CELL TRANSFORMATION RNDr. Jan Šrámek [email protected] 2013 Seminar lesson of cell and...

CELL TRANSFORMATION

RNDr. Jan Šrámek

[email protected]

2013 Seminar lesson of cell and molecular biologie

What is it today about

Cell transformation Characteristics of tranformed cells Mechanisms of transformation Carcinogenes Tumors and their classification Cancer therapy

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Cell transformation

What is cell transformation?

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Cell transformation

What is 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)

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Characteristics of tranformed cells

What are typical transformed cells characteristics?

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What are typical transformed cells characteristics? Independence on stimulatory cytokines Loss of ‘anchorage dependence’

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What are typical transformed cells characteristics? Independence on stimulatory cytokines Loss of ‘anchorage dependence’ Capability of non-regulated clonal growth and loss of contact

inhibition

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

apoptosis Inability to differentiate Ability of induction of angiogenesis Different cell surface molecules and chromosomal reconstruction

(CD44, annexiny, etc.) Genetic instability

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

apoptosis Inability to differentiate Ability of induction of angiogenesis Different cell surface molecules and chromosomal reconstruction

(CD44, annexiny, etc.) Genetic instability

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

Mechanisms of cell transformation

1) What is the nature of cell transformation mechanisms?2) What are oncogenes and anti-oncogenes, name some.3) How many mutations are approximately needed to cause

cell tranformation? 4) What are the most common genetic changes (types)

during the cell transformation process?5) What is hypothetical probability of cancer development in

human?6) What can positively affect cancer incidence?7) What can negatively affect cancer incidence?

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What are typical transformed cells characteristics?Multistage process (cancer incidence correlates with age)

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Multistage process

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

Cancer cell

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


What are typical transformed cells characteristics?Multistage process (cancer incidence correlates with age)Non-returnable process

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What are typical transformed cells characteristics?Multistage process (cancer incidence correlates with age)Non-returnable processUnder selection stress

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Process under the selection stress18


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

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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-

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

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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-

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

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Carcinogens10-8

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Immune system10-8

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Theory of immune survailence

What is the nature of theory of immune survailence?

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Theory of immune survailence

What is the nature of theory of immune survailence?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.

Cancer incidance markedly higher after passing 40 years.

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Cancer is a consequance of immune system failure in cancer cells elimination.

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

Change of quality × change of quantity

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

(c-abl)

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Philadelphia chromosome

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Regulation domain Tyrosin-kinase domain

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 and strong promotor. Responsible for many types of leukemia.

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

Simultaneously, it has markedly higher expression rate (according to Bcr promotor) marked change of quantity.

Point mutation of c-ras gene

Ras

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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

Carcinogens

1. What are carcinogens and how do they affect?

2. Which tree main classes of carcinogens do we know?

3. Name some of each basic type of carcinogen.

4. By which mechanism chemical carcinogens cause cell transformation?

5. What cause ion radiation?

6. What cause UV radiation?

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Carcinogens

Cause genetic changes via interactions with DNA leading to cell

transformation.

Factors causing cell transformation: Chemical Physical Biological

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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 transition

HSO-3: deamination of C on U transition

NH2OH

H2N-O-CH3

Alkyl agent: alkylsulfates, N-alkyl-N-nitrosamines (nitrates). Alkyl C, T and G block or

change base pairing, cause between- and interchain crossbonds block of replication

and transcription

Psoralenes: intercalar agens, furocumarin, 8-metoxypsoralene

Pre-carcinogens: metabolic activation via specific enzymes (cyt. P450) is necessary

N-acetyl-2-aminofluoren (AAF), Benzo(a)pyren, Aflatoxins, Nitrates and others

Cause transitions, transversions, bases modifications or covalentely bind to DNA

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Physical carcinogens

Ionize radiation: dsDNA breaks → chromosomal translocations. 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.

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crossbonds

Breaks

crossbond

strand

strand

Biological carcinogens

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1. Describe the mechanism of cancer cell development in a consequance of the effect of oncogene virus?


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1. Describe the mechanism of cancer cell development in a consequance of the effect of oncogene virus?

2. Which viruses (name examples) can affect as 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)

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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)

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

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

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

devil (Sarcophilus harrisii)

Histiocytes

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Tumor

1) What is a tumor and what is its function in an organism?2) How many transformed cells is sufficient for tumor

development?3) Where are tumors in an organism mostly developed?4) What is the diference between benign and malign tumor?5) How do we classify tumors?6) What is metastasis and how is developed?

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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)

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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 secondary tumors.

Tumors classification I41

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

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According to affected organ (tissue):Breast carcinomaColorectal carcinomaCervical carcinomaGland carcinomaStomach cancerOvarian carcinomaLeukemiaAnd many others

Tumors classification III

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Process of metastasis development

Primary tumor

Secondary tumor - metastasis

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Cancer therapy

1. What are basic approaches in cancer therapy?2. Which chemoterapeutics do we know and what is common

mechanism of their effect?3. What other preparates can be used?4. What is gene therapy?

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Cancer therapy

Classical approach:Local therapy – surgical strike, local radiation ( radiation)Systematical approach (combinated with surgical strike and radiation) – ChemotherapyChemotherapy: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-CSFBiological therapy (targeted therapy, [Gene therapy])‘Antisense’ oligonucleotides: target to specific oncogenesTransfection: functional anti-oncogenes….

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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

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Biologicac 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

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Monoclonal antibodies

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Biologicac therapy


High prices


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Differential therapy51

Cancer cell

Specific drug

Differentiated cell

Differentiated cell

Progenitor cell

Mutations

Developmental line

Biologicac therapy


High prices


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Biologicac therapy


High prices


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Anti-angiogenesis therapy

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a) Cancer cells induce angiogenesis b) Vessel reacts by protrusion

c) Tumor have nutrition and growth and release cells into the circulation

d) anti-angiogene agens (white circles) block angiogenesis

Biologicac therapy


High prices


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Antisense oligonucleotides

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Biologicac therapy


High prices


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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

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Thank you for your attention Next seminar lesson: Mechanisms of effect of oncogenes and

tumor-supresor-genes (no. 130)

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CELL TRANSFORMATION RNDr. Jan Šrámek [email protected] 2013 Seminar lesson of cell and...

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