Standard and quantitative analysis of cyclin E threshold by cyclin E/DNA multiparameter flow cytometry

To examine the effects of triptolide (TPL) on T-cell leukemia cells and identify their underlying mechanisms. The cytotoxicity of TPL was assessed by MTT assay. Cell apoptosis was determined using annexin V and DAPI staining and analyzed by flow cytometry or fluorescence microscopy. The activation of caspase pathways and the expression of nuclear factor κB (NF-κB) p65 were examined by Western blotting. Differences in microRNA (miRNA) expression in Molt-4 and Jurkat cells before and after TPL treatment were identified using microarrays and real-time RT-PCR, respectively. TPL 20-160 nmol/L treatment potently inhibited cell growth and induced apoptosis in T-cell lymphocytic leukemia cell lines. Molt-4 and Jurkat cells, however, were more sensitive to TPL than L428 and Raji cells. After 24 h of treatment, bortezomib abrogated the growth of Molt-4 and Jurkat cells with an IC(50) of 15.25 and 24.68 nmol/L, respectively. Using Molt-4 cells, we demonstrated that treatment 20-80 nmol/L inhibited the translocation of NF-κB p65 from the cytoplasm to the nucleus and that phosphorylated NF-κB p65 in nuclear extracts was down-regulated in a dose-dependent manner. Similar results were also seen in Jurkat cells but not in L428 cells, as these cells are resistant to TPL and bortezomib (a NF-κB inhibitor). Twenty-three miRNAs were differentially expressed after TPL treatment. Functional analysis revealed that TPL treatment could inhibit expression of miR-16-1* and that transfection of miR-16-1* led to significantly decreased apoptosis induced by TPL. Our in vitro studies suggest that TPL might be an effective therapeutic agent for treatment of T-cell lymphocytic leukemia and that its cytotoxic effects could be associated with inhibition of NF-κB and down-regulation of miR-16-1*.

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

BackgroundHigh throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure.ResultsAll scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range.ConclusionsIt is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

Dual Inhibition of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin with NVP-BEZ235 as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia.

The multisubunit proteasome complex is the principal mediator of nonlysosomal protein degradation. The proteasome subunit varies minimally between cells with the exception of LMP2, LMP7, and LMP10 subunits in rodent and human cells. LMP2 and LMP7 subunits are encoded by the human lymphocyte antigen region, and they optimize proteolytic mediated antigen presentation. The proteasome is also important for the function of transcription factor nuclear factor-kappaB (NF-kappaB). It is required for NF-kappaB subunits p50 and p52 generation and catalyzes degradation of phosphorylated IkappaBalpha. These proteasome-mediated reactions have now been shown to be defective in T2 cells, a human lymphocyte cell line that lacks both LMP2 and LMP7. Although T2 cells contain normal expression of p100 and p105, the abundance of p50 and p52 was greatly reduced. Tumor necrosis factor-alpha (TNF-alpha) induced normal phosphorylation of IkappaBalpha but failed to induce degradation of phosphorylated IkappaBalpha. Both DNA binding assays and luciferase assays revealed that TNF-alpha-induced NF-kappaB activation is defective in T2 cells. Unlike parental cells, T2 cells were susceptible to TNF-alpha-induced apoptosis. These data indicate human leukocyte antigen-linked proteasome subunits are essential for NF-kappaB activation and protection of cells from TNF-alpha-induced apoptosis.

Jurkat human lymphoblastoid cells were incubated in increasing concentrations of doxorubicin (0.05, 0.1 and 0.15 μM) to induce cell death, and their expression of cyclin A, B1 and D1 was evaluated by flow cytometry (cell cycle progression, Annexin V assay, percentages and levels of each of the cyclins), transmission electron microscopy (ultrastructure) and confocal fluorescence microscopy (expression and intracellular localization of cyclins). After low-dose doxorubicin treatment, Jurkat cells responded mainly by G2/M arrest, which was related to increased cyclin B1, A and D1 levels, a low level of apoptosis and/or mitotic catastrophe. The influence of doxorubicin on levels and/or localization of selected cyclins was confirmed, which may in turn contribute to the G2/M arrest induced by the drug.

Infiltrating T lymphocyte in tumors is correlated with the cancer prognosis. Clinical data showed the accumulation of infiltrating T lymphocyte in peritumoral area is not good for the prognosis, which could reduce the efficiency of contact-dependent lysis of tumor cells. Tumor cells could secrete extracellular matrix (ECM) to impede T lymphocyte infiltration. In this study, we demonstrate that ECM produced by tumors can directly regulate T cell migration. We performed the transmigration of T cells through the tumor cell monolayers or tumor ECM in the transwell system. The result showed that the transmigration rates of primary T cells and lymphoma cell lines Jurkat and Molt-4 cells through the cell monolayers or ECM of glioma cell line U-87MG, U-118MG, and U373MG cells were consistently lower than that through the cell monolayers or ECM of breast cancer cell line MCF-7 and hepatoma cell line HepG2. According to timelapse microscopy observation, the morphological changes of Jurkat T cells on tumor ECM were classified into round, rough, and amoeba-like shape. The low transmigration rate of Jurkat T cells was mainly due to the reduction of ameba-like shape formation. By analyzing the componets of tumor ECM, we found the tenascin-C expression was only detected in glioma cell lines but not in MCF-7 and HepG2 cells. Knocking down the tenascin-C gene using the shRNA strategy converted glioma cells to a transmigration-permissive phenotype for T cell transmigration and ameba-like shape formation. Exogenous tenascin-C protein reduced the amoeba-like shape formation and transmigration of T cells through MCF-7 and HepG2 cell monolayers. By immunoflurosecent staining, we found that high level of tenascin-C was visualized in glioma tumor tissues. CD3+ T cells were detected in the boundary tumor area stained strong positive for tenascin-C. Similar phenomenon was also found in tenascin-C-overexpressed B16F10 tumor model. In addition, tenascin-C could reduce the LPS-induced leukocyte infiltration in air pouch of mouse model. Further, we found that kinase activites of PI3K, ERK, and PKA are required for the Jurkat cell transmigration by the treatments of LY294002, U0126, and H-89 inhibitors, respectively. However, Jurkat T cells showed delayed ERK activation when they were in contact with the cell monolayer or ECM of glioma cells as compared with those in contact with HepG2 and MCF-7 cells. Meanwhile, amoeba-like shape of Jurkat cells on MCF-7 ECM was recuded upon U0126 treatment. Interestingly, ERK activation was no more delayed when Jurkat cells contacted with the cell monolayer of tenascin-C-dowenregulated U-118MG cells. Besides, phospho-ERK was colocalized with polymerized F-actin at the leading edge of amoeba-like moving Jurkat. In conclusion, glioma cells can actively paralyze T cell migration by the expression of tenascin-C, representing a novel immune suppressive mechanism achieved through tumor ECM.

Impact of Minimal Residual Disease (MRD) By Multiparameter Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) on Outcome: Results of Newly Diagnosed Transplant-Eligible Multiple Myeloma (MM) Patients Enrolled in the Forte Trial

Utilizing Multiparametric Flow Cytometry to Identify Patients with Primary Plasma Cell Leukemia at Diagnosis

Complete response after acute myeloid leukemia (AML) therapy historically requires a decrease in bone marrow (BM) blasts to <5% by morphologic assessment. However, accumulating data indicate that measurable residual disease by more sensitive multiparameter flow cytometry (MFC) is a better predictor of outcome than traditional morphologic evaluation. In most cases, morphology alone cannot differentiate between regenerative and neoplastic blasts, raising the question of its added value to more specific MFC. The objective of this study was to assess the value morphologic evaluation adds to MFC for the detection of residual AML in the post-chemotherapy (induction or consolidation) setting. A search of our pathology database identified 47 consecutive nontransplant BM evaluations (41 total patients) in the post-chemotherapy setting between January and March 2020. The morphology portion for each case was reviewed by three independent hematopathologists for evaluation of adequacy (>200 cells), blast count, trilineage hematopoiesis, dysplastic features, focal/patchy disease, and any non-hematopoietic or alternative diagnoses that would not have been discovered by the associated MFC study. The MFC data for each case was separately reviewed by two hematopathologists. Six of the 47 cases showed >5% blasts by morphology (range 8%-70%); in all six cases, >5% abnormal blasts were also identified by MFC (range 23%-78%). Of cases with <5% blasts by morphology (defined as negative by morphology), MFC detected >5% abnormal blasts in four cases and <5% abnormal blasts in five cases (range 0.007-14%). One of these additional nine cases by MFC was identified to have focally clustered blasts by morphology (7% blasts by MFC, <5% overall by morphology). Two of the nine additional cases by MFC were deemed to have inadequate aspirates by morphology. In total, four of the 47 morphology cases had inadequate aspirate smears (9%). Of these four cases, three had adequate particle preparations. Eleven cases overall had corresponding core biopsies submitted, of which five were inadequate (45%). Morphologic examination identified seven cases with dysplastic features; abnormal blasts were identified in four of these cases by MFC (all >5% blasts). In the remaining three cases with dysplasia, ancillary molecular studies supported molecular evidence of residual disease in one case (NPM1 mutation and FLT3-ITD positivity by PCR). No unexpected carcinomas or other findings that would not be detected by the associated MFC study were identified. In conclusion, in our study, morphologic evaluation did not definitively identify any additional cases of residual/recurrent AML that were not identified by MFC. Morphologic evaluation detected dysplasia without abnormal blasts in three cases; however, the significance was unclear without supportive molecular studies. Reducing the number of cases for morphologic evaluation could save resources and improve turnaround time if MFC can provide the relevant details to assess response.

Multiparameter Flow Cytometry Identifies Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes: Correlation with Cytomorphology and Cytogenetics.

Confirming diagnosis of myelodysplastic syndromes (MDS) is often challenging. Standard diagnostic methods are cytomorphology (CM) and cytogenetics. Multiparameter flow cytometry (MFC) is upcoming in MDS diagnostic work up, comparability and investigator experiences are critical. Myeloid nuclear differentiation antigen (MNDA) in myelomonocytic cells might be expressed more weakly in patients with MDS. The analysis of MNDA may thus improve diagnostic capabilities of MFC in MDS. Staining methods and antibody combinations for MFC in MDS are outlined, giving details for interpretation of results in regard to dyspoiesis. MFC results are correlated with CM and CG and with survival data. Use of MNDA in MDS diagnostics was evaluated in 239 patients with MDS, AML, other cytopenic conditions, and in 30 negative controls. Strong correlation between findings in CM and MFC was found; MFC results correlated well with those of CG. Patients with higher grades of dysplasia in MFC had shorter overall survival. Percentages of granulocytes and monocytes with diminished MNDA expression (%dimG, %dimM) were higher in patients with MDS and AML. Mean fluorescence intensity (MFI) of MNDA in monocytes was lower in MDS and AML. Cut-off values for %dimG (12%) and %dimM (22%) as well as for MFI in monocytes (72) were defined discriminating between MDS and non-MDS. MFC adds significant information on dyspoiesis in the diagnostic work up for MDS and provides prognostic information. MNDA expression can be assessed by MFC and may facilitate evaluation of dyspoiesis when added to MDS MFC panels. © 2015 International Clinical Cytometry Society.

Bone marrow segmentation and radiomics analysis of [18F]FDG PET/CT images for measurable residual disease assessment in multiple myeloma

MRD Evaluation By PET/CT According to Deauville Criteria Combined with Multiparameter Flow Cytometry in Newly Diagnosed Transplant Eligible Multiple Myeloma (MM) Patients Enrolled in the Phase II Randomized Forte Trial

Minimal Residual Disease After Allogeneic Stem Cell Transplantation: A Comparison Among Multiparameter Flow Cytometry, Wilms Tumor 1 Expression and Chimerism Status (Complete vs Low-Level Mixed Chimerism) in Patients with Acute Leukemia