Monoubiquitinated Fanconi anemia D2 (FANCD2-Ub) is required for BCR-ABL1 kinase-induced leukemogenesis.

Fanconi D2 (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) in response to DNA double-strand breaks (DSBs) to stimulate repair of these potentially lethal DNA lesions. FANCD2-Ub was upregulated in CD34+ chronic myeloid leukemia (CML) cells and in BCR-ABL1 kinase-positive cell lines in response to elevated levels of reactive oxygen species (ROS) and DNA cross-linking agent mitomycin C. Downregulation of FANCD2 and inhibition of FANCD2-Ub reduced the clonogenic potential of CD34+ CML cells and delayed BCR-ABL1 leukemogenesis in mice. Retarded proliferation of BCR-ABL1 positive FANCD2-/- leukemia cells could be rescued by FANCD2 expression. BCR-ABL1 positive FANCD2-/- cells accumulated more ROS-induced DSBs in comparison with BCR-ABL1 positive FANCD2+/+ cells. Antioxidants diminished the number of DSBs and enhanced proliferation of BCR-ABL1 positive FANCD2-/- cells. Expression of wild-type FANCD2 and FANCD2(S222A) phosphorylation-defective mutant (deficient in stimulation of intra-S phase checkpoint, but proficient in DSB repair), but not FANCD2(K561R) monoubiquitination-defective mutant (proficient in stimulation of intra-S phase checkpoint, but deficient in DSB repair) reduced the number of DSBs and facilitated proliferation of BCR-ABL1 positive FANCD2-/- cells. We hypothesize that FANCD2-Ub has an important role in BCR-ABL1 leukemogenesis because of its ability to facilitate the repair of numerous ROS-induced DSBs.

[Evaluation of the usefulness of various PCR method variations and nucleic acid hybridization for CMV infection in immunosuppressed patients]. / Ocena przydatnosci róznych wariantów metody PCR oraz metody hybrydyzacji kwasów nukleinowych w wykrywaniu zakazenia CMV u pacjentów z immunosupresja.

In diagnosis of CMV infection various laboratory methods are used. The methods based on detection of viral nucleic acids have been introduced routinely in many laboratories. The aim of this study was to compare nucleic acid hybridisation method and various variants of PCR methods with respect to their ability to detect CMV DNA. The studied material comprised 60 blood samples from 19 patients including 13 renal transplant recipients and 6 with acute leukaemia. The samples were subjected to hybridisation (Murex Hybrid Capture System CMV DNA) and PCR carried out in 3 variants: with one pair of primers (single PCR), nested PCR and Digene SHARP System with detection of PCR product using a genetic probe in ELISA system. The sensitivity of the variants ranged from 10(0) particles of viral DNA in nested PCR to 10(2) in single PCR. The producer claimed the sensitivity of the hybridisation test to be 3 x 10(5) and it seems to be sufficient for detection of CMV infection. The obtained results show that sensitivity of hybridisation was comparable to that of single PCR and the possibility of obtaining quantitative results makes it superior, on efficacy of antiviral therapy, especially in monitoring CMV infection in immunossuppressed patients and in following the efficacy of antiviral treatment.

[Preliminary treatment results of relapsed or refractory acute leukemia using two and three drug regimens]. / Wstepne wyniki leczenia ostrych bialaczek opornych i nawrotowych przy zastosowaniu schematów dwu--oraz trzylekowych.

36 patients with relapsed (29) or refractory (7) acute lymphoblastic or nonlymphoblastic leukaemia received regimens employing 1-3 courses of mitoxantrone (or idarubicin), intermediate doses of cytarabine and etoposide. Complete remission (CR) was achieved in 30% of patients (5/15 ALL, 6/21 AML, 5 cases of refractory and 6 of relapsed leukaemia). Duration of CR was 3-6+ months (3 patients are still alive). Toxicity of the treatment was acceptable, however 5 patients with severe granulocytopenia died from sepsis.

[Diagnosis of CMV infections in patients diagnosed with acute leukemia]. / Diagnostyka zakazen CMV u pacjentów z rozpoznaniem ostrej bialaczki.

The purpose of this paper was to estimate the best method of CMV diagnosis in leukemic patients. Materials from 9 patients (serum, heparinized blood and urine) were investigated by serological methods (ELISA and Western blot), for the presence of specific antigens and virus capable of replication, and also by genetic methods (hybridization and PCR). It seems, that the best method for CMV diagnosis in leukemic patients is hybridization performed in quantitation variant, and that DNA CMV level of approximately 20 pg/ml of blood was not linked to symptomatic infection.

[Unclassified chronic myeloproliferative Ph(-); i(17q); +8 syndrome with mixed myelo-megakaryoblastic crisis--case report]. / Niesklasyfikowany przewlekly zespól mieloproliferacyjny Ph(-); i(17q); +8 z transformacja mielo-megakarioblastyczna--opis przypadku.

We present a case of a 17-year old patient with extreme hepatosplenomegaly, hyperthrombocytosis, hyperleucocytosis and the presence of myelo- and megakaryoblasts in the peripheral blood film. Numerous complications that occurred in the course of the disease made cytostatic treatment difficult. Since Ph chromosome and hybrid gene bcr/abl were absent, the diagnosis of unclassified chronic myeloproliferative syndrome in the phase of blast crisis was established. Immunophenotyping confirmed a mixed myelo- megakaryoblastic character of the crisis. In the differential diagnosis other myeloproliferative syndromes were taken into account including i(17q) syndrome. The patient died after a 13-month observation due to neoplasm progression and sepsis.

[A relationship between the breakpoint of the bcr gene and some hematologic parameters in patients with chronic myelogenous leukemia]. / Zaleznosc miedzy miejscem zlamania genu bcr a niektórymi parametrami hematologicznymi u chorych na przewlekla bialaczke szpikowa.

We examined the hybrid bcr/abl mRNA present in 59 patients with chronic myeloid leukemia, using the reverse transcription method and polymerase chain reaction. Bcr/abl gene was found in 98% of patients. 60% of patients had b3a2 type of translocation, 40% type b2a2. Patients with b3a2 type had higher platelet count at diagnosis presentation than patients with b2a2. Other hematological data were similar in both groups.

Enhanced erythropoietin secretion in hepatoblastoma cells in response to hypoxia.

Erythropoietin (Ep) levels in spent culture media of a Hep G2 human hepatoblastoma cell line were measured by radioimmunoassay (RIA), fetal mouse liver erythroid colony formation (FMLC), and the exhypoxic polycythemic mouse assay (EHPCMA). The Hep G2 cells at high density produced approximately 700 mU/ml Ep when measured with the RIA. On the other hand, the Ep levels when assayed in EHPCMA and FMLC were 50 and 2,600 mU/ml, respectively. The bioactivity in FMLC was completely neutralized by an antibody to purified human recombinant Ep, indicating that the erythropoietic activity in the Hep G2 spent culture medium was immunologically equivalent to Ep. Ep levels in the medium from low-density Hep G2 cells in 5% O2 and 1% O2 were 2.5- and 4-fold greater, respectively, than that of 20% O2. In contrast, hyperoxia (40% O2) significantly inhibited Ep production. A significant increase in Ep secretion was also observed when the cells were incubated with cobaltous chloride (2 X 10(-6) -2.5 X 10(-4) M). Tunicamycin (0.5 micrograms/ml), which inhibits N-linked glycosylation, significantly reduced the enhancement of Ep secretion induced by hypoxia (1% O2) without affecting cell growth. Forskolin and cholera toxin, each of which increased the levels of cyclic AMP in the Hep G2 cells by 40-fold, produced a significant (P less than 0.05) further increase in Ep secretion in the presence of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythropoietin production in exhypoxic polycythemic mice.

Our present study was undertaken to determine the serum erythropoietin concentration (radioimmunoassay), hematocrit, red cell mass, and body weight of mice exposed to hypoxia in a hypobaric chamber (0.42 atm, 22 h/day) for 14 days and during the 10 posthypoxic days at ambient pressure to clarify the correlation of the red cell mass and erythropoietin production during hypoxia. The mean serum erythropoietin titer was 326.23 +/- 77.04 mU/ml after 2 days, reached the highest level after 3 days (452.2 +/- 114.5 mU/ml), then gradually declined to a level of 36.5 +/- 11.4 mU/ml after 14 days of hypoxia, and was undetectable during the 10-day posthypoxic period. The hematocrit values were significantly increased from 41.09 +/- 0.50% at day 0 to 51.65 +/- 1.08% after 3 days and to 72.20 +/- 1.53% after 14 days of hypoxia. The red cell mass (calculated from initial body weight) increased from 3.24 +/- 0.1 ml/100 g at day 0 to 7.32 +/- 0.46 ml/100 g after 14 days of hypoxia and declined to 6.66 +/- 0.53 ml/100 g at the end of the 10-day posthypoxic period. The mice lost weight while they were in the hypobaric chamber and showed a significant increase in body weight during the 10-day posthypoxic period. These studies support the concept that chronic intermittent hypoxia causes an early increase, followed by a rapid decline, in erythropoietin production, which is correlated with the gradual increase in red cell mass.

Possible involvement of phospholipase activation in erythroid progenitor cell proliferation.

The possible role of phospholipase activation in erythroid progenitor (colony-forming units erythroid; CFU-E) cell proliferation was investigated using a variety of inhibitors of phospholipase activity. Quinacrine and alpha-p-dibromoacetophenone (BPB), both nonselective phospholipase inhibitors, were tested for their effects on CFU-E-derived colony formation. Both drugs significantly inhibited colony formation in the presence or absence of added erythropoietin (Epo). For quinacrine, the concentration causing 50% inhibition (IC50) was 1 microM, whereas for BPB the IC50 was 25 microM. A more selective inhibitor of phospholipase A2, 7,7-dimethyleicosadienoic acid (DEDA), was also tested and the IC50 was 250 microM in the presence of Epo. The addition of phospholipase A or C did not significantly affect CFU-E colony formation, although arachidonic acid increased CFU-E formation as did 12-hydroperoxyeicosatetraenoic acid (12-HPETE), whereas 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and 15-HPETE had no effect. These findings suggest an important role for phospholipase activation in erythroid progenitor cell proliferation.