Tracy Criswell

Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation

Over the past 15 years, a wealth of information has been published on transcripts and proteins ‘induced’ (requiring new protein synthesis) in mammalian cells after ionizing radiation (IR) exposure. Many of these studies have also attempted to elucidate the transcription factors that are ‘activated’ (i.e., not requiring de novo synthesis) in specific cells by IR. Unfortunately, all too often this information has been obtained using supralethal doses of IR, with investigators assuming that induction of these proteins, or activation of corresponding transcription factors, can be ‘extrapolated’ to low-dose IR exposures. This review focuses on what is known at the molecular level about transcription factors induced at clinically relevant (⩽2 Gy) doses of IR. A review of the literature demonstrates that extrapolation from high doses of IR to low doses of IR is inaccurate for most transcription factors and most IR-inducible transcripts/proteins, and that induction of transactivating proteins at low doses must be empirically derived. The signal transduction pathways stimulated after high versus low doses of IR, which act to transactivate certain transcription factors in the cell, will be discussed. To date, only three transcription factors appear to be responsive (i.e. activated) after physiological doses (doses wherein cells survive or recover) of IR. These are p53, nuclear factor kappa B(NF-κB), and the SP1-related retinoblastoma control proteins (RCPs). Clearly, more information on transcription factors and proteins induced in mammalian cells at clinically or environmentally relevant doses of IR is needed to understand the role of these stress responses in cancer susceptibility/resistance and radio-sensitivity/resistance mechanisms.

Delayed activation of insulin-like growth factor-1 receptor/Src/MAPK/Egr-1 signaling regulates clusterin expression, a pro-survival factor.

Secretory clusterin protein (sCLU) is a general genotoxic stress-induced, pro-survival gene product implicated in aging, obesity, heart disease, and cancer. However, the regulatory signal transduction processes that control sCLU expression remain undefined. Here, we report that induction of sCLU is delayed, peaking 72 h after low doses of ionizing radiation, and is dependent on the up-regulation of insulin-like growth factor-1 as well as phosphorylation-dependent activation of its receptor (IGF-1 and IGF-1R, respectively). Activated IGF-1R then stimulates the downstream Src-Mek-Erk signal transduction cascade to ultimately transactivate the early growth response-1 (Egr-1) transcription factor, required for sCLU expression. Thus, ionizing radiation exposure causes stress-induced activation of IGF-1R-Src-Mek-Erk-Egr-1 signaling that regulates the sCLU pro-survival cascade pathway, important for radiation resistance in cancer therapy.

Repression of IR-inducible clusterin expression by the p53 tumor suppressor protein.

The clusterin (CLU) protein has been reported to have both cytoprotective and cytotoxic activities. Previous data from our lab suggest that the secretory form of CLU (sCLU) is cytoprotective and induced after very low, nontoxic doses of ionizing radiation (IR: >0.02 Gy), while a nuclear form is cytotoxic.1 Cells must presumably suppress sCLU to stimulate cell death, however, factors regulating the stress-inducible expression of sCLU have not been elucidated. Here we demonstrate that p53 can suppress sCLU induction responses. A variety of cytotoxic agents stimulated sCLU expression and DNA damage was sufficient but not necessary for induction. IR-stimulated CLU promoter activity, with concomitant increases in CLU mRNA and protein, showed that CLU induction was delayed with maximal expression observed 48-96 h post-treatment. Expression of the human papillomavirus E6 protein in MCF-7 breast or RKO colon cancer cells enhanced basal CLU levels. Isogenically matched HCT116 colon cancer cell lines that differed only in p53 or p21 status, confirmed a role for p53 in the transcriptional repression of sCLU. Loss of functional p53 in HCT116:p53-/- cells augmented CLU de novo synthesis after IR exposure. Repression of sCLU protein levels by p53 may be important for the cascade of p53-mediated events leading to cell death after IR or other cytotoxic agent exposure.

Modulation of NFκB activity and E-cadherin by the type III transforming growth factor β receptor regulates cell growth and motility

Transforming growth factor β is growth-inhibitory in non-transformed epithelial cells but becomes growth-promoting during tumorigenesis. The role of the type I and II receptors in tumorigenesis has been extensively studied, but the role of the ubiquitously expressed type III receptor (TβRIII) remains elusive. We developed short hairpin RNAs directed against TβRIII to investigate the role of this receptor in breast cancer tumorigenesis. Nontumorigenic NMuMG mouse cells stably expressing short hairpin RNA specific to mouse TβRIII (NM-kd) demonstrated increased cell growth, motility, and invasion as compared with control cells expressing shRNA to human TβRIII (NM-con). Reconstitution of TβRIII expression with rat TβRIII abrogated the increased growth and motility seen in the NM-kd cells. In addition, the NM-kd cells exhibited marked reduction in the expression of the adherens junction protein, E-cadherin. This loss of E-cadherin was due to increased NFκB activity that, in turn, resulted in increased expression of the transcriptional repressors of E-cadherin such as Snail, Slug, Twist, and Sip1. Finally, NMuMG cells in which TβRIII had been knocked down formed invasive tumors in athymic nude mice, whereas the control cells did not. These data indicate that TβRIII acts as a tumor suppressor in nontumorigenic mammary epithelial cells at least in part by inhibiting NFκB-mediated repression of E-cadherin.

Knockdown of the Transforming Growth Factor-β Type III Receptor Impairs Motility and Invasion of Metastatic Cancer Cells

The transforming growth factor-beta (TGF-beta) signaling pathway plays dual roles in epithelial cell tumorigenesis. TGF-beta is initially growth inhibitory, but as tumorigenesis progresses, TGF-beta becomes prometastatic. Although the role of the types I and II TGF-beta receptors is fairly well established, the role of the ubiquitously expressed TGF-beta type III receptor (TbetaRIII) in tumorigenesis is less defined. To examine the role of TbetaRIII in breast cancer cells, we stably expressed short hairpin RNAs specific to TbetaRIII in MDA-231 human breast cancer cells and mouse mammary carcinoma cells expressing the polyomavirus middle T oncogene (PMTLuc). MDA-231 and PMTLuc cells with down-regulated TbetaRIII expression (231-kd; PMTLuc-kd) exhibited decreased growth rate, motility, and invasion into Matrigel, as well as an increase in apoptosis, compared with control cells. MDA-231 xenografts established in nude mice metastasized, whereas tumors made by 231-kd cells did not. Nuclear factor-kappaB (NF-kappaB) activity, which is known to regulate cell growth and motility, was lower in the MDA-231 and PMTLuc knockdown cells compared with control cells. Transfection of an expression vector encoding constitutively active IKK2 into the 231-kd cells restored the ability of TbetaRIII-deficient cells to invade Matrigel and decreased their basal level of apoptosis. These data indicate that TbetaRIII differentially regulates cell growth, motility, and invasion in tumorigenic MDA-231 and PMTLuc cells and that these growth changes occur through the modulation of NF-kappaB activity.

Clusterin: a protein with multiple functions as a potential ionizing radiation exposure marker

Abstract Secretory clusterin (sCLU), a multifunctional glycoprotein, is induced in response to ionizing radiation (IR) at low and high doses. We are, therefore, developing a cellular biodosimetry system utilizing the CLU promoter-luciferase-neo® genetic construct and a bioluminescent imaging system (BLIS) for the real-time detection of CLU induction as a function of low doses of IR, using both tissue culture and animal systems. In order to develop such a biodosimeter, endogenous sCLU levels in tissues of animals must be characterized using Western blot analyses to ensure that the luciferase reporter gene induction matches induction responses, in terms of dose–responses and time-courses. Whole body 0.1–5 Gy gamma-irradiation of C57BL/6J mice induced CLU levels 2–3 days post-IR in thymus, spleen, and bone marrow. Interestingly, sCLU was not induced in liver, brain, or colon. These data indicate a tissue-specific, and a rather delayed time-course of sCLU induction, matching sCLU IR responses in human MCF-7:WS8 breast cancer cells in culture. To develop a biodosimeter, an MCF-7 1403 cell line expressing the luciferase gene regulated by the 1403 bp CLU promoter was generated, wherein CLU promoter expression mimicked the endogenous CLU gene. In contrast to standard luciferase assays using a luminometer, significant changes in CLU promoter expression in MCF-7 1403 cells were detected after 10 cGy using BLIS, which were comparable with endogenous sCLU protein expression. In summary, sCLU is induced in response to low-dose IR both in vitro using human tissue culture and in vivo in mouse proliferating tissues. Examination of CLU promoter-driven luciferase levels in irradiated MCF-7 1403 cells by BLIS is a promising approach for applications in biodosimetry.