Fixed drug combination (levosulpiride and rabeprazole)-induced atypical Parkinsonian's disorders with associated anxiety and low-lying depression.

We hereby describe a rare case of levosulpiride-induced atypical parkinsonism presenting with sluggish movements, atypical kinetic tremors (tremors with voluntary movement), periorbital tremors, dystonia, difficulty in speech and coordination, postural imbalance, with additional features of difficulty in swallowing and drooling with associated recent onset psychiatric disturbances such as anxiety and low-lying depression. The dechallenge of levosulpiride and medications for associated anxiety and low-lying depression caused a complete remission of the disease within 2 ½ months.

Impaired DNA Repair Fidelity in a Breast Cancer Patient With Adverse Reactions to Radiotherapy.

We tested the hypothesis that differences in DNA double-strand break (DSB) repair fidelity underlies differences in individual radiosensitivity and, consequently, normal tissue reactions to radiotherapy. Fibroblast cultures derived from a radio-sensitive (RS) breast cancer patient with grade 3 adverse reactions to radiotherapy were compared with normal control (NC) and hyper-radiosensitive ataxia-telangiectasia mutated (ATM) cells. DSB repair and repair fidelity were studied by Southern blotting and hybridization to Alu repetitive sequence and to a specific 3.2-Mbp NotI restriction fragment on chromosome 21, respectively. Results for DNA repair kinetics using the NotI fidelity assay showed significant differences (P < 0.001) with higher levels of misrepaired (misrejoined and unrejoined) DSBs in RS and ATM compared with NC. At 24-h postradiation, the relative fractions of misrepaired DSBs were 10.64, 23.08, and 44.70% for NC, RS, and ATM, respectively. The Alu assay showed significant (P < 0.05) differences in unrepaired DSBs only between the ATM and both NC and RS at the time points of 12 and 24 h. At 24 h, the relative percentages of DSBs unrepaired were 1.33, 3.43, and 12.13% for NC, RS, and ATM, respectively. The comparison between the two assays indicated an average of 5-fold higher fractions of misrepaired (NotI assay) than unrepaired (Alu assay) DSBs. In conclusion, this patient with increased radiotoxicity displayed more prominent misrepaired than unrepaired DSBs, suggesting that DNA repair fidelity is a potential marker for the adverse reactions to radiotherapy. More studies are required to confirm these results and further develop DSB repair fidelity as a hallmark biomarker for interindividual differences in radiosensitivity.

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 gamma-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, gamma-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 gamma-H2AX phosphorylation can be used as a predictive marker of radioresponse.

Radiosensitivity is predicted by DNA end-binding complex density, but not by nuclear levels of band components.

BACKGROUND AND PURPOSE: We previously determined that the density of a rapidly migrating DNA end-binding complex (termed 'band-A') predicts radiosensitivity of human normal and tumor cells. The goal of this study was first to identify the protein components of band-A and to determine if the protein levels of band-A components would correlate with band-A density and radiosensitivity. PATIENTS AND METHODS: DNA end-binding protein complex (DNA-EBC) protein components were identified by adding antibodies specific for a variety of DNA repair-associated proteins to the DNA-EBC reaction and then noting which antibodies super-shifted various DNA-EBC bands. Band-A levels were correlated with SF2 for a panel of primary human fibroblasts heterozygous for sequence-proven mutations in BRCA1 or BRCA2. The nuclear protein levels of band-A components were determined in each BRCA1 heterozygote by western hybridization. RESULTS: DNA-EBC analysis of human nuclear proteins revealed 10 identifiable bands. The density of the most rapidly migrating DNA-EBC band correlated closely with both BRCA-mutation status and radiosensitivity (r(2)=0.85). This band was absent in cells with homozygous mutations in their ataxia-telangiectasia-mutated protein (ATM) genes. This band was also completely supershifted by the addition of antibodies to ATM, Ku70, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53. However, the intranuclear concentrations of these proteins did not correlate with either the SF2 or DNA-EBC density. Neither BRCA1 or BRCA2 could be detected in band-A. CONCLUSIONS: DNA-EBC analysis of human nuclear extracts resulted in 10 bands, at least six of which contained ATM. The density of one of the DNA-EBCs predicted the radiosensitization caused by BRCA haploinsufficiency, and this band contains Ku70, ATM, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53 but not BRCA 1 or 2. The density of band-A was independent of the nuclear concentration of any of its known component.

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.

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 (Delta-mtDNA(4977)), takes place. The Delta-mtDNA(4977) 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 Delta-mtDNA(4977) was assessed by polymerase chain reaction (PCR). Significant levels of Delta-mtDNA(4977) 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.

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

DNA-PK and ATM are required for radiation-enhanced integration.

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.