Induced repair of DNA double-strand breaks at the G1/S-phase border

Exposure of human cells to ionizing radiation at the G1/S-phase border of the cell cycle leads to the production of repair patches of 3 nucleotides, representing the constitutive repair response, and very long repair patches (VLRP) of at least 150 nucleotides, representing an induced response. We examined the type of DNA damage that may signal this induced repair response using two chemicals that produce subsets of the damage induced by ionizing radiation. Treatment of cells at the G1/S-phase border with bleomycin, which produces a high proportion of DNA double-strand breaks, also leads to the production of VLRP of at least 130 nucleotides. In contrast, when cells were treated with hydrogen peroxide, which produces base modifications and single-strand breaks, no VLRP were observed. Thus it would appear that DNA double-strand breaks are the signal that leads to the induction of the VLRP. We also examined the relationship between the induced repair response and DNA replication. When cells are treated with hydroxyurea, under conditions that inhibit more than 98% of the DNA synthesis, prior to exposure to 5 Gy, repair patches of 3 and 150 nucleotides are found. This indicates that the longer repair patches are not a result of aberrant DNA replication. However, when cells are treated with the DNA polymerase inhibitor aphidicolin in combination with hydroxyurea and cytosine arabinoside, no induced long patches are found. These results indicate that DNA polymerase alpha, delta or epsilon is required for the synthesis of the VLRP.     

X radiation causes a persistent induction of reactive oxygen species and a delayed reinduction of TP53 in normal human diploid fibroblasts

Multiple genetic changes are required for the development of a malignant cell. The frequency of such changes in cancer cells is higher than can be explained through random mutation, and it was proposed that a subpopulation of cells develop a persistent mutator phenotype. Evidence for such a phenotype has been observed in mammalian cells after treatment with ionizing radiation. The mechanism that promotes this effect has not been defined, but proposed explanations include increased levels of reactive oxygen species (ROS) in irradiated cells and their progeny. The tumor suppressor TP53 is of prime importance in coordinating the cellular response to damage, and it has been suggested to have a role in regulating the cellular redox state. We investigated the persistence of induced levels of ROS in normal diploid human cells for 1 month after X-ray exposure and the role of TP53 in this oxidant response. X radiation induced an oxidant response that persisted for 2 weeks after exposure in cells with normal TP53 function. ROS levels in cells with abrogated TP53 function were decreased in magnitude and duration. X radiation caused a primary transient induction of TP53 followed by a reinduction of TP53 5 days after irradiation. This reinduction persisted for at least 2 days and coincided with the largest induction of apoptosis. The persistently elevated levels of ROS and delayed reinduction of TP53 reported here are further evidence of the delayed effects of ionizing radiation and add to the growing number of such observations.     

Enhancement of radiation cytotoxicity by UCN-01 in non-small cell lung carcinoma cells

Thoracic ionizing radiation is a standard component of combined-modality therapy for locally advanced non-small cell lung cancer. To improve low 5-year survival rates (5- 15%), new strategies for enhancing the effectiveness of ionizing radiation are needed. The kinase inhibitor UCN-01 has multiple cell cycle effects, including abrogation of DNA damage-induced S- and G(2)-phase arrest, which may limit DNA repair prior to mitosis. To test the hypothesis that therapy-induced cell cycle effects would have an impact on the efficacy of a combination of UCN-01 plus ionizing radiation, the cell cycle responses of the non-small cell lung cancer cell lines Calu1 (TP53-null) and A549 (wild-type TP53) to 2 Gy ionizing radiation were correlated with clonogenic survival after irradiation plus UCN-01. Irradiated cells were exposed to UCN-01 simultaneously and at 3-h increments after irradiation. In Calu1 cells but not A549 cells, sequence-dependent potentiation of radiation by UCN-01 was observed, with maximal interaction occurring when UCN-01 was administered 6 h after irradiation. This coincided with the postirradiation time with the greatest depletion of cells from G(1). Abrogation of G(2) arrest was observed regardless of TP53 status. The role of TP53 was investigated using siRNA to achieve gene silencing. These studies demonstrated that radiation plus UCN-01 was more effective in cells with diminished TP53 activity, associated with a reduced G(1) checkpoint arrest. These studies indicate that simultaneous elimination of multiple DNA damage-induced checkpoints in G(1), S and G(2) may enhance the effects of radiation and that drug scheduling may have an impact on clinical efficacy.     

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

Wortmannin增强细胞辐射敏感性的分子机制

 Ｗ ｏｒｔｍ ａｎｎｉｎ（ Ｗ Ｏ Ｒ Ｔ）是 Ｐ Ｉ３ Ｋ 家族激酶特异抑制剂,对 ｐ５３ 野生型及突变型细胞的辐射敏感性均有提高．为了阐明 Ｗ Ｏ Ｒ Ｔ 的辐射增敏机制,通过免疫沉淀及免疫印迹法分析了 Ｗ Ｏ Ｒ Ｔ对辐射引起的细胞 Ｇ２／ Ｍ 转换中关键分子 ｃｄｃ２ 酪氨酸脱磷酸化延迟的影响;通过流式细胞术分析了 Ｗ Ｏ Ｒ Ｔ 对辐射引起的细胞 Ｇ２ 期延迟、细胞凋亡的影响;经报告基因转染的方法分析了 Ｗ Ｏ Ｒ Ｔ对宿主细胞对辐射损伤报告基因修复的影响;发现 Ｗ Ｏ Ｒ Ｔ 可促进受照细胞 ｃｄｃ２ 酪氨酸脱磷酸化、减弱辐射引起的细胞 Ｇ２ 期延迟、增强细胞凋亡并抑制损伤 Ｄ Ｎ Ａ 修复．提示 Ｗ Ｏ Ｒ Ｔ 辐射增敏是通过干扰细胞 Ｇ２ 期检查点调控、抑制损伤 Ｄ Ｎ Ａ 修复和促进细胞凋亡等多种途径实现的．     

Induction of telomerase activity by irradiation in human lymphoblasts

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693-697 (2001). Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres (+1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.     

Epidermal growth factor and ionizing radiation up-regulate the DNA repair genes XRCC1 and ERCC1 in DU145 and LNCaP prostate carcinoma through MAPK signaling

This work examined the importance of radiation-induced and ligand-induced EGFR-ERK signaling for the regulation of DNA repair proteins XRCC1 and ERCC1 in prostate carcinoma cells, DU145 (TP53(mut)), displaying EGFR-TGFA-dependent autocrine growth and high MAPK (ERK1/2) activity, and LNCaP (TP53(wt)) cells expressing low constitutive levels of ERK1/2 activity. Using quantitative RT-PCR and Western analyses, we determined that ionizing radiation activated the DNA repair genes XRCC1 and ERCC1 in an ERK1/2-dependent fashion for each cell line. After irradiation, a rapid increase followed by a decrease in ERK1/2 activity preceded the increase in XRCC1/ERCC1 expression in DU145 cells, while only the rapid decrease in ERK1/2 preceded the increase in XRCC1/ERCC1 expression in LNCaP cells. Administration of EGF, however, markedly increased the up-regulation of phospho-ERK, ERCC1 and XRCC1 in both cell lines. Although the EGFR inhibitor tyrphostin (AG-1478) and the MEK inhibitor PD90859 both attenuated EGF-induced levels of the ERCC1 and XRCC1 protein, PD98059 blocked the induction of ERCC1 and XRCC1 by radiation more effectively in both cell lines. Inhibition of ERK at a level that reduced the up-regulation of DNA repair led to the persistence of apurinic/apyrimidinic (AP) sites of DNA damage and increased cell killing. Taken together, these data imply a complex control of DNA repair activation that may be more generally dependent on MAPK (ERK1/2) signaling than was previously noted. These data provide novel insights into the capacity of the EGFR-ERK signaling to modulate DNA repair in cancer cells and into the functional significance of this signaling.     

Parthenolide sensitizes cells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.     

The level of induced DNA double-strand breaks does not correlate with cell killing in X-irradiated mitotic and G1-phase CHO cells

The neutral (pH 9.6) filter elution technique was used to evaluate DNA damage induced in CHO cells irradiated at mitosis or in G1-phase under various incubation and postirradiation treatment conditions. Mitotic and G1/S border cells were more sensitive to radiation than G1 cells with respect to cell killing, but showed similar (G1/S) or lower (M) DNA elution dose--response curves. Similar cell survival and DNA/elution dose--response curves were obtained with plateau-phase cultures containing mainly G1-cells, as well as with G1 cells obtained after division of mitotic cells in either fresh or conditioned medium. However, survival of plateau-phase cells could be modified substantially by delayed-plating or postirradiation treatment with araA. These results, together with previously published observations, indicate that induction of DNA dsb cannot be invoked as an explanation for the variations in radiosensitivity observed through the cycle, or as an explanation for the formation of the survival curve shoulder. It is proposed that repair and fixation of radiation-induced DNA damage, expressed at the cell survival level as repair and fixation of alpha-PLD, are responsible for these effects.     

Poly(ADP-ribose) polymerase-1 is a positive regulator of the p53-mediated G1 arrest response following ionizing radiation

Poly(ADP-ribose) polymerase-1 (PARP-1) and the p53 tumor suppressor protein are both involved in the cellular response to genotoxic stress. Upon binding to the site of DNA strand breakage, PARP-1 is activated, leading to rapid and transient poly(ADP-ribosyl)ation of nuclear proteins using NAD+ as substrate. To investigate the role of PARP-1 in the p53 response to ionizing radiation in human cells, PARP-1 function was disrupted in wild-type p53 expressing MCF-7 and BJ/TERT cells using two strategies: chemical inhibition with 1,5-dihydroxyisoquinoline, and trans-dominant inhibition by overexpression of the PARP-1 DNA-binding domain. Although a number of proteins can catalyze poly(ADP-ribosyl)ation in addition to PARP-1, we show that PARP-1 is the only detectable active species in BJ/TERT and MCF-7 cells. 1,5-Dihydroxyisoquinoline treatment prior to ionizing radiation delayed and attenuated the induction of two p53-responsive genes, p21 and mdm-2, and led to suppression of the p53-mediated G1-arrest response in MCF-7 and BJ/TERT cells. Trans-dominant inhibition of PARP-1 by overexpression of the PARP-1 DNA-binding domain in MCF-7 cells also led to a delay and attenuation in p21 induction and suppression of the p53-mediated G1 arrest response to ionizing radiation. Hence, inhibition of endogenous PARP-1 function suppresses the transactivation function of p53 in response to ionizing radiation. This study establishes PARP-1 as a critical regulator of the p53 response to DNA damage.     

DNA lesions that signal the induction of radioresistance and DNA repair in yeast

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.     

Suppression of apoptosis and clonogenic survival in irradiated human lymphoblasts with different TP53 status

The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G2/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.     

Sister chromatid exchanges occur in G2-irradiated cells

DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or mis-repaired DSBs can lead to chromosomal aberrations and cell death. The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. Following S-phase-dependent genotoxins, HR events give rise to sister chromatid exchanges (SCEs), which can be detected cytogenetically in mitosis. Here, we describe that HR occurring in G2-irradiated cells also generates SCEs in ~50% of HR events. Since HR of IR-induced DSBs in G2 is a slow process, SCE formation in G2-irradiated cells requires several hours. During this time, irradiated S-phase cells can also reach mitosis, which has contributed to the widely held belief that SCEs form only during S phase. We describe procedures to measure SCEs exclusively in G2-irradiated cells and provide evidence that following IR cells do not need to progress through S phase in order to form SCEs.     

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.     

Cidofovir administered with radiation displays an antiangiogenic effect mediated by E6 inhibition and subsequent TP53-dependent VEGF repression in HPV18+ cell lines

Therapeutic administration of the antiviral agent cidofovir with radiation markedly enhanced the antitumor effect of ionizing radiation in cells of two HPV18+ human cervical carcinoma cell lines. Although this potent radiosensitizing effect was associated with repression of the viral oncoproteins E6/ E7 and restoration of TP53 as shown previously, additional mechanisms may be involved. In the present study, we investigated the antiangiogenic effect of the combination of cidofovir and radiation in cells of two HPV18+ cervical cancer cell lines, HeLa and ME180, and assessed the molecular mechanisms associated with the antiangiogenic effect observed. Cells were exposed to cidofovir (10 microg/ml) and irradiated (1-9 Gy). The angiogenic response was studied in vitro by a matrigel invasion assay. Modulations of E6, TP53 and VEGF mRNA and protein levels were studied by real-time RT-PCR, Western blot analysis and ELISA, respectively. Then a double RNA interference approach was used to analyze the connection between E6/TP53 and VEGF. The combination of cidofovir and radiation had a potent antiangiogenic effect. It induced E6 inhibition, restoration of TP53, and reduction of the proangiogenic phenotype of HPV18+ cells associated with VEGF inhibition. A siRNA strategy showed an anti-VEGF action of the combination mediated directly by E6 inhibition and TP53 restoration, since E6 siRNA inhibited VEGF whereas co-transfection with E6 and TP53 siRNA abrogated the anti-VEGF effect. This study showed that the combination of cidofovir with ionizing radiation has an antiangiogenic effect associated with VEGF inhibition subsequent to E6 inhibition and TP53 restoration.     

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H2O2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.     

Ionizing radiation enhances the expression of the nonsteroidal anti-inflammatory drug-activated gene (NAG1) by increasing the expression of TP53 in human colon cancer cells

The induction of apoptosis in cells of human colon cancer cell lines after gamma irradiation was investigated to determine whether apoptosis was mediated by TP53 and the subsequent expression of its downstream target, the NSAID-activated gene (NAG1). HCT116 (TP53(+/+)), HCT15 (TP53 mutant) and TP53 null HCT116 (TP53(-/-)) cells were irradiated with gamma rays, and apoptosis was measured at various times after irradiation. In HCT116 TP53(+/+) cells, apoptosis was increased after irradiation; the increase was dependent on the time after treatment and the dose of gamma rays. However, in HCT15 TP53 mutant cells and HCT116 TP53(-/-) cells, there were no remarkable changes in apoptosis. The expression of TP53 protein in HCT116 cells was increased after irradiation and was followed by an increase in the expression of NAG1 protein. In contrast, the expression of NAG1 protein in TP53 mutant cells and TP53(-/-) cells was not increased by the radiation treatment, suggesting that NAG1 was required for apoptosis. The expression of NAG1 increased apoptosis in HCT116 cells, but radiation treatment did not further increase apoptosis. The transfection of a NAG1 siRNA into HCT116 cells suppressed radiation-induced apoptosis and inhibited the induction of NAG1 protein without altering the expression of TP53. a NAG1 luciferase promoter construct that included both of the TP53 binding sites, was activated by radiation in dose-dependent manner, while the promoters lacking one or both of the TP53 binding sites in the NAG1 promoter activity either was less responsive or did not respond. The findings reported here indicate that gamma radiation activates the TP53 tumor suppressor, which then increases the expression of NAG1. NAG1 mediates the induction of apoptosis in human colorectal cells.     

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.