2 The Cell Cycle There are two major parts in the cell cycle: Interphase: G1 = gap1 S = DNA synthesis G2 = gap2Mitosis: MThere are two essential functions of the cell cycle:To ensure that each chromosomal DNA molecule is replicated only once per cycleTo ensure that the identical replicas of each chromosome are distributed equally to the two daughter cells
4 The Cell Cycle The cell cycle is under genetic control A fundamental feature of the cell cycle is that it is a true cycle: it is not reversibleMany genes are transcribed during the cell cycle just before their products are neededMutations affecting the cell cycle have helped to identified the key regulatory pathways
5 The Cell CycleProgression from one phase to the next is propelled by characteristic protein complexes, which are composed of Cyclins and cyclin-dependent protein kinases (CDK)Expression of mitotic cyclins E, A, and B are periodic, whereas cyclin D is expressed throughout the cell cycle in response to mitosis stimulating drugs (mitogens)The cyclin-CDK complexes phosphorylate targeted proteins, changing dramatically their activity
7 The Retinoblastoma Protein The retinoblastoma (RB) protein controls the initiation of DNA synthesis.RB maintains cells at a point in G1 called the G1 restriction point or start by binding to the transcription factor E2F, until the cell has attained proper sizeIf the cycling cell is growing properly and becomes committed to DNA synthesis, several cyclin-CDK complexes inactivate RB by phosphorylationAfter cell enters S phase E2F becomes phosphorylated as well and loses its ability to bind DNA
9 The Cell CycleThe progression from G2 to M is controlled by a cyclin B-CDK2 complex know as maturation-promoting complexProtein degradation (proteolysis) also helps regulate the cell cycle. The anaphase-promoting complex (APC/C), which is a ubiquitin–protein ligase responsible for adding the 76-amino-acid protein ubiquitin to its target proteins and marking them for destruction in the proteasomeProteolysis eliminates proteins used in the preceding phase as well as proteins that would inhibit progression into the next
10 CheckpointsCells monitor their external environments and internal state and functionsCheckpoints in the cell cycle serve to maintain the correct order of steps as the cycle progresses; they do this by causing the cell cycle to pause while defects are corrected or repairedCheckpoints in the cell cycle allow damaged cells to repair themselves or to self-destruct
11 Three Main Checkpoints A DNA damage checkpointA centrosome duplication checkpointA spindle checkpoint
13 A DNA Damage Checkpoint A DNA damage checkpoint arrests the cell cycle when DNA is damaged or replication is not completed.In animal cells, a DNA damage checkpoint acts at three stages in the cell cycle: at the G1/S transition, in the S period and at the G2/M boundaryThe p53 transcription factor is a key player in the DNA damage checkpoint.
14 A DNA Damage Checkpoint In normal cells, level of activated p53 is very lowProtein Mdm2 keeps p53 inactivated by preventing phosphorylation and acetylation of p53 and by exporting p53 from the nucleusDamaged DNA leads to activation of p53 and its release from Mdm2
15 A DNA Damage Checkpoint Activated p53 triggers transcription of a number of genes - p21, s, Bax, Apaf1, Maspin,GADD45DNA damage detected in G1 blocks cell G1/S transitionDNA damage in S phase reduces processivity of DNA polymerase and gives the cell time for repairProcessivity: number of consecutive nucleotides that replicate before polymerase detaches from templateDNA damage detected in S or G1 arrests cells at G2/M transition
17 A DNA Damage Checkpoint DNA damage also triggers activation of a pathway for apoptosis = programmed cell deathWhen the apoptotic pathway is activated, a cascade of proteolysis is initiated that culminates in cell suicideThe proteases involved are called caspases
18 Centrosome Duplication Checkpoint Monitors spindle formationFunctions to maintain the normal complement of chromosomesSometimes coordinates with the spindle checkpoint and the exit from mitosis
19 The Spindle Checkpoint Monitors assembly of the spindle and its attachment to kinetochoresThe kinetochore is the spindle-fiber attachment site on the chromosomeIncorrect or unbalanced attachment to the spindle activates spindle checkpoint proteins, triggers a block in the separation of the sister chromatids by preventing activation of the anaphase-promoting complex (APC/C)
21 CancerCancer cells have a small number of mutations that prevent normal checkpoint functionCancer is not one disease but rather many diseases with similar cellular attributesAll cancer cells show uncontrolled growth as a result of mutations in a relatively small number of genesCancer is a disease of somatic cells
22 Cancer1% of cancer cases are familial: show evidence for segregation of a gene in pedigree99% are sporadic: the result of genetic changes in somatic cellsWithin an organism, tumor cells are clonal, which means that they are descendants from a single ancestral cell that became cancerous.
23 Cancer Cells vs. Normal Cells In normal cells, cell-to-cell contact inhibits further growth and division, a process called contact inhibitionCancer cells have lost contact inhibition: they continue to grow and divide, and they even pile on top of one another
24 Cancer Cells vs. Normal Cells Even in the absence of damage, normal cells cease to divide in culture after about 50 doublings = cell senescenceSenescence of normal cells is associated with a loss of telomerase activity: the telomeres are no longer elongated, which contributes to the onset of senescence and cell deathCancer cells have high levels of telomerase, which help to protect them from senescence, making them immortal
25 Key Mutational Targets Many cancers are the result of alterations in cell cycle control, particularly in control of the G1-to-S transitionThese alterations also affect apoptosis through their interactions with p53The major mutational targets for the multistep cancer progression are of two types:Proto-oncogenesTumor-suppressor genes
26 Key Mutational Targets The normal function of proto-oncogenes is to promote cell division or to prevent apoptosisThe normal function of tumor-suppressor genes is to prevent cell division or to promote apoptosis
27 OncogenesOncogenes are derived from normal cellular genes called proto-oncogenesOncogenes are gain-of-function mutations associated with cancer progressionOncogenes are gain-of-function mutations because they improperly enhance the expression of genes that promote cell proliferation or inhibit apoptosis
28 OncogenesMdm2:Amplification of Mdm2 gene and overexpression of Mdm2 protein leads to inactivation of p53 geneAmplification of Mdm2 gene has been found in many tumors of adipose tissue, soft tissue sarcomas, osteosarcoma, and esophageal carcinomaCyclin D and CDK4:Amplification and overexpression leads to unscheduledentry to S phaseAmplification and/or overexpression has been found in many esophageal carcinomas, bladder and breast cancers
29 Oncogenes Growth-factor receptors: Cellular growth factors stimulate growth by binding to a growth-factor receptor at the cell membraneThe binding activates a signal transduction pathway that acts through Ras, cyclin D, and its partner CDKs.Amplification and overexpression of the gene that encodes the receptor for epidermal growth factor (EGFR) has been found in many malignant astrocytomas, glioblastomas, breast and ovarian cancers, head and neck cancers and melanomas.
30 OncogenesRas:The Ras protein acts as a switch in stimulating cellular growth in the presence of growth factorsCertain mutant Ras proteins lack GTPase activity and remain in the form of Ras–GTP. The signal for cellular growth is transmitted constitutively--unrestrained growth and division take placeFig
31 Tumor-Suppressor Genes Tumor-suppressor genes normally negatively control cell proliferation or activate the apoptotic pathwayLoss-of-function mutations in tumor-suppressor genes contribute to cancer progression.
32 Tumor-Suppressor Genes Loss of function of p53 eliminates the DNA checkpoint that monitors DNA damage in G1 and SThe damaged cells survive and proliferate and their genetic instability increases the probability of additional genetic changes and thus progression toward the cancerous statep53 proves to be nonfunctional in more than half of all cancersMutant p53 proteins are found frequently in melanomas, lung cancers, colorectal tumors, bladder and prostate cancers, and astrocytomas.
33 Tumor-Suppressor Genes Loss of p21 function results in renewed rounds of DNA synthesis without mitosis, and the level of ploidy of the cell increases. Mutations in the p21 gene occur in some prostate cancers.p16/p19ARF:The p16 and p19ARF proteins are products of the same gene transcribed from different promoters The p16 product can inhibit the cyclin D–Cdk4 complex and help control entry into S phase The gene is deleted in many gliomas, mesotheliomas, melanomas, nasopharyngeal carcinomas, biliary-tract and esophageal carcinomas.
34 Tumor-Suppressor Genes RB:The retinoblastoma protein controls the transition from G1 to S phase by controlling the activity of the transcription factor E2F Loss of RB function frees E2F hence excessive rounds of DNA synthesis are continuously being initiated.Loss of RB function is found in melanomas, small-cell lung carcinoma, osteosarcoma and liposarcomas
35 Tumor-Suppressor Genes Bax:The Bax tumor-suppressor protein promotes apoptosisLoss of Bax function is found particularly in gastric adenocarcinomas and in colorectal carcinomas associated with microsatellite instability because of defective mismatch repair. Cells that are defective in mismatch repair are prone to undergo replication slippage leading to deletions or additions of nucleotides in runs of short tandem repeats
36 Tumor-Suppressor Genes Bub l:Bub1 is a protein that is primarily involved in the spindle checkpointA subset of colon cancers show chromosomal instability. Some of these unstable lines are defective in Bub1.
37 Familial CancersMutations that predispose to cancer can be inherited through the germ lineThe presence of this mutation predisposes the individual to cancer, because it reduces the number of additional somatic mutations necessary for a precancerous cell to progress to malignancy
38 Li–Fraumeni SyndromeThe Li–Fraumeni syndrome shows clear autosomal dominant inheritance. However, the affected individuals have a range of different tumors and often have more than one, including osteosarcoma, leukemia, breast cancer, lung cancer, soft-tissue sarcoma, and brain tumorsA large fraction of Li–Fraumeni families show segregation for a mutation in the p53 gene.A situation analogous to the human Li–Fraumeni syndrome has been created in mice by experimental knockout (loss of function) of the p53gene via the germ-line transformation
40 RetinoblastomaRB protein in animal cells holds cells at restriction point by binding to and holding E2FAlfred Knudson in 1971 suggested that loss of the wildtype allele of a tumor-suppressor gene might be the triggering event at the cellular level for tumors in heterozygous genotypes
41 RetinoblastomaKnudson suggested that genesis of a tumor in familial cases of RB required a “single hit” in a somatic cell, whereas genesis of a tumor in sporadic cases required “two hits”RB is inherited in pedigrees as a simple Mendelian dominant. But Knudson’s hypothesis implied that even in familial cases, there must be another mutational event that triggers tumor development.Analysis of genetic markers around the gene in tumor cells revealed that the triggering event is the loss of the wildtype RB1 allele
42 RetinoblastomaAt the organismic level, mutant gene is dominant. At the cellular level, it is recessive.Several mechanisms can uncover the mutant allele: chromosome loss, mitotic recombination, deletion, and inactivating nucleotide substitutionsUncovering of the recessive allele by various mechanisms is called loss of heterozygosityRetinoblastoma (RB) is an inherited cancer syndrome associated with loss of heterozygosity in the tumor cells.
43 Defects in DNA RepairGenetic instability clearly contributes to the origin of tumor cellsSome inherited cancer syndromes result from defects in processes of DNA repairInherited skin cancer syndromes are called xeroderma pigmentosumXeroderma pigmentosum cells are defective in nucleotide excision repairIndividuals with this syndrome are very sensitive to ultraviolet light
44 Acute LeukemiasAcute leukemias are malignant diseases of the bone marrow, spleen, and lymph nodes associated with uncontrolled proliferation of leukocytes and their precursors in the bone marrowAcute leukemias do not arise as a consequence of alterations in cell cycle regulation or checkpoints, nor are they familialUp to 60% of acute leukemias result from a chromosomal translocation that fuses a transcription factor with a leukocyte regulatory sequence
45 Acute Leukemias The translocations are of the two types: promoter fusion—the coding region for a gene that encodes transcription factor is translocated near an enhancer for an immunoglobulin heavy-chain gene or a T-cell receptor genegene fusion—found more frequently in acute leukemia than a promoter fusion.the translocation breakpoints occur in introns of genes for transcription factors in two different chromosomes. The result is a fusion gene called a chimeric gene composed of parts of the original gene