Sheikh Ismail

Histone Deacetylase Inhibitors Radiosensitize Human Melanoma Cells by Suppressing DNA Repair Activity

Purpose: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. Experimental Design: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. Results: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined γ-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, γ-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. Conclusions: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that γ-H2AX phosphorylation can be used as a predictive marker of radioresponse.

Accumulation of the common mitochondrial DNA deletion induced by ionizing radiation

Point mutations and deletions in mitochondrial DNA (mtDNA) accumulate as a result of oxidative stress, including ionizing radiation. As a result, dysfunctional mitochondria suffer from a decline in oxidative phosphorylation and increased release of superoxides and other reactive oxygen species (ROS). Through this mechanism, mitochondria have been implicated in a host of degenerative diseases. Associated with this type of damage, and serving as a marker of total mtDNA mutations and deletions, the accumulation of a specific 4977‐bp deletion, known as the common deletion (Δ‐mtDNA4977), takes place. The Δ‐mtDNA4977 has been reported to increase with age and during the progression of mitochondrial degeneration. The purpose of this study was to investigate whether ionizing radiation induces the formation of the common deletion in a variety of human cell lines and to determine if it is associated with cellular radiosensitivity. Cell lines used included eight normal human skin fibroblast lines, a radiosensitive non‐transformed and an SV40 transformed ataxia telangiectasia (AT) homozygous fibroblast line, a Kearns Sayre Syndrome (KSS) line known to contain mitochondrial deletions, and five human tumor lines. The Δ‐mtDNA4977 was assessed by polymerase chain reaction (PCR). Significant levels of Δ‐mtDNA4977 accumulated 72 h after irradiation doses of 2, 5, 10 or 20 Gy in all of the normal lines with lower response in tumor cell lines, but the absolute amounts of the induced deletion were variable. There was no consistent dose–response relationship. SV40 transformed and non‐transformed AT cell lines both showed significant induction of the deletion. However, the five tumor cell lines showed only a modest induction of the deletion, including the one line that was deficient in DNA damage repair. No relationship was found between sensitivity to radiation‐induced deletions and sensitivity to cell killing by radiation.

Adenoviral-mediated mda-7 expression suppresses DNA repair capacity and radiosensitizes non-small-cell lung cancer cells.

The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentiation. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells – A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radiosensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electrophoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs.

Predicting Radiosensitivity Using DNA End-Binding Complex Analysis

Previous reports have suggested that measuring radiosensitivity of normal and tumor cells would have significant clinical relevance for the practice of radiation oncology. We hypothesized that radiosensitivity might be predicted by analyzing DNA end-binding complexes (DNA-EBCs), which form at DNA double-strand breaks, the most important cytotoxic lesion caused by radiation. To test this hypothesis, the DNA-EBC pattern of 21 primary human fibroblast cultures and 15 tumor cell lines were studied. DNA-EBC patterns were determined using a modified electrophoretic mobility shift assay and were correlated with radiosensitivity, as measured by SF2. DNA-EBC analysis identified a rapidly migrating ATM-containing band (identified as “band-A”) of which the density correlated with SF2 (0.02 ≤ SF2 ≤ 0.41) in primary fibroblasts (r2 = 0.77). The DNA-EBC pattern of peripheral blood lymphocytes was identical to that of fibroblasts. In addition, band-A density correlated with SF2 (0.35 ≤ SF2 ≤ 0.80) in 15 human tumor cell lines (r2 = 0.91). Densitometry of other bands, or total DNA-EBC binding, correlated more poorly with SF2 (r2 < 0.45). These data indicate that DNA-EBC analysis may be a practical, clinically relevant predictor of tumor and primary cell radiosensitivity.

DNA-PK and ATM are Required for Radiation-Enhanced Integration

Abstract Nimura, Y., Ismail, S. M., Chen, D. J. and Stevens, C. W. DNA-PK and ATM are Required for Radiation-Enhanced Integration. Radiat. Res. 157, 562–567 (2002). Ionizing radiation is known to improve transfection of exogenous DNA, a process we have termed radiation-enhanced integration. Previous observations have demonstrated that Ku proteins are critical for radiation-enhanced integration. Since Ku proteins form the DNA-binding domain of DNA-PK and since DNA-PK is important in nonhomologous DNA end joining, it was hypothesized that DNA-PK function might be important for radiation-enhanced integration. The ATM protein has been shown to be important in the recognition of a variety of types of DNA damage and to associate with DNA-PK under certain conditions. It was thus hypothesized that ATM might also play a role in radiation-enhanced integration. To test these hypotheses, radiation-enhanced integration was measured in hamster cells that are defective in the catalytic subunit of DNA-PK and in human cells containing mutant ATM. Radiation-enhanced integration was not detected in any of the cell lines with mutant PRKDC (also known as DNA-PKcs), but it was present in cells of the same lineage with wild-type PRKDC. Radiation-enhanced integration was defective in cells lacking kinase activation. ATM-deficient cell lines also showed defective radiation-enhanced integration. These data demonstrate that DNA-PK and ATM must both be active for radiation-enhanced integration to be observed.

Identification of proteins in the hamster DNA end‐binding complex

Purpose: To identify the protein components of the DNA end‐binding complex in hamster cells. Materials and methods: DNA end‐binding complexes were identified as follows. Nuclear extracts from Chinese hamster ovary cells (0.5–1.0 µg protein/lane) were incubated with 0.5 ng 32P‐labelled probe (144 bp) for 20 min at room temperature in the presence of 1 µg closed circular pUC18 plasmid, a non‐specific competitor in a final volume of 20 µl. The electrophoretic mobility of the protein–DNA complexes was analysed by electrophoresis in 5% polyacrylamide gels subjected to autoradiography. Antibodies to various DNA repair‐associated proteins were added to the DNA end‐binding complex reaction and a supershift identified DNA end‐binding complex components. These were confirmed by Western analysis of purified DNA end‐binding complex contents. Results: Using both supershift and Western analysis, the following proteins were identified in the DNA end‐binding complex: Ku70, Ku80, DNA‐dependent protein kinase catalytic subunit, DNA ligase IV, X‐ray cross complementing protein 4, meiotic recombination protein 11 (Mre11), Werners syndrome protein, Blooms syndrome protein, p53, poly(ADP‐ribose) polymerase, replication protein A (RPA) 14, and RPA32, ataxia telangiectasia mutant, c‐Abl, Rad50, Nijmegen breakage syndrome protein 1 (NBS1), and DNA ligase III were not detected in the binding complex by any assay. Using a combination of electro‐elution and autoradiography, it was estimated that the single DNA end‐binding complex contains at least 15 proteins whose molecular weights of the DNA end‐binding proteins ranged from 620 to 12 kDa. Conclusions: A combination of both a supershift assay and Western analysis of the DNA end‐binding complexes has identified 12 of at least 15 proteins present in the DNA end‐binding complex of Chinese hamster ovary cells. This protein complex contains Mre11, but not Rad50 or NBS1, suggesting that under some conditions, Mre11 might function independently of Rad50 and NBS1.

Nucleotide excision repair proteins and their importance for radiation-enhanced transfection.

Purpose: Irradiated cells transfect more efficiently than unirradiated cells because of a radiation‐induced increase in plasmid integration. However, the molecular mechanism is unclear. Because of recent observations that nucleotide excision repair (NER) proteins can be involved in certain types of recombination in yeast, it was hypothesized that NER proteins might play a role in this radiation‐enhanced integration. Materials and methods: Hamster and human cells with inactivating mutations in NER genes were irradiated at doses from 0 to 6 Gy and then immediately transfected with a linearized selectable marker plasmid. Transfection‐enhancement ratios (TERs) were calculated as the ratio of the number of drug‐resistant colonies in unirradiated cells to the number of transfectants in irradiated cells, corrected for cytotoxicity from radiation. Results: Transfection into unirradiated rodent cells was unaffected by NER mutation status. Transfection into unirradiated human cells, however, was increased by NER mutation. The TERs were 5 and 100 for CHO and primary human fibroblasts, respectively, after exposure of the cells to 6 Gy. Mutations in ERCC1, XPA, XPB, XPC, XPF, XPG and CSB dramatically reduced TER. Mutations in ERCC1, XPC, XPF, XPG and CSB suppressed transfection so that the TER was significantly below 1. Conclusions: The mechanism of radiation‐enhanced plasmid integration was distinct from that of plasmid integration in unirradiated cells, and NER gene products were critical for enhanced integration to occur.