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Cancer Etiology

1. Introduction 2. Chemical Factors in Carcinogenesis 3. Physical Factors in Carcinogenesis4. Viral Oncogenesis5. Genetic Predisposition

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肿瘤（ tumor ）

在致瘤因素作用下，细胞基因失去对细胞增殖、分化和死亡的正常调控，导致组织细胞不断增生而形成的新生物。

良性肿瘤（ benign tumor ）恶性肿瘤（ malignant tumor ）

Introduction

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肿瘤发病率和死亡率

2010 年国际抗癌联盟（ UICC ）： 2008 年全世界 1270 万新增癌症患者，死亡人数 760 万。

《 2010 中国卫生统计年鉴》 ：2009 年中国恶性肿瘤成为首位死因。每年新发癌症病人约 200 万，死亡人数约 150 万。肺癌、肝癌、结直肠癌、乳腺癌、膀胱癌死亡率及其构成明显上升。肺癌成为我国首位恶性肿瘤死亡原因。

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Hallmarks of cancer (Weinberg, Cell, 2000)

Figure 1. The Hallmarks of Cancer. This illustration encompasses the six hallmark capabilities originally proposed in our 2000 perspective. The past decade has witnessed remarkable progress toward understanding the mechanistic underpinnings of each hallmark. (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)

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Hallmarks of cancer (Weinberg, Cell, 2011)

. (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)

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The Hallmarks of Cancer Self-sufficiency in growth signals

Cancer cells do not need stimulation from external signals (in the form of growth factors) to multiply.

Insensitivity to anti-growth signals

Cancer cells are generally resistant to growth-preventing signals from their neighbours.

Tissue invasion and metastasis

Cancer cells can break away from their site or organ of origin to invade surrounding tissue and spread (metastasis) to distant body parts.

Limitless reproductive potential

Non-cancer cells die after a certain number of divisions. Cancer cells escape this limit and are apparently capable of indefinite growth and division (immortality).

Sustained angiogenesis

Cancer cells appear to be able to kickstart this process, ensuring that such cells receive a continual supply of oxygen and other nutrients.

Evading apoptosis

Apoptosis is a form of programmed cell death, the mechanism by which cells are programmed to die after a certain number of divisions or in the event they become damaged. Cancer cells characteristically are able to bypass this mechanism.

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Deregulated metabolism

Most cancer cells use abnormal metabolic pathways to generate energy, a fact appreciated since the early twentieth century with the postulation of the Warburg hypothesis, but only now gaining renewed research interest.

Evading the immune system

Cancer cells appear to be invisible to the body’s immune system.

Unstable DNA

Cancer cells generally have severe chromosomal abnormalities, which worsen as the disease progresses.

Inflammation

Recent discoveries have highlighted the role of local chronic inflammation in inducing many types of cancer.

(Hanahan, D.; Weinberg, R. A. (2011). "Hallmarks of Cancer: The Next Generation". Cell 144 (5): 646–674. doi:10.1016/j.cell.2011.02.013 )

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Chemical Carcinogenesis

Multi-stage Theory of Chemical Carcinogenesis Classification of chemical carcinogens Mechanisms of Chemical Carcinogenesis DNA Damage Induced by Ultimate Carcinogens DNA Repair

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Multi-stage Theory of Chemical Carcinogenesis

Initiation -----------Genetic events

Chemical Carcinogens (Direct and Indirect Carcinogens)

Promotion -------Epigenetic events

Tumor promoters

– Murine skin carcinogenesis model:

• A single dose of polycyclic aromatic hydrocarbon (PAH, initiator)

• Repeated doses of croton oil (promoter)

Malignant conversion

Progression ------Genetic and epigenetic events

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Initiation

• Irreversible genetic damage:

A necessary, but insufficient prerequisite for tumor initiation

• Activation of proto-oncogene, inactivation of a tumor suppressor gene, and etc

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Promotion • Promotion: Selective expansion of initiated cells, which

are at risk of further genetic changes and malignant conversion

• Promoters are usually nonmutagenic, not carcinogenic alone, often do not need metabolic activation, can induce tumor in conjuction with a dose of an initiator that is too low to be carcinogenic alone

• Chemicals capable of both initiation and promotion are called complete carcinogens: benzo[a]pyrene and 4-aminobiphenyl

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Malignant conversion • The transformation of a preneoplastic cell into

that expresses the malignant phenotype• Further genetic changes• Reversible• The further genetic changes may result from

infidelity of DNA synthesis• May be mediated through the activation of

proto-oncogene and inactivation of tumor-suppressor gene

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Progression

• The expression of malignant phenotype, the tendency to acquire more aggressive characteristics, Metastasis

• Propensity for genomic instability and uncontrolled growth

• Further genetic changes: the activation of proto-oncogenes and the inactivation of tumor-suppressor genes

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• Activation of proto-oncogenes:– Point mutations: ras gene family, hotspots– Overexpression:

• Amplification

• Translocation

• Loss of function of tumor-suppressor genes: usually a bimodal fashion– Point mutation in one allele– Loss of second allele by deletion, recombinational

event, or chromosomal nondisjunction

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Gene-environmental interactions

• The metabolism of xenobiotics by biologic systems– Individual variation – The competition between activation and detoxication

• The alteration of genes by xenobiotics

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Classification of chemical carcinogens

1. Based on mechanisms(1) Genotoxic carcinogen (DNA-reactive)• Direct-acting: intrinsically reactive

N-methyl-N’-nitro-N-nitrosoguanidine (MNNG),

methyl methanesulfonate (MMS),

N-ethyl-N-nitrosourea (ENU), nitrogen and sulfur mustards • Indirect-acting: require metabolic activation by cellular enzyme to form the DNA-

reactive metabolite (members of the cytochrome P450 family) benzo[]pyrene, 2-acetylaminofluorene, benzidine, Aflatoxin B1, B2.

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(2) Epigenetic carcinogens

• Promotes cancer in ways other than direct DNA damage/ do not change the primary sequence of DNA

• Alter the expression or repression of certain genes and cellular events related to proliferation and differentiation

• Promoters, hormone modifying agents, peroxisome proliferators, cytotoxic agents, and immunosuppressors

• Organochlorine pesticides, [saccharin], estrogen, cyclosporine A, azathioprine

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2. Based on sturcture(1) Nitrosamines (NA)

MNNG, MMS (direct carcinogen)

(2) Polycyclic aromatic hydrocarbons (PAH)

Benzo(a)pyrene (indirect carcinogen)

(3) Aromatic amines (AA)

2-acetylaminofluorene, benzidine (indirect carcinogen)

(4) Aflatoxin (AF)

(5) Inorganic elements and their compounds: arsenic, chromium,

and nickel are also considered genotoxic agents

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Mechanisms of Initiation in Chemical Carcinogenesis

(1) DNA damages:Pro-carcinogen metabolic activation (Phase I and II) Ultimate carcinogen (electrophiles) Interaction with macromolecules (nucleophiles) DNA damage, mutations, chromosomal aberrations, or cell death

(2) Epigenetic changes

(3)Activation of oncogenes; inactivation of tumor suppressor genes

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(1) Alkylating agents are electrophilic compounds with affinity for

nucleophilic centers in organic macromolecules.[Fu D, Calvo JA, Samson LD. Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat Rev Cancer. 2012 Jan 12;12(2):104-20. doi: 10.1038/nrc3185.]

(2) These agents can be either monofunctional or bifunctional.

---Monofunctional alkylating agents have a single reactive group and

thus interact covalently with single nucleophilic centers in DNA (although varied).

such as MNNG

---Bifunctional alkylating agents have two reactive groups, and each molecule is potentially able to react with two sites in DNA.

Interstrand DNA cross-link: the two sites are on opposite polynucleotide strands;

Intrastrand cross-link: on the same polynucleotide chain of a DNA duplex.

such as Nitrogen and sulfur mustard, mitomycin, cis-platinum

Direct Chemical Carcinogens

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Numerous potential reaction sites for alkylation have been identified in all four bases of DNA (not all of them have equal reactivity:

MNNG N-Methyl-N-nitroso-N'-nitroguanidine

---Monofunctional alkylating agents

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---Bifunctional alkylating agents

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Indirect Chemical Carcinogensand Their Phase I Metabolic derivatives

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BPDE binds DNA covalently, resulting in bulky adduct damage

BPDE intercalates into dsDNA non-covalently, leading to conformational abnormalities

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Types of DNA Damage Induced by Ultimate Carcinogens

• DNA Adduct Formation• DNA Break Single Strand Break Double Strand Break • DNA Linkage DNA-DNA linkage DNA-protein Linkage• Intercalation

Bulky aromatic-type adducts, Alkylation (small adducts),

Oxidation, Dimerization, Deamination

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Repair systems• Direct DNA repair/ Direct reversal :

– DNA alkyltransferase (O6-alkylguanine-DNA alkyl transferase)

– One enzyme per lesion

• Base excision repair (BER)– small adducts, – overlap with direct repair – glycosylase to remove the adducted base

DNA Repair

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• Nucleotide excision repair (NER): – involves recognition, preincision, incision, gap-filling,

and ligation, – large distortions – strand specific, the transcribed strand is preferentially

repaired – xeroderma pigmentosum (XP): NER deficiency

• Mismatch repair (MMR) – transition mispairs are more efficiently repaired (G-T

or A-C) than transversion mispairs – microenvironment influences efficiency – similar to NER – involves the excision of large pieces of the DNA

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• Double-strand breaks (DSBs) – homologous recombination – non-homologous end joining (NHEJ): DNA-PK

• Postreplication repair – a damage tolerance mechanism – occurs in response to replication of DNA on a

damaged template – the gap

• either filled through homologous recombination with parental strand

• or insert an A residue at the single nucleotide gap

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Translesion DNA synthesis

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1.DNA damage blocks the progression of the replication fork.

2.PCNA plays a central role in recruiting the TLS polymerases (translesion DNA synthesis) and effecting the polymerase switch from replicative to TLS polymerase (low stringency DNA polymerases).

3. TLS polymerases carry out TLS, either singly or in combination, past different types of DNA damage.

4.Such regulation must ensure that (1) the specialized polymerases act only when needed, and (2) that polymerases act only at the right location in DNA.

5.TLS evolved in mammals as a system that balances gain in survival with a tolerable mutational cost, and that disturbing this balance causes a potentially harmful increase in mutations, which might play a role in carcinogenesis.

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Classification of TLS polymerases

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• All the Y-family polymerases are localized in the nucleus, and during S phase,

polη, ι, and Rev1 relocate to replication factories with the polymerase sliding

clamp PCNA, and other proteins associated with DNA replication.

• There are three examples of TLS reactions in which a specialized DNA

polymerase bypasses its cognate DNA lesion with higher efficiency and higher

fidelity than any other polymerase in the cell:

---Polh and the UV light-induced CPD (cyclobutane pyrimidine dimers);

---Polk and benzo[a]pyrene-guanine (major tobacco smoke-induced DNA lesion);

---Polh and cisplatin-GG (an adduct produced by a drug used in cancer chemotherapy).

• They operate at low speed, low processivity and with low fidelity.

• Their active sites adopt a much more open structure than replicative polymerases, they are less stringent and can accommodate altered bases in their active sites.

• Y-family polymerases lack a 3’-5’ exonuclease activity, which is an integral part of all replicative polymerases and performs a proofreading function.

• Each Y family polymerase differs in substrate specificity.

Characteristics of TLS polymerases

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1. Polη • Polη was discovered as the protein deficient in the variant form of the skin cancer-prone

genetic disorder xeroderma pigmentosum (XP). • Most XP patients are deficient in the ability to remove UV photoproducts from their

DNA by nucleotide excision repair (NER), but about 20% have problems in replicating their DNA after UV irradiation because of defectiveness of polη gene.

• Polη carrys out TLS past CPD (cyclobutane pyrimidine dimers) photoproducts generated by exposure to sunlight. XP variant cells have an elevated UV-induced mutation frequency.

2. Polκ

Polκcan carry out TLS past DNA containing benzo[a] pyrene-guanine adducts.

3. Rev1

Rev1 has a restricted DNA polymerase activity that is confined to the incorporation of one or two molecules of dCMP regardless of the nature of the template nucleotide.

• Rev1 interacts with multiple TLS polymerases, notably Polη, Polκ, Polι, Polλ, and the REV7 (subunit of Polζ).

• Rev1 protein may be specifically involved in polymerase switching during TLS.

4. Polι

5. Polζ is a heterodimer containing the Rev3 catalytic subunit and the Rev7 regulatory subunit.

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Cellular responses evoked by DNA damaging agents are very complex events

• Responses may triggered by the signals originated from: genomic and mitochondrial DNA damage malfunction of signaling molecules endoplasmic reticulum stress others• Networks between different signaling pathways • Cellular responses are the comprehensive and integrated

consequences

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Hormones and the etiology of cancer

• Major carcinogenic consequence of hormone exposure: cell proliferation

• The emergence of a malignant phenotype depends on a series of somatic mutation

• Germline mutations may also occur• How to get exposure: contraceptives, hormone

replacement therapy, or during prevention of miscarriage

• Epidemiological studies

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Hormone-related cancer

• Estrogen and breast cancer

• Endometrial cancer: Estrogen replacement therapy

• Ovarian cancer: follicle stimulating hormone

• Prostate cancer and androgen

• Vaginal adenocarcinoma: in utero diethylstilbestrol (DES) exposure

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Other hormone-related cancers

• Cervical cancer: OC use might increase the risk, still a lot complicating factors

• Thyroid cancer: the pituitary hormone thyroid stimulating hormone (TSH)

• Osteosarcoma: incidence associates with the pattern of childhood skeleton growth; and hormonal activity is a primary stimulus for skeleton growth

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Physical factors in carcinogenesis

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Physical carcinogens

– Corpuscular radiations

– Electromagnetic radiations

– Ultraviolet lights (UV)

– Low and high temperatures

– Mechanical traumas

– Solid and gel materials

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Ionizing radiation (IR)

• Penetrate cells, unaffected by the usual cellular barriers to chemical agents

• IR: a relatively weak carcinogen and mutagen • The initial critical biologic change is damages to DNA• It takes place in a matter of the order of a microsecond

or less

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Electromagnetic fields (EMF)

Remains controversial: • Minimal increase in relative risk of brain tumor

and leukemia in electric utility workers• Also relatively increased risk for acute

lymphoblastic leukemia by EMF exposure during pregnancy or postnatally

• However, some studies lend no support for this proposition

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Ultraviolet (UV)

• Sunlight and skin cancer• Well established for basal and squamous cell cancers• Some controversy remains for melanoma• Nonmelanoma skin cancers are the most common

cancer in the US (45%)• Usually occurs at the age of 50 – 60

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Sunlight spectrum and wavelength• UVA (320-400)

– photocarcinogenic– weakly absorbed in DNA and protein– active oxygen and free radicals

• UVB (290-320) – overlaps the upper end of DNA and protein absorption spectra – mainly responsible through direct photochemical damage

• UVC (240-290) – not present in ambient sunlight – low pressure mercury sterilizing lamps– experimental system

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Shielding us from the sun

• Ozone: shorter than 300 nm cannot reach the earth’s surface

• UVA and UVB: only a minute portion of the emitted solar wavelengths ( 0.0000001%)

• Skin:

– melanin pigment

– keratin layers

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Xeroderma pigmentosum (XP)( 着色性干皮病 )

• Autosomal recessive disease, 1/250,000• Obligate heterozygotes (parents): asymptomatic• Homozygotes: skin and eyes, even neurologic

degeneration • Onset at 1-2 year of age• 2,000 times higher frequency for cancer• 30-year reduction in lifespan

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• 7 complementation groups, with various reduced rates for excision repair

• An 8th, the XP variant, has a defect in replication of damaged DNA (polymerase )

• Groups A and D are very sensitive to UV killing• Group C is the largest group, or called the

common/classic form, only shows skin disorders, preferentially repairs transcriptionally active genes

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Viral Oncogenesis

• RNA Oncovirus (Retrovirus)

• DNA Oncovirus

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RNA Oncovirus

Retroviruses: ssRNA viruses

Reverse transcriptase

Oncogenes

Rous sarcoma in chickens (RSV): in 1911

Human T-cell lymphotropic virus (HTLV-I,II)

Human immunodeficiency virus (HIV)

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Classification of retrovirus

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Structure of RNA Oncovirus

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Genome of RNA Oncovirus and Gene Products

Genome of Human T-cell Leukemia virus (HTLV)

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Life cycle1. Receptor binding and membrane fusion 2. Internalization and uncoating 3. Reverse transcription of the RNA genome to form double-stranded

linear DNA 4. Nuclear entry of the DNA 5. Integration of the linear DNA into host chromosomal DNA to form the

provirus 6. Transcription of the provirus to form viral RNAs

7. Splicing and nuclear export of the RNAs

8. Translation of the RNAs to form precursor proteins

9. Assembly of the virion and packaging of the viral RNA genome

10. Budding and release of the virions

11. Proteolytic processing of the precursors and maturation of the virions

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Replication of RNA Oncovirus

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Mechanisms of Oncogenesis Induced by RNA Oncovirus

• Transducing Retrovirus v-onc

• cis-Activating Retrovirus c-onc

• trans-Activating Retrovirus tax trans-acting x p40tax

rex repressive expression x p27rex, p21rex

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• Oncogene transduction– Acutely transforming in vivo and in vitro– Transform cells by the delivery (transduction) of an

oncogene from the host cell (v-onc) to a target cell– Cause the formation of polyclonal tumors– Most of this group of viruses are replication defective

(the requirement of a helper virus) – Examples: RSV (v-src); Abelson murine leukemia virus (v-Abl)

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•Insertional activation

– Long latent periods, Less efficient– Do not induce transformation of cells in vitro– Usually are replication competent– No oncogenes– Tumors are usually monoclonal– Provirus (LTR) is found within the vincity of a proto-

oncogene (c-myc)– Examples: lymphoid leukosis virus;

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•Grow stimulation and two-step oncogenesis

– The defective spleen focus-forming virus (SFFV) and its helper, the Friend murine leukemia virus (Fr-MuLV)

– Induce a polyclonal erythrocytosis in mice– Require the continued viral replication– A mutant env protein gp55 of SFFV binds and

stimulated the erythropoietin receptor, thus inducing erythroid hyperplasia

– Fr-MuLV or SFFV integration inactivates p53

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• Transactivation

– HTLV-1 and 2– Like cis-activation group: replication competent, carries no

oncogene, induces monoclonal leukemia, and latent– Like transducing group: can immortalize cells in vitro, has no

specific integration site– Unique 3’ genomic structure: the X region; Encodes at least

three proteins: Tax (p40), Rex (p27, p21)– Tax is the focus

– Transactivate the viral LTR, results in a 100- to 200-fold increase in the rate of proviral transcription

– Transactivate cellular enhancers and promoters, including genes for IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), c-fos, and others.

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•Immunodeficiency

• AIDS patients have an extraordinary increased rate of developing high-grade lymphomas and Kaposi’s sarcoma (KS)

• Probably secondary• However, Tat protein of HIV (the transactivating

protein) may induce KS-like lesions in mice.

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Endogenous retroviruses• Exo or endo: somatic vs germline• 0.5-1% mammalian genome is composed of retroviral

proviruses• Some properties:

– Most are defective– Great variations between species or within– Variable level of expression– Generally not pathogenic– The potential to induce disease is notable

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DNA Oncovirus

Papilloma virus

Polyoma virus

Adenovirus

Herpes virus: EB virus

Hepatitis B virus

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Mechanism of Oncogenesis Induced by DNA Oncovirus

Transforming proteins 1. HPV E6 interact with P53 E7 interact with RB

2. Adenovirus E1a interact with RB E1b

3. Polyoma virus SV40 Large T interact with RB Py virus Large and Middle T

Transcription activators 1. EB virus EBNA-2 and LMP 2. HBV p28 X protein

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Gene Map and Function of HPV

ORF Function

E1 Virus proliferationE2 Regulation of transcriptionE5 、 E6 、 E7 Cell transformationL1 、 L2 Encoding capsid proteinE4 Encoding late cytosolic proteinE3 、 E8 Unkown

E5: activates growth factor receptorE6: ubiquitin-mediated degradation of p53E7: binds and inactivates unphosphorylated pRb

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Genome of Adenovirus

Transformaing genes:E1A: Encoding intranuclear 26 and 30 kD phosphorylated proteinsE1B: Encoding a 19 kD protein located in nuclear and plasma membranes

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Gene Map of Polyoma and SV40 Virus

Transforming Genes SV40 virus: Large T Polyoma virus: Large and Middle T

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Genome of EB Virus

EBNA (EB virus Nuclear Antigen) EBNA-1 Immortalization of cell EBNA-2 trans-acting transcription activator EBNA-3 Function unknownLP: Leader Protein RNA ProcessingLMP: Latent Membrane Protein Activation of NF-κBTP: Terminal Protein Function unknown

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Genome and Products of HBV

Transforming gene: X gene X protein activates gene transcription via XRE

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Genetic Predisposition • Hereditary Cancer• Tumor Genetic Susceptibility

---Tumor susceptibility genes:Cytochrome P450 family, DNA repair genes, Tumor suppressor genes

• Immunity• Hormones and metabolism• Psychological factors• others

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Localization and presumed functions of proteins encoded by inherited cancer genes

(shown in magenta)

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Implications for Tumor Biology and Therapy

• Determinants of the Success of anticancer Agents

Cell cycle checkpoints, DNA repair, and Apoptosis• Genetic Regulation of Tumor Cell death in Response to

Anticancer Therapy• Improving the Therapeutic Ratio by regulation Cell

Death and Senescence