1. When mammalian cells are subjected to stress signals, oxygen deficiency, radiation, DNA damage, or Chemo- therapeutic drugs, p53 is activated, leading to p53 mediated induction of programmed cell death or cell cycle arrest, or both. Introduction

2. Apoptosis Apoptosis or programmed cell death is a normal physiological cell suicide program that is highly conserved among all animals. This regulated process of cell death plays a critical role during embryogenesis, tissue homeostasis and remodelling, and serves to remove unwanted cells such as tumor cells, cells with irreparable DNA damage or those infected with viruses.

3. Morphological changes in cells undergoing apoptosis Cell dehydration (shrinkage) Chromatin condensation Nuclear fragmentation Loss of plasma membrane microvilli Apoptotic bodies

4. Dysregulation of the cell cycle Loss of cell cycle checkpoints is a hallmark of human cancers. Alterations in components of the cell cycle machinery and checkpoint signaling pathways occur in most human tumors. Genetic modifications result in the dysregulaton of oncogens and tumor suppressor genes, which has important implications for the optimization of current therapeutic regimens and the selection of novel cell-cycle targets.

10. Alterations in checkpoint signaling proteins Mutation of p53 is the most observed genetic lesion in human tumors. Directly related to regulatory mechanisms such as the bcl-2 family. Caspase cascade, specially active caspase 3.

11. REGULATION OF APOPTOSIS DETECTED BY MULTIPARAMETER FLOW CYTOMETRY Undergoing apoptotic cell death is possible to detect by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we have adopted the TUNEL, active caspase 3, cleaved PARP, Bcl-2, Bax, Bcl-xl, p53, Fas, Fas-L and CD40 to flow cytometry which enables the simultaneous detection of cellular DNA content, cell cycle, and DNA fragmentation by multiparametric analysis.

12. MULTIPARAMETER FLOW CYTOMETRY

13. AT-1; DU-145; and PC-3 prostate carcinoma cell lines: apoptotic marker

16. a bc d e f Fig. 3 Ehemann et al.

17. f.

19. SPONTANEOUS APOPTOSIS IN BREAST CANCER USING MULTIPARAMETER FLOW CYTOMETRY 1700 human breast carcinomas were screened. In forty cases (2.3%) we detect a hypodiploid sub -G0/1 apoptotic peak with DNA-indices between 0.2 – 0.8. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. The tumor specimens with apoptosis presented 95% ductal invasive, and 5% lobular invasive carcinomas. A correlation (r 2 = 0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells

21. SPONTANEOUS APOPTOSIS IN BREAST CANCER USING MULTIPARAMETER FLOW CYTOMETRY DNA-fragmentation was correlated with the corresponding cell cycle phases. Most of the TUNEL-positive cells contained a DNA-index close to that of cells in G2-phase/ late S-phase of the aneuploid stemline. The high proliferation index corresponds well (r 2 = 0.807) with the increased amount of TUNEL positive cells.

22. TUNEL POSITIVE CELLS IN MAMMARY BREAST CANCER

23. Conclusions Coordinated detection of DNA content, cell cycle, apoptosis, and cell cycle perturbation are essential. This method can be a powerful tool to assess divergent cell cycle effects in clinical relevant research settings. This may prove extremely useful in evaluating response to therapy in patient undergoing chemotherapy. Loss of cell cycle checkpoints is a hallmark of cancers. Alteration in components of the cell cycle machinery and checkpoint signaling pathways occur in most tumors.