Requirement of Pax6 for the integration of guidance cues in cell migration

The intricate patterns of cell migration that are found throughout development are generated through a vast array of guidance cues. Responding integratively to distinct, often conflicting, migratory signals is probably crucial for cells to reach their correct destination. Pax6 is a master transcription factor with key roles in neural development that include the control of cell migration. In this study, we have investigated the ability of cells derived from cortical neurospheres from wildtype (WT) and Pax6-/- mouse embryos to integrate diverging guidance cues. We used two different cues, either separately or in combination: substratum nanogrooves to induce contact guidance, and electric fields (EFs) to induce electrotaxis. In the absence of an EF, both WT and Pax6-/- cells aligned and migrated parallel to grooves, and on a flat substrate both showed marked electrotaxis towards the cathode. When an EF was applied in a perpendicular orientation to grooves, WT cells responded significantly to both cues, migrating in highly oblique trajectories in the general direction of the cathode. However, Pax6-/- cells had an impaired response to both cues simultaneously. Our results demonstrate that these neurosphere derived cells have the capacity to integrate diverging guidance cues, which requires Pax6 function

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid‐containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development

Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the evelopment of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.PEDECIBA. ANII

Estudio de estrés celular y metabolismo en un modelo de microambiente tumoral in vitro en cáncer oral

Objetivos. El microambiente tumoral temprano es determinante para la evolución tumoral. Para estudiar in vitro la progresión tumoral, necesitamos desarrollar modelos que recapitulen las propiedades del microambiente tumoral. Una de las principales características de dicho microambiente es la falta de oxígeno. En este estudio, hemos usado un modelo de microambiente hipóxico que hemos desarrollado previamente, con el objetivo de estudiar los efectos de este microambiente en células HaCaT que expresan los oncogenes del HPV-18, y que pueden usarse como modelo de una etapa intermedia de la carcinogénesis oral. Métodos. Utilizamos la variante que desarrollamos del método de hipoxia inducida por cubreobjetos para generar un microambiente tumoral temprano in vitro. Visualizamos hipoxia y estrés oxidativo con sondas fluorescentes específicas. Por otra parte realizamos medidas de parámetros bioquímicos del microambiente tumoral mediante un radiómetro ABL800 Flex, y también medimos parámetros metabólicos de las células HaCaT control y con oncogenes virales mediante el analizador de flujo metabólico Seahorse XF24. Resultados. Observamos niveles de estrés oxidativo cambiantes de acuerdo al nivel de hipoxia registrado. También observamos que en condiciones de hipoxia ocurre una dismin ución de pH y un aumento en la presión de CO2 en relación a la normoxia. Asimismo, observamos diferencias en parámetros metabólicos entre HaCaT con oncogenes y HaCaT control. Conclusiones. Nuestros resultados indican que un modelo de microambiente tumoral in vitro produce estrés hipóxico y oxidativo,y que está acompañado de cambios en varios parámetros ambientales, que en conjunto pueden tener una profunda influencia en el proceso de carcinogénesis oral

Expresión de EP-CAM (MOC-31) en germen dental y Ameloblastoma

Objetivos: Ep-CAM, una glicoproteína transmembrana expresada en la mayoría de los epitelios en condiciones normales, tiene diversas funciones en estos tejidos, incluida la adhesión celular, proliferación, diferenciación y regulación del ciclo celular, migración y señalización intracelular. También se encuentra sobreexpresado en la mayoría de las neoplasias malignas, participando en la iniciación, progresión y diseminación metastásica del tumor. La expresión y los roles de esta proteína en las neoplasias orales, particularmente en los tumores odontogénicos, aún no se han establecido. El objetivo de este estudio consistió en analizar la expresión de esta proteína en ameloblastoma y germen dental. Métodos. La expresión de Ep-CAM (MOC-31) se evaluó mediante inmunohistoquímica en gérmenes dentales (TG) (n = 16) y ameloblastomas (AM) (n = 60). Resultado: La mayoría de los gérmenes dentales expresaron MOC-31 (81,3%). El 16,7% de los casos de AM mostraron expresión de MOC-31. No se observó correlación entre la expresión de proteínas y el sexo, la edad, las variantes clínicas o los subtipos histológicos. Conclusiones. Nuestros resultados sugieren que la sobreexpresión de Ep-CAM en TG en comparación con AM podría estar relacionada con procesos de odontogénesis. La ausencia de expresión en AM podría estar relacionada con el comportamiento biológico de esta neoplasia. Se necesitan ulteriores enfoques experimentales que refuercen estos hallazgos iniciales

Lamin A/C expression silencing leads to transformation.

<p><b>(A)</b> Lamin A/C was monitored by WB after shRNA depletion by SK-N-SH-lamin-A/C-shRNA or SK-N-SH-scramble-shRNA. Actin was used as a loading control. <b>(B)</b> Cumulative cell numbers of SK-N-SH-lamin-A/C-shRNA and SK-N-SH-scramble-shRNA, respectively. Three independent experiments were performed in triplicate (n = 9) using cells at less than eight passages, and error bars represent the s.d. Statistical significance was assessed using Student’s t-test;(p<0.01). Representative images showing differential cell growth on day 8 of the experiment. Scale bar, 100μm. <b>(C)</b> Wounding assay of confluent cell layers of SK-N-SH-lamin-A/C-shRNA or SK-N-SH-scramble-shRNA.Number of cells that migrated into a delimited wound area after 12h is plotted. Cells in three defined area per group per experiment were quantified in three independent experiments with three technical replicates. Error bars represent the s.d. Statistical significance was assessed using Student’s t-test; (*p<0.01). Representative images; scale bar, 100μM. <b>(D)</b> Quantification of the Matrigel chamber migration assay for SK-N-SH-lamin-A/C-shRNA and SK-N-SH-scramble-shRNA. Error bars represent the s.d. (n = 9). Statistical significance was assessed by Student’s t-test; (**p<0.01). Scale bar, 10μM. <b>(E)</b> SK-N-SH-lamin-A/C increased the number of colonies in soft agar compared with the SK-N-SH-scramble-shRNA control. The number of colonies per well was counted and plotted. Three independent experiments (n1 = 3) with 3 replicates per experiment (n2 = 9) were performed. Error bars represent the s.d. (n = 9). Statistical significance was assessed using Student’s t-test (**p<0.01). Scale bar, 100μM.</p

Methylation analysis of the Lamin A/C promoter.

<p><b>RNA and protein analysis (A)</b> Schematic of the Lamin A/C CpG Island around the transcription start site (TSS) (long black arrow). CpG dinucleotides are represented as black bars.The locations of bisulphite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as white and black arrows, respectively. The bisulphite sequencing PCR (BSP)results are shown for 10 individual clones for each cancer cell line studied and primary normal human fetal brain neuronal progenitor cells(PNP) cells and IVD αs negative and positive controls, respectively.Two chromatograms are shown to compare one methylated and one unmethylated cell line. <b>(B)</b> Methylation-specific PCR (MSP) of the Lamin A/C gene in neuroblastoma cell lines. The presence of a PCR band under lane M indicates methylated genes, while the presence of a PCR band under lane U indicates unmethylated genes. <b>(C)</b> RT-PCR analysis of Lamin A/C expression. <b>(D)</b> Treatment with the demethylating agent 5-aza reactivated Lamin A/C gene expression in the methylated neuroblastoma SK-N-DZ cell line at the RNA level. <b>(E-F)</b> Treatment with 5-aza-2’ deoxycytidine (5-aza) reactivated Lamin A/C gene expression in the methylated neuroblastoma SK-N-DZ cell line at the protein level evaluated by WB and IF, respectively. GAPDH and actin were used as loading controls for RNA and protein expression, respectively. Lamin A/C is observed in green and DNA in blue (DAPI). Scale bar, 10μM.</p

Silencing of Lamin A/C in unmethylated neuroblastoma cells induces changes in different cytoskeletal components.

<p>Immunofluorescence staining showing changes in SK-N-SH-lamin-A/C-shRNA compared with SK-N-SH-scramble-shRNA in <b>(A)</b> β-actin filaments, <b>(B)</b> F-actin filaments. <b>(C)</b> Vimentin filaments, and <b>(D)</b> α-tubulin. Lamin A/C is shown in green; β-actin, F-actin, vimentin, and α-tubulin are shown in red.DNA is stained in blue (DAPI). Scale bar, 10μM.</p

Progerin introduction in SK-N-DZ cells induces changes in cytoskeletal components and mechanical properties.

<p><b>(A)</b> Immunofluorescence staining showing Progerin. <b>(B)</b> Changes in β-actin, F-actin, vimentin filaments, and α-tubulin after Progerin introduction. Progerin is shown in green; β-actin, F-actin, vimentin, and α-tubulin are shown in red; and DNA is stained in blue (DAPI). Scale bar, 10μm. <b>(C)</b> Representative AFM images of height measurement in SK-N-DZ-ev cells. In red and blue height profile across lines in different cells from height AFM image are shown Scale bar, 10μm <b>(D)</b>The AFM tip is positioned directly above the lamellar region. Scale bar 10μm <b>(E)</b> Normalized histogram of the data obtained from both groups for the lamellar region. Each histogram was fitted to a Gaussian curve to obtain the mean value and the standard deviation of Young’s Modulus. Three independent experiments (n1 = 3) with 3 replicates per experiment (n2 = 9) were performed. A Student’s t-test confirmed that the difference was significant (**p<0.01).</p