Various Stages Of Apoptosis Research Articles

Detection of Live Circulating Tumor Cells by a Class of Near-Infrared Heptamethine Carbocyanine Dyes in Patients with Localized and Metastatic Prostate Cancer

Tumor cells are inherently heterogeneous and often exhibit diminished adhesion, resulting in the shedding of tumor cells into the circulation to form circulating tumor cells (CTCs). A fraction of these are live CTCs with potential of metastatic colonization whereas others are at various stages of apoptosis making them likely to be less relevant to understanding the disease. Isolation and characterization of live CTCs may augment information yielded by standard enumeration to help physicians to more accurately establish diagnosis, choose therapy, monitor response, and provide prognosis. We previously reported on a group of near-infrared (NIR) heptamethine carbocyanine dyes that are specifically and actively transported into live cancer cells. In this study, this viable tumor cell-specific behavior was utilized to detect live CTCs in prostate cancer patients. Peripheral blood mononuclear cells (PBMCs) from 40 patients with localized prostate cancer together with 5 patients with metastatic disease were stained with IR-783, the prototype heptamethine cyanine dye. Stained cells were subjected to flow cytometric analysis to identify live (NIR+) CTCs from the pool of total CTCs, which were identified by EpCAM staining. In patients with localized tumor, live CTC counts corresponded with total CTC numbers. Higher live CTC counts were seen in patients with larger tumors and those with more aggressive pathologic features including positive margins and/or lymph node invasion. Even higher CTC numbers (live and total) were detected in patients with metastatic disease. Live CTC counts declined when patients were receiving effective treatments, and conversely the counts tended to rise at the time of disease progression. Our study demonstrates the feasibility of applying of this staining technique to identify live CTCs, creating an opportunity for further molecular interrogation of a more biologically relevant CTC population.

Morphologic variation of circulating tumor cells in metastatic breast cancer patients visualized by immunofluorescent stain pattern.

e21079 Background: Isolation of circulating tumor cells (CTCs) using microfiltration is a growing utility in the field of CTC detection. The microfiltration approach can be used on peripheral blood as a non-invasive liquid biopsy for cancer detection and subtyping. We present a utilization of the CellSieve microfilter to subtype CTCs based on immunofluorescent staining pattern of cytokeratin filamentation and EpCAM surface marker expression. Our initial study on CTCs in patient blood indicates that disseminated CTC populations have high rates of phenotypic heterogeneity. Further detailed molecular analysis and patient tracking of these phenotypes may lead to individualized patient assessment based on CTC characterization. Methods: 7.5 mL of whole blood collected from metastatic breast cancer patients were diluted in a fixative solution. An 8 µm CellSieve precision microfilter was placed into a filter holder and the samples were passed through the filter (~ 90 seconds). The cells captured on the filter were fixed, permeabilized, and stained with DAPI, cytokeratin (FITC), EpCAM (PE), and CD45 (Cy5). Cells without CD45 staining were classified by their morphology, nuclear integrity and the presence of cytokeratin and EpCAM staining. Results: In our initial assessment, patient samples were found to have a number of phenotypic CTC subtypes. Cytokeratin filamentation was clearly seen on a number of CTCs while other cells presented with spotted patterns, implying CTCs in various stages of apoptosis. Later stage apoptosis, with segmented nuclear signature, was also seen in various samples. Cell clusters and cells in division were DAPI positive, while EpCAM positivity was negligible, 0-3 cells/sample, correlating with established data from the CellSearch CTC assay. Conclusions: In addition to enumeration, phenotypic variation of CTCs may be a valuable tool for the personalized care of cancer patients. We have shown that individual breast cancer patients have overlapping phenotypes of CTCs circulating in their peripheral blood. We have begun categorizing patients based on these phenotypes and plan to correlate them with overall prognosis.

MTOR and its link to the picture of Dorian Gray - re-activation of mTOR promotes aging.

The limited dividing potential of normal cells leads to replicative cellular senescence that is defined by irreversible loss of proliferative potential, adoption of characteristic morphology and expression of typical biomarkers [1,2]. Cellular senescence acts as a barrier to malignant cell transformation in vivo [3] and may contribute to organismal ageing [1,2,4]. The p53 tumor suppressor is a major determinant of cellular senescence [5]. It plays a crucial role in the integration of stress signaling and coordination of cellular responses to stress. Depending on the kind of stress stimuli, stress strength and cellular context, activation of tumor suppressor p53 can induce reversible quiescence, cellular senescence and apoptosis [6]. The function of wt p53 is extremely complex and, of course, has to be explained within the context of the expression of distinct p53 isoforms, levels of p53-induced micro-RNA and a complex network of p53-interacting proteins. Two p53 isoforms (Δ133p53 and p53β), and in particular their mutual interaction, seem to act as endogenous regulators of cellular senescence in normal human fibroblasts [7]. The relevance of these findings to in vivo events reflects the fact that increases in Δ133p53 expression and reductions in p53β expression have been observed in vivo in colon adenomas with senescent phenotypes [8]. The cancer-protective function of wt p53 is also related to its master function in the regulation of various stages of apoptosis [9]. However, wt p53 plays a dual role in the control of cells' suicide; depending on stress strength it can either prevent or induce programmed cell death. While the apoptosis-promoting function of p53tumor suppressor protein has been intensively scrutinized and is indisputable, the pro-survival function of wt p53 (mediated inter alia via TIGAR, its downstream target) has been less explored and due to some controversies remains an object of debate. However, p53 tumor suppressor has been shown to respond to metabolic changes and to influence metabolic pathways through several mechanisms. In response to a lack of nutrients, p53 becomes activated through the activation of AMP-activated protein kinase (AMPK) and the inhibition of AKT. p53 protein further induces AMPK (both directly and indirectly through the sestrins) and activates the expression of tuberous sclerosis 2 (TSC2), resulting in the inhibition of mTOR, a cytoplamic kinase that transfers signals induced by growth factors to cellular machinery promoting cell proliferation and survival. However, cross-talk between p53 and the mTOR signaling pathways is more complex. Recent studies provide evidence regarding the decision-making role of these pathways in the choice between p53-mediated cellular quiescence and senescence. The paradoxical function of p53, on the one hand repressing cellular senescence by promoting quiescence, and on the other inducing senescence, has been systematically addressed and rigorously studied by M. Blagosklonny and colleagues [10-11]. Demidenko et al. [10] have shown that suppression of cellular senescence by wt p53 is associated with p53-induced quiescence and requires p53 transactivation and inhibition of mTOR. Conversely, the activation of mTOR by depletion of TSC2, a negative regulator of mTOR, favors senescence in normal cells [11]. These observations seem to explain why limited activation of p53 may prolong the lifespan of mice [12] and correlate with observations of age-related decline in p53 function. In this issue, Leontieva and Blagosklonny further explore the crosstalk between p53 and mTOR in normal cells and highlight the role of the functional status of mTOR in the transition of cells from quiescence to senescence [13]. To separate these activities the authors induced quiescence prior to the induction of p53, using either the DNA-damaging drug etoposide or nutlin-3a, which induces Mdm2-mediated degradation but does not damage DNA. After removal of nutlin-3a and serum refeeding, nutlin-3a-treated cells entered the cell cycle and divided, whereas etoposide-treated cells did not proliferate after etoposide removal and serum addition. However, etoposide-treated cells re-entered cell cycling if co-treated with rapamycin, implying that functional mTOR is essential for permanent loss of proliferative potential. Thus, this study provides evidence that activation of mTOR in quiescent cells is a decision-making factor in the transition to cellular senescence.

Mechanisms of apoptosis of the human squamous cell carcinoma cell line A431 induced by triptolide

Objective To study the mechanisms of apoptosis of the human squamous cell carcinoma cell line A431 induced bv triptolide.Methods Cultured A431 cells were treated with triptolide at difierent concentrations(1,10,100 mg/L)for various durations(12,18,24,48 hours).Various stages of apoptosis of A431 cells were quantified using flow cytometry and dual staining with Annexin V-FITC and propidium iodide (PI).The expressions of Caspase 3,8 and 9 in triptolide-treated or untreated A431 cells were examined by microspectrophotometry.Results As flow cytometry and fluorescence microscopy revealed,early apoptosis of A431 cells was observed 12 hours after treatment with triptolide,and typical apoptosis was noticed at 24 hours.Moreover,the apoptosis rate was positively correlated with the treatment concentration and durations of trilptolide.The activities of Caspase 3,8 and 9 commenced to increase at 12 hours,peaked at 24 hours (Caspase 3 and 9)or 18 hours (Caspase 8)after triptolide treatment,and the top activities expressed as absorbence value were 0.723,0.850,1.127 for Caspase 3,1.072,1.091 and 1.191 for Caspase-9,0.641,0.780 and 0.910 for Caspase 8 after treatment with triptolide of 1,10 and 100 mg/L,respectively,which were significantly higher than those in untreated control cells at corresponding time points (all P<0.05).Conclusion Triptolide could induce the apoptosis of A431 cells,and the induction effect seems to be concentration-and time-dependent. Key words: Carcinoma; squamous cell; Triptolide; Tumor cells; cultured; Apoptosis; Caspase 3; Caspase 8; Caspase 9

Predicting Responses on the Death Pathway

Multiple signaling pathways can influence whether a cell commits to the cell death program known as apoptosis. For many years, it has been possible to categorize signals as contributing to the "gas" or to the "brakes." However, predicting the biological outcome of multiple signals that apply some gas here and a stomp on the brakes there has remained a challenge. Janes et al. applied a systems–level approach to this problem and created a model to analyze coupling between almost 8000 measurements of signaling parameters in cultured cells with about 1500 measures of the various stages of apoptosis in cells treated with various combinations of cytokines. The model allows the cellular apoptotic response to be correctly predicted under a variety of conditions. K. A. Janes, J. G. Albeck, S. Gaudet, P. K. Sorger, D. A. Lauffenburger, M. B. Yaffe, A systems model of signaling identifies a molecular basis set for cytokine–induced apoptosis. Science 310 , 1646–1653 (2005). [Abstract] [Full Text]

Apoptotic rate in patients with myelodisplastic syndrome treated with modulatory compounds of pro-apoptotic cytokines.

Excessive apoptosis has a central role in ineffective hematopoiesis in myelodysplastic syndrome (MDS). The aim of the study was to quantify apoptosis and Bcl-2 expression in patients with MDS and to use these parameters in the evaluation of treatment efficacy with compounds modulating proapoptotic cytokines. Bone marrow (BM) samples from eight MDS patients were studied: four with refractory anemia and four with refractory anemia with ringed sideroblasts. Two patients with Hodgkin disease without BM determination were studied for control. Therapy consisted in administration of pentoxyphylline, dexamethasone and ciprofloxacin. Biochemical assay of apoptosis and Bcl-2 was performed using annexin V-biotin conjugate antibody and anti-human Bcl-2 antibody respectively, followed by streptavidine-peroxidase conjugate, and peroxidase substrate. Ultrastructural investigation of BM samples was performed with standard electron microscopy techniques. Most of BM hematopoietic cells in the MDS patients had ultrastructural features of various stages of apoptosis including chromatin condensation and margination, cytoplasm condensation and budding of nuclear and plasma membranes to produce apoptotic bodies. Bcl-2 expression showed an inverse correlation with the rate of the apoptotic process. Periodic evaluation of these two parameters has shown an increase of Bcl-2 expression and a decrease of apoptotic rate in patients who had responded to the treatment. Response to the treatment was appreciated in accordance with their transfusion needs. Treatment efficiency diminished in time. The rate of apoptosis was inversely correlated with the level of Bcl-2 expression. These results confirm the importance of the apoptotic process evaluation in monitoring MDS treatment.

The dynamics of morphological changes during in vitro aging of bovine virgin mammary gland neutrophils

The present study was an in vitro analysis of the dynamics of bovine mammary gland neutrophil apop&amp;shy;tosis based on the detection of morphological changes. The neutrophils were isolated from mammary glands of five virgin heifers. The mammary glands were lavaged, the suspensions were then bacteriologically examined, and total and differential cell counts were made. The cells were cultivated in vitro for 4 hours. After 2, 3 and 4 hours of cultivation, they were panoptically stained, and the proportions of apoptotic neutrophils and trypan blue positive neutrophils were determined. Neutrophil apoptosis and impaired cytoplasmic membrane integrity of neutrophils were already observed in the mammary gland lavages (11.9% and 0.8%, respectively). During the cultivation, a progressive increase in the number of apoptotic neutrophils in various stages of apoptosis &amp;ndash; karyopyknosis, zeiosis and apoptotic bodies &amp;ndash; was observed. Karyopyknotic neutrophils represented a dominant part of the apoptotic neutrophil population in the course of the whole cultivation. The most intensive increase was observed in zeiosis, whereas the levels of apoptotic bodies remained the same. After 4 hours of cultivation, 31.7% apoptotic neutrophils and 9.8% trypan blue positive neutrophils (i.e. Secondary necrotic cells) were found. The results of this work show that spontaneous apoptosis and secondary neutrophil necrosis must be taken into account during in vitro cultivations of bovine mammary gland neutrophils.

DNA fragmentation and morphological changes in apoptotic human lymphocytes

Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that ⩾50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density.The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.

Morphologic evidence of apoptosis in childhood acute myeloblastic leukemia treated with high-dose methylprednisolone.

We have previously demonstrated that various subtypes of AML children respond to high-dose methylprednisolone (HDMP; 20-30 mg/kg/day) which could induce in vivo differentiation of myeloid leukemic cells to mature granulocytes. In this study we have evaluated whether apoptosis occurs in AML cells of patients treated by HDMP using morphological criteria. For light and electron microscopic examination bone marrow aspirates were obtained four days and two weeks after methylprednisolone (30 mg/kg/day) treatment from two children with newly diagnosed AML (AML-M3 and AML-M4). In both patients maturation of leukemic cells has previously been reported four days (in patient with AML-M3) and two weeks (in patient with AML-M4) after HDMP treatment. Electron microscopy revealed the characteristic ultrastructural changes of various stages of apoptosis four days after HDMP treatment in a case with AML-M3. Morphologic evidence of apoptosis induced by HDMP were also detected on Wright-stained and toluidine blue stained semithin sections of BM preparations in a patient with AML-M4 and AML-M3 respectively. These findings suggest that HDMP which could induce in vivo terminal differentiation in myeloid leukemic cells is also able to induce apoptosis in patients with AML. The possibility of HDMP-induced apoptosis should be evaluated in a larger series of patients with AML and other types of malignant tumors.

Macrophage expression of interleukin-1 in lymphoepithelioid cell ("Lennert's") lymphoma.

Macrophage activation was studied in three cases of a genuine form of T cell non-Hodgkin's lymphoma particularly rich in epithelioid histiocytes, the so-called "lymphoepithelioid cell lymphoma" or "Lennert's lymphoma". Host tumor infiltrating macrophages actively produced Interleukin-1 as demonstrated by immunohistochemistry. The activated histiocytes also contained intracytoplasmic tumor cells which were either intact or at various stages of apoptosis. We postulate that in Lennert's lymphoma, tumor cells are capable of activating host macrophages. Initial macrophage activation is followed by IL-1 production with recruitment of additional macrophages accounting for the characteristic histological appearance of this tumor. The activated macrophages are also engaged in a phagocytic antitumoral response. Future studies should investigate if this host response can be potentiated.

Behavior of Nuclear Matrix Proteins during Camptothecin-Induced Apoptosis in HL-60 Human Leukemia Cells

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis, of the toad, Bufo bufo

In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the "surplus" of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (Kb) and the rate of desquamation (Kd), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as "dark cells" and "omega-figures" were found to correlate relatively well with the Kb/Kd-ratio. A subsequent ultrastructural analysis - on a cell-by-cell basis - of "dark cells" showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of "omega-figures" combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.