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Abstract: During both maturation and function, neutrophils are subjected to reorganization of the actin cytoskeleton. Among the molecules that influence cytoskeletal architecture, the amount and subcellular localization of phosphoinositides, regulated by specific kinases and phosphatases, may play a crucial role. In neutrophils, PLC-β2 is a major phosphoinositide-dependent phospholipase C isoform activated in response to chemoattractants, even though its role in the modifications of cell morphology and motility that occur during the inflammatory process has not been fully elucidated. In APL-derived promyelocytes induced to complete their maturation program, we have found that the expression levels of PLC-β2 positively correlate with the degree of the reached neutrophil differentiation. Here, we demonstrate that PLC-β2 modulates the migration capability of promyelocytes induced to differentiate with ATRA. In differentiating cells, the association of PLC-β2 with actin, mediated by the PH domain, seems crucial for catalytic activity. We conclude that phosphodiesterase activity of PLC-β2 on the actin-associated PIP2 may be responsible, by modifying the phosphoinositide pools, for the modifications of cytoskeleton architecture that take place during motility of differentiating promyelocytes. [Copyright &y& Elsevier]

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K + channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2- b ]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xl bright and pAkt bright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38 bright cells followed apoptosis (IC 50 : 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo.

Background: The malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-β2 induces the conversion of CD133 high to CD133 low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype., Methods: A magnetic step-by-step cell isolation with antibodies directed against CD133 and/or EpCAM was performed on the TNBC-derived MDA-MB-231 cell line. In the same cell model, PLC-β2 was over-expressed or down-modulated and cell proliferation and invasion capability were evaluated by Real-time cell assays. The surface expression of CD133, EpCAM and CD44 in the different experimental conditions were measured by multi-color flow cytometry immunophenotyping., Results: A CD133 + /EpCAM + sub-population with high proliferation rate and invasion capability is present in the MDA-MB-231 cell line. Over-expression of PLC-β2 in CD133 + /EpCAM + cells reduced the surface expression of both CD133 and EpCAM, as well as proliferation and invasion capability of this cellular subset. On the other hand, the up-modulation of PLC-β2 in the whole MDA-MB-231 cell population reduced the number of cells with a CD44 + /CD133 + /EpCAM + stem-like phenotype., Conclusions: Since selective targeting of the cells with the highest aggressive potential may have a great clinical importance for TNBC, the up-modulation of PLC-β2, reducing the number of cells with a stem-like phenotype, may be a promising goal for novel therapies aimed to prevent the progression of aggressive breast tumors.

Like most human neoplasm, breast cancer has aberrations in signal transduction elements that can lead to increased proliferative potential, apoptosis inhibition, tissue invasion and metastasis. Due to the high heterogeneity of this tumor, currently, no markers are clearly associated with the insurgence of breast cancer, as well as with its progression from in situ lesion to invasive carcinoma. We have recently demonstrated an altered expression of the beta2 isoform of the phosphoinositide-dependent phospholipase C (PLC) in invasive breast tumors with different histopathological features. In primary breast tumor cells, elevated amounts of this protein are closely correlated with a poor prognosis of patients with mammary carcinoma, suggesting that PLC-beta2 may be involved in the development and worsening of the malignant phenotype. Here we demonstrate that PLC-beta2 may improve some malignant characteristics of tumor cells, like motility and invasion capability, but it fails to induce tumorigenesis in non-transformed breast-derived cells. We also report that, compared with the G(0)/G(1) phases of the cell cycle, the cells in S/G(2)/M phases show high PLC-beta2 expressions that reach the greatest levels during the late mitotic stages. In addition, even if unable to modify the proliferation rate and the expression of cell cycle-related enzymes of malignant cells, PLC-beta2 may promote the G(2)/M progression, a critical event in cancer evolution. Since phosphoinositides, substrates of PLC, are involved in regulating cytoskeleton architecture, PLC-beta2 in breast tumor cells may mediate the modification of cell shape that characterizes cell division, motility and invasion. On the basis of these data, PLC-beta2 may constitute a molecular marker of breast tumor cells able to monitor the progression to invasive cancers and a target for novel therapeutic breast cancer strategies.

Objective: Erythroid differentiation is a process characterized by modulation of different proteins including phosphoinositide-related enzymes such as protein kinase C (PKC) isoforms. Because in different cell lines PKC-alpha and PKC-delta have been reported to be involved in the mechanisms controlling proliferation and differentiation, the aim of this study was to examine the relative involvement of these PKC isoforms in the development of CD235a+ erythroid cells from human healthy hematopoietic progenitors., Materials and Methods: Erythroid differentiation from human primary hematopoietic progenitor cells was achieved by adopting the human erythroblasts mass amplification culture. Expression and activity of PKC isoforms and their relationship with proliferation and differentiation were investigated by morphologic analysis, reverse-transcriptase polymerase chain reaction, Western blotting, multiparametric flow cytometry, and transfection experiments., Results: PKC-alpha was found expressed and phosphorylated in cells undergoing both proliferation and differentiation, although PKC-delta, largely expressed and activated during proliferation, was evidently downregulated during differentiation. Overexpression of PKC-delta-CAT scarcely influenced the development of glycophorin-A (CD235a)+ erythroid cells from hematopoietic progenitors, although overexpression of PKC-alpha-CAT strongly induced the development of CD235a+ erythroid cells. On the other hand, in PKC-alpha-CAT-transfected cells, pharmacologic inhibition of PKC-delta further increased the number of CD235a+ cells, although inhibition of PKC-alpha resulted in an evident impairment of the development of CD235a+ erythroid cells., Conclusions: Our results indicate that the suppression or at least a strong downregulation of PKC-delta, concomitant to PKC-alpha expression and activity, might be a cofactor to be further investigated and might be involved in the events regulating erythropoietin-induced erythroid differentiation from human primary hematopoietic progenitor cells.

A selective involvement of protein kinase C-zeta (PKC-zeta) in the events regulating cell proliferation has been recently proposed. Here we report a flow cytometric method allowing the simultaneous association of intracellular PKC-zeta expression or phosphorylation with each cell cycle phase. Current methods for flow cytometry analysis were applied to several cell lines and compared to the method developed in our laboratory. The latter includes 2% paraformaldehyde (PFA), as fixing agent, a permeabilization/saturation step by means of a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.05% NP-40, 0.25% lambda-carrageenan and 0.02% NaN3, followed by labelling with a primary antibody (PKC-zeta or P-PKC-zeta) and with the appropriate FITC-conjugated secondary antibody. Cells processed by such a method disclosed no substantial modification of light scattering features with respect to live cells. In addition, stainability with anti-PKC-zeta or anti-P-PKC-zeta antibodies was well preserved while stoichiometric staining of DNA with PI enabled accurate cell cycle analysis. Results show that a distinct up-regulation of P-PKC-zeta in G2/M phase occurs. The method here described, therefore, represents a simple, reproducible and conservative assay for a simultaneous assessment of intracellular PKC or P-PKC modulations within each cell cycle phase.

The differentiation therapy in treatment of acute promyelocytic leukemia (APL), based on the administration of all-trans retinoic acid (ATRA), is currently flanked with the use of As2O3, a safe and effective agent for patients showing a resistance to ATRA treatment. A synergy between ATRA and As3O3 was also reported in inducing granulocytic differentiation of APL-derived cells. We have demonstrated that phospholipase C-beta2 (PLC-beta2), highly expressed in neutrophils and nearly absent in tumoral promyelocytes, largely increases during ATRA treatment of APL-derived cells and strongly correlates with the responsiveness of APL patients to ATRA-based differentiating therapies. Here we report that, in APL-derived cells, low doses of As3O3 induce a slight increase of PLC-beta2 together with a moderate maturation, and cooperate with ATRA to provoke a significant increase of PLC-beta2 expression. Remarkably, the amounts of PLC-beta2 draw a parallel with the differentiation levels reached by both ATRA-responsive and -resistant cells treated with ATRA/As2O3 combinations. PLC-beta2 is not necessary for the progression of tumoral promyelocytes along the granulocytic lineage and is unable to overcome the differentiation block or to potentiate the agonist-induced maturation. On the other hand, since its expression closely correlates with the differentiation level reached by APL-derived cells induced to maturate by drugs presently employed in APL therapies, PLC-beta2 represents indeed a specific marker to test the ability of differentiation agents to induce the release of the maturation blockade of tumoral myeloid precursors.

Despite the identification of many putative biomarkers in breast cancer, a specific pattern of proteins to be used as a prognosticator is not well defined. A growing body of evidence supports the role of phospholipase C (PLC) in the invasion and metastasis of different tumors, including breast cancer. To assess whether the expression of specific PLC isoforms correlates with malignancy-related features of human breast tumors and, hence, could have prognostic significance, an immunohistochemical analysis of PLC-beta2 was performed on tissue microarrays and the relationship between PLC-beta2 expression and biological and clinico-pathological factors was assessed. The analysis of 77 samples of breast tumors with different histotypes revealed that PLC-beta2 is highly expressed in a large majority of the analyzed cancer tissue, particularly ductal and lobular carcinomas, in comparison with normal breast. The expression of PLC-beta2 in primary tumors correlated with size, proliferation index and final grade, while no significant relationship was observed with nodal status or estrogen receptor levels, or with the expression of tumor suppressor p53. Remarkably, high PLC-beta2 levels in primary tumors predict an unfavourable prognosis, suggesting the contribution of this protein to the progression of human mammary carcinomas. Our data indicate that PLC-beta2 expression correlates highly with breast cancer malignancy and suggest that it can be included, as an independent marker, among the prognostic indicators in current use.

In this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients.