title: S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy.
abstract: 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.
authors.affiliation: Department of Gastrointestinal Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: H
authors.initials: H
authors.lastname: Konishi
authors.forename: G
authors.initials: G
authors.lastname: Steinbach
authors.forename: N H
authors.initials: NH
authors.lastname: Terry
authors.forename: K
authors.initials: K
authors.lastname: Fujita
authors.forename: J J
authors.initials: JJ
authors.lastname: Lee
authors.forename: A
authors.initials: A
authors.lastname: Ruifrok
authors.forename: D
authors.initials: D
authors.lastname: Spaulding
authors.forename: P M
authors.initials: PM
authors.lastname: Lynch
authors.forename: J A
authors.initials: JA
authors.lastname: Dubin
authors.forename: M
authors.initials: M
authors.lastname: Andreeff
authors.forename: A M
authors.initials: AM
authors.lastname: Goodacre
authors.forename: T
authors.initials: T
authors.lastname: Hattori
authors.forename: W N
authors.initials: WN
authors.lastname: Hittelman
mesh_terms: 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*
chemical_list: D006657:Histones; D012333:RNA, Messenger