p53 Levels, Cell Cycle Kinetics and Radiosensitivity in Two SV40 Transformed Wi38VA13 Fibroblast Strains

Strahlenther Onkol 2001 · No. 12 © Urban & Vogel662

Strahlentherapieund Onkologie Original Article

p53 Levels, Cell Cycle Kinetics and Radiosensitivity inTwo SV40 Transformed Wi38VA13 Fibroblast Strains Frank Werner, Friedo Zölzer, Christian Streffer1

Background: The tumor suppressor protein p53 which can mediate an ionizing radiation-induced G1 arrest in mammalian cells,forms complexes with SV40 large T antigen (l-T-Ag). We have analyzed the p53 levels, the capability to undergo a G1 arrest andthe radiosensitivity of two SV40 transformed fibroblast strains differing in their large T antigen expression.Material and Methods: One of the two strains (VA13F) is the commercially available form of Wi38VA13, the other (VA13E) arosespontaneously from the original one in our laboratory. Their p53 levels were measured by means of flow cytometry (FCM) andWestern blot (WB) with two p53 antibodies (Ab-3, clone PAb240; Ab-6, clone DO-1; both Oncogene Science). Cell cycle distribu-tions were determined flow cytometrically after BrdU labeling at regular time intervals after exposure to 250 kV X-rays. Ra-diosensitivity was assessed in a clonogenicity assay.Results: The p53 levels of the two strains corresponded to their large T antigen expression, presumably due to complex formationbetween the two proteins. The strain with a high p53 level did not show a G1 arrest and had a relatively high radiosensitivity,whereas the strain with a low p53 level showed a significant G1 arrest and a lower radiosensitivity.Conclusion: These results suggest that 1. complex formation between the large T antigen and p53 reduces the latter’s function-ality; 2. in these two strains the G1 arrest is one of the factors determining radiosensitivity.

Key Words: p53 level · p53 antibodies · Radiosensitivity · Large T antigen · G1 arrest

Strahlenther Onkol 2001;177:662–9DOI 10.1007/s00066-001-0860-0

p53-Niveau, Zellzykluskinetik und Strahlenempfindlichkeit in zwei SV40-transformierten Wi38VA13- Fibroblastenstämmen

Hintergrund: Das Tumorsuppressorprotein p53, das in Säugerzellen einen strahleninduzierten G1-Block vermitteln kann, bildetKomplexe mit dem großen T-Antigen von SV40. Wir haben die p53-Niveaus, die Fähigkeit zum Aufbau eines G1-Blocks und dieStrahlenempfindlichkeit zweier SV40-transformierter Fibroblastenstämme analysiert, die sich in der Expression des großen T-An-tigens unterscheiden.Material und Methoden: Bei einem der beiden Stämme (VA13F) handelte es sich um die kommerziell erhältliche Form vonWi38VA13, der andere (VA13E) entstand in unserem Labor spontan aus dem ursprünglichen Stamm. Die p53-Niveaus wurden mitHilfe von Durchflusszytometrie und Western Blot bestimmt, wobei zwei p53-Antikörper zum Einsatz kamen (Ab-3, Klon PAb240und Ab-6, Klon DO-1, beide von Oncogene Science). Die Zellzyklusverteilungen wurden in regelmäßigen Zeitabständen nach Be-strahlung mit 250 kV Röntgenstrahlung durchflusszytometrisch (nach BrdU-Markierung) analysiert. Der Koloniebildungstest dien-te zur Untersuchung der Strahlenempfindlichkeit.Ergebnisse: Die p53-Niveaus der beiden Stämme entsprachen der jeweiligen Expression des großen T-Antigens, wahrscheinlichwegen der Komplexbildung zwischen den beiden Proteinen. Der Stamm mit dem hohen p53-Niveau zeigte keinen G1-Block undhatte eine relativ hohe Strahlenempfindlichkeit, während der Stamm mit dem niedrigen p53-Niveau einen signifikanten G1-Blockaufwies und eine geringere Strahlenempfindlichkeit. Schlussfolgerung: Diese Ergebnisse deuten darauf hin, dass 1. die Komplexbildung zwischen dem großen T-Antigen und p53 dieFunktionalität des letzteren Proteins beeinträchtigt und 2. bei diesen beiden Stämmen die Strahlenempfindlichkeit wesentlichdurch den G1-Block mitbestimmt wird.

Schlüsselwörter: p53-Niveau · p53-Antikörper · Strahlenempfindlichkeit · Großes T-Antigen · G1-Block

Received: January 24, 2001; accepted: August 22, 2001

1 Institut für Medizinische Strahlenbiologie, Universitätsklinikum Essen, Germany.

Werner F, et al. Radiosensitivity in Wi38VA13 Fibroblast Strains

663Strahlenther Onkol 2001 · No. 12 © Urban & Vogel

Introduction The p53 tumor suppressor protein is involved in cell cycle con-trol, apoptosis and DNA repair [2, 7, 10, 13]. Changes of theactive p53 protein level occur after radiation, probably due toposttranslational modifications or conformational changes.p53 binds to DNA, turning transcription on and off for a num-ber of downstream effector genes (e.g. p21 [WAF1], GADD45and mdm-2). The radiation induced G1 arrest which resultsfrom the increased expression of these genes in most if not allp53 wild-type cells is thought to give the cell additional time torepair DNA damage before entry into the S phase [22].

The large T antigen (l-T-Ag) of the SV40 virus inducesprofound changes in the phenotype of infected cells leading togrowth characteristics typical of immortalized or establishedcell lines. It is also known to interact with the p53 protein. Intransformed cells both l-T-Ag and p53 are extensively phos-phorylated. Due to the increased extent of phosphorylation,p53 shows a slightly higher electrophoretic mobility in SV40transformed cells. SV40 l-T-Ag induces or activates proteinkinases (for example p34) which phosphorylate p53. There isalso a coincidence of enhanced phosphorylation and stabiliza-tion of p53 after infection and transformation of cells withSV40 [23]. The majority of l-T-Ag molecules form tight non-covalent complexes with p53 thus increasing the latter’s life-time [20]. More than 90% of the T antigen in transformed cellsis located in the cell nucleus, and a small but significant frac-tion is bound to the cytoplasmatic membrane. p53 can associ-ate with l-T-Ag at both of these locations [4].

The study of l-T-Ag and p53 as a model system may offerthe possibility of a better understanding of the role of p53 incell cycle arrest and progression. We used two substrains ofthe well-known Wi38VA13 fibroblasts which differ in theirintracellular l-T-Ag concentration. One of them, Wi38VA13strain F, is the commercially available form, derived from theoriginal Wi38 cells by transformation with SV40. The otherstrain, Wi38VA13 strain E, arose spontaneously in our labora-tory and was later found to have a reduced detectable level ofl-T-Ag.

In these investigations it was studied whether these twoWi38VA13 strains, 1. have different detectable p53 levels,2. have different abilities to undergo a G1 arrest, 3. have dif-ferent survival rates after X-rays.

Materials and Methods Cell Culture Conditions and Treatment

Wi38VA13 derived from a human fetal fibroblast cell line withepitheloid morphology. Strain Wi38VA13F is commerciallyavailable (ATCC CCL-75.1), whereas Wi38VA13E arosefrom this cell line spontaneously in our laboratory. Bothstrains were grown in Minimal Essential Medium with Eagle’ssalts (MEM, Sigma) supplemented with 20% foetal calf serum(FCS, Sigma). They were subcultured two times a week andregularly checked for mycoplasms. For the harvesting proce-dure, the medium was removed, the cells were washed in phos-

phate buffered saline (8 g/l NaCl, 0.2 g/l KCl, 0.1 g/l MgCl2 �6 H2O, 2.16 g/l NaHPO4 � 7 H2O, PBS, Biochrom) and thenharvested using a trypsin-ethylenediamine-tetraacetate solu-tion (EDTA, 0.5 g/l porcine trypsin, 0.2 g/l ethylenediamine-tetraacetate, pH 7.5, Sigma). The number of cells was deter-mined using a cell counter (Coulter Counter, Model ZM,Coulter Electronics GmbH, Krefeld).

All experiments were started with cells taken from a 24-h-old exponentially growing culture. For the flow cytometric de-termination of p53 and large T antigen levels, cells were fixedin 96% ethanol. For the Western blotting experiments the cellswere dissolved in tris(hydroxymethyl)aminomethane-sucrosebuffer with protease inhibitors (50 mM tris(hydroxymethyl)aminomethane, 250 mM sucrose, 1 mM phenylmethysulfonylfluoride, 1 mM EDTA, 1 mM ethylen glycol-bis(�-aminoethylether)-tetraacetic acid, 1 µM leupeptin, pH 7.4). For cell cycledistribution experiments, cells were incubated under low serumconditions (0.5% FCS) for 6 days before further processing sothat they were 70–90% confluent. Irradiation was carried outwith an X-ray unit at 1 Gy/min (Stabilipan, Siemens, 240 kV,0.5 mm Cu filter, 15 mA) before addition of fresh medium(MEM with 20% FCS). For the colony forming assay cells wereirradiated and plated at appropriate densities in culture dishes.

Flow Cytometric Measurements of Large T Antigen and p53

Fixed cells (96% ethanol) were centrifuged to remove theethanol, then washed with PBS (pH 7.4) and divided into twoaliquots. The first was used for incubation (1 h at 4 °C) with anantibody against p53 or the large T antigen (1 µg/200 µl) andthe second for a control antibody (same IgG isotype). Twoprimary antibodies against p53 were used: 1. Ab-3, clonePAb240; 2. Ab-6, clone DO-1. All antibodies were obtainedfrom Oncogene Science.

After washing with PBS-(0.1%)NaN3 the cells were incu-bated with 200 µl of the secondary antibody (anti-mouse IgGFITC conjugated [Sigma], diluted 1 : 40 in PBS-[0.1%]NaN3-[5%]FCS) for 1 h at 4 °C. After staining with propidium iodide(2.5 � 10-5 M, Serva) for 30 min at room temperature the flowcytometric measurements were performed with a BectonDickinson FACScan using an argon laser at 488 nm. For analy-sis, green fluorescence of FITC (Figure 1, Y-axis, which repre-sents p53 immunoreactivity) was plotted against red fluores-cence of propidium iodide (Figure 1, X-axis representingDNA content). Mean Y values of the FITC fluorescence withthe specific anti-p53 antibody and with the unspecific negativecontrol antibody were determined. The specific p53 expres-sion was calculated as a fluorescence index (Fi) [29]:

Fi = specific fluorescence minus unspecific fluorescence

unspecific fluorescence

– unspecific fluorescence determined by incubation with acontrol antibody,

– samples with Fi > 0.5 were regarded p53 positive.

664 Strahlenther Onkol 2001 · No. 12 © Urban & Vogel


Western Blot Analysis of p53The constitutive level of p53 protein was estimated with thehelp of Western blots. Cell lysates were harvested in SDS/PAGE sample loading buffer (pH 6.8).

The proteins were separated by polyacrylamide gel(10%) electrophoresis and transferred to nitrocellulose. Theprotein transfer was carried out in a blotting buffer (25 mMTris, 192 mM glycine, 20% v/v methanol, pH 8.2) for 12 h at30 V in a transblot tank (Bio-Rad). For p53 detection, theblots were blocked with PBS containing 5% BSA (Serva) and0.05% Tween 20 (Serva) and probed with 10 ml anti-p53 Ab

(2 µg/ml; Ab-3, clone PAb240; Ab-6, clone DO-1, OncogeneScience).

After washing with buffer (PBS-[0.1%]Tween 20) themembranes were incubated with 10 ml of a diluted (1 : 20 inwashing buffer) horseradish peroxidase-labeled secondaryantibody (Amersham). After washing again in PBS containing0.05% Tween 20, specific complexes were detected using theenhanced chemiluminescence technique according to themanufacturer’s recommendations (ECL, Amersham) [5]. Theautoradiographic films (Amersham) of the Western blots werescanned with a Scan Jet Plus scanner (Hewlett Packard).

Colony Formation Assay Cells (2.5 � 105) were cultured with 5 ml MEM with 20% FCSfor 24 h in culture flasks (5 ml, 25 cm2). After radiation thecells were trypsinized, counted and plated in appropriate den-sities on culture dishes (3.5 cm, 3 ml MEM with 20% FCS).Three replicate plates were used for each X-ray dose. Fixation(90% methanol) and staining (0.5% crystal violet solution) ofcolonies were carried out 14 days later.

Cell Cycle Kinetics Cells were cultured for 6 days under low serum conditions in5 ml MEM with 0.5% FCS. At different times after treatment(irradiation and addition of fresh medium), 50 µl BrdU(1 mM) were added to the flasks, the cells were incubated foranother 30 min after which cells were trypsinized and fixed in96% ethanol. Centrifuged cells were washed in 0.9% NaCland incubated in pepsin solution (0.5% pepsin (1 000 U/g) in0.055 N HCl, pH 1.8) for 10 min at 37 °C. After washing againin 1 ml PBS, 1 ml HCl (2 N) was added for 30 min at room tem-perature in order to partially denaturate DNA. The cell nu-clei were then washed once with 1 ml NaCl (0.9%) and oncewith 1 ml PBS-Tween (0.05%) and incubated with anti-BrdUmouse IgG (1 : 20 in PBS-[0.05%]Tween, Becton Dickinson)for 30 min at 4 °C in the dark. After washing twice in PBS-(0.05%)Tween-(1%)BSA the cell nuclei were incubatedwith FITC-conjugated goat anti-mouse IgG (1:100 in PBS-(0.05%)Tween-(1%)BSA), again for 30 min at 4 °C in thedark. Finally, the nuclei were washed again in PBS-(0.05%)Tween, resuspended in PBS and stained with 2.5 � 10-5 M pro-pidium iodide (in 0.1 M Tris, 0.1 M NaCl, pH 7.5). Green(FITC) and red (PI) fluorescence after 488 nm laser excitationwas recorded in a FACScan flow cytometer (Becton Dickin-son). Data were collected with the Becton Dickinson FAC-Scan software program and displayed as two-parameter dotplots. Evaluation of cell cycle fractions was carried out withappropriate windows on the dot plots.

Statistical Analysis All data (fluorescence indices, cell cycle fractions, survivingfractions) representaveragevaluesof three independentexper-iments with corresponding standard deviations. Statistical asso-ciations between parameters were assessed by Student’s t test.

Figure 1. Flow cytometric measurements of p53. Scattergrams of twoWi38VA13 strains (A and B) with a specific p53 antibody (clone DO-1)and an unspecific control antibody. Strain Wi38VA13F demonstratedsignificantly higher levels of p53 as compared with strain Wi38VA13E.Abbildung 1. Durchflusszytometrische Bestimmung von p53. Ergeb-nisse der beiden Wi38VA13-Stämme (A und B) mit einem p53-spezifi-schen Antikörper (Klon DO-1) und einem unspezifischen Kontrollanti-körper. Der Stamm Wi38VA13F zeigte signifikant höhere p53-Niveausals der Stamm Wi38VA13E.

A Wi38VA13F

p53 level

control antibody

p53 specificantibody

DNA content

B Wi38VA13E

p53 level p53 specificantibody

control antibody

DNA content


Results Flow Cytometric Measurements (FCM) and p53 Levels

Figure 1 depicts typical scattergrams of Wi38VA13F and E(24 h old cultures), for the binding of the DO-1 antibody show-ing a large difference between the fluorescence levels of thecontrol antibody (unspecific fluorescence) and the specificanti-p53 antibody (specific fluorescence) for the Wi38VA13Fstrain, but only a small difference in the fluorescence levels forthe Wi38VA13E strain. For the Wi38VA13F we calculated aFi-value of 22.28 and for the strain Wi38VA13E a Fi-value of0.78. A more detailed analysis also showed that there was nosignificant difference between the fluorescence intensitieswith respect to p53 antibody binding during the various cellcycle phases (G1, S, G2).

Flow cytometric measurements revealed clear p53 detec-tion in both Wi38VA13 strains not only with the antibodyclone DO-1, but also with clone PAb240. The fluorescence in-dices (Fi) were greater than 0.5 for all four combinations (twocell strains and two antibodies). However, the calculated Fi ofthe Wi38VA13F strain was 4.5-fold lower with the clone

PAb240 antibody than with the clone DO-1 antibody (p =0.0006), whereas for the Wi38VA13E strain the PAb240 valuewas 6.2 fold higher than the DO-1 value (p = 0.045).

Thus, with the clone DO-1 antibody we observed a 28.6-fold, highly significant difference in the Fis between the two cellstrains (p = 0.0001). No significant difference between the twostrains was found with the clone PAb240 antibody (p = 0.481).

It is interesting to note at this point that the FCM deter-mination of large T antigen expression revealed a similar dif-ference between the two strains as was seen for p53 with theDO-1 antibody. The Fi was 0 for Wi38VA13E, but 17.4 forWi38VA13F (details not shown).

Western Blot (WB) and p53 Level Exponentially growing cells were fixed after 24 h and analyzedfor constitutive p53 levels. For both Wi38VA13 cell lines weprepared separate staining samples with an unspecific controlantibody and a specific p53 antibody (Ab-6, clone DO-1). Alot of unspecific binding was observed on the blots (Figures 2aand 2b).


Figure 2a – Abbildung 2a Figure 2b – Abbildung 2b

Figures 2a and 2b. Western blots of p53 detection. Blots of two Wi38VA13 strains with a specific p53 antibody (clone DO-1) and an unspecific con-trol antibody. Blots of strain Wi38VA13F (a) and blots of strain Wi38VA13E (b).Abbildungen 2a und 2b. Western Blots zum Nachweis von p53. Ergebnisse der beiden Wi38VA13-Stämme mit einem p53-spezifischen Antikörper(Klon DO-1) und einem unspezifischen Kontrollantikörper. Blots für den Stamm Wi38VA13F (a) und für den Stamm Wi38VA13E (b).

Wi38VA13Funspecific antibody

p53 antibody clone DO-1

53 kd

Wi38VA13Eunspecific antibody

p53 antibody clone DO-1

53 kd

The largest difference between unspecific and specific sig-nal strength in the area of 53 kDa was observed for the blots ofWi38VA13F, while there was only a slight difference in p53signal strength for the blots of Wi38VA13E (Figure 2). Theseresults are consistent with our flow cytometric measurements(Figure 3).

Cell Cycle Progression Analysis The cell cycle distribution was analyzed 0–30 h after 6 daysserum starvation, irradiation and addition of fresh medium.Normally the serum starvation would be expected to reducethe percentage of S phase cells (Figure 4) and to increase thepercentage of G1/G0 cells (Figure 5). Our results show thatindeed strain E cells were mostly in a quiescent state (only10% S phase cells). In contrast, strain F cells were in a non-quiescent state (42% S phase cells) even after 6 days of serumstarvation.

The strain E cells started to enter into S phase about8–10 h after the exchange of the medium. The observed rise inS phase fraction (between 10 and 16 h after addition of freshmedium) was delayed by 2–4 h after irradiation with 4 Gy X-rays. No significant change of the number of S phase cells andno radiation-induced cell cycle delay (G1 block) was observedwith the strain F cells.

Correspondingly the G1/G0 phase cells decreased around10 h and later in the cells of the strain E but no change oc-curred in the cells of the strain F. After irradiation the de-crease of G1/G0 cells was delayed in the strain E (see Figure 5).



p53

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Wi38VA13E

Wi38VA13F

4.86 4.96

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22.28

clone PAb240 clone DO-1antibody

Figure 3. p53 fluorescence index by flow cytometric measurements.p53 fluorescence index of two Wi38VA13 strains with two p53 anti-bodies (clone PAb240 and clone DO-1). Results are represented asmean with corresponding standard deviation of three independentexperiments. Abbildung 3. p53-Fluoreszenzindex aus durchflusszytometrischenMessungen für die beiden Wi38VA13-Stämme. Zwei p53-Antikörperwurden verwendet (Klon Pab240 und Klon DO-1). Die Ergebnisse sinddargestellt als Mittelwerte mit den entsprechenden Standardabwei-chungen aus drei verschiedenen Experimenten.

60

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ls [%

]

0 5 10 15 20 25 30

time [h] after radiation and addition of fresh medium

Wi38VA13EWi38VA13 E-4GyWi38VA13FWi38VA13F-4Gy

Figure 4. Percentage of S phase cells of two Wi38VA13 strains after 6days of serum starvation and treatment with radiation and additionof fresh medium. Error bars represent mean values ± standard devia-tion of three independent experiments. Abbildung 4. Anteil der S-Phase-Zellen bei den beiden Wi38VA13-Stämmen nach 6 Tagen Serumhungerung, Bestrahlung und Medium-wechsel. Die Fehlerbalken repräsentieren Mittelwerte ± Standardab-weichung aus drei verschiedenen Experimenten.

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se c

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[%]

0 5 10 15 20 25 30time [h] after radiation and addition of fresh medium

Wi38VA13EWi38VA13 E-4GyWi38VA13FWi38VA13F-4Gy

Figure 5. Percentage of G1/G0 phase cells of two Wi38VA13 strains af-ter 6 days of serum starvation and treatment with radiation and addi-tion of fresh medium. Error bars represent mean ± standard deviationof three independent experiments.Abbildung 5. Anteil der G1/G0-Phase-Zellen bei den beiden Wi38VA13-Stämmen nach 6 Tagen Serumhungerung, Bestrahlung und Medium-wechsel. Die Fehlerbalken repräsentieren Mittelwerte ± Standardab-weichung aus drei verschiedenen Experimenten.

Cell Survival after Irradiation The survival curves of the two Wi38VA13 strains after X-rayexposure are shown in Figure 6. It is obvious that theWi38VA13 strain F has a higher radiosensitivity than theWi38VA13 strain E (the strain with low detectable p53 levels).

Discussion p53 Detection by FCM and WB

It has been shown by several authors that it is possible to meas-ure p53 by FCM [1, 8, 28]. It is usually assumed that an in-creased immunoreactivity is due to the presence of mutantp53. The detection of wild-type p53 by FCM is controversial.The protein is present at measurable levels in fibroblasts andin cell lines transformed with viruses, while it is observed atlower levels and difficult to measure in other normal cells, e.g.lymphocytes, and in established tumor cell lines without a mu-tation in the p53 gene. Our studies suggest that the detectabil-ity depends on the type of antibody used and that a combina-tion of two p53 antibodies, in particular Ab-6 (clone DO-1)and Ab-3 (clone PAb240), may be of advantage providingmore detailed information than the use of one single antibody.

The monoclonal antibody Ab-3 (clone PAb240) reactswith amino acids 212–217 of human p53 [26]. The providerclaims that there is a selective reactivity for different mutantsunder certain conditions. In Western blots, however, clonePAb240 antibody is supposed to react with both mutant andwild-type p53. There are also indications that clone PAb240antibody recognizes its epitope in different conformations andeven after denaturation or in case of mutations [12]. With this

antibody we did not find significant differences in the Fis forthe two Wi38VA13 strains.

The monoclonal antibody Ab-6 (clone DO-1) recognizesthe p53 protein at the amino acids 21 and 25 at the amino ter-minal end of the protein. The provider in this case suggeststhat clone DO-1 antibody reacts with both wild- type and mu-tant p53. Other authors mention that the clone DO-1 antibodyreacts with a denaturation stable determinant of p53 [19]. Us-ing Ab-6, we obtained extremely low Fis for the Wi38VA13Estrain and a 28 times higher Fi for the Wi38VA13F strain. Thisis in strong contrast to the results obtained with Ab-3.

We therefore asked the obvious question: Which form ofp53 is detectable with these antibodies, uncomplexed (free),complexed or both? The relative amounts of free and com-plexed p53 are determined by both the p53 and the l-T-Agconcentrations [25]. Around 50% of the l-T-Ag is complexedwith p53 in SV 40 transformed fibroblasts (different from theones used here) [18]. On the other hand, 70% of the p53 pro-tein seem to be free and 30% bound in a complex with l-T-Agin Wi38VA13 cells (presumably identical to our F substrain)[27]. The free p53 is obviously quite stable in these cells [9],whereas in normal cells its half life is very short and it is there-fore virtually undetectable [11]. In NIH 3T3 cells, only a sub-set of cells expressing l-T-Ag shows a high p53 level, whereasanother subset does not, indicating that complex formationand/or degradation depend on more than just the relative con-centrations of the two components [33].

There is evidence that the antibody Ab-3 (clone PAb240)recognizes only p53 which is not complexed [32]. This is un-derstandable in view of the fact that the p53 binding sites forboth the antibody (amino acids 212–217) and the l-T-Ag(amino acids 100–200) are very near to each other. If it is real-ly only the free p53 which is recognized by Ab-3, we have toconclude that the two cell substrains used here contain similaramounts of this form of the protein. They do, however, differin the amount of p53 detectable by the other antibody used.

As the binding site of Ab-6 is at the amino terminal of theprotein (amino acids 21–25) it can be assumed that it recog-nizes any form of p53 (free as well as complexed). The differ-ence between the two substrains can then be attributed to the16-fold difference in l-T-Ag expression as mentioned above,which is presumably also reflected in the p53-l-T-Ag complexconcentration.

Functional vs Unfunctional p53It has been shown in a number of studies that p53 is function-ally inactivated in its complexed form, due to a blockage of thesequence of the specific DNA binding site by the l-T-Ag [15].In the protein conformation identified by Ab-3 (clonePAb240), however, the p53 protein seems to be functional, asthe antibody can be used to transiently release the suppressoreffect of p53, allowing for cell proliferation [30]. Milner [24]1991 mentioned that the clone PAb240 identified a p53 wild-type protein which functions as suppressor of cell proliferation.



100

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ract

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(%)

0 2 4 6 8

Wi38VA13E

Wi38VA13F

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Figure 6. X-ray survival curves of two Wi38VA13 strains. Mean val-ues ± standard deviation of three independent experiments. Abbildung 6. Röntgen-Überlebenskurven der beiden Wi38VA13-Stäm-me. Mittelwerte ± Standardabweichung aus drei verschiedenen Expe-rimenten.



This, again, is in support of the idea that Ab-3 recognizesmainly, if not exclusively, free p53. If these considerations arecorrect, i.e. if our two substrains contain similar amounts offunctional p53, they might be expected to show more or lessthe same radiation response. In particular, treatment inducedchanges in cell cycle progression should be similar. From thedata presented above, however, it is obvious that the twosubstrains differ very strongly in their proliferation behav-ior. There are at least three possible explanations for this ob-servation: 1. The assumption that Ab-3 recognizes only free p53 could be

wrong. If at least part of the complexed protein was also de-tected by this antibody, the similarity between the results forthe two substrains would just be coincidental. One couldthink of the Western blot analysis (which is done under con-ditions destroying complexes between p53 and l-T-Ag) as ameans to clarify the question, but unfortunately the sensi-tivity of the method was very low with the antibody Ab-3.We do not, however, favor this possibility because the evi-dence for the specificity of Ab-3 seems quite strong.

2. The assumption that complexed p53 was functionally inacti-vated could be wrong. This seems rather unlikely in view ofthe overlap between the binding sites for DNA and the l-T-Ag which was already mentioned.

3. There could be indirect effects of the complexed p53 oneither the function of the free protein or its inducibility byradiation. For instance, one could think of interactions withother proteins involving the amino or carboxy terminal ofp53 rather than the l-T-Ag binding site. In this way, a highconcentration of complexed p53 may be able to interferewith certain feed-back loops and exert an influence on theprotein’s posttranslational modification in the case of DNAdamage and/or on downstream effectors of p53. The retino-blastoma protein, for instance, is known to form complexeswith the large T antigen as well [16, 31].

p53 Genotype, G1 Arrest and Radiosensitivity One of the main functions of the p53 is to control the G1 cellcycle checkpoint, delaying entry into S phase after DNA dam-age and allowing more time for DNA repair. This function hasbeen characterized by the term “guardian of the genome”[21]. Our data suggest a direct influence of p53 protein expres-sion and G1 delay on the radiosensitivity of these two strains toradiation.

The strain E with low but apparently active level of p53showed a G1 delay (see Figure 4) and was less radiosensitivethan strain F (see Figure 6), which did not undergo this type ofdelay. Probably the modified VA13E strain is comparable withthe original untransformed strain Wi38 which had a p53 wtprotein and showed a G1 arrest [14]. Wi38 cells were arrestedat the G0 phase by serum starvation and progressed into thecell cycle after serum stimulation [17]. The VA13F cells, onthe other hand, were not arrested at the G0/G1 phase by serumstarvation. The degree of quiescence was also very low in

transformed Wi38VA13 cells presumably identical to our sub-strain F [6].

As to the relationship between G1 delay and radiosensi-tivity, there is far less agreement in the literature than wouldbe expected on the basis of the “guardian of the genome” con-cept. Of 36 studies, only three found an increased radiosensi-tivity in p53 mutant cell lines unable to arrest in G1 [3]. Also,as summarized by Bristow et al [3], six of nine studies of com-plexed p53 protein showed a decreased radiosensitivity (nostudy with an increase). This is not in agreement with our re-sults. On the other hand, Yamagishi et al [34] showed that hu-man radioresistant cell lines became radiosensitive after intro-duction of l-T-Ag. This study supports the connection betweenradioresistance and cell cycle block in G1 phase in the sameway as do the data presented here.

We conclude that only free (active) wild-type p53 is de-tectable with Ab-3 (clone PAb240) in Wi38VA13 strain E andF, whereas complexed (probably inactive form) as well as freewild-type p53 is detectable with Ab-6 (clone DO-1) in strain F.

Similar levels of free p53 seem to exist in E and F. Possibly,the complexed form has indirect effects on the function of freeprotein and its inducibility by radiation, leading to radiationinduced G1 delay and high radioresistance in strain E, but notin strain F.

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p53 Levels, Cell Cycle Kinetics and Radiosensitivity in Two SV40 Transformed Wi38VA13 Fibroblast Strains