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Pigments such as melanin are most often associated with the surface of an organism, providing functions such as coloration and protection from UV radiation. However, the internal organs of some species also contain melanin. Internal melanin may also perform protective functions when cellular stress is experienced. We tested liver tissue of two tree lizard species that experienced introgression of their mitochondria to see whether melanin was present and whether it was at higher concentrations in the types of lizards thought to be under cellular stress. Previous work found that mitochondria from livers of hybrid tree lizards (those with introgressed mitochondrial DNA) had higher levels of ATP production at higher temperatures than both parental species and showed indications of dysfunction. Therefore, we predicted that if internal melanin functions to mediate the impact of damaging by-products of metabolism, such as excessive reactive oxygen species (ROS), melanin would be highest in livers of lizards with introgressed mitochondria. To test this, we used a melanin-specific stain on liver tissue sections and measured melanin concentration with spectrophotometry of heavy-membrane fractions from whole-liver homogenates of both parental species and their hybrids with introgressed mitochondria. Slides of liver sections treated with a melanin-specific stain revealed that hybrids contained melanin and had significantly higher levels of it than either parental species. Spectrophotometry gave the same result. This distribution of internal melanin matches the pattern expected if melanin functions as a compensatory mechanism to deal with higher ATP production and the subsequent high levels of ROS expected in these hybrid lizards. Future studies will examine this mechanistically.

With support from the American Society for Biochemistry and Molecular Biology (ASBMB), a community of biochemistry and molecular biology (BMB) scientist-educators has developed and administered an assessment instrument designed to evaluate student competence across four core concept and skill areas fundamental to BMB. The four areas encompass energy and metabolism; information storage and transfer; macromolecular structure, function, and assembly; and skills including analytical and quantitative reasoning. First offered in 2014, the exam has now been administered to nearly 4000 students in ASBMB-accredited programs at more than 70 colleges and universities. Here, we describe the development and continued maturation of the exam program, including the organic role of faculty volunteers as drivers and stewards of all facets: content and format selection, question development, and scoring. Teagle Foundation; ASBMB Published version This work was funded by the Teagle Foundation and the ASBMB. The authors thank the reviewers of this Essay for their thoughtful comments. We also thank Cheryl Bailey for her leadership as chair of the ASBMB Education and Professional Development Committee. We deeply appreciate the dedication of everyone involved in question development and exam scoring, especially ASBMB Education Fellows: Benjamin Alper, Rafael Alvarez-Gonzalez, Michele Alves-Bezerra, Ellen Anderson, Cindy Arrigo, Christina Arther, Suzanne Barbour, Ana Maria Barral, J. Ellis Bell, Jessica Bell, Paul Black, Michael Borland, Cory Brooks, Benjamin Caldwell, Kevin Callahan, Zachary Campbell, Danielle Cass, Vidya Chandrasekaran, Joseph Chihade, Brian Chiswell, Brian Cohen, Brooks Crickard, Laura Danai, Amy Danowitz, Nicole Davis, Gergana Deevska, Rebecca Dickstein, Edward Ferroni, Kirsten Fertuck, Emily Fogle, Geoffrey Ford, Kristin Fox, Rene Fuanta, Scott Gabriel, Allison Goldberg, Joy Goto, Thomas Goyne, David Gross, Nicholas Grossoehme, Bonnie Hall, Orla Hart, Curtis Henderson, Jennifer Hennigan, Doba Jackson, Henry Jakubowski, Sara Johnson, Carol A. Jones, Kelly Keenan, Malik Keshwani, Youngjoo Kim, Melissa Kosinski-Collins, Cheryl Kozina, Anne Kruchten, Michael Latham, Jim Lawrence, Stefanie Leacock, Watson Lees, Eric Lewis, Robley Light, Debra Martin, Betsy Martinez-Vaz, John May, Michael Mendenhall, Pamela Mertz, Florencia Meyer, Natalie Mikita, Stephen Mills, Rachel Milner, Rebecca Moen, Sarah Mordan-McCombs, Dana Morrone, Christopher Myer, Alexis Nagengast, Amjad Nasir, Venkatesh Nemmara, Ellie Nguyen, James Nolan, Daniel O'Keefe, Amy Parente, Mary Peek, John Penniston, Joseph Provost, Aswathy Rai, Supriyo Ray, Nancy Rice, John Richardson, Karen Rippe, Jennifer Roecklein-Canfield, Niina Ronkainen, Melissa Rowland-Goldsmith, Robin Rylaarsdam, John Santalucia, Kara Sawarynski, Marcia Schilling, Kristopher Schmidt, Jessica Schrader, David Segal, Cheryl Sensibaugh, Shameka Shelby, Joshua Slee, Alyson Smith, Dheeraj Soni, Claudia B. Spani, Amy Springer, Evelyn Swain, Uma Swamy, Blair Szymczyna, Ann Taylor, Cassidy Terrell, Candace Timpte, Pam Trotter, Sonia Underwood, Melanie Van Stry, Carrie Vance, Quinn Vega, Sarah Wacker, John Weldon, Scott Witherow, Michael Wolyniak, Ann Wright, Chuan Xiao, Yujia Xu, Philip Yeagle, Laura Zapanta, Nicholas Zeringo, Xiao-Ning Zhang, and Jing Zhang. Finally, our sincere thanks to participating institutions and students for their commitment to BMB teaching and learning.

The COVID-19 pandemic has challenged undergraduate instructors and students in an unprecedented manner. Each has needed to find creative ways to continue the engaged teaching and learning process in an environment defined by physical separation and emotional anxiety and uncertainty. As a potential tool to meet this challenge, we developed a set of curricular materials that combined our respective life science teaching interests with the real-time scientific problem of the COVID-19 pandemic in progress. Discrete modules were designed that are engaging to students, implement active learning-based coursework in a variety of institutional and learning settings, and can be used either in person or remotely. The resulting interdisciplinary curriculum, dubbed "COVID-360," enables instructors to select from a menu of curricular options that best fit their course content, desired activities, and mode of class delivery. Here we describe how we devised the COVID-360 curriculum and how it represents our efforts to creatively and effectively respond to the instructional needs of diverse students in the face of an ongoing instructional crisis.

Mitochondria play a key role in the ecology and evolution of species through their influence on aerobic metabolism. Mitochondrial DNA (mtDNA) and nuclear genomes must interact for optimal functioning of oxidative phosphorylation to produce ATP, and breakdown of coadaptation components from each may have important evolutionary consequences for hybridization. Introgression of mitochondria in natural populations through hybridization with unidirectional backcrossing allows the testing of coadaptation of mitochondria to different nuclear backgrounds. We compared the function of mitochondria isolated from two species of Urosaurus lizards and hybrid populations. Due to past introgression, hybrids contain the nuclear genome of the "hot-adapted" species (U. graciosus) but the mtDNA of the less heat-tolerant species (U. ornatus). It was found that the function of the parental forms of mitochondria had significantly diverged with the hot-adapted species. There was significant genotype × genotype × environment interactions for mitochondrial membrane potential and genotype × genotype interactions for ATP production. Membrane potential decreased less at a higher temperature, while ATP production was higher at both temperatures in introgressed mitochondria. Oxygen consumption was lower in U. graciosus than in U. ornatus parental-type mitochondria, indicating a likely response to living in hotter environments. Respiratory control ratio values, which provide an indication of the functional quality of isolated mitochondria, were lower in introgressed mitochondria than in parental U. ornatus types, indicating a negative impact on biological function in introgressed mitochondria.

The Vision and Change in Undergraduate Biology Education reports cite the critical role of professional societies in undergraduate life science education and, since 2008, have called for the increased involvement of professional societies in support of undergraduate education. Our study explored the level of support being provided by societies for undergraduate education and documented changes in support during the Vision and Change era. Society representatives responded to a survey on programs, awards, meetings, membership, teaching resources, publications, staffing, finances, evaluation, and collaborations that address undergraduate faculty and students. A longitudinal comparison group of societies responded to surveys in both 2008 and 2014. Results indicate that life science professional societies are extensively engaged in undergraduate education in their fields, setting standards for their discipline, providing vetted education resources, engaging students in both research and education, and enhancing professional development and recognition/status for educators. Societies are devoting funding and staff to these efforts and engaging volunteer leadership. Longitudinal comparison group responses indicate there have been significant and quantifiable expansions of undergraduate efforts in many areas since 2008. These indicators can serve as a baseline for defining, aligning, and measuring how professional societies can promote sustainable, evidence-based support of undergraduate education initiatives.

Endocrine disruptors, such as 17β-estradiol (E2) and estriol (E3), are ubiquitously found in the environment and their presence has gained recognition as a significant health risk. Sorption of estrogens by dissolved organic matter (DOM) influences their concentration and effects. Although there is some experimental evidence suggesting that sorption occurs through hydrophobic interactions, there is not a complete understanding of this association. Therefore, we sought to more extensively characterize binding between humic acid (HA), a major component of DOM, with E2 and E3 using nuclear magnetic resonance spectroscopy (NMR). Our results indicate that binding between these estrogens and HA occurs primarily at the aromatic ring, and takes place even at relatively low HA concentration. Specific interactions between E2 and HA were confirmed by assessing binding via ELISA assay, and the effect of binding on estrogenic activity was studied via a yeast bioassay. Together, these studies build upon previously theorized interactions, yielding a more comprehensive model of binding between estrogens and HA, as well as demonstrate the value of the confluence of biochemical assay methods with analytical NMR techniques.

Pathological events are well characterized in amyotrophic lateral sclerosis (ALS) mouse models, but review of the literature fails to identify a specific initiating event that precipitates disease pathology. There is now growing consensus in the field that axon and synapses are first cellular sites of degeneration, but controversy exists over whether axon and synapse loss is initiated autonomously at those sites or by pathology in the cell body, in nonneuronal cells or even in nonmotoneurons (MNs). Previous studies have identified pathological events in the mutant superoxide dismutase 1 (SOD1) models involving spinal cord, peripheral axons, neuromuscular junctions (NMJs), or muscle; however, few studies have systematically examined pathogenesis at multiple sites in the same study. We have performed ultrastructural examination of both central and peripheral components of the neuromuscular system in the SOD1(G93A) mouse model of ALS. Twenty percent of MNs undergo degeneration by P60, but NMJ innervation in fast fatigable muscles is reduced by 40% by P30. Gait alterations and muscle weakness were also found at P30. There was no change in axonal transport prior to initial NMJ denervation. Mitochondrial morphological changes are observed at P7 and become more prominent with disease progression. At P30 there was a significant decrease in excitatory axo-dendritic and axo-somatic synapses with an increase in C-type axo-somatic synapses. Our study examined early pathology in both peripheral and central neuromuscular system. The muscle denervation is associated with functional motor deficits and begins during the first postnatal month in SOD1(G93A) mice. Physiological dysfunction and pathology in the mitochondria of synapses and MN soma and dendrites occur, and disease onset in these animals begins more than 2 months earlier than originally thought. This information may be valuable for designing preclinical trials that are more likely to impact disease onset and progression.

Apoptosis is a form of programmed cell death that is controlled at the mitochondrion by the BCL-2 family of proteins. While much has been learned about the structure and function of these proteins over the past two decades, the important goal of predicting cell fate decisions in response to toxic stimuli is largely unrealized. BH3 profiling is a functional approach that can be used to predict cellular responses to stimuli based on measuring the response of mitochondria to perturbation by a panel of BH3 domain peptides. BH3 profiling has proven useful in identifying and understanding cellular dependence on individual anti-apoptotic proteins like BCL-2 or MCL-1. Consequently, it can also be used to predict cellular response to chemotherapy agents such as ABT-737 that target these individual proteins.

Sepsis is a system-wide inflammatory response to infection in the blood, which can be caused by any type of pathogen such as bacteria or fungi. During severe sepsis, end-organs often shut down as the body goes into shock, causing irreparable damage and eventual fatality. Even though sepsis is the third highest cause of death in the world, only one third of Americans have heard of the condition. Such unawareness reflects the deficit in knowledge about of how end-organ damage that results from sepsis. Consequently, most current treatments for sepsis address symptoms rather than the cause. While the effects of sepsis on immune cells have been thoroughly examined, the effect on other organs, such as the kidneys, has not been elucidated. In septic patients, a condition caused by excessive cell death resulting in massive tissue damage called acute kidney injury (AKI), becomes considerably more dangerous in septic patients, doubling the mortality rate of AKI. Programmed cell death, otherwise known as apoptosis, is a natural form of cell death, and is only damaging when it behaves in an irregular manner such as not being triggered in cancer cells or excessively occurring after ischemia-reperfusion. Apoptosis is one mechanism that is thought to be responsible for AKI during sepsis, and therefore studying apoptotic regulatory proteins may provide insight into how renal cell death occurs during sepsis-induced AKI (SI-AKI). Furthermore, unraveling the molecular mechanism of kidney cell death could lead to the development of more effective treatments or preventative measures for this widespread condition.

Cancer is a disease of accumulated genetic mutations that alter a cell’s function. One of the common cellular functions affected in cancer cells is the apoptotic pathway. Apoptosis, also known as programmed cell death, is a regulatory biologic function controlled by protein interactions within the BCL-2 protein family. Normally, cells use this pathway to induce cell death when they detect an error in the cell’s functioning that cannot be fixed. In multiple types of cancers, including prostate cancer, there is often an excess of pro-survival BCL-2 proteins. Consequently, these cells have an inability to induce apoptosis, leading to an increase in cancer cell survival, sometimes even in the presence of chemotherapeutic chemicals. This research investigates the potential difference in BCL-2 proteins between metastatic prostate cancer cells and premetastatic prostate cancer cells. Specifically, changes in cancer cell response to chemotherapy after inhibiting antiapoptotic BCL-2 proteins via small-molecule inhibitors are being evaluated. We hypothesize that chemoresistant cancer cells are sensitized to BCL-2 inhibitors by first priming their mitochondria with a traditional chemotherapeutic agent. “Mitochondrial priming” is bringing the antiapoptotic BCL-2 proteins to their apoptotic threshold (1). To accomplish these aims, metastatic prostate cancer cell lines PC3, LnCap C4/2, and DU145 as well as a normal prostate cancer cell line, PNT2, were used. We first sought to compare the level of BCL-2 family proteins among the different cell types. PC3 cells have significantly higher levels of BCL-2, BCL-xL, and BAD as detected by Western blot and quantified by densitometry, corroborating findings from other labs. Next, cells were treated with 5uM etoposide or 100nM docetaxel for 24 hours or 48 hours, and viability was assessed by MTT and apoptosis via Annexin V-FITC. We found that cell viability was significantly decreased in LnCap and DU145 cells at 24 hours, which can be attributed specifically to apoptosis. Moreover, increasing amounts of apoptosis occurred after 48 hours of treatment. Comparatively, PNT2 and PC3 cells have significantly less apoptosis induced by either etoposide or docetaxel treatment. Apoptosis was further confirmed by caspase-3 activation as well as the ability to block apoptosis by pretreatment with the caspase inhibitor Boc-D-fmk in LnCap cells. Additionally, overexpression of BCL-xL in LnCap cells abrogated etoposide-induced apoptosis. Therefore BCL-xL is a potential target for priming. Next, we focused on enhancing the apoptotic effects of etoposide and docetaxel by combing their use with ABT-737, which is a pan BCL-2 antiapoptotic inhibitor. Evaluating the effects of these drugs on various prostate cancerous cell lines in comparison to the effects on a normal prostate cell line may provide insight into their clinical usefulness and relevance in combination treatments. Cells were treated with 0, 100nM, 500nM, 1uM, 2.5uM, or 5uM ABT-737 for 24 hours and apoptosis was assessed via Annexin V-FITC. Preliminary results suggest that on its own, ABT-737 has little effect on LnCap cells (~10%) and no effect on PC3, DU-145, or PNT2 cells. However, pretreating LnCap cells for 24 hours with etoposide as low as 100nM followed by 5 hours exposure to 500nM ABT-737 can induce twice as much apoptosis. These results suggest that etoposide “primes” cells and then the BCL-2 inhibitor pushes them over the edge towards apoptosis. Since less etoposide (100nM) in the combined treatment was effective, as opposed to 500nM when given alone, it may be possible to administer lower amounts of clinically established chemotherapeutics. However, preliminary results also show a ~30% increase in apoptosis in DU145 cells and 50% increase in the normal prostate cell line (PNT2) after combination treatment of 50nM docetaxel for 48 hours followed by 5 hours exposure to 1uM ABT-737. These results suggest that even though lower levels of chemotherapeutics may be effective at killing prostate cancer cells when paired with ABT-737, this combination treatment may be just as harmful to healthy prostate cells. Ongoing studies are also focused on combination treatments with ABT-199, which is selective for antiapoptotic BCL-xL. Reference: 1. Chonghalie TN, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011:334(6059):1129-33. Citation Format: Kaitlyn Walsh, Rachel Wilson, Mary E. McManamy, Victoria Del Gaizo Moore. Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis? [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A075.

Cancer cells acquire disruptions in normal signal transduction pathways and homeostatic mechanisms that would trigger apoptosis in normal cells. These abnormalities include genomic instability, oncogene activation, and growth factor independent proliferation. Therefore, cancer cells likely require a block in apoptosis in order to survive. Overexpression of the antiapoptotic protein BCL-2 provides a block in apoptosis that is frequently observed in cancer cells. We have developed methods for the detection and analysis of BCL-2 dependence and here apply them to acute lymphoblastic leukemia (ALL). BH3 profiling, a mitochondrial assay that classifies blocks in the intrinsic apoptotic pathway, indicated a dependence on BCL-2 of both ALL cell lines and primary samples. This dependence predicted that BCL-2 would be complexed with select pro-death BH3 family proteins, a prediction confirmed by the isolation of BCL-2 complexes with BIM. Furthermore, the BH3 profiling and protein analysis predicted that ALL cell lines and primary cells would be sensitive to ABT-737 as a single agent. Finally, BH3 profiling and protein studies accurately predicted a relative degree of sensitivity to BCL-2 antagonism in cell lines. The ALL cells studied exhibit BCL-2 dependence, supporting clinical trials of BCL-2 antagonists in ALL as single agents or combination therapies.

Antiapoptotic B cell leukemia/lymphoma 2 (BCL2) family proteins are expressed in many cancers, but the circumstances under which these proteins are necessary for tumor maintenance are poorly understood. We exploited a novel functional assay that uses BCL2 homology domain 3 (BH3) peptides to predict dependence on antiapoptotic proteins, a strategy we call BH3 profiling. BH3 profiling accurately predicts sensitivity to BCL2 antagonist ABT-737 in primary chronic lymphocytic leukemia (CLL) cells. BH3 profiling also accurately distinguishes myeloid cell leukemia sequence 1 (MCL1) from BCL2 dependence in myeloma cell lines. We show that the special sensitivity of CLL cells to BCL2 antagonism arises from the requirement that BCL2 tonically sequester proapoptotic BIM in CLL. ABT-737 displaced BIM from BCL2’s BH3-binding pocket, allowing BIM to activate BAX, induce mitochondrial permeabilization, and rapidly commit the CLL cell to death. Our experiments demonstrate that BCL2 expression alone does not dictate sensitivity to ABT-737. Instead, BCL2 complexed to BIM is the critical target for ABT-737 in CLL. An important implication is that in cancer, BCL2 may not effectively buffer chemotherapy death signals if it is already sequestering proapoptotic BH3-only proteins. Indeed, activator BH3-only occupation of BCL2 may prime cancer cells for death, offering a potential explanation for the marked chemosensitivity of certain cancers that express abundant BCL2, such as CLL and follicular lymphoma.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Sepsis, a condition that affects 750,000 people per year, is characterized as a severe inflammatory response to infection. Patients exhibit highly increased concentrations of circulating cytokines as a result of their immune response. Consequently, end-organ failure observed in many septic patients has been linked to these increased cytokine levels, with tissue damage resulting, at least in part, via apoptosis. Specifically, acute kidney injury (AKI) induced by sepsis is a serious problem, and is poorly characterized. This study aims to determine the cellular mechanism of apoptosis during sepsis using an in vitro model of sepsis-induced AKI. Cultured kidney cells were treated with either cytokines secreted from THP1 cells exposed to LPS, or recombinant cytokines. Then, MTT assays, at various times post-exposure, were used to measure changes in cell viability. Furthermore, flow cytometry of whole cells stained with Annexin-V-FITC determined specifically the amount of apoptosis incurred. Our results show a ...

Summary We show that the antiapoptotic proteins BCL-2, BCL-XL, MCL-1, BFL-1, and BCL-w each bear a unique pattern of interaction with a panel of peptides derived from BH3 domains of BH3-only proteins. Cellular dependence on an antiapoptotic protein for survival can be decoded based on the pattern of mitochondrial sensitivity to this peptide panel, a strategy that we call BH3 profiling. Dependence on antiapoptotic proteins correlates with sequestration of activator BH3-only proteins like BID or BIM by antiapoptotic proteins. Sensitivity to the cell-permeable BCL-2 antagonist ABT-737 is also related to priming of BCL-2 by activator BH3-only molecules. Our data allow us to distinguish a cellular state we call "primed for death," which can be determined by BH3 profiling and which correlates with dependence on antiapoptotic family members for survival.

Diabetic cardiomyopathy (DCM) is a complication of diabetes that affects heart function and contributes to mortality. Chronic, elevated glucose levels negatively effects heart tissue and has been i...

PTEN loss and constitutive activation of the PI3K signaling pathway have been associated with advanced androgen-independent prostate cancer. PTEN-deficient prostate cancer C42Luc cells survive in serum-free media and show relative resistance to apoptosis even in the presence of the PI3K inhibitor ZSTK474. Yet, when ZSTK474 is combined with the translation inhibitor cycloheximide, C42Luc cells undergo apoptosis within 6 hours. We identified dephosphorylation of BAD (Bcl2-associated death promoter) as a main apoptosis-regulatory molecule downstream from PI3K, and loss of MCL-1 (Myeloid cell leukemia -1) as a major target of cycloheximide. The combination of MCL-1 knockdown and expression of phosphorylation-deficient mutant BAD2SA is sufficient to trigger rapid apoptosis in prostate cancer cells. These results establish the mechanism for the synergistic induction of apoptosis by the combination of a PI3K inhibitor and of a protein synthesis inhibitor in PTEN-deficient prostate cancer cells.

Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively kills tumor cells. Here we show that clinical response to these drugs correlates with, and may be partially governed by, the pretreatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from proapoptotic BH3 domains of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial priming might enhance the efficacy of cytotoxic agents.

A mechanism for circumventing apoptosis prevalent in many cancer cells is the overexpression of antiapoptotic BCL-2 family members. Upregulated expression of BCL-2 may be required to permit ongoing death signaling without a cellular response. Therefore, antagonizing BCL-2 function may cause death in many cancer cells. The selection for expression of BCL-2 or other antiapoptotic proteins during oncogenesis may derive from these proteins' ability to bind and sequester proapoptotic BH3-only proteins. This situation may be advantageous from a therapeutic viewpoint because cancer cells may be distinguished from normal cells by being primed with death signals. There are several strategies currently under investigation that may lead to improved treatment of many cancers by taking advantage of these differences.

The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT·GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction.

CLL cells consistently express BCL-2 at a high level. Using a compound demonstrated to antagonize BCL-2 function, ABT-737, we show that CLL cells in short-term primary culture are uniformly sensitive to single-agent BCL-2 antagonism, with EC50’s in the 10 nM range. To understand the mechanism of CLL sensitivity to this compound, we studied mitochondria from these primary CLL cells. We employed a panel of peptides derived from the BH3 domain of pro-death BH3-only proteins, certain of which selectively inhibit BCL-2 function in vitro. We demonstrate that those with activity against BCL-2 in vitro induce cytochrome c release, a hallmark of the mitochondrial dysfunction during apoptosis. A non-malignant cellular model of BCL-2 dependence revealed that binding of the pro-apoptotic protein BIM to BCL-2 correlated with sensitivity to BCL-2 inhibitors. We likewise discovered that BCL-2 binds and sequesters BIM in CLL cells. In contrast to the BCL-2 dependent CLL cells, we examined myeloma cell lines with defined MCL-1 dependence and relative BCL-2 independence. These cell lines were 100 fold less sensitive to ABT-737. Furthermore, in mitochondrial assays only BH3 peptides which interact with MCL-1 induce cytochrome c release. These experiments reveal that cellular requirement for an anti-apoptotic protein function can be deduced from a functional mitochondrial requirement determined using our panel of BH3 peptides. In CLL, and perhaps other malignancies, it appears that BCL-2 maintains survival by sequestering pro-apoptotic proteins like BIM. Inhibition of this sequestration provokes cell death ex vivo, even with a single agent BCL-2 inhibitor. Reliance on BCL-2 for survival may be an Achilles heel for CLL and other cancers.