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Relationship between intrinsic radiation sensitivity and metastatic potential: Difference between spontaneous and experimetal metastatic potential
225
Proceedings of the 36th Annual ASTRO Meeting
125 ~53 DEPENDENT
SENSITMTY TO RADIAlTON IN A TRANSPLANTED TUMOR MODEL
David E. Fisher’, Stefan Bodis 1,2, Scott W. Lowej, H. Earl Ruley‘t, David E. Housman3, Tyler Jacks3 1 Dana-Farber Cancer Institute, Boston, MA 02115; 2 Joint Center for Radiation Therapy, Boston, MA 02115; 3 Department of Biology, MIT, Cambridge, MA 02139; 4 Vanderbilt University School of Medicine, Nashville, TN 37232 Purpose: ~53 is the most frequently mutated gene in human cancer. It has recently been associated with key roles in cell cycle progression and apoptosis. In vitro, lack of ~53 correlated with loss of apoptotic response to DNA damaging agents. The current study was designed to examine the behavior of defined oncogene transformed cells which either contain or lack wild type ~53. Specifically, we asked whether ~53 modulates radio- and chemosensitivity of molecularly defined solid tumors in animals and if so, whether apoptosis was occurring. Materials and Methods: Mouse embryonic fibroblasts (derived from homozygous knockout animals or ~53 wildtype littermates) were transformed by ElA and Ras in vitro and injected after early passage into the rear flanks of athymic nude mice. Tumor growth was monitored by the NC1 formula and upon reaching a critical size was followed by treatments with TBI or chemotherapy. Treatment effects were monitored by serial size measurements. Presence of apoptosis was examined by morphology within tissue sections from treated tumors. Results: Tumors lacking ~53 appeared earlier and grew faster than p53+ tumors. The “take” rate was very high (100%) for ~53 negative tumors and significantly more variable for p53+ tumors (20&O%, depending on cell clone), based on injection of identical cell numbers. Established p53+ tumors maintained normal ~53 protein levels. Once established, exponential tumor growth continued to large sizes for both groups in untreated animals. Responses to single doses of TBI with 5 or 7 Gy were strikingly different in the two groups. Tumors with wild type ~53 shrunk in a dose dependent fashion to ~10% pretreatment size within 5 days whereas ~53 negative tumors continued to grow with essentially unaltered kinetics. Similar results were obtained with adriamycin treatment. Relapses occurred for the treated p53+ tumors, but substantial responses could be obtained with repeated treatments. Interestingly, the magnitude and duration of these secondary responses decreased with subsequent cycles. Histological examination of treated p53+ tumors revealed abundant apoptosis which was lacking in p53tumors. Conclusions: Solid tumor biology was examined and directly compared using these transformed cell populations which contain defined molecular lesions. Response to radiation and chemotherapy was strikingly ~53 dependent. Whereas mice with p53+ tumors responded dramaticallv, those with ~53 negative ones disnlaved essentially unaltered growth kinetics with doses as high as 12 Gv. The mechanism accounting for this differ&&l &sitivity appe&s*to involve the induction &f apoptosis which, for the agen; tested, isapparently mediated by a ~53 dependent pathway. The increased refractoriness of p53+ tumors to repetitive treatments offers a potential model to examine molecular causes of resistance (e.g. accumulation of ~53 mutations, etc.). More aggressive growth properties of p53- tumors suggest that as an isolated variable ~53 may profoundly influence growth rate and propensity for establishing de novo tumors (e.g. metastatic potential). Studies are underway utilizing these reagents to examine strategies capable of restoring treatment responses to ~53 negative tumors such as fractionated radiotherapy and cell cycle specific drugs.
126 MUTANT P53 INCREASES RADIOTHERAPY
RADIORESISTANCE
IN TEANSFECTED
EAT EMBRYO
FIBROBLASTS:
IMPLICATIONS
FOR
Robert Bristow, Anne Jang, James Peacock, Stephen Chung, Samuel Ben&no1 and Richard P. Hill Departments of Radiation Oncology and Research, The Princess Margaret Hospital, 500 Sherboume Street, Toronto, Ontario, CANADA M4X lK9 PURPOSE: It has been suggested that abrogation of normal cell cycle checkpoints through intereference with wild type pS3 or retinoblastoma gene product function may alter the cellular response to ionizing radiation. Human anogenital carcinomas (ie. cervix) have been shown to contain mutant ~53. HPV16 and activated H-ras sequences alone, or in combination with each other. Clinical resistance to current radiation treatment protocols may therefore be based on the aberrant expression of such oncoproteins. METHODS: To investigate this hypothesis, rat embryo fibroblasts (REF) were transfected using calcium phosphate with a variety of plasmids containing mutant forms of the ~53 gene alone, or in combination with plasmids encoding the H-ras (pJH6.6) and HPV E7 (pJ4G16.E7) sequences. Mutant forms of the p53 gene can cooperate both with the H-ras gene, and the human papilloma virus HPV16E7 gene to render such cells tumorigenic. Positive transfectant clones were selected through use of a neomycin marker, and chamckrixed as to tbeir relative radiosensitivity (assays of clonogenicity and apoptosis) , cell cycle chamcteristics using flow cytometry, tumorigenicity, spontaneous metastatic ability, and levels of oncopmteiu expression. Clonogenic survival assays were performed both in agar and on plastic. RESULTS; H-raptransfected REF clones expressing high levels of activated ~21”’protein did not show radimesistance when compared to REF cells transfected with the neomycin plasmid alone (mean SF,, of 0.40 vs. 0.54 respectively). cell lines uan&cmd with the mutant p53 gene alone lost their ability to undergo a Gl delay following hradiadou, but on average were not significantly more kstant than REF/neo cells (mean SF, = 0.57). Double (H-ras/p53pro193) and triple (I-I-ras/p53pro-193/E7) transfectants were. radios&stant (mean SF, > 0.85) and unifosmly tumorigenic in SClD mice. Based on Western blot analyses, expression of the mutant p53-pro193 onqrotein was observed to conelate with radioresistauce, but not spontaneous metastasis in transfected RE!Fcells. Further experiments have shown that intrinsic radiosensitivity did not correlate to changes
226
Radiation Oncology, Biology, Physics
Volume 30, Supplement 1
in the status of radiation-induced Gl delay, although REF transfectants clones which expressed mutant ~53 protein did loat the ability to ariest intbeGlphaseaftcr4Gy. NoneoftheuntrensfectedortransfectedREFce~lintsunderwcntapoptwisafferimdiation(~srangingfnnn2-10 Gy). Additional transfections utilizing plasmids encoding a variety of diffemnt murine and human ~53 mutant alleles (deleticm-44, ala-143, val-135. ser-273, asn-190, asp-236, ser-135) suggest that the acquisition of the radiomsistant pm may be limited to specitic ~53 mutant proteins. Wbether such mutant proteins arz acting in a negatively dominant manner through interaction with wild type ~53 protein, l)r are positively acting (ie. demonstrate “gain of function”) is the basii of current experimentation. CONCLUSIONS: In summary, increased radiamaistance in terms of surviving clonogens after inadiadon occurs only when high levels of the mutant p53allekanex~saed.Thisradiaesisernccis~~ntofthestatusof tbeG1am?storrelatinapopto&inourpaneloftransfectedREFcelI lines.As clinically aggressive ano@tal tumors may contain both WV-E!7 and mutant ~53 sequences, the present REF cell lines pnn+ick a model for further studies into -in-mediated radio&smnce and malignancy.
127 Relationship Between Intrinsic Radiation Sensitivity and Metastatic Potential: Difference Between Sponfaneour and E.qwimenfuZ Metastatic Potential Anne M. Lewisl, Jay Doty2, Alphonse Taghianl, Su Meil, and Francisco S. Pardo Dept. Radiation Oncology, Massachusetts General Hospital, and Dept .of Physiology, Tufts University, Boston, Massachusetts Background: Tumor growth and angiogenesis is a prerequisite for human tumor progression and metastasis. The probability of tumor radiosensitivity and local connol by therapeutic irradiation, however, does not necessarily correlate with its metastatic potential. We investigated the possibilty of a relationship between intrinsic radiation sensitivity and the spontanteous and experimental metastatic potential of genetically well. chamcterized cell lines. Metho& Rat embryo cells @EC) were transfected with the following oncogenes, and where appropriate, with corresponding selection markers:pCMVneopEJ66ras, pEJ6.6ras/v-myc, pElalZs,pElalk, PEJ6.6ras/Elal3s, PEJ6.6ras/Ela12s, p53CX3, ~53 CSN. Individual transfectant clones and corresponding pooled cellular populations wete propagated in selective medium. In vitro cellular radiation sensitivity was determined via clonogenic assays, a minimum of three, by standard techniques and individual SF2 and D parameters determir& Tumorigenicity was defined as the number oftumors forming pa one week unit of time following the injection of 1X105-1X106 cells injected into the axillary pouch of thtce different suains of immunedeficient mice. Animals were sacrificed once resultant tumors reached a size of 2.1 cm in transverse diameter. Fordeterminati on of spontaneous metastatic potential, lungs were harvested and scored visualI for metastases after overnight fixation . into the tail veins of in picric acid For de termination of experimental metastatic potential, cell numbers between 1X104-1X103 were Injected litter-matched sibling mice in pamllel to the tumorigencity/spontaneous metastatic potential studies. Reaulta: Radiobiologic studies indicated similar levels of radiation sensitivity among REC. mock-transfected REC, ElA12s, Ela 13s, and combined pEla 13s ras transfectants. pEJ6.6 ras and combined eye transfectants, together with p53CX3 pooled cellular populations demonstrated increases in radiation n%istance when compared to the pooled radiobiologic data from untmnsfected and mock-transfected corresponding pooled cellular populations@<.O5, twc+tailed test.SF2,B). REC, Elal2s Ela 13s, and wild type ~53 transfectants were relatively radiation sensitive and non- mmorieenic. uElal3sras and ~53 Q(3 transfectants were tumoriaenic but demonstrated relativelv low excerimental metastatic potential, and no sponta&ous ~&tastatic potent&. Ras/myc transfectants demons&d the highest levels of spon&eour &astatic potential followed by ras alone and one allelic variant of mutant p53(p<.o5, Fischer Exact Test). Ras, ras/myc and ~53 CX3 tmnsfectants, however, demonstrated almost identical levels of eqwimenmZ metastaticpotential on lung colonization assays. Con&dons: A good correlation exists between the intrinsic radiationsensitivity and the spontaneous metastatic potential of transfected REC. The highest levels of radiation resistance in v&o and spo~raneo~ metastatic potential in vivo were found among REC transfected with ras/myc, followed by ms alone, and an allelic variant ofmutant~53. The degree of sponzam metastadc potential does not necessarily cormlate with the degree of ~rimental metas&& potential on lung colonization assay. This discnpaacy between experimental and spontaneous metastatic potential is most probably a by-product of methodologic differences in metastatic potential assays. Mechanisms of on*mic activation and methodologic peculiarities with nspect to the quantification of metastatic potential, together with preliminary data on expression of the putative tumor suppressor gene, nm23, will be presented.
Proceedings of the 36th Annual ASTRO Meeting
125 ~53 DEPENDENT
SENSITMTY TO RADIAlTON IN A TRANSPLANTED TUMOR MODEL
David E. Fisher’, Stefan Bodis 1,2, Scott W. Lowej, H. Earl Ruley‘t, David E. Housman3, Tyler Jacks3 1 Dana-Farber Cancer Institute, Boston, MA 02115; 2 Joint Center for Radiation Therapy, Boston, MA 02115; 3 Department of Biology, MIT, Cambridge, MA 02139; 4 Vanderbilt University School of Medicine, Nashville, TN 37232 Purpose: ~53 is the most frequently mutated gene in human cancer. It has recently been associated with key roles in cell cycle progression and apoptosis. In vitro, lack of ~53 correlated with loss of apoptotic response to DNA damaging agents. The current study was designed to examine the behavior of defined oncogene transformed cells which either contain or lack wild type ~53. Specifically, we asked whether ~53 modulates radio- and chemosensitivity of molecularly defined solid tumors in animals and if so, whether apoptosis was occurring. Materials and Methods: Mouse embryonic fibroblasts (derived from homozygous knockout animals or ~53 wildtype littermates) were transformed by ElA and Ras in vitro and injected after early passage into the rear flanks of athymic nude mice. Tumor growth was monitored by the NC1 formula and upon reaching a critical size was followed by treatments with TBI or chemotherapy. Treatment effects were monitored by serial size measurements. Presence of apoptosis was examined by morphology within tissue sections from treated tumors. Results: Tumors lacking ~53 appeared earlier and grew faster than p53+ tumors. The “take” rate was very high (100%) for ~53 negative tumors and significantly more variable for p53+ tumors (20&O%, depending on cell clone), based on injection of identical cell numbers. Established p53+ tumors maintained normal ~53 protein levels. Once established, exponential tumor growth continued to large sizes for both groups in untreated animals. Responses to single doses of TBI with 5 or 7 Gy were strikingly different in the two groups. Tumors with wild type ~53 shrunk in a dose dependent fashion to ~10% pretreatment size within 5 days whereas ~53 negative tumors continued to grow with essentially unaltered kinetics. Similar results were obtained with adriamycin treatment. Relapses occurred for the treated p53+ tumors, but substantial responses could be obtained with repeated treatments. Interestingly, the magnitude and duration of these secondary responses decreased with subsequent cycles. Histological examination of treated p53+ tumors revealed abundant apoptosis which was lacking in p53tumors. Conclusions: Solid tumor biology was examined and directly compared using these transformed cell populations which contain defined molecular lesions. Response to radiation and chemotherapy was strikingly ~53 dependent. Whereas mice with p53+ tumors responded dramaticallv, those with ~53 negative ones disnlaved essentially unaltered growth kinetics with doses as high as 12 Gv. The mechanism accounting for this differ&&l &sitivity appe&s*to involve the induction &f apoptosis which, for the agen; tested, isapparently mediated by a ~53 dependent pathway. The increased refractoriness of p53+ tumors to repetitive treatments offers a potential model to examine molecular causes of resistance (e.g. accumulation of ~53 mutations, etc.). More aggressive growth properties of p53- tumors suggest that as an isolated variable ~53 may profoundly influence growth rate and propensity for establishing de novo tumors (e.g. metastatic potential). Studies are underway utilizing these reagents to examine strategies capable of restoring treatment responses to ~53 negative tumors such as fractionated radiotherapy and cell cycle specific drugs.
126 MUTANT P53 INCREASES RADIOTHERAPY
RADIORESISTANCE
IN TEANSFECTED
EAT EMBRYO
FIBROBLASTS:
IMPLICATIONS
FOR
Robert Bristow, Anne Jang, James Peacock, Stephen Chung, Samuel Ben&no1 and Richard P. Hill Departments of Radiation Oncology and Research, The Princess Margaret Hospital, 500 Sherboume Street, Toronto, Ontario, CANADA M4X lK9 PURPOSE: It has been suggested that abrogation of normal cell cycle checkpoints through intereference with wild type pS3 or retinoblastoma gene product function may alter the cellular response to ionizing radiation. Human anogenital carcinomas (ie. cervix) have been shown to contain mutant ~53. HPV16 and activated H-ras sequences alone, or in combination with each other. Clinical resistance to current radiation treatment protocols may therefore be based on the aberrant expression of such oncoproteins. METHODS: To investigate this hypothesis, rat embryo fibroblasts (REF) were transfected using calcium phosphate with a variety of plasmids containing mutant forms of the ~53 gene alone, or in combination with plasmids encoding the H-ras (pJH6.6) and HPV E7 (pJ4G16.E7) sequences. Mutant forms of the p53 gene can cooperate both with the H-ras gene, and the human papilloma virus HPV16E7 gene to render such cells tumorigenic. Positive transfectant clones were selected through use of a neomycin marker, and chamckrixed as to tbeir relative radiosensitivity (assays of clonogenicity and apoptosis) , cell cycle chamcteristics using flow cytometry, tumorigenicity, spontaneous metastatic ability, and levels of oncopmteiu expression. Clonogenic survival assays were performed both in agar and on plastic. RESULTS; H-raptransfected REF clones expressing high levels of activated ~21”’protein did not show radimesistance when compared to REF cells transfected with the neomycin plasmid alone (mean SF,, of 0.40 vs. 0.54 respectively). cell lines uan&cmd with the mutant p53 gene alone lost their ability to undergo a Gl delay following hradiadou, but on average were not significantly more kstant than REF/neo cells (mean SF, = 0.57). Double (H-ras/p53pro193) and triple (I-I-ras/p53pro-193/E7) transfectants were. radios&stant (mean SF, > 0.85) and unifosmly tumorigenic in SClD mice. Based on Western blot analyses, expression of the mutant p53-pro193 onqrotein was observed to conelate with radioresistauce, but not spontaneous metastasis in transfected RE!Fcells. Further experiments have shown that intrinsic radiosensitivity did not correlate to changes
226
Radiation Oncology, Biology, Physics
Volume 30, Supplement 1
in the status of radiation-induced Gl delay, although REF transfectants clones which expressed mutant ~53 protein did loat the ability to ariest intbeGlphaseaftcr4Gy. NoneoftheuntrensfectedortransfectedREFce~lintsunderwcntapoptwisafferimdiation(~srangingfnnn2-10 Gy). Additional transfections utilizing plasmids encoding a variety of diffemnt murine and human ~53 mutant alleles (deleticm-44, ala-143, val-135. ser-273, asn-190, asp-236, ser-135) suggest that the acquisition of the radiomsistant pm may be limited to specitic ~53 mutant proteins. Wbether such mutant proteins arz acting in a negatively dominant manner through interaction with wild type ~53 protein, l)r are positively acting (ie. demonstrate “gain of function”) is the basii of current experimentation. CONCLUSIONS: In summary, increased radiamaistance in terms of surviving clonogens after inadiadon occurs only when high levels of the mutant p53allekanex~saed.Thisradiaesisernccis~~ntofthestatusof tbeG1am?storrelatinapopto&inourpaneloftransfectedREFcelI lines.As clinically aggressive ano@tal tumors may contain both WV-E!7 and mutant ~53 sequences, the present REF cell lines pnn+ick a model for further studies into -in-mediated radio&smnce and malignancy.
127 Relationship Between Intrinsic Radiation Sensitivity and Metastatic Potential: Difference Between Sponfaneour and E.qwimenfuZ Metastatic Potential Anne M. Lewisl, Jay Doty2, Alphonse Taghianl, Su Meil, and Francisco S. Pardo Dept. Radiation Oncology, Massachusetts General Hospital, and Dept .of Physiology, Tufts University, Boston, Massachusetts Background: Tumor growth and angiogenesis is a prerequisite for human tumor progression and metastasis. The probability of tumor radiosensitivity and local connol by therapeutic irradiation, however, does not necessarily correlate with its metastatic potential. We investigated the possibilty of a relationship between intrinsic radiation sensitivity and the spontanteous and experimental metastatic potential of genetically well. chamcterized cell lines. Metho& Rat embryo cells @EC) were transfected with the following oncogenes, and where appropriate, with corresponding selection markers:pCMVneopEJ66ras, pEJ6.6ras/v-myc, pElalZs,pElalk, PEJ6.6ras/Elal3s, PEJ6.6ras/Ela12s, p53CX3, ~53 CSN. Individual transfectant clones and corresponding pooled cellular populations wete propagated in selective medium. In vitro cellular radiation sensitivity was determined via clonogenic assays, a minimum of three, by standard techniques and individual SF2 and D parameters determir& Tumorigenicity was defined as the number oftumors forming pa one week unit of time following the injection of 1X105-1X106 cells injected into the axillary pouch of thtce different suains of immunedeficient mice. Animals were sacrificed once resultant tumors reached a size of 2.1 cm in transverse diameter. Fordeterminati on of spontaneous metastatic potential, lungs were harvested and scored visualI for metastases after overnight fixation . into the tail veins of in picric acid For de termination of experimental metastatic potential, cell numbers between 1X104-1X103 were Injected litter-matched sibling mice in pamllel to the tumorigencity/spontaneous metastatic potential studies. Reaulta: Radiobiologic studies indicated similar levels of radiation sensitivity among REC. mock-transfected REC, ElA12s, Ela 13s, and combined pEla 13s ras transfectants. pEJ6.6 ras and combined eye transfectants, together with p53CX3 pooled cellular populations demonstrated increases in radiation n%istance when compared to the pooled radiobiologic data from untmnsfected and mock-transfected corresponding pooled cellular populations@<.O5, twc+tailed test.SF2,B). REC, Elal2s Ela 13s, and wild type ~53 transfectants were relatively radiation sensitive and non- mmorieenic. uElal3sras and ~53 Q(3 transfectants were tumoriaenic but demonstrated relativelv low excerimental metastatic potential, and no sponta&ous ~&tastatic potent&. Ras/myc transfectants demons&d the highest levels of spon&eour &astatic potential followed by ras alone and one allelic variant of mutant p53(p<.o5, Fischer Exact Test). Ras, ras/myc and ~53 CX3 tmnsfectants, however, demonstrated almost identical levels of eqwimenmZ metastaticpotential on lung colonization assays. Con&dons: A good correlation exists between the intrinsic radiationsensitivity and the spontaneous metastatic potential of transfected REC. The highest levels of radiation resistance in v&o and spo~raneo~ metastatic potential in vivo were found among REC transfected with ras/myc, followed by ms alone, and an allelic variant ofmutant~53. The degree of sponzam metastadc potential does not necessarily cormlate with the degree of ~rimental metas&& potential on lung colonization assay. This discnpaacy between experimental and spontaneous metastatic potential is most probably a by-product of methodologic differences in metastatic potential assays. Mechanisms of on*mic activation and methodologic peculiarities with nspect to the quantification of metastatic potential, together with preliminary data on expression of the putative tumor suppressor gene, nm23, will be presented.