Silver-Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues.

In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome-specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non-affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.

Differences between BCL2-break positive and negative follicular lymphoma unraveled by whole-exome sequencing.

Depending on disease stage follicular lymphoma (FL) lack the t(14;18) in ~15-~50% of cases. Nevertheless, most of these cases express BCL2. To elucidate mechanisms triggering BCL2 expression and promoting pathogenesis in t(14;18)-negative FL, exonic single-nucleotide variant (SNV) profiles of 28 t(14;18)-positive and 13 t(14;18)-negative FL were analyzed, followed by the integration of copy-number changes, copy-neutral LOH and published gene-expression data as well as the assessment of immunoglobulin N-glycosylation sites. Typical FL mutations also affected t(14;18)-negative FL. Curated gene set/pathway annotation of genes mutated in either t(14;18)-positive or t(14;18)-negative FL revealed a strong enrichment of same or similar gene sets but also a more prominent or exclusive enrichment of immune response and N-glycosylation signatures in t(14;18)-negative FL. Mutated genes showed high BCL2 association in both subgroups. Among the genes mutated in t(14;18)-negative FL 555 were affected by copy-number alterations and/or copy-neutral LOH and 96 were differently expressed between t(14;18)-positive and t(14;18)-negative FL (P<0.01). N-glycosylation sites were detected considerably less frequently in t(14;18)-negative FL. These results suggest a diverse portfolio of genetic alterations that may induce or regulate BCL2 expression or promote pathogenesis of t(14;18)-negative FL as well as a less specific but increased crosstalk with the microenvironment that may compensate for the lack of N-glycosylation.

UV-induced apoptosis in skin equivalents: inhibition by phorbol ester and Bcl-2 overexpression.

To determine the role of apoptosis in epidermal homeostasis and to identify its regulators in skin, we have developed and characterised a physiologically relevant in vitro model of epidermal apoptosis. First, we show that keratinocyte cell death can be induced by ultraviolet irradiation within the stratified epidermis of the skin equivalent in an in vivo-like manner. DNA fragmentation and changes in the patterns of expression of p53 and Bcl-2 suggest that the mechanisms operating in UV-induced apoptosis in the skin equivalent are controlled by these factors. Secondly, we demonstrate that apoptosis in this model is amenable to modulation by exogenous factors present in the culture medium, such as phorbol ester, and by tranfected genes, as shown by overexpression of bcl-2. These studies show that the skin equivalent is a valuable model in which to determine the controllable steps of the apoptotic pathway independently of the immune system and to correlate apoptosis to the physiologic state of the keratinocyte.

Apoptosis: the skin from a new perspective.

In this review we present skin biology from the perspective of apoptosis. We stress that apoptosis acts as an important homeostatic and defence mechanism in the developing and mature epidermis. Programmed cell death functions in establishing the architecture of the human epidermis and its appendages during development by deletion of stage-specific cells and in the adult epidermis by elimination of excess and abnormal cells. Arguments are presented to support the hypothesis that known regulators of keratinocyte growth may act as survival factors which suppress the cell death pathway. Surviving cells continue to divide until they encounter anti-proliferative factors. Then, unless cells are severely injured and die of necrosis, they will terminally differentiate to death or will die by apoptosis. The mechanisms controlling keratinocyte maturation are co-ordinated with cell position within the epidermal strata. Inappropriate regulatory signals or response of a cell inappropriate to its state will activate apoptosis. Parallels between terminally differentiating keratinocytes and apoptotic cells imply that terminal differentiation and apoptosis proceed along the same death pathway. For terminally differentiating cells, however, this pathway is more elaborate because it allows expression of tissue- and differentiation-specific genes. A model is presented that integrates apoptosis and keratinocyte growth and differentiation.

The mutational pattern of primary lymphoma of the central nervous system determined by whole-exome sequencing.

To decipher the mutational pattern of primary CNS lymphoma (PCNSL), we performed whole-exome sequencing to a median coverage of 103 × followed by mutation verification in 9 PCNSL and validation using Sanger sequencing in 22 PCNSL. We identified a median of 202 (range: 139-251) potentially somatic single nucleotide variants (SNV) and 14 small indels (range: 7-22) with potentially protein-changing features per PCNSL. Mutations affected the B-cell receptor, toll-like receptor, and NF-κB and genes involved in chromatin structure and modifications, cell-cycle regulation, and immune recognition. A median of 22.2% (range: 20.0-24.7%) of somatic SNVs in 9 PCNSL overlaps with the RGYW motif targeted by somatic hypermutation (SHM); a median of 7.9% (range: 6.2-12.6%) affects its hotspot position suggesting a major impact of SHM on PCNSL pathogenesis. In addition to the well-known targets of aberrant SHM (aSHM) (PIM1), our data suggest new targets of aSHM (KLHL14, OSBPL10, and SUSD2). Among the four most frequently mutated genes was ODZ4 showing protein-changing mutations in 4/9 PCNSL. Together with mutations affecting CSMD2, CSMD3, and PTPRD, these findings may suggest that alterations in genes having a role in CNS development may facilitate diffuse large B-cell lymphoma manifestation in the CNS. This may point to intriguing mechanisms of CNS tropism in PCNSL.

Genomic and epigenomic co-evolution in follicular lymphomas.

Follicular lymphoma (FL) with a t(14;18) is a B-cell neoplasm clinically characterized by multiple recurrencies. In order to investigate the clonal evolution of this lymphoma, we studied paired primary and relapse tumor samples from 33 patients with recurrent non-transformed t(14;18)-positive FL. We reconstructed phylogenetic trees of the evolution by taking advantage of the activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) active in the germinal center reaction using sequences of the clonal VHDHJH rearrangements of the immunoglobulin heavy chain (IGH) locus. Mutational analysis of the IGH locus showed evidence for ongoing somatic mutation and for counter-selection of mutations affecting the BCR conformation during tumor evolution. We further followed evolutionary divergence by targeted sequencing of gene loci affected by aberrant SHM as well as of known driver genes of lymphomagenesis, and by array-based genome-wide chromosomal imbalance and DNA methylation analysis. We observed a wide spectrum of evolutionary patterns ranging from almost no evolution to divergent evolution within recurrent non-transformed t(14;18) FL. Remarkably, we observed a correlation of the magnitude of evolutionary divergence across all genetic and epigenetic levels suggesting co-evolution. The distribution of coding mutations in driver genes and the correlation with SHM suggest CREBBP and AID to be potential modifiers of genetic and epigenetic co-evolution in FL.

Keratinocytes regulate melanocyte number in human fetal and neonatal skin equivalents.

To determine if keratinocytes influence melanocyte number and position in the developing epidermis we have experimentally recombined keratinocytes and melanocytes from epidermis of different stages of differentiation in the skin equivalent (SE) system. Previously we showed that developmental differences in the position and number of melanocytes characteristic of the epidermis in vivo were preserved in fetal and neonatal skin equivalents. In the present study we have combined cultured fetal or neonatal keratinocytes with age-matched or non-age-matched cultured melanocytes on the dermal equivalent. The ratio of basal keratinocytes to melanocytes (BK/M) present in multiple high-power fields was determined after localization of melanocytes by staining with the melanocyte-specific monoclonal antibody, HMB-45. The BK/M ratio in SE composed of neonatal keratinocytes and either fetal (n = 4) or neonatal (n = 5) melanocytes was 26.2 and 21.5, respectively. The BK/M ratio in SE composed of fetal keratinocytes and either fetal (n = 8) or neonatal (n = 5) melanocytes was 9.2 and 7.7, respectively. In each case, the BK/M ratio was dependent on the keratinocytes rather than the melanocytes. With either type of melanocyte, ratios in SE composed of neonatal keratinocytes were significantly greater than those with fetal keratinocytes. These results establish that keratinocytes regulate the BK/M ratio in this model and suggest that developmental differences between fetal and neonatal keratinocytes may be responsible for determining melanocyte numbers in the epidermal-melanin unit in vivo. The precise mechanisms that control the organization and number of melanocytes in the epidermis are unknown although keratinocytes may interact with melanocytes via growth factors, cell surface molecules, or other factors related to proliferation and differentiation of the epidermis.

Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents.

There is evidence that epidermal keratinocytes play a critical role in melanocyte position and differentiation in the epidermis, although little is known about the molecular mechanisms involved. We have used an in vitro skin equivalent as a model system in which to study keratinocyte/melanocyte interactions in both fetal and neonatal skin. Because the skin equivalent model has been shown to closely simulate the morphologic and biochemical features of differentiated epidermis we hypothesized that the factors that influence melanocyte position and differnetiation would also function in this system. Localization of melanocytes in skin equivalents, using the monoclonal antibody HMB-45, established that melanocytes in fetal skin equivalents are grouped and distributed both basally and suprabasally, whereas melanocytes in neonatal skin equivalents are singly distributed among basal epidermal keratinocytes, similar to the distributions of fetal and neonatal melanocytes, respectively, in vivo. Similarly, in fetal and neonatal skin equivalents the patterns of expression of a number of melanoma/melanocyte-associated antigens closely parallels that seen in vivo. These results suggest that the skin equivalent model is an excellent system in which to study the dynamic factors that regulate melanocyte migration, proliferation, and differentiation during ontogeny and post-natal differentiation of the skin.

Cell death by apoptosis in epidermal biology.

Homeostasis in continually renewing tissues is maintained by a tightly regulated balance between cell proliferation, cell differentiation, and cell death. Until recently, proliferation was thought to be the primary point of control in the regulation of normal tissue kinetic homeostasis and as such has been the major focus of both understanding the etiology of disease and developing therapeutic strategies. Now, physiologic cell death, known as apoptosis (a-pop-to' sis, a-po-to' sis [Thomas CL (ed.): Taber's Cyclopedic Medical Dictionary. F.A. Davis, Co., Philadelphia, 1989)] has gained scientific recognition as an active regulatory mechanism, complementary, but functionally opposite, to proliferation with important roles in shaping and maintaining tissue size and prevention of disease. In this review we will describe the concept of apoptosis and discuss possible molecular mechanisms of its regulation that may have implications for skin biology.

Induction of TR4 orphan receptor by retinoic acid in human HaCaT keratinocytes.

Human TR4 orphan receptor (TR4) can modulate the transcriptional activity of the reporter gene containing an AGGTCA direct repeat-hormone response element. Here we studied the potential role of TR4 in human HaCaT keratinocytes. Using a chloramphenicol acetyl-transferase reporter gene assay, it was shown that TR4 can suppress retinoic acid-induced transactivation by 47.3% in human HaCaT keratinocytes. Electrophoretic mobility shift assay indicated that this suppression may be due to TR4 binding with higher affinity to the retinoic acid response element than retinoid receptors. Western blot analysis further suggested that retinoic acid can increase the expression of TR4 protein in human HaCaT keratinocytes, indicating that TR4 acts as a negative feedback modulator for retinoic acid action. Interestingly, TR4 expression is increased in normal human keratinocytes when substituting a low calcium medium with a high calcium medium. Together, our data suggested, for the first time, that an orphan receptor, such as TR4, may play an important part in retinoid-mediated signaling pathways in human keratinocytes, providing a new insight into keratinocyte biology.

Localization of the gene for Darier disease to a 5-cM interval on chromosome 12q.

Darier disease is an autosomal dominant abnormality of epidermal differentiation characterized clinically by the presence of hyperkeratotic papules on the skin and histologically by the loss of cell cohesion and by disorderly keratinization. Two groups recently found evidence that the gene whose mutations underlie this disease is located at chromosome 12q23-q24.1, a site on chromosome 12 that clearly is distal to the type II keratin gene cluster. We report here evidence for sublocalization to a 5-cM region of that site in an additional ten families of European and Middle Eastern ancestry with a combined lod score in excess of 20.

Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for BCL-2.

Normal human keratinocytes synthesize and release nerve growth factor (NGF) and express both the low- and the high-affinity NGF receptor. Because NGF has been shown to rescue certain cell types from programmed cell death, we investigated the role of endogenous NGF in preventing keratinocyte apoptosis. We report here that apoptosis is induced in normal human keratinocytes in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K252, a specific inhibitor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was assessed by DNA laddering, electron microscopy, and in situ nick end labeling technique. In anti-NGF-treated keratinocytes, the apoptotic process starts at 96 h, and is maximal at 120 h. After K252 treatment, apoptosis starts at 48 h and peaks at 120 h. Because the product of the bcl-2 proto-oncogene protects many cell types from apoptosis, we measured the levels of this protein in apoptotic keratinocytes. We found that both K252 and anti-NGF antibody strikingly downregulate bcl-2 expression, starting at 72 h. Furthermore, HaCat keratinocytes stably transfected with a plasmid containing bcl-2 cDNA fail to undergo apoptosis when treated with K252. These findings show that autocrine NGF acts as a survival factor for human keratinocytes in vitro through its high-affinity NGF receptor, possibly by maintaining constant levels of Bcl-2.

Localization of the Darier disease gene to a 2-cM portion of 12q23-24.1.

Positional cloning with microsatellite markers allowed further localization of the Darier disease gene to a 2-cM interval of chromosome 12, 12q23-24.1, between the polymorphic loci D12S234 and D12S129. A region this size is suitable for construction of a contig to identify the Darier disease gene. Use of a polymorphic intronic marker for nitric oxide synthetase 1 gene, which maps to the same chromosomal area as the Darier gene, allowed exclusion of that gene as the Darier disease gene.

Bcl-2 overexpression in the HaCaT cell line is associated with a different membrane fatty acid composition and sensitivity to oxidative stress.

Different mechanisms have been proposed for the activity of the Bcl-2 proto-oncogene product. A bona fide antioxidant activity and a pro-oxidant setting up of the cell have been suggested using different experimental models, yet many uncertainties exist about the biochemical mechanism of Bcl-2 action. In the present paper, we report the characterization of the cellular response to mild oxidative stress of a cultured cell line of immortalized keratinocytes (HaCaT), overexpressing the Bcl-2 oncogene product. A sublethal oxidative stress was induced by 1 h treatment with 200 microM tert-butyl-hydroperoxide (t-BOOH). Following peroxide treatment, the formation of reactive oxygen species was lower in Bcl-2 expressing cells, suggesting a better capacity to counter oxidative stress. Total Superoxide Dismutase activity was induced by oxidative t-BOOH treatment in bcl-2 transfected cells, which also accumulated less damage to membrane lipids and proteins, as assessed by TBA-RS and carbonyl formation respectively. On the other hand, the formation of 4-hydroxy-nonenal, a more specific marker of peroxidative damage to polyunsaturated fatty acids, was higher in bcl-2 transfected cells than in control cells. Bcl-2 over-expression was also associated with significant changes in the fatty acid composition of cell membranes. Transfected cells presented a higher proportion of mono-unsaturated fatty acids and omega6 poly unsaturated fatty acids and a lower proportion of penta-enoic PUFA, thus resulting in a higher unsaturation index with respect to control cells. Changes in protein kinase C activity were also associated to bcl-2 expression, possibly resulting from the differences in membrane fatty acid composition. These data may be an important background for the understanding of Bcl-2 involvement in the control of apoptotic response as well as in the induction of antioxidant cell defenses against oxidative stress.

Localized Darier disease. Implications for genetic studies.

BACKGROUND: Darier disease is an uncommon genodermatosis characterized by the symmetrical eruption of keratotic reddish-brown papules occurring in the seborrheic areas of the body. A unilateral, or localized, variant has been identified. We report 4 new cases of localized Darier disease and review the English-language literature. The implications of these cases on future genetic studies are also discussed. OBSERVATIONS: Localized Darier disease occurred with equal frequency in males and females. The average age at onset was 27 years. The most frequent site of involvement was the trunk (40% [16/40]). This condition was aggravated by sunlight, heat, or sweating in 42% (19/40) of reported cases, and 38% (15/40) of the patients responded to treatment with topical tretinoin. CONCLUSIONS: Many of the clinical features of localized Darier disease suggest that it is a genetic mosaic of generalized Darier disease. Further studies of localized Darier disease may therefore prove to be instrumental in the search for the Darier disease gene.

Induced G2/M arrest and apoptosis in human epidermoid carcinoma cell lines by semisynthetic drug Ukrain.

Exposure of ME180 and A431 carcinoma cells to Ukrain (NSC-631570), a novel semisynthetic drug from Chelidonium majus L, results in cell growth inhibition which is concomitant with reversible G2/M cell cycle arrest and apoptosis at doses as low as 7 microM. In contrast, the same drug concentrations were not affective towards normal human keratinocytes. In order to investigate whether cell cycle control mechanisms are effected in response to Ukrain, we analyzed cell cycle distribution and levels of cyclins and cyclin-dependent kinases in drug treated carcinoma cells. We found alterations in levels of mitotic cyclins A and B1, and cyclin-dependent kinases CDK1 and CDK2, after treatment. We also observed an upregulation of CDK inhibitor p27 in both cancer cell lines which may lead to the G2/M cells accumulation.

Bcl-2 overexpression protects human keratinocyte cells from Ukrain-induced apoptosis but not from G2/M arrest.

Exposure of ME180 and A431 carcinoma cells to Ukrain (NSC-631570), a semisynthetic compound consisting of alkaloids isolated from Chelidonium majus L. (Papaveracea), results in cell cycle arrest at the G2/M phase. Ukrain selectively inhibits growth of ME180 and A431 cells at a concentration range from 3.5 microM to 7.0 microM and induces apoptosis. In contrast, normal human keratinocytes showed no difference in the kinetics of progression through the cell cycle in response to this compound. We found that at a concentration of 7.0 microM of this drug Bcl-2 protein overexpression protected HaCaT cell line keratinocytes against apoptosis induced by Ukrain but did not prevent G2/M arrest. Following exposure of normal keratinocytes to Ukrain, we detected an increase in Bcl-2 protein levels and a significant change in protein modification as suggested by observation of its different isoform with shifted electrophoretic mobility. Bcl-2 protein expression and its isoform distribution did not change substantially in ME180 and A431 carcinoma cells. We also suggest that drug-induced mitotic arrest and apoptosis represent dual Ukrain action on cell cycle progression machinery and Bcl-2-involved program cell death in the cell.

Induced apoptosis in human prostate cancer cell line LNCaP by Ukrain.

Exposure of LNCaP prostate cancer cells to Ukrain (NSC-631570), a novel semisynthetic drug from Chelidonium majus L., results in cell growth inhibition which is concomitant with apoptosis. After 24 h treatment with 3.5 microM of Ukrain as many as 73% cells were found in the G2/M phase. However, at higher drug concentrations (7 microM and 17.5 microM) the changes in cell phase distribution were less dramatic but cell accumulation in the G2/M phase was still evident. The rate of apoptotic cells rose steadily with increased drug concentration in a dose-dependent manner and reached 20% at a dosage of 17.5 microM. To investigate whether the cell cycle control mechanisms are affected in response to Ukrain, we analyzed the expression levels of some cyclins, cyclin-dependent kinases (CDK) and apoptosis-related proteins in drug treated cancer cells. Western blot experiments revealed alterations in levels of CDK1 and CDK2, after treatment. Up-regulation of the CDK inhibitor p27 was observed, which may lead to G2/M cell accumulation, but no substantial changes in expression of Bcl-2 and Bax proteins were found.

Positioning of small particles by an ultrasound field excited by surface waves.

A method for the controlled positioning of small particles in one or two dimensions by an ultrasound field excited by a surface wave is presented. Particles of a diameter between 10 and 100 microm placed on a surface can be concentrated at certain locations and moved over the surface. In other approaches it is possible to let the particle levitate freely in the fluid. However for the use of ultrasonic positioning in for example microassembling it is necessary to move particles over a surface as well as to let them levitate over the surface. Physical principle: A two- or three-dimensional ultrasound field is excited in a fluid filled gap between a rigid surface at the bottom and a vibrating surface of a solid at the top. The height of the gap varies between 0.1 and 2 mm. A one-dimensional sinusoidal vibration of the upper surface excites a two-dimensional ultrasound field in the fluid. Particles that are arbitrarily distributed on the lower surface will be concentrated in lines by the ultrasound field. First the calculation of the field of forces on particles in the fluid layer is presented. Then the dispersion relation of a vibrating plate which is in contact with a fluid on one side is derived. The technical setup will be introduced. Finally the experiments are shown and compared to the theoretical results.

Micro-manipulation of small particles by node position control of an ultrasonic standing wave.

For the controlled positioning of small particles with ultrasound a standing wave in a fluid is used. The standing wave is implemented in a resonator, that consists of a fluid filled tube and two piezoelectric transducers on each end. A one-dimensional model of a piezo-device including the fluid-loading on one side and a backside support is introduced. This model allows the calculation of the transmitted wave as a function of the applied electric voltage and the incident wave. In addition, when an electrical impedance is connected to the piezo-device, the reflection coefficient can be varied in amplitude and phase, so that the parameters of the reflected wave can be controlled completely. The resonator itself, consisting of a piezo-device on each end and the fluid between, is included in the model. Several methods to shift the nodes of the standing wave in the resonator are investigated and the ability to position particles is discussed.