Cell Biological Changes Research Articles

A challenging epigenetic message: telomerase activity is associated with complex changes in lifestyle

As an outcome of The 2009 Nobel Prize in Physiology or Medicine, a connection has been highlighted between the length of telomeres and epigenetic effects, such as intensive changes in lifestyle and nutrition as well as behavioural and psychological factors. In this review, the various elements of molecular, cell biological, nutritional and lifestyle changes are introduced and discussed.

Kinetics of the cell biological changes occurring in the progression of DNA damage-induced senescence.

Cellular senescence is characterized by cell-cycle arrest accompanied by various cell biological changes. Although these changes have been heavily relied on as senescence markers in numerous studies on senescence and its intervention, their underlying mechanisms and relationship to each other are poorly understood. Furthermore, the depth and the reversibility of those changes have not been addressed previously. Using flow cytometry coupled with confocal microscopy and Western blotting, we quantified various senescence-associated cellular changes and determined their time course profiles in MCF-7 cells undergoing DNA damage-induced senescence. The examined properties changed with several different kinetics patterns. Autofluorescence, side scattering, and the mitochondria content increased progressively and linearly. Cell volume, lysosome content, and reactive oxygen species (ROS) level increased abruptly at an early stage. Meanwhile, senescence associated β-galactosidase activity increased after a lag of a few days. In addition, during the senescence progression, lysosomes exhibited a loss of integrity, which may have been associated with the accumulation of ROS. The finding that various senescence phenotypes matured at different rates with different lag times suggests multiple independent mechanisms controlling the expression of senescence phenotypes. This type of kinetics study would promote the understanding of how cells become fully senescent and facilitate the screening of methods that intervene in cellular senescence.

Lunar phase cycle and psychiatric hospital emergency visits, inpatient admissions and aggressive behavior

Introduction We examined the correlation between lunar cycles and the number of psychiatric emergency visits, patients’ gender, aggressive behavior, need for inpatient admission, legal referral and need for involuntary chemical restraint in a period of one calendar year. Method Charts of all psychiatric emergency room patients were reviewed retrospectively. Arabic calendar lunar months were divided based on three different models: Two 15 days sections, three 10 days, and six 5 days parts in the third model. Results 5431 patients were seen in the psychiatric emergency service (PES) during a one year period. We did not find a relationship between the visit time (day or night), gender, and legal referral in relation to moon cycles. A higher number of visits during full moon period which was not statistically significant ( P > 0.05) was found. Highest frequency of inpatient admissions was discovered in the first and last parts of the three part model and in the first and last segments of the six part model followed by second and fifth parts ( P-values: 0.000). Conclusion This study revealed a slight increase in number of visits to psychiatric emergency room during full moon periods. In addition a highly significant increase in the severity of illness and aggressive behaviors and agitation in the beginning and end of the moon cycles were noticed. Some of the underlying possible causes such as changes in intra/extracellular fluids, neural and endocrine cell functions, hemodynamic and biological responses and changes in social dynamics and support are discussed.

Workflow-Based Software Environment for Large-Scale Biological Experiments

High-content screening (HCS) technologies are becoming increasingly used in both large-scale drug discovery and basic research programs. These automated imaging and analysis technologies enable the researcher to elucidate the complex biology that underlies the functions of genes, proteins, and other biomolecules at the cellular level. HCS combines the power of automated digital microscopy and advanced software-based image analysis algorithms to detect and quantify biological changes in cells and tissues. This technology is a particularly powerful tool when used to interrogate the cellular effects of exogenously applied agents such as RNAi and/or small molecules. HCS allows for the evaluation of cellular perturbations that occur both at the level of the single cell and within cellular populations. In a multivariate approach, multiple cellular parameters are collected, allowing for more complex analysis. However, in these scenarios, data flow and management still represent substantial bottlenecks in HCS projects. HCS data include a diversity of information from multiple sources such as details pertaining to screening libraries (e.g., siRNA and small molecules), image stacks acquired from automated microscopes (of which there may be up to several million), and the image analysis data. From this, postprocessing algorithms are required to generate statistical, quality control bioinformatic information and ultimately a final hit list. To accomplish these individual tasks, numerous tools can be used to perform each analytical step; however, management of the entire information flow currently requires the use of commercially available proprietary software, the scope of which is often limited, or bespoke customized scripts. In this article, the authors introduce an open-source research tool that allows for the management of the entire data flow of the HCS data chain, by handling and linking information and providing many powerful postprocessing and visualization tools.

Effect of treadmill exercise on Purkinje cell loss and astrocytic reaction in the cerebellum after traumatic brain injury

The cerebellum is one of the brain areas, which is selectively vulnerable to forebrain traumatic brain injuries (TBI). Physical exercise in animals is known to promote cell survival and functional recovery after brain injuries. However, the detailed pathologic and functional alterations by exercise following an indirect cerebellar injury induced by a TBI are largely unknown. We determined the effects of treadmill exercise on survival of Purkinje neurons and on a population of reactive astrocytes in the gyrus of lobules VIII and IX of the cerebellum after TBI. The rats were divided into four groups: the sham-operation group, the sham-operation with exercise group, the TBI-induction group, and the TBI-induction with exercise group. Cell biological changes of Purkinje neurons following indirect cerebellar injury were analyzed by immunohistochemistry. TBI-induced loss of calbindin-stained Purkinje neurons in the posterior region of the cerebellum and TBI also increased formation of reactive astroyctes in both the granular and molecular layers of the cerebellar posterior region. Treadmill exercise for 10 days after TBI increased the number of calbindin-stained Purkinje neurons and suppressed formation of reactive astroyctes. The present study provides the possibility that treadmill exercise may be an important mediator to enhance survival of Purkinje neurons in TBI-induced indirect cerebellar injury.

Chondrocyte Rather Than Osteoblast Conversion of Vascular Cells Underlies Medial Calcification in Uremic Rats

To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure. Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for 4 weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro-computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After 4 weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor-related protein 6 and beta-catenin, molecules of the wingless-type MMTV integration site family member (Wnt)/beta-catenin pathway induced during osteoblast differentiation, was suppressed. In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.

2009 Nobel Prize in Medicine and an interesting message: telomerase activity is associated with lifestyle

A 2009. évi orvosi Nobel-díjat a telomerák és a telomeráz enzim felfedezéséért és az ezekkel kapcsolatos úttörő kutatásokért három, Amerikában dolgozó tudós kapta megosztva. Nem régóta az is ismert, hogy bizonyos életmód- és táplálkozásbeli változások (Ornish-féle életmódváltozás), magatartásbeli, pszichológiai tényezők, valamint a telomerarövidülés között bizonyított kapcsolat létezik. Az összefoglalóban ezen összefüggések molekuláris, sejtbiológiai, életmódbeli és magatartási vonatkozásait, valamint ezek lehetséges összefüggéseit mutatják be.

S1634 Bone Morphogenetic Protein-4 Has an Important Role in the Transformation of Normal Esophageal Cells to an Intestinal Phenotype in Barrett's Esophagus via CDx2 Pathway

Background & Aims: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. A recent study has shown that bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor-β superfamily, is uniquely overexpressed in BE. The mechanisms of the BMP signaling pathway in the metaplastic transformation in BE, however, remain unclear. The aim of this study was to examine the role of the BMP signaling pathway in the metaplastic transformation of normal squamous cells into columnar cells. Methods: The expression of BMP signaling related molecules, including BMP-4, ID2, SMAD4 in biopsy samples was assessed. Samples were taken fromBE (16 cases), with or without reflux esophagitis, and fromnormal esophagus (12 cases) and assayed by immunohistochemistry and real-time PCR. In addition, the In Vitro effects of acid and/or bile acid on BMP-4 expression were analyzed in HET1A, an immortalized human esophageal squamous cell line, and OE33, a human esophageal adenocarcinoma cell line. These cells were treated with recombinant human BMP-4 (10-100 ng/ mL), and cell biological changes were examined by immunoblot analysis and real-time PCR. Cell proliferation was assessed by MTS assay. Results: Immunohistochemistry of biopsy specimens demonstrated that the expression of BMP-4 protein was observed in all the cases of BE, but not in normal squamous epithelium. Consistent with the immunohistochemistry results, BMP-4 mRNA expression was 13 fold greater in BE as compared with normal squamous epithelium. Up-regulation of BMP-4 expression was more evident in BE with reflux esophagitis. In Vitro, acid (pH4) and bile acid exposure synergistically induced BMP4 expression in both HET1A and OE33 cells. Treatment of these cells with recombinant BMP-4 resulted in increased expression of the intestine-specific transcription factor, CDX2. Blocking BMP signalling by preincubating cells with the BMP antagonist Noggin before treatment with BMP-4, resulted in decreased expression of CDX2. In addition, Noggin also blocked the proliferation of OE33 cells. Conclusion: These results suggest that the BMP signaling pathway is activated in BE, and that BMP-4 has an important role in the process of transformation of squamous epithelium toward a mucosa that resembles columnar metaplasia. The results suggest that transformation occurs via a CDX2-dependent pathway, and that this process might be promoted in the inflamed condition. We propose that the premalignant metaplastic development of BE is mediated by BMP-4, and that inhibition of BMP-4 signaling offers a novel therapeutic approach for this disease.

Physiological responses of polar benthic algae to ultraviolet radiation

Abstract Stratospheric ozone depletion and the concomitant increase in ultraviolet (UV) B radiation at the earth's surface represent major threats to polar marine ecosystems. Whereas in coastal rocky shore environments macroalgae constitute an assemblage of particular significance to ecosystem function, benthic diatoms dominate microphytobenthic assemblages, which typically grow on shallow-water sediments as highly productive and stabilising phototrophic biofilms. This review summarises present knowledge on how UV radiation affects the physiology of polar benthic algae with an emphasis on cell biological and structural changes, molecular targets and repair mechanisms, induction of reactive oxygen species and antioxidative strategies, photosynthesis and growth, photoprotective mechanisms, interactive effects between UV radiation and other abiotic factors, and finally ecological consequences. Although available data indicate that there are specific characteristics and adaptations in polar benthic micro- and macroalgae that explain their ecological success and limits under environmentally extreme conditions, much more research is needed to understand the underlying mechanisms. In particular, more ecosystem approaches and studies on interactive effects, as well as modern genomic, proteomic and metabolomic approaches could help address all open questions and depict a more holistic picture.

Clinical stem cell therapies for severe autoimmune diseases

Severe autoimmune diseases (ADs) are a major source of disability and reduced quality of life and may result in shortened life expectancy, particularly in treatment-resistant patients. For several decades, allogeneic and autologous haematopoietic stem cell transplantation (HSCT) has been the focus of scientific investigation as a potential means of delivering 'one-off' intensive treatment in severe ADs. Improvements in the clinical safety of HCST were followed by its increasing use in recent years as an experimental treatment for severe and resistant ADs. European and North American registries have accumulated between one and two thousand procedures. Retrospective analyses and prospective studies have demonstrated the feasibility, safety and initial efficacy data in various ADs. Profound cell biological changes induced by HSCT leading to stabilization or reversal of organ damage have been characterized. These have also shed light on basic disease mechanisms and support investigation of more specific cellular therapy in ADs. There is clear potential for harnessing a profound immunological effect through HSCT. However, there is a need for an ongoing balance against evolving non-transplant treatments for ADs. Ideally, these issues should be resolved in phase III studies, in which HSCT approaches are compared with the best comparator.

The multiple mechanisms of multidrug resistance and cellular pH.

Multidrug resistance (MDR) describes a variety of strategies that tumour cells develop to evade the cytotoxic effects of anti-cancer drugs. Tumour cells that have become MDR show decreased cellular sensitivity to the drug employed in chemotherapy as well as a broad spectrum of drugs without obvious common targets or structural homology. MDR is a major obstacle to the successful treatment of tumours. Our understanding of multidrug resistance is limited, in part, by our limited knowledge of what makes tumour cells more sensitive to chemotherapeutics. Drug resistance may subvert the same mechanisms that make tumour cells hypersensitive or use molecular modifications unrelated to the mechanisms that lead to hypersensitivity in tumour cells. Indeed, multidrug resistance is likely to be the consequence of a multitude of mechanisms. Some drug-resistant and drug-sensitive mammalian cell lines have shown aberrations in regulation of cellular pH. These changes can contribute to some of the physiological and cell biological changes that are observed both in drug-sensitive and MDR tumour cell lines. As such, at least in some tumour cell lines, the changes of pH contribute both to the physiology of transformation and the physiology of drug-resistance. The observation that these variations from normal pH are not universally observed in either tumour or MDR cells suggests that these mechanisms cannot be generalized to all transformed or MDR cells. It remains to be resolved to what extent these changes are a consequence of studying cell lines grown in vitro or if they reflect the in situ physiology of human tumours. There are no detectable changes in cytosolic or organellar pH in cells transfected with P-glycoprotein, a plasma membrane-based protein implicated in drug resistance. Thus, there are likely to be multiple mechanisms contributing to multiple drug-resistance.

Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the “Shen Zhou IV” spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

신생아와 노인 유래 섬유아세포의 노화과정에서의 세포학적 성질의 비교

이 논문에서는 신생아와 노인 유래의 섬유아세포들의 노화의 특징들을 비교하여 사람의 나이와 세포의 수명 및 세포 형질의 관계에 대해 연구하였다. 본 연구의 결과는 비록 한가지의 노인세포에 대해 얻어진 것이기는 하지만 다음과 같은 세 가지 중요한 가능성을 제시한다. 첫째로, 노인에서 유래한 섬유아세포의 증식속도가 신생아 유래의 세포에 비해서 느릴 가능성이 있다. 이러한 결과는 실제로 노인 신체에 존재하는 세포가 신생아에 존재하는 세포에 비해 낮은 속도로 증식할 가능성을 시사하는 것으로서, 노인에서 관찰되는 조직실질의 감소 원인을 설명하는 자료가 될 수 있겠다. 둘째로, 노인 유래 섬유아세포의 early passage 세포가 신생아 유래의 세포의 early passage 세포와 동일하게 낮은 수준의 SA <TEX>${\beta}-Gal$</TEX> 활성, autofluorescence, lysosome 함량, 그리고 활성산소 수준을 갖고 있었다. 이 점은, early passage 때의 세포가 보이는 형질이 신체에 존재하는 세포의 상황과 크게 다르지 않다고 가정할 때, 노인 신체의 조직에 존재하는 세포들이 신생아의 세포와 유사한 상태로 존재할 가능성을 시사하는 것이다. 즉, 노인 신체에서는 in vitro 노화세포에서 나타나는 수준의 세포노화가 일어나 있지 않다는 것이다. 셋째, 노인세포가 노화했을 때는 신생아세포의 경우와 거의 동일한 수준의 활성산소, lysosome, SA <TEX>${\beta}-Gal$</TEX> activity 증가를 보이고 있었는데, 이는 노인 유래의 세포가 in vitro 배양 시 신생아 유래의 세포보다 더 심하거나 또는 빠른 산화적 손상이나 세포학적 변화를 겪지는 않는다는 것을 보여주는 것으로서, 세포가 보유한 항산화적 기능이 노인이 되면서 크게 약화되지는 않음을 시사하고 있다. 결론적으로 노인 유래의 세포는 세포증식 속도를 제외하면 대체로 신생아 때의 상태와 동일한 세포 내 상태를 갖고 있다고 결론 내릴 수 있겠다. Normal somatic cells proliferate for a limited number of doublings in culture and then enter an irreversible growth-arrest state called replicative senescence. Replicative senescence has been believed a reason for the limited cellular turnover and deterioration of tissue function in aged animals. However, there is no experimental evidence supporting this assumption. Furthermore, cells from aged person have been poorly characterized with an exception of the cases of T cells. In this study, we examined cell biological changes occurring in replicative senescence of fibroblast strains originated from a new-born (NHF-NB) and a 87 year old man (NHF-87). NHF-87 (and the cells from a 75-year old) proliferated to smaller population doublings and with longer doubling times than NHF-NB did. At early passages, NHF-87 exhibited a low senescence-associated <TEX>${\beta}-Gal$</TEX> (SA <TEX>${\beta}-Gal$</TEX>) activity and lipofuscin level, typical markers for cellular senescence. Furthermore, they maintained low levels of lysosome and reactive oxygen species (ROS). All of these levels increased dramatically in the late passage NHF-87 quite similarly as those in the late passaged NHF-NB did. These results indicate that most cells originated from the aged maintain a phenotype of the cells originated from new-born donors and undergo replicative senescence with the same kinetics as that of the cells from new-born. It is also indicated that not SA <TEX>${\beta}-gal$</TEX> activity but cell proliferation rate may be qualified as a biomarker for cells aged in vivo.

Filament formation in Saccharomyces cerevisiae — a review

Many yeasts can produce filamentous elongated cells identifiable as hyphae, pseudohyphae or invasive filaments. Filament formation has been understood as a foraging response that occurs in nutrient-poor conditions. However, fusel alcohols were observed to induce filament formation in rich nutrient conditions in every yeast species examined. Fusel alcohols, e.g., 3-methyl-1-butanol (3Me-BuOH; 'isoamyl alcohol'), 2-methyl-1-propanol (isobutyl alcohol), (-)-2-methyl-1-butanol ('active amyl alcohol'), 2-phenylethanol and 3-(2-hydroxyethyl)indole (tryptophol) (the end products of leucine, valine, isoleucine, phenylalanine and tryptophan catabolism, respectively) are the end products of amino acid catabolism that accumulate when nutrients become limiting. Thus, yeast responds to its own metabolic by-products. Considerable effort was made to define the cell biological and biochemical changes that take place during 3Me-BuOH-induced filamentation. In Saccharomyces cerevisiae filaments contain significantly greater mitochondrial mass and increased chitin content in comparison with yeast-form cells. The global transcriptional response of S. cerevisiae during the early stages of 3Me-BuOH-induced filament formation has been described. Four ORFs displayed very significant (more than 10-fold) increases in their RNA species, and 12 ORFs displayed increases in transcription of more than 5-fold. The transcription of five genes (all of which encode transporters) decreased by similar amounts. Where examined, the activity of the proteins encoded reflected the transcriptional pattern of their respective mRNAs. To understand this regulation, studies were performed to see whether deletion or overexpression of key genes affects the ability to filament and invade solid YEPD medium. This has led to identification of those proteins that are essential for filament formation, repressors and those which are simply not required. It also leads to the conclusion that 3Me-BuOH-induced filament formation is not a foraging response but a response to reduced growth rate.

Bone Morphogenetic Protein 4 Expressed in Esophagitis Induces a Columnar Phenotype in Esophageal Squamous Cells

Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. It is proposed that one of the possible mechanisms is dedifferentiation of squamous epithelium into columnar epithelium. The pathophysiology through which this metaplasia occurs is unknown. A recent study by serial analysis of gene expression showed that bone morphogenetic protein 4 (BMP-4) is uniquely expressed in BE. In this study, the role of the BMP pathway in the metaplastic transformation of normal squamous cells into columnar cells was examined. Tissues from patients with esophagitis and BE and in an esophagitis-BE rat model were examined for the activation of the BMP pathway. Short-term cultures of primary normal squamous esophageal cells were treated with BMP-4, and cell biological changes were examined by Western blot analysis, immunohistochemistry, and microarrays. In both human and rat tissues, the BMP pathway proved to be activated in esophagitis and BE. Upon incubation of squamous cell cultures with BMP-4, the cytokeratin expression pattern showed a shift that was consistent with columnar epithelium. Involvement of the BMP pathway was suggested by up-regulation of Phosphorylated-Smad 1/5/8 (P-Smad 1/5/8) that was effectively blocked by Noggin, a BMP antagonist. Comparison of the gene expression profiles of squamous cells, BMP-4-treated squamous cells, and BE cells showed a significant shift in the profile of the BMP-4-treated squamous cells toward that of the cultured BE cells. These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.

Experimental study on molecular mechanism of nm23-H1 gene transfection reversing the malignant phenotype of human high-metastatic large cell lung cancer cell line

nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors. The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods. The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01). Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.

AKT1 Provides an Essential Survival Signal Required for Differentiation and Stratification of Primary Human Keratinocytes

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF

We recently published a review in this journal describing the design, hybridisation and basic data processing required to use gene arrays to investigate vascular biology (Evans et al. Angiogenesis 2003; 6: 93-104). Here, we build on this review by describing a set of powerful and robust methods for the analysis and interpretation of gene array data derived from primary vascular cell cultures. First, we describe the evaluation of transcriptome heterogeneity between primary cultures derived from different individuals, and estimation of the false discovery rate introduced by this heterogeneity and by experimental noise. Then, we discuss the appropriate use of Bayesian t-tests, clustering and independent component analysis to mine the data. We illustrate these principles by analysis of a previously unpublished set of gene array data in which human umbilical vein endothelial cells (HUVEC) cultured in either rich or low-serum media were exposed to vascular endothelial growth factor (VEGF)-A165 or placental growth factor (PlGF)-1(131). We have used Affymetrix U95A gene arrays to map the effects of these factors on the HUVEC transcriptome. These experiments followed a paired design and were biologically replicated three times. In addition, one experiment was repeated using serial analysis of gene expression (SAGE). In contrast to some previous studies, we found that VEGF-A and PlGF consistently regulated only small, non-overlapping and culture media-dependant sets of HUVEC transcripts, despite causing significant cell biological changes.

Genome-scale analysis of lung cancer progression.

Among cancers, lung cancer is the single biggest killer in the US. It is estimated that lung cancer was responsible for 171900 newly diagnosed cases of cancer in the US in 2003, and for 157200 deaths. Over many years, however, there has been little improvement in the clinical outcome of lung cancer, and any improvement in the incidence or mortality from lung cancer can largely be attributed to smoking cessation and not to the success of therapy. The histopathology of lung cancer reveals that it is a disease with many faces. Lung cancer is often nonresponsive to traditional therapy, leaving few, if any, alternatives in the management of the advanced stages of the disease. The molecular pathogenesis of lung cancer, only recently illuminated, involves numerous molecular and cell biological changes revealing a very complex disease progression. Large-scale mRNA expression analysis has been recently used to classify lung cancers molecularly. These techniques have been used successfully to differentiate lung cancer histotypes based on patterns of genes expressed. The use of protein analysis to this end has also been attempted, with limited correlation with RNA experiments. This likely reflects the limited sensitivity of the technologies and complex, poorly understood post-synthesis protein modifications. In any event, there have been great strides made in understanding the nature of lung cancer from a molecular perspective; these effects represent a great advancement in the diagnosis and prognosis of lung cancer. Moreover, these advances may lead to the improvement of patient survival by guiding the choice of more efficacious therapy.

Basement membrane integrity and keratinization in healthy and ulcerated bovine hoof tissue.

Damage to, or deterioration of, the keratinized horn tissue of the bovine hoof claw culminates ultimately in the development of solear ulceration. We have observed abnormal keratin distribution at the site of solear ulceration in the bovine claw that may be due to alteration of the positional cues of the keratinocytes. In this study we have characterized key cell biological changes associated with ulceration in the claw that may precipitate abnormal keratinization. Loss of basement membrane at sites of ulceration was found by immunofluorescent detection of laminin and integrins. In other tissues, basement membrane breakdown results from degradation by matrix metalloproteinases (MMPs). Similarly, elevated levels of MMPs 2 and 9 were observed in ulcerated bovine claw tissue both by zymography and, quantitatively, by assay of enzyme activity. In the sole of claws that contained an ulcer, tissue distal to the ulcer site also had elevated MMP 2 when compared with healthy sole tissue from the same animals, as did sole tissue of claws recovering from ulceration. Tissue inhibitor of metalloproteinase 2 (TIMP 2) was detected by ELISA in healthy tissue. TIMP 2 tended to be lower in diseased tissue distal to ulcer sites, and was significantly lower in ulcerated tissue. MMP 2 was located by immunofluorescence in the dermal and basal epidermal region of sole tissue, in the region of the basement membrane. Increased punctate staining of material in the dermis was associated with ulcerated material. ELISA of TIMP 2 in tissue extracts enriched for dermis or epidermis confirmed that the inhibitor was located predominantly in the dermis. To investigate a possible causal relationship between basement membrane anchorage and epidermal keratinization, the effect of function-blocking antibodies to laminins and integrins was tested in tissue explant cultures prepared from healthy sole tissue. Anti-integrin antibody treatment had no effect on either protein or DNA synthesis. In contrast, in the presence of anti-laminin antibody, protein synthesis was decreased in a concentration-dependent manner, a significant effect being observed at the highest concentration after treatment for 24 h. At this concentration, DNA synthesis was also decreased after 48 h of culture, an effect that may be relevant to a hibernal reduction in claw cell turnover, and the associated seasonal vulnerability of cows to claw damage. The results provide evidence for basement membrane disruption at ulcer sites, and an increased potential for disruption in the diseased claw, and a causal link between this and abnormal epidermal keratinization. Basement membrane disruption is in turn associated with reciprocal changes in MMPs and their inhibitors, favouring extracellular proteolysis. Whether MMP activation is the primary cause of dermal-epidermal deterioration and, if so, how MMP activation is triggered, remains to be determined.