Radiosensitivity and transcription factor NF-kappaB inhibition-progress and pitfalls.

Cellular responses to ionizing radiation include activation of signal transduction cascades that may originate at the plasma membrane, cytoplasm, or nucleus. The cell’s success in dealing with radiation “stress” determines its survival or death. Activation of transcription factor NFkB, an immediate early response after exposure to ionizing radiations, functions to protect cells from apoptosis (programmed cell death), but the role of NFkB in mitotic cell death has not been fully defined (1,2). NF-kB is constitutively activated in lymphoid cells, immortal ataxia telangiectasia (AT) fibroblasts, and other cells (3–6). Activation of NF-kB after ionizing radiation requires intact ATM gene function in AT fibroblasts(7). Because AT fibroblasts exhibit dysregulated NFkB activation and extreme radiation sensitivity, it is reasonable to ask if radiation responses of tumor cells can be modified by inhibiting NFkB. NF-kB/Rel transcription factors are activated by a variety of different signals in pathways that converge on the phosphorylation and degradation of I kBs, inhibitors of NFkB. This results in the unmasking of the nuclear localization signals that lead to translocation of NFkB/Rel dimers into the nucleus. Five known mammalian NFkB/Rel proteins include c-Rel, p65 (RelA), RelB, p50 (NFkB1), and p52 (NFkB2) that bind DNA as homodimers or heterodimers (8). It should also be noted that p50–p65/I kB is not the only complex relevant to NF-kB activation. The p50–p65 heterodimer can interact with other IkB family members, and NFkB can also exist in other homodimer or heterodimer complexes, such as p50–p50 and p65–p65 (8). Furthermore, the I kB family consists of three members—I kBa, IkBb, and IkB«. In addition, the carboxylterminal regions of the precursors for p50 and p52, p105 and p100, respectively, can also function as inhibitory proteins. These inhibitors respond differentially to NFkB-inducing signals in a cell type-dependent and stimulus-dependent manner in vivo (9). In this issue of the Journal, Pajonk et al. (10) report that activation of NFkB does not determine the intrinsic radiosensitivity of cancer cells. Cells were transduced with an adenoviral vector expressing a mutant form of I kBa, an inhibitor of NF-kB. The resultant clonogenicity of cells transduced with mutant IkBa was markedly reduced to 7.4% (compared with 29.5% for control cells), and the apoptotic index was 90%. Therefore, subsequent radiation survival experiments were done on relatively small subsets of cells, whereas biochemical assays were done on mostly apoptotic cells. It is unusual to see publication of essentially negative studies in the Journal. However, there is potential value if definitive data can provide direction for other scientists in the field. Clinically, intrinsic radiation sensitivity has been implicated in tumor curability (11). The ability to manipulate radiation sensitivity offers a potential strategy for improving the therapeutic ratio in cancer treatment. Therefore, an examination of the experimental results and data interpretation underlying the negative radiobiologic conclusions of Pajonk et al. (10) are appropriate. 1) Are PC3 and HD-MyZ cells resistant to radiation? Pajonk et al. (10) state that the two cell lines were chosen because they have high constitutive levels of NFkB that might confer relatively high resistance to radiation. Strictly speaking, radiation resistance is an imprecise term that may include various aspects of cellular responses to ionizing radiation (12). On the other hand, radiation sensitivity is defined by the steepness of the terminal slope of the cellular radiation survival curve (single-hit multitarget model). ADo value (the dose required to decrease survival by 1/ e) can be determined for relative quantitation of radiation sensitivities. Do values for PC3 and HD-MyZ cells are estimated to be 1.0–1.1 Gy [Fig. 3 in (10)], which is quite sensitive for tumor cell lines and is comparable in sensitivity to AT heterozygote fibroblasts (13). Disruption of signal transduction pathways of resistant cells has been reported to result in radiosensitization (14), but it may be difficult to make already sensitive cells more sensitive. An alternative interpretation of the constitutively activated NFkB levels observed in PC3 and HD-MyZ cells may be related to apoptotic stresses rather than reflecting potential resistance. This is consistent with subsequent observations of Pajonk et al. (10) that 90% of these cells die of apoptosis when mutant I kB-a is expressed, inhibiting protective functions of activated NFkB. 2) Does the I kBa superrepressor product inhibit radiationinduced NFkB activation in the small subset of transduced cells that do not undergo apoptosis? Gel-shift experiments are offered by Pajonk et al. (10) showing DNA binding by NF-kB to be present in cytosolic extracts from control cells but not from Ad5-I kB-transduced cells that were either given 30 Gy of ionizing radiation or not. These experiments were performed 48 hours after transduction of cells and are interpreted to show that the mutated I kB gene product inhibits constitutive NFkB activity and radiation-induced activation. An alternative interpretation of these data may be that cytosolic extracts obtained from still viable control cells differ from those obtained from the Ad5-I kB-transduced cells that were mostly dead. The substantial disparity of surviving cell fractions makes the reported biochemical comparisons in survivors unreliable. Furthermore, the use of cytosolic extracts from cells undergoing apoptosis must include consideration of possible effects of caspases targeting components of NFkB (15).