title: Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis.
abstract: BACKGROUND
  To objectively measure changes in nuclear morphology and cell distribution following induction of apoptosis.
  
  
  METHODS
  A spontaneously immortalized retinal pigment epithelial cell line (ARPE-19) was cultured for three days in DMEM/F12 with 10% fetal bovine serum followed by 24 hours incubation in staurosporine to induce apoptosis. Cells that were not incubated in staurosporine served as control. Caspase-3 expression in apoptotic cells was demonstrated by quantitative immunofluorescence. Nuclei were counterstained with DAPI. Assessments of nuclear morphology and cell distribution were performed using ImageJ software. Statistical analyses included Student's t-test and Pearson's correlation coefficient. Nearest neighbor analysis was used to assess cell nuclei distribution.
  
  
  RESULTS
  Caspase-3 expression in staurosporine-incubated cells increased by 471% ± 182% compared to control (P=0.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68% ± 5%; P<0.001) and nuclear circumference (78 ± 3%; P<0.001), while nuclear form factor was larger (110% ± 1%; P<0.001). Cell nuclei from the staurosporine-group (R=1.12 ± 0.04; P<0.01) and the control (R=1.28 ± 0.03; P<0.01) were evenly spaced throughout the cultures, thereby demonstrating a non-clustered and non-random cell distribution. However, the staurosporine-incubated group had a significantly lower R-value compared to the control (P=0.002), which indicated a move towards cell clustering following induction of apoptosis. Caspase-3 expression of each individual cell correlated significantly with the following morphological indicators: circumference of the nucleus divided by form factor (r=-0.475; P<0.001), nuclear area divided by form factor (r=-0.470; P<0.001), nuclear circumference (r=-0.469; P<0.001), nuclear area (r=-0.445; P<0.001), nuclear form factor (r=0.410; P<0.001) and the nuclear area multiplied by form factor) (r=-0.377; P<0.001).
  
  
  CONCLUSIONS
  Caspase-3 positive apoptotic cells demonstrate morphological features that can be objectively quantified using freely available ImageJ software. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression.
  The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3271993311662947.
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway. j.r.eidet@gmail.com.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Eidet
authors.forename: Lara
authors.initials: L
authors.lastname: Pasovic
authors.forename: Rima
authors.initials: R
authors.lastname: Maria
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Jackson
authors.forename: Tor P
authors.initials: TP
authors.lastname: Utheim
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D053144:Cell Nucleus Shape / Q000187:drug effects; D053145:Cell Nucleus Size / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D008856:Microscopy, Fluorescence; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D012984:Software; D019311:Staurosporine / Q000494:pharmacology
chemical_list: C505407:CASP3 protein, human; D053148:Caspase 3; D019311:Staurosporine