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Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.

It is becoming increasingly clear that the cell nucleus is a highly structurized organelle. Because of its tight compartmentalization, it is generally believed that a framework must exist, responsible for maintaining such a spatial organization. Over the last twenty years many investigations have been devoted to identifying the nuclear framework. Structures isolated by different techniques have been obtained in vitro and are variously referred to as nuclear matrix, nucleoskeleton or nuclear scaffold. Many different functions, such as DNA replication and repair, mRNA transcription, processing and transport have been described to occur in close association with these structures. However, there is still much debate as to whether or not any of these preparations corresponds to a nuclear framework that exists in vivo. In this article we summarize the most commonly-used methods for obtaining preparations of nuclear frameworks and we also stress the possible artifacts that can be created in vitro during the isolation procedures. Emphasis is placed also on the protein composition of the frameworks as well as on some possible signalling functions that have been recently' described to occur in tight association with the nuclear matrix.

Human metaphase chromosomes were isolated and digested in situ with HaeIII restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed by antibodies conjugated either with fluorescein isothiocyanate or 1-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 488 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination with the red fluorescence of propidium iodide, and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non-fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence, of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences after in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of HaeIII restriction enzyme that cut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located. In chromosomes, the fluorescent reaction pattern showed large areas of labeling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like bands actually are multiple symmetrical spots localized on sister chromatids. In addition, some chromosomes, and in particular 1 and 9, displayed a C-negative banding due to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.

Macroautophagy, usually referred to as autophagy, is a degradative pathway wherein cytoplasmatic components such as aggregated/misfolded proteins and organelles are engulfed within double-membrane vesicles (autophagosomes) and then delivered to lysosomes for degradation. Autophagy plays an important role in the regulation of numerous physiological functions, including hematopoiesis, through elimination of aggregated/misfolded proteins, and damaged/superfluous organelles. The catabolic products of autophagy (amino acids, fatty acids, nucleotides) are released into the cytosol from autophagolysosomes and recycled into bio-energetic pathways. Therefore, autophagy allows cells to survive starvation and other unfavorable conditions, including hypoxia, heat shock, and microbial pathogens. Nevertheless, depending upon the cell context and functional status, autophagy can also serve as a death mechanism. The cohort of proteins that constitute the autophagy machinery function in a complex, multistep biochemical pathway which has been partially identified over the past decade. Dysregulation of autophagy may contribute to the development of several disorders, including acute leukemias. In this kind of hematologic malignancies, autophagy can either act as a chemo-resistance mechanism or have tumor suppressive functions, depending on the context. Therefore, strategies exploiting autophagy, either for activating or inhibiting it, could find a broad application for innovative treatment of acute leukemias and could significantly contribute to improved clinical outcomes. These aspects are discussed here after a brief introduction to the autophagic molecular machinery and its roles in hematopoiesis.