Mechanisms of carcinogenesis and modern anticancer therapies

obligatory courses

Students are obliged to complete the 1st and the 2nd year of the first level studies (Bachelor's) before starting the subject.

The student should know basic techniques used in biochemistry as electrophoretical techniques, principles of the work with enzymes. Students should include laboratory classes from Biochemistry, as well as know the techniques of cell cultures.

Classroom

Cancer epidemiology as well as environmental and endogenous factors, which favor formation of tumors. Mechanisms of induction of mutations as causative factor of carcinogenic process will be discussed, as well as epigenetic mechanisms (DNA methylation, miRNA). DNA repair mechanisms will be presented as well as tumor cell characteristics, stages of carcinogenesis, major critical genes, dysfuction of which triggers carcinogenic process (oncogenes, tumor suppressor genes), apoptosis, proteolysis, angiogenesis and metastasis. Cancer stem cells. Role of viruses in cancer induction. Diagnostics and the basis of modern treatment methods; personal therapy.

Lecture:

Cancer epidemiology. Factors favoring cancer induction: environment pollution, genetics. Characteristics of cancer cell. Critical genes, oncogenes, tumor suppressor genes and stabilizing genes, their role in carcinogenesis. Mutation theory of carcinogenesis, classic models: initiation, promotion, progression. Metabolic activation and detoxification of xenobiotics: polymorphism of metabolic genes as a marker of susceptibility to cancer. DNA lesions as an origin of mutations. Exogenous and internal factors damaging DNA: UV, ionizing radiation, environmwntal carcinogens, diet, inflammations, infections, oxidative stress, enzymatic DNA methylation. Replicative and transcriptional mutagenesis; family of low fidelity DNA polymerases – characteristics and their role in induction of mutations and cancer. DNA repair: direct reversal, postreplicative mismatch repair, excision repair, recombination repair. The role DNA repair proteins in the formation of antibodies. Modern theory of carcinogenesis. Disorders in Signal transduction. Cell cycle and the role of its disorders in the ability to gain unlimited number of cell divisions. Apoptosis and its role in carcinogenesis. DNA repair and cell signaling.

Angiogenesis – formation of new blood vessels. Metastasis. Tumor growth in a new location, relationship with growth factors and adhesion proteins. The reasons of metastasis to defined organs. Human disease related to malfunctioning of DNA repair: cancer (Xeroderma pigmentosum, Bloom syndrome, Ataxia telangiectasia, Fanconi anemia), developmental and neurodegenerative disorders (Cockayne syndrome, Trichotiodystrophy), aging (Werner syndrome). Viral carcinogenesis: oncogenic viruses (DNA, RNA), human oncogenic viruses (cervix cancer – HPV 16, 18; leukemia – HTLV 1, 2; hepatitis – HBV, HCV). Epigenetic theory of carcinogenesis: methylation and demethylation of gene promoters, the role of DNA repair and Tet proteins; histone acetylation and methylation; miRNA. Cancer stemlike cells: characteristics, markers, potential inhibitors. Modern cancer diagnostics. Methods based on PCR techniques (PCR, LTR, RT-PCR, QTR-PCR), techniques based on restriction enzymes. Classic methods of cancer therapy: radiotherapy, chemotherapy, modern targeted therapy, new targets in cancer treatment, synthetic lethality, modern carriers of anticancer drugs.

Lecture includes 30 h and is completed with the examination. The time necessary to prepare for the examination is individual, not lower than 30 h.

Laboratory classes:

Laboratory classes 60 h (10 classes, 6 h each)

1. DNA damage as a source of mutations and cancer; DNA repair pathways

- Induction of DNA damage by alkylating agents, and oxidizing using the model of the plasmid DNA and DNA repair enzymes

- Induction of damage in mammalian cells, wild type and inactive DNA repair systems - survival test (Alamar Blue)

- Secondary DNA damage; Modification of the single-stranded M13 phage DNA with dimethylsulfate

2. Secondary DNA damage.

- Depurination of alkylated DNA bases and opening the imidazole ring; repair, biological properties

3. Use of the lac operon to determine the types of mutations;

-Study mutagenic specificity of UV radiation, oxidation and alkylation agents using strains Miller.

4. Tests to detect environmental mutagens

- SOS Chromotest, demonstration of sister chromatid exchanges and preneoplastic lesions in the large intestine

5. Direct DNA repair without affecting DNA integrity - Oxidative dealkylation - AlkB protein; test participation proteins AlkB and system BER repair etenoadduktów mutants of E. coli.

6. Polimorfizm genów naprawy DNA jako czynnik ryzyka rozwoju choroby nowotworowej.

Badanie polimorfizmu genów OGG1, OH-1, GST

7. Cellular DNA damage response mechanisms:

- Observation of replication inhibition in mammalian cells – DNA fiber technique (movie and show of microscopic slides with DNA fibers – analysis of sites of DNA replication inhibition)

- DNA damage response (DDR) – DDR activation – phosphorylation of ATM, ATR, Chk1, H2AX (Western analysis; optional observation of γH2AX foci in cells treated with Fenton reagent)

- Analysis of minority mutations in critical genes (APC, RAS) in colon cancer using MSSCP method

8. Modern and experimental therapies: induction of synthetic lethality by PARP1 inhibitors in wild type cells and cells defective in DNA repair proteins (BER, HR, NHEJ)

9. Combination therapies: Synergistic effect of drugs (camptothecin, TRAIL, CK2 inhibitors). Survival of cancer cell lines using standard MTT test. Determination of the percentage of cells survival In response to therapeutic molecules with different mechanism of action. Analysis of synergistic activity of studied molecules.

10. New methods of introducing drugs into the cell – penetrating peptides

Introduction of CPP cassette into recombined protein is a new method used during construction of drugs, which therapeutic target is located inside the cell. During this class students will incorporate into the cell recombined protein possessing attached sequence YGRKKRRQRR deriving from TAT protein, and identification of incorporated protein inside cell.

1.Bal J (red) Biologia molekularna w medycynie. Wydawnictwo Naukowe PWN, Warszawa, 2013.

2. M. Wojtukiewicz i E. Sierko (red) LECZENIE CELOWANE W ONKOLOGII I HEMATOONKOLOGII, Via Medica, Gdańsk, 2013.

3.Friedberg E, DNA Repair,

4.Tudek B, Wewnątrzkomórkowe utlenianie i alkilacja DNA - mechanizmy i skutki. w Koroniak H, Barciszewski J (red) "Na pograniczu Chemii i Biologii", t IV, Wydawnictwa Naukowe UAM, Poznań 2000, str. 201-229.

5.Rzeszowska-Wolny J, Drobot L, Sygnalizacja i zmiany funkcjonowania komórki po uszkodzeniu DNA. W: Koroniak H, Barciszewski J (red) "Na pograniczu Chemii i Biologii", t IV, Wydawnictwa Naukowe UAM, Poznań 2000, str. 231-252.

6.Sugimura T, Wakabayashi K, Nakagama H, Nagao M (2004) Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci. 94(4): 290-299.

7.Jagerstad M, Skog K (2005) Genotoxicity of heat-processed foods, Mutation res., 574: 156-172.

8.Goodman M (2002) Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu Rev.Biochem., 71 : 17-50.

9. Śliwińska-Hill U, Trocha J: Najnowsze terapie przeciwnowotworowe, Post.Farm. 2011 nr 1; s.14-18

Once you have mastered the material covered in lecture and exercises student of Biotechnology (medical biotechnology):

-demonstrates knowledge of the current state of knowledge in the main sectors of biotechnology, has knowledge of: the latest research, discoveries and their medical uses with particular reference to the mechanism of cancerogenesis (S3_W01),

-knows the types of anticancer therapies (S3_W01),

-demonstrates knowledge of the bases of research planning, modern techniques of data collection and the use of various research tools on mechanisms of carcinogenesis and cancer therapies (S3_W02),

-has knowledge of independent planning and conducting experimental work, analyzing the results in a form suitable for discussion, assessment or publication (S3_U03),

-demonstrates the ability of critical analysis and selection of information, especially from electronic sources (S3_K01),

-shows responsibility for the entrusted scope of research and for the work of its own laboratory and others (S3_K03),

-is able to properly assess the risks from the use of research techniques and create the conditions for safe operation (S3_K03),

-appreciates the importance of statistical tools and bioinformatics in the description of the results of experimental work and processes in nature (S3_K01),

-has a habit of using objective sources of scientific information (S3_K01).

Once you have mastered the material covered in lecture and exercises student of Biology (molecular biology):

- Demonstrates knowledge of the current state of knowledge in the main sectors of biotechnology, has knowledge of: the latest research discoveries and their applications in medicine with special emphasis on the mechanism of cancerogenesis (S2_W01),

- Knows the types of anticancer therapies (S2_W01),

-demonstrates knowledge of the bases of research planning, modern techniques of data collection and the use of various research tools on mechanisms of carcinogenesis and cancer therapies (S2_W02),

- Has knowledge of independent planning and conducting experimental work, analyzing the results in a form suitable for discussion, assessment or publication (S2_U03)

-demonstrates the ability of critical analysis and selection of information, especially from electronic sources (S2_K01),

-shows responsibility for the entrusted scope of research and for the work of its own laboratory and others (S2_K03),

-is able to properly assess the risks from the use of research techniques and create the conditions for safe operation (S2_K03),

-appreciates the importance of statistical tools and bioinformatics in the description of the results of experimental work and processes in nature (S2_K01),

-has a habit of using objective sources of scientific information (S2_K01).

Assessment exercises:

- Attendance at a minimum of 85% of classes;

- Participation in classes, demonstrating knowledge, skills, and social competencies acquired during the course (as described in the syllabus as subject-specific learning outcomes);

- Preparation of reports describing the results of all exercises and their interpretation;

- Preparation of a presentation on a selected topic related to the subject matter of the classes (evaluated by the course instructors based on the completeness of the presented information and its substantive value).

The final laboratory grade consists of the test grade (50%) and the presentation grade (50%)

Assessment lecture:

The exam is a multiple choice test. The condition for passing the exam is to get more than half of the points.

Not applicable