Corrigendum to "Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation".

[This corrects the article DOI: 10.1155/2019/5879616.].

Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation.

The recent introduction of the "precision medicine" concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326- EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

Comparative proteomic profiling of Hodgkin lymphoma cell lines.

Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies.

Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia.

Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions.

Cryopreservation effects on Wharton's Jelly Stem Cells proteome.

Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tissue engineering. However, the freeze-thaw process strongly contributes to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation and altered physical cellular properties. Our previous proteomics investigation was carried out on Wharton's Jelly Stem Cells (WJSCs) having similar properties to adult mesenchymal stem cells and thus representing a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJSCs proteome in different experimental conditions: fresh primary cell culture and frozen cell. To analyze changes in protein expression of WJSCs undergoing different culturing procedures, we performed a comparative proteomic analysis (2DE followed by MALDI-TOF MS/MS nanoESI-Q-TOF MS coupled with nanoLC) between WJSCs from fresh and frozen cell culturing, respectively. Frozen WJSCs showed qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defence mechanism and metabolism, that could ensure freeze-thaw survival. The results of this study could play a key role in elucidating possible mechanisms related to maintaining active proliferation and maximal cellular plasticity and thus making the use of WJSCs in cell therapy safe following bio-banking.

Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Characterization, GFP gene Nucleofection, and allotransplantation in injured tendons of ovine amniotic fluid-derived stem cells.

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ∼63% and ∼37%, while cell recoveries were ∼10% and ∼22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achille's tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.

Achilles tendon regeneration can be improved by amniotic epithelial cell allotransplantation.

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14(-), CD31(-), CD45(-), CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial-temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-ß1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells.

Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling.

Novel shift of Jak/Stat signalling characterizes the protective effect of aurintricarboxylic acid (ATA) from tumor necrosis factor-alpha toxicity in human B lymphocytes.

Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-alpha was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors Stat3 and Stat5b. In this study we demonstrate that the balance of survival signals in the presence of TNF-alpha was altered by the addition of a salicylate compound, the endonuclease inhibitor aurintricarboxylic acid (ATA). Apoptosis effected by TNF-alpha alone was suppressed by ATA and this event was paralleled by phosphorylation and nuclear translocation of Jak2, Stat2, Stat4 and NF-kB, along with inhibition of caspase activation. These results confirm that among the different cellular responses evoked by TNF-alpha in human B cells, recruitment of Jak/Stat proteins and possible related gene modulation represent contributing factors and address the issue of the development of potential therapeutic strategies aimed at the control of systemic or local effects produced by TNF-alpha.

Inositide-modifying enzymes: a cooperative role in regulating nuclear morphology during differentiation of myeloid cells.

Differentiation and functional response of mature myeloid cells require cytoskeleton remodelling in a dynamic system that involves subcellular organization and regional signalling. Within the myeloid lineage, neutrophils constitute a cell type in which different cell compartments, and predominantly the nucleus, undergo distinctive large changes involving actin reorganization. In the context of the progressive elucidation of the nuclear structure and composition that has been achieved in the last two decades, it is now clear that the nucleus possesses an ordered and dynamic skeletal structure which shares many properties with the cytoskeleton, and the full set of substrates and enzymes that participate in the inositol lipid metabolism. Consolidated evidence indicate that the changes in cytoskeleton assembly are regulated also by phosphoinositides in a way dependent on their local concentration and availability. Indeed, enzymes able to affect the amount and phosphorylation of inositol lipids can play fundamental roles in determining the architectural transitions of the cell. The expression pattern and the changes of activity of PLC and PI 3-K in the nucleus during differentiation of tumoral myeloid precursors suggest that these enzymes play a crucial role in modifying the intranuclear pool of phosphoinositides, which in turn induce the changes in nucleoskeleton associated to granulocytic maturation. It can be speculated that defective control of nucleoskeleton assembly is one of the causes of dysregulated cell maturation or differentiative block in the course of myeloid leukemias. Inositide modifying enzymes can thus be regarded as potential targets for molecularly designed therapeutic intervention on hematological malignancies.

Fhit protein expression in human gastric cancer and related precancerous lesions.

The FHIT gene is altered in several types of tumors and abnormal expression of Fhit protein have also been reported in some preneoplastic lesions. We have determined the Fhit expression on histological samples of 26 patients affected by preneoplastic lesions who developed a gastric cancer within 2 years. The expression of the Fhit protein was always present in all preneoplastic lesions, while the Fhit protein immunostaining was distributed unevenly in 10 cases and completely lost in 6. The complete loss of Fhit expression only in areas of neoplastic low differentiation suggests that FHIT gene takes part in late gastric carcinogenesis.

Phospholipase C delta2 expression characterizes the neoplastic transformation of the human gastric mucosa.

The expression, cellular distribution, and activity of PIP(2)-specific phospholipase C (PLC) in healthy human gastric-mucosa cells have been recently studied in our laboratories and a direct evidence for an almost exclusive expression of PLC beta isoforms, with the exception of PLC beta4, has been provided. These results addressed our attention to possible modification of PLC expression and activity during neoplastic transformation of the human gastric mucosa. In the present article we present results indicating that PLC delta2 is markedly expressed in type II intestinal metaplasia and in the adenocarcinoma whereas traces of other PLC isoforms were sometime detected. Interestingly, we found that type I intestinal metaplasia was in the majority of the cases PLC delta2-negative, but when expressed, this type of metaplasia generally considered as benignant, always evolved toward neoplastic transformation. These results therefore readdress the question of surveillance of the patients with type I intestinal metaplasia and suggest that PLC delta2 expression might be a possible marker of gastric malignant transformation.

Requirement of tyrosine-phosphorylated Vav for morphological differentiation of all-trans-retinoic acid-treated HL-60 cells.

Our previous data demonstrated that cellular and nuclear tyrosine-phosphorylated Vav associate with phosphoinositide 3-kinase during all-trans-retinoic acid-dependent granulocytic differentiation of HL-60 cells. In this study, aimed to analyze the mechanism by which Vav is recruited and activated, we report that the Src homology 2 domain of Vav interacts with tyrosine-phosphorylated proteins in a differentiation-dependent manner. Two adaptor proteins, Cbl and SLP-76, were identified, showing a discrete distribution inside the cells, with Cbl absent from the nuclei and SLP-76 particularly abundant in the nuclear compartment. Of note, Vav interacts with the tyrosine kinase Syk, which is also present in the nuclear compartment and may phosphorylate Vav in vitro when cells differentiate. Inhibition of Syk activity by piceatannol prevents both in vitro and in vivo Vav tyrosine phosphorylation, its association with the regulatory subunit of phosphoinositide 3-kinase, and the nuclear modifications typically observed during granulocytic differentiation of this cell line. These findings suggest that tyrosine-phosphorylated Vav and its association with phosphoinositide 3-kinase play a crucial role in all-trans-retinoic acid-induced reorganization of the nucleoskeleton, which is responsible for the changes in nuclear morphology observed during granulocytic differentiation of HL-60 cells.

Histochemical and biochemical analysis of phospholipase C isoforms in normal human gastric mucosa cells.

The expression and activity of PIP2-specific phospholipase C (PLC) in healthy human gastric mucosa cells were investigated by means of Western blotting, immunohistochemistry and in vitro activity assays. The results provide direct evidence for an almost exclusive expression of the PLC beta family and at the same time supply a cellular cartography of each represented isoform of this family. In this context, the putative roles of each isoform in the signaling events regulating the gastric mucosa metabolic machinery are discussed. These data provide a unique map of the specific expression and cellular distribution of the most represented PLC isoforms in healthy human gastric mucosa cells, which may constitute a reference point in future studies aimed at highlighting possible cytochemical and biochemical hallmarks of metaplastic or malignant transformation.

Stromal derived factor-1 alpha (SDF-1 alpha) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway.

Stromal-derived factor-1alpha (SDF-1alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF-1alpha-mediated Jurkat cell apoptosis was observed in serum-free or serum-containing cultures, peaked at SDF-1alpha concentrations of 10-100 ng/ml, required 3 days to take place, and was completely blocked by the z-VAD-fmk tripeptide caspase inhibitor. Although SDF-1alpha did not modify the expression of TNF-alpha or that of TNF-RI and TNF-RII, it increased the expression of surface Fas/APO-1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF-1alpha to induce apoptosis was inhibited by an anti-CD95 Fab' neutralizing antibody. These findings suggest a role for SDF-1alpha in the homeostatic control of CD4+ T-cell survival/apoptosis mediated by the CD95-CD95L pathway.

Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738-29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non-canonical 3-phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC-zeta nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3-kinase (PI 3-K) activity and its product, phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P(3)]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3-K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3-K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P(3). Maximal translocation of PKC-zeta from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG-sensitive, PKC-alpha. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, was unable to abrogate nuclear translocation of the DAG-insensitive PKC-zeta. These data suggest that a nuclear increase in PI 3-K activity and PtdIns(3,4,5)P(3) production are necessary for the subsequent nuclear translocation of PKC-zeta. Furthermore, they point to the likelihood that PKC-zeta is a putative nuclear downstream target of PI 3-K during NGF-promoted neural differentiation.-Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4, 5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

Spatial distribution of lamin A and B1 in the K562 cell nuclear matrix stabilized with metal ions.

When the nucleus is stripped of most DNA, RNA, and soluble proteins, a structure remains that has been referred to as the nuclear matrix, which acts as a framework to determine the higher order of chromatin organization. However, there is always uncertainty as to whether or not the nuclear matrix, isolated in vitro, could really represent a skeleton of the nucleus in vivo. In fact, the only nuclear framework of which the existence is universally accepted is the nuclear lamina, a continuous thin layer that underlies the inner nuclear membrane and is mainly composed of three related proteins: lamins A, B, and C. Nevertheless, a number of recent investigations performed on different cell types have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nuclear matrix. In most cell types investigated, the nuclear matrix does not spontaneously resist the extraction steps, but must rather be stabilized before the application of extracting agents. In this investigation, by immunochemical and morphological analysis, we studied the effect of stabilization with different divalent cations (Zn(2+), Cu(2+), Cd(2+)) on the distribution of lamin A and B1 in the nuclear matrix obtained from K562 human erythroleukemia cells. In intact cells, antibodies to both lamin A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. The fluorescent lamin A pattern detected in Cu(2+)- and Cd(2+)-stabilized nuclei was markedly modified, whereas Zn(2+)-incubated nuclei showed an unaltered pattern of lamin A distribution. By contrast, the distribution of lamin B1 in isolated nuclei was not modified by the stabilizing cations. When chromatin was removed by nuclease digestion and extraction with solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix. Our results indicate that when metal ions are used as stabilizing agents for the recovery of the nuclear matrix, the distribution of both lamin A and lamin B1 in the final structures, corresponds to the pattern we have very recently reported using different extraction procedures. This observation strengthen the concept that intranuclear lamins may act as structural components of the nuclear matrix.

Monocytic differentiation of HL-60 cells is characterized by the nuclear translocation of phosphatidylinositol 3-kinase and of definite phosphatidylinositol-specific phospholipase C isoforms.

Immunochemical and immunocytochemical data indicate that nuclei of HL-60 cells contain different enzymes involved in the phosphoinositide cycle, such as PI 3-K and the phosphatidylinositol-specific PLC isoforms beta3, gamma1 and gamma2. These enzymes translocate differently to the nuclear fraction when HL-60 cells are treated with differentiating doses of vitamin D3: PI 3-K translocated progressively to the nucleus in parallel with full differentiation until 96 hours. PLC beta3 increased until 72 hours of treatment and then lowered its intranuclear amount and PLC gamma1 was unchanged at all the examined times. PLC gamma2 nuclear translocation increased progressively until 96 hours of vitamin D3 administration. A fourth PLC isozyme, beta2, present in the cytoplasm of untreated cells, translocates to the cytoplasm after vitamin D3 addition and reaches the highest concentration at the end of monocytic differentiation. Terminal monocytic differentiation was characterized at the nuclear level by high levels of PI 3-K and PLC gamma2 and by the novel expression of PLC beta2. We then observed that the xi isoform of PKC, constitutively present in nuclei of HL-60 cells, translocated to the nucleus when cells were induced to differentiate along the monocytic lineage, but the nuclear translocation of PKC xi was blocked as a consequence of PI 3-K inhibition by Wortmannin. These findings indicate that the main components of the noncanonical and canonical inositol lipid signal transduction pathways, including PI 3-K, PLC beta2 and beta3, PLC gamma2, undergo nuclear translocation and may therefore play a relevant role during monocytic differentiation at the nuclear level. Furthermore, PKC xi nuclear translocation appears to be related to PI 3-K activity.

Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoisomerase IIalpha may act as structural components of the nuclear matrix.