CELL CYCLE CONTROL AND CANCER Prof T Ranga Rao MD Biochemistry Department Guntur Medical College,GUNTUR
The steps of cellular growth and division that we observe today in our own cells is there in the original eukaryotic cells since time immemorial
Outline
Cell cycle control Biochemical
Cancer Progressing from biochemical to genetic to clinical
Three biologists share 2001’s Nobel Prize in Physiology or Medicine for helping to unlock the secrets of the cell cycle,
Leland H. Hartwell, USA Paul M. Nurse, UK R. Timothy Hunt, UK
Main phases of a cell cycle Two chromatids per chromosome
G2
M One chromatid per chromosome
S
One chromatid per chromosome
G1
The alternative to cell cycling G2 M
S G1 G0
G0 cells are not dividing, but instead will be differentiated
The major cell cycle checkpoints The G2 checkpoint: Has all DNA completed division?
The M checkpoint: Are chromosomes aligned in the equatorial plane?
G2
M
S
All three checkpoints: Is the environment favorable?
G1
The G1 checkpoint: Is the cell large enough?
The integration at Cyclin dependent Kinase (CDK) Has this phosphate been removed Is cyclin present
Has this phosphate been added P P
CDK activity is “ON” only if the answer is “yes” to all questions
CDK-molecules and cyclins drive the cell from one phase to the next. The CDK-molecules can be compared with an engine and the cyclins with a gear box controlling whether the engine will run in the idling state or drive the cell forward in the cell cycle.
The importance of Cyclins • Different Cyclins increase in abundance at different stages of the cell cycle • Each Cyclin Dependent Kinase (CDK) interacts with one or two specific Cyclin(s)
CANCER
Growth Invasion Spread
90 % sporadic 10% familial
Cancer: General Etiology and Pathogenesis
Cancer • Cells in culture and in vivo exhibit contactinhibition through feedback mechanism. • Cancer cells lack contact inhibition feedback mechanisms. Clumps or foci develop.
Specific Cellular Functions in Cancer: Genetic Alterations Genetic Instability DNA Repair
CANCER Tumor Suppressor Genes Interstitial Deletion Inactivating Mutation Hypermethylation
Oncogenes Gene Amplification Gene Overexpression Activating Mutation
Pathways leading to cancer
Alterations of Specific Cellular Functions in Cancer DNA Repair
Tumor Suppressor Genes
Oncogenes
Activation Inactivation Proliferation
CANCER
Definitions • A tumor suppressor gene has a normal function in the cell, but can be inactivated. The normal function is putting a brake on the cell cycle in a regulated fashion. • A proto-oncogene has a normal function in the cell, but can be activated into an oncogene. The normal function is to promote cell division in a regulated fashion.
Point mutations are often the initial events in cancer development • Point mutations lead to changes in amino acid sequence • Point mutations occur as result of DNA replication or (repair of) DNA damage • Especially DNA polymerases repairing DNA damage are error prone
Some of the factors involved in G1 checkpoint control Apoptosis
p16 p53 p21
? K4 CD
E2F G1
M
S G2
pRB
?
Cyclin D
CDKActivation &Cancer
Papilloma virus-Cancer
Papilloma virus -- interaction with p53 and pRB Apoptosis
p16
E6 p53 p21
p53 degradation
? K4 CD
E2F
M S
Cyclin D
pRB pRB inactivation relieves E2F of inhibition
G1
G2
E7
?
Papilloma virus proteins
Tumor suppressors include p53, p16, p21, and pRB Apoptosis
p16 p53 p21
? K4 CD
E2F G1
M S
G2
pRB
?
Cyclin D
The role of p53 Apoptosis
p16
p53 p21
? K4 CD
E2F G1
M
S G2
pRB
?
Cyclin D
P53 and the cell cycle
P21 is a CDK inhibitor
P53 arrests the cell cycle primarily by upregulating p21 which inactivates CDK/cyclin. P53 can also activate apoptosis
p53 - the most commonly mutated gene in cancers • Mutations in p53 almost always somatic, very rarely inherited • If DNA is damaged, p53 gene product stops cell division through the pRB pathway • If DNA and the pRB gene is damaged, p53 will induce apoptosis (programmed cell death)
p53 - continued • p53 mutated cell can therefore proceed through S-phase with damaged DNA • Radiation treatment of tumors damages DNA. • Tumor cells in which p53 is mutated survives Radiation better giving the patient a poorer prognosis.
p53 - continued • p53 is most often classified as a tumor suppressor gene • Unlike other tumor suppressors, p53 has been found with dominant negative mutations. In these instances, one mutation in one of the alleles is enough to produce a phenotype
Tumor suppressor genes • Normally, one functional allele in a cell is enough to have full function • Therefore, Knudsons two-hit hypothesis on inactivation of the second allele
Knudsons two-hit hypothesis • Knudsons two-hit hypothesis relates to tumor suppressor genes. • It states that in some cancers, like the retinoblastoma, two hits are necessary before the cells start uncontrolled divisions. In the inherited cases, hit number one is found in all cells. In sporadic cases, hit number one is found in one or few somatic cell, and hit number two happens at least in one cell.
Knudsons two-hit hypothesis (2) • Familial cancer shows phenotype only if a second hit does happen. • Well known familial cancers show dominant inheritance with penetrance of approx. 60-90%. • How can the penetrance be that high? • High penetrance means that loss of the second allele happens with a high probability in one or few of the many cells in the body.
Tumor suppressors in summary • TS gene products acts as brakes on the cell cycle. Loss of both alleles leads to loss of the brake action, therefore leading to increased cell divisions. • TS genes can also be involved in DNA repair and apoptosis, with loss of both alleles leading to increased cell divisions.
Where mitogens enter the picture Apoptosis
p16 p53 p21
? K4 CD
E2F
?
Cyclin D
pRB
G1
M S
G2
Mitogens
Examples of mitogens • Insulin • Epidermal growth factor (EGF), platelet derived GF (PDGF), fibroblast GF (FGF), insulin-like GF (IGF), etc.
Response of the insulin receptor kinase (IRK) to ligand binding • Heterotetramer (2a, 2b) • Insulin binding leads to change in structure (different from other RTKs) • Conformation change activates b-subunit TK activity • b subunit phosphorylates Tyr residues on cytoplasmic domains as well as downstream substrates (IRS)
Three-dimensional structures of the insulin receptor tyrosine kinase (IRK) IRK conformational change upon activation loop phosphorylation. The N-terminal lobe of IRK is colored white and the C-terminal lobe is colored dark grey. The activation loop (green) contains autophosphorylation sites Y1158, Y1162 and Y1163, and the catalytic loop (orange) contains the putative catalytic base, D1132. Also shown are the unbound/bound ATP analog and tyrosine-containing substrate peptide (pink). [Hubbard, EMBO J. 16, 5572 (1997)]
Once Tyr-Phosphorylated, the IRK activity trigerrs a number of signaling pathways
• Phosphatidylinositol 3hydroxy kinase, makes PIP2,PIP3 • Grb2, Sos, activates Ras • Activation of PI-PLC
Fibroblast Growth Factor Receptor Tyrosine Kinase • The fibroblast growth factor receptor (FGFR) family have been linked widely to the development of cancer and disease. • Dimeric assembly of 2 FGF2:FGFR1 complexes. FGF2 is colored orange, Iglike domain 2 of FGFR1 is colored green, and Ig-like domain 3 of FGFR1 is colored cyan. [Plotnikov et al., Cell 98, 641 (1999)]
Growth factor signaling • Binding to external parts of receptors • activation of second messengers by either – integral receptor kinase – dissociation of trimeric G-proteins – activation of other associated enzyme (mostly a kinase)
• Further levels in the cascade
Growth factor signaling (2) • Activation of a transcription factor and subsequent changes in gene regulation is the most important end-point of signaling. • Steroid hormones reach this level directly, as they bind to receptors that themselves are transcription factors. Any gene encoding any of these proteins have the potential for being a protooncogene
Proto-oncogenes vs Oncogenes Proto-oncogenes are the normal cellular gene from which the viral oncogenes are derived. Mechanism of oncogenic activation: Mutation and/or Overexpression Slow transforming retroviruses demonstrate that overexpression of proto-oncogenes also plays a role in retroviral oncogenesis.
Mechanisms of oncogene activation
Oncogenes of Acutely Transforming Retroviri src myc erb A, erb B myb ets rel H-ras K-ras abl raf fos fms fes sis
Rous sarcoma virus Avian myelocytomatosis virus Avian erythroblastosis virus Avian myeloblastosis virus Avian erythroblastosis virus Avian reticuloendotheliosis virus Harvey rat sarcoma virus Kirsten murine sarcoma virus Abelson murine leukemia virus Murine sarcoma virus Mouse osteosarcoma virus Feline sarcoma virus Feline sarcoma virus Simian sarcoma virus
Chicken Chicken Chicken Chicken Chicken Turkey Rat Mouse Mouse Mouse Mouse Cat Cat Monkey
Types of proteins encodes by oncogenes
The classical pathway growth factor receptor > Grb2 > SOS > RAS > MAPKKK (RAF) > MAPKK (MEK) > MAPK
The RAS protein • RAS binds either GTP or GDP • RAS containing GTP is active, RAS-GDP is inactive • RAS can hydrolyze GTP to GDP when triggered by GTPase-Activating Proteins (GAPs) • RAS exchanges GDP for GTP when interacting with a GDPrelease protein (GNRP)
The RAS example • Several mitogenic proteins (growth factors) bind to receptors with tyrosine kinase activity, thereby causing receptor autophosphorylation • The phospho-tyrosine gets bound by the adapter GRB2 which binds GTP-exchange protein SOS. • SOS thereafter is active in exchanging GDP to GTP on RAS
The RAS example (2) • Active GTP-RAS binds temporarily to RAF(MAPKKK), releasing it from inhibitory proteins. • Free RAF protein binds to and phosphorylates MEK (MAPKK). • P-MEK binds to and phosphorylates MAP Kinase on both tyrosine and threonine thereby activating it
Words • MEK is MAP-ERK Kinase, it phospholylates both tyrosine and serine • MAP is Mitogen Activated Protein • ERK is Extracellular signal-Regulated Kinases
MAP Kinase • P-MAP Kinase phosphorylates multiple targets differing among cell types. • The targets include transcription factors such as SRF, indirectly increasing the levels of FOS and JUN (together = AP-1). • AP-1 increases Cyclin-D production.
Parallel pathways • A number of parallel pathways exist. • Several of these have a Jun N-terminal Kinase (JNK) as the final activated kinase • Each of these phosphorylates isoforms of Jun, the partner of Fos in AP-1. P-Jun is more stable than native Jun, thereby increasing the effective concentration of this transcription factor.
Oncogenes – RAS Proteins • RAS is a protein binding either GDP or GTP, hydrolyzing the latter to the former when induced by interaction with a GTPase Activating Protein(GAP). • Mutations in RAS often impair the ability to hydrolyze GTP, freezing RAS in the active state. • HRAS, KRAS and NRAS are three examples of RAS genes.
Oncogenes – RAS Proteins(2) • HRAS involved in carcinoma of colon, lung, pancreas and others. • KRAS involved in melanoma, thyroid carcinoma, acute myeloblastic leukemia (AML), etc.. • NRAS involved in myeloma and leukemia, and in neuroblastoma. Less commonly activated than KRAS in myeloma and leukemia cancers.
Oncogenes in summary • Oncogenes are very rarely inherited • Oncogenes are the activated, cancercausing forms of proto-oncogenes • Any gene performing one of the steps from growth factor binding to gene transcription that leads to cell division could become an oncogene
Clinical observations • Inherited cancers occur earlier in life than sporadic cancers • Inherited cancers show more foci (independent neoplasms) than sporadic cancers – E.g., breast cancer with bilateral occurrence indicates an inherited case
• More than one case of a rare cancer in a family indicates segregation of a familial cancer mutation
Knowledge of cancer mutations: any help? • Yes! Recent news have claimed that cheap, fast genotyping of patient tumors combined with knowledge of which medicine work best for which combination of mutations, will become the next big step forward in cancer treatment. • The cost of such a tailored treatment will be even higher than current costs.
Summary • Tumor suppressors are inactivated – Both alleles are inactivated in the cancer cell
• Oncogenes are activated • Inherited cancer involves inheritance of one mutated tumor suppressor allele • Inherited cancer shows dominant inheritance with reduced penetrance
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