Search PubMed⌕ Search

Biomedical subjects

David A Boothman

Publications and source records attributed to David A Boothman.

At least 19 recordsLinked to original sources
PubMed2006

Calcium-dependent modulation of poly(ADP-ribose) polymerase-1 alters cellular metabolism and DNA repair.

After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD(+) and ATP depletion and cell death. Mechanisms of PARP-1-mediated cell death and downstream proteolysis remain enigmatic. beta-lapachone (beta-lap) is the first chemotherapeutic agent to elicit a Ca(2+)-mediated cell death by PARP-1 hyperactivation at clinically relevant doses in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. Beta-lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca(2+)-dependent gamma-H2AX formation and PARP-1 hyperactivation. Subsequent NAD(+) and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca(2+) chelation protects cells. Interestingly, Ca(2+) chelation abrogates hydrogen peroxide (H(2)O(2)), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca(2+) appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage, which alters cellular metabolism and DNA repair.

Adenosine Triphosphate↗
PubMed2006

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors.

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, beta-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.

Anticarcinogenic Agents↗
PubMed2006

Mornings with Art, lessons learned: feedback regulation, restriction threshold biology, and redundancy govern molecular stress responses.

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.

Apoptosis↗
PubMed2006

Modulating beta-lapachone release from polymer millirods through cyclodextrin complexation.

Beta-lapachone (beta-lap) is a novel anticancer agent that kills tumors overexpressing the NADPH: quinone oxidoreductase enzyme. However, poor aqueous solubility and low bioavailability hinder its therapeutic applications. Herein we describe the development of poly(D,L-lactide-co-glycolide) (PLGA) polymer millirods for local delivery of beta-lap. The objective was to investigate the use of beta-lap inclusion complexes with cyclodextrins (CDs) to control beta-lap release kinetics from PLGA millirods. Differential scanning calorimetry was performed to measure drug/polymer interactions, complexation efficiency with different CDs, and complex/polymer interactions. beta-Lap was found to have a solid-state solubility of 13% in PLGA. beta-Lap dissolution in PLGA matrix lowered the glass transition temperature of PLGA from 44 to 31 degrees C, and led to a slow release of beta-lap (8.8+/-1.2% release after 22 days). For beta-lap and CD interactions, increasing complexation efficiency was observed in the order of alpha-CD, gamma-CD, and beta-CD. beta-Lap complexation with hydroxypropyl-beta-cyclodextrin (HPbeta-CD) prevented drug dissolution in PLGA, and led to fast release (79.6+/-2.1% after 2 days). Sustained drug release was achieved when beta-lap was complexed with alpha-CD or gamma-CD. These data demonstrate the ability to tailor beta-lap release kinetics via CD complexation, providing exciting opportunities for the use of beta-lap-millirods for intratumoral drug delivery.

Antineoplastic Agents↗
PubMed2006

Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems.

We describe the development of multifunctional polymeric micelles with cancer-targeting capability via alpha(v)beta(3) integrins, controlled drug delivery, and efficient magnetic resonance imaging (MRI) contrast characteristics. Doxorubicin and a cluster of superparamagnetic iron oxide (SPIO) nanoparticles were loaded successfully inside the micelle core. The presence of cRGD on the micelle surface resulted in the cancer-targeted delivery to alpha(v)beta(3)-expressing tumor cells. In vitro MRI and cytotoxicity studies demonstrated the ultrasensitive MRI imaging and alpha(v)beta(3)-specific cytotoxic response of these multifunctional polymeric micelles.

Cell Line, Tumor↗
PubMed2005

Heat-induced up-regulation of NAD(P)H:quinone oxidoreductase potentiates anticancer effects of beta-lapachone.

PURPOSE: The purpose of the present study was to evaluate the efficacy of mild hyperthermia to potentiate the anticancer effects of beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) by up-regulating NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells. EXPERIMENTAL DESIGN: Effects of beta-lapachone alone or in combination with mild heating on the clonogenic survival of FSaII fibrosarcoma cells of C3H mice and A549 human lung tumor cells in vitro was determined. Effects of heating on the NQO1 level in the cancer cells in vitro were assessed using Western blot analysis for NQO1 expression, biochemical determination of NQO1 activity, and immunofluorescence microscopy for NQO1 expression. Growth of FSaII tumors in the hind legs of C3H mice was determined after treating the host mice with i.p. injection of 45 mg/kg beta-lapachone followed by heating the tumors at 42 degrees C for 1 hour every other day for four times. RESULTS: Incubation of FSaII tumor cells and A549 tumor cells with beta-lapachone at 37 degrees C reduced clonogenic survival of the cells in dose-dependent and incubation time-dependent manner. NQO1 level in the cancer cells in vitro increased within 1 hour after heating at 42 degrees C for 1 hour and remained elevated for >72 hours. The clonogenic cell death caused by beta-lapachone increased in parallel with the increase in NQO1 levels in heated cells. Heating FSaII tumors in the legs of C3H mice enhanced the effect of i.p.-injected beta-lapachone in suppressing tumor growth. CONCLUSION: We observed for the first time that mild heat shock up-regulates NQO1 in tumor cells. The heat-induced up-regulation of NQO1 enhanced the anticancer effects of beta-lapachone in vitro and in vivo.

Animals↗
PubMed2005

Interactions between self-assembled polyelectrolyte shells and tumor cells.

Layer-by-layer self-assembled polyelectrolyte shells are a new class of micro/nanocapsules with unique physicochemical properties for potential applications in drug/gene delivery. The objective of this study was to investigate the interactions of polyelectrolyte shells ( approximately 1 mum in diameter) with MCF-7 breast cancer cells and identify key parameters that affect such interactions. Tailoring of surface properties of polyelectrolyte shells was achieved by choosing different outermost layer materials, including cationic polymers, anionic polymers, and lipid bilayers. Different surface compositions led to a wide range of electrostatic potentials from -46 to +47 mV in phophate-buffered saline buffer. Confocal microscopy studies showed that the polyelectrolyte shells were internalized into the cell cytoplasm, but not into the nuclei. Correlation of cell uptake with shell surface compositions was complicated by the adsorption of serum proteins on the surface of polyelectrolyte shells, particularly polycation-coated shells. To prevent protein adsorption, poly(ethylene glycol) (PEG) grafted poly(ethyleneimine) (PEI) copolymers (1:1, 1:5, 1:10 graft ratios) were synthesized and introduced on the shell surface. Shells coated with PEI-PEG copolymers effectively reduced protein adsorption whereas PEI-PEG copolymers with lower graft ratios achieved higher cell uptake efficiency after 24 h of incubation with MCF-7 cells.

Biocompatible Materials↗
PubMed2005

Development of beta-lapachone prodrugs for therapy against human cancer cells with elevated NAD(P)H:quinone oxidoreductase 1 levels.

beta-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces beta-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to beta-lapachone. Thus, beta-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non-small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of beta-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to beta-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to beta-lapachone. Once converted, beta-lapachone derivatives caused NQO1-dependent, mu-calpain-mediated cell death in human cancer cells identical to that caused by beta-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect beta-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of beta-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to beta-lapachone. The use of beta-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

Cell Division↗
PubMed2005

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.

Cell Line, Tumor↗
PubMed2005

Efficacy of beta-lapachone in pancreatic cancer treatment: exploiting the novel, therapeutic target NQO1.

NAD(P)H:quinone oxidoreductase (NQO1) is elevated in human pancreatic cancers. We hypothesized that beta-lapachone, a novel 1,2-naphthoquinone with potential antitumor activity in cancer cells expressing elevated levels of NQO1, would induce cytotoxicity in pancreatic cancer cells, wherein this two-electron reductase was recently found elevated. beta-lapachone decreased clonogenic cell survival, metabolic cell viability, and anchorage- independent growth in soft agar. The cytotoxic in vitro effects of beta-lapachone were inhibited with coadministration of dicumarol, a specific inhibitor of NQO1. In preestablished human pancreatic tumor xenografts in nude mice, beta-lapachone demonstrated greater tumor growth inhibition when given intratumorally compared to when complexed with cyclodextrin to increase its bioavailability. Due to the poor prognosis of patients with pancreatic cancer and the limited effectiveness of surgery, chemotherapy, and radiation therapy, treatment regimens based on sound, tumor-specific rationales are desperately need for this disease.

Aged↗
PubMed2005

Susceptibility of cancer cells to beta-lapachone is enhanced by ionizing radiation.

PURPOSE: To reveal the interaction between beta-lapachone (beta-lap) and ionizing radiation (IR) in causing clonogenic death in cancer cells and to elucidate the potential usefulness of beta-lap treatment in combination with radiotherapy of cancer. METHODS AND MATERIALS: FSaII tumor cells of C3H mice were used. The cytotoxicity of beta-lap alone or in combination with IR in vitro was determined using clonogenic survival assay method. The IR-induced changes in the expression and the enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1), a mediator of beta-lap cytotoxicity, were elucidated and the relationship between the NQO1 level and the sensitivity of cells to beta-lap was investigated. The combined effect of IR and beta-lap to suppress tumor growth was studied using FSaII tumors grown subcutaneously in the thigh of C3H mice. RESULTS: beta-Lap caused clonogenic death of FSaII tumor cells in vitro in a dose- and time-dependent manner. When cells were treated first with beta-lap and then exposed to IR in vitro, the resultant cell death was only additive. On the contrary, exposing cells to IR at 2.5 Gy first and then treating the cells with beta-lap killed the cells in a synergistic manner. Importantly, the 2.5 Gy cells were sensitive to beta-lap as long as 10 h after irradiation, which was long after the sublethal radiation damage was repaired. Irradiation of FSaII cells in vitro with 2.5 Gy significantly increased the expression and enzymatic activity of NQO1. The growth delay of FSaII tumors caused by an intraperitoneal injection of beta-lap in combination with 20 Gy irradiation of tumor was significantly greater than that caused by beta-lap or 20 Gy irradiation alone. CONCLUSION: The sensitivity of cells to beta-lap is dependent on NQO1 activity. IR caused a long-lasting increase in NQO1 activity in cancer cells, thereby sensitizing cells to beta-lap and treatment of experimental mouse tumors with IR and beta-lap suppressed tumor growth in a synergistic manner. The combination of beta-lap and radiotherapy is a potentially effective regimen for the treatment of human cancer.

Animals↗
PubMed2004

DNA mismatch repair-dependent response to fluoropyrimidine-generated damage.

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.

Adenosine Triphosphatases↗
PubMed2004

IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects.

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.

Animals↗
PubMed2004

Role of DNA mismatch repair in apoptotic responses to therapeutic agents.

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.

Alkylating Agents↗
PubMed2003

A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage.

The phenomenon of damage tolerance, whereby cells incur DNA lesions that are nonlethal, largely ignored, but highly mutagenic, appears to play a key role in carcinogenesis. Typically, these lesions are generated by alkylation of DNA or incorporation of base analogues. This tolerance is usually a result of the loss of specific DNA repair processes, most often DNA mismatch repair (MMR). The availability of genetically matched MMR-deficient and -corrected cell systems allows dissection of the consequences of this unrepaired damage in carcinogenesis as well as the elucidation of cell cycle checkpoint responses and cell death consequences. Recent data indicate that MMR plays an important role in detecting damage caused by fluorinated pyrimidines (FPs) and represents a repair system that is probably not the primary system for detecting damage caused by these agents, but may be an important system for correcting key mutagenic lesions that could initiate carcinogenesis. In fact, clinical studies have shown that there is no benefit of FP-based adjuvant chemotherapy in colon cancer patients exhibiting microsatellite instability, a hallmark of MMR deficiency. MMR-mediated damage tolerance and futile cycle repair processes are discussed, as well as possible strategies using FPs to exploit these systems for improved anticancer therapy.

Animals↗
PubMed2003

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 (< or =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-kappaB), 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.

Animals↗
PubMed2003

Synthesis and functional analyses of nuclear clusterin, a cell death protein.

Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis.

Amino Acid Sequence↗