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Synergism of myc with ras in the induction of radioresistance
148
Radiation
Oncology,
Biology,
Physics
October
1989, Volume
17, Supplement
1
all animals obtained a single whole body irradiation of 5.0 Gy to suppress residual NK- and transplantation, macrophage-related immunity. Groups of 10 to 12 animals were stratified according to tumor size (median treatment volume: 200~1) and randomly allocated to 4 photonand 2 fast neutron single dose regimens including one control group for each treatment. The dose rates of the 6oCo source and the d(l4)+Be neutrons were 2.9 Gy/min and 0.5 Gy/min. respectively. The tumors were rendered acutely hypoxic by clamping the tumor bed 10 min prior to Total doses ranged from 8 to 40 Gy for photons and 3 to 10 Gy for fast neutrons, and during irradiation. respectively. The endpoint of the experiments was the specific growth delay (SGD). In terms of an isoeffective SGD of 2.0 DT’s, total photonand neutron doses in the range of 3.3 to 36.7 Gy and No significant correlation was found between the DT’s of the xeno1.3 to 8.9 Gy, respectively, were necessary. grafts and their radiosensitivities or values of the relative biological effectiveness (RBE). The ranking of the tumors according to the growthand SGD agreed with a correlation coefficient of 0.9. At SGD’s of 1.0 and 2.0 The RBE’s remained constant or decreased with DT’s, RBE’s ranged from 1.7 to 6.7 and 1.5 to 6.1, respectively. increasing effect level. The RBE values were corrected for the oxygen enhancement ratio (OER) at a SGD level of to be about 1.6 and 1.2 with respect to photons and neutrons in the EL5-line. 1 and 2 DT’s, which was determined The corrected RBE-values at a SGD level of 1.0 (2.0) DT’s were in the range of 1.1 (1.0) to 4.5 (4.11, respectia significant gain for at least 4 vely. Thus assuming an RBE of 3.0 as to be adequate for normal soft tissues, out of 10 tumor lines was observed. These results indicate a potential usefulness of fast neutrons for clinical radiation therapy of soft tissue tumors as well as the considerable heterogeneity of the radiation response despite homogeneous hypoxia.
65 SYNERGISM OF MYC WITH RAS IN THE INDUCTION OF RADIORESISTANCE Marisa C. Weiss’, Ruth J. Muschel*, Vincent J. Bakanauskas l, Brian Endlich3, Clifford Ling3, Martha Sack2 and W. Gillies McKennal. Depts of Radiation Oncology1 and Pathology and Laboratory Medicine 2, University of Pennsylvania, Philadelphia, PA and Dept of Radiation Oncology3, UCSF School of Medicine, San Francisco, CA.
Resistance of tumors to irradiation or chemotherapeutic agents is thought to be one of the reasons why patients who present with early malignancies may fail to be cured. Much is now known about the molecular mechanisms that underlie drug resistance but until recently little was known about genetic contributions to radiation resistance. Some evidence now links oncogenes, particularly the ras family of oncogenes, to radiation resistance but heterogeneity between tumors and cell lines has complicated this analysis. Primary rat embryo cells (RX) have been chosen as a model system in which the effects on radiation resistance of the Hras oncogene could be studied on a uniform genetic background. These cells offer several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any pre-existing mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of a second oncogene on the appearance of the radioresistant phenotype. The primary cells and cells immortalized spontaneously or with the myc oncogene were compared to a series of independent celI lines transformed by ras alone or ras cotransfected with myc. The results show that the activated Hras oncogene is associated with radiation resistance in primary rat cells after transformation. The effect of the oncogene by itself is small but is significant at p c 0.02 when compared by Student’s t test. However, the oncogene myc, which has no effect on radiation resistance by itself, has a synergistic effect on radiation resistance with Hras (p c 0.01). There appear to be differences in the phenotype of radiation resistance associated with with these two forms of transfectants. Thus, radiation resistance seen with I-has by itself is characterized by a change in the B component of the radiation survival curve. Little or no change is seen within the shoulder region of the radiation survival curve. Radiation resistance seen in Hras plus myc transformants is also characterized by an increase in the slope of the curve at high doses but there is also a large effect on the a component within the shoulder region of the radiation survival curve. These studies led to the following conclusions; (1) the radioresistant phenotype is not due to pre-existing genetic heterogeneity in the cells prior to transfection; (2) the radiation resistant phenotype of cells transformed by Hras is seen to a greater degree in cells which also contain the myc oncogene; (3) the myc oncogene may play an important role in the phenotype of radiation resistance at low doses which is within the range most critical for clinical practice. The most important questions raised by these studies is whether the transfected oncogenes actually induce a change in the cells’ phenotype by their expression or whether these radioresistant cells represent different epigenetic or differentiation states of the cells. These could pm-exist transfection and be selected for in the transformation procedure or they could occur after oncogene transfection. To attempt to answer these questions we have now performed a series of sequential transfections. That is by first transfecting one oncogene, verifying the phenotype obtained, then hansfecting a second oncogene we can ask whether there is now a second change in cellular phenotype. The results of this analysis will be presented. Preliminary data suggest that karyotypic changes in the cells, which must have happened after transfection since we start with diploid cells, are important features which have to be considered in addition to the oncogene composition.
66 EXPRESSION
OF THE SRC ONCOGENE
DOES
Anna B. Hill, Ti Lin, Dina Millikin, Departments of Radiation Tucson, AZ, 85724
Oncology
NOT ALTER RADIOSENSITIVITY Sharon
L. Olson,
and Microbiology
David S. Shimm
and Immunology,
University
of Arizona
Cancer
Center,
Recent work in mouse NIH 3T3 and Chinese hamster cell lines has implicated the activated rd~ oncogene, whose gene product is a G-protein located in the plasma membrane, in contributing to radioresistance. Another
Radiation
Oncology,
Biology,
Physics
October
1989, Volume
17, Supplement
1
all animals obtained a single whole body irradiation of 5.0 Gy to suppress residual NK- and transplantation, macrophage-related immunity. Groups of 10 to 12 animals were stratified according to tumor size (median treatment volume: 200~1) and randomly allocated to 4 photonand 2 fast neutron single dose regimens including one control group for each treatment. The dose rates of the 6oCo source and the d(l4)+Be neutrons were 2.9 Gy/min and 0.5 Gy/min. respectively. The tumors were rendered acutely hypoxic by clamping the tumor bed 10 min prior to Total doses ranged from 8 to 40 Gy for photons and 3 to 10 Gy for fast neutrons, and during irradiation. respectively. The endpoint of the experiments was the specific growth delay (SGD). In terms of an isoeffective SGD of 2.0 DT’s, total photonand neutron doses in the range of 3.3 to 36.7 Gy and No significant correlation was found between the DT’s of the xeno1.3 to 8.9 Gy, respectively, were necessary. grafts and their radiosensitivities or values of the relative biological effectiveness (RBE). The ranking of the tumors according to the growthand SGD agreed with a correlation coefficient of 0.9. At SGD’s of 1.0 and 2.0 The RBE’s remained constant or decreased with DT’s, RBE’s ranged from 1.7 to 6.7 and 1.5 to 6.1, respectively. increasing effect level. The RBE values were corrected for the oxygen enhancement ratio (OER) at a SGD level of to be about 1.6 and 1.2 with respect to photons and neutrons in the EL5-line. 1 and 2 DT’s, which was determined The corrected RBE-values at a SGD level of 1.0 (2.0) DT’s were in the range of 1.1 (1.0) to 4.5 (4.11, respectia significant gain for at least 4 vely. Thus assuming an RBE of 3.0 as to be adequate for normal soft tissues, out of 10 tumor lines was observed. These results indicate a potential usefulness of fast neutrons for clinical radiation therapy of soft tissue tumors as well as the considerable heterogeneity of the radiation response despite homogeneous hypoxia.
65 SYNERGISM OF MYC WITH RAS IN THE INDUCTION OF RADIORESISTANCE Marisa C. Weiss’, Ruth J. Muschel*, Vincent J. Bakanauskas l, Brian Endlich3, Clifford Ling3, Martha Sack2 and W. Gillies McKennal. Depts of Radiation Oncology1 and Pathology and Laboratory Medicine 2, University of Pennsylvania, Philadelphia, PA and Dept of Radiation Oncology3, UCSF School of Medicine, San Francisco, CA.
Resistance of tumors to irradiation or chemotherapeutic agents is thought to be one of the reasons why patients who present with early malignancies may fail to be cured. Much is now known about the molecular mechanisms that underlie drug resistance but until recently little was known about genetic contributions to radiation resistance. Some evidence now links oncogenes, particularly the ras family of oncogenes, to radiation resistance but heterogeneity between tumors and cell lines has complicated this analysis. Primary rat embryo cells (RX) have been chosen as a model system in which the effects on radiation resistance of the Hras oncogene could be studied on a uniform genetic background. These cells offer several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any pre-existing mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of a second oncogene on the appearance of the radioresistant phenotype. The primary cells and cells immortalized spontaneously or with the myc oncogene were compared to a series of independent celI lines transformed by ras alone or ras cotransfected with myc. The results show that the activated Hras oncogene is associated with radiation resistance in primary rat cells after transformation. The effect of the oncogene by itself is small but is significant at p c 0.02 when compared by Student’s t test. However, the oncogene myc, which has no effect on radiation resistance by itself, has a synergistic effect on radiation resistance with Hras (p c 0.01). There appear to be differences in the phenotype of radiation resistance associated with with these two forms of transfectants. Thus, radiation resistance seen with I-has by itself is characterized by a change in the B component of the radiation survival curve. Little or no change is seen within the shoulder region of the radiation survival curve. Radiation resistance seen in Hras plus myc transformants is also characterized by an increase in the slope of the curve at high doses but there is also a large effect on the a component within the shoulder region of the radiation survival curve. These studies led to the following conclusions; (1) the radioresistant phenotype is not due to pre-existing genetic heterogeneity in the cells prior to transfection; (2) the radiation resistant phenotype of cells transformed by Hras is seen to a greater degree in cells which also contain the myc oncogene; (3) the myc oncogene may play an important role in the phenotype of radiation resistance at low doses which is within the range most critical for clinical practice. The most important questions raised by these studies is whether the transfected oncogenes actually induce a change in the cells’ phenotype by their expression or whether these radioresistant cells represent different epigenetic or differentiation states of the cells. These could pm-exist transfection and be selected for in the transformation procedure or they could occur after oncogene transfection. To attempt to answer these questions we have now performed a series of sequential transfections. That is by first transfecting one oncogene, verifying the phenotype obtained, then hansfecting a second oncogene we can ask whether there is now a second change in cellular phenotype. The results of this analysis will be presented. Preliminary data suggest that karyotypic changes in the cells, which must have happened after transfection since we start with diploid cells, are important features which have to be considered in addition to the oncogene composition.
66 EXPRESSION
OF THE SRC ONCOGENE
DOES
Anna B. Hill, Ti Lin, Dina Millikin, Departments of Radiation Tucson, AZ, 85724
Oncology
NOT ALTER RADIOSENSITIVITY Sharon
L. Olson,
and Microbiology
David S. Shimm
and Immunology,
University
of Arizona
Cancer
Center,
Recent work in mouse NIH 3T3 and Chinese hamster cell lines has implicated the activated rd~ oncogene, whose gene product is a G-protein located in the plasma membrane, in contributing to radioresistance. Another