Reduced Induction of P53 Protein by T-Irradiation in Ataxia Telangiectasia Cells without Constitutional Mutations in Exons 5, 6, 7, and 8 of the p53 Gene

Nargis Nasrin, Mohammed Kunhi, Michael Einspenner, Sultan AI-Sedairy, and Mohammed Hannan

ABSTRACT: Atax/a telangiectasia (AT) is an autosomal recessive disease of childhood with several pheno- typic characteristics. One of the hallmarks of this syndrome is its hypersensitivity to ionizing rodiat/on, which is believed to be due to defects in DNA repair/processing. In addition to radio-resistant DNA synthe- sis, both fibroblasts and lymphoblastoid cell lines derived from these patients have been shown to have an impaired G1 arrest and prolonged G2 accumulation of cells indicating a defect in the regulation of cell cycle after irradiation. Since the (tumor suppressor) p53 protein has been reported to participate in the regulation of G1 arrest after irradiation, the possibility of p53 gene mutation and deregulating cell cycle in AT needed to be examined. We used the PCR amplification and DNA sequencing methods to detect mutations in the hyperrnutable exons (5-8) ofgermline p53 in fibroblast cells from 3 AT homozygotes. No mutation was found in any of these exons. In order to determine the role of the p53 protein in G~ ar- rest, its levels were measured before and after ~,-irradiation by flow cytometry in both AT and normal cells. Radiation-induced p53 protein levels in the AT cells varied from 6 to 60% compared to the normal cells, indicating a reduced induction of the protein in AT. These results suggest that mutation in the AT gene affects the p53 induction by irradiation and may, thus, alter the cell cycle regulation in the AT patients.

INTRODUCTION

Ataxia telangiectasia (AT) is a fatal recessive disease with many phenotypic characteristics. The disorder is usually characterized by cerebellar ataxia, oculocutaneous telangiec- tasia, immunodeficiency, sinopulmonary infection, and chromosomal instability [1, 2]. AT patients are highly sus- ceptible to malignancies, particularly leukemia and lym- phoma [3]. Fibroblasts and lymphocytes from AT patients show hypersensitivity to ionizing irradiation [4]. Painter et al. [5] showed that resistance to the inhibition of DNA syn- thesis following ionizing irradiation could be another marker for AT, indicating a defect in the regulation of cell cycle. Mu- tation of genes regulating the completion of different cell cycle stages would have an effect on genetic stability. The demonstration of a lack of GI-S arrest and a prolonged G2 delay in AT cells after exposure to DNA damaging agents not

From the Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Sand/ Arabia.

Address reprint requests to: Dr. Nargis Nasrin, Department of Biological and Medical Research (MBC-03), King Faisal Specialist Hospital and Research Centre, P.O. Box No. 3354, Riyadh 11211, Saudi Arabia.

Received July 30, 1993; accepted February 10, •994.

14 Cancer Genet Cytogenet 77:14-18 (1994) 0165-4608/94/$07.00

only confirmed the participation of AT gene(s) in cell cycle control but also accounts for their increased radiosensitiv- ity [6, 7].

In response to DNA damage cell cycle is regulated at least in two stages, i.e., GI-S and G2-M transitions. A delay at these stages allow the repair of induced DNA damage in cells and, thus, protects them against lethal and chromosomal ef- fects. Recent studies have shown that the tumor suppressor gene Tp53 (henceforth termed the p53 gene) is involved in GI-S checkpoint in normal cells while tumor cells carrying mutated p53 were found to be deficient in such a cell cycle arrest [7-9]. In support of these observations Kastan and his colleagues [7] have shown that, following irradiation, there was no increase in p53 protein in fibroblasts from AT cells of complementation group A while there was only a 2-fold induction of the protein in AT cells of complementation group C as compared to a 7-fold increase in normal cells. While these studies suggested a role of p53 protein in the regulation of cell cycle in AT cells, there is no information as to whether or not the p53 gene is mutated in AT patients. We, therefore, examined this possibility by sequencing the hypermutable exons 5, 6, 7, and 8 of the p53 gene in 3 classi- cal ATs in which the alteration in cell cycle progression and the level of radiation induced p53 protein were also mea- sured in comparison with the control (cells from a healthy subject).

© 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

Reduced Induction of p53 Protein 15

MATERIALS AND METHODS

Cells and T-Irradiation Skin fibroblast cell strains were developed from cutaneous biopsies obtained from a (Saudi) healthy subject and 3 AT [homozygotes) patients by growing the skin explants in min- imal essential medium (MEM) supplemented with Earl~s salts, penicillin (100 U/mL), streptomycin (100 ~tg/mL), gluta- mine (2 mM), and 10% fetal bovine serum (FBS) in 25-cm 2 tissue culture flasks. The flasks were incubated at 37°C in a humidified (80%) atmosphere with 5% Co 2, 95% air. The cells were grown to confluence. Confluent cells were tryp- sinized and plated on 60-mm dishes at a density of 300,000 cells/dish. Twenty-four hours after plating, both AT and nor- mal cells were irradiated with a 137Cs source (Gamma Cell, Atomic Energy of Canada, Ltd} at a dose rate of 7.5 Gy/min- ute. Two hours after irradiation, cells were harvested and processed to examine the level of p53 expression by flow cytometry [FACScan). To demonstrate the cell cycle changes, AT and normal cells were harvested 17 hours after exposure to 0 and 4.0 Gy T-rays, stained with ethidium bromide, and the histograms were analyzed by FACScan.

DNA Extraction High-molecular-weight DNA was prepared from fibroblast cells from both AT patients and the control. Cells were tryp- sinized and suspended in DNA extraction buffer containing 10 mM Tris-HC1 (pH 7.5), 50 mM NaC1, and I mM EDTA (pH 8.0), 0.5% SDS. The cell suspension was digested with pro- teinase K (200 ~tg/mL} overnight at 50°C, and the genomic DNA was extracted by phenol and chloroform and precipi- tated with ethanol.

PCR Analysis Using Specific Primers Amplifications were performed with 500 ng of genomic DNA in 50 ~tL volume. The primers used to amplify exons 5-8 of the p53 gene were purchased commercially (Research Ge- netics} and the sequences are given in Table 1. Thirty cycles were used for amplifications consisting of 30 seconds at 94°C, 30 seconds at 55°C for annealing, and 1 minute at 72°C for extension. The amplified exons were analyzed by 2% agarose gel.

Flow Cytometric Assay for p53 Expression p53 antibody (Ab-2) used in this study was purchased from Oncor Science. The method of flow cytometric analysis (FAC-

Table 1 Primers used in PCR

-?

i . . . . 1 . . . . i ' , ,

2 s Q

Figure 1 Effects of ?-irradiation on the alteration in cell cycle progression in normal and ataxia telangiectasia (AT) cells. Control (O) and irradiated cells (4 Gy) were harvested 17 hours after irradi- ation.

Scan) of the p53 protein was essentially same as that of Kastan et al. [7]. Briefly, cells were fixed by dropwise addition of ice cold 70% methanol and incubation for 5 minutes at - 70°C. Cells were then washed 1× with phosphate-buffered

saline (PBS) and resuspended with Ab-2 antibody (50 Ixg/mL conc) in PBS. The cells were incubated for I hour at 4°C with occasional shaking, then washed 2 × in PBS, and further in- cubated with goat-anti-mouse IgG2a fluorescein isothiocya- nate (FITC) (diluted 1:100 in PBS) for 30 minutes at room tem- perature, washed 2 x with PBS. Nonspecific blocking serum (2% fetal bovine serum, FBS) was present during each anti- body incubation and washing solution. Finally, cells were resuspended in 1 mL of PBS, 10 ~tL of 1 mg/mL RNase, and 10 ~tL of 10 mg/mL ethidium bromide at least 30 minutes be- fore being analyzed on FACScan (Becton Dickinson, Sun- nyvale, CA). Cells incubated with FBS plus second antibody served as control for background fluorescence. Relative lev- els of p53 protein were evaluated by determining the corrected p53 green fluorescence. (Corrected p53 fluorescence equals the difference between the green fluorescence of the p53 Ab-2 antibody plus goat-anti-mouse IgG2a FITC and goat-anti- mouse IgG2a FITC alone.) Finally, the difference in p53 pro-

Gene Exons Length (bp) Primer sequence

p53 5 184 - 15'CTG-CTC-ACC-sATC-GCT-ATC-TG-3'

6 113

7 110

8 137

+ ~5q'CC-TAC-AGT-ACT-CCC-CTG-CC-3'

- 15'CAA-ACC-AGA-CCT-CAG-GCG-GC-3'

+ ~ 5' -GAT-TGC-TCT-TAG-GTC-TGG-CC-3'

-~5"-TGT-CGA-GGG-TGG-CAA-GTG-GC-3"

+ ~ 5'-TCT-CCT-AGG-TTG-GCT-CTG-AC-3'

5'-CCT-GCT-TGC-TTA-CCT-CGC-TT-3'

+ ) 5~FCC-TGA-GTA-GTG-GTA-ATC-TA- 3 •

16 N. Nasrin et al.

Table 2 Percentage of cells in different stages of cell cycle after being exposed to 0 and 4 Gy

O rad 400 rad

Normal Ga 54.5 89.3 S 34.0 5.7 G2 + M 11.5 5.0

AT G1 68.8 65.6 S 20.9 19.9 G2 + M 10.3 14.6

tein levels in unirradiated and irradiated (4 Gy) cells was evaluated by KS Stats (Kolmogrov-Smirnov statistics op- tion) [10].

the normal cells the AT cells used in our study were not ar- rested in Ga after irradiation confirming the cell cycle de- fect in the latter.

RESULTS AND DISCUSSION

Alteration of Cell Cycle Progression in AT and Normal Cells after y-Irradiation We examined 3 fibroblast cell strains generated from homozy- gous AT patients. (Homozygosity was determined by in- creased radiosensitivity to chronic y-irradiation.) Figure 1 shows the DNA histogram of normal and AT cells before and after irradiation. The percentage of cells in different stages is shown in Table 2. There is a marked decrease of cells en- tering S phase in normal cells while there is no change in AT cells, following irradiation. These data show that unlike

p53 Protein Level in Normal and AT Cells after Irradiation The data from flow cytometric analysis (both histograms and KS-Stats curves) of p53 protein expression with and without irradiation are illustrated in Figure 2A and B for normal cells and Figure 3A-F for AT cells. These results clearly showed the occurrence of increased p53 protein expression as a re- sult of irradiation in normal cells. Although a radiation- induced expression of p53 protein was also observed in 2 of 3 AT cell strains (Fig. 3A-D), the degree of induct ion was much less compared to the normal cells. This difference is

Figure 2 Level of p53 proteins in normal cells exposed to v-irradiation. (A) Flow cytometric analysis of p53 expres- sion (binding of Ab-2 antibodies) 2 hours after exposure to 0 tad ( - - ) or 400 rad (...). Both ( ) and (-..) represent fluorescence histograms after staining with anti-p53 conjugated with fluorescein isothiocyanate (FITC). A shift to the right of the histogram indicates the induction of p53 protein. Both histograms were standardized against blank where normal cells were incubated only with FITC-conjugated anti-p53 antibody. (B) Summation curves were com- puted by KS-Stats curve plot from the two histograms (0 and 4 Gy) of the normal cells. D represents the maximum differences between two summation curves. D/S (n) is a value that is indicative of the similarity of the two curves compared. The vertical axis defines the cell number and the horizontal axis (FL-1) indicates p53 FITC expression.

A

. . . . I"'F' ' " '1 ' ' ' ' I P . , . ~ . ' ' I . . . . I(~EI 2 ~ 0

r l |

p~ FITC

B

I. b P | l l 3

2 b P9114

10c~

,=3

I' '''|''''

FLI p53 FITC Range Analyzed: 0 to 255 Channel of Maximum Difference: D: 0.50 D/s(n): 33.30

1 2 8

NORMAL

A B 2QB

I . . . . I ' ' ' ' l ' ' ' ' l ' ' ' ~ l . . . . I B ~ 2BB

p" RTC rL,

l{l{! I. ) Pl= I I I I t

I ~ I I ~ l l l l r l

Q

p" RTC IGB

FLI

Range Analyzed: 0 to 255 Channel of Maximum Difference: D: 0.31 D/s(n}: 14.94

2 0 0

105

ISm

C

c1 IQm 213Q FLI

p~ FITC

D

1 ~ t . ) ~s3Bit

2 ~ IIIJlllIPJL

$

' ' ' ' 1 . . . . I . . . . I ' ' ' ' | ' ' ' ' Q H a ~ l 2~1

p~ FITC rL 1

Range Analyzed: 0 to 255 Channel of Maximum Difference: D: 0.21 D/s(n}: 9.84

118

$0 ) , ) l i t - I | 1

4 ) l | ! - i i i

E

$ . . ' . . • J •

• I •

;, %, • %

. . . . , . . . . I . . . . I ' ' ' : J : ' ' ' 10B 2BB

FL I

p'~ FITC

A T - 2

F

) . ) I | 1 - I l l

I ) I | I I - I l l

z

.7

] /

t t

t

/

~ ' ' ' ' 1 . . . . I ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1

)53FITC rL,

Range Analyzed: 0 to 255 Channel of Maximum Difference: D: 0.03 D/s(n): 0 . 9 5

64

AT - 3

Figure 3 Flow cytometric analysis of p53 protein expression and comparison of two summation curves {as described in Fig. 2} 2 hours after exposure of 3 AT cell strains. (A, C, E) The histogram analysis of p53 expression; {B, D, F) KS-Stats plot from the AT cells.

18 N. Nasrin et al.

illustrated by the D values derived from KS-Stats curves (Figs. 2B and 3B, D, F). D value represents the max imum differ- ence between two summat ion curves (0 and 4 Gy). In case of normal cells, the D value was 0.5, whereas in 3 AT cell strains, they were 0.3, 0.21, and 0.03 only. Compared to the normal cells the level of p53 protein induct ion in AT cells varied between 6 and 60%. It was interesting to note that one of the AT cell strains (Fig. 3E and F) showing low level of constitutive expression of p53 protein had a negligible in- duct ion of the protein after irradiation. The differences of expression in p53 protein levels in AT cell strains could be explained by either a variation in mutational defects or differ- ent signal t ransduct ion pathways of AT genes. Whi le this manuscript was in preparation, Jonveaux and Berger reported the lack of mutat ion in p53 gene (exons 5-8) of AT patients analyzed by the single strand conformation po lymorph i sm (SSCP) technique [11]. These authors, however, d id not se- quence the ampli f ied exons. To further rule out the possibi l- ity of mutat ions in exons 5-8 of the p53 gene in down- regulat ion of its expression after irradiation, these exons in all 3 AT cell strains in the present s tudy were sequenced by the Sanger dideoxy method. No germ line mutat ion in the p53 gene was found in any of the AT cases when compared with the wi ld- type sequences of the corresponding exons (data not shown).

These results suggested that the mutant AT gene prod- uct(s) down-regulated the inducibi l i ty of p53 wi ld- type pro- tein after i rradiat ion and, thus, per turbed the cell cycle regu- lation. This observation supports the notion that a posit ive interaction between the wild- type AT and p53 genes is neces- sary for cell cycle regulation. Further studies are needed to exclude the possibi l i ty of mutat ions in other exons of the p53 gene that could alter its function wi th respect to the cell cy- cle. However, our results are in agreement wi th those of Jon- veaux and Berger, suggesting that the increased susceptibil- ity to cancer in AT patients is not due to mutat ions in the hypermutable exons of p53.

While our observations suppor t the notion that mutated AT gene(s) may down-regulate the expression or stabiliza- t ion of the wi ld- type p53 protein and thereby minimize the G2 arrest, caut ion should be exercised in general izing the positive contr ibut ion of p53 gene mutat ions per se toward enhancement of cel lular radiosensit ivity. Despite the role of p53 protein in the regulation of the cell cycle (i.e., G 1 arrest), recent studies have repor ted that the loss of wi ld- type p53 function, contrary to expectation, resulted in an increased radiotolerance in numerous cancer cell l ines [12-14]. These f indings were consistent wi th the observation in the Li- Fraumeni syndrome in which a mutat ion in the p53 gene is believed to be responsible for increased radioresis tance and cancer p red i spos i t ion [15, 16]. In view of the controver- sial reports on a correlation between altered p53 gene or gene function and cellular radiosensitivity, it would be worthwhile to address this problem by examining the manner in which

the p53 gene interacts wi th other genes involved in the regu- lat ion of cell cycle, response to genotoxic stress, and DNA repairabili ty.

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