Biomedical subjects

Timothy J Kinsella

Publications and source records attributed to Timothy J Kinsella.

25 records · Page 2Linked to original sources

PubMed2003

Synthesis and functional analyses of nuclear clusterin, a cell death protein.

Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis.

Amino Acid Sequence↗

PubMed2003

Using intraoperative radiation therapy--a case study.

The introduction of a mobile linear accelerator in the OR has made intraoperative radiation therapy (IORT) more plausible. An IORT treatment can deliver a single high dose of radiation to a tumor or tumor bed after surgical resection or surgical exposure of high risk areas. This article details a case study in which IORT was used on a patient with sigmoid carcinoma and the procedure outcomes.

Adenosarcoma↗

PubMed2003

Normal tissue tolerance to intraoperative radiotherapy.

Much experimental evidence has been accumulated assessing the tolerance of various tissues to IORT, and much of the tolerance data have resulted from the use of canine models. Guidelines of IORT tissue tolerance established in experimental models have been used in the clinical application of IORT at numerous institutions. Although the radiotolerance of differing tissues can vary among species, sufficient clinical experience has accumulated to validate the canine tissue tolerance model as representative of human tissue responses to IORT. Cellular effects from radiation principally stem from direct damage to DNA, and thus proliferating tissues are among the most radiosensitive, with arrested or abnormal cell division. These tissues can manifest striking early toxicity, reflecting the rate of cell division that is affected by the radiation. Irradiation of nonproliferating or slowly proliferating tissues may show little or no early toxicity, but late effects can be manifested to considerable and varying degrees. In much of this late toxicity, pathologic changes develop from progressive ischemia, brought about by the gradual obliteration of small blood vessels. Irradiated endothelium often becomes replaced by a thickened fibrous layer, which, in small vessels, leads to occlusion and ischemic necrotic changes in the supplied tissue. In larger vessels, fibrosis can lead to wall weakening and aneurysmal dilatation, rupture, or thrombosis. The common denominator, then, of radiation damage to many tissues is related to vascular effects. Although the tolerance to IORT-induced toxicity can vary considerably among tissues, doses ranging to 25 Gy can generally be tolerated without significant toxicity. Vital areas where IORT dose must be carefully monitored include critical vasculature, gastrointestinal viscera, ureter, significant motor or sensory nerve trunks, and central nervous system structures. Higher doses can generally be delivered safely to anatomic areas at risk for tumor that are at a distance from sensitive organs or tissues. The general principle providing the rationale of IORT should always be practiced: maximize the radiation dose to the tumor and tumor-harboring tissues while minimizing dose exposure to surrounding normal tissues.

Animals↗

PubMed2003

Intraoperative radiotherapy in the multimodality approach to bone and soft tissue cancers.

IORT is a powerful tool for the management of cancer and has been shown to improve local control in the treatment of sarcomas in one prospective, randomized, controlled trial. IORT allows dose escalation in areas where escalation with EBRT would cause potentially excessive acute or late normal tissue toxicity. It has a unique toxicity profile but remains safe and effective when used in appropriately selected patients. In addition, IORT is becoming more widely available at different cancer centers and offers great flexibility in treatment planning for a wide variety of malignancies. As IORT use becomes more common, special care will be required to ensure that it is used by a multidisciplinary team of radiation oncologists and surgeons to ensure the best results for patients.

Adult↗

PubMed2003

Real-time inverse planning for Gamma Knife radiosurgery.

The challenges of real-time Gamma Knife inverse planning are the large number of variables involved and the unknown search space a priori. With limited collimator sizes, shots have to be heavily overlapped to form a smooth prescription isodose line that conforms to the irregular target shape. Such overlaps greatly influence the total number of shots per plan, making pre-determination of the total number of shots impractical. However, this total number of shots usually defines the search space, a pre-requisite for most of the optimization methods. Since each shot only covers part of the target, a collection of shots in different locations and various collimator sizes selected makes up the global dose distribution that conforms to the target. Hence, planning or placing these shots is a combinatorial optimization process that is computationally expensive by nature. We have previously developed a theory of shot placement and optimization based on skeletonization. The real-time inverse planning process, reported in this paper, is an expansion and the clinical implementation of this theory. The complete planning process consists of two steps. The first step is to determine an optimal number of shots including locations and sizes and to assign initial collimator size to each of the shots. The second step is to fine-tune the weights using a linear-programming technique. The objective function is to minimize the total dose to the target boundary (i.e., maximize the dose conformity). Results of an ellipsoid test target and ten clinical cases are presented. The clinical cases are also compared with physician's manual plans. The target coverage is more than 99% for manual plans and 97% for all the inverse plans. The RTOG PITV conformity indices for the manual plans are between 1.16 and 3.46, compared to 1.36 to 2.4 for the inverse plans. All the inverse plans are generated in less than 2 min, making real-time inverse planning a reality.

Algorithms↗

PubMed2003

DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells.

PURPOSE: The DNA mismatch repair (MMR) system plays an important role in mediating cell death after treatment with various types of chemotherapeutic agents, although the molecular mechanisms are not well understood. In this study, we sought to determine what signal is introduced by MMR after 6-thioguanine (6-TG) treatment to signal a G(2)-M arrest leading to cell death. EXPERIMENTAL DESIGN: A comparison study was carried out using an isogenic MMR(+) and MMR(-) human colorectal cancer RKO cell system, which we established for this study. Cells were exposed to 6-TG (3 micro M x 24 h) and then harvested daily for the next 3-6 days for growth inhibition assays. Cell cycle effects were determined by flow cytometry, and DNA strand breaks were measured using pulsed-field gel electrophoresis and alkaline Comet assays. RESULTS: We first established MMR(+) RKO cell lines by transfection of human MutL homologue 1 (hMLH1) cDNA into the hMLH1-deficient (MMR(-)) RKO cell line. The ectopically expressed hMLH1 protein restored a MMR-proficient phenotype in the hMLH1(+) transfectants, showing a significantly increased and prolonged G(2)-M arrest followed by cell death after 6-TG exposure, compared with the vector controls. The MMR-mediated, 6-TG-induced G(2)-M arrest started on day 1, peaked on day 3, and persisted to day 6 after 6-TG removal. We found that DNA double-strand breaks were comparably produced in both our MMR(+) and MMR(-) cells, peaking within 1 day of 6-TG treatment. In contrast, single-strand breaks (SSBs) were more frequent and longer lived in MMR(+) cells, and the duration of SSB formation was temporally correlated with the time course of 6-TG-induced G(2)-M arrest. CONCLUSIONS: Our data suggest that MMR mediates 6-TG-induced G(2)-M arrest by introducing SSBs to signal a persistent G(2)-M arrest leading to enhanced cell death.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Overexpression of the R2 subunit of ribonucleotide reductase in human nasopharyngeal cancer cells reduces radiosensitivity.

PURPOSE: Ribonucleotide reductase is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleotide triphosphates, which are utilized in both DNA synthesis and DNA repair. We reported previously that RR enzyme activity and R2 (catalytic subunit of RR) protein levels were increased after exposure to ionizing radiation (IR) in growth-arrested human tumor cells, suggesting that R2 protein expression regulates RR activity to allow for IR damage repair. Using isogenic human nasopharyngeal carcinoma cells in this study, we examine the relationship of overexpression of either the R1 regulatory subunit or the R2 catalytic subunit of RR to the cellular response of IR damage. MATERIALS AND METHODS: We used three isogenic human nasopharyngeal cancer cell lines previously derived by Zhou et al, including KB, the parental tumor cell line; KB/M1, an R1 protein-overexpressing clone stably transfected with human R1 complementary DNA; and KB/M2, a R2 protein-overexpressing clone stably transfected with human R2 complementary DNA. We initially characterized these isogenic human tumor cell lines in exponential growth for R2 protein expression, RR enzyme activity, and R2 protein changes during the cell cycle by flow cytometry. Subsequently, the IR response in these cell lines was determined by clonogenic survival, cell cycle changes occurring after IR, and an analysis of IR DNA damage determined by pulsed field gel electrophoresis. The effect of combining IR and hydroxyurea, a RR (R2) inhibitor, was also studied in KB and KB/M2 cells. RESULTS: KB/M2 cells were found to have 4.5-fold higher R2 protein expression and a threefold higher RR enzyme activity in exponential growth than KB and KB/M1. Although R2 protein levels increased at the G1/S transition in all cell lines, KB/M2 cells also demonstrated consistently higher R2 protein levels throughout the cell cycle. Using a linear-quadratic analysis of IR clonogenic survival data, KB/M2 cells were more radioresistant than KB and KB/M1 cells, including both decreased alpha and decreased beta values, a finding that correlates with increased reparable IR damage. KB/M2 cells also show a reduced G2 cell cycle arrest and fewer DNA double strand breaks 18 hours after IR (6 Gy). Exposure of KB/M2 cells to hydroxyurea (300 microM) after exposure to IR restored in vitro radiosensitivity in a manner similar to that found in KB and KB/M1 cells. DISCUSSION: An increase in R2 protein levels and RR activity in KB/M2 cells results in IR resistance, which appears mediated by enhanced IR damage repair during G2. R1 protein overexpression in these isogenic human tumor cells (KB/M1) did not affect RR activity or IR response.

Antineoplastic Agents↗