exam 3 cancer biology

Lecture 10

• -  Heterokaryon: 2 isolated cells with their own DNA- treated with PEG (which mimics a membrane)

• -  Normal cells are tumorigenic, and are seen as dominant

- Single copy of gene results in phenotype

- Cancer cells are nontumorigenic, and are seen as recessive
- Both copies of gene must be affected to result in phenotype

• -  Both copies must be mutated in order to be cancerous

• -  Predicted dominant as cancerous and then found them as recessive- which suggested

  the idea of oncogenes

• -  Evidence for the existence of antioncogenes

  • -  Biological regulation suggested the existence of proteins that oppose the actions of oncogenes (RASGAPS)

  • -  Much easier to inactivate in comparison to hyperactivate

• -  Evidence against the antioncogenes

- Would have to inactivate both copies of a gene. Seemed unlikely given the time scale.

- Children with bilateral (familial) Rb have a high risk of developing non-retinal tumors - Germline mutations in Rb gene leads to predisposition of cancer

• -  Recessive mutation but dominant look is an anti oncogene

• -  The Knudson “two hit” hypothesis

  • -  Idea of inheritance

  • -  Bilateral found more often

  • -  spontaneous - both copies mutated

  • -  Familial: inherited gene

• -  Retinoblastoma is inherited as dominant, and recessive at cellular level

  • -  Deletion at cellular level

  • -  Chromosome 13: deletion of some part of chromosome

• -  Loss of Heterozygocity (LOH)

  - Function of one allele, while other is inactivated

  - Mechanisms that inactivate second copy

• -  One mutation leads to an increase in second mutation

  - Leads to loss of heterozygousity

  - Mitotic recombination

• -  Mitotic recombination

- Crossing over: results in exchange of genetic information, occurs during meiosis

• -  Gene conversion can also inactivate the second copy of gene- resulting in

  heterozygousity

  • -  Occurs in response to double strand breaks

  • -  Affected DNA is only about 200-1000 nucleotides in length

• -  Gene conversion mechanisms

- Double strand breaks: when this happens, the protein complex binds to the end

of strands, but it is error prone. OR, use the completed strand as a template but

this is risky because of loss of heterozygosity and any imperfections need

passed on

• -  Gene for hereditary retinoblastoma assigned to human chromosome 13 linkage to

  esterase1

  • -  Finding retinoblastoma gene

  • -  Confirm loss of heterozygousity

  • -  Exists as isoform A and B

• -  Heterozygous for inactivating: viable

• -  Homozygous for inactivation: lethal

• -  Rb is a tumor suppressor gene

- Form an osteosarcoma

• -  Cloning an Rb gene was done by chromosome walking, which used the libraries ECORI

  as the probe, and BAMHI as the recovery fragment.

• -  Rescue experiment

- Saos
- Cancer cells detective for Rb

• -  Add back WT copy of Rb via transfection

• -  Restore Rb function so cells behave healthily

• -  Tumor suppressor genes can be mapped using SNPs

  • -  Change in nucleoides change in mutation

  • -  These changes have no consequences - SNP

• -  Use primers to look for single changes

  • -  PCR

  • -  Alleles A and B

• -  Rb is a critical regulator of cell cycle progression

  • -  PRB is a nuclear protein that regulates transition from G1 to S phases called the R point

  • -  If RB inhibits, cells will not stop dividing

• -  Cell cycle:

- G1 phase (GAP1)

• -  Longest time in cell cycle

• -  Cell deciding whether or not to divide again

• -  Cellular cues

  • -  Restriction point

  • -  Rb regulates restriction point- prevents cell from going past

    restriction point

• -  S (Synthesis)

  - Replication

• -  G2 phase (GAP2)

- Checking that dna is replicated properly - M phase (mitosis)

• -  Separating

• -  Chromosomes dividing

• -  Reform nuclear membrane

Lecture 11

• -  You can observe the stages S and M in the cell cycle

• -  CDKs

  • -  Ser/Thr kinase that is present throughout the cell

  • -  Activity is cyclical

  • -  Humans have 20

• -  Cyclin

- Regulatory- binding to CDK controls the kinase activity of CDK

• -  Distinct CDKs associate with different cyclins to trigger the cell cycle

  • -  G1: cycD, CDK4, CDK6

  • -  G1/S: cycA, CDK2

  • -  S: cycA, CDK2

  • -  G2: CycA, CDK1, CDC2

  • -  M: cycB, CDK1, CDC2

• -  M-CDK can phosphorylate many proteins involved in meiosis and regulation

  • -  Lamins: nuclear envelope disassembly

  • -  Proteins: condensing chromosomes

  • -  Proteins: spindle

  • -  Kinetochore proteins

• -  Mechanisms of CDK regulation

  • -  Abundance of cyclins

  • -  CDK phosphorylation

  • -  Binding to CKIs (inhibitory protein)

• -  Abundance of cyclins:

  • -  Cyclins need to appear (transcriptional)

  • -  Cyclins need to disappear (ubiquitination)

• -  Binding to CDK causes conformational changes rhat allow kinase to bind to subtares - PSTAIRE- direct contact with cyclin, alters T-loop

• -  CDK activity is regulated by positioning of the PSTAIRE helix and the T loop - Activation loop rearranges
  - Allows substrate to bind
  - Cyclin binding isnt efficient

• -  All cyclins have similar conformations that mirror alpha helices

  • -  29 human cyclins

  • -  Mrail sequences are important for binding substrates of CDK/Cyclin complex

• -  Cyclin Domains

  • -  Cyclin box: binds to CDK

  • -  PEST domain: amino acids rich in proline, glutamic acid, ser, thr. All of them are

    important for cyclin development

• -  G2 cyclin (A/B): have destruction box instead of PEST domain

  • -  Destruction box: sequence important for cyclin degradation

  • -  Cytoplastic retention sequence: retains cyclin in cytosol. Phosphorylation by ERK

    converts into nuclear import sequence

  • -  Cyclin box: sequence important to binding to CDK

- Ubiquitin: 76 amino acid long proteins that can be conjugated to other proteins. Highly conserved

- Conjucation happens between Gly and Lys

• -  Mono-ubiquitination: regulates protein activity

• -  Poly-ubiquitination: chains frequently target proteins for degradation

• -  E1: ubiquitin activating enzyme, regulates ATP

• -  E2: ubiquitin conjugating enzyme: transfers ubiquitin to substrate

• -  E3 proteon complex that confers specificity to target

  • -  SCF complex made of: SKP, Cullin and f-box

  • -  Anaphase promoting complex

• -  Ub chains can form on different lys residues

  • -  K48 linked: destroyed in proteasome. Has a closed conformation

  • -  K63: done trigger degradation but is used as an organizer. Open conformation

• -  Cyclins eliminated by ub

- Mediated by ub-dependent proteolytic system
- Enzymes that ubiquitinates cyclins are also controlled by cell cycle

- Activated by CDKs, has a built in delay - Cyclins are degraded by different E3 ligases

- Cyclin E (G1/S) is degraded by SCF complex E3 ligase

• -  The anaphase promoting complex has two specificity factors: CDH1 and CDC20

• -  APC/C uses CDH1 as a specificity factor to degrade cyclin B

• -  Mechanisms of CDK regulation

  • -  Abundance of cyclin

  • -  CDK phosphorylation

  • -  Binding to CK1 inhibitory protein

    Lecture 12

• -  Binding cyclin is not sufficient to activate CDKs

  • -  Pushes PSTAIRE

  • -  Revamps activation loop

• -  Cyclin A bound to CDK2

• -  Cyclin E bound to CD2

• -  Activation phosphorylation is catalyzed vy CAKs

- CAK phosphorylates CDK- results in activating phosphorylation

• -  Wee1 kinase

  • -  Adds inhibitory phosphorus and holds kinase active

  • -  Must bind to ATP- inhibitory. Prevents reaction in CDC

  • -  The phosphorylation by Wee1 Tyr kinase blocks CDKs active site

  • -  Phosphorylates T14 and Y15

• -  Regulation

  • -  Inhibitory phosphorus- phosphatase removes inhibitory phosphorus to activate

    CDK

  • -  Removes inhibitory phosphorus to make CDK active

• -  CAK phosphorus and Thr160 inserted into cationic pocket

• -  Opening the activation loop stabilizes inactive conformation, then ATP can phosphorylate mechanism.

  • -  Further stabilizes CDK in active configuration

  • -  CAK adds phosphorus to Thr160

• -  Inhibitory phosphorylation is also involved in CDK regulation

- Wee1/CDC25 switch event is regulated by substrates and extrinsic signals

• -  CDC25: phosphatase that removes inhibitory block- held inactive

• -  Full activation of CDKs require both phosphorylation and de phosphorylation

• -  A positive feedback mechanism further increases m-cdk (cyclin B-CDK1) activity

  • -  Results in burst of MCDK activity at end of interphase

  • -  Getting rid of cyclin inhibits pathway

• -  Phosphorylating and activating CDC25 works on inactive complexes. The cell-cell

  control system can arrest the cycle at various checkpoints

• -  CDC25 in various points to pause cell cycle and repair DNA

• -  The G0 phase makes nondividing unfavorable

• -  CDK inhibitor proteins (CKIs) can halt the cell cycle progression by binding to complex

  and inhibiting activity

• -  2 classes of CDK inhibition (CKIs)

- INK4 family binds to CAK4/6, blocking cyclin D binding
- INK4 binds to active CDK/cyclin complex- distorts cyclin binding sites,

reducing affinity for cyclins. Releases cyclins and distorts ATP - cip/kip family blocks active site of multiple CDKs

• -  CKIs inhibit CDK activity

• -  CDK6 distorts ATP binding site, compromising catalytic activity

• -  KIP1: obstruct ATP binding site, compromising catalytic activity

• -  CIP/KIP: cyclin remains bound, binds to whole complex

• -  CKIs must be degraded for cell cycle progression to occur, via the SCF complex

• -  SCF complex

• -  FBOX is responsible for p27

• -  Mechanisms of CDK regulation

  • -  Abundance of cyclin

  • -  CDK phosphorylation

  • -  Binding to CKI

• -  Cyclin/CDK binding

  • -  G1: cyclinD is bound to CDK4/6, cyclinE to CDK2

  • -  S: cyclin A is bound to CDK2

• -  G2: Cyclin A is binding to CDK1

• -  M: cyclin B is binding to CKI

• -  Mitogens promote cell cycle progression by simulating cyclin D transcription

• -  G1 mechanisms

  • -  CDK4/6 kinase phosphorylates Rb

  • -  Hyperphosphorylates, one or more phosphorus added to RB

  • -  Allows selective transcription, promotes cyclin E

  • -  As Rb binds to E2F, prevents from transcribing genes

• -  The G1 phase decides whether or not now is the right time to divide using:

  • -  Mitogens (signaling molecules that promote cell division)

  • -  Anti-mitogens (signaling molecules that prevent cell division)

  • -  If the cell does not recieve any signals- goes through anoikis

• -  Mitogens and anti-mitogens promote signaling from Ras and MAPk, results in Cjun/Fos

• -  One of the targets of cyclin D and CDK4/6 is PRb

• -  Rb protein regulates restriction point by inactivating E2F

• -  Rb is in the pocket protein family

• -  The pocket protein family is made of - N terminal

  - C terminal
  - Middle sequence called the pocket terminus
  - Consists of A lobe and B lobe, linked by spacer - Similar to cyclin fold

• -  E2F binds to the groove between two lobes of pocket domain

• -  Things that bind to the pocket are

  - E2F

  - Oncoproteins

• -  PRb contains at least 16 different sites for cyclin/CDK phosphorylation

- Some of the phosphorylation events maintains binding of PRb with E2F, whereas other phosphorylations cause disruption of PRb1- E2F interaction

• -  Phosphorylation of Ser567 in PRb is predicted to disrupt E2F binding

• -  Phosphorylation of Rb is also thought to result in several distinct conformations of PRb

  that disrupt E2F binding

• -  The phosphorylation status of Rb is important for its different functions

• -  The mechanisms of how Rb regulates is still debated

- Hypophosphorylation: results in selective transcription

- Hyperphosphorylation
- PRbs function relates to a family of transcription factors called E2Fs

- E2Fs are needed for transcription of genes that are essential for cell to enter cell cycle

• -  The DNA binding domain contacts the DNA segment in promoter region

• -  Transcriptional activation domain recruits RNA polymerase II

• -  NLS accounts for how many proteins need translated

  • -  Transcription happens in nucleus

  • -  Translation in extracellular space

- NLS localizes E2F to nucleus by traveling through nuclear pore complex

• -  E2Fs have more than 100 target genes, mostly involved in the first steps of DNA

  replication

  • -  One of the targets is CycE gene

  • -  Transcription of CycE starts a positive feedback loop

• -  Cyclin E has additional roles like licensing ORI for dna replication

• -  CyclinE/CDC2 targets CDC6, helicase loading protein

• -  CyclinA has roles in both S and G2S phases

  • -  S phase

    • -  As cyclin a increases, cyclin E decreases. Cyclin A binds to CDK2

    • -  Cyclin A and CDK2 regulate transitioning into dna replication

    • -  Dna replication only occurs once from each origin of replication

  • -  G2/M phase

    • -  Cyclin A/CDK1 helps with cyclin B, CDK1 activation and stabilization

    • -  Cyclin A mutants in drosophila have delayed activation of M-CDK

• -  CDK1 and cyclin B play important roles in regulating different stages of mitosis

  • -  CDK/CyclinB phosphorylate nuclear lamins to disassemble nuclear envelope

  • -  CyclinB/CDK1 regulate events in anaphase such as sister chromatid separation

• -  M-CDK (cyclinB/CDK1)

- Promotes anaphase (pulling apart sister chromatids)

- APC/C
- E3 ubiquitin ligase

• -  Securin: inhibitor of separate

• -  Separase: protease- cuts proteins and breaks peptide bonds

• -  Securin is a chaperone for separase and helps with proper protein folding

• -  Sepaease only folds correctly when bound to securin

• -  Securin gets destroyed when ubiquitinated, but separase has already folded correctly.

  MCDK becomes inactive and dephosphorylates sepharase.