Spheroid architecture strongly enhances miR-221/222 expression and promotes oxidative phosphorylation in an ovarian cancer cell line through a mechanism that includes restriction of miR-9 expression.

BACKGROUND: Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. METHODS AND RESULTS: We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. CONCLUSIONS: The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.

Curcumin Loaded Dendrimers Specifically Reduce Viability of Glioblastoma Cell Lines.

Glioblastoma (GB) is a deadly and aggressive cancer of the CNS. Even with extensive resection and chemoradiotherapy, patient survival is still only 15 months. To maintain growth and proliferation, cancer cells require a high oxidative state. Curcumin, a well-known anti-inflammatory antioxidant, is a potential candidate for treatment of GB. To facilitate efficient delivery of therapeutic doses of curcumin into cells, we encapsulated the drug in surface-modified polyamidoamine (PAMAM) dendrimers. We studied the in vitro effectiveness of a traditional PAMAM dendrimer (100% amine surface, G4 NH2), surface-modified dendrimer (10% amine and 90% hydroxyl-G4 90/10-Cys), and curcumin (Cur)-encapsulated dendrimer (G4 90/10-Cys-Cur) on three species of glioblastoma cell lines: mouse-GL261, rat-F98, and human-U87. Using an MTT assay for cell viability, we found that G4 90/10-Cys-Cur reduced viability of all three glioblastoma cell lines compared to non-cancerous control cells. Under similar conditions, unencapsulated curcumin was not effective, while the non-modified dendrimer (G4 NH2) caused significant death of both cancerous and normal cells. By harnessing and optimizing the components of PAMAM dendrimers, we are providing a promising new route for delivering cancer therapeutics. Our results with curcumin suggest that antioxidants are good candidates for treating glioblastoma.

Changes in Nup62 content affect contact-induced differentiation of cultured myoblasts.

Differentiation of cultured skeletal myoblasts is induced by extrinsic signals that include reduction in ambient mitogen concentration and increased cell density. Using an established murine myoblast cell line (C2C12), we have found that experimental reduction of the nucleoporin p62 (Nup62) content of myoblasts enhances differentiation in high-mitogen medium, while forced expression of Nup62 inhibits density-induced differentiation. In contrast, differentiation of myoblasts induced by low-mitogen medium was unaffected by ectopic Nup62 expression. Further analyses suggested that Nup62 content affects density-induced myoblast differentiation through a mechanism involving activation of p38 MAP kinase. Nuclear pore complex (NPC) composition, in particular changes in NUP62 content, may be altered during viral infection, differentiation, and in neoplastic growth. The results support a functional role for changes in Nup62 composition in NPCs and density-induced myogenic differentiation, and suggest a link between loss of Nup62 content and induction of an intracellular stress signaling pathways.

Role for NUP62 depletion and PYK2 redistribution in dendritic retraction resulting from chronic stress.

Genetic evidence suggests cell-type-specific functions for certain nucleoporins, and gene expression profiling has revealed that nucleoporin p62 (NUP62) transcripts are decreased in the prefrontal cortex of major depressives. Chronic stress, which can precipitate depression, induces changes in the architecture and plasticity of apical dendrites that are particularly evident in the CA3 region of the hippocampus. Genetically targeted translating ribosome affinity purification revealed a selective reduction in translated Nup62 transcripts in CA3 of chronically stressed mice, and the Nup62 protein content of nuclei extracted from whole hippocampus was found to be decreased in chronically stressed rats. In cultured cells, phosphorylation of a FAK/proline-rich tyrosine kinase 2 (PYK2) consensus site in the alpha-helical domain of NUP62 (human Y422) is shown to be associated with shedding of NUP62 from the nuclear pore complex (NPC) and/or retention of NUP62 in the cytoplasm. Increased levels of phospho-Y425 Nup62 were observed in cytoplasmic fractions of hippocampi from chronically stressed rats, and immunofluorescence microscopy revealed redistribution of activated Pyk2 to the perinuclear region of stressed pyramidal neurons. Depletion of Nup62 from cultured embryonic day 18 rat hippocampal and cortical neurons resulted in simplification and retraction of dendritic arbors, without disruption of axon initial segment integrity. Thus, at least two types of mechanisms--one affecting expression and the other association with the NPC--could contribute to loss of NUP62 from CA3 pyramidal neurons during chronic stress. Their combined actions may account for the enhanced responsiveness of CA3 apical dendrites to chronic stress and may either be pathogenic or serve to protect CA3 neurons from permanent damage.

Nuclear distributions of NUP62 and NUP214 suggest architectural diversity and spatial patterning among nuclear pore complexes.

The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin.

Alterations in nuclear pore architecture allow cancer cell entry into or exit from drug-resistant dormancy.

Phenotypic diversity arises in tumors just as it does in developing organisms, and tumor recurrence frequently manifests from the selective survival of divergent drug-resistant cells. Although the expanding tumor cell population may be successfully targeted, drug-resistant cells may persist and sustain the tumor or enter dormancy before igniting a future relapse. Herein, we show that partial knockdown of nucleoporin p62 (NUP62) by small-interfering RNA confers cisplatin resistance to cultured high-grade ovarian carcinoma cells. Treatment with NUP62 small-interfering RNA and cisplatin leaves resistant cells in a state of dormancy; some dormant cells can be induced to proliferate by transient induction of NUP62 expression from an ectopic expression construct. In addition to suggesting functional links between nuclear pore complex architecture and cancer cell survival, the culture system provides a novel experimental window into the dynamics of tumor cell drug resistance and dormancy.

Fiber-types of sarcomeric proteins expressed in cultured myogenic cells are modulated by the dose of myogenin activity.

The myogenic basic helix-loop-helix regulatory factors (MRFs) maintain commitment of proliferating cells to the skeletal myogenic lineage, and contribute to activation of transcription of muscle-specific genes in myocytes and muscle fibers. A clear role for any or all of the MRFs in muscle fiber-type determination, however, has not emerged from expression or genetic studies. During fetal, neonatal and adult life, diversification of muscle fiber types, and the dynamics of slow or fast fiber type adaptation and growth, are controlled by exogenous factors, including innervation, work load, and hormonal signaling. In contrast, stereotypical development of muscle fibers preferentially expressing slow or fast isoforms of sarcomeric proteins in the embryo occurs in the absence of these factors, and appears to be mediated both by input from the surrounding interstitial milieu, as well by cell autonomous mechanisms. We report here that diversification of myogenic cells in culture towards the expression of fast or slow skeletal muscle fiber types can be determined by the activity and dose of at least one MRF, myogenin. The dose of myogenin is modulated by two parameters: the phosphorylation state of the transcriptional activation domains, and the level of expression. Low doses of myogenin promoted a fast phenotype, whereas higher doses promoted a slow phenotype, and further studies suggested that diversification is mediated by both transcriptional and post-transcriptional mechanisms. The potential for dose or numeration signaling by basic helix-loop-helix regulators has been revealed by studies in Drosophila melanogaster, while the present results support the notion that this mechanism may be more commonly employed to generate subdiversity among developing cell types.

Induction of autophagy in axonal dystrophy and degeneration.

Autophagy is a highly regulated cellular mechanism for the bulk degradation of cytoplasmic contents. It has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. However, the regulation of autophagy in neurons and its role in neuronal and axonal pathology are not yet understood. Using transgenic mice producing green fluorescent protein-tagged autophagic marker microtubule-associated protein light chain 3 (GFP-LC3), we provide molecular evidence for the induction of autophagy in axonal dystrophy and degeneration in Purkinje cells of the Lurcher mice, a model for excitotoxic neurodegeneration. We show that the excitotoxic insult of Lurcher mutation triggers an early response of Purkinje cells involving accumulation of GFP-LC3-labeled autophagosomes in axonal dystrophic swellings (a hallmark of CNS axonopathy). In brain, LC3 interacts with high affinity with the microtubule-associated protein 1B (MAP1B). We show that MAP1B binds to LC3 of both cytosolic form (LC3I) and lipidated form (LC3II). Moreover, in cell culture, overexpression of MAP1B results in reduced LC3II levels and number of GFP-LC3-labeled autophagosomes; phosphorylated MAP1B is associated with GFP-LC3-labeled autophagosomes. Furthermore, in brain, phosphorylated MAP1B accumulates in axonal dystrophic swellings of degenerating Purkinje cells and binds to LC3 at increased level. Therefore, the MAP1B-LC3 interaction may participate in regulation of LC3-associated autophagosomes in neurons, in particular at axons, under normal and pathogenic conditions. We propose that induction of autophagy serves as an early stress response in axonal dystrophy and may participate in the remodeling of axon structures.

Functional interaction of Puralpha with the Cdk2 moiety of cyclin A/Cdk2.

Puralpha is a sequence-specific single-stranded nucleic acid-binding protein and a member of the highly conserved Pur family. Puralpha has been shown to colocalize with cyclin A/Cdk2 and to coimmunoprecipitate with cyclin A during S-phase. Here we show that this interaction is mediated by a specific affinity of Puralpha for Cdk2. In pull-down assays GST-Puralpha efficiently binds Cdk2 and Cdk1, binds Cdk4 less efficiently, and does not display binding to Cdk6. Puralpha stimulates several-fold the phosphorylation in vitro of histone H1 by cyclin A/Cdk2, produced from baculovirus constructs. Double chromatin immunoprecipitation using antibodies to Cdk2 and Puralpha reveals that both proteins colocalize in HeLa cells to DNA segments upstream of the c-MYC gene. Pur family member Purgamma colocalizes with Cdk2 to a specific DNA segment in this region.

A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer's disease.

Among the earliest invariant neuropathological changes in Alzheimer's disease (AD) is the degeneration of vulnerable hippocampal CA1 and subicular pyramidal neurons. Semaphorin 3A (Sema3A) is a secreted protein that functions in signaling growth cone collapse, chemorepulsion and neuronal apoptosis during early development of the central nervous system. In this report we show that accumulation of an internalized form of Sema3A is associated with degeneration of neurons in vulnerable fields of the hippocampus during AD. Accumulation of Sema3A overlaps the appearance of phosphorylated MAP1B and tau in many neurons, suggesting that Sema3A signaling at some level may be coupled to these previously identified cytoskeletal markers of neurodegeneration. Consistent with this, we isolated and partially characterized a multiprotein complex from the hippocampus of patients with AD that contains phosphorylated MAP1B, collapsin-response mediator protein 2 (CRMP-2), Plexins A1 and A2, and a processed form of Sema3A. A model is presented in which aberrant release of Sema3A from expressing neurons in the subiculum during AD results in the internalization and transport of Sema3A from this field to CA1. Within the context of the myriad of potential insults that contribute to Alzheimer's and other neurodegenerative diseases, the bioactivity of Sema3A may contribute either directly to neurodegeneration by inducing neuronal collapse, or indirectly by abrogating the recovery capabilities of adult neurons faced with these insults.

Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway.

Signals from several receptor tyrosine kinases are transduced by activation of the Ras family of GTP-binding proteins. Activation of Ras initiates a kinase cascade that culminates in activation of the mitogen-activated protein kinases (MAPKs). The MAPKs include the c-jun NH(2)-terminal protein kinases (JNKs) and extracellular signal-regulated kinases (ERKs), both of which phosphorylate Elk-1/TCF, a factor that activates transcription of the c-fos gene. In this report, we identify a novel 19 kDa gene product as a negative regulator of signaling through the ERK1/2 pathway. While these studies were in progress, the human homologue of this gene was characterized as diphosphoinositol polyphosphate phosphohydrolase (DIPP1) [EMBO J. 17 (1998) 6599], a phosphohydrolase that converts diphosphate groups on diphosphoinositol polyphosphates to monophosphates. Ectopic expression of murine DIPP1 (muDIPP1) blocked activation of the c-fos promoter by the ERK1/2 pathway. Inhibition of signal transduction through the ERK1/2 pathway by muDIPP1 occurs at or downstream from activation of MEK. In vitro kinase studies suggest that muDIPP1 is not a direct inhibitor of MEK or ERK activity, although, ectopic expression at near physiological levels results in attenuation of ERK phosphorylation in vivo. Interestingly, a site mutant of muDIPP1 lacking phosphohydrolase activity blocked signaling through the ERK1/2 pathway with greater efficiency than wild-type muDIPP1. This result suggests that inhibition of signaling through the ERK1/2 pathway is a distinct function of muDIPP1 that is not dependent on, but may be regulated by, its activity as a phosphohydrolase.

Expression of p63 in papillary thyroid carcinoma and in Hashimoto's thyroiditis: a pathobiologic link?

p63 proteins are p53 homologs that are postulated to regulate squamous stem cell commitment. An immunohistochemical survey of p63 expression in normal thyroid and in reactive, neoplastic, and inflammatory thyroid disorders was performed. Sections from routinely fixed and processed archival thyroidectomy specimens were pretreated with citric acid, pH 6.0, for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as a chromagen, and then were counterstained with hematoxylin. The results showed that p63 expression was negative in normal thyroid tissue, nodular goiters, and oncocytic follicular adenomas. Positivity was rare and weak in follicular adenomas. p63-positive foci were commonly found in Hashimoto's thyroiditis (1 or more foci in 78.8% of cases), but rare in Graves' disease. Twenty-seven of 33 papillary thyroid carcinomas (81.8%) displayed p63-positive foci. Staining was uncommon in follicular carcinomas and rare in medullary carcinomas. One case of insular carcinoma was p63-positive. All squamoid structures were p63-positive; p63-positive structures morphologically consistent with solid cell nests were also identified. Based on the results of this study, we conclude that p63 is commonly expressed in papillary thyroid carcinoma and in Hashimoto's thyroiditis. Given the debated association of papillary thyroid carcinoma with Hashimoto's thyroiditis, it is possible that p63 expression may be a potential pathobiologic link between the two disorders. The finding of p63 in benign squamoid nests supports a possible interrelationship between these structures and both Hashimoto's thyroiditis and papillary carcinoma. The high percentage of papillary carcinomas with p63-positive foci appears to distinguish papillary carcinoma from other neoplasms originating in the thyroid.

Immunohistochemical visualization of histone H1 phosphorylation in squamous intraepithelial lesions of the gynecologic tract.

Immunohistochemical staining was performed on gynecologic tract squamous intraepithelial lesions using a novel phosphorylation-specific monoclonal antibody (designated 12D11) that detects histone H1 when phosphorylated at a cyclin-dependent kinase (CDK)-responsive epitope. Findings were compared to immunostaining by MIB-1, an extensively studied antibody probe of proliferation. Routinely fixed and processed archival sections were subjected to distinct antigen retrieval and staining protocols for each antibody and were processed for immunodetection of either Ki-67 (with MIB-1) or phosphohistone H1, using a streptavidin-biotin kit and diaminobenzidine as chromagen. For 12D11 staining, antigen retrieval was performed at pH 4.0, and the antibody incubation buffer was supplemented with 1.0 M NaCl. Both 12D11 and MIB-1 stained parabasal cells in normal squamous epithelium. Staining by 12D11 and MIB-1 of cells in progressively higher strata was found to correlate with the severity of lesions. The mean proportion of positively stained cells was higher in MIB-1-stained sections than in 12D11-stained sections in normal squamous epithelium and in all grades of squamous intraepithelial lesions. We conclude that the changes in expression patterns of CDK-phosphorylated histone H1 in the spectrum of gynecologic squamous intraepithelial lesions are similar to staining patterns obtained with the proliferation probe MIB-1. The differing proportion of cells stained by MIB-1 and 12D11 suggests that phosphohistone H1 may be a useful alternative proliferation marker that detects a different subpopulation of cycling cells in premalignant squamous lesions.

P63 in pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors.

p63 is a p53-homologous nuclear protein that appears to play a crucial role in regulation of stem cell commitment in squamous and other epithelia. In this study, p63 expression was examined in benign lung and in neoplasms of pulmonary origin. Eighty sections from routinely fixed and processed archival bronchoscopic biopsy or lobectomy specimens were pretreated with citric acid (pH 6.0) for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as chromagen, and were counterstained with hematoxylin. In normal lung, p63 intensely stained nuclei of bronchial reserve cells but did not stain ciliated cells, alveolar epithelial cells, or nonepithelial cells. The lower strata of squamous metaplastic bronchial epithelium stained positively. All squamous-cell carcinomas stained positively (n = 30). In some well-differentiated carcinomas, staining was found at the periphery of tumor nests but was negative in central zones showing squamous maturation. Poorly differentiated carcinomas showed very high proportions (80% to 100%) of p63-positive nuclei. All small-cell carcinomas were p63 negative (n = 9). Staining of bronchioloalveolar carcinomas (n = 7) and adenocarcinomas (n = 23) was variable: some tumors showed no detectable staining, others showed heterogeneously positive staining. Adenosquamous carcinomas (n = 5) displayed a unique basalar staining pattern. Carcinoid tumors were almost entirely negative (n = 5). We conclude that p63 is expressed in benign bronchial stem cells, in neoplastic cells with either squamous differentiation or squamous differentiating potential, and in a subpopulation of adenocarcinomas. p63 immunostaining may also aid in some histopathologic distinctions, such as in small biopsies where the differential diagnosis is poorly differentiated squamous carcinoma versus small-cell carcinoma. A stem cell biology-based classification system for squamous carcinomas is proposed.

Monoclonal antibody specific for histone H1 phosphorylated by cyclin-dependent kinases: a novel immunohistochemical probe of proliferation and neoplasia.

Monoclonal antibody 12D11 (MAb 12D11) has been shown to bind histone H1 isolated from human placenta and other tissues but not histone H1 that has been digested with bacterial alkaline phosphatase. We show here that phosphorylation of phosphatase-treated histone H1 with cyclin dependent-kinase (CDK) restores binding by MAb 12D11. We conclude that MAb 12D11 selectively binds histone H1 that has been phosphorylated by CDKs, and we have investigated the use of MAb 12D11 as an immunohistochemical probe of CDK activity in situ. Previous immunofluorescence studies have revealed strong nuclear staining by MAb 12D11 in proliferating cultured cells and the absence of staining in terminally differentiated cells. Immunohistochemical staining of frozen and formalin-fixed, paraffin-embedded sections of benign tissues with MAb 12D11 was nuclear and confined to recognized foci of cell proliferation. In lymphoid germinal centers, MAb 12D11 preferentially stained large lymphoid cells with a relative lack of staining in small cleaved cells, contrasting with a lack of cell size discrimination observed with the monoclonal antibody proliferation probe, MIB-1. Tumor tissues displayed strong albeit heterogeneous staining of malignant cells by MAb 12D11, with little or no staining observed in surrounding nonneoplastic stromal cells. Differential staining by MAb 12D11 of invasive and in situ carcinoma suggest applications in prognostication. MAb 12D11 may also be useful in identification of tumors more likely to respond to therapeutic CDK inhibitors.