cell bio exam 4

cytoskeleton

comprised of microtubules, microfilaments (f-actin), and intermediate filaments

• dynamic

• diseases

Stretch experiment

1. stretch each one

   1. actin

      1. not very stretchy, but can withhold a stronger force

   2. intermediate filaments

      1. proportional-stretching increases with force

   3. microtubules

      1. stretching a lot, but force is less before it breaks

         1. sensitive to force

actin filaments

• microfilaments

• actin binds ATP

• form rigid gels, networks, and linear bundles

• related assembly from a large number of locations

• polarized

• tracks for myosins

• contractile machinery and network at the cell cortex

• ex: f-actin: filaments, g-actin: globular

• highly dynamic

• 7-9 nm

F-actin

1. up to 10% of total protein (muscle cells)

2. G-actin (building block): mw=42,000

   1. requires ATP and Mg

3. Humans

   1. alpha-actin

      1. used in contractile structures

   2. beta-actin

      1. used for moving

   3. gamma-actin

      1. stress fibers

4. f-actin can be decorated with myosin S1 head

   1. pointed end (- end) and barbed end (+ end)

F-actin polymerization in test tube experiment

1. purify g-actin and put in test tube

2. goes through nucleases stage

   1. g-actin units come together initially and are first scaffold

3. elongation phase

   1. g-actin gets longer

4. steady state

   1. g-actin doesn’t get any longer

   2. not static though because g-actin is constantly adding and subtracting at both ends

Treadmilling

• critical concentration (g-actin) at -/+ ends where there is no net addition

• critical concentration of + end=0.12, - end=0.60

• nucleus treadmills and moves down f-actin

cytochalasin D

• inhibits polymerization at the + end

• fungal product

Phalloidin

• death cap mushroom

• promotes polymerization

• commonly used as fluorescent statin

Thymosin beta4

• sequestering protein of g-actin

  • 40% of g-actin is soluble

• regulate g-actin polymerization

  • increase g-actin pool

Profilin

• ATP/ADP exchanger

• binds to g-protein with ADP and recharges it with ATP

• regulates g-actin polymerization

Cofilin

• severing protein

• dissociate from - end

Formin

• facilitator protein

  • facilitates nucleation (formation of a new structure)

    • nucleates the assembly of actin into filaments

    • binds to actin subunits at the + end

    • speeds up nuclease stage

• a dimer and nucleating protein

Capping protein

• prevents polymerization from occurring

  • ex: cap z caps the + end

  • ex: tropomodulin caps the - end

Optical tweezer microscope

• look at step size and force

• measure force of a myosin molecule and its association with actin

  • do this by measuring the displacement of an actin filament relative to myosin molecule

Listeria

• bacteria that can cause food poisoning and death

• due to bacteria listeria monocytogenes

• mother can pass listeria bacteria across placenta to unborn child

  • f-actin motile

    • actA facilitates addition of g-actin to + end

      • now listeria is motile

    • endocytotic vesicles too

    • opsonization

      • antibodies covering bacteria

      • requires antibodies and f-actin

• can cause eye problems

Arc 2/3

• moves the endocytotic vesicle

• driven by f-actin like listeria

• can be analyzed in cell free systems with rhodamine labeled transferrin and fluorescein-labeled actin

Diaphanous gene

• actin assembly defect in hair cells

• diaphanous gene defect

  • by age 30, completely deaf

  • inherited hearing problems

  • microvilli (have f-actin) structure is critical to hearing

    • this disease is a type of formin defect

      • can’t keep hairs straight and narrow

intermediate filaments

ex: desmosomes connect to intermediate filaments

• IF subunits don’t bind a nucleotide

• great tensile strength

  • “rope”

• assembled onto preexisting laments

• unpolarized

• no motors

• cell and tissue integrity

• 70 intermediate filament genes

  • keratin filaments: skin, epithelial cells

  • neural filaments: in axons

  • lamins: kargoskeleton

    • provides structure for nucleus

• 10nm

• least dynamic; static

• intermediate filament associated proteins (IFAP) keep IF connected

Epidermolysis bullosa simplex

• disease due to defective keratin filaments in body

  • birth defect

• skin sheds off

• under skin, there is a regenerative layer which these cells differentiate and move up to become epidermis

  • in EB, there is stress in the regenerative layer because keratin filaments can’t hold it together so skin falls right off

Mark Eisenberg

• developed Ortec company to treat EB

  • create substitute skin using cells from infants foreskin

VYJUVEK

• FDA approved drug to treat dystrophic EB

  • this type of EB has no type 7 collagen

• gene therapy that works to restore type 7 collagen

  • delivers new COL7A1 gene

    • restores ability for cells to make functional type 7 collagen protein and form anchoring fibrils

microtubules

• ab-tubulin dimer binds GTP

  • molecular weight=2×50,000

  • alpha-non exchangeable GTP

  • beta-exchangeable GTP

• largest out of the 3 categories

  • typical microtubule has 13 protofilaments in a singlet

• exhibits treatmilling

• rigid and not easily bent

• regulated assembly from a small number of locations

• polarized

• tracks for kinesins and dyneins

• organization and long-range transport of organelles

• 25 nm

• star-like array

• highly dynamic-assemble and disassemble quickly

  • GTP cap controls this

    • little cap=disassembles

  • assembly, catastrophe, disassemble, rescue

• ex: mitotic spindle

microtubules and organelle movement

• camouflage

• experiment:

  1. take fish pigment cell with melanosomes

     1. vesicles containing pigment

  2. decrease cAMP and add microtubule inhibitor

  3. no longer microtubules present and melanosomes are no dispersed but congregated in the center

     1. shows that microtubules are very important in the movement of pigments through cells

MAPs

• microtubules associated proteins

• bind tenaciously

• govern microtubule function

• experiment:

  1. axon in vitro

  2. add tau antisense to one axon and MAP2 antisense to another axon

  3. Tau

     1. shortens axon

  4. MAP2

     1. promotes axonal cargo spreading, slows down and speeds up cargo movement

     2. regulates cargo entry into axons

Tau

• involved in both Alzheimer’s and Parkinson’s

  • Tau is missing

• neurofibrillary tangles

  • tau congregates together and microtubule destabilizes

• microtubular stabilizing protein

  • promotes axonal transport and axonal growth

XMAP215 + CLASP

• stabilize microtubules

• support polymerization

Kinesin 13 + Stathmin

• cause depolymerization

Katanin

• severs long microtubules during neuronal development

  • embryos

Colchicine

• depolymerizes microtubules

• gout + cancer

taxotere/taxol

• promotes polymerization disrupting the mitotic spindle triggering dividing cells to commit apoptosis

• can also block Bcl-2

Molecular motors

• used by microtubules to move things from - to + or opposite direction

• kinesin moves in anterograde

  • nucleus to synapse

  • away from cell to axon

• dynein moves in retrograde

  • synapse to nucleus

  • away from axon to the cell

Kartageners syndrome

• ciliary dyskinesia

• has to do with molecular motos

• defect in dynein arms in cilia

  • lots of mucus in lungs

    • results in growth of bad things accumulating in the lungs, causing more infections

Dr. Father Alfred Cioffi

• Catholic priest with 2 PhDs

Uses of stem cells

1. increased understanding of how diseases develop

2. cure diseases

   1. stem cell therapy

   2. covid

      1. mesenchymal stem cells shown through studies that they can dampen immune response in a number of different disease cells

3. drug safety

   1. human hepatocytes to test for drugs but can now use human iPSCs

4. generate new stem cells to replace or aid diseased or damaged cells

   1. stem cell therapy

5. research how certain cells develop into cancer

   1. cancer stem cells

6. regenerative medicine applications

7. fix genetic diseases

   1. CRISPR-CAS9

8. tissue engineering

   1. organ on a chip

9. clean meat industry

   1. differentiate stem cell into muscle cell and make hamburger

Wolly mammoth meatball

• Vow company

• tried to generate meat from stem cells

  • 1 gene from wolly mammoth was inserted into sheep cells

Stem cells in space

• testing stem cells because they can behave differently with no gravity

  • how to generate capillaries

• study how human heart tissue functions in space with heart organoids

Immunomodulatory entity

• mesenchymal stem cells

  • can decrease immune and inflammatory responses in covid patients

Stem cell

• cell that can renew or differentiate

  • have many more replication cycles than typical somatic cell

  • controlled by stem cell niche

• number of doublings influences by source and type

  • hESC and iPSCs are immortal

    • adult sources

    • 100-200 doublings

• maintain stem cell population

  • make one differentiated cell and regenerate stem cell

Differentiation

• cells become more specialized

• fibroblast: produce collagen

• hepatocyte: detoxification

• can be partial or full

  • need molecular metrics to compare, for example, one iPSC generated hepatocyte to another iPSC generated hepatocyte

• restricted lineage

  • often called “progenitor”

  • limited to only one or two types of cells while others are totipotent

Transdifferentiation

• ability of differentiated cells to become another without going through an embryonic step

  • unlike iPSCs

  • via transcription factors, microRNAs

    

Dedifferentiation and redifferentiation

• ability of a cell to become more embryonic-like and differentiate into another cell type in vivo

• eastern red spotted newt

  • can regenerate lost eye and limb

• can do this in a lab using reversine

Stem cell niche

• stem cell microenvironment

• critical to controlling cell division vs differentiation

• complex includes neighboring cells, ECM, local growth factors, and physical environment

Potency

1. totipotent

   1. all cell types

   2. highest level of “stemness”

2. pluripotent

   1. many cell types

   2. restricted level of “stemness”

3. unipotent

   1. one cell type

   2. lowest level of “stemness”

Blastocycst

• late pre-implantation stage embryo

• hESCs originate from inner cell mass

Fusogenic

• love to fuse with each other

• problem with stem cells because the end up with tetraploid cell and can end up being a cancer cell because it drops chromosomes/genes

• when stem cells are injected into patients mechanical stress can cause fusion

Chimera test

• designed to prove pluripotency

• legal with mice but not humans

  • can never prove that any human stem cell derived or isolated in the lab is truly totipotent

1. label test stem cell with GFP

2. implant GFP labeled stem cell in blastocyst and then implant chimeric embryo into surrogate mother

   1. can do this because stem cell mixes in group and is differentiated to do what it needs to do

3. track GFP labeled stem cell in all tissues and organs of newborn

4. may get progeny that are all green meaning cells differentiated into a variety of cell types

Bioethics

• the norms of conduct

• relative term and country dependent

Four categories of stem cells

1. adult stem cell

   1. most popular=adipose-derived mesenchymal stem cells

2. fetal stem cells

   1. amniotic, umbilical cord, placental

3. embryonic stem cells

   1. hESCs and hPSCs

   2. hESCs in clinical trials-blastocysts

4. induced pluripotent stem cells

   1. not in clinical trials in the US

Biodistribution and homing

• to understand how/if stem cells can find their way in stem cell therapy patient

• ability of stem cells to find “home” in targeted tissue

• damaged or compromised tissue released factors that causes endogenous MSCs to home to damaged site

  • transplanted female hearts in male patients upon autopsy have male cardiomyocites

    • demonstration of endogenous stem cell homing and repair

    • transplant heart seen as damaged cardiac tissue and can recruit stem cells to repair it

Shinya Yamanaka and James Thompson

• 2012 nobel prize for iPSCs

• start with adult cells and reprogramming factors to generate/induce adult cells to become pluripotent

  • embryonic so not transdifferentiation, but iPSCs

• iPSCs can make any tissue cell type we need

• doing this procedure means we can make patient specific for testing new drugs or fixing them with crispr-cas9

STAP

• stimulus triggered acquisition of pluripoteny

• has now been retracted- fradulent

• idea is by giving cells a short acid shock, you can get them to generate into STAP to be differentiated

  • did chimera test and whole thing turned green

    • claimed that STAP stem cells could be totipotent because they appear everywhere

Therapeutic cloning

• production of embryonic stem cells for the use in replacing/repairing damage

• somatic cell nuclear transfer

  • start with egg, take out egg nucleus, put into somatic nucleus and generate embryo into blastocyst

    • take out inner cell mass and use as stem cell

• under lab conditions

• creating embryonic stem cells to treat diabetes and alzheimer’s

Reproductive cloning

• production of genetically identical individuals

• under uterine conditions

  • take blastocyst and put in surrogate female

• important for harvesting stem cells that can be used to study embryonic development

SCID mouse

• severe combined immunodifficiency mice

• have no immune system

  • no B and T cells

  • important because the immune system can’t attack the stem cells

• used for determining if an injected stem cell can differentiate in vivo into a multitude of tissue and cell types in vivo

• used to determine if a candidate human cancer cell can generate tumors in vivo

Somatic cell nuclear transfer

• injection of somatic nucleus into an egg

• John Gurdon was the first person to clone

• can now clone animals from this

• Dolly the Sheep, frog, “little nicky”

  • Sir Ian Wilmut cloned Dolly

    • Dolly has a lot of medical problems but other identical clones were fine

• important to development of iPSCs because it was obvious that cytoplasmic factors could reprogram a somatic nucleus

  • showed that SCNT nucleus could create an entire functioning animal due to cytoplasmic factors in egg

Cloning primates

• cloned rhesus monkeys survived over two years

• identical genetics

  • better for drug testing because lack of genetic variability

Challenges with SCNT

• 1000s of SCNT are required for one implantable embryo

• some researchers are still attempting human SCNT designed for therapeutic cloning because hESCs could serve as an autograpft

Parthogenesis

• generation of an animal or cell without sperm/fertilization

  • “virgin birth”

• Loeb

  • unfertilized sea urchin eggs were induced to undergo parthogenesis by changing osmolarity of the surrounding medium

    • made adult sea urchin

  • unfertilized star fish eggs were also induced by using dilute acid

• hPSCs

  • ISCO company

    • claim they can cure many different diseases

  • benefits:

    • only 200 to 300 eggs required to generate hPSCs that could match anyone in the world

  • limitations:

    • all alleles would be homozygous because there is no sperm

    • not FDA approved

    • is it ethical to create human embryos?

iPSCs

• Shinya Yamanaka and James Thomson

  • published separate research but on the same day

1. take fully differentiated somatic cell

2. introduce four reprogramming factors

3. transit embryonic cell is created which can be grown in large numbers

4. embryonic cell is cultured and differentiates into various cells that can be implanted or repaired and then differentiated

• somatic human cells can be converted to true stem cells with only 4 additional genes

• clues to iPSCs came from research on embryonic master genes and SCNT

  • mastergenes: OCT 4, SOX 2, and nanog

  • cytoplasmic factors telling cell what it can be

  • some way to convince nucleus to do this

  • SCID mouse and inject stem cells into mouse and found they differentiated into teratoma (embryonic master tumor)

    • stem cells have long telomeres

      • rare tumor-teratocarcinoma occurs in humans from stem cells

        • fully formed teeth and digits

      • long telomeres=teratocarcinoma

• Cellular Dynamics Inc

  • iPSCs for sale

Promises of iPSCs

• basic research on differentiation

• can make patient specific cells of individuals carrying genetic defects

  • useful for drug development

• source of cells in the future for stem cell therapy

  • not yet FDA approved

• have proven useful in tissue engineering organoids

Maturation phase transitent reprogramming

• “time jump” skin cells by 30 years

• used the same 4 Yamanaka reprogramming factors but waited only 13 days, not 50

Liver regeneration in mice

• didn’t generate teratomas or other cancers

• only one day protocol

Pros + Cons of SCNT

• could be used for autologous transplant if FDA approved

• no US federal law banning therapeutic or reproductive cloning but some states forbid it

  • but is it ethical? a human embryo is being created

Pros + Cons of Pathogenesis

• can match to a world population-only 300 eggs required

• all alleles are homozygous

• allogeneic not autologous like SCNT unless female donated egg

• is it ethical? a human embryo is being created

Pros + Cons of iPSCs

• potential for teratocarcinomas

• no human embryo is being created

• can be autologous or allogeneic

• more pluripotent than adipose derived mesenchymal stem cells and easier to procure

Tumorigenicity

• safety issue with stem cells

• stem cells have long telomeres and can divide many more times than normal cells

  • propensity to form tumors such as teratocarcinomas

• one clinical trial started in Japan overseen by the RIKEN Institute was stopped after only one patient due to this concern

Immunogenicity

• safety concern with stem cells

• propensity to trigger immune responses

• the more frequent the stem cell injections the higher the chance of immune rejection complications that can include anaphylaxis

• autologous as well as allogenic can launch an immune resposne

Inappropriate differentiation

• safety concern with stem cells

• risk of stem cells differentiating into cells that were not intended and not native to target organ

• a woman injected with human mesenchymal stem cells near her eye ended up with bone tissue growing inside her eyelids

Cord blood

• MSCs and hematopoietic stem cells

  • hematopoiesis: ability of cells to differentiate into every cell type in blood

• another source of stem cells

• procedure is when baby is born, cord blood cells stored in liquid nitrogen

• Private

  • for profit organization

  • donor pays initial fee and a maintenance fee

  • cells not available to the public

  • better if there is a genetic disease in the family and multiple members require the cells

• Public

  • not for protfit

  • available to the public through the National Marrow Donor Program through which cord blood is matched

Robert Horwitz: C. elegans

• model organism

• easy to grow in agar plates

• non pathogenic

• translucent-can optically section through organism

• stable mutant C. elegans are available for study

• important in cell differentiation

  • all cells have been coded and differentiation predicted

  • cell division/differentiation patterns can be predicted and always follow the same pattern

• many gene like apoptotic genes have mammalian homologs

  • excellent model to study apoptosis

• comprised of a limited number of cells

• roundworm

• first microRNA (miRNA) discovered

  • nobel prize to Victor Ambros and Gary Ruvkun

Development of C. elegans

• PAR proteins

  • establish polarity

• Heterochronic mutant

  • key to discovering lin-4 RNA

    • first miRNA ever discovered

Xenoboths

• first reproducing stem cell-fueled living “xenobots”

  • mobile and reproduce

• made from frogs

Nucleus

• first seen with Carl Zeiss microscope

• not all cells have nuclei

  1. mammalian RBC

     1. only way to get concave shape is to not have a nucleus

  2. skin epidermis

     1. regenerative layer is nucleated, but as they move up to stratum corneum, they are not nucleated

        1. in a highly ordered process

  3. lens of eye

     1. lens epithelial cells differentiate into lens fibers which don’t have nuclei

Nuclear envelope

• consists of 2 membranes

• connected to the ER

  • ER gives rise to the new nucleus at the end of mitosis

• nuclear pores

  • most common protein complex

  • can pass proteins via diffusion

    • less than 62,500 daltons

  • histones=20,000 daltons

  • non histones includes transcription factors, etc

    • greater than 62,500 daltons

Nucleoplasmin

• example of protein in nucleus that exceeds 62,500

• find in eggs of X. laevis (African Clawed Toad)

  • 10% of all proteins are nucleoplasmins

• first molecular chaperone ever discovered

• pentamer

  • each part is 33,000 D (x5)=165,000 D

• gene stability

• transcriptional regulation

• requires ATP to move through nuclear pores

  • receptor present

Lamins

• nuclear dissolution and reassembly

  • maturation promoting factor (MPF) causes nuclear dissolution via lamin phosphorylation

• intermediate filaments

• part of the karyosteleton

• lamins A, B, and C

  • all about 60-70,000 D

• connect to chromatin in chromosomes

• have hydrophobic region which is critical to anchor them to envelope

Progeria

• defects in Lamin A assembly

• precocious aging disease

• Lamin A protein attached to farnesyl inserted into nuclear lamina and leaves, but in Progeria, farnesyl is not clipped off so nuclear lamina is defective

  • Lamina A can’t integrate causing nuclei to look squished

Lonafarnib

• farnesyltransferase inhibitor

• drug for progeria

Nucleus in mitosis

• nuclear envelope dissolution

  • completely disappears

• early mitosis

  • nuclear envelope and lamins

  • goes into late mitosis and lamins A, B, C just float in space after dissolution of nuclear envelope

• dissolution is caused by MPF which phosphorylates lamin B

  • triggers nucleus dissolution

  • cell fusion experiment

    • fusing M cells and G1 cells

      • something in mitosis cell causes dissolution of nuclear envelope and something with the chromosomes

Cell cycle

• first proposed in 1953 by Howard and Pele

  • looked at broad bean

• you come from one cell

• cell cycle is embryogenesis

• no mistakes allowed

• cell death is balanced with cell division

  • ex: human skin with highly ordered event of enucleating regenerative layer

  • ex: RBCs

    • born and then die

    • half life is 120 days

    • 2.5×10% RBCs/second are generated

G0

• quiescent period

  • resting stage

• cells with no intention to divide

G1

• about 9 hr of a 24 hr cycle

• Arthur Pardee worked with 3T3 cells

  • commonly used for cell cycle and oncogene studies because they are east to convert from normal to cancer cells

  • isolated from mouse embryo tissue

  • cells in G1 need PDGF and insulin

  • in G1, there are early and late response genes

    • mRNA levels of early genes increase and decrease over time, while late just increase under normal conditions

    • under experimental conditions using protein synthesis inhibitors involved in the degradation of early response genes

      • early maintain increased level then straight across

      • late response genes do not increase and just stay at the bottom

Cell syncrony

• to have all cells in the same cell cycle compartment

  • amino acid deprivation

    • stall in G1 because of growth factors

  • serum deprivation

    • stall in G1

  • protein synthesis inhibitors

    • stall in G1

  • microtubule inhibitors

    • stall in M

    • nocodazole

  • DNA synthesis inhibitors

    • stall in S

• eventually, cells fall out of synchrony over some days because G1 is extremely variable in time

G1 checkpoint

• checkpoint 1

  • check fidelity because cycle must be perfectly accurate to see if anything needs to be fixed

    • “START” in yeast

• checkpoint 2

  • Pardee/restriction point

  • at G1-S border

  • “go-no go” point

Cyclins

• controls cell cycle transit between G1, S, G2, M

  • act as a signal for the cell to pass to the next cycle stage

• first discovered using synchronized cells for a natural system

  • Ruderman and Hunt

  • used sea urchin embryos

    • once cell is fertilized they go through synchronous series of steps all going through the cell cycle

  • used SDS gels to find proteins that were cyclin A and B with cell cycle compartment

  • Cyclin B-CDK 1 phosphorylates lamin B which causes the dissolution of nuclear envelope

    • Cyclin B only or CDK do nothing on their own; they have to be partnered together so lamin B can be phosphorylated by cyclin B

      

• Gloucester Marina Genomics Institute (GMGI)

  • do work on red sea urchins that have tumor suppression genes

    • can live 200 years

Q: Is cyclin D required for G1 cell cycle transit?

1. experiment: use blocking antibody

   1. add growth factors to get cells to go through G1 out of stall state and add BRDU (same as 3H-thymidine)

   2. cyclin D blocking antibody is added so whole activity of complex is blocked

      1. therefore, cyclin D is required for G1 passage

Cell cycle kinetics

• G1

  • synchronize cells so all are G0

  • G0-G1

    • 3H-thymidine or BRDU

      • at some point, we see first appearance of radioactively tagged cells at G1-S border (BRDU positive cells)

    • can now figure out time in G1

• S phase

  • use random cycling

  • use 3H-thymidine or BRDU

    • see lots of cells and some are positive

    • count percentage in S and multiply by total cell cycle time

  • gives rough cell cycle time of C

• G2 phase

  • use random cycling

  • use 3H-thymidine

    • after 30 mins look for radioactive M cells

• M phase

  • shortest is 30 mins

  • use random cycling

  • count percentage of mitotic figures and multiply by total cell cycle time

    • when cell goes into mitosis they are really small and retractile

• found that there was no difference in timing for cancer and normal cells

S phase

• DNA synthesis

• bidirectional

• 10 hours of 24 hr cycle

• DNA doubles here

• MCM helicase (minichromosome maintenance)

G2 phase

• cell verifies that all of the DNA has been correctly duplicated and all DNA errors have been corrected

• chromosome condensation is initiated

• early organization of the cell cytoskeleton

• mitotic CDKs initiate activity

M phase

• very short-30 mins

• when cells go through mitosis, they shut down everything like protein trafficking

• at the end of mitosis new nuclear envelope comes from ER which is why it is continuous with it

  • very complex process

• nuclear pores are reformed

MPF

• maturation promoting factor

  • X. laevis

• mitosis phase factor

  • contributed to dissolution

• MPF=MPF=mitotic cyclin (cyclin B) + CDK

Ruth Sager

• found tumor suppressant genes

  • cell cycle checkpoints: p53

• take a normal cell and a cancer cells and fuse them together to get a heterokaryon and a hybridoma

• hybridoma has a normal phenotype and over time the cancer phenotype showed up

  • tumor suppressor genes were lost and could not control cancer

    • showing phenotype

• discovered everything in budding yeast

P53

• guardian of the genome

• tumor suppressor gene

• unstable transcription factor but can be phosphorylated by ATM/R and stabilized

  • checks the fidelity of DNA

• then P21 is phosphorylated which blocks CDK

• p53 fixes DNA problems if it is mild

• if serious, the cell commits apoptosis

Cancer

• the process by which a cell loses its ability to control its cell cycle

• #2 killer

• 200 types of cancers based on histological identification

Challenges of cancer

• a typical cancer cell has 5000 mutations

  • how do we handle this

1. identify the driver mutation

   1. the mutations responsible for the normal to cancer transition

   2. most cancer cells have average of 5

   3. non driver mutations=passenger mutations

2. determine how to best intervene with driver mutations

   1. drivers are the most important so have to determine which is driver and which is passenger

3. the best treatment modality selectively targets cancer cells only

   1. not possible unless CAR-T therapy

4. billions from NIH have been spent trying to understand the basis of cancer

5. understanding the molecular basis of select cancers doesn’t immediately translate into a cure

• early onset cancer is on the rise

  • younger people appear to be aging faster than older people

• over 200+ histologically different cancers

• cancer scam drugs

Model organism: zebra fish used to study genes that cause melanoma

Normal vs Cancer cells

Not Transformed-Normal	Transformed-Cancer
- Dont grow in soft agar (semi-solid medium) - leads to anoikis (apoptosis as a result of not suspending cells)	- Grow in semi solid medium - can generate clone
Typical karyotype (23 sets of 2 chromosomes)	Often demonstrate aneuploidy (extra or missing chromosomes)
Have normal set of microRNAs (miRNAs)	Have many unique miRNAs
Secrete few extracellular proteases	Secrete more proteases (metastasis) - Melanoma secretes proteases across basal lamina with collageneous
Usually larger: 10-15 microns - normal cells are stuck in G0	Usually smaller: 3-5 microns - uncontrolled division
Lower nuclear/cytoplasmic ratio	Higher nuclear/cytoplasmic ratio
Cytosketelton more organized	Cytoskeleton less organized
Extensive/normal ECM	Little ECM (no fibronectin secreted)
Growth to a single-cell layer	Multilayer forms
Doesn’t cause tumors when injected - injected into recipient animal: SCID mouse bc immune cells attack tumors	Causes tumors when injected - SCID mouse generates tumors
Serum-dependent growth - 10% fetal calf serum	Serum-independent  - depends on cell type → ability to secrete own growth factors like PDGF
Secretes few growth factors	Can secrete lots of growth factors
Telomeres shorten with each division	Telomeres stay the same length
25-50x doublings	Unlimited cell division in vitro bc long telomeres and can repair them
Few genetic defects	Genetic defects in p53, RAS, etc. - common driver mutations
Normal glycolysis	Warburg effect - only make ATP out of glycolysis - faster way to make ATP rather than going thru all of oxidative phosphorylation
Cell membrane permeability is normal	Cell membrane 10x more permeable
Don’t typically secrete angiogenesis factors such as VEGF (vascular endothelial growth factor)	Can secrete angiogenesis factors: VEGF - this is the invasion of blood cells
RTKs are normal (regulated)	RTKs can be aberrant (constitutive)
Apoptosis normal	Innate apoptosis can be inhibited

What causes cancer?

1. Viruses

   1. originally thought to be the primary cause of cancer

   2. Peyton Rous discovered the Rous Sarcoma virus

      1. showed that Rous sarcoma virus could cause animal cells to become cancerous

         1. it is a retrovirus that uses reverse transcriptase to make copy of DNA that it inserts into its host eukaryotic cell

      2. scientists found that chickens and other species appeared to have normal gene

         1. c-src which is a proto-oncogene that controls cell division

      3. v-src is an oncogene which controls division but is constitutively regulated (always turned on)

   3. SV40

      1. DNA virus that causes cancer

      2. have large T antigen that can bind retinoblastoma (rb) and p53

      3. both are tumor suppressor proteins

         

   4. HPV

      1. large group of viruses

      2. can cause warts and cancer-cervical cancer

      3. Gardisil is the first vaccine against any viral based cancer

         1. prevents up to 90% of HPV infections

   5. Epstein Barr virus

      1. can cause Hodgkins Lymphoma and Burkitt’s Lymphoma

2. Radiation

   1. UV light

   2. generates thymine-thymine dimers

   3. can be corrected but if too many or one isn’t corrected it could lead to skin cancer

      1. learned from WWII that radiation can cause cancer

         1. two years after bomb dropped-leukemia in children

            

3. Chemical mutagens

   1. cause base substitutions, DNA cross linking, chromosome breakage

      1. EMS-used in lab to make random cell mutants

      2. tobacco smoke has mutagens and tumor promoters

         1. over 60 carcinogens

4. defective cell cycle genes (cyclins)

   1. tumor suppressor genes work through cyclins

      1. so if the genes are ok but one cyclin is defective, then the cell cycle may not pause and fix errors

   2. cell cycle genes-Cyclin D

      1. defects can cause breast cancer

      2. cyclin D1 is overamplified in greater than 50% of breast cancers

5. defective tumor suppressor genes

   1. defects in genes that oversee the fidelity of the cell cycle such as p53

6. defective caretaker genes

   1. DNA correction or repairs enzymes

   2. xeroderm pigmentosum

      1. sensitivity to sun and UV light

7. defects in apoptosis pathway

   1. ex: too much bcl-2

   2. some viruses such as SV40 inhibit apoptosis by sequestering Rb and p53 tumor suppressor genes

8. defects in growth factor signaling pathway

   1. mutant Ras

      1. g-protein that is the effector of many growth factor receptors can be constitutively turned on

         1. RasD found in most human tumors

      2. loss of TGF beta signaling pathway

         1. antigrowth factor

      3. defective growth factor receptors that are constitutive

         1. many are derivatives of naturally occuring RTK

9. defective telomeres or telomerase

   1. mitotic clock

   2. cancer cells require non-shortening telomeres

10. cancer stem cells

    1. arise from normal cells or tumors

       1. many produce their own stem cells

    2. less than 1% of the tumor cell population

    3. they can both self renew as well as differentiate into a heterogenous tumor population

    4. they are thought to be able to differentiate into a heterogenous non tumor cell population

    5. defined via FACS and cell surface marker, CD133

       1. CD133 is accepted as definitive metric to identify cancer stem cells

    6. influences by tumor microenvironment

    7. reason why some tumors metastasize or reemergence after remission

    8. often resistant to chemo radiation/drug regime that is effective against associated tumor

11. chromosomal translocation

    1. ex: Burkitt’s lymphoma

    2. Bcr-abl fusion protein which is a protein kinase

12. DNA amplification

    1. numerous copies exist leading to overproduction of encoded protein

    2. minute chromosomes

       1. small, independent mini chromosome-like structures

13. Overproduction or mutation of nuclear transcription factors

    1. C-MYC

       1. increased proliferation

       2. diagnostic

          1. if there are a lot there is a poor outlook

    2. C-Fos

       1. increase drug resistance

       2. increase stem cell activity

Oncogene

• defined as a gene that can cause cancer and is often a deviant or variant form of the normal, proto-oncogene

• Robert Weinberg-MIT

  • discovered the first tumor suppressor gene: Rb

    • transfer of DNA from host tumor cell to a non transformed cell can confer oncogenesis

    • DNA from a human bladder cell carcinoma can transform mouse 3T3 cells

      • thus there is a gene that can cause cancer and isn’t species specific

• cellular oncogenes or proto-oncogenes are normal genes that regulate cell division

  • oncogenes are a form of the proto-oncogene that can cause cancer

    • mutated and increase in abundance

    • Ras is a proto-oncogene that is a GTPase controlling cell growth; rasD is an oncogene

  • one of the earliest discovered was v-src, a cancer form of c-src

• more than a single oncogene is necessary for cancer to develop

  • Ha-ras oncogene transforms 3T3 cells which are mouse embryo fibroblasts

    • can’t transform REF unless placed in soft agar

• there are several types of proteins that participate in controlling cell growth and proliferation

  • mutations at many of these points can lead to cancer

• the conversion of a proto-oncogene to an oncogene is considered a “gain of function” mutation

  • point mutations

    • change in a single base pair that results in a constitutively active protein product

  • chromosomal translocation

    • fuses two genes to produce a hybrid gene encoding a chimeric protein whose activity is constructive

    • brings a growth regulatory gene under the control of a different promoter that causes inappropriate expression of the gene

    • leads to production of oncoproteins

Examples of oncogenes

• special focus in new oncoprotein and ErbB oncoprotein

• many oncogenes associated with cancers are RTK

  • Her2 can be converted to an oncogene called neu oncogene by a single point mutation

    • now constitutive

  • overproduction of Her2 can lead to cancer

    • Herceptin is being tested as a drug to treat this cancer

• Trk oncogene

  • chromosomal translocation results in replacement of most extracellular domain of normal trk protein with nonmuscle tropomyosin

  • constitutively active and found in cytosol, not plasma membrane where normal one would be found

• viral activators or proteins act as oncoproteins

  • activation of the erythropoietin receptor

    • SFFV induces erythroleukemia-a tumor of erythroid progenitors

    • Gp55 is a SFFV envelope glycoprotein that induces formation of excessive numbers of erythrocytes

Multi-hit model of cancer induction

• the incidence of cancer increases with time

  • the older you get, the higher the chance of developing cancer

• multi hit model

  • cancer accumulates over time because numerous genetic errors accumulate

  • most cancers have an average of 5 driver mutations that take time to accumulate

• most cancers arise from single mutated cells

  • can be verified by examining female tumors

    • females are a mosaic of cells with one X chromosome inactivated can distinguish histologically

  • if tumor did not originate from single cell, then the tumor would consist of a mosaic of X-activated cells

    • this isn’t the case as tumors in women all have the same X chromosome inactivated

• mice experiments with mutated MYC and Ras show the effects of both mutations are synergistic

  • MYC is required for the growth of many tumors

• cancer can be due to a sequence of mutations

  • colon cancer is the best known

    • multi hit model of colon cancer takes time

      • colonoscopies should now start at age 45 not 60

      • explains why you get colonoscopies every 5 years

Defective tumor suppressor

• people with inherited defects in tumor suppressor genes have a propensity for cancer

• ex: Rb

  • first tumor suppressor gene discovered by Robert Weinberg

  • characterized by retinal tumors that can be bilateral

    • occurs in childhood and develops from neural precursor cells

    • 1/200,000 children affected

    • 60% not inherited, 40% inherited

    • enucleation as treatment

• ex: p53

  • Li-Fraumeni syndrom

    • defective p53 that leads to many cancers resulting in a 25x greater chance of having cancer compared to normal population

  • ex: BRCA-1

    • women who have defective BRCA1 have 60% possibility of developing breast cancer by age 50 compared to 2% for normal population

• cancer can be caused due to heterozygosity (LOH)

  • normal allele is lost, so no mutant allele is expressed

  • this can occur due to mis-segregation of mitotic recombination