Development of transformed cell

Seminar of Molecular and Cell Biology

Mgr. Jan Šrámek

jan.sramek@lf3.cuni.cz

Syllabi

• Cell transformation• Characteristic of tranformed cells• Mechanisms of transformation• Cancerogenes• Tumors nad their classification• Cancer therapy

Cell transformation

• Process of transformation of normal cell that react to feedback homeostatic mechanisms to cell with autonomous growth and ability of invasion.

• All cancer cell are transformed cell

• But! Not all transformed cells are cancer cells (e.g. cells of cell cultures)

Characteristic of tranformed cells

• Independence on stimulatory cytokines• Loss of ‘anchorage dependence’• Capability of non-regulated clonal growth and loss of contact

inhibition• Immortality (no dependence on ‘lifespan limit’) and resistance to

apoptosis• Inability to differentiate• Increase activity of telomerase• Ability of angiogenesis• Different cell surface molecules and chromosomal reconstruction• Genetic instability• (ability to survive in host organism)

Abnormal proliferation in space and time represents basic characteristic of transformed (tumor) cells.

Mechanisms of transformation

• Multistage process (cancer incidence correlates with age)

• Non-returnable process

• Under selection stress

• Spontaneous x induced

• Genetic changes (mutations):

▫ Cancer incidence is 10-8 (includes 4 mutations, spontaneous mutation incidence is 10-6 per one cell division, number of cell divisions in human life is 1016; 10(-

6)x4/1016 = 10-8) 1 human per 100 milion x reality

▫ Influence of other factor: mutagenes, immune system

Multistage processsequential acumulation of genetic changes (4–7 mutations), according to dozens of different genes

1. mutation 2. mutation 3. mutation 4. mutation

Cancer cell

Mechanisms of transformation

• Multistage process (cancer incidence correlates with age)

• Non-returnable process

• Under selection stress

• Spontaneous x induced

• Genetic changes (mutations)

▫ Cancer incidence is 10-8 (includes 4 mutations, spontaneous mutation incidence is 10-6 per one cell division, number of cell divisions in human life is 1016; 10(-

6)x4/1016 = 10-8) 1 human per 100 milion x reality

▫ Influence of other factor: mutagenes, immune system

Process under the selection stress

Mechanisms of transformation

• Multistage process (cancer incidence correlates with age)• Non-returnable process• Under selection stress• Spontaneous x induced• Genetic changes (mutations)

▫ Cancer incidence is 10-8 (includes 4 mutations, spontaneous mutation incidence is 10-6 per one cell division, number of cell divisions in human life is 1016; 10(-

6)x4/1016 = 10-8) 1 human per 100 milion x reality▫ Influence of other factor: carcinogens, immune system

Carcinogens10-8

Ca

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Immune system10-8

Ca

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Theory of immune survailence

Cancer incidence is higher due to loss of immune system efficiency

▫ Majority of cancer cells is eliminated by immune system in organism (Tc-lymphocytes).

▫ Sooner, mean lifespan was about 35–40 years. Nowadays, mean lifespan increased markedly in western countries.

▫ Maximum efficiency of immune system is between 30–40 years of life.

The main role of genetic changes▫ Accumulation of genetic changes (mutations)▫ Primary role of oncogenes and antioncogenes (tumor-supressor

genes)▫ Change of function (quality) and/or level of expression (quantity)

of onco-/antioncogenes via: Point mutations Deletions Chromosomal translocations Gene amplifications

(c-ras) (c-myc, c-myb, N-ras)

(c-abl)

(c-ras) (c-myc, c-myb, N-ras)

(c-abl)

Reciprocal translocation between the 9th and 22nd chromosome.

Fusion of Bcr (22nd Chromosome) and of Abl genes (proto-oncogene [tyrosin kinase] of 9th chromosome).

Bcr protein is extensively produced in lymphocytes and has unclear function.

Bcr/Abl fusion protein p210 has encrease tyrozin-kinase activity (no regulation domain) no regulation of signaling.

Responsible for many types of leukemia.

Philadelphia chromosome

Regulation domain Tyrosin-kinase domain

Ras protein has GTP function

Signaling molecule

Mutation of c-ras gene leads to continuous activation of Ras protein increase expression of proteins stimulating cell division tumor development

Point mutation of c-ras gene

Ras

Carcinogens

Cause genetic changes via interactions with DNA leading to celltransformation.

Factors causing cell transformation:• Chemical• Physical • Biological

Chemical carcinogens

Bases analogs: 5-bromuracil (BU) – supersede T base transition Agens modifying bases:HNO2: deamination of C on U, A on hX, G on X transitionHSO-

3: deamination of C on U transitionNH2OHH2N-O-CH3 Alkyl agent: alkylsulfates, N-alkyl-N-nitrosamines (nitrates). Alkyl C, T and G block orchange base pairing, cause between- and interchain crossbonds block of replicationand trancriptionPsoralenes: intercalar agens, furocumarin, 8-metoxypsoralene

Pre-carcinogens: metabolic activation via specific enzymes (cyt. P450) is necessaryN-acetyl-2-aminofluoren (AAF), α-Nafthylamine, Benzo(a)pyren, Aflatoxins, Nitrates andothers

Cause transitions, transversions, bases modifications or covalentely bind to DNACause 80 % of all human tumors

Examples from history

• 1761: John Hill – polyps in snuffers of tabacco• 1775: Sir Percival Pott – scrotal carcinoma in chimney sweepers

(first documented work disease), long-term exposition to carcinogens (asbest, benzen, nitrosamins….)

• 1795: Samuel von Soemmering – smoking of pipe as a carcinogen.

Sir Percival Pott

Samuel von Soemmering

Benzo(a)pyren activation

P450

P450

Tumorigenesis of aromatic hydrocarbons

„fjord“ „bay“

Non-planarDiolepoxidesReact with DNA (A)More carcinogenic

PlanarLess reactive Less carcinogenic

α-Nafthylamine activation

NH2

NH2

NH-OH

OH

NH2

OR

Bladder

NH-OR

• Used in weld industry• Strong carcinogen having latence 15-20 years• In liver tranformed to carcinogen, than conjugated to glykoside• Hydrolysis in bladder: release of active carcinogen bladder carcinomas

Physical carcinogens• Ionize radiation:

▫ dsDNA breaks.▫ generate crossbonds (covalent bond between antiparalel

nucleotids)▫ base modifications (8-hydroxyguanin, 5-hydroxymetyluracil…).

• UV radiation: atom excitation generate thymin dimers block of replication and transcription processes.

crossbonds

Breaks

crossbond

strand

strand

Biological carcinogens

• Viruses: ▫ Oncogene RNA viruses: retroviruses (classical oncogenes)

HIV – probably supporting function only, Kaposi sarcoma… Human lymphotropic virus type I and II (HLTV-1, HLTV-2) – T-

leukemia, lymphomas HCV – hepatocarcinomas

▫ Oncogene DNA viruses: Papovaviruses (HPV) – anogenital tumors Herpesviruses - Epstein-Barr virus (EBV) – lymphomas (BL, HD),

nasopharingeal carcinomas (NPC); and others (HCMV; HSV-2; KSHV)

Hepadnaviruses - hepatitis B virus (HBV) - hepatocarcinomas Adenoviruses (animals)

HPV• Virus genome is circular dsDNA (8 kb)• Protein E7

▫ Inhibition of Rb-proteinu▫ Inactivation of p21Cip and p27Kip

▫ Abolishes inhibition effect of TGF- on growth of cells▫ Causes development of multiple centrosomes

• Protein E6 ▫ p53 degradation (using of ubiquitin ligase E6AP)▫ interact with Bak (inhibition of apoptosis)▫ activate hTERT expression (activation of telomerase)

• Genome is integrated in several places of host genome• Integration is specific according to genome of the virus –

leads to disorder of protein E2 expression (regulate E6 and E7 expression)

Effect of Papillomaviruses (Papovaviruses) proteins E6 and E7 on cell transformation

Effect of Papillomaviruses (Papovaviruses) proteins E6 and E7 on cell transformation

invasion

Loss of polarity

Unlimited replication capacity

proliferation

Apoptosis, cell cycle arrest

Loss of cell adhesion

Biological carcinogens

•„Infectionous cancer“:▫ Dogs CTVT (Stickers sarcoma) ▫ Non-viral parasitic cancer of Swan cells (DFTD) of Tasmanian

devil (Sarcophilus harrisii)

Histiocytes

Tumor

• Structure consists of cancer and connective cells that are under the controle of cancer cells (stroma and blood cells).

• No physiological function in an organism.

• Its growth is not in conformity with surrounding tissue and organism homeostasis

• Developed in places with high proliferating activity that are simultaneously the most displayed to carcinogenes, i.e. mainly epithels (skin, lung, digestive tract, but breast gland)

• The only one transformed cell is sufficient to develope tumor! (clonal character)

According to its infiltration ability:•Benign: solid bordered structure, located in one place, slow proliferation, symtoms of local character.•Malign: infiltrate surrounding tissues and using blood and lymphatic system the whole body, in „infected“ tissues produce secondary tumors (metastases)•primary x secondary tumors.

Tumors classification I

According to type of source cells:•Carcinomas – tumors of epithelial cells (ca 89 % of human tumors)

•Sarcomas – solid tumors developed from supporting or connective cells (tissue) – muscle, bone, cartilage (ca 2 % of human tumors)

•Leukemias and lymphomas – developed from hematopoietic cells and immune cells (ca 8 % of human tumors)

•Gliomas – developed from nerve tissue (ca 1 % of human tumors)

Tumors classification II

According to affected organ (tissue):•Breast carcinoma•Colorectal carcinoma•Cervical carcinoma•Gland carcinoma•Stomach cancer•Ovarian carcinoma•Leukemia•And many others

Tumors classification III

Process of tumor development

Process of metastasis developmentPrimary tumor

Secondary tumor - metastasis

Cancer therapyClassical approach:Local therapy – surgical strike, local radiation ( radiation)Systematical approach (combinated with surgical strike and radiation) – Chemotherapy

Chemotherapy:Cytotoxic agents: cyclophosphamide, cisplatinum, methotrexate, doxorubicin(interaction with DNA)Cytostatics: Vinca alcaloides (vinblastine, vincristine)

Application of cytokines:Inhibitors of cell proliferation and inductors of apoptosis: Interferones, TNFSupport influence of cytokines: IL-2, GM-CSF

Biological therapy (targeted therapy, [Gene therapy])‘Antisense’ oligonucleotides: target to specific oncogenesTransfection: functional anti-oncogenes….

Biologicac therapy (targeted therapy)

Use defensive capacity of immune system and/or targeted drugs(modificators of immune response) – cause specificaly only (or

mainly)on specific type of cells (e.g. cancer cells or immune cells):

• Blocks, restore or reduce precesses that are responsible for tumor progression

• Marks cancer cells to be well recognizable by immune system • Improve ability of some immune cells (T-lymphocytes, macrophags) to

destroy cancer cells• Changes growing ability of cancer cells• Blocks or restore processes responsible for cell tranformation• Improves ability of organism to repair or replace damaged cells injured by

other types of cancer therapy • Blocks cancer cells spreading

Biological therapy (targeted therapy)

Preparation:▫ Monoclonal antibodies▫ Differenciacal therapy▫ Inhibitors of proteasom, tyrosinkinases ▫ Anti-angiogene therapy▫ Antisense oligonucleotides

High prices

Limited usage depending on these criterias:▫ Tumor subtype ▫ Other types of cancer therapies are non-effective ▫ Undesirable effect of other types of cancer therapies

Monoclonal antibodies

Biological therapy (targeted therapy)

Preparation:▫ Monoclonal antibodies▫ Differenciacal therapy▫ Inhibitors of proteasom, tyrosinkinases ▫ Anti-angiogene therapy▫ Antisense oligonucleotides

High prices

Limited usage depending on these criterias:▫ Tumor subtype ▫ Other types of cancer therapies are non-effective ▫ Undesirable effect of other types of cancer therapies

Differential therapy

Cancer cell

Specific drug

Differentiated cell

Differentiated cell

Progenitor cell

Mutations

Developmental line

Biological therapy (targeted therapy)

Preparation:▫ Monoclonal antibodies▫ Differenciacal therapy▫ Inhibitors of proteasom, tyrosinkinases ▫ Anti-angiogene therapy▫ Antisense oligonucleotides

High prices

Limited usage depending on these criterias:▫ Tumor subtype ▫ Other types of cancer therapies are non-effective ▫ Undesirable effect of other types of cancer therapies

Inhibition of proteasome

Biological therapy (targeted therapy)

Preparation:▫ Monoclonal antibodies▫ Differenciacal therapy▫ Inhibitors of proteasom, tyrosinkinases, ▫ Anti-angiogenesis therapy▫ Antisense oligonucleotides

High prices

Limited usage depending on these criterias:▫ Tumor subtype ▫ Other types of cancer therapies are non-effective ▫ Undesirable effect of other types of cancer therapies

Cancer cells release angiogenesis factors (VEGF)

Vessel reaction

Tumor has nutrition support and cangrow and invade throught blood system

Using of angiogene drugs blocksangiogenesis effect

Anti-angiogenesis therapy

Biological therapy (targeted therapy)

Preparation:▫ Monoclonal antibodies▫ Differenciacal therapy▫ Inhibitors of proteasom, tyrosinkinases, ▫ Anti-angiogene therapy▫ Antisense oligonucleotides

High prices

Limited usage depending on these criterias:▫ Tumor subtype ▫ Other types of cancer therapies are non-effective ▫ Undesirable effect of other types of cancer therapies

Antisense oligonucleotides

Biological therapy (targeted therapy)

High prices

Limited usage depending on these criterias: Tumor subtype Other types of cancer therapies are non-effective Undesirable effect of other types of cancer therapies

Biological therapy (targeted therapy)

Suitable types of tumors for biological therapy:• Kidney tumors • Prostate tumors • Intestine tumors • Lung tumors • Breast gland tumors • Female genital tumors • Melanoma • Kaposi’s sarkoma

Thank you for your attention