Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations

Prostate. 2007 Sep 15;67(13):1384-96. doi: 10.1002/pros.20620.


Background: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic.

Methods: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture.

Results: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells.

Conclusions: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AC133 Antigen
  • Adult Stem Cells / cytology*
  • Adult Stem Cells / metabolism
  • Adult Stem Cells / pathology
  • Antigens, CD / biosynthesis
  • Benzimidazoles / chemistry*
  • Cell Fractionation / methods
  • Cell Growth Processes / physiology
  • Cell Line
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Flow Cytometry
  • Fluorescent Dyes / chemistry
  • Glycoproteins / biosynthesis
  • Humans
  • Immunohistochemistry
  • Immunophenotyping
  • Leukocyte Common Antigens / biosynthesis
  • Male
  • Microscopy, Confocal
  • Peptides
  • Prostatic Hyperplasia / metabolism
  • Prostatic Hyperplasia / pathology*
  • Prostatic Neoplasms / metabolism
  • Prostatic Neoplasms / pathology*


  • AC133 Antigen
  • Antigens, CD
  • Benzimidazoles
  • Fluorescent Dyes
  • Glycoproteins
  • PROM1 protein, human
  • Peptides
  • Leukocyte Common Antigens
  • bisbenzimide ethoxide trihydrochloride