostomas, medulloblastoma

5q21

Regulation of level of transcriptional activator β-catenin

TP53

Li-Fraumeni

Sarcomas, breast cancer

Brain tumours, leukemia, adrenocortical carcinoma, others

17q13

Response to DNA damage and other cellular stresses

WT1

Familial Wilms Tumour

Wilms tumour

WAGR syndrome (Wilms tumour, aniridia, genitourinary abnormalities, mental retardation), Denys-Drash syndrome

11p13

Transcriptional regulation, e.g., of apoptotic factors

NF1

Neurofibromatosis type 1

Neurofibroma

Neurofibrosarcoma, brain tumors

17q11

GTPase-activating protein, regulating RAS proteins

NF2

Neurofibromatosis type 2

Acoustic neuromas

Meningioma, glioma ependymoma

22q12

Links cell membrane proteins to cytoskeleton . Cell adhesion and coordination of growth factor receptor signalling.

ATM

Ataxia telangiectasia

Lymphoma

Cerebellar ataxia immunodeficiency, breast cancer in heterozygotes (?)

11q22

Cell cycle arrest (via p53) DNA repair (via BRCA1/MRE11/NBS1)

MEN1

Multiple endocrine neoplasia type 1

MEN 1

Parathyroid hyperplasia, pituitary adenoma, pancreatic islet cell tumors

11q13

Incompletely understood, but transcriptional regulation likely

RET

Multiple endocrine neoplasia type 2

MEN 2

Medullary thyroid cancer plus in MEN 2A: pheochromocytoma, parathyroid hyperplasia. In MEN 2B: pheochromocytoma, mucosal neuroma

10q11

Receptor tyrosine kinase

BRCA1 BRCA2

Familial breast cancer

Breast cancer

Ovarian cancer

BRCA1

BRCA2

Double strand break repair of DNA

VHL

Von Hippel-Lindau disease

Renal cancer, pheochromocytoma

Retinal angiomas, hemangioblastomas

3p25

Indirect regulator of transcription of hypoxia-inducible genes

THE CELL CYCLE

Survival of an organism is dependent on the accurate transmission of genetic information from one cell to progeny cells. Such faithful transmission requires accuracy in the replication of DNA, precision in chromosome distribution, and the ability to survive spontaneous and induced DNA damage. The accurate distribution of a fully replicated, intact genome perhaps defines the purpose of the cell cycle, which is a complex, organized series of events pertaining to the production of daughter cells (Nurse, 2000). Depending on the type of signal received, cells have three choices, they can progress through the cell cycle to complete division, enter cellular senescence or they can undergo apoptosis.

The cell cycle is divided into four distinct phases, each of which must be complete before entering the next, G1, S, G2, M phase, at G0 cells can opt out of the cell cycle (cellular senescence). Progression through the cell cycle is not only regulated by a series of checkpoints (section ????) but is also controlled by varying levels of cyclins and cyclin dependent kinases (CDKs). The CDKs are known as the master regulators of the cell cycle and form complexes with the cyclins to allow the cell cycle to proceed from one phase to the next (Yata & Esashi, 2009). The phases of the cell cycle are illustrated in figure ???, also shown are the various cyclins and their associated kinases (cdks) at the stages at which they are thought to act. Many of the genes that are required for cell cycle control are mutant in cancer cells (Nurse, 2000).

Figure ???? CDKs and the cell cycle. Representation of some of the mammalian CDKs involved in progression throughout the different phases of the cell cycle. Some of these kinases are required for DNA replication (S-phase) whereas other participate in the preparation for chromosome segregation during mitosis.

DNA checkpoints control cell cycle progression

DNA checkpoints can be perceived as the brakes in the cell cycle machinery. They

function to halt cell cycle progression, thus safe-guarding completion of one phase before

another can begin. In cancer cells, typically these checkpoints are defective. DNA replication occurs despite damage being present, mitotic entry occurs with unrepaired damage and, in addition to this, chromosome segregation is inefficient. This results in genomic instability and results in cellular malignancy. There are four DNA integrity checkpoints which respond directly to changes in DNA structure; G1, SdNTP-M, intra-S and the G2-M checkpoint.

G1/S checkpoint - At this stage cells make a commitment to enter into the mitotic cycle. Cdk2/cyclin E levels control this checkpoint and entry into S phase is inhibited where there is unrepaired DNA damage which leads to apoptosis.

The intra-S checkpoint functions to ensure that S phase events are complete before entry into mitosis. Mitosis can only occur if all chromosomal DNA has been replicated, with the simultaneous establishment of sister chromatid-cohesion and centrosome duplication.

G2/M checkpoint - At the G2-M transition, cells initiate the mitotic process of chromosome condensation, mitotic spindle assembly and cytokinesis. Before cells pass the late G2 checkpoint to enter mitosis two criteria must be met: first the cell must be a critical size, and second the chromosomes must be intact. Entry into mitosis depends on the phosphorolation status of Cdk1/cyclin B; controlled by the phosphatase Cdc25C. Incomplete DNA replication or unrepaired damage initiates signals which activate Cdc25C inhibitors, thus preventing Cdk1 from becoming active.

The spindle (or mitotic) checkpoint - Many of the events of mitosis are dependent on the formation of the mitotic spindle, a structure composed of microtubules designed to coordinate the chromosomal movements of eukaryotic cell division. During S phase, not only is the DNA duplicated but so is the spindle pole body (SPB). The anaphase promoting complex / cyclosome (APC)/C controls the metaphase to anaphase transition. The APC/C is a cell cycle regulating ubiquitin ligase responsible for mediating the proteolysis of the mitotic cyclins, cyclin A and B and indirectly cohesin. APC/C attaches multiple ubiquitin residues to target substrates for degradation by the 26S proteasome. Kinetochores are attached to both the chromosome centromeres and the spindle microtubule. When kinetochores attach to spindle fibres tension is generated across centromers as result of the fibres pulling the chromosome attached kinetochores towards themselves and the sister chromatid cohesin opposing the pulling force. To ensure accurate segregation with the correct chromosome complement, the kinetochores which are not attached to the spindle microtubule secrete a signal that inhibits the APC. The chromosomes will separate only if the correct degree of tension is generated. If there is a defective signal, chromatids can separate before all of them have been correctly attached to spindle fibers resulting in cells receiving an inaccurate chromosome complement (Riedel et al., 2006).

The decision to enter another round of division occurs in G1; in terms of carciongenesis this is particularly important. Three key tumour suppressor genes control entry into G1 of the cell cycle, these are RB1, TP53, CDKN2A genes.

RB1 retinoblastoma

The RB1 gene, identified as having a role in retinoblastoma.

A two-year-old with leukokoria in the left eye. A Leukokoria is a white reflex, or white pupil, instead of the normal red reflex and is the most common presenting sign in retinoblastoma.

The RB1 gene localises to 13q14.1-q14.2.1, and encodes a 100kDa nuclear protein existing in two related isoforms, p107 and p130, both of which function in the Rb pathway. A complex formed by cyclin D and two cyclin dependent kinases (Cdk4 or Cdk6) phosphorylate pRB in turn deactivating pRB and releasing the E2F transcription factor. E2F stimulates the transcription of genes involved in S phase progression. pRB prevents the cell from replicating damaged DNA by preventing progression from G1 into S phase In cells with loss of function mutations in the RB gene E2F is inappropriately inactivated causing uncontrolled cell proliferation even in the presence of DNA damage.

TP53 and Li Fraumeni syndrome

TP53 is a tumour suppressor gene and is known as the guardian of the genome, the gene localises to 17p13.1. Somatic mutations in TP53, account for the most common single genetic changes in cancer and the gene is the major target of mutations in cancer. Tumour cells with absent or non-functional p53 may continue to replicate damaged DNA and do not undergo apoptosis. The MDM2 oncogene, expressed in sarcomas controls levels of p53. Both protein products are involved in negative feedback regulation whereby the Mdm2 protein (also a transcriptional target of p53) ubiquitylates p53 for degradation. p53 is phosphorylated when the cell undergoes stress from e.g. DNA damage, once phosphorylated, it is no longer a substrate for Mdm2, levels of p53 subsequently increase and p53 dependent genes e.g. p21WAF/CIP1 (an inhibitor of Cdk2) and the genes involved in apoptosis (PUMA, BAX, NOXA) are transcribed, figure ???

Prior to DNA synthesis, the p53 protein ensures that the integrity of DNA is intact and if it is not then it responds in one of two ways; either initiating cycle arrest or by inducing apoptosis. The p53 response begins with the MRN complex which is a hetero-trimeric protein complex consisting of Mre11, Rad50 and Nbs1. DNA damage is initially detected by the 3056 amino acid ATM protein kinase which is activated by double strand breaks. Upon activation the protein phosphorylates p53, NBS1 (Nibrin), CHEK2 and BRCA1. A related kinase ATR, performs a similar function in respect of stalled replication forks.

Mutations in p53 cause Li-Fraumeni syndrome a rare autosomal dominant hereditary disorder increasing susceptibility to cancer. Li-Fraumeni syndrome is linked to germline mutations in the p53 gene and affected individuals are at risk for a wide range of malignancies, in particular breast cancer, where it accounts for approximately 1% of cases. It also has an involvement in brain tumors, acute leukemia and soft tissue sarcomas.

Once Nibrin is phosphorylated it forms the MRN complex which localises to sites of damage and is proposed to recruit further repair enzymes for e.g. BRCA1. CHEK2 is a mediator kinase and upon phosphorylation by ATM it communicates this signal in turn to other substrates including p53.

CDKN2A

Cyclin dependent kinase inhibitor 2A (CDKN2A) also known as MTS and INK4A localised at 9p21, uses two alternative promoters and first exons to encode two key regulatory proteins which are structurally unrelated. p16INK4A is translated from exons 1α, 2 and 3 and p14ARF from exons 1β, 2 and 3 with an alternative reading frame (ARF) for exons 2 and 3. Both proteins are active in the p53 ad RB pathway. Mutations in p16INK4A are seen in families affected with multiple melanomas. p14ARF intercedes G1 arrest by sequestering Mdm2 this causes an increase of the p53 protein. Loss of p14ARF, leads to enhanced Mdm2 levels causing unnecessary and excessive destruction of p53 and in turn loss of cell cycle control.

p53 and pRB are key players in a common pathway and it has been proposed that tumour cells need to inactivate both pRB and p53 for a cell to bypass cell cycle checks and void triggering apoptosis. Homozygous deletions of the CDKN2A gene have the ability to inactivate pRB and p53 to cause tumourogenesis.

p53 and RB interact with the products of the CDKN2A gene. Both p53 and RB are important components of two main tumour-suppressor pathways mediating a cells response to oncogenic stimuli. In the p53 pathway, a DNA damage signal induces the ARF gene which increases levels of p53 by reducing Mdm2. A reduction in Mdm2 prevents the degradation and inactivation of p53. P53 has both transactivation and transrepression activity and so controls the transcription of numerous genes. p53 targets WAF1 a CDK inhibitor and BAX a promoter of the apoptotic pathway. In the RB pathway, p16INK4A is a CDK inhibitor and this prevents the phosphorylation of pRB which is required to inactivate pRB during the G1 phase of the cell cycle. RB recruits transcription factors and chromatin remodelling proteins and in this way can control the expression of numerous genes. In particular, it inhibits E2F activity; E2F has a role in the transcription of several genes required for progression through the G1 and S phases of the cell cycle.

DNA repair

Everything discussed so far about the cell cycle in this introduction has obviated one basic requirement, that the integrity of a cells genome be maintained. Although these mechanisms exist to ensure everything goes smoothly, damage to DNA is unavoidable.

The process of DNA repair could be considered an umbrella term encompassing a spectrum of processes whereby a cell identifies and corrects damage to DNA molecules encoding the genome. Damage to a cell can occur as a consequence of defects in DNA metabolism such as errors in replication, or environmental factors such as ionizing radiation. DNA repair processes must be constantly active to repair damaged DNA immediately, and prevent genomic instability (Friedberg, 2008).

DNA damage can arise in many ways, at an exogenous level through environmental agents such as UV-C and ionizing radiation, or endogenously by reaction with genotoxic chemicals that are by-products of normal cellular metabolism. DNA is also susceptible to damage caused from chemicals such as polycyclic hydrocarbons in smoke. Chemical damage can cause a multitude of DNA adducts, oxidised bases, alkylated phosphotriesters and DNA cross-linking.

In normal cells, signals initiate DNA repair pathways Defective repair mechanisms can also contribute to the development of cancer, this will be discussed in the following sections.

Repair pathways

DNA repair systems are highly conserved throughout all organisms from both prokaryotic cell systems to eukaryotes. Many common repair pathways are present within cells such as the mismatch repair pathway (MMR), nucleotide excision repair (NER), base excision repair (BER), translesion DNA synthesis (TLS) and homologous recombination.

Mismatch repair (MMR) pathway and Hereditary Nonpolyposis Colorectal Cancer (HNPCC)

Mismatches in DNA arise by spontaneous or induced base modifications, by replication errors, or by heteroduplex formation during recombination. MMR systems have to complete at least three processes which include recognising the mismatched base pairs, determining which base in the mismatch is the incorrect one and, finally, excising the incorrect base and carry out repair synthesis.

HNPCC accounts for about 5% to 8% of all patients affected with colorectal cancers. It is difficult to diagnose because there are no premalignant polyps present. The Amsterdam criteria II are strict diagnostic guidelines produced to aid in the diagnosis of HNPCC. These are:

Exclusion of familial polyposis and verification of tumour types by pathological examination

Presence of HNPCC - related cancer in at least three relatives. One of whom should be a first degree relative of the other two.

Two or more successive generations affected

At least one of the affected family members should be less than 50 years old at the time of diagnosis.

Family studies using linkage analysis identified several causative genes for HNPCC, neither of which have been linked to tumour suppressor genes, these are all part of the MMR pathway and are listed in Table ????

E-coli

Human

Chromosomal Location

Frequency in HNPCC

MutS

MSH2

2p21

35%

MSH3

5q14.1

0%

MSH6

2p16

5%

MutL

MLH1

3p22.2

60%

MLH3

14q24.3

0%

PMS2

7p22

Few cases reported

Sequence homology of the protein products indicated there was similarity between the bacterial mismatch repair proteins. Mutations in the MMR genes not only impair the mismatch repair pathway but also cause regions called microsatellites to get longer or shorter; this is known as microsatellite instability (MSI). Approximately 90% of HNPCC tumours display MSI. Genetic testing for HNPCC involves looking at MMR genes for MSI. In the absence of MSIs it is unlikely that MMR genes are the cause of the tumour. Microsatellite instability is seen in 10-15% of colorectal, endometrial and ovarian carcinomas but only occasionally in other tumours.

The majority of individuals affected with HNPCC are heterozygous for a loss of function mutation, almost always involving MLH1 or MSH2. Normal cells of these individuals have a functional mismatch repair system and do not show the MIN+ phenotype. For the development of a tumour, the second copy of the gene must be lost in a manner similar to described for retinoblastoma. An alternative explanation for the loss of the second copy of the gene can be attributed to promoter methylation. This is more applicable to the MLH1 gene.

Mammalian MMR involves genes homologous to the E. Coli MutS and MutL (I) and (II) and (III) Heterodimers of hMSH2/6(named hMutSa) focus on mismatches and single-base loops. hMSH2/3 dimers (hMutSb) recognize insertion/deletion loops. Heterodimeric complexes of the hMutL-like proteins hMLH1/hPMS2 (hMutLa) and hMLH1/hPMS1 (hMutLb) interact with MSH complexes and replication factors. Strand discrimination may be based on contact with the nearby replication machinery. A number of proteins are implicated in the excision of the new strand past the mismatch and resynthesis steps, including polδ/;, RPA, PCNA, RFC, exonuclease 1, and endonuclease FEN1.

Nucleotide excision repair (NER) and Xeroderma Pigmentosa

This repair pathway is highly conserved between eukaryotes and is involved in the removal of chemical adducts such as UV damage, and also the repair of alkylation damage (Yonemasu, 1997; Lehmann, 2000; Memisoglu & Samson, 2000). Proteins involved in the NER pathway "slide" along the double stranded DNA surface looking for irregularities. Once identified, the proteins recruit the transcription factor TFIIH, whose helicase subunits, using ATP as an energy source, partially unwind the double helix to form a bubble. This involvement of the RNA polymerase II component is called transcription coupled repair. An endonuclease cuts the damaged bases, releasing them, and the gap is filled by DNA polymerase activity, and DNA ligase seals the nick.

There are two NER pathways of 5 steps. Global-genome-NER (GG-NER) and transcription coupled repair (TCR). In total, 25 or more proteins participate in NER and these individual components of the NER pathway are assembled step wise at the site of a lesion. After a single repair event, the entire complex is disassembled. The GG-NER specific complex XPC-hHR23B screens first on the basis of disrupted base pairing and not lesions. (I) In TCR, the ability of a lesion (whether of the NER- or BER-type) to block RNA polymerase seems critical. The stalled polymerase must be displaced to reveal the site of damage for repair; this requires two TCR-specific factors: CSB and CSA. (II) The stages of GG-NER and TCR which follow may be similar. The multi-subunit transcription factor TFIIH is composed of XPB and XPD helicases these open ~30 base pairs of DNA around the site of damage. (III) It has been proposed that XPA confirms the presence of damage by looking for abnormal structures within the backbone and if no mutation is detected NER is abandoned. The single-stranded-binding protein RPA (replication protein A) stabilizes the open intermediate by binding to the undamaged strand. (IV)The use of subsequent factors, allows very high damage specificity. XPG and ERCC1/XPF are endonucleases and, respectively cleave the 3' and 5' of the opened stretch only in the damaged strand respectively. This generates a 24-32-base oligonucleotide with the site of damage. (V)The regular DNA replication machinery then completes the repair by filling the gap.

Base excision repair (BER) and susceptibility to colon cancer

As well as using NER, alkylation damage can also be removed via this pathway. BER initiation occurs by the action of DNA glycosylases that recognise abnormal DNA bases and cleave the glycosidic bond linking the base to the sugar phosphate backbone. Following cleavage, the base is released to leave an apurinic/apyrimidinic (AP) site. An AP endonuclease Flap endonuclease 1 nicks the damaged DNA on the 5' side of the AP site to create a free 3'-OH. This allows DNA polymerase to synthesis a new DNA strand to fill the gap which is then sealed with DNA ligase (Memisoglu & Samson, 2000)

Mutations in the DNA glycosylase encoded by the MYH gene have been linked to incidences of colon cancer.

The BER reaction operates across the genome. However, some BER lesions block transcription, and in this case the problem is dealt with by the TCR pathway described, including TFIIH, XPG, stimulating some glycosylases and possibly the remainder of the NER apparatus. There are nine steps. 2(I Glycosylases cleave the damaged base from the sugar-phosphate backbone (II) this produces a basic site. (III) When BER is initiated from a SSB, Poly(ADP-ribose) polymerase (PARP), which binds to and is activated by DNA and polynucleotide kinase (PNK) may be important to protect and cut the ends for repair synthesis. (IV) (V) (VI) DNA polβ performs a one nucleotide gap-filling reaction. This polymerase also has lysis activity and removes the terminal baseless sugar residue, this is then followed by sealing of the nick using XRCC1-ligase3 complex. (VII, VIII, IX) The XRCC1 scaffold protein interacts with most of the above BER core components indicating the importance of its in role in protein exchange. The long-patch repair mode involves DNA polβ, polδ; and proliferating cell nuclear antigen (PCNA) for repair synthesis (2-10 bases) as well as the FEN1 endonuclease to remove the displaced DNA flap and DNA ligase 1 for sealing

Translesion DNA synthesis

The methods described for repair have all involved in some way where the damage bases have been excised. Cells can also repair their damaged during the replication process. DNA polymerases possess an intrinsic exonuclease activity and can "proof read" their DNA as synthesis proceeds. When a polymerase encounters a replication blockage it pauses excises the misincorporated base and then continues to synthesis. In addition to regular DNA polymerases cells have specialised DNA polymerases which evolved to bypass blockages. This process is called translesion DNA synthesis (TLS) and allows strand extensions across template lesions (Friedberg et al., 2005)

Mechanisms of replicational bypass of DNA lesions indicated by X. In the DNA template the lesion may be bypassed by the replication apparatus in two different ways: DNA polymerase switch (upper strand) and template switch (lower strand). In the DNA polymerase switch, the regular DNA polymerase (in this case polδ/;, carrying out leading-strand synthesis) stops at the site of the damage. The particular translesion polymerase (pol-κ), or a combination of these polymerases,takes over synthesis from the normal polymerase until the site of site of DNA damage is bypassed. After the lesion is bypassed the normal polymerase takes over again, however, this process is high in error. In the template switch, the regular DNA polymerase (in this case polα, responsible for lagging-strand synthesis) is arrested at a damaged site. The resulting gap in the newly synthesized strand is filled in using the undamaged, newly synthesized leading strand via recombinational strand exchange (or alternatively by fork regression and annealing of the new strand, not shown). This mechanism may involve specific factors as well as members of the RAD52 family implicated in homologous recombination repair. In principle, this mode of lesion bypass is error-free. Note that in both of these processes the lesion remains and that the two scenarios may apply to both strands.

Homologous recombination (HR) for repair of double strand breaks (DSBs) and BRCA1 and BRCA2

The best template for repair is one which is as identical to the original chromosome as possible; this is the basis for homologous recombination. DSBs are the most detrimental lesions that a cell's DNA can endure. Left un-repaired severe chromosomal defects can predispose cells to cancer, such as translocation events giving rise to the Philadelphia chromosome t (9;22) (q34;q11) seen in chronic myeloid leukaemia (Bonthron et al., 1998), or mutations in the BRCA1/BRCA2 breast cancer genes which are involved in the repair of DSBs via HR (Liu & West, 2002).

The BRCA1 protein is part of a multiprotein complex called the BRCA1-associated genome surveillance complex (BASC) and is involved in DNA damage and repair. BRCA1 is also involved in recombination, chromatin remodelling and transcriptional control. BRCA2 is another breast cancer susceptibility protein and despite bearing no structural similarities to BRCA1 also loads the recombination protein RAD51 onto single-stranded DNA at damaged sites. These proteins will be discussed in more detail in ?????

Ionizing radiations such as X rays and γ - radiation cause DSBs in DNA. Cells have evolved two systems to repair these types of lesions, NHEJ and HJ. The first involves joining the non homologous ends of two DNA molecules. The process is error prone even if the two fragments were to come from the same chromosome. Since the two ends are simply ligated together several base pairs are lost at the joining point.

The second mechanism is HR and occurs when the damaged chromosome can "copy" the information from its identical partner to repair itself. The process of HR is illustrated in Figure 1.13. After a DSB occurs in the DNA, an exonuclease cleaves the ends to leave 5' single-stranded ends (step 1). A single strand binding protein RAD51 catalyzes the invasion of one strand from one homologue into the other. The 3' end of the invading strand is extended by a DNA polymerase which causes the parental strand to form a D-loop (light blue) (step 2). Once the strand is long enough, base-pairing occurs between the complementary bases of the broken non-invading strand (pink) and the D-loop, and DNA synthesis happens (step 4). The two newly synthesised 3' ends are ligated to the 5' ends generated in step 1. This generates two Holliday structures in the paired molecules (step 5). Repair is finalised when the strands are cleaved at the positions shown by the arrows, and depending on how cleavage takes place either recombinant chromosomes with cross over events (CO) arise or there are no cross over (NCO) events. Ligation of the alternative 5' and 3' ends at each cleaved Holliday structure generates two recombinant chromosomes that each contain the DNA of one parental DNA molecule on one side of the break point (pink and red) and the DNA of the other parental molecular (light blue and dark blue). Each chromosome also contains a heteroduplex located near the initial breakpoint; here one strand from one parent is base-paired to the complementary strand of the other parent (pink or red base-paired to dark or light blue).

Double strand break repair (DSBR) by homologous recombination.

The double helix of each homologous chromosome is represented as two parallel lines with sister chromatids coloured accordingly. Polarity of the strands is indicated by arrowheads at their 3' ends. The upper molecular has a DSB, which is digested to generate 3' single stranded ends (1). A single strand binding protein mediates strand invasion (2) which is then extended by DNA polymerases until the displaced single-stranded DNA (light blue) base pairs with the other (pink) (3), this 3' end is then extended with the DNA polymerase (4), the ends are ligated (5) and then depending on how the phosphodiester bond is cleaved either cross over (CO) events take place or no cross overs take place (NCO). Figure adapted from Heyer, 2004.