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Michael R. Horsman - One of the best experts on this subject based on the ideXlab platform.
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relative biological effectiveness of carbon ions for tumor control acute skin damage and late radiation induced fibrosis in a Mouse model
Acta Oncologica, 2015Co-Authors: Brita Singers Sorensen, Michael R. Horsman, Jens Overgaard, Jan Alsner, Thomas Friedrich, Marco Durante, Michael Scholz, Niels BasslerAbstract:ABSTRACTBackground. The aim of the present study was to compare the biological effectiveness of carbon ions relative to x-rays between tumor control, acute skin reaction and late RIF of CDF1 mice.Material and methods. CDF1 mice with a C3H Mouse mammary carcinoma implanted subcutaneously on the foot of the right hind limb were irradiated with single fractions of either photons, or 12C ions using a 30-mm spread-out Bragg peak. The endpoint of the study was local control (no tumor recurrence within 90 days). For the acute skin reaction, non-tumor bearing CDF1 mice were irradiated with a comparable radiation scheme, and monitored for acute skin damage between Day 7 and 40. Late RIF was assessed in the irradiated mice.Results. The TCD50 (dose producing tumor control in 50% of mice) values with 95% confidence interval were 29.7 (25.4–34.8) Gy for C ions and 43.9 (39.2–49.2) Gy for photons, with a corresponding Relative biological effectiveness (RBE) value of 1.48 (1.28–1.72). For acute skin damage the MDD50 (do...
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relationship between radiobiological hypoxia in a C3H Mouse mammary carcinoma and osteopontin levels in Mouse serum
International Journal of Radiation Biology, 2005Co-Authors: Slavka Lukacova, Jan Alsner, Jens Overgaard, A A Khalil, Michael R. HorsmanAbstract:Purpose: To investigate the possible relationship between radiobiological hypoxia in a C3H Mouse mammary carcinoma and osteopontin (OPN) levels measured in Mouse serum.Material and methods: Experiments were performed in CDF1 mice that were either non-tumour bearing or with different sized tumours implanted in the right rear foot. Osteopontin levels in extracted Mouse blood serum and tissue from the transplanted tumours were measured using an ELISA assay. The tumour oxygenation status was estimated using the Eppendorf Histograph and the fraction of oxygen partial pressure (pO2) values ≤5 mm Hg (HF5) was calculated. Necrosis was measured in haematoxylin and eosin-stained sections. Tumour hypoxia was increased by placing animals in a low-oxygen (10%) environment. Single radiation doses (240 kV x-rays) were given locally to tumours under ambient or clamped conditions and response assessed using a tumour control assay.Results: Serum OPN levels increased linearly with increasing tumour volume and this increase ...
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tumour specific enhancement of thermoradiotherapy at mild temperatures by the vascular targeting agent 5 6 dimethylxanthenone 4 acetic acid
International Journal of Hyperthermia, 2004Co-Authors: Rumi Murata, Michael R. HorsmanAbstract:The effect of combining the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) with both radiation and hyperthermia treatments was investigated in a transplanted C3H Mouse mammary carcinoma and a normal Mouse tissue. Tumours were grown on the right rear foot of female CDF1 mice and treated when sized 200 mm3. The foot skin of non-tumour-bearing CDF1 mice was used to assess normal tissue damage. Radiation and hyperthermia were given locally to the tumour/skin of restrained non-anaesthetized animals. DMXAA (20 mg/kg) was dissolved in saline and injected intraperitoneally 1 h after irradiating and then heating started 3 h later. The endpoints were local tumour control within 90 days or the development of moist desquamation in skin between 11 and 23 days after treatment. The radiation dose (+/- 95% confidence intervals) producing local tumour control in 50% of treated animals was 53 (51-55) Gy for radiation alone. This value was significantly (Chi-squared test; p < 0.05) decreased to 47 (42-52) Gy by DMXAA and to 47 (44-51) Gy by heating (41.5 degrees C/60 min) 4 h after irradiation. Combining both DMXAA and heating further reduced this to 30 (26-35) Gy. When the heating temperature was decreased to 40.5 degrees C, the effect of the triple combination was decreased but was still significant compared with radiation + DMXAA or radiation + hyperthermia. However, this enhancement disappeared at 39.5 degrees C. Radiation damage of normal foot skin was not enhanced by combining DMXAA and hyperthermia at 41.5 degrees C. In conclusion, adding DMXAA to thermoradiotherapy at 40.5-41.5 degrees C significantly improved local tumour control without enhancing normal tissue damage. Thus, including a vascular targeting agent in a mild thermoradiotherapy treatment regimen is a useful approach that may lead to a re-evaluation of the use of hyperthermia in cancer treatment.
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preclinical studies on how to deal with patient intolerance to nicotinamide and carbogen
Radiotherapy and Oncology, 2004Co-Authors: Michael R. Horsman, Jens OvergaardAbstract:Abstract Background and purpose Accelerated radiation carbogen nicotinamide (ARCON) therapy is generally well tolerated, but some patients experience intolerance to nicotinamide and carbogen (95% O 2 +5% CO 2 ). This study investigated the effect of reducing both the nicotinamide dose and carbogen CO 2 content on radiation response. Materials and methods A C3H Mouse mammary carcinoma grown in the right rear foot of female CDF1 was used and treated when at 200 mm 3 . Nicotinamide was intraperitoneally injected 20 min prior to irradiation. Carbogen (CO 2 content of either 2 or 5%, balance O 2 ) breathing was started 5 min before, and continued during, additional treatments. Radiation was given locally to tissues of restrained non-anaesthetised mice either as a single or fractionated (10 fractions in 12 days) schedule. The endpoints were local tumour control at 90 days, development of moist desquamation 11–23 days after treatment of normal foot skin, or tumour oxygenation measured with the Eppendorf electrode. Results The TCD50 values in this tumour following single or fractionated radiation treatment were 52 and 71Gy, respectively. Carbogen (5% CO 2 content) breathing with every radiation treatment in the fractionated schedule significantly (Chi-squared test; P 2 content of the gas breathing was varied from 0% (i.e. 100% O 2 ) to 2 or 5% (balance O 2 ), although a CO 2 content of 2% did give a smaller enhancement of radiation-induced normal skin damage than 5%. Conclusions Both the nicotinamide dose, but not the frequency, and carbogen CO 2 content may be reduced in patients experience intolerance without any significant loss of sensitisation.
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vascular targeting effects of zd6126 in a C3H Mouse mammary carcinoma and the enhancement of radiation response
International Journal of Radiation Oncology Biology Physics, 2003Co-Authors: Michael R. Horsman, Rumi MurataAbstract:Abstract Purpose The aim of this study was to investigate the pathophysiologic effects induced by the novel vascular targeting agent ZD6126 in a C3H Mouse mammary carcinoma and to evaluate the agent's ability to inhibit tumor growth either when given alone or in combination with radiation. Methods and materials A C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was treated when at 200 mm 3 in size. ZD6126 was dissolved in saline and injected intraperitoneally. Blood perfusion was measured using the RbCl extraction procedure, tumor oxygen (pO 2 ) status was assessed with the Eppendorf electrode, and tumor necrosis was estimated from histologic sections. Radiation (240-kV X-rays) was locally administered to tumors of restrained nonanesthetized mice, and response was assessed using a tumor growth assay. Results ZD6126 induced a significant dose- and time-dependent decrease in tumor perfusion, reaching a maximal 70% reduction around 3 h after injecting 150–300 mg/kg. However, full recovery was seen within 6 h. A 200 mg/kg dose significantly decreased tumor oxygenation status at 3 h (median pO 2 decreased from 7 to 3 mm Hg; % pO 2 values ≤2.5 mm Hg increased from 30% to 55%) and by 24 h had significantly increased necrotic fraction from 14.5% to 25.2%. This ZD6126 dose resulted in a small, yet significant, 1.4 days inhibition of tumor growth when given alone. It also enhanced the tumor response to radiation, giving rise to a significant 1.3-fold increase in the slope of the radiation dose–response curve. Of the normal tissues, only muscle (at 3 h) and spleen (at 6 h) showed any significant reduction in perfusion after injecting 200 mg/kg, but these transient decreases were only 32% and 49%, respectively. Conclusions Our preclinical studies clearly demonstrate a tumor-specific reduction in blood perfusion by ZD6126. Although these changes were transient, they were sufficient to increase tumor necrosis, inhibit tumor growth, and enhance radiation response.
Jens Overgaard - One of the best experts on this subject based on the ideXlab platform.
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relative biological effectiveness of carbon ions for tumor control acute skin damage and late radiation induced fibrosis in a Mouse model
Acta Oncologica, 2015Co-Authors: Brita Singers Sorensen, Michael R. Horsman, Jens Overgaard, Jan Alsner, Thomas Friedrich, Marco Durante, Michael Scholz, Niels BasslerAbstract:ABSTRACTBackground. The aim of the present study was to compare the biological effectiveness of carbon ions relative to x-rays between tumor control, acute skin reaction and late RIF of CDF1 mice.Material and methods. CDF1 mice with a C3H Mouse mammary carcinoma implanted subcutaneously on the foot of the right hind limb were irradiated with single fractions of either photons, or 12C ions using a 30-mm spread-out Bragg peak. The endpoint of the study was local control (no tumor recurrence within 90 days). For the acute skin reaction, non-tumor bearing CDF1 mice were irradiated with a comparable radiation scheme, and monitored for acute skin damage between Day 7 and 40. Late RIF was assessed in the irradiated mice.Results. The TCD50 (dose producing tumor control in 50% of mice) values with 95% confidence interval were 29.7 (25.4–34.8) Gy for C ions and 43.9 (39.2–49.2) Gy for photons, with a corresponding Relative biological effectiveness (RBE) value of 1.48 (1.28–1.72). For acute skin damage the MDD50 (do...
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relationship between radiobiological hypoxia in a C3H Mouse mammary carcinoma and osteopontin levels in Mouse serum
International Journal of Radiation Biology, 2005Co-Authors: Slavka Lukacova, Jan Alsner, Jens Overgaard, A A Khalil, Michael R. HorsmanAbstract:Purpose: To investigate the possible relationship between radiobiological hypoxia in a C3H Mouse mammary carcinoma and osteopontin (OPN) levels measured in Mouse serum.Material and methods: Experiments were performed in CDF1 mice that were either non-tumour bearing or with different sized tumours implanted in the right rear foot. Osteopontin levels in extracted Mouse blood serum and tissue from the transplanted tumours were measured using an ELISA assay. The tumour oxygenation status was estimated using the Eppendorf Histograph and the fraction of oxygen partial pressure (pO2) values ≤5 mm Hg (HF5) was calculated. Necrosis was measured in haematoxylin and eosin-stained sections. Tumour hypoxia was increased by placing animals in a low-oxygen (10%) environment. Single radiation doses (240 kV x-rays) were given locally to tumours under ambient or clamped conditions and response assessed using a tumour control assay.Results: Serum OPN levels increased linearly with increasing tumour volume and this increase ...
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intravascular contrast agent enhanced mri measuring contrast clearance and tumor blood volume and the effects of vascular modifiers in an experimental tumor
International Journal of Radiation Oncology Biology Physics, 2005Co-Authors: Lise Entze, Jens Overgaard, Pete Vestergaardpoulse, Thomas Nielse, Atle Jornerud, Kare C Ileysaebo, Michael R Horsma, Leif OstergaardAbstract:Purpose To examine the feasibility of using the MRI blood pool agent NC100150 for evaluation of tumor blood volume (TBV) estimates by both dynamic contrast-enhanced MRI (DCE-MRI) and susceptibility contrast MRI assays in an experimental tumor. Contrast agent clearance (K trans ; depends on perfusion and permeability) from the DCE-MRI time curves was estimated, and changes in TBV and K trans were measured after administration of two drugs that reduce perfusion by different mechanisms. Methods and materials The DCE-MRI experiments were simulated with expected physiologic values for the C3H Mouse mammary carcinoma. The C3H tumor was examined by DCE-MRI and susceptibility contrast MRI with NC100150 (NC100150 Injection, Clariscan; Amersham Health, Oslo, Norway) after treatment with either hydralazine or combretastatin (Oxigene, Boston, MA). Results Simulations showed that reliable estimates of changes in TBV and K trans could be performed with DCE-MRI. Hydralazine was shown to reduce TBV as measured by both assays and to reduce K trans . Dynamic contrast-enhanced MRI also suggested that TBV and K trans were reduced in combretastatin-treated tumors, and the TBV reduction was confirmed by susceptibility contrast MRI. Data suggested the drug to affect mainly the total TBV, whereas microvessels as such seemed less altered. Conclusion The study supports the use of the combined DCE-MRI and susceptibility contrast MRI assay with a blood pool agent in characterizing tumors and their response to treatment.
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preclinical studies on how to deal with patient intolerance to nicotinamide and carbogen
Radiotherapy and Oncology, 2004Co-Authors: Michael R. Horsman, Jens OvergaardAbstract:Abstract Background and purpose Accelerated radiation carbogen nicotinamide (ARCON) therapy is generally well tolerated, but some patients experience intolerance to nicotinamide and carbogen (95% O 2 +5% CO 2 ). This study investigated the effect of reducing both the nicotinamide dose and carbogen CO 2 content on radiation response. Materials and methods A C3H Mouse mammary carcinoma grown in the right rear foot of female CDF1 was used and treated when at 200 mm 3 . Nicotinamide was intraperitoneally injected 20 min prior to irradiation. Carbogen (CO 2 content of either 2 or 5%, balance O 2 ) breathing was started 5 min before, and continued during, additional treatments. Radiation was given locally to tissues of restrained non-anaesthetised mice either as a single or fractionated (10 fractions in 12 days) schedule. The endpoints were local tumour control at 90 days, development of moist desquamation 11–23 days after treatment of normal foot skin, or tumour oxygenation measured with the Eppendorf electrode. Results The TCD50 values in this tumour following single or fractionated radiation treatment were 52 and 71Gy, respectively. Carbogen (5% CO 2 content) breathing with every radiation treatment in the fractionated schedule significantly (Chi-squared test; P 2 content of the gas breathing was varied from 0% (i.e. 100% O 2 ) to 2 or 5% (balance O 2 ), although a CO 2 content of 2% did give a smaller enhancement of radiation-induced normal skin damage than 5%. Conclusions Both the nicotinamide dose, but not the frequency, and carbogen CO 2 content may be reduced in patients experience intolerance without any significant loss of sensitisation.
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combretastatin a 4 disodium phosphate a vascular targeting agent that improves that improves the anti tumor effects of hyperthermia radiation and mild thermoradiotheraphy
International Journal of Radiation Oncology Biology Physics, 2001Co-Authors: Rumi Murata, Jens Overgaard, Michael R. HorsmanAbstract:Abstract Purpose: To investigate the effect of combining the vascular targeting drug combretastatin A-4 disodium phosphate (CA4DP) with hyperthermia, radiation, or mild thermoradiotherapy in a transplanted C3H Mouse mammary carcinoma. Methods and Materials: The C3H mammary carcinoma was grown on the rear foot of female CDF1 mice and treated when at 200 mm 3 in size. CA4DP was dissolved in saline and injected i.p. Hyperthermia and/or radiation were locally given to tumors in restrained nonanesthetized mice. Tumor response was assessed using either a tumor growth or a tumor control assay. Mouse foot skin was used to assess normal tissue damage. Results: CA4DP significantly enhanced thermal damage in this tumor model. This effect was independent of drug doses between 25–400 mg/kg, but was strongly dependent on the time interval between drug injection and heating, with the greatest improvement seen when CA4DP preceded the heating by 1 h or less. There was also a suggestion of a temperature dependency with a 1.9-fold increase in heat damage at 42.5°C and a 2.6-fold increase at 41.5 and 40.5°C. Heat-induced normal tissue damage was also enhanced by combining CA4DP with heat, but the degree of enhancement was less than that seen in tumors. CA4DP (25 mg/kg) significantly increased radiation-induced local tumor control and this was further enhanced by combining CA4DP with mild temperature (41.5°C, 60 min) heating. Conclusions: CA4DP improved the anti-tumor effect of hyperthermia, especially at mild temperatures. More importantly, it also increased the tumor response to mild hyperthermia and radiation, which suggests that CA4DP may ultimately have an important application in clinical thermoradiotherapy.
Rumi Murata - One of the best experts on this subject based on the ideXlab platform.
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tumour specific enhancement of thermoradiotherapy at mild temperatures by the vascular targeting agent 5 6 dimethylxanthenone 4 acetic acid
International Journal of Hyperthermia, 2004Co-Authors: Rumi Murata, Michael R. HorsmanAbstract:The effect of combining the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) with both radiation and hyperthermia treatments was investigated in a transplanted C3H Mouse mammary carcinoma and a normal Mouse tissue. Tumours were grown on the right rear foot of female CDF1 mice and treated when sized 200 mm3. The foot skin of non-tumour-bearing CDF1 mice was used to assess normal tissue damage. Radiation and hyperthermia were given locally to the tumour/skin of restrained non-anaesthetized animals. DMXAA (20 mg/kg) was dissolved in saline and injected intraperitoneally 1 h after irradiating and then heating started 3 h later. The endpoints were local tumour control within 90 days or the development of moist desquamation in skin between 11 and 23 days after treatment. The radiation dose (+/- 95% confidence intervals) producing local tumour control in 50% of treated animals was 53 (51-55) Gy for radiation alone. This value was significantly (Chi-squared test; p < 0.05) decreased to 47 (42-52) Gy by DMXAA and to 47 (44-51) Gy by heating (41.5 degrees C/60 min) 4 h after irradiation. Combining both DMXAA and heating further reduced this to 30 (26-35) Gy. When the heating temperature was decreased to 40.5 degrees C, the effect of the triple combination was decreased but was still significant compared with radiation + DMXAA or radiation + hyperthermia. However, this enhancement disappeared at 39.5 degrees C. Radiation damage of normal foot skin was not enhanced by combining DMXAA and hyperthermia at 41.5 degrees C. In conclusion, adding DMXAA to thermoradiotherapy at 40.5-41.5 degrees C significantly improved local tumour control without enhancing normal tissue damage. Thus, including a vascular targeting agent in a mild thermoradiotherapy treatment regimen is a useful approach that may lead to a re-evaluation of the use of hyperthermia in cancer treatment.
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vascular targeting effects of zd6126 in a C3H Mouse mammary carcinoma and the enhancement of radiation response
International Journal of Radiation Oncology Biology Physics, 2003Co-Authors: Michael R. Horsman, Rumi MurataAbstract:Abstract Purpose The aim of this study was to investigate the pathophysiologic effects induced by the novel vascular targeting agent ZD6126 in a C3H Mouse mammary carcinoma and to evaluate the agent's ability to inhibit tumor growth either when given alone or in combination with radiation. Methods and materials A C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was treated when at 200 mm 3 in size. ZD6126 was dissolved in saline and injected intraperitoneally. Blood perfusion was measured using the RbCl extraction procedure, tumor oxygen (pO 2 ) status was assessed with the Eppendorf electrode, and tumor necrosis was estimated from histologic sections. Radiation (240-kV X-rays) was locally administered to tumors of restrained nonanesthetized mice, and response was assessed using a tumor growth assay. Results ZD6126 induced a significant dose- and time-dependent decrease in tumor perfusion, reaching a maximal 70% reduction around 3 h after injecting 150–300 mg/kg. However, full recovery was seen within 6 h. A 200 mg/kg dose significantly decreased tumor oxygenation status at 3 h (median pO 2 decreased from 7 to 3 mm Hg; % pO 2 values ≤2.5 mm Hg increased from 30% to 55%) and by 24 h had significantly increased necrotic fraction from 14.5% to 25.2%. This ZD6126 dose resulted in a small, yet significant, 1.4 days inhibition of tumor growth when given alone. It also enhanced the tumor response to radiation, giving rise to a significant 1.3-fold increase in the slope of the radiation dose–response curve. Of the normal tissues, only muscle (at 3 h) and spleen (at 6 h) showed any significant reduction in perfusion after injecting 200 mg/kg, but these transient decreases were only 32% and 49%, respectively. Conclusions Our preclinical studies clearly demonstrate a tumor-specific reduction in blood perfusion by ZD6126. Although these changes were transient, they were sufficient to increase tumor necrosis, inhibit tumor growth, and enhance radiation response.
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combretastatin a 4 disodium phosphate a vascular targeting agent that improves that improves the anti tumor effects of hyperthermia radiation and mild thermoradiotheraphy
International Journal of Radiation Oncology Biology Physics, 2001Co-Authors: Rumi Murata, Jens Overgaard, Michael R. HorsmanAbstract:Abstract Purpose: To investigate the effect of combining the vascular targeting drug combretastatin A-4 disodium phosphate (CA4DP) with hyperthermia, radiation, or mild thermoradiotherapy in a transplanted C3H Mouse mammary carcinoma. Methods and Materials: The C3H mammary carcinoma was grown on the rear foot of female CDF1 mice and treated when at 200 mm 3 in size. CA4DP was dissolved in saline and injected i.p. Hyperthermia and/or radiation were locally given to tumors in restrained nonanesthetized mice. Tumor response was assessed using either a tumor growth or a tumor control assay. Mouse foot skin was used to assess normal tissue damage. Results: CA4DP significantly enhanced thermal damage in this tumor model. This effect was independent of drug doses between 25–400 mg/kg, but was strongly dependent on the time interval between drug injection and heating, with the greatest improvement seen when CA4DP preceded the heating by 1 h or less. There was also a suggestion of a temperature dependency with a 1.9-fold increase in heat damage at 42.5°C and a 2.6-fold increase at 41.5 and 40.5°C. Heat-induced normal tissue damage was also enhanced by combining CA4DP with heat, but the degree of enhancement was less than that seen in tumors. CA4DP (25 mg/kg) significantly increased radiation-induced local tumor control and this was further enhanced by combining CA4DP with mild temperature (41.5°C, 60 min) heating. Conclusions: CA4DP improved the anti-tumor effect of hyperthermia, especially at mild temperatures. More importantly, it also increased the tumor response to mild hyperthermia and radiation, which suggests that CA4DP may ultimately have an important application in clinical thermoradiotherapy.
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potentiation of the anti tumour effect of hyperthermia by combining with the vascular targeting agent 5 6 dimethylxanthenone 4 acetic acid
International Journal of Hyperthermia, 2001Co-Authors: Rumi Murata, Jens Overgaard, Michael R. HorsmanAbstract:The potential of the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) to enhance the effect of hyperthermia was investigated in a C3H Mouse mammary carcinoma grown in the feet of female CDF1 mice and in normal foot skin. DMXAA, when injected intraperitoneally in restrained non-anaesthetized animals, reduced tumour perfusion, as measured using the RbCl extraction procedure, and increased necrosis in histological section, but these effects were dependent on the drug dose and time interval. At a dose of 20mg/kg, it significantly enhanced the thermal damage of this tumour, when given 1h or more before the start of heating, as assessed by a tumour growth assay. This enhancement became larger with increasing interval between the two treatments. No thermo-potentiation was seen at doses of 10mg/kg or lower. These combined effects seem to be associated with the tumour vascular shut-down by DMXAA. Thermal potentiation by DMXAA was also dependent on the heating temperature, with a greater enhanc...
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combretastatins novel vascular targeting drugs for improving anti cancer therapy combretastatins and conventional therapy
Advances in Experimental Medicine and Biology, 2000Co-Authors: Michael R. Horsman, Rumi Murata, T Breidahl, F U Nielsen, R J Maxwell, H Stodkiledjorgensen, Jens OvergaardAbstract:Combretastatins are a new class of compounds that appear to have anti-tumour activity as a result of specifically targeting the vasculature of tumours. The aim of this study was to investigate the potential of combretastatin A-4 disodium phosphate (CA4DP) to induce vascular effects in a C3H Mouse mammary carcinoma, and to see if the anti-tumour response could be improved by combining the drug with conventional anti-cancer therapies. It was found that CA4DP (250 mg/kg) significantly decreased tumour perfusion within 30 minutes after injection and maintained this decrease for several hours, although there was a return to normal by 24 hours. Similar changes were seen in the tumours bioenergetic and oxygenation status. The drug also significantly increased tumour necrosis and had a small inhibitory effect on tumour growth. It was also able to enhance the tumour response to radiation and hyperthermia, when given at the same time or 30 minutes after the radiation and hyperthermia, respectively. Giving the drug 1 hour after cisplatin injection only resulted in a tumour response that was no greater than additive. These results confirm the anti-vascular effects of CA4DP and demonstrate its potential to enhance the anti-tumour activity of conventional therapy.
Jaquelin P Dudley - One of the best experts on this subject based on the ideXlab platform.
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C3H Mouse mammary tumor virus superantigen function requires a splice donor site in the envelope gene
Journal of Virology, 2000Co-Authors: Farah Mustafa, Mary M Lozano, Jaquelin P DudleyAbstract:Mouse mammary tumor virus (MMTV) is transmitted from the milk of infected mothers to the gut of susceptible offspring (33). MMTV infects B cells in the guts of newborn mice, and these cells express the virally encoded superantigen (Sag) at the plasma membrane in association with the major histocompatibility complex (MHC) class II protein (1, 28). Sag is a type II transmembrane protein that is required for efficient transmission of milk-borne MMTV from the gut to the mammary gland (12, 17, 23). The Sag-MHC complex is recognized by entire classes of T cells bearing particular T-cell receptor (TCR) β chains (18, 28). Recognition of Sag by specific T cells leads to cell proliferation and/or release of cytokines, and the released cytokines recruit additional B and T lymphocytes that are infected by MMTV (27). B-cell-deficient mice or mice lacking Sag-reactive T cells cannot be efficiently infected by MMTV (4, 12). Ultimately, viral infection of the mammary gland is necessary to allow MMTV release into the milk. Mice that lack B cells or Sag-specific T cells also are defective in the spread of MMTV within the mammary gland (14). Thus, Sag is required for generation of a reservoir of virally infected B and T lymphocytes that are involved in MMTV transmission and viral spread to the mammary tissues. The regulation of MMTV sag expression is controversial. Early studies indicated that Sag is translated from a singly spliced mRNA that initiates at the U3/R border of the viral long terminal repeat (LTR) from the predominant U3 promoter (21, 41). This U3 promoter also drives expression of the viral structural genes, gag, pol, and env (for a review, see reference 10) (Fig. (Fig.1).1). Cloning and sequencing revealed that this sag mRNA uses a single splice donor site in the leader region that also is used for the generation of spliced envelope mRNA (32). The splice acceptor site for this sag mRNA is located in the envelope region just upstream of the 3′ LTR (21). Multiple start codons are located near the 5′ end of this mRNA, and mutagenesis experiments have suggested that the first or second codons can suffice for functional Sag production in cell culture (8). Subsequently, at least three other potential sag mRNAs have been described (Fig. (Fig.1).1). One of these mRNAs uses the same splice donor and acceptor sites as those described earlier, except that this transcript initiates approximately 500 bp upstream of the standard viral RNAs (16). Another transcript initially was identified as a phorbol ester-inducible, cyclosporine-suppressible RNA in a T-cell lymphoma (11, 31, 38). This sag-specific RNA is initiated from an intragenic promoter within the envelope-coding region; the transcript uses a unique splice donor site within the envelope region and the same splice acceptor site as the other two sag RNAs. Most recently, a different sag promoter has been described within the envelope region (2). This promoter was shown to be active in transient transfection experiments with reporter gene constructs. The resulting sag mRNA appears to be unspliced and initiated within 100 bp of the 3′ LTR (2). FIG. 1 Diagram of the MMTV sag-specific mRNAs and the location of the splice donor mutation. (A) Schematic representation of the MMTV genome and reported MMTV sag mRNAs. The boxes on the MMTV proviral genome show the positions of the indicated open reading frames ... Using PCR assays, our experiments have shown that all of the spliced sag transcripts are detectable in lymphocytes of BALB/c mice that contain three endogenous MMTVs, Mtv-6, Mtv-8, and Mtv-9 (9, 24). However, only the spliced sag transcript from the envelope promoter was detectable in cells infected in vitro or in vivo with milk-borne C3H MMTV. Similarly, deletion mutants of a C3H MMTV-derived infectious molecular clone (39) suggested that an intragenic envelope promoter and an enhancer in the pol gene were responsible for sag expression (34, 35). To test whether the spliced mRNA from the env promoter is required for C3H MMTV sag expression, we used the C3H-derived infectious molecular clone to construct a mutant with alterations in the splice donor site within the envelope region that is unique to this transcript. Stable transfections of this mutant or a control frameshift mutant with an alteration within the sag coding region that has been shown to abolish Sag function (13) produced virus-expressing cell lines. Both the splice donor and frameshift mutants induced some mammary tumors with long latency and sporadic T-cell deletion after direct injection of transfected cells. However, neither the frameshift nor the splice donor mutant was transmissible to susceptible third-litter progeny through the milk-borne route. These experiments show that the spliced mRNA from the envelope region is necessary and sufficient for efficient C3H MMTV milk-borne transmission.
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Mutational and Functional Analysis of the C-Terminal Region of the C3H Mouse Mammary Tumor Virus Superantigen
Journal of Virology, 1998Co-Authors: Thomas J. Wrona, Mary M Lozano, Awadh A. Binhazim, Jaquelin P DudleyAbstract:Mouse mammary tumor virus (MMTV) is transmitted through the germ line as integrated proviral DNA (endogenous viruses) or through maternal milk to susceptible offspring (exogenous or milk-borne virus) (10). Milk-borne MMTV infects B cells in gut-associated lymphoid tissue (29), where superantigen (Sag) is presented at the cell surface as a type II transmembrane glycoprotein in conjunction with major histocompatibility complex (MHC) class II protein (25, 32). The Sag-MHC complex interacts with particular variable regions of the beta chain (Vβ) of the T-cell receptor (TCR) on the surface of T cells, causing these cells to release cytokines and to proliferate (20, 25). The release of cytokines stimulates neighboring B and T cells to divide, creating additional target cells for MMTV integration and expanding the pool of cells that previously have been infected (20, 25). The infected lymphoid cells then act as a reservoir for infection of the mammary gland when this tissue begins development during puberty. Recent results have shown that both T cells and B cells are required for MMTV transmission from infected milk in the gut to the mammary gland (3, 14, 21). Other experiments have shown that injection of MMTV-infected CD4+ or CD8+ T cells as well as infected B cells will transfer viral infection to susceptible mice (60). All known MMTVs encode Sag within the U3 region of the long terminal repeat (LTR) (5); this region also specifies many of the viral transcriptional regulatory sequences (6, 23, 36, 37, 42, 47). Expression of the endogenous MMTV Sag proteins results in the deletion of reactive T cells through the process of negative selection in the thymus (25), whereas expression of Sag protein from milk-borne virus is believed to result in stimulation and proliferation of cognate T cells followed by a gradual deletion of these cells (40). Thus, the complement of endogenous MMTV strains determines whether reactive T cells are available for exogenous MMTV infection (21). Indeed, previous experiments have shown that expression of the exogenous C3H MMTV sag gene from the germ line of transgenic animals is sufficient to prevent infection by milk-borne C3H virus (14). Sag is a type II transmembrane protein that contains a small N-terminal intracellular domain (32) and a large extracellular C terminus that interacts with the Vβ portion of the TCR (8). Sequence comparisons of the Sag proteins from several MMTV strains showed that there was a high degree of sequence identity between Sag molecules in two regions called polymorphic regions I (amino acids 164 to 198) and II (from amino acid 288 to the C terminus) (67). C-terminal variability in polymorphic regions I and II correlated well with observations that certain Sag molecules reacted with particular TCRs (67); e.g., the C3H and GR exogenous Sags reacted with T cells bearing Vβ14 chains (7). Moreover, experiments performed by Yazdanbakhsh et al. showed that substitution of the polymorphic region II of endogenous Mtv-1 Sag (Vβ3 reactive) for the polymorphic region II of Mtv-7 Sag (Vβ6 reactive) allowed the recombinant Sag to react with Vβ6+ T cells in stable transfection assays (67). The reciprocal experiment confirmed that polymorphic Sag region II (30 to 40 amino acids) at the C terminus is sufficient to specify interactions with certain TCR Vβ chains (67); however, the C-terminal half of Sag is insufficient to allow Sag function (34). Alignment of C-terminal Sag amino acids (Fig. (Fig.1)1) has been used to construct phylogenetic trees to predict relatedness among various MMTV strains (5). FIG. 1 Comparison of C-terminal sequences of MMTV Sag molecules and their TCR Vβ specificities. Amino acid identities are shown by dots compared to the C3H MMTV sequence. Sequence information was obtained from previous reports (1, 2, 17, 24, 26– ... Although the C-terminal region of Sag has been shown to specify interactions with the TCR (67), little is known about the amino acid requirements within this region for Sag function. Therefore, a series of BglII linker substitutions and deletions were created within the region encoding the C terminus of Sag, and these substitution mutants were transferred into a cloned, infectious MMTV provirus (50) for in vivo analysis of C3H Sag function and effects on MMTV transmission and tumorigenicity. These experiments showed that virtually all C-terminal amino acid substitutions abolished Sag function and that sag mutant viruses failed to induce tumors in injected mice. However, mutants that lacked Sag function, but had overlapping mutations in a negative regulatory element (NRE) affecting MMTV transcription (4, 37), retained the ability to induce mammary tumors in mutant-injected animals. All mutants, except one that affected the C-terminal three amino acids and retained partial Sag function (HPA242), lost the ability to be transmitted through milk to susceptible offspring. Thus, virtually any mutation within the C-terminal region alters Sag function.
Edward H Birkenmeier - One of the best experts on this subject based on the ideXlab platform.
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intracisternal a particle element transposition into the murine beta glucuronidase gene correlates with loss of enzyme activity a new model for beta glucuronidase deficiency in the C3H Mouse
Molecular and Cellular Biology, 1998Co-Authors: B Gwynn, Mark S. Sands, Kira Lueders, Edward H BirkenmeierAbstract:The severity of human mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, depends on the relative activity of the enzyme β-glucuronidase. Loss of β-glucuronidase activity can cause hydrops fetalis, with in utero or postnatal death of the patient. In this report, we show that β-glucuronidase activity is not detectable by a standard fluorometric assay in C3H/HeOuJ (C3H) mice homozygous for a new mutation, gusmps2J. These gusmps2J/gusmps2J mice are born and survive much longer than the previously characterized β-glucuronidase-null B6.C-H-2bm1/ByBir-gusmps (gusmps/gusmps) mice. Northern blot analysis of liver from gusmps2J/gusmps2J mice demonstrates a 750-bp reduction in size of β-glucuronidase mRNA. A 5.4-kb insertion in the Gus-sh nucleotide sequence from these mice was localized by Southern blot analysis to intron 8. The ends of the inserted sequences were cloned by inverse PCR and revealed an intracisternal A-particle (IAP) element inserted near the 3′ end of the intron. The sequence of the long terminal repeat (LTR) regions of the IAP most closely matches that of a composite LTR found in transposed IAPs previously identified in the C3H strain. The inserted IAP may contribute to diminished β-glucuronidase activity either by interfering with transcription or by destabilizing the message. The resulting phenotype is much less severe than that previously described in the gusmps/gusmps Mouse and provides an opportunity to study MPS VII on a genetic background that clearly modulates disease severity.
