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Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.

Background Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, characterized high rates of tumor protein 53 (p53) mutation and limited targeted therapies. Despite being clinically advantageous, direct targeting of mutant p53 has been largely ineffective. Therefore, we hypothesized that there exist pathways upon which p53-mutant TNBC cells rely upon for survival. Methods In vitro and in silico drug screens were used to identify drugs that induced preferential death in p53-mutant breast cancer cells. The effects of the glutathione peroxidase 4 (GPX4) inhibitor ML-162 was deleniated using growth and death assays, both in vitro and in vivo. The mechanism of ML-162 induced death was determined using small molecule inhibition and genetic knockout. Results High-throughput drug screening demonstrated that p53-mutant TNBCs are highly sensitive to peroxidase, cell cycle, cell division, and proteasome inhibitors. We further characterized the effect of the Glutathione Peroxidase 4 (GPX4) inhibitor ML-162 and demonstrated that ML-162 induces preferential ferroptosis in p53-mutant, as compared to p53-wild type, TNBC cell lines. Treatment of p53-mutant xenografts with ML-162 suppressed tumor growth and increased lipid peroxidation in vivo. Testing multiple ferroptosis inducers demonstrated p53-missense mutant, and not p53-null or wild type cells, were more sensitive to ferroptosis, and that expression of mutant TP53 genes in p53-null cells sensitized cells to ML-162 treatment. Finally, we demonstrated that p53-mutation correlates with ALOX15 expression, which rescues ML-162 induced ferroptosis. Conclusions This study demonstrates that p53-mutant TNBC cells have critical, unique survival pathways that can be effectively targeted. Our results illustrate the intrinsic vulnerability of p53-mutant TNBCs to ferroptosis, and highlight GPX4 as a promising target for the precision treatment of p53-mutant triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 15–20% of breast cancer cases in the United States, lacks targeted therapeutic options, and is associated with a 40–80% risk of recurrence. Thus, identifying actionable targets in treatment-naïve and chemoresistant TNBC is a critical unmet medical need. To address this need, we performed high-throughput drug viability screens on human tumor cells isolated from 16 patient-derived xenograft models of treatment-naïve primary TNBC. The models span a range of TNBC subtypes and exhibit a diverse set of putative driver mutations, thus providing a unique patient-derived, molecularly annotated pharmacologic resource that is reflective of TNBC. We identified therapeutically actionable targets including kinesin spindle protein (KSP). The KSP inhibitor targets the mitotic spindle through mechanisms independent of microtubule stability and showed efficacy in models that were resistant to microtubule inhibitors used as part of the current standard of care for TNBC. We also observed subtype selectivity of Prima-1Met, which showed higher levels of efficacy in the mesenchymal subtype. Coupling pharmacologic data with genomic and transcriptomic information, we showed that Prima-1Met activity was independent of its canonical target, mutant p53, and was better associated with glutathione metabolism, providing an alternate molecularly defined biomarker for this drug.

A reliable pipeline to identify DNA methylation signatures in CSF ctDNA could serve as potential biomarkers for MB patients., Medulloblastoma (MB), the most common form of pediatric brain malignancy, has a low frequency of oncogenic mutations but pronouncedly abnormal DNA methylation changes. Epigenetic analysis of circulating cell-free tumor DNA (ctDNA) by liquid biopsy offers an approach for real-time monitoring of tumor status without tumor dissection. In this study, we identified 6598 differentially methylated CpGs in both MB tumors and the ctDNA isolated from matched cerebrospinal fluid (CSF) compared with normal cerebellum, which could be used to detect MB tumor occurrence and determine its subtype. Furthermore, DNA methylation changes in serial CSF samples could be used to monitor the treatment response and tumor recurrence. Integrating our data with large public datasets, we identified reliable MB DNA methylation signatures in ctDNA that have potential diagnostic and prognostic values. Our study sets the stage for exploiting epigenetic markers in CSF to improve the clinical management of patients with MB.

Smart plugs are useful devices for measuring the appliance load, but intrusive. It has long been the goal of energy companies and researchers to monitor the load of all household appliances in a non-intrusive manner, using only a single smart meter. We show that deep neural networks can be extremely effective in this regard. Automatic feature learning can pick out distinctive load-dependent shapes in the time series power data, enabling identification of appliance signatures. We find that properly arranged deep neural networks are capable of multi-class appliance classification, outperforming a traditional multiclass classification algorithm. We evaluate on the public PLAID dataset, and compare results with features extracted from sampling frequencies in the 1Hz-1kHz range. We show that adding features extracted from high frequency sampling significantly improves classification performance over data obtained at typical smart meter frequencies.

Background Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. Methods A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients. Results We identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24− in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival. Conclusions Taken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Background: P53 is the most commonly mutated protein in cancer with more than 40% of all cancer patients harboring a p53 mutation. Up to 85% of triple-negative breast cancers (TNBCs), aggressive breast cancers characterized by the absence of ER, PR, and Her2 receptors, have p53 mutations. P53 is a tumor suppressor that helps balance cellular growth and death after a genomic insult. Due to the rate of p53 mutation in cancer, the targeting of p53 in a clinical setting is an attractive option. However, currently there are no available p53-targeted drugs. We hypothesized that molecular pathways exist, that when inhibited, induce death or suppress growth of p53-mutant breast cancer cells, without affecting p53 wild-type breast cells. To investigate this hypothesis, we conducted a high-throughput drug screen to identify drugs that induce death or suppress growth of p53-mutant TNBCs. Methods: A library of 453 drugs with diverse and known mechanisms of action was used to perform high-throughput drug screening through collaboration with Texas A&M’s Center for Translational Cancer Research. An ATP luminescence assay was used to test eight TNBC cell lines with distinct p53 mutations in this screen. We then tested a subset of these drugs, identified to have an effect on p53-mutant TNBC, on the growth of five p53 wild-type breast cancer cell lines. Cell death assays to differentiate between growth suppression and death-inductive phenotypes are currently underway. Results: 145 drugs were identified that induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Of these drugs, 16 drugs were found to differentially suppress the growth of p53-mutant breast cancer cells, as compared to p53-wild type breast cancer cells. Further analysis of these drugs identified proteasome, apoptosis, and iron-dependent cell death pathways as potential p53-mutant specific targets in TNBCs. Conclusion: A screen of molecularly annotated drugs was performed in p53-mutant and p53-wild type breast cancer cells to identify drugs that preferentially kill or suppress the growth of p53-mutant breast cancers. These studies identified 145 drugs that were able to induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Testing these drugs in p53 wild-type breast cancer cell lines demonstrated that several of the identified drugs suppress growth or induce death preferentially in p53-mutant, as compared to p53 wild-type, breast cancers. These drugs affect specific cellular pathways such as proteasomal degradation, ferroptosis, and apoptosis pathways, which appear to be critical for the growth and survival of p53-mutant breast cancers. Further work will elucidate the interplay between p53-mutation and the identified cellular pathways. These studies will provide the basic science foundation for the development of new drugs for the treatment of p53-mutant breast cancer. This work was supported by a generous gift from the John Charles Cain Family. Citation Format: William Miska Tahaney, Reid Powell, Nghi Nguyen, Lakshmi Reddy Bollu, Jing Qian, Abhijit Mazumdar, Clifford Stephan, Peter JA Davies, Powel H Brown. Identification of drugs that induce the death or suppress the growth of p53-mutant breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-08.

Primary and acquired drug resistance imposes a major threat to achieving optimized clinical outcomes during cancer treatment. Aberrant changes in epigenetic modifications are closely involved in drug resistance of tumor cells. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on the CRISPR interference technology, we identified the second intron of IncRNA, PVT1, as a unique bona fide gained enhancer that restored MYC transcription independent of BRD4 recruitment in leukemia. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYC locus in BETi-resistant leukemia cells. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies., Epigenetic changes can drive drug resistance in cancer. Here, the authors show that in BET inhibitor resistant leukaemia cells, genome-wide enhancer remodelling drives therapeutic resistance and targeting enhancer plasticity may overcome this resistance.

Graphical abstractDisplay Omitted Highlights? PSI represents concepts and relations in hyperdimensional space. ? PSI is used to infer discovery patterns from known therapeutic relationships. ? These patterns are used to recover therapeutic relationships for a held-out disease set. ? PSI outperforms a co-occurrence based approach in this regard. ? PSI searches efficiently across large networks of relevant relationships.. In this paper we utilize methods of hyperdimensional computing to mediate the identification of therapeutically useful connections for the purpose of literature-based discovery. Our approach, named Predication-based Semantic Indexing, is utilized to identify empirically sequences of relationships known as "discovery patterns", such as "drug x INHIBITS substance y, substance y CAUSES disease z" that link pharmaceutical substances to diseases they are known to treat. These sequences are derived from semantic predications extracted from the biomedical literature by the SemRep system, and subsequently utilized to direct the search for known treatments for a held out set of diseases. Rapid and efficient inference is accomplished through the application of geometric operators in PSI space, allowing for both the derivation of discovery patterns from a large set of known TREATS relationships, and the application of these discovered patterns to constrain search for therapeutic relationships at scale. Our results include the rediscovery of discovery patterns that have been constructed manually by other authors in previous research, as well as the discovery of a set of previously unrecognized patterns. The application of these patterns to direct search through PSI space results in better recovery of therapeutic relationships than is accomplished with models based on distributional statistics alone. These results demonstrate the utility of efficient approximate inference in geometric space as a means to identify therapeutic relationships, suggesting a role of these methods in drug repurposing efforts. In addition, the results provide strong support for the utility of the discovery pattern approach pioneered by Hristovski and his colleagues.

The idea that immunotherapy for tau pathology could be effective would seem to require acceptance of the notion that this spreads from cell to cell with an extracellular intermediate that is accessible to an antibody. Passive immunotherapy appears to be the major approach, and it has been reported that several different monoclonal antibodies to tau are effective at limiting the development of pathology in a number of different tau transgenic mice. There are at least four different mechanisms by which this might be achieved, and these are not mutually exclusive. The special problems inherent in attempting to use active immunization strategies for treatment of tau pathology are discussed, but such efforts seem ill-advised at this time. The major issue remaining is whether the mouse experiments will predict efficacy of tau immunotherapy in humans, and the next few years should answer this question.

In rodents, a brief neonatal exposure of the developing reproductive tract to the xenoestrogen, diethylstilbestrol (DES) reprograms developing tissues to increase susceptibility to tumorigenesis in adult animals, including uterine adenocarcinoma. Progression from a normal endometrium to carcinoma occurs via the intermediate stage of endometrial hyperplasia. We previously reported that endometrial hyperplasia in postmenopausal women is linked to abnormal insulin-like growth factor-I (IGF-I) signaling. To identify early events involved in the development of hyperplasia in the endometrium, we examined expression and activation of IGF-I pathway components in endometrium of rats exposed to DES. By 5 months of age, 36/60 (60%) of rats exposed to DES on days 3-5 after birth developed endometrial hyperplasia compared to 0% of vehicle-treated controls. Consistent with activation of a mitogenic signaling pathway, Ki67-positive cells increased in DES-exposed endometrium despite compromised ovarian function and hypoestrogenic milieu characteristic of DES-exposed animals. The endometrium of DES-exposed rats overexpressed IGF-II and insulin receptor substrate-1 (IRS-1) and exhibited elevated Akt expression and activation (as judged by phosphorylation) and mTOR signaling (phosphorylation of S6) compared to vehicle-treated endometrium. In contrast to vehicle-treated endometrium, in which negative feedback to IRS-1 was observed (phosphorylation of S636/639), negative feedback to IRS-1 was absent in DES-exposed endometrium. These data support a central role for IGF-I signaling in the development of both human and rodent endometrial hyperplasia. Furthermore, both global activation of IGF-IR signaling and abrogation of negative feedback to IRS-1 appear to be reprogrammed by DES in endometrial hyperplasia, implicating for the first time loss of negative feedback to IRS-1 in development of a preneoplastic lesion.

Retinoid X receptor (RXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metazoan life. A class of nuclear receptors requires RXR as heterodimerization partner for their function. This places RXR in the crossroad of multiple distinct biological pathways. This and the fact that the debate on the endogenous ligand requirement for RXR is not yet settled make RXR still an enigmatic transcription factor. Here, we review some of the biology of RXR. We place RXR into the evolution of nuclear receptors, review structural details and ligands of the receptor. Then processes regulated by RXR are discussed focusing on the developmental roles deduced from studies on knockout animals and metabolic roles in diseases such as diabetes and atherosclerosis deduced from pharmacological studies. Finally, aspects of RXR's involvement in myeloid differentiation and apoptosis are summarized along with issues on RXR's suitability as a therapeutic target.

Background/Aims: The normal architecture and function of the kidney are maintained by a critical turnover of the extracellular matrix (ECM). Although increased synthesis of ECM leads to progression of renal failure, the role of degradation of ECM by metalloproteinases in progressive nephropathies is unclear. Recently, in an animal model of membranous glomerulonephritis (Heymann nephritis), visceral epithelial cell (VEC) injury has been found to rapidly increase matrix metalloproteinase-9 (MMP-9) synthesis by these cells during the period of maximal proteinuria. Because injury to glomerular VECs may be an important initiating factor in the pathogenesis of focal segmental glomerulosclerosis (FSGS), especially in HIV-associated nephropathy (HIVAN), we determined the expression of MMP-9 and its inhibitors in renal biopsies of patients with HIVAN. Methods: RNA was isolated from cortical renal tissue of 6 patients with HIVAN. Only those biopsies which displayed characteristic findings of HIVAN including collapsing FSGS, cystic dilatation of the tubules, and proliferation of VECs were used. As controls, we obtained renal cortical tissue from normal areas of kidneys removed due to malignancies. MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expressions were determined using real-time quantitative reverse transcription-polymerase chain reaction. In all 6 patients with HIVAN, MMP-9 and TIMP-1 were overexpressed. We localized MMP-9 protein to the glomeruli with immunohistochemical detection. Conclusion: MMP-9 is overexpressed in the glomeruli of patients with HIVAN and may be involved in the pathogenesis of the disease.

This study examines the possible role of estrogen in regulating the expression of the human CYP3A subfamily: CYP3A4, CYP3A5, CYP3A7, and CYP3A43. To accomplish this goal, mRNA was quantified from human livers and endometrial samples, and total CYP3A protein levels were evaluated by Western immunoblot analysis of the liver samples. The human endometrial samples were from premenopausal and postmenopausal women. The premenopausal endometrium was either in the proliferative or secretory phase, whereas for the postmenopausal endometrium samples, the women had been treated with either a placebo or estropipate, an estrogen substitute. After analyses, CYP3A4 mRNA was shown to have lower hepatic expression in females than in males. In the endometrium, CYP3A4 and CYP3A43 are down-regulated by estrogen, whereas CYP3A5 is expressed at higher levels during the secretory phase. CYP3A7 was not detected in the endometrium. In addition, the CYP3A subfamily showed increased mRNA expression in the liver as age increased. The expression levels of total CYP3A protein and total CYP3A mRNA showed good correlation. Despite apparent regulation of CYP3A4 mRNA expression by estrogen, the effects of estrogen may be overshadowed by additional regulators of gene expression.

To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR alpha, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation.

The advent of gene array technology brings the ability to classify disease states to the molecular level by examining changes in all mRNAs expressed in cells or tissues. Comparing changes in gene expression patterns between normal and diseased cells and/or tissues has elucidated unique subsets of genes identifiable to a specific disease. Already, new subclassifications of specific cancers have been discovered, belying that genomic profiling can uniquely distinguish a specific disease state and tissue of origin. This technology bestows the ability to examine global changes occurring in a cell or tissue(s), thereby allowing the elucidation of alterations in dysregulated biological, biochemical, and molecular events leading to disease states such as diabetes, hypertension, infertility, obesity, osteoporosis, and atherosclerosis. Furthermore, genomic profiling will lead to new molecular targets for the development of drug therapeutics. Futuristically, one could envision personalized patient therapies based upon identification of specific aberrant signaling pathways that can be targeted for drug therapy.

Cyclin D1 overexpression is a valuable marker for the diagnosis of mantle cell lymphoma (MCL). We used a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method to quantify levels of cyclin D1, CD20, and cyclophilin A mRNA in manually microdissected, paraffin-embedded tissue sections using an ABI 7700 qRT-PCR system. The study group included 21 cases of MCL and 37 cases of other types of B-cell non-Hodgkin’s lymphoma. Cyclin D1 mRNA copy number was normalized to CD20 and cyclophilin A mRNA and evaluated statistically by analysis of variance. The relative cyclin D1 levels were similar whether normalized to CD20 or cyclophilin A, indicating that CD20 levels are stable and can be used as a B-cell-specific normalizer. Statistically significant differences were found in the median levels of cyclin D1 mRNA (expressed as % CD20 mRNA) among cases of MCL (87.6), small lymphocytic lymphoma (9.9), follicular lymphoma (2.4), diffuse large B-cell lymphoma (5.9), marginal zone B-cell lymphoma (39.8), and Burkitt lymphoma (7.1) (P < 0.05). We conclude that qRT-PCR can be used to quantify cyclin D1 mRNA levels in archival tissue sections. Normalization of cyclin D1 to a B-cell-specific marker more accurately reflects overexpression by MCL than other methods that normalize using constitutively expressed mRNA species.

We recently found that alternative transcripts of tissue transglutaminase (tTG or TG2) were present in hippocampal brain regions of Alzheimer’s disease (AD), but not in control, nondemented, age-matched brains. Since antecedent non-severe trauma has been implicated in AD and other neurodegenerative diseases, such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), we were interested in whether alternative transcripts might be detected in a model of neurotrauma, controlled-contusion spinal cord injury (SCI) in the rat. Implicated in diverse roles from growth and differentiation to apoptotic cell death, only bifunctional tTG, of the nine member TG family, has dual catalytic activities: guanine trinucleotide (GTP) hydrolyzing activity (GTPase), as well as protein cross-linking. These functions imply two physiological functions: programmed cell life and death. These may have profound roles in the nervous system since studies in cultured astrocytes found tTG short (S) mRNA transcripts induced by treatment with injury-related cytokines. In the developing rat spinal cord, tTG activity is concentrated in ventral horn alpha motoneurons, but neither studies of spinal cord tTG gene expression, nor evaluation of the GTP-regulated isoforms in tissues, have been reported. We now report increased tTG protein and gene expression occurring rapidly after SCI. In parallel, novel appearance of a second, short form transcript, in addition to the normal long (L) isoform, occurs by 8 h of injury. Up-regulation of tTG message and activity following neural injury. with appearance of a truncated GTP-unregulated S form, may represent new approaches to drug targets

Background: We have previously shown that several metabolic genes are downregulated in the failing human heart. We now tested the hypothesis that mechanical unloading might reverse this process. Methods: Clinical data and myocardial tissue were collected from 14 failing hearts paired for the time of implantation and explantation of a left ventricular assist device (LVAD) and compared to 10 non-failing hearts. Transcript levels for key regulators of energy metabolism and for atrial natriuretic factor (ANF) were measured by real-time quantitative RT-PCR. Results: The expression of the glucose transporter 1 and 4 (GLUT1, GLUT4), muscle carnitine palmitoyl transferase-1 (mCPT-1), and uncoupling protein 3 (UCP3) were downregulated by up to 80% in the failing heart. Although LVAD treatment improved clinical markers of heart failure (decrease of left ventricular diastolic dimension and normalization of serum sodium), only UCP3 expression reversed to non-failing transcript levels following mechanical unloading. Conclusions: LVAD treatment only partially reverses depressed metabolic gene expression in the failing human heart. Reversal of depressed UCP3 expression may be an important mechanism for reducing the formation of oxygen-derived free radicals. Further studies are necessary to define the effects of mechanical unloading on post-transcriptional mechanisms.

Chemotherapeutic drugs are known to eliminate cancer cells by inducing apoptosis. Tissue transglutaminase (tTG), a frequent player in apoptotic processes, is markedly induced in drug-resistant cancer cells. To better understand the action of apoptosis-inducing drugs, our study elucidates changes in the expression of tTG in the early phase of cell death, before the downstream events of apoptosis. We demonstrate that HepG2 cells uniformly induce both tTG mRNA and enzyme activity upon treatment with cisplatin, doxorubicin, and bleomycin, chemotherapeutic agents with different modes of action. The expression of fas ligand, caspase3 and baxalpha changes differentially or remain unaffected. tTG expression did not change significantly after administration of either the peroxisome proliferator activated receptor-alpha agonist WY14643 or the retinoid X receptor-specific analog LG 100268. However, both compounds blocked drug-induced tTG induction without affecting the extent of cell death. The pleiotropic cytokine interleukin-6 effectively rescued hepatoma cells from apoptosis while tTG induction still took place, along with the induction of antiapoptotic transcripts bcl-x(L), gp130, and her2/neu. These results suggest that the induction of tTG, although present in drug-induced apoptosis, is pharmacologically dissociable from the early, initiating events of apoptosis. Blocking the induction of tTG during drug-induced cell death may alleviate limiting side effects of anticancer agents, including fibrosis and neuropathies.

Cell-cell contact of myeloma-derived cell lines (MDCL) or fresh myeloma cells with bone marrow stromal cells (BMSC) is known to induce interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1) production by a marrow stromal cell line. To determine if other BMSC transcripts are altered during cell-cell contact between BMSC and tumor cells, we have used cell lines ARH77 and U266 in an in vitro model. Using mRNA differential display and reverse transcriptase-polymerase chain reaction (RT-PCR), it was determined that a total of 141 transcripts were either upregulated or downregulated in the BMSC on contact with cell membrane from cell lines ARH77 and U266. Induction of two of these transcripts, interleukin-6 (IL-6) and gp130 in the BMSC by ARH77 cell membranes was studied in greater detail. Real-time PCR was used to quantitate transcript levels of gp130, IL-6, and 36b4, a housekeeping gene. Cycloheximide (CHX) alone increased both gp130 and IL-6 transcripts in the BMSC. In addition, CHX caused a superinduction of these transcripts in BMSC exposed to ARH77 cell membranes. The induction of gp130 was independent of the increase in IL-6 mRNA. Upregulation of gp130, a component of the membrane receptors for the IL-6 superfamily, can have profound effects on the response of BMSC to the IL-6 superfamily of cytokines.

Novel strategies in the treatment of heart failure include mechanical unloading with a left ventricular assist device. Although first considered as a bridge to cardiac transplantation, this surgical treatment may improve cardiac function in patients with heart failure, even after removal of the device. The molecular adaptation of the heart to unloading remains largely unknown. Most of the enzymes involved in the regulation of myocardial energetics (including contractile proteins, ion pumps, and metabolic enzymes) exist in “fetal” and “adult” isoforms. It is known that cardiac hypertrophy due to increased work load in vivo involves a switching from the normally expressed adult isoform to the fetal isoform. Our work has now shown that the same pattern occurs in the unloaded heart. In both conditions, this switching is accompanied by the reexpression of growth factors and proto-oncogenes. The functional improvement of the failing heart after mechanical unloading may in part be the result of a reexpression of fetal genes.

We previously reported that mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck et al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Approximately 10% of the RARbeta-/- /RARgamma-/- mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley et al., 1995, Genes Dev. 9, 2795-2807; Luo et al., 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression.

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.

Tissue transglutaminase is a multifunctional enzyme that accumulates to high levels in cells undergoing apoptosis. Retinoids act as an acute and direct regulator of tissue transglutaminase gene transcription. The studies reported here were carried out to elucidate the molecular mechanisms involved in the regulation of tissue transglutaminase expression. We have isolated and characterized the mouse tissue transglutaminase gene promoter and 3.8 kb of 5'-flanking DNA. A large fragment of the promoter that includes both the core promoter and 3.8 kb of 5'-flanking DNA shows retinoid-dependent transcriptional activity when stably transfected into HeLa cells. In these stably transfected HeLa cells both the endogenous tissue transglutaminase gene and transfected mouse tissue transglutaminase promoter are activated by all-trans retinoic acid and by retinoic acid receptor (RAR)-specific and retinoid X receptor (RXR)-specific retinoids. In embryos made transgenic with a transglutaminase promoter-beta-galactosidase reporter gene, the transgene shows specific patterns of expression during limb development. The transglutaminase transgene is expressed in cartilage, the cells of the apical ectodermal ridge, and in regions of apoptotic cell death of the interdigital mesenchyme. It appears that cis-acting elements responsible for the complex retinoid regulation, tissue- and apoptosis-specific expression are embedded within the proximal 3.8 kb of DNA flanking the 5'-end of the mouse tissue transglutaminase gene.

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through ϵ(γ-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

Stably transfected Balb-C 3T3 fibroblasts (clone 5), overexpressing a catalytically active tissue transglutaminase, showed a basal adenylate cyclase activity lower than control cells (clone 1). Several modulators of the adenylate cyclase activity (forskolin, Mn2+ and pertussis toxin) showed the existence of a marked negative control on the adenylate cyclase activity present in clone 5 cells. Very interestingly, this same marked negative control was also found in a Balb-C 3T3 fibroblast clone stably transfected with a mutagenized human tissue transglutaminase (mut277 cys > ser) virtually devoid of transglutaminase catalytic activity (clone Ser). Conversely, a significant increase of the adenylate cyclase activity was observed in bovine aortic endothelial cells after the lowering of tissue transglutaminase expression levels by the transfection of an eukaryotic expression vector containing the gene for tissue transglutaminase in antisense orientation. All these findings suggest a possible role for type II tissue transglutaminase as a negative modulator of the adenylate cyclase activity in different cell types, beside its transglutaminase enzyme activity.

We have investigated the role of DNA methylation in the regulation of the expression of the human tissue transglutaminase gene. Studies on the methylation of the transglutaminase promoter in normal and neoplastic human cells demonstrated that the promoter is methylated in vivo and hypomethylation of the promoter is correlated with constitutive gene expression. Demethylation of the promoter in vivo by treatment of the cells with 5-azacytidine increased transglutaminase expression and hypermethylation of the promoter in vitro suppressed its activity. These studies suggest that alternations in DNA methylation may be one of the mechanisms regulating the tissue-specific expression of the tissue transglutaminase gene.

This study was undertaken to delineate a possible role for tissue transglutaminase (tTG), an enzyme that catalyzes protein cross-linking, in hepatic fibrogenesis. Rats were treated with CCl4 solution and then killed at different stages of liver injury and fibrogenesis. Liver tTG mRNA levels were markedly increased as early as 6 h after the first injection, peaked at 4 days and 1 wk, and remained increased for 8 wk. The enzymatic activity of tTG was increased in livers of rats treated with CCl4, in a fashion that paralleled the Northern blot results. Cell isolation experiments indicated that all hepatic cell types synthesize tTG mRNA. Increased binding to the nuclear factor-kappaB (NF-kappaB) motif of the tTG promoter was found in the nuclear extracts prepared from CCl4-treated samples. These data demonstrate an increase in tTG gene expression during hepatic injury and fibrosis, suggesting a possible role for this enzyme in stabilizing the fibrotic bands during hepatic fibrogenesis. Moreover, increased NF-kappaB binding to the tTG promoter may represent one of the mechanisms by which cell injury induces tTG transcription and thus potentiates the process of fibrogenesis.

Retinoids induce terminal differentiation and subsequent apoptosis in the human myeloid leukemia (HL-60) cell line. We have previously shown that in HL-60 cells, ligand activation of retinoic acid receptors (RARs) is sufficient to induce differentiation but ligand activation of retinoid X receptors (RXRs) is necessary for the retinoid-induced apoptosis of these cells. In the present studies we have characterized the effect of retinoids on the expression of two apoptosis-linked gene products, BCL-2 and tissue transglutaminase. BCL-2 is a membrane-associated protein whose expression has been linked to the suppression of apoptosis in many cells. Tissue transglutaminase is a protein cross-linking enzyme that accumulates in many cells undergoing apoptotic cell death. Our data suggest that ligand activation of RARs in HL-60 cells results in a global suppression of BCL-2 expression whereas ligand activation of both RARs and RXRs triggers the selective accumulation of tissue transglutaminase in the apoptotic HL-60 cells.

Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.RXR) heterodimers or RXR homodimers. Optimal induction is achieved with retinoid receptor panagonists; partial activation can also be achieved with either RAR-specific or RXR-specific retinoids. Retinoid-dependent activation of the tissue transglutaminase promoter depends on both a proximal regulatory region containing sequences highly conserved between the human and the mouse tissue transglutaminase promoters and a distal region that includes a 30-base pair retinoid response element (mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two canonical and one non-canonical) in a DR7/DR5 motif that bind both RAR*RXR heterodimers and RXR homodimers. These studies suggest that retinoid-dependent expression of the mouse tissue transglutaminase gene is mediated by a versatile tripartite retinoid response element located 1.7 kilobases upstream of the transcription start site.

Tissue transglutaminase belongs to a family of calcium-dependent enzymes, the transglutaminases that catalyze the covalent cross-linking of specific proteins by the formation of epsilon (gamma-glutamyl)lysine isopeptide bonds. The goal of this study has been the isolation and characterization of the human tissue transglutaminase gene promoter. Genomic DNA clones, spanning the 5' region of the gene, were isolated and the structure of the 5'-end of the human tissue transglutaminase gene was determined. 1.74 kilobases of flanking DNA were sequenced and were found to contain a TATA box element (TATAA), a CAAT box element (GGACAAT), a series of potential transcription factor-binding sites (AP1, SP1, interleukin-6 response element), and a glucocorticoid response elements. Transient transfection experiments showed that this DNA fragment included a functional promoter, which is constitutively active in multiple cell types.

We report that CYP3a13 gene, located on mouse chromosome 5, spans 27.5 Kb and contains 13 exons. The transcription start site is 35 bp upstream of the coding region and results in a 109 bp 5' untranslated region. CYP3a13 promoter shows putative binding sites for retinoid X receptor, pregnane X receptor, and estrogen receptor. CYP3a13 shows a broad tissue distribution with predominant expression in liver. Although CYP3a13 shares 92% nucleotide identity with the female-specific rat CYP3A9, its expression does not exhibit sexual dimorphism. Ligand activation of peroxisomal proliferator-activated receptor-gamma and retinoid X receptor inhibit expression of CYP3a13 at the transcription level in a tissue-specific manner. Another novel finding is hepatic induction of CYP3a13 by dexamethasone occurring only in pregnane X receptor null mice. We also report that pregnane X receptor is essential to maintain robust in vivo basal levels of CYP3a13 in contrast to CYP3a11. CYP3a13 protein expressed in vitro can metabolize clinically active drugs ethylmorphine and erythromycin, as well as benzphetamine. We conclude that CYP3a13 is regulated differentially by various nuclear receptors. In humans this may lead to altered drug metabolism, as many of the newly synthesized ligands/drugs targeted toward these nuclear receptors could influence CYP3A gene expression.

Retinoids elicit their biological actions by activating nuclear receptors that regulate gene transcription. There are six known retinoid receptors which belong to the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families. We report RXR-active stilbene retinoid analogs and discuss the structural features that impart RAR and RXR activation properties to compounds of this class.

Cores collected in a shelf to basin transect from northeastern Australia document the stratigraphic development of late Quaternary deposits. Low sea-level (to − 120 m) slope sedimentation is dominated by prograding fluvial deposits with wave-dominated deltas forming on the shelf edge and upper slope. The onset of sea-level transgression into the excavated shelf river channels on the outer shelf resulted in estuarine sedimentation along an indented coastline (15,000–11,000 yrs B.P.), reducing terrigenous sediment supplies to the slope. On the outer shelf, Halimeda muddy sandy gravels were deposited over a Pleistocene limestone erosional surface from about 10,500 yrs B.P. Transgression submerged the limestone outcrops (and estuaries) of the middle shelf where coral reef complexes formed; the outer and middle parts of the shelf thus became dominated by carbonate sedimentation while the inner shelf received fluvial input mixed with locally derived carbonates. The sequence is bounded by an unconformity, clearly marked by erosional down-cutting. The results of the study are combined with those of previous workers to develop transgressive and continued high sea-level system tract models. The latter (continued high sea level) model is used to extrapolate present high sea-level sedimentation patterns into the future, providing a model for comparison with ancient sequences which may also have formed in such a depositional environment.

The Alpha Retinoic Acid Receptor (RARalpha) is a Retinoic acid A receptor subtype expressed ubiquitously in embryonic and post-embryonic stages. RARalpha especially plays an important role in cardiac development due to cardiac malformation and cardiac failure related deaths in RARalpha KO mice.

The Beta Retinoic Acid Receptor (RARbeta) is a Retinoic acid A receptor subtype expressed in embryonic and post-embryonic stages. RARbeta especially plays an important role in development of central nervous system. Additionally, RARbeta expression is down-regulated in several forms of cancer.

Purpose: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer. Experimental Design: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry. Results: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to Conclusions: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.

Cellular differentiation is governed by changes in gene expression, but at the same time, a cell's identity needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mechanisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced differentiation. Here we report that the induction of an intermediary or "primed" state of myeloid differentiation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcription. Moreover, inhibition of methyltransferase activity, pre-acetylation, or activation of the enzyme PAD4 attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration mechanism during cell differentiation and, as such, may provide epigenetic memory.

Rexinoids and thiazolidinediones (TZDs) are two classes of nuclear receptor ligands that induce insulin sensitization in diabetic rodents. TZDs are peroxisome proliferator-activated receptor gamma (PPARgamma) activators, whereas rexinoids are selective ligands for the retinoid X receptors (RXRs). Activation of both the insulin receptor substrates (IRSs)/Akt and the c-Cbl-associated protein (CAP)/c-Cbl pathways are important in regulating insulin-stimulated glucose transport. We have compared the effects of a rexinoid (LG268) and a TZD (rosiglitazone) on these two signal pathways in skeletal muscle of diabetic (db/db) mice. The results we have obtained show that treatment of db/db mice with either LG268 or rosiglitazone for 2 weeks results in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Treatment with LG268 increases insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation in skeletal muscle without affecting the activity of the CAP/c-Cbl pathway. In contrast, rosiglitazone increases the levels of CAP expression and insulin-stimulated c-Cbl phosphorylation without affecting the IRS-1/Akt pathway. The effects of LG268 on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Ser(307) phosphorylation. Taken together, these data suggest that rexinoids improve insulin sensitivity via changes in skeletal muscle metabolism that are distinct from those induced by TZDs. Rexinoids represent a novel class of insulin sensitizers with potential applications in the treatment of insulin resistance.

Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes peptide cross-linking and polyamination reactions, and also is a signal-transducing GTPase. tTG protein content and enzymatic activity are upregulated in the brain in Huntington's disease and in other neurological diseases and conditions. Since mouse models are currently being used to study the role of tTG in Huntington's disease and other neurodegenerative diseases, it is critical that the level of its expression in the mouse forebrain be determined. In contrast to human forebrain where tTG is abundant, tTG can only be detected in mouse forebrain by immunoblotting a GTP-binding-enriched protein fraction. tTG mRNA content and transamidating activity are approximately 70% lower in mouse than in human forebrain. However, tTG contributes to the majority of transglutaminase activity within mouse forebrain. Thus, although tTG is expressed at lower levels in mouse compared with human forebrain, it likely plays important roles in neuronal function.

Objective: Implantation of a left ventricular assist device (LVAD) in the failing human heart initiates structural and functional changes termed reverse remodeling. Mechanical unloading improves cardiac adrenergic responsiveness and lipid metabolism, processes regulated by caveolar function. We tested the hypothesis that mechanical unloading alters the expression of caveolins and these changes are linked to altered expression of markers of reverse remodeling. Methods: Paired human myocardial samples were obtained from patients who received an LVAD as a bridge to cardiac transplantation. Transcript levels were measured using real-time Q-RT-PCR in RNA prepared from 34 pairs of formalin-fixed myocardial tissue blocks. Caveolin-1 and -3 protein levels were determined from frozen tissue ( n = 5) by Western blots. Caveolin-3 localization was demonstrated by immunohistochemistry. Results: Caveolin-1 protein levels were upregulated in all LVAD-patients after mechanical unloading ( P = 0.002). Caveolin-1 mRNA was increased in 76% of the patients ( n = 34, P

A cDNA encoding for the human tissue transglutaminase gene has been used to identify the chromosomal localization of the corresponding structural gene. The precise chromosomal and subregional localizations have been established by using in situ fluorescence mapping with a recombinant lambda-Zap phage containing the full cDNA coding sequence. The study showed that the human tissue transglutaminase gene is localized on chromosome 20 and, more precisely, within the band 20q12. To date, this is the third member of the transglutaminase gene family to be mapped. Human factor XIIIa (plasma transglutaminase), human keratinocyte transglutaminase (type I), and human tissue transglutaminase (type II) genes, although codifying for homologous enzymes, are localized on three different chromosomes.

Background: Experimental and clinical data suggest that the loss of membrane receptor tyrosine kinase (RTK) activity in cardiac myocytes results in increased frequency of apoptotic cell death and progression of heart failure. The goal of our study was to examine the expression characteristics of RTKs in ventricular myocardium obtained from patients before and after mechanical unloading. Methods: We extracted RNA from paired formalin-fixed, paraffin-embedded left ventricular tissue blocks obtained at the time of left ventricular assist device (LVAD) implantation and explantation from a cohort of 36 patients (median age 51 years). The duration of LVAD support ranged from 1 to 314 days (median 95 days), 17 patients had ischemic and 19 non-ischemic cardiomyopathy at the time of LVAD implantation. Using real-time reverse transcription–polymerase chain reaction (RT-PCR) we quantitated transcripts for atrial natriuretic factor (ANF) and tumor necrosis factor-alpha (TNF-α), markers of heart failure, and the RTKs Her2/neu, Her4 and gp130, regulators of cardiac cell survival. Results: In patients undergoing mechanical unloading, ANF and TNF-α mRNA levels were independently suppressed. Her2/neu, along with Her4 was upregulated, mostly in cases of ischemic cardiomyopathy, whereas gp130 levels decreased. Post-LVAD transcript levels of Her2 correlated tightly with gp130 in patients with non-pathologic entry values of gp130. Duration of treatment and age were also determining factors in the change of expression of these genes. Conclusion: Real-time quantitative (Q)-RT-PCR can be used to quantitate gene expression in archival myocardial tissue blocks. Mechanical unloading leads to a re-adjustment of RTK transcript levels, but not their reverting to control values in heart failure patients.

Increasing interest and awareness of protein aggregation as being implicated in neurodegenerative processes has developed in recent years. One novel mechanism for this may be transglutaminase (TGase)-mediated protein crosslinking, that is involved in a variety of natural processes ranging from the stabilization of fibrin clots to production of the epidermal cell envelope and the fluid barrier of the skin. TGases are also implicated in both function and dysfunction of the central (CNS) and peripheral (PNS) nervous systems. The most ubiquitously expressed member of the TGase family, known as tissue TGase (tTG) or TG2, which, in addition to catalyzing the production of epsilon-lysine to gamma-glutaminyl isodipeptide bonds, serves a dual function as the G-protein Galpha(h) and is both expressed and active in PNS and CNS. It differs from other members of the TGase gene family in this regard and may implicate it in 'switches' from life or trophic signaling to those associated with apoptosis. In this regard, recent data indicate that one or more TGases are involved in neurodegenerative disorders such as the Qn/CAG repeat disorders, as well as Alzheimer's and Parkinson's diseases. As do many genes, particularly those highly expressed in the CNS, tTG undergoes alternative processing. Elevated expression and alternative splicing, resulting in a short (S) isoform of tTG with more active crosslinking activity, are associated with increased neuronal loss in affected regions in the demented brain. Our recent and novel data indicate that tTG mRNA, protein, and TGase activity are elevated in certain neurodegenerative diseases, and are accompanied by transcription of this S splice variant that results in unregulated crosslinking, unique to neurodegenerative disorders.

In order to understand the mechanism for insoluble neurotoxic protein polymerization in Alzheimer's disease (AD) brain neurons, we examined protein and gene expression for transglutaminase (TGase 2; tissue transglutaminase (tTG)) in hippocampus and isocortex. We found co-localization of tTG protein and activity with tau-positive neurofibrillary tangles, whereas mRNA and sequence analysis indicated an absolute increase in tTG synthesized. Although apoptosis in AD hippocampus is now an established mode of neuronal cell death, no definite underlying mechanism(s) is known. Since TGase-mediated protein aggregation is implicated in polyglutamine ((CAG)(n)/Q(n) expansion) disorder apoptosis, and expanded Q(n) repeats are excellent TGase substrates, a role for TGase in AD is possible. However, despite such suggestions almost 20 years ago, the molecular mechanism remained elusive. We now present one possible molecular mechanism for tTG-mediated, neurotoxic protein polymerization leading to neuronal apoptosis in AD that involves not its substrates (like Q(n) repeats) but rather the unique presence of alternative transcripts of tTG mRNA. In addition to a full-length (L) isoform in aged non-demented brains, we found a short isoform (S) lacking a binding domain in all AD brains. Our current results identify intron-exon "switching" between L and S isoforms, implicating G-protein-coupled signaling pathways associated with tTG that may help to determine the dual roles of this enzyme in neuronal life and death processes.

C. Depre, M. E. Young, J. Ying, H. Singh Ahuja, Q. Han, N. Garza, P. J. A. Davies and H. Taegtmeyer. Streptozotocin-induced Changes in Cardiac Gene Expression in the Absence of Severe Contractile Dysfunction. Journal of Molecular and Cellular Cardiology (2000) 32, 985–996. Diabetes mellitus alters energy substrate metabolism and gene expression in the heart. It is not known whether the changes in gene expression are an adaptive or maladaptive process. To answer this question, we determined both the time-course and the extent of the alteration of gene expession induced by insulin-deficient diabetes. Transcript analysis with real-time quantitative polymerase chain reaction (PCR) was performed in rat hearts 1 week (acute group) or 6 months (chronic group) after administration of streptozotocin (55 mg/kg). In the acute group, insulin-dependent diabetes induced a 55–70% decrease of both glucose transporter 1 (GLUT1) and GLUT4 transcripts, a slight decrease of liver-specific carnitine palmitoyltransferase I (CPT I), and no change in muscle-specific CPT I. The uncoupling protein UCP-3 increased three-fold, with no change in UCP-2. These metabolic alterations were accompanied by an isoform switching from the normally expressed α myosin heavy chain (MHC) to the fetal isoform β MHC mRNA, by a 50% decrease of cardiac α -actin mRNA, a 30% decrease of the sarcoplasmic Ca++-ATPase mRNA, and a 50% decrease of muscle creatine kinase (P