Loss of PTEN-assisted G2/M checkpoint impedes homologous recombination repair and enhances radio-curability and PARP inhibitor treatment response in prostate cancer.

Here we report that PTEN contributes to DNA double-strand break (DSB) repair via homologous recombination (HR), as evidenced by (i) inhibition of HR in a reporter plasmid assay, (ii) enhanced sensitivity to mitomycin-C or olaparib and (iii) reduced RAD51 loading at IR-induced DSBs upon PTEN knockdown. No association was observed between PTEN-status and RAD51 expression either in-vitro or in-vivo in a tissue microarray of 1500 PTEN-deficient prostate cancer (PC) samples. PTEN depletion and sustained activation of AKT sequestered CHK1 in the cytoplasm, thus impairing the G2/M-checkpoint after irradiation. Consistently, AKT inhibition recovered the G2/M-checkpoint and restored HR efficiency in PTEN-depleted cells. We show that, although PTEN loss correlates with a worse prognosis, it may predict for improved response of PC patients to radiotherapy. Further, we provide evidence for the use of PTEN as a biomarker for predicting the response to PARP inhibitors as radiosensitizing agents in prostate cancer. Collectively, these data implicate PTEN in maintaining genomic stability by delaying G2/M-phase progression of damaged cells, thus allowing time for DSB repair by HR. Furthermore, we identify PTEN-status in PC as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor alone or combined with radiotherapy.

p53 modulates homologous recombination at I-SceI-induced double-strand breaks through cell-cycle regulation.

Inhibition of homologous recombination (HR) is believed to be a transactivation-independent function of p53 that protects from genetic instability. Misrepair by HR can lead to genetic alterations such as translocations, duplications, insertions and loss of heterozygosity, which all bear the risk of driving oncogenic transformation. Regulation of HR by wild-type p53 (wtp53) should prevent these genomic rearrangements. Mutation of p53 is a frequent event during carcinogenesis. In particular, dominant-negative mutants inhibiting wtp53 expressed from the unperturbed allel can drive oncogenic transformation by disrupting the p53-dependent anticancer barrier. Here, we asked whether the hot spot mutants R175H and R273H relax HR control in p53-proficient cells. Utilizing an I-SceI-based reporter assay, we observed a moderate (1.5 × ) stimulation of HR upon expression of the mutant proteins in p53-proficient CV-1, but not in p53-deficient H1299 cells. Importantly, the stimulatory effect was exactly paralleled by an increase in the number of HR competent S- and G2-phase cells, which can well explain the enhanced recombination frequencies. Furthermore, the impact on HR exerted by the transactivation domain double-mutant L22Q/W23S and mutant R273P, both of which were reported to regulate HR independently of G1-arrest execution, is also exactly mirrored by cell-cycle behavior. These results are in contrast to previous concepts stating that the transactivation-independent impact of p53 on HR is a general phenomenon valid for replication-associated and also for directly induced double-strand break. Our data strongly suggest that the latter is largely mediated by cell-cycle regulation, a classical transactivation-dependent function of p53.

[Determination of DNA damage in vitro]. / Bestimmung der DNA-Schädigung in vitro.

Ionising irradiation acts primarily via induction of DNA damage, among which double-strand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of double-strand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about double-strand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

No correlation between radiosensitivity or double-strand break repair capacity of normal fibroblasts and acute normal tissue reaction after radiotherapy of breast cancer patients.

The aim was to study the relationship between cellular radiosensitivity or double-strand break (dsb) repair capacity of skin fibroblasts and the extent of acute reaction after radiotherapy for breast cancer. The study was performed with 25 breast cancer patients submitted to the radiotherapy unit of the Egyptian National Cancer Institute after conserving surgery. Dermal fibroblasts, established from skin biopsies, were used to determine the cellular radiosensitivity via colony assay and the capacity of dsb repair by constant-field gel electrophoresis. Acute reactions were scored using the Radiation Therapy Oncology Group (RTOG) classification. The spectrum of acute reactions varied from grade 1 to 4, whereby most patients developed a grade 1 reaction after total doses ranging between 46 and 70 Gy. Skin fibroblasts showed a pronounced variation in both cellular radiosensitivity expressed as the mean inactivation dose (Dbar) (coefficient of variation, CV=25%) as well as in the number of residual dsb (CV=33%) with no significant correlation between these two endpoints (r2=0.20, p=0.14). Both parameters did not correlate with the extent of acute reaction of the respective patient. The data obtained indicate that the sensitivity of fibroblasts measured either by colony assay or by dsb repair capacity is not a major parameter determining the extent of acute reaction after radiotherapy of breast cancer patients.

Mechanism of radiosensitization by hyperthermia (> or = 43 degrees C) as derived from studies with DNA repair defective mutant cell lines.

All biochemical and cytogenetic data on radiosensitization by heat treatment at and above 43 degrees C indicate that inhibition of DNA repair plays a central role. There are several DNA repair pathways involved in restoration of damage after ionising irradiation and the kinetics of all of them are affected by heat shock. This, however, does not imply that the inhibition of each of these pathways is relevant to the effect of heat on cellular radiosensitivity. The current review evaluates the available data on heat radiosensitization in mutant or knockout cell lines defective in various DNA repair proteins and/or pathways. The data show that thermal inhibition of the non-homologous end-joining pathway (NHEJ) plays no role in heat radiosensitization. Furthermore, limited data suggest that the homologous recombination pathway may also not be a major heat target. By deduction, it is suggested that inhibition of base damage repair (BER) could be the crucial step in radiosensitization by heat. While a lack of mutant cell lines and redundancy of the BER pathway have hampered efforts toward a conclusive study, biochemical and correlative evidence support this hypothesis.

Human and rodent cell lines showing no differences in the induction but differing in the repair kinetics of radiation-induced DNA base damage.

PURPOSE: To compare the induction and repair of radiation-induced base damage in human and rodent cell lines. MATERIAL AND METHODS: Experiments were performed with two human (normal fibroblasts HSF1 and tumour HeLa cells) and two rodent (mouse L929 and hamster CHO-K1) cell lines. Base damage was determined with the alkaline comet assay combined with the repair enzyme formamidopyrimidine-glycosylase (Fpg). Proteins were detected by Western blot. RESULTS: The induction of Fpg-sensitive sites was measured in human and rodent cell lines for doses up to 8 or 5 Gy, respectively. Comets were analysed in terms of tail moments, which were transformed into Gy-equivalents. The amount of Fpg-sensitive sites increased linearly with doses up to 4 Gy, whereby the ratio of single-strand breaks (ssb) to Fpg-sensitive sites was nearly identical for human and rodent cells with ssb:Fpg-sensitive sites=1:0.41+/-0.07 and 1:0.45+/-0.05, respectively. For doses exceeding 4 Gy, the amount of Fpg-sensitive sites did not increase further, indicating a dose limit up to which the comet assay can be used to detect Fpg-sensitive sites. Repair of Fpg-sensitive sites was studied for an X-ray dose of 4 Gy. For all four cell lines, the repair was measured to be completed 24 h after irradiation, but with pronounced differences in the kinetics. In both rodent cell lines, 50% of Fpg-sensitive sites were removed after t((1/2))=25+/-10 min in contrast to t((1/2))=80+/-20 min in the two human cell lines. The two species also differed in the level of polymerase ss with, on average, a three- to fivefold higher level in rodent cells compared with human cells. CONCLUSIONS: Repair of radiation-induced Fpg-sensitive sites was much faster in rodent than in human cells, which might result from the higher level of polymerase ss found in rodent cells.

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks.

Nine human tumour cell lines (four mammary, one bladder, two prostate, one cervical, and one squamous cell carcinoma) were studied as to whether cellular radiosensitivity is related to the number of initial or residual double-strand breaks (dsb). Cellular sensitivity was measured by colony assay and dsb by means of constant- and graded-field gel electrophoresis (CFGE and GFGE, respectively). The nine tumour cell lines showed a broad variation in cellular sensitivity (SF2 0.17-0.63). The number of initial dsb as measured by GFGE ranged between 14 and 27 dsb/Gy/diploid DNA content. In contrast, normal fibroblasts raised from skin biopsies of seven individuals showed only a marginal variation with 18-20 dsb/Gy/diploid DNA content. For eight of the nine tumour cell lines, there was a significant correlation between the number of initial dsb and the cellular radiosensitivity. The tumour cells showed a broad variation in the amount of dsb measured 24 h after irradiation by CFGE, which, however, was not correlated with the cellular sensitivity. This residual damage was found to be influenced not only by the actual number of residual dsb, but also by apoptosis and cell cycle progression which had impact on CFGE measurements. Some cell line strains were able to proliferate even after exposure to 150 Gy while others were found to degrade their DNA. Our results suggest that for tumour cells, in contrast to normal cells, the variation in sensitivity is mainly determined by differences in the initial number of dsb induced.

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements.

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G(0) fibroblasts were either preheated (45 degrees C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 x 10(-3) dsb/Gy/MB: Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12-16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.

Hyperthermic radiosensitization: mode of action and clinical relevance.

PURPOSE: To provide an update on the recent knowledge about the molecular mechanisms of thermal radiosensitization and its possible relevance to thermoradiotherapy. SUMMARY: Hyperthermia is probably the most potent cellular radiosensitizer known to date. Heat interacts with radiation and potentiates the cellular action of radiation by interfering with the cells' capability to deal with radiation-induced DNA damage. For ionizing irradiation, heat inhibits the repair of all types of DNA damage. Genetic and biochemical data suggest that the main pathways for DNA double-strand break (DSB) rejoining, non-homologous end-joining and homologous recombination, are not the likely primary targets for heat-induced radiosensitization. Rather, heat is suggested to affect primarily the religation step of base excision repair. Subsequently additional DSB arise during the DNA repair process in irradiated and heated cells and these additional DSB are all repaired with slow kinetics, the repair of which is highly error prone. Both mis- and non-rejoined DSB lead to an elevated number of lethal chromosome aberrations, finally causing additional cell killing. Heat-induced inhibition of DNA repair is considered not to result from altered signalling or enzyme inactivation but rather from alterations in higher-order chromatin structure. Although, the detailed mechanisms are not yet known, a substantial body of indirect and correlative data suggests that heat-induced protein aggregation at the level of attachment of looped DNA to the nuclear matrix impairs the accessibility of the damaged DNA for the repair machinery or impairs the processivity of the repair machinery itself. CONCLUSION: Since recent phase III clinical trials have shown significant benefit of adding hyperthermia to radiotherapy regimens for a number of malignancies, it will become more important again to determine the molecular effects underlying this success. Such information could eventually also improve treatment quality in terms of patient selection, improved sequencing of the heat and radiation treatments, the number of heat treatments, and multimodality treatments (i.e. thermochemoradiotherapy).

Effect of heat on dsb repair in G1- and S-phase studied in the human HeLa S3 cell line.

PURPOSE: The aim of this study was to investigate the relation between double-strand breaks and thermal radiosensitization in dependence on cell-cycle position. MATERIALS AND METHODS: The experiments were performed with the human tumour cell line HeLa S3. Cells synchronized in G1- and S-phase were exposed to X-rays alone or in combination with prior heating at 44 degrees C for 20 min. Cell kill was determined by means of colony forming assay, double-strand breaks (dsb) using constant-field gel electrophoresis and apoptotic cell death was scored using the fraction of detached cells. RESULTS: In both cell-cycle phases heating at 44 degrees C for 20 min prior to irradiation resulted in an increased cellular radiosensitivity, whereby the thermal enhancement ratio (TER) was significantly higher in S- than in G1-phase cells with TER=2.1 and 1.2, respectively. Prior heating at 44 degrees C did not affect the number of radiation-induced dsb but was found to modify their repair as measured for a X-ray dose of 40 Gy. In both cell cycle phases dsb repair kinetics measured after irradiation alone could be described by a fast and a slow component with the majority of dsb being repaired with fast kinetics. Prior heating at 44 degrees C was found to have only a minor effect on these half-times but mainly to affect the number of slowly rejoined dsb. In G1-phase cells the number of slowly rejoined dsb measured 300 min after irradiation was enhanced by a factor of 1.8 and in S-phase cells even by a factor of 3.2. Fraction of apoptotically dying cells was low after X-irradiation alone but was clearly enhanced after combined treatment, which was especially pronounced for S-phase cells. CONCLUSIONS: The pronounced thermal radiosensitization found for S-phase cells was attributed to the heat-mediated increase in the number of slowly rejoined dsb and partly also to the enhanced fraction of apoptotically dying cells when compared to G1-phase cells.

The Bowman-Birk protease inhibitor enhances clonogenic cell survival of ionizing radiation-treated nucleotide excision repair-competent cells but not of xeroderma pigmentosum cells.

PURPOSE: The radioprotective effect of the Bowman-Birk protease inhibitor (BBI) was previously shown to result from a TP53 dependent mechanism. Whether this effect involves specific DNA repair mechanisms is now tested. MATERIAL AND METHODS: Normal human fibroblasts were pre-treated with BBI before exposure to X-rays, UVB or to chemical agents (bleomycin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cisplatin). These agents were chosen because of their ability to induce different spectra of DNA damage. The radiometric agent bleomycin primarily induces double-strand breaks (dsb), which are repaired by recombination; MNNG results in alkylated bases which are repaired by base excision repair (BER); cisplatin results in DNA-crosslinks which are repaired mainly by nucleotide excision repair (NER); and finally UVB generates thymine dimers and thymine-cytosine-6-4 products which are also repaired by NER. Cell survival was analysed by colony formation assay and DNA dsb by constant field gel electrophoresis. The combined effect of BBI and X-rays was also tested for XP-fibroblasts, which are defective in NER. RESULTS: For normal human fibroblasts the radioprotective effect of BBI was clearly found by using a delayed plating procedure. The radioprotective effect was found to be unrelated to an altered induction or repair of radiation-induced DNA dsb. Pretreatment with BBI did not affect cell killing after exposure to bleomycin or MNNG, but resulted in a significant protection of cells exposed to cisplatin or UVB. These results indicate that pre-treatment with BBI did not alter recombination repair or BER, but was able to modify NER. The latter finding was supported by the observation made for XP-cells, where pretreatment with BBI failed to result in radioprotection after exposure to ionizing radiation. CONCLUSIONS: On the basis of these data it is proposed that the radioprotective effect of BBI is the result of an improved nucleotide excision repair mechanism.

Relationship between DNA double-strand breaks, cell killing, and fibrosis studied in confluent skin fibroblasts derived from breast cancer patients.

PURPOSE: To investigate the relationship between DNA double-strand breaks (dsbs), cell killing, and fibrosis using skin fibroblasts derived from breast cancer patients who received postmastectomy radiotherapy. METHODS AND MATERIALS: Experiments were performed with 12 lines of normal skin fibroblasts derived from recurrence-free breast cancer patients. Cells were irradiated in confluence and cell survival was determined either after immediate or delayed (14 h) plating using a colony-forming assay. Dsbs were measured by constant-field gel electrophoresis. The "excess risk of fibrosis" was previously scored by Johansen et al. (IJRB 1994;66:407-412). RESULTS: The 12 cell lines showed a typical spectrum of radiosensitivity. The mean value of surviving fraction after 3.5 Gy (SF3.5) was 0.063 for immediate and 0.174 for delayed plating with a coefficient of variation (CV) of 44 and 39%, respectively. There was also a broad variation in the extent of recovery from potentially lethal damage (RPLD), which was not correlated with the immediate sensitivity. The number of initial dsbs as well as the half-times of dsb repair showed little variation, whereas there were considerable differences in the number of residual dsbs (CV = 29%). The number of residual dsbs after 100 Gy was correlated significantly only with SF3.5 after delayed (r2 = O.59; p = 0.006) but not after immediate plating (r2 = 0.21, p = 0.16). There was also no significant relationship between residual dsbs and the "excess risk of fibrosis" determined for the respective patients. CONCLUSION: It is shown that the number of residual dsbs measured in confluent human fibroblast lines can be used to predict the cellular radiosensitivity after delayed but not after immediate plating and also not to predict the excess risk of fibrosis of the respective breast cancer patients.

Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts.

The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines. The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non-homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5-7. The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 GY, ranging from 0.03 to 0.28. These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs. We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated. In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines. Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines. Obviously, in this respect, other proteins/pathways appear to be more relevant.

DNA-repair, cell killing and normal tissue damage.

BACKGROUND: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. MATERIAL AND METHODS: Review. RESULTS: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. CONCLUSION: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly.

Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines.

PURPOSE: To test the relationship between cell killing and non-repaired DNA strand breaks both in repair proficient and deficient cell lines. MATERIALS AND METHODS: Five of the cell lines used are repair competent (CHO, CHO K1, rat rhabdomyosarcoma R1H, mouse balb and normal human fibroblasts), while four display a reduced repair capacity (scid, xrs1, xrs5, AT). Cell survival was determined by colony formation assay. The total number of strand breaks was measured by the alkaline unwinding technique and the numbers of double-strand breaks by constant-field gel electrophoresis. RESULTS: The nine cell lines showed a broad spectrum in radiosensitivity with SF2 values ranging from 0.018 to 0.58. The cell lines did not vary in the number of induced strand breaks, neither for all strand breaks nor for double-strand breaks alone. In contrast, there was a large variation in the number of non-repaired strand breaks measured 24 h after irradiation. Comparison of cell killing with the number of non-repaired breaks measured after a dose of 90 Gy showed no correlation for single-strand breaks (r2=0.29) but a fairly good correlation for double-strand breaks (r2=0.87). This correlation was found to hold both for repair proficient and deficient cell lines. CONCLUSIONS: The results obtained strongly suggest that the number of non-repaired double-strand breaks measured 24h after irradiation can be used as an indicator of cellular radiosensitivity.

Comparison between the alkaline unwinding technique and neutral filter elution using CHO, V79 and EAT cells.

Induction and repair of DNA strand breaks were measured in X-irradiated CHO, V79 and EAT cells using either the alkaline unwinding or the neutral filter elution techniques. After irradiation on ice, the cells were incubated at 37 degrees C for various times to allow for repair. The repair curves obtained were quite similar and the fast initial decline was always characterized by a half-time of 6-8 min and the final slow phase by a half-time of about 200 min independently of the technique used. The curves were found to differ only in the relative fraction of damage repaired during the fast or slow phase. From the similarity of the half-times it is concluded that the fast phase recorded by alkaline unwinding reflects the repair of single-strand breaks but also the repair of fast rejoining double-strand breaks. Comparing the known ratio of induced single- and double-strand breaks with the measured ratio of fast and slowly rejoining strand breaks as derived from the kinetics, it can be further concluded that the slow phase measured by alkaline unwinding covers the repair of both slowly rejoining double strand breaks as well as slowly rejoining single strand lesions.