Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Control of cell cycle progression by phosphorylation of cyclin-dependent kinase (CDK) substrates

The eukaryotic cell cycle is a fundamental evolutionarily conserved process that regulates cell division from simple unicellular organisms, such as yeast, through to higher multicellular organisms, such as humans. The cell cycle comprises several phases, including the S-phase (DNA synthesis phase) and M-phase (mitotic phase). During S-phase, the genetic material is replicated, and is then segregated into two identical daughter cells following mitotic M-phase and cytokinesis. The S- and M-phases are separated by two gap phases (G1 and G2) that govern the readiness of cells to enter S- or M-phase. Genetic and biochemical studies demonstrate that cell division in eukaryotes is mediated by CDKs (cyclin-dependent kinases). Active CDKs comprise a protein kinase subunit whose catalytic activity is dependent on association with a regulatory cyclin subunit. Cell-cycle-stage-dependent accumulation and proteolytic degradation of different cyclin subunits regulates their association with CDKs to control different stages of cell division. CDKs promote cell cycle progression by phosphorylating critical downstream substrates to alter their activity. Here, we will review some of the well-characterized CDK substrates to provide mechanistic insights into how these kinases control different stages of cell division.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

CDCA3 regulates the cell cycle and modulates cisplatin sensitivity in non-small cell lung cancer

Cisplatin-based regimens are currently the most effective chemotherapy for non-small cell lung cancer (NSCLC). Cisplatin forms DNA crosslinks to stall DNA replication and induce apoptosis. However, intrinsic and acquired chemoresistance is a major therapeutic problem. We have identified ‘cell division cycle associated protein 3’ (CDCA3) as a novel protein that may prove useful in delaying or preventing cisplatin resistance in NSCLC. CDCA3 functions as part of an ubiquitin ligase complex to degrade the endogenous cell cycle inhibitors. While a role for CDCA3 in disease is emerging with elevated expression noted in oral squamous cell carcinoma, little else is known about CDCA3 or whether this protein may prove useful clinically.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Overview of mesenchymal stem cells

Stem cells are unprogrammed cells which possess plasticity and self renewal capability. The term of stem cell was first used to describe cells committed to give rise to germline cells, and to describe proposed progenitor cells of the blood system [1]. A unique feature of stem cell is to remain quiescent in vivo in an uncommitted state. They serve as reservoir or natural support system to replenish cells lost due to disease, injury or aging. When triggered by appropriate signals these cells divide and may become specialized, committed cells; however being able to control this differentiation process still remains one of the biggest challenge in stem cell research [2]. The cell division of stem cells is a distinct aspect of their biology, since this division may be either symmetric or asymmetric. Symmetric division takes place when the stem cells divides and forms two new daughter cells. Asymmetric division is thought to take place only under certain conditions where stem cells divides and gives rise to a daughter cell which remains primitive and does not proliferate, and one committed progenitor cell, which heads down a path of differentiation. Asymmetric division of stem cells helps reparative process, and also ensures that the stem cells pool does not decrease, whereas symmetric division is responsible for stem cells undergoing self renewal and proliferation. The factors which prompt the stem cells to undergo asymmetric division are, however, not well understood, but it is clear that the delicate balance between the self renewal and differentiation is what maintains tissue homeostasis.

Lucky draw in the gene raffle

The Cancer Genome Project intends to search every human gene for cancer-related mutations. Its first success is the discovery of such mutations in the BRAF gene.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Cyclin A/CDK2 regulates multiple G2 phase pathways

The cell cycle is a carefully choreographed series of phases that when executed successfully will allow the complete replication of the genome and the equal division of the genome and other cellular content into two independent daughter cells. The inability of the cell to execute cell division successfully can result in either checkpoint activation to allow repair and/or apoptosis and/or mutations/errors that may or may not lead to tumourgenesis. Cyclin A/CDK2 is the primary cyclin/CDK regulating G2 phase progression of the cell cycle. Cyclin A/CDK2 activity peaks in G2 phase and its inhibition causes a G2 phase delay that we have termed 'the cyclin A/CDK2 dependent G2 delay'. Understanding the key pathways that are involved in the cyclin A/CDK2 dependent G2 delay has been the primary focus of this study. Characterising the cyclin A/CDK2 dependent G2 delay revealed accumulated levels of the inactive form of the mitotic regulator, cyclin B/CDK1. Surprisingly, there was also increased microtubule nucleation at the centrosomes, and the centrosomes stained for markers of cyclin B/CDK1 activity. Both microtubule nucleation at the centrosomes and phosphoprotein markers were lost with short-term treatment of CDK1/2 inhibition. Cyclin A/CDK2 localised at the centrosomes in late G2 phase after separation of the centrosomes but before the start of prophase. Thus G2 phase cyclin A/CDK2 controls the timing of entry into mitosis by controlling the subsequent activation of cyclin B/CDK1, but also has an unexpected role in coordinating the activation of cyclin B/CDK1 at the centrosome and in the nucleus. In addition to regulating the timing of cyclin B/CDK1 activation and entry into mitosis in the unperturbed cell cycle, cyclin A/CDK2 also was shown to have a role in G2 phase checkpoint recovery. Known G2 phase regulators were investigated to determine whether they had a role in imposing the cyclin A/ CDK2 dependent G2 delay. Examination of the critical G2 checkpoint arrest protein, Chk1, which also has a role during unperturbed G2/M phases revealed the presence of activated Chk1 in G2 phase, in a range of cell lines. Activated Chk1 levels were shown to accumulate in cyclin A/CDK2 depleted/inhibited cells. Further investigations revealed that Chk1, but not Chk2, depletion could reverse the cyclin A/CDK2 dependent G2 delay. It was confirmed that the accumulative activation of Chk1 was not a consequence of DNA damage induced by cyclin A depletion. The potential of cyclin A/CDK2 to regulate Chk1 revealed that the inhibitory phosphorylations, Ser286 and Ser301, were not directly catalysed by cyclin A/CDK2 in G2 phase to regulate mitotic entry. It appeared that the ability of cyclin A/CDK2 to regulate cyclin B/CDK1 activation impacted cyclin B/CDK1s phosphorylation of Chk1 on Ser286 and Ser301, thereby contributing to the delay in G2/M phase progression. Chk1 inhibition/depletion partially abrogated the cyclin A/CDK2 dependent G2 delay, and was less effective in abrogating G2 phase checkpoint suggesting that other cyclin A/CDK2 dependent mechanisms contributed to these roles of cyclin A/CDK2. In an attempt to identify these other contributing factors another G2/M phase regulator known to be regulated by cyclin A/CDK2, Cdh1 and its substrates Plk1 and Claspin were examined. Cdh1 levels were reduced in cyclin A/CDK2 depleted/inhibited cells although this had little effect on Plk1, a known Cdh1 substrate. However, the level of another substrate, Claspin, was increased. Cdh1 depletion mimicked the effect of cyclin A depletion but to a weaker extent and was sufficient at increasing Claspin levels similar to the increase caused by cyclin A depletion. Co-depletion of cyclin A and Claspin blocked the accumulation of activated Chk1 normally seen with cyclin A depletion alone. However Claspin depletion alone did not reduce the cyclin A/CDK2 dependent G2 delay but this is likely to be a result of inhibition of S phase roles of Claspin. Together, these data suggest that cyclin A/CDK2 regulates a number of different mechanisms that contribute to G2/M phase progression. Here it has been demonstrated that in normal G2/M progression and possibly to a lesser extent in G2 phase checkpoint recovery, cyclin A/CDK2 regulates the level of Cdh1 which in turn affects at least one of its substrates, Claspin, and consequently results in the increased level of activated Chk1 observed. However, the involvement of Cdh1 and Claspin alone does not explain the G2 phase delay observed with cyclin A/CDK2 depletion/inhibition. It is likely that other mechanisms, possibly including cyclin A/CDK2 regulation of Wee1 and FoxM1, as reported by others, combine with the mechanism described here to regulate normal G2/M phase progression and G2 phase checkpoint recovery. These findings support the critical role for cyclin A/CDK2 in regulating progression into mitosis and suggest that upstream regulators of cyclin A/CDK2 activation will also be critical controllers of this cell cycle transition. The pathways that work to co-ordinate cell cycle progression are very intricate and deciphering these pathways, required for normal cell cycle progression, is key to understanding tumour development. By understanding cell cycle regulatory pathways it will allow the identification of the pathway/s and their mechanism/s that become affected in tumourgenesis. This will lead to the development of better targeted therapies, inferring better efficacy with fewer side effects than commonly seen with the use of traditional therapies, such as chemotherapy. Furthermore, this has the potential to positively impact the development of personalised medicines and the customisation of healthcare.

Escape from apoptosis after prolonged serum deprivation is associated with the regulation of the mitochondrial death pathway by Bcl-x(l)

Bcl-x(l) and Bax play important roles in the regulation of apoptosis. This study investigated the involvement of the mitochondrial death pathway and the role of Bcl-x(l) and Bax in the escape from apoptosis after prolonged serum deprivation in Madin-Darby canine kidney (MDCK) cells. Low level apoptosis and basal activity of the mitochondrial death pathway were detectable in normal cell growth. In serum deprivation, mitosis was partially suppressed, and the mitochondrial activity was stimulated. The level of apoptosis continuously rose over 48 h. This rise was concomitant with the increasing presence of cytochrome c in cytosol. However, both apoptosis and cytosolic cytochrome c fell dramatically at 72 h. Elevation of whole cell Bcl-x(l) and redistribution of Bcl-x(l) protein from cytosol to the membrane at 48 h and 72 h was observed. Redistribution of Bax protein from the membrane to cytosol occurred at 24 h, and remained steady to 72 h. Bax/Bcl-x(l) coimmunoprecipitation by anti-Bax antibody showed reduced Bax/Bcl-x(l) interaction at the membrane at 72 h, but not at 24 or 48 h. These results suggest that apoptosis upon serum withdrawal results from the leakage of cytochrome c to cytosol. Amelioration of the leakage of cytochrome c and apoptosis requires not only the increase of Bcl-x(l)/Bax ratio, but also the release of Bcl-x(l) from Bax at the membrane.

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Understanding chromosome disorders and their implications for special educators

More children are now being diagnosed with chromosome abnormalities. Some chromosome disorder syndromes are relatively well known; while others are so rare that there is only limited evidence about their likely impact on learning and development. For educators, a basic level of knowledge about chromosome abnormalities is important for understanding the literature and communicating with families and professionals. This paper describes chromosomes, and the numerical and structural anomalies that can occur, usually spontaneously during early cell division. Distinctive features of various chromosome syndromes are summarised before a discussion of the rare chromosome disorders that are labelled, not with a syndrome name, but simply by a description of the chromosome number, size and shape. Because of the potential within-group variability that characterises syndromes, and the scarcity of literature about the rare chromosome disorders, expectations for learning and development of individual students need to be based on the range of possible outcomes that may be achievable.

Modeling of protein signaling networks in clinical proteomics

Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomicend points in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states.One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor micro environment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination,clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens.