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Cataract induction after total body irradiation and bone marrow transplantation: Dose-effect relationship
288
1. .I. Radiation
Oncology
l Biology
l Physic\
Volume 48, Number 3. Supplement.
2000
membrane for Western blotting. Blots were incubated with antibodies ngain\t the non-homologous end-joining (NHEJ) proteins Mrel I, RadSO. or NBSI. and protein hands were detected and later quantitatcd using a chemiluminescent detection system. Results: We found that Mrel I. RadSO, and NBS I (components of a multi-protein complex involved in NHEJ of DNA DSBs) are tranalocated from the nucleus to the cytoplasm when human LJ- I melanoma cells are heated for 15 min at 455°C. or when cells are heated after irradiation with 12 Gy of x-rays. The magnitude of migration of these proteins to the cytoplasm was dependent upon post-treatment incubation time at 37” C. and both the kinetic? and magnitude of translocation were dependent upon whether or not cells were irradiated prior to heating. No such translocation was observed when cell\ were treated with Iradiation alone. When cells were irradiated just prior to heating. the translocation occurred earlier (migration to the cytoplasm began during heating). Additionally, a phosphorylated form of NBS I was detected in the cytoplasmic fraction of heated irradiated cells after translocation. Tran\location ofRadS0 and NBS1 (but not Mrel I) was inhibited when heated irradiated cells were incubated in the presence of leptomycin B (which inhibit\ CRMI-mediated nuclear export). Conclusion: We propose that heat-induced redistribution or alterations of critical NHEJ proteins lead to inhibition repair after irradiation. and may be implicated. at least in part, as a mechanism for heat-radiosensitization.
2047
Diminished
DNA repair and elevated mutagenesis
P. M. Cilafer. J. Yuan, S. Rockwell.
L. Narayanan
Yclle Lirlil’rr.$,’ School 0f Mrtlicine.
NC,\\,Htn’rrr, CT
in mammalian
of DNA
cells exposed to hypoxia and low pH
Purpose: The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH and nutrient deprivation. It has been proposed that this unique tissue environment may. itself. constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could intluence cellular repair of DNA damage. 3Materials and Methods: An assay for DNA repair was established based on reactivation of UV-damaged plasmid DNA. This type of assay, termed ‘host cell reactivation’ (HCR). has been employed in a variety of studies to assess DNA repair in viva. DNA substrates containing pre-formed damage are introduced into cells, and the ability of the cells to repair the damage in the transfected DNA is probed under various conditions. In our experiments, plasmid pGL3. which encodes il luciferase gene, was damaged by UV irradiation at a dose of 5000 J/mZ. This damaged plasmid DNA. or an equal amount of undamaged luciferase plasmid DNA. was transfected into mouse fihroblast cells which were immediately placed under either normal culture conditions or under the hypoxic and acidic conditions to be tested. Repair and removal of the transcription-blocking UV lesions allow\ subsequent expression of the luciferase reporter gene. which is assayed as a measure of repairsells in viva. Mutagenesis in cells grown under either nol-moxie or hypoxic conditions was also ahhayed using a chrorno\omally integrated lambda shuttlc vector containing the supFG1 mutation reporter gene. Cells were exposed to UV at a dose of 3J/m2, and were immediately placed under either standard culture conditions or hypoxia at pH 6.5 for 24 hours. Cells were returned to the standard conditions for I week of growth in culture, and mutations occurring in the supFG1 reporter gene were assayed by shuttle vector revcue from the cell DNA. Results: Cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA repair compared to control cells grown under standard culture conditions. with repair levels only 40% to 50% of that in the control cells. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesi\ compared to those maintained in standard growth conditions after irradiation (53.2 X 10-S versus 28.0 X IO-S). Conclusions: Taken together. the results suggest that cellular repair functions may he impaired under the conditions of the tumor microenvironment, causing hypermutahility to DNA damage. This alteration in repair capacity may constitute iul important mechanism underlying the genetic instability of cancer. The concept that the conditions of the tumor microenvironment can inhibit DNA repail- and consequently promote genetic instability. furthermore, provides a hasi? for understanding the observation that very hypoxic tumors follow a more aggressive clinical cou~~se.
2048
<:a taract
induction
M. L. Van Kempen-Harteveld.’ ‘Ur~ivemit~ Mrdicrrl Ckwolw. /m/y
Center.
after total hody irradiation Y. Belkacemi,’
Uttwht,
and bone marrow
H. B. Kal,’ M. Labopin.”
Nr~h~rlrrrrtl,v, *Hopitcd
transplantation:
Dose-effect
relationship
F. Frassoni’
Ter~on. Pnvis. FW~KV trml ~‘Istitnto N~~;iowIe
Ricrrcrr
Ctrrwm,
Purpose: Data of 1063 patients collected by the European Group for Blood and Marrow Transplantation (EBMT) were analyzed. The purpose was to determine the radiobiological parameter5 illp (tissue-specific parameter In the linear-quadratic. LQ. concept) and p (rate of repair of sublethal damage) from the EBMT data to derive a dose-effect relatiomhip for cataract induction after total body irradiation (TBI) and bone marrow transpl:lntation (BMT) for acute leukemia. Materials and Methods: Data were analyzed using the LQ-concept. Incidence of cataract was the endpoint used. Subgroups were constructed with respect to allogeneic or autologous BMT. with or without heparin and/or steroids use. Extrapolated tolerance doses (ETD) weI-e calculated for each patient for different sets of values for alp and I*. ETD=D[I +kR/(a/F)] with k=2[ I -(I --exp(-FT))/(wT)]/p.. D is the total dose, R is dose rate and T is the treatment time per fraction. Cataract incidences per ETD interval were calculated with the Kaplan-Meier method. Results: The use of ETD instead of total dose enabled incidence\ of cataract to be plotted in a re;tsonahly consistent way. With alp values of I to 2 Gy and a k-value of 0.5/h to ().75/h satisfactory dose-response curves were obtained. Using the same procedure the influence of age, steroids and heparin can be determined. Conclusion: The alp and p-values found ilre characteristic for late respondin g tissues: thus, cataract formation tits in the category late effects. For a specific TBI scheme the incidence of cataract can now be approximated and countermeasures to limit the cataract incidence by partly shielding the eyes can be made.
1. .I. Radiation
Oncology
l Biology
l Physic\
Volume 48, Number 3. Supplement.
2000
membrane for Western blotting. Blots were incubated with antibodies ngain\t the non-homologous end-joining (NHEJ) proteins Mrel I, RadSO. or NBSI. and protein hands were detected and later quantitatcd using a chemiluminescent detection system. Results: We found that Mrel I. RadSO, and NBS I (components of a multi-protein complex involved in NHEJ of DNA DSBs) are tranalocated from the nucleus to the cytoplasm when human LJ- I melanoma cells are heated for 15 min at 455°C. or when cells are heated after irradiation with 12 Gy of x-rays. The magnitude of migration of these proteins to the cytoplasm was dependent upon post-treatment incubation time at 37” C. and both the kinetic? and magnitude of translocation were dependent upon whether or not cells were irradiated prior to heating. No such translocation was observed when cell\ were treated with Iradiation alone. When cells were irradiated just prior to heating. the translocation occurred earlier (migration to the cytoplasm began during heating). Additionally, a phosphorylated form of NBS I was detected in the cytoplasmic fraction of heated irradiated cells after translocation. Tran\location ofRadS0 and NBS1 (but not Mrel I) was inhibited when heated irradiated cells were incubated in the presence of leptomycin B (which inhibit\ CRMI-mediated nuclear export). Conclusion: We propose that heat-induced redistribution or alterations of critical NHEJ proteins lead to inhibition repair after irradiation. and may be implicated. at least in part, as a mechanism for heat-radiosensitization.
2047
Diminished
DNA repair and elevated mutagenesis
P. M. Cilafer. J. Yuan, S. Rockwell.
L. Narayanan
Yclle Lirlil’rr.$,’ School 0f Mrtlicine.
NC,\\,Htn’rrr, CT
in mammalian
of DNA
cells exposed to hypoxia and low pH
Purpose: The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH and nutrient deprivation. It has been proposed that this unique tissue environment may. itself. constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could intluence cellular repair of DNA damage. 3Materials and Methods: An assay for DNA repair was established based on reactivation of UV-damaged plasmid DNA. This type of assay, termed ‘host cell reactivation’ (HCR). has been employed in a variety of studies to assess DNA repair in viva. DNA substrates containing pre-formed damage are introduced into cells, and the ability of the cells to repair the damage in the transfected DNA is probed under various conditions. In our experiments, plasmid pGL3. which encodes il luciferase gene, was damaged by UV irradiation at a dose of 5000 J/mZ. This damaged plasmid DNA. or an equal amount of undamaged luciferase plasmid DNA. was transfected into mouse fihroblast cells which were immediately placed under either normal culture conditions or under the hypoxic and acidic conditions to be tested. Repair and removal of the transcription-blocking UV lesions allow\ subsequent expression of the luciferase reporter gene. which is assayed as a measure of repairsells in viva. Mutagenesis in cells grown under either nol-moxie or hypoxic conditions was also ahhayed using a chrorno\omally integrated lambda shuttlc vector containing the supFG1 mutation reporter gene. Cells were exposed to UV at a dose of 3J/m2, and were immediately placed under either standard culture conditions or hypoxia at pH 6.5 for 24 hours. Cells were returned to the standard conditions for I week of growth in culture, and mutations occurring in the supFG1 reporter gene were assayed by shuttle vector revcue from the cell DNA. Results: Cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA repair compared to control cells grown under standard culture conditions. with repair levels only 40% to 50% of that in the control cells. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesi\ compared to those maintained in standard growth conditions after irradiation (53.2 X 10-S versus 28.0 X IO-S). Conclusions: Taken together. the results suggest that cellular repair functions may he impaired under the conditions of the tumor microenvironment, causing hypermutahility to DNA damage. This alteration in repair capacity may constitute iul important mechanism underlying the genetic instability of cancer. The concept that the conditions of the tumor microenvironment can inhibit DNA repail- and consequently promote genetic instability. furthermore, provides a hasi? for understanding the observation that very hypoxic tumors follow a more aggressive clinical cou~~se.
2048
<:a taract
induction
M. L. Van Kempen-Harteveld.’ ‘Ur~ivemit~ Mrdicrrl Ckwolw. /m/y
Center.
after total hody irradiation Y. Belkacemi,’
Uttwht,
and bone marrow
H. B. Kal,’ M. Labopin.”
Nr~h~rlrrrrtl,v, *Hopitcd
transplantation:
Dose-effect
relationship
F. Frassoni’
Ter~on. Pnvis. FW~KV trml ~‘Istitnto N~~;iowIe
Ricrrcrr
Ctrrwm,
Purpose: Data of 1063 patients collected by the European Group for Blood and Marrow Transplantation (EBMT) were analyzed. The purpose was to determine the radiobiological parameter5 illp (tissue-specific parameter In the linear-quadratic. LQ. concept) and p (rate of repair of sublethal damage) from the EBMT data to derive a dose-effect relatiomhip for cataract induction after total body irradiation (TBI) and bone marrow transpl:lntation (BMT) for acute leukemia. Materials and Methods: Data were analyzed using the LQ-concept. Incidence of cataract was the endpoint used. Subgroups were constructed with respect to allogeneic or autologous BMT. with or without heparin and/or steroids use. Extrapolated tolerance doses (ETD) weI-e calculated for each patient for different sets of values for alp and I*. ETD=D[I +kR/(a/F)] with k=2[ I -(I --exp(-FT))/(wT)]/p.. D is the total dose, R is dose rate and T is the treatment time per fraction. Cataract incidences per ETD interval were calculated with the Kaplan-Meier method. Results: The use of ETD instead of total dose enabled incidence\ of cataract to be plotted in a re;tsonahly consistent way. With alp values of I to 2 Gy and a k-value of 0.5/h to ().75/h satisfactory dose-response curves were obtained. Using the same procedure the influence of age, steroids and heparin can be determined. Conclusion: The alp and p-values found ilre characteristic for late respondin g tissues: thus, cataract formation tits in the category late effects. For a specific TBI scheme the incidence of cataract can now be approximated and countermeasures to limit the cataract incidence by partly shielding the eyes can be made.