A Different View for an Old Disease: NEDDylation and Other Ubiquitin-Like Post-Translational Modifications in Chronic Lymphocytic Leukemia.

Despite the enormous amount of molecular data obtained over the years, the molecular etiology of chronic lymphocytic leukemia (CLL) is still largely unknown. All that information has enabled the development of new therapeutic approaches that have improved life expectancy of the patients but are still not curative. We must increase our knowledge of the molecular alterations responsible for the characteristics common to all CLL patients. One of such characteristics is the poor correlation between mRNA and protein expression, that suggests a role of post-translational mechanisms in CLL physiopathology. Drugs targeting these processes have indeed demonstrated an effect either alone or in combination with other aimed at specific pathways. A recent article unveiled an increment in ubiquitin-like modifications in CLL, with many protein members of relevant pathways affected. Interestingly, the inhibition of the NEDD8-activating protein NAE reverted a substantial number of those modifications. The present review gets the scarce data published about the role of NEDDylation in CLL together and establishes connections to what is known from other neoplasias, thus providing a new perspective to the underlying mechanisms in CLL.

Elevated FANCA expression determines a worse prognosis in chronic lymphocytic leukemia and interferes with p53 function.

Chronic lymphocytic leukemia (CLL) is characterized by a failure in the mechanisms of apoptosis that leads to an accumulation of mature B cells in peripheral blood, bone marrow, and lymphoid organs. The molecular basis of CLL remains unknown. Certain cytogenetic and molecular markers determine a bad prognosis in CLL. Fanconi anemia complementation (FANC) proteins have been related to chromosomal instability and alterations in the mechanisms of p53 activation, control of cell cycle, and apoptosis. We investigated the role of certain FANC proteins in CLL. Our data identified a group of patients with CLL with high expression of FANCA in peripheral B-CLL cells and we established its relationship with the deletion of 11q23 and a worse prognosis. When we investigated the molecular mechanisms of this bad prognosis, we observed a reduction in the expression of 2 p53 target genes, p21 and ∆Np73, in CLL primary cells transfected with FANCA. Functional studies demonstrated an impairment of p53 by FANCA. Moreover, we obtained evidence of a cooperation between FANCA and the NEDD8-interacting protein NUB1L in the destabilization of p53. For the first time, FANCA is reported as a bad prognosis marker by a mechanism other than its role in the Fanconi anemia-breast cancer DNA repair pathway.-Bravo-Navas, S., Yáñez, L., Romón, Í., Pipaón, C. Elevated FANCA expression determines a worse prognosis in chronic lymphocytic leukemia and interferes with p53 function.

Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

While working with G418-resistant stably transfected cells, we realized the neomycin resistance (NeoR) gene, which encodes the aminoglycoside-3'-phosphotransferase-IIa [APH(3')-IIa], also confers resistance to the nucleoside analog fludarabine. Fludarabine is a cytostatic drug widely used in the treatment of hematologic and solid tumors, as well as in the conditioning of patients before transplantation of hematopoietic progenitors. We present evidence that NeoR-transfected cells do not incorporate fludarabine, thus avoiding DNA damage caused by the drug, evidenced by a lack of FANCD2 monoubiquitination and impaired apoptosis. A screening of other nucleoside analogs revealed that APH(3')-IIa only protects against ATP purine analogs. Moreover, APH(3')-IIa ATPase activity is inhibited by fludarabine monophosphate, suggesting that APH(3')-IIa blocks fludarabine incorporation into DNA by dephosphorylating its active fludarabine triphosphate form. Furthermore, overexpression of the catalytic subunit of the eukaryotic kinase PKA, which is structurally related to APHs, also provides resistance to fludarabine, anticipating its putative utility as a response marker to the drug. Our results preclude the use of Neo marker plasmids in the study of purine analogs and unveils a new resistance mechanism against these chemotherapeuticals.-Sánchez-Carrera, D., Bravo-Navas, S., Cabezón, E., Arechaga, I., Cabezas, M., Yáñez, L., Pipaón, C. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

∆Np73 is capable of inducing apoptosis by co-ordinately activating several BH3-only proteins.

Inactivation of p53 is one of the most relevant events in human cancer, since it allows transformed cells to escape their own proliferation control and leave them irresponsive to drugs that aim to damage their DNA. When p53 falls, other members of its family may become targets to attack tumoural cells. p73 has shown capacity to mediate these attacks. However, its N-terminal truncated isoforms have been associated with oncogenesis due to their capacity to act as dominant negatives of p53 and the transactivation (TA) isoforms of p73. We previously found a relationship between the overexpression of N-terminus-truncated p73 isoform (∆Np73) and that of the proapoptotic gene Bcl-2-interacting killer (BIK). In the present report we demonstrate that ∆Np73-α has the capacity to induce apoptosis through the co-ordinated activation of a group of genes harbouring GC-rich elements in their regulatory regions. ∆Np73-α synergizes with specificity protein (Sp1) on these elements but the overall response of these genes probably depends on the additional presence of consensus p53 elements. We explore the domains of ∆Np73-α involved in this transactivation capacity and found divergences with the previously described functions for them. Moreover, we found that the transforming mutation V12 of HRas impairs this transactivation capacity of ∆Np73-α, further supporting the anti-tumoural function of this later. Our data add complexity to the action of p73 on the induction of apoptosis and tumourogenesis, opening new interpretations to the expression profile of p73 isoforms in different human neoplasias.

Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells' sensitivity to DNA interstrand crosslinking drugs.

Progressive bone marrow failure starting in the first decade of life is one of the main characteristics of Fanconi anemia. Along with the bone marrow failure, this pathology is characterized by congenital malformations, endocrine dysfunction and an extraordinary predisposition to develop cancer. The fact that hematopoietic progenitor cells from subjects with Fanconi anemia are sensitive to both DNA-interstrand crosslinking agents and inflammatory cytokines, which are aberrantly overproduced in these patients, has led to different explanations for the causes of the bone marrow failure. We analyzed STAT1 expression in lymphoblastoid cell lines derived from patients with Fanconi anemia group A and correlated this with aspects of the Fanconi anemia phenotype such as sensitivity to genotoxic agents or to inhibitory cytokines. We provide evidence of overexpression of STAT1 in FANCA-deficient cells which has both transcriptional and post-translational components, and is related to the constitutive activation of ERK in Fanconi anemia group A cells, since it can be reverted by treatment with U0126. STAT1 phosphorylation was not defective in the lymphoblasts, so these cells accumulated higher levels of active STAT1 in response to interferon gamma, probably in relation to their greater sensitivity to this cytokine. On the other hand, inhibition of STAT1 by genetic or chemical means reverted the hypersensitivity of Fanconi anemia group A lymphoblasts to DNA interstrand crosslinking agents. Our data provide an explanation for the mixed sensitivity of Fanconi anemia group A cells to both genotoxic stress and inflammatory cytokines and indicate new targets for the treatment of bone marrow failure in these patients.

BIK (NBK) is a mediator of the sensitivity of Fanconi anaemia group C lymphoblastoid cell lines to interstrand DNA cross-linking agents.

FA (Fanconi anaemia) is a rare hereditary disorder characterized by congenital malformations, progressive bone marrow failure and an extraordinary predisposition to develop cancer. At present, 15 genes have been related to this condition and mutations of them have also been found in different types of cancer. Bone marrow failure threatens the life of FA patients during the first decade of their life, but the mechanisms underlying this process are not completely understood. In the present study we investigate a possible imbalance between the expression of pro- and anti-apoptotic proteins as a cause for the hypersensitivity of FANCC (FA, complementation group C)-deficient cells to genotoxic stress. We found a BIK (Bcl-2 interacting killer) over-expression in lymphoblastoid cell lines derived from FA-C patients when compared with their phenotypically corrected counterparts. This overexpression has a transcriptional basis since the regulatory region of the gene shows higher activity in FANCC-deficient cells. We demonstrate the involvement of BIK in the sensitivity of FA-C lymphoblasts to interstrand DNA cross-linking agents as it is induced by these drugs and interference of its expression in these cells preserves their viability and reduces apoptosis. We investigate the mechanism of BIK overexpression in FANCC-deficient cells by analysing the activity of many different signalling pathways in these cells. Finally, we provide evidence of a previously undescribed indirect epigenetic regulation of BIK in FA-C lymphoblasts mediated by ΔNp73, an isoform of p73 lacking its transactivation domain that activates BIK through a proximal element in its promoter.

Association of the aromatase gene alleles with BMD: epidemiological and functional evidence.

BMD has a strong heritable component. Estrogen activity depends on the aromatization of androgenic precursors in nongonadal tissues both in postmenopausal women and men. Therefore, aromatase is an appealing candidate gene to explain, in part, the genetic component of BMD. In fact, an association between aromatase polymorphisms and BMD has been previously reported in some relatively small groups. In this study, we explored the relationship between several SNPs in the aromatase region and hip BMD in 1163 postmenopausal women. We found significant differences across genotypes, particularly in older women. The BMD differences between homozygous women with opposing genotypes were 4.2% in the whole group and 7.3% in women >67 yr of age. Body weight was significantly associated with BMD also, but there was no evidence for a statistically significant interaction between body weight and aromatase polymorphisms. Electrophoretic mobility shift assays suggested the binding of the CEBPss transcription factor to the C/G rs1062033 locus, located approximately 12 kb upstream of the translation start site. Experiments of transient transfection of osteoblastic cells with luciferase reporters showed differences in the transcriptional activity of alleles C and G at this locus, with different responses to the co-transfection of a CEBPss expression vector. Furthermore, evidence for differential allelic expression was found in bone tissue samples. In conclusion, polymorphisms in a 12-kb region of the aromatase gene are associated with BMD in postmenopausal women, particularly during the late postmenopausal period. In vitro functional studies point to rs1062033 as a true regulatory polymorphism.

Elevated levels of IL-1beta in Fanconi anaemia group A patients due to a constitutively active phosphoinositide 3-kinase-Akt pathway are capable of promoting tumour cell proliferation.

FA (Fanconi anaemia) is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present manuscript we describe an anomalous high level of the proinflammatory cytokine IL-1beta (interleukin-1beta) present in the serum of FA patients. The elevated levels of IL-1beta were completely reverted by transduction of a wild-type copy of the FancA cDNA into FA-A (FA group A) lymphocytes. Although the transcription factor NF-kappaB (nuclear factor-kappaB) is a well established regulator of IL-1beta expression, our experiments did not show any proof of elevated NF-kappaB activity in FA-A cells. However, we found that the overexpression of IL-1beta in FA-A cells is related to a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway in these cells. We provide evidence that the effect of Akt on IL-1beta activation is mediated by the inhibition of GSK3beta (glycogen synthase kinase 3beta). Finally, our data indicate that the levels of IL-1beta produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these results demonstrate that the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1beta through an NF-kappaB-independent mechanism and that this overproduction activates the proliferation of tumour cells.

Deficiency of the NF-kappaB inhibitor caspase activating and recruitment domain 8 in patients with rheumatoid arthritis is associated with disease severity.

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-kappaB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-kappaB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-kappaB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-alpha and the p65 subunit of NF-kappaB. Moreover, TNF-alpha and overexpression of p65 induced the formation of NF-kappaB-CARD8 promoter complexes. Thus, CARD8 may control NF-kappaB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-kappaB transcriptional activity, and abrogates the binding of NF-kappaB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-kappaB target genes (cIAP, A1), in response to TNF-alpha. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-kappaB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

Transcriptional activation of the proapoptotic bik gene by E2F proteins in cancer cells.

BH3-only proteins are required for execution of apoptotic cell death. We have found that one of these proteins, Bik, is strongly induced in cancer cells treated with chemotherapeutic agents. Furthermore, we showed that chemotherapy-induced expression of bik is independent of p53. Consistent with its pro-apoptotic activity, blockade of bik expression reduces the adriamycin-mediated apoptotic cell death. We also found that the bik gene is transcriptionally activated by E2F proteins. Consistently, adriamycin induces the E2F-bik pathway. In addition, E2Fs transactivate bik by a p53-independent mechanism. Thus, our data indicate that transcriptional regulation of bik contributes to the efficient apoptotic response to chemotherapeutic agents.

A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

Defective binding of transcriptional repressor ZEB via DNA methylation contributes to increased constitutive levels of p73 in Fanconi anemia cells.

Little is known about the molecular mediators of the Fanconi anemia (FA) pathway involved in the machinery that maintains genomic integrity. Here, we report that the levels of p73 and its target genes, are increased in cells derived from FA patients belonging to complementation group A (FA-A). Moreover, functional correction of FA-A cells by gene transfer reduces the expression of p73. We also demonstrate that DNA methylation contributes to increased levels of p73 in FA-A cells by hampering the binding of the transcriptional repressor ZEB to an intronic regulatory region of the p73 gene. Together, our data may help explain the susceptibility of these cells to DNA damaging agents.

Ipaf is upregulated by tumor necrosis factor-alpha in human leukemia cells.

Ipaf has been associated with apoptosis, cytokine processing, and nuclear factor-kappaB activation. Here, we describe that Ipaf is highly expressed in myelomonocytic cells and that the mRNA levels of Ipaf progressively increase during differentiation of CD34(+) progenitors to granulocytes and monocytes. Additionally, treatment with tumor necrosis factor-alpha and exposure to UV radiation induced the transcriptional activation of Ipaf in human leukemia HL-60 cells. Thus, Ipaf may contribute to modulate the response of myeloid cells to genotoxic and pro-inflammatory stimuli.

Jun N-terminal kinase activity and early growth-response factor-1 gene expression are down-regulated in Fanconi anemia group A lymphoblasts.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome characterized by cellular sensitivity to genotoxic agents. In recent years, FA proteins have been associated with different molecules involved in signal transduction, which has raised the interest in FA-dependent signaling pathways. Here, we report that the c-Jun N-terminal kinase (JNK) fails to phosphorylate in response to UV radiation and treatment with mitomycin C in FA lymphoblast cells derived from type A patients (FA-A). Furthermore, defective kinase activity seems to be specific for JNK, because extracellular signal-regulated kinase (ERK) responded to the proper stimuli in FA-A cells. We also demonstrate that the early growth-response factor-1 (Egr-1), a JNK downstream target gene that is normally induced by genotoxic stress, is not upregulated in UV-treated FA-A cells. Moreover, FA-A cells are more sensitive to apoptosis than control lymphoblasts. Both JNK and Egr-1 may be part of a pathway triggered by FA proteins, because functional correction of FA-A cells by gene transfer restores, at least in part, JNK activation and Egr-1 expression after UV exposure. Together, our data suggest that activation of JNK and expression of Egr-1 gene in B lymphoblasts mediate a cellular response to genotoxic agents that may be induced by FA proteins.

Egr-1 induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone: combinatorial regulation by transcription factors cyclic adenosine 3',5'-monophosphate regulatory element binding protein, serum response factor, sp1, and early growth response factor-1.

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within -164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at -64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the -164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

Mitogen-activated protein kinase routes as targets in the action of diaza-anthracene compounds with a potent growth-inhibitory effect on cancer cells.

1,8-Diaza-anthracene-tetraones are novel intermediates in the synthesis of the antifolate antibiotic diazaquinomycin A that was found before to have potent antitumor activity. Three of them (CV65, CV66, and CV70) were found to inhibit growth of a panel of several human tumor cell lines. The IC50s ranged from 0.05 to 1.5 microM and are comparable with that of doxorubicin. Among the three drugs, CV70 showed the highest cytotoxic activity. The growth-inhibitory action of these compounds was unrelated to the p53 status of the cells. At micromolar concentrations, all three compounds induced apoptosis, CV70 being the most proapoptotic. The incubation of HeLa cells with CV65, CV66, and CV70, at concentrations between 10 and 20 microM, inhibited the activation of c-Jun NH2-terminal kinase by various stimuli and prevented growth factor-induced extracellular signal-regulated kinase (ERK) 5 activation. At least one drug, CV65, also inhibited p38. This was surprising because proapoptotic antitumor drugs activate stress signaling pathways. Activation of ERK1/ 2 by growth factors or phorbol esters was unaffected by preincubation of cells with CV compounds. In vitro, CV compounds inhibit the enzyme quinone reductase but not c-Jun NH2-terminal kinase or ERK5. Because doxorubicin also inhibits quinone reductase, we conclude that the inhibitory effect of CV compounds on stress signaling kinases is not a direct effect on the kinases and is likely attributable to upstream elements of the activation cascades.

Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B activation.

Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-kappaB, a transcription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn's disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34(+) hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14(+) (monocytes), CD15(+) (granulocytes), and CD40(+)/CD86(+) (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNFalpha resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-kappaB binding element that was required for transcriptional activity in response to TNFalpha. Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative IkappaBalpha blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNFalpha or lipopolysaccharide, both p50 and p65 subunits of NF-kappaB were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-kappaB, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.

Induction of chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI ) gene expression is mediated by ETS factor binding sites.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI, or NR2F1) is an orphan nuclear receptor that plays a major role in the development of the nervous system. We show here that three ETS response elements in the COUP-TFI promoter mediate its transcription. A reporter gene containing these ETS binding sites is activated by Ets-1, while the same reporter with point mutations on all three ETS response elements is not. We also show that Ets-1 binds to these response elements and that other ETS factors also transactivate the COUP-TFI promoter. In addition, COUP-TFI is coexpressed with some ETS factors in the mouse embryo. These results indicate that members of the ETS family can activate COUP-TFI gene expression.