Heterogeneity Of Apoptosis Research Articles

Temporal Heterogeneity in Apoptosis Determined by Imaging Flow Cytometry.

Apoptotic process is highly heterogeneous, and a long-standing question is how many parameters define time and reversibility of the apoptotic response at a population and single-cell levels. Cell death analysis applications have greatly expanded since the introduction of flow cytometry. Classical approach for evaluation of apoptosis is en masse analysis of cells treated with different stimuli, but these methods cannot demonstrate heterogeneity in the population. Single-cell heterogeneity is now usually assessed by multicolor fluorescence microscopy; however obtaining reasonable statistics is time consuming and laborious. Therefore we combined flow cytometry, imaging flow cytometry, and fluorescent microscopy to characterize at a single-cell and population level sequence of apoptotic events induced by a variety of treatments (Vorobjev, Barteneva, J Histochem Cytochem 63:494-510, 2015). We show that simultaneous use of membrane potential dye TMRE, caspases 3/7 sensor, Annexin V and nuclear staining along with morphological parameters demonstrate heterogeneity of the whole process and is a valuable method for quantitative study of the apoptosis execution. Imaging flow cytometry allowed us to analyze correlation between TMRE, caspases 3/7, and Annexin V staining and morphological characteristics providing valuable information on the process of apoptotic execution. Importantly, comparisons of different data sets obtained by three methods allowed us to achieve temporal resolution of the whole process superior to that had been obtained by only one method.

Radiation-induced apoptosis varies among individuals and is modified by sex and age

Purpose: Although there are considerable data on mechanisms of radiation-induced apoptosis in vitro and in animal models, little is known about functional variation in these pathways in humans. We sought to develop a tractable system to evaluate this.Materials and methods: Peripheral blood mononuclear cells were isolated from 90 healthy volunteers, divided into two aliquots, one irradiated with a 5 Gy dose and the other sham-treated (0 Gy), and assessed for damage-induced apoptosis after 24 hours. To investigate reproducibility, 10 individuals spanning the entire radiation-induced apoptotic range were tested three times each, with 3–6 months between replicates.Results: We observed surprising heterogeneity in apoptosis among individuals, ranging from 21–62%. Biological replicates from a single individual, however, were completely concordant, suggesting the variability observed across individuals is not the result of stochastic or short-term effects. We found significantly higher radiation-induced apoptosis in males than in females (Mean: 41.0% vs. 30.7%; p < 3.5 × 10−7). Moreover, advancing age was associated with decreasing radiation-induced apoptosis in males (p = 0.01) but not females (p = 0.82).Conclusions: Our results provide evidence that the function of cellular pathways crucial for stress-induced apoptosis varies by sex and could decline with age in humans.

Abstract 429: Damage-induced apoptosis in primary human lymphocytes is a genetic program modified by gender and age.

Abstract Apoptosis is a core pathway deregulated in hematologic and other cancers. Although there are considerable functional and genetic data on the regulation of apoptosis in vitro and in model organisms, little is known about how these pathways are modified in primary cells freshly isolated from people, who are continually subjected to a variety of different and often unquantified environmental stimuli. We hypothesize that these exposures may have important and as yet unappreciated modifying effects on apoptosis that may have implications for cancer risk or response to therapy. To test this, we isolated peripheral blood mononuclear cells (PBMCs) from 90 normal healthy individuals and treated them with 5 Gy ionizing radiation (IR). Damage-induced apoptosis was measured at 24 hours after IR by TUNEL. We observed surprising heterogeneity in apoptosis among individuals, ranging from 21-65 percent. To determine whether this diversity in response was programmed or stochastic, we repeated the experiment 3 times in 10 individuals spanning the entire IR-induced apoptotic range and found results to be completely reproducible within individuals, suggesting that it resulted from a genetic program. We then assessed the association with individual characteristics. Whereas neither race nor age was associated with IR-induced apoptosis, gender was significantly associated (Mean: 44.8% vs 32.9%; p = 1.02 x 10-8). Moreover, when stratified by gender, age was significantly associated with IR-induced apoptosis in males (p = 0.026) but not females (p = 0.94). Thus, we conclude that IR-induced apoptosis in freshly isolated human PBMCs results from an intrinsic genetic program modified by age, but only within males. Genetic models to explain this will be discussed. Citation Format: Mark A. Applebaum, Andrew Skol, Gareth Bond, Kenan Onel. Damage-induced apoptosis in primary human lymphocytes is a genetic program modified by gender and age. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 429. doi:10.1158/1538-7445.AM2013-429

Low Probability Activation of Bax/Bak Can Induce Selective Killing of Cancer Cells by Generating Heterogeneity in Apoptosis

Biomimetic pro-apoptotic agents (e.g., BH3 mimetics) have been shown to activate the intrinsic death pathway (Type 2 apoptosis) selectively in cancer cells, a mechanism that can be key to developing successful anti-cancer therapy. This work reports mathematical modeling and computer simulations to explore the mechanisms for cancer cell apoptosis. The results indicate that a combination of low probability Bid-Bax type reaction along with overexpressed reactant molecules allows specific killing of cancer cells. Low-probability activation of Bax also emerges as a basis for inherent cell-to-cell variability in apoptotic activation. Variations in Bcl-2 to Bax ratio within a cancer cell population can further affect intrinsic fluctuations generated due to the stochastic Bid-Bax reaction. Such heterogeneity in apoptosis resistance can also provide a mechanism for the origin of cells with higher tumorigenic potential (cancer stem-like cells). The implications of our results for cancer therapy, such as in minimizing stochastic fluctuations in cancer cell death, are discussed.

Heterogeneity of Apoptosis and Growth Fraction within Superficial Spreading Type Early Gastric Cancer

Heterogeneity of Apoptosis and Growth Fraction within Superficial Spreading Type Early Gastric Cancer

Heterogeneity of apoptosis and proliferative activity within superficial spreading type early gastric cancer

Using the in situ DNA nick end-labeling method and immunohistochemistry, the indices of apoptosis (AI) and Ki-67 labeling index (LI) among 3 different portions within the tumor (the central surface vs. the lateral margin or deepest portion) were determined and compared in 14 superficial spreading type early gastric cancers. The AI was the lowest and the LI remained high in the lateral margin compared with the central surface, whereas the AI remained high and the LI was the lowest in the deepest portion. This heterogeneity of apoptosis and proliferation within the tumor might be related to the predominantly horizontal growth of superficial spreading type early gastric cancer.