BIOL516 EXAM 1

lectures 01-08

Why is cancer considered to be multiple diseases?

it affects different tissues, organ systems, symptoms. Mutations and microenvironment also play a role.

what is Incidence?

the rate of diagnosis of the disease in the human population per year.

includes diagnoses and deaths, but does not DOUBLE count them (ie. deaths are from undiagnosed people)

Incidence| the older the population, _________

the higher the incidence

cancer results from ________________

an accumulation of biological insults over a lifetime

What is Mortality?

the rate of death from cancer in the human population per year

What other factors can contribute to mortality?

race, health insurance, access to healthcare, biology (eg. triple negative common in black women), etc

What is Survival?

number of patients with a potentially fatal cancer that are alive 5 years after diagnosis

Observed survival rate

% of group of patients alive after 5 years

expected survival rate

% of a control group alive after 5 years

relative survival rate

obs/exp

what are the 3 main ways to attenuate cancer?

Prevention, Screening, Therapeutics

what is a basket trial

target a specific genetic mutation regardless of tumor type (many cancers, many organs, ONE genetic mutation)

what is an umbrella trial?

different treatments based on different mutations for the SAME cancer.

what is the benefit of a basket trial?

increases enrollment for rare genetic mutations

what are cancer hallmarks?

distinctive and complementary capabilities that enable tumor growth and metastasis.

what are common sites of loss of function and gain of function mutations, respectively?

tumor suppressors, oncogenes

what is neoplasia?

formation of new abnormal tissue

Neoplasm

heritably altered, relatively autonomous growth of tissue

what is anaplasia?

losing normal characteristics

5 Key Characteristics of Cancer: 1

high nuclear to cytoplasm ratio

5 Key Characteristics of Cancer: 2

variable size and shape

5 Key Characteristics of Cancer: 3

high mitotic %

5 Key Characteristics of Cancer: 4

loss of specialized function

5 Key Characteristics of Cancer: 5

altered DNA content; abnormal chromosomes/genomic instability

what is aneuploidy?

abnormal number of chromosomes

Characeristics of cancer tissue

disorganized, histologically different from neighboring tissues

presence of necrotic tissue (massive cell death from hypoxia/anoxia)

poorly defined tissue boundary, invasive cells

loss of cell polarity

what is the difference between benign and malignant tumors?

cancer has altered properties as cells AND tissues

benign tumors are encapsulated, slow growing, localized, resembles normal tissue

malignant tumors are not encapsulated (invade), disorganized, rapid growth, dissimilar to normal tissue

classifications of neoplasms; staging

clinical and pathologic stages

what is the clinical stage of neoplasms?

based on info obtained before surgery (ie. imaging, symptoms)

what is the pathologic stage of neoplasms?

based on info obtained after surgery and microscopic evaluation by a pathologist

what is the TNM system?

parameters to determine tumor stage; Tumor, regional lymph Nodes, distant Metastasis

what is the T stage of TNM?

tumor

T0 = no tumor

T1-T4 = size of tumor

what is the N in TNM?

lymph nodes;

N0 = no cancer in nodes

N1-N3 = number and location of lymph nodes that contain cancer

what is the M in TNM?

metastasis

M0 = no mets

M1 = metastasis

Physical carcinogens

ionizing radiation

UV radiation

particulates (asbestos!)

explain how asbestos causes cancer.

asbestos causes inflammation which can cause secondary mutations

biological carcinogens

bacteria

parasites

viruses

how do bacteria and parasites contribute to cancer?

inflammation

how do viruses contribute to cancer?

genetics

chemical carcinogens

N-hydroxy-AAF

describe how AAF contributes to carcinogenicity

AAF is a procarcinogen that is metabolized by the body into an ultimate carcinogen, N-hydroxy-AAF

what is a DNA adduct?

a new molecular entity formed from the reaction of two formerly separate entities

types of adducts

ethylation, methylation, alkylation, OR the addition of bulky groups to DNA

VERY damaging

what is a common target for DNA adduct formation?

guanine

DNA adduct formation is influenced by _____

1. how efficiently a chemical is metabolically activated

2. the reactivity of the ultimate carcinogen produced

3. the persistence of the adduct in DNA

what mutations are commonly formed from adducts and how?

base changes or frameshift; adducts are misread by polymerase

bulky adducts can result in ______

DNA breaks, translocations, chromosome duplications

cell types with high levels of _____ are less likely to suffer permanent damage from adducts

dna repair enzymes

describe the discovery from the AMES test

mutagens are carcinogens; target of activity was DNA

Describe the Ames test

combine test compound with liver enzymes to convert the procarcinogen into the ultimate carcinogen, plate on a medium in which WT cells would not survive, colonies that grew contain mutation to allow growth

what DNA repair mechanism corrects DNA replication errors?

Mismatch repair (MMR)

what DNA repair mechanism replaces bulky adducts and pyrimidine dimers?

Nucleotide excision repair (NER)

what DNA repair mechanism replaces defective bases?

Base-excision repair; uses PARP for single strand breaks to replace the base

what DNA repair methods repair double strand breaks?

which is more accurate?

homologous recombination (HR) and nonhomologous end joining (NHEJ)

homologous recombination

when can HR occur in the cell cycle?

S/G2 only

when can NHEJ occur during the cell cycle?

everything except S/G2

what are initiating agents?

genotoxic, mutagens, can either directly react with dna or indirectly react (converted to reactant) before reacting

what are promoting agents?

non-genotoxic, stimulate initiated cells to grow

ex. asbestos, alcohol, inflammation, hormones…

steps in the development of a malignant neoplasm

1. organ development

2. initiation

3. progression

4. metastasis

describe step 1 of mal. neoplasm development (organ development)

imperfect dna replication, otherwise unaffected (phenotypically)

describe step 2 of mal. neoplasm development (initiation)

driver gene mutations are acquired that forms a precursor lesion

describe step 3 in the mal. neoplasm development (progression)

primary tumor formed from multiple mutations; after promotion

why do some mutated cell groups become malignant from the primary tumor and not others?

some tumors acquire different mutations that have a survival advantage.

describe step 4 of mal neoplasm development (metastasis)

tumor types (all kinds of mutations) disseminate and travel to other body parts

morphological changes that occur during apoptosis (6)

1. condensation of chromatin

2. cytoplasmic condensation (cell shrivels)

3. membrane blebbing: small ‘packets’ of cell contents, still connected to main cell

4. fragmentation of nuclear contents

5. encapsulation into apoptotic bodies: fragments are contained in little bubbles to prevent cell contents from dumping into environment. protects neighboring cells. cell content dumping otherwise could lead to a large immune response & inflammation

   1. apoptotic bodies are cleared away by phagocytosis

Intrinsic Apoptotic Pathway

what are some intrinsic lethal stimuli?

what is important to note about them?

DNA damage, oncogene activation, stress

they are the primary driver of mutation

Intrinsic Apoptotic Pathway

is BCL2 pro or anti-apoptotic?

anti-apoptotic

Intrinsic Apoptotic Pathway

what does BH3 bind to?

BCL2 and BAX, depends on a balance

Intrinsic Apoptotic Pathway

what is BAX?

pro-apoptotic protein that binds to BH3 to become active

Intrinsic Apoptotic Pathway

how will BH3 decide which protein to bind with?

higher concentration of the protein, BAX or BCL2, will have dibs on BH3

Intrinsic Apoptotic Pathway

what happens when BH3 binds to BCL2?

to BAX?

prevention of apoptosis

stimulation of apoptosis

Intrinsic Apoptotic Pathway

what is BAX’s role in the pro-apoptotic pathway?

what is the significance?

BAX localizes to the outer mitochondrial membrane and forms pores

pores result in Mitochondrial Outer Membrane Permeability (MOMP) which kicks off apoptosis

Intrinsic Apoptotic Pathway

what is significant about MOMP?

MOMP releases cytochrome C from the mitochondria, which is the ‘point of no return’ for the apoptotic pathway

Intrinsic Apoptotic Pathway

what is needed to initiate MOMP? hint: what family of proteins & what about them

a balance between BCL2 family proteins is critical for the initiation of MOMP

Intrinsic Apoptotic Pathway

describe the anatomy of an apoptosome

receptor for cytochrome C, APAF1 protease, Pro-Caspase9

Intrinsic Apoptotic Pathway

describe the role of the apoptosome in the pathway

when the receptor of the apoptosome binds cytochrome C, APAF1 (a protease) cleaves pro-caspase9, making an active caspase 9.

Intrinsic Apoptotic Pathway

what does active caspace9 do?

active caspase9 cleaves and activates caspases3/6/7 (the executioners) to execute apoptosis

Intrinsic Apoptotic Pathway

why are there so many steps in the apoptotic pathway?

less chance of whole pathway failing if one protein is mutated

allows cells to try and recover

Intrinsic Apoptotic Pathway

Is BCL2 overexpression enough to drive tumor formation? why or why not?

it is not enough. most cells are not actively proliferating. Overexpression of BCL2, which can inhibit the apoptosis pathway should there be damage, would not contribute because most cells barely use the pathway. however, if BCL2 overexpression is present in conjunction with other factors, it will accelerate tumor progression.

Intrinsic Apoptotic Pathway

if BCL2 is overexpressing, what treatment can be used and what is the method of action?

venetoclax - venetoclax binds to BCL2 so it cannot sequester BH3 from BAX, which allows BAX to initiate the apoptotic pathway.

Methods to Detect Cell Death

DNA laddering

apoptosis will show banding on a gel in succession because the DNA is cleaved between histones in the programmed apoptosis pathway

necrosis shows a long smear on a gel - nonspecific cleavage of DNA

Methods to Detect Cell Death

caspases

active Cas9 cleaves pro-caspase3 into the active form, active caspase3 cleaves PARP, which repairs single strand breaks, to prevent repair to DNA and allow apoptosis to proceed

Methods to Detect Cell Death

TUNEL assay

idek

lecture 04 slide 21

Methods to Detect Cell Death

fluorescent dye

dye cannot cross membranes in viable cells. non-viable cells will have perforations in the membrane to allow dye in. cells that fluoresce = dead

Methods to Detect Cell Death

flow cytometry

Annexin V - binds to phosphatidyl serine (PS)

PI - not cell permeable

Flippase - transmembrane protein that prevents PS from crossing the membrane

Methods to Detect Cell Death

describe Live, Early apoptotic, and Late apoptotic phases WRT flow cytometry

Live: PS is only found on the inner membrane side. No Annexin V. No PI.

Early Apoptosis: active cas3 cleaves Flippase; PS crosses to outer side of membrane. Annexin V binds to PS on membrane. No PI.

Late Apoptosis: PI is able to enter the cell. PI glows green, dead cells will glow green.

what is G0?

cell cycle arrest - cell are not actively dividing

what is G1?

growth - cellular contents (not chromosomes) are duplicated

what is S phase?

DNA synthesis - chromosomes are duplicated

what is G2 phase?

growth and prep for mitosis - checks for chromosome duplication error & makes repairs

where are the 3 main checkpoints in the cell cycle?

G1/S checkpoint

G2/M checkpoint

M/G1 checkpoint

what does G1/S checkpoint look for in cells?

+ sufficient number of organelles, growth factors activated

- damaged genome, ATP deficiency

what does G2/M checkpoint look for in cells?

+ genome is completely replicated, large cell volume

- dna damage

what does M/G1 checkpoint look for in cells?

+ daughter cells have equally distributed chromosomes

- chromatids not properly assembled on mitotic spindle

what is the restriction point in the cell cycle? which phase?

the restriction point is the ‘point of no return.’ cells are committed to completing the cell cycle regardless of extracellular signals.

G1

cell cycle important facts:

1. it is sequential and unidirectional

2. controlled by chemical signals that diffuse freely between cytoplasm and nucleus

3. later stages are dominant over early stages

a cell in G1 fuses with a cell in S phase. What phase will the fused cell be in?

S phase

a G2 cell and S phase cell are fused. what is the result?

the G2 nucleus does not go into mitosis right away. Rather, S nucleus catches up to G2 phase first, then the fused cell enters mitosis.

Describe maturation promoting factor in oogenesis

a soluble factor in the cytoplasm drives the oocyte into meiosis

is MPF level constant in each phase?

no, it oscillates