GIM 201 Lesson 2 - Cell Cycle PDF

The mammalian cell cycle The mammalian growth-and-division cycle is divided into four phases: G1, the first growth period of the cell cycle,...

The mammalian cell cycle The mammalian growth-and-division cycle is divided into four phases: G1, the first growth period of the cell cycle, during interphase, the cell responds to its environment and synthesizes nucleotides and organelles S, during which DNA is replicated G2, second growth period of the cell cycle, rapid cell growth and protein synthesis during which the cell readies itself for mitosis M, Mitosis – cell division G0 is a period in the cell cycle in which cells exist in a quiescent state Cell Cycle Checkpoints Checkpoints impose quality control to ensure that a cell has properly completed all the requisite steps of one phase of the cell cycle before it is allowed to enter into the next phase DNA damage checkpoints: entrance into S phase is blocked if genome is damaged During S phase, DNA replication halted if genome is damaged Entrance to M phase blocked if DNA replication is not completed. During Mitosis, Anaphase blocked if chromatids are not properly assembled on mitotic spindle.  Cell Cycle Checkpoints The cell faces a number of points in the cell cycle where it has to satisfy certain molecular requirements before it is permitted to continue along the cell cycle. SAC Loading… G1 G2 CHECKPOINT CHECKPOIN CHECKPOIN T T  The mammalian cell cycle WHAT REGULATES ENTRY IN EACH CELL CYCLE PHASE?  Cell cycle control Different CDK complexes regulate entry and exit of different cell cycle phases cyclin cyclin cyclin E A B cyclin D Loading… cyc cyc cyc cyc A B D E CDK CDK CDK CDK 2 1 6 2 cyc D CDK 4 The cell cycle as governor of growth and proliferation  Restriction point Responsiveness to extracellular signals during the cell cycle only in a discrete window that begins at the onset of G1 and ends just before the end of G1. The end of this time window is designated the restriction point (R), which denotes when a cell must make a commitment to advance through the cell cycle, remain in G1 or retreat from the active cell cycle to G0. Rb family plays a major role in controlling the restriction point (R)!  Cell cycle dependent phosphorylation of Rb RB is hypo-phosphorylated on small number serine/threonine residues by CDK4/6 As cell cycle moves through the R point becomes hyper-phosphorylated by CDK2 Exit from mitosis (M), protein phosphatase type 1 (PP1) removes phosphate groups  Control of ’R’ point by mitogens Mitogens: factors that induce cell proliferation, regulate cell cycle entry Restriction points is regulated by Rb phosphorylation (not expression!) by CDKs ! E2F and Rb interaction is regulated by phosphorylation When Rb binds E2F, it blocks their transcription activating domains, other proteins also bind to this complex and repress transcription. After passing R point, the Rb protein becomes hyper- phosphorylated and then released E2Fs, allowing them to activate transcription of genes involved in late G1. As cell enters the S Phase, the E2Fs are inactivated/ degraded. CDK Inhibitors: CDKI CDK inhibitors block the actions of CDKs at various points in the cell cycle. The four CDKI (p16,p15, p18, Loading… p19) are specialized to inhibit the cyclin D-CDK4/6 complexes that are active in early and mid-G1. p57, p27 and p21 inhibit the remaining complexes throughout the cycle.  CDK Inhibitors: CDKI p21 and p27 can stimulate CDK4/6 while inhibiting CDK2  CDK Inhibitors: CDKI At the ‘R’ point all CDK4/6 and CDK2 are bound to p21/p27 But p21/p27 have higher affinity to CDK4/6 As more CDK4/6-cycD complexes form, p21/p27 gets displaced from CDK2 to bound CDK4/6!  Senescence What defines senescence? Lack of cell division Irreversible growth arrest – senescence vs quiescence Cells are still metabolically alive Phase I: active cell division Phase II: exponential growth Phase III: senescence The proliferative capacity of primary human fibroblasts taken from embryos Cells remain viable but are unable to proliferate again  The Hayflick limit Alexis Carrol French surgeon and Nobel laureate who stated cells in culture were immortal and claimed to have grown chicken heart cells in culture for 34 years. In fact the culture medium was probably supplying fresh chicken stem cells… Leonard Hayflick Couldn’t repeat these findings. In 1961 published studies on fibroblasts in culture, that could proliferate up to 50 times before becoming senescent. This idea that normal cells in culture can only divide a set number of times before division stops is now called “The Hayflick limit”  But how cells achieve immortality? - Telomeres Telomere shortening causes: End to end fusions of chromosomes Karyotype chaos and widespread death by apoptosis Cellular crisis after telomeres are critically shortened  Telomeres Red: mitotic chromosomes Green: telomere marker Figure 10.11 The Biology of Cancer (© Garland Science 2007) Normal metaphase chromosomes TRF2 -/- cells with multiple chromosome fusions TRF2 is critical for telomere maintenance TRF2 is a component of the shelterin  Telomeres structure Telomeres are 6-nucleotide repeated G-rich sequences on the 3’ strand Repeats can average 5-10 kilobases Repeated sequence in humans  Telomeres structure Telomeres form a capped end structure that is associated with a T-loop and multiple protein complexes No free dsDNA end Telomeres – protein complexes Telomeric DNA is protected from degradation by a group of physically associated proteins: the shelterin complex This is present in multiple copies per telomere and also is responsible for maintaining the structure of the T-loop Telomere end replication problem Ineffective copying of the ends of chromosomes during S-phase of the cell cycle An RNA primer initiates the ‘lagging strand’ synthesis The nucleotide sequence at the tip of the template strand is not properly copied Exonucleases within cells may also erode telomeres Critically – telomeres may lose 50 – 100 base pair of DNA each cell division  Telomere shortening The length of telomeric DNA shortens as cells proliferate Telomeres shorten from 10-15kb (in germ cells) to 3-5kb after 50-60 population doublings  Telomeres and DNA damage Figure 10.14a The Biology of Cancer (© Garland Science 2007) HOW CAN TELOMERE SHORTENING BE PREVENTED?  Telomerase Telomerase (TERT) is a reverse transcriptase which is unique in carrying around its own RNA template Defence against telomere shortening TERT is the limiting factor and can be used to immortalise cells  Telomerase Eukaryotic chromosomes are linear and so use the telomerase enzyme to counteract the chromosome shortening due to DNA replication Telomerase is expressed in embryonic stem cells and some adult stem cells but it absent (or very low) in most somatic cells  Telomerase and cancer Cancer cells often escape crisis by expressing telomerase Telomerase activity is detectable in 85 to 90% of human tumour cell samples The telomerase holoenzyme contains two subunits: human Telomerase Reverse Transcriptase (hTERT) catalytic subunit and hTR RNA subunit hTERT expression is critical – this is the limiting factor in cells Expression of TERT in HEK cells makes them immortal  Telomeres are repetitive nucleotide sequences at the ends of linear chromosomes. In humans, telomeres consist of repeats of the sequence TTAGGG. These structures play a crucial role in maintaining chromosome stability and protecting the ends of chromosomes from being recognised as double-strand breaks, which could otherwise trigger unwanted DNA repair processes. Structure of Telomeres TTAGGG Repeats: The telomeric DNA consists of thousands of tandem repeats of the sequence TTAGGG in vertebrates. Telomere Loop (T-loop): The telomere folds back on itself to form a loop structure, protecting the chromosome end. Shelterin Complex: A protein complex that binds to telomeres to protect them from being recognised as DNA damage. The shelterin complex includes proteins such as TRF1, TRF2, POT1, and TIN2.  Functions of Telomeres Protection of Chromosome Ends: Telomeres prevent the ends of chromosomes from being mistaken for broken DNA by forming the protective T-loop. This prevents chromosome ends from undergoing inappropriate repair mechanisms such as end-to-end fusions. Prevention of DNA Loss: During DNA replication, the enzyme DNA polymerase cannot fully replicate the 3' ends of linear chromosomes, leading to progressive shortening with each cell division. Telomeres buffer Loading… this loss of genetic material by ensuring the important coding sequences remain intact. Regulation of Cellular Aging: Telomeres shorten with each round of cell division due to the "end- replication problem." When telomeres become too short, cells enter a state called senescence (a permanent growth arrest) or undergo apoptosis (programmed cell death). This process limits the number of divisions a cell can undergo, a concept known as the Hayflick limit.  Telomerase and Telomere Maintenance Telomerase Enzyme: Telomeres are maintained by the enzyme telomerase, a reverse transcriptase that extends telomeres by adding repeats to the 3' end of the DNA strand. Telomerase is composed of two key components: i. TERT (Telomerase Reverse Transcriptase): The protein component that synthesises new DNA. ii. TERC (Telomerase RNA Component): Provides the RNA template that directs the addition of the TTAGGG repeats. Activity in Different Cells: iii. Germ cells and stem cells express high levels of telomerase to maintain long telomeres and ensure unlimited replication potential. iv. Somatic cells express low or undetectable levels of telomerase, leading to gradual telomere shortening as these cells divide. v. Cancer cells often reactivate telomerase, allowing them to bypass the senescence limit and divide indefinitely, contributing to uncontrolled growth and tumorigenesis.  Telomeres, Aging, and Disease Telomere Shortening and Aging: As telomeres shorten, cells lose their ability to divide. Short telomeres are associated with aging and age-related diseases, such as cardiovascular disease and weakened immune function. Telomere length is considered a biomarker of cellular aging. Telomeres and Cancer: Cancer cells often evade the normal regulatory mechanisms of cellular aging by upregulating telomerase, allowing them to maintain telomere length and continue dividing. Targeting telomerase is an emerging strategy for cancer therapy. Dyskeratosis Congenita: A genetic disorder caused by mutations in telomerase components (e.g., TERT, TERC) that results in premature telomere shortening. Affected individuals experience bone marrow failure, skin abnormalities, and increased cancer risk.  Mitosis Mitosis serves fundamental functions such as growth, tissue repair, and asexual reproduction in single-celled organisms. The primary objective is to produce two daughter cells that are genetically identical to the parent cell, thus maintaining the integrity of the genetic material across cell generations. Mitosis involves a single division that includes the following phases: i. Prophase: Chromosomes condense, nuclear envelope breaks down, spindle apparatus forms. ii. Metaphase: Chromosomes align at the cell equator. iii. Anaphase: Sister chromatids separate and move toward opposite poles. iv. Telophase and Cytokinesis: Chromosomes decondense, nuclear envelopes reform, and the cell divides.  Meiosis Meiosis consists of two sequential stages, Meiosis I and Meiosis II, each with similar phases but distinct outcomes: i. Meiosis I: Prophase I: Homologous chromosomes pair and exchange segments (crossing over). Metaphase I: Paired homologous chromosomes line up along the cell equator. Anaphase I: Homologous chromosomes separate, reducing the chromosome number by half. Telophase I: Cells divide to form two haploid cells. ii. Meiosis II: Resembles mitosis where sister chromatids in each cell separate, leading to four genetically unique haploid cells.  Metaphase Chromosomes Cytogenetic analyses are almost always based on examination of chromosomes fixed during mitotic metaphase. During that phase of the cell cycle, DNA has been replicated and the chromatin is highly condensed. The two daughter DNAs are encased in chromosomal proteins forming sister chromatids, which are held together at their centromere. The centromere is the structure where the mitotic spindle attaches prior to segregation. Genetic crossover Chromosomes line up next to each other during synapsis and allow crossing over of genetic information, via chiasmata. This exchange of genetic information is critical in allowing an increase in the possible combinations of genetic information in the gametes. Each diploid spermatogonium produces four haploid sperm cells Each diploid oogonium produces three polar bodies and one haploid ovum Conclusion: The cell cycle is strictly controlled with checkpoints in place to ensure that it does not progress if there are problems with replication. Cyclin dependent kinases and cyclins play key role. The tumour suppressor, retinoblastoma, is critical in controlling progression through the restriction point, where the cell cycle changes from responding to its environment to being totally autonomous. Telomeres play a vital role in protecting the ends of DNA, and must be lengthened by telomerase in order for cells to overcome the Hayflick limit. Mitosis and meiosis show distinct differences that allow generation of either faithfully copied diploid cells (mitosis) or genetic crossover leading to haploid gametes with differing genomes (meiosis).