Novel somatic mutations in heterotrimeric G proteins in melanoma.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) mediate signals between G-protein coupled receptors and their downstream pathways, and have been shown to be mutated in cancer. In particular, GNAQ was found to be frequently mutated in blue nevi of the skin and uveal melanoma, acting as an oncogene in its mutated form. To further examine the role of heterotrimeric G proteins in malignant melanoma, we performed a comprehensive mutational analysis of the 35 genes in the heterotrimeric G protein gene family in a panel of 80 melanoma samples. Somatic alterations in a G protein subunit were detected in 17% of samples spanning 7 genes. The highest rates of somatic, non-synonymous mutations were found in GNG10 and GNAZ, neither of which has been previously reported to be mutated in melanoma. Our study is the first systematic analysis of the heterotrimeric G proteins in melanoma and indicates that multiple mutated heterotrimeric G proteins may be involved in melanoma progression.

Requirements for T lymphocyte migration in explanted lymph nodes.

Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 microm/s and a partial pressure of O(2) (pO(2)) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO(2), but also decreased the speed of locomotion in the deep paracortex. Although CCR7(-/-) naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Galpha(i)-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO(2), tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.

Targeting the molecular effects of a hypoxic tumor microenvironment.

Tumor hypoxia is a serious and enduring problem for traditional solid tumor therapies. Many scientists continue to explore methods to improve or exploit tumor oxygenation; more recently, scientists have also focused on altering the molecular effects of hypoxia. These cellular responses to hypoxia and the resulting physiological effects, with a focus on angiogenesis, invasion/metastases, apoptosis, and metabolism, are examined. Recent efforts to mitigate or exploit these molecular pathways alone and in conjunction with traditional therapies are also explored. Current experimental results suggest that targeting multiple downstream molecular pathways of hypoxia will be more effective than targeting a single molecular pathway of hypoxia, and careful planning is necessary in scheduling these new therapies to optimize their effects in combination with traditional therapies.

Effects of fluctuating oxygenation on tirapazamine efficacy: Theoretical predictions.

PURPOSE: To examine the effects of fluctuating oxygen levels on the hypoxic cytotoxin tirapazamine (TPZ) using theoretical predictions. METHODS AND MATERIALS: Tirapazamine's pharmacokinetic and pharmacodynamic oxygen dependence has previously been characterized in vitro. Here, a one-dimensional theoretical model was used to examine the effects of fluctuating hypoxia on metabolized TPZ concentration, assuming sinusoidally fluctuating oxygen levels. TPZ concentration is changing according to published experimental data. Simulations of experimentally observed time-courses of perivascular pO2 were also conducted. RESULTS: The predicted pharmacodynamic effect of TPZ was increased with fluctuating (vs. constant) hypoxia at all frequencies (1-30 min period) and all amplitudes (1-15 mm Hg). Additionally, fluctuating oxygen resulted in more metabolized TPZ near the oxygen source as compared with the steady-state condition of the same overall average pO2. CONCLUSIONS: Fluctuating pO2 reduced the concentration of metabolized TPZ at distances farther from the source, thereby limiting its ability to reach and kill the most hypoxic cells. These results suggest that the kinetics of fluctuating oxygenation should be taken into account when considering drug designs that involve oxygen-sensitive agents.

Oxygen regulation of tumor perfusion by S-nitrosohemoglobin reveals a pressor activity of nitric oxide.

In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). It has been shown that SNO-Hb behaves as a nitric oxide (NO) donor at low oxygen tensions. This property, in combination with oxygen transport capacity, suggests that SNO-Hb may have unique potential to reoxygenate hypoxic tissues. The present study was designed to test the idea that the allosteric properties of SNO-Hb could be manipulated to enhance oxygen delivery in a hypoxic tumor. Using Laser Doppler flowmetry, we showed that SNO-Hb infusion to animals breathing 21% O2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO2 (100% O2) slowed the release of NO bioactivity from SNO-Hb (ie, prolonged the plasma half-life of the SNO in Hb), preserved tumor perfusion, and raised the blood pressure. In contrast, native Hb reduced both tumor perfusion and heart rate independently of the oxygen concentration of the inhaled gas, and did not elicit hypertensive effects. Window chamber (to image tumor arteriolar reactivity in vivo) and hemodynamic measurements indicated that the preservation of tissue perfusion by micromolar concentrations of SNO-Hb is a composite effect created by reduced peripheral vascular resistance and direct inhibition of the baroreceptor reflex, leading to increased blood pressure. Overall, these results indicate that the properties of SNO-Hb are attributable to allosteric control of NO release by oxygen in central as well as peripheral issues.

Tumor-dependent kinetics of partial pressure of oxygen fluctuations during air and oxygen breathing.

The primary purpose of this study was to examine the kinetics of partial pressure of oxygen (pO2) fluctuations in fibrosarcoma (FSA) and 9L tumors under air and O2 breathing conditions. The overall hypothesis was that key factors relating to oxygen tension fluctuations would vary between the two tumor types and as a function of the oxygen content of the breathing gas. To assist in the interpretation of the temporal data, spatial pO2 distributions were measured in 10 FSA and 8 9L tumors transplanted into the subcutis of the hind leg of Nembutal-anesthetized (50 mg/kg) Fischer 344 rats. Recessed-tip oxygen microelectrodes were inserted into the tumor, and linear pO2 measurements were recorded in 50-microm steps along a 3-mm path, and blood pressure was simultaneously measured via femoral arterial access. Additionally, pO2 was measured at a single location for 90 to 120 minutes in FSA (n=11) or 9L tumors (n=12). Rats were switched from air to 100% O2 breathing after 45 minutes. Temporal pO2 records were evaluated for their potential radiobiological significance by assessing the number of times they crossed a 10-mm-Hg threshold. In addition, the data were subjected to Fourier analysis for air and O2 breathing. FSA and 9L tumors had spatial median pO2 measurements of 4 and 1 mm Hg, respectively. 9L had more low pO2 measurements < or =2.5 mm Hg than did FSA, whereas between 2.5 and 10 mm Hg this pattern was reversed. Pimonidazole staining patterns in FSA and 9L tumors supported these results. Temporal pO2 instability was observed in all experiments during air and O2 breathing. Threshold analyses indicated that the 10 mm Hg threshold was crossed 2 to 5 times per hour, independent of tumor type. However, the magnitude of 9L pO2 fluctuations was approximately eight times greater than FSA fluctuations, as assessed with Fourier transform analysis (Wilcoxon, P < 0.005). O2 breathing significantly increased median pO2 in FSA from 3 to 8 mm Hg (P < 0.005) and caused a significant increase in frequency and magnitude of pO2 fluctuations. One hundred percent O2 breathing had no effect on 9L tumor pO2, and it decreased the magnitude of pO2 fluctuations with borderline significance. These results show that these two tumors differ significantly with respect to spatial and temporal oxygenation conditions under air and O2 breathing. Fluctuations of pO2 of the type reported herein are predicted to significantly affect radiotherapy response and could be a source for genetic instability, increased angiogenesis, and metastases.