Prof J.E.Brown Password: CCL

Outline of Lectures  Anthracyclines, Dactinomycin; Bleomycin, Mitomycin; Procarbazine and their mechanisms of action  Drugs inhibiting microtubule formation eg: vinblastine, vincristine; Microtubule enhancers eg: Paclitaxel  Nucleotide biosynthesis; Cytidine analogues, fluorinated pyrimidines, purine analogues  Hormone antagonists - Use of and mechanism of action of anti- oestrogens such as tamoxifen, anti-androgens and adrenal hormone synthesis inhibitors. 2

Lecture 2  Cell Cycle  Mitosis  Mitotic Spindles  Drugs inhibiting microtubule formation: vinblastine, vincristine;  Microtubule stabilisers e.g. taxanes including paclitaxel, docetaxel 3

4 4 Cell Cycle  Cells replicate in a characteristic way and rate  Yeast cells can divide every 2 hours, most mammalian cells take hours  DNA replication is main limiting factor  DNA replicated in special part of cell cycle termed S (for synthetic) phase  Mitosis (cell division) occurs during M Phase  There are two G (gap) phases (G 1 and G 2 ) when DNA and other cell macromolecules are synthesised  There is also a quiescent or resting (or G 0 ) phase where there is minimal activity  These resting cells are a problem in cancer treatment, since they are not vulnerable to cancer drugs

5 Phases of Cell Cycle (Example shown has 16 hour cycle)

6 Mitosis  Cell division is subdivided into 6 stages, 5 of which are termed mitosis  1 Prophase G 2 to M transition DNA and macromolecules have duplicated Chromatin condenses into chromosomes Chromosomes exist a two sister chromatids joined at the centromere Mitotic spindle (microtubules) forms from tubulin Nucleolus starts to disappear  2 Prometaphase Disruption of nuclear envelope Mitotic spindle now enters nuclear area Kinetochore microtubules appear Chromosomes are pulled towards division point  3 Metaphase Chromosomes halfway between spindle poles Each chromosome is held in tension at metaphase plate by kinetochore fibres

7 Mitosis (Ctd.)  4 Anaphase Metaphase may take considerable time When triggered, anaphase is rapid, with chromosomes pulled towards their mitotic poles All chromatids are pulled at same speed (1 µm per minute) Kinetochore fibres shorten Spindle fibres elongate as chromosomes move apart Lasts only a few minutes  5 Telophase Daughter chromatids arrive at the poles Kinetochore fibres disappear Polar fibres elongate further New nuclear envelope forms around daughter chromatids Chromatin formed from chromosomes Nucleolus reappears  Cytokinesis is where cytoplasm divides and membrane separates daughter cells

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10 Prophase (in plant cell, tubules stained red)

11 Metaphase

12 Anaphase

13 Telophase

Mitotic Spindle  Chromosome segregation in mitosos depends on the mitotic spindle  The mitotic spindle is based on a bipolar array of microtubules  These are polarized protein polymers with one end (minus end) embedded in a spindle pole and the other end ( plus end) pointing outward from the pole. 14

Mitotic Spindle ctd.  The plus ends from one pole overlap with plus ends from another, resulting in an antiparallel array in the spindle midzone  Microtubules are highly dynamic polymers that continuously grow and shrink, and is regulated by many different proteins that bind to the sides or ends of microtubules  These include the motor proteins, which can travel along microtubules and have important roles in the assembly and stability of the microtubule array and the movement of chromosomes on the spindle.  Each chromatid has a kinetochore, a multiprotein complex that attaches the chromatid to microtubules, which are connected to a spindle pole  In addition, proteins in the kinetochore help generate forces that drive chromosome movement  Motors and microtubule-regulatory proteins in the chromatid arms also help regulate microtubule growth and spindle assembly. 15

16 Important Vinca Alkaloids

Vinca Alkaloids  Vincristine and vinblastine are alkaloids found in the Madagascar periwinkle, Catharanthus roseus  These were formerly classified as Vinca rosea, which led to these compounds becoming called Vinca alkaloids  Vindesine and vinorelbine are semisynthetic derivatives of vinblastine  All Vinca alkaloids work by inhibiting mitosis in metaphase, hence are M-phase specific  They bind tubulin, and inhibit formation of the mitotic spindles  Vincristine has a serum half-life of about 85 hours.  Used mainly to treat acute leukaemia, rhabdomyosarcoma, neuroblastoma, Hodgkin's disease and other lymphomas  The typical dose in 1.4 mg per square metre of body surface  Vinca alkaloids can cause damage to the peripheral nervous system, and neurotoxicity is the dose limiting factor  Vinorelbine is currently in Phase II clinical trials as a treatment for ovarian cancer 17

18 Structures of Paclitaxel (taxol) and Docetaxel (taxotere)

Taxanes  Paclitaxel (formerly called taxol) was isolated it from the bark of the Pacific yew tree, Taxus brevifolia  Paclitaxel is an important anticancer agent which is M-phase specific  It inhibits cell replication by binding to and hyper- stabilising microtubule polymers  May also act by sequestering free tubulin, prompting apoptosis (programmed cell death)  Paclitaxel is used to treat lung, ovarian, breast cancer, head and neck cancer, and advanced forms of Kaposi's sarcoma  The National Cancer Institute (US) collected Taxus bark - 28kg of crude extract had been isolated from almost 1,200kg of bark, although this ultimately yielded only 10g of pure material!  Taxus brevifolia takes 10 years to grow to maturity  Currently, all paclitaxel production uses a Taxus plant cell fermentation (PCF) biotechnology process  Common side-effects include nausea and vomiting, loss of appetite, change in taste, thinned or brittle hair, pain in the joints of the arms or legs 19