The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: evidence of hypoxia-induced potentially lethal damage.

PURPOSE: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill. MATERIALS AND METHODS: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N(2) gas containing 0, 0.1 or 0.5% O(2). Cell survival was determined by colony formation assay immediately after 0-96 h hypoxia. To reoxygenate medium were replaced fresh following 48 or 72 h in hypoxia and cells were incubated in aerobic environment for 2-24 h before survival assay. RESULTS: When incubated in hypoxia, cells lost reproductive capability ∼ exponentially as a function of time in hypoxia, and depending on the O(2) concentration. DNKu70 rendered cells more prone to hypoxia-induced cell-kill. Following reoxygenation cell survival increased rapidly but without detectable cell proliferation during first 24 hours. This evinced hypoxia-induced potentially lethal damage (PLD) that was repairable upon reoxygenation. DNKu70 did not significantly inhibit this repair. CONCLUSION: Hypoxia-induced cell lethality was facilitated by DNKu70, but substantially repaired upon reoxygenation. This may have negative impact on the effect of reoxygenation in cancer therapy.

Hypoxia-inducible factor-1 drives annexin A2 system-mediated perivascular fibrin clearance in oxygen-induced retinopathy in mice.

Oxygen-induced retinopathy (OIR) is a well-characterized model for retinopathy of prematurity, a disorder that results from rapid microvascular proliferation after exposure of the retina to high oxygen levels. Here, we report that the proliferative phase of OIR requires transcriptional induction of the annexin A2 (A2) gene through the direct action of the hypoxia-inducible factor-1 complex. We show, in addition, that A2 stabilizes its binding partner, p11, and promotes OIR-related angiogenesis by enabling clearance of perivascular fibrin. Adenoviral-mediated restoration of A2 expression restores neovascularization in the oxygen-primed Anxa2(-/-) retina and reinstates plasmin generation and directed migration in cultured Anxa2(-/-) endothelial cells. Systemic depletion of fibrin repairs the neovascular response to high oxygen treatment in the Anxa2(-/-) retina, whereas inhibition of plasminogen activation dampens angiogenesis under the same conditions. These findings show that the A2 system enables retinal neoangiogenesis in OIR by enhancing perivascular activation of plasmin and remodeling of fibrin. These data suggest new potential approaches to retinal angiogenic disorders on the basis of modulation of perivascular fibrinolysis.

Radiosynthesis of [(131)I]IAZGP via nucleophilic substitution and its biological evaluation as a hypoxia marker - is specific activity a factor influencing hypoxia-mapping ability of a hypoxia marker?

INTRODUCTION: The hypoxia marker IAZGP, 1-(6-deoxy-6-iodo-beta-d-galactopyranosyl)-2-nitroimidazole, has been labeled with (123)I/(124)I/(125)I/(131)I via iodine-radioiodine exchange, which gives the radiotracer in a specific activity of 10-90 MBq/micromol. We synthesized the same radiotracer possessing several hundred to thousand times higher specific activity (high-SA IAZGP) via nucleophilic substitution and compared its biological behavior with that of conventionally produced IAZGP (low-SA IAZGP) to determine if specific activity is a factor influencing cell uptake kinetics, biodistribution and intratumor microregional localization of the radiotracer. METHODS: High-SA [(131)I]IAZGP was prepared by substitution of the tosyl functionality with [(131)I]iodide. In vitro uptake of high- and low-SA [(131)I]IAZGP by HCT8 and HT29 cells was assessed in normoxic and hypoxic conditions. Biodistribution and intratumor localization of high- and low-SA [(131)I]IAZGP were determined by injection into HT29 tumor-bearing mice. RESULTS: The nucleophilic substitution reaction proceeded efficiently in acetonitrile at 150 degrees C, giving the final product in an average yield of 42% and an average specific activity of 30 GBq/micromol. In vitro, high-SA [(131)I]IAZGP was incorporated into the tumor cells with similar kinetics and oxygen dependence to low-SA [(131)I]IAZGP. In HT29 tumor-bearing mice, biodistributions of high- and low-SA [(131)I]IAZGP were equivalent. Ex vivo autoradiography revealed heterogeneous intratumor localization of high-SA [(131)I]IAZGP corresponding closely to distributions of other exogenous and endogenous hypoxia markers. Comparable microregional distribution patterns were observed with low-SA [(131)I]IAZGP. CONCLUSIONS: Radiolabeled IAZGP produced via nucleophilic substitution is validated as an exogenous hypoxia marker. Specific activity does not appear to influence the in vivo hypoxia-mapping ability of the radiotracer.

In vivo 19F magnetic resonance spectroscopy and chemical shift imaging of tri-fluoro-nitroimidazole as a potential hypoxia reporter in solid tumors.

PURPOSE: 2-Nitro-alpha-[(2,2,2-trifluoroethoxy)methyl]-imidazole-1-ethanol (TF-MISO) was investigated as a potential noninvasive marker of tissue oxygen levels in tumors using (19)F magnetic resonance spectroscopy (MRS) and (19)F chemical shift imaging. EXPERIMENTAL DESIGNS: In vitro data were obtained using high-performance liquid chromatography on tumor cells incubated under varying oxygen conditions to determine the oxygen-binding characteristics. In vivo data were obtained using a well-characterized hypoxic murine breast tumor (MCa), in addition to studies on a rat prostate tumor model (R3327-AT) implanted in nude mice. Detection of intratumor (19)F signal from TF-MISO was done using MRS for up to 10 h following a 75 mg/kg i.v. injection. Localized distribution of the compound in the implanted MCa tumor has been imaged using slice-selective two-dimensional chemical shift imaging 6 h after injection. RESULTS: The in vitro results showed that TF-MISO preferentially accumulates in cells incubated under anoxic conditions. The in vivo (19)F MR spectral features (line width and chemical shift) were recorded as a function of time after injection, and the results indicate that the fluorine atoms are indeed sensitive to changes in the local environment while still providing a detectable MR signal. Ex vivo spectra were collected and established the visibility of the (19)F signal under conditions of maximum hypoxia. Late time point (>6 h) tumor tissue concentrations, as obtained from (19)F MRS, suggest that TF-MISO is reduced and retained in hypoxic tumor. The feasibility of obtaining TF-MISO tumor distribution maps in a reasonable time frame was established. CONCLUSIONS: Based on the results presented herein, it is suggested that TF-MISO has the potential to be a valid magnetic resonance hypoxia imaging reporter for both preclinical hypoxia studies and hypoxia-directed clinical therapy.

Detecting changes in tumor hypoxia with carbonic anhydrase IX and pimonidazole.

We have used immunohistochemistry to examine the dynamics of tumor hypoxia.. Expression of CAIX is known to be influenced by tumor hypoxia, and this protein has been shown to be an endogenous hypoxia marker in several models. However, due to its long half-life, it could also be present in oxygenated tissue that had recently been hypoxic. To investigate this issue we have compared CAIX expression to the exogenous hypoxia marker, pimonidazole using HT29 (human colorectal cancer) xenografts. We manipulated tumor hypoxia with carbogen and hydralazine, treatments that respectively increased and decreased tumor oxygenation. (Carbogen was given 75 minutes and hydralazine 30 minutes before sacrifice). In tumors from the control group, CAIX and pimonidazole exhibited similar (though not identical) spatial distribution, and for both markers, the fraction of the section staining positively was similar (13.2% and 12.6% respectively). The mice treated with hydralazine showed a significant increase in pimonidazole accumulation (37.2%, p = 0.03), though the CAIX positive fraction was unchanged (14.2%). In contrast, in the carbogen group pimonidazole staining decreased to 3% (p = 0.01) though CAIX expression was again unaltered. These results suggest that comparison of CAIX and pimonidazole will allow for the detection of reoxygenation.

Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM.

BACKGROUND: Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) [Cu-ATSM] is a potential marker for tumor hypoxia that has been under evaluation for clinical use. In this study, we examined the mechanisms underlying the uptake of (64)Cu in cells incubated with (64)Cu-ATSM. METHODS: The in vitro uptake of (64)Cu was determined as a function of oxygenation conditions and incubation time with (64)Cu-ATSM using four and two tumor cell lines of human origin and rodent origin, respectively. Additionally, the rate of (64)Cu efflux and Cu-ATSM metabolism was determined. RESULTS: (64)Cu accumulation is rapid during the first 0.5-1 h of incubation. It is highest in anoxic cells but is also significant in normoxic cells. After this initial period, the level of intracellular (64)Cu varies depending on the cell line and the oxygenation conditions and, in some circumstances, may decrease. During the first 0.5-1 h, the ratio of (64)Cu levels between anoxic and normoxic cells is approximately 2:10 and that between hypoxic (0.5% O(2)) and normoxic cells is approximately 1:2.5, depending on the cell line. These ratios generally decrease at longer times. The (64)Cu-ATSM compound was found to be metabolized during incubation in a manner dependent on oxygenation conditions. Within 2 h under anoxic conditions, (64)Cu-ATSM could no longer be detected, although 60-90% of the amount of (64)Cu added as (64)Cu-ATSM was present in the medium. Non-ATSM (64)Cu was taken up by the cells, albeit at a much slower rate. Efflux rates of (64)Cu were found to be cell line dependent and appeared to be inversely correlated with the final (64)Cu uptake levels under anoxic conditions. CONCLUSION: The uptake and retention of (64)Cu and their relation to oxygenation conditions were found to be cell line dependent. Given the complexities in the oxygen dependence and cell line-dependent kinetics of uptake and retention of Cu following exposure to Cu-ATSM, the clinical utility of this compound may be disease site specific.

Assessment of regional tumor hypoxia using 18F-fluoromisonidazole and 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) positron emission tomography: Comparative study featuring microPET imaging, Po2 probe measurement, autoradiography, and fluorescent microscopy in the R3327-AT and FaDu rat tumor models.

PURPOSE: To compare two potential positron emission tomography (PET) tracers of tumor hypoxia in an animal model. METHODS AND MATERIALS: The purported hypoxia imaging agents (18)F-fluoromisonidazole (FMISO) and (64)Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) were compared by serial microPET imaging of Fisher-Copenhagen rats bearing the R3327-AT anaplastic rat prostate tumor. Probe measurements of intratumoral Po(2) were compared with the image data. At the microscopic level, the relationship between the spatial distributions of (64)Cu (assessed by digital autoradiography) and tumor hypoxia (assessed by immunofluorescent detection of pimonidazole) was examined. (18)F-FMISO and (64)Cu-ATSM microPET images were also acquired in nude rats bearing xenografts derived from the human squamous cell carcinoma cell line, FaDu. RESULTS: In R3327-AT tumors, the intratumoral distribution of (18)F-FMISO remained relatively constant 1-4 h after injection. However, that of (64)Cu-ATSM displayed a significant temporal evolution for 0.5-20 h after injection in most tumors. In general, only when (64)Cu-ATSM was imaged at later times (16-20 h after injection) did it correspond to the distribution of (18)F-FMISO. Oxygen probe measurements were broadly consistent with (18)F-FMISO and late (64)Cu-ATSM images but not with early (64)Cu-ATSM images. At the microscopic level, a negative correlation was found between tumor hypoxia and (64)Cu distribution when assessed at early times and a positive correlation when assessed at later times. For the FaDu tumor model, the early and late (64)Cu-ATSM microPET images were similar and were in general concordance with the (18)F-FMISO scans. CONCLUSION: The difference in behavior between the R3327-AT and FaDu tumor models suggests a tumor-specific dependence of Cu-ATSM uptake and retention under hypoxic conditions.

A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia.

PURPOSE: Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. METHODS: The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFP fusion gene. The expression of this reporter gene can be assessed with the 124I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (124I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped 124I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of 124I-FIAU was also compared with that of an exogenous hypoxic cell marker, 18F-fluoromisonidazole (FMISO). RESULTS: Our results showed that 124I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of 124I-FIAU and 18F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between 124I-FIAU and 18F-FMISO. CONCLUSION: This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.