TY - JOUR
T1 - S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy
AU - Konishi, Hideyuki
AU - Steinbach, Gideon
AU - Terry, Nicholas H.A.
AU - Fujita, Kinya
AU - Lee, J. Jack
AU - Ruifrok, Arnout
AU - Spaulding, Dave
AU - Lynch, Patrick M.
AU - Dubin, Joel A.
AU - Andreeff, Michael
AU - Goodacre, Angela M.
AU - Hattori, Takanori
AU - Hittelman, Walter N.
PY - 1997
Y1 - 1997
N2 - Proliferating cells have a restricted three-dimensional spatial distribution within the crypt, which is the proliferative unit of the colon. Accurate quantitative and spatial analyses of S phase cells in the colon have therefore been limited by histological techniques. To overcome these limitations, S phase cells in microdissected intact colonic crypts of control, modified starved, and refed rats were labeled by historic H3 in situ hybridization and analyzed by confocal microscopy. High-resolution digital images of the crypt cell nuclei stained with cyanine nucleic acid and of the labeled S phase cells were produced from confocal microscopic optical crypt sections. The S phase labeling index (LI) per whole crypt significantly (P ≤ 0.001) discriminated the proliferative differences between control, modified starved, and refed rats and correlated (r = 0.92) with the LI determined from histological crypt sections of the same rats. The variance component of the LI attributable to differences between whole crypts, 0.44 (95% confidence interval, 0.38-0.51), was considerably smaller than that attributable to differences between histological crypt sections, 6.07 (95% confidence interval, 5.18-6.96). Confocal microscopy and histone H3 in situ hybridization of intact three-dimensional crypts enables precise in vitro quantitation and spatial analysis of the total and S phase crypt cells.
AB - Proliferating cells have a restricted three-dimensional spatial distribution within the crypt, which is the proliferative unit of the colon. Accurate quantitative and spatial analyses of S phase cells in the colon have therefore been limited by histological techniques. To overcome these limitations, S phase cells in microdissected intact colonic crypts of control, modified starved, and refed rats were labeled by historic H3 in situ hybridization and analyzed by confocal microscopy. High-resolution digital images of the crypt cell nuclei stained with cyanine nucleic acid and of the labeled S phase cells were produced from confocal microscopic optical crypt sections. The S phase labeling index (LI) per whole crypt significantly (P ≤ 0.001) discriminated the proliferative differences between control, modified starved, and refed rats and correlated (r = 0.92) with the LI determined from histological crypt sections of the same rats. The variance component of the LI attributable to differences between whole crypts, 0.44 (95% confidence interval, 0.38-0.51), was considerably smaller than that attributable to differences between histological crypt sections, 6.07 (95% confidence interval, 5.18-6.96). Confocal microscopy and histone H3 in situ hybridization of intact three-dimensional crypts enables precise in vitro quantitation and spatial analysis of the total and S phase crypt cells.
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M3 - Article
C2 - 9232341
AN - SCOPUS:16944364152
SN - 1055-9965
VL - 6
SP - 531
EP - 536
JO - Cancer Epidemiology Biomarkers and Prevention
JF - Cancer Epidemiology Biomarkers and Prevention
IS - 7
ER -