S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy.
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 histone 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.
Konishi
H
H
Department of Gastrointestinal Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA.
Steinbach
G
G
Terry
N H
NH
Fujita
K
K
Lee
J J
JJ
Ruifrok
A
A
Spaulding
D
D
Lynch
P M
PM
Dubin
J A
JA
Andreeff
M
M
Goodacre
A M
AM
Hattori
T
T
Hittelman
W N
WN
D000818:Animals; D002455:Cell Division / Q000235:genetics*; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000473:pathology; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D006657:Histones / Q000235:genetics*; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D007413:Intestinal Mucosa / Q000473:pathology; D008297:Male; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011916:Rats, Inbred F344; D016196:S Phase / Q000235:genetics*
D006657:Histones; D012333:RNA, Messenger