Selective and Reversible Ligand Assembly on the DNA and RNA Repeat Sequences in Myotonic Dystrophy.

Small molecule targeting of DNA and RNA sequences has come into focus as a therapeutic strategy for diseases such as myotonic dystrophy type 1 (DM1), a trinucleotide repeat disease characterized by RNA gain-of-function. Herein, we report a novel template-selected, reversible assembly of therapeutic agents in situ via aldehyde-amine condensation. Rationally designed small molecule targeting agents functionalized with either an aldehyde or an amine were synthesized and screened against the target nucleic acid sequence. The assembly of fragments was confirmed by MALDI-MS in the presence of DM1-relevant nucleic acid sequences. The resulting hit combinations of aldehyde and amine inhibited the formation of r(CUG)exp in vitro in a cooperative manner at low micromolar levels and rescued mis-splicing defects in DM1 model cells. This reversible template-selected assembly is a promising approach to achieve cell permeable and multivalent targeting via in situ synthesis and could be applied to other nucleic acid targets.

Syndapin I Loss-of-Function in Mice Leads to Schizophrenia-Like Symptoms.

Schizophrenia is associated with cognitive and behavioral dysfunctions thought to reflect imbalances in neurotransmission systems. Recent screenings suggested that lack of (functional) syndapin I (PACSIN1) may be linked to schizophrenia. We therefore studied syndapin I KO mice to address the suggested causal relationship to schizophrenia and to analyze associated molecular, cellular, and neurophysiological defects. Syndapin I knockout (KO) mice developed schizophrenia-related behaviors, such as hyperactivity, reduced anxiety, reduced response to social novelty, and an exaggerated novel object response and exhibited defects in dendritic arborization in the cortex. Neuromorphogenic deficits were also observed for a schizophrenia-associated syndapin I mutant in cultured neurons and coincided with a lack of syndapin I-mediated membrane recruitment of cytoskeletal effectors. Syndapin I KO furthermore caused glutamatergic hypofunctions. Syndapin I regulated both AMPAR and NMDAR availabilities at synapses during basal synaptic activity and during synaptic plasticity-particularly striking were a complete lack of long-term potentiation and defects in long-term depression in syndapin I KO mice. These synaptic plasticity defects coincided with alterations of postsynaptic actin dynamics, synaptic GluA1 clustering, and GluA1 mobility. Both GluA1 and GluA2 were not appropriately internalized. Summarized, syndapin I KO led to schizophrenia-like behavior, and our analyses uncovered associated molecular and cellular mechanisms.

Intravesical Liposomal Tacrolimus Protects against Radiation Cystitis Induced by 3-Beam Targeted Bladder Radiation.

PURPOSE: We primarily determined whether the small animal radiation research platform could create a rat radiation cystitis model via targeted bladder irradiation (phase I). The response to treating early phase radiation cystitis in rats with transurethral catheter instillation of liposomal tacrolimus was also examined (phase II). MATERIALS AND METHODS: In phase I 16 adult female Sprague Dawley® rats were used. Metabolic urination patterns were analyzed before and after exposure to 20, 30 or 40 Gy radiation. In phase II irradiated rats were randomly assigned to receive a single instillation of saline or liposomal tacrolimus. RESULTS: The 40 Gy radiation dose induced statistically significant reductions in the intermicturition interval compared to the lower radiation doses. By approximately 20 minutes 40 Gy radiation caused a significant decrease in the mean intermicturition interval (p < 0.0001). Histological analysis revealed degenerative epithelial changes and urothelial swelling with evidence of pseudocarcinomatous epithelial hyperplasia. Therefore, 40 Gy were chosen for the phase II efficacy study. There was no measurable change in total voided urine volume after irradiation, or after liposomal tacrolimus or saline instillation. Liposomal tacrolimus significantly increased the post-irradiation intermicturition interval by approximately 30 minutes back to baseline (p < 0.001). CONCLUSIONS: The radiation cystitis rat model showed a dose dependent decrease in the intermicturition interval without inducing short-term skin or gastrointestinal damage. This study demonstrates that liposomal tacrolimus may be a promising new intravesical therapy for the rare, serious condition of radiation cystitis.

White-nose syndrome, winter duration, and pre-hibernation climate impact abundance of reproductive female bats.

White-nose syndrome (WNS) is an infectious disease that disrupts hibernation in bats, leading to premature exhaustion of fat stores. Though we know WNS does impact reproduction in hibernating female bats, we are unsure how these impacts are exacerbated by local climate factors. We compiled data from four southeastern U.S. states and used generalized linear mixed effects models to compare effects of WNS, pre-hibernation climate variables, and winter duration on the number of reproductive females in species across the range of WNS susceptibility. We predicted we would see a decline in the number of reproductive females in WNS-susceptible species, with the effect exaggerated by longer winter durations and pre-hibernation climate variables that lead to reductions in foraging. We found that the number of reproductive females in WNS-susceptible species was positively correlated with pre-hibernation local climate conditions conducive to foraging; however, WNS-susceptible species experienced an overall decline with the presence of WNS and as winter duration increased. Our long-term dataset provides evidence that pre-hibernation climate, specifically favorable summer weather conditions for foraging, greatly influences the reproduction, regardless of WNS status.

Organ growth without cell division: somatic polyploidy in a moth, Ephestia kuehniella.

Organ growth depends on cell division and (or) cell growth. Here, we present a study on two organs whose growth depends entirely on cell growth, once they are formed in the embryo: Malpighian tubules and silk glands of the flour moth, Ephestia kuehniella . Between first and last larval instar, the volume of Malpighian tubule cells increases by a factor of ∼1800 and that of silk gland cells by a factor of ∼3100. We determined the number of endocyles required to reach these stages by Feulgen cytometry. Cells of Malpighian tubules were in the 2C stage in first instar larvae and reached 1024C after 9 endocycles in last instar larvae (1C = 0.45 pg DNA). Silk gland cells already reached a DNA content of 8C-16C in first instar larvae and attained up to 8192C in last instar larvae after a total of 12 endocycles. The nuclei were small and more or less spherical in first instar larvae, but they were huge, flat, and bizarrely branched in last instar larvae. We consider branching as a compensatory adaptation to improve molecular traffic between nucleus and cytoplasm in these excessively large and highly polyploid cells (i) by reducing the mean distance between nucleus and cytoplasm and (ii) by enlarging the surface-to-volume ratio of these nuclei.

Synthesis and minimum inhibitory concentrations of SK-03-92 against Staphylococcus aureus and other gram-positive bacteria.

Staphylococcus aureus causes hundreds of thousands of infections and thousands of deaths per year in the United States. The emergence of methicillin-resistant S. aureus (MRSA), including community-associated methicillin-resistant S. aureus (CA-MRSA), has added to the problem. As MRSA continue to evolve, they are becoming resistant to more classes of antibiotics. In the past 20 years, only three new antibiotics have been approved for human use (linezolid, daptomycin, and tigecycline), and resistance to these three drugs has already emerged. New antibiotics are needed, and we have developed a promising drug candidate that may be applicable to treating MRSA, among other gram-positive bacterial infections. We have identified a novel synthetic drug, coded SK-03-92, that shows broad in vitro efficacy against a variety of gram-positive bacterial strains that include a number of strains of S. aureus. Besides the activity against gram-positive bacteria, this new drug also exhibits activity against Mycobacterium strains.

Dynamic Covalent Template-Guided Screen for Nucleic Acid-Targeting Agents.

Trinucleotide repeat diseases such as myotonic dystrophy type 1 (DM1) and Huntington's disease (HD) are caused by expanded DNA repeats that can be used as templates to synthesize their own inhibitors. Because it would be particularly advantageous to reversibly assemble multivalent nucleic acid-targeting agents in situ, we sought to develop a target-guided screen that uses dynamic covalent chemistry to identify multitarget inhibitors. We report the synthesis of a library of amine- or aldehyde-containing fragments. The assembly of these fragments led to a diverse set of hit combinations that was confirmed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in the presence of DM1 and HD repeat sequences. Of interest for both diseases, the resulting hit combinations inhibited transcription selectively and in a cooperative manner in vitro, with inhibitory concentration (IC50) values in the micromolar range. This dynamic covalent library and screening approach could be applied to identify compounds that reversibly assemble on other nucleic acid targets.

TMPRSS2/ERG fusion gene expression alters chemo- and radio-responsiveness in cell culture models of androgen independent prostate cancer.

PURPOSE/OBJECTIVES: The androgen regulated transmembrane serine protease (TMPRSS2) and ETS transcription factor (ERG) gene fusion is a strong prognostic factor for disease recurrence following prostatectomy. Expression of TMPRSS2/ETS-related gene (ERG) fusion gene transcripts is linked with tumor proliferation, invasion, and an aggressive phenotype. The aim of this study was to define the effect of TMPRSS2/ERG fusion gene expression on chemo- and radiosensitivity in prostate tumor cell lines. MATERIALS/METHODS: Clonogenic survival of PC3 and DU145 cells stably expressing TMPRSS2/ERG Types III and VI fusion genes was measured after X-irradiation (0-8 Gy) and Paclitaxel. Cell cycle changes and DNA double-strand break induction and repair were assessed. Differential gene expression was measured by microarray analysis. ERG signaling pathway interactions were studied using Ariadne Pathway Studio. RESULTS: Expression of the TMPRSS2/ERG fusions in PC3 cells increased radiation sensitivity and decreased paclitaxel sensitivity. Increased radiosensitivity was associated with persistent DNA breaks 24 hr post-irradiation, down-regulation of genes involved in DNA repair and mitosis and up-regulation of ETV, an ETS transcription factor. However, DU145 Types III and VI demonstrated a different sensitivity phenotype and gene expression changes. Pathway analysis of ERG signaling further illustrated the variation between the PC3 and DU145 cell lines containing TMPRSS2/ERG fusions. CONCLUSIONS: The effect of TMPRSS2/ERG gene fusions had differing effects on radiosensitivity and chemosensitivity depending on cell line and fusion type. Further work is needed with clinical samples to establish whether TMPRSS2/ERG gene fusions affect radio- and chemosensitivity in vivo.

A Novel Minor Groove Binder as a Potential Therapeutic Agent for Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder that is inherited in an autosomal dominant manner. DM1 originates in a (CTG⋅CAG) repeat expansion in the 3'-UTR of the dystrophia myotonic protein kinase (DMPK) gene on chromosome 19. One of the transcripts, r(CUG)exp , is toxic in various ways. Herein we report a rationally designed small molecule with a thiazole peptidomimetic unit that can serve as a minor groove binder for the nucleic acid targets. This peptide unit linked to two triaminotriazine recognition units selectively binds to d(CTG)exp to inhibit the transcription process, and also targets r(CUG)exp selectively to improve representative DM1 pathological molecular features, including foci formation and pre-mRNA splicing defects in DM1 model cells. As such, it represents a new structure type that might serve as a lead compound for future structure-activity optimization.

Versatile Target-Guided Screen for Discovering Bidirectional Transcription Inhibitors of a Trinucleotide Repeat Disease.

Myotonic dystrophy type 1 originates from d(CTG·CAG) repeats that undergo aberrant expansion during normal processing because the d(CTG) repeat forms stable hairpin structures. Bidirectional transcription of d(CTG·CAG) yields two RNA transcripts that undergo repeat-associated non-ATG (RAN) translation to form homopolymeric proteins. Thus, both the r(CUG) transcript and the r(CAG) transcript are known to be toxic. We report a pairwise fragment-based, target-guided approach to screen for proximity-induced click dimers formed on the nucleic acid template. This screen uses an azide/alkyne clickable fragment library of nucleic acid-binding ligands incubated in parallel, pairwise reactions as an alternative to our previously reported one-pot screening method. MALDI-TOF mass spectroscopy was used to detect template assisted click products. Hit compounds inhibited the in vitro transcription of d(CTG·CAG)90 bidirectionally with IC50 values in the low micromolar range. This approach may be broadly applicable to other trinucleotide repeat diseases and in targeting other disease-associated nucleic acid sequences.

A new class of potential anti-tuberculosis agents: Synthesis and preliminary evaluation of novel acrylic acid ethyl ester derivatives.

Novel acrylic acid ethyl ester derivatives were synthesized and evaluated as potential agents against Mycobacterium species. A versatile and efficient copper-catalyzed coupling process was developed and used to prepare a library of substituted acrylic acid ethyl ester analogs. Minimum inhibitory concentration assays indicated that two of these compounds 3 and 4 have greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Moreover, members of this new class of compounds appear to exhibit a specific anti-mycobacterial effect and do not inhibit the growth of the other Gram-positive or Gram-negative species tested.

New classes of Gram-positive selective antibacterials: inhibitors of MRSA and surrogates of the causative agents of anthrax and tuberculosis.

An antimicrobial phenolic stilbene, (E)-3-hydroxy-5-methoxystilbene, 1 was recently isolated from the leaves of Comptonia peregrina (L.) Coulter and shown to possess inhibitory activity against several Gram-positive bacteria, including isolates of methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium bovis BCG, and avirulent Bacillusanthracis (Sterne strain), among others. These results prompted the design and synthesis of two new classes of compounds, phenoxystyrenes and phenothiostyrenes, as analogs of the natural antimicrobial stilbene. These and additional stilbenoid analogs were synthesized using new, efficient, copper-mediated coupling strategies. Minimum inhibitory concentration (MIC) antimicrobial assays were performed on all compounds prepared. These preliminary structure-activity relationship studies indicated that both new classes of synthetic analogs, as well as the stilbenes, show promising activity against Gram-positive bacteria when at least one phenolic moiety is present, but not when absent. The potencies of the phenolic phenoxystyrenes and phenothiostyrenes were found to be comparable to those of the phenolic stilbenes tested.

Targeting CD74 in multiple myeloma with the novel, site-specific antibody-drug conjugate STRO-001.

STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.

Transition in survival from low-dose hyper-radiosensitivity to increased radioresistance is independent of activation of ATM Ser1981 activity.

PURPOSE: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G(2)-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. METHODS AND MATERIALS: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 microg/mL chloroquine, 15 microM genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eight cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G(2)-phase cells entering mitosis, using histone H3 phosphorylation analysis. RESULTS: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G(2)-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. CONCLUSION: Overcoming HRS is reliant on the function of the early G(2)-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.

Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination.

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

Modeling of chronic radiation-induced cystitis in mice.

PURPOSE: Radiation cystitis (RC), a severe inflammatory bladder condition, develops as a side-effect of pelvic radiation therapy in cancer patients. There are currently no effective therapies to treat RC, in part due to the lack of preclinical model systems. In this study, we developed a mouse model for RC and used a small animal radiation research platform (SARRP) to simulate the targeted delivery of radiation as used with human patients. METHODS AND MATERIALS: To induce RC, C3H mice received a single radiation dose of 20Gy delivered through two beams. Mice were subjected to weekly micturition measurements to assess changes in urinary frequency. At the end of the study, bladder tissues were processed for histology. RESULTS: Radiation was well tolerated as no change in weight was observed in the weeks post treatment, and there was no hair loss at the irradiation sites. Starting at 17 weeks post treatment, micturition frequency was significantly higher in irradiated mice versus control animals. Pathological changes include fibrosis, inflammation, urothelial thinning and necrosis. At a site of severe insult, we observed telangiectasia, absence of Uroplakin-3 and E-cadherin relocalization. CONCLUSIONS: We developed a RC model that mimics the human pathology and functional changes. Furthermore, radiation exposure attenuates the urothelial integrity long-term allowing for potential continuous irritability of the bladder wall from exposure to urine. Future studies will focus on the underlying molecular changes associated with this condition and investigate novel treatment strategies.

Pulsed Radiation Therapy With Concurrent Cisplatin Results in Superior Tumor Growth Delay in a Head and Neck Squamous Cell Carcinoma Murine Model.

PURPOSE: To assess the efficacy of 3-week schedules of low-dose pulsed radiation treatment (PRT) and standard radiation therapy (SRT), with concurrent cisplatin (CDDP) in a head and neck squamous cell carcinoma xenograft model. METHODS AND MATERIALS: Subcutaneous UT-SCC-14 tumors were established in athymic NIH III HO female mice. A total of 30 Gy was administered as 2 Gy/d, 5 d/wk for 3 weeks, either by PRT (10 × 0.2 Gy/d, with a 3-minute break between each 0.2-Gy dose) or SRT (2 Gy/d, uninterrupted delivery) in combination with concurrent 2 mg/kg CDDP 3 times per week in the final 2 weeks of radiation therapy. Treatment-induced growth delays were defined from twice-weekly tumor volume measurements. Tumor hypoxia was assessed by (18)F-fluoromisonidazole positron emission tomography imaging, and calculated maximum standardized uptake values compared with tumor histology. Tumor vessel density and hypoxia were measured by quantitative immunohistochemistry. Normal tissues effects were evaluated in gut and skin. RESULTS: Untreated tumors grew to 1000 mm(3) in 25.4 days (±1.2), compared with delays of 62.3 days (±3.5) for SRT + CDDP and 80.2 days (±5.0) for PRT + CDDP. Time to reach 2× pretreatment volume ranged from 8.2 days (±1.8) for untreated tumors to 67.1 days (±4.7) after PRT + CDDP. Significant differences in tumor growth delay were observed for SRT versus SRT + CDDP (P=.04), PRT versus PRT + CDDP (P=.035), and SRT + CDDP versus PRT + CDDP (P=.033), and for survival between PRT versus PRT + CDDP (P=.017) and SRT + CDDP versus PRT + CDDP (P=.008). Differences in tumor hypoxia were evident by (18)F-fluoromisonidazole positron emission tomography imaging between SRT and PRT (P=.025), although not with concurrent CDDP. Tumor vessel density differed between SRT + CDDP and PRT + CDDP (P=.011). No differences in normal tissue parameters were seen. CONCLUSIONS: Concurrent CDDP was more effective in combination PRT than SRT at restricting tumor growth. Significant differences in tumor vascular density were evident between PRT and SRT, suggesting a preservation of vascular network with PRT.

Effect of Irradiation on Tumor Microenvironment and Bone Marrow Cell Migration in a Preclinical Tumor Model.

PURPOSE: To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. METHODS AND MATERIALS: Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2 Gy/d for a total dose of 20 Gy using a 5 days on, 2 days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40 Gy in 4 weeks using the same 2-Gy/d regimens. RESULTS: Over the 2 weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31 ± 0.02 mm(3)/d and 0.55 ± 0.04 mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5% ± 3%), hypoxia (10% ± 3.5%), vascular endothelial growth factor (2.3% ± 1%), and stromal-derived factor-1α (2.5% ± 1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8% ± 2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6% ± 0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. CONCLUSIONS: Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.

The Effects of Pulsed Radiation Therapy on Tumor Oxygenation in 2 Murine Models of Head and Neck Squamous Cell Carcinoma.

PURPOSE: To evaluate the efficacy of low-dose pulsed radiation therapy (PRT) in 2 head and neck squamous cell carcinoma (HNSCC) xenografts and to investigate the mechanism of action of PRT compared with standard radiation therapy (SRT). METHODS AND MATERIALS: Subcutaneous radiosensitive UT-SCC-14 and radioresistant UT-SCC-15 xenografts were established in athymic NIH III HO female mice. Tumors were irradiated with 2 Gy/day by continuous standard delivery (SRT: 2 Gy) or discontinuous low-dose pulsed delivery (PRT: 0.2 Gy × 10 with 3-min pulse interval) to total doses of 20 Gy (UT14) or 40 Gy (UT15) using a clinical 5-day on/2-day off schedule. Treatment response was assessed by changes in tumor volume, (18)F-fluorodeoxyglucose (FDG) (tumor metabolism), and (18)F-fluoromisonidazole (FMISO) (hypoxia) positron emission tomography (PET) imaging before, at midpoint, and after treatment. Tumor hypoxia using pimonidazole staining and vascular density (CD34 staining) were assessed by quantitative histopathology. RESULTS: UT15 and UT14 tumors responded similarly in terms of growth delay to either SRT or PRT. When compared with UT14 tumors, UT15 tumors demonstrated significantly lower uptake of FDG at all time points after irradiation. UT14 tumors demonstrated higher levels of tumor hypoxia after SRT when compared with PRT as measured by (18)F-FMISO PET. By contrast, no differences were seen in (18)F-FMISO PET imaging between SRT and PRT for UT15 tumors. Histologic analysis of pimonidazole staining mimicked the (18)F-FMISO PET imaging data, showing an increase in hypoxia in SRT-treated UT14 tumors but not PRT-treated tumors. CONCLUSIONS: Differences in (18)F-FMISO uptake for UT14 tumors after radiation therapy between PRT and SRT were measurable despite the similar tumor growth delay responses. In UT15 tumors, both SRT and PRT were equally effective at reducing tumor hypoxia to a significant level as measured by (18)F-FMISO and pimonidazole.