Evidence for the expression of radiation-induced potentially lethal damage being a p53-dependent process

PURPOSE: To test the hypothesis that the expression of potentially lethal damage (PLD) is a p53-dependent process. MATERIALS AND METHODS: Previously reported data on radiation sensitivity, DNA double-strand break rejoining, PLD expression and repair (PLDR) were analyzed for a group of 12 human tumor cell lines and three human diploid fibroblast cell lines. Seven of these cell lines had normal p53 gene expression while the other eight were functionally p53-deficient. None of the cell lines was sensitive to radiation-induced apoptosis. RESULTS: Cell lines with a normal p53 expression were more sensitive to radiation, but only when sensitivity was measured in plateau-phase cultures under conditions where PLDR was minimized. Mutation or functional inactivation of p53 by HPV E6-transformation led to a more radioresistant phenotype under these conditions as well as a significant reduction in PLDR. PLDR was inversely proportional to the percentage of radiation-induced DNA double-strand breaks rejoined in 1 h in the p53 normal cell lines. CONCLUSIONS: These results suggest that the expression of PLD is primarily a p53-dependent process. In the absence of functional p53 gene expression, the effects of PLD are minimized. These observations help clarify the role of p53 in tumor response to radiation therapy because they suggest that the effects of alterations in p53 are highly dependent on the microenvironment of the tumor, i.e. whether conditions allow for PLDR.     

Evidence for the expression of radiation-induced potentially lethal damage being a p53 -dependent process

Purpose : To test the hypothesis that the expression of potentially lethal damage (PLD) is a p 53 -dependent process. Materials and methods : Previously reported data on radiation sensitivity, DNA double-strand break rejoining, PLD expression and repair (PLDR) were analyzed for a group of 12 human tumor cell lines and three human diploid fibroblast cell lines. Seven of these cell lines had normal p53 gene expression while the other eight were functionally p53 -deficient. None of the cell lines was sensitive to radiation-induced apoptosis. Results : Cell lines with a normal p53 expression were more sensitive to radiation, but only when sensitivity was measured in plateau-phase cultures under conditions where PLDR was minimized. Mutation or functional inactivation of p53 by HPV E6 transformation led to a more radioresistant phenotype under these conditions as well as a significant reduction in PLDR. PLDR was inversely proportional to the percentage of radiationinduced DNA double-strand breaks rejoined in 1h in the p53 normal cell lines. Conclusions : These results suggest that the expression of PLD is primarily a p53 -dependent process. In the absence of functional p53 gene expression, the effects of PLD are minimized. These observations help clarify the role of p53 in tumor response to radiation therapy because they suggest that the effects of alterations in p53 are highly dependent on the microenvironment of the tumor, i.e. whether conditions allow for PLDR.     

The relationship between potentially lethal damage repair and intrinsic radiosensitivity of human cells

The intrinsic radiosensitivity of exponentially growing cells (exp) was compared to that of immediately plated plateau phase cells (ip) using published data on 60 human cell lines (27 fibroblast lines and 33 tumour cell lines). The values for alpha, D and S2 are not significantly different for the two groups; beta is significantly higher in ip cells. This produces a smaller alpha/beta ratio in ip cells than in exp cells. The influence of potentially lethal damage (PLD) repair was assessed by comparing the radiosensitivities of ip cells and plateau phase cells with delayed plating (dp). The published data for 81 human cell lines (48 fibroblasts and 33 tumour lines) were used. PLD repair was found to lead to a decrease in alpha and an increase in D and S2, whereas neither beta nor the alpha/beta ratio changed significantly. The relationship between PLD repair and intrinsic radiosensitivity was assessed by repair capacity and the repair ratio. The fitted relationship is a bell-shaped curve with a maximum at 2.2 Gy for repair capacity. The fitted curve predicts that repair capacity is zero at a D up of 0.28 Gy and at 4 Gy. Thus, PLD repair is a reasonable reflection of intrinsic radiosensitivity up to 2.2 Gy. Above 2.2 Gy, the relationship is reversed: the greater the radioresistance, the lower the PLD repair.     

Potentially lethal damage repair by total and quiescent tumor cells following various DNA-damaging treatments.

After continuous labeling of proliferating (P) cells with 5-bromo-2'-deoxyuridine (BrdU) for 5 days, SCC VII tumor-bearing mice received various kinds of DNA-damaging treatments: gamma-ray irradiation, tirapazamine (TPZ, hypoxia-specific cytotoxin) administration, or cisplatin injection. From 0.5 to 72 hr after treatment, tumors were excised, minced, and trypsinized. Single tumor cell suspensions were incubated for 48 hr with a cytokinesis-blocker, cytochalasin-B. Then, the micronucleus (MN) frequency for BrdU-unlabeled cells, quiescent (Q) cells at treatment, was determined using immunofluorescence staining for BrdU. The MN frequency for total (P+Q) cells was obtained from tumors that were not pretreated with BrdU labeling. The sensitivity to each DNA-damaging treatment was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). Treatment with gamma-rays or cisplatin resulted in a larger MN frequency in total cells than in Q cells. In contrast, TPZ treatment produced a smaller MN frequency in total cells than in Q cells. Regardless of the treatment used, Q cells showed greater repair capacities than total cells. However, TPZ caused much smaller repair capacity in both total and Q cells, compared with gamma-rays or cisplatin. Gamma-rays and cisplatin produced similar repair patterns. Differences in sensitivity between total and Q cells and repair patterns of the two cell populations were thought to depend on differences between the two cell populations in the toxicity of the DNA-damaging treatment and distribution pattern of the anticancer agent.     

Functional inactivation of p53 by HPV-E6 transformation is associated with a reduced expression of radiation-induced potentially lethal damage

PURPOSE: To examine the effect of functional loss of p53 on radiation sensitivity and potentially lethal damage repair (PLDR). MATERIALS AND METHODS: Radiation sensitivity and PLDR were examined in an isogenic pair of human tumour cell lines created by HPV-E6 transformation. RESULTS: Inactivation of p53 by E6 transformation resulted in a cell line that was more resistant to killing by radiation but showed little enhancement in survival (PLDR) when plateau-phase cells were held non-cycling after radiation exposure. Holding p53-normal cells in plateau-phase after radiation exposure not only led to enhanced survival, but also to a reduction in the proportion of cells that blocked in G1 subsequent to release. CONCLUSIONS: These results suggest that p53 expression influences that component of radiation sensitivity associated with PLDR.     

Conditioned medium from plateau-phase cells. Effect on growth of proliferative cells and on repair of potentially lethal radiation damage.

In vitro cell cultures grown into plateau phase may be more suitable than exponentially growing cells as a model for tumor cell growth in vivo. Conditioned medium extracted from cells in plateau phase inhibits growth of log-phase cultures of either the same or a different cell line. Attempts to demonstrate that this inhibition is due to a heat-labile diffusible substance have failed. Conditioned medium extracted from plateau-phase cultures was effective in enhancing repair of potentially lethal damage following x irradiation. Cell survival is significantly increased following a six-hour postirradiation culture in conditioned medium as opposed to complete fresh medium.     

Rapid repair of potentially lethal damage in normal and ataxia telangiectasia cell lines

Potentially lethal damage repair (PLDR) was investigated in two normal and three ataxia telangiectasia (AT) human-skin fibroblast cell lines cultured in vitro. Using plateau-phase cells, both the time kinetics and the amount of repair were measured after irradiation. PLDR depended on both dose and survival level, as previously seen in rodent cells. Human cells differed from rodent cells in PLDR speed and the ability to discern two components within the repair response. Fast repair had a t1/2 of approximately 5-7 min while the slow response occurred over hours. AT cells had demonstrable PLDR in contrast to previous studies. Quantitatively, the proportion of fast and slow repair was similar for each dose in either normal or AT cells. However, AT cells had lower levels of both types of repair. When analyzing PLDR in human cells, differences in the rate of repair between human and rodent cells must be taken into account.     

Similarities in the repair kinetics of sublethal and potentially lethal X-ray damage in log phase Chinese hamster V79 cells

We have compared the repair kinetics of X-ray-induced sublethal damage (SLD) and potentially lethal damage (PLD) in V79 cells during postirradiation incubation in growth medium at 37 degrees C. Kinetics of SLD repair were studied by dose fractionation (D1 + D2 = 2 + 2, 4 + 2, 4 + 4, 10 + 4 and 10 + 7 Gy). Kinetics of PLD repair were studied by incubating cells with 0.5 M hypertonic saline (HS) for 20 min at various times after irradiation. The repair of SLD, after 2, 4 and 10 Gy, was complete in about 11, 23 and 62 min, respectively. This repair time was independent of the magnitude of dose D2 (test dose). The repair of PLD, after 2, 4 and 10 Gy, was also complete in about 9, 23 and 61 min, respectively. These data indicate that: (1) the kinetics of repair of SLD for a given dose are independent of the magnitude of the test dose D2; (2) the time taken to complete the repair (repair time) is dependent on dose D1, i.e. the larger the dose D1 the longer the repair time; (3) for a given dose, the repair times assayed either by dose fractionation or by HS treatment are similar; and (4) similarities in the repair times may imply that SLD and PLD are the same. The relationship between the repair kinetics and the shape of the shoulder on fractionated survival curves is discussed.     

The effect of recovery from potentially lethal damage on the determination of repair and repopulation in a murine tumour

Repair and repopulation following X irradiation of clamped-off murine anaplastic MT tumours was investigated using the established method of (Dn-D1)/(n-1). Repair was complete in 4 h, similar in extent to that reported in other tumours, and within the range of that reported for normal tissues. Subsequent repopulation commenced after 4 days and was equivalent to 1.8 Gy/day recovered dose, corresponding to a clonogenic cell number doubling time of 1.8 days. However, estimates of repair and repopulation may have been in error because the chronically hypoxic cells in this tumour alone have the ability to recover from potentially lethal damage (PLD) and so are more radioresistant than cells rendered acutely hypoxic by clamping. Because of this, even clamping off tumours at irradiation does not render all cell populations equally radioresistant, and so reoxygenation between fractions could result in an underestimate of repair and repopulation. Further, the differing sensitivity between acutely and chronically hypoxic cells renders the apparent OER a function of dose (i.e., oxygen not truly dose-modifying to chronically hypoxic cells). Consequently it is incorrect to assume a constant OER in order to compare repair in tumours irradiated under hypoxic conditions with that in normal tissues irradiated under aerobic conditions. It will be argued here that in the case of the present tumour neither reoxygenation nor the choice of OER will have qualitatively altered the conclusion reached from the conventional method.     

Radiosensitivity throughout the cell cycle and repair of potentially lethal damage and DNA double-strand breaks in an X-ray-sensitive CHO mutant

The response to ionizing radiation of synchronized and plateau-phase populations of xrs-5 cells was studied at the DNA and cellular level. Induction and repair of DNA double-strand breaks (dsb) were measured by the non-unwinding filter elution technique. Biphasic survival curves were obtained throughout the cell cycle as well as in the plateau phase, suggesting the presence in the cultures of a radiation-sensitive and a radiation-resistant subpopulation. Delayed plating of plateau-phase xrs-5 cells did not significantly modify cell survival indicating a deficiency in the repair of potentially lethal damage (PLD). Post-irradiation treatment with araA further indicated a deficiency in the repair of a form of PLD sensitive to this drug in the radiation-sensitive subpopulation. The radiosensitivity of synchronized populations decreased as cells progressed through S. Cells in late S were found to be most resistant to radiation, whereas cells irradiated in G2 + M had a sensitivity similar to that of cells irradiated in G1. The decrease in radiosensitivity in S was tentatively attributed to an increase in the proportion of radioresistant cells in the cell population. Preliminary analysis of the results obtained, assuming the presence of two subpopulations showing an exponential response as a function of dose, indicated no change in the D0 values of the radiation-sensitive and the radiation-resistant component throughout the cell cycle. The D0 value of the radiation-resistant component was similar to the D0 of repair-proficient CHO cells. At no stage in the cell cycle were shoulder-type survival curves observed. Transient expression of the xrs-5 repair gene may underlie the observed variation of radiosensitivity throughout the cell cycle. This gene expression could occur either at random, in a subpopulation of cells independent of cell cycle phase, or in a specific manner related to gene replication and the hemimethylated state transiently found immediately thereafter. It is proposed that the resistant tail observed in the survival curves throughout the cell cycle is due to the former phenomenon, and the decreased radiosensitivity in late S to the latter. xrs-5 cells were found to be deficient in dsb repair in the plateau phase of growth, as well as in G1 and mid-S phase. The latter observation indicates that the reduction in radiation sensitivity in S is not associated with a modulation in the ability of cells to repair dsb.     

Repair of potentially lethal radiation damage in acute and chronically hypoxic tumor cells in vivo.

The ability of animal tumor cells to repair potentially lethat damage was studied in vivo. Fifth-generation isotransplants of a spontaneous mouse squamous-cell carcinoma were irradiated under tourniquet-induced hypoxia or in air. Tumors were removed either immediately or 6 hours after irradiation and dose-response curves were determined by TD50 assays. Repair was attributed to cells in the hypoxic cell component for animals irradiated in air. Extensive repair was also noted for those irradiated under typoxic conditions. Implications of these results are discussed.     

G2-block after irradiation of cells with different p53 status

Background

Although it is clear that functional p53 is not required for radiation-induced G₂ block, certain experimental findings suggest a role for p53 in this context. For instance, as we also confirm here, the maximum accumulation in the G₂ compartment after X-ray exposure occurs much later in p53 mutants than in wild types. It remains to be seen, however, whether this difference is due to a longer block in the G₂ phase itself.

Material and methods

We observed the movement of BrdU-labeled cells through G₂ and M into G₁. From an analysis of the fraction of labeled cells that entered the second posttreatment cell cycle, we were able to determine the absolute duration of the G₂ and M phases in unirradiated and irradiated cells.

Results

Our experiments with four cell lines, two melanomas and two squamous carcinomas, showed that the radiation-induced delay of transition through the G₂ and M phases did not correlate with p53 status.

Conclusion

We conclude that looking at the accumulation of cells in the G₂ compartment alone is misleading when differences in the G₂ block are investigated and that the G₂ block itself is indeed independent of functional p53.     

Modification of potentially lethal damage repair by some intrinsic intra- and extracellular agents: I. Proteinases and proteinase inhibitors

The effects of nine intra- and extracellular proteinases and six proteinase inhibitors on the repair of potentially lethal damage (PLDR) induced by gamma-rays in plateau-phase V79 cells were examined. It was demonstrated that these agents, which are intrinsic factors produced within mammalian cells, can modify PLDR activity. A stimulatory effect on PLDR was seen with calf liver neutral proteinase, and to a lesser extent, with inhibitor pepstatin A. Other proteinases which belong to serine, cysteine and aspartic superfamilies, as well as proteinase inhibitors, inhibited PLDR to different degrees. The effects of some of these agents, present during the PLDR period, on the rate of tritiated thymidine incorporation into the acid-insoluble cell fraction was also examined. They can modify the DNA synthesis of cells when subcultured from plateau phase for the assessment of colony-forming ability. There is no clear evidence that the effects observed are entirely attributed to the alteration of cellular proliferative processes. It seems more likely that many serine and cysteine proteinases and their inhibitors can adversely affect the PLDR process by modulating the activity of proteinase(s) and other enzymes involved more directly in PLDR because of interrelationships of the entire intracellular proteinase system.     

Effect of cisplatin on the recovery from sublethal and potentially lethal radiation damage in cell culture

The effect of Cisplatin upon the recovery from sublethal damage during fractionated irradiation and upon the recovery from potentially lethal damage after a single dose has been investigated. In three mammalian cell lines, Cisplatin did not influence the recovery from sublethal and potentially lethal damage. There were differences among the three cell lines in their ability to recover from radiation damage. However, a significant quantitative difference between the recovery from sublethal and potentially lethal damage in any one cell line could not be found.     

Role of p53 mutations, protein function and DNA damage for the radiosensitivity of human tumour cells

PURPOSE: The tumour suppressor protein p53 is considered to have an impact on the radiosensitivity of tumour cells. However, this concept does not easily translate to the tumour sensitivity in the clinics. The aim of the present study was to determine whether a functional or dysfunctional p53 is associated with a sensitive or resistant phenotype. It was further studied whether DNA damage might be an additive factor by which p53 has impact on cell survival. MATERIALS AND METHODS: Nine human tumour cell lines were studied for p53 mutation by direct sequencing of exons 4-9. Regulation of p53 and p21(cip1/waf1) protein was assessed by immunoblotting and cell cycle effects by combining 5-bromodeoxyuridine incorporation and flow cytometry. RESULTS AND CONCLUSION: Three strains (RT112, Du145, SCC4451) were found to have a missense-mutation in the core domain and one did not express p53 at all (HeLa), presumably due to HPV18 infection. Immunoblots of these cells showed neither a regulated p53 nor p21 expression. The cells did not arrest in G1 phase after X-irradiation but did arrest in G2/M. All cells expressing wild-type protein (LNCaP, T47D-B8, MCF-7 and sublines BB and Bus) showed an intact p53 and p21 regulation and a modest arrest in both G1 and G2/M. Thus, in contrast to other studies, all tumour cells investigated showed either a typical p53wt or mutant (mut) pattern. Protein function was compared with cell survival and DNA damage, as assessed previously. p53 wild-type cells were on average 1.3-times (n.s.) more radiosensitive than mutant cells, but there was a considerable overlap between both groups. Further, the 1.3-fold enhanced resistance of cells lacking wild-type p53 was paralleled by a 1.3-fold lower number of induced double-strand breaks. The results suggest that p53 could have impact on chromatin compaction and thus effect DNA damage induction and radiosensitivity of tumour cells.     

辐射联合外源性野生型p53基因对不同p53状态的黑色素瘤细胞系的生物学效应

目的研究辐射结合腺病毒（Ad CMV）载体介导的p53基因转导对不同p53状态的人黑色素瘤细胞系基因转移效率、凋亡和辐射敏感性的影响。方法用复制缺陷的重组腺病毒载体（AdCMV-p53）介导入p53基因转导1Gy X射线预照射的黑色素瘤细胞系A375（wt p53）和WM983a（mu p53），RT-PCR检测mRNA水平，流式细胞仪测定细胞周期阻滞及外源性P53蛋白表达情况，Tunel法检测细胞凋亡，克隆形成率测定辐射后细胞存活率。用携带报道基因的复制缺陷重组腺病毒载体AdCMV-GFP作为对照。结果1Gy X射线照射可较高地增加AdCMV-p53对A375和WM983a细胞系的基因转导效率，转导的外源性野生型p53可在两种细胞中高效表达，并诱导细胞周期G1期阻滞；单纯转导p53对A375（wt p53）细胞无明显诱导凋亡和生长抑制效应，但可部分诱导WM983a（mu p53）细胞凋亡；而转导p53基因48h后给予X射线辐射，两种细胞的克隆存活率较其对照组均明显减低，外源性p53基因对WM983a（mu p53）细胞的辐射增敏作用较A375（wt p53）细胞明显。结论外源性野生型p53基因过表达可增加黑色素瘤细胞系A375和WM983a的辐射敏感性，但对WM983a细胞系的辐射增敏作用高于A375细胞系。表明p53是基因治疗黑色素瘤较好的候选基因。     

Repair of potentially mutagenic damage, sensitive to anisotonic treatment, in actively growing Chinese hamster cells

Using exponentially growing Chinese hamster V79 cells we studied the effect of anisotonic phosphate-buffered saline (PBS) treatment on the frequency of mutation following X-irradiation. Induction of mutants was studied at the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT). When cells were X-irradiated and immediately followed by 0.5 M NaCl/PBS treatment for 20 min at 37 degrees C, cell survival was significantly decreased and a concomitant increase in the number of mutants was observed, indicating that postirradiation treatment with anisotonic salt solution caused conversions of potentially lethal damage and potentially mutagenic damage to lethal and mutagenic damage, respectively. The repair kinetics of potentially mutagenic damage expressible by 0.5 M NaCl/PBS after 5.86 Gy of X-rays were similar to those of potentially lethal damage. The repair of both types of damage was essentially complete within 40 min after X-irradiation.