Watson Lectures Flashcards
(42 cards)
Pre-Molecular Era (Important Concepts)
- Cancer is a disease of the cells
- Cancer cells have become immortalized
- Properties of cancer cells are transmitted from parental to daughter cells.
- Cancer involves changes in DNA (the vehicle of heredity)
How can you detect Oncogenes in Human Tumours?
1) DNA Transfections.
To detect oncogenes in human tumors, DNA from tumor cells is prepared and co-precipitated using a phosphate buffer and calcium ions, forming a calcium phosphate-DNA complex. This DNA is then introduced into NIH/3T3 cells, a line of mouse fibroblasts, which are cultured for two weeks. Transformed NIH/3T3 cells, which grow distinctively as foci among untransformed cells, indicate the presence of oncogenic sequences in the tumor DNA.
How does Ras oncogenes get activated? Where are they found?
- They get activated by Point Mutations.
- Ras mutations in tumours usually involve coodns 12, 13, 59, and 61.
- Mutated ras is found in 50% of colorectal cancers and 95% of pancreatic cancers.
- plays a major role in tumour maintenance.
Ras GTPase
1) Exists in 2 states.
- Active: GTP bound.
- Inactive: GDP bound.
2) Ras is a crucial regulator of:
- cell shape
- motility
- growth
What are some different characteristics found in normal cells and tumour cells?
1) Density-dependent inhibition of growth:
normal = present
tumour = absent
2) growth factor requirements
normal = high
tumour = low
3) anchorage dependence
normal = present
tumour = absent
4) proliferative life span
normal = finite
tumour = indefinite
5) contact inhibition of movement
normal = present
tumour = absent
6) adhesiveness
normal = high
tumour = low
7) morphology
normal = flat
tumour = rounded
What is cancer caused by? And what are the 2 categories of Cancer Research?
- Cancer is caused by the accumulation of mutations that result in the activation of oncogenes and the inactivation of tumour suppressors.
2 categories:
- tumour suppressor genes (inactivated)
- proto-oncogene becomes an oncogene (activated)
Onocogenes
- frequently amplified in human tumours
- can be identified as alterations in chromosome structure
- take the karyotype (all chromosomes of cancers) and visualize them.
- oncogene amplification can be identified as alterations in chromosome structure.
Proto-oncogene amplification
Multiple copies of proto-oncogenes have been found in various tumours.
1) homogeneous staining regions (HSRs): Areas in chromosomes where amplified genes are integrated, resulting in uniform staining during karyotyping.
2) double minute chromosomes (DMs): Circular extrachromosomal DNA fragments that carry amplified oncogenes and segregate with cell division.
In studies with 3T3 fibroblasts, DMs were analyzed, revealing three genes—MDM1, MDM2, and MDM3—on these structures. The MDM2 gene was identified as responsible for the transformation phenotype, demonstrating its role as an oncogene.
function of p53 & its negative regulator
- MDM2 is a negative regulator of p53. (promote cancer growth)
- p53 act as checkpoint for entry into S phase.
lack of nucleotides, UV radiation, ionizing radiation, oncogene signalling, hypoxia, blockage of transcription, all lead to activation of p53, which will activate cell cycle arrest/senescence/return to proliferation, DNA repair, block of angionensis, apoptosis. (inhibit cancer growth - tumour suppressor gene)
What is the main oncogene present in breast cancer?
- 20-25% of breast cancer have amplification of the HER2 RTK (EGFR family)
- HER2 is a prognostic factor and therapeutic target
- a prognostic factor provides information regarding patient outcome at time of diagnosis and provides information on the likelihood of response to a therapeutic.
- HER2 is important for breast cancer growth and is accessible as a cell surface receptor.
One thing that is important for HER2 and other receptor tyrosine kinases, they are on the cell surface of cells, can identify therapies such as monoclonal antibodies that target signal
What is the personalized medicine that will target HER2 (breast cancer oncogene)?
Herceptin or Pertuzumab (monoclonal antibody 2C4, similar strategy to herceptin)
will block HER2.
HER2 monoclonal antibodies:
- inhibited proliferation of HER2 over expressing tumour cells.
- inhibited in vivo growth of HER2 tumours in nude mice.
- Genentech developed a humanized monoclonal antibody (Herceptin)
- HER2 amplification in breast cancer - Chromosome 17q. (can visualize using DNA - southern blot, RNA - northern blot, protein (immunoprecipitation)).
Identifications of Oncogenes at Chromosomal Breakpoints
- Creation of the Philadelphia chromosome. Found in >95% of CML patients.
- chromosome 9 and 22 contain the genes c-abl and bcr at their breakpoint, respectively.
- formation of bcr-abl chimeric gene.
- formation of bcr-abl chimeric RNA.
- formation of bcr-abl chimeric protein.
- Bcr-Abl rearrangement present in 98% of CML.
How do we target Bcr-Abl (CML)?
- Abl is a tyrosine kinase (cytosolic - not transmembrane - hence not suitable to target mAb type therapies). Instead, develop small molecule inhibitors to the ATP binding pocket of the tyrosine kinase domain.
- the small molecule, Gleevec, is defined to fit into that ATP binding pocket of the Bcr-Abl tyrosine kinase, this competes with access to the activate site.
- Gleevec “personalized medicine” for CML.
How can we test the efficacy of potential small molecule inhibitors?
- must develop in vitro screens.
In normal BaF3 pre-B cells, growth requires IL-3, and they die when IL-3 is removed. However, BaF3 cells transduced with Bcr-abl continue to grow without IL-3, showing that Bcr-abl acts as an oncogene; Gleevec is not toxic to normal cells but specifically inhibits cells with Bcr-abl.
Patients in the acute blast crisis phase of CML often develop resistance to Gleevec due to mutations in the ATP-binding pocket of the Abl-kinase domain. These mutations occur in critical amino acids for Gleevec binding, leading to the need for alternative inhibitors targeting these resistant mutants.
Why did alternative inhibitors, for example for CML, need to start being developed?
- Patients in acute blast crisis of CMLoften develop resistance to Gleevec
caused by mutations in the ATP
binding pocket of Abl-kinase domain. - Many of the mutations in patients are
found within the same amino acids in
Abl kinase-those critical for binding
Gleevec.
Hence can start to develop
alternate inhibitors that would target
the mutants!
What are some strengths and weaknesses of anti-receptor antibodies versus low-molecular-weight tyrosine kinase inhibitors as anti-cancer agents?
1) Target:
Small molecule: tyrosine kinase domain
Antibody: receptor ectodomain
2) Specificity:
Small molecule: +++
Antibody: ++++++
3) Binding:
Small molecule: most are rapidly reversible
Antibody: receptor internalized, only slowly regenerated
4) Dosing:
Small molecule: oral daily
Antibody: intravenous, weekly
5) Distribution in tissues:
Small molecule: more complete
Antibody: less complete
6) Toxicity:
Small molecule: rash, diarrhea, pulmonary
Antibody: rash, allergy
7)Antibody-dependent cellular cytotoxicity:
Small molecule: no
Antibody: possibly
Some translocations don’t lead to a new chimeric protein, but uncouples the gene from its regulating elements. What is an example?
- The Myc gene can become deregulated through various mechanisms, disrupting its normal function.
- Myc is a type of protein in the basic helix-loop-helix family.
- It forms heterodimers (pairs) with other proteins to bind DNA and regulate genes.
- Myc-Max: Promotes transcription of genes containing an “E box” in their promoters.
- Mad-Max: Represses the same genes.
- As cells differentiate, Mad protein levels increase.
- This displaces Myc from the complex, reducing Myc-Max activity.
- The decrease in Myc-Max activity promotes differentiation and slows proliferation.
- Myc regulates genes that control the cell cycle and proliferation, including:
- Cyclin D2 and CDK4: Promote cell cycle progression.
- Cul1 and E2F: Important for DNA replication and S phase entry.
- Myc-Max complexes promote progression through the S phase of the cell cycle.
Identification & Isolation of Tumour Suppressor Genes
- Usually multiple genetic changes in any single tumor.
- tumor suppressors are usually recessive.
Retinoblastoma (Rb)
1) Role of Rb Gene:
Rb gene prevents uncontrolled cell division (tumor suppressor).
Loss of Rb function predisposes cells to tumor development.
2) Familial Retinoblastoma:
Zygote carries 1 defective Rb copy; all cells have only 1 functional copy.
A somatic mutation in a retinal cell eliminates the functional Rb copy, leading to tumor formation.
Patients are prone to bilateral tumors (both eyes).
Higher risk of second tumors later in life.
3) Sporadic Retinoblastoma:
Both Rb copies must be inactivated by 2 sporadic mutations in the same retinal cell.
Rare; typically causes a single tumor in one eye.
4) Mechanisms of Rb Loss:
Mitotic recombination: Exchange of genetic material during cell division.
Gene conversion: One allele is replaced by another, eliminating the functional Rb copy.
5) History of Rb Research:
1914–1984: Rb patients studied; bilateral cases showed higher second tumor risks.
1986: Rb gene cloned using loss of heterozygosity (LOH) in chromosome 13 from tumor tissue.
Gene walking mapped deletions to pinpoint Rb gene location.
6) Key Facts:
Familial Rb: Germline mutation + somatic mutation.
Sporadic Rb: Two somatic mutations required.
Role of cyclin D1, Cdk4 and Rb in cell cycle progression
- Rb controls cell cycle progression by switching between active and inactive states through phosphorylation.
- In M/G1 phase, Rb is dephosphorylated (active) and stops cells from progressing.
- In early G1 phase, Cyclin D-CDK4/6 partially phosphorylates Rb (hypophosphorylation), starting to inactivate it.
- At the restriction point (R), Cyclin E-CDK fully phosphorylates Rb (hyperphosphorylation), fully inactivating it and allowing entry into S phase.
- Rb and p53 act as checkpoints to prevent uncontrolled cell division.
- If Rb is lost, cells bypass control, leading to continuous cell cycle progression.
What are different hallmarks of cancer and why are they useful?
- they are useful because by understanding the biology oncogenes and tumour suppressors the field has found common hallmarks that are frequently found in cancer.
1) Sustaining proliferative signalling.
2) Evading growth.
3) Activating invasion and metastasis.
4) Enabling replicative immortality.
5) Inducing angiogenesis.
6) Resisting cell death.
What is the definition of Genome and what is meant by Genomics?
Genome: fusion of the words gene and chromosome.
Genomics involves the quantitative analysis of DNA, RNA, and epigenetic using technologies such as microarray and sequencing.
Cytogenetic Tools/Genomic Methods and Technologies (DNA)
- Cytogenetic methods were the first tools that enabled genomic analysis of chromosomes.
- First genomic maps were constructed by staining chromosomes with Giemsa (G-banding).
- Dark regions have low GC content and are gene-poor, while light regions are gene-rich and have high GC content.
1) Can see on a karyogram.
2) Spectra karyotyping (SKY) uses fluroescently labeled DNA probes to colour chromosomes allowing detection of translocations, deletions and amplifications.
3) Array comparative genomic hybridization (CGH):
- can measure genome-wide copy number aberrations at high-resolution to detect amplifications and deletions.
- Use probes to look at different regions of the genome and compare the DNA we get from tumours vs DNA we get from normal blood, can look at all the copy number alterations by comparing to a reference set (which would be normal DNA).
4) Affymetrix SNP arrays (for copy number)
(what was used in the cancer research atlast project)
What are some problems with Genomic Methods and Technologies (RNA)?
1) Northern analysis of gene expression.
- Amount.
- Limited to few known genes/scalability.
2) PCR based analysis of gene expression.
- limited to known genes/scalability.
3) Microarray of gene expression.
- thousands of known and unknown genes analyzed at one time.
- nanoscale technology.
