CHAPTER 13 Molecular Genetics of the Cell Cycle and Cancer Cancer results from mutations that overcome the normal limits to the number of cell divisions that can take place before a cell dies 13 1 The cell cycle is under genetic control The spindle pore is organized around a small region of clear cytoplasm near the centrosomes Centrioles are more particular in appearance both are microtubule organizing centers Centrosome duplication begins late in G1 and is complemented during S phase Completion of DNA replication marks the beginning of G2 Many genes are transcribed during the cell cycle just before their product is needed Red indicates overexpression and green indicates under expression Enzymes needed for synthesis of the trinucleotide precursors of DNA and for DNA replication are made in G1 immediately prior to their use in S phase Mutations affecting the cell cycle have helped to identify the key regulatory pathways Temperature sensitive cell division cycle cdc mutants are typically wildtype at 23 degrees Celsius the permissive temperature but unable to complete the cell cycle at 36 degrees Celsius the restrictive temperature One mutant designated cdc13 that causes a block an arrest at the G2 M boundary Cyclins and cyclin dependent protein kinases propel the cell through the cell cycle All eukaryotes regulate progression through the cell cycle by means of cyclin CDK complexes Active cyclin CDK complexes result in the activation of a transcription factor that leads to transcription of the next cyclin needed in the cell cycle cause transcriptional activation of genes other than cyclins Phosphorylation may activate one enzyme but inactivate another Phosphate enzymes dephosphorylate proteins that the cyclin dependent kinases have phosphorylated The retinoblastoma protein controls the initiation of DNA synthesis Cyclin A combines with Cdk2 in S phase and with Cdc2 in late S and G2 phase Cyclin B combines with Cdc2 and Cyclin E combines with Cdk2 Cyclin D combines with Cdk4 and Cdk6 A mitogen is a chemical or treatment that stimulates the mitotic cell cycle The normal role of the RB protein is to maintain cycling cells at a point in G1 called the G1 restriction point RB holds transcription factor E2F in an inactive form Phosphorylation of RB eliminates its ability to bind the E2F transcription factor E2F becomes active and stimulates transcription of genes and translation of enzymes for DNA synthesis including DNA polymerase cyclin E cyclin A and Cdk2 After entering S phase cyclin A Cdk2 phosphorylates E2F and inhibits its binding to DNA This cyclinA Cdk2 is required throughout S phase to keep RB phosphorylated The progression from G2 to M is controlled by a cyclin B Cdc2 complex maturation promoting factor Protein degradation also helps regulate the cell cycle Proteolysis protein degradation eliminates proteins used in previous phase keeps cycle moving in one direction Exit from mitosis requires the destruction of cyclin B caused by anaphase promoting complex APC C which is an ubiquitin protein ligase that adds ubiquitin to target proteins 13 2 Checkpoints in the cell cycle allow damaged cells to repair themselves or to self destruct Defects in centrosome duplication or segregation arrest cells at the G2 M transition Nicks in DNA block the initiation of replication and arrest cells at the G1 S transition Blocked movement of replication fork arrests cells at the G2 M transition Double stranded DNA breaks arrest cells in the G2 M transition A chromosome that is not attached at the spindle blocks the onset of anaphase Checkpoints in the cell cycle serve to maintain the correct order of steps with respect to each other as the cycle progresses they do this by causing the cell cycle to pause while defects are corrected or repaired The three principal checkpoints are DNA damage centrosome duplication and spindle The p53 transcription factor is a key player in the DNA damage checkpoint DNA damage checkpoints occur at G1 S S and G2 M Normally p53 activation levels are low and are kept low by Mdm2 In order to function p53 must be activated by Phosphorylation and acetylation Mdm2 binds to p53 to prevent phosphorylation and also exports p53 out of nucleus GADD45 binds to proliferating cell nuclear antigen PCNA blocking its role as a processivity factor for DNA polymerase and hence blocking DNA replication functions directly in DNA repair Bax acts as a positive regulator of apoptosis The G1 s checkpoint is mediated by activated p21 Caspases are proteases involved in apoptosis P53 can activate apoptosis pathway by activating Bax and Apaf1 and blocking Bcl2 Bcl2 is an inhibitor of apoptosis Activation of oncogenes or cancer genes increases the level of activated Bcl2 which prevents apoptosis and allows the growing of cells indefinitely Increased Bax due to p53 activation results in apoptosis increased Bcl2 due to oncogene activation blocks apoptosis and cells grow indefinitely Balanced normal cells The centrosome duplication checkpoint and the spindle checkpoint function to maintain the normal complement of chromosomes Activation of cyclin B Cdc2 kinase is correlated with centrosome duplication and formation of the spindle When Cdc20 is activated APC C is also activated Failure of any checkpoint results in genetic instability 13 3 Cancer cells have a small number of mutations that prevent normal checkpoint function Cancer is a disease of somatic cells 1 is familial segregation of gene 99 sporadic Cancer cells are characterized by loss of growth factor dependence insensitivity to anti growth signals evasion of apoptosis immortality ability to metastasize and sustained angiogenesis Many cancers evolve from cell cycle alterations in G1 S transition The normal function of proto oncogenes is to promote cell division or to prevent apoptosis the normal function of tumor suppressor genes is to prevent cell division or to promote apoptosis Proto oncogenes normally function to promote cell proliferation or to prevent apoptosis Oncogenes are gain of function mutations associated with cancer progression They are derived from normal cellular genes called proto oncogenes PROTO ONCOGENES cyclin D and Cdk4 overexpression from gene amplification promotes unscheduled entry into S phase Ras activated when bound to GTP overexpression leads to enhanced transduction and proliferation Tumor suppressor genes normally act to inhibit cell proliferation or to promote apoptosis Loss of function mutations in tumor