Cell Cycle Regulation, Apoptosis, and Cancer PDF

Cell divides it will processed to the next CELL CYCLE REGULATION, APOPTOSIS AND stage of S CANCER ○ Some cells opt to enter a G0 stage...

Cell divides it will processed to the next CELL CYCLE REGULATION, APOPTOSIS AND stage of S CANCER ○ Some cells opt to enter a G0 stage ○ Some cells might go back to the cell cycle, induced to divide by certain factors ○ Some cells that opt to stage in G0 to undertake terminal differentiation - S stage / phase DNA synthesis / DNA replication takes place during this phase Not only DNA synthesis is taking place ○ Includes proteins that are being synthesized, a lot of genes being transcribed and translated to form the different proteins that are - Fertilized sea urchin eggs in various phases of mitosis needed in various stages - DNA molecules = blue - G2 phase - Microtubules = gold Cell prepares to undergo cell division - Stem cells divide over a short period of time VARIATIONS IN THE OCCURRENCE OF CELL CYCLE 1. Cells that undergo continuous cell division to replace cells that die: - Epithelial cells of mucosal layer of stomach, intestines, body cavities - Epithelial cells of stratum germinativum of skin / stratum basale in the epidermis of the skin - Hematopoietic stem cells (stem cells in the bone marrow that are in blood cell formation) - Spermatogonia (undergoes continuous cell division throughout adult life) 2. Do not divide after maturation - Mature nerve tissue - Cell cycle - Mature muscle tissues - Shortest period is the M phase - Mature red blood cells Cell undergoes cell division 3. Divide when induced Can be mitosis in the somatic cells also in - Liver cells in mature liver the young germ cells - Lymphocytes Can be meiosis in the germ cells as they undergo maturation - Cell cycle involved in the meiosis process can have cell variations Involves 2 cell divisions - G1 phase Vary in length (in some cells in can be very long or very short) Cell grows in size and different metabolites and macromolecules and organelles are synthesize as it prepares to enter the next round of cell division - There are variation in the cell cycles of cells in the same organism depending on the stage of development - DNA replication that takes place in the S phase, DNA replication occurs in the presence of the origin of replication Eukaryotic organism has a number of origins of replication in the chromosome and each of these are starting points of DNA - The fertilized egg keeps on dividing without having replication so that there are several replicon the daughter cells to grow in size before the ○ Replicon = replication unit succeeding cell division - In the S phase, the replicons are all activated at the - From a big cell, you will have small cells that are same time so that DNA replication in the cell takes formed place very rapidly - There is a minimum size that a cell should have - No G1 in cleaving embryo cells - How is this prevented from occurring? In the fertilized egg / zygote, we have a big - Target of Rapamyscin (TOR) / mechanistic Target amount of the protoplasm. The cell is a big of Rapamycin (mTOR) cell so that after one cell division, the Rapamycin is a compound isolated from daughter cells do not have to grow in size certain bacteria anymore before they divide again Rapamycin targets the molecule TOR, it They keep dividing without having the results in the inhibition of cell proliferation protoplasm increase in mass in the eukaryotic organism G2 may be very short - Role of TOR in the regulation of cell growth The TOR can activate molecules that can result in the increase in protein synthesis, resulting in the increase of cell mass leading to cell division On the other hand, the molecule also activates the entry of the cell into the S phase Both of these will result to the grow of the cell and the division of the cell - It prevents the cell from becoming too small - Cleavage of a fertilized egg - Zygote (fertilized egg) undergoes cell division. After fertilization the next stage is cleave where in the cells undergo a series of mitotic division - The cell is quite big, after the first division to 2 daughter cells (they don't have to grow in size anymore) - They will continue to divide until there are smaller cells that are formed - Group of cells called the morula - Followed by the blastula stage - Focused on the transition points in the latter part of - There is a limit to the small size that a cell can attain G1, at the latter part of G2, and within the M phase There are various conditions that have to met by the cell before the cell is allowed to proceed to the next phase - In the restriction point, the cell has to be checked to - The soluble fibers that are involved in the transition ensure that there are growth factors in the medium from one phase to the next were found to be Products of cell’s in the body, they are not associated with the transition of the cell from G2 to nutrients coming from the outside the M phase The cells should also have the appropriate nutrients, attained the correct cell size, if there are DNA damages, it should be corrected - G2-M transition point The cell is checked for the cell size, presence of DNA damage whether DNA replication has been completed or not - If you were to compare the different factors checked in transition point G1 and G2, they have common factors (cell size and DNA damage), however in the restriction point, there are additional factors (growth factors and nutrients) - In G2, the cells have to be checked whether the DNA replication have been completed In G2 the cell would have already had - Soluble factors that are key to the transition of the completed the S phase cells from one phase to another - Within the M phase, there is a Metaphase-Anaphase (a) Utilized cell fusion experiments transition wherein the cells have to be checked Cell in the S phase fused with a cell in G1 whether there are spindle fibers attached to the phase kinetochore of the sister chromatids before the cell is Fusion can be done through the participation allowed to proceed to the anaphase of certain viruses or with treatment of the - Done at an appropriate time (factor in the transition) cells with the polyethylene glycol - Cell should be able to respond to external factors The result of the cell fusion produces the (growth factors and nutrients) hybrid cell and if we were to look at the activities of the cells, the S phase cell is expected to still undergo DNA replication However, the cell in the G1 phase was induced to enter the S phase even if it was not prepare to enter the S phase yet because G2-M TRANSITION there were soluble factors found in the S phase that were taken up by the cell in the G1 phase (b) Instead of using the cell in the S phase, they made use of the cell in the M phase fused with the G1 phase In the hybrid cell, the cell in the M phase continues to be in the M phase, however the cell in the G1 phase was induced to also undertake mitosis Even if it was not prepared to do so, it has not pass through the S1, no sister chromatids, the cell has not replicated yet But it has undergone condensation already, which is a feature of a cell undergoing mitosis DNA undergoes condensation in the prophase stage Nuclear membrane undergo disassembly Formations of the asters Features of a cell undergoing mitosis  There are soluble factors in the cell - When cell is fused in the M phase, soluble factors in undergoing mitosis which induce the cell in the M phase cells induce the cell in the S phase to the G1 phase to also enter the cell division also enter mitosis stage - Pulverized chromosome fragments Replicating DNA were induced to undergo condensation Replicating DNA molecules are very susceptible to degradation When they are induced to undertake condensation, it results in their fragmentation and pulverization - Chromosomes in the S phase cells were induced with soluble factors found in the cell in the M phase to also undertake mitosis as shown by induction to undertake condensation - Figure shows the chromosomes of the cells that were fused together - Chromosomes of the cell in the M phase Condensed They have sister chromatids - Chromosomes of the cell in the G1 phase They did not undergo the S phase yet, we do not see sister chromatids - G2M transition Since it was fused with a cell undergoing The first soluble factors that were mitosis, the soluble factors in the cells discovered found to be those that are undergoing mitosis induce the cell in the G1 involved in G2M transition phase to also undergo mitosis - The experiment shows how soluble factors involved Since the chromosomes did not undergo in that transition point were discovered gene replication, are also condensed already - Involves 2 cells Occurs during the prophase of mitosis Mature egg cell Primary oocyte (1) Cytoplasm is extracted from mature egg cell - Homologous chromosomes are not together anymore - The cell in undergoing meiosis 2 - Meiosis 1 Anaphase stage Homologous chromosomes are separated from each other - The cells aligned in Metaphase 2 - Sister chromatids are the ones to be separated from each other in Anaphase 2 (2) Extracted cytoplasm is injected into the primary oocyte - In the other cell (primary oocyte), in the ovaries, cell cycle is arrested generally in the Prophase of meiosis 1 - When bacterial from the cytoplasm of a mature egg cell, were inoculated to the primary oocyte, which has been arrested in Prophase 1 - Chromosomes of another hybrid cell - It induce the primary oocyte to undertake meiosis - Cell that is in the M phase, fused with a cell in the S (3) Meiosis is triggered phase - In the S phase cell, the DNA is being replicated  - Meiosis 1 because the homologous chromosomes are Towards the later part of mitosis, the still together concentration of cyclin drops - When it enters Anaphase 1, homologous As the cell enters another cell cycle, the chromosomes will separate from each other concentration of the mitotic cyclin increases (4) mature egg cell results through the different phases and it reaches - In Anaphase 2, sister chromatids will separate from its peak / M phase each other When M phase is about to be completed, the concentration drops - In the cytoplasm of the mature egg cell, you will have Cyclin concentration ↑ = activity of MPF↑ soluble factors that induce primary oocyte to also Cyclin concentration ↓ = activity of MPF↓ enter meiosis - Concentration of mitosis cyclin goes up and down - Soluble factors = maturation promoting factor - Concentration of the mitotic cyclin dependent kinase (MPF) / mitotic promoting factor is constant of the different phases of the cell cycle Involved in the transition of the cell from the G2 to mitosis; induce the cell to undergo mitosis A complex formed by 2 molecules 2 types of molecules that make the MPF: ○ Kinase (enzyme that catalyzes the addition of a phosphate to another molecule) Not functional by itself Function only with the presence of cyclin Cyclin dependent kinase Maturation promoting factor (MPF): mitotic - Different types of cyclin that are involved in the cyclin-dependent kinase different phases of the cell cycle (Cdk) - mitotic cyclin - MPF comprise of the mitotic cyclin (now called cyclin B) - Mitotic cyclin = cyclin B complex - Concentration of cyclin B increases when the cell is ○ Mitotic cyclin (cyclin B) about to enter the M phase because it is vital for the Concentration varies progression from the G2 to the M phase - Types of cyclin D1, D2, E, and A - There are different phases in the cell cycle would involve different types of cyclin - MPF activities vs. concentration of the cyclin that is shown in blue lines - Different stages / phases in the cell cycle indicated up - Y axis - relative concentration of the mitotic cyclin Concentration goes up and down - Mitotic cyclin Involved in the transition from G2 to M Induce the entry of the cell into mitosis, the cyclin concentration increase when nearing the M phase - G2-M transition - DNA replication checkpoint and DNA Cells in the G2 phase are prepared to enter damage checkpoint (in G2): phosphatase the M phase inhibited Checked for cell size, DNA damage, and DNA replication MITOTIC-CDK CYCLE When they pass the different checkpoints, they can now enter the M phase MPF - complex involved in the transition from G2 to M phase ○ Comprised of the mitotic cyclin dependent kinase and mitotic cyclin - Activation of mitotic Cdk-mitotic cyclin (cyclin B) complex Kinase cannot function with the absence of - Activated mitotic cyclin and mitotic Cdk complex cyclin (MPF) (1) Mitotic Cdk and mitotic cyclin form a complex Involved in the termination of mitosis (2) Inhibiting kinases - Concentration of the mitotic Cdk is constant all - 2 steps of phosphorylation which are done throughout the different stages of cell cycle by kinases - Mitotic cyclin molecule is absent in certain stages of - Phosphate that they add to the Cdk result in cell cycle the inactivation of this complex Concentration goes up towards the end of - Source of phosphate: ATP molecules G2 because is now needed in the formation (3) Activating kinase of MPF - Addition of another phosphate At the end of mitosis, it is degraded so that - Kinase that does that is called the activating mitosis is terminated and the next stages can kinase proceed Phosphate is added to the side in No mitotic cyclin in other stages as they are Cdk which eventually will lead to degraded the activation of the complex Cdk and cyclin complex, having 2 ACTIVE MITOTIC CDK-CYCLIN inactivating phosphate attached and ACTIVATE/INACTIVATES THROUGH 1 activating phosphate PHOSPHORYLATION Complex is still inactive 1. Nuclear envelope breakdown Still has inactive phosphate - Nuclear envelope of the cell undergoing attached to the Cdk mitosis/meiosis undergoes disassembly (4) Phosphatase - Spindle fibers have to attach to the - Enzyme that catalyzes the removal of chromosome found inside the nucleus and phosphate molecule sister chromatid have to travel to both poles - Removes the 2 inhibiting phosphate - Nuclear envelope is made out of 2 molecules leaving only the activating membranes phosphate attached to the Cdk - Inner nuclear membrane - Complex is now active, maturation Lined with nuclear lamina promoting factor Made up of protein subunits called - Serves to stimulate more phosphatases, lamin proteins activation of new complexes are faster  Active MPF causes the - Activity of the activated mitotic Cdk-cyclin phosphorylation of these lamin complex / activated MPF in the degradation of proteins proteins cyclin as well as securin When they are phosphorylated, - Anaphase stage they now become inactive When DNA is replicated during the S phase Nuclear lamina undergoes nuclear to form sister chromatids, molecules called degradation and nuclear membrane cohesin are attached to the sister chromatids undergo disassembly Remain attached to each other in prophase 2. Chromosome condensation and metaphase, they separate during - Chromosome undergoes condensation in the anaphase prophase stage Mitosis / Anaphase 2 in meiosis 2 - Active MFP kinase catalyzes the - When cell is not yet in anaphase stage, sister phosphorylation of the molecules called chromatids are still together condensin - When they are ready to enter the anaphase from - When it is phosphorylated, it is activated metaphase, we have the enzyme called separase / 3. Mitotic spindle formation separin - Mitotic spindle made out of microtubules Will catalyze the degradation of the cohesin are formed such that the sister chromatids are separated - Active MPF phosphorylates the microtubule from each other during anaphase associated proteins (AMP) They will brought to opposite poles so that - Activates the formation or the assembly of each of the daughter cells will have the the microtubules to form the mitotic spindle complete chromosomal material fibers / asters at the polar region of the cell - There should be a check so that this cohesin is not 4. Targeted protein degradation (cyclin, securin via degraded before the cell is ready to enter the APC/cyclosome) anaphase - Stimulate / activate the degradation of - The enzyme separase / separin is inhibit by a certain protein molecule called securin - Degradation of cyclin towards the end of - When the cell is now ready to enter anaphase, this mitosis so that the mitosis process is securin has to be degraded so that the separase will be terminated activate to do its function of destroying the cohesin - When there is no cyclin anymore towards - Activated MPF the end of mitosis because they are degraded The kinase catalyzes the addition of with the participation of the MPF, the phosphate to a molecule called Cdc 20 nuclear membrane will assembly, Cdc 20 is an activator of chromosomes undergo condensation and Anaphase-promoting complex (APC) microtubules undergo disassembly to the ○ An ubiquitin ligase tubulin subunits ○ Ligase is an enzyme that catalyzes the addition of a molecule (to tie) ○ Allow the attachment of protein molecule called ubiquitin to a target) ○ The securin molecule APC catalyzes the addition of ubiquitin molecules to securin Securin has ubiquitin molecules attached to it, it is marked for degradation by the proteasome Proteasome look for proteins that have ubiquitin attached to it Securin is marked for degradation by the proteasome Securin is not able to inhibit the separin / separase, such that the separin is not activated to catalyze the degraded of cohesin  allowing the sister chromatids to separate - Ubiquitin ligase = Anaphase-promoting complex from each other during anaphase (APC) - APC will catalyze the addition of ubiquitin to the target - Another side of the E3 is the target protein (cyclin, mitotic cyclin, and securin) which will be ubiquitinated - Proteasome recognizes that these molecules are targeted for degradation CHECKPOINTS I. Control by G1 Cdk-cyclin: Entry into S phase RESTRICTION POINT (START) 1. Mitotic Cdk-cyclin added PO4 to CDC, an activator of APC - Activated mitotic Cdk-cyclin allows the addition of phosphate groups to CDC20 molecule, wants it to be phosphorylated to become activated - Activates its target APC 2. Activated APC is an ubiquitin ligase, attached ubiquitin to: a. Securin and b. Mitotic cyclin - Once activated, binds ubiquitin molecules to securin and mitotic cyclin 3. Ubiquitinated screen and mitotic cyclin are marked for degradation by proteasome 4. Theses molecules are marked for degradation by the proteasome - Transition from G1 to S - There are several factors to be checked: Growth factors Nutrients Cell size DNA damage - Complex involved is the G1 Cdk-cyclin - Ubiquitin is a very small molecule - Ubiquitin is first attached to the ubiquitin-activating enzyme (E1) E1 attached to the ubiquitin E1 conjugated with E2 - Ubiquitin conjugating enzyme (E2) Allows the attachment of ubiquitin to its target through ubiquitin ligase - E1 attached to ubiquitin, then E1 attached to E2, E2 is bound to E3 (ubiquitin ligase) - Role of G1 Cdk-cyclin of the cell from G1 to the S phase - If the cell is not ready to enter the S phase then you Promotes cell proliferation have a protein called retinoblastoma protein - Growth factors are produced by certain cells of the When the gene coding for the body retinoblastoma protein has a mutation, it - If you have the correct growth factors interacting results in the occurrence of the cancer with the respective receptors, it will lead to the retinoblastoma activation of the Ras pathway Everyone has the retinoblastoma gene / - Once Ras pathway is activated, produces proteins that protein if you do not have the mutation are needed in the succeeding cycle (synthesis of - When the cell is still in the G1 and not ready to enter cyclin, E2f, and Cdk) the S phase, the rb inhibits the activity of E2F Allows the cell to leave G1 and enter the S E2F - transcription factor phase Molecule that sits on a region of the gene called the response element It regulates the transcription of that gene - E2F is a transcription factor that will upregulate, cause increase expression of genes coding for proteins needed in the S phase - Cell will now be allowed to enter the S phase because the cell has the proteins needed for the S phase - In the presence of a growth factor, there is a pathway called the Ras pathway Involves in the cell proliferation, cause cells to proliferate - Ras pathway is activated, there are molecules that are needed for the transition to the S phase that are - REVIEW OF EVERYTHING THAT WAS synthesize DISCUSSED UP G1 Cdk-cyclin - G2 to M phase - Kinase from G1 Cdk-cyclin can phosphorylate the Rb Mitotic Cdk - concentration is constant protein then it results in the inactivation of the Rb throughout the different phases of the cell - Rb proteins is inactive through phosphorylation, the cycle E2F is now activate and sits on the response element *dotted lines* - mitotic Cdk is not activated found in the genes coding for proteins that are yet required in the S phase Mitotic cyclin - not present during the other - Cell is now allowed to enter the S phase phases but builds up only towards the G2 to - There is a P21 molecule that inhibits G1 Cdk, the M transition cell does not progress from G1 to S Mitotic cyclin and mitotic Cdk as a complex If there is cell damage, P21 is synthesized *purple and blue* - inhibit by adding and the cell is arrested in G1 to allow the phosphates to molecules of those with repair of the damage addition of an activating phosphate 1 molecule of that and the phosphatase will remove the 2 molecules of the inhibiting phosphates Active MPF / active mitotic Cdk-cyclin complex ○ Allow the preparation of the cells to enter the M phase ○ Activity of the active MPF in allowing the disassembly of the nuclear envelope by phosphorylating the lamin proteins ○ Condensation of the DNA molecule by phosphorylating condensin and - Ras pathway activating condensin  ○ Formation of spindle fibers made Active molecules called Ataxia up of microtubules by telangiectasia mutated (ATM) / Rad-3 phosphorylating the microtubule related (ATR): protein kinases associated proteins ○ Individuals with mutation in the ○ Anaphase-promoting complex that gene causes the condition called leads to the degradation of the ataxia telangiectasia cyclin ○ Unsteady posture and unbalanced ○ Towards the end of the mitosis, the state; nerve cells in the cerebellum MPF is not active anymore because are affected the mitotic cyclin is not present ○ Occurrence of the dilatation of the anymore blood vessels of the eye, face, and ○ Allow the cell to form the nuclear other parts of the body envelope for the DNA to undergo ○ Important in addressing the decondensation for the presence of DNA damage microtubules to undergo - Normal conditions (no DNA damage): in the cell, we disassembly so that mitosis is have the synthesize of the molecule P53 terminated and the cell can proceed Guardian of the cell to the next stage of the cell cycle Involved in apoptosis - G1 to S phase No requirement to undergo the cell cycle Complex involved is the G1 Cdk-cyclin Does not have to undergo apoptosis Active G1 Cdk-cyclin phosphorylation the P53 is degraded Rb protein when the cell is ready to enter the ○ There is another ubiquitin ligase S phase Mdm2 Once the Rb is phosphorylated, it becomes ○ Catalyze the addition of ubiquitin inactive to P53 and proteasome will Activate the E2F, which is a transcription recognize P53 and undergo factor that will allow the synthesis of degradation proteins that are needed for DNA ○ We don't want active P53 replication, that way the cell enters from G1 - In the case of the presence of DNA damage, the to S phase ATM/ATR protein kinases are activated and add phosphates to checkpoint kinases (phosphorylated CHECKPOINTS other kinases) II. G1-S and G2-M: DNA damage checkpoint These kinases will phosphorylate the P53 - 2 transition points wherein the cell is Getting ATP using as the source of the checked before it is allowed to proceed to phosphate the next phase It cannot be acted on by the Mdm2 Transition from G1 to S P53 has to go to work (should not be Transition from mitosis to G2 destroyed) Kinases will add phosphate groups to P53 As it is phosphorylated, Mdm2 cannot cause the addition of ubiquitin to P53 P53 will not be degraded by the proteasome It can now function to address the problem of DNA damage Transcription factor (P53, ubiquitin ligase (Mdm2)) P53 is a transcription factor It will sit on the response element of certain genes so that it will be expressed There is upregulation in the expression of that gene Gene that is coding for the P21 - Damaged DNA molecule Activate certain proteins called MRN  P21 is a protein that inhibits the G21 ○ To prepare it for DNA synthesis Cdk-cyclin so that it will not phosphorylate during G1 phase — ORC, MCM, the retinoblastoma protein (Rb protein) helicase loaders attached to If Rb protein is not phosphorylated, the E2F origin of replication forming a remains inhibited, cannot upregulate the pre-replication complex gene coding for proteins that are needed for ○ After licensing done in G1 phase the S phase (cell remains in G1) Cell cycle arrest in G1 because of the (turn off) — by CdK and Gemini presence of P21 (produced during S phase) - There are cases wherein the P53 as a transcription CdK — how does it prevent factor will sit on the response element of the gene another round of synthesis — coding for the molecule called puma (P52 blocks relicensing and upregulated modulator of apoptosis) inhibits ORC, helicase Proapoptotic protein loaders and activate DNA Enhances apoptotic to take place resulting in synthesis programmed cell death Geminin — blocks binding Eliminating a cell with DNA damage that of MCM cannot be repaired You do not want to have a damaged cell ○ DNA undergo S phase and - Bcl-2 is a protein that inhibits apoptosis mitosis is completed and it Results to the death of the cell become active again during this time, CdK and CHECKPOINTS geminin were degraded Checkpoints III. G1 and S phase — DNA replication licensing IV. G2-M:DNA replication checkpoint translation of G2 to M-phase require checking of cell for completion of DNA replication Licensing of DNA replication during Eukaryotic cell Mitotic cyclins — involved in transition of cycle G2 to M phase Two events ○ addition of 2 inhibiting phosphate ○ licensing ensure that DNA is and molecule of activating replicated during S phase (in G1 phosphatase — inactive stage) need enzyme phosphatase ○ DNA gets replicated once per cycle (DNA replication checkpoint Green/G1 (licensing system is active) — and DNA damage checkpoint forming a pre replication complex and enter in G2 — if there are defects S phase it will be inhibited) — How is licensing done? remove two molecules of ○ DNA should have origin of inhibits phosphate leaving an replication activating phosphate  Mitotic CdK complex — What happens to chromosome don’t have spindle stimulates more process of fibers in kinetochore phosphate Wait signals — from Mad and Bub families and accumulate at unattached kinetochores ○ if attached — they block Cdc 20 and cannot activate APC Cdc20 cannot activate anaphase promoting complex — if inactive, securin molecules will not be V. Spindle checkpoint degrades and enzymes separase and cohesin in sister chromatids will not separate them checks if chromosome has spindle fibers — need to be attach to kinetochores during anaphase each of sister Examples of cancers due to defects in cell cycle chromatids is brought opposite poles regulation 1. Oncogene: its presence can trigger development of cancer Carried by viruses infecting man and animals From mutation of normal cellular genes (proto oncogenes) Oncogenes – encode proteins stimulate excessive cell proliferation and promote cell survival by inhibiting apoptosis held together through cohesin molecules centromere has protein called kinetochore Plasma membrane GTP-binding proteins Cell undergo cell division and the new KRAS nuclei formed – with chromosomes from Ras (protein product) ras pathway is both cancer and normal cells present and overactivity takes place; it will ○ Hybrid cell with normal growth maintained to be on and lead to excess control and some chromosomes are proliferation of cell loss (with tumor suppressor Point mutation (oncogene origin) gene) and manifested with cancer HRAS cell with uncontrolled growth NRAS BCL2 Bcl-2 (defective) inhibits apoptosis P53 upregulate synthesis of PUMA (activates apoptosis by inhibits Bcl-2) Inactive Bcl-2 – greater inhibition of apoptosis and defective genes are not remove and malignant cells are produced MDM2 Adds ubiquitin to P53 to be degraded Presence of Mdm2 – abnormal addition of ubiquitin on P53 (become unavailable to activate apoptosis – malignant cells produced) Gatekeeper gene APC 2. Tumor suppressor gene: Its loss or Standards for adenomatous polyposis coli – inactivation can lead to cancer affect Wnt signaling pathway Relates to function of inhibiting cell RB proliferation Retinoblastoma protein – If become non functional – E24 (upregulate expression of gene coding for proteins needed in S phase’ cell is not ready to enter S phase RB14 inhibit E24 and not allow it to enter)  Even if cell is not ready to enter S phase – E24 can still function as transcription factor upregulation P53 If the gene is inactivated – no apoptosis Cancer due to changes in Tumor suppressor genes 1. RB tumor suppressor gene Viral oncogene act on retinoblastoma protein Results in hereditary The effects of serum deprivation on growth of retinoblastoma – eye cancer in normal and transformed cells children - Cancer cells will grow with or without the Non Hereditary retinoblastoma – presence of growth factors unlike normal retina, lung, breast, bladder cells RB protein is the target for E7 Serum – liquid part of blood is loss proteins (oncogene of human ○ It is rich in growth factors papilloma virus) Broken line Blue – normals cells; not allow to cell cycle Blue line – normal cells with growth factors; complete cell cycles (high # of cells) Red – Broken line red – cancer cells without growth factors Red – cancer cells with growth factors Cancer Typically requires two successive mutations one in each of two copies of genes on two homologous chromosomes ○ If one lang (cancer is not expressed, it will be normal cell) But in oncogene – it only requires one mutation from an allele resulting in Hereditary – short manifestation of tumor suppressor gene ○ Normal egg and sperm (RB gene) which fertilizes the egg and mitosis happened resulting for the RB gene presence in all the cells – then there is a second copy with both RB gene in the alleles Non hereditary – long  ○ Normal egg and sperm and result in normal zygote; mitosis results in normal but one has mutation; second mutation – all cells have the RB gene; resulting in the manifestation of RB gene in both alleles 2. P53 tumor suppressor gene – TP53 in humans Li-Frraumeni syndrome (Cancer) ○ Due to mutation or viral oncogene, hereditary/non Wnt pathway (induced cell proliferation) hereditary, cancer in lung, Normal cell – No proliferation skin,breast Frizzled receptor and LRP6 (coreceptor) ○ Target for E7 protein for because it is not activated (no Wnt protein destruction – product of in receptor) oncogene of human ○ There is destruction complex in cell papilloma virus (Axin, APC, GSK3) – it can phosphorylate the Beta-catenin and Ubiquitin is attached to it Normal cell– Cell proliferation Wnt protein interaction with receptor – activate binding of Axin then no destruction complex (from first pic) will happen – Beta catenin will not be phosphorylated enter nucleus and bind to TCF and upregulate HPV is naked virus with double stranded synthesis coding for proteins resulting in DNA – causes genital warts leading to cell proliferation cervical cancer Cancer cell – Cell Proliferation - RB protein sits in E2F and inhibits function Defected APC and it will not be produced of E2F transcription factor so it will not and destruction complex will be absent upregulate expression of gene coding for and even if there is a Wnt protein; proteins needed in S phase and it will not B-catenin will not be phosphorylated and enter S phase from G1 ubiquitin will not be degraded and not enter ○ E7 product – bind to RB and it will nucleus to have a function like in normal cell not bind to E2F and enter S phase – always on resulting in uncontrolled even if it is not checked and lead to growth of cancer cancer cells ○ E6 product – targets and bind to Apoptosis (AY-PAW-TOE-SIS) P53 resulting the addition of Ubiquitin and P53 is degraded and apoptosis is not activated 3. APC tumor suppressor gene Adenomatous polyposis coli Inherited familial AP and in inherited and non inherited colon cancer and involved in Wnt pathway  Cell starting apoptosis Necrosis Inactivation of focal adhesion kinase– Extensive vacuolation of cytoplasm disruption of cell adhesion (loses its shape Mitochondrial swelling and cytoplasm will shrink); undergo Dilation of ER degradation of the cell Rupture of plasma membrane Caspase– activated DNAse cytoskeletal Cell lysis without formation of vesicles changes Cellular content are liberated into ○ With blebs (circle) intracellular space: damage to neighboring Phosphatidyl-serine in outer leaflet – cell evoke inflammatory responses scramblase Increase intracellular Ca2+ Remnants of dead cell ingested by Independent of caspase (in apoptosis), and phagocytic cell instead, cytosolic calpains Caspases Conditions Hydrolytic enzymes; cysteine-dependent ○ Ischaemia, hypoxia, stroke, aspartate-directed proteases alzheimer's disease, huntington, Cysteine in active site; cleaves proteins at parkinson’s and amyotrophic lateral aspartate-containing site sclerosis Initiator (2,8,9,10); Executioner (3,6,7) Apoptosis Targets of Caspases 1. For proper development Target protein kinases –inactivation of FAK Tadpole tail Cleaves Lamins (molecules forming nuclear Tissue in fingers and toes of fetus lamina of nuclear envelope) Endometrium at start of Proteins of cytoskeleton menstruation Removal of surplus cell in synapses of neurons in brain 2. To destroy unwanted cells that affect integrity of organism Cells infected with viruses induced by cytotoxic T lymphocytes Macrophages with phagocytized bacteria – have to be eliminated via apoptosis Cells with DNA damage Cells at end of life span (RBC,WBC)  Extrinsic pathway of apoptosis ○ plasma membrane undergo apoptosis – with cell death receptor (CD95 Fas receptor and ligand) ○ Cell death signals – carried by cytotoxic T cells and want to eradicate cells with virus Attach to receptors like FAS Receptor to undergo trimerization and activates adaptor (FADD) to be attach to receptor and recruit Procaspase - 8, activated to be Caspase 8 and activated executioner Procaspase 3 and form caspase 3 (executioner caspase) which complete process of apoptosis Intrinsic pathways of apoptosis – receptor in plasma membrane not involved ○ Trigger activate death promoting protein (Bax or Bad) counteract activity of Bcl-2 (inhibits apoptosis – antiapoptotic protein) – prevent inhibition of apoptosis by Bcl-2 – How does apoptosis happen? allows release of Cytochrome c form mitochondrion 3 molecules form complex called Apoptosome which activate initiator Procaspase 9 to become Caspase 9 and act to activate executioner caspase 3 so apoptosis is effective (cytochrome C, Apaf-1, Procaspase-9) Triggers (oxidative stress, lack of growth) lead P53 to  transcription of genes producing pro apoptotic protein like Death promoting protein and PUMA inhibit Bcl-2 Some reference – lack of survival factors (lack of growth) is the third mechanism by apoptosis can be activated but others are not