MCB: Kurssammanfattning Flashcards
Oncogene - procedure to cancer
Gain of function
Tumor Suppressor gene - procedure to cancer
Loss of function
Cancer cases/year
About 10million
The emerging hallmarks (7)
- Deregulating cellular energetics
- Sustaining proliferative signalling
- Evading growth suppressor
- Enabling replicative immortality
- Activating invasion and metastasis
- Inducing angiogenesis
- Resisting cell death
Non-neoplastic proliferation
Controlled and reversible
- Hypertrophy: Size
- Hyperplasia: Number
- Metaplasia: Change. Still at reversible state
- Dysplasia: Disordered. Often irreversible
Neoplastic proliferation
Uncontrolled and irreversible
Benign tumors: Are localized and non invasive. Not a cancer (besides when growing in your brain). For colon cancer it is common to have benign legions for 10 years, after which it might switch to cancer cells.
Cancerous: Malignant cells.
Attributes of cancer cells
- Immortal
- Transformed
- Motile
- Invasive
- Metastatic
- No longer obey “rules” of controlled growth;
- Contact inhibition
- Need for solid support
- Requirement of growth factors
Tumor cell amount that is
a) Smallest detectable
b) Usually detected at
c) Maximal size still compatible with life
a) 10^9
b) 10^10
c) 10^12
Biological properties of benign neoplasms
a) Structure
b) Mitoses
c) Growth
d) Growth rate
e) Growth duration
f) Encapsulation
g) Metastasis
h) Effect on host
a) Structure - Resemblance to normal cells (well differentiated)
b) Mitoses - Few
c) Growth - Usually purely expansive
e) Growth duration - May cease growing
f) Encapsulation - ususally
g) Metastasis - None
h) Effect on host - Slight harm, due to location or complication
Biological properties of malignant neoplasms
a) Structure
b) Mitoses
c) Growth
d) Growth rate
e) Growth duration
f) Encapsulation
g) Metastasis
h) Effect on host
a) Structure - Abnormal; less similarity to normal cells
b) Mitoses - Relatively common
c) Growth - Invasive
d) Growth rate - Rapied
e) Growth duration - Rarely cease growing
f) Encapsulation - Rarely
g) Metastasis - Frequent
h) Effect on host - Significant harm, due to invasion and metastasis
Exception of malignant neoplasms
Basal cell carcinoma
- The most common cancer, highly invasive but almost never metasies.
Exception of benign neoplasms
Benign brain, blood vessel and airway tumors can kill patients still because they cause obstruction of these organs
Placement of neoplastic growth affecting tumor
a) Continually replacing cells
b) Non-renewing cells with regenerative capacity
c) Essentially non-replacing cells
a) Continually replacing cells - most common placement of cancer. For example male germline, hematopoietic tissue, surface epithelium.
b) Non-renewing cells with regenerative capacity. Liver, kidney, connective tissue. Quite common placement for cancer
c) Essentially non-replacing cells - very unusal placement for cancer. For example neurons, muscles, female germline.
Difference in general doubling time between embryonal tumours and adenocarcinoma
Embryonal tumours have growth fraction at 90% and adenocarcinoma at 6%
Carcinoma
Cancer formed in epithelial tissue or in tissue lining internal organs, epidermal tissue. >90% of tumors origin from epithelial tissues.
Sarcoma
Tumor formed from mesenchymal cell types, 1% of all cancer. Cancer of connective tissue origin
Lymphomas
Arise in the lymph nodes and tissue of the immune system
Lymphomas: 5% of all cancer
Leukemias
Cancer of the white blood cells
Staging malignant neoplasms
- T score
- N score
- M score
- T score: Based upon tumor size (1-4)
- N score: Extent of lymph node involvement, tells if it has begun to spread or not. (0-3)
- M score: Indicates whether distant Metastases are present (0 or 1)
Staging malignant neoplasms
Stage levels
1-4, progressively more and more metastases and invasion and size
Grading malignant neoplasms (1-4)
1 - Well differentiated
2 - Moderately differentiated
3 - Poorly differentiated
4 - Nearly anaplastic
Different ways to do lab work to see possible tumors (5)
- Classical biopsy
-Tumor tissue array
-Laser capture microdissection
-Microarray
-Single cell RNA seq
CODEX protein staining
Ab acts as reporter probe and is linked to a barcode and we can have for example 3 different reporters.
Able to do bulk single cell to see biomarkers and how they are distributed. Hardest part of technique is linking barcodes to biomarkers.
Liquid biopsy
Less invasive than normal biopsy.
Normally much less tumor cells in blood –> high sensitivity needed. Also methylated DNA is looked at, can be used to predict tumor source
Sanger seq has highest sensitivity.
Cancer genetics according to Theodor Boveri
Boveri gave the chromosomal theory of heredity. Showed that fertilization of sea-urchin eggs by two sperm eggs gave multiple cell poles that cause unequal segregation of chromosomes
- Specific chromosomes gave rise to particular phenotypes
- Genetic imbalance can result in death
- Individual chromosomes must carry different information
Predicted the cell cycle and much of the biology of cancers.
Point mutation
Subsitution of one DNA base pair to another
Missense : substitution of one aa
Nonsense: base substitution –> premature stop codon
Splicing effect: Base substitution near intron-exon junctions.
Translocation
Hybrid chromosome result of joining one part of chromosome to another. Gives
- Structurally changed gene version and its protein
- Gene under new trancriptional contorl
Gene amplification
Often give increase levels of mRNA and protein expression
Amplified genes may form two types of microscoplcally abnormal chr. config.:
DM : Double minutes, small paired extrachromosomal chromatin bodies segregating randomly during mitosis
HSR: Homogenously staining regions, expanded chromosomal regions that do not exhibit normal banding patterns
Deletion
Single base - complete chromosomes
Large deletions inactivate all the genes included within it
In frame deletion: Consequence depend on funciton of deleted segment
Out of frame deletion: Consequence is frameshift - premature stop codon
Insertion
Single base to kbps
Less common as mutation events in cancer genes
In frame insertions
Out of frame insertion - consequence is frame shift - premature stop codon
Passenger mutation
Occurring but does not give an advantage to the cells. Genetic changes that do not confer any selective advantage in cancer developement.
Driver mutation
Can make a difference, giving more proliferation. Genetic changes that give selective advantages to clones during cancer developement.
Mutator phenotype
mutation in one of the enzymes present used to repair the cells Accumulation speeds up.
Somatic mutations
Mutations that are acquired as opposed to inherited
Mutational signature
Pattern of mutations produced by a mutational process
Mutational portrait
Total genetic changes observed in the cancer genome: sum of all mutational signatures occurring in a lifetime
Microsatellite instability (MIN)
Variation in length of homopolymeric regions (particularly poly(A) sequences)
– Variation in the length of dinucleotide and trinucleotide repeat regions
– Associated with defective Mismatch Repair (MMR)
Chromosome instability (CIN)
– Abnormal variation in gross chromosome number (aneuploidy)
* Loss of chromosomes can be balanced (duplication of the remaining WT chromosome) or result in loss of heterozygosity (LOH)
– Increased rate of chromosomal alterations
* 10-100 fold increased rate of chromosome gain or loss and similar rates for LOH at specific genomic sites.
– The molecular origin of CIN is less well established
* Abnormal centrosome function in cancer cells can cause chromosome instability
– Centrosome overduplication resulting in multifocal spindles and grossly abnormal mitoses
* Improper behavior of the mitotic spindle can be due to defects in mitotic checkpoints
Regulation of mitosis
In the metaphase, microtubules will attach to the chromosome. Many proteins are involved in the fine tuning of this process.
In the prometaphase the chromosomes ae not yet aligned correctly and you cannot start pulling apart the chromatids yet. CDC20 and APC/C complex will drive the process of chromosomal segregation.
If bound to MAD2 it is inactive.
Cells with MAD2 downregulation
Premature anaphase entry. 50% of MAD2 removed in order to induce.
Gives chromosome loss
Securin negative cells
Separain-securin forms a complex to help cleave aligned the cohesins correctly during the metaphase. If not working then incorrect cutting of chromosome
Chromothripsis
chromosome shattering. Cancer genomes usually have strange things such as reshuffled genomes in cancer. Chromosomal breakage leads to non-homologus end joining wich lead to the loss of the cell.
The adenomatous Polyposis Coli (APC) protein
Cancerous syndrome in human. Very large protein, that is very different from the Anaphase Promoting Complex.
Large protein with different domains. Some parts b-cathein degradation proteins. Microtubule binding domain binding to kinetochore of chromosomes.
- tumor suppressor gene, keeps genes away from being expressed unless it has a signal that signifies it.
- APC mutant cells have no connection to microtubules
- Gene for colon cancer
- Germline mutation cause FAP
- Very important for Wnt signalling pathway
Wnt signalling pathway
The pathway is simple. APC is part of a complex with B-catenin and axin/conduction. Allows phosphorylated B-catenin to be ubiquitylated. Cytoplasmic B-cathein will be degraded in the absence of Wnt signal
Endogenous source of DNA damage
- Oxidative agents
side effect of its own mitochondrial energy metabolism. Reactive oxygen species (ROS) attack DNA. Much more oxidation of DNA when you have a higher metabolic rate - Spontaneous disintegration of DNA
Hydrolysis of nucleotides - abasic sites
Deamination of cytosine, adenine, guanine and 5-methylcytosine converts these bases to miscoding Uracil, Hypoxanthine, Xanthine and Thymine - Radiation
UV, Gamma and X-ray - Food constitutents
Aromatic amines - causes DNA damage and is highly mutagenic - Chemical agents
Mustard gas, genotoxic compounds present in cigarette smoke
*Physical carcinogens
Asbestos - leads to inflammatory condition that will cause colleteral damage
*Aflatoxin
Important to store food safely to reduce risk
*Inflammation can be mutagenic
-Caused by for example hepatitus B and C, HPV
-Chronic inflammation in lungs of smokers
Ames test
Test to estimate if a product is carcinogenic or not. Take for instance rat liver cells and homogenize them. Then add test compound that you assume could be cancerous. Then you add this to Salmonella which cannot grow on a medium without histidine. If the product is mutagenic, a mutation will happen leading to histidin being produced; hence Salmonella will grow. Still used today.
Mechanisms to revert DNA injuries
- DNA alkyltransferases: Single repair proteins that directly revert certain injuries
The easiest and most perfect DNA repair mechanism is mediated by O6-alkylguanine-DNA alkyltransferase - Nucleotide excision-repair (NER): Deals with whole class of helix distorting lesions which generally obstruct transcription, Lesions of exogenous origin, Most versatile in lesion recognition, Only one DNA strand is affected
- Base excision-repair (BER): Small chemical alterations of bases are targeted by BER, Lesions of endogenous origin, Only one DNA strand is affected, Mutations arisen due to metabolic action.
- Mismatch Repair (MMR): Repairs base mismatches, single base loops, insertion and deletion loops (mainly replication errors) , Used to solve replication errors. DNA polymerase has issues with repeated codons.
- Homologous recombination, Double strand breaks (DSB) - both strands are affected (most difficult to repair. Seems to dominate during S and G2 (DNA is already replicated). HDR or NHEJ
- End joining; DSB repair, Most active in G1 (no second copy available) – much less accurate
Xeroderma pigmentosum
Disease inherited strictly autosomal recessive. Median age of skin cancer onset is 8 years. The incidence of basal cell carcinoma is 1000 fold to unaffected individuals. A mutation in XP that makes you unable to handle UV damage. The repair system is not working.
Xeroderma pigmentosum V (XPV)
A variant of XP that is clinically indistinguishable from the normal XP. The XPV gene is the DNA polymerase η that has a error rate 6000x that of high fidelity DNA pol and can bypass DNA lesions and incorporates nucleotides opposite of DNA damage
HDR
Homology directed repair (HDR)
A crucial role during the normal cell-devision cycle (repairing strand breaks arising during DNA replication). Have for certain neurogenesis defects been seen in DSB repair mutants.
NHEJ
Nonhomologous end-joining (NHEJ)
Error-prone repair process. Responsible for the rejoining of DSB during V(D)J recombination.
SSCP - Single stranded confirmation polymorphism
Identify coding regions, mix WT and mutated DNA and denaturate. When DNA is kept ss you can see the base complimentary interaction.
Radioactivity is used to detect mutations because sporadic mutations will not be identified by Sanger since only a part of the fragments will be mutated. Mutation has to be present in more than 50% of the cells to be detected by Sanger. With radioactivity you have an enormous sensitivity.
Mutation detection by denaturing high performance liquid chromatography (dHPLC)
If you would run the heteroduplexes on a gel you would see mobility shifts.
+of the triethylammonium ion interacts electrostatistically with -charge of DNA. Determines precisely amount dsDNA present.
For heteroduplexes, they don’t binds as strong to the column and will come out much earlier.
The WAVE system is a fully automated dHPLC system that uses UV and/or fluorescence to detect DNA. SYBR green for example is very sensitive. It is a computer controlled automated data acquisition.
The technique is very precise is detecting mutations.
Melting Curve Analysis
qPCR, can use intercalating dyes to detect DNA, such as SYBR green. One big disadvantage with SYBRgreen is if you add too much then it will be toxic to TAG polymerase.
?Heteroduplexes melt easier than homoduplexes, but if you have too little SYBR green you wont be able to detect it.
4 things leading to malignant transformation
- RNA virus oncogene
- Cellular proto-oncogene
- DNA virus oncogene
- Cellular tumor suppressor gene
Different functions of cellular oncogenes
- Signalling molecule
- Signal receptor
- Intracellular transducers
- Apoptotic proteins
- DNA repair proteins
- Cell cycle control proteins
- Transcription factors
EGF in cancer
v-erbB is the EGF receptor with extracellular domain missing
The EGF receptor is heavily amplified in many cancers. Just a single mutation from Val to Glu make the ligand receptor Her2 independent and it starts acting as a oncopotein.
Cloning oncogenes from human tumors
- Transfect mouse with DNA from human tumor cells
- Culture for 2 weeks
- Focus of transformed NIH/3T3 cell growing among untransformed cells
- Extract DNA, transform new mouse cells
- Second cycle
- Extract genomic DNA
- Introduce into phage vector
- Plate phage on E.coli
- Put replica on filter paper using alu probe
- Oncogene
Human tumor viruses
Viral infections are linked to at least 15% of all malignant tumors in humans - the second most common identified risk factor for human cancer after tobacco smoking.
Both RNA and DNA viruses are linked to various forms of human malignancies. Retroviruses are rare cancer causes in humans. Oncogenic DNA viruses are important causative agent in human cancer
The amount of virus-associated cancers may further increase with the improvement of viral detection techniques.
