Cell-cycle perturbations

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

After washout, edotecarin or SN-38 produced analogue cell-cycle perturbations after 7 hours of recovery (G and S delay). While in blocked cells after treatment with edotecarin, effects persisted up to 24 hours of 30 nM and up to 72 hours at 300 nM, whereas cells treated with SN-38 at both doses restarted cycling after 24 hours of recovery. 

Horber DH, et al. Cytotoxicity, cell cycle perturbations and apoptosis in human tumor cells by lipophilic N" -alkyl-l-P-D-arabinofuranosylcytosine derivatives and the new heteronucleoside phosphate dimer arabinocytidylyl-(5 5 )-N" -octadecyl-l-P-D-ara-C. J Cancer Res Clin Oncol 2000 126 311. 

The simultaneous measurement of BrdU incorporation and DNA content confers sensitivity and versatility in detection of cell cycle perturbations in response drugs or radiation and tracing the lineage of a cell within the cell cycle. The use of computer-generated windows facilitates the analysis of any population of cells within any phase of the cell cycle and is not restricted to the mitotic window, as is the case with 3HTdR/autoradiography. 

To incorporate BrdU m vitro into monolayers or cell suspensions, the cells are incubated with 10-20 pM BrdU for 10—20 min Concentrations as small as 1 iM can be detected, whereas concentrations >50 xM may cause cell cycle perturbations. The BrdU is thoroughly washed out by two washes in PBS or medium. It is important to keep everything at 37°C for cell kinetic studies to avoid any perturbations caused by lowering the temperature. 

Coronnello M, Marcon G, Carotti S, Caciagli B, Mini E, Mazzei T, Orioli P, Messori L (2000) Cytotoxicity, DNA damage, and cell cycle perturbations induced by two representative gold(III) complexes in human leukemic cells with different cisplatin sensitivity. Oncol Res 12 361-370... 

Fig. 3. The cell-cycle perturbations that occur as a consequence ofDNA damage induced by cisplatin. The dark box represents the time period during which cells arrest at various phases of the cell cycle with the intent to repair the damage. Once the DNA is repaired, cells may recover and continue to grow. The dotted arrows imply that caffeine and UCN-01 can overcome S- and G2-phase arrest and drive the cells into a lethal mitosis.

Experiments in this laboratory identified apoptosis as a consequence of the action of cisplatin and many other anticancer agents [50-52], Many authors have subsequently misquoted these results when suggesting that cisplatin kills by apoptosis, and that suppression of apoptosis is a mechanism of resistance. It should be evident by now that apoptosis is better defined as a consequence of the mechanism of action of cisplatin and a failure of the mechanisms of resistance. Apoptosis is certainly not an alternative to the formation of DNA cross-links, nor to the cell-cycle perturbations that result these are still essential events in the initiation phase of apoptosis. The mechanisms of resistance to cisplatin still include reduced drug accumulation, reduced DNA platination, and altered DNA repair. However, apoptosis provides a framework for understanding the complete pathway from initial insult to eventual death of a cell. It provides the realization that there are additional factors that influence cell survival and death. Expression of Bcl-2 family members or changes in signal transduction pathways impact... 

By measuring DNA content and BUdR presence simultaneously, the technique is very sensitive in detecting cell cycle perturbations, owing to drugs... 

Weller EM, Kubbies M and Nusse M (1995). Induction of cell cycle perturbations by tear gas 2-chlorobenzylidene malononitrile (CS) in synchronously and asynchronously proliferating mammalian cells. Cytometry, 19, 334-342. 

The abnormal cell killing and cell-cycle perturbation effects of Ho33342 on A-T cells can be attributed to the dose-modifying effect (in terms of DNA damage) of the chromatin anomaly. Experiments to determine the effects of inhibition of poly(ADP-... 

The cytotoxic effects of the synthetic actinocin derivatives were investigated by examination of the drug-induced apoptosis and cell cycle perturbations in a human leukemia MOLT-3 cell line [88]. 

Erba, E. Ubezio, P Broggini, M. Ponti, M. D Incalci, M. DNA damage, cytotoxic effect and cell-cycle perturbation of Hoechst 33342 on L1210 cells in vitro. Cytometry 1988, 9, 1-6. 

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