Cancer Research Meeting Calendar Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 1008-1014, March 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Syljuåsen, R. G.
Right arrow Articles by Little, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Syljuåsen, R. G.
Right arrow Articles by Little, J. B.
[Cancer Research 59, 1008-1014, March 1, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Loss of Normal G1 Checkpoint Control Is an Early Step in Carcinogenesis, Independent of p53 Status1

Randi G. Syljuåsen, Bozena Krolewski and John B. Little2

Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have described a diminished radiation-induced G1 arrest in some wild-type (wt) p53 human tumor cell lines compared to normal human fibroblasts. However, the significance of this finding was unclear, particularly because tumor cell lines may have accumulated additional genetic changes after long periods in culture. Because malignant transformation of individual cells is thought to be an early step in carcinogenesis, we have used a model system of normal and transformed mouse fibroblast 10T1/2 cell clones to examine whether loss of G1 checkpoint control may be an early event in tumor development and to study the relationships between G1 arrest, radiosensitivity, and genetic alterations. Twelve transformed clones were established from type III foci induced by irradiation of normal 10T1/2 cells and were compared with six clones derived from wt 10T1/2 cells. Three of the transformed clones expressed mutant p53; two of these had the same point mutation at codon 132 (exon 5), and one had a point mutation at codon 135. The remaining transformed and normal clones had wt p53 status. The radiosensitivity of transformed clones, as measured by a clonogenic assay, was similar to that of normal clones; the three clones with mutant p53 did not differ from the others. There was no relationship between G1 arrest and radiosensitivity. Normal 10T1/2 cell clones showed a transient G1 arrest lasting ~9 h after 6 Gy of irradiation. This G1 arrest was either absent or markedly reduced in all of the transformed clones, regardless of p53 status. These results suggest that diminished G1 checkpoint control is an early event in the process of carcinogenesis that is associated with the malignant transformation of individual cells and is independent of p53 status.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has long been known that mammalian cells can arrest in the G1 phase of the cell cycle in response to ionizing radiation (1) . However, it remains unclear whether there is a systematic difference between normal and neoplastic cells in the capacity to undergo such G1 arrest, and what role, if any, G1 checkpoint control may play in determining radiosensitivity or in the process of radiation-induced carcinogenesis.

A prolonged first cycle G1 arrest after irradiation was initially described in normal human diploid fibroblasts (2) . In experiments in which human diploid fibroblasts were synchronized in the G0/G1 phase before irradiation by contact inhibition or serum starvation and the progression of cells into S phase was monitored by continuous labeling with [3H]thymidine, a transient delay in the progression of cells from G1 to S phase was observed, and a fraction of the cells appeared irreversibly arrested in G1 (3) . Both the G1 delay and the prolonged G1 arrest were completely abolished in human fibroblasts transfected with the E6 papilloma virus gene to abrogate p53 function (4 , 5) . In addition, E6-transfected cells were more resistant to cell killing by radiation (6 , 7) .

Interestingly, similar experiments with 14 human tumor cell lines demonstrated a much reduced capacity to undergo a G1 arrest after irradiation compared to normal human diploid fibroblasts (8) . G1 arrest was completely absent in the tumor cell lines with mutant p53 status. The tumor lines expressing wt3 p53 showed either no G1 arrest or a minor arrest that was much smaller than that observed in human diploid fibroblasts (8) . Similar results were obtained with wt p53 tumor cells synchronized in early G1 phase by the mitotic shake-off technique (9) and with cells irradiated during exponential growth (10) . These results suggested that tumor cells generally have a reduced capacity to undergo G1 arrest after irradiation compared to normal cells, regardless of the p53 status of the tumor. This is consistent with the generally accepted concept that tumor cells have multiple genetic alterations, many of them involved in cell cycle control during the G1 phase.

On the other hand, other laboratories have reported a prolonged p53-dependent first-cycle G1 arrest after irradiation of some human tumor cell lines (11, 12, 13) . However, most of these studies used nonsynchronized exponentially growing tumor cells and infrequent sampling at late times (16–20 h) after irradiation, raising the question of whether it was first- or second-cycle G1 arrest that was measured (discussed in Ref. (9) ). A pronounced first-cycle prolonged G1 arrest has been reported in one human glioma cell line (14) .

Whatever the controversy between previous reports, one weakness of studies with established human tumor cell lines is that many of them have been carried for long periods, often years, in culture. Tumor cells are known to be genetically unstable, and such long-term passage in vitro could result in the accumulation of additional genetic alterations. The reduced G1 arrest observed in human tumor cell lines compared to early-passage normal human diploid fibroblasts could, thus, be due to a difference between late-passage established cell lines versus early-passage primary cells, rather than being a true difference between the normal and neoplastic state. Another complication with such a comparison is that most of the human tumor cells studied do not arise from fibroblasts, and they are also not derived from the same patient.

To clarify this issue and to explore the relationships between G1 arrest, radiosensitivity, and genetic alterations in neoplastic cells, we have used a model system of normal and transformed mouse 10T1/2 fibroblast cell clones. Malignant transformation of individual cells is thought to represent an early stage in carcinogenesis. The advantage of this system is that the transformed cells can be directly compared with the normal cells from which they originated. The transformed cell clones developed over a 6-week period after irradiation, leading to minimal differences between the normal and transformed cells resulting from passaging alone. Furthermore, because radiation is used to induce transformation, the transformed clones are heterogeneous with respect to genetic alterations, which makes this a suitable system to study the effects of p53 status. Using this model system, we compared radiation-induced G1 arrest in normal and transformed clones and explored the relationship between G1 arrest, radiosensitivity and p53 status. All of the transformed clones examined showed the G1 arrest to be absent or markedly reduced, suggesting that diminished G1 checkpoint control is an early event in the process of carcinogenesis, independent of p53 status.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture Conditions.
Cells from C3H 10T1/2 clone 8 mouse fibroblasts (15) were maintained at 37°C/5% CO2 in Eagle’s basal medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (Rehatuin, Intergen), 50 units/ml penicillin, and 50 µg/ml streptomycin.

Transformation.
Transformed clones of irradiated 10T1/2 cells were established as described previously (16) . Briefly, confluent 10T1/2 cells (passage 8–10) were irradiated with 6 Gy of X-irradiation (220-kV and 15-mA X-rays; dose rate, 0.8 Gy/min) or 2-Gy {alpha}-irradiation (3.7-MeV {alpha}-particles from a 238Pu source; dose rate, 0.099 Gy/min) and immediately seeded at low densities to yield ~300 viable cells per dish. The cells went through ~13 rounds of cell division before the cultures entered the confluent, density-inhibited phase of growth. Culture medium was changed twice a week until cells became confluent and once a week thereafter. After 6 weeks, transformed clones (type III foci) overlying the confluent monolayer were isolated from the dishes and expanded in cell culture. Normal clones were isolated from nonirradiated 10T1/2 cells. All clones were tested for the ability to form colonies in soft agar.

Cell Doubling Times.
A total of 104 cells were seeded with 2 ml of culture medium to 10 dishes (diameter = 35 mm). On subsequent days thereafter, cells from one dish were trypsinized and the number of cells was counted using a Coulter particle counter (Coulter Limited Electronics, United Kingdom). Culture medium in the remaining dishes was changed twice a week. The exponential part of the growth curve was fitted to a straight line using the method of least squares, and cell doubling times were determined as ln2/k, where k is the slope. Saturation densities were obtained from the last points of the growth curves.

Colony Formation in Soft Agar.
Plastic dishes (60 mm) were precoated with 5 ml of medium containing 0.5% agarose (type II; Sigma) and 20% fetal bovine serum. Cell suspensions at 104 cells/1.5 ml were made in medium containing 0.2% agarose and 10% heat-inactivated fetal bovine serum, and 1.5 ml of this was spread over the precoated dish. Subsequently, cells were fed once a week with 1 ml of medium (without agarose). After 2 months, the number of colonies was scored by viewing the dishes through a microscope and counting four to eight separate areas marked on each dish. Pictures were taken with an Olympus PM20 camera.

G1 Arrest.
G1 arrest was measured by the cumulative labeling indices method (3 , 8) . Cells were synchronized in the G0/G1 phase before irradiation by holding cells at confluence and waiting 3 days after the last medium change. After exposure to 6 Gy of X-irradiation (160-kV and 18-mA X-rays; dose rate, 0.75 Gy/min), cells were immediately subcultured at low density with medium containing 1 µCi/ml [3H]thymidine (20 Ci/mmol; New England Nuclear, Boston, MA). Dishes of irradiated and control cells were fixed in 100% ethanol at appropriate times thereafter. Standard autoradiographic techniques were used; 200 cells were scored on each dish to determine the cumulative labeling indices.

Clonogenic Assay.
Confluent cells were irradiated (X-ray) and immediately suspended and reseeded at low density to measure colony-forming ability. Duplicates with two different cell numbers were plated for each radiation dose. After 14 days, cells were fixed in 70% ethanol, stained with crystal violet, and counted. Colonies containing more than 50 cells were scored as survivors.

Western Analysis.
Confluent cells were irradiated with 6 Gy (X-rays), and samples were collected 3 h later. Briefly, cells were pelleted and lysed in immunoprecipitation assay buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1%SDS, 1 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA]. After centrifugation, protein quantification was carried out by the Bradford method (Bio-Rad). The protease inhibitors aprotinin, leupeptin, and pepstatin were added (1 µg/µl). Seventy µg of protein were loaded to each lane in 12% polyacrylamide gels. The antibodies used were anti-p53 (Ab-7) and anti-p21Waf1 (Ab-6) from Oncogene Science.

DNA Extraction.
DNA extraction was performed using a modified version of the salting-out method (17) . Briefly, cells were pelleted and resuspended in 300 µl of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM EDTA, pH 8.0) supplemented with 30 µl of 10% SDS and 15 µl of 10 mg/ml proteinase K. After overnight digestion at 37°C, 150 µl of saturated NaCl was added. Samples were agitated for 15 s and centrifuged at 13000 rpm for 10 min. The supernatant containing DNA was transferred to a new tube, and DNA was precipitated by 2.5 volumes of ethanol.

PCR-SSCP Analysis.
The presence of mutations in the mouse p53 gene was determined by PCR-SSCP analysis and subsequent sequencing. Exons 5–8 were amplified by PCR using the following primer set (Refs. 18 and 19 ; purchased from Life Technologies, Inc.): exon 5, 5'-GAC ACC TGA TCG TTA CTC GG-3' (upstream) and 5'-GGA GGC TGC CAG TCC TAA CC-3' (downstream); exon 6, 5'-GGT TAG GAC TGG CAG CCT CC-3' (upstream) and 5'-GTC AAC TGT CTC TAA GAC GC-3' (downstream); exon 7, 5'-GAC TTC ACC TGG ATC CTG TG-3' (upstream) and 5'-CTA ACC TAA CCT ACC ACG CG-3' (downstream); and exon 8, 5'-TTC TTA CTG CCT TGT GCT GG-3' (upstream) and 5'-TGA AGC TCA ACA GGC TCC TC-3' (downstream). The PCRs were performed in a 40-µl volume containing: 500 ng of sample DNA; 50 pmol of upstream and downstream primer; 6.3 µCi of [35S]dATP (1250 Ci/mmol; New England Nuclear, Boston, MA); 4 units of AmpliTaq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT); 1.25 µmol of dATP; 12.5 µmol of dCTP, dGTP, and dTTP; 2 mM MgCl2; and 4 µl of 10x PCR buffer II (Perkin-Elmer). The PCR amplification was performed using a programmable thermal controller (MJ Research Inc.) with 35 cycles of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C, and a final step of 10 min at 72°C. After PCR, 5 µl of the final PCR product were mixed with 5 µl of SSCP dilution buffer (0.1% SDS-10 mM EDTA) and 10 µl of Stop Solution (United States Biochemical; 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). Samples were heated to 90°C for 3–4 min and analyzed on a 6% polyacrylamide gel with 10% glycerol. The gel was run at room temperature for 16 h at 6 W and exposed to film for 2–4 days.

DNA Sequencing.
DNA sequencing was performed using the Sequenase PCR Product Sequencing Kit (United States Biochemical) with the same primer set as that used for the PCR-SSCP analysis. The products were analyzed on a 6% polyacrylamide denatured sequencing gel. The gel was run at 1500 V for 3–5 h, dried, and exposed to film for 3–5 days for autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth Characteristics of Normal and Transformed Clones.
Cells from the normal 10T1/2 clones formed a flat, density-inhibited monolayer at confluence and were not able to grow in soft agar (Fig. 1Citation and Table 1Citation ), characteristic of a normal phenotype. The cell doubling times of the normal clones were ~20 h. In contrast, cells from transformed clones that were established from type III foci of irradiated 10T1/2 cells grew to higher densities at confluence and formed colonies in soft agar (Fig. 1Citation and Table 1Citation ), consistent with a transformed phenotype. It is interesting to note the large variations between individual transformed clones in both the cloning efficiency and size and shape of colonies in soft agar as well as the saturation densities and cell doubling times (Fig. 1Citation and Table 1Citation ). This heterogeneity may be due to differences in genetic alterations between the transformed clones.



View larger version (91K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Growth in soft agar of cells from normal and radiation-transformed 10T1/2 clones. A total of 104 cells were plated in 60-mm dishes in soft agar medium, and photos were taken 2 months later. A, normal 10T1/2 cell clones. B, transformed 10T1/2 cell clones.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Characteristics of normal and transformed clones

 
One of the clones, clone 6, which originally was established from a type III focus, failed to form colonies in soft agar and grew to a saturation density similar to that of normal clones (Fig. 1Citation and Table 1Citation ). Clone 6 may thus have reverted back to the normal phenotype, as has been described previously (20) . This clone was included in our study as a normal control clone, which differs from the other normal clones in that it has survived a radiation dose similar to the transformed clones.

p53 Status.
Nonirradiated normal 10T1/2 clones showed low levels of p53 and p21 protein, which were increased 2.5–5.0- and 1.5–3.0-fold, respectively, at 3 h after a dose of 6 Gy of irradiation (Fig. 2)Citation , consistent with a normal, functional p53 gene. Similarly, nine of the transformed cell clones showed a functional p53 gene, with increased p53 and p21 protein levels after exposure to 6 Gy (examples shown in Fig. 2Citation ). The remaining three transformed clones (clones 7, 15, and 17), however, showed very high levels of p53 protein and no regulation of p53 or p21 levels after irradiation (example shown in Fig. 2Citation ), suggesting that p53 was mutant in these clones. This was confirmed by PCR-SSCP analysis of exons 5–8 of the p53 gene and subsequent sequencing. The same point mutation with a single base shift from C->G at codon 132 (exon 5) was found in clones 7 and 17, and a point mutation with a single base shift from G->C at codon 135 (exon 5) was found in clone 15 (Fig. 3)Citation . No evidence of mutation was found by sequencing of the parental wt cells.



View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Examples of Western analysis of p53 and p21 protein levels at 3 h after exposure to 6 Gy. A, normal 10T1/2 cell clones. B, transformed 10T1/2 cell clones. Induction of p53 and p21 protein expression by radiation was quantified by densitometry in all clones (ratios of 2.5–5.0 for p53 and ratios of 1.5–3.0 for p21), except for clone 7, which had a mutation in the p53 gene (ratio 243 1). Symbols are: -, nonirradiated cells; and +, cells that were exposed to 6 Gy.

 


View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Mutations in the p53 gene in three radiation-transformed 10T1/2 cell clones, as revealed by sequencing of a small region of exon 5. Transformed clones 7 and 17 had the same single base shift from C->G at codon 132, leading to an amino acid change from cysteine to tryptophan. Transformed clone 15 had a single base shift from G->C at codon 135, leading to an amino acid change from alanine to proline.

 
G1 Arrest in Normal versus Transformed Clones.
To accurately measure first cycle G1 arrest in the normal and transformed cell clones, cells were synchronized in G0/G1 by confluence holding and subsequent subculture to low density. The cumulative progression of cells into the S phase was monitored by uptake of [3H]thymidine. Fig. 4Citation shows the results from representative experiments. Parental 10T1/2 cells showed a transient G1 delay lasting ~9 h after exposure to 6 Gy, as measured by the delay time at 50% of the maximum cumulative labeling index (Fig. 4A)Citation . Transformed clone 9 with wt p53 status showed a G1 delay of ~3 h (Fig. 4B)Citation , and transformed clone 7 with mutant p53 status showed no G1 delay (Fig. 4C)Citation . The results of similar measurements of G1 delay in the whole panel of normal and transformed clones are summarized in Fig. 5Citation . All of the normal clones showed a transient G1 delay lasting ~9 h after 6 Gy of irradiation. In comparison, G1 delay was markedly reduced in the transformed clones. The three transformed clones with mutant p53 and two of the clones with wt p53 lacked an X-ray induced G1 arrest, whereas the remaining transformed clones showed short delays, again with some variation among the individual clones.



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. G1 delay measurements by the cumulative labeling index method. The cumulative number of cells that have entered S phase is shown as a function of time after irradiation of cells synchronized in G0/G1 and subsequently released. The G1 delay times after 6 Gy as determined by comparison of control and irradiated cultures at 50% of the maximum labeling index (dashed lines with arrows) are as follows. A, normal parental 10T1/2 cells: 9 h. B, transformed clone 9 (wt p53): 3 h. C, transformed clone 7 (mut p53): 0 h. •, controls; {circ}, 6 Gy of X-rays.

 


View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Summary of G1 delay times after 6 Gy irradiation in all of the normal and transformed 10T1/2 cell clones. G1 delay times were measured at 50% of maximum labeling index (see Fig. 4Citation ). *, transformed clones with mutated p53.

 
Relationship between G1 Arrest, p53 Status, and Radiosensitivity.
Standard clonogenic survival assays were performed after irradiation. Survival curves derived from the mean survival levels at each radiation dose for normal and transformed clones are shown in Fig. 6Citation . Although the mean survival levels for transformed clones were slightly higher, no significant difference between normal and transformed clones is evident, in agreement with two previous reports (21 , 22) . The Ps were 0.17 (2 Gy), 0.25 (4 Gy), and 0.34 (6 Gy). The results for the individual clones are presented in Fig. 7Citation . Interestingly, the three transformed clones with mutant p53 were no more radioresistant than the clones expressing wt p53. To better assess any eventual relationship between G1 arrest and radiosensitivity, G1 delay times were plotted as a function of radiosensitivity (Fig. 8)Citation . From Fig. 8Citation , it is clear that there was no relationship between G1 arrest and radiosensitivity in this model system of normal and transformed mouse cells.



View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Radiosensitivity of normal and transformed 10T1/2 cell clones as determined by a clonogenic assay. Data points, mean survival levels at each radiation dose for the 6 normal (•) and 12 transformed ({circ}) clones; bars, SE.

 


View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Radiosensitivity of individual normal and transformed 10T1/2 cell clones, as determined by a clonogenic assay. The survival parameter D10, which is the radiation dose yielding 10% survival as derived from the complete survival curve, is shown. *, transformed clones with mutated p53. Columns, means of two to four independent experiments; bars, SE.

 


View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. The relationship between G1 arrest and radiosensitivity. G1 delay times are plotted as a function of the survival parameter D10.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study indicate that the radiation-induced first-cycle G1 arrest is markedly diminished in transformed as compared to normal mouse 10T1/2 cells, regardless of p53 status. Because the transformed cells arise from the same normal cells in this system, they come from the same genetic and histology background as the normal clones. In light of a model for tumor development involving multiple steps during the progression from an initiation event to a fully malignant invasive tumor (23) , the changes we have observed most likely correspond to the time around tumor initiation. The results, thus, strongly support the conclusion that loss of normal G1 checkpoint control may be an early event in tumor development, independent of p53 status. This finding is consistent with previous reports of a diminished G1 arrest after irradiation in wt p53 human tumor cell lines (8, 9, 10) . Tumor cells may thus be defective in G1 cell cycle control in response to ionizing radiation as well as to a variety of growth control signals (24 , 25) .

Radiation-induced G1 arrest is known to be mediated, at least in part, by p53-dependent transcriptional activation of the cyclin-dependent kinase inhibitor p21 (26 , 27) . Nine of the transformed clones in our study showed a functional p53 gene in terms of p53 and p21 protein induction. The lack of a normal G1 arrest in these clones indicates that p21 may not be functioning properly, perhaps due to other genetic alterations that occurred during transformation. It is of interest in this context that p21 appeared to be constitutively overexpressed in the transformed as compared with normal clones, and that its induction by radiation was weaker (Fig. 2)Citation .4 Elevated constitutive levels of p21 protein have also been described in many wt p53 human tumors (28) . It is not known how this might relate to the reduced capacity for a G1 arrest. Clearly, further studies will be needed to determine the exact mechanisms by which transformed cells expressing wt p53 show a reduced G1 arrest; however, these may vary among individual clones because they likely have different genetic alterations.

The results presented in Fig. 8Citation showed no relationship between G1 arrest and radiosensitivity in transformed mouse cells. Thus, the extent of G1 arrest alone cannot determine radiosensitivity, although it may play a role in some cases, such as in human normal fibroblasts (6 , 7) . Furthermore, we found no relationship between p53 status and radiosensitivity in the transformed mouse cells. This is in agreement with previous reports from our group and others in which no relationship between p53 status and radiosensitivity was found in most human tumor cell lines (8 , 29 , 30) . Other studies have reported that some tumors with mutant p53 tend to be more radioresistant (31, 32, 33) . The lack of a strict relationship between p53 status and radiosensitivity in tumor cells may be because of multiple genetic alterations, some of which may lead to enhanced radioresistance, whereas others enhance radiosensitivity. In contrast, there is a clear relationship between abrogating p53 function by E6 transfection and enhanced radioresistance in normal human fibroblasts where no other genetic alterations are present (6 , 7) . This is consistent with preliminary findings in p53-/- mouse embryonic fibroblasts.5

We found p53 mutations in 3 of 12 (25%) radiation-induced transformed clones, consistent with a previous screening where p53 mutations were found in 31% of transformed clones (16) . Interestingly, two of the clones had the same point mutation at codon 132; the third clone had a point mutation very close by at codon 135. Both these mutations are located in a part of the DNA-binding region of the p53 gene that is highly conserved among species (34) . In fact, Cys-132 in the mouse p53 gene is a homologue to Cys-135 in the human p53 gene (34) , which is known as a relative hotspot for mutations in human cancer (35) . Thus, radiation-induced p53-mutations in the 10T1/2 mouse model system may be highly relevant with respect to p53-mutations in human cancer. One important issue to address for future studies with this model system is at what time these p53 mutations occur and whether they are likely to be a cause or a consequence of the transformation process. Apparently, two events must have occurred during the 6 week period transformed foci developed: mutation of one allele and loss of heterozygosity at the other.

In conclusion, we have shown that radiation-induced G1 arrest is reduced in transformed as compared to normal mouse 10 T1/2 cells, regardless of p53 status. We also found no relationship between G1 arrest and radiosensitivity or p53 status and radiosensitivity in this model system. These results suggest that diminished G1 checkpoint control may be an early event in the genesis of a tumor, one that is independent of p53 status.


   ACKNOWLEDGMENTS
 
We thank Drs. Edouard I. Azzam, Hatsumi Nagasawa, and Yongjia Yu for expert technical advice and for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Research Grant CA-47542 and NIH Center Grant ES-00002. Back

2 To whom requests for reprints should be addressed, at Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. Phone: (617) 432-1184; Fax: (617) 432-0107; E-mail: gbraga{at}sph.harvard.edu Back

3 The abbreviations used are: wt, wild-type; SSCP, single-strand conformation polymorphism. Back

4 B. Krolewski. Overexpression of p21 protein in radiation transformed mouse 10 T1/2 cell clones, manuscript in preparation. Back

5 R. G. Syljuåsen, unpublished data. Back

Received 9/ 9/98. Accepted 1/ 4/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Okada S. Radiation effects on cell cycle progress through the life cycle. Radiation Biochemistry, Vol. I: 189-246, Academic Press New York 1970.
  2. Little J. B. Delayed initiation of DNA synthesis in irradiated human diploid cells. Nature (Lond.), 218: 1064-1065, 1968.[Medline]
  3. Nagasawa H., Little J. B. Comparison of kinetics of x-ray-induced cell killing in normal, ataxia telangiectasia and hereditary retinoblastoma fibroblasts. Mutat. Res., 109: 297-308, 1983.[Medline]
  4. DiLeonardo A., Linke S. P., Clarkin K., Wahl G. M. DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev., 8: 2540-2551, 1994.[Abstract/Free Full Text]
  5. Li C. Y., Nagasawa H., Tsang N. M., Little J. B. Radiation-induced reversible G(0)/G(1) block is abolished in human diploid fibroblasts transfected with the human papilloma virus E6 gene: implication of the p53-Cip1/WAF1 pathway. Int. J. Oncol., 6: 233-236, 1995.
  6. Tsang N. M., Nagasawa H., Li C., Little J. B. Abrogation of p53 function by transfection of HPV16 E6 gene enhances the resistance of human diploid fibroblasts to ionizing radiation. Oncogene, 10: 2403-2408, 1995.[Medline]
  7. Linke S. P., Clarkin K. C., Wahl G. M. p53 mediates permanent arrest over multiple cell cycles in response to {gamma}-irradiation. Cancer Res., 57: 1171-1179, 1997.[Abstract/Free Full Text]
  8. Li C. Y., Nagasawa H., Dahlberg W. K., Little J. B. Diminished capacity for p53 in mediating a radiation-induced G1 arrest in established human tumor cell lines. Oncogene, 11: 1885-1892, 1995.[Medline]
  9. Nagasawa H., Keng P., Maki C., Yu Y., Little J. B. Absence of a radiation-induced first-cycle G1-S arrest in p53+ human tumor cells synchronized by mitotic selection. Cancer Res., 58: 2036-2041, 1998.[Abstract/Free Full Text]
  10. Nagasawa H., Li C. Y., Maki C. G., Imrich A. C., Little J. B. Relationship between radiation-induced G1 phase arrest and p53 function in human tumor cells. Cancer Res., 55: 1842-1846, 1995.[Abstract/Free Full Text]
  11. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res., 51: 6304-6311, 1991.[Abstract/Free Full Text]
  12. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA, 89: 7491-7495, 1992.[Abstract/Free Full Text]
  13. O’Connor P. M., Jackman J., Bae I., Myers T. G., Fan S., Mutoh M., Scudiero D. A., Monks A., Sausville E. A., Weinstein J. N., Friend S., Fornace A. J. J., Kohn K. W. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res., 57: 4285-4300, 1997.[Abstract/Free Full Text]
  14. Yount G. L., Haas-Kogan D. A., Vidair C. A., Haas M., Dewey W. C., Israel M. A. Cell cycle synchrony unmasks the influence of p53 function on radiosensitivity of human glioblastoma cells. Cancer Res., 56: 500-506, 1996.[Abstract/Free Full Text]
  15. Reznikoff C. A., Bertram J. S., Brankow D. W., Heidelberger C. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res., 33: 3239-3249, 1973.[Abstract/Free Full Text]
  16. Krolewski B., Little J. B. Application of denaturing gradient gel blots to detect p53 mutations in x-ray-transformed mouse C3H 10T1/2 clones. Mol. Carcinog., 7: 190-196, 1993.[Medline]
  17. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16: 1215 1988.[Free Full Text]
  18. Terhune P. G., Memoli V. A., Longnecker D. S. Evaluation of p53 mutation in pancreatic acinar cell carcinomas of humans and transgenic mice. Pancreas, 16: 6-12, 1998.[Medline]
  19. Bienz B., Zakut-Houri R., Givol D., Oren M. Analysis of the gene coding for the murine cellular tumour antigen. p53. EMBO J., 3: 2179-2183, 1984.[Medline]
  20. Paquette B., Wagner R., Little J. B. In vitro reversion of transformed phenotype in mouse C3H/10T1/2 cells: modification in genomic 5-methylcytosine content. Int. J. Onc., 8: 727-734, 1998.
  21. Crompton N. E., Emery G. C., Shi Y., Sigg M., Blattmann H. Radiation-induced genetic instability: no association with changes in radiosensitivity or cell cycle checkpoints in C3H 10T1/2 mouse fibroblasts. Radiat. Environ. Biophys., 36: 255-259, 1998.[Medline]
  22. Raaphorst G. P., Vadasz J. A., Azzam E. I., Sargent M. D., Borsa J., Einspenner M. Comparison of heat and/or radiation sensitivity and membrane composition of seven x-ray-transformed C3H 10T1/2 cell lines and normal C3H 10T1/2 cells. Cancer Res., 45: 5452-5456, 1985.[Abstract/Free Full Text]
  23. Fearon E. R., Vogelstein B. A genetic model for colorectal tumorigenesis. Cell, 61: 759-767, 1990.[Medline]
  24. Kadohama T., Tsuji K., Ogawa K. Indistinct cell cycle checkpoint after UV damage in H-ras-transformed mouse liver cells despite normal p53 gene expression. Oncogene, 9: 2845-2852, 1994.[Medline]
  25. Sherr C. J. Cancer cell cycles. Science (Washington DC), 274: 1672-1677, 1996.[Abstract/Free Full Text]
  26. El-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817-825, 1993.[Medline]
  27. Dulic V., Kaufmann W. K., Wilson S. J., Tlsty T. D., Lees E., Harper J. W., Elledge S. J., Reed S. I. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell, 76: 1013-1023, 1994.[Medline]
  28. Jung J. M., Bruner J. M., Ruan S., Langford L. A., Kyritsis A. P., Kobayashi T., Levin V. A., Zhang W. Increased levels of p21WAF1/Cip1 in human brain tumors. Oncogene, 11: 2021-2028, 1995.[Medline]
  29. Slichenmyer W. J., Nelson W. G., Slebos R. J., Kastan M. B. Loss of a p53-associated G1 checkpoint does not decrease cell survival following DNA damage. Cancer Res., 53: 4164-4168, 1993.[Abstract/Free Full Text]
  30. Huang H., Li C. Y., Little J. B. Abrogation of p53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation. Int. J. Radiat. Biol., 70: 151-160, 1996.[Medline]
  31. Lowe S. W., Bodis S., McClatchey A., Remington L., Ruley H. E., Fisher D. E., Housman D. E., Jacks T. p53 status and the efficacy of cancer therapy in vivo. Science (Washington DC), 266: 807-810, 1994.[Abstract/Free Full Text]
  32. McIlwrath A. J., Vasey P. A., Ross G. M., Brown R. Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity. Cancer Res., 54: 3718-3722, 1994.[Abstract/Free Full Text]
  33. Fan S., El-Deiry W. S., Bae I., Freeman J., Jondle D., Bhatia K., Fornace A. J. J., Magrath I., Kohn K. W., O’Connor P. M. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res., 54: 5824-5830, 1994.[Abstract/Free Full Text]
  34. Soussi T., de Fromentel C. C, May P. Structural aspects of the p53 protein in relation to gene evolution. Oncogene, 5: 945-952, 1990.[Medline]
  35. Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54: 4855-4878, 1994.[Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
H. Fung, P. Liu, and B. Demple
ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis
Mol. Cell. Biol., December 15, 2007; 27(24): 8834 - 8847.
[Abstract] [Full Text] [PDF]

Home page
 Neuro Oncol DukeHome page
A. Balasubramaniam, P. Shannon, M. Hodaie, N. Laperriere, H. Michaels, and A. Guha
Glioblastoma multiforme after stereotactic radiotherapy for acoustic neuroma: Case report and review of the literature
Neuro-oncol, October 1, 2007; 9(4): 447 - 453.
[Abstract] [Full Text] [PDF]

Home page
 Radiat Prot DosimetryHome page
S. G. Andreev, Y. A. Eidelman, I. V. Salnikov, and I. K. Khvostunov
Mechanistic modelling of genetic and epigenetic events in radiation carcinogenesis
Radiat Prot Dosimetry, December 1, 2006; 122(1-4): 335 - 339.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
Y. Dai, M. Rahmani, X.-Y. Pei, P. Khanna, S. I. Han, C. Mitchell, P. Dent, and S. Grant
Farnesyltransferase inhibitors interact synergistically with the Chk1 inhibitor UCN-01 to induce apoptosis in human leukemia cells through interruption of both Akt and MEK/ERK pathways and activation of SEK1/JNK
Blood, February 15, 2005; 105(4): 1706 - 1716.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Wharton
Repression of G0/G1 Traverse in Human Fibroblasts Exposed to Low Levels of Ionizing Radiation
J. Biol. Chem., October 15, 2004; 279(42): 43667 - 43674.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. E. Whang, C. Tran, C. Henderson, R. G. Syljuasen, N. Rozengurt, W. H. McBride, and C. L. Sawyers
c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency
PNAS, May 9, 2000; 97(10): 5486 - 5491.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. B. Little
Radiation carcinogenesis
Carcinogenesis, March 1, 2000; 21(3): 397 - 404.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
N. H. Chehab, A. Malikzay, M. Appel, and T. D. Halazonetis
Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53
Genes & Dev., February 1, 2000; 14(3): 278 - 288.
[Abstract] [Full Text]

Home page
 Cold Spring Harb Symp Quant BiolHome page
L.B. SCHULTZ, N.H. CHEHAB, A. MALIKZAY, R.A. DITULLIO, E.S. STAVRIDI, and T.D. HALAZONETIS
The DNA Damage Checkpoint and Human Cancer
Cold Spring Harb Symp Quant Biol, January 1, 2000; 65(0): 489 - 498.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Syljuåsen, R. G.
Right arrow Articles by Little, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Syljuåsen, R. G.
Right arrow Articles by Little, J. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online