The mitochondrial protease ClpP is a druggable target that controls VSMC phenotype by a SIRT1-dependent mechanism.

Vascular smooth muscle cells (VSMCs), known for their remarkable lifelong phenotypic plasticity, play a pivotal role in vascular pathologies through their ability to transition between different phenotypes. Our group discovered that the deficiency of the mitochondrial protein Poldip2 induces VSMC differentiation both in vivo and in vitro. Further comprehensive biochemical investigations revealed Poldip2's specific interaction with the mitochondrial ATPase caseinolytic protease chaperone subunit X (CLPX), which is the regulatory subunit for the caseinolytic protease proteolytic subunit (ClpP) that forms part of the ClpXP complex - a proteasome-like protease evolutionarily conserved from bacteria to humans. This interaction limits the protease's activity, and reduced Poldip2 levels lead to ClpXP complex activation. This finding prompted the hypothesis that ClpXP complex activity within the mitochondria may regulate the VSMC phenotype. Employing gain-of-function and loss-of-function strategies, we demonstrated that ClpXP activity significantly influences the VSMC phenotype. Notably, both genetic and pharmacological activation of ClpXP inhibits VSMC plasticity and fosters a quiescent, differentiated, and anti-inflammatory VSMC phenotype. The pharmacological activation of ClpP using TIC10, currently in phase III clinical trials for cancer, successfully replicates this phenotype both in vitro and in vivo and markedly reduces aneurysm development in a mouse model of elastase-induced aortic aneurysms. Our mechanistic exploration indicates that ClpP activation regulates the VSMC phenotype by modifying the cellular NAD+/NADH ratio and activating Sirtuin 1. Our findings reveal the crucial role of mitochondrial proteostasis in the regulation of the VSMC phenotype and propose the ClpP protease as a novel, actionable target for manipulating the VSMC phenotype.

Metabolic regulation of the proteasome under hypoxia by Poldip2 controls fibrotic signaling in vascular smooth muscle cells.

The polymerase delta interacting protein 2 (Poldip2) is a nuclear-encoded mitochondrial protein required for oxidative metabolism. Under hypoxia, Poldip2 expression is repressed by an unknown mechanism. Therefore, low levels of Poldip2 are required to maintain glycolytic metabolism. The Cellular Communication Network Factor 2 (CCN2, Connective tissue growth factor, CTGF) is a profibrogenic molecule highly expressed in cancer and vascular inflammation in advanced atherosclerosis. Because CCN2 is upregulated under hypoxia and is associated with glycolytic metabolism, we hypothesize that Poldip2 downregulation is responsible for the upregulation of profibrotic signaling under hypoxia. Here, we report that Poldip2 is repressed under hypoxia by a mechanism that requires the activation of the enhancer of zeste homolog 2 repressive complex (EZH2) downstream from the Cyclin-Dependent Kinase 2 (CDK2). Importantly, we found that Poldip2 repression is required for CCN2 expression downstream of metabolic inhibition of the ubiquitin-proteasome system (UPS)-dependent stabilization of the serum response factor. Pharmacological or gene expression inhibition of CDK2 under hypoxia reverses Poldip2 downregulation, the inhibition of the UPS, and the expression of CCN2, collagen, and fibronectin. Thus, our findings connect cell cycle regulation and proteasome activity to mitochondrial function and fibrotic responses under hypoxia.

Endothelial Poldip2 regulates sepsis-induced lung injury via Rho pathway activation.

AIMS: Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2. METHODS AND RESULTS: Endothelial-specific Poldip2 knock-out mice (EC-/-) and their wild-type littermates (EC+/+) were injected with saline or lipopolysaccharide (18 mg/kg) to model sepsis-induced lung injury. At 18 h post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leucocyte infiltration. Poldip2 EC-/- mice showed reduced lung leucocyte infiltration in BAL (0.21 ± 0.9×106 vs. 1.29 ± 1.8×106 cells/mL) and lung tissue (12.7 ± 1.8 vs. 23 ± 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNFα 2.23 ± 0.39 vs. 4.15 ± 0.5-fold, IκBα 4.32 ± 1.53 vs. 8.97 ± 1.59-fold), neutrophil chemoattractant gene expression (CXCL1 68.8 ± 29.6 vs. 147 ± 25.7-fold, CXCL2 65 ± 25.6 vs. 215 ± 27.3-fold) and a marker of endothelial activation (VCAM1 1.25 ± 0.25 vs. 3.8 ± 0.38-fold) in Poldip2 EC-/- compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knock-down of Poldip2 (5 ± 0.5 vs. 17.5 ± 3-fold for permeability, 1.5 ± 0.4 vs. 10.9 ± 1.3-fold for proportion of disrupted VE-cadherin). Poldip2 knock-down altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94 ± 0.05 vs. 1.29 ± 0.01 of relative RhoA activity) accompanied by redistribution of active-RhoA staining to the centre of the cell. CONCLUSION: Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.

Metabolic adaptation in hypoxia and cancer.

The ability of tumor cells to adapt to changes in oxygen tension is essential for tumor development. Low oxygen concentration influences cellular metabolism and, thus, affects proliferation, migration, and invasion. A focal point of the cell's adaptation to hypoxia is the transcription factor HIF1α (hypoxia-inducible factor 1 alpha), which affects the expression of specific gene networks involved in cellular energetics and metabolism. This review illustrates the mechanisms by which HIF1α-induced metabolic adaptation promotes angiogenesis, participates in the escape from immune recognition, and increases cancer cell antioxidant capacity. In addition to hypoxia, metabolic inhibition of 2-oxoglutarate-dependent dioxygenases regulates HIF1α stability and transcriptional activity. This phenomenon, known as pseudohypoxia, is frequently used by cancer cells to promote glycolytic metabolism to support biomass synthesis for cell growth and proliferation. In this review, we highlight the role of the most important metabolic intermediaries that are at the center of cancer's biology, and in particular, the participation of these metabolites in HIF1α retrograde signaling during the establishment of pseudohypoxia. Finally, we will discuss how these changes affect both the development of cancers and their resistance to treatment.

Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice.

Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.

Time-resolved photoelectron spectroscopy of adenosine and adenosine monophosphate photodeactivation dynamics in water microjets.

The excited state relaxation dynamics of adenosine and adenosine monophosphate were studied at multiple excitation energies using femtosecond time-resolved photoelectron spectroscopy in a liquid water microjet. At pump energies of 4.69-4.97 eV, the lowest ππ* excited state, S1, was accessed and its decay dynamics were probed via ionization at 6.20 eV. By reversing the role of the pump and probe lasers, a higher-lying ππ* state was excited at 6.20 eV and its time-evolving photoelectron spectrum was monitored at probe energies of 4.69-4.97 eV. The S1 ππ* excited state was found to decay with a lifetime ranging from ∼210 to 250 fs in adenosine and ∼220 to 250 fs in adenosine monophosphate. This lifetime drops with increasing pump photon energy. Signal from the higher-lying ππ* excited state decayed on a time scale of ∼320 fs and was measureable only in adenosine monophosphate.

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.

Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

Treatment of veterans with mental health symptoms in VA primary care prior to suicide.

OBJECTIVE: We describe Veterans Affairs (VA) primary care received by veterans with mental health symptoms in the year prior to suicide to identify opportunities to improve care. METHOD: Death certificate data from 11 states were linked to VA national patient care data for veterans who died by suicide in 2009 and had received VA care. We identified 118 age-, sex- and clinician-matched case-control pairs (suicide decedents and living controls) with mental health symptoms. Using McNemar's chi-square and paired t tests, we compare primary care follow-up received during the year prior to death. RESULTS: Cases and controls received similar primary care clinician follow-up and treatment for mental health symptoms. Cases were less likely than controls to fill 90 or more total days of an antidepressant during the year (P=.02), despite no differences in prescription orders from clinicians (P=.05). Cases and controls were equally likely to fill 90 or more consecutive days of an antidepressant (P=.47). Across both groups, 48% (n=113) received assessment for suicidal ideation in primary care. CONCLUSION: We identified two areas to improve primary care for veterans at risk for suicide: monitoring antidepressant treatment adherence and improving suicidal ideation assessment and follow-up for veterans with mental health symptoms.

Transforming growth factor ß inhibits platelet derived growth factor-induced vascular smooth muscle cell proliferation via Akt-independent, Smad-mediated cyclin D1 downregulation.

In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor ß (TGFß) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFß and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFß completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFß. TGFß had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFß on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFß. siRNA against Smad4 completely reversed the inhibitory effect of TGFß on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFß-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFß via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.

In vitro approach to predict post-translational phosphorylation response to mixtures.

While exposure to chemical mixtures is an everyday reality, an understanding of their combined effects, and any potential prediction thereof, is extremely limited. Realistic exposures potentially consist of hundreds to thousands of chemicals per day, but even relatively simple binary mixture interactions can be inherently difficult to predict based upon the lack of temporal and spatial mechanisms for the individual constituents. To this end, we explore the concept of capitalizing on downstream convergence of intracellular signal transduction to experimentally simplify the means of determining xenobiotics that, when combined, could result in increased or unexpected toxicity. In a proof of principle study, we exposed HepG2 cells to deguelin, a natural isoflavonoid, alone and in combination with KCN, and determined the relative post-translational phosphorylation responses to several key proteins related to mitochondrial outer membrane permeabilization. Dose-dependent phosphorylation activity provides a clear identification of threshold response to low-level exposures, and crosstalk amongst selected proteins correctly forecasts mixtures interactions that may lead to increased toxicity. We then used Bliss Independence to determine if the experimentally measured mixture phosphorylation responses could be predicted with individual responses. Independence accurately predicted mixture interactions for deguelin and KCN (87.5%). To more fully exhaust independence as a model for determining potential pharmacodynamic interactions, we exposed HepG2 cells to deguelin and staurosporine, a broad kinase inhibitor; independence accurately predicted these mixture responses (77.5%). In this study, we demonstrate the potential of a new in vitro approach for the prediction of toxic mixtures interactions that is fundamentally driven by the interdependence of signal transduction and apoptosis.

Role of coronin 1B in PDGF-induced migration of vascular smooth muscle cells.

RATIONALE: The type I subclass of coronins, a family of actin-binding proteins, regulates various actin-dependent cellular processes, including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined. OBJECTIVE: The goal of the present study was to define the mechanism by which coronins regulate platelet-derived growth factor (PDGF)-induced VSMC migration. METHODS AND RESULTS: Coronin 1B (Coro1B) and 1C (Coro1C) were both found to be expressed in VSMCs at the mRNA and protein levels. Downregulation of Coro1B by siRNA increases PDGF-induced migration, while downregulation of Coro1C has no effect. We confirmed through kymograph analysis that the Coro1B-downregulation-mediated increase in migration is directly linked to increased lamellipodial protraction rate and protrusion distance in VSMC. In other cell types, coronins exert their effects on lamellipodia dynamics by an inhibitory interaction with the ARP2/3 complex, which is disrupted by the phosphorylation of Coro1B. We found that PDGF induces phosphorylation of Coro1B on serine-2 via PKCε, leading to a decrease in the interaction of Coro1B with the ARP2/3 complex. VSMCs transfected with a phosphodeficient S2A Coro1B mutant showed decreased migration in response to PDGF, suggesting that the phosphorylation of Coro1B is required for the promotion of migration by PDGF. In both the rat and mouse, Coro1B phosphorylation was increased in response to vessel injury in vivo. CONCLUSIONS: Our data suggest that phosphorylation of Coro1B and the subsequent reduced interaction with ARP2/3 complex participate in PDGF-induced VSMC migration, an important step in vascular lesion formation.

Exploring the boundaries of additivity: mixtures of NADH: quinone oxidoreductase inhibitors.

The activity of mitochondrial complex I of the electron transport chain (ETC) is known to be affected by an extraordinarily large number of diverse xenobiotics, and dysfunction at complex I has been associated with a variety of disparate human diseases, including those with potentially environmentally relevant etiologies. However, the risks associated with mixtures of complex I inhibitors have not been fully explored, and this warrants further examination of potentially greater than additive effects that could lead to toxicity. A potential complication for the prediction of mixture effects arises because mammalian mitochondrial complex I has been shown to exist in two distinct dynamic conformations based upon substrate availability. In this study, we tested the accepted models of additivity as applied to mixtures of rotenone, deguelin, and pyridaben, with and without substrate limitation. These compounds represent both natural and synthetic inhibitors of complex I of the ETC, and experimental evidence to date indicates that these inhibitors share a common binding domain with partially overlapping binding sites. Therefore, we hypothesized that prediction of their mixtures effects would follow dose addition. Using human hepatocytes, we analyzed the effects of these mixtures at doses between 0.001 and 100 µM on overall cellular viability. Analysis of the dose-response curves resulting from challenge with all possible binary and ternary mixtures revealed that the appropriate model was not clear. All of the mixtures tested were found to be in agreement with response addition, but only rotenone plus deguelin and the ternary mixture followed dose addition. To determine if conformational regulation via substrate limitation could improve model selection and our predictions, we tested the models of additivity for the binary and ternary mixtures of inhibitors when coexposed with 2-deoxy-d-glucose (2-DG), which limits NADH via upstream inhibition of glycolysis. Coexposure of inhibitors with 2-DG did facilitate model selection: Rotenone plus pyridaben and the ternary mixture were in sole agreement with dose addition, while deguelin plus pyridaben was in sole agreement with response addition. The only ambiguous result was the agreement of both models with the mixture of rotenone plus deguelin with 2-DG, which may be explained by deguelin's well-known affinity for protein kinase B (Akt) in addition to complex I. Thus, our findings indicate that predictive models for mixtures of mitochondrial complex I inhibitors appear to be compound specific, and our research highlights the need to control for dynamic conformational changes to improve our mechanistic understanding of additivity with these inhibitors.

Effect of Iyengar yoga practice on fatigue and diurnal salivary cortisol concentration in breast cancer survivors.

PURPOSE: To examine the effect of regular Iyengar yoga practice on measures of self-perceived psychosocial function and diurnal salivary cortisol secretion in stage II-IV breast cancer survivors (n = 18). DATA SOURCES: Women were randomly assigned to attend yoga practice for 90 min twice weekly for 8 weeks (n = 9) or to a wait-listed, noninterventional control group (n = 9). Traditional Iyengar yoga routines that progressively increased in difficulty as participants gained strength and flexibility were used. At baseline and after the 8-week study period, women completed self-report instruments to document various aspects of psychosocial and physical functioning, and collected salivary samples for cortisol analysis four times during the day for two consecutive days. CONCLUSIONS: The yoga group had lower morning and 5 p.m. salivary cortisol and improved emotional well-being and fatigue scores. IMPLICATIONS FOR PRACTICE: Breast cancer survivors are at risk for chronic psychosocial distress that may alter activity of the hypothalamic-pituitary-adrenal axis, resulting in aberrant regulation of cortisol secretion and increased risk of immune dysfunction and cancer progression. Regular yoga practice may be a low-risk, cost-effective way to improve psychosocial functioning, fatigue, and regulation of cortisol secretion in breast cancer survivors. These findings require validation with a larger randomized study.

Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells.

Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.

Measurement of glut-1 expression using tissue microarrays to determine a race specific prognostic marker for breast cancer.

BACKGROUND: African-American women (AAW) have more adverse tumor characteristics than non-African American women (non-AAW) and there is a need for a specific predictive marker for AAW recurrence. Tumors with higher grade and proliferative activity are associated with overexpression of the glucose transporter (Glut-1). An examination of Glut-1 expression relative to recurrence and no recurrence was conducted on both groups to determine if it predicts prognosis and could predict a race specific prognosis. METHODS: A breast cancer data set containing clinical information including race and biological characteristics was generated between 1991 and 1996. Tissue samples were selected from this group with similar characteristics in both racial groups for a retrospective analysis of Glut-1 expression using tissue microarrays (TMAs). Mean Glut-1 expression for intensity and percent of tumor cells staining was determined using standard immunohistochemistry. RESULTS: Clinical and biological differences were noted in the original data set between AAW and non-AAW. No significant difference between races was noted in mean Glut-1 values using a subset which had similar characteristics. The mean Glut-1 did not predict recurrence but a rounded score did indicate that higher levels of Glut-1 expression was indicative of a lower disease free survival (p = 0.063). The actual average mean for AAW with no recurrence was significantly lower than any other group (p = 0.04). Conclusions. TMA analysis for Glut-1 expression may be useful to predict disease free survival but it does not predict race specific recurrence.

Development of Herceptin resistance in breast cancer cells.

BACKGROUND: Herceptin, a humanized antibody to HER-2, is now utilized in the clinic for metastatic breast cancer treatment. The response rate for HER-2+ patients is only 30% and little is known as to mechanisms of resistance. The mechanism of Herceptin action is also unknown but has been related to cell cycle inhibition. METHODS: The effects of Herceptin and other antibody treatments were determined by cell counting and cell cycle analysis. HER-2 and p27 expression levels were analyzed by flow cytometry and levels of activated AKT were compared by Western blot analysis. Cellular HER-2 and p27 expression was measured by immunofluorescence. RESULTS: Herceptin treatment of BT-474 cells results in inhibition of cell growth and arrest in the G1 phase. The efficacy of growth arrest was not directly correlated to the binding affinity of antibodies to Her-2. Our laboratory has developed cell lines that are resistant to Herceptin treatment. In resistant cell lines, binding of antibodies is not hindered. However, Herceptin has completely lost the ability to inhibit cell proliferation. Yet, the mouse isotype 4D5 maintains significant inhibitory activity upon Herceptin-resistant clones. CONCLUSIONS: Herceptin binds effectively to Her-2 on the cell surface of Herceptin-resistant cell lines and the level of Her-2 expression on the cell surface is not downregulated. Herceptin resistance is not due to downregulation of levels of AKT protein expression, although, phosphorylation of AKT is enhanced in resistant lines and could have a role in resistance. Resistance appears to correlate with the loss of nuclear expression of the cyclin-dependent kinase inhibitor, p27, as defined by immunofluorescence and flow cytometry studies and cdk-2 binding studies.

Tuberculous mediastinal mass presenting with stridor in a 3-month-old child.

A 3-month-old boy with a history of intermittent stridor was found to have obstructive emphysema on chest x-ray. Further investigations found a mediastinal mass compressing the carina and left mainstem bronchus. The mass was excised and found to be of tuberculous origin.

Parasitic omphalopagus complicated by omphalocele and congenital heart disease.

Conjoined twins occur in approximately one of every 50,000 to 200,000 births. Atypical or parasitic conjoined twins result from the embryonic death of one twin, which leaves behind body parts vascularized by the primary twin. Omphalopagus refers to conjoined twins joined at the level of the umbilicus. The authors report a case of atypical omphalopagus involving an acardiac, acephalic, parasitic twin and a host twin with a large omphalocele, transposition of the great arteries, and aortic coarctation. The authors briefly review reported cases of parasitic omphalopagus and emphasize the role of intensive neonatal care, preoperative planning, and staged surgical intervention in the successful management of complicated variants.