Cell cycle checkpoint evasion and protracted cell ...

int. j. radiat. biol 1999, vol. 75, no. 9, 1137± 1147

Cell cycle checkpoint evasion and protracted cell cycle arrest in X-irradiated small-cell lung carcinoma cells P. J. SMITH†*, M. WILTSHIRE†, S. F. CHIN‡, P. RABBITTS‡ and S. SOUE`S§

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(Received 12 February 1999; accepted 21 April 1999) Abstract. Purpose: To determine the longevity and dose-dependence of acute X-irradiation-induced cell cycle perturbations in a panel of seven small-cell lung carcinoma (SCLC) cell lines (CORL32B, COR-L51B, COR-L88B, COR-L96C, COR-L103, CORL266B, COR-L279), assessed for TP53 tumour suppressor gene status and showing characteristically long population doubling periods. Materials and methods: Cell lines were screened for abnormalities in TP53. Cell cycle arrest and nuclear fragmentation were determined by ow cytometry under culture conditions that minimized the propensity of SCLC cells to form multicellular aggregates. A faster growing SCLC cell line (NCI-H69) and two breast tumour cell lines were used as controls. Results: NCI-H69 and ve of the COR-SCLC cell lines showed clear evidence of TP53 abnormalities and the cycle arrest responses of the breast tumour cell lines established the eÚ ects of TP53 mutation on G1 /S checkpoint loss. All SCLC lines, at 24 h after low dose irradiation, showed abrogation of the G1 /S checkpoint together with a range of expression of a protracted G2 /M delay. G 2 /M delay progressed in all panel cell lines up to 48 h post-irradiation while NCI-H69 showed signi cant recovery for the dose range 75–600 cGy. Only NCI-H69 and one panel line showed dose-dependent progression to complete nuclear DNA fragmentation. Conclusions : The culture method permits the measurement of cell cycle eÚ ects that re ect the TP53 status of SCLC cells. G1 /S checkpoint failure, long-term radiation-induced G2 arrest, highly muted apoptotic responses and delayed recovery appear to be typical responses of the recently derived COR-SCLC lines. The results imply that low levels of unrepaired DNA damage, induced at clinically relevant doses, can persist for days in SCLC cells with long cell cycle traverse times, and can remain capable of checkpoint activation with implications for S phase-targeted therapies.

1. Introduction Small-cell lung cancer (SCLC) accounts for 25% of all lung cancers and is frequently fatal. Despite the apparent initial responsiveness to therapy the disease can be widely metastatic even when apparently localized to the lung and intrathoracic lymph * Author for correspondence; e-mail: [email protected] † Department of Pathology, University of Wales College of Medicine, Heath Park, CardiÚ CF4 4XN, UK. ‡ MRC Centre, Hills Road, Cambridge CB2 2QH, UK. § Laboratoire de Biologie Cellulaire, UFR Biome´dicale des Saints Pe`res, Universite´ Rene´ Descartes—Paris V, 45 rue des Saints Pe`res, F-75270 Paris Ce´ dex 06, France.

nodes. Radiotherapy improves both local control of the tumour and patient survival for limited stage SCLC (for reviews, see Williams and Turrisi 1997, Wagner 1998). There continue to be concerted attempts to improve therapeutic outcome by the adoption of combined protocols for the care of limited stage disease (Saijo 1992, Schnabel and Schmitt 1993, Williams and Turrisi 1997) and through the appreciation that local control of disease is necessary for eÚ ective treatment (Wagner 1998). Future progress for the treatment of SCLC will probably require therapies based on the speci c molecular and biological characteristics of SCLC, including its autocrine growth regulation. Indeed, although SCLC may evade normal growth controls by the loss of the proliferation inhibitor pRB (Schauer et al. 1994), ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are operative in a manner diÚ erent from other lung cancers (Ravi et al. 1998). The challenge will be to integrate novel therapies, targeted on these diÚ erences, with current radiation and chemotherapeutic approaches (Wagner 1998, Roth 1998). The probability of treatment-resistant cells arising in a SCLC tumour will be a function of the cell number and time. An appreciation of SCLC population dynamics is clearly important if the development of resistance to chemotherapy and radiotherapy is to be minimized by the use of eÚ ective treatment modalities early in the treatment programme for limited stage SCLC (Coy et al. 1994). There remain outstanding issues, in terms of the radiation component of combined modalities, not least the appropriate fractionation schedule and dose selection. This paper addresses the dynamic changes that take place in Xirradiated SCLC cultures to determine whether they adhere to the predicted responses of p53-defective tumours and to assess the contribution of cell death to the initial shifts in cell cycle distribution. The longevity of the perturbations and the cell cycle disposition of surviving cells will be important given the phase selectivity of agents used in combined modalities (Gorczyca et al. 1993).

International Journal of Radiation Biology ISSN 0955-3002 print/ISSN 1362-3095 online © 1999 Taylor & Francis Ltd http://www.tandf.co.uk/JNLS/rab.htm http://www.taylorandfrancis.com/JNLS/rab.htm


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The tumour suppressor gene TP53 is a fundamental element in the dynamics of cell cycle arrest and cell death responses to DNA damage. TP53 mutations/abnormalities are found in ~ 70% of SCLC cases, potentially representing an early marker for the detection of neoplasia (Bierrer and Brown 1992, Winter et al. 1992, Greenblatt et al. 1994). In cells with functional p53-dependent pathways, ionizing radiation triggers both G1 and G2 arrests of cells (Kastan et al. 1992, Kuerbitz et al. 1992, Maity et al. 1994). The arrest of cells at the G1 /S phase checkpoint requires the accumulation of wild-type p53 protein and the activation of the expression of various genes involved in DNA repair or the inhibition of cyclin-dependent kinases (Kastan et al. 1992, Kuerbitz et al. 1992, Perry et al. 1993, El-Deiry et al. 1993, 1994). It has been hypothesized that the function of this delay is to provide additional time for repair of DNA before the cell enters critical periods of the cell cycle, such as DNA synthesis in S phase or chromosome condensation in G2 phase (for a review, see Murnane 1995). Cells lacking p53 or its downstream eÚ ector p21 (WAF1) fail to maintain a G2 arrest following c-irradiation (Bunz 1998). Sustained G2 arrest in cells expressing wild-type p53 is associated with nuclear localization of CDC2 rather than inactivation via inhibitory phosphorylation. Nuclear localization of CDC2 can be promoted in a p53 null background by forced expression of p21 (Winters 1998). Thus the G1 and G2 checkpoints are interrelated and share initiators and eÚ ectors. Apoptosis (Arends 1990) is an important factor in describing population responses to radiation (Muschel and McKenna 1997) and is related in part to p53 function. While wild-type p53 cells undergo apoptosis after exposure to radiation, p53 mutant cells show delays in the progression to apoptosis (Lowe et al. 1993b). Dysfunction of p53-dependent pathways can allow tumour cells to evade the checkpoint controls and apoptosis, thereby gaining a selective advantage in attempting to overcome therapy (Lowe et al. 1993a). Indeed, in general cancers with TP53 mutations tend to have a worse prognosis for responses to chemotherapy than those showing wild-type alleles (Callahan 1992). The analysis of cell cycle kinetics and DNA damage-induced cycle arrest is problematic in SCLC due to the tendency of cultured cells to aggregate and form multicellular spheroids adding physical and microenvironmental constraints to proliferative potential. Whether these multicellular spheroids, without vascular components, represent an eÚ ective tumour model for SCLC is not known. We have attempted to reduce the eÚ ects of spheroid formation on SCLC proliferative potential in vitro, using early

passage SCLC cell lines with known TP53 status, to permit the analysis of cell death and cell cycle arrest induced by acute X-irradiation. 2. Materials and methods 2.1. Cell lines and cell culture The human breast tumour cell line MCF7 was maintained in Dulbecco’s minimal essential medium (Gibco, BRL, Life Technologies, Paisley, UK) supplemented with 10% foetal calf serum, 100 IU/ml penicillin, 100 mg/ml streptomycin and 20 mm glutamine and were incubated at 37ß C in an atmosphere of 5% CO 2 in air. The human breast tumour cell line T-47D was maintained in RPMI 1640 medium (Gibco) supplemented as above. Monolayer cultures were detached by two washes with PBS, one with PBS containing 0.025% trypsin and 0.02% EDTA, and incubation at 37ß C for 3–5 min before cell cycle analysis. NCI-H69, a human SCLC cell line was kindly donated by Dr D. N. Carney (NDI, Bethesda, MD, USA). The other SCLC cell lines described in this study, all with COR designations, were derived from clinical specimens and originally established in culture and kindly provided by Dr P. Twentyman (Baillie-Johnson et al. 1985). All seven COR-SCLC cell lines displayed typical SCLC cytology (BaillieJohnson et al. 1985). Each cell line was grown as a suspension culture (except COR-L88B, which grew loosely attached), in RPMI medium supplemented with 10% foetal calf serum, 2 mm glutamine, 100 mg/ml streptomycin, 100 units/ml penicillin and incubated at 37ß C in an atmosphere of 5% CO2 in air. Typically, static cultures were grown in 75- or 2 150-cm asks in 25 or 50 ml media volumes respectively, and cells were resuspended daily by gentle pipetting. Cultures were resuspended by aspiration using a Pasteur pipette and cell concentrations determined using a Coulter counter, (Coulter Electronics Ltd, Luton, UK). 2.2. X-irradiation Irradiations were performed in normal medium and culture asks using a 250 kV machine (Pantak, Windsor, UK) operating at 15 mA with ltration of 2.32 mm copper half-value thickness and a dose-rate of 0.685 Gy/min. 2.3. Cell cycle analysis and cell sorting Cells were stained for 10 min with ethidium bromide (50 mg/ml) plus 0.125% Triton X-100 and 0.5 mg/ml ribonuclease A, before ow cytometric


SCLC cell cycle delay analysis as described by Epstein et al. (1988). Samples were processed by data-analysis algorithms to display DNA uorescence and 90ß light-scatter contour plots (Watson et al. 1987). The metaphase population tends to slightly understain, resulting in a G2 /M peak, which is biased towards S phase DNA content, contributing to an overestimation of S phase. Thus mitotic cells were identi ed by their reduced 90ß light scatter versus DNA content. DNA frequency distribution histograms were analysed to yield percentage of cells in G1 , S phase, G2 (Watson et al. 1987) and combined with the mitotic fraction to yield relative frequencies. Veri cation of the morphological characteristics of each fraction was achieved by sorting cells using a FACS Vantage cell sorter (Becton Dickinson, UK Ltd, Cowley, UK). Preparations were examined directly by transmission/ uorescence imaging on a BioRad 1024-MP confocal imaging system operating in single photon excitation mode with the 488-nm line derived from a krypton–argon laser. 2.4. Apoptosis detection and confocal imaging DNA fragmentation was detected using the in situ 7 terminal deoxytransferase (TdT) assay. Brie y, 10 cells were washed in PBS and xed in 70% ethanol for 30 min. 10 ml cell suspension was dropped onto polylysine-treated slides and incubated overnight in a humidi ed chamber. Slides were washed in PBS, distilled water and air-dried. Preparations were incubated with 12.5 units TdT enzyme (Promega Corporation, Madison, WI, USA), in TdT buÚ er plus 1% uorescein-conjugated dUTP (Amersham Life Sciences Ltd, Little Chalfont, UK) for 1 h at 37ß C in a humidi ed chamber. Finally, preparations were washed with PBS, distilled water and stained with propidium iodide (PI) at 0.04 mg/ml for 2 min at room temperature. Preparations were rinsed, air dried and mounted in Vectashield medium (Vector Laboratories Ltd, Peterborough, UK). Apoptotic cells, showing TdT and PI positivity, were visualized using a confocal dual channel uorescence microscope (MRC600; Biorad Microsciences, Hemel Hempsted, UK). The confocal system was also used to visualize cells stained with acridine orange and those derived from cell cycle analysis preparations. 2.5. Identication of mutations in the TP53 gene Mutations in exons 4–8 in the TP53 gene were identi ed as described by Chung et al. (1995). DNA was isolated from the SCLC cell lines shown in table 2 and exons 5–8 were individually ampli ed using oligonucleotides in the introns adjacent to the

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exon–intron boundaries. a P-dCTP was incorporated and the products were subjected to gel electrophoresis followed by autoradiography. If this SSCP analysis failed to reveal any band changes, exon 4 was also examined in the same way. When an extra band or a band shift was detected, the exon involved was sequenced. 3. Results 3.1. SCLC cell lines: biological characteristics and p53 status SCLC cell lines are often grown in agitated suspension culture, conditions which favour the formation of spheroids usually containing > 100 cells per aggregate. Figure 1a shows a typical NCI-H69 spheroid in which internal cavities are apparent and cells show a range of morphologies including apoptotic characteristics. Spheroid development can result in many cells in the aggregates not being in cycle and therefore not suitable for kinetic measurements. Cultures containing large established spheroids can prove diÝ cult to reduce to single-cell suspensions even with the use of protease digestion. By growing cells in static culture, but with regular resuspension by aspiration (i.e. static/resuspension culture; see Materials and methods), cells could be maintained in actively growing culture but with smaller and less rmly compacted aggregates ( gure 1b). The preparation method for cell cycle analysis selected does not require cell washing and, therefore, there is no loss of degraded cells and nuclear fragments. Upon preparation of nuclei for ow cytometry aspects of nuclear structure are retained ( gure 1c; mean diameter of NCI-H69 nuclei of 18.0Ô 2.0 mm). Such preparations can be dispersed easily with reasonable preservation of apoptotic ( gure 1d) and mitotic ( gure 1c) nuclei. Cell cycle distributions of static/resuspension SCLC cultures are in the ranges: 35–55% in G1 phase, 31–42% in S and 13–27% in G2 /M (table 1). None of the SCLC cell lines exhibited < 45% of cells in S+ G2 /M phases, suggesting that most cell lines maintained high fractions in cycle but with variable population doubling times. CORL96C (35.3%) presented the lowest and COR-L88B (54.6%) the highest percentage of cells in G1 . As previously reported for other SCLC cell lines (Giaccone et al. 1992) each COR-SCLC cell line had a slow growth rate, the doubling times ranging from 58 to 202 h as determined by growth curve analysis. Distinctly fast growing NCI-H69 cells (58Ô 10 h) were used as the experimental control in all experiments. In another survey of 13 SCLC lines, six were found to grow slowly (mean doubling time 99 h)

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Figure 1. Laser scanning confocal uorescence images of cultured SCLC cells showing apoptotic (ap) and mitotic (mt) nuclear morphologies. (a) Spheroid of NCI-H69 cells grown in agitated suspension culture, stained with acridine orange and showing cavities. (b) Small aggregates of COR-L32B cells grown in static/resuspension culture and acridine orange-stained at 24 h following 450 cGy X-radiation exposure. (c) Ethidium bromide-stained nuclei of Triton-X permeabilized unirradiated NCI-H69 cells, derived from static/resuspension culture and showing stable nuclei. (d) Ethidium bromide-stained nuclei of Triton-X permeabilized unirradiated COR-L279 cells, derived from static/resuspension culture and resuspended to provide full dispersal of cell aggregates before ow cytometric analysis. Image sizes (mm): (a) 779Ö 519; (b) 182Ö 121; (c) 182Ö 121; (d) 545Ö 364.

Table 1.

Cell line

a

Characteristics of human tumour cell lines. a

Cell cycle distribution (%Ô

SE)

G1

G2 /M

S

Breast carcinoma cell lines MCF-7 48.0 Ô T47D 60.8 Ô

9.3 3.2

34.0 Ô 0.4 28.4 Ô 5.1

18.1 Ô 10.9 Ô

8.9 1.8

SCLC cell lines NCI-H69 COR-L32B COR-L51B COR-L88B COR-L96C COR-L103 COR-L266B COR-L279

2.8 0.9 2.1 4 4.1 4.2 3.3 2.3

38.3 Ô 32.6 Ô 42.3 Ô 32.2 Ô 37.6 Ô 37.8 Ô 35.2 Ô 31.2 Ô

19.6 Ô 16.8 Ô 13.5 Ô 13.1 Ô 27.0 Ô 14.2 Ô 20Ô 18.3 Ô

1.7 0.8 1.1 0.4 3.7 1.5 2.1 0.9

42.1 Ô 51.1 Ô 44.3 Ô 54.6 Ô 35.3 Ô 48.1 Ô 44.9 Ô 50.5 Ô

1.3 1.0 2 4.5 1.4 4.7 1.9 2.1

Nuclei with reduced DNA b (% gated events)

Tdt+ ve nuclei (% cells)

nd nd

nd nd

15.5 Ô 42.4 Ô 37.7 Ô 41.0 Ô 47.4 Ô 37.2 Ô 29.7 Ô 20.2 Ô

4.4 14.7 10.6 10.3 13.7 7.2 5.5 4.9

8.1Ô 1.4 22.7 Ô 13.0 7.6Ô 3.6 5.6Ô 2.2 7.9Ô 3.4 5.0Ô 2.5 3.7Ô 5.1 15.9 Ô 8.3

MeanÔ SE ( n > 5) for gated cells with normal DNA content (type 1 and type 2 cells; see text) as determined by ow cytometry. MeanÔ SE (n> 4) for type 3 cells with reduced DNA content. nd, Not determined. b

SCLC cell cycle delay and had lower S-phase percentages (mean 32%), while the remaining seven cell lines grew quickly (mean doubling time 45 h) and had higher S-phase percentages (mean 44%) (Matsumoto et al. 1991). In the present study the COR-SCLC cell lines showed a comparable range of values with an average S-phase percentage of 35.6Ô 4.0% and a mean doubling time of 128Ô 42 h.


3.2. SCLC cell lines: p53 status The results are summarized in table 2, revealing that ve of the seven COR-SCLC cell lines show TP53 tumour suppressor gene abnormalities. Parallel ELISA assays for detecting the expression of both mutant and wild-type p53 protein, using the mouse monoclonal MAb 1801 antibody, indicated that p53 protein was undetectable in NCI-H69 and CORL266B cells (data not shown). There was no detectable mutation in exons 4–8 of TP53 in COR-L32B or COR-L96C. In the case of COR-L96C there remains a possibility of p53 alteration since both immunocytochemistry and ELISA assays suggest stabilization of the protein (data not shown). COR-L32B cells presented an abnormality in exon 4 involving no amino acid change (table 2). Attempts to detect p53 expression by ow cytometry after staining of xed COR-L32B cells with PAb 421 ( pantropic) or PAb 240 (mutant speci c) polyclonal antibodies suggested a low level stabilization of p53 protein that did not react with PAb 240. Given that COR-L32B was derived from a patient originally Table 2. Cell line

p53 status of human tumour cells lines. Mutation site in TP53

Breast carcinoma cell lines MCF-7 wild-type T47D exon 6 SCLC cell lines NCI-H69 exon 5 at 171 stop COR-L32B exon 4 at 124 COR-L51B exon 5 at 131 COR-L88B exon 5 at 157 COR-L96C no mutations in exons 4–8 COR-L103 exon 7 at 234

p53 status wild-type mutant stop mutation mutation; no amino acid change mutant; heterozygous mutant; homozygous wild-type?

missense mutant; heterozygous COR-L266B exon 6 at 213 mutation; no amino acid change exon 8 at 226 stop mutation COR-L279 D15bp in exon 5 small deletion at 5 ¾ end of exon 5 destroys splice junction (homozygous)

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classi ed as having a poorly diÚ erentiated squamous cell carcinoma (Baillie-Johnson et al. 1985), and that there is no evidence of a chromosome 3 abnormality (PR; unpublished data), it is reasonable to question the true SCLC status of COR-L32B and whether it presents p53 dysfunction. 3.3. Spontaneous and radiation-induced cell death The present authors have observed that ow cytometry of nuclear preparations of Triton-X permeabilized irradiated and control SCLC cultures showed three types of nuclei characterized by 90ß laser light scatter (SSC) and ethidium bromide staining. Type 1 cells comprising intact nuclei with normal light scatter and DNA content. Type 2 cells representing intact nuclei with abnormal morphology, identi ed after cell sorting, in the earlier phases of apoptosis with normal DNA content but high SSC ( pulse height analysis). Lastly, type 3 cells, characteristic of later stage apoptotic cells and fragmenting nuclei, with reduced DNA content and reduced SSC ( pulse width analysis). Nuclear fragmentation can be seen in gure 1d representing the precursors of type 3 cells. Sorting of type 1 and 2 nuclei con rmed the characteristics of normal or apoptotic nuclei respectively ( gure 2c and f ). Parallel experiments using p53 wild-type human B cell lymphoma cells capable of rapid cellular commitment to apoptosis indicated that the time-dependent increase in type 2 cells preceded the increase in cells with TdT-positive nuclei (data not shown). The fraction of late stage apoptotic cells in control cultures diÚ ered between cell lines (table 1). Table 1 also shows the relationship between this direct assessment of culture integrity and TdT-positive cell frequency. As expected the frequency of type 3 cells is greater than that of TdT-positive cells since the type 3 fraction may incorporate large fragments of shattered cells and the TdT preparation protocol tends to increase the probability of fragment loss. There was no clear relationship, as determined by linear regression, between the two parameters for all SCLC cell lines (r = 0.14). However, the majority of cell lines contained ve times as many late stage type 3 apoptotic cells as TdT-positive cells. These ndings are consistent with the results of parallel experiments (data not shown), suggesting that the SCLC cell lines show prolonged engagement of the apoptotic process with the generation of high molecular weight 50 kb DNA cleavage and reduced release of small molecular weight fragments ( < 5 kb) upon detergent lysis of cells. Both NCI-H69 and COR-L279 cell line showed an increase in type 3 cells in response to higher

P. J. Smith et al.


1142

Figure 2. Laser scanning confocal uorescence (d–f ) and transmission (a–c) images of nuclei sorted according to DNA content and light scatter characteristics. (a, d) G1 cells with normal light scatter (type 1 cells; see text). (b, e) G2 cells with normal light scatter (type 1 cells). (d, f ) G1 cells with abnormally high light scatter (type 2 cells). All images derived from ethidium bromidestained nuclei of Triton-X permeabilized unirradiated NCI-H69 cells derived from static/resuspension culture. Scale bar in (d) = 10 mm.

( > 450 cGy) radiation doses (table 3, gure 3a) while no signi cant increase could be detected in CORL32B, COR-L51B, COR-L88B, COR-L103 and COR-L266B cultures ( gure 3a and b). It is suggested that the majority of the SCLC cell lines shows resistance to apoptotic commitment in keeping with the TP53 mutant status of those cell lines. Table 3.

Time-dependent accumulation of X-ray-induced apoptotic cells in NCI-H69 cell cultures.

X-ray dose (cGy) 75 150 300 450 600 a

Change in frequency (% treatedÕ % control) for a post-irradiation incubation period of: 24 h 1.5Ô 4.2Ô 5.2Ô 5.4Ô 8.4Ô

0.1 1.1 1.0 1.6 1.0 Õ

48 h 2.7Ô 1.6 0.9Ô 4.1 8.4Ô 3.6 20.9 Ô 8.1 21.3 Ô 7.6

a

Õ

Õ

96 h 4.8Ô 1.2Ô 19.0 Ô 33.8 Ô 45.8 Ô

1.8 9.1 11.2 5.4 3.8

Data are meanÔ SE for the frequency of type 3 cells (see text) for ve independent experiments; absolute value for unirradiated control cultures = 15.5Ô 4.4% gated cells.

3.4. Cell cycle checkpoint status To establish the characteristics of p53 wild-type and p53 mutant responses to X-irradiation, two human breast cancer cell lines, included in the US National Cancer Institute (NCI) tumour cell line panel and of known TP53 status (MCF7, TP53 wildtype; T-47D, exon 6 mutant), were used as controls. Unirradiated cell cycle distributions are given in table 1, while gure 4 shows the lack of eÚ ective G1 emptying by MCF7 cells compared with the > 50% emptying shown by T-47D cells at 24 h after 450 cGy X-radiation. The G1 emptying in T-47D is associated with a rst cycle dose-dependent arrest in G2 showing saturation at 450 cGy. In both cell types the fraction of cells in S phase remained essentially unchanged. Figure 5 shows the changes in cell cycle distribution of irradiated NCI-H69 cells with a postirradiation incubation period. In this analysis a distinction was made between G2 and M cells (Epstein et al. 1988). NCI-H69 clearly shows the T-47D-like response with eÚ ective dose-dependent emptying of G1 , no accumulation of cells in M or S phase, and evidence that the fraction of cells delayed in G2


SCLC cell cycle delay

1143

Figure 3. EÚ ect of X-irradiation dose on the frequency of SCLC nuclei/cells with abnormal nuclear light scatter and DNA content characteristics (type 3 cells) determined at 48 h post-irradiation. NCI-H69, D ; COR-L32B, ] ; COR-L51B, * ; COR-L88B, x ; COR-L96C, E ; COR-L103, _ ; COR-L266B, + ; COR-L279, y . Data ( Ô SE) derived from three to ve experiments.

Figure 4. X-ray-induced cell cycle perturbations determined at 24 h post-irradiation of human breast cancer cell lines demonstrating p53 wild-type (MCF7) and p53 mutant (T-47D) responses. G1 , D ; G2 /M, ] ; S phase, * . Data are meanÔ SD for three experiments.

Figure 5.

Time-dependent changes in cell cycle perturbations of NCI-H69 cells following acute X-irradiation. G1 , D ; G 2 , y S phase, * . Data are meanÔ SD for three to ve experiments.

; M, x

;


1144

P. J. Smith et al.

decreases after 24 h post-irradiation incubation. Concomitant treatment with nocodazole (100 nm ) to block progression through mitosis revealed that 450 cGy exposure almost emptied the G1 compartment (G1 < 5%), but a 600 cGy dose was necessary to prevent all cells from cycling through to mitosis (data not shown). The panel of seven COR-SCLC cell lines were screened for changes in G1 and G2 /M fractions for 24 and 48 h post-irradiation incubation periods. The results are shown in gure 6 with the NCI-H69 data reproduced for comparison. All COR-SCLC cell lines showed a dose-dependent G1 emptying eÚ ect accompanied by a G2 /M accumulation, although reduced responses ( < 20% peak G2 accumulation at 24 h) were observed for COR-L32B and COR-L103. The eÚ ects observed at 24 h appeared to saturate at 300–450 cGy with NCI-H69 showing maximum arrest. At 48 h post-irradiation incubation there is evidence of recovery in COR-L266B and CORL51B for the 75–150 cGy low dose range similar to that observed in NCI-H69. 4. Discussion Genetic analyses con rmed the TP53 mutation in NCI-H69, and show TP53 abnormalities in CORL51B, COR-L88B, COR-L103, COR-L266B and COR-L279. This included the COR-L88B line derived from a patient in relapse after SCLC chemotherapy. COR-L32B, derived from a patient originally classi ed as having a poorly diÚ erentiated squamous cell carcinoma, provided no rm evidence of TP53 abnormalities. All of the SCLC cell lines showed the phenotypic marker of G1 /S checkpoint evasion following acute X-irradiation demonstrated by a breast tumour mutant TP53 control cell line. Ancillary studies on the COR-L96C line, presenting an inconclusive genetic analysis, suggest p53 dysfunction consistent with the nding of the loss of G1 /S checkpoint control. In the case of COR-L103 eÚ ective G1 emptying did occur but the long population doubling time (202Ô 43 h) make rm conclusions diÝ cult. The rst cycle G2 arrests observed were protracted, and continued after 24 h incubation in some lines while others displayed evidence of recovery which cannot be attributed to selective loss via apoptosis. Given slow SCLC culture growth rates, consistent with clinical measurements (Shibamoto et al. 1998), the determination of cell cycle perturbations is problematic with the additional possibility that cell cycle arrest due to nutrient depletion could lead to an overestimation of kinetic parameters in vitro (Tinnemans et al. 1993, 1995). One solution is to use

mitotic blocking agents, although these can result in cell damage, leakage through the mitotic block and are less suitable for long-term analyses. the present authors initial attempts to follow cell cycle changes in cultures of fully formed spheroids did not produce readily interpretable results and prompted the use of the static/resuspension method. This approach has allowed us to assign a p53 mutant-like radiation response to NCI-H69 and at least six of the seven COR-SCLC lines. Comparing the results obtained for NCI-H69 and COR-L279, there is a clear diÚ erence in the cycle perturbations occurring between the 24 and 48 h incubation periods. NCI-H69 appears to demonstrate recovery while COR-L279 continues to express late arrest. In the absence of cell loss, release of a cell from G2 arrest would theoretically result in a supply of two cells to G1 . Thus recovery from pre-mitotic arrest should be accompanied by an apparently greater reduction in the ‘G1 emptying eÚ ect’ during the 24–48 h incubation period. This was not observed and for radiation doses > 300 cGy COR-L32B, COR-L88B, CORL266B and COR-L279 showed continued accumulation of cells in G2 /M at 24–48 h with maintenance of an emptied G1 fraction. Interestingly COR-L51B showed a similar response but with highly dosedependent recovery. Previous studies have shown that SCLC cells can undergo programmed cell death upon failure to progress through the cell cycle (Tallet et al. 1996) while a range of reports has highlighted the importance of the extent and longevity of post G1 /S checkpoint arrest in de ning the delayed apoptotic responses of p53-defective human cells (Allday et al. 1995, Canman et al. 1995, Guillouf et al. 1995, Bernhard et al. 1996). The TP53 defective CORSCLC cell lines appear to have a protracted execution of apoptosis, which have been measured here in terms of changes in light scatter characteristics to minimize the manipulative procedures involved. NCI-H69 and COR-L279 appear similar with respect to background and radiation-induced apoptotic cells. Thus, the diÚ erences in arrest recovery shown by NCI-H69 and COR-L279 would have to involve the loss of arrested cells in the case of NCI-H69 and non-arrested cells in COR-L279. Further studies are underway to determine cell cycle-dependent cell death rates. Di Leonardo et al. (1994) studied the p53-dependent G1 arrest in several normal human diploid broblast strains and p53-de cient cell lines treated with 10–600 cGy c-radiation and concluded that p53 helps maintain genetic stability in such cells by mediating a permanent cell cycle arrest through longterm induction of p21 WAF1/Cip1 and that low

Figure 6.

G1 emptying and G2 /M arrest of SCLC cells 24 h (open symbols) and 48 h (closed symbols) after treatment with X-irradiation. G1 , E ; D , G2 /M; _ ] . Data are meanÔ SD for three to nine experiments, except for COR-L96C for which one experiment is shown.


, S phase,

SCLC cell cycle delay 1145

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levels of unrepaired DNA damage are responsible for arrest. In the current study arrest was evident in COR-SCLC cell lines up to 48 h postirradiation even in the low dose ( < 300 cGy) range. The implication is that low levels of unrepaired DNA damage, induced at clinically relevant doses, can persist for days in cells with long cell cycle traverse times and can remain capable of checkpoint activation. Interestingly the NCI-H69 cell line, long established in culture, showed the most eÚ ective recovery from arrest at the higher doses, suggesting that this line may have gained an additional capacity to overcome cycle arrest or show enhanced lesion resolution through long-term selection in a background of genetic instability. The present study indicates the feasibility of obtaining cell kinetics data on slow growing SCLC cells which re ects their p53 status with pre-mitotic DNA damage-sensing checkpoint activation detectable at doses as low as 75 cGy. Preliminary studies have also indicated diÚ erences between cell lines in the COR-SCLC panel in their sensitivity to S phase arrest by the anti-cancer agent etoposide, a candidate agent for combined modalities. The delay in onset of the full apoptotic programme, protracted G2 arrest and variable expression of S phase delay would act to complicate the design of combined modalities or radiation fractionation schedules. It is predicted that targeted therapies requiring active S phase traverse may be of reduced eÝ cacy in combination with radiation if these protracted eÚ ects are not taken into consideration. An important step would be to develop methods for the de-convolution of data sets in which delay, arrest, cell loss and recovery may be contributing simultaneously to the changes in cell cycle age dispositions over protracted periods. Acknowledgements This work was supported in part by a research grant from the UK Medical Research Council. References Allday, M . J., Inman, G . J., C r awford, D . H. and Farrell,

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