Biomedical subjects

Michael D Story

Publications and source records attributed to Michael D Story.

8 recordsLinked to original sources

PubMed2004

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.

Adenoviridae↗

PubMed2004

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.

Adolescent↗

PubMed2004

A Ser49Cys variant in the ataxia telangiectasia, mutated, gene that is more common in patients with breast carcinoma compared with population controls.

BACKGROUND: Mothers of children who have ataxia telangiectasia have been reported to be at increased risk for development of breast carcinoma. To test whether sequence variants in the ataxia telangiectasia, mutated, gene (ATM) are associated with breast carcinoma, the authors compared the frequency of ATM cDNA sequence changes in patients with breast carcinoma with the corresponding frequency in control patients. METHODS: The authors sequenced ATM cDNA from 91 patients with breast carcinoma and compared the frequencies of sequence changes in these patients with the corresponding frequencies in a control sample of 940 individuals with no history of malignant disease. RESULTS: Thirty-five patients with breast carcinoma had one or more single-base changes in ATM. Three genetic variants were found in at least two patients. These variants resulted in Asp1853Asn, Pro1054Arg, or Ser49Cys amino acid substitutions in the ATM protein. The Ser49Cys variant was more common in patients with breast carcinoma than in the control patients, with respective frequencies of 6.7% (5 of 75 patients) and 1.3% (12 of 940 patients; P=0.006; Fisher two-sided exact test). The subgroup of patients with bilateral breast carcinoma had a Ser49Cys frequency of 11.8% (2 of 17 patients), which again was significantly different from what was observed in the control group (P=0.024; Fisher two-sided exact test). The allele frequencies of the other two variants were not different between case patients and control patients. CONCLUSIONS: Patients with breast carcinoma, particularly those with bilateral disease, were more likely to have a variant in the ATM gene that resulted in a Ser49Cys substitution in the gene product. Additional studies are needed to evaluate the potential functional consequences of the Ser49Cys substitution and confirm the relevance of this variant in the development of breast carcinoma.

Adult↗

PubMed2004

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 (r(2) = 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 (r(2) = 0.91). Densitometry of other bands, or total DNA-EBC binding, correlated more poorly with SF2 (r(2) < 0.45). These data indicate that DNA-EBC analysis may be a practical, clinically relevant predictor of tumor and primary cell radiosensitivity.

Cell Line↗

PubMed2004

Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response.

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.

Animals↗

PubMed2003

Chromosomal fragility syndrome and family history of radiosensitivity as indicators for radiotherapy dose modification.

Beside a few known radiosensitive syndromes, a patient's reaction to radiotherapy is difficult to predict. In this report we describe the management of a pediatric cancer patient presented with a family history of radiosensitivity and cancer proneness. Laboratory investigations revealed a chromosomal fragility syndrome and an increased cellular radiosensitivity in vitro. AT gene sequencing revealed no mutations. The patient was treated with reduced radiation doses to avoid the presumed increased risks of toxicity to normal tissues. The patient tolerated well the treatment with no significant acute or late radiation sequelae. Five years later, the patient remains both disease and complications free. While an accurate laboratory test for radiosensitivity is still lacking, assessments of chromosomal fragility, cell survival and clinical medicine will continue to be useful for a small number of patients.

Cerebellar Neoplasms↗

PubMed2002

Cyclin B1 overexpression and resistance to radiotherapy in head and neck squamous cell carcinoma.

Radiotherapy (RT) and surgery are the cornerstones in treating head and neck squamous cell carcinoma (HNSCC). RT is effective in the initial treatment of early to intermediate stage HNSCC but less effective for locally advanced disease, some cases of which are best managed using the combination of surgery and RT. Although various clinical/pathologic parameters have been used to classify patients according to their likelihood of responding to RT, they have generally low predictive value. We have shown previously that cyclin B1 is associated with a poor clinical outcome in HNSCC patients. In this study, we investigate the potential role of cyclin B1 in assessing RT response in patients with HNSCC. Tumor specimens obtained from 80 patients participated in a prospective Phase III clinical trial addressing the dose and fractionation regimen of postoperative RT were analyzed for cyclin B1 expression by immunohistochemistry. Patients were classified according to currently accepted clinical/pathologic parameters into three risk groups, i.e., low, intermediate, and high risk, and received surgery alone, surgery plus intermediate-dose RT, and surgery plus high-dose RT, respectively. The median follow-up duration was 4.9 years. Cyclin B1 overexpression was noted in 38 of the 80 (47%) HNSCC tumors. Interestingly, 11 of the 38 patients (29%) with cyclin B1-overexpressing tumors experienced local or nodal recurrence compared with only 3 of 42 patients (7%) having carcinomas with no or weak cyclin B1 expression (P = 0.01). When locoregional control was used as the end point for the high-risk group, patients whose tumors showed cyclin B1 overexpression had a statistically significant higher tumor recurrence and metastasis compared with patients whose tumors showed no cyclin B1 overexpression (P = 0.01). Our results indicate that tumors overexpressing cyclin B1 may be resistant to RT, and cyclin B1 may be an indicator of the risk of locoregional recurrence and metastasis in patients having HNSCC receiving RT.

Adult↗

PubMed2002

Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks.

Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both gamma-H2AX and 53BP1 after DNA damage. Mutation of Thr2609 to Ala leads to radiation sensitivity and impaired DSB rejoining. These findings establish that Ku-dependent phosphorylation of DNA-PKcs at Thr2609 is required for the repair of DSBs by NHEJ.

Amino Acid Sequence↗