Correction to: Xenopus LAP2ß protein knockdown affects location of Lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

The original publication of this paper contains a mistake.

N-acetyl cysteine alleviates oxidative stress and protects mice from dilated cardiomyopathy caused by mutations in nuclear A-type lamins gene.

Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscular and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. We showed here an increase in oxidative stress levels in the hearts of mice carrying LMNA mutation, associated with a decrease of the key cellular antioxidant glutathione (GHS). Oral administration of N-acetyl cysteine, a GHS precursor, led to a marked improvement of GHS content, a decrease in oxidative stress markers including protein carbonyls and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our novel results provide therapeutic insights into LMNA cardiomyopathy.

Xenopus LAP2ß protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

Xenopus LAP2ß protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2ß fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2ß antibody. Knockdown of the XLAP2ß protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

Lamin A/C-dependent interaction with 53BP1 promotes cellular responses to DNA damage.

Lamins A/C have been implicated in DNA damage response pathways. We show that the DNA repair protein 53BP1 is a lamin A/C binding protein. In undamaged human dermal fibroblasts (HDF), 53BP1 is a nucleoskeleton protein. 53BP1 binds to lamins A/C via its Tudor domain, and this is abrogated by DNA damage. Lamins A/C regulate 53BP1 levels and consequently lamin A/C-null HDF display a 53BP1 null-like phenotype. Our data favour a model in which lamins A/C maintain a nucleoplasmic pool of 53BP1 in order to facilitate its rapid recruitment to sites of DNA damage and could explain why an absence of lamin A/C accelerates aging.

Do lamins influence disease progression in cancer?

For nearly 60 years, diagnosis of cancer has been based on pathological tests that look for enlargement and distortion of nuclear shape. Because of their involvement in supporting nuclear architecture, it has been postulated that the basis for nuclear shape changes during cancer progression is altered expression of nuclear lamins and in particular lamins A and C. However, studies on lamin expression patterns in a range of different cancers have generated equivocal and apparently contradictory results. This might have been anticipated since cancers are diverse and complex diseases. Moreover, whilst altered epigenetic control over gene expression is a feature of many cancers, this level of control cannot be considered in isolation. Here I have reviewed those studies relating to altered expression of lamins in cancers and argue that consideration of changes in the expression of individual lamins cannot be considered in isolation but only in the context of an understanding of their functions in transformed cells.

Generation and characterization of multipotent stem cells from established dermal cultures.

Human multipotent skin derived precursor cells (SKPs) are traditionally sourced from dissociated dermal tissues; therefore, donor availability may become limiting. Here we demonstrate that both normal and diseased adult human dermal fibroblasts (DF) pre-cultured in conventional monolayers are capable of forming SKPs (termed m-SKPs). Moreover, we show that these m-SKPs can be passaged and that cryopreservation of original fibroblast monolayer cultures does not reduce m-SKP yield; however, extensive monolayer passaging does. Like SKPs generated from dissociated dermis, these m-SKPs expressed nestin, fibronectin and versican at the protein level. At the transcriptional level, m-SKPs derived from normal adult human DF, expressed neural crest stem cell markers such as p75NTR, embryonic stem cell markers such as Nanog and the mesenchymal stem cell marker Dermo-1. Furthermore, appropriate stimuli induced m-SKPs to differentiate down either mesenchymal or neural lineages resulting in lipid accumulation, calcification and S100ß or ß-III tubulin expression (with multiple processes). m-SKP yield was greater from neonatal foreskin cultures compared to those from adult DF cultures; however, the former showed a greater decrease in m-SKP forming capacity after extensive monolayer passaging. m-SKP yield was greater from adult DF cultures expressing more alpha-smooth muscle actin (αSMA). In turn, elevated αSMA expression correlated with cells originating from specimens isolated from biopsies containing more terminal hair follicles; however, αSMA expression was lost upon m-SKP formation. Others have shown that dissociated human hair follicle dermal papilla (DP) are a highly enriched source of SKPs. However, conversely and unexpectedly, monolayer cultured human hair follicle DP cells failed to form m-SKPs whereas those from the murine vibrissae follicles did. Collectively, these findings reveal the potential for using expanded DF cultures to produce SKPs, the heterogeneity of SKP forming potential of skin from distinct anatomical locations and ages, and question the progenitor status of human hair follicle DP cells.

The role of DNA damage in laminopathy progeroid syndromes.

Progeroid laminopathies are characterized by the abnormal processing of lamin A, the appearance of misshapen nuclei, and the accumulation and persistence of DNA damage. In the present article, I consider the contribution of defective DNA damage pathways to the pathology of progeroid laminopathies. Defects in DNA repair pathways appear to be caused by a combination of factors. These include abnormal epigenetic modifications of chromatin that are required to recruit DNA repair pathways to sites of DNA damage, abnormal recruitment of DNA excision repair proteins to sites of DNA double-strand breaks, and unrepairable ROS (reactive oxygen species)-induced DNA damage. At least two of these defective processes offer the potential for novel therapeutic approaches.

Conserved cysteine residues in the mammalian lamin A tail are essential for cellular responses to ROS generation.

Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence.

The accumulation of un-repairable DNA damage in laminopathy progeria fibroblasts is caused by ROS generation and is prevented by treatment with N-acetyl cysteine.

Fibroblasts from patients with the severe laminopathy diseases, restrictive dermopathy (RD) and Hutchinson Gilford progeria syndrome (HGPS), are characterized by poor growth in culture, the presence of abnormally shaped nuclei and the accumulation of DNA double-strand breaks (DSB). Here we show that the accumulation of DSB and poor growth of the fibroblasts but not the presence of abnormally shaped nuclei are caused by elevated levels of reactive oxygen species (ROS) and greater sensitivity to oxidative stress. Basal levels of ROS and sensitivity to H(2)O(2) were compared in fibroblasts from normal, RD and HGPS individuals using fluorescence activated cell sorting-based assays. Basal levels of ROS and stimulated levels of ROS were both 5-fold higher in the progeria fibroblasts. Elevated levels of ROS were correlated with lower proliferation indices but not with the presence of abnormally shaped nuclei. DSB induced by etoposide were repaired efficiently in normal, RD and HGPS fibroblasts. In contrast, DSB induced by ROS were repaired efficiently in normal fibroblasts, but in RD and HGPS fibroblasts many ROS-induced DSB were un-repairable. The accumulation of ROS-induced DSB appeared to cause the poor growth of RD and HGPS fibroblasts, since culture in the presence of the ROS scavenger N-acetyl cysteine (NAC) reduced the basal levels of DSB, eliminated un-repairable ROS-induced DSB and greatly improved population-doubling times. Our findings suggest that un-repaired ROS-induced DSB contribute significantly to the RD and HGPS phenotypes and that inclusion of NAC in a combinatorial therapy might prove beneficial to HGPS patients.

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/-) cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope.

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2ß were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2ß was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2ß was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains.

Lamins as cancer biomarkers.

Lamins are multifunctional proteins that are often aberrantly expressed or localized in tumours. Here, we endeavour to assess their uses as cancer biomarkers: to diagnose tumours, analyse cancer characteristics and predict patient survival. It appears that the nature of lamin function in cancer is very complex. Lamin expression can be variable between and even within cancer subtypes, which limits their uses as diagnostic biomarkers. Expression of A-type lamins is a marker of differentiated tumour cells and has been shown to be a marker of good or poor patient survival depending on tumour subtype. Further research into the functions of lamins in cancer cells and the mechanisms that determine its patterns of expression may provide more potential uses of lamins as cancer biomarkers.

Lamin A: a putative colonic epithelial stem cell biomarker which identifies colorectal tumours with a more aggressive phenotype.

Abnormalities in the expression, distribution and structural organization of A-type lamins are most commonly associated with a spectrum of inherited disorders which predominantly affect mesenchymal lineages, collectively known as laminopathies. However, a new role for lamin A has been discovered in the progression of a common epithelial cancer. CRC (colorectal cancer) patients expressing lamin A/C in their tumour tissue were found to have a 2-fold greater risk of CRC-related mortality compared with patients with lamin A/C-negative tumours. Consequently, lamin A/C is a prognostic biomarker in CRC. In vitro studies suggest that lamin A is an upstream regulator of a pathway linking actin dynamics to loss of cell adhesion, leading to enhanced cell motility and consequently increased invasive potential within a tumour. The finding that lamin A is a putative colonic epithelial stem cell biomarker suggests that the poor outcome associated with lamin A/C-positive tumours may be reflective of a more stem-cell-like phenotype. The present review discusses the link between lamin A expression and tumour progression in one of the commonest causes of cancer-related death in the Western world.

Lamin A/C is a risk biomarker in colorectal cancer.

BACKGROUND: A-type lamins are type V intermediate filament proteins encoded by the gene LMNA. Mutations in LMNA give rise to diverse degenerative diseases related to premature ageing. A-type lamins also influence the activity of the Retinoblastoma protein (pRb) and oncogenes such a beta-catenin. Consequently, it has been speculated that expression of A-type lamins may also influence tumour progression. METHODOLOGY/PRINCIPAL FINDINGS: An archive of colorectal cancer (CRC) and normal colon tissue was screened for expression of A-type lamins. We used the Cox proportional hazard ratio (HR) method to investigate patient survival. Using CRC cell lines we investigated the effects of lamin A expression on other genes by RT-PCR; on cell growth by FACS analysis; and on invasiveness by cell migration assays and siRNA knockdown of targeted genes. We found that lamin A is expressed in colonic stem cells and that patients with A-type lamin-expressing tumours have significantly worse prognosis than patients with A-type lamin negative tumours (HR = 1.85, p = 0.005). To understand this finding, we established a model system based upon expression of GFP-lamin A in CRC cells. We found that expression of GFP-lamin A in these cells did not affect cell proliferation but did promote greatly increased cell motility and invasiveness. The reason for this increased invasiveness was that expression of lamin A promoted up-regulation of the actin bundling protein T-plastin, leading to down regulation of the cell adhesion molecule E-cadherin. CONCLUSIONS: Expression of A-type lamins increases the risk of death from CRC because its presence gives rise to increased invasiveness and potentially a more stem cell-like phenotype. This report directly links A-type lamin expression to tumour progression and raises the profile of LMNA from one implicated in multiple but rare genetic conditions to a gene involved in one of the commonest diseases in the Western World.

Colonic and colorectal cancer stem cells: progress in the search for putative biomarkers.

The maintenance of healthy colonic crypts is dependent on the integrity of the adult epithelial stem cells located within them. Perturbations in stem cell dynamics are generally believed to represent the first step towards colorectal tumorigenesis. Experimental manipulation of intestinal stem cells has greatly increased our understanding of them, but further progress has been slowed due to the absence of a reliable stem cell biomarker. In this review we discuss the candidate colonic stem cell biomarkers which have been proposed. Furthermore, we investigate the putative biomarkers for so-called colorectal cancer stem cells, a highly aggressive subpopulation of cells considered to drive tumour development.

Adult stem cell maintenance and tissue regeneration in the ageing context: the role for A-type lamins as intrinsic modulators of ageing in adult stem cells and their niches.

Adult stem cells have been identified in most mammalian tissues of the adult body and are known to support the continuous repair and regeneration of tissues. A generalized decline in tissue regenerative responses associated with age is believed to result from a depletion and/or a loss of function of adult stem cells, which itself may be a driving cause of many age-related disease pathologies. Here we review the striking similarities between tissue phenotypes seen in many degenerative conditions associated with old age and those reported in age-related nuclear envelope disorders caused by mutations in the LMNA gene. The concept is beginning to emerge that nuclear filament proteins, A-type lamins, may act as signalling receptors in the nucleus required for receiving and/or transducing upstream cytosolic signals in a number of pathways central to adult stem cell maintenance as well as adaptive responses to stress. We propose that during ageing and in diseases caused by lamin A mutations, dysfunction of the A-type lamin stress-resistant signalling network in adult stem cells, their progenitors and/or stem cell niches leads to a loss of protection against growth-related stress. This in turn triggers an inappropriate activation or a complete failure of self-renewal pathways with the consequent initiation of stress-induced senescence. As such, A-type lamins should be regarded as intrinsic modulators of ageing within adult stem cells and their niches that are essential for survival to old age.

NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes.

In vertebrates, the nuclear envelope (NE) assembles and disassembles during mitosis. As the NE is a complex structure consisting of inner and outer membranes, nuclear pore complexes (NPCs) and the nuclear lamina, NE assembly must be a controlled and systematic process. In Xenopus egg extracts, NE assembly is mediated by two distinct membrane vesicle populations, termed NEP-A and NEP-B. Here, we re-investigate how these two membrane populations contribute to NPC assembly. In growing stage III Xenopus oocytes, NPC assembly intermediates are frequently observed. High concentrations of NPC assembly intermediates always correlate with fusion of vesicles into preformed membranes. In Xenopus egg extracts, two integral membrane proteins essential for NPC assembly, POM121 and NDC1, are exclusively associated with NEP-B membranes. By contrast, a third integral membrane protein associated with the NPCs, gp210, associates only with NEP-A membranes. During NE assembly, fusion between NEP-A and NEP-B led to the formation of fusion junctions at which >65% of assembling NPCs were located. To investigate how each membrane type contributes to NPC assembly, we preferentially limited NEP-A in NE assembly assays. We found that, by limiting the NEP-A contribution to the NE, partially formed NPCs were assembled in which protein components of the nucleoplasmic face were depleted or absent. Our data suggest that fusion between NEP-A and NEP-B membranes is essential for NPC assembly and that, in contrast to previous reports, both membranes contribute to NPC assembly.

Filaments made from A- and B-type lamins differ in structure and organization.

Lamins are intermediate filament proteins and the major component of the nuclear lamina. Current views of the lamina are based on the remarkably regular arrangement of lamin LIII in amphibian oocyte nuclei. We have re-examined the LIII lamina and propose a new interpretation of its organization. Rather than consisting of two perpendicular arrays of parallel filaments, we suggest that the oocyte lamina consists of parallel filaments that are interconnected in register to give the impression of a second set of perpendicular filaments. We have also used the oocyte system to investigate the organization of somatic lamins. Currently, it is not feasible to examine the organization of somatic lamins in situ because of their tight association with chromatin. It is also difficult to assemble vertebrate lamin filaments in vitro. Therefore, we have used the oocyte system, where exogenously expressed somatic B-type and A-type lamins assemble into filaments. Expression of B-type lamins induces the formation of intranuclear membranes that are covered by single filament layers. LIII filaments appear identical to the endogenous lamina, whereas lamin B2 assembles into filaments that are organized less precisely. Lamin A induces sheets of thicker filaments on the endogenous lamina and significantly increases the rigidity of the nuclear envelope.