Characterization of Seven Kidney Tumors by Flow Cytometry: Analysis of Cell Cycle, DNA Content and P-Glycoprotein Expression

1992 ◽  
Vol 21 (1) ◽  
pp. 39-42 ◽  
Author(s):  
G. Lizard ◽  
P. Roignot ◽  
L. Dusserre-Guion ◽  
F. Morlevat ◽  
D. Michiels-Marzais ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Content ◽  
Flow Cytometry Analysis ◽  
Kidney Tumors ◽  
P Glycoprotein

scholarly journals Preliminary Characterization of a Monoclonal Antibody (AS-2) against Cell Cycle Related Proteins

1998 ◽  
Vol 17 (2) ◽  
pp. 93-101
Author(s):  
Stefano Nigro ◽  
Anna Rapallo ◽  
Angela Di Vinci ◽  
Elio Geido ◽  
Roberto Orecchia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Dna Content ◽  
Staining Pattern ◽  
Isolated Nuclei ◽  
Erythroleukemia Cell ◽  
Related Proteins

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described.AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

scholarly journals The PAF1c Subunit Cdc73 Is Essential for Hematopoiesis and Displays Differential Gene Regulation in MLL-AF9 Driven Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1280-1280
Author(s):  
Nirmalya SAHA ◽  
James Ropa ◽  
Lili Chen ◽  
Hsiang-Yu Hu ◽  
Maria Mysliwski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Flow Cytometry Analysis ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
A Cell

Abstract The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is a multi-protein complex composed of Paf1, Cdc73, Leo1, Ctr9, Rtf1 and WDR61, which have all been shown to play a role in disease progression and different types of cancer. Previous reports demonstrated that the PAF1c is required for MLL-fusion driven acute myeloid leukemia. This is due, in part, to a direct interaction between the PAF1c and wild type MLL or MLL fusion proteins. Importantly, targeted disruption of the PAF1c-MLL interaction impairs the growth of MLL-fusion leukemic cells but is tolerated by normal hematopoietic stem cells. These data point to differential functions for the PAF1c in normal and malignant hematopoietic cells that may be exploited for therapeutic purposes. However, a detailed exploration of the PAF1c in normal hematopoiesis is currently lacking. Here, we utilize a mouse genetic model to interrogate the role of the PAF1c subunit, Cdc73, in the development and sustenance of normal hematopoiesis. Using hematopoietic-specific constitutive and conditional drivers to express Cre recombinase, we efficiently excise floxed alleles of Cdc73 in hematopoietic cells. VavCre mediated excision of Cdc73 results in embryonic lethality due to hematopoietic failure. Characterization of the hematopoietic system demonstrated that cKit+ hematopoietic stem and progenitor cells (HSPC) are depleted due to Cdc73 knockout. We next investigated the role of Cdc73 in adult hematopoiesis using Mx1Cre mediated excision. Conditional knockout of Cdc73 in the adult hematopoietic system leads to lethality within 15 days of Cdc73 excision while no phenotype was observed in heterozygous Cdc73fl/wt controls. Pathological examination of bones in these mice showed extensive bone marrow failure. Flow cytometry analysis revealed that cKit+ HSPCs in adult mice are ablated following loss of Cdc73. Bone marrow transplantation assays demonstrated a cell autonomous requirement of Cdc73 for HSC function in vivo. To perform cellular characterization of HSPCs upon Cdc73 KO, we optimized excision conditions to capture cKit+ HSPCs with excised Cdc73 but before their exhaustion. Flow cytometry analysis demonstrated that Cdc73 KO leads to a cell cycle defect. Cdc73 excision leads to a 2.5 fold increase in the accumulation of HSPCs in the G0 phase of cell cycle with a reduction in the proliferative phases. This is accompanied with an increase in cellular death as indicated by Annexin V staining. Together, these data indicate that Cdc73 is required for cell cycle progression and HSPC survival. To understand the molecular function of Cdc73, we performed RNAseq analysis to identify genes regulated by Cdc73 in HSPCs. We observed 390 genes are upregulated and 433 genes are downregulated upon loss of Cdc73. Specifically, Cdc73 excision results in upregulation of cell cycle inhibitor genes such as p21 and p57, consistent with the cell cycle defect observed following Cdc73 excision. Further, when comparing our results to leukemic cells, we uncovered key differences in Cdc73 gene program regulation between ckit+ hematopoietic cells and MLL-AF9 AML cells. Loss of Cdc73 in leukemic cells leads to downregulation of genes associated with early hematopoietic progenitors and upregulation of myeloid differentiation genes consistent with previous studies. Interestingly, we observed a more even distribution of expression changes (non-directional) within these gene programs following Cdc73 inactivation in HSPCs. Most importantly, while loss of Cdc73 in MLL-AF9 AML cells leads to a profound downregulation of the Hoxa9/Meis1 gene program, excision of Cdc73 in HSPCs results in a modest non-directional change in expression of the Hoxa9/Meis1 gene program. This was attributed to no change in Hoxa9 and Meis1 expression in HSPCs following excision of Cdc73, in contrast to MLL-AF9 cells where these pro leukemic targets are significantly downregulated. Together, these data indicate an essential role for the PAF1c subunit Cdc73 in normal hematopoiesis but differential roles and context specific functions in normal and malignant hematopoiesis, which may be of therapeutic value for patients with AMLs expressing Hoxa9/Meis1 gene programs. Disclosures No relevant conflicts of interest to declare.

scholarly journals MICROPROPAGATION AND PLOIDY STABILITY OF Lippia lacunosa Mart. & Schauer: AN ENDANGERED BRAZILIAN MEDICINAL PLANT

2019 ◽  
Vol 6 (1) ◽  
pp. 1-7
Author(s):  
Diego Pandeló José ◽  
José Marcello Salabert De Campos ◽  
Lyderson Facio Viccini ◽  
Emilly Ruas Alkimim ◽  
Marcelo De Oliveira Santos
Keyword(s):  
Flow Cytometry ◽  
Medicinal Plant ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Naphthalene Acetic Acid ◽  
Flow Cytometry Analysis ◽  
Ploidy Levels

Lippia lacunosa is a Brazilian savanna plant that belongs to the Verbenaceae family. It has been used in folk medicine as a treatment for different diseases. This species represents an endangered Brazilian medicinal plant, and this is the first report documenting a reliable protocol for the in vitro propagation and regeneration of L. lacunosa. Axenic explants were cultivated in MS medium containing different concentrations of naphthalene acetic acid (NAA) to induce root growth. The mean shoot length and the number of roots were highest with 0.06 mg·L-1 NAA. The highest number of buds in shoot regeneration was induced with 2 mg·L-1 6-benzylaminopurine (BA). To obtain a long-term culture, the dwarf shoots were elongated on MS media containing 0.5 mg·L-1 BA alternated with MS containing 2 mg·L-1 BA every 40 days. In the present protocol, the long-term shoots retained the ability to root even after long periods of BA treatment. In addition, we evaluated the nuclear DNA content and ploidy levels, including the occurrence of endopolyploidy, in long-term micropropagated plant leaves using flow cytometry analysis. The plants propagated in vitro over several years possessed nuclear DNA contents ranging from 2.940 to 3.095 pg, and no differences in DNA content were found among in vitro plants or between these plants and the control (L. lacunosa from a greenhouse with a DNA content of 3.08 pg). The flow cytometry analysis also demonstrated that there was no polyploidization. The present study will be useful for biotechnological approaches and provides the first estimate of the nuclear DNA content of this species using flow cytometry.

Urine cell flow cytometry analysis of PD-L1 expression and DNA content for bladder cancer.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 466-466
Author(s):  
Pin-I Chen ◽  
Alice (Xiaoyang) Wang ◽  
Mustafa Deebajah ◽  
Shaheen Alanee ◽  
Bruce Kendrick Patterson
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Bladder Cancer ◽  
Epithelial Cells ◽  
Cancer Patient ◽  
Cancer Patients ◽  
Dna Content ◽  
Urine Samples ◽  
Normal Donor ◽  
Bladder Cancer Patient

466 Background: Bladder cancer is the fifth most common cancer in the United States. PD-1/PD-L1, a pathway used by cancer cells to evade immune response, correlates with bladder cancer severity and has emerged as a target in bladder cancer treatment. Chromosomal instability is also a prominent feature associated with the development of bladder cancer. A method to unbiasedly analyze PD-L1 expression and DNA content in cells from urine samples will help us better understand bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in urine samples were pelleted, fixed/permeabilized (in incellMAX, IncellDx Inc.) and stained with antibodies against pan-cytokeratin (CK), CD45, PD-L1 and a cell cycle dye. The stained samples were analyzed by a flow cytometer alongside stained control cells. Results: Fifty bladder cancer patient and 15 normal donor urine samples were collected and tested with this assay. We could distinguish epithelial cells (pan-CK+) and white blood cells (WBCs, CD45+) in urine samples and obtain PD-L1 expression and DNA content information simultaneously from these cell populations. The patient samples showed a significantly higher percentage of WBCs with substantial PD-L1 expression. The percentage of PD-L1 positive epithelial cells was not distinguishable between normal donor and patient samples. However, increased post G1 epithelial cells ( > 5%) were observed in a majority of bladder cancer patients, with around 25% of samples showing a DNA index above 1.05. In addition, a comparison of urine collection fixatives showed that incellMAX-fixed samples had the best single cell recovery and DNA content measurement, as shown by lower cell cycle dye staining variability (lower rCV). Statistically significant differences were found between cancer patients and normal samples. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples. Comparing urine samples from bladder cancer patients and normal yielded statistically significant differences that could provide valuable information for bladder cancer patient management.

Abstract 218: Nuclear Remodeling Following Mechanical Circulatory Support

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Jun Luo ◽  
Stephen Farris ◽  
Deri Helterline ◽  
April Stempien-Otero
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Content ◽  
Left Ventricular ◽  
Ventricular Assist Devices ◽  
Circulatory Support ◽  
Ventricular Assist ◽  
Imaging Flow Cytometry ◽  
Nuclear Number ◽  
Nuclear Remodeling

Rationale: Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates the analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox. Objective: We used a novel technique-imaging flow cytometry-to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart. Methods and Results: Cardiomyocytes were isolated from 8 subjects undergoing primary heart transplantation and 15 subjects following unloading with left ventricular assist device (LVAD, mean unloading time 13.7 ± 9.1 months). Myocyte size, nuclear number and size, DNA content (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Myocyte size and nuclear morphology was not significantly different between the groups. However, DNA content per nucleus was significantly decreased (P < 0.01) and the correlation between nuclear size and DNA content lost. The frequency of the cell cycle markers, Ki67 and phospho-histone3 (H3P) were not increased after hemodynamic unloading. Conclusions: Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.

Flow Cytometry Analysis in Voided Urine of Patients with Bladder Cancer

1993 ◽  
Vol 60 (2) ◽  
pp. 162-166
Author(s):  
A. Lotto ◽  
G. Carluccio ◽  
A. Calisti ◽  
A. Disperati ◽  
E. Capuzzo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Bladder Cancer ◽  
Quantitative Evaluation ◽  
Dna Content ◽  
Urine Cytology ◽  
Urinary Cytology ◽  
Flow Cytometry Analysis ◽  
Excellent Correlation ◽  
First Diagnosis

Flow cytometry is known to be able to give a quantitative evaluation of the DNA of cellular populations (grade of ploidy), as well as to estimate the percentages of phases (S + G2M) providing useful information about the pathology in question and its aggressivity. This method has been applied in diagnosing patients with bladder cancer, using their voided urine and comparing with urine cytology. Our data, from 59 patients, indicate flow cytometry utility in diagnosing bladder cancer; in fact there is an excellent correlation between the urinary cytology and the DNA content in cytometry which increases in higher grade bladder cancer. The sensitivity of CFM is in the range of 92% to 94%, and is superior to that of conventional voided urine cytology (range 64% to 84%). It is felt that cytofluorometric analysis permits a reliable evaluation of voided urine, not only at first diagnosis, but especially during follow-up.

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