Carlos Richard

Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells

We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undifferentiated state and to undergo drug-mediated apoptosis.

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°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.

Myc Inhibits p27-Induced Erythroid Differentiation of Leukemia Cells by Repressing Erythroid Master Genes without Reversing p27-Mediated Cell Cycle Arrest

ABSTRACT Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn2+) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G1 arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation.

Scedosporium inflatum infection in immunocompromised haematological patients

We report four cases of Scedosporium inflatum (S. inflatum) infection in severely immunocompromised haematological patients. Six well‐documented cases of S. inflatum disseminated infection in haematological patients have been reported: four in Australia and two in Spain. Their clinical and pathological characteristics are heterogenous, particularly in the Australian cases. However, the clinical and pathological profile emerging from our and other Spanish cases is homogenous and very similar to the clinico‐pathological spectrum of other disseminated mycoses, including Aspergillus and S. apiospermum. The optimal treatment of S. inflatum infection is unknown and the outcome in haematological patients is very poor. Eight patients died despite systemic antifungal treatment.

The Potential of BORIS Detected in the Leukocytes of Breast Cancer Patients as an Early Marker of Tumorigenesis

Purpose:Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-testis antigen gene family. These genes are normally expressed only in spermatocytes but abnormally activated in different malignancies, including breast cancer. The aim of this study was to investigate the expression of BORIS in the leukocytes of breast cancer patients and the correlation between BORIS levels and clinical/pathologic variables. Experimental Design: Leukocytes were obtained from whole blood of 87 breast cancer patients and 52 donors not diagnosed with cancer. BORIS protein was detected in leukocytes by immunohistochemical staining; the immunoreactivity score (IRS) of each sample was determined. Additionally, BORIS expression was assessed by Western blot analysis and real-time reverse transcription-PCR. Results: We describe significantly high levels of BORIS (IRS = 4.25 ± 0.034) in a subpopulation of leukocytes, the neutrophil polymorphonuclear granulocytes, in 88.5% of breast cancer patients. Increased IRS for BORIS in these patients correlated with increased tumor size. In comparison, 19.2% samples from the control group were BORIS positive with only very low levels of BORIS (IRS = 0.25 ± 0.009). Conclusion: We report here the novel finding of BORIS expression in polymorphonuclear granulocytes of breast cancer patients. This tumor-related occurrence is a phenomenon not observed in donors with injuries and immune and inflammatory diseases. Detection of BORIS in a high proportion of patients with various types of breast tumors indicates that BORIS can be a valuable early blood marker of breast cancer. We conclude that BORIS represents a new class of cancer biomarkers different from those currently used in medical practice.

Allogeneic bone marrow transplantation versus intensification chemotherapy for acute myelogenous leukaemia in first remission: a prospective controlled trial.

In 1982 we began a prospective controlled trial to assess the effectiveness of allogeneic bone marrow transplantation and intensive post‐remission chemotherapy for patients with acute myelogenous leukaemia in first complete remission. Fourteen patients, 3–45 years of age, who had an HLA‐identical sibling donor, received bone marrow transplantation. Twenty‐five patients who either lacked an HLA‐identical sibling or were over 45 years of age received intensive consolidation chemotherapy including high‐dose cytosine arabinoside with or without adriamycin.

Myeloid Leukemia Cell Growth and Differentiation Are Independent of Mitogen-activated Protein Kinase ERK1/2 Activation*

The mitogen-activated protein kinase ERK1/2 pathway is essential in the control of cell proliferation and differentiation in most cellular systems. As such, it has been considered a potential target for antineoplastic therapy. For this purpose, we have examined the role of ERK activation in myeloid leukemia cell growth and differentiation. Using a representative set of myeloid leukemia cell lines, we show that cell proliferation was not accompanied by increases on ERK1/2 activation, and mitogenic stimulation did not enhance ERK activity. Moreover, abolition of ERK function by the inhibitor PD98059 or by a dominant inhibitory mutant ERK2 had no significant effects on proliferation. With the aid of various differentiation inducers, we found that within the same cell line, differentiation to a given lineage could occur with and without ERK1/2 activation, depending on the stimulus. Also, a differentiator could have the same effect in the presence or absence of ERK stimulation, depending on the cell line. ERK inhibition did not affect the differentiation elicited by stimuli whose effects were accompanied by ERK activation. Finally, constitutive ERK activity was also ineffective on proliferation and differentiation. Thus, our results indicate that ERK1/2 activation is not an essential requirement for leukemic cell growth and differentiation.

Pneumococcal pericarditis with cardiac tamponade in a patient with chronic graft-versus-host disease

We report a case of pneumococcal pericarditis in a 13-year-old boy following allogeneic BMT from an HLA-identical unrelated donor. The post-transplant course was complicated by chronic GVHD which led to reinstitution of immunosuppressive therapy. Eight months after BMT the patient developed pericarditis with cardiac tamponade, and Streptococcus pneumoniae was isolated in the pericardiocentesis fluid. This is the first reported case of pneumococcal pericarditis after BMT. Although pericardial effusions after allogeneic BMT are often sterile and related to conditioning therapy or associated with chronic GVHD, rapid microbiological investigation and empirical treatment with antibiotics are necessary. Prophylaxis for pneumococcal infection in patients with chronic GVHD is recommended.

THE USEFULNESS OF 1,25‐DIHYDROXY‐VITAMIN D3 (1,25(OH)2 vit D3) IN THE TREATMENT OF IDIOPATHIC MYELOFIBROSIS

In a recent annotation published in this journal, McCarthy (1 98.5) reviewed the causes and underlying mechanism of the deposition of collagen in bone marrow and discussed the possibility of using 1,25(OH)2 vit D3 to control such deposition and its ability to reverse myelofibrosis. He had published this suggested form of treatment previously (McCarthy ef al , 1984). The theory is that the active metabolite of vitamin D3 is able to inhibit the proliferation of megakaryocytes which usually promote collagen synthesis by releasing PDGF: in addition. that it can facilitate the reabsorption and increase the catabolism of collagen by being able to promote differentiation of CFIJ-GM in macrophages and monocytes which producc collagenase. Following the original suggestion (McCarthy et al, 1984), other authors (Arlet et al, 1984) published the results of treating three patients. One of these had myelofibrosis associated with chronic myelomonocytic leukaemia (CMML), one had myelofibrosis associated with posthypoparathyroid thrombocythacmia and one had ‘acute myelofibrosis’; there was apparent improvement in all the patients, and particularly in thc third, after treatment with 1 .2.5(OH)2 vit D 3 was started. We treated four patients in whom a diagnosis of idiopathic myelofibrosis had been made previously with 1,25(OH), vit D3 at a dose of 2.5 pg/d for 6 months. These patients had becri followed up for 1-7 years before treatment. None of these patients were transfusion dependent. Haematological data before and after treatment is given in Table 1. In addition, the collagen content of a bone marrow biopsy was assessed by two separate observers. In order to prevent the onset of secondary hypercalcaemia a calcium-deficient diet was given and fluid intake was increased; serum calcium, phosphorus and creatinine and urinary calcium and

Primary lymphoplasmacytoid lymphoma of the trachea with immunoglobulin G paraprotein.

A primary tracheal lymphoma with immunoglobulin G (IgG)‐associated monoclonal serum paraprotein treated with surgery and chemotherapy is reported. As far as we know this is the first lymphoplasmacytoid lymphoma reported in the tracheobronchial tree and the first with a serum and tissue IgG monoclonal paraprotein. Differential diagnosis must be made essentially with extramedullary plasmacytoma and mucosa‐associated lymphoid tissue lymphoma. CD‐45RB strong positivity and the absence of lymphoepithelial lesions may help to differentiate lymphoplasmacytoid lymphoma from them. We expand the spectrum of lymphoid lesions with plasmacytoid features that can occur in the tracheobronchial tract.