Analysis Of DNA Content In Cells Research Articles

Simple, fast, cost-efficient, reliable, and highly automated DNA content analysis of cells in adherent cultures.

The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double-stranded DNA. This is a laborious and time-consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample-processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well-proven commercial tool-The BD Cycletest™ Plus DNA Reagent Kit-primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.

A Comparative Study of the Antiproliferative Effect of Kohlrabi and Green Cabbage on Colorectal Cancer Cell Lines In Vitro

Experimental studies showed that phytochemicals from Brassicaceae Cruciferae vegetables possess anticarcinogenic properties. Two members of this family, namely cabbage (Brassica oleracea, group Capitata) and kohlrabi (Brassica oleracea, group Gongylodes) seem to share similar anticarcinogenic characteristics. The aim of this study is to compare the effect of the ethanolic extracts of the edible parts of these two plants on the proliferation and apoptosis of HT‐29 and Caco‐2 colorectal cancer cell lines. The cytotoxicity of the extracts was assayed using the Trypan Blue Exclusion method and the IC50 was determined to be higher in kohlrabi compared to cabbage. Consequently, WST‐1 assay was used to assess the effect of these plant extracts on the proliferation of colorectal cancer cell lines: both extracts exhibited a sharp anti‐proliferative effect in a dose and time dependent manner. Analysis of cell DNA content by flow cytomtery revealed the ability of both kohlrabi and cabbage extracts to induce cell cycle arrest in HT‐29 cells at the G phase and S phase in similar patterns. Furthermore both extracts promoted apoptosis in colorectal cancer cell lines. The apoptotic pathway seems to involve the pro‐apoptotic protein Bax and the anti‐apoptotic protein Bcl‐2. As a conclusion, kohlrabi should be considered, like cabbage, among the potent anti‐carcinogenic cruciferous vegetables.

αII-Spectrin Is Critical for Cell Adhesion and Cell Cycle

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

The Selective Protein Kinase CB Inhibitor, Enzastaurin, Induces In Vitro and In Vivo Antitumor Activity in Waldenstrom's Macroglobulinemia.

Background: Waldenstrom's Macroglobulinemia is an incurable lymphoplasmacytic lymphoma with limited options of therapy. We have previously demonstrated upregulation of PKCβ protein in WM using protein array techniques, and confirmed increased expression in WM using immunohistochemistry. PKCβ regulates cell survival and growth, as well as migration and homing in many B-cell malignancies. We therefore hypothesized that inhibition of PKCβ will induce cytotoxicity in WM.Methods: In this study, we examined the effect of serial dilutions of the PKCβ enzastaurin (2.5 uM to 20 uM) on WM cell lines (BCWM1 and WM-WSU), IgM secreting low-grade lymphoma cell lines (MEC-1, RL), as well as primary CD19+ WM cells and WM cells adherent to bone marrow stromal cells (BMSCs), which induce resistance to conventional therapy. Cytotoxicity was measured by MTT assay and inhibition of cell proliferation was determined by thymidine uptake assay. Apoptosis was measured by flow cytometry using Annexin V and DAPI staining at 48 h. Cell DNA content analysis was performed using DAPI staining on fresh cells. Cell signaling pathways targeted by enzastaurin were determined using immunoblotting at 6 h (2.5 to 10 uM) and at 7.5 uM (10 min to 12 h). The effect of enzastaurin in vivo was determined using a subcutaneous WM model in SCID mice. Enzastaurin was given by oral gavage (80mg/kg twice daily).Results: Enzastaurin demonstrated time and dose-dependent inhibition of PKCβ in WM cells. It induced a significant decrease of proliferation at 24 and 48 h in all cell lines tested with an IC50 of 2.5 to 10 uM, even in the presence of DOPPA, a specific PKCβ stimulator. Similar effects were demonstrated in primary CD19+ WM cells, with no cytotoxicity on peripheral blood mononuclear cells indicating selective toxicity on malignant cells. Enzastaurin induced dose-dependent apoptosis at 24 and 48 h with induction of caspases 3, 8, 9 and PARP cleavage as well as a decrease in Bcl-xL. Analysis of cell DNA content confirmed apoptosis at low doses of enzastaurin (5 uM). To further determine the mechanism of action of enzastaurin in WM, we examined downstream molecules. It significantly inhibited AKT phosphorylation and AKT kinase activity, as determined by inhibition of phosphorylation of GSKα/β fusion protein. In addition, enzastaurin inhibited p-MARCK, and ribosomal p-S6. Enzastaurin overcame resistance induced by co-culture of WM cells with bone marrow stromal cells. In addition, enzastaurin (2.5 to 5 uM) in combination with bortezomib (2.5 to 10 nM), another active agent in WM, demonstrated strong synergistic activity using the Calcusyn software for synergy. Given that PKCβ regulates migration and homing of B-cells, we next determined the effect of enzastaurin on in vitro migration of WM cells. In the transwell migration assay, enzastaurin inhibited migration in a dose-dependent fashion (p=0.041). Finally, in vivo animal studies demonstrated significant inhibition of WM tumor growth in the enzastaurin treated mice (n=11), compared to control mice (n=8) (p=0.028).Conclusion: Enzastaurin has significant antitumor activity in WM in vitro and in vivo, providing the framework for clinical trials evaluating enzastaurin as a new therapeutic agent in patients with WM.

Hypodiploidy, Ki-67 growth fraction and prognosis of surgically resected lung cancers.

One hundred and thirty-seven lung cancer patients (123 non-small-cell lung cancers (NSCLC), 10 small-cell lung cancers (SCLC) and four carcinoid tumours) who underwent surgery in an attempt at complete resection were prospectively entered in a study whose aim was to determine the prognostic significance of a hypodiploidy or a multiploidy pattern of tumour cell DNA content and a high immunohistochemical reactivity of Ki-67, a nuclear antigen related to the cell cycle. Indirect immunoperoxidase reactivity of Ki-67 on frozen tumour tissue sections was evaluated both visually, using a classical semiquantitative scale, and by means of a computer-assisted image processor. Cell DNA content analysis was done using static computer-assisted cytometry on tumour cytological prints stained by the pararosaline Feulgen-Schiff technique. The ploidy was characterised for each tumour by DNA index (DI), percentage of hypodiploid cells and type of DNA content histogram (near diploid, hyperdiploid, hypodiploid and multiploid). Ki-67 immunostaining was negative in 64 tumours (48%) and positive in 69 (52%). DNA histogram classification disclosed 57 (42%) near diploid tumours. Among the 80 (58%) aneuploid tumours, 16 were hypodiploid, 44 hyperdiploid and 20 multiploid. The prevalence of both a positive Ki-67 immunostaining and an aneuploid DNA histogram differed according to histology as SCLC demonstrated a higher frequency of both features when compared with NSCLC and carcinoid tumours. On the other hand, Ki-67 immunostaining and ploidy did not significantly differ according to degree of differentiation, nodal status and Mountain's stage grouping. The percentage of cells in the hypodiploid modal DNA was significantly higher for tumours which demonstrated a high Ki-67 immunostaining, suggesting a link between growth fraction and DNA content abnormalities. In univariate analysis, survival did not differ significantly according to either the Ki-67 immunohistochemical reactivity or the DNA index. Patients with a hypodiploid tumour had a shorter survival than patients with other DNA histogram patterns but, owing to the low frequency of hypodiploidy, this difference did not reach statistical significance. In Cox's proportional hazard model, an SCLC histology, an advanced tumour status, a positive nodal status and a hypodiploid tumour (hazard ratio: 2.070; 95% confidence interval 1.041-4.116) were significant determinants of survival. We conclude that hypodiploidy in lung cancer is a distinct DNA content abnormality as it contributes significantly to prognosis. Neither visually assessed nor computer-generated Ki-67 immunostaining measurements significantly determine prognosis.

Differential effects of transforming growth factor‐β and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)

DNA content of rectal scrapings from individuals at low and high risk for the development of colorectal cancer. A feasibility study.

Scrapings of superficial rectal mucosa were collected from 31 patients with colorectal carcinoma, 66 patients with sporadic adenoma, and 53 control subjects with no personal or family history of colorectal cancer. The DNA ploidy level and proliferative patterns of each specimen were analyzed by flow cytometry (FCM). A GMS index, calculated as the ratio of G2 + M:S, was found to be significantly lower in control subjects than in any of the high-risk groups studied. Aneuploidy was more prevalent in rectal scrapings from cancer patients and adenoma patients than in those from control subjects. Aneuploid cell populations were detected in apparently normal rectal scrapings from two control subjects. Some high-risk individuals (i.e., cancer patients and patients with adenomas and a family history of cancer) exhibited higher proportions of tetraploid (designated G2/M) cells and a higher G2/M:S phase ratio than control subjects. The results accumulated thus far show that the rectal scraping procedure is safe and easy to perform. Our limited findings give hope that the DNA content analysis of cells obtained by rectal scraping may eventually prove useful in mass screening for colorectal cancer risk. However, definitive evaluation will require further refinement and elaboration of analytic technique and testing on more patients at various levels of predetermined risk.

DNA content of rectal scrapings from individuals at low and high risk for the development of colorectal cancer. A feasibility study

Scrapings of superficial rectal mucosa were collected from 31 patients with colorectal carcinoma, 66 patients with sporadic adenoma, and 53 control subjects with no personal or family history of colorectal cancer. The DNA ploidy level and proliferative patterns of each specimen were analyzed by flow cytometry (FCM). A GMS index, calculated as the ratio of G2 + M:S, was found to be significantly lower in control subjects than in any of the high-risk groups studied. Aneuploidy was more prevalent in rectal scrapings from cancer patients and adenoma patients than in those from control subjects. Aneuploid cell populations were detected in apparently normal rectal scrapings from two control subjects. Some high-risk individuals (i.e., cancer patients and patients with adenomas and a family history of cancer) exhibited higher proportions of tetraploid (designated G2/M) cells and a higher G2/M:S phase ratio than control subjects. The results accumulated thus far show that the rectal scraping procedure is safe and easy to perform. Our limited findings give hope that the DNA content analysis of cells obtained by rectal scraping may eventually prove useful in mass screening for colorectal cancer risk. However, definitive evaluation will require further refinement and elaboration of analytic technique and testing on more patients at various levels of predetermined risk.