Novel aspects of live intestinal epithelial cell function revealed using a custom time-lapse video microscopy apparatus.

Many aspects of cell physiology, including migration, membrane function, and cell division, are best understood by observing live cell dynamics over time using video microscopy. To probe these phenomena in colon epithelial cells using simple components with a limited budget, we have constructed an inexpensive (<$410) self-contained apparatus, consisting of a closed-loop, feedback-controlled system regulated by a PID (proportional-integrative-derivative) controller contained within a 0.077 m3 insulated acrylic box. Temperature, humidity, pH, and proliferative capacity of colon epithelial cells in this system mimic those in a standard tissue culture incubator for over four days. Our system offers significant advantages over existing cost-prohibitive commercially available and custom-made devices because of its very low cost, use of PID temperature control, lack of reliance on constant infusion of external humidified, heated air or carbon dioxide, ability to directly measure cell culture medium temperature, and combination of exquisite cellular detail with minimal focus drift under physiological conditions for extended periods of time. Using this apparatus, coupled with an inverted microscope equipped with phase contrast optics and a programmable digital camera, we have observed many events in colon epithelial cells not visible by static imaging, including kinetics of normal and abnormal mitoses, dynamic membrane structures, intracellular vesicle movements, and cell migration. © 2018 International Society for Advancement of Cytometry.

Phase II trial of bortezomib alone or in combination with irinotecan in patients with adenocarcinoma of the gastroesophageal junction or stomach.

PURPOSE: To determine the effectiveness of bortezomib plus irinotecan and bortezomib alone in patients with advanced gastroesophageal junction (GEJ) and gastric adenocarcinoma. We also sought to explore the effect of these therapeutics on tumor and normal gene expression in vivo. METHODS: Forty-one patients with advanced GEJ (89 %) or gastric (11 %) adenocarcinoma received bortezomib (1.3 mg/m(2) days 1, 4, 8, 11) plus irinotecan (125 mg/m(2) days 1, 8) every 21 days as first line therapy (N = 29), or bortezomib alone as second line therapy (N = 12). The trial was designed to detect a 40 % response rate for the combination, and 20 % response rate for bortezomib alone. Affymetrix HU133A gene chip arrays were used for gene expression studies. RESULTS: Objective response occurred in 3 of 29 patients (10 %, 95 % confidence intervals [CI] 2 %, 27 %) treated with bortezomib plus irinotecan, and in 1 of 12 patients (8 %, 95 % CI 0 %, 39 %) with bortezomib alone. Due to the limited number of responders, there were no significant correlations with response found in the gene expression profiles of 12 patients whose tumors were sampled before and 24 h after therapy with bortezomib alone (N = 2) or the combination (N = 10). CONCLUSIONS: We conclude that bortezomib is not effective for the treatment of advanced adenocarcinoma of the GEJ or stomach, whether used alone or in combination with irinotecan, in an unselected patient population.

Mybl2, downregulated during colon epithelial cell maturation, is suppressed by miR-365.

Altered profiles of gene expression reflect the reprogramming of intestinal epithelial cells during their maturation along the crypt-luminal axis. To focus on genes important in this process, and how they in turn are regulated, we identified 14 transcripts commonly downregulated in expression during lineage-specific maturation of the immortalized cell lines Caco-2 (absorptive), HT29Cl16E (goblet), and HT29Cl19A (secretory) induced by contact inhibition of growth or the short-chain fatty acid butyrate. One such gene, Mybl2 (Myb-related protein B), has been linked to the stem cell phenotype, and we report is also markedly suppressed in maturing cells along the crypt-luminal axis in vivo. Mybl2 is not significantly downregulated transcriptionally during colon cell maturation, but we identified a potential micro-RNA (miRNA)-binding sequence in the Mybl2 3'-untranslated region that mediates reporter gene suppression in differentiating colon cells. Accordingly, miRNAs predicted to bind this functional target are upregulated in differentiating colon epithelial cells in vitro and in vivo; expression of one of these, hsa-miR-365 (but not hsa-324-5p), suppresses Mybl2 protein expression in proliferating Caco-2 cells. These data demonstrate that miRNA silencing plays an important role in regulating gene expression in maturing colon epithelial cells, and that utilizing a target-centered approach, rather than profiling global miRNA expression, can identify physiologically relevant, functional miRNAs.

MYBL2, a link between proliferation and differentiation in maturing colon epithelial cells.

Multiple signals, controlling both proliferation and differentiation, must be integrated in the reprogramming of intestinal epithelial cells during maturation along the crypt-luminal axis. The v-myb family member Mybl2, a molecule implicated in the development and maintenance of the stem cell phenotype, has been suggested to play an important role in proliferation and differentiation of several cell types and is a gene we have found is commonly regulated in several systems of colon cell maturation both in vitro and in vivo. Here we show that siRNA silencing of Mybl2 in proliferating Caco-2 cells increases expression of the cell-cycle regulators cdk2, cyclin D2, and c-myc and decreases expression of cdc25B and cyclin B2 with a consequent 10% increase of cells in G2/M and a complementary 10% decrease in G1. Mybl2 occupies sequences upstream of transcriptional start sites of cyclin D2, c-myc, cyclin B2, and cdc25B and regulates reporter activity driven by upstream regions of cdk2, cyclin D2, and c-myc. These data suggest that Mybl2 plays a subtle but key role in linking specific aspects of cell-cycle progression with generation of signals for differentiation and may therefore be fundamental in commitment of intestinal epithelial cells to differentiation pathways during their maturation.

GATA-1 directly regulates p21 gene expression during erythroid differentiation.

Lineage-determination transcription factors coordinate cell differentiation and proliferation by controlling the synthesis of lineage-specific gene products as well as cell cycle regulators. GATA-1 is a master regulator of erythropoiesis. Its role in regulating erythroid-specific genes has been extensively studied, whereas its role in controlling genes that regulate cell proliferation is less understood. Ectopic expression of GATA-1 in erythroleukemia cells releases the block to their differentiation and leads to terminal cell division. An early event in reprogramming the erythroleukemia cells is induction of the cyclin-dependent kinase inhibitor p21. Remarkably, ectopic expression of p21 also induces the erythroleukemia cells to differentiate. We now report that GATA-1 directly regulates transcription of the p21 gene in both erythroleukemia cells and normal erythroid progenitors. Using reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we show that GATA-1 stimulates p21 gene transcription by binding to consensus binding sites in the upstream region of the p21 gene promoter. This activity is also dependent on a binding site for Sp1/KLF-like factors near the transcription start site. Our findings indicate that p21 is a crucial downstream gene target and effector of GATA-1 during red blood cell terminal differentiation.

Reprogramming leukemia cells to terminal differentiation and growth arrest by RNA interference of PU.1.

Malignant transformation often leads to both loss of normal proliferation control and inhibition of cell differentiation. Some tumor cells can be stimulated to reenter their differentiation program and to undergo terminal growth arrest. The in vitro differentiation of mouse erythroleukemia (MEL) cells is an important example of tumor cell reprogramming. MEL cells are malignant erythroblasts that are blocked from differentiating into mature RBC due to dysregulated expression of the transcription factor PU.1, which binds to and represses GATA-1, the major transcriptional regulator of erythropoiesis. We used RNA interference to ask whether inhibiting PU.1 synthesis was sufficient to cause MEL cells to lose their malignant properties. We report here that transfection of MEL cells with a PU.1-specific short interfering RNA oligonucleotide causes the cells to resume erythroid differentiation, accumulate hemoglobin, and undergo terminal growth arrest. RNA interference directed at specific, aberrantly expressed transcription factors may hold promise for the development of potent antitumor therapies in other hematologic malignancies.

PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure.

Transcriptional repression mechanisms are important during differentiation of multipotential hematopoietic progenitors, where they are thought to regulate lineage commitment and to extinguish alternative differentiation programs. PU.1 and GATA-1 are two critical hematopoietic transcription factors that physically interact and mutually antagonize each other's transcriptional activity and ability to promote myeloid and erythroid differentiation, respectively. We find that PU.1 inhibits the erythroid program by binding to GATA-1 on its target genes and organizing a complex of proteins that creates a repressive chromatin structure containing lysine-9 methylated H3 histones and heterochromatin protein 1. Although these features are thought to be stable aspects of repressed chromatin, we find that silencing of PU.1 expression leads to removal of the repression complex, loss of the repressive chromatin marks and reactivation of the erythroid program. This process involves incorporation of the replacement histone variant H3.3 into nucleosomes. Repression of one transcription factor bound to DNA by another transcription factor not on the DNA represents a new mechanism for downregulating an alternative gene expression program during lineage commitment of multipotential hematopoietic progenitors.

MAPK-mediated phosphorylation of GATA-1 promotes Bcl-XL expression and cell survival.

In the interleukin 3-dependent hematopoietic cell line Ba/F3, inhibition of mitogen-activated protein kinase, a member of the MAPK/c-Jun N-terminal kinase/stress-activated protein kinase kinase family that plays an important role in cell growth and death control, rapidly leads to severe apoptosis. However, most of the antiapoptotic substrates of MAPK remain to be identified. Here we report that, upon interleukin-3 stimulation of Ba/F3 cells, the transcription factor GATA-1 is strongly phosphorylated at residue serine 26 by a MAPK-dependent pathway. Phosphorylation of GATA-1 increases GATA-1-mediated transcription of the E4bp4 survival gene without significantly changing the DNA-binding affinity of GATA-1. Further characterization of GATA-1 phosphorylation site mutants revealed that the antiapoptotic function of GATA-1 is strongly dependent upon its phosphorylation at the Ser-26 position and is probably mediated through its up-regulation of Bcl-X(L) expression. Taken together, our data demonstrate that MAPK-dependent GATA-1 phosphorylation is important for its transactivation of the E4bp4 gene, Bcl-X(L) expression and cell survival. Therefore, GATA-1 may represent a novel MAPK substrate that plays an essential role in a cytokine-mediated antiapoptotic response.

FGF-2 antagonizes the TGF-beta1-mediated induction of pericyte alpha-smooth muscle actin expression: a role for myf-5 and Smad-mediated signaling pathways.

PURPOSE: Although the FGF and TGF-beta families are known to play an important role in regulating vascular endothelial and smooth muscle cell behavior, the influence of these matrix-binding growth factors on microvascular pericyte morphogenesis is not well understood. The current study was undertaken to examine the molecular mechanisms that mediate the effects of the endothelium-produced growth regulators FGF-2 and TGF-beta1 on retinal pericyte proliferation and contractile phenotype. METHODS: Using purified retinal pericytes, a series of assays were implemented, including RT-PCR, DNA binding, immunoprecipitation, electrophoretic mobility shift, and indirect immunofluorescence, in an attempt to elucidate the FGF/TGF-beta1 signaling cascades that mediate retinal microvascular cell growth and contractile phenotype. RESULTS: Treatment of retinal pericytes with FGF-2 and heparin stimulated nearly a log order increase in proliferation, whereas removal of FGF-2 or addition of TGF-beta1 caused withdrawal from the growth cycle, inducing a smooth-muscle-like contractile phenotype, as indicated by upregulation of alpha-smooth muscle actin (alpha-SMA). This switch from a growth-potentiated to a growth-arrested state followed induction of the transcriptional regulator myf-5, as well as the nuclear translocation of myf-5 and Smad2. CONCLUSIONS: Several critical features of the endothelial cell-extracellular matrix-pericyte molecular signaling axis were elucidated in the study that are likely to be responsible for regulating retinal microvascular morphogenesis during normal development, as well as the pathologic angiogenesis accompanying several ocular disorders, including diabetic retinopathy and age-related macular degeneration.

Mechanisms of normal and tumor-derived angiogenesis.

Often those diseases most evasive to therapeutic intervention usurp the human body's own cellular machinery or deregulate normal physiological processes for propagation. Tumor-induced angiogenesis is a pathological condition that results from aberrant deployment of normal angiogenesis, an essential process in which the vascular tree is remodeled by the growth of new capillaries from preexisting vessels. Normal angiogenesis ensures that developing or healing tissues receive an adequate supply of nutrients. Within the confines of a tumor, the availability of nutrients is limited by competition among actively proliferating cells, and diffusion of metabolites is impeded by high interstitial pressure (Jain RK. Cancer Res 47: 3039-3051, 1987). As a result, tumor cells induce the formation of a new blood supply from the preexisting vasculature, and this affords tumor cells the ability to survive and propagate in a hostile environment. Because both normal and tumor-induced neovascularization fulfill the essential role of satisfying the metabolic demands of a tissue, the mechanisms by which cancer cells stimulate pathological neovascularization mimic those utilized by normal cells to foster physiological angiogenesis. This review investigates mechanisms of tumor-induced angiogenesis. The strategies used by cancer cells to develop their own blood supply are discussed in relation to those employed by normal cells during physiological angiogenesis. With an understanding of blood vessel growth in both normal and abnormal settings, we are better suited to design effective therapeutics for cancer.