TIG3 Tumor Suppressor-Dependent Organelle Redistribution and Apoptosis in Skin Cancer Cells

TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis

Toward Predicting Acute Myeloid Leukemia Patient Response to 7 + 3 Induction Chemotherapy via Diagnostic Microdosing

Acute myeloid leukemia (AML) is a rare yet deadly cancer of the blood and bone marrow. Presently, induction chemotherapy with the DNA damaging drugs cytarabine (ARA-C) and idarubicin (IDA), known as 7 + 3, is the standard of care for most AML patients. However, 7 + 3 is a relatively ineffective therapy, particularly in older patients, and has serious therapy-related toxicities. Therefore, a diagnostic test to predict which patients will respond to 7 + 3 is a critical unmet medical need. We hypothesize that a threshold level of therapy-induced 7 + 3 drug-DNA adducts determines cytotoxicity and clinical response. We further hypothesize that in vitro exposure of AML cells to nontoxic diagnostic microdoses enables prediction of the ability of AML cells to achieve that threshold during treatment. Our test involves dosing cells with very low levels of 14C-labeled drug followed by DNA isolation and quantification of drug-DNA adducts via accelerator mass spectrometry. Here, we have shown proof of principle by correlating ARA-C- and DOX-DNA adduct levels with cellular IC50 values of paired sensitive and resistant cancer cell lines and AML cell lines. Moreover, we have completed a pilot retrospective trial of diagnostic microdosing for 10 viably cryopreserved primary AML samples and observed higher ARA-C- and DOX-DNA adducts in the 7 + 3 responders than nonresponders. These initial results suggest that diagnostic microdosing may be a feasible and useful test for predicting patient response to 7 + 3 induction chemotherapy, leading to improved outcomes for AML patients and reduced treatment-related morbidity and mortality

Diagnostic Microdosing Approach to Study Gemcitabine Resistance

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic "microdoses" of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of 14C-labeled gemcitabine for 4-24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity

Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications

Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system

<div><p>ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. <i>In vitro</i> analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published <i>in vitro</i> binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the <i>in vitro</i> characterization of ErbB heterodimers and disease-relevant mutants.</p></div

Diagnostic Microdosing Approach to Study Gemcitabine Resistance

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic “microdoses” of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of ¹⁴C-labeled gemcitabine for 4–24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity

NLP assemblies contain EGFR monomers, dimers, and multimers.

<p>Empty and EGFR-NLPs were separated by SDS-PAGE with (+β-ME/heat) and without denaturing conditions (-β-ME/heat) to observe EGFR multimers and analyzed by immunoblotting with anti-pY4G10, anti-EGFR, and anti-ApoA1 antibodies. Similar results were observed in each of three biological replicates. Numbers indicate band intensity relative to EGFR-NLPs normalized to ApoA1 signal.</p

NLP-associated EGFR interacts with EGFR-targeted therapeutic agents.

<p><i>A</i>, NLP incorporated EGFR was dephosphorylated via phosphatase treatment (CIP) then allowed to autophosphorylate in the absence or presence of indicated tyrosine kinase inhibitors (M, mubritinib; A, afatinib; C, canertinib; L, lapatinib) followed by immunoblotting with anti-pY4G10 and anti-EGFR antibodies. Similar results were observed in each of three biological replicates. *Autophosphorylation is observed without additional EGF due to EGF being present in the fetal bovine serum and in the initial assembly mixture. <i>B</i>, Association and dissociation of 100 nM of empty or EGFR-NLP to biotinylated EGF on the sensors with and without 1 μM unlabeled EGF or cetuximab. Data points indicate the mean of two technical duplicates and are representative of results from two biological replicates. <i>C</i>, Association and dissociation curves of 100 nM EGFR-NLPs binding to biotinylated EGF on the sensors in the presence of increasing levels of Cetuximab. Cetuximab concentrations were titrated by a 1.5-fold dilution to the concentrations displayed to the right of the graph. Curves were normalized by subtracting the 0 μM Cetuximab curve. <i>D</i>, Log plot of cetuximab inhibiting the binding of EGFR-NLPs to biotinylated EGF on the sensors. Data points indicate the mean of two technical duplicates and are representative of results from two biological replicates.</p