Dr. Timothy F. Lane Jonsson Comprehensive Cancer Center, Department of Biological Chemistry

1. Dr. Lane LECTURE #1 1 267A:Cell Cycle I Dr. Timothy F. Lane Jonsson Comprehensive Cancer Center, Department of Biological Chemistry Office: 549 BSRB email: tlane@mednet.ucla.edu Syncitial Divisions in Drosophila embryo. From Bill Sullivan UCSC These notes are posted on the www page! http://bio.research.ucsc.edu/people/sullivan/images.html

2. Goals Features of Cell Cycles and some basic methodology Source of materials: Lectures 1-5 follow the course produced by Dr. Herschman (2006-07) Lecture 6 - T.B.D.

3. The two “musts” of cell division: 1. Cells must replicate their DNA 2. Cells must segregate the DNA to the progeny cells in mitosis Syncitial Divisions in Drosophila embryo. From Bill Sullivan UCSC http://bio.research.ucsc.edu/people/sullivan/images.html

4. INTERPHASE How do we study the cell cycle? Mitotic cells can be identified under a microscope. Microscopic studies carried out early in the 20th century described mitotic cells and identified a period of “supposed” quiescence called interphase. How do we identify other features of cycling cells? Mitotic cells

5. What was happening in 1950? Cell cycle: Model: ? Question: When is nuclear material synthesized? http://whatdidyoueat.typepad.com/what_did_you_eat/2006/05/braised_fava_be.html Howard and Pelc (1951) [Exptl Cell Res 51: 2: 178]

6. Alma Howard’s experiment: Results Expt #1: no incorporation into M cells, incorporation into some interphase cells. Interpretation: Interphase contains a period of DNA synthesis that is completed before M. interphase cell Mitotic cell Model: Expt 1] Label with 32P, autoradiograph immediately Howard and Pelc (1951) [Exptl Cell Res 51: 2: 178]

7. Alma Howard’s experiment: Results Expt #2: • For 2-4 hours, no mitotic cells are labeled! • After six hours, some mitotic cells are labeled; Interpretation: There exists a period of several hours prior to M in which no DNA synthesis occurs. Cells labeled for >6 hours passed through a period of DNA synthesis, after which a “Gap” period where no DNA synthesis occurred. interphase cell Mitotic cell Model: = 2-4hr gap before labeled Mitotic cells appear. Expt 2] Label with 32P, add colchicine, wait 2 hrs. Howard and Pelc (1951) [Exptl Cell Res 51: 2: 178]

8. Alma Howard’s experiment: Results Expt #2: When [32P] was added for 1hr, then removed for 6-8 hours, some of the cells entering mitosis were not labeled. Interpretation: This implies there is a period between mitosis and the beginning of DNA synthesis when no DNA synthesis occurs, another Gap. interphase cell Mitotic cell Model: Expt 3] Label with 32P, add colchicine, wait longer (6-8 hrs). http://www.nature.com/nature/journal/v426/n6968/full/426759a.html Howard and Pelc (1951) [Exptl Cell Res 51: 2: 178]

9. Today, we label cells with dNTP precursors like [3H]-thymidine (TdR) or analogs like BrdU. Schematic of cultured fibroblasts grown on TC plastic. 30 Minute Pulse with 3H-TdR, Autoradiograph Immediately

10. Today, we label cells with dNTP precursors like [3H]-thymidine. Result #1 Some interphase cells labeled ( ), some not ( ). 30 Minute Pulse with 3H-TdR, Autoradiograph Immediately

11. Today, we label cells with dNTP precursors like [3H]-thymidine. Result #2 No labeled mitoses 30 Minute Pulse with 3H-TdR, Autoradiograph Immediately

12. Can we get cell cycle information from such an experiment? Expt 4: Prepare a group of identical plates containing your favorite cell type. Each plate contains randomly growing cells distributed (randomly) around the cell cycle. PLAN: Add tritiated thymidine for 30 minutes Wash out the labeled thymidine Add colchicine, to prevent microtubule polymerization At intervals (e.g., one hour) fix a plate and count Collect the following data 1. The number of mitotic cells 2. The number of radiolabeled mitotic cells

13. Can we get cell cycle information from such an experiment? Expt 4: Data: 1. The number of mitotic cells 2. The number of radiolabeled mitotic cells Number of mitotic cells RESULT: there is a steady increase in mitotic cells following colchicine treatment, finally reaching a plateau as all cells pass into M. time

14. As time passes, some mitoses become labeled 30 Minute Pulse with 3H-TdR, add Colchicine, Autoradiograph at later times.

15. Can we get cell cycle information from such an experiment? Expt 4: Data: 1. The number of mitotic cells 2. The number of radiolabeledmitotic cells Number of mitotic cells Number of 3H-thymidine labeled mitotic cells RESULT: there is a delay in the appearance of labeled mitotic cells following colchicine treatment of 3H-Thy pulse-labeled cells time

16. Expt 4: Data: 1. The number of mitotic cells 2. The number of radiolabeledmitotic cells Number of mitotic cells Number of 3H-thymidine labeled mitotic cells RESULT: there is a delay in the appearance of labeled mitotic cells following colchicine treatment of 3H-Thy pulse-labeled cells time

17. Results Expt #4: Interpretation: M we are looking at DNA Synthesis Gap M M Lag equals a Gap following S, before cells enter M.

18. A further incr. in # of unlabeled mitotic cells, until a steady state is reached. 30 Minute Pulse with 3H-TdR, add Colchicine, Autoradiograph at later times.

19. Expt 4: Data: 1. The number of mitotic cells 2. The number of radiolabeledmitotic cells Number of mitotic cells Number of 3H-thymidine labeled mitotic cells RESULT: a plateau of labeled Mitotic cells is reached. The plateau is reached prior to the plateau in total M cells. time

20. Results Expt #4: Interpretation: Gap DNA Synthesis Gap M M The time of increased labeling equals S

21. Results Expt #4: Interpretation: Gap DNA Synthesis Gap M M S G2 G1 The additional time until the total number of M cell plateaus represents the GAP prior to S.

22. Expt 4: Data: 1. The number of mitotic cells 2. The number of radiolabeledmitotic cells Number of mitotic cells Number of 3H-thymidine labeled mitotic cells RESULT: A time line for each stage of the cell cycle can be obtained. time G2 S G1

23. G1 M G2 S Current representations of the “Cell Cycle” Features: S has discrete beginning and end. S is separated from M by Gaps. S, G2 and M periods of the cycle are are of fairly consistent lengths. Differences in cycle time are usually due to alterations in G1. In non-dividing tissues most. cells are frozen in G1, or "Go". Go

24. G1 M G2 S Current representations of the “Cell Cycle” Features: The “Generation Time” is the length of a complete cell cycle M---M. GT = G1 + S + G2 + M GT = Generation time

25. G1 M G2 S DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: The classic method is known as labeled mitoses. count labeled mitoses after a pulse with a DNA label (eg [3H] TdR) 2. M can be determined as the percent of cells that appear as mitotic figures in a randomly growing population. S-phase cells are labeled after a short “pulse” with tritiated thymidine

26. G1 M G2 S DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: The classic method is known as labeled mitoses. count labeled mitoses after a pulse with a DNA label (eg [3H] TdR) 2. M can be determined as the percent of cells that appear as mitotic figures in a randomly growing population. S-phase cells are labeled after a short “pulse” with tritiated thymidine

27. DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of Labeled Mitoses: M = GT / (%M) (%M is determined microscopically) Number of labeled mitoses G2 S G2 + M + G1 Time

28. DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of micro-fluorimetry (FMF) or FACS: Capillary Flow Cells stained with DNA intercalating fluorescent dye: Examples: DAPI Hoescht 33258 Propidium Iodide Topro3 Excitation (lasers) Emission Intensity is proportional to amount of die bound: G1 = 2n S = 2-4n G2 = 4n

29. DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of micro-fluorimetry (FMF), Flow cytometry, FACS: Cell cycle analysis by flow cytometry requires: A single cell suspension of the cells of interest. Cells must be permeable (detergent, fixation) DNA in cells can be stained with a fluorescent dye DNA probes like Propidium Iodide are STOICHIOMETRIC allow quantitative assessment of DNA content Basic protocol - fix, wash twice, stain with DNA-binding dye (remove RNA)

30. DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of micro-fluorimetry (FMF) or FACS:

31. # of Events Increase in Fluorescence Intensity DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of micro-fluorimetry (FMF) or FACS: G1(2n) G2 (4n) S Derek Davies, Cancer Research UK

32. DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of micro-fluorimetry (FMF) or FACS: G1(2n) # of Events G2 (4n) S Increase in Fluorescence Intensity Derek Davies, Cancer Research UK

33. S G2 G1 M DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: Method of micro-fluorimetry (FMF) or flow cytometry: When coupled with FACS, cells can be isolated for biochemical analysis. # of Events Increase in Fluorescence Intensity Derek Davies, Cancer Research UK

34. G1 M G2 S MAJOR features of the cell cycle are “conserved” in all eukaryotes: Conserved Features: Replication of DNA (S) Chromosome segregation (M) Highly accurate duplication of DNA (3.2pg, 7ft of W/C DNA in humans)

35. A large number of eukaryotic systems are used to study various aspects of cell cycle machinery: Systems: Yeast Many invertebrate embryos (clams, sea urchins) Frog oocytes Mammalian cells in culture. Fibroblasts (Thulberg et al) Xenopus (J. Smith www) Fly (W. Sullivan www) Yeast (Kerry Bloom www) We will draw heavily on mammalian, Xenopus, and yeast experiments in this class. (Biochemisty and genetics)

36. We can “synchronize” populations of cells at various points in the cell cycle. R=Restriction Point G1 Starving cells leads to G1 arrest (R) (serum, isoleucine, or PO4 ) M Several agents can be used to block S (HU, nucleotide analogues, aphidicolin, TdR). G2 S

37. G2 G2 G2 DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: 0 hrs Method of micro-fluorimetry (FMF) or flow cytometry: S phase block Agents like HydroxyUrea (HU), nucleotide analogues, aphidicolin fluorodeoxyuridine (FdU) methotrexate uridine (MU) TdR (more in a minute) 24 hrs Derek Davies, Cancer Research UK

38. G2 G2 G2 DETERMINING THE CELL CYCLE PARAMETERS G1, S, G2 and M: 0 hrs Method of micro-fluorimetry (FMF) or flow cytometry: G2 phase block Agents like DHAQ ICRF (chelating agent) Polo Kinase inhibitors 24 hrs Derek Davies, Cancer Research UK

39. M M We can “synchronize” populations of cells at various points in the cell cycle. Position of Block Single Thymidine Block: General Method Treat cells w/ .25mM Thymidine for 24 hours (or a timed greater than G2+M+G1) Release by washing 2x, then proceeding as required for experiment. DNA synthesis inhibitor Cell Number Time Release and count cells at intervals after release Benefits: Provides moderately pure S cells. Very gentle, tolerated by many types of cells.

40. We can “synchronize” populations of cells at various points in the cell cycle. Position of Block Double Thymidine Block: General Method Treat cells w/ .25mM Thymidine for a timed greater than G2+M+G1 wash 2x and culture for a time greater than S, less than G2+ M+ G1. Treat cells w/ .25mM Thymidine for a timed greater than G2+M+G1 Release by washing 2x, then proceeding as required for experiment. 2nd round Thymidine M M Cell Number Time Cells released following 1st block Release and count cells at intervals after release Benefits: Provides very pure S cells. Very gentle, tolerated by many types of cells.

41. Mitotic cells We can “synchronize” populations of cells at various points in the cell cycle. Mitotic Selection (aka Mitotic Shake-off) : General Method Gently wash off loose cells and collect them. Increase yield by treating with Nocodazole (-tubule inhibitor) Theory Adherent tissue culture cells adhere to the plate, but “round up” as they enter M. This in an opportunity to isolate them. Benefits: Provides relatively pure M/G1 cells. Very gentle, no biochemical manipulation.

42. We can “synchronize” populations of cells at various points in the cell cycle. Comparison of non-toxic methods : Single TdR block G1/S and S Double TdR block G1/S Mitotic selection M/G1

43. We can “synchronize” populations of cells at various points in the cell cycle. Centrifugal Elutriation : General Method Single cell suspension. Special centrifuge configuration that allows collection of samples during the run!. Theory Cells increase their size as they go through the cycle M< G1 < S < G2. Benifits: Very large numbers/volumes of cells. Can enrich for many stages. Excellent for blood/yeast Yeast Cell cycle mutants: J Bahler (Sanger Center)

44. G1 Cell number S G2 Control early mid-E mid-L late fluorescence intensity We can “synchronize” populations of cells at various points in the cell cycle. Centrifugal Elutriation : Confirmation of separtation by FMF

45. RATE AMOUNT 2X cpm 1X G1 S G2 M CHALLENGES IN CELL CYCLE: DNA SYNTHESIS What initiates DNA synthesis? Is the signal positive or negative? How is synthesis restricted? How is chromatin regulated during the cycle? Is DNA synthesized in a random or ordered fashion? Is DNA synthesized early in one cycle always synthesized early? Questions:

46. QUESTIONS CONCERNING DNA SYNTHESIS: Is DNA Synthesized sequentially or randomly? In 1960s there was cytochemical evidence suggest that "early replicating" and "late replicating" DNAs exists. (X-chromosome, others) Mueller and Kajiwara asked “Is DNA that is synthesized early in one cell cycle synthesized early the next cell cycle? • PLAN: Synchronize HeLa cells with a double-thymidine block at G1/S (S = 6hr). • Release from block, add [3H]-TdR for 3 hours (ie, during the first half of S). • Then chase with "cold" thymidine media. • Grow the [3H]-TdR labeled cells for a few generations. (loose synchrony). • Then resynchronize with a second double-TdR block at G1/S. • Release in the presence of BUdR for 3 hours. • Isolate DNA, shear the DNA, and centrifuge on CsCl gradient. • Follow radioactivity and density. OUTCOMES: If early replicating DNA is resynthesized early, then [3H] will always go with the BUdR density label. If DNA synthesis is random, then [3H] will be present in all fractions Mueller and Kajiwara, BBA 114: 108 (1966)

47. QUESTIONS CONCERNING DNA SYNTHESIS: Is DNA Synthesized sequentially or randomly? 3H TdR BUdR Let grow several generations, then re-synchronize, label with BUdR for three hours Synchronize cells, release into 3H-TdR for three hours Mueller and Kajiwara, BBA 114: 108 (1966)

48. Optical density 3H TdR HL LL Density QUESTIONS CONCERNING DNA SYNTHESIS: Is DNA Synthesized sequentially or randomly? RESULT: [3H] AND BUdR co eluted. CONCLUSION: DNA SYNTHESIZED EARLY IN ONE CYCLE IS ALSO SYNTHESIZED EARLY IN THE NEXT CYCLE Mueller and Kajiwara, BBA 114: 108 (1966)