Olle Sangfelt

Control of Iron Homeostasis by an Iron-Regulated Ubiquitin Ligase

Iron Sensor Intracellular iron is an essential cofactor for many proteins, but can also damage macromolecules, so its levels are carefully controlled. Cellular iron homeostasis is mediated by iron regulatory proteins that regulate the expression of genes involved in iron uptake and storage. However, it is not clear how cells sense iron bioavailability (see the Perspective by Rouault). Using different approaches, Salahudeen et al. (p. 722, published online 17 September) and Vashisht et al. (p. 718, published online 17 September) have identified the F-box protein FBXL5 as a human iron sensor. FBXL5 is part of an E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins and thereby cellular iron levels. It contains a hemerythrin domain that binds iron and acts as an iron-dependent regulatory switch, causing the degradation of FBXL5 under low iron conditions. This alternative pathway for the regulation of iron homeostasis has implications for both normal cellular physiology and disease. A vertebrate hemerythrin domain in an E3 ubiquitin ligase complex senses and regulates cellular iron levels. Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.

Mechanisms of Interferon-alpha induced apoptosis in malignant cells.

Interferon alpha (IFNα) has been used in the treatment of several types of cancer for almost 30 years, yet the mechanism(s) responsible for its anti-tumoral action remains unknown. A variety of cellular responses, including inhibition of cell growth and induction of apoptosis are induced by IFNs, and apoptotic induction by this cytokine has been proposed to be of importance for both its anti-tumoral in addition to its anti-viral responses. The aim of the present study was to delineate the pathways activated during IFNα-induced apoptosis in malignant cell lines. We found that apoptosis induced by IFNα was associated with activation of caspases-1, -2, -3, -8 and -9 and that this activation was a critical event. Caspase-3 activation was dependent on activity of caspases-8 and -9, moreover, activation of caspase-8 seems to be the upstream event in IFNα-induced caspase cascade. We also found loss of mitochondrial membrane potential as well as release of cytochrome c post IFN-treatment, clearly implicating the involvement of mitochondria in IFN-mediated apoptosis. Furthermore, IFNα-induced apoptosis was found to be independent on interactions between the Fas-receptor and its ligand. These studies form the basis for further investigations aiming to improve IFN therapy and the development of future strategies to overcome the IFN resistance observed in some malignancies.

Molecular mechanisms underlying interferon-α-induced G0/G1 arrest: CKI-mediated regulation of G1 Cdk-complexes and activation of pocket proteins

One prominent effect of IFNs is their cell growth-inhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the effect of IFN-α treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-αs cell growth-inhibitory effects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are significantly reduced. In addition to p21/p15 induction, IFN-α regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is first an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-α not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-α response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-α causes no detectable changes in p27 expression and, furthermore, no effects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-α treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes.

DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-15a and miR-16-1

The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.

Induction of Cip/Kip and Ink4 cyclin dependent kinase inhibitors by interferon-α in hematopoietic cell lines

One prominent effect of IFNs is their cell growth inhibitory activity. The exact molecular mechanism behind this inhibition of proliferation remains to be elucidated. Possible effectors for IFN-induced growth inhibition are the recently discovered cyclin-dependent kinase inhibitors. The effect of IFN-α treatment on the members of the Ink4 and Cip/Kip families of Cdk inhibitors was investigated in three hematopoietic cell lines Daudi, U-266 and H9. Two of these cell lines, Daudi and U-266, respond to IFN-α by G1 arrest, whereas the H9 cell line is not growth arrested by IFN-α. We show that a p53-independent upregulation of p21 mRNA occurs following IFN-α treatment in all three cell lines. In Daudi and U-266 cells, the mRNA induction is accompanied by an increase in p21 protein, followed by an increased binding of p21 to Cdk2 and a subsequent decrease in Cdk2 activity, temporally coinciding with G1 arrest. In both these cell lines, there was also an increased binding of p21 to Cdk4. In contrast, p21 protein was not expressed in H9 cells, despite high levels of p21 mRNA following IFN-α treatment. In U-266 cells, IFN-α increased not only p21 but also p15 mRNA and protein levels, followed by an increased association of p15 with Cdk4. Furthermore, IFN-α treatment caused a four- to sixfold induction of the p16 E1β transcript in U-266 cells. Expression levels of the other Ink4 and Cip/Kip Cdk inhibitors were not induced by IFN treatment in any of the cell lines. We conclude that IFN-α can act as a potent regulator of Cdk-inhibitor expression, correlating with decreased Cdk activity and cell growth inhibition. One mechanism for resistance to IFN may be loss of the ability of cells to upregulate these proteins.

Mutation of hCDC4 Leads to Cell Cycle Deregulation of Cyclin E in Cancer

hCDC4, the gene that encodes the F-box protein responsible for targeting cyclin E for ubiquitin-mediated proteolysis, has been found to be mutated in a number of primary cancers and cancer-derived cell lines. We have observed that functional inactivation of hCDC4 does not necessarily correlate with elevated levels of cyclin E in tumors. Here we show, however, that hCDC4 mutation in primary tumors correlates strongly with loss of cell cycle regulation of cyclin E. Similarly, a breast carcinoma-derived cell line mutated for hCDC4 exhibits cell cycle deregulation of cyclin E, but periodic expression is restored by reintroducing hCDC4 via retroviral transduction. Conversely, small interfering RNA-mediated silencing of hCdc4 deregulates cyclin E with respect to the cell cycle. These results indicate that hCdc4 function is an absolute prerequisite for cell cycle regulation of cyclin E levels, and loss of hCdc4 function is sufficient to deregulate cyclin E.

Involvement of the Ink4 proteins p16 and p15 in T-lymphocyte senescence.

Little is known about the molecular background to senescence in T-lymphocytes. In fibroblast systems replicative senescence has been shown to correlate with a number of changes in the expression of the proteins normally regulating progression through the G1 phase of the cell cycle, and recently the Ink4 inhibitor p16 was implicated as a central regulator of replicative senescence in human fibroblasts. It has, however, been claimed that p16 is not expressed in T-lymphocytes. In the present study we have analysed G1 regulating proteins in ageing human T-lymphocytes. We show that PHA and IL-2 stimulated T-lymphocytes cease to proliferate after around 20 population doublings, these cells can not thereafter be restimulated to growth, and were also found to exhibit markers for senescence. We found that T-lymphocytes accumulate p16 and p15 protein during successive population doublings and display high levels of these proteins as they enter into replicative senescence. There was also an increased binding of p16 to the Cdk6 kinase in senescent cells, and a decreased Cdk6 as well as Cdk2 kinase activity. The levels of other G1 regulating proteins were also altered in the senescent cells, such as slightly elevated levels of p21/WAF1, and downregulation of Cdk2 and cyclinD3. The levels of p27/Kip1 is down regulated in proliferating cells but rise to approximately 15% of the levels in un-stimulated quiescent cells. As a high proportion of T-cell childhood acute lymphoblastic leukaemias have deletions of both p15 and p16, our data suggest that inactivation of these genes makes it possible for leukemic cells to avoid senescence.

MiRNA-27a controls FBW7/hCDC4-dependent cyclin E degradation and cell cycle progression

The F-box protein FBW7/hCDC4 is a tumor suppressor that acts as the substrate recognition component of an SCF ubiquitin ligase that targets numerous oncoproteins for proteasomal degradation. In this study, we investigated whether FBW7 is regulated by microRNAs, using a screen combining bioinformatic analysis, luciferase reporters and microRNA libraries. The ubiquitous miR-27a was identified as a major suppressor of FBW7 and in line with this, miR-27a prohibited ubiquitylation and turnover of the key FBW7 substrate cyclin E. Notably, we found that miR-27a only suppresses FBW7 during specific cell cycle phases, relieving its negative impact at the G1 to S-phase transition, prior to cyclin E protein degradation. We also demonstrate that attenuation of FBW7 by miR-27a overexpression leads to improper cell cycle progression and DNA replication stress, consistent with dysregulation of cyclin E expression. Finally, in the context of human cancer, miR-27a was discovered to be generally overexpressed in pediatric B-ALL and its expression to be inversely correlated with that of FBW7 in hyperdiploid cases of B-ALL. These data provide evidence for microRNA-mediated regulation of FBW7, and highlight the role of miR-27a as a novel factor fine-tuning the periodic events regulating cell cycle progression.

How does interferon exert its cell growth inhibitory effect

Abstract: The interferons (IFNs) have become accepted therapy in a range of haematological and non‐haematological malignancies. The mechanism behind IFNs antitumour action is, however, unclear. Interferons (IFNs) are capable of modulating a variety of cellular responses. One prominent effect of IFNs is their cell growth inhibitory activity, which has also been suggested to be of major importance in their antitumour action. In the present review we will discuss the cellular events leading to a decreased number of cells following IFN treatment, the molecular mechanisms underlying these phenomena, and the importance of these effects in a clinical perspective.

The Fbxw7/hCdc4 tumor suppressor in human cancer.

Fbxw7/hCdc4 is a member of the F-box family of proteins, which function as interchangeable substrate recognition components of the SCF ubiquitin ligases. SCF(Fbxw7/hCdc4) targets several important oncoproteins including c-Myc, c-Jun, cyclin E1, and Notch, for ubiquitin-dependent proteolysis. Recent studies have shown that FBXW7/hCDC4 is mutated in a variety of human tumor types, suggesting that it is a general tumor suppressor in human cancer. Alteration of Fbxw7/hCdc4 function is linked to defects in differentiation, cellular proliferation, and genetic instability. In this review, we summarize what is known about Fbxw7/hCdc4-mediated degradation in the regulation of cellular proliferation and discuss how alteration of its function contributes to human tumorigenesis.