Activation of diverse pathways to apoptosis by 125IdUrd and γ‐photon exposure

Purpose: To delineate the mechanisms underlying induction of apoptosis in malignant cells irradiated by DNA‐incorporated iodine‐125 or γ‐photons.

Materials and methods: Human tumor cells (RKO, LS174T, TE671, and MCF7) were irradiated by DNA‐incorporated 5‐[¹²⁵I]iodo‐2′‐deoxyuridine (¹²⁵IdUrd) or by γ‐photons. Clonogenic survival was determined by the colony‐forming assay. Caspase‐3 induction was measured with a fluorogenic substrate assay, and DNA fragmentation was determined by ligation‐mediated polymerase chain reaction. DNA arrays were used to assess the expression of the B‐cell lymphoma/leukaemia‐2 (Bcl‐2) family and related genes in RKO cells and in caspase‐3‐gene‐defective MCF7 cells.

Results: After ¹²⁵IdUrd or γ‐photon exposure, the highest induction of caspase‐3 was observed in the radiation‐sensitive cell lines (RKO and LS174T). DNA fragmentation was prominent in the radiosensitive cells and undetectable in TE671 (¹²⁵IdUrd and γ‐photons) and MCF7 (¹²⁵IdUrd only) cells. Exposure of RKO and MCF7 cells to ¹²⁵I decay led to up‐regulation of several pro‐apoptotic and anti‐apoptotic Bcl‐2 family genes whereas γ‐irradiation produced minimal activation.

Conclusions: Apoptosis generated by a DNA‐incorporated Auger electron emitter is induced through the mitochondrial/caspase‐3‐mediated pathway and correlates with cellular radiosensitivity. Apoptosis caused by γ‐radiation can be signaled without activation of Bcl‐2 family genes, and DNA fragmentation occurs with or without caspase‐3 activation.     

5-[123I/125I]iodo-2'-deoxyuridine in metastatic lung cancer: radiopharmaceutical formulation affects targeting.

This study assesses targeting of lung metastases in mice with the radioiodinated thymidine analog 5-[(123)I/(125)I]iodo-2'-deoxyuridine ((123)I-IUdR/(125)I-IUdR), formulated with varying amounts of tributyltin precursor and injected intravenously. METHODS: Six- to 8-wk-old C57BL/6 mice were injected intravenously with B16F10 melanoma cells. Two weeks later, when lung tumors were established, the animals were injected intravenously with (125)I-IUdR synthesized using 1, 35, 100, 150, 200, or 250 microg 5-tributylstannyl-2'-deoxyuridine (SnUdR) in the presence of an oxidant. Nontumor-bearing mice were also injected with these formulations and served as control animals. Twenty-four hours later, the animals were killed, and the radioactivity associated with the lungs and other tissues was measured in a gamma-counter. The percentage injected dose per gram tissue (%ID/g) and tumor-to-nontumor ratios (T/NT ratios) were calculated. Phosphor imaging was done on lungs from tumor-bearing and nontumor-bearing mice injected with (125)I-IUdR formulated with each tin precursor concentration. Scintigraphy was also performed 3 and 24 h after intravenous injection of (123)I-IUdR. RESULTS: The %ID/g (125)I-IUdR was higher in lungs of tumor-bearing animals than in lungs of control animals. Although the increase in SnUdR present led to a small but statistically significant decrease in the radioactive content of normal lungs, a 3-fold increase was observed in the lungs of tumor-bearing animals with radiopharmaceutical formulated with 100 microg SnUdR (5 microg per mouse). This enhancement in radioactive uptake by the lungs led to approximately 14-fold increases in T/NT ratios. Phosphor imaging ((125)I-IUdR) of lungs as well as scintigraphy ((123)I-IUdR) of whole animals substantiated these findings. CONCLUSION: The formulation for the synthesis of radio-IUdR that leads to the highest %ID/g in tumor and the best T/NT ratio has been identified. Further studies are required to determine the factors responsible for specific enhancement in IUdR tumor uptake.     

Functional MRI of the Brain in Women with Overactive Bladder: Brain Activation During Urinary Urgency

OBJECTIVES: To identify abnormal function of the limbic cortex (LC) in response to urinary urgency among patients with Overactive Bladder (OAB) using brain functional MRI (fMRI) METHODS: 5 OAB subjects and 5 Controls underwent bladder filling and rated urgency sensations while fMRI measured activation in discrete volumes (voxels) within the brain. Changes in brain activation were related to bladder distension and individual subject's rating of urgency via multiple regression analysis. Beta weights from regression equations were converted into percent signal change (PSC) for each voxel and PSC compared to the null hypothesis using T-tests. Significance threshold of P<.05 was applied along with a cluster size threshold of.32 ml (5 voxels). RESULTS: OAB patients showed increased brain activation in LC, specifically the insula (IN) and Anterior Cingulate Gyrus (ACG), associated with increased urgency. Urgency sensations during low volumes were associated with bilateral IN activation in OAB subjects (7,621 voxels right IN, 4,453 voxels left IN, mean beta weights .018 +/- .014 and .014 +/- .011) Minimal activation was present in Controls (790 voxels right IN, beta weight =.010 +/- .007). Urgency sensations during high volumes were associated with bilateral ACG activation in OAB subjects (2,304 voxels right IN, 5,005 voxels left IN, mean beta weights of 005 +/- .003 and 004+/-.003) without activation in Controls. CONCLUSIONS: Urinary urgency in patients with OAB is associated with increased activation of the LC. This activation likely represents abnormal processing of sensory input in brain regions associated with emotional response to discomfort.     

Computational modeling and experimental evaluation of a novel prodrug for targeting the extracellular space of prostate tumors

We are developing a noninvasive approach for targeting imaging and therapeutic radionuclides to prostate cancer. Our method, Enzyme-Mediated Cancer Imaging and Therapy (EMCIT), aims to use enzyme-dependent, site-specific, in vivo precipitation of a radioactive molecule within the extracellular space of solid tumors. Advanced methods for data mining of the literature, protein databases, and knowledge bases (IT.Omics LSGraph and Ingenuity Systems) identified prostatic acid phosphatase (PAP) as an enzyme overexpressed in prostate cancer and secreted in the extracellular space. Using AutoDock 3.0 software, the prodrug ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-P)) was docked in silico into the X-ray structure of PAP. The data indicate that IQ(2-P) docked into the PAP active site with a calculated inhibition constant (K(i)) more favorable than that of the PAP inhibitor alpha-benzylaminobenzylphosphonic acid. When (125)IQ(2-P), the radioiodinated form of the water-soluble prodrug, was incubated with PAP, rapid hydrolysis of the compound was observed as exemplified by formation of the water-insoluble 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone ((125)IQ(2-OH)). Similarly, the incubation of IQ(2-P) with human LNCaP, PC-3, and 22Rv1 prostate tumor cells resulted in the formation of large fluorescent IQ(2-OH) crystals. No hydrolysis was seen in the presence of normal human cells. Autoradiography of tumor cells incubated with (125)IQ(2-P) showed accumulation of radioactive grains ((125)IQ(2-OH)) around the cells. We anticipate that the EMCIT approach will enable the active in vivo entrapment of radioimaging and radiotherapeutic compounds within the extracellular spaces of primary prostate tumors and their metastases.     

Computational and biological evaluation of quinazolinone prodrug for targeting pancreatic cancer

Our concept of enzyme-mediated cancer imaging and therapy aims to use radiolabeled compounds to target hydrolases over-expressed on the extracellular surface of solid tumors. A data mining approach identified extracellular sulfatase 1 (SULF1) as an enzyme expressed on the surface of pancreatic cancer cells. We designed, synthesized, and characterized 2-(2'-sulfooxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-S)) as well as its radioiodinated form ((125) IQ(2-S)) as a prodrug with potential for hydrolysis by SULF1. IQ(2-S) was successfully docked in silico into three enzymes - homolog of SULF1, alkaline phosphatase, and prostatic acid phosphatase. The incubation of (125) IQ(2-S) and (125) IQ(2-P) with the three enzymes in solution confirms the docking results and enzyme selectivity for the analogs. The hydrolysis of both radioactive compounds produces the water-insoluble, fluorescent product 2-(2'-hydroxyphenyl)-6-[(125) I]iodo-4-(3H)-quinazolinone ((125) IQ(2-OH)). The in vitro incubation of (127) IQ(2-S) and (127) IQ(2-P) with pancreatic, ovarian, and prostate cancer cells expressing studied hydrolases also results in their hydrolysis and the precipitation of (127) IQ(2-OH) fluorescent crystals on the cell surface. To our knowledge, these findings are the first to report the targeting of a radioactive substrate to SULF1 and that this prodrug may be potentially useful in the imaging ((123) I/(124) I/(131) I) and radiotherapy ((131) I) of pancreatic cancer.     

Induction of apoptosis in human tumor cells after exposure to Auger electrons: comparison with gamma-ray exposure

To clarify the contribution of apoptosis to cell death in four human solid tumor cell lines, clonogenic cell survival (indicator of radiosensitivity) and induction of caspase-3 (CASP-3)/caspase-3-like proteases (CASP-3LP) and the production of DNA fragmentation (markers for apoptosis) were studied in RKO, LS174T, MCF7 and TE671 cells exposed to DNA-incorporated Auger-electron-emitting ¹²⁵I (5-[¹²⁵I]iodo-2′-deoxyuridine) or γ-radiation. Clonogenic survival was assessed by colony-forming assay, CASP-3/CASP-3LP induction with a fluorogenic substrate and DNA fragmentation by ligation-mediated polymerase chain reaction. For ¹²⁵I, log dose–survival curves had no shoulder [high-linear-energy-transfer (LET)-like] and decreased exponentially at different rates in various cell lines. Induction of CASP-3/CASP-3LP in radiosensitive RKO and LS174T cells was threefold greater than that in radioresistant TE671 and MCF7 cells. Nucleosomal laddering in ¹²⁵I-radiosensitive cell lines was dose-dependent, and no laddering was detected in radioresistant lines. For γ-radiation, the survival curve for LS174T cells was monoexponential and that for the other lines exhibited a distinct shoulder (low-LET-like). The most radiosensitive cell line, LS174T, showed the highest induction of CASP-3/CASP-3LP, and the most radioresistant line, TE671, showed the lowest induction. Although DNA laddering was not detectable in TE671 cells, it was observed in other lines, being most prominent in LS174T cells. We conclude that apoptosis initiated by DNA-incorporated ¹²⁵I is dose-dependent, correlates with cell radiosensitivity and takes place through a CASP-3-mediated pathway, whereas that after γ-irradiation probably occurs via a CASP-3-independent pathway and/or a CASP-3-mediated pathway and does not correlate with cell radiosensitivity.     

Rodeo related large animal injury: is protective head-gear warranted?

To compare rodeo associated large animal injuries to large animal associated trauma from other aetiologies in order to determine whether mandatory protective head-gear during rodeo is warranted.

Retrospective analysis related to injury involving large animal admissions between 1 January 1990 and 31 December 1995.

The setting is at the University of New Mexico Health Science Center, a level 1 trauma centre.

All patients admitted with Injury Severity Scores of 1 or higher following large animal associated injuries.

There were 140 admissions for which mechanism of injury was known. Thirty-nine occurred during rodeo competition and 101 occurred during other activities. Bovine associated activities were the aetiology in 34 (87%) of rodeo related injuries while equine related activities were the aetiology in 97 (96%) of non-rodeo related injuries (P<0.001). Rodeo related injuries involved the head and neck in five patients (13%) compared to 42 patients (42%) in non-rodeo activities (P=0.001). Mean Regional Injury Severity Score head and neck was 0.4 for injured rodeo riders and 1.5 for injured non-rodeo riders (P<0.001). Mean admission Glascow Coma Scale was 14.9 for rodeo-injured patients and 13.3 for non-rodeo-injured patients (P<0.001). Total ISS was significantly lower for rodeo injured patients (9.1 vs. 11.7, P=0.03). No rodeo injured patient died as a result of head injury.

Mechanism of injury, ISS head, GCS, total ISS, and outcome differ between rodeo and non-rodeo injuries. While routine helmet use during non-rodeo events appears justified, mandatory use of helmets in rodeo events is unwarranted. Orthotics to protect the chest and abdomen are more likely to reduce morbidity and mortality for rodeo participants.