© 1997 American Heart Association, Inc.
Articles |
Cellular Radiosensitivity, Radioresistant DNA Synthesis, and Defect in Radioinduction of p53 in Fibroblasts From Atherosclerosis Patients
From the Department of Biological and Medical Research (N.N., P.S.M., M.K., D.S., S.A.-S., M.A.H.), and the Department of Cardiovascular Diseases (L.A.M.), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
Correspondence to Dr Mohammed A. Hannan, Biological and Medical Research (MBC-03), King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.
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Abstract |
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Abstract Earlier studies have suggested that both cancer and atherosclerosis may follow a common pathway in the early stage of development and share certain risk factors. One report indicated that the gene responsible for the radiosensitive, cancer-prone, multisystem disorder ataxia telangiectasia (AT) may increase the risk of developing ischemic heart disease. The present studies were carried out to find similarities, if any, between atherosclerosis patients and AT homozygotes or heterozygotes (ATHs) in their cellular/molecular response to ionizing radiation, which acts as a carcinogen as well as an atherogen. Fibroblast cell strains developed from healthy subjects and from AT homozygotes, ATHs, and atherosclerosis patients were compared for (1) survival, by the colony-forming assay and (2) DNA synthesis inhibition after irradiation, determined by [3H]thymidine incorporation, cell cycle distribution, and the expression of p53 and p21 proteins, analyzed by flow cytometry. Fibroblasts from the atherosclerosis patients as a group, compared with the healthy subjects, showed enhanced sensitivity to chronic (low-dose-rate) irradiation. A majority of the cell strains representing atherosclerosis patients exhibited varying degrees of radioresistant DNA synthesis (RDS), with roughly 33% showing an AT-like and the rest an ATH-like response. All cell strains with an AT-like and one quarter with an ATH-like RDS were found to be defective in the radioinduction of both p53 and p21 proteins, which are concerned with cell cycle regulation. An absence of G1 arrest after irradiation was observed in cell strains lacking a radioinduced expression of p53 and p21. Cellular/ molecular defects leading to increased radiosensitivity, reduced induction of p53/p21, and cell cycle deregulation found to be associated with cancer-prone disorders such as AT may constitute important risk factors for atherosclerosis as well.
Key Words: atherosclerosis • radiosensitivity • cell cycle defect • radioresistant DNA synthesis • radioinduced p53 and p21 proteins
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Introduction |
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Several studies have suggested that both cancer and AS originating through a multifactorial process may involve a common pathway in the early stage of development and share certain risk factors.1 This hypothesis is supported by the observations that atherosclerotic plaques, like tumors, can be induced by mutagens/carcinogens and show the evidence of both monoclonal origin and somatic mutations.2 3 4 5 Among various carcinogenic agents, ionizing radiation has been reported to be a proven atherogen.6 7 8 Therefore, it is expected that increased radiosensitivity, as well as the mechanisms deregulating cellular/molecular response to agents like radiation, which are associated with enhanced susceptibility to carcinogenesis,9 may also be predisposing factors for AS. Indeed, an epidemiological study by Swift and Chase10 suggested that the gene for the multisystem disorder AT, which is characterized by increased radiosensitivity, RDS, and susceptibility to malignancies, could also be a risk factor for ischemic heart disease. However, very little experimental research has been carried out to demonstrate any genetic/phenotypic similarity in the cells from cancer-prone individuals and patients with AS to define the biological basis of the connection between cancer and AS. In a preliminary study,11 we found that cultured skin fibroblasts from five patients with coronary AS exhibited a moderate increase in radiosensitivity (like ATHs) and RDS, indicating an abnormality in the regulation of the cell cycle. These observations warranted further studies with body cells from a larger number of patients to determine their possible association with enhanced radiosensitivity and RDS as well as to analyze the factors affecting the mechanism(s) of cell proliferation control in these patients.
In the present study, cultured skin fibroblasts from 19 AS patients were compared with those from 3 normal subjects, 2 AT homozygotes, and 3 ATHs for their response to irradiation with respect to colony-forming ability and inhibition of DNA synthesis. As ionizing radiation is well known for the induction of gene products such as p53, concerned with cell cycle progression,12 13 we also examined the cell strains from AS patients exhibiting RDS for radioinduction of the protein p53 and its transactivatable gene product p21, which have been linked to G1 arrest and inhibition of cyclin E/CDK2 complexes after DNA damage.14
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Cell Cultures
Cutaneous biopsies were obtained from both patients and healthy subjects (on consent). The clinical profile of the AS patients is given in Table 1
. Seventeen of the 19 AS patients (age range, 32 to 69 years) included in this study underwent angioplasty and 2 underwent coronary artery bypass surgery. Of the AS patients, 4 had previous angioplasty, 10 had previous myocardial infarction, and 6 had non–insulin-dependent diabetes. Coronary atherectomy was performed in 4 of these patients because of restenosis within 1 year of angioplasty. In this cohort of patients, there were no interrelated individuals. The 3 healthy subjects (2 males and 1 female) serving as controls had an age range of 26 to 52 years. Primary fibroblast cultures were developed by growing the skin explants in minimum essential medium supplemented with Earle's salts, penicillin (100 U/mL), streptomycin (100 µg/mL), glutamine (2 mmol/L), and 15% FBS in 25-cm2 tissue-culture flasks incubated at 37°C in a humidified atmosphere with 5% CO2, 95% O2.
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Radiation Survival Analysis
Relative sensitivity of different fibroblast strains to chronic irradiation was determined by the colony-survival assay as described previously.11 Briefly, early-passage (ie, 3 to 5) skin fibroblast cells from different subjects were grown in Ham's F-12 containing the same supplements as in minimum essential medium. Confluent cell cultures in 100-mm dishes were irradiated inside a CO2 incubator using a 137Cs source (Gamma Cell 1000, Atomic Energy of Canada, Ltd) at a dose rate of 0.007 Gy/min. After irradiation, the growth medium was renewed, and the dishes containing the cells were left in the CO2 incubator overnight. Cells from different irradiated dishes were then trypsinized, harvested, diluted, and seeded (200 to 20 000 per plate), together with a feeder layer of 50 Gy radiation-inactivated normal human fibroblasts (60 000 per plate). After 3 weeks of incubation, with a weekly change of growth medium, macroscopic colonies (with more than 50 cells per colony) were scored as survivors. PBS was used to wash the cells, which were then stained with crystal violet for scoring the colonies. Percent survival was calculated on the basis of colony counts from at least four dishes for each radiation dose point compared with the respective control. The D37 and D50 values (radiation doses resulting in 37% and 50% survival) were estimated from the survival curves without curve fitting to compare the relative sensitivity of the cell strains originating from different groups of subjects. In our earlier studies,11 we established that cells grown from the same strain on different occasions produced similar radiation survival data (with an acceptable limit of variation). In the present study, therefore, we report data from single experiments with each cell strain. Separate biopsies from the same patient have not been studied.
Postirradiation DNA Synthesis Measurement
Log-phase cells from 12 patients with coronary AS, 2 AT homozygotes, 2 ATHs, and 3 healthy subjects were exposed to a single dose (4 Gy) of radiation at a dose rate of 7.5 Gy/min. After 30 minutes of postirradiation incubation, [3H]thymidine (specific activity, 5 µCi/mL) was added (1 µCi per dish) to the cells, which were incubated for an additional 2 hours. Cells were then washed three times with PBS, trypsinized, and harvested on glass fiber filters using a skatron harvester for counting radioactivity with a liquid scintillation spectrometer. Inhibition of DNA synthesis was expressed as percent of counts per minute in irradiated cell DNA compared with that in the unirradiated controls. The uptake of [3H]thymidine measured the level of postirradiation DNA synthesis per cell proliferation and thus indicated the level of RDS or cell cycle defect in cell strains from patients compared with the healthy subjects.
Flow Cytometric Assay for p53 and p21 Proteins
The expression of p53 before and after irradiation of cells was measured by the procedure described previously.15 Briefly, confluent cells were trypsinized and plated on 100-mm dishes at a density of 4x105 cells per dish. Twenty-four hours after plating, cells from both the normal subjects and the AS patients were irradiated at a dose of 4 Gy with a 137Cs source at a dose rate of 7.5 Gy/min. Three hours after irradiation, the cells were harvested and processed for determining the level of p53 expression by flow cytometry. The p53 expression was detected by indirect immunofluorescence using mouse anti-human p53 monoclonal antibody (Ab-2) and FITC-conjugated (goat) anti-mouse secondary antibody (Oncogene Science). Cells were fixed by dropwise addition of ice-cold 70% methanol and incubated for 5 minutes at -70°C. Cells were then washed once with PBS and resuspended with p53 antibody (50 µg/mL) in PBS. The cells were incubated for 1 hour at 4°C with occasional shaking, then washed twice with PBS, further incubated with FITC-conjugated (goat) anti-mouse secondary antibody (diluted 1:100 in PBS) for 30 minutes at room temperature, and again washed twice with PBS. Nonspecific blocking serum (2% FBS) was present during each antibody incubation and washing solution. Finally, cells were resuspended in 1 mL of PBS, 10 µL of 1 mg/mL RNase, and 10 µL of 10 mg/mL ethidium bromide at least 30 minutes before being analyzed on FACScan (Becton Dickinson). Cells incubated with FBS plus second antibody served as control for background fluorescence. Relative levels of p53 protein were evaluated by determining the corrected p53 green fluorescence. Corrected p53 fluorescence equals the difference between the green fluorescence of the primary antibody plus FITC-conjugated second antibody and FITC-conjugated second antibody alone. Finally, the difference in p53 protein levels between unirradiated and irradiated (4 Gy) cells was evaluated by K-S stat (Kolmogorov-Smirnov Statistics option).16
The above method using cells growing for 24 hours before irradiation did not prove to be helpful in obtaining consistent results for p21. Therefore, a slightly modified method was followed, which involved harvesting the confluent cells, irradiating them in suspension, incubating them (at a density of 4x105 per plate) for 20 and 27 hours, and then analyzing the levels of both p53 and p21 at the same postirradiation time points by flow cytometry. Except for the difference in cell culture used for irradiation, all other procedures remained the same as described above.
Cell Cycle Distribution Analysis
Cells were harvested (using the cell-culture procedure for the detection of p53 and p21 proteins) 24 hours after irradiation (unirradiated cells grown for 24 hours serving as control) and fixed by a dropwise addition of 70% ethanol that had been kept at -20°C. After 20 minutes, cells were washed with PBS. Cells were then incubated with RNAse (50 µg/mL), stained with ethidium bromide (25 µg/mL), and analyzed in FACScan for DNA contents. On the basis of the DNA contents, different compartments were marked representing cells in G0/G1, S, and G2/M phases.
PCR-SSCP Analysis of p53 Mutation
DNA samples from cell strains showing RDS and a lack of radioinduced p53 were amplified for the exons 5 through 8 of the p53 gene by PCR, using the primers described earlier.17 The PCR-amplified products were analyzed for SSCP, indicating possible mutation in exons 5 through 8 following the procedure described by Orita et al.18
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Cell Survival Analysis for Radiosensitivity
Typical survival curves obtained after chronic irradiation of cell strains representing a normal subject, an AT homozygote, an ATH, and two AS patients are illustrated in Fig 1
. An examination of individual survival curves of cell strains belonging to each group of subjects showed a considerable interstrain difference. Therefore, the D50 and D37 values were calculated from each survival curve (without curve fitting), and the ranges of these values for cell strains originating from different groups were compared. These data, presented in Table 2 (columns 2 and 3), showed that the ranges of D50 and D37 values for the AS patients were lower than those of the normal subjects, indicating an enhanced cellular radiosensitivity of the AS patients as a group. As indicated by these values, the cells from the AT homozygotes, as expected, were found to be the most radiosensitive, while those from the ATH cases occupied an intermediate position between AT homozygotes and normal subjects. A comparison of both individual survival curves and the D37 values thereof showed that the level of radiosensitivity of most of the AS patients overlapped with that of the ATH cases, while a few exhibiting considerably greater or less sensitivity than ATH cases were also noted.
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Postirradiation DNA Synthesis Inhibition and RDS
The levels of postirradiation [3H]thymidine incorporation observed in cell strains from 12 AS patients compared with those from 3 normal subjects, 2 AT homozygotes, and 3 ATHs are shown in Table 1
(column 4). It was interesting to note that the cells from 4 (33%) of the 12 AS patients showed RDS similar to the AT homozygotes, while 6 (50%) of the 12 exhibited a response mostly overlapping with that of ATHs but clearly being intermediate between AT homozygotes and normal subjects. The level of postirradiation DNA synthesis inhibition in 3 (25%) of 12 AS patients appeared to be similar to that of the normal subjects. These data suggested that RDS similar to what is found in cells from AT homozygotes and ATHs is quite common in AS patients.
Flow Cytometric Analysis of Radioinduced p53 and p21 Proteins
An analysis of preirradiation and postirradiation levels of the proteins p53 and p21 was carried out in eight cell strains from AS patients, four showing an AT-like and four an ATH-like RDS. First, cells grown to confluence were harvested and replated at a density of 4x105 per plate, incubated for 24 hours, and then irradiated. The flow cytometric analysis carried out 3 hours postirradiation showed no radioinduction of p53 in three and a considerably reduced induction in one of the four cell strains (AS) showing an AT-like RDS compared with the cell strains from the healthy subjects. On the other hand, only one of four cell strains from AS patients with an intermediate level of RDS showed a lack of radioinduced p53, while the rest showed a normal response. Fig 2 illustrates the typical data of flow cytometric analysis for p53 expression before and after irradiation in cells from one normal subject and one AS patient.
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Both p53 and p21 proteins were then analyzed in cells that were harvested from the confluent state, irradiated in suspension, replated (4x105 cells per plate), and incubated for 6, 12, 20, and 27 hours. No radioinduction of either p53 or p21 protein was detected at 6 and 12 hours postirradiation in any of the cell strains representing the normal subjects and AS patients (data not shown). The earliest radioinduction of these proteins in normal cells could be detected 20 hours after irradiation. At this time point, no radioinduction of the two proteins was observed in three cell strains from AS patients showing an AT-like RDS and the one showing an ATH-like RDS. An analysis carried out 27 hours postirradiation produced similar results (data not shown). The results of flow cytometric analysis in cells from one normal subject, one AT homozygote, and one AS patient are illustrated in Fig 3A through 3C, showing the absence of radioinduction of p53 and p21 in the AS patient and reduced induction in the AT homozygote cells compared with the usual response in the normal cells.
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PCR-SSCP Analysis for p53 Gene Mutation in Cells From AS Patients Showing Defect in Radioinduction of the p53 Protein
To find whether or not a mutation in the p53 gene would be responsible for the lack of radioinduced expression/stability of the p53 protein, the PCR-SSCP analysis was carried out on DNA of the three cell strains from AS patients exhibiting RDS and no radioinduced p53/p21. No mutation was found in exons 5 through 8 of the p53 gene in any of the cell strains (data not shown).
24-Hour Postirradiation Cell Cycle Distribution
Two of the cell strains from AS patients showing RDS as well as an absence of radioinduced p53/p21 were examined by flow cytometry for cell cycle distribution 24 hours after irradiation, with the same cell-culture procedure used for the detection of p53 and p21. Both the cell strains showed a postirradiation S-phase activity similar to that of the AT homozygotes, while it was negligible in the control cells (healthy subjects). Fig 4 compares the levels of S-phase activity in cell strains from a healthy subject, an AT homozygote, and an AS patient. The two-dimensional dot plots (Fig 4) indicated a reduction of cells in the S-phase resulting from irradiation in the case of the healthy subject only (control) but did not show a clear evidence of G1 arrest. We therefore estimated the percents of cells as distributed in different phases of the cell cycle in the three cell strains 24 hours postirradiation from the flow cytometry DNA histograms. These quantitative data, presented in Table 3, showed that in the control, 89% of the cells were in G1 24 hours after irradiation compared with 69% in unirradiated plates. On the other hand, there was no radiation-induced G1 arrest in cells from the AS patient and relatively little arrest in the AT homozygote cells.
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Discussion |
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The data obtained by the colony-survival assay are consistent with our earlier findings11 that increased cellular sensitivity to low-dose-rate irradiation is associated with AS patients. Although the majority of the AS patients overlapped with ATHs for their sensitivity to irradiation, a few patients exhibited an AT-like increased radiosensitivity without any physical symptoms of AT. In view of the suggestion10 that the carriers of the AT gene have an increased risk of ischemic heart disease and our findings that cells from the AS patients show an ATH-like radiosensitivity, it would be tempting to conclude that most of our AS patients were ATHs. However, such a conclusion must be deferred until a molecular probing shows the existence of mutant AT gene(s) in the cells of AS patients or further studies rule out the involvement of gene(s) other than AT causing increased radiosensitivity in these patients. Nevertheless, the association of increased radiosensitivity with AS patients as well as with certain cancer patients and cancer-prone disorders19 20 does support the hypothesis that AS and cancer may share some common risk factors.1 Therefore, both the underlying mechanisms and the consequences of radiosensitivity must be further studied in relation to the genetic predisposition and the pathogenetic events leading to AS as well as carcinogenesis.
Genetic alterations leading to abnormal expression of oncogenes, tumor suppressor genes, and deregulation of cell cycle control mechanisms are believed to be involved in the development of cancer.21 22 The classic cancer-prone disorder AT has been the focus of various studies aimed at understanding some of these abnormalities. It has been reported that the wild-type AT gene product is required for the activation of the signal-transduction pathway leading to the expression of p53 and p21 proteins concerned with cell cycle regulation (G1 arrest) in response to DNA damage. These proteins have therefore been found to be downregulated in cells from both AT homozygotes and ATHs.15 23 24 When we measured the levels of p53 after irradiation in eight cell strains from AS patients, four showing an AT-like and four showing an ATH-like RDS, we observed a defect in the radioinduction of the tumor suppresser protein p53 in 50% of the cell strains. The cell strains defective in p53 expression/stability also showed an absence of radioinduction of p21, which is consistent with the belief that p53 is generally responsible for p21 transactivation.25 Our data showing a defect in the radioinduction of these proteins in 50% of the cell strains studied indicated that such a defect is common in the AS patients. Furthermore, the cells showing RDS and a lack of p53 induction exhibited no radiation-induced G1 arrest relative to those from a healthy subject, confirming an abnormality in their cell cycle control mechanisms. The failure to detect any mutation in exons 5 through 8 of the p53 gene in the cell strains from AS patients showing a defect in radioinduction of the p53 protein and a lack of G1 arrest may be interpreted to mean that either mutation occurred in exons other than 5 through 8 or in other genes involved in the regulation of p53 expression and cell cycle checkpoints. Usually, mutations in the p53 gene are associated with stable accumulation of the p53 protein,26 whereas we observed a lack of radioinduction of the protein in the cells from AS patients. Thus, it seems plausible that a defect in gene(s) upstream of p53, whether AT or not, is responsible for our observations. Such defective gene products, radioinducible or not, may occur in the AS patients, leading to degradation/instability of p53, which plays a key role in cell cycle regulation. A premature degradation of p53 and p21 found in a Burkitt's lymphoma cell line (EW36) was considered to be responsible for a defect in G1 arrest,27 suggesting that such phenomena may occur in other cases. Also, the literature indicates that a defect in the radioinduction of certain protein kinases may be responsible for the destabilization of the p53 protein.28 Further studies will aim at identifying which genes/gene function other than AT, if any, are defective in the AS patients showing increased cellular radiosensitivity and uncontrolled DNA synthesis after irradiation. Such genes altering the expression of p53 and consequently deregulating cell proliferation control mechanisms may determine susceptibility to AS.
The mechanism through which a defect in p53 induction and/or increased radiosensitivity found in normal body cells would contribute to AS needs to be fully understood. Speir et al29 found that an interaction of the cytomegalovirus protein with p53 led to the functional loss of the latter, resulting in an uncontrolled growth of SMCs and causing restenosis after angioplasty. This observation, as well as the findings that a deletion of another cell cycle controlling gene, ie, rb, resulted in abolishing growth arrest in skeletal muscle cells and SMCs,30 31 strongly implicated cell cycle deregulation in the development of AS. Like cancer, therefore, AS may be a disease of abnormal cell proliferation. Thus, inherent defects in cell proliferation controlling factors observed in normal body cells may be a reflection of defective SMC proliferation after exposure to environmental stress or genotoxic agents. Studies on SMCs and noninvolved body cells such as fibroblasts/blood lymphocytes from the same AS patients showing similar defect(s) in cell proliferation control processes will further clarify the role of certain gene products, particularly p53 in AS.
In our study, it was interesting to note a fair correlation between a moderate enhancement in cellular sensitivity to low-dose-rate irradiation and RDS accompanied with a lack of p53/p21 radioinduction. However, the degree of RDS did not correlate with the level of radiosensitivity, and certainly not all cell strains from AS patients showing an AT-like RDS were as radiosensitive as AT homozygotes. Conflicting reports exist on the relationship between p53 accumulation, radiosensitivity, and cell cycle regulation, ie, G1 arrest.32 33 Some investigators found that mutation in the p53 gene or accumulation of the p53 protein resulted in radioresistance in tumor cells or transformed cell lines.34 35 36 37 The difference between our data and those of others could be explained by the fact that we examined cell survival after low-dose-rate irradiation and analyzed p53 accumulation in normal fibroblasts after high-dose-rate irradiation, in which the mechanisms of both p53 accumulation and cell death may be affected by different factors. A tissue- specific absence of p53 radioinduction has been reported,38 as well as differential effects of various gene expression on cellular resistance to low-dose-rate irradiation.39 Also, a low-dose-rate specific radioinduction of apoptotic cell death and certain gene products has been reported.40 41 The studies showing radioresistance resulting from mutations or accumulation of p53 were conducted after high-dose-rate irradiation. The cell strains we isolated from AS patients showing increased sensitivity to low-dose-rate irradiation and exhibiting an absence of radioinduced p53/p21 proteins as well as G1 arrest will serve as valuable tools for further studies on mechanisms underlying cellular response to DNA damage and cell cycle regulation and their impact on the development of AS.
Finally, further studies on body cells from the asymptomatic relatives of the AS patients would confirm whether or not these patients carried common genetic factor(s) resulting in increased radiosensitivity and/or postirradiation DNA synthesis abnormality. Alternatively, certain metabolic anomalies associated with AS could affect cellular response to irradiation. Seeking such a correlation was difficult with this small cohort of patients. However, an attempt was made to find a possible relationship between the criteria of abnormal response to irradiation (D37 values and reduction in DNA synthesis inhibition) and any of the clinical/metabolic parameters listed in Table 1 by using a bivariate correlation, 
2 test, and t test (Wilcoxon rank sum test). This analysis revealed a significant (P=.056) association of lower D37 values (increased cellular sensitivity to irradiation) only with the AS patients who had previous myocardial infarction. Once again, a characterization of the specific metabolic/ genetic factor(s) enhancing cellular radiosensitivity in the myocardial infarction patients will require further studies.
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Acknowledgments |
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This work was supported by the King Faisal Specialist Hospital and Research Centre Advisory Council project No. 94-0005P. The authors thank Dr Bashir A. Khan of the Department of Biomedical Statistics and Scientific Computing for the statistical analysis.
Received June 12, 1996; accepted October 2, 1996.
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