XIII Cell and Tissue Kinetics

1. XIII Cell and Tissue Kinetics

2. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

3. The Cell Cycle The average interval between successive mitoses or divisions is called the cell cycle or mitotic cycle time (Tc) Howard and Pelc were the first to subdivide the mitotic cycle by the use of a labeled DNA prescursor. Mitosis (M) is the only event that can be distinguished by light microscope. The DNA synthetic phase (S) may be identified by autoradiography. The intervals of apparent inactivity are G1 and G2.

4. The proportion of cells in mitosis is called the “mitotic index”. MI = lTM/TC Proportion of labeled cells is called the “Labeling Index”. LI = lTS/TC Cells cannot be distributed uniformly in time around the cell cycle because they double in number during mitosis. The simplest assumption is that they are distributed as an exponential function of time.

5. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

6. Cell-labeling techniques • Autoradiography • [3H] Thymidine, Howard and Pelc, 1953 • Dye/staining • 5-Bromodeoxyuridine • Flow-cytometry • Propidium Iodide

7. Measurement of cell cycle parameters by 3H-thymidine Mouse Corneal Cells Percentage of mitotic cells that carry a radioactive label indicates the percentage of labeled mitoses. The cell preparation was flash labeled some hours before with tritiated thymidine, which was taken up by cells in S. By the time the autoradiograph was made, the cell marked LM had moved around the cycle into mitosis; this is an example of labeled mitotic figure. Other cells in mitosis are not labeled (UM).

8. A plot of percentage of labeled cells as function of time Percent-labeled mitoses curve for an idealized cell population in which all of the cells have identical mitotic cycle times. Typical percent-labeled mitoses curve obtained in practice for the cells of a tissue or tumor. It differs from the idealized curve in that the only points that can be identified with precision are the peaks of the curve and the 50% levels. The first peak is symmetric, and the second peak is lower than the first because the cells of a population have a range of cell cycle times. TG1 = Tc- (Ts + TG2 + TM) Mitotic Index = TM / Tc

9. In-vivo Labeling Index Percent-labeled mitoses curve for two transplantable rat sarcomas with widely different growth rates. The tumor in the upper panel has a gross doubling time of 22 hours, compared with 190 hours for the tumor in the lower panel. Percent-labeled mitoses curve for an EMT6 mouse tumor. Top: The distribution of cell cycle times consistent with the damped labeled mitoses curve, obtained by computer analysis of the data and the mathematic model.

11. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

12. Measurement by flow cytometry, DNA staining and BrdU Pulsed Photo Cytometry • Excited the stained DNA with a laser beam and subsequently detected using a fluorescence detector. • As per the DNA content, the cells are distributed in different phases of cell cycle.

13. The flow cytometric analysis of cellular bromodexyuridine (BrdUrd) and DNA content for cells stained with fluorescein (linked to BrdUrd) and propidium iodide (linked to DNA) using a single biopsy specimen taken 4 to 8 hours after the injection of a tracer amount of a thymidine analogue (BrdUrd or Iododeoxyurine). The LI is the green fluorescence. Ts is mean red fluorescence of S cells relative to G1 and G2 cells. The DNA content in G2 is double that in G1.

14. Autoradiography / Dye staining • Pulse labeling

15. Autoradiograph / Bromodeoxyuridine staining

16. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

17. Cell cycle synchronization techniques and uses A synchronized culture is one where cells pass through the division cycle as a relatively uniform cohort and represent, at different time points, cells of different cell cycle ages. In passing through the cell cycle a newborn eukaryotic cell first passes through the G1 phase (absence of DNA synthesis), then S phase (period of DNA synthesis), then G2 phase (absence of DNA synthesis), with division occurring at M phase (mitosis). If many cells in a culture approximate this pattern as a group, these cells would be called a synchronized culture. Cooper, 2003

18. Cell cycle synchronization techniques • Mitotic shake-off Terasima and Tolmach • Use of drug/pharmacological agents Hydroxyurea (Adverse cellular perturbations) • Serum deprivation • Contact inhibition • Centrifugal elutriation • Fluorescence activated cell sorting Batch treatments (i.e., treatments that affect all cells equally) • Many methods related to serum starvation may be grouped as methods that arrest cells at a specific point in the G1 phase, sometimes referred to as the ‘restriction point.’ • Other synchronization methods such as the double-thymidine block method or hydroxyurea inhibition affect DNA synthesis and are proposed to arrest cells in S phase. • A third class of batch synchronization methods arrests cells at mitosis. The classical inhibitor for such a process is nocodazole.

19. Mode of action of HU

20. HU effect on Vicia root tip

21. Serum starvation Serum starvation or low serum concentration is believed to arrest cells at a particular point in the cell cycle. The arrested cells with a G phase amount of DNA are proposed to be arrested at this point in the G1 phase or in what is generally called the G0 phase. Upon restoration of serum, these arrested cells are assumed to pass synchronously through the cell cycle. Cooper, 2003

22. Membrane-elution Exponentially growing cells bound to a membrane (Cells grow normally on the membrane) At division, one of the daughter cells remains attached to the membrane while the other daughter cell is released into the eluate In a typical membrane-elution synchronized culture, one can produce three clear synchronized divisions with clear plateaus between the doublings in cell number. Temperature and medium are maintained constant throughout the procedure. DNA content distributions as well as size distributions indicate that the cells eluted from the membrane-elution apparatus reflect the normal pattern of growth during the division cycle. The newborn cells are appropriately small (equivalent in size to the cells at the low-size end of the size distribution of the exponential culture) and these newborn cells have a G1 phase DNA content. The membrane- elution method therefore shows that without any starvation, one can have a narrowing in the size distribution and a narrow DNA content that reflects the normal cell cycle. Cooper, 2003

23. Cell cycle synchronization uses • Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phase-dependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations. Davis et al ., 2001

24. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

25. Effect of cell cycle phase on radiosensitivity Chinese hamster cells

26. Radiation response in different phases of cell cycle Chinese hamster cells Most sensitive: G2 and M Most resistant: Late S Intermediate: G1 and early S

27. Synchronized human kidney cells show a differential survival depending on cell cycle phase in which they are irradiated. Cells are most sensitive to irradiation during mitosis and in G2, less sensitive in G1, and least sensitive during the latter part of S phase. Pawlik and Keyomarsi, 2004

28. Radiation response and the length of G1 • Longer G1 leads to resistance in early G1 followed by enhanced sensitivity in the end of G1.

29. Radiation response in-vivo Age-response

30. Summary • Cell cycle phases: M, G1, S and G2 • CDK family regulates the phases of cell cycle • Fastest cycling cells: crypt cells (9-10 hrs) • Slowest cycling cells: mouse skin stem cells (200hrs) • M and G2 are radiosensitive and late S is radioresistant • Long G1 results in a second resistant peak in early G1 • Radiation induces G2 arrest through molecular checkpoint genes • The age-response for crypt cells in tissue is similar to that of cells in culture

31. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

32. Cell cycle arrest and redistribution following irradiation Several chemotherapeutic drugs and ionizing radiation (IR) can arrest the cells in different phases of the cell cycle leading to cell cycle phase redistribution and may lead to partial synchronization with time. For example: IR can retard the rate of progression of proliferating cell populations through various phases of the cell cycle causing cells to accumulate in G2 phase and keeping cells from undergoing mitotic division. Split or fractionated radiation may therefore be more efficacious by inducing a transient cell cycle arrest after which a secondary RT fraction is administered exactly at the height of cell accumulation in the most radiosensitive cell cycle phase (G2/M). This suggests that the redistribution of cells in a particular phase would determine the response of an initially asynchronous population to fractionated RT.

33. Cell cycle • Measurement of cell cycle parameters by 3H-thymidine • Measurement by flow cytometry, DNA staining and BrdU • Cell cycle synchronization techniques and uses • Effect of cell cycle phase on radiosensitivity • Cell cycle arrest and redistribution following irradiation • Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors • Tissue kinetics • Stem, progenitor, differentiated cells • Growth fraction • Cell loss factor • Volume doubling times • Tpot • Growth kinetics of clinical and experimental tumors

34. Cell cycle checkpoints, cyclins, cyclin dependent kinase inhibitors Molecular checkpoint genes

35. Genes regulate cell cycle phases • Cyclin-dependent kinases • Cyclins A, B, D1, K1, K4 and E • Rb Blockers • p53 mediated p21 inhibits CDKs

36. Cell Cycle Regulation The current concept of the cycle and its regulation by protein kinases, activated by cyclins.

37. Progression through the cell cycle from one phase to the next is governed by protein kinases, activated by cyclins. In mammals, cyclins A through H have been described: each cyclin protein is synthesized at a discrete phase of the cell cycle. Cyclin levels oscillate with phase of cycle, as shown schematically in this figure

38. DNA damage response reactions in mammalian cells The four responses (DNA repair, transcriptional response, DNA damage checkpoints, and apoptosis) may function independently, but frequently a protein primarily involved in one response may participate in other responses. Sancar et al., 2004

39. Components of the DNA damage checkpoints in human cells Current model of the mammalian DNA damage and replication checkpoints. A line ending with an arrowhead indicates activation. A line ending with a bar indicates inhibition. Li and Zou, 2005 The damage is detected by sensors that, with the aid of mediators, transduce the signal to transducers. The transducers, in turn, activate or inactivate other proteins (effectors) that directly participate in inhibiting the G1/S transition, S-phase progression, or the G2/M transition. Sancar et al., 2004

40. Sancar et al., 2004

41. A simplified scheme of cell-cycle checkpoint pathways induced in response to DNA damage (here DSBs), with highlighted tumor suppressors shown in red and proto-oncogenes shown in green. The proximal checkpoint kinases ATM and ATR phosphorylate diverse components of the network, either directly (red ‘P’) or through the transducing kinases CHK2 and CHK1 (black ‘P’). (For simplicity, some candidate damage sensors and several ATM/ATR and CHK1/CHK2 substrates have been omitted.) The BRCA1 protein also contributes to cell-cycle arrest and DNA repair by homologous recombination, whereas p53 controls genes involved in cell death and DNA-repair mechanisms. The cell-cycle phase and the duration of the blockade affected by the effector pathways are indicated, including the potential permanent arrest (senescence), as mediated by p53. The global checkpoint network regulated by ATM/ATR and CHK2/CHK1 also affects cellular responses other than cell cycle progression, including DNA repair, transcription, chromatin assembly and cell death. Kastan and Bartek, 2004

42. The G1/S checkpoint DNA damage is sensed by ATM after double-strand breaks or by ATR, Rad17-RFC, and the 9-1-1 complex after UV-damage. ATM/ATR phosphorylates Rad17, Rad9, p53, and Chk1/Chk2 that in turn phosphorylates Cdc25A, causing its inactivation by nuclear exclusion and ubiquitin-mediated degradation. Phosphorylated and inactivated Cdk2 accumulates and cannot phosphorylate Cdc45 to initiate replication. Maintenance of the G1/S arrest is achieved by p53, which is phosphorylated on Ser15 by ATM/ATR and on Ser20 by Chk1/Chk2. Phosphorylated p53 induces p21WAF-1/Cip1 transcription, and p21WAF-1/Cip1 binds to the Cdk4/CycD complex, thus preventing it from phosphorylating Rb, which is necessary for the release of the E2F transcription factor and subsequent transcription of S-phase genes. p21WAF-1/Cip1 also binds to and inactivates the Cdk2/CycE complex, thus securing the maintenance of the G1/S checkpoint. Sancar et al., 2004

43. The ATM-regulated intra-S-phase checkpoint In response to double-strand breaks induced by ionizing radiation, ATM triggers two cooperating parallel cascades to inhibit replicative DNA synthesis. ATM, through the intermediacy of MDC1, H2AX, and 53BP1, phosphorylates Chk2 on Thr68 to induce ubiquitin-mediated degradation of Cdc25A phosphatase. The degradation locks the S phase–promoting Cyclin E/Cdk2 in its inactive, phosphorylated form and prevents the loading of Cdc45 on the replication origin. ATM also initiates a second pathway by phosphorylating NBS1 of the M/R/N complex, as well as SMC1, BRCA1, and FANCD2. Sancar et al., 2004

44. The ATR-mediated intra-S-phase checkpoint The ATR-ATRIP complex, Rad17-RFC, the 9-1-1 complex, and Claspin are independently recruited to RPA(A)- coated single-stranded regions of the stalled replication fork. ATR then phosphorylates Chk1 and other substrates, and activated Chk1 phosphorylates Cdc25A, which leads to inactivation of Cdk2/Cyclin E complex. Single-strand DNA gaps can also be sensed by ATR and following ATR activation, origin firing and consequently DNA replication is inhibited through downregulation of Cdc7/Dbf4 protein kinase activity. Sancar et al., 2004

45. The G2/M checkpoint The ionizing radiation- (ATM) and UV damage responsive sensor proteins (ATR-ATRIP, Rad17-RFC, and 9-1-1) are recruited to the damage site. The mediator proteins such as MDC1, BRCA1 and/or 53BP1 communicate the DNA damage signal to Chk1 and/or Chk2, thereby regulating the Cdc2/CyclinB, Wee1, and Cdc25A proteins that are crucial for the G2/M transition by changing their expression, phosphorylation, and cellular localization. Sancar et al., 2004

46. The Replication Checkpoint (S/M Checkpoint) The replication checkpoint is the process by which mitosis is inhibited while DNA replication is ongoing or blocked. In both the G2/M and replication checkpoints, the ATR-Chk1-Cdc25 signal transduction pathway is utilized to inhibit mitosis, although the initiating signals for the two checkpoints are different. Ongoing replication or replication forks blocked by DNA damage or nucleotide starvation initiate the replication checkpoint. Evidence from in vitro studies with Xenopus egg extracts indicates that the initiating signal is a component of the replication fork, and experiments with various replication inhibitors suggested that the signal might actually be the RNA primer of Okazaki fragments. However, more recent experiments have raised questions about the true identity of the replication checkpoint signal, as the inhibitors used to assign it to RNA primers inhibited the entire replisome assembly. The replication checkpoint has been observed in all model systems, including yeast and mammalian cells. Surprisingly, however, recent evidence indicates that in mice the replication checkpoint activated by hydroxyurea and aphidicolin is independent of ATM and ATR, but the UV- and ionizing radiation induced replication checkpoint is dependent on these damage sensor/signal transducer kinases, raising the possibility that replication forks and DNA damage during S phase may inhibit mitosis by different signaling mechanisms. Sancar et al., 2004

47. Schematic Representation of the DNA damage and replication checkpoint in yeast and mammals. Arrows represent either biochemical or genetic evidence for a connection. Regulators of the DNA damage response are indicated in red. Regulators that act only in the replication/intra-S-phase checkpoint are indicated in blue. Kinases that are used by both branches of the pathway are in green. McGowan, 2002

48. Cyclin dependent kinase inhibitor; p21 Schematic model of the p21CIP1 protein and its key functional activities mediated by binding to different partner proteins. Apparently, p21CIP1 can localize to different cellular compartments including the nucleus, the cytoplasm, and the centrosome. Samuel et al., 2002

49. Links to cancer and genetic instability Loss of cell cycle checkpoint control has emerged as a central cause of genetic instability. Consequently, chances that these unstable cells progress to cancer are increased. This notion has several important implications: 1. Since checkpoints may determine the ultimate response (arrest vs. apoptosis), the integrity of these checkpoints influences the susceptibility of cells to DNA damage. This is relevant either to the cells’ fate after accumulation of undesired DNA damage or to the cells’ sensitivity to desired damage during chemo-/radiotherapy. 2. Exploring the early checkpoint defects in cancerous or pre-cancerous lesions may serve as a prognostic or, in certain tissues, as an additional diagnostic marker. 3. Known defects of pivotal checkpoint genes may help to predict treatment outcome or to design more specific therapeutic strategies. In addition, checkpoint components which are defective in certain cancer cells may be targeted during therapy to enhance the anti-tumor effect, e.g., by preventing arrest and/or by forcing cells into apoptosis. Work is in progress to develop novel therapeutic strategies with an increased therapeutic index. 4. Strategies could be considered to restore missing or dysfunctional checkpoints in order to provide additional time for DNA repair and delay the onset of cancer. 5. Since some of the components that are involved in the DNA damage checkpoint are also involved in other cellular regulatory activities, e.g., during senescence, differentiation, or certain immunological responses, this could lead to cross-signaling into other pathways and might permit new strategies to influence related cellular functions. Samuel et al., 2002

50. Kastan and Bartek, 2004