2005 S-nitrosohemoglobin modifies blood flow in rat mammary adenocarcinoma

Proceedings

Results: between survival

Hallahan Vanderbilt

ASTRO

281

Meeting

Patients in CF (conventional fractionation) and AF (accelerated fractionation) showed no evidence of differences the two arms in tumor control (log rank = 2.62, p=O.l), disease-free survival (log rank=3.06, p=O.O8), overall (log rank=0.38 , p=O.54) and acute toxicity (p 0.12).

Conclusion: from 45-48

2004

of the 41st Annual

Similar local tumor control with similar acute toxicity days to 38 days (66 Gy133 fractions in both arms).

was observed

ICAMMOUSE

ALTER

DE: Geng

AND P-SELECTIN LUNG L, Dugger

University,

Nashville,

NULL

MUTATIONS

despite

THE

the reduction

RADIATION

of overall

RESPONSE

treatment

time

IN THE

.I TN,

USA

Purpose: Irradiation of the lung induces an inflammatory response and fibrosis. There is some controversy over the relationship between inflammation and fibrosis following lung irradiation. We studied this relationship in knockout mice lacking genes required for inflammation. We have found that several cell adhesion molecules are induced by radiation in the pulmonary vascular endothelium. These include ICY&-l and P-selectin. We studied the physiologic response of the lungs in mice with the ICAMand P-selectin null mutations by use of dynamic pulmonary compliance, respiratory rates and histology following thoracic irradiation. Methods: Endotrachial intubation was performed using a dissecting microscope. Plethysmography was performed as described (Hershenson, 1992). Trachial intubation was achieved by use of a metal cannula (22 gauge) following tracheostomy using a dissecting microscope. Animals were mechanically ventilated using a tital volume of 8 ml/kg at a frequency of 140 breaths per minute using a Harvard Apparatus volume-controlled rodent ventilator (South Natick, MA). Changes in plethysmographic pressure measured relative to a Whiter reference chamber by means of differential pressure transducer (Honeywell Microswitch, Freeport, IL) reflected changes in lung volume. Dynamic compliance was calculated breath by breath using software designed for this application (Mouse PRC Software, Lakeshore Technologies, Chicago, IL). Animals were also observed for the onset of tachypnea. The lethal dose for 50% of mice (LD50) was determined over 6 and 12.months. Lungs were fixed in stain to determine pulmonary fibrosis at the time of respiratory insufficiency or at 12 months. Results: C57BL6 mice treated with 14 Gy showed diminished pulmonary compliance compared to untreated controls. Wild type mice showed baseline dynamic compliance compared to ICAMknockouts 0.051 +/- ,009. Irradiated C57BL6 mice showed compliance to 0.038 +/- 0.004 (P=O.O2). ICAM knockout mice showed no significant 0.042 +/- ,004 (P=O.3). Methylcholine was used to further study the change in dynamic pulmonary compliance in irradiated knockouts by 35% +/- 2% as compared to a 53% (P=O.OOl). The LD50 for wild type C57BL6 mice was 16 Gy. The LD50 for ICAMThe LD50 for P-selectin knockout mice was 12.5 Gy (P=O.Ol). Histologic evaluation of diminished pulmonary fibrosis, but fibrosis was not absent. Histologic evaluation of platelet aggregation and extravasation of blood components into the alveoli.

at 5 weeks following irradiation as of 0.054 ml/cm H20 +/- .003 as a significant reduction in pulmonary reduction in pulmonary compliance to compliance. Methylcholine reduced reduction in irradiated wild type mice knockout mice was 18 Gy (P=O.O5). lungs show that ICAM knockouts have P-selectin knockouts show enhanced

Conclusion: ICAMand P-selectin are induced by irradiation in pulmonary endothelium. Knockout of ICAMreduces inflammatory infiltration of irradiated lung resulting in improved pulmonary compliance and increased LD50. P-selectin null mutation results in increased radiation sensitivity associated with increased platelet aggregation and extravasation of blood components into the alveoli. These finding show that radiation-induced molecules contribute to both tissue injury and tissue repair. Therefore, the role of radiation-induced molecules in the radiation response must be evaluated before clinical studies are designed to eliminate each of these responses. This work was supported by NIH grants CA58508 and CA70937.

2005 Kaz

S-NITROSOHEMOGLOBIN

A’, Snyder

Duke University Kingdom’

S’, Braun Medical

MODIFIES

R’, Rosner Center,

G ‘, Pulowski

Durham,

NC,

USA’;

BLOOD

FLOW

J’, Bonaventura Gray

Laboratory

IN RAT

.I’, Stamler Research

MAMMARY .I’, Tozer Trust,

ADENOCARCINOMA

G’, Wilson Northwood,

HZ, Dewhirst Middlesex,

M’ United

Purpose: Use of stroma free hemoglobin to improve tumor oxygenation can be complicated by avid nitric oxide (NO) binding which can lead to reduced tumor blood flow and oxygenation (TBF). The mechanism of this effect relates to preferential vasoconstriction that occurs in tumor arterioles, leading to vascular steal to surrounding normal tissue (1). S-nitrosohemoglobin (SNOHb) is formed in the lung at low concentrations and plays a dominant role in control of peripheral vasomotor tone, since NO unloading is allosterically controlled by the oxygenation state of Hb (2). In this study we hypothesized that we could avoid tumor arteriolar constriction and reduction in TBF by using SNOW, since NO unloading peripherally may prevent tumor arteriolar constriction. Two doses of SNOHb were tested, one that is near physiologic levels normally encountered in vivo and the other in the range needed for therapeutic purposes. Materials and Methods: Female Fischer 344 rats with 1 cm tumors transplanted in the flank or 3-4 mm diameter skin window chamber tumors were anesthetized with nembutal (50 mg/kg). Mean arterial pressure and heart rate were measured via femoral arterial transducer and tumor blood flow with laser Doppler flowmetry. Tumor arteriolar diameter was measured in skin window chambers using intravital microscopy. Animals received either 12.9 (low dose) or 200 mg/kg (high dose) SNOHb, either iv. or i.a. while breathing either room air or 100% 0,. Results: The low dose of SNOHb in air breathing animals had no effect on MAP or HR, but TBF was reduced in both flank tumors and skin window tumors. Tumor arteriolar diameter was also reduced in the latter. When the animals breathed 100% oxygen, there was a slight pressor effect for both iv. and i.a. administration, but there was no effect on tumor blood flow or arteriolar diameter. The high dose of SNOHb had no effect on MAP or HR in either air or 100% oxygen breathing conditions. However, in both cases, TBF was reduced.

282

I. J. Radiation

Oncology

Biology

l

l

Physics

Volume

4.5, Number

3 Supplement

1999

Conclusion: The reduction in TBF during air breathing for the low dose SNOHb, suggest that normally NO is unloaded in arterial vessels upstream from tumor arterioles, leading to vasodilation and vascular steal. When animals breathe 100% oxygen, NO unloading upstream is minimized, and blood flow is maintained. The reduction in TBF at the high dose of SNOHb, regardless of the breathing air, suggests that regulation of peripheral tone by SNOHb is normally under control by very low molar quantities of SNOHb. With the higher dose of SNOHb, there is enough release of NO upstream from the tumor arterioles to still lead to vascular steal, even though its release is minimized by breathing high oxygen content gas. These results are consistent with the currently held theories regarding the activity of SNOHb in the peripheral circulation (2). These results suggest that minimization of vasoactive effects of stroma free hemoglobin solutions might be best achieved by using combinations of oxyhemoglobin and SNOW. Work supported by grants from the NIWNCI (CA 40355) the Hughes Foundation, NATO (Travel award awarded to MWD) and the Gray Laboratory Trust. References: 1. Hahn, J. S., Braun, R. D., Dewhirst, M. W., Shan, S., Snyder, S. A., Taube, J. M., Ong, E. T., Rosner, G. L., Dodge, R. K., Bonaventura, J., Bonaventura, C., DeAngelo, J.. and Meyer, R. E. Stroma-free human hemoglobin A decreases R3230Ac rat mammary adenocarcinoma blood flow and oxygen partial pressure, Radiat Res. 147: 185-94, 1997. 2. Jia, L., Bonaventura, C, Bonaventura, J, Stamler, JS S-nitrosohemoglobin: a dynamic activity of blood involved in vascular conlrol, Nature. 380: 221-226, 1996.

2006 Dubray

INFLUENCE OF OVERALL AFTER RADIOTHERAPY

BM’,

van Limbergen

Institut Curie, Paris, Houston, TX USA3

TREATMENT ALONE

E’, Thames

France’;

University

Purpose: To quantify the effect of tumor cancer treated by irradiation alone.

HD’,

TIME

van den Bogaert

Hospital

Gasthuisberg,

volume,

radiotherapy

ON LOCAL W’,

CONTROL

van des Schueren

Leuven,

Belgiun?;

dose and protraction

OF BREAST

CANCER

EZ

M.D.

Anderson

Cancer

on local control

Center,

rates m operable

breast

Patients and methods: Retrospective study of 221 operable Tis. TI -T3, NO-N1 breast carcinomas treated by exclusive radiotherapy between 1967 and 1984 at the University Hospital of Leuven, Belgium. This series was selected because the patients were treated with large variations in total dose (from 45 to 96 Gy) and overall treatment time (from 5 weeks to several months). Multivariate analyses of actuarial local control probabilities were performed by means of the proportional hazards and the mixture models. Results: Forty (18%) patients 10 years were 83% (Standard local failure being 96 months total dose of irradiation, and tumor volume, the dose Dprolif with the proportional hazards

recurred in the treated breast. The actuarial probabilities of being free of local relapse at 5 and Deviation 3%) and 79% (S.D. 3%) respectively, the mean follow-up duration for patients without (S.D. 54 months). Multivariate analyses showed a decreased risk of local failure with increasing an increased risk in patients with larger tumors and longer treatment times. After adjustment for to compensate for radiotherapy protraction was 0.22 Gy/day [95% confidence limits: 0.17,0.28] model, and 0.24 Gy/day [0.13, 0.681 with the mixture model.

Conclusion: Our results indicate exclusive irradiation in operable

2007 Olkowski Emory

FUNCTIONAL RADIATION

a moderate but significant breast cancers.

RESPONSE

OF HUMAN

detrimental

NATURAL

effect

KILLER

of longer

(NK)

treatment

CELLS

times

on local control

after

TO IONIZING

ZL University

School

of Medicirze,

Atlanta,

GA, USA

Purpose: Although lymphocytes are considered as one of the most radiosensitive cells in human, cancer patients undergoing radiation therapy show only transient lymphocytopenia and impaired response to mitogens in vitro. Furthermore, many patients lack clinical symptoms of impaired immunocompetence after radiation therapy. To clarify these contrasting findings, radiation response of human natural killer (NK) cells, crucial in the immune surveillance and destruction of tumor cells was investigated. Cytotoxic function of NK cells was used as a biological endpoint in the measurement of their radiosensitivity. The repair of DNA damage in irradiated NK cells was evaluated to explain their relative radioresistance. Methods and Materials: Peripheral blood lymphocytes obtained from five consenting healthy donors were cultured in AIM-V media supplemented with recombinant Interleukin 2 (rIL-2) for 5-21 days to sufficiently expand cytotoxic cell cultures. Lymphocytes were phenotyped on days 0, 5, 14 and 21 in cell cultures to determine the percentage of NK cells and cytotoxic T-lymphocytes in irradiated population. Upon completion of cell cultures, lymphocytes were exposed to a single dose of 1.0; 5.0, 13.0, 17.0. 25.0, 40.0, 60.0, 70.0, and 90.0 Gy of gamma radiation from the Cesium-137 source at dose rate 1.03 Gy/min. Viability of lymphocytes was measured by a Trypan Blue exclusion test before and after exposure to radiation. NK activity was measured utilizing a standard Chromium-51 release assay with K-562 target cells. Recombinant DNA repair was investigated by the sister chromatid exchange (SCE) assay and a capacity for AP sites repair was measured by AP endonuclease activity, utilizing a synthetic substrate. Results: Lymphocytes viability remained high (96.98%) throughout the experiment. The percentage of NK cells. i-IL-2 receptor+ cells and cytotoxic T Lymphocytes increased significantly in cell cultured with rIL-2 for 21 days. At the same time, cytotoxic activity of cultured NK cells gradually increased from 55 lytic units (LU) at day 0 of cell culture to 960 LU at day 21. Lymphocytes cultured for 14 and 21 days and exposed to radiation started to show a decline in NK activity at the dose level of 13 Gy and 17 Gy respectively, when compared to nonirradiated controls. However, the NK activity of cells incubated with rIL-2 for 2ldays and exposed to 90 Gy was still present at about 50% of that found in non-irradiated controls. SCE rate and the AP endonuclease activity was significantly higher in irradiated cells, when compared to controls.