Maintenance of Genomic Integrity and the Development of Cancer

1. Maintenance of Genomic Integrity and the Development of Cancer

2. Cell genomes are threatened by errors made during DNA replication • The stability of genome is under constant challenge by a variety of agents and processes: • The replication of DNA is subjected to a low but significant level of errors. • incorporation of chemically different nucleotide precursors • The nucleotides within DNA molecules undergo chemical changes spontaneously. • DNA molecules may be attacked by various mutagenic agents, including endogenous and exogenous agents.

3. Cell genomes are under constant attack from endogenous biochemical processes • Endogenous biochemical processes may make greater contributions to genome mutation than do exogenous mutations. • Chemical damage of DNA molecules through the actions of hydrogen and hydroxy ions that are present at low concentration (~ 10-7 M) at neutral pH.

4. Spontaneous depurination or ADENINE By some estimates, as many as 10,000 purine bases are lost by depurination each day in a mammalian cell.

5. Spontaneous base deamination C → T transition C → T transition CpG

6. The deamination of 5-methylcytocine represents a major source of point mutations in human DNA. • By one estimate, 63% of the point mutations in the genomes of tumors of internal organs arise in CpG sequences. Among mutant p53 alleles, about 30% seem to arise from CpG sequences present in the wild-type p53 alleles.

7. Oxidation • Generation of a variety of intermediates as O2 is progressively reduced to H2O in mitochondria: O2 + e- → O2.- + e- → H2O2+ e-→.OH + e-→ H2O superoxide hydrogen hydroxy ion peroxide radical reactive oxygen species (ROS) • Oxidants arisen as byproducts of various O2-utilizing enzymes, including those in peroxisomesand from spontaneous oxidation of lipids. • Inflammation provides an important source of the oxidants, e.g., NO, O2.-, H2O2, OCl-(hypochlorite).

8. Oxidation of bases in the DNA ROS ↓

9. Methylation of bases in the DNA

10. The oxidation, depurination, deamination, and methylation, which together may alter thousands of bases per cell genome each day, greatly exceeds the amount of damage created by exogenous mutagenic agents in most tissues.

11. Inflammation can have both mitogenic and mutagenic consequences • The phagocytic cells destroy infected cells in part by releasing oxidants - NO, O2.- , H2O2, and OCl-. • These oxidants act as mutagens on the genomes of nearby cells (nitration, oxidation, deamination, and halogenation). • The DNA of inflamed and neoplastic tissues have been found to carry increased concentrations of 8-oxo-dG, one of the primary products of DNA oxidation.

12. Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular genome Rat cells suffer about 10-fold more oxidative hits per cell per day in their genomes than do human cells because they have about a 7-fold greater metabolic rate.

13. Cell genomes are under occasional attack from exogenous mutagens and their metabolites • X-rays – ionizing radiation • generate ionized, chemically reactive molecules • create s.s. and d.s. breaks in the double helix • UV radiation – more common source of environmental radiation than X-rays • form thymidine dimers • Chemicals – many are electrophilic • alkylating agents are mutagens which are capable of • attaching alkyl groups covalently to the DNA bases • form DNA adducts

14. Products of UV irradiation cyclobutane pyrimidine dimers pyrimidine (6-4) pyrimidinone (60% T-T, 30% C-T, 10% C-C dimers)

15. In benign skin lesions and basal cell carcinomas of the skin, many of the mutant p53 alleles carry a dipyrimidine substitution.

16. Methylation of bases by alkylating agents

17. Cytochrome P-450 (CYP) enzymes oxidize procarcinogens to ultimate carcinogens a polycyclic aromatic hydro-carbon (PAH) present in coal tar and tobacco smoke Cytochrome-P450s are involved in the biosynthesis or degradation of steroid hormones, cholesterol, bile acids, and fatty acids. In addition, they aid the oxidation and detoxification of drugs and carcinogens.

18. Formation of DNA adducts chemically reactive epoxide group ultimate carcinogens can also link to O6 or N7 6 7

19. Activation of aflatoxin B1 (AFB1) by cytochrome P-450

20. Heterocyclic amines (HCA) are generated from meats which are cooked at high temperature 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine (PhIP) is the principal HCA in the human diet.

21. Oxidation of PhIP and the formation of DNA adduct

22. Cells deploy a variety of defenses to protect DNA molecules from attack by mutagens • Physical shield • skin and the melanin pigment • detoxifying enzymes • superoxide dismutase (SOD) & catalase • free-radical scavengers • vitamin C, vitamin E, bilirubin • glutathione-S-transferases (GSTs) reacting with electrophilc compounds

23. Melanin pigment shields keratinocyte nuclei from UV radiation supranuclearcap (parasol or sun umbrella) keratinocyte nucleus

24. Superoxide dismutases (SOD) • The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. • It is an important antioxidant defense in nearly all cells exposed to O2. • In humans, 3 forms of SOD are present : • SOD1 – Cu-Zn-SOD (in cytoplasm) • SOD2 – Mn-SOD (in mitochondria) • SOD3 – Cu-Zn-SOD (extracellular)

25. Mice lacking SOD2 die several days after birth with massive oxidative stress. • Mice lacking SOD1 develop a wide range of pathologies, including hepatocellular carcinoma, an acceleration of age-related muscle mass loss, an earlier incidence of cataracts and a reduced lifespan. • In humans, mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS), a form of motor neuron disease. • Action of catalase : 2 H2O2 → 2 H2O + O2

26. Effect of glutathione-S-transferase (GST) reactive epoxide 90% of human prostate adenocarcinomas show a shutdown of GST-π expression due to methylation of the promoter of the GST gene. a tripeptide

27. Inter-individual differences in carcinogen activation seem to contribute to cancer risk and responses to therapy • cytochrome-encoding Cyp1A1 • glutathione-S-transferase M1 (GSTM1) • N-acetyltransferase 1 (NAT1) - breast cancer (help to convert heterocyclic amines into active mutagens) - lung cancer

28. Repair enzymes fix DNA that has been altered by mutagens • If genotoxic chemicals are not intercepted before they attack DNA, mammalian cells have a backup strategy for minimizing the genetic damage caused by these potential carcinogens.

29. Cell genomes are threatened by errors made during DNA replication • During DNA replication, the DNA molecules are especially vulnerable to breakage in the single-stranded portions of the molecule near the replication fork that have not been undergone replication

30. A cell has two major strategies for detecting and removing the miscopied nucleotides arising during DNA replication. • Proofreading by DNA polymerases • DNA repair by mismatch repair (MMR) enzymes

31. Proofreading by DNA polymerases δ

33. Proofreading by DNA polymerase δand cancer incidence in mice D400A mutation: change of the #400 a.a. from D (aspartic acid) to A (alanine) in the proofreading domain of DNA polymerase δ Deaths of the mutant homozygotes were all due to malignancies.

34. Mismatch repair (MMR) enzymes detect mistakes in newly synthesized DNA strand • Two components of the MMR apparatus, MutS and MutL, collaborate to remove mismatched DNA: • MutS scans the DNA • for mismatches. • MutLthen scans the DNA • for single-strand nicks, • which identify the strand • that has recently been • synthesized.

35. The action of mismatch repair system is critical in regions of the DNA that carry repeated sequences (microsatellite sequence). • Mononucleotide repeats: AAAAAAA • Dinucleotide repeats: AGAGAGAG • Repeats of greater sequence complexity A defective MMR system will result in the expansion or shrinkage of microsatellite sequences, known as microsatellite instability (MIN).

36. A TGF-β receptor gene affected by microsatellite instability The type II TGF-β receptor is frequently inactivated in human colon cancers, which carry defects in mismatch repair genes and exhibit microsatellite instability (MIN).

37. Cells deploy a wide variety of enzymes to accomplish the very challenging task of restoring normal DNA structure. • Mismatch repair (MMR) enzymes largely focused on detecting nucleotides of normal structure that have been incorporated into the wrong positions. • Other repair mechanisms detect nucleotides of abnormal chemical structure. • dealkylating enzymes • base-excision repair (BER) • nucleotide-excision repair (NER) • error-prone repair

38. DNA alkyltransferase removes methyl or ethyl adducts from the O6 position of guanine (ethylnitrosourea) O6-methylguanine-DNA methyltransferase or O6-alkylguanine DNA alkyltransferase (AGT) or DNA alkyltransferase

39. The MGMTgene is silenced by promoter methylation in 40% of gliomas and colorectal tumors, and in 25% of non-small-cell carcinomas, lymphomas, and head and neck carcinomas. • The loss of this DNA repair function in certain tissues favors increased rates of mutation and hence accelerated tumor progression.

40. Base-excision repair (BER) cleave the glycosyl bond linking the altered base and the deoxyribose apurinic/apyrimidinic endonuclease

41. Base-excision repair (BER) tends to repair lesions in the DNA that derive from endogenous sources such as the reactive oxygen species (ROS) and depurination events. • BER seems to concentrate on fixing lesions that do not create structural distortions of the DNA double helix. • For example, when U is mistakenly incorporated into the DNA, it is removed by the enzyme uracil DNA-glycosylase and soon replaced with a C.

42. Nucleotide-excision repair (NER) NER is accomplished by a large multiprotein complex composed of almost ~20’s subunits. PCNA:proliferation-cell nuclear antigen RPA:single-strand DNA-binding protein

43. Nucleotide-excision repair (NER) focuses largely on repairing the lesions created by exogenous agents, such as UV photons and chemical carcinogens. • NER enzymes can recognize and remove helix-distorting alterations (e.g., bulky base adducts) created by polycyclic aromatic hydrocarbons (PAH), heterocyclic amines, aflatoxin B1, and pyrimidine dimers formed by UV radiation. • For example, following exposure to UV radiation, cultured human cells can repair ~ 80% of their pyrimidine dimers in 24 hrs.

44. Error-prone repair • Error-prone DNA synthesis occurs when a DNA replication fork is advancing during replication and encounters a still-unrepaired DNA lesion. • The replication apparatus must “guess” which of the 4 nucleotides is appropriate for incorporation.

45. Inherited defects in nucleotide-excision repair (NER) and mismatch repair (MMR) lead to specific cancer susceptibility • NER defect:Xeroderma pigmentosum (XP) • MMR defect:Hereditary non-polyposis colon cancer (HNPCC)

46. Hereditary non-polyposiscolon cancer (HNPCC) : • a familial cancer syndrome • comprising 2 to 3% of all colon cancer cases • Some HNPCC patients have increased susceptibility to endometrial, stomach, ovarian, and urinary tract carcinoma in addition to colon carcinomas. • germline mutations in the genes encoding mismatch repair (MMR) proteins

47. A variety of other DNA repair defects confer increased cancer susceptibility • Almost 50% of all identified familial breast cancers involve germline transmission of a mutant BRCA1 or BRCA2 allele. • By some estimates, 70 to 80% of all familial ovarian cancers are due to mutant germline alleles of BRCA1 or BRCA2.