Exam 3
8 oxo guanosine
8-oxo-guanosine is a potentially carcinogenic lesion - if you prevent its repair in mice, their risk of cancer will increase ◦ make a mutant mouse able to repair Beta-oxo-dG = increase in risk of cancer ▪ good evidence ◦ high levels of Beta-oxo-dG expression patients vs those without it ▪ reduce survival ▪ Beta-oxo-dG promote progression ▪ Beta-oxo-dG 1- endogenous exposure Loss of expression of enzymes involved in repairing 8-oxodeoxyguanosine reduces breast cancer survival - evidence that 8-oxo-dG promoted progression
NSAIDS
Acetaminophen/Tylenol is not an NSAID - it is analgesic but not anti-inflammatory Steroidal anti-inflammatory drugs do not prevent cancer
stem cell asymmetry
A characteristic property of a stem cell is the ability to divide asymmetrically Asymmetric division allows stem cells to protect a conserved DNA strand
DNA polymerase δ
A mutation of DNA pol δ that eliminates its proofreading activity and increases cancer development in mice ◦ show the polymerase mutation of no 5'-> 3' activity decreases survival ◦ Poly = important safe guard
EpCAMhigh/CD44+
A single-cell EpCAMhigh/CD44+ cell can initiate a tumor and generate cells with the KRT20 differentiation marker
breast cancer stem cells
A small foci of potential breast cancer stem cells stained with an antibody to a cell surface marker the detects cancer stem cells The cancer stem cells may be the source of most cells in the lesion Non-stem cells of the tumor may be less proliferative and less dangerous
alfatoxin B
Aflatoxin B is formed by molds that grow on improperly stored grains It is activated in the liver, where detoxifying pathways compete with DNA reactivity DNA reactivity consistent with the AGG to AGT mutations found in the p53 gene in individuals exposed to aflatoxin B Cytochrome P-450s perform the phase 1 metabolism sticks oxygen to increase solubility. Phase 2 detoxification enzymes follow
base excision repair
Base excision repair (BER) - used to repair small DNA adducts like 8-oxo-dG Requires a specific glycosylase that recognizes the damaged base and clips it out (leaving the phosphate back bone intacts) DNA is then cleaved adjacent to the AP site (apurinic/apyrimidinic site) and the strand is replaced
benzoapyrene
Benzo[a]pyrene from tobacco smoke is metabolized to BPDE in the lung BPDE binds to guanine, causes it to be read as a "T" during DNA replication This leads to a G to T transversion ◦ tobacco ◦ benzo[a]pyrene (Procarcinogen) - bunch of CH on it ▪ in body ▪ not water soluble so difficult to get rid of through urine ◦ body increase its solubility by sticking O on it ▪ enzyme CYP1A1 ◦ End up with bento[a]pyrenediolpoxide ▪ now a DNA reactive ready molecule
Brca1 and Brca2
Brca1 and Brca2 tumor suppressor genes Mutations in Brca1 or 2 are associated with 50% of familial forms of breast cancer, and ~80% of familial forms of ovarian cancer Brca1 shows up at stalled replication forks in cells treated with hydroxyurea (hydroxyurea inhibits deoxyribose nucleotide synthesis) Brca1 co-localizes with γ-H2AX(loaded onto chromatin at sites of DNA damage), a protein involved in DNA repair
Cd44 and CD24
Breast cancer cells are sorted for CD44 and CD 24 expression (membrane protein "markers") Cells with high CD44 and low CD24 expression are highly tumorigenic, whereas the majority of breast cancer cells do not form tumors The CD44 high/CD24 low cells may be enriched for cancer stem cells CD44 and CD 24 are cell surface proteins that help purify a cell population that was representative of a cancer stem cell -cells with high levels of 24 but low 44 might be indicative of cancer stem cells -44 and 24 were stained with 2 different fluorescent markers
Xeroderma pigmentosa
Cancer control after DNA damage: lessons learned from the cancer syndrome Xeroderma pigmentosa - inherited defects in the NER pathway Dry skin with many freckles 1000-fold increased risk of skin cancer Cancer development in early childhood (~8 years)
cancer initiation, promotion, and progression
Cancer initiation - genetic mutation(s) that disrupts cell growth control Cancer promotion - environmental agents that promote survival and expansion of mutated cells. (Agents that can override endogenous growth control and immune-mediated pathways.) Cancer progression - the accumulation of additional genetic and epigenetic changes towards malignancy
how do cells protect gDNA from contamination
Cells have developed efficient mechanisms to protect their genomic DNA from damage These mechanisms include: shielding stem cells from DNA damage repairing damaged DNA detoxifying mutagens Cancer cells frequently lose some of these safeguards, and can acquire a mutator phenotype
methods of protecting genomic DNA
Cells have developed efficient mechanisms to protect their genomic DNA from damage These mechanisms include: shielding stem cells from DNA damage repairing damaged DNA detoxifying mutagens Cancer cells frequently lose some of these safeguards, and can acquire a mutator phenotype
NIH 3T3 experiments
Certain sets of oncogenes can work together to transform a normal cell These experiments were first done with low-passage rat embryonic fibroblasts or baby hamster kidney cells (NOT NIH 3T3 CELLS) NIH 3T3 cell line-partially transformed from the start and immortal -single oncogene transformation Normal cells-multiple oncogenes needed The use of low-passage primary rodent cell lines revealed cooperating oncogenes It seems that one gene needs to encode a cytoplasmic proteins, and the other, a nuclear protein Oncogenes appear to collaborate in unique ways Note that ras alone will sometimes promote growth arrest or senescence, which is counteracted by the cooperating oncogene
combined MMTV regulated myc and ras oncogenes
Combined MMTV-regulated myc and ras oncogenes leads to rapid mammary tumor development
Dclk1
Dclk1 in small intestine In normal crypts the stem cell is LGR5 expressing stem cell Within the tumor the cancer stem cell is an LGR5 and Dclk1 positive cell
Dclk1 vs Lgr5 lineage tracing
Dclk1 marks tumor stem cells in ApcMin/+ mice Tumors also stain blue Lgr5 is the stem cell giving rise to cells in the normal tissue and in the tumor tissue Dclk1 cells most of the tissue is not stained consistent with Dclk1 not being expressed by stem cells, the tumors although turn completely blue thus every cells has arisen Dclk1 from the time of Tamoxifen exposure A new Dclk1+Lgr5+ cell appears at the base of adenomas Is this a new type of stem cell for the tumor?
diphtheria toxin and Dclk1
Diptheria toxin ablation shows Dclk1 cells important for maintaining tumors Dclk1 is restricted to the tumor stem cells can have entire tumor express the diphtheria toxin receptor to determine what would happen if knock out all Dclk1 stem cells -activated expression of diphtheria toxin receptor in the tumor cells and can eliminate the entire lesion with a diphtheria toxin A new Dclk1+Lgr5+ cell appears to be the "tumor stem cell"
errors during DNA replciation
Errors during DNA replication can be minimized: Many DNA polymerases also have proof-reading activity They have 5' -> 3' polymerase activity, and 3'-> 5' exonuclease activity to correct mistakes ◦ DNA poly - high fidelity ◦ first line of defense - hardwire poly with 5' -> 3' activity ▪ backs up if error is detected
estrogen
Estrogen is a tumor promoter that stimulates cell proliferation in the mammary tissue Fewer menstrual cycles lower the risk of breast cancer Removal of ovaries can reduce breast cancer risk
TNF secretion
Evidence has been obtained that TNF secretion by inflammatory cells and transcription factor NF-κB activation in transformed cells plays an important role in linking inflammation and carcinogenesis
smoking and ethanol
Example of a cancer initiator and promoter in a human cancer: a promoter that induces cell turnover Smoking and ethanol consumption combine to increase head and neck cancer risk 100-fold Cigarette smoke contains many carcinogens, so it can clearly act as an initiator Ethanol is not DNA reactive. It is however cytotoxic and will kill cells when present at high concentrations (i.e., when consumed in a distilled form).The increased rate of cell division to repair the tissue potentially promotes cancer development.
phase 2 enzymes affected by diet
Example: Induction of GSTs in human intervention studies by cruciferous vegetables glutathione s transferase increased after these vegetables are consumed Reactive compounds derived from cruciferous vegetables stimulate expression of the phase II enzymes isothyiocynate-high levels in cruciferous vegetables which increases glutathione transferase activity
exogenous DNA damaging agents
Exogenous DNA damaging agents: chemical and physical damage of DNA by environmental agents is responsible for most cancers (How do we know this?) X-radiation is ionizing radiation. X-rays can hit water and make ROS. A direct hit to DNA will cause strand breakage. UV radiation is a more common source of DNA damage, particularly to skin cells. Common DNA photoproducts include: cyclobutane dimers and 6-4 photoproducts between adjacent pyrimidines ◦ radiation - environmental exposure ◦ gamma radiation - DNA molecule destroyed the chain broken ▪ electron get kicked up to higher energy level = increase the radio activism of molecule cell has bypass poly that reads quickly so why common for these to get
flow cytometry and fluorescence activated cell sorting
Flow cytometry and Fluorescence Activated Cell Sorting (FACS) - Cells are stained with fluorescent antibodies (of different colors) - Cells are passed single file through an aperture where their fluorescence is measured In FACS, a voltage gradient can be used to sort the cells into different tubes
glutathione conjugation
Glutathione S-transferase is a Phase II detoxifying enzyme It links glutathione to the Xenobiotic/carcinogen, which facilitates its excretion in the urine or bile inside cell high Ms of glutathione and keeps sulfhydryls reduced (high reduced environment inside cells) glutathione s transferase finds xenobiotics and takes glutathione to make conjugate with it for detoxification and excretion
HBV induced inflammation
HBV-induced inflammation as a tumor promoter: HBV-infection - increases liver cancer risk ~7-fold Aflatoxin exposure (a DNA damaging carcinogen) - increases liver cancer risk ~3-fold Exposure to both increases liver cancer risk by ~60-fold
HNPCC mutations
HNPCC (hereditary nonpolyposis colon cancer) - mutations in MMR proteins Cancers show microsatellite expansions/contractions in tumor suppressor genes The classic example is the mutation in TGF-β receptor II in HNPCC colon cancers - a caretaker mutation leads to a gatekeeper mutation usually mutation in micro satellite because a dna slip during replication leading to a mutated receptor, epithelial cells in colon lose growth sensing receptors
hepatitis B and C
Hepatitis B and hepatitis C viral infection of the liver increases the risk of hepatocellular carcinoma almost 100-fold Infection leads to a heavy immune and inflammatory cell infiltration
heterocyclic amines
Heterocyclic amines (HCAs) like PhIP are derived from broiled and charred meats HCAs are also metabolized to carcinogens by a cytochrome P450 PhIP and other HCAs may contribute to the elevated risk of western cancers, such as prostate cancer
p450 phase 1 enzymes
Cytochrome P450 enzymes are phase I enzymes Phase II enzymes conjugate metabolites to small molecules for secretion in the urine or bile chemical groups reduce activity of molecule and put tag on the molecule so body knows how to get rid of it
expansion of LGR5 progeny through adenoma
Retracing studies show an expansion of a LGR5 progeny through an adenoma Lgr5EGFP-Ires-CreERT2/Apcfl/fl with the confetti locus treated twice with tamoxifen Start with entirely red crypt and retrace a small portion of the cell has turned from red to blue and those cells take over the lesion which indicates a subpopulation of cells in the lesions which provide a source of cells for the rest of the tissue
satellite sequences
Satellite sequences are DNA repeats found in the genome that are 100 bp or larger Microsatellites are single, di- or tri-basepair repeats DNA polymerase can "slip" when replicating these repeats, creating insertions or deletions
transcriptome analysis of colon cancers
Single cells isolated from the tissue can be subjected to RNA-seq and other transcriptome studies Colon cancers were made into single cell suspension and isolated into single cells and transcriptome was analyzed Done with normal tissue which shows mature cells, amplifying cells, and stem cells Single cell analysis of adenoma and cancer show some degree of differentiation
immature and mature cell types in colon cancer
Single-cell PCR gene-expression analysis of cells from a colon cancer reveals cell populations mirroring those observed in normal tissue The cells in the cancer cell differentiate into different cell types that resemble the normal tissue in both mature and immature cell types
MMR proteins
Sometimes the proofreading activity of DNA polymerase fails However, the cells can express DNA mismatch repair (MMR) proteins to fix mistakes MMR proteins recognize and replace misincorporated bases They also recognize and repair base deletions and insertions that occur on microsatellite sequences
lineage tracing and CSC concept
Lineage tracing and other experimental approaches using mouse models support CSC concept Lineage tracing -can use Cre recombinase and express that in cell -when Cre is expressed it takes out Lox stop and get expression of the reporter which is always on even if Cre is turned off Equipotent-all cells should mix in tumor in equal numbers, a stem cell in lesion would make only labeled stem cells grow out and populate tissue
lineage tracing of Lgr5 stem cells
Lineage tracing of Lgr5 stem cells in adenomas indicates that each adenomatous crypt has its own stem cell Lgr5EGFP-Ires-CreERT2/Apcfl/fl mice with the confetti locus. Low level tamoxifen administration traces LGR5-CSCs with four different colors Before tamoxifen treatment - no fluorescence After a short tamoxifen exposure, a fluorescent protein will be expressed A second tamoxifen treatment will switch the color Have a stop cassette and different fluorescent proteins -give Tamoxifen treatment which gives recombination event to turn cells either green, yellow, red, blue -give to Confetti mouse this burst of colors and trace where they go -individual cells in the lesion recombine to give one of the colors -individual crypts in the adenoma turn single colors because the individual crypts are driven by idividual stem cells that have been induced to recombine one of the proteins, consistent with stem cells driivng expansion of the lesions If all cells were equal to form an adenoma than the crypts would be single colored and derived from a single cell Second tamoxifen treatment switches the fluorescent reporter again to do retracing
lineage tracing and DCLK1
Lineage tracing shows that DCLK1 is expressed only in short-lived Tuft cells Lineage tracing Cre recombinase is fused to an estrogen receptor and in cells that are expressing this the cre recombinase will take out stop casette and express lac z When tamoxifen any cells expressing cre recombinase the cells will be blue because cell will be spliced out and cause lac expression Bcl1 expressing cre recomibinase will only turn cells from a BcR1 cell to stian blue
breast, inflammatory breast, GBM and colorectal markers
Markers used to enrich cancer stem cells / cancer initiating cells from solid tumors Breast cancers: CD44+ CD24low Inflammatory breast cancers: ALDH+ CD44+ CD24- GBM and colorectal: CD133+ Are these really cancer stem cells, or just cells that survive tumor dissociation and FACS sorting? Stem cell number estimates can differ significantly when different conditions are used to enumerate them. CSC stem cell counts will increase when more severely immune compromised animals are used. Are the isolated cells simply less immunogenic in the mouse? Would want to find single # of stem cells regardless of the variables but the numbers differ depending on the kind of mouse
Mdr null mice
Mdr null mice develop liver inflammation that ultimately leads to cancer Inflammation and cancer development can be suppressed by the NSAID ibuprofen Inflammatory cells secrete cytokines and other inflammatory mediators that promote the survival and proliferation of transformed cells NSAIDs decrease the odds of getting esophageal cancer by almost 50%
self-renewing stem cells
Most tissues are composed of rare, long-lived stem cells, that give rise to short-lived differentiated cells. Evidence has been obtained that mutations in the long-lived stem cells give rise to cancer. When differentiated cells incur a mutation, they will usually die off before additional mutations occur.
mutations can arise from
Mutations can arise from: Errors during DNA replication Spontaneous base changes (resulting from chemical instability) Endogenous and exogenous DNA damaging agents
mutations in Xeroderma pigmentosa
Mutations in one of 8 genes can lead to the Xeroderma Pigmentosa (XP) phenotype Cell fusion experiments were performed to identify 8 different complementation groups Affected individuals inherit two mutant/null alleles of an XP gene cell with same mutation and fused together remains sensitive to UV light cells with different defects and fused together they are resistant to UV light
Conclusions about multi-step tumorigenesis
Mutations incurred in the stem cell population of are more likely to give rise to a lesion. Tissues with high numbers of proliferative stem cells may be at elevated risk of cancer development. Increasing the number of stem cells in the tissue is likely to increase the risk of cancer development. Cancers are composed of cells that vary in tumor initiating potential and differentiation status. The stem cells of a tumor have properties distinct from normal stem cells. Selective targeting of cancer stem cells is likely to be a highly effective approach to cancer treatment.
transgenic mice
Oncogene cooperation demonstrated in vivo using transgenic mice The MMTV promoter (derived from the mouse mammary tumor virus) expresses the transgenic oncogenes specifically in mouse mammary tissue promoter
two general models of tumors
Stem cell is long-lived cell -gives rise to other cells in the tissue In treating cells want to focus on cancer stem cells that give rise to the lesion so the TA and differentiated cells don't form
Stem cells
Stem cells - serve as a safe haven for genomic DNA They are usually buried within the tissue, shielded from DNA damaging agents They replicate their DNA and divide infrequently, so there is a lower chance that they will acquire a mutation ◦ prevent steam cells form doing too much proliferation ◦ most tissues are set up in which stem cell divide frequently ◦ transit amplifying are busier ▪ more potent to pick up a mutation ▪ programmed not to last very long ▪ in tissue for short period of time, little longer of resin ◦ stem cell population ▪ hang on for aligner period than transit
structure of crypts
Structure of normal tissue: Intestinal cells proposed to be derived from LGR5-expressing stem cells (ISCs) at the crypt base Base of tissue protects the cells from the environment Lgr5 expression restricted to crypt base columnar cells These are thought to be the stem cells of the tissue.
HNPCC
Patients with HNPCC have mutated MMR proteins Their tumors will show contractions or expansions of microsatellites - called microsatellite instability (MSI) BAT25 is a microsatellite on chromosome 4 ◦ familial syndrome HPNCC (heredity non-polynercosis syndrome?) ◦ mutated and or methylated MMR protein ◦ micro satellite region is variable ▪ effect is due to loss of MMR ability ▪ to analyze look at the contraction of micro satellite areas
dna repair pathways
Preventing base pair changes Base demethylation Nucleotide excision repair (NER) Base excision repair (BER) Mismatch repair (MMR) Strand break repair/preventing chromosome fragmentation Non-homologous end joining (NHEJ) Homologous recombination repair (HR)
5 pathways to transform a human cell
Primary human cells are very difficult to transform More safeguards have probably evolved in humans because humans are larger (more cell targets) and live longer (longer exposure) than rodents 5 pathways must be targeted in cell culture experiments to transform a human cell - this is close to the theoretical estimates (Note: in these transfection experiments, only oncogenes are used)
TPA activation of protein kinase C
TPA goes in middle of pathway and activates protein kinase C -alone is enough to activate protein kinase C Causes cell proliferation and survival signals
PKC
TPA stimulates cell proliferation by activating protein kinase C PKC normally activated by diacylglycerol (DAG) produced transiently following physiological stimuli - TPA provides a chronic signal
mechanism of phase 2 gene activation
The Keap1 complex degrades the Nrf2 transcription factor in unexposed cells - Reactive molecules (from the diet) bind and inactivate Keap1, resulting in Nrf2 stabilization - Nrf2 binds the antioxidant response element (ARE) of target genes (with Maf) to activate transcription Nrf2-TF that binds to antioxidant response element (ARE) found in the promoter of phase 2 regulators of enzymes Keap1 keeps Nrf2 off through ubiquitin ligation leading to its degradation under resting conditions Keap1 has sulhydryl groups to react with xenobiotics and ubiquinates nrf2 for phase 2 genes Different compounds bind to different Cys residues on Keap1 to activate Nrf2
DNA damage can result from
The chemical reactivity of the DNA base The physiological generation of reactive molecules For a mutation to occur, the damaged base must lead to a base-pair change, either through base conversion or base mis-reading by the DNA polymerase
2 step mouse skin cancer model
The classic 2-step mouse skin cancer model Permutations of the mouse 2-step skin cancer model helped define the differences between an initiator and a promoter DMBA is a genotoxic initiator (which induces a stable genetic change) TPA is a non-genotoxic promoter
cancer stem cells
The concept is based partly on experiments using membrane protein markers to sort out cancer cells into different populations - some populations are more tumorigenic in nude mice than others Antibody binds to cancer stem cell marker and label the antibody with a fluorescent marker or secondary antibody -use flow cytometry -take solid tumor, proteases to make single cell suspension, add fluorescent antibodies to label the cells that are expressing the cell surface marker and use a fluorescence-activated cell sorter to sort the cells out into different pools based on the intensity of the expression
Are and estrogen receptor
The timing of Cre can also be controlled using CreER Cre recombinase fused to Estrogen Receptor only enters the nucleus when Tamoxifen is present Cancer mutations can be induced in specific cell types at different times (by injecting tamoxifen) Cell stays in cytoplasm until Tamoxifen is present for transport into the nucleus Can control what cells the Cre is expressed in through manipulation of promoters and the time that it is activated
TPA and inflammation
There are numerous examples of inflammation serving to promote carcinogenesis In fact, TPA functions in part by promoting inflammation Neutrophils invading skin tissue of PKC transgenic mice treated with TPA
Coxibs
Traditional NSAIDs inhibit both COX1 and COX2 Coxibs are COX2-specific inhibitors. Coxibs include Vioxx and Celebrex. Coxibs take advantage of the fact that COX2 is only expressed in inflamed or cancerous tissues PGE2 binds receptors on cancer cells to promote proliferation/survival PGE2 promotes angiogenesis The coxib celecoxib induces apoptosis and reduces proliferation in a mouse cancer model
stem cell mutations drive carcinogenesis
Transgenic expression of a ras oncogene in skin stem cells causes skin cancer, whereas expression in differentiated skin cells does not Apc mutation in intestinal stem cells leads to tumor development, whereas this mutation in villus cells generates a small, non-progressing lesion In chronic myelogenous leukemia, many cells of the hematopoetic lineage can contain the Bcr-Abl fusion ◦ APC mutation higher in tissue outside of stem cell compartment do not get tumor
transit amplifying cells
Transit-amplifying cells are the most at-risk cells for mutation, but their life-time is limited Intestinal cell: 5-7 days Skin cell: 20-30 days
tumor progression
Tumor progression - a clone of transformed cells breaches numerous growth barriers to become malignant Progression is the result of sequential mutations and epigenetic alterations For most cancers, this process takes many years
inflammation
Tumor promoters often function by directly stimulating cell proliferation (particularly mutated cells). Tumor promoting agents can also function by inducing inflammation (recruiting white blood cells to the tissue). This can promote cell proliferation, survival and angiogenesis. Chronic inflammation may also suppress the immune response in the tissue. Inflammation is also tumor-promoting Inflammation generally characterized by the infiltration of cells from the blood stream. Inflammatory cells damage tissue/increase cell turnover and stimulate angiogenesis, and ultimately promote tumor development
aspirin vs Cox2
Unfortunately, Cox2 inhibitors cut colon adenoma risk in half, but increase cardiovascular death almost 10-fold "Chemoprevention" of cancer is risky business
Are/Lox system
Using the Cre/Lox system to induce mutations in selected cell types - LoxP sites are engineered into a target gene (e.g., a tumor suppressor gene). - Cre is expressed in specific cell types to induce a mutation in that cell type only. A great experimental system to study cancer initiation.
XP-V
XP-V encodes a specialized DNA polymerase that incorporates AA across from TT cyclobutane dimers Enzyme is needed to deal with the high level of photoproduct generated from UV exposure Mutation of this enzyme responsible for some Xeroderma pigmentosa cases XPA through G are involved in a large multiprotein complex used in nucleotide excision repair (NER) XPV encodes DNA polymerase η, which specializes in incorporating AA across from TT photoproducts
glucosinolates and isothiocyanates
rats exposed ot dmba carcinogen for mice on controlled diet or diet with high levels of phase 2 inducers -on controlled diet high levels of tumors, tumor inhibited with compounds
Cyto p450
usually the first line of detoxification Substrates of Human Cytochrome P450 Isoenzymes - many Cyp P450 enzymes to handle all the potential substrates Cytochrome P450 enzymes are present on the smooth ER -many parts of it are hydrophobic and stay near lipids thus thats where p450 mostly is
top of crypt differentiation
Colon cancers that express "top-of-crypt" differentiation markers show better survival Subdivided cancers into cells either expressing high levels of mature cell markers or low levels of mature cell markers Stem cell can differentiate into mature cells If lesion can't differentiate into mature cells than that is more dangerous lesion
aspirin
Additional evidence for the link between inflammation and cancer comes from epidemiological studies: Nonsteroidal anti-inflammatory drugs like aspirin decrease cancer risk at multiple tissues One aspirin a day Lung cancer risk: 0.68 Breast cancer risk: 0.7 Colorectal cancer: 0.35 (young men)
alkylating agents
DNA alkylating agents are electrophiles that modify bases and cause them to be misread by the DNA polymerase A number of carcinogens are DNA alkylating agents - one example is mustard gas Interestingly, some DNA alkylating agents are effective for chemotherapy ◦ a lot environmental compounds that can stick methyl group on DNA Mistakes during replication and spontaneous base changes (due to chemical instability of bases) can contribute to genomic instability Environmental agents make an even larger contribution to genomic changes that lead to cancer
DNA alkylating agents
DNA alkylating agents are electrophiles that modify bases and cause them to be misread by the DNA polymerase O6-methyl-G is read as T as end product
demaniation
DNA bases can also spontaneously deaminate and change the DNA sequence
mismatch vs nuceltodie excision vs base excision
DNA repair pathways: MMR, NER and BER Mismatch repair - base misincorporation Nucleotide excision repair - bulky adducts, helix distortion Base excision repair - small lesions -strands aren't cut but specific base is cut out and repaired
chemoprevention
Dr. Michael Sporn coined the term "cancer chemoprevention" in 1976 based on cancer prevention studies with vitamin A and vitamin A derivatives
COX-1 vs COX-2
Immunohistochemical analysis of COX-2 expression and localization in normal mouse colon and tumor tissue. Immunohistochemical analysis of COX-2 expression and localization in normal mouse colon, ACF and tumor tissue. (A) Normal colon section from a vehicle-treated control mouse. Note the low level of staining for COX-2 in columnar epithelial cells and within the lamina propria. (B) Large dysplastic ACF induced by AOM. Note the marked enhancement in staining for COX-2 within the ACF relative to adjacent normal-appearing colonic crypts. (C) Polypoid adenocarcinoma induced by AOM. Note the increased staining for COX-2 within the tumor crypts. (D) Negative control tumor section immunostained with 10% goat serum in place of the primary COX-2 antibody showing no positive staining. Cox 1 is found in normal tissue as a maintenance cyclooxygenase COX-2 is inducible and found in cancer tissue or in inflamed tissue
interaction between initiator and promoter
Interaction between initiator and promoter Promoters promote the proliferation and survival of mutated/initiated cells Promoters often are general stimulators of cell proliferation If oncogene activated and cell goes into senescence but if have strong growth promoting signal can keep initiated cells alive and proliferating to undergo mutations
O-6-methylguanine-DNA methyltransferase
MGMT is an "enzyme" dedicated to removing methyl groups from DNA -not reused like normal enzyme
MutS and MutL
MMR proteins like MutS and MutL can bind these insertions and deletions They then scan the DNA to find a nick - the nick indicates the newly synthesized strand - so the new / incorrect strand can be repaired Repair involves strand removal and replacement ◦ MutS can see where helix is no longer base pairing ◦ MutL look for closet strand break and remove the stand all the way to the lesion and get a repair synthesis
pro-carcinogens
Most carcinogens enter the body as non-reactive molecules known as pro-carcinogens To eliminate these agents, cells first make them water soluble by adding electronegative oxygen to them A family of enzymes known as the cytochrome P450s perform this catalysis However, addition of oxygen will make some molecules highly reactive carcinogens that will attach to DNA
phase 2 in liver
Most cytochrome P450 and phase 2 enzyme metabolism occurs in the liver Blood from the small intestine flows through the liver Some of the pro-carcinogens may be missed by the liver, or the metabolized compounds may find their way to a distal organ
NF-kB activation
NF-κB activation involves a phosphorylation-dependent degradation of the IκB inhibitor Signals include TNF, interleukins and other inflammatory cytokines Also oxidative stress, UV radiation and other cell stressors Rel A-p50 = NFkB IKB (inhibitor kappa B) in non-stimulated cells Complex is found in the cytoplasm of resting cells -inflammatory cytokines I kappa B kinase gets phosphorylated and U10 ligase chops NFkB so it can now go into the nucleus
NSAID mechanism
NSAIDs suppress inflammation and cancer by inhibiting cyclooxygenase (COX) activity and prostaglandin production Prostaglandins induce inflammation and promote proliferation and survival of transformed cells inflammatory signal which activates phospholipase 2 which releases arachidonic acid -activates prostaglandin pathway shown as PG -PGE2 important prostaglandin which stimulates cell proliferation, survival and angiogenesis -keeps level of tumor promoter low
TAP tumor promoter
Non-mutagenic factors that promote the formation of cancer are called "tumor promoters" TPA (also known as PMA) is one of the first identified tumor promoter It was indentified in a mouse skin cancer model Tumor promotion: the increase of cancer development in the absence of DNA damage Tumor progression: the accumulation of genetic mutations that increase cancer development and growth Lesions- series of mutations Promotion-environmental factors Not genotoxic Favor growth and survival of mutated cells TPA not genotoxic but can turn a small lesion into a larger tumor or malignant cancer
CD133
Not all cells from colon cancers are able to form a tumor: Rare CD133 cells are more tumorigenic CD133 is a cancer stem cell marker for colon cancers -most of cells have low levels of 133 but populations with high levels of 133 can be sorted by fluorescent activated cell sorting, inject into animals can get many tumors forming quickly in the mouse -CD133 negative cells will never form a cancer
stem cell buried
Note the buried location of the stem cells They are also protected in the tissue by a mucus layer secreted by goblet cells in the tissue Animals that are unable to secrete mucus (MUC2 -/- mice) spontaneously develop tumors ◦ stem cell are buried and protected by other cells in the tissue ◦ evidence that protective covering is important ▪ can make knock-out mouse that doesn't make so much protection that mouse will spontaneously generate multiple tumors
nucleotide excision repair
Nucleotide excision repair (NER) occurs when the DNA helix is distorted, usually by a bulky or UV photoproducts (such as TT dimers) strand with bulky adduct is cut on both sides and removed by excision which is filled in by repair polymerase system
oxidation of guanosine
Oxidation of guanosine can lead to the formation of 8-oxo-guanosine If un-repaired, 8-oxo-guanosine can be read as a "T" by DNA polymerase, leading to a G to A transition (G to T or C would be a transversion) ◦ G base more sensitive bases ▪ will get oxide make Beta-oxo-dG
depurination
Purines bases are prone to depurination leaving an abasic site that will require repair This reaction is promoted at lower pH Pyrimidines are more stable ▪ G common to common off making it abasic
ROS
◦ ROS damage DNA ◦ series of reduction of O2 to get H2O ◦ hydroxyl radical is most reactive
lymphocytes and myeloid cells
◦ stem cell present in bone marrow ◦ either lymphoid pathway or myeloid pathway ▪ indication the mutation happen in stem cell population ◦ recall Bcr-Abl - fusion oncogene in M-cyl
