Table of Contents
Molecular Analyses of Adaptive Survival Responses (ASRs): Role of ASRs in Radiotherapy1
David A. Boothman2 Ph.D., Eric Odegaard, B.S., Chin-Rang Yang, Ph.D., Kelly Hosley, Ph.D., and Marc S. Mendonca3 Ph.D.
Department of Human Oncology, K4/626 Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792.
Tel.: (608) 262-4970; Fax: (608) 263-8613, e-mail: firstname.lastname@example.orgRadiation and Cancer Biology Laboratory, Department of Radiation Oncology, 975 West Walnut St., IB-346, Indiana University School of Medicine, Indianapolis, IN 46202 1Supported by grant # DE-FG02-93ER6l707 to D.A.B. from the U.S. Department of Energy, and in part by Departmental grants awarded to M.S.M., Indiana University School of Medicine.
Adaptive survival responses (ASRs4), whereby cells demonstrate a survival advantage when exposed to very low doses of ionizing radiation (IR) four to twenty four hours prior to a high dose challenge, were first reported over 15 years ago. These responses were linked to hormesis, which implied that exposure to low levels of IR may be beneficial to the cell. We postulate that increased survival does not necessarily mean that the treatment is beneficial.
Studies at the molecular level indicate that ASRs are the result of misregulated cell cycle checkpoint responses, occurring in the G1 phase of the cell cycle after IR. Specific gene products (i.e., PCNA, cyclin D1, cyclin A, XIP8, xip5 and xip13) appear to control these cell cycle checkpoint responses. Certain neoplastic cells show potent ASRs because they bypass checkpoints which would otherwise lead to apoptosis or other forms of cell death (possibly necrosis), and/or these cancer cells lack genetic factors, such as specific caspases (cysteine aspartate-specific proteases), that control apoptosis. Alterations in these cell cycle checkpoints or apoptotic responses may also occur during IR-induced stress responses in normal cells, at critical times (10-18 days posttreatment) following IR. One IR-induced protein, XIP8, may be a critical controlling factor at this point where delayed-onset apoptosis occurs. Additionally, we have shown that the presence or absence (i.e., SCID cells) of nonhomologous DNA double strand break repair did not seem to influence ASRs, suggesting that ASRs may be caused by signal transduction stress responses.
ASRs may be beneficial to survival, however, the consequence(s) of that survival may be dire. For example, many neoplastic cells exhibited far greater ASRs than normal cells. Additionally, ASRs were induced by as little as 1 cGy and were enhanced by repeated exposures of low level radiation. The implications for radiotherapy are that when a patient arrives for port film imaging during the course of therapy, the dose-rate, overall level of exposure, and time between port film exposure and high dose IR treatment become potentially important factors for improved efficacy of treatment of certain cancers. Further research is warranted to determine what molecular factors are most important for ASRs, and current work is focusing on XIP8.
Adaptive Survival Responses (ASRs) to ionizing radiation (i.e., X- or gamma-rays) are phenomena whereby the harmful effects of a high dose challenge of ionizing radiation can be mitigated if cells are first exposed to a single (or repeated) low dose(s) of ionizing radiation (1-3). Cells exhibiting ASRs demonstrate increased survival and lowered chromosome aberrations, possibly due to increased DNA repair processes. An ASR to radiation was first reported in 1984 using human lymphocytes (4, 5). When cultured with [3H]thymidine as a source of low-level radiation and subsequently exposed to 150 cGy, the amount of chromatid aberrations was approximately two-fold less than the sum of aberrations caused by [3H]thymidine or X-rays alone. Similar results were found using low doses of X-rays (1 or 5 cGy) (6). Adaptation required at least 4 h to appear, lasted at least three cell cycles, and only occurred when cells were adapted in G1- or S-phase, but not G0 (7). This response was not due to differential cell cycle stage-sensitivity or a radiosensitive sub-population of lymphocytes (8). Pretreatment of cells with a low dose of X-rays followed by exposure to DNA damaging agents, such as bleomycin (which can induce double-strand breaks) (9, 10) or mitomycin C (which can induce DNA cross-links) (9), led to cross-adaptation (i.e., reduced chromosomal aberrations). This cross-adaptation was not observed, however, for the alkylating agent, methylmethane sulfonate (which indirectly induces single-strand breaks in DNA via monofunctional methylation) (9). The adaptive response could be blocked by incubation of lymphocytes with the protein synthesis inhibitor, cycloheximide (11), or the poly(ADP-ribose) polymerase (PARP) inhibitor, 3-aminobenzamide (8). The frequency of mutations at the hprt locus was reduced by approximately 70% in adapted lymphocytes (12). ASRs have been demonstrated in a wide variety of systems, including V79 Chinese hamster cells (13), C57 black 6 mice (14, 15), human skin fibroblasts (16), and C3H10T1/2 cells (17).
Evidence for Adaptive Survival Responses (ASRs) in Human Cells. Various human normal and neoplastic cells were exposed to low priming doses of IR (5 cGy/day) over 4 days. Primed and unprimed growth-arrested human cells were then exposed to a high challenging dose of IR on the 5th day, and colony-forming assays were used to assess survival as described (18, 19). Out of eleven normal human cells and human tumor cell lines tested, only two cell lines, U1-Mel and HEp-2, showed significant survival enhancements (i.e., ASRs) over their unprimed counterparts following a 5 Gy challenging dose of IR (Table 1 gives a partial listing of cell lines tested). SCID, CB17, 100E and 50D rodent cell lines also demonstrated ASRs, but were found to express mutant p53. ASRs in HEp-2 and U1-Mel cells were prevented by actinomycin D or cycloheximide, indicating that both new transcription and protein synthesis were required (16). ASRs were not observed in any normal human fibroblasts tested, nor in many other neoplastic cells (16). All rodent cells tested demonstrated ASRs (Table 1).
Influence of Priming Doses on ASRs. We then investigated the possibility that different cells might require different priming doses for ASRs. Confluence-arrested normal or cancer cells were treated with various priming doses of IR (0.01 to 50 cGy), then challenged with a single high equitoxic dose of IR. Again, ASRs were observed only in U1-Mel and HEp-2 cells, but only after a very specific range (1-10 cGy) of doses. None of the normal human fibroblasts or other human cancer cell lines demonstrated ASRs (16). Finally, primed HEp-2 or U1-Mel cells were challenged at various times (one to five days) after their final priming dose to estimate how long ASRs were maintained. ASRs were sustained for two to three days, but were absent within five days post-IR in HEp-2 and U1-Mel cells.
Identification of Transcripts Whose Levels Are Altered During ASRs. In order to find out why U1-Mel and HEp-2 cells demonstrated ASRs and other human cells did not, we decided to investigate the molecular changes and expression of key proteins that could be affected by low dose IR priming treatments. Two transcripts, xip5 and xip13 (a 0.5 kb transcript related to xip12), gradually accumulated after priming doses of IR in both HEp-2 and in U1-Mel cells, but were not noted in other human normal or other cancer cells. Treatment of U1-Mel cells with actinomycin D at 5 µg/ml for 4 h after each exposure to IR prevented their expression (16). Recently, a third transcript/protein (i.e., XIP8) has been shown to increase with IR (20), it has been linked to DSB repair, and may control apoptosis following high doses of IR (Yang et al., Unpublished data). Interestingly, this transcript was not induced by low dose IR treatments used to establish ASRs in U1-Mel and HEp-2 cells and its induction was suppressed following high challenging doses in these cells under conditions where they exhibited ASRs. We theorize that XIP8 may play a role(s) in IR-induced delayed-onset apoptosis which occurs following high dose IR, and this transcript/protein may be suppressed during ASRs, leading to a lower level of programmed cell death (21) as described (22).
Since changes in cyclin levels have been reported after IR (23), these transcripts were also examined. Cyclin A transcript levels accumulated in HEp-2 and U1-Mel cells during low priming doses (1-10 cGy X 4 days) and did not accumulate in cells exposed only to 5 Gy. Cyclin D1 mRNA levels increased after only one 1-10 cGy treatment, however, its levels did not continually increase with each priming dose. Similar increases in cyclin D1 levels were observed after 5 cGy or 5.0 Gy. Cyclin A and cyclin D1 transcripts increased three-fold and two-fold, respectively, in primed cells exposed to 4.5 Gy compared to unprimed cells treated with IR alone. Cyclin B levels remained unchanged in all cells examined. Similar changes in cyclin D1 and A transcript levels were found in HEp-2 cells (16).
Analyses of Protein Changes During ASRs in Human Cells. We then examined alterations in protein levels (in whole cell or in isolated nuclei) after priming or high challenging doses of IR. Consistent with changes in transcript levels, nuclear protein levels of cyclin A increased during low dose priming exposures and after a high dose of IR in U1-Mel and HEp-2 cells, but not in the other human normal, neoplastic, or cancer-prone cells (Table 1). Interestingly, we noted high constitutive nuclear protein levels of both PCNA and cyclin D1 in primed or unprimed human U1-Mel and HEp-2 cells. Much lower levels, or no constitutive levels of these two proteins were observed in any of the other human normal cells examined. Increases in nuclear p53 protein levels were noted only after high dose IR (challenging) exposures of all human cells examined, including HEp-2 and U1-Mel cells; these data suggest that all cells examined expressed wild-type p53 (24). Similarly, X-ray-inducible protein #8 was not induced after low dose IR treatments (16, 20). Thus, p53 or XIP8 stress responses do not appear to be activated during ASRs in cells competent for developing this temporary radioresistant state. These data indicated to us that a combination of increased cyclin A protein levels, constitutively elevated PCNA and cyclin D1 levels, and a lack of p53- or XIP8-dependent apoptotic responses may be key factors in the ability of certain human cancer cells to display ASRs.
Cell Cycle Analysis of Primed Cells. We then determined if primed U1-Mel cells were progressing into a later portion of G1, which would explain increases in cyclin A and lack of increase in thymidine kinase transcript levels (16). We replated primed or unprimed U1-Mel cells at low density and followed their re-entry into the cell cycle using flow cytometry. Northern blot analysis of an S-phase specific transcript (i.e., thymidine kinase), and measurements of [3H]thymidine-labeled nuclei were also monitored. We found that confluence-arrested, primed U1-Mel cells entered S-phase within 6-8 h, whereas confluence-arrested unprimed U1-Mel cells entered S-phase in 12-14 h. These data indicate that primed human U1-Mel cells were at an advanced, but apparently arrested, state compared to a similar population of unprimed, growth-arrested human U1-Mel cells. We called this state an A (for Adaptive) cell cycle checkpoint (25).
Delayed On-set Apoptosis Following IR: Influence of ASRs. In a series of separate investigations, we have also begun to monitor the influence of ASRs on delayed on-set apoptosis after IR. To date, we have characterized an interesting delayed-onset apoptotic response using CGL1 cells; a HeLa X normal human fibroblast cell line which can be neoplastically transformed following IR (21, 26-28). The appearance of neoplastically transformed foci in this system was delayed, beginning at day 9 post-irradiation. Mendonca et al. (29) noted that neoplastic transformation correlated well with a rather dramatic reduction in plating efficiency (PE). It was recently found that after 7 Gy, both foci initiation and reduced PE correlated with the emergence of apoptosis in the progeny of the irradiated cells, but only after 10 to 12 cell divisions (Mendonca et al., In Preparation). Delayed apoptosis began around day 8 post-irradiation and lasted for ~10 days. Classic apoptotic morphology, viewed by TUNEL & Annexin V assays, were evidence of genomic DNA degradation. Cleavage of PARP correlated with the above apoptotic events.
Interestingly, a delayed (over a week postirradiation) induction/stabilization of p53 and the XIP8 proteins also correlated with the onset of delayed apoptosis (Mendonca and Boothman et. al., Unpublished Data). Both proteins have been implicated in apoptotic responses. It has been proposed that a delayed build-up of genomic DNA damage or loss of genetic material over time (10-12 cell divisions post-irradiation) has two relevant outcomes: (a) cell death due to the delayed induction of a p53-dependent and/or XIP8-dependent apoptotic response; or (b) neoplastic transformation of a minor subset of survivors which have lost fibroblast chromosomes #11 and #14 [tumor suppressor loci for this system, (26)], perhaps resulting in a loss of apoptotic regulatory factors. We are now examining the effects of ASRs on this coordinate delayed-onset apoptosis (i.e., reduction in PE) and neoplastic transformation process.
ASRs Require New Protein Synthesis. Although new protein and RNA synthesis appear to be required for ASRs [e.g., we (Table 1) and others (11) have shown that CHM or actinomycin D treatments block ASRs], activation of common transcription factors was not noted during low dose IR priming reactions (30). Two-dimensional gel electrophoretic analyses of adapted lymphocytes showed increased expression of a number of proteins (10, 31). Induced proteins may have a direct (i.e., p53 or XIP8) or indirect role in DNA repair and/or apoptosis. Alternatively, the induction of new proteins during ASRs may be involved in cell cycle control or prevention of apoptosis. The role of damage-induced proteins in delayed-onset apoptosis has yet to be defined, although considerable evidence is building for a role of p53 and possibly XIP8 in such processes. For example, p53 levels have been found to increase in response to X-rays, presumably to arrest the cell cycle and allow for apoptosis if the cell is severely damaged (32). XIP8 may play a similar role (Yang et al., Unpublished Data). The absence or actual suppression of these proteins during ASRs in certain neoplastic cells may be one mechanism whereby cells establish this temporary radioresistant state and increase survival following high dose challenging events.
Cyclin D1, whose mRNA levels increased during ASRs, but whose protein levels were constitutively elevated, is associated with various cyclin-dependent kinases (cdk2, 4 and 5), proliferating cell nuclear antigen (PCNA), and DNA polymerase-alpha. Increases in cyclin A and constitutively high levels of PCNA and cyclin D1 were noted in cells proficient at ASRs (16). Interestingly, cyclin D1, PCNA, DNA polymerase-alpha and cyclin A may participate in a DNA repair complex (33). Thus, our data indicate a possible correlation between the expression of PCNA/cyclin D1/cyclin A proteins and ASRs in human cells. Only HEp-2 and U1-Mel cells demonstrated ASRs. These cell lines over-expressed cyclin D1 and PCNA, and have notable increases in low dose IR-inducible cyclin A levels during the low priming doses of IR required for ASRs.
Our results are consistent with a low dose-inducible adaptive survival (A) cell cycle checkpoint in G1 (16). U1-Mel or HEp-2 cells were initially arrested in G0 or in early G1. Upon repeated exposure to low doses of radiation, cells progress to an A (Adaptive) point near the G1/S border of the cell cycle, where multiple cell cycle checkpoints are thought to exist (34). This was corroborated by our cell cycle release data in which low dose primed U1-Mel cells were 4-6 h advanced in the cell cycle compared to non-irradiated human U1-Mel cells (16). Gene transcripts which build up slowly in response to low priming doses of IR (e.g., xips5 and 13) may produce proteins that stimulate cell cycle progression or move cells to a new cell cycle checkpoint at which point they may induce a radioresistant phenotype. Unfortunately, we do not yet have antibodies to xips5 and 13, and we are having difficulties screening for full-length cDNAs corresponding to these two transcripts. We theorize that once low dose IR primed cells escape from their initial growth and low serum arrested state, they move to another cell cycle checkpoint at which cyclin A can be expressed. The constitutive elevation of cyclin D1 and PCNA proteins in these human neoplastic cells probably contributes to their abrogation of one cell cycle checkpoint and may contribute to arresting the cell at the A point after multiple low dose priming exposures. At the A point, we hypothesize that cells may then stimulate or establish various DNA repair complexes (possibly containing cyclin D1, PCNA and DNA polymerase-alpha) which are not inducible in the initial G0/G1-arrested state, but are eventually required, in combination with the inducible levels of cyclin A, to establish ASRs. Further research is warranted in this under-funded area of radiation research.
Relevance to Radiotherapy. ASRs may be relevant to two aspects of radiotherapy: the radioresponsiveness of the primary tumor and the generation of secondary tumors many cell generations later. The fact that low dose IR can induce a shift from one cell cycle checkpoint presumably through the generation of key regulatory proteins, strongly suggests that the administration of low doses of radiation prior to a high dose challenge can induce a temporary radioresistant state. Such a state may result in a dramatic reduction in curing the primary tumor. During radiation therapy it is standard practice to apply one or several doses of low level radiation in order to visualize the tumor prior to therapy. These port films, which utilize less that 1 cGy per treatment, may be sufficient to induce an ASR in vivo, although such doses did not induce ASRs in vitro. We have not studied the molecular changes in ASRs occurring in vivo using human tumor xenografts, but we anticipate that lower radiation levels will probably establish ASRs, since IR-induced stress responses can be visualized in vivo with far less IR doses than in vitro (Davis and Boothman et. al., Unpublished Data). The potential adverse effects of ASRs on radiotherapy efficacy should be examined given the present knowledge of molecular changes occurring in vitro (16).
ASRs may also be a major effector in IR-induced carcinogenesis and the formation of secondary cancer cells from exposed normal cells, numerous cell generations post-IR treatment. Substantial data are accumulating regarding the interplay between genomic instability, delayed on-set apoptosis, and the emergence of neoplastically transformed cells [Mendonca et al., Submitted, and refs. (21, 26, 29)]. One IR-induced protein, XIP8, may play a role(s) in all three of the above processes. The protein is induced by low or high doses of IR, it appears to play a role in apoptosis, and has recently been linked to DNA double strand break repair (Yang et al., Unpublished Data); the mechanism by which this protein interacts with the DNA repair machinery will be published shortly (Yang and Boothman et. al., In Preparation). We recently found that this protein accumulated at the same time IR-induced, p53-dependent delayed on-set apoptosis was activated, prior to the emergence of neoplastically transformed cells (Mendonca and Boothman et. al., In Preparation). Further studies using XIP8 knockout animals, which now have been generated, will be required to see if this protein protects against, or causes, apoptosis in cells that were treated with low or high doses of IR. The appearance of proteins which regulate cell cycle checkpoints (i.e., cyclins A and D1 and PCNA) or apoptosis (i.e., p53 or XIP8) following IR could result in reduced efficacy of treatment and/or the emergence of secondary tumors some generations posttreatment. For example, if XIP8 causes apoptotic responses during low dose IR-induced ASRs or after high dose IR treatments, then its down-regulation or lack of induction in cells competent for ASRs may avert apoptosis caused by a subsequent high dose IR challenge. Evidence for suppression of apoptotic responses during ASRs have been demonstrated (22). Further research on the exact role of XIP8 in apoptotic responses during ASRs or after high dose IR is warranted.
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