Cell Cycle Arrests and Radiosensitivity of Human Tumor Cell Lines: Dependence on Wild-Type P53 for Radiosensitivity'

[CANCERRESEARCH54.3718â€”3722.July15. 19941 Advances in Brief

Cell Cycle Arrests and Radiosensitivity of Human Tumor Cell Lines: Dependence on Wild-Type p53 for Radiosensitivity'

Amanda J. Mcllwrath, Paul A. Vasey, Gillian M. Ross, and Robert Brown2

CRC Department of Medical Oncology, CRC Beatson Laboratories, Switchback Roa4 Garscube Estate, Bearsden Glasgow G61 JBD fA. J. M., P. A. V., R. B.J, and Radiotherapy Research Unit, Institute of Cancer Research, Sutton 5M2 5NG fG. M. R.J, United Kingdom

Abstract p53 is transfected into a human colon tumor cell line, such that a radiation-induced p53-dependent G1 arrest is abrogated, the cells do Loss of p53 function has been shown to cause increased resistance to not change their radiosensitivity (1 1). The lack of effect on radiosen ionizing radiation in normal murine cells; however, the role of p53 in sitivity in this line may be due to expression of other resistance radloresistance of human tumor cells is less clear. Since wild-type p53 mechanisms which overwhelm any impact on sensitivity of modulat function is required for radiation-induced G1 arrest, we measured G1 ing p53 function. We have used a similar approach to investigate the arrest in 12 human tumor cell lines that have a wide range of radiosen sitivities (surviving fraction at 2 Gy, 0.11â€”0.8). We observed a significant effect on radiation sensitivity of losing the radiation-induced G1 arrest correlation between the level of ionizing radiation-induced G1 arrest and in a radiosensitive human ovarian cell line. radiosensitivity. Cell lines having G1 arrest are more radiosensitive. There is no correlation between maximal G2 arrest and radiosensitivity. Expres. Materials and Methods sion of a dominant-negative mutant of p53 (codon 143, Val to Ala) in Cell Culture, Radiation Treatment, and Flow Cytometry. The cell lines transfectants of the radiosensitive human ovarian cell line A2780 abro used in the present study are shown in Table 1. All cell lines were maintained gates the radiation-induced G1 arrest. Such mutant p53 transfectants are as monolayers in RPMI 1640 with 10% fetal calf serum and grown at 37Â°Cin more resistant to ionizing radiation than the parental line and vector 95% air/5% CO2. Exponentially growing cells were irradiated with y-rays at alone transfectants, as measured by clonogenic assays. These results sup room temperature using a @â€˜Â°Cosourcedelivering 2Gy/min. DNA synthesis was port the concept that wild-type p53 function Is required for sensitivity of assessed by incorporation of BrdUrd3 and flow cytometric analysis as de tumor cells to DNA-damaging agents, such as ionizing radiation, and that scribed previously (1, 9). Exponentially growing cells were @yirradiated, at the loss of p53 function in certain human tumor cells can lead to resistance various times medium containing 10 @iMBrdUrdwas added, and the cells were to ionizing radiation. incubated for 4 h at 37Â°C.Thecells were harvested and fixed; after denatur ation of the DNA with 2 N HC1, cells were incubated with an anti-BrdUrd Introduction mouse MAt, (Dako). Bound complexes were detected with goat anti-mouse fluorescein isothiocyanate (Sigma, Poole, United Kingdom), stained overnight Damage to the DNA of proliferating cells by ionizing radiation at 4Â°Cwithpropidium iodide, and analyzed by flow cytometry using a Coulter causes an arrest of mammalian cells in the G1 and G2 phases of the (Hialeah, FL) EPICS Profile Analyzer. cell cycle (1 , 2). Absence of such cell cycle arrests has been shown to Immunoassays. Cell extracts were prepared by lysing exponentially grow be associated with hypersensitivity of eukaryotic cells to ionizing ing cells in 1% Nonidet P-40, 500 m@iNaCI, 50 mM Tris (jH 7.5), and I mM radiation (1â€”3).Certain types of tumor, such as neuroblastomas and dithiothreitol in the presence of protease inhibitors. Protein concentrations testicular germ cell tumors, are exquisitely sensitive to radiotherapy as were determined by the Bio-Rad (Richmond, CA) protein assay. Immuno well as chemotherapy, in comparison to the majority of tumors (4). blotting was carried out as described previously (9) using the p53 antibody, Furthermore, cells derived from such tumors retain a sensitive phe pAB2 (Oncogene Sciences, Manhasset, NY) and visualized by enhanced notype in vitro (5, 6). This leads to the hypothesis that radiosensitive chemiluminescence; the intensity of the autoradiographic signal was quantified tumors may have genetic defects in cell cycle arrest equivalent to by laser densitometry. For EUSA analysis, a sandwich immunoassay was used to measure p53 levels in cell extracts as described previously (12) with those present in radiation-sensitive mutants of yeast or mammalian anti-p53 MAb 240 (13) as the solid-phase reagent. Levels of p53 were cells (1, 7). In the present study, we have analyzed G1 and G2 cell quantitated by reference to a calibration curve using purified recombinant p53. cycle arrests induced by ionizing radiation in human tumor cell lines DNA Transfections and Drug Sensitivity Assays The plasmid pCS3- with a wide range of radiosensitivities. We show that G@arrest but not SCX3 (14) containingmutantp53 complementaryDNA (codon 143, Val to G2 arrest correlates with radiosensitivity. Since radiation-induced G1 Ala) expressed from a CMV promoter and vector alone without insert were arrest in mammalian cells has been shown to require wild-type p.5.3 transfected into the cell line A2780 (9). Expression of p53 in individual clones expression (1), we have examined expression of p53 in more detail in was assayed by ELISA as described above. For clonogenic drug sensitivity @ these lines. Lack of G1 arrest and loss of p53 function has been assays, cells were seeded into 10-cm2 plates and 24 h later were irradiated correlated with acquired or intrinsic resistance of cells to DNA with -y-rays from a @Â°Cosource.After incubation of the plates for 10 days, damaging agents such as ionizing radiation and cisplatin (8, 9). Since colonies were stained, and those greater than 200 cells were counted. wild-type p53 has been shown to be required for ionizing radiation Results induced cell death by apoptosis (10), this may explain why loss of p53 function as measured by loss of G1 arrest correlates with resistance. Radiation-induced Cell Cycle Arrests. Table 1 summarizes the However, it has been shown that when a dominant-negative mutant of cell lines used in the present study. The sensitivities of the cell lines to ionizing radiation have been determined by cbonogenic assays. Received 4/26/94; accepted 6/1/94. Shown in Table 1 are the respective surviving fraction of clonogenic The costs of publication of this article were defrayed in part by the payment of page cells after 2 Gy irradiation (SF2Gy). Cell lines that were analyzed charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. â€˜Thisworkwas supported by the Cancer Research Campaign (United Kingdom) and 3 The abbreviations used are: BrdUrd, bromodeoxyuridine; MAb, monoclonal anti the Scottish Hospitals Endowment Research Trust. body; ELISA, enzyme-linked immunosorbent assay; SF2Gy, the surviving fraction at 2 To whom requests for reprints should be addressed. 2 Gy. 3718

arrestCell Table 1 Radiosensitivity and cellcycle

240g ELISARadiosensitivelineOriginâ€•SF2Gyb2 Gy%5CESFâ€•% 2 GyG2â€• ESVFold p531MAb

HX142 1 23 2 NB1 Neuroblastoma 0.15 45 35 228 319 3.2 1.5 SUSA Teratoma 0.16 43 53 476 345 2.1 10.4 SK-N-SH Neuroblastoma 0.21 24 24 153 153 6.8 1.6 A2780 Ovarian Adeno. 0.22 23 23 259 259 4.5 4.2 GCF27Neuroblastoma Teratoma0.1 0.2710 4126 41145 1881 1881 2.04 2.8 Radioresistant U251 Glioma 0.56105562914631.012.40.5779140275316NDâ€•o0.58162741352921.4170.6291721652861.2350.6694871331591.5630.77101911802090.9170.8931002003711.216 MGH-U1 Bladder TCC MOG-G-CCM Glioma RT112 BladderTCC MOG-G-UV Glioma IP-SB18 Glioma @ Glioma

a Type of tumor from which cell line was derived. Adeno., adenocarcinoma. b The surviving fraction of clonogenic cells after 2 Gy irradiation.

C Percentage of cells incorporating BrdUrd 24 h after irradiation compared to untreated cells. d Percentage of maximal increase in cells with 4N DNA content after irradiation compared to untreated cells. e Cells irradiatedto give approximately equivalent clonogenic surviving fraction(ESF) of 0.2â€”0.3. 1Themaximumfoldincreasein p53proteinafter2 Gy irradiationcomparedtountreatedcells.Levelsof p53weredetectedbyWesternimmunoblottingusingMAb1801and quantitated by laser densitometry.

g @53 protein (ng/mg) cell extract detected by ELISA using MAb 240 which recognizes a p53 epitope exposed when p53 is in mutant conformation (13). The levels of p53 were quantitated by comparison with a calibration curve using recombinant p53 and using a concentration of cell extract which is in the linear range of the ELISA. h ND, p53 protein not detected.

were categorized as radiation sensitive if they had a SF2Gy value of in the radiosensitive lines is not due to inhibition of cell cycle less than 0.35. Conversely, radioresistant lines were defined as having progression deriving from the G2 arrest. a SF2Gy value of greater than 0.35. The radiosensitive lines (SF2Gy, Accumulation of p53 in Radiosensitiveand Radioresistant 0. 11â€”0.27)were derived from neuroblastoma, testicular and ovarian Lines. As shown in Fig. 2, all of the radiosensitive lines had increased carcinoma, tumor types that can be highly responsive to radiotherapy levels of p53 protein 8 h after irradiation with 2 Gy y-rays. On the in vivo (4, 5). The radioresistant lines (SF2Gy, 0.56â€”0.8)were de other hand, the radioresistant lines showed little significant difference rived from glioma and bladder carcinoma, tumors that respond poorly in levels of p53 protein after irradiation at any of the time points or relapse rapidly after radiotherapy (5, 6). examined (Table 1). However, all of the radioresistant lines showed At various times after -y-irradiation, the percentage of cells in S markedly higher levels of p53 protein than the sensitive lines, with the (BrdUrd positive) and at G2/M (4N DNA content) of the cell cycle has exception of the MGH-U1 cell line, which has no detectable p53 been measured compared to control, untreated cells. The maximal protein before or after irradiation. The high levels of p53 observed in decrease in the percentage of S cells was observed 24 h after irradi the radioresistant lines may be indicative of these lines containing ation. All of the radiosensitive lines showed markedly more inhibition nonfunctional p53 protein, which has a longer half-life due to mis of DNA synthesis after irradiation with 2 Gy than the radioresistant lines sense mutation in the p53 protein or stabilization due to the binding to (Table 1). The radiosensitive cell lines showed more than 50% maximal other cellular proteins (15). Many of the missense mutations that inhibition of DNA synthesis after irradiation with 2 Gy, whereas the increase the p53 half-life also cause a conformational change in the radioresistant lines showed little or no inhibition of replicative DNA p53 protein, which can be detected by cross-reaction with p53 anti synthesis. The level of inhibition of DNA synthesis significantly corre bodies such as MAb 240 (13). The ability of MAb 240 to cross-react bated with SF2Gy, using a Pearson's correlation (r = 0.808; P < 0.01; with p53 from the radiosensitive and radioresistant lines has been Fig. IA). As well as comparing the cells using equidoses of irradiation, quantified by ELISA (Table 1). the cell lines were compared using doses of irradiation that gave approx All of the radioresistant lines show high levels of expression of p53 imately equivalent surviving fractions of 0.2â€”03(Table 1). Significant as quantified by ELISA using MAb24O, except for the line MGH-U1 correlation is observed between SF2Gy and the percentage of S cells at which had undetectable levels. Two of these lines, T98G and U251, 24 h after irradiation using a dose of radiation that gave equivalent are known to express missense mutants of p.5.3 (16). The radiosensi survival (r = 0.795; P < 0.01; Fig. 1B). These results show that the tive lines all express p53 that can be detected by ELISA, in particular, radiosensitive cell lines all have marked G1 arrest after irradiation, the SuSa cell line, but all of these sensitive lines have less p53 protein whereas the radioresistant lines have little or no G1 arrest. in a mutant conformation, as detected by cross-reaction with All of the lines show G2 arrest as measured by the increase in cell MAb24O, than the radioresistant lines. Two of the radiosensitive lines number with a 4N DNA content after irradiation (Table 1). Although (A2780 and SK-N-SH) are known to only express wild-type p53 (9, a range in the maximal level of G2 arrest observed for each line is 17). The cross-reaction detected in the radiosensitive cell lines appears observed after irradiation (35 to 376% increase in G2 cells after to be p53-specific since HL-60 cells, which have the p53 gene deleted irradiation compared to untreated cultures), there was no significant (18), show no cross-reaction, and A2780 cells transfected with mutant correlation between SF2Gy values and the percentage level of G2 p53 give higher levels of p53 cross-reaction (data not shown). arrest observed using either 2 Gy or equivalent survival doses (Fig. 1, Effects of Mutant p53 on G1 Arrest and Radiosensitivity after C and D). Therefore, the presence of G1 arrest but not G2 arrest Transfectioninto a RadiosensitiveCell Line ExpressingWild appears to correlate with the radiosensitivity of these lines. There was Type p53. A p53 complementary DNA containing a mutation at codon also no significant correlation between the level of G2 arrest and the 143, leading to a substitution of alanine for valine, expressed from a percentage reduction in S cells, implying that the G1 arrest observed CMV promoter, (14) was transfected into the radiosensitive human 3719

200 - A. râ€”O.SO$,p'cO.Ol 200 - B. râ€”O.795,p

U 150- 150- I .5 U

.5 U m @ 100- U' @_ U III z U U Lu U' @ Lu U a. Lu 50 - a. 50 0 0 0 0@ Fig. 1. Radiation-induced cell cycle arrest and 0

@@@ radiosensitivity. A, the surviving fraction at 2 Gy 0 â€” I (SF2Gy) plotted against the percentage of BrdUrd @@ positive cells 24 h after 2 Gy irradiation. B, SF2Gy 0@ I I plottedagainstthe percentageofBrdUrd-positive 0.0 0.2 0.4 0.6 0.8 1.0 cells 24 h after irradiation with a dose giving 0.2â€” 0.0 0.2 0.4 0.6 0.8 1.0 0.3 surviving clonogenic fraction. C, SF2Gy plot SF2Oy tedagalnstthemaximalpercentageofcellswith4N SF2Gy DNA after 2 Gy irradiation.D, SF2Gyplotted 500 - C. râ€”O.29,p-NS. 500 - D. r-O.414, p-N8. againstthe maximalpercentageof cellswith 4N 0 DNA 24 h after irradiation with a dose giving U 0.2â€”0.3survivingclonogenicfraction.0, radiosen sitive lines; U, radioresistant lines. The Pearson's correlation coefficients (r) and the respective P 400- 400- valuesareshown.NS,notsignificant. U 0 0 U @ 300- U 5300- @ 0 U â€˜U 0 ULu 0 ULu @ 200- U @200- U a. 0 UU U Do 100- 100-

@ 0- . â€˜ . , 0 II 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 SF2Oy SF2Gy

ovarian cell line A2780. The A2780 cells express only wild-typep53 (9), Using a linear quadratic model to fit the surviving fraction after and irradiation of the parental A2780 cell line and vector-alone transfec irradiation, the vector-alone transfectants have a parameters of 0.18 tants showed that these lines possessed a G1 arrest and inhibited DNA and 0.47 with SEs of 0.12 and 0.18, respectively, and fi parameters of synthesis after irradiation (Fig. 3A). Two of the A2780 mutant p5.3 0.26 and 0.23 with SEs of 0.05 and 0.07, respectively. The mutant p53 transfectants lost the G1 arrest and continued to incorporate BrdUrd after transfectants have a parameters of 0.19 and 0.05 with SEs of 0.07 and irradiation. These two mutant p5.3 transfectantS expressed increased 1ev 0.08, respectively, and @3parametersofO.09 and 0.12 with SEs of 0.02 els of p53 as detected by MAb24O EUSA, whereas one mutant p5.3 and 0.03, respectively. Thus, the mutant p53 transfectants of A2780 transfectant, which still possessed a G@arrest, did not show increased p53 cells were significantly more resistant to an acute exposure to ionizing expression by EUSA compared to vector-alone controls (data not radiation in the dose range 1â€”3(ly than vector-alone controls or the shown). This demonstrates that, in this ovarian cell line, expression of a parental A2780 cells, and the difference in sensitivity is particularly mutant p53 can abrogate G1 arrest in a dominant-negative manner, apparent by a reduction in the f3 parameter of the fit to a linear confirming that wild-type p53 ftmction is necessary for the G@arrest in quadratic model. Therefore, abrogation of a radiation-induced G1 these cells. arrest in these radiosensitive cells decreases their radiosensitivity. Radiation sensitivities of the mutant p53 transfectants, which have been shown to lose the G@arrest, and vector-alone controls were Discussion compared by clonogenic assays after irradiation with an acute dose of -y-rays. As shown in Fig. 3B, the mutant p53 transfectants of A2780 We have shown a correlation between intrinsic radiosensitivity of are more resistant to ionizing radiation than vector-alone transfectants. human tumor cell lines and the level of G1 arrest induced by ionizing 3720

Radiosensitive lines:

SKNSH HX142 NB1 SuSa GCT27 A2780 F- 1F@ 11 II â€˜F ir â€” + â€” + â€” + â€” +-. + â€” +

@@@ Fig. 2. p53 levels after irradiation. The levels of â€¢ â€¢ â€¢ P53 p53 were detected by immunoblotting in the cell â€˜. lines 8 h after treatment with 2 Gy â€˜y-rays(+) or after no treatment (â€”). Radioresistant lines: CCM U251 UVW T98G Ui SBI8 RT112 @ F__ iF 1l_ 1 F_ 1 I@_@ IF 1F__ 1

radiation. Cells possessing a G1 arrest are more sensitive to ionizing radiation than cells lacking such an arrest. Similar correlations have been made in Burkitt's lymphoma cell lines and in ovarian lines ISO - A. selected for resistance in vitro to DNA-damaging agents (8, 9). We observed no correlation with the level of G2 arrest in the lines, R --- although this does not exclude the possibility that, for any given line, â€˜U G2 arrest may affect sensitivity. We have used cell lines that have a I @ wide range of radiosensitivity (SF2Gy, 0.1 1 to 0.8) and which include @100. U- - cell lines derived from tumors which are particularly radiosensitive. This may explain why correlation between radiosensitivity and p53 mutation has not been observed in previous studies in other tumor lines with narrower sensitivity ranges (19), although it is also now clear that p53 function can be disrupted by factors other than muta a. 50 - tions in the p53 gene (15). Indeed, measuring G1 arrest induced by DNA-damaging agents such as ionizing radiation may be one of the most effective means of assaying p53 function. It has previously been shown that mammalian radiosensitive mutant cell lines often lack G1 arrest (3). Furthermore, increased accumula 0-- tion of p53 protein after DNA damage can be defective in certain cell III lines derived from individuals who are sensitive to DNA-damaging 0 10 20 30 40 50 agents (1, 7). It has been suggested that radiosensitive tumors may TIME (HOUMS) possess similar types of genetic changes to these radiosensitive mu tants (1, 7). However, we observed no evidence of this in the radio 1.0 sensitive tumor lines examined, as all possess a G1 arrest and all increase levels of p53 after irradiation. Expression of mutant p53 (codon 143, Val to Ala) in the radiosen 0.0 sitive cell line A2780, which normally only expresses wild-type p53, causes the cells to lose the radiation-induced G1 arrest. These mutant p53 transfectants are more resistant than the parental or vector-alone â€¢1.0 transfectants to an acute exposure of ionizing radiation as measured by clonogenic assay. These results do not show that absence of a G1 arrest confers resistance, rather they show a correlation between p53 .E -2.0 function (as measured by G1 arrest) and radiosensitivity. The loss of p53 function may have other effects on the cells, including loss or reduction in the kinetics of apoptosis induced by ionizing radiation. -3.0 This possibility would be in agreement with the observations that genetic inactivation of the p5.3 gene in transgenic mice causes in -4.0 creased resistance of normal cells to ionizing radiation-induced apop tosis (10). However, abrogation of a G1 arrest in colon tumor cells by 0.0 1.0 2.0 3.0 4.0 expression of a dominant negative mutant ofp53 (1 1) has been shown DOSE (Gy) previously to have no effect on radiosensitivity. This may be due to Fig. 3. Radiation-induced G1 arrest and radiosensitivities of A2780 p53 transfectants A, colon cells having other resistance mechanisms operating that over the percentageof BrdUrd-positivecells after irradiationwith 2 Gy. Points means of at least whelm any effect of loss of p53 function. Such mechanisms may two independent experiments counting a minimum of 20,000 events@B, the surviving fraction ofclonogeniccells,expressedas naturallog,afterirradiation.Linesare fittedusinga two-order include increased bcl-2 expression and expression of other genes polynomial regression; bars SD. U vector-alone transfectants ofA2780 cells; U, mutantp53 which modulate apoptosis (20). This implies that the impact of p53 (codon 143, Val to Ala) transfectants; @,A2780parental cells. function on radiosensitivity will vary between different tumor types, depending on the expression of other components of a DNA damage inducible apoptotic pathway. 3721

The fact that radiosensitive cell lines possess functional p53 paral ing UV or ionizing radiation: defects in chromosome instability syndromes? Cell, 75: lels observations in human tumors. The majority of neuroblastoma 765â€”778,1993. 8. O'Connor, P. M., Jackman, J., Jondle, D., Bhatia, K., Magrath, I., and Kohn, K. W. and testis tumors are highly responsive to both radiotherapy and Role of the p53 tumour suppressor gene in cell cycle arrest and radiosensitivity of chemotherapy and have been shown to express wild-typep53 (21, 22). Burkitt's lymphoma cell lines. Cancer Res., 53: 4776â€”4780, 1993. This would predict that tumors that have mutant p53 will be radiore 9. Brown,R.,Clugston,C.,Burns,P.,Edlin,A.,Vasey,P.,Vojtesek,B.,andKaye,S.B. Increased accumulation of p53 in cisplatin-resistant ovarian cell lines. Int. J. Cancer, sistant; however, because p53 function can be abrogated by a number 55: 1â€”7,1993. of mechanisms, the presence of wild-type p53 will not necessarily 10. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1994 American Association for Cancer Research. Cell Cycle Arrests and Radiosensitivity of Human Tumor Cell Lines: Dependence on Wild-Type p53 for Radiosensitivity

Amanda J. McIlwrath, Paul A. Vasey, Gillian M. Ross, et al.

Cancer Res 1994;54:3718-3722.

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