Nucleoside Analogs Radiosensitize G0 Cells by Activating DNA End Resection and Alternative End-Joining.

Alternative end-joining (alt-EJ) is a DNA end resection-dependent, error-prone pathway utilized by vertebrate cells to repair DNA double-strand breaks (DSBs), but its engagement is linked to chromosomal translocations and genomic instability. Here, we report that when proliferating cells are exposed to ionizing radiation, treatment with nucleoside analogs (NAs) causes strong radiosensitization by increasing engagement of alt-EJ, while at the same time suppressing homologous recombination (HR) in S- and G2phase cells. This NA-mediated pathway shift may reflect a passive compensatory engagement of alt-EJ following HR suppression that is specific for S- and G2-phase cells, and/or the direct activation of alt-EJ throughout the cell cycle. To distinguish between these possibilities, we utilize here a cell culture model that exploits genetic and cell cycle-dependent inactivation of DSB repair pathways, to exclusively study alt-EJ and its modulation by NAs in murine and human cell lines. To this end, we allow LIG4-/--deficient cells to accumulate in G1/G0 phase by transfer to serum-deprived media and obtain cells deficient in c-NHEJ owing to the genetic LIG4 knockout, deficient in HR owing to the absence of S- or G2-phase cells, and compromised in their ability to carry out alt-EJ owing to their accumulation in G0. We find that in these cells irradiation and treatment with the NA, ß-arabinofuranosyladenine (araA), and to a lesser degree with other NAs, promptly activates suppressed alt-EJ that now functions at levels approximating those of c-NHEJ in wild-type cells. Results at high dose (20 Gy) generated using pulsed-field gel electrophoresis (PFGE) are corroborated by results at low dose (1 Gy) generated by scoring 53BP1 foci. Strikingly, araA treatment activates a normally undetectable DNA-end-resection at DSBs, which requires ATR activity, but proceeds unimpeded after CtIP knockdown. Treatment with araA increases the formation of chromosomal aberrations and enhances radiation-induced cell killing. The results support direct stimulation of resection by NAs and alt-EJ as a mechanism of their documented radiosensitizing potential. We propose that this stimulation also occurs in repair-proficient cells and that it occurs throughout the cell cycle. It may therefore be harnessed to develop protocols combining NAs with radiation to treat human cancer.

RAC2 promotes abnormal proliferation of quiescent cells by enhanced JUNB expression via the MAL-SRF pathway.

Radiation-induced lung injury (RILI) occurs most often in radiotherapy of lung cancer, esophageal cancer, and other thoracic cancers. The occurrence of RILI is a complex process that includes a variety of cellular and molecular interactions, which ultimately result in carcinogenesis. However, the underlying mechanism is unknown. Here we show that Ras-related C3 botulinum toxin substrate 2 (RAC2) and transcription factor jun-B (JUNB) were upregulated in non-small cell carcinoma (NSCLC) tissues and were associated with poor prognoses for NSCLC patients. Ionizing radiation also caused increased expression of RAC2 in quiescent stage cells, and the reentry of quiescent cells into a new cell cycle. The activity of the serum response factor (SRF) was activated by RAC2 and other Rho family genes (RhoA, ROCK, and LIM kinase). Consequently, JUNB acted as an oncogene and induced abnormal proliferation of quiescent cells. Together, the results showed that RAC2 can be used as a target gene for radiation protection. A better understanding of the RAC2 and JUNB mechanisms in the molecular etiology of lung cancer will be helpful in reducing cancer risks and side effects during treatment of this disorder. Our study therefore provides a new perspective on the involvement of RAC2 and JUNB as oncogenes in the tumorigenesis of NSCLC.

Transmission of persistent ionizing radiation-induced foci through cell division in human primary cells.

Unrepaired DNA double-strand breaks (DSBs) induced by ionizing radiation are associated with lethal effects and genomic instability. After the initial breaks and chromatin destabilization, a set of post-translational modifications of histones occurs, including phosphorylation of serine 139 of histone H2AX (γH2AX), which leads to the formation of ionizing radiation-induced foci (IRIF). DSB repair results in the disappearance of most IRIF within hours after exposure, although some remain 24h after irradiation. Their relation to unrepaired DSBs is generally accepted but still controversial. This study evaluates the frequency and kinetics of persistent IRIF and analyzes their impact on cell proliferation. We observed persistent IRIF up to 7 days postirradiation, and more than 70% of cells exposed to 5Gy had at least one of these persistent IRIF 24h after exposure. Moreover we demonstrated that persistent IRIF did not block cell proliferation definitively. The frequency of IRIF was lower in daughter cells, due to asymmetric distribution of IRIF between some of them. We report a positive association between the presence of IRIF and the likelihood of DNA missegregation. Hence, the structure formed after the passage of a persistent IRI focus across the S and G2 phases may impede the correct segregation of the affected chromosome's sister chromatids. The ensuing abnormal resolution of anaphase might therefore cause the nature of IRIF in daughter-cell nuclei to differ before and after the first cell division. The resulting atypical chromosomal assembly may be lethal or result in a gene dosage imbalance and possibly enhanced genomic instability, in particular in the daughter cells.

Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation.

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.

Radiosensitivity and relative biological effectiveness based on a generalized target model.

By considering both cellular repair effects and indirect effects of radiation, we have generalized the traditional target model, and made it have a linear-quadratic-linear characteristic. To assess the repair capacity-dependent radiosensitivity and relative biological effectiveness (RBE), the generalized target model was used to fit the survival of human normal embryonic lung fibroblast MRC-5 cells in the G0 and G1 phases after various types of radiations. The fitting results indicate that the generalized target model works well in the dose ranges considered. The resulting calculations qualitatively show that the parameter ratio (a/V) in the model could represent the cellular repair capacity. In particular, the significant linear correlations between radiosensitivity/RBE and cellular repair capacity are observed for different slopes of the linear regression curves. These results show that the radiosensitivity and RBE depend on the cellular repair capacity and can be regulated by linear energy transfer. These analyses suggest that the ratio a/V in the generalized target model can also be used for radiation damage assessment in radiotherapy.

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay).

PURPOSE: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. MATERIALS AND METHODS: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. RESULTS: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. CONCLUSIONS: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.

Msi RNA-binding proteins control reserve intestinal stem cell quiescence.

Regeneration of the intestinal epithelium is driven by multiple intestinal stem cell (ISC) types, including an active, radiosensitive Wnthigh ISC that fuels turnover during homeostasis and a reserve, radioresistant Wntlow/off ISC capable of generating active Wnthigh ISCs. We examined the role of the Msi family of oncoproteins in the ISC compartment. We demonstrated that Msi proteins are dispensable for normal homeostasis and self-renewal of the active ISC, despite their being highly expressed in these cells. In contrast, Msi proteins are required specifically for activation of reserve ISCs, where Msi activity is both necessary and sufficient to drive exit from quiescence and entry into the cell cycle. Ablation of Msi activity in reserve ISCs rendered the epithelium unable to regenerate in response to injury that ablates the active stem cell compartment. These findings delineate a molecular mechanism governing reserve ISC quiescence and demonstrate a necessity for the activity of this rare stem cell population in intestinal regeneration.

Gene expression of immediate early genes of AP-1 transcription factor in human peripheral blood mononuclear cells in response to ionizing radiation.

Ionizing radiation (IR) is considered ubiquitous in nature. The immediate early genes are considered the earliest nuclear targets of IR and are induced in the absence of de novo protein synthesis. Many of these genes encode transcription factors that constitute the first step in signal transduction to couple cytoplasmic effects with long-term cellular response. In this paper, coordinated transcript response of fos and jun family members which constitute activator protein 1 transcription factor was studied in response to IR in human peripheral blood lymphocytes at the G0 stage. Gene expression was monitored 5 min, 1 h and 4 h post-irradiation with Co60 γ-rays (dose rate of 0.417 Gy/min) and compared with sham-irradiated controls. When gene expression was analyzed at the early time point of 5 min post-irradiation with 0.3 Gy, the studied samples showed two distinct trends. Six out of ten individuals (called 'Group I responders') showed transient, but significant up-regulation for fosB, fosL1, fosL2 and c-jun with an average fold change (FC) ≥1.5 as compared to sham-irradiated controls. The Students's t test p value for all four genes was ≤0.001, indicating strong up-regulation. The remaining four individuals (called Group II responders) showed down-regulation for these same four genes. The average FC with 0.3 Gy in Group II individuals was 0.53 ± 0.22 (p = 0.006) for fosB, 0.60 ± 0.14 (p = 0.001) for fosL1, 0.52 ± 0.16 (p = 0.001) for fosL2 and 0.59 ± 0.28 (p = 0.03) for c-jun. The two groups could be clearly distinguished at this dose/time point using principal component analysis. Both Group I and Group II responders did not show any change in expression for three genes (c-fos, junB and junD) as compared to sham-irradiated controls. Though a similar trend was seen 5 min post-irradiation with a relatively high dose of 1 Gy, the average FC was lower and change in gene expression was not statistically significant (at p < 0.05), except for the down-regulation at fosL2 for Group II individuals (mean FC = 0.70 ± 0.15, p = 0.008). Both groups of individuals did not show any differential change in expression (FC ~ 1.0) for most loci at the late time points of 1 and 4 h, neither with 0.3 Gy nor with 1 Gy.

Research on radiotherapy at different times of the day for inoperable cervical cancer.

PURPOSE: To investigate the radiation effects and acute damage in inoperable cervical cancer patients irradiated at different times as well as the underlying mechanisms. METHODS: 67 patients were randomized to a morning group (MG, 9:00 - 11:00 AM) and an evening group (EG, 9:00 - 11:00 PM) and both received external beam radiotherapy (RT) (50 Gy in 25 fractions) at different times. Brachytherapy (36 - 42 Gy in 6 - 7 fractions) was also performed to enhance the radiation response twice every week in all patients at the same time. Clinical therapeutic effects and acute toxicities were evaluated after RT. Flow cytometry was analyzed before and after RT. RESULTS: Patients' response to radiation was similar in the two groups. Incidences of overall and high-grade (III - IV) diarrhea in the MG vs. the EG were 75.0% vs. 57.6% and 12.5% vs. 6.1%, respectively. The incidence of severe hematological toxicity in the EG was significantly increased compared to the MG group. Cell apoptosis in the EG was significantly higher at 9:00 - 11:00 PM than that at 9:00 - 11:00 AM after RT. No significant differences were found in Gap Phase 0/Gap Phase 1 (G0/G1), Gap Phase 2/Metaphase Phase (G2/M), and Synthesis Phase (S) phase between different times and groups, nor were expressions of Per1, Per2, and Clock. But expressions of Per1, Per2, and Clock were significantly negative with G2/M phase and positively correlated with cell apoptosis. CONCLUSION: RT at different time intervals results in similar efficacy. However, RT in the morning reduces severe hematological toxicity. Radiation responses may be associated with circadian genes by influence of cell cycles and apoptosis.
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Cytogenetic evidence that DNA topoisomerase II is not involved in radiation induced chromsome-type aberrations.

ICRF-187 (Cardioxane™, Chiron) is a catalytic inhibitor of DNA topoisomerase II (Topo II), proposed to act by blocking Topo II-mediated DNA cleavage without stabilizing DNA-Topo II-"cleavable complexes". In this study ICRF-187 was used to evaluate the potential involvement of DNA topoisomerase II in the formation of the radiation-induced chromosome-type aberrations in the G0 phase of the cell cycle in human lymphocytes from three healthy male donors. This is based on many evidences that DNA topoisomerases are involved in DNA recombination, mainly of illegitimate type (non-homologous) both in vitro and in vivo. The results obtained clearly indicated that ICRF-187 did not induce per se any chromosomal damage. When challenged with the non-catalytic Topo II poison VP-16 (etoposide), which acts by stabilizing the "cleavable complex" generating "protein concealed" DSB's and thus chromosomal aberrations, it completely abolished the significant induction of chromosome-type aberrations and formation of dicentric chromosomes. This indicates that ICRF-187 acts effectively as catalytic inhibitor of Topo II. On the other hand, when X-ray treatments were challenged with ICRF-187 using experimental conditions as for VP-16 treatments, no modification of the incidence of chromosome-type aberrations and dicentric chromosomes was observed. On this basis, we conclude that Topo II is not involved in the formation of X-ray-induced chromosome-type aberrations and dicentric chromosomes in human lymphocytes in the G0 phase of the cell cycle.

Radiation resistance: Cancer stem cells (CSCs) and their enigmatic pro-survival signaling.

Despite the fact that radiation therapy is a highly effective therapeutic approach, a small intratumoral cell subpopulation known as "cancer stem cells" (CSCs) is radiation-resistant and possesses specific molecular properties protecting it against radiation-induced damage. The exact mechanisms of this radioresistance are still not fully elucidated, but they relate to these cells' enhanced DNA repair capacities and their low intracellular ROS concentrations, resulting from their up-regulation of ROS scavengers. The low ROS content is accompanied by disturbances in cell cycle regulation, so it can be assumed that either CSCs are quiescent or dormant themselves, or that this cell population consists of at least two cell subpopulations: the normally and the slowly proliferating cells (quiescent or dormant cells). Slowly dividing CSCs show concomitant dysregulation of the signaling molecules mediating both cell cycle progression and maintenance of cell stemness. Despite a massive accumulation of data concerning the mechanisms underlying DNA damage response in CSCs, it represents a challenge to researchers in the era of personalized medicine to elucidate the role of intracellular ROS and of signaling pathways associated with the radiation resistance of these cells; there is a clear need to understand the molecular mechanisms helping CSCs to survive radiation exposure.

The bystander cell-killing effect mediated by nitric oxide in normal human fibroblasts varies with irradiation dose but not with radiation quality.

PURPOSE: To investigate the dependence of the bystander cell-killing effect on radiation dose and quality, and to elucidate related molecular mechanisms. MATERIALS AND METHODS: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method. RESULTS: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ≤ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations. CONCLUSION: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.

Cytogenetic low-dose hyperradiosensitivity is observed in human peripheral blood lymphocytes.

PURPOSE: The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low-dose exposures. However, the low-dose hyperradiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low-dose region. HRS is more prominent in the G2 phase of the cell cycle than in the G0/G1 or S phases. Here we provide the first cytogenetic mechanistic evidence of low-dose HRS in human peripheral blood lymphocytes using structural chromosomal aberrations. METHODS AND MATERIALS: Human peripheral blood lymphocytes from 2 normal healthy female donors were acutely exposed to cobalt 60 γ rays in either G0 or G2 using closely spaced doses ranging from 0 to 1.5 Gy. Structural chromosomal aberrations were enumerated, and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. RESULTS: HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5- to 3.5-fold higher for doses ≤0.4 Gy than for doses >0.5 Gy. CONCLUSIONS: These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

Role of base excision repair genes and proteins in gamma-irradiated resting human peripheral blood mononuclear cells.

Ionising radiation induces several isolated and clustered DNA lesions in human cells. Depending on the type of lesions, DNA repair pathways get activated to maintain the integrity of the genome. Base excision repair (BER) pathway is known to repair single-strand breaks and base damages through short- and long-patch genes and proteins. In the present study, attempt has been made to study the role of BER genes and proteins in resting human peripheral blood mononuclear cells (PBMCs) exposed to gamma radiation. Venous blood samples were collected from 20 random and healthy individuals with written informed consent. Dose-response and time-dependent changes at the level of DNA damage, transcription and protein expression were studied in PBMC. Dose-response studies were done in PBMC exposed between 0.1 and 2.0 Gy, whereas time-dependent changes in post-irradiated PBMC were studied up to 240 min. Our results have shown a significant (P ≤ 0.05) dose-dependent increase in the percentage of DNA in tail (%T) among the individuals studied. At transcriptional level, LIGASE3, MBD4 and LIGASE1 showed significant up-regulation (P ≤ 0.05) at 4h compared to 0h. Short-patch BER proteins such as OGG1 and LIGASE3 showed significant increase (P ≤ 0.05) in expression at lower doses (<0.6 Gy), whereas long-patch BER proteins such as MBD4, FEN1 and LIGASE1 showed an increase in expression at higher doses (1.0 and 2.0 Gy), suggesting dose-dependent and pathway-specific role of BER proteins in human PBMCs at G0/G1. In conclusion, BER genes and proteins play an active role in repairing radiation-induced DNA damage in resting PBMC, which has important biological significance in terms of DNA repair process in humans.

Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB.

Pharmacologic stabilization of HIF-1α increases hematopoietic stem cell quiescence in vivo and accelerates blood recovery after severe irradiation.

UNLABELLED: Quiescent hematopoietic stem cells (HSCs) preferentially reside in poorly perfused niches that may be relatively hypoxic. Most of the cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIFs). To investigate the effects of hypoxia on HSCs, we blocked O2-dependent HIF-1α degradation in vivo in mice by injecting 2 structurally unrelated prolyl hydroxylase domain (PHD) enzyme inhibitors: dimethyloxalyl glycine and FG-4497. Injection of either of these 2 PHD inhibitors stabilized HIF-1α protein expression in the BM. In vivo stabilization of HIF-1a with these PHD inhibitors increased the proportion of phenotypic HSCs and immature hematopoietic progenitor cells in phase G0 of the cell cycle and decreased their proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. This effect was independent of erythropoietin, the expression of which was increased in response to PHD inhibitors. Finally, pretreatment of mice with a HIF-1α stabilizer before severe, sublethal 9.0-Gy irradiation improved blood recovery and enhanced 89-fold HSC survival in the BM of irradiated mice as measured in long-term competitive repopulation assays. The results of the present study demonstrate that the levels of HIF-1α protein can be manipulated pharmacologically in vivo to increase HSC quiescence and recovery from irradiation. KEY POINTS: HIF-1α protein stabilization increases HSC quiescence in vivo. HIF-1α protein stabilization increases HSC resistance to irradiation and accelerates recovery.

Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates γ-histone 2AX (γ-H2AX) levels upon ionizing radiation.

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.

Chromosomal radiosensitivity in Ukrainian breast cancer patients and healthy individuals.

AIM: Recent studies showed that increased chromosomal damage induced by ionizing radiation is observed among patients with different tumor types. The aim of the study was evaluation of chromosomal radiosensitivity in breast cancer (BC) patients (n = 37) and healthy women (n = 44). METHODS: Chromosomal radiosensitivity was assessed with G0 and G2 assay. For G0 assay lymphocytes were exposed in vitro to 1,5 Gy of X-rays before culture setting. For G2 assay lymphocytes were irradiated with 0,5 Gy of X-rays after 47 h of incubation. RESULTS: Significant differences in mean scores both of G0 and G2 assay between breast cancer patients and controls were observed indicating the increased chromosomal radiosensitivity of lymphocytes of cancer patients. 11% of healthy women and 38% of BC patients were determined to be radiosensitive with G2 assay. CONCLUSION: Obtained results support the concept of association between elevated individual G2 chromosomal radiosensitivity and predisposition to BC.

Simultaneous inhibition of EGFR and PI3K enhances radiosensitivity in human breast cancer.

PURPOSE: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. METHODS AND MATERIALS: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used to investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. RESULTS: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF(SF2)) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF(SF2) at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. CONCLUSIONS: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN.