Evolving treatment strategies for patients newly diagnosed with chronic myeloid leukemia: the role of second-generation BCR-ABL inhibitors as first-line therapy.

In patients with chronic myeloid leukemia (CML), disease in the initial chronic phase (CP) and subsequent progression are driven by the oncogenic activity of the BCR-ABL fusion kinase. Imatinib, a tyrosine kinase inhibitor of BCR-ABL, has been the mainstay of first-line therapy for CML for 10 years. Although patients with CML-CP respond well to imatinib, those who have delayed reductions in leukemic burden during imatinib therapy, such as not achieving a complete cytogenetic response (CCyR) by 12 months, have an increased risk of disease progression. It has been recognized, with 8 years of observation, that patients who achieve an early major molecular response (MMR) on imatinib have a very low probability of disease progression. Recent randomized phase 3 trials have shown that first-line treatment with dasatinib or nilotinib-more potent BCR-ABL inhibitors-results in significantly higher rates and more rapid achievement of CCyR and MMR in comparison with standard-dose imatinib. These trials suggest that CML treatment can be improved with more potent BCR-ABL inhibition during initial therapy, but further follow-up is needed to confirm that the improved response rates with dasatinib and nilotinib are maintained long term.

Angiogenesis in acute and chronic leukemias.

Angiogenesis plays a key role in the growth of solid tumors. More recently, accumulating evidence has linked angiogenesis to the pathophysiology of leukemias. Different investigators have shown evidence of increased angiogenesis in the bone marrows of patients with acute and chronic leukemias. Elevated levels of angiogenic factors have also been reported in leukemic patients. A potential role for VEGF as an autocrine growth factor in AML has been suggested. Studies on the role of angiogenesis and VEGF as prognostic factors in leukemia require confirmation. The role of angiogenesis inhibitors in the treatment of leukemia is currently under investigation.

Identification and characterization of a novel gene that is upregulated in leukaemia cells by nitric oxide.

Nitric oxide (NO) inhibits growth and induces differentiation in acute myeloid leukaemia (AML) cells. To identify genes associated with these processes, we studied the effect of NO on AML gene expression using the technique of Representational Difference Analysis. Exposure of HL-60 cells to the NO donor DETA-NO for 24 h induced the expression of a novel gene that was named rno (regulated by nitric oxide). Treatment of HL-60 cells with dimethyl sulphoxide induced expression of rno, but treatment with Vitamin D3 or all-trans retinoic acid did not. Upregulation of rno by NO was cGMP independent. Northern blot analysis indicated that constitutive expression of the novel gene was limited to leucocytes. Three isoforms of rno were identified. An rno cDNA clone was obtained by screening a human leucocyte library. The nucleotide sequence of the open reading frame shared significant homology with that of the human ribonuclease/angiogenin inhibitor (RI). The predicted amino acid sequence indicated that, like RI, rno is leucine and cysteine rich and is comprised of a series of repetitive elements (leucine-rich repeats) that may mediate macromolecular interactions. Enhancement of expression of rno may be a component of the process by which differentiation and growth inhibition of leukaemia cells is induced by NO.

Esterase-sensitive nitric oxide donors of the diazeniumdiolate family: in vitro antileukemic activity.

We have designed a novel prodrug class that is stable in neutral aqueous media but releases bioactive nitric oxide (NO) on metabolism by esterase. Diazeniumdiolates of structure R(2)N-N(O)=N-OR', in which R' = Na, were reacted with BrCH(2)OAc to convert the spontaneously NO-releasing salts 1a (R(2)N = diethylamino) and 1b (R(2)N = pyrrolidino) to prodrugs 2a (AcOM-DEA/NO) and 2b (AcOM-PYRRO/NO), respectively, where R' = CH(2)OAc. In contrast to anions 1a and 1b (half-lives in pH 7.4 phosphate at 37 degrees C of 2 min and 3 s, respectively), 2a and 2b showed only minimal decomposition after 16 h under these conditions. Very rapid hydrolysis occurred in the presence of porcine liver esterase, however, with free anion 1a being observed as an intermediate in the esterase-induced generation of NO from 2a. The potential utility of this prodrug class is illustrated with a comparison of 1 and 2 as antiproliferative agents in NO-sensitive human leukemia cell lines HL-60 and U937. While the 72-h IC(50)'s for 1a and 1b (which generate NO throughout the medium) in HL-60 cell cultures were >600 microM, those of 2a and 2b were 8.3 and 6.4 microM, respectively. This result is consistent with our hypothesis that 2 is selectively hydrolyzed to 1 and thence to NO intracellularly. For U937 cells, the 72-h IC(50) for both 2a and 2b was 53 microM. By contrast, relatively high antiproliferative IC(50)'s (>100 microM in U937 cells) were observed for analogues in which R' = CH(2)CH(2)SC(O)Me, from which acetyl and 2-mercaptoethyl groups must be successively cleaved to free the NO-releasing diazeniumdiolate function. Within 24 h at initial concentrations of 50 microM, 2a and 2b induced apoptosis in 50% and 57% of the HL-60 cells, respectively (35% and 40% of the U937 cells, respectively). The data reveal significant in vitro antileukemic activity on the part of these novel compounds. Moreover, their substantial ease-of-handling advantages over the anionic diazeniumdiolates from which they are derived suggest their use as convenient agents for probing the biological roles of NO.

Evidence of increased angiogenesis in patients with acute myeloid leukemia.

Angiogenesis plays a key role in solid tumor growth. The purpose of this work was to study angiogenesis in acute myeloid leukemia (AML). We stained bone marrow samples from 20 adult patients with untreated AML and 20 normal controls using endothelial cell markers (ULEX-E and von Willebrand factor [vWF]). The number of vessels per millimeter length of bone marrow core biopsy specimen was scored by light microscopy. Using ULEX-E staining, AML marrows had (average +/- SEM) 8.3 +/- 3.6 vessels/mm (range, 3.7-19.3), whereas normal marrows had 4.3 +/- 1.8 vessels/mm (range, 1.6-7.9). A similar difference was noted using vWF staining (8.6 +/- 3.0 vessels/mm vs 4. 9 +/- 2.2 vessels/mm in AML vs normal bone marrows, respectively). The differences between the numbers of vessels/mm in AML and normal marrows were highly significant (P <.0001 for both ULEX-E and vWF staining). When analyzed by FAB category, there was no difference in the average number of vessels/mm among the different subgroups of AML. Using reverse transcriptase polymerase chain reaction, we observed that the HL-60 and U937 human AML cell lines and 4 of 4 freshly isolated AML cells from untreated patients expressed mRNA for vascular endothelial growth factor (VEGF). Both cell lines as well as all fresh AML isolates tested expressed VEGF protein. Basic fibroblast growth factor was expressed only in HL-60 cells and in only 3 of 4 fresh AML samples. These observations suggest that angiogenesis may play a role in the pathogenesis of AML. Inhibition of angiogenesis could constitute a novel strategy for the treatment of AML. (Blood. 2000;95:309-313).

Schedule and concentration-dependent induction of apoptosis in leukemia cells by nitric oxide.

Nitric oxide (NO) has potent antiproliferative properties. In previous work we have shown that NO inhibits growth, induces differentiation and modulates gene expression in acute nonlymphocytic leukemia (ANLL) cells. The goal of this work was to determine whether the rate of NO delivery affected its growth inhibition of ANLL cells. We also wanted to determine whether the NO inhibition of ANLL cell growth is associated with the induction of apoptosis. We treated HL-60 and U937 cells with three compounds that generate the same amount of NO but at different rates. MAMA-NO, PAPA-NO and DETA-NO have half-lives of NO delivery of 2 and 30 min, and 20 h, respectively. The compound with the longest t(1/2) of NO delivery (DETA-NO) was the most potent inhibitor of leukemia cell and colony growth. Furthermore, the NO-induced growth inhibition was associated with apoptosis in a rate and concentration-dependent fashion.

A functional tethered ligand thrombin receptor is present on human hematopoietic progenitor cells.

The processing of inflammatory signals occurs through a variety of mechanisms; the recent descriptions of the tethered ligand receptor for thrombin (JA Hoxie et al., J Biol Chem 268:13756, and TK Vu et al., Cell 64:1057) provide a novel route and mechanism for cellular activation after inflammation and thrombosis. Using standard flow-cytometric techniques, it has been shown that the tethered ligand receptor is found on a number of terminally differentiated hematopoietic cells including platelets, lymphocytes, and monocytes. In this paper, we show that the CD34+ subset of hematopoietic stem cells bears the tethered ligand receptor on its surface; in addition, stimulation of this receptor with the agonist peptide SFLLRN results in a dose-dependent increase in intracellular calcium levels. We also show that culturing bone marrow mononuclear cells in the presence of thrombin or the tethered ligand receptor agonist peptide results in a statistically significant increase in colony-forming units-erythroid and -granulocyte/macrophage (CFU-E and CFU-GM). Although more work is needed to establish the exact mechanism of this effect, our results suggest that activation of the tethered ligand thrombin receptor may modulate the proliferative responses of CD34+ hematopoietic progenitor cells.

Differential effects of nitric oxide on erythroid and myeloid colony growth from CD34+ human bone marrow cells.

Nitric oxide (NO) is a reactive molecule with numerous physiologic and pathophysiologic roles affecting the nervous, cardiovascular, and immune systems. In previous work, we have demonstrated that NO inhibits the growth and induces the monocytic differentiation of cells of the HL-60 cell line. We have also demonstrated that NO inhibits the growth of acute nonlymphocytic leukemia cells freshly isolated from untreated patients and increases monocytic differentiation antigens in some. In the present work, we studied the effect of NO on the growth and differentiation of normal human bone marrow cells in vitro. Mononuclear cells isolated from human bone marrow were cultured in semisolid media and treated with the NO-donating agents sodium nitroprusside (SNP) or S-nitroso-acetyl penicillamine (SNAP) (0.25 to 1 mmol/L). Both agents decreased colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) formation by 34% to 100%. When CD34+ cells were examined, we noted that these cells responded to SNP and SNAP differently than did the mononuclear cells. At a concentration range of 0.25 to 1 mmol/L, SNP inhibited the growth of CFU-E by 30% to 75%. However, at the same concentration range, SNP increased the number of CFU-GM by up to 94%. At concentrations of 0.25 to 1 mmol/L, SNAP inhibited the growth of CFU-E by 33% to 100%. At a concentration of 0.25 mmol/L, SNAP did not affect CFU-GM. At higher concentrations, SNAP inhibited the growth of CFU-GM. Although SNP increased intracellular levels of cGMP in bone marrow cells, increasing cGMP in cells by addition of 8-Br-cGMP (a membrane permeable cGMP analogue) did not reproduce the observed NO effects on bone marrow colonies. These results demonstrate that NO can influence the growth and differentiation of normal human bone marrow cells. NO (generated in the bone marrow microenvironment) may play an important role modulating the growth and differentiation of bone marrow cells in vivo.

Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Nitric oxide modulation of the growth and differentiation of freshly isolated acute non-lymphocytic leukemia cells.

Freshly isolated acute non-lymphocytic leukemia (ANLL) cells were treated with the nitric oxide (NO)-liberating compounds sodium nitroprusside or S-nitrosoacetyl penicillamine and analyzed for viability, growth, and differentiation at 3-5 days. NO decreased the viability and the growth of freshly isolated ANLL cells in vitro. NO treatment significantly increased expression of CD14 in blast cells from patients with M5 ANLL, and increased at least one differentiation parameter in M4 or M5 cells. It had little or no effect on parameters of differentiation in other ANLL cells. We conclude that in vitro culture with NO decreases the growth and viability of most freshly isolated ANLL cells. NO also induces the differentiation of ANLL cells with a monocytic phenotype.

A case of retrograde jejunoduodenal intussusception caused by a feeding gastrostomy tube.

A case is presented of migration of a gastrostomy feeding tube (Foley type) through the pylorus with duodenal obstruction and subsequent retrograde intussusception. Although feeding tube migration is not uncommon, retrograde intussusception of the jejunum into the duodenum is rare. In this case, surgery was required with resection of a segment of necrotic bowel. Recommendations are given for treatment of this unusual complication with emphasis on its avoidance through use of a retaining bar or disc at the tube's skin exit site.

Nitric oxide modulation of human leukemia cell differentiation and gene expression.

Nitric oxide (NO) functions as an intercellular messenger molecule in such varied contexts as neurotransmission, immune regulation, and the control of vascular tone. We report that NO, delivered as purified gas or released from the pharmacologic NO donors sodium nitroprusside or 6-morpholino-sydnonimine, caused monocytic differentiation of cells of the human myeloid leukemia cell line HL-60 and altered gene expression. The treated cells stopped proliferating, became spread and vacuolated, had increased expression of nonspecific esterase and the monocyte marker CD14, and displayed increased capacity to produce hydrogen peroxide. Furthermore, these treated cells had increased steady-state expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), but decreased expression of mRNA for the proto-oncogenes c-myc and c-myb. The increase in TNF-alpha and IL-1 beta mRNA levels was due (at least in part) to a new transcription of these specific mRNAs. NO elaborated in the bone marrow microenvironment may have a role in normal and malignant hematopoietic cell growth and differentiation.