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Journal Article

Animals; Antineoplastic Agents; Apoptosis; Bioware; Caspase 3; Cell Line, Tumor; Female; Firefly Luciferin; Humans; Luminescent Agents; MDA-MB-231-D3H2LN cells; Mice; Mice, SCID; SKOV3-luc-D3 cells; Molecular Imaging; Neoplasms; Oligopeptides; Taxoids

Apoptosis is a highly regulated process of programmed cell death essential for normal physiology. Dysregulation of apoptosis contributes to the development and progression of various diseases, including cancer, neurodegenerative disorders, and chronic heart failure. Quantitative noninvasive imaging of apoptosis in preclinical models would allow for dynamic longitudinal screening of compounds and facilitates a more rapid determination of therapeutic efficacy. In this study, we report the in vivo characterization of Z-DEVD-aminoluciferin, a modified firefly luciferase substrate that in apoptotic cells is cleaved by caspase-3 to liberate aminoluciferin, which can be consumed by luciferase to generate a luminescent signal. In two oncology models, namely SKOV3-luc and MDA-MB-231-luc-LN, at 24, 48, and 72 h after treatment with docetaxel, animals were injected with Z-DEVD-aminoluciferin and bioluminescent images were acquired. Significantly more light was detected at 24 (P<0.05), 48 (P<0.01), and 72 h (P<0.01) in the docetaxel-treated group compared with the vehicle-treated group, with caspase-3 activation at these time points confirmed using immunohistochemistry. Importantly, whereas significant differences between groups were detected as early as 24 h after treatment by molecular imaging, caliper measurements were unable to detect a difference for 4-5 additional days. Taken together, these data show that in vivo imaging of apoptosis using Z-DEVD-aminoluciferin could provide a sensitive and rapid method for early detection of drug efficacy, which could potentially be used by numerous therapeutic programs.

Journal Article

Androgens; Animals; Animals, Genetically Modified; Apoptosis; Bioware; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Glutathione S-Transferase pi; Humans; In Situ Nick-End Labeling; Male; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; PC-3M-luc; Prostatic Neoplasms; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering

Prostate cancers generally acquire an androgen-independent growth capacity with progression, resulting in resistance to antiandrogen therapy. Therefore, identification of the genes regulated through this process may be important for understanding the mechanisms of prostate carcinogenesis. We here utilized androgen-dependent/independent transplantable tumors, newly established with the 'transgenic rat adenocarcinoma in prostate' (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen-independent prostate cancers compared with the androgen-dependent tumors, glutathione S-transferase pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared with LNCaP (androgen dependent) as determined by semiquantitative reverse transcription-polymerase chain reaction analysis. To investigate the roles of GST-pi expression in androgen-independent human prostate cancers, GST-pi was knocked down by a small interfering RNA (siRNA), resulting in significant decrease of the proliferation rate in the androgen-independent PC3 cell line. In vivo, administration of GST-pi siRNA-atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TdT mediated dUTP-biotin nick-end labering (TUNEL)-positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen-independent human prostate cancer cells.

Journal Article

Animals; Bioware; carcinoma, renal cell; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Firefly Luciferin; HT-29-luc-D6 cells; Humans; Kidney Neoplasms; Luminescence; Luminescent Agents; Male; Mice; Mice, SCID; Models, Biological; Tumor Burden

PURPOSE Bioluminescent imaging permits sensitive in vivo detection and quantification of cells engineered to emit light. We developed a bioluminescent human renal cancer cell line for in vitro and in vivo studies. MATERIAL AND METHODS The 2 human renal cell carcinoma cell lines SN12-C and SN12-L1 were stably transfected to constitutively express luciferase using a retroviral shuttle. The bioluminescent signal was correlated with tumor cell numbers in vitro. Parental and transfected cells were compared by growth kinetics and histology. Tumor burden after heterotopic injection in immune deficient mice was monitored up to 39 days. The kinetics of the bioluminescent signal was evaluated for 1 to 60 minutes following luciferin injection. RESULTS Bioengineered renal cancer cell lines stably expressed luciferase. The growth kinetics of the cells in vitro and the histology of tumors resulting from implantation of these cells were unaffected by retroviral transfection with the luciferase gene. As few as 1,000 cells could be reliably detected. The intensity of the bioluminescent signal correlated with the number of tumor cells in vitro. Photon emission in vivo and ex vivo correlated significantly with tumor weight at sacrifice. After intraperitoneal injection of luciferin there was a time dependent change in the intensity of the bioluminescent signal with maximum photon emission at 20 minutes (optimal 17 to 25). CONCLUSIONS Luciferase transfected human renal cancer lines allow reliable, rapid, noninvasive and longitudinal monitoring of tumor growth in vivo. The ability to assess tumor development in vivo with time is economical and effective compared to end point data experiments.

Journal Article

Animals; Anthrax; Bacillus anthracis; Bioware; Disease Models, Animal; Gastrointestinal Diseases; Inhalation Exposure; Luciferases; Luminescence; Luminescent Measurements; Lymph Nodes; Mice; Mice, Inbred BALB C; Nasal Cavity; Organisms, Genetically Modified; Peyer's Patches; Pharynx; pXen-5; Skin; Spores, Bacterial

Bacillus anthracis causes three forms of anthrax: inhalational, gastrointestinal, and cutaneous. Anthrax is characterized by both toxemia, which is caused by secretion of immunomodulating toxins (lethal toxin and edema toxin), and septicemia, which is associated with bacterial encapsulation. Here we report that, contrary to the current view of B. anthracis pathogenesis, B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues. Furthermore, we found that Peyer's patches in the mouse intestine are the primary site of bacterial growth after intragastric inoculation, thus establishing an animal model of gastrointestinal anthrax. All routes of infection progressed to the draining lymph nodes, spleen, lungs, and ultimately the blood. These discoveries were made possible through the development of a novel dynamic mouse model of B. anthracis infection using bioluminescent non-toxinogenic capsulated bacteria that can be visualized within the mouse in real-time, and demonstrate the value of in vivo imaging in the analysis of B. anthracis infection. Our data imply that previously unrecognized portals of bacterial entry demand more intensive investigation, and will significantly transform the current perception of inhalational, gastrointestinal, and cutaneous B. anthracis pathogenesis.

Journal Article

Animals; Antibiotics, Antitubercular; Biofilms; Bioware; Colony Count, Microbial; Diagnostic Imaging; DNA-Directed RNA Polymerases; Luminescent Measurements; Mice; Rifampin; Staphylococcal Infections; Staphylococcus aureus; Xen29

Eradication of Staphylococcus aureus biofilms after rifampin treatment was tested in a mouse model of device-related infection by using biophotonic imaging. Following treatment, the bioluminescent signals decreased to undetectable levels, irrespective of the age of the biofilm. After the final treatment, the signals rebounded in a time-dependent manner and reached those for the untreated mice. Readministration of rifampin was unsuccessful in eradicating reestablished infections, with the rifampin MICs for such bacteria being increased and with the bacteria having point mutations in the rpoB gene.