The low molecular weight (LMW) isoforms of cyclin E provide a novel mechanism of cell cycle deregulation

Cyclin E, in complex with cyclin dependent kinase 2 (CDK2), is a positive regulator of G1 to S phase progression of the cell cycle. Deregulation of G1/S phase transition occurs in the majority of tumors. Cyclin E is overexpressed and post-translationally generates low molecular weight (LMW) isoforms in breast cancer, but not normal cells. Such alteration of cyclin E is linked to poor prognosis. Therefore, we hypothesized that the LMW isoforms of cyclin E provide a novel mechanism of cell cycle de-regulation in cancer cells. Insect cell expression system was used to explore the biochemical properties of the cyclin E isoforms. Non-tumorigenic (76NE6) and tumorigenic (T47D) mammary epithelial cells transfected with the cyclin E isoforms and breast tumor tissue endogenously expressing the LMW isoforms were used to study the biologic consequences of the LMW isoforms of cyclin E. All model systems studied show that the LMW forms (compared to full-length cyclin E) have increased kinase activity when partnered with CDK2. Increases in the percentage of cells in S phase and colony formation were also observed after overexpression of LMW compared to full-length cyclin E. The LMW isoforms of cyclin E utilize several mechanisms to attain their hyper-activity. They bind CDK2 more efficiently, and are resistant to inhibition by cyclin dependent kinase inhibitors (CKIs) as compared to full-length cyclin E. In addition, the LMW isoforms sequester the CKIs from full-length cyclin E abrogating the overall negative regulation of cyclin E. Despite their correlation with adverse biological consequences, the direct role of the LMW isoforms of cyclin E in mediating tumorigenesis remained unanswered. Subsequent to LMW cyclin E expression in 76NE6 cells, they lose their ability to enter quiescence and exhibit genomic instability, both characteristic of a tumor cell phenotype. Furthermore, injection of 76NE6 cells overexpressing each of the cyclin E isoforms into the mammary fat pad of nude mice revealed that the LMW isoforms of cyclin E yield tumors, whereas the full-length cyclin E does not. In conclusion, the LMW isoforms of cyclin E utilize several mechanisms to acquire a hyperactive phenotype that results in deregulation of the cell cycle and initiates the tumorigenic process in otherwise non-transformed mammary epithelial cells. ^

Aggressive vs. conservative phototherapy for infants with extremely low birth weight.

BACKGROUND: It is unclear whether aggressive phototherapy to prevent neurotoxic effects of bilirubin benefits or harms infants with extremely low birth weight (1000 g or less). METHODS: We randomly assigned 1974 infants with extremely low birth weight at 12 to 36 hours of age to undergo either aggressive or conservative phototherapy. The primary outcome was a composite of death or neurodevelopmental impairment determined for 91% of the infants by investigators who were unaware of the treatment assignments. RESULTS: Aggressive phototherapy, as compared with conservative phototherapy, significantly reduced the mean peak serum bilirubin level (7.0 vs. 9.8 mg per deciliter [120 vs. 168 micromol per liter], P<0.01) but not the rate of the primary outcome (52% vs. 55%; relative risk, 0.94; 95% confidence interval [CI], 0.87 to 1.02; P=0.15). Aggressive phototherapy did reduce rates of neurodevelopmental impairment (26%, vs. 30% for conservative phototherapy; relative risk, 0.86; 95% CI, 0.74 to 0.99). Rates of death in the aggressive-phototherapy and conservative-phototherapy groups were 24% and 23%, respectively (relative risk, 1.05; 95% CI, 0.90 to 1.22). In preplanned subgroup analyses, the rates of death were 13% with aggressive phototherapy and 14% with conservative phototherapy for infants with a birth weight of 751 to 1000 g and 39% and 34%, respectively (relative risk, 1.13; 95% CI, 0.96 to 1.34), for infants with a birth weight of 501 to 750 g. CONCLUSIONS: Aggressive phototherapy did not significantly reduce the rate of death or neurodevelopmental impairment. The rate of neurodevelopmental impairment alone was significantly reduced with aggressive phototherapy. This reduction may be offset by an increase in mortality among infants weighing 501 to 750 g at birth. (ClinicalTrials.gov number, NCT00114543.)

Vitamin A supplementation in very low birth weight infants

PURPOSE: In United States, the percentage of Extremely Low Birth Weight (ELBW) born for year 2006 was 0.8% (approximately 32,000 babies) & Very Low Birth Weight (VLBW) 1.48% (1). ELBW babies account for nearly half (49%) of the infant mortality for United States. Very Low birth weight infants are at a significant risk for high mortality and morbidity due to their multi system involvement and predisposition to lung prematurity and impaired immune function. One of the common causes cited is Vitamin A deficiency (2, 3).The purpose of this study is to look at published literature on Vitamin A supplementation in very low birth weight (VLBW) infants. ^ RESEARCH DESIGN: Systematic review of literature of published articles meeting the pre-defined criteria. ^ PROCEDURE: Studies included in this review were those which looked at very low birth weight infants defined as birth weight<1500gms. All experimental studies were reviewed. Studies looking at the effect of Vitamin A supplementation in comparison with a placebo or by itself in varying dosing regimens as an intervention were reviewed. Vitamin A deficiency and its manifestations were of interest. We used key words such as "very low birth weight", "mortality", "Vitamin A", "retinol" and "supplementation" in our search. ^ RISKS & POTENTIAL BENEFITS: We do not see any potential risks associated with this study. The potential benefit is recommendation for future studies based on the review of literature available currently. ^ IMPORTANCE OF KNOWLEDGE THAT MAY REASONABLY BE EXPECTED TO RESULT: The systematic review of literature of all experimental studies in VLBW infants showed uniform correlation of parenteral Vitamin A dosing and high plasma concentrations achieved. The recommended dosage for use is 5000 IU 3 times/week given intramuscularly for 4 weeks to prevent CLD. Higher doses have not shown benefit, with a potential for toxicity, while lower doses are inadequate. There is no role of use of Vitamin A in closure of patent ductus arteriosus & reducing mortality. However, it is important to state that the number of studies done so far is limited with small sample sizes. There is a need in the future for experimental studies to ascertain the role of Vitamin A to improve outcomes in VLBW. Atleast, one more RCT should be conducted using the dosage recommended above to make this a standard practice.^

Computational selection of inhibitors of Abeta aggregation and neuronal toxicity.

Alzheimer's disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-beta protein (Abeta). Disease symptoms can be alleviated, in vitro and in vivo, by 'beta-sheet breaker' pentapeptides that reduce plaque load. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related beta-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Abeta. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features and a conformation similar to the active peptides were selected, ranked by docking and biochemical parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Abeta aggregation at 2-3microM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Abeta on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD.

LMW-E MEDIATES MAMMARY TUMORIGENESIS BY DEREGULATING ACINAR MORPHOGENESIS & GENERATING CANCER STEM CELLS

Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in breast cancer generates two low molecular weight (LMW) isoforms that exhibit both enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E disrupts normal mammary acinar morphogenesis and serves as the initial route into breast tumor development. We first demonstrate that LMW-E overexpression in non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice significantly more than overexpression of full-length cyclin E and requires CDK2- associated kinase activity. Further in vivo passaging of these tumors augments LMW-E expression and tumorigenic potential. When subjected to acinar morphogenesis in vitro, LMW-E mediates significant morphological disruption by generating hyperproliferative and multi-acinar complexes. Proteomic analysis of patient tissues and tumor cells with high LMW-E expression reveals that the activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E expression predicts poor patient survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness. These tumor cells also enrich for cells with CSC phenotypes such as increased CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo population also shows positive correlation with LMW-E expression in both the tumor cell line model and breast cancer patient samples (p<0.0001 & p=0.0435, respectively). Combination treatment using doxorubicin and salinomycin demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in those with full-length cyclin E expression. Finally, ProtoArray microarray identifies Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression results in enrichment for CSCs. Collectively, these data emphasize the strong oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible therapeutic strategies to treat breast cancer patients with high LMW-E expression.

Traumatic brain injury (TBI) produces cytoskeletal protein derangements within cortical neurons that can be attenuated by protease inhibition

TBI produces a consistent and extensive loss of neurofilament 68 (NF68) and neurofilament 200 (NF200), key intermediate cytoskeletal proteins found in neurons including axons and dendrites, in cortical samples from injured brain. The presence of low molecular weight NF68 breakdown products (BDPs) strongly suggest that calpain proteolysis at least in part contributes to neurofilament (NF) protein loss following injury. Furthermore, one and two-dimensional gel electrophoresis analyses of NF BDPs obtained from in situ and in vitro tissue also implicated the involvement of calpain 2 mediated proteolysis of neurofilaments following TBI. Immunohistochemical examination of derangements in cytoskeletal proteins following traumatic brain injury in rats indicated that preferential dendritic rather than axonal damage occurs within three hours post-TBI. Although proteolysis of cytoskeletal proteins occurred concurrently with early morphological alterations, evidence of proteolysis preceded the full expression of evolutionary histopathological changes. Furthermore, cytoskeletal immunofluorescence alterations were not restricted to the site of impact. Confocal microscopic investigations of NF68 and NF200 immunofluorescence within injured cortical neurons revealed alterations in neurofilament assembly in the absence of NF derangements detectable at the light microscopic level ($<$15 minutes post-TBI). Collectively immunohistochemistry studies suggest that derangements to neuronal processes are biochemical and evolutionary in nature, and not due solely to mechanical shearing. Importantly, a systemically administered calpain inhibitor (calpain inhibitor 2) significantly reduced NF200, NF68, and spectrin protein loss as well as providing marked preservation of NF proteins in neuronal somata, dendrites, and axons at 24 hours post-TBI. ^

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Molecular characterization of a gene required for entry into S phase of the mitotic cell cycle in the yeast {\it Saccharomyces cerevisiae\/}

A strain of Saccaromyces cerevisiae (SC3B) with a temperature sensitive defect in the synthesis of DNA has been isolated. This defect is due to a single recessive mutation in a gene named INS1 required for the initiation of S phase. Arrested cells carrying the ins1$\sp{ts}$ allele are defective in the completion of G1 to S phase transition events including SPB duplication or separation, initiation of DNA synthesis, normal control of budding, and bud neck stability. The mutation and a gene which complements the mutation were mapped to chromosome IV. The complementing gene was proved to be the wild type allele of the temperature sensitive mutation by genetic linkage of an integrated clone. A very low abundance 4.2 kb RNA message was observed in the strain SC3B which increased greatly in this strain transformed with a multiple copy plasmid carrying the complementing clone. The wild type gene was sequenced and found to encode a 1268 amino acid protein of with a molecular weight of 142,655 Daltons. Computer assisted searches for similar DNA sequences revealed no significant homology matches. However, searches for protein sequence homology revealed a protein (the DIS3 gene product of S. pombe) with a similar sequence over a 534 amino acid stretch to the predicted INS1 gene product. A later search revealed a near identical sequence for a gene (SRK1) also isolated from S. cerevisiae. ^

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^