Study Notes Of The Lectures PDF

Lecture 1 1) 8 a) high mitotic index, morphological changes and disorganized cytoskeleton...

Lecture 1 1) 8 a) high mitotic index, morphological changes and disorganized cytoskeleton b) Loss of cell-contact inhibition c)loss of growth regulation d)Loss of dependence on anchorage for growth, anchorage independent e)Altered gene expression f) malignant tumours - cancerous/metastasize Benign tumours - not cancerous/don't metastasize g) Invasion h) altered energy metabolism- aerobic glycolysis known as the Warburg effect 2) for a) can use eosin staining too look at cytoskeleton for breast cancer, b) use scanning electron microscopy to observe the pile up of cells because of the loss of cell contact inhibition, c) growth factors within a petri dish, d) proliferate into spherical colonies with attachment to solid substrate ie petri dish, e)gene expression arrays, f) inject cells through tail of mouse see if the cells grow in the lungs if yes then malignant, g) Matrigel Invasion Assay, h) can use a PET scan to see if tumours have accumulated radioactive glucose 3) Multi-step process: in experimental liver and skin carcinogenesis ( normal cells are transformed into cancer cells), ALSO in colon cancer that involves several mutational hits to change normal cells into cancerous ones 4) Different types of cancers are caused by different agents. WHY? Accessibility of target tissues to those agents, and the nature of the cells for example the mammary cells are rich in steroid receptor highly replicative white bloods cells LECTURE 2 1) the key chemical features are the electrophilicity, binding to negative charged biomolecules 2) - Initiation: Interact with DNA by single base mutation, tends to be irreversible by the DNA repair mechanism Promotion: does induced proliferation, to produce benign lesions called Papillomas(SKIN), Nodules (LIVER), Polyps(COLON), it is reversible, and they don't form electrophilic species and don't bind DNA. affect signal transduction and cell cycle. KNOW EXAMPLES ARE: TPA activates protein kinase c pathway, and PB decrease TGFb and p53 Progression: invasive and metastasize, chromosomes are damaged, translocated, and deleted. Further mutations to oncogenes activation and tumour suppressor genes inactivation 3) divided into 2 phases:  phase 1: goal is to introduce functional group to the carcinogenic, common enzyme cytochrome P450, ◦ oxidation of C,N, S atoms to produce: ‣ hydroxylated metabolites for phase 2 OR ‣ unstable epoxides carcinogenic phase 2: UDP-glucuronosyltransferases, sulfotransferases, methyltransferases, glutathione-s- transferases. acetyltransferases ◦ play roles in: ‣ detoxifying: introduction bulky water soluble group onto phase 1 metabolite AND ‣ activate pro-carcinogens: metabolites unstable, can breakdown spontaneously to DNA reactive electrophiles 4) types OF ELECTROPHILES Alkylating agents: by oxidation of C atoms resulting in Carbonim ion or Aliphatic epoxide Aryl(aromatic) aminating agents: oxidation or reduction of N atoms on aromatic amines or amides making aryl nitrenium (THIS IS JUST A POSITIVE CHARGE ON N) Aralkylating agents: polycyclic aromatic hydrocarbons in aromatic epoxide 5) DNA adducts formed when electrophilic molecules react chemically with DNA bases Depends on the: - How ionic an electrophile is and, - The atom which it reacts with 6) time for people being susceptible to cancer: long latent period btw exposure and clinical detection of tumour difficult to quantify type and amt of exposure exposure to multiple chemicals and other cancer causing agents genetic variety/forms low exposure to carcinogen so can't detect them 7) Transformed fibroblast assay: Ames Test: used to test mutagencey chems ID as carcinogens expt animals and cell cultures and are mutagenic mutagenic but not necessary carcinogenic ◦ b/c DNA repair mechanics and metabolism ◦ chem potential to be carcinogenic b/c mutagenic ACTUAL TEST: mutant salmonella that can't synthesize HIS and can't grow in HIS deficient medium, mutagens result in the reversion of mutation back to HIS independence and can grow in it. The number of  revertant colonies is the measure of mutagenic potency LECTURE 3 1) Oncogenes: any gene encodes transforming cells in culture or induce cancer in animals Proto-Oncogenes: progenitors of oncogenes Tumour suppressor genes(TSG): inhibiting transformation of cells in culture, or cancer growth in animals 2) Gene deletion/mutation, Gene amplification, Chromosomal translocation 3) Gene mutation/ deletion example: single point mutation of p21 ras gene on 12th codon Gene amplification: separate chromosomes called double minutes or homogeneously staining regions, which causes over expression at mRNA and protein levels leads to malignant phenotype Chromosome translocation: Philadelphia chromosome: c-abl gene from chromo 9 to chromo 22 ◦ RET proto-oncogene; TRY kinase domain is truncated and fused w/ other genes -> novel fusion proteins constitutive kinase activity 4) myc gene is involved in growth and differentiation and is tightly regulated in normal cells, so once it is mutated within the cells (cancerous) it allows high levels of mRNA and proteins 5) evidence of multiple oncogenes activation:  In HL-60 leukaemia amplified myc gene and activated N-ras gene Burkitt's Lymphoma contain rearranged myc gene activated n-ras gene colon cancer: at least 6 different genetic alterations further evidence w/in gene transfer experiments: ◦ needed both the rearranged myc gene and activated rase gene to show immortalization and morphological transformation of fibroblasts which when injected into animals caused cancer 6) TSG first discovered in: In somatic cell hybrid studies: when malignant and normal cells result in a hybrid which then the malignant lost its characteristics -> normal cell has Tumour suppressor proteins Consistent deletion of specific regions of particular chromosomes in certain hereditary cancers: ◦ suggest loss of chromosomal DNA for development in cancer; repeated loss of certain chromosomes in specific types of cancer i.e. Wilms' tumour lost of 11p13 7) pRb: regulates from G1 to S phase, by regulating the E2F transcription factors, when mutated this will prevent it from binding to E2F and the cell will go into S phase without any restriction control p53: by the mutation of p53 it will effect : p21: will inhibit cdks and slows G1 -> S but when p53 damage the is failure of DNA damage and cells have the potential to become malignant, because there is no longer a slow down as p21 wont be active from p53 GADD45: inhibitor of cell replication/growth pro-apoptotic proteins: with p53 increase the activation of transcription bax genes which leads to  apoptosis but when damaged there is a decrease in apoptosis the ability of radiation/chemo to induce cell death in cancer cells is decreased, that meaning the cancer becomes insensitive to radiation NF1: Normally converts RAS into inactive form; shut downs RAS signalling. With the loss of NF1 function continuously activated RAS which leads to excessive down stream signalling pathways further to morphological changes Viruses and Cancer 1) a) Esptein-Barr virus (EBV) b) Human papilloma virus (HPV) c) Human T-lymphotropic virus (HTLV 1 and 2) d) hepatitis B and C (HBV and HCV) e) Kaposi's sarcoma-associated herpesvirus (KSHV) f) Merkel cell polyomavirus (MCV) 2) - uses synthetic machinery of infected host cells - 3) HPV: E5 membrane protein interacts with growth factor receptors and activates of MAPK pathway E7 binds to pRb and degrades pRB E6 targets p53 and ubiquited p53 and degrades it HBV: HBV has 3 glycoproteins L, M, and S L interacts with HBV receptor hepatocyte plasma membrane once recircularized direct RNA synthesis for viral protein synthesis(C,S,P,X) and of RNA pre-genome, which is a template for the viral DNA replication viral polymerase is reverse transcriptase that makes - strand then an endoribonuclease cleaves RNA pre-genome into small RNA fragments for + strand (unsure about this part babes)  disruption of cyclin A and ⍺-RAR genes HBx up-regulates stimulate transcription of genes in growth HBx sequesters p53 4) RNA viruses can cause cancer by carrying oncogenes, insertional mutagenesis, or activating genes important for growth. The virus enters the cell and sheds its envelope then uses reverse transcriptase to make DNA/RNA then a DNA/DNA double helix. This DNA copy is integrated into the host chromosome and transcription occurs. This forms many copies of RNA which are translated for the assembly of many new virus particles, each containing reverse transcriptase for continued amplification. Example of RNA virus in action would be HCV: RNA not transcribed into DNA does not integrate into host chromosome core proteins have oncogenic potential that activates ◦ JNK signal transduction pathway ◦ Up-regulate NF-kb activity ◦ inactivate p53  1) 2) 3) a) the dextran-coated charcoal method: disadvantage is large amts of material required B) immuno-cytochemical method: advantaged C) immuno-histochemistry: advantaged includes direct correlation of histology with receptor status D) Quantitative PCR : advantaged for very small amount of material 4) 5) clinal expansion: from a premalignant cell or stem cell Or adaptive/epigenetic response: normal cell regression or premalignat cell  1) Cyclins and cyclin-dependent kinases CDKS: require the binding of cyclins to activate with a phosphorylation by CAK, WEE1 another phosphorylation inactive, cdc25 to activate remove 2nd phosphate, Cyclins: oscillate, G1-> S phase regulation by D,E,A Cycline D cdks 4/6 G0-> cell cycle, cyclin E with cdk 2 cyclin A/cdk2 after cyclin E/cdk 2 activation for initation through S phase cdk1 inactive till G2/M phase, activation by cdc25 and CAK CDK Inhibitory proteins: KIP family (kinase inhibitory protein): p21, p27, p57, binds to and inhibit cdks 1-6 INK4 (inhibitory of cdk4) specifically inhibits cyclin D-dependent kinases 4 and 6, destabilize the association btw cyclin D and cdk 4/6 3) myc: transcription factor, with max role in cell proliferation, expression enter cell cycle w/o growth factors 4) Cyclin E1: gene amplification, and translocation Clcyin D1 gene amplification and translocation common in many cancers ex lung Cyclin A: over expressed in liver and breaks cancers cdc25A/B over expression good indicators of poor prognosis with breast cancer  1) 2) Regulators of apoptosis: stimulators (agonists) Apaf-1: form oligomers, to procaspase 9 to its processing activation, with cyt c and dATP Inhibitors (antagonists) Bcl-2: protect cells from apoptosis, inhibit cytochrome c release proteases Caspases: cys proteases in inactive form, two classification initiators 8,9,10 and executioners 3,6,7 4) a) alteration in membrane structure: fluorescence or flow cytometer b) release of cyt c into cytosol: Immunohistochemistry c) processing of caspases and their substrates: western blot d) fragmentation of DNA: dna laddering, TUNEL assay 5) Loss of expression of caspase 8: associated with amplification N-myc oncogene Loss of expression of Apaf-1: melanomas Over expression of Bcl-2: varies in cancerm 6)a) Need a caspase inhibitor, inhibit Cas 8 so RIP1 can down the Necrosis pathway, if cas 8 not inhbit goes down the apoptosis pathway instead b)Drugs that induce Necrosis always an inflammatory cell death, cell death in tumour may also capable of initiating anti-tumour immunity