Illuminating the dark depths inside coral.

The ability to observe in situ 3D distribution and dynamics of endosymbionts in corals is crucial for gaining a mechanistic understanding of coral bleaching and reef degradation. Here, we report the development of a tissue clearing (TC) coupled with light sheet fluorescence microscopy (LSFM) method for 3D imaging of the coral holobiont at single-cell resolution. The initial applications have demonstrated the ability of this technique to provide high spatial resolution quantitative information of endosymbiont abundance and distribution within corals. With specific fluorescent probes or assays, TC-LSFM also revealed spatial distribution and dynamics of physiological conditions (such as cell proliferation, apoptosis, and hypoxia response) in both corals and their endosymbionts. This tool is highly promising for in situ and in-depth data acquisition to illuminate coral symbiosis and health conditions in the changing marine environment, providing fundamental information for coral reef conservation and restoration

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage

Nuclear envelope structural defects cause chromosomal numerical instability and aneuploidy in ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Despite our substantial understanding of molecular mechanisms and gene mutations involved in cancer, the technical approaches for diagnosis and prognosis of cancer are limited. In routine clinical diagnosis of cancer, the procedure is very basic: nuclear morphology is used as a common assessment of the degree of malignancy, and hence acts as a prognostic and predictive indicator of the disease. Furthermore, though the atypical nuclear morphology of cancer cells is believed to be a consequence of oncogenic signaling, the molecular basis remains unclear. Another common characteristic of human cancer is aneuploidy, but the causes and its role in carcinogenesis are not well established.</p> <p>Methods</p> <p>We investigated the expression of the nuclear envelope proteins lamin A/C in ovarian cancer by immunohistochemistry and studied the consequence of lamin A/C suppression using siRNA in primary human ovarian surface epithelial cells in culture. We used immunofluorescence microscopy to analyze nuclear morphology, flow cytometry to analyze cellular DNA content, and fluorescence <it>in situ </it>hybridization to examine cell ploidy of the lamin A/C-suppressed cells.</p> <p>Results</p> <p>We found that nuclear lamina proteins lamin A/C are often absent (47%) in ovarian cancer cells and tissues. Even in lamin A/C-positive ovarian cancer, the expression is heterogeneous within the population of tumor cells. In most cancer cell lines, a significant fraction of the lamin A/C-negative population was observed to intermix with the lamin A/C-positive cells. Down regulation of lamin A/C in non-cancerous primary ovarian surface epithelial cells led to morphological deformation and development of aneuploidy. The aneuploid cells became growth retarded due to a p53-dependent induction of the cell cycle inhibitor p21.</p> <p>Conclusions</p> <p>We conclude that the loss of nuclear envelope structural proteins, such as lamin A/C, may underlie two of the hallmarks of cancer - aberrations in nuclear morphology and aneuploidy.</p

Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies

From proteomic analysis to potential therapeutic targets: functional profile of two lung cancer cell lines, A549 and SW900, widely studied in pre-clinical research

Lung cancer is a serious health problem and the leading cause of cancer death worldwide. The standard use of cell lines as in vitro pre-clinical models to study the molecular mechanisms that drive tumorigenesis and access drug sensitivity/effectiveness is of undisputable importance. Label-free mass spectrometry and bioinformatics were employed to study the proteomic profiles of two representative lung cancer cell lines and to unravel the specific biological processes. Adenocarcinoma A549 cells were enriched in proteins related to cellular respiration, ubiquitination, apoptosis and response to drug/hypoxia/oxidative stress. In turn, squamous carcinoma SW900 cells were enriched in proteins related to translation, apoptosis, response to inorganic/organic substances and cytoskeleton organization. Several proteins with differential expression were related to cancer transformation, tumor resistance, proliferation, migration, invasion and metastasis. Combined analysis of proteome and interactome data highlighted key proteins and suggested that adenocarcinoma might be more prone to PI3K/Akt/mTOR and topoisomerase IIα inhibitors, and squamous carcinoma to Ck2 inhibitors. Moreover, ILF3 overexpression in adenocarcinoma, and PCNA and NEDD8 in squamous carcinoma shows them as promising candidates for therapeutic purposes. This study highlights the functional proteomic differences of two main subtypes of lung cancer models and hints several targeted therapies that might assist in this type of cancer.publishe

Lamin A/C deficiency is an independent risk factor for cervical cancer

In the past, cervical cancer has been linked to Human Papilloma Virus (HPV) infection. Previously, we found that pre-neoplastic breast and ovarian lesions may be associated with lamin A/C deficiency, resulting in abnormal nuclear morphologies and chromosomal instability. Ultimately, these phenomena are thought to lead to cancer. Here, we assessed lamin A/C deficiency as an indicator for the risk to develop cervical cancer. The expression of lamin A/C was assessed by Western blotting in cervical uterine smears (CUS) of 76 adult women from Benin concomitant with nuclear morphology assessment and HPV genotyping using microscopy and PCR-based assays, respectively. In vitro analyses were performed to uncover the mechanism underlying lamin A/C expression alterations observed in vivo. The presence of cervical intra-epithelial neoplasia (CIN) was assessed by colposcopy. Normal lamin A/C expression (group A) was observed in 39% of the CUS, weak lamin A/C expression (group B) was observed in 28% of the CUS and no lamin A/C expression (group C) was observed in 33% of the CUS tested. Infection with oncogenic HPV was found to be significantly higher in group C (36%) than in groups A (17%) and B (14%). Two years after our first assessment, CIN was observed in 20% of the women in group C. The in vitro application of either a histone deacetylase inhibitor (trichostatin) or a protein kinase inhibitor (staurosporine) was found to restore lamin A/C expression in cervical cancer-derived cells. Lamin A/C deficiency may serve as an independent risk factor for CIN development and as an indicator for preventive therapy in cervical cancer