Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker.
Aneuploidy is associated with spontaneous abortions, birth defects, and many types of human cancers. Currently there are few assays developed for the efficient detection of aneuploidy in vivo. However, with the recent availability of chromosome-specific DNA probes for the rat, fluorescence in situ hybridization (FISH) techniques could be used for the rapid and sensitive detection of aneuploidy in different tissue and cell types. In order to develop a system that can detect alterations in chromosome number in rat cells in vitro, we treated cultured rat lymphocytes with three aneugens-noscapine hydrochloride (0-150 microM) and vincristine and vinblastine sulfate (0-0.06 microM). 5-Bromo-2-deoxyuridine (BrdU; 1 microM) was added to the culture medium to allow proliferating and non-proliferating cells to be distinguished. To test this assay under in vivo conditions, 21-day-old male Sprague-Dawley rats were subcutaneously implanted with osmotic pumps that delivered BrdU (approximately 12 mg/kg per day) continuously. These rats were administered vinblastine sulfate (0, 0.5 and 1mg/kg) by intraperitoneal injection. The rat lymphocytes and hepatocytes incorporating BrdU were detected by immuno-fluorescent labeling, and FISH with a rat chromosome 4 probe was performed on the labeled and unlabeled cells. Highly significant increases in hyperdiploidy were seen in the replicating rat lymphocytes treated with noscapine, vincristine or vinblastine in vitro and in the rat hepatocytes treated with vinblastine in vivo. In contrast, no significant increase in hyperdiploidy was observed in the non-replicating cells. These results demonstrate that this BrdU-enhanced FISH assay with chromosome-specific rat probes can be used to efficiently detect numerical chromosomal aberrations in vitro and in vivo in slowly or moderately replicating rat tissues. The combination of BrdU-labeling and FISH allows the scoring of hyperdiploidy to be focused on the actively replicating cells, thereby increasing the sensitivity of the FISH technique.
Balakrishnan
S
S
Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521, USA.
Payawal
J
J
Schuler
M J
MJ
Hasegawa
L
L
Eastmond
D A
DA
D000782:Aneuploidy; D000818:Animals; D015415:Biomarkers; D001973:Bromodeoxyuridine / Q000032:analysis / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008214:Lymphocytes / Q000187:drug effects / Q000502:physiology; D008297:Male; D009665:Noscapine / Q000009:adverse effects; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014747:Vinblastine / Q000009:adverse effects; D014750:Vincristine / Q000009:adverse effects
D015415:Biomarkers; D014750:Vincristine; D014747:Vinblastine; D009665:Noscapine; D001973:Bromodeoxyuridine