Cell Proliferation and Apoptosis (Advanced Methods)

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Counts of apoptotic cells and apoptotic bodies were performed using 40x objective.

Cell Proliferation and Apoptosis

All identified apoptosis in the tissue were counted and the apoptotic index AI was defined as the total number of apoptotic cells and apoptotic bodies in at least tumoral cells. Clusters of minute apoptotic bodies within the epithelium were scored as one, and the relatively few apoptotic bodies that were shed into the lumen were not scored because they are likely to persist for an indefinite period of time. Areas of marked lymphocytic infiltration or necrosis were not used for counting.

Expression of p53, bcl-2 and Ki67 were determined by immunohistochemistry, using avidin-biotin-peroxidase complex staining method. The sections were microwaved in 10 mM sodium citrate buffer pH 6,0 at 10 min intervals for a total of 20 min. The sections were washed with PBS and incubated with a biotinylated secondary antibody for 30 minutes, followed by incubation with streptavidin-biotin-peroxidase complex Dako for further 30 minutes, at room temperature.

Sections known to express high levels of p53, bcl-2 and Ki were included as positive controls, while negative control slides omitted the primary antibody. Ki labelling index was determined by observing nuclei in areas of the section with the highest labelling rates, and the percentage of Ki labelled nuclei was used for analyses. The significance of associations was determined by the chi-square test or the Fisher's exact test.

The t Student test was done to compare the levels of the parameters studied between the two groups. The Spearman correlation was done between the markers studied. Seventeen were of intestinal type and five of diffuse type according to the Lauren's classification.

Comet assay - apoptosis assay

Apoptotic cells showed nuclear condensation and eosinophilic cytoplasm forming a clear halo or fragmentation of the nucleus, corresponding to apoptotic bodies with no predominant localisation within tumor tissue. Occasionally, apoptotic cells were shed into the glandular lumen.

Lymphocytes that infiltrate the gastric epithelium can be differentiated of apoptotic bodies by noting that the cytoplasm of lymphocytes is scanty and not strongly eosinophilic and that the nuclei are hyperchromatic, but their chromatin pattern is still clearly discernible. The GC tissue has a higher number of apoptotic bodies when compared to the metaplasia intestinal tissue 0. The GC tissue and the IM shown nuclear staining for Ki67, with a variable numbers of nuclei in both tissues, ranging from 0. No nuclear bcl-2 staining was found in this series of tissues.

Small lymphocytes were also very often positive. This difference was not significant. Immunoreactivity for p53 was localised exclusively in the nuclei of positive cells. Ki67 index and bcl-2 expression was similar in p53 positive or negative tumors.

Methods in apoptosis: a four-way battle to the death

No difference among the number of apoptosis was observed among these stages. In this study we evaluated the presence of the apoptotic bodies, the Ki67LI, the expression of p53 and bcl-2 in GC tissue and IM, a premalignant lesion. Cell proliferation is essential in normal cell life and turnover.

However, when it is excessive, it potentiates the action of any carcinogen that targets DNA and may lead to a neoplasia. Apoptosis in the other hand, plays an opposite role in human cell populations. Alteration of the balance between proliferation and apoptosis results in a disturbance of tissue homeostasis and is associated with cancer. Apoptosis is the programmed cell death and was first described by KERR et al.

Evaluation of the extent of apoptosis is a potentially useful measure of a tumor cell kinetics and biologic behavior.

Cell viability, cytotoxicity, and apoptosis assay guide | Abcam

Apoptotic bodies have a different morphology from the other cells and can be differentiated from lymphocytic infiltration and from tumor cell necrosis. Apoptotic bodies were observed in GC as well as in IM, but the number of apoptotic cells was higher in the cancer tissue. This indicates that the failure of the apoptotic process leads to an increase survival of cells with DNA damage. The mechanism may be involved in GC growth. In individuals with normal mucosa, apoptosis seems to be rare and this number increases as the process of multistep carcinogenesis progress from atrophic gastritis, metaplasia intestinal, dysplasia and cancer 12, When the cancer tissues where compared, advanced GC had lower number of apoptosis 17, In these assay we did not find differences between the numbers of apoptosis among the stages, probably due to the short number of tissues with cancer studied.

Apoptosis can occur by the action of p53 at the end of the G1 and G2 phases. Immunohistochemical staining with the monoclonal antibody DO-7 is positive for wild-type p53 as well as for mutant-type p53; however, it is commonly believed that most positive cells represent mutant p53, since the half-life of the wild-type p53 protein is very short This indicated that the mutation of p53 might be a late event in gastric carcinogenesis. Although most of the GC tissue was positive for p53, the AI was higher in this group than in MI, confirming the hypothesis that apoptosis can be induced via a p53 gene independent pathway In the present study, GC and IM tissues demonstrated positive bcl-2 staining.

In the GC patients, bcl-2 was expressed in early GC more frequently than in advanced stage. Others authors 24 published a higher percentage of bcl-2 in early GC when compared to the advanced GC. Some studies observed that bcl-2 was negative in normal gastric mucosa, increase in metaplasia and decrease in GC 12, The aberrant expression of bcl-2 protein may be associated with the early process 10 of the carcinogenic sequence before other carcinogenic events such mutation of p53 was acquired. Perhaps the increased expression of bcl-2 in IM allows prolonged cell survival that allows for persistence and possible progression of a pre-malignant by accumulating genetic abnormalities.

Although bcl-2 was a strong inhibitor of apoptosis, it probably could not induce the cancer alone. Some investigators showed an inverse relationship between p53 and bcl-2 expression in non-Hodgkin's lymphoma 18 , breast carcinoma 8. In this study we also observed an inverse, but not significant correlation. The positivity of bcl-2 was higher in the metaplasia and probably is involved in the progression of carcinogenesis. The p53 was negative in metaplasia and positive in GC, mostly in stage IV, suggesting a late event in this cancer. The bcl-2 gene is rearranged in many diffuse B-cell lymphomas.

Blood ; Apoptosis and Bcl-2 expression in gastric carcinomas: correlation with clinicopathological variables, p53 expression, cell proliferation and prognosis. Int J Oncol ; Apoptosis and disease.

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Lancet ; Pathol Res Pract ; Expression of p53, inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions: correlation with clinical features. BMC Cancer ; Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki Am J Pathol ; Immunohistochemical analysis of bcl -2 and p53 expression in breast carcinomas: their correlation with Ki growth fraction.

Virchows Arch ; Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell-death. Nature ; Frequent occurrence of apoptosis is an early event in the oncogenesis of human gastric carcinoma. Estimative of incidence and mortality of cancer in Brasil, [homepage on the Internet]. Murray A. Recycling the cell cycle: cyclins revisited. Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.

Sci Rep. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods. Quantitative live imaging of endogenous DNA replication in mammalian cells. J Biomol Screen. Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

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In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins. Mol Cell.


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