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Int J Radiat Oncol Biol Phys. 2012 Feb 28. [Epub ahead of print]
Inhibiting the Aurora B Kinase Potently Suppresses Repopulation During Fractionated Irradiation of Human Lung Cancer Cell Lines.

Sak A, Stuschke M, Groneberg M, Kübler D, Pöttgen C, Eberhardt WE.
Department of Radiotherapy, West German Cancer Centre (WTZ), University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Abstract
PURPOSE:
The use of molecular-targeted agents during radiotherapy of non-small-cell lung cancer (NSCLC) is a promising strategy to inhibit repopulation, thereby improving therapeutic outcome. We assessed the combined effectiveness of inhibiting Aurora B kinase and irradiation on human NSCLC cell lines in vitro.
METHODS AND MATERIALS:
NSCLC cell lines were exposed to concentrations of AZD1152-hydroxyquinazoline pyrazol anilide (AZD1152-HQPA) inhibiting colony formation by 50% (IC50(clone)) in combination with single dose irradiation or different fractionation schedules using multiple 2-Gy fractions per day up to total doses of 4-40 Gy. The total irradiation dose required to control growth of 50% of the plaque monolayers (TCD50) was determined. Apoptosis, G2/M progression, and polyploidization were also analyzed.
RESULTS:
TCD50 values after single dose irradiation were similar for the H460 and H661 cell lines with 11.4 ± 0.2 Gy and 10.7 ± 0.3 Gy, respectively. Fractionated irradiation using 3 × 2 Gy/day, 2 × 2 Gy/day, and 1 × 2 Gy/day schedules significantly increased TCD50 values for both cell lines grown as plaque monolayers with increasing radiation treatment time. This could be explained by a repopulation effect per day that counteracts 75 ± 8% and 27 ± 6% of the effect of a 2-Gy fraction in H460 and H661 cells, respectively. AZD1152-HQPA treatment concomitant to radiotherapy significantly decreased the daily repopulation effect (H460: 28 ± 5%, H661: 10 ± 4% of a 2-Gy fraction per day). Treatment with IC50(clone) AZD1152-HPQA did not induce apoptosis, prolong radiation-induced G2 arrest, or delay cell cycle progression before the spindle check point. However, polyploidization was detected, especially in cell lines without functional p53.
CONCLUSIONS:
Inhibition of Aurora B kinase with low AZD1152-HQPA concentrations during irradiation of NSCLC cell lines affects repopulation during radiotherapy. Thus, concomitant Aurora B kinase inhibition and irradiation may be a promising strategy for fast repopulating tumors, which are difficult to cure by dose escalation based on conventional fractionation.




Eur J Haematol. 2011 Jul;87(1):10-9. doi: 10.1111/j.1600-0609.2011.01626.x.
Investigating the role of CD38 and functionally related molecular risk factors in the CLL NOD/SCID xenograft model.
Aydin S, Grabellus F, Eisele L, Möllmann M, Hanoun M, Ebeling P, Moritz T, Carpinteiro A, Nückel H, Sak A, Göthert JR, Dührsen U, Dürig J.
Department of Hematology, University of Duisburg-Essen Medical School, Essen, Germany.
Abstract
We explored the role of CD38 and functionally associated molecular risk factors in a recently described chronic lymphocytic leukemia (CLL) nonobese diabetic/ severe combined immunodeficient xenograft model. Intravenous injection of peripheral blood mononuclear cells from 73 patients with CLL into 244 mice resulted in robust engraftment of leukemic cells into the murine spleens detected 4 wks after transplantation. Leukemic cell engraftment correlated significantly (P < 0.05) with markers reflecting disease activity, e.g., Binet stage and lymphocyte doubling time, and the expression of molecular risk factors including CD38, CD49d, ZAP-70, and IgVH mutational status. Increased engraftment levels of CD38+ as compared to CD38- CLL cells could be attributed, in part, to leukemic cell proliferation as evidenced by combined immunostaining of murine spleen sections for Ki-67 and CD20. In short-term (24 h) homing assays, CD38+ CLL cells migrated more efficiently to the bone marrow of the recipient animals than their CD38- counterparts. Finally, CD38 expression by the leukemic cells was found to be dynamic in that it was regulated not only by elements of the murine microenvironment but also by co-engrafting non-malignant human T cells. This model could be useful for evaluating the biological basis of CLL growth in the context of the hematopoietic microenvironment as well as preclinical testing of novel compounds.




Semin Radiat Oncol. 2010 Oct;20(4):223-31.

Use of γH2AX and other biomarkers of double-strand breaks during radiotherapy.

Sak A, Stuschke M.

Department of Radiotherapy, University Hospital Essen, Essen, Germany. ali.sak@unidue.de

Abstract

The rapid phosphorylation of histone H2AX at serine 139 (γH2AX) serves as a sensitive marker for DNA double-strand breaks induced by ionizing radiation or other genotoxic agents. The potential clinical applications of γH2AX detection in tissues from cancer patients during fractionated radiotherapy and the sensitivity for detection of in vivo drug effects on radiation-induced DNA damage responses (DDRs) are discussed. The quantification of γH2AX foci in the nuclei of peripheral blood lymphocytes allows estimation of the applied integral body dose by conformal radiotherapy to tumors in different sites of the body. The limits of precision of biodosimetry in peripheral blood lymphocytes with a γH2AX assay shortly after radiation exposure are shown. The high sensitivity of the in vitro radiation dose-γH2AX foci response allows monitoring of drug effects on DDR pathways after in vivo drug exposure and in vitro irradiation. Drugs are under clinical investigation that modify radiation-induced damage response. If interindividual or intertumoral differences in drug sensitivity exist, the measurement of radiation-induced foci formation and resolution after in vivo drug exposure and in vitro or in vivo irradiation of a cellular probe can serve as a functional assay that may predict the individual gain of a combination therapy. Validation by prospective studies is needed.




Clin Cancer Res.

Long-term in vivo effects of cisplatin on gamma-H2AX foci signaling in peripheral lymphocytes of tumor patients after irradiation.

Sak A, Grehl S, Engelhard M, Wierlemann A, Kaelberlah HP, Erichsen P, Pöttgen C, Groneberg M, Stuschke M.

Department of Radiotherapy, University Hospital Essen, Essen, Germany.

PURPOSE: This study determined the effects of cis-diamminedichloroplatinum(II) on radiation-induced foci formation of gamma-H2AX and Rad51 in lymphocytes. EXPERIMENTAL DESIGN: Twenty-eight cancer patients were irradiated for intrathoracic, pelvic, or head and neck tumors and received simultaneous cisplatin containing chemotherapy. The effect of cisplatin on radiation-induced gamma-H2AX and Rad51 foci as a response to ionizing radiation-induced DNA double-strand breaks was measured in lymphocytes after in vivo and in vitro radiochemotherapy. The role of DNA-dependent protein kinase and ataxia-telangiectasia mutated kinase in gamma-H2AX signaling, the consequences of altered gamma-H2AX foci formation on double-strand break end joining, was studied. RESULTS: Cisplatin decreased the number of induced gamma-H2AX foci in lymphocytes after in vivo or in vitro irradiation by 34% +/- 6% at days 0 to 3 after cisplatin (P < 0.0001) and remained significant until day 6. The variation in this cisplatin effect from patient to patient was larger than the retest error within the same patient (P = 0.01). The cisplatin effect was not accompanied by an inhibition of end joining of double-strand break as analyzed using gel electrophoresis of DNA under neutral conditions. Cisplatin also decreased radiation induced Rad51 foci formation in lymphocytes after stimulation of proliferation with phytohemagglutinin by 47% +/- 6% (P < 0.0001). CONCLUSION: Cisplatin has long-term effects on the early double-strand break response of gamma-H2AX and Rad51 foci formation after ionizing radiation. Inhibition of sensing and processing of double-strand break by gamma-H2AX and Rad51 foci formation are important mechanisms by which cisplatin can alter the radiation response.

 

Mol Cancer Ther. 2008 Jul;7(7):1772-81.

Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair.

Toulany M, Kehlbach R, Florczak U, Sak A, Wang S, Chen J, Lobrich M, Rodemann HP.

Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Eberhard-Karls University Tuebingen, Roentgenweg 11, 72076 Tuebingen, Germany.

We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59 CJ-OH (API; 1-5 micromol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G(1) cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the gamma-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.

 

 Cell Prolif. 2008 Aug;41(4):660-70. Epub 2008 Jun 19.

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines.

Sak A, Fegers I, Groneberg M, Stuschke M.

Department of Radiotherapy, University Hospital Essen, Essen, Germany. ali.sak@uni-due.de

OBJECTIVES: This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G(2)/M phases and consecutive cell survival. MATERIALS AND METHODS: Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. RESULTS: By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G(2) phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G(2) accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly (P < 0.01) increased the fraction of radiation-induced G(2) arrested cells 30-56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by gamma-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. CONCLUSIONS: These results indicate that separase has additional targets involved in regulation of G(2) to M progression after DNA damage. Prolonged G(2) phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.

 

Int J Radiat Biol. 2007 Oct;83(10):639-52.

gamma-H2AX foci formation in peripheral blood lymphocytes of tumor patients after local radiotherapy to different sites of the body: dependence on the dose-distribution, irradiated site and time from start of treatment.

Sak A, Grehl S, Erichsen P, Engelhard M, Grannass A, Levegrün S, Pöttgen C, Groneberg M, Stuschke M.

Department of Radiotherapy, University Hospital Essen, Essen, Germany. ali.sak@uni-duisburg-essen.de

PURPOSE: To evaluate the relationship between an estimated integral total body radiation dose delivered and phosphorylated histone H2AX protein (gamma-H2AX) foci formation in peripheral blood lymphocytes of cancer patients. MATERIAL AND METHODS: gamma-H2AX formation was quantified as the mean number of foci per lymphocyte (N(meanH2AX)) and the percentage of lymphocytes with > or =n foci. The integrated total body radiation dose was estimated from the dose volume histogram of patient's body corrected for the proportion of the body scanned by computed tomography for 3D treatment planning. RESULTS: There was a strong linear correlation between the mean number of gamma-H2AX foci per lymphocyte in the peripheral blood sample and integrated total body radiation dose (r = 0.83, p < 0.0001). The slope of the relationship was dependent on the site of body irradiated. In comparison to chest irradiation with a slope of 8.7 +/- 0.8 foci Gy(-1), the slopes for brain, upper leg and pelvic sites were significantly shallower by -4.7, -4.3, and -3.8 Gy(-1), respectively (p < 0.0001), while the slope for upper abdomen irradiation was significantly larger by 9.1 +/- 2.6 Gy(-1) (p = 0.0007). There was a slight time effect since the start of radiotherapy on the slopes of the in vivo dose responses leading to shallower slopes (-1.5 +/- 0.7 Gy(-1), p = 0.03) later (> or =10 day) during radiotherapy. After in vitro irradiation, lymphocytes showed 10.41 +/- 0.12 foci per Gy with no evidence of inter-individual heterogeneity. CONCLUSIONS: gamma-H2AX measurements in peripheral lymphocytes after local radiotherapy allow the estimation of the applied integral body dose. The site and time dependence have to be considered.

 

 Br J Cancer. 2005 Mar 28;92(6):1089-97.

Targeting of Rad51-dependent homologous recombination: implications for the radiation sensitivity of human lung cancer cell lines.

Sak A, Stueben G, Groneberg M, Böcker W, Stuschke M.

Department of Radiotherapy, University Hospital Essen, 45122 Essen, Germany. ali.sak@uni-essen.de

The aim of the present work was to study the role of Rad51-dependent homologous recombination in the radiation response of non-small-cell lung cancer (NSCLC) cell lines. A dose- and time-dependent increase in the formation of Rad51 and gamma-H2AX foci with a maximum at about 4 and 1 h after irradiation, followed by a decrease, has been found. The relative fraction of cells with persisting Rad51 foci was 20-30% in radioresistant and 60-80% in radiosensitive cell lines. In comparison, a higher fraction of residual Dsb was evident in cell lines with nonfunctional p53. Transfection with As-Rad51 significantly downregulates radiation-induced formation of Rad51 foci and increases apoptosis, but did not influence the rejoining of DNA double-strand breaks. Interestingly, wortmannin, a well-known inhibitor of nonhomologous end-joining, also inhibits Rad51 foci formation. In general, there was no correlation between the clonogenic survival at 2 Gy and the percentage of initial Rad51 or gamma-H2AX foci after ionising radiation (IR). The most reliable predictive factor for radiosensitivity of NSCLC cell lines was the relative fraction of Rad51 foci remaining at 24 h after IR. Although most of the Rad51 foci are co-localised with gamma-H2AX foci, no correlation of the relative fraction of persisting gamma-H2AX foci and SF2 is evident.

 

Cancer Gene Ther. 2003 Dec;10(12):926-34.

Increased radiation-induced apoptosis and altered cell cycle progression of human lung cancer cell lines by antisense oligodeoxynucleotides targeting p53 and p21(WAF1/CIP1).

Sak A, Wurm R, Elo B, Grehl S, Pöttgen C, Stüben G, Sinn B, Wolf G, Budach V, Stuschke M.

Department of Radiotherapy, University Essen, 45122 Essen, Germany. ali.sak@uni-essen.de

Lung cancer is difficult to control locally by radiotherapy and is known to have frequently p53 mutations. Previous results have shown that non-small-cell lung cancer (NSCLC) cell lines with nonfunctional p53 have a higher fraction of radiation-induced apoptosis and that apoptosis follows after the release from the G2/M arrest. The aim of the present work was to study whether inhibition of the p53 response in NSCLC cell lines can modulate the G2/M arrest and the induction of apoptosis after ionizing radiation. Antisense oligodeoxynucleotides (As-ODNs) were used to inhibit the p53 response in the cell lines H460 and A549 with functional p53. In addition, H661 with nonfunctional p53 was used. The results have shown that As-ODNs targeting mRNA of p53 and p21 downregulate radiation-induced expression of p53 and p21(WAF1/CIP1). Delayed apoptosis (35.7+/-4.2% in H460, 1.2+/-0.4% in A549 and 72.2+/-6.5% in H661) was observed after cell cycle progression beyond the G2 block, either in the late G2 phase of the same cell cycle being irradiated (H661) or in the G1 phase of the subsequent cell cycle (H460, A549). As-p53 significantly decreased the fraction of G2/M-arrested cells in H460 cells and increased radiation-induced apoptosis at 96 hours by 17.9+/-8.5 and 9.1+/-3.3% to 53.6+/-7.4 and 10.8+/-2.9% in H460 and A549 cells (P<.01), respectively, but had no effect in H661 cells with nonfunctional p53. In addition, As-p21 decreased the fraction of G2-arrested A549 and H460 cells and increased apoptosis by 23.8+/-5.2 and 31.6+/-7.3% to 59.4+/-3.1 and 32.8+/-7.3%, respectively (P<.01). In conclusion, these data show that radiation-induced G2 arrest is decreased in NSCLC cells and radiation-induced apoptosis is increased when p53-responsive pathways are blocked via As-ODN targeting p53 or p21(WAF1/CIP1) mRNA. In view of the fact that p53 and p21 As-ODN had similar effects on radiation-induced apoptosis normalized by their ability to inhibit radiation-induced p21 expression, we concluded that p21 is an important trigger of late ionizing radiation-induced apoptosis.

  

Cancer Res. 2002 Nov 15;62(22):6621-4.

Selective inactivation of DNA-dependent protein kinase with antisense oligodeoxynucleotides: consequences for the rejoining of radiation-induced DNA double-strand breaks and radiosensitivity of human cancer cell lines.

Sak A, Stuschke M, Wurm R, Schroeder G, Sinn B, Wolf G, Budach V.

Department of Radiotherapy, University Essen, 45122 Essen, Germany. ali.sak@uni-essen.de

The inhibition of DNA-dependent protein kinase activity with antisense-oligodeoxynucleotide (As-ODN) and its consequences for the rejoining of DNA-double-strand breaks (Dsbs) and radiation sensitivity was studied in human non-small cell lung cancer (NSCLC) cell lines. Cells were transfected with As-ODNs specific for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In comparison, cells were treated with Wortmannin, a potent but nonspecific inhibitor of DNA-PK activity. As-ODN efficiently reduced the kinase activity with an IC50 of about 100-200 IC50 of Wortmannin was at approximately 5-10 micro M. Treatment of cells with 300 nM As-ODN increased the fraction of residual Dsb at 4 h after irradiation by a factor of 4.4, 2.6, and 1.7 in A549, H460, and H661 cells, respectively. The respective values after treatment with 20 micro M Wortmannin were 5.3, 4.3, and 2.2. Inhibition of DNA-PK activity by As-ODN and Wortmannin also decreased the surviving fraction of the NSCLC cell lines. These data show that kinase activity of DNA-PKcs can be specifically inhibited with As-ODN as effective as Wortmannin and results in marked inhibition of DNA-Dsb rejoining and radiosensitization of NSCLC cell lines.

 

Int J Radiat Biol. 2002 Sep;78(9):807-19.

Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint.

Stuschke M, Sak A, Wurm R, Sinn B, Wolf G, Stüben G, Budach V.

Department of Radiotherapy, Humboldt-University, Charité, Berlin, Germany. martin.stuschke@uni-essen.de

PURPOSE: To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. MATERIALS AND METHODS: Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. RESULTS: Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. CONCLUSIONS: These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.

 

 Int J Radiat Biol. 1998 Jan;73(1):35-43.

Repair of ionizing radiation induced DNA double-strand breaks (dsb) at the c-myc locus in comparison to the overall genome.

Sak A, Stuschke M.

Department of Radiotherapy, University of Essen, Germany.

PURPOSE: The aim of this study was to determine the repair of radiation-induced DNA double-strand breaks (dsb) in actively transcribed regions and in the overall genome. MATERIALS AND METHODS: Pulsed-field gel electrophoresis was performed on Sfi I restriction enzyme digested DNA, labelled with a c-myc probe and on non-specifically 14C-labelled DNA of the adenocarcinoma cell line COLO320HSR after 400 Gy irradiation with 7 MeV electrons and repair incubation. RESULTS: At the 130 kbp c-myc locus 68 +/- 5% of all dsb induced at a dose of 400 Gy were repaired by a fast mechanism with a repair half time of 9.4 +/- 3.2 min in comparison to the overall genome where all dsb induced at 400 Gy were repaired with a half time of 86 +/- 23 min. The fraction of residual dsb was about 30% higher in the c-myc locus than in the overall genome. CONCLUSIONS: This study demonstrates intragenomic heterogeneity in half times of dsb repair with faster repair at the c-myc locus. In addition, differences in the residual dsb were found to represent region specific heterogeneity in residual damage or to possibly be attributed to the different assays used. The approach with gene probing can distinguish between correct and incorrect rejoining of dsb within the resolution of the experiments (< or = 20 kbp), in contrast to the assay at the overall genome.

 

Int J Radiat Biol. 1996 Jun;69(6):679-85.

Induction of DNA double-strand breaks by ionizing radiation at the c-myc locus compared with the whole genome: a study using pulsed-field gel electrophoresis and gene probing.

Sak A, Stuschke M, Stapper N, Streffer C.

Department of Radiotherapy, University of Essen, Germany.

Ionizing radiation-induced double-strand breaks (dsb) in a human colon carcinoma-derived cell line COLO320HSR were determined from the fragment size distribution of non-specifically labelled DNA and Sfi I restriction enzyme-digested DNA uniformly labelled with a c-myc probe. The dose-effect relation for the induction of DNA dsb was linear with no significant difference between slopes for the curves in the whole genome (7.2 +/- 0.3 x 10(-9) dsb/bp/Gy) and in the 130 kbp restriction fragments containing c-myc (6.5 +/- 0.5 x 10(-9) dsb/bp/Gy). The size distribution of the c-myc fragments showed deviations from the random-breakage model, indicating heterogeneity of dsb induction at this locus.

 

Int J Cancer. 1995 Jul 4;62(1):58-62.

Radiation-induced apoptosis in human sarcoma and glioma cell lines.

Stapper NJ, Stuschke M, Sak A, Stüben G.

Department of Radiotherapy, University of Essen, Germany.

Six human soft-tissue sarcoma and 14 glioma cell lines, exhibiting considerable differences in radioresponsiveness and histological grade of differentiation of the parental tumour, were examined with respect to apoptosis development after irradiation with 60Co gamma-rays. After test doses of 6 and 25 Gy, significant changes characteristic of apoptosis occurring within 6 to 30 hr were exhibited by only 2 differentiated sarcoma cell lines, EL7 and ESS2. The characteristic internucleosomal fragmentation of DNA was detected as early as 6 hr after exposure of subconfluent monolayer cultures to 6 Gy. It was limited to cells that had detached from the culture plate, whereas adherent cells showed random degradation of DNA, namely after higher doses (25Gy) or longer incubation times (30 hr). As assessed by fluorescence microscopy of unfixed cultures stained with Hoechst 33342 and propidium iodide, the proportion of cells showing apoptotic bodies in non-irradiated controls was < 0.1% and 0.3% for EL7 and ESS2, respectively. The dose-response relationship for apoptosis was determined at 9 hr post-irradiation. After 2 Gy, the percentage of apoptotic cells was elevated to 3.4% in EL7 and 4.5% in ESS2 cultures. Saturation was obtained above 6 Gy, with 8.4% apoptosis in EL7 and 15% in ESS2 after 25 Gy. Taken together, rapid ionizing-radiation-induced apoptosis seems to be limited to a subgroup of sarcomas and is unlikely to occur in gliomas.

 

Mol Endocrinol. 1993 Aug;7(8):1061-71.

Recombinant activation domains of virion protein 16 and human estrogen receptor generate transcriptional interference in vitro by distinct mechanisms.

Pfitzner E, Sak A, Ulber V, Ryffel GU, Klein-Hitpass L.

Institut für Zellbiologie (Tumorforschung) Universitätsklinikum, Essen, Germany.

Overexpression of transcriptional activators in transfection assays may inhibit their own activity or interfere with trans-activation by different sequence-specific transcription factors. In this study we show that this phenomenon of transcriptional interference (squelching) can be mimicked in vitro by adding recombinant activation domains to nuclear extracts. We demonstrate that the acidic activation domain of virion protein 16 interferes both with basal transcription from TATA-box promoters and promoters activated by various trans-activators in two different mammalian cell-free transcription systems. This suggests that virion protein 16 interacts with and thereby sequesters a basal transcription factor. In contrast the recombinant activation function 2 (AF-2) of human estrogen receptor does not affect basal promoter activity but inhibits TATA promoters activated by human progesterone receptor (hPR) or Sp 1 as well as the beta-globin and adenovirus major late promoter. By analyzing the effects of AF-2 on DNA binding of hPR and Sp1 we found that AF-2 inhibits the DNA binding activity of hPR, but not Sp1. Our data suggest that the recombinant AF-2 squelches Sp1 trans-activation by sequestering a common coactivator(s), whereas hPR function might be inhibited due to competition for a common cofactor stabilizing hPR dimers or through the formation of inactive heterodimers between AF-2 and hPR.

 
 
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