Novel CTCF binding at a site in exon1A of BCL6 is associated with active histone marks and a transcriptionally active locus.

BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin.

MYC oncogene in myeloid neoplasias

MYC is a transcription factor that regulates many critical genes for cell proliferation, differentiation, and biomass accumulation. MYC is one of the most prevalent oncogenes found to be altered in human cancer, being deregulated in about 50 % of tumors. Although MYC deregulation has been more frequently associated to lymphoma and lymphoblastic leukemia than to myeloid malignancies, a body of evidence has been gathered showing that MYC plays a relevant role in malignancies derived from the myeloid compartment. The myeloid leukemogenic activity of MYC has been demonstrated in different murine models. Not surprisingly, MYC has been found to be amplified or/and deregulated in the three major types of myeloid neoplasms: acute myeloid leukemia, myelodysplastic syndromes, and myeloproliferative neoplasms, including chronic myeloid leukemia. Here, we review the recent literature describing the involvement of MYC in myeloid tumors (AU)

MYC antagonizes the differentiation induced by imatinib in chronic myeloid leukemia cells through downregulation of p27(KIP1.).

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and dasatinib. In the CML-derived K562 cell line, low concentrations of imatinib induce proliferative arrest and erythroid differentiation. We found that imatinib upregulated the cell cycle inhibitor p27(KIP1) (p27) in a time- and -concentration dependent manner, and that the extent of imatinib-mediated differentiation was severely decreased in cells with depleted p27. MYC (c-Myc) is a transcription factor frequently deregulated in human cancer. MYC is overexpressed in untreated CML and is associated to poor response to imatinib. Using K562 sublines with conditional MYC expression (induced by Zn(2+) or activated by 4-hydroxy-tamoxifen) we show that MYC prevented the erythroid differentiation induced by imatinib and dasatinib. The differentiation inhibition is not due to increased proliferation of MYC-expressing clones or enhanced apoptosis of differentiated cells. As p27 overexpression is reported to induce erythroid differentiation in K562, we explored the effect of MYC on imatinib-dependent induction of p27. We show that MYC abrogated the imatinib-induced upregulation of p27 concomitantly with the differentiation inhibition, suggesting that MYC inhibits differentiation by antagonizing the imatinib-mediated upregulation of p27. This effect occurs mainly by p27 protein destabilization. This was in part due to MYC-dependent induction of SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation.

[Conservative treatment of the pectus carinatum]. / Tratamiento conservador del pectus carinatum.

INTRODUCTION: Pectus carinatum (PC) is a deformity that involves the protrusion of the anterior chest wall. It is 10 times less frequent than pectus excavatum. It has a progressive growth and is more common with men. There are two different types, the lower or condrocorporal which is the most common one, and the upper or condromanubrial. Most of the time there are no cardio-respiratory symptoms. OBJECTIVE: We present our experience in the orthopedic treatment of the pectus carinatum. METHOD: Retrospective review of patients treated in our hospital from 2002 until 2009. Patients were treated with observation, aerobic exercises, postural change and/or compression braces. Literature review was performed of the treatment for this pathology. RESULTS: 18 patients have been diagnosed with PC, 16 were men and 2 women. All were treated in a nonoperative way. Only 11 of them used a compression brace. We missed two follow-ups and another has just yet begun to achieve proper results. All the rest have had excellent results with nonoperative treatment. None of them have had a surgical treatment. CONCLUSION: The PC is a disease that most often is a cosmetic problem, with no impact on a cardio-respiratory level. Classically it has been a surgical entity. In our experience we have found that the orthopedic method is an effective alternative, safe and with a significant reduction in morbidity. But we need the collaboration of the patient to accept and maintain continuity in the use of the prostheses.

[Drawing of blood and anemia of the critical patient]. / Extracciones de sangre y anemia del paciente crítico.

UNLABELLED: OBJECTIVE. To determine the hemoglobin level variations in non-bleeding patients, its possible relationship with the blood volume drawn and number of extractions. METHODS: An observational, prospective study conducted from April to August 2007. Hemoglobin values during the ICU stay, blood volume drawn in each phlebotomy, fluid balance, APACHE II and other demographic variables were determined. RESULTS: One hundred and twenty four patients were studied. Of these, 59.7% experienced a mild decrease of hemoglobin levels (< 2g/dl) whereas 21.8% presented a more severe reduction (> 2g/dl). These decreases were correlated with the blood volume withdrawn and number of phlebotomies performed (r= 0.557, p < 0.000). There was no significant relationship between fluid balance and decrease in hemoglobin. CONCLUSION. Anemia in critical ill patient seems to be related to blood volume drawn and number of phlebotomies.

Gene expression regulation and cancer

Gene expression is mostly controlled at the level of the transcription initiation. The transcription control regions of protein-encoding genes include: the core promoter, where RNA polymerase II binds, the proximal and distal promoter, responsible for gene expression regulation, and the enhancers and silencers. Chromatin represents an additional level of regulation of gene expression. The switching between inactive and active chromatin is closely related to the activity of histone-modifying enzymes and chromatin-remodelling complexes. Transcriptional activation of a gene requires the binding of specific transcription factors to regulatory DNA elements, the opening of the chromatin, the binding of Mediator, and the assembly of the preinitiation complex with RNA polymerase and RNA synthesis initiation. Transcription factors ultimately transduce the proliferation signals elicited by growth factors. Moreover, many human oncogenes encode for transcription factors, and some of them are prevalent in particular neoplasias (e.g., MYC, MLL, PML-RARa). Also, some of the most prominent tumor suppressors (e.g. p53) are transcription factors (AU)

[Gastric heterotopia and cleft palate. Deformation sequence or syndromic association?]. / Heterotopia gástrica y fisura palatina. Secuencia deformativa o asociación sindrómica?

Gastric heterotopia is more frequently in small intestine, gall bladder, biliary tract, Meckel's diverticulum, colon and rectum, but it can be found in other locations. It is especially rare in oral cavity. We only find one case that concurs with cleft palate. We present the case of a neonate with gastric heterotopia and cleft palate in addition to other congenital malformations not related at first. We make a brief revision of literature, showing the pathogenesis of heterotopia and its possible association with the cleft palate.

[Presurgical orthopedic treatment for cleft lip and palate]. / Uso de la ortopedia prequirúrgica en pacientes con fisura palatina: nuestra experiencia.

INTRODUCTION: A multidisciplinary approach with several specialits allows a complete treatment for Cleft Lip and Palate. We show our experience in presurgical orthopedic treatment in these patients, their advantages, their problems and the results. MATERIAL AND METHODS: Since 1999 presurgical orthopedy has been applied to 12 patients (3 bilateral cleft lip and palate and 9 unilateral cleft lip and palate). This approach was applied when there was a long distance between the alveolar segments. A palate mould and the location of Latham's appliance have been made in the operating room under general anesthesia. The patients were controlled by the orthodoncist and Latham's appliance was removed when cleft lip was closed. RESULTS: Latham's appliance was kept for 4-7 weeks with once a week controls until the distance between the maxillary segments was less than 1 mm; in bilateral cases of cleft lip and palate the premaxilla was moved between lateral segments. Then, lip closure and nasoplasty was made and, sometimes, an obturador was placed. CONCLUSIONS: Latham's appliance permit to achieve a perfect alignment of alveolar segments decreasing the soft tissues tension and facilitating the lip surgery, thus, a better aesthetic and functional results can be achieved. A more anatomic position of palate can be made and easier future orthopedic treatments are possible.

Diagnóstico prenatal de hemangioma cutáneo fetal / Antenatal diagnosis of fetal cutaneous hemangioma

Los hemangiomas cutáneos son la malformación congénita más frecuente en el recién nacido; sin embargo, los casos descritos en la bibliografía son escasos. Presentamos en este artículo un caso clínico de un hemangioma cutáneo fetal de localización abdominal diagnosticado prenatalmente mediante ultrasonografía en el tercer trimestre y confirmado posteriormente tras el nacimiento. Asimismo evaluamos las distintas posibilidades diagnósticas ante un hallazgo de estas características, así como su posible manejo terapéutico (AU)

Variability in the levels of PML-RAR alpha fusion transcripts detected by the laboratories participating in an external quality control program using several reverse transcription polymerase chain reaction protocols.

BACKGROUND AND OBJECTIVES: The detection of PML-RAR by reverse transcription (RT) polymerase chain reaction (PCR) in acute promyelocytic leukemia (APL) patients who are in hematologic remission influences therapeutic decision making in several trials. In the light of this, the Spanish group has recently designed an external quality assessment program (EQAP) of RT-PCR detection of PML-RAR, which includes a study of sensitivity of the participating laboratories. DESIGN AND METHODS: Eighteen laboratories were involved in the program. Ten laboratories followed the method of Biondi et al., 5 employed that of Borrow et al. and the 3 remaining used other protocols. The sensitivity was studied in five rounds of quality control. The first two shipments consisted of dilutions of NB4 RNA into non-APL RNA. The third round consisted of serial dilutions of the NB4 cell line into HL60 cells. The fourth and five rounds consisted of plasmid dilutions containing the bcr1 and bcr3 PML-RAR isoforms. RESULTS: The results showed that the distinct methods allow detection of the PML-RAR hybrid up to a dilution of 10(-4), and exceptionally, up to 10(-5). The laboratories following the method of Biondi et al. usually detected the 10(-3) dilution and less frequently the 10(-4) one, whereas those using other methods usually detected PML-RAR transcript in the 10(-4) dilution, and less commonly in the 10(-5) dilution. However, each of the PCR methods used by EQAP participating laboratories successfully detected at least 50 copies of PML-RAR alpha fusion transcript in plasmid dilution controls. INTERPRETATION AND CONCLUSIONS: The results point to heterogeneous sensitivity amongst participating laboratories. This may reflect differences in methodology, although variations in sample quality may also account for discrepant findings.

c-Myc inhibits CD11a and CD11c leukocyte integrin promoters.

The c-Myc transcription factor is an important regulator of cell growth and differentiation, and its gene repression ability seems to play a key role in Myc-mediated cellular transformation. Since Myc overexpression has been associated with reduced expression of beta1 and beta2 integrins, we have investigated the role of c-Myc on CD11a and CD11c transcription. c-Myc inhibited CD11a and CD11c integrin promoter activity in co-transfection experiments, and similar repression was obtained in cells where c-Myc expression (KmycB) or activity (Rat-1 c-MycER) is inducible. The c-Myc repression on the CD11c promoter was independent of the USF-binding site (USF-150), other putative Myc-binding elements, or the integrity of the initiator (Inr)-like sequence present at the major transcriptional start site. Analysis of deletion and mutant promoter constructs revealed that, in the absence of additional upstream cisacting elements, an AP-1-binding site at -60 (AP1-60) is required for c-Myc repressor activity. The c-Myc repressor activity on both integrin promoters was abrogated by deletion of c-Myc residues 106-143, a domain involved in Inr-dependent transcriptional repression. These results demonstrate a direct effect of c-Myc on integrin gene transcription and suggest the existence of a c-Myc-dependent mechanism for coupling leukocyte integrin expression to the cell proliferative state.

c-Myc antagonizes the effect of p53 on apoptosis and p21WAF1 transactivation in K562 leukemia cells.

c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two different methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53Vall35 mutant, which adopts a wild-type conformation at 32 degrees C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc significantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21WAF1, Bax and cytomegalovirus promoters. The impairment of p21WAF1 transactivation by c-Myc was confirmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21WAF1 mRNA protein were significantly reduced by c-Myc, while Bax levels were unaffected. Consistently, c-Myc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer.

H-, K- and N-Ras inhibit myeloid leukemia cell proliferation by a p21WAF1-dependent mechanism.

Mutated ras genes are frequently found in human cancer. However, it has been shown that oncogenic ras inhibits growth of primary cells, through pathways involving p53 and the cell cycle inhibitors p16INK4a and p19ARF. We have analysed the effect of the ectopic expression of the three mammalian ras genes on the proliferation of K562 leukemia cells, which are deficient for p53, p16INK4a, p15INK4b and p19ARF genes. We have found that high expression levels of both wild-type and oncogenic H-, K- and N-ras inhibit the clonogenic growth of K562 cells. Induction of H-rasV12 expression in K562 transfectants retards growth and this effect is accompanied with an increase of p21WAF1 mRNA and protein levels. Furthermore, p21WAF1 promoter is activated potently by oncogenic ras and less pronounced by wild-type ras. This induction is p53-independent since a p21WAF1 promoter devoid of the p53 responsive elements is still activated by Ras. Finally, inhibition of p21WAF1 expression by an antisense construct partially overcomes the growth inhibitory action of oncogenic H-ras. Altogether, these results indicate that the antiproliferative effect of ras in myeloid leukemia cells is associated to the induction of p21WAF1 expression and suggest the existence of p19ARF and p16INK4a-independent pathways for ras-mediated growth inhibition.

Surgery of subglottic stenosis in neonates and children.

The excellent management of patients in the different Intensive Care Units has decreased mortality but, as a side effect, we have to treat an increasing number of patients with airway problems secondary to prolonged intubation. The clinical records of patients diagnosed of acquired or congenital subglottic stenosis (SE) between 1990 and 1995 were retrospectively reviewed. Types of treatment included conservative, endoscopic, and open surgery: anterior cricoid split (ACS), anterior laryngotracheoplasty (ALTP) and anteroposterior laryngotracheoplasty (APLTP). 46 patients had SE: 7 congenital and 39 acquired. According to Cotton's classification 13 had grade I, 16 grade II, 12 grade III and none grade IV. Eleven of twelve cases treated conservatively did well (92%); one out of six patients managed endoscopically required further surgery (7%); good results were obtained in 5 of 7 cases treated by ACS (71 %); 8 out of 9 patients treated by ALTP did well (89%) and 7 out of 8 managed by APLTP had good results (87.5%). One iatrogenic suture dehiscence required further surgery. There is no statistical difference in the complication rate between patients treated conservatively and those treated by open surgery, while the mean hospital stay was higher in the latter (p < 0.05). An appropriate surgical technique should be offered to those patients with SE who do not do well with conservative management, since these techniques have yielded good results with a low rate of complications. Long-term follow-up shows the absence of recurrence.

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.

Translocation (10;11)(p13;p15) in an infant acute myeloid leukemia with MLL gene rearrangement.

Molecular rearrangements of the MLL gene at the 11q23 region have been identified in most cases of infant leukemia, regardless of the phenotype. We present a case of acute myeloid leukemia which coexpressed myeloid and lymphoid markers in a 12-month-old girl. Karyotype analysis revealed the presence of a thus far unreported translocation t(10;11)(p13;p15). Although no 11q23 abnormalities were cytogenetically detectable, an MLL gene molecular rearrangement was found.

Apoptosis and mitotic arrest are two independent effects of the protein phosphatases inhibitor okadaic acid in K562 leukemia cells.

Treatment of human myeloid leukemia K562 cells with the serine/threonine protein phosphatases inhibitor okadaic acid induced mitotic arrest followed by apoptosis in a synchronized manner. The effect was observed at drug concentrations that inhibited the protein phosphatase type 2A but not type 1. We investigated whether apoptosis was a consequence of the preceding mitosis arrest or was induced independently by okadaic acid. We found that (1) apoptosis, but not mitotic arrest, was inhibited in cells with constitutive expression of Bcl-2; (2) pretreatment of cells with the DNA synthesis inhibitor hydroxyurea blocked the mitotic arrest but not the apoptosis mediated by okadaic acid; (3) down-regulation of c-myc gene was associated with apoptosis, but not with mitotic arrest; and (4) inhibition of protein synthesis abrogated mitotic arrest, but not apoptosis. The results suggest that inhibition of protein phosphatase 2A by okadaic acid provokes mitotic arrest and apoptosis of leukemia cells by independent mechanisms.

Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells.

CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels.

Preliminary experience in external quality control of RT-PCR PML-RAR alpha detection in promyelocytic leukemia.

To standardize the results obtained in PML/RAR alpha RT-PCR detection by laboratories of hospitals involved in the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) LPA-96, designed for the treatment of acute promyelocytic leukemia (APL), cDNA samples obtained by reverse transcription of RNA from bone marrow samples of patients with APL were sent to participating laboratories. During the first year of this external quality assessment trial nine samples were tested by a maximum of 12 laboratories. The control gene was satisfactorily amplified in 90% of the samples (62 of 69 samples), supporting the adequacy of the cDNA to be used as control sample. There was an 83% concordance between laboratories for PML/RAR alpha detection with similar results for the type of PML/RR alpha rearrangements. However, 17% disagreement still remained, attributable to low sensitivity or inadequacy of methods followed. The results stressed the need for implementation of an external quality assessment scheme to ensure the standardization of the results.