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The possible prognostic value of the position of the breakpoint within the M-BCR in patients with Phl+ CML is still being debated. We analyzed the DNA rearrangements and the transcript types of 244 patients and tried to correlate the data obtained with prognostic features, defined according to Sokal's risk index, and with chronic phase and/or survival duration. The exact location of the breakpoint, either 5' or 3' to the Hind III restriction site within the M-BCR was identified. Moreover, the exact M-BCR subregion was also identified. As a whole, 150 pts were rearranged in the 5' part and 94 in the 3' part of the M-BCR. No correlation was observed between the site of rearrangement on the one hand and the Sokal's prognostic index and survival, on the other. Transcript analysis was performed in 130 patients; 59 carried an a2b2 and 69 an a2b3 pattern. Two patients carried both transcripts. Of the patients rearranged in the 5' area, according to Southern blotting, 29.2% showed an a2b3 transcript. Therefore, R...

The characterisation of leukemic cell autofluorescence during differentiation, induced by 12-O-tetradecanoylphorbol 13-acetate and all-trans retinoic acid, was performed by autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy. We have found that a dependence exists between the cell autofluorescence pattern and the degree of cell differentiation. When cells differentiate, their autofluorescence emission changes, following the morphological and functional rearrangement of cell structures. A decrease in emission intensity and a different distribution of endogenous fluorophores are observed. Thus, autofluorescence monitoring on living cells is a potentially useful tool for in vitro study of the differentiation processes. Furthermore, different maturation steps can be distinguished on the basis of the cell fluorescence pattern, leading the way to future application of the technique in diagnostics.

T-lymphocytes (LYs) from normal and IFN-gamma knockout mice were activated by anti-CD3 and anti-CD28 antibodies and cultured in inserts in the presence of interleukin (IL)-12 (IL-12-activated LYs) or not (activated LYs). Their ability to modulate the genetic programs of two tumor lines growing at the bottom of transwells was evaluated. cDNA gene expression array, reverse transcription-PCR, and protein expression showed that LPS, transcription termination factor 1, transforming growth factor, and fibroblast growth factor genes were up-modulated by factors other than IFN-gamma released by activated LYS: The high levels of IFN-gamma released by normal IL-12-activated LYs up-modulated the expression of STAT1, IRF-1, LMP2, LMP7, monokine induced by IFN-gamma, monocyte chemoattractant protein 1, and angiopoietin 2 genes but down-modulated the expression of vascular endothelial growth factor. PA28, IFN-inducible protein 10, inducible NO synthetase, and macrophage-inhibitory protein 2 genes were up-modulated by factors released only by IL-12-activated LYs apart from IFN-gamma. The opposite modulations of vascular endothelial growth factor expression and of angiopoietin 2, monokine induced by IFN-gamma, IFN-inducible protein 10, and inducible NO synthetase by IL-12-activated LYs fit in well with the inhibition of angiogenesis that characterizes the antitumor activity of IL-12. T-LYs thus modify a tumor's behavior so that it becomes a party to its own inhibition.

Human normal and malignant T cells cease to proliferate, down-modulate Bcl-2 expression, and undergo apoptosis when cultured in the presence of NO-donor compounds (sodium nitroprusside and NOC12) for 48 h. At 72 h, cells that evade apoptosis start to proliferate again, overexpress both chains of the IFN-gammaR, and thus become susceptible to apoptosis in the presence of IFN-gamma. By contrast, in the presence of IFN-gamma, no apoptosis, but an increase of proliferation was displayed by control cultures of T cells not exposed to NO and not overexpressing IFN-gammaR chains. The NO-induced cell surface overexpression of IFN-gammaR chains did not affect the transduction of IFN-gamma-mediated signals, as shown by the expression of the transcription factor IFN regulatory factor 1 (IRF-1). However, transduction of these signals was quantitatively modified, because IFN-gamma induces enhanced levels of caspase-1 effector death in NO-treated cells. These findings identify NO as one of the environmental factors that critically govern the response of T cells to IFN-gamma. By inducing the overexpression of IFN-gammaR chains, NO decides whether IFN-gamma promotes cell proliferation or the induction of apoptosis.

We have previously shown that the contrasting ability of interferon-gamma (IFN-gamma) either to stimulate the proliferation of malignant T cells or to induce their apoptosis is determined by the low and high intensity of IFN-gamma receptor (IFN-gamma R) expression, respectively. High IFN-gamma R expression is a marker for the T cell stress that precedes apoptosis. In this paper, we show that a 12- to 24-h culture of three human malignant T-cell lines displaying distinct differentiation stages (ST4, PF382, and Jurkat) in medium supplemented with four chemotherapy drugs (etoposide, cisplatin, cytarabine, and daunomycin) up-modulates their IFN-gamma R expression followed by their apoptosis after 24-48 h later. Increased IFN-gamma R expression (by at least an order of magnitude) was observed in 30 to 90% of cells during exposure to pharmacologic drug concentrations. Timely combination of chemotherapy drugs with IFN-gamma may thus provide a more effective way of inhibiting the progress of human malignant T cells through synergistic induction of their apoptosis.

To find out how physiologically secreted IFN-gamma controls either the proliferation or the apoptosis of human T lymphocytes, the kinetics of expression of the alpha- and beta-chains of its receptor (IFN-gamma R) were sequentially followed on T lymphocytes first activated with PHA and then cultured in the presence of IL-2, and related to the kinetics of expression of Fas, Bcl-2, and IL-2R p55 chain. Both IFN-gamma R chains were poorly expressed on the membrane of resting T lymphocytes. Following their stimulation with PHA, IFN-gamma R alpha but not IFN gamma R beta-chain up-modulated before T lymphocyte entry into the S phase, and then IFN-gamma R alpha down-modulated when they passed through the S and G2/M. The ensuing proliferative response was inhibited by an anti-IFN-gamma R alpha mAb that impeded the binding of IFN-gamma. When PHA-activated T lymphoblasts were cultured for 16 days with IL-2, IFN-gamma R alpha expression increased, whereas that of the beta-chain remained barely detectable. Fas and Bcl-2 were both highly expressed. When these T lymphoblasts were restimulated by PHA, OKT3, or Staphylococcus enterotoxin beta-pokeweed mitogen, both chains up-modulated and most cells underwent apoptosis in a way apparently independent of Bcl-2, but not of Fas. This apoptosis, too, was prevented by the anti-IFN-gamma R alpha mAb. Physiologically secreted IFN-gamma is thus involved in the activation of resting T lymphocytes and in the apoptosis of reactivated lymphoblasts. However, high expression of IFN-gamma R beta took place when IFN-gamma induced apoptosis, but not when it induced proliferation. In conclusion, a correlation exists between differential expression of the IFN-gamma R beta-chain and the delivery by IFN-gamma of proliferative or apoptotic signals.

The heterodimeric interferon (IFN)-gamma receptor (IFN-gammaR) is formed of two chains. Here we show that the binding chain (IFN-gammaR1) was highly expressed on the membranes of T, B, and myeloid cells. Conversely, the transducing chain (IFN-gammaR2) was highly expressed on the surfaces of myeloid cells, moderately expressed on B cells, and poorly expressed on the surfaces of T cells. Differential cell membrane expression of IFN-gammaR2 determined the number of receptor complexes that transduced the IFN-gamma signal and resulted in a different response to IFN-gamma. After IFN-gamma stimulation, high IFN-gammaR2 membrane expression induced rapid activation of signal transducer and activator of transcription-1 (STAT-1) and high levels of interferon regulatory factor-1 (IRF-1), which then triggered the apoptotic program. By contrast, low cell membrane expression resulted in slow activation of STAT-1, lower levels of IRF-1, and induction of proliferation. Because the forced expression of IFN-gammaR2 on T cells switched their response to IFN-gamma from proliferative to apoptotic, we concluded that the surface expression of IFN-gammaR2 determines whether a cell stimulated by IFN-gamma undergoes proliferation or apoptosis.

Chronic myeloid leukemia (CML) is a disease characterized by the presence of a unique molecular marker, i.e. the fusion gene bcr-abl and its mRNA and protein products. This marker permits minimal residual disease follow-up after bone marrow transplantation (BMT) through cytogenetic or molecular analysis. Although the reverse transcriptase polymerase chain reaction (RT-PCR) method is largely employed, the clinical value and impact of a positive RT-PCR as a herald of hematological relapse has not yet been definitively ascertained.In order to verify the frequency of bcr-abl positivity in CML patients who underwent alloBMT, we performed serial two-step RT-PCR on 63 peripheral blood and bone marrow specimens obtained at different times after non T-cell-depleted BMT from 16 CML patients treated in our Institution. After amplification, RT-PCR products were always checked by liquid hybridization with a specific probe. Median molecular follow-up after BMT was 38 months (range 2-144 months).None of the patients studied presented clinical hematological relapse after BMT. Six out of sixteen patients were found to be positive for bcr-abl. PCR positivity appeared in 4/6 patients more than one year post-BMT and in 2/6 patients within one year post BMT. In both instances PCR was an isolated finding in 5/6 patients and reverted to negativity in subsequent analysis; only one case was PCR-positive twice. It is noteworthy that RT-PCR positivity appeared in five patients presenting acute or chronic graft versus host disease (GVHD) and in one patient who had received MUD-BMT.In our cohort of patients, transient bcr-abl positivity had no clinical relevance and was also found in MUD-BMT without heralding hematological relapse. Our observations further stress the importance of applying only quantitative PCR methods during the post BMT follow-up of CML patients.

BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.

Leukemia cell motility and transendothelial migration into extramedullary sites are regulated by angiogenic factors and are considered unfavorable prognostic factors in acute leukemias. We have studied cross talk among (1) the vascular endothelial growth factor receptor-1, FLT-1; (2) the human eag-related gene 1 (hERG1) K+ channels; and (3) integrin receptors in acute myeloid leukemia (AML) cells. FLT-1, hERG1, and the β1 integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for FLT-1 signaling activation and AML cell migration. Both effects were inhibited when hERG1 channels were specifically blocked. A FLT-1/hERG1/ β1 complex was also observed in primary AML blasts, obtained from a population of human patients. The co-expression of FLT-1 and hERG1 conferred a pro-migratory phenotype to AML blasts. Such a phenotype was also observed in vivo. The hERG1-positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, hERG1 expression in leukemia patients correlated with a higher probability of relapse and shorter survival periods. We conclude that in AML, hERG1 channels mediate the FLT-1-dependent cell migration and invasion, and hence confer a greater malignancy. © 2007 by The American Society of Hematology.

An important target in the understanding of the pathogenesis of acute myeloid leukemias (AML) relies on deciphering the molecular features of normal and leukemic hemopoietic progenitors. In particular, the analysis of the mechanisms involved in the regulation of cell proliferation is decisive for the establishment of new targeted therapies. To gain further insight into this topic we report herein a novel approach by analyzing the role of HERG K(+) channels in the regulation of hemopoietic cell proliferation. These channels, encoded by the human ether-a-gò-gò-related gene (herg), belong to a family of K(+) channels, whose role in oncogenesis has been recently demonstrated. We report here that herg is switched off in normal peripheral blood mononuclear cells (PBMNC) as well as in circulating CD34(+) cells, however, it is rapidly turned on in the latter upon induction of the mitotic cycle. Moreover, hergappears to be constitutively activated in leukemic cell lines as well as in the majority of circulating blasts from primary AML. Evidence is also provided that HERG channel activity regulates cell proliferation in stimulated CD34(+) as well as in blast cells from AML patients. These results open new perspectives on the pathogenetic role of HERG K(+) channels in leukemias.