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Introduction
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.
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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.

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Morphological changes in cells undergoing
apoptosis

• Cell dehydration (shrinkage)
• Chromatin condensation
• Nuclear fragmentation
• Loss of plasma membrane microvilli
• Apoptotic bodies

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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.

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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.

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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.
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MULTIPARAMETER FLOW CYTOMETRY
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AT-1 DU-145 and PC-3 prostate carcinoma cell
lines apoptotic marker
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Fig. 3 Ehemann et al.
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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 (r2 0.78) was found
between apoptotic cells in sub-G0/1-peak measured
by DNA-cytometry and TUNEL positive cells
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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 (r2 0.807) with the increased
amount of TUNEL positive cells.
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TUNEL POSITIVE CELLS IN MAMMARY BREAST CANCER


 
         
     



 
 
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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.