HA14-1 selectively induces apoptosis in Bcl-2-overexpressing leukemia/lymphoma cells, and enhances cytarabine-induced cell death.

The Bcl-2 oncoprotein is commonly overexpressed in hematological malignancy, where it promotes the survival of neoplastic cells. Recently, a small molecule (HA14-1) was reported to bind the surface pocket of Bcl-2 that mediates antiapoptotic interactions, triggering apoptosis in a Bcl-2-transfected cell line. We investigated the activity of this compound in a panel of malignant hematopoietic cell lines. Consistent with its proposed role as a Bcl-2 inhibitor, HA14-1 was most cytotoxic in lines expressing high levels of Bcl-2. In addition, at lower concentrations (5-12.5 muM), the compound predominantly triggered apoptosis. However, at concentrations two-fold higher than this and above, increasing primary necrosis was observed, suggesting the onset of interactions supplementary to Bcl-2 inhibition. In experiments on primary cells, 25 muM HA14-1 induced extensive apoptosis in acute leukemic blasts, but also suppressed normal hematopoietic colony formation to <50% of baseline. Importantly, low-concentration HA14-1 (5 muM) was nontoxic to normal colony-forming cells, whereas it enhanced the cytotoxicity of the antileukemia drug cytarabine in Bcl-2-positive lymphoblastic leukemia cells. In conclusion, our results indicate that HA14-1 at low concentration selectively triggers apoptosis in malignant hematopoietic cells that overexpress Bcl-2. Agents of this class may have particular utility in combination with cytotoxic chemotherapy drugs.

Combined morphological and interphase fluorescence in situ hybridization study in multiple myeloma of Chinese patients.

To gain insight into the real incidence of the numeric chromosomal aberrations and the cell lineage involvement of the neoplastic process in multiple myeloma (MM), we examined 18 Chinese MM patients by May-Grunwald-Giemsa (MGG) staining and fluorescence in situ hybridization using three DNA centromeric probes specific for chromosomes 3, 7, and 9. In this investigation, cytogenetic abnormalities were detected in plasma cells (PCs), myeloid cells (MCs), and lymphoid cells (LCs) in all of the MM patients studied. This is the first demonstration of the cytogenetic aberration involved in the myeloid series. Furthermore, the MCs and PCs of 16 MM patients had the same aneuploidies in one or more of the chromosomes analyzed. These data suggest that the neoplastic transformation of MM may occur early in the hematopoietic development. Chromosomal aberrations involving mainly subclones and considerable cellular heterogeneity with gain of a variety of copy numbers of the same chromosome were demonstrated within PCs, which may possibly be the result of an underlying defect of PCs in the control of their number of chromosomes. Whereas PCs showed evidence suggestive of increased polyploidization, MCs and LCs, which exhibited similar chromosomal patterns as the former, rarely did. Thus, the clonal evolution from LC to PC, if that happens in MM, is characterized by chromosomal instability favoring growth of tumor cells with polysomies and polyploidies.

Fatal Penicillium citrinum pneumonia with pericarditis in a patient with acute leukemia.

We report here a case of fatal Penicillium citrinum infection. The patient, who suffered from acute myeloid leukemia, developed signs and symptoms typical of fungal pneumonia and pericardial tamponade after undergoing standard induction chemotherapy. Despite attaining complete remission of her leukemia, the patient succumbed 8 weeks after presentation. At autopsy, multiple nodular cavitary pulmonary lesions with invasion by fungal hyphae were found. Pericardial and lung tissue obtained at autopsy grew P. citrinum, a fungus ubiquitous in the environment but seldom reported as a pathogen. The microbiological findings were consistent with the histopathological features and confirmed this as a case of true P. citrinum infection causing fatal pulmonary and pericardial complications in an immunocompromised host.

Frequent hypermethylation of p16 and p15 genes in multiple myeloma.

Both p16 and p15, encoded by the genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases (CDK4/6) and the upstream regulators of Rb function. In hematopoietic malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid, and more particularly from B-lineage malignancies except multiple myeloma (MM). To investigate whether these genes are inactivated by deletions, mutations, and hypermethylation of the 5' CpG islands, we examined 12 MM patients by Southern hybridization and polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. No deletions nor mutations of the p16 and p15 genes were found. However, hypermethylation was observed in 75% for p16 and 67% for p15 in our group of MM patients. Such high frequencies of involvement of these genes in MM make them hitherto the most common genetic abnormalities in this disease. Concomitant hypermethylation, uncommon thus far in the literature of the study of these genes, is a rather common phenomenon, occurring in 67% of our patient group. Moreover, hypermethylation of p16/p15 was associated with blastic disease and concomitant hypermethylation of both genes may be pathogenetically related to plasmacytoma development. These results indicate that these genes are important in MM pathogenesis. Here we report, for the first time in the literature, the high incidences of p16 and p15 alterations in MM, not by homozygous deletions or mutations, but solely by hypermethylation of the 5' CpG islands, which may be a specific mechanism in this disease.

Monoclonal gammopathy of unknown significance and malignant paraproteinemia in Hong Kong.

The incidence of multiple myeloma is lower in Southeast Asia than in the West. However, there are few reports on the overall incidence of paraproteinemia and its disease-associations in the Chinese. Therefore, the authors have correlated the laboratory features with the eventual clinical diagnosis in patients with paraproteinemia in a Hong Kong general/teaching hospital. Over 18 months, 1,600 patients were investigated for the presence of paraproteinemia. Paraproteinemia was detected in 157 (10%) patients. In 11 patients, investigations could not be completed. The remaining 146 patients were subjected to detailed clinical, radiologic, and laboratory investigations. Eighty-seven (59.6%) had monoclonal gammopathy of unknown significance (MGUS), 44 (30.1%) myeloma and 15 (10.3%) other lymphoproliferative disorder (LPD). There was no significant difference in the paraprotein concentration, frequency of hypogammaglobulinemia of Bence Jones proteinuria (BJP) or concentration of nonparaprotein immunoglobulin (Ig) between the myeloma and LPD groups. The overall kappa:lambda light chains ratios were 2.9, 1.6 and 3.3 in the MGUS, MM and LPD groups, respectively. Polyclonal Ig elevation was rare with myeloma (4.5%) but was detected in 33% of patients with LPD (P < .02) and 40% of those with MGUS (P < .0001). Biclonal (and one triclonal) gammopathy was detected in 11.5% of patients with MGUS, 11% with LPD and 4.5% with myeloma. In the MGUS group, infection was the commonest associated clinical disorder (29.3%). Moreover, 70% of patients with biclonal gammopathy and MGUS had an infection. Five of 15 patients with LPD had a T-cell malignancy, including 3 lymphomas and 2 large granular cell leukemias. Only one patient had primary systemic amyloidosis. It is concluded that the high frequency of biclonality and its association with infection, of paraproteinemia in association with T-cell malignancy and of kappa light chains in the MGUS and LPD groups are at variance with reports from the West and probably reflect local differences.

Upper airway obstruction caused by acquired inhibitor to factor VIII.

Bleeding caused by inhibitors to factor VIII is a rare medical emergency requiring immediate specialist investigation and management. Urgent initiation of therapy with high dose factor VIII concentrates may be life saving. Successful management of acute upper airway obstruction from uncontrolled haemorrhage into the oropharyngeal tissues should be achieved with fibreoptic guided nasotracheal intubation.

Acute monoblastic leukaemia with conjunctival tumours.

We describe an unusual occurrence of bilateral conjunctival tumours in a 25-year-old woman. This was the first sign of relapse of acute monoblastic leukaemia. There was also both marrow relapse and subsequent skin infiltration. No central nervous system involvement was detected. The tumours appeared as pink raised lesions in the upper conjunctivae of both eyes. They were not associated with pain or visual impairment. Conjunctival tumour biopsy revealed a dense mononuclear cell infiltration. Complete remission (conjunctival tumours, skin infiltration and bone marrow) was attained following systemic chemotherapy in combination with intrathecal chemotherapy.

Endothelial cell platelet-activating factor primes neutrophil responses: amplification of endothelial activation by neutrophil products.

We have shown that platelet-activating factor (PAF) primes neutrophil (PMN) responses and enhances their ability to damage endothelial cells. Furthermore, thrombin-stimulated endothelial cells which produce PAF can augment and prime PMN superoxide production, elastase release and adhesion to endothelium. We wondered whether these marginated neutrophils (PMN) themselves, or their release products, might feedback and further amplify endothelial cell activation. To measure cellular activation, we assessed changes in endothelial cell intracellular calcium [( Ca2+]i) in endothelial monolayers loaded with Fura-2, and PAF production by [3H]acetate incorporation into phospholipid. Resting PMNs induced no change in [Ca2+]i, while N-formyl-L-methionine-L-leucyl-L-phenylalanine stimulated PMN and their lysosomal products caused a 25% increase in endothelial cell calcium. Sonicates of PMN produced a much larger increase in [Ca2+]i than activated PMN; the effect of PMN sonicates could in part be reduced by the serine protease inhibitor, alpha 1 antitrypsin. In contrast, purified neutrophil elastase did not alter endothelial cell [Ca2+]i. Despite hydrogen peroxide's ability to increase [Ca2+]i, catalase failed to inhibit the PMN-induced rise in [Ca2+]i. Since polyanionic heparin inhibited the PMN sonicate rise in calcium, a cationic protein released by PMN was thought to be responsible. The cationic primary granule enzyme, cathepsin G, duplicates the rise in [Ca2+]i seen with PMN sonicates. Furthermore, PAF production increased threefold in response to neutrophil sonicates. Thus, during inflammation, when coagulation and inflammatory cells are activated the endothelium responds by priming PMNs and promoting margination.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin-treated endothelium primes neutrophil functions: inhibition by platelet-activating factor receptor antagonists.

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or "prime" polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginated PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10(-7) M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021, when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF--a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial/PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.

Lupus anticoagulant induces a selective defect in thrombin-mediated endothelial prostacyclin release and platelet aggregation.

A patient with microvascular thrombosis and thrombocytopenia was found to have a high-titre lupus anticoagulant. The biological effects of the patient's lupus anticoagulant were studied using whole patient serum and plasma. Staph Protein A eluate, and affinity-purified lupus anticoagulant. The latter was isolated by immunoadsorption of serum onto cardiolipin/phosphatidylserine/cholesterol liposomes. Each source of lupus anticoagulant demonstrated 'anticoagulant' activity, defined as prolongation of a modified kaolin clotting time, and contained antibody which bound to endothelial monolayers. Each interfered with thrombin-mediated prostacyclin release from endothelial cells, but had no effect on arachidonate-induced prostacyclin release. In addition, the lupus anticoagulant selectively blocked platelet aggregation in response to thrombin, but not in response to arachidonate, ADP or epinephrine. Lupus anticoagulant also reduced thrombin-stimulated shifts in cytosolic calcium. Thrombin-mediated membrane inositol metabolism and total thrombin binding to endothelium were unaffected by lupus anticoagulant, and another endothelial anticoagulant function related thrombin binding. Protein C activation by thrombomodulin, was not altered. We conclude that the binding of lupus anticoagulant to endothelial cells and platelets does not prevent all thrombin signalling events, but does interrupt prostacyclin production.

Measurement of intracellular calcium concentration in intact monolayers of human endothelial cells.

Intracellular calcium ([Ca++]i) plays an important role in signal transduction and cell activation. The measurement of [Ca++]i in intact monolayers of human umbilical vein endothelial cells with the fluorescent calcium-sensitive probe fura 2 has been evaluated. Monolayers provide a more physiologic cell preparation than suspensions and allow a greater variety of experimental manipulation. Basal [Ca++]i was 117 +/- 5 nmol/L, with a range from 40 to 280 nmol/L that was not affected by cell age (days of primary culture) or degree of confluence. Thrombin in concentrations of 0.005 to 5 NIH units/ml produced a dose-dependent increase in [Ca++]i up to a maximum of 1500 +/- 147 nmol/L; this increase was shown to depend in part on the concentration of extracellular calcium. The presence of antithrombin III at physiologic concentrations abolished responses to 0.5 NIH units/ml thrombin but had no effect on 5 NIH units/ml. The potential of this technique was demonstrated further by our ability to examine [Ca++]i responses in endothelial cells following infection with herpes simplex virus type 1, a virus implicated in vascular injury. After 18 hours' infection, the response to both thrombin and histamine was dramatically reduced despite a normal resting [Ca++]i. It is concluded that this method may be useful for detecting early and subtle changes in endothelial cell function under a variety of physiologic and pathologic conditions.

Endogenous and exogenous adenosine inhibit granulocyte aggregation without altering the associated rise in intracellular calcium concentration.

The effects of adenosine, adenosine deaminase (ADA), and an irreversible ADA inhibitor 2'-deoxycoformycin (DCF) on granulocyte aggregation in response to four different stimuli: the synthetic chemotaxin N-formyl-met-leu-phe (FMLP), zymosan-activated plasma (ZAP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were studied. Adenosine inhibited granulocyte aggregation in response to 10(-7) mol/L FMLP in a dose-dependent fashion; inhibition in the presence of 1 mumol/L adenosine was 25% +/- 3% (SD) and was 50% (the maximal inhibition observed) with 1 mmol/L adenosine. Quantitatively similar results were obtained when ZAP or A23187 was used as the aggregant but the response to PMA was not affected. ADA not only reversed the inhibition due to adenosine but actually augmented the aggregation to FMLP by 118% +/- 9%. Similar results were obtained with ZAP and A23187 but not with PMA. These effects of ADA depended on its enzymatic activity as they could be blocked by preincubation with DCF. Fluorescent measurement of intracellular calcium in fura-2 loaded granulocyte suspensions established that neither adenosine nor ADA affected subsequent FMLP-stimulated calcium responses. Adenosine, therefore, may inhibit granulocyte responsiveness by blocking signal transduction at a point after calcium entry/mobilization but before activation of protein kinase C. Furthermore, the augmentation of responses seen with ADA suggests that endogenous adenosine may be a physiologic autocrine regulator of granulocyte function.