Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

RECYCLING OF RESTING CELLS IN THE JB-1 ASCITES TUMOUR AFTER TREATMENT WITH 1-β-D-ARABINOFURANOSYLCYTOSINE (Ara-C)

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of growth of the murine JB-1 ascites tumour (i.e. 10 days after 2–5 × 10⁶ cells i.p.) large fractions of non-cycling cells with G₁ and G₂ DNA content (Q₁ and Q₂ cells) are present, and the fate of these resting cells was investigated after treatment with l-β-d-arabinofuranosylcytosine (Ara-C). The experimental work consisted of growth curves, percentage of labelled mitoses curves after continuous labelling with ³H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with ³H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6–8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with ³H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with ³H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q_t cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. These results indicate recycling of resting cells first with G₂ and later with G_x DNA content, which contribute to the regrowth of the tumours.     

Optimal conditions of fixation for immunohistochemical staining of proliferating cell nuclear antigen in tumour cells and its cell cycle related immunohistochemical expression

Abstract. Comparison of the results of immunohistochemical expression, such as proliferating cell nuclear antigen (PCNA) in archival material of tumours, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain stable and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fNation, processing and staining of samples using animal-implanted MBT-2 cells derived from chemical-induced mouse bladder carcinoma and PC10, a monoclonal antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temperature before fixation for less than 12 h, fixation in formaldehyde was less than 48 h, and paraffin-embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PCNA positive rates obtained under these conditions was compared with the bromodeoxyuridine (BrdUrd) labelling indices. Although the average PCNA positive rate was significantly higher than the BrdUrd labelling index (P?0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In order to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Occurrence of PCNA positive G₁ cells was expected at a half-life of the PCNA-protein of 20 h and a tumour cell doubling time of about 24h. Thus, the percentage of PCNA positive nuclei in a conventionally paraffin-embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of the biological malignant potential of tumours.     

Cell cycle analysis of tumour-derived cultures ofCrepis capillaris: A kinetic analogy with proliferation of animal tumour cells

Summary Analysis of the cell cycle by three methods has revealed unusual kinetics of proliferation in tumour derived suspensions ofCrepis capillaris. The different methods of analysis yield different estimates of cycle phase durations, and such discrepancies have been explained in terms of low growth fractions with rapid total cycle traverse. Specifically, confidence in the estimation of G₂ duration by the fraction of labelled mitosis analysis, and comparison with shorter G₂ estimates obtained by the two other methods, suggests that cells drop out in G₁. However, cells which do not drop out of the proliferative compartment traverse G₁ extremely rapidly. Extremely short cell cycle durations in which the G₁ phase is virtually non-existent are uncharacteristic of plant cell suspension cultures, in which the G₁ phase has previously been shown to be extended as compared with meristematic root tip cells. A model has been proposed in which a central core of rapidly dividing cells continuously loses cells into a subpopulation of resting or G₀ cells with the G₁ DNA content. Similarities between plant and animal tumours with respect to cell growth and division are discussed.     

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G₂/M phases, whereas the G₁ phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G₂/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G₂/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G₂/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

Evidence for quiescent S- and G2-phase cells in human colorectal carcinomas: a flow cytometric study with the Ki-67 antibody

The expression of certain antigens specific for proliferating cells can be determined simultaneously with cell cycle distribution by means of two-dimensional flow cytometry. In this way, a tumour's growth potential is characterized more precisely than with any one parameter alone. Here we describe such simultaneous measurements of DNA content and labelling with the Ki-67 antibody that distinguishes between cycling and non-cycling cells. Having overcome a number of technical problems we were able to analyse material from 29 biopsies of human colorectal tumours. In a number of cases, Ki-67 negative cells were found with a DNA-content of G_0/1 only, whereas all cells with an S- or G₂-phase DNA-content were Ki-67 positive. There were other cases in which cells with an S- and G₂-phase DNA-content had obviously become quiescent (Ki-67 negative), sometimes even outnumbering the proliferating (Ki-67 positive) cells in the respective compartments of the cycle. Generally, however, when Ki-67 negative and positive subpopulations were analysed separately it was found that the former had a significantly lower (S + G₂)-phase fraction than the latter. There was evidence for a correlation between Ki-67 index and (S + G₂)-phase fraction at least in the subgroup of aneuploid tumours. Neither of the two parameters was correlated with stage according to Duke's classification or tumour size. However, a positive correlation was found between the fraction of unlabelled S- and G₂-phase cells and tumour size as reflected in the T category.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY I. AN IN VITRO STUDY USING MURINE TUMOUR CELL LINES

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G₁, S and (G₂+ M) DNA content in a population. As this method gives the relative (G₂+ M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G₂+ M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with the increase in the (G₂+ M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.     

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G₀/G₁ phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G₂/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G₂, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of “decoy, trap and shoot” chemotherapy.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY

A new method for the evaluation of cell production rates combining flow cytometry (FCM) and the stathmokinetic method using vineristine sulphate (VS) has been used for the analysis of three aneuploid ascites tumours at different stages of growth. Using this technique it was possible to estimate the well-known decrease in cell production rates of ageing ascites tumours. The percentage of normal host cells in the aneuploid tumours studied was easily determined by FCM prior to the calculation of the tumour cell-production rates. A correlation was found between the percentage of tumour cells in the S phase and the tumour cell-production rate. This correlation is probably explained by the gradual transfer of proliferating cells in S phase to resting G₁ and G₂ phases with increasing tumour age.     

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G₁/G₀ arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours. Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G₁/G₀ arrest. Lastly, in XX germ cells we observed a down‐regulation of genes involved in both G₁‐ and G₂‐phases of the cell cycle consistent with their entry into meiosis. Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.     

Tumor-targeting Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle as visualized by FUCCI imaging and become sensitive to chemotherapy

Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. Time-lapse imaging demonstrated that tumor-targeting Salmonella typhimurium A1-R (A1-R) decoyed cancer cells in monolayer culture and in tumor spheres to cycle from G₀/G₁ to S/G₂/M, as demonstrated by fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging. A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice also decoyed quiescent cancer cells, which were the majority of the cells in the tumors, to cycle from G₀/G₁ to S/G₂/M, thereby making them sensitive to cytotoxic agents. The combination of A1-R and cisplatinum or paclitaxel reduced tumor size compared with A1-R monotherapy or cisplatinum or paclitaxel alone. The results of this study demonstrate that A1-R can decoy quiescent cancer cells to cycle to S/G₂/M and sensitize them to cytotoxic chemotherapy. These results suggest a new paradigm of bacterial-decoy chemotherapy of cancer.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro

Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [3H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G0/G1, S, and G2/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [3H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [3H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G2 tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

L-Canavanine as a radiosensitization agent for human pancreatic cancer cells

This study evaluated the in vitro effect of L-canavanine on cell cycle progression in the two human pancreatic cancer cells lines PANC-1 and MIA PaCa-2. After 72 h of exposure to L-canavanine, the percentage of cells in the radiosensitive G₂/M phase of the cell cycle increased 6-fold in PANC-1 cells and 4-fold in MIA PaCa-2 cells, when compared to untreated cells. The capacity of L-canavanine to redistribute cells into the G₂/M phase of the cell cycle was both concentration- and time-dependent. Since many drugs that cause cells to accumulate in the G₂/M phase of the cell cycle are effective radiosensitization agents, the potential of L-canavanine to synergistically enhance the effects of ionizing radiation also was evaluated. The interaction between these treatment modalities was quantified using the median-effect equation and combination index analysis. L-Canavanine was found to be synergistic with radiation when either PANC-1 or MIA PaCa-2 cells were exposed to L-canavanine for 72 h prior to irradiation. These results suggest that L-canavanine in combination with radiation may have clinical potential in the treatment of pancreatic cancer.     

Structural characterization andin vivo immunosuppressive activity of neuroblastoma GD2

Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, G_D2. G_D2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of G_D2 in vivo were then determined in an established murine model. G_D2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus G_D2-treated mice;p<0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2×10⁶ cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as G_D2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.     

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU‐145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU‐145 and LNCaP cells. The IC₅₀ values decreased 1.5‐fold and 1.3‐fold in the DU‐145 and LNCaP cells respectively. In the DU‐145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl‐2 expression. Treatment with both drugs in DU‐145 cells led to an increase in sub‐G₁ arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G₂/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU‐145 through apoptosis and in LNCaP cells through cell cycle arrest (G₂/M arrest).     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Modelling the flow of cytometric data obtained from unperturbed human tumour cell lines: Parameter fitting and comparison

In this paper we firstly present three alternative formulations of a mathematical model for human tumour cell lines unperturbed by cancer therapy. The model counts the number density of cells in each phase of the cell cycle over time where cells are differentiated by their DNA content. Data are available from the Auckland Cancer Society Research Centre, Auckland, New Zealand, in the form of DNA histograms or profiles from 11 different human tumour cell lines (i.e. in vitro) unperturbed by cancer therapy. We then apply one (computationally fast) formulation of the model and discover that although in general different combinations of parameter values give rise to very different DNA profiles it is possible that different combinations of parameter values give rise to virtually identical profiles. Experimental estimates of the rate of transition from the G ₁-phase (growth) to the S-phase (DNA synthesis) enable us to uniquely determine other model parameters of interest that give the least square error between the model and data. We finally apply our model to each of the 11 different cell lines and compare cell cycle phase transit times. Although the DNA histograms of each of the cell lines have similar shapes these cell lines have different combinations of transit times to each other, which could explain why they often react very differently when exposed to anti-cancer therapies during laboratory experiments. An understanding of the in vitro situation may give an insight into why some human cancer patients do not respond to cancer therapy. An erratum to this article is available at .