Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.     

Valproic acid decreases the reparation capacity of irradiated MOLT-4 cells

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.     

In vivo radioprotection by 5-aminosalicylic acid

The radioprotective effect of 5-aminosalicylic acid (5ASA) was investigated in mouse bone marrow. The present study was aimed at investigating the radioprotective effect of pre-irradiation treatment with 5ASA against a range of whole-body lethal (8-11 Gy) and sublethal (1-4 Gy) doses of gamma-radiation (RT) in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 5ASA at a dose of 25mg/kg body weight (b. wt.) 30 min before lethal RT increased survival, giving a dose modification factor (DMF) of 1.08. Injection of 5ASA (25 mg/kg b. wt.) 60 or 30 min before or within 15 min after 3 Gy whole body RT resulted in a significant decrease in the radiation-induced aberrant metaphases, at 24 h post-irradiation. Maximum effect was seen when the drug was administered 30 min before irradiation. 5ASA (25 mg/kg b. wt.) significantly reduced the number of aberrant metaphases and the different types of aberrations at all the radiation doses (1-4 Gy) tested, giving a DMFs of 1.43 for number of aberrant metaphases. 5ASA pretreatment also significantly enhanced the endogenous spleen colonies in mouse exposed to 11 Gy RT. Pretreatment with 5ASA, protected plasmid DNA (pGEM-7Zf) against breakage induced by RT and Fenton reactants. Using nanosecond pulse radiolysis technique, the bimolecular rate constant of the reaction of 5ASA with hydroxyl radical was found to be 6.7x10(9)M(-1)s(-1). The p53 and p21 protein levels of bone marrow and spleen were evaluated to identify the specific molecular mechanisms. Both p53 and p21 increased 24h after 6 Gy irradiation, while treatment with 5ASA inhibited this RT-induced increase. Therefore, the present data suggest that 5ASA pretreatment decreases death caused by RT-induced gastrointestinal and hemopoeitic syndromes. The proposed mechanism of radioprotection by 5ASA is through the inhibition of damage to DNA, lipids, and proteins; and prevention of RT-induced increased expression of p53 and p21.     

Radiation-induced gastric epithelial apoptosis occurs in the proliferative zone and is regulated by p53, bak, bax, and bcl-2

Unlike the small intestine and colon where gamma-radiation-induced apoptosis has previously been well characterized, the response of murine gastric epithelium to gamma-radiation has not been investigated in detail. Apoptosis was therefore assessed on a cell positional basis in gastric antral and corpus glands from adult male mice following gamma-radiation. Maximum numbers of apoptotic cells were observed in both antrum and corpus at 48 h and at radiation doses greater than 12 Gy. However, the number of apoptotic cells observed in the gastric epithelium was much lower than observed in the small intestine or colon after similar doses of radiation. Hematoxylin and eosin, caspase 3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling detected similar numbers and cell positional distributions of apoptotic cells, hence hematoxylin and eosin was used for subsequent studies. The highest numbers of apoptotic cells were observed at cell positions 5-6 in the antrum and cell positions 15-18 in the corpus. These distributions coincided with the distributions of PCNA-labeled proliferating cells, but not with the distributions of H(+)-K(+)-ATPase-labeled parietal cells or TFF2-labeled mucous neck cells. Decreased numbers of apoptotic gastric epithelial cells were observed in p53-null, bak-null, and bax-null mice compared with wild-type counterparts 6 and 48 h after 12 Gy gamma-radiation. Significantly increased numbers of apoptotic gastric epithelial cells were observed in bcl-2-null mice compared with wild-type littermates 6 h after 12 Gy gamma-radiation. Radiation therefore induces apoptosis in the proliferative zone of mouse gastric epithelium. This response is regulated by the expression of p53, bak, bax, and bcl-2.     

Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more susceptible to increase ∼25% cell death than the p53-null HCT116 cells after treatment with 100 μM celecoxib for 24 h. Transfection with a small interfering RNA of p53 reduced the celecoxib-induced cytotoxicity in the RKO (p53-wild type) colorectal cancer cells. Celecoxib (80-100 μM for 24 h) significantly increased total p53 proteins and the phosphorylated p53 proteins at serine-15, -20, -46, and -392 in RKO cells. However, the phospho-p53 (serine-15, -20, and -392) proteins were presented on the nuclei of cells but the phospho-p53 (serine-46) protein was located on the cytoplasma of apoptotic cells following treatment with celecoxib. Interestingly, the p53 up-regulated modulator of apoptosis (PUMA) protein, which located on the mitochondria, was induced by celecoxib in the p53-functional colorectal cancer cells but not in the p53-mutational cells. Together, this study provides the first time that celecoxib induces the various phosphorylated sites of p53 and activates p53-PUMA pathway, which potentiates the apoptosis induction in human colorectal cancer cells.     

Dose-dependent biphasic accumulation of TP53 protein in normal human embryo cells after X irradiation

The effects of various doses of X radiation on the kinetics of accumulation of TP53 protein (formerly known as p53) were examined in normal human embryo cells. We found that the rate of accumulation of TP53 protein was biphasic at X-ray doses between 1 and 4 Gy, while monophasic accumulation was observed after X irradiation with doses higher than 6 Gy. The first phase of accumulation was detected within 1 h after irradiation, and a second phase of accumulation was detected between 6 and 12 h after irradiation. The induction of CDKN1A (formerly known as p21(WAF1/CIP1)) and MDM2 proteins was also biphasic after doses of 4 Gy or less, while monophasic accumulation was observed after 6 Gy or higher. We found that the proteasome inhibitor ALLN increased the constitutive level of TP53 protein, and no change was observed in the TP53 level after X irradiation when cells were treated with ALLN. These results indicate that the dose-dependent accumulation of TP53 is due to an inhibition of TP53 degradation, and that the induction of MDM2 might be responsible in part for the different kinetics of accumulation of TP53.     

Gamma-radiation-induced ATM-dependent signalling in human T-lymphocyte leukemic cells, MOLT-4

ATM kinase (ATM) is essential for activation of cell cycle check points and DNA repair in response to ionizing radiation (IR). In this work we studied the molecular mechanisms regulating DNA repair and cell death in human T-lymphocyte leukemic cells, MOLT-4. Apoptosis was evaluated by flow-cytometric detection of annexin V. Early apoptotic cells were determined as sub-G1 cells and late apoptotic cells were determined as APO2.7-positive ones. Proteins involved in ATM signalling pathway were analysed by Western-blotting. We observed a rapid (0.5 h) phosphorylation of ATM declining after 6 h after irradiation by all the doses studied (1.5, 3.0, and 7.5 Gy). Checkpoint kinase-2 (Chk-2) was also phosphorylated after 0.5 h but its phosphorylated form persisted 4, 2, and 1 h after the doses of 1.5, 3.0, and 7.5 Gy, respectively. The amount of p53 protein and its form phosphorylated on Ser-392 increased 1 h after irradiation (1-10 Gy). The lethal dose of 7.5 Gy caused an immediate induction and phosphorylation of p53 after 0.5 h post-irradiation. At the time of phosphorylation of p53, we found simultaneous phosphorylation of the oncoprotein Mdm2 on Ser-166. Neither ATM nor its downstream targets showed a dose-dependent response after 1 h when irradiated by the doses of 1-10 Gy. MOLT-4 cells were very sensitive to the effect of IR. Even low doses, such as 1.5 Gy, induced apoptosis 16 h after irradiation (evaluated according to the cleavage of nuclear lamin B to a 48-kDa fragment). IR-induced molecular signalling after exposure to all the tested doses was triggered by rapid phosphorylation of ATM and Chk-2. Subsequent induction of p53 protein and its phosphorylation was accompanied by concomitant phosphorylation of its negative regulator, oncoprotein Mdm2, and followed by induction of apoptosis.     

The dynamics of the rats higher nervous activity disturbances after total influence of electrons and gamma-rays in doses 5-100 Gy

The dynamics of using of stabilized motor defensive conditioned reflex of active avoidance in "shuttle-box" in rats after total influence of high energy electrons and of gamma-rays in doses 5-100 Gy were investigated. The quality structure of higher nervous activity disturbances after the influence of these kinds of ionizing radiation was identical. Therefore the tendency to disturbances aggravating after the electron radiation influence in the periods of the initial depression and of relatively normalization was revealed, especially after the irradiation in dose 50 Gy. The effective compensation of the functional disturbances in the central nervous system at the first 5-10 min after irradiation was after influence of electron radiation in doses about 30 Gy and after the influence of gamma-radiation in doses about 50 Gy. The irradiation of rats in doses 10 Gy and 5 Gy caused qualitative different dynamics of radiation disturbances in rats higher nervous activity. The differences in rats higher nervous activity after influence of electron and of gamma-radiation in these doses did not manifest distinctly.     

Low-dose-rate radiation attenuates the response of the tumor suppressor TP53

Acute low-dose irradiation (0.1-1 Gy, 1.33 Gy/min) of cells of a human glioblastoma cell line, A-172, induced a dose-dependent monophasic accumulation of TP53 (formerly known as p53) and CDKN1A (formerly known as WAF1). In contrast, chronic gamma irradiation (0.001 Gy/min) produced a clear biphasic response of accumulation TP53 with the first peak at 1.5 h (0.09 Gy) and the second peak at 10 h (0.54 Gy). Significantly, when the cells were preirradiated with a chronic dose of gamma irradiation for 24 h (1.44 Gy) or 50 h (3 Gy), they no longer responded to an acute challenging dose to produce a dose-dependent response of the TP53 pathway. These findings suggest that chronic irradiation at low dose rate alters the TP53-dependent signal transduction pathway. Wearing away of the TP53 pathway by chronic exposure to radiation may have important implications for radiation protection.     

The Influence of Ionizing Radiation on Proneness to Audiogenic Seizure and Behavior in Krushinsky–Molodkina Rats

Biophysics - We studied the effects of whole body gamma-radiation (4 Gy, dose rate 0.6 Gy/min) and proton-beam head irradiation (with an energy of 150 meV, 4 Gy and a dose rate of 0.8 Gy/min) on...     

Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability

Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express about 50 gammaH2AX foci per cell (i.e., equivalent to the number of breaks produced by about 2Gy) showed similar numbers of dsbs as C33A cells that exhibit fewer than three foci per cell. The possibility that differences in numbers of these endogenous foci could be explained by genomic instability perhaps related to misrepair was examined. For 17cell lines selected from the panel of NCI-60 tumor cells previously characterized for karyotypic complexity [A.V. Roschke, G. Tonon, K.S. Gehlhaus, N. McTyre, K.J. Bussey, S. Lababidi, D.A. Scudiero, J.N. Weinstein, I.R. Kirsch, Karyotypic complexity of the NCI-60 drug-screening panel, Cancer Res. 63 (2003) 8634-8647], there was a significant trend (r=0.6) for cell lines with greater numbers of structural or numerical chromosomal rearrangements to show a higher background expression of gammaH2AX. Moreover, cells from this panel with wild-type p53 showed a significantly lower background level of gammaH2AX than cells with mutant p53. To confirm the importance of p53 expression, endogenous and radiation-induced gammaH2AX expression were analyzed using four isogenic SKOV3 cell lines varying in p53 function. Again, higher gammaH2AX expression was found in SKOV3 cell lines expressing mutant p53 compared to wild-type p53. HFL-1 primary lung fibroblasts showed a progressive increase in gammaH2AX as they moved towards senescence, confirming the importance of telomere instability in the development of at least some gammaH2AX foci. Therefore, the explanation for high endogenous levels of gammaH2AX in some tumor cells appears to be multifactorial and may be best described as a consequence of chromatin instability.     

Effect of preliminary chronic irradiation and alpha-tocopherol on the frequency of chromosome aberrations in mouse bone marrow cells, induced by exposure to acute gamma-irradiation

The incidence of chromosome aberrations in bone marrow cells of femur did not exceed the spontaneous one in CBA mice exposed, during 70 days, to gamma-radiation at dose--rates of 33.7-35.8 nA/kg and cumulative dose of 2.75 Gy. A single acute exposure of intact animals to a dose of 2.98 Gy increased significantly the mutation level. Preirradiation with small doses increased the resistance of hereditary structures to sublethal radiation doses. Exogenous alpha-tocopherol (0.06 mg/20 g mass) protected the genetic apparatus of cells from total-body irradiation and was an additional factor decreasing the mutation level after acute exposure of mice at the background of long-term irradiation with small doses.     

DNA double-strand breaks in human lymphocytes after single irradiation by low doses of pulsed X-rays: non-linear dose-response relationship

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. A pulsed mode of irradiation is even more problematic in terms of predicting the radiation-induced response in cells. Thus, the aim of this paper was to study and analyze the effects of dose and frequency of pulsed X-rays on the frequency of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes in vitro. Analysis of radiation-induced gammaH2AX and 53BP1 repair foci was used to assess the DNA damage in these cells. The dose-response curve of radiation-induced foci of both proteins has shown deviations from linearity to a higher effect in the 12-32 mGy dose range and a lower effect at 72 mGy. The dose-response curve was linear at doses higher than 100 mGy. The number of radiation-induced gammaH2AX and 53BP1 foci depended on the frequency of X-ray pulses: the highest effect was registered at 13 pulses per second. Moreover, slower repair kinetics was observed for those foci induced by very low doses with a nonlinear dose-response relationship.     

Effect of somatostatin on repair of ionizing radiation-induced DNA damage in pituitary adenoma cells GH3

Ionizing radiation and somatostatin analogues are used for acromegaly treatment to achieve normalization or reduction of growth hormone hypersecretion and tumor shrinkage. In this study, we investigated a combination of somatostatin (SS14) with ionizing radiation of (60)Co and its effect on reparation of radiation-induced damage and cell death of somatomammotroph pituitary cells GH3. Doses of gamma-radiation 20-50 Gy were shown to inhibit proliferation and induce apoptosis in GH3 cells regardless of somatostatin presence. It has been found that the D(0) value for GH3 cells was 2.5 Gy. Somatostatin treatment increased radiosensitivity of GH3 cells, so that D(0) value decreased to 2.2 Gy. We detected quick phosphorylation of histone H2A.X upon irradiation by the dose 20 Gy and its colocalization with phosphorylated protein Nbs-1 in the site of double strand break of DNA (DSB). Number of DSB decreased significantly 24 h after irradiation, however, clearly distinguished foci persisted, indicating non repaired DSB, after irradiation alone or after combined treatment by irradiation and SS14. We found that SS14 alone triggers phosphorylation of Nbs1 (p-Nbs1), which correlates with antiproliferative effect of SS14. Irradiation also increased the presence of p-Nbs1. Most intensive phosphorylation of Nbs1 was detected after combined treatment of irradiation and SS14. The decrease of the number of the DSB foci 24 h after treatment shows a significant capacity of repair systems of GH3 cells. In spite of this, large number of unrepaired DSB persists for 24 h after the treatment. We conclude that SS14 does not have a radioprotective effect on somatomammotroph GH3 cells.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions

Exposure of cells to hypertonic medium after X-irradiation results in a 3-4-fold increase in the phosphorylation of histone H2AX (gammaH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3-4-fold increase in H2AX phosphorylation in the presence of 0.5M Na(+). However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5M Na(+). Instead, 0.5M Na(+) caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the gammaH2AX foci.     

Structural and functional changes in various cells following radiation exposure]

Structural and functional changes in thymocytes, splenocytes and hepatocytes, taking place under gamma-irradiation of cellular suspensions were investigated. It was found that splenocytes begin to lose their functional activity and structural features at a dose of 2 Gy. Increasing the irradiation dose leads to the acceleration of these processes. Thymocytes lose rapidly their functional activity at 2 Gy, but they maintain their structure and membrane parameters even after irradiation with a dose of 10 Gy. In comparison with other cell types, hepatocytes exhibit the greatest stability under the influence of gamma-radiation.     

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.     

p53 mutations in tumors derived from irradiated human thyroid epithelial cells

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.