Novel molecular targets for tumor radiosensitization Molecular radiation biology and oncology workshop: Translation of molecular mechanisms into clinical radiotherapy (2000)

A scientific workshop sponsored by the Radiation Effects Branch of the National Cancer Institute was held March 8 and 9, 1999, in Rockville, Maryland. To promote scientific collaborations and the exchange of new technologies, the workshop brought together investigators from three geographically close medical centers that recently were awarded P01 grants in radiation oncology to explore new avenues of translating novel molecular mechanisms of cellular radiosensitization into clinical trials. Scientists from Georgetown University (GU), Medical College of Virginia (MCV), and University of Pennsylvania (UP) presented their work in four scientific sessions followed by general discussions. Dr. Kristoffer Valerie (MCV), scientific coorganizer with Dr. Rupert Schmidt-Ullrich (MCV), opened the workshop by welcoming everyone. Dr. Ruth Muschel (UP) chaired Session I and also gave the first presentation, “The Mechanism of Radiation Induced G2 Block and Its Relationship to Radiosensitivity.” Dr. Muschel's studies on the molecular factors affecting the G2 delay have shown that the G2 delay induced by radiation has a multicomponent effect involving altered cyclin B expression, CDC2 kinase activation, and cellular localization. She has expanded her studies on drugs in conjunction with radiation and induction of G2 delay, in particularly in response to different etoposide agents. Dr. Muschel presented data demonstrating that the Cyclin B/Cdk2 complex is inhibited by the phosphatase Cdc25 through the cell cycle checkpoint protein Chk-1 and that radiation results in a decrease of CyclinB/Cdk2 histone H1 kinase activity. Inducible expression of Cyclin B1 demonstrated that increased levels of Cyclin B1 do not alter cell cycle progression at lower doses of 2 to 4 Gy, but abrogate the G2 delay after larger doses. Furthermore, p34cdc2 is excluded from the nucleus after Cyclin B1 induction and irradiation with 3 Gy. In studies on the mechanisms underlying the G2 delay in collaboration with Dr. Timothy Yen (UP), she has cloned several human homologs of the chk-1 gene, which have not been previously identified. She has developed specific antibodies to these proteins and shown that these proteins co-localize with ATM, the protein mutated in ataxia telangiectasia, suggesting that these proteins may be substrates for ATM. The next presentation was given by Dr. Rupert Schmidt-Ullrich (MCV) on “Genetic Manipulation of EGFR Expression and Radiosensitivity of Human Carcinoma Cells.” He showed data supporting the idea that disruption of EGFR function can be exploited for the radiosensitization of autocrine growth-regulated human mammary and squamous carcinoma cells. EGFR is activated by single ionizing radiation (IR) exposures in the 1–5 Gy dose range. As is known for growth factor ligands, such as EGF, IR stimulates the cytoprotective signal transduction pathway involving mitogen-activated protein kinase (MAPK) which mediates cellular proliferation responses. The demonstration that IR induces proliferation through activation of the EGFR/MAPK cascade does not only identify the mechanism of IR-induced accelerated repopulation but also identifies a novel mechanism of cellular radiosensitization through disruption of EGFR function. Data were presented that this can be accomplished through tetracycline-inducible expression of a dominant-negative EGFR-CD533, lacking the entire cytoplasmic domain. Alternatively, the tyrphostin PD183035 represents an alternative novel pharmacological approach. The inhibition of IR-induced activation of EGFR and its downstream effector MAPK could be linked to 5- to 7-fold radiosensitization in a newly developed in vitro assay combining repeated 2 Gy IR exposures with cell proliferation. As a first step towards gene therapeutic exploitation, more preliminary data demonstrated that effective disruption of EGFR function with cellular radiosensitization can be accomplished when human carcinoma cells are infected with an adenoviral vector expressing EGFR-CD533. The third presentation in this session was by Dr. Vincente Notario (GU) on “Mechanisms of Radiation Response and ADP-Ribose Metabolism.” Using Ewing's sarcoma cells, Dr. Notario showed that poly(ADP)ribose polymerase (PARP) is activated by DNA damage and that this is an early marker of apoptosis. Poly(ADP)-ribosylation provides a negative charge to molecules, including PARP, which most likely modulates the interaction of proteins. Poly(ADP-ribose) polymerase (PARP) is a chromatin-associated enzyme that catalyzes the transfer of the ADP-ribose moiety from NAD on to itself and other nuclear acceptor proteins. The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA, and is modulated by the level of automodification. It has been suggested that PARP-dependent protein modification plays a functional role in important cellular processes such as DNA replication, recombination, repair, cell cycle regulation, cell differentiation, and neoplastic transformation. However, despite numerous studies on the function of PARP in mammalian cells and recent advances in the molecular genetics of the PARP gene, very little is known about the mechanisms for regulation of PARP gene transcription. PARP expression is maintained at relatively low levels in most human tissues, suggesting the existence of intrinsic mechanisms for the autoregulation of endogenous content of PARP protein. In contrast, EWS cells accumulate PARP mRNA, protein, and polymerase activity at levels that, with the exception of yeast, would cause the death of other cell types. Therefore, EWS cells represent a unique model to investigate PARP transcriptional regulation with regard to the identification of the transcription effectors responsible for the unusually high levels of PARP in these primitive neuroectodermal tumor cells. To understand the molecular mechanisms underlying PARP up-regulation, we cloned and analyzed the 5'- flanking region of the PARP gene from EWS cells by deletion analysis and CAT activity assays, and compared with the 5' sequences of the PARP gene from normal cells. Nucleotide sequence analysis of the cloned EWS fragment revealed no remarkable differences with the sequences reported for PARP promoter regions isolated from normal human cells, suggesting that the enhanced PARP levels in EWS cells relative to normal cells could be caused by transcriptional up-regulation of the PARP promoter rather than to sequence differences within the PARP 5'- regulatory region. The PARP promoter encompasses five putative Ets recognition sites, denoted E1-E5, that match perfectly the binding core motif reported for Ets proteins A/C GGA A/T. Of particular importance is the fact that these Ets-binding motifs are located within the regions containing positive and negative control elements. These results suggested the potential involvement of members of the Ets DNA-binding proteins in the transcriptional regulation of the expression of the PARP gene. In fact, there is a coordinated up-regulation of both PARP and Ets-l, relative to cells of other human tumor types expressing lower levels of PARP. Transient coexpression of Ets-l in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of Ets in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with an Ets-l antisense cDNA construct. Antisense-mediated down-regulation of endogenous Ets-l resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for Ets regulation of PARP expression levels, and implicate Ets transcription factors in the radiation response of EWS cells. Ongoing studies are directed toward identifying whether other members of the ETS family, including Fli-l, may play regulatory roles on PARP in EWS cells, and to evaluate the contribution of the various Ets DNA-binding motifs to PARP regulation. The final presentation in this session was given by Dr. Eric Bernard (UP) on “The Involvement of Ras Signal Transduction in the Cell Cycle Response to Radiation.” Drs. McKenna and Bernhard are studying the underlying mechanism of the ras oncogene on radiation sensitivity. Mutation in ras is common (50%–90%) in a variety of cancers with some resulting in constitutively active Ras. Activation of Ras contributes to the intrinsic radioresistance of certain tumors. Ras is posttranslationally modified by farnesylation, which is essential for its activation. Drs. McKenna and Bernard examined the effects of farnsyltranferase inhibitors on the radiation response of tumor cells in cells expressing both H and K ras oncogenes. Significantly, they have shown that to inhibit K-Ras function, not only farnesyltranferase needs to be inhibited but also geranyl geranyl transferase, and that inhibition of both enzymes results in significant radiosensitization. Inhibitors of prenyltransferase, such as FTI-277 and FTI-276, increase clonogenic survival of human T24 carcinoma cells, which express mutant H-Ras, in vitro, but do not affect survival of SV40-transformed cells, which do not express Ras. There is a growth delay in T24 tumors treated with 6 Gy and FTI-277. Signal transduction pathways involved in the mechanism of ras alteration of radiosensitivity have also been studied. Contrary to the findings by others that the MAP kinase pathway is involved in radiosensitization, Drs. McKenna and Bernard have conclusively shown that MAP kinase activation is not involved in induction of radiation resistance by Ras. Inhibition of MAP kinase activity has no effect on the survival of cell lines expressing oncogenic or wild type Ras. MAPK is inhibited by FTI-277 in T24 cells and MAPK activity is induced in RT4 cells but not in T24 cells. There is no decrease in survival by inhibiting the MAPK pathway by using the MEK1 inhibitor PD98059. This is consistent with results using Ras effector loop domain mutants that lead to a similar conclusion. All together, these studies have demonstrated that farnesyltranferase inhibitors are potentially very useful agents to manipulating Ras-induced radiosensitivity. Dr. Timothy Yen (Fox Chase Cancer Center and the University of Pennsylvania) chaired the second scientific session and gave the first presentation, “Role of Ataxia-Telangiectasia in Checkpoint Control.” Dr. Yen discussed results examining the properties of the ATM protein mutated in the disease ataxia telangiectasia. The ATM protein is a large 3,056 amino acid protein that is associated with chromatin (80%) and with the nuclear membrane (20%). ATM protein levels are unaffected by the cell cycle in different cell lines and irradiation does not affect ATM steady state protein levels. Dr. Yen has been able to directly isolate ATM in a multicomponent complex from Hela cells, and to purify this in sufficient quantities to examine ATM interacting proteins by protein sequence determination. In particular, his studies are focused on the ability of ATM to phosphorylate Cdc25c and he is currently working on the determining the sites of phosphorylation of this protein. Furthermore, a subunit of replicating protein A, RPA-34, is phosphorylated by ATM in a DNA-dependent (ss and ds) fashion. RPA-34 phosphorylation is mainly on Ser/Thr residues, but no significant enhancement of ATM protein kinase activity is observed after IR. RPA-34 may also be phosphorylated by ATM in vivo. Dr. Yen also discussed the results of yeast two-hybrid experiments done in collaboration with Dr. Muschel that has identified two proteins, p73 and p79, that associate with ATM. These proteins are related to the Chk1/Rad53 protein kinases and have been named ChkI. He has also, in collaboration with Dr. El-Deiry (UP), shown that ATM is a necessary component in the induction of killer/DR5 via the tumor suppressor p53. The second presentation, by Dr. Anatoly Dritschilo (GU), was “Molecular Studies of Radiation Resistant Tumor Cells.” Dr. Dritschilo reviewed data from his group examining the radiosensitivities and properties of numerous immortalized and primary AT cell lines and strains that constitutes one of the largest cell repositories in the country. The third presentation in this session was given by Dr. Mira Jung (GU) on the “Mechanisms of Radiation Sensitivity in AT Cells.” Ionizing radiation-induced NF-κB activation occurs after both small and large doses of radiation. However, the radiation specific signaling pathways involved is not known. Previously, Dr. Jung's group has reported that the regulation of NF-κB is impaired in cells from patients with the complex human genetic disease, ataxia-telangiectasia (AT), which exhibits an exquisite sensitivity to ionizing radiation and abnormalities in cellular regulation processes. The mutated ATM gene found in AT patients is believed to be involved in a number of functions that lead to and Furthermore, ATM in The signaling pathway through which ionizing radiation induces NF-κB activation is not an protein of mediates the activation of NF-κB in response to various including and other Dr. Jung has identified an ionizing radiation-induced signaling pathway through NF-κB that is of is in response to but is in response to ionizing radiation in cells from with of ATM radiation-induced of Furthermore, phosphorylation of by ATM kinase is increased in control and transfected AT cells after ionizing radiation These data provide support for a novel ionizing radiation-induced signaling pathway for activation of NF-κB and a molecular for the sensitivity of AT patients to AT cells abnormalities in ionizing radiation signaling involved in cell However, the mechanisms underlying in response to IR are not known. studies have shown that activation of NF-κB is for cell Expression of a negative of AT from induced by radiation but in normal Furthermore, the to may of These data that regulation of NF-κB may play an important role in cell death induced by DNA damage in AT cells. Dr. Jung has also identified IR-induced that in AT cells showed an of about within of to IR. This was with protein of PARP, and control and a AT cell not these revealed increased and within of However, of activation of and but not induction after irradiation with Dr. (GU) chaired Session and also the first and PARP in Radiation Induced There is a for a of and other nuclear proteins at the early of apoptosis. The modification of nuclear proteins plays an important role in DNA of as as in other which DNA such as apoptosis. Dr. has found that a of occurs early in different cell immortalized from showed this early and a cells from PARP of PARP cells with PARP these cells to apoptosis. PARP and to be essential for by in the death experiments revealed that, together with a variety of other nuclear proteins, to in human a increase in the of was also of to by was observed with the of activation, and DNA the of cell The decrease in to also with the induction of the gene, These results that may play a role in the regulation of function. PARP also the expression and promoter activity of expression of has been shown to lead to and apoptosis. The of PARP by has been to of NAD and which are to be for in apoptosis. The role of PARP in was in human cells stably transfected with a vector a PARP Expression of this mutant PARP increased the of both and in cells by the for the of activation, and DNA as as the of DNA of PARP also accelerated the not to the as in cells expressing the mutant PARP. results were with cells from PARP and with mutant or PARP These effects of the mutant and enzymes be caused by PARP activation an NAD These results that PARP activation and have active and complex roles in apoptosis. the of a more for the interaction of PARP with both proteins and DNA, we have also the of the of to directly binding to various of preliminary new work was on the of to investigate whether PARP alter by with increased in the resulting from in DNA and further support for a role for PARP in the of The second in this session was by Dr. (MCV) on of Ionizing Human cell lines and have been isolated that the proteins and These cells resistance to damage and induced by including drugs and ionizing radiation. the of induced by Gy in cells was of that observed in their cells were and to an that and or a kinase inhibitor that the DNA damage was The increase in was by a in clonogenic suggesting that of by these agents to the of cell Studies are currently to whether and by including a in the membrane potential and of into the The third presentation was given by Drs. and (UP) on in DNA Cells.” Dr. the was to the Dr. El-Deiry his studies on in DNA damage cells. He has shown that an inhibitor of can inhibit the dependent up-regulation of killer/DR5 mRNA, and to his studies on the regulation of killer/DR5 by including studies of novel DNA binding sites both in the regulatory regions of killer/DR5 and in He has also to mutants of the within killer/DR5 to understand the function of this protein. He is to isolate interacting proteins with which may be involved in the signaling of cell Dr. of killer/DR5 a potential mechanism by which may in response to DNA damage and this potentially significant into the mechanism of the in the regulation of cell the second Dr. Kristoffer Valerie (MCV) chaired the session and gave the first presentation, with in with group has recently demonstrated that of cells in vitro with an expressing the kinase gene and of infected cells to which is into cell DNA as the cells more to radiation compared with cells. Cell with increased of apoptosis. Dr. Valerie has the for into by and of by a positive and can as as in the In a significant effect of the compared with is observed using tumors in Dr. Valerie has shown that the from an cells to radiation in with the However, relatively high of are to significant radiosensitization. levels than the lower the of this genetic to cells in vivo. To the lower of in an expressing a mutant isolated for its more sensitivity to than was Using this was demonstrated that of cells and to or cells more to lower to 3 of the drugs than after with expressing the cells more to lower doses of radiation than cells infected with an expressing This is also by an increased of apoptosis. In we that of cells with an expressing a mutant the cells to radiation after to at lower than for These results that this mutant may in of gene The second presentation in this session was given by Dr. (MCV) on the of by Transduction of function are a common in and are believed responsible for the genetic and resistance to IR that the of patients such tumors. most express most of in the of protein The activation and that transduction of could be to proliferation and the sensitivity of cells to IR. Studies at examining the activity of mechanisms in and human cells, and the ability of expression to do in these A significant increase in and expression in and human cells after IR that these cells are for p53. studies of human cells these cells to express mutant p53. other cell lines to be in the expressing and mutant have been by sequence analysis and similar functional with an adenoviral vector containing the gene demonstrated transduction of cells in vitro, and studies with an adenoviral vector containing the gene. and a increase in expression was after transduction of cells, no increase in expression was after transduction with studies have demonstrated the of of expression in and tumors by cells were with the adenoviral vector or adenoviral vector and to IR and for clonogenic transduction with the gene caused no in the radiosensitivity of these cells, but of the gene significant decrease in clonogenic particularly at doses Gy for radiation To effects on of cells were analyzed by radiation a increase in caused by transduction can be The of doses of IR these to a response after and particularly in the of Gy. in cells tumors similar to control cells, but the cells than with control cells. of radiation to further survival in this transduction with by positive has as a gene for radiosensitivity of tumors. Dr. findings enhanced radiosensitivity of cells expressing after of with an that this may be to human of The third presentation was by Dr. Timothy (GU) on and Tumor Radiation kinase is a enzyme of the cellular and is essential for of to Dr. has found that kinase expression is a in cells. that expression are more relative to the cells. The of is particularly at the dose levels in The in survival can be for by a in the ability of the cells to of kinase was into the kinase cells, radioresistance was and damage was also All other including DNA repair, potentially damage repair, G2 and cell cycle similar the cell These data that the kinase enzyme or its cellular gene may be an therapeutic to increase radiosensitivity the of Dr. to the mechanism of kinase and identify potential therapeutic on molecular of has been linked to a radiation signal transduction pathway by and has been shown to play a role both and downstream in this He is currently working on the that tumor cells caused by on which in processes. He has shown that are in tumor cells, and that these cells do by apoptosis. He is to directly the to activation of apoptosis. He has also to role of itself in cellular radioresistance by at the effect of large of on the downstream of cell cycle and apoptosis. He has shown that induction of is by but not However, is is These results support the that to may be the signal for activation, and that inhibition of may tumor cell radiosensitivity. The protein be an important for levels to be by Dr. group is currently as a of this pathway and antisense to inhibit radiation-induced activation of and radiation-induced apoptosis. He is the potential of these to tumors in in a The and radiation signal transduction processes is an important only does provide a mechanism for the effect of but also to other proteins in the such as as potential for tumor radiosensitization. These may to be therapeutic than they control the mechanisms by which cells radiation The final presentation in this and the workshop was given by Dr. (GU) on Manipulation of Tumor Cell Dr. group has demonstrated the in of and antisense as a radiation inhibited protein expression in vivo. or ionizing radiation (IR) caused inhibition of tumor growth in but not tumor A of and IR to significant and tumor for at after the of tumor revealed a significant of cells containing chromatin in group as compared with single and control These data the potential of antisense in the of on the studies to the clinical of antisense as a radiation The workshop was a that several important in molecular radiation oncology and a number of cellular that are for that may radiation A of and was presented by investigators from the three medical A were identified as potential to clinical in the very The activity of the Ras involved in radiation can be by using specific inhibitors as demonstrated by Drs. Bernard and the expression of the protein kinase has also been shown by a number of to antisense that expression could potentially be to radioresistance of human squamous carcinoma as shown by Dr. the of using the tumor suppressor or the gene in with the are also that can be to as demonstrated by Drs. and This work was by by and awarded by the National Cancer