Novel Putative Fragile Sites Observed in Feline Fibroblasts Treated with Aphidicolin and Fluorodeoxyuridine.

Abstract

ABSTRACT. Fragile sites are non-randomly distributed chromosomal breaks and gaps observed in the cells cultivated under certain conditions. Feline fragile sites were analyzed using skin fibroblast strains after the treatments with aphidicolin and fluorodeoxyuridine in combination with caffeine. Three aphidicolin-induced fragile sites (A1q21, C2q13 and E1p21) as well as a folate-sensitive site (B1q14) were observed in all the 3 fibroblast strains tested for each treatment group. The loci in A1q21 and B1q14 are very close to that reported previously for peripheral blood lymphocytes and lung cells. Two chromosomal break points in C2q13 and E1p21 seem to be new fragile sites. Fifteen candidates for feline fragile sites were also assigned their locations in feline chromosomes. Both the incidence and distribution of feline fragile sites in skin fibroblasts seem to be different at least in part from those in lymphocytes. -KEY WORDS: aphidicolin, chromosomal break, feline, fluorodeoxyuridine, fragile site.J. Vet. Med. Sci. 60 (7): [809][810][811][812][813] 1998 in peripheral blood lymphocytes. For the study on feline fragile sites, however, lymphocytes are less suited, because the culture often suffer a heavy loss of lectin-activated cells by aggregation with platelets. In human, the fragile sites in skin fibroblasts treated with aphidicolin differ in both frequency and distribution as compared with those observed in lymphocytes [10,11]. In the present study, we have investigated novel fragile sites induced in feline skin fibroblasts, because the banding pattern of their chromosomes have been well-characterized [12]. MATERIALS AND METHODSFeline fibroblast strains: Small patches of skin were excised from foot pads of four healthy female domestic cats under the sterile condition. The skin fragments were minced with scissors in the Ca 2+ -and Mg 2+ -free Dulbecco's phosphate buffered saline (PBS(-)), and cell suspension obtained was inoculated into 25 cm 2 plastic culture flasks containing Eagle's minimum essential medium (Nissui) supplemented with MEM sodium pyruvate solution (GIBCO), nonessential amino acid solution (GIBCO), and 10% inactivated fetal bovine serum (Hyclone). The flasks were incubated in the fumidified atmosphere containing 5% CO 2 at 37˚C and medium was replenished periodically until the growth of the fibroblasts reached confluence.Drug treatment: Single-cell suspension was prepared from a confluent culture of fibroblasts by trypsinization and inoculated into a plastic dish containing 10 ml of the growth medium. The dish was then incubated under the condition mentioned above for 2 days. Drug treatment was carried out by the procedure described by Yunis and Soreng with slight modifications [30]. Briefly, the log-phase culture of fibroblast strains FFB2, FFB3 and FFB4 were treated with Fragile sites are non-random chromosomal breaks and gaps observed in the cells under the folate-deficient condition [25] as well as those treated with particular mutagen, carcinogen and clastogens [31]. A great number of p...

Induction of Fragile Sites by Fluorodeoxyuridine and Cafeine Accompanies with Misincorpolation of Endogenous Uridine Nucleotide into DNA fo Feline Fibroblasts.

“…Small patches of skin were excised from foot pads of a healthy female domestic cat under the sterile condition [13]. The skin fragments were minced with scissors in the Ca 2+ -and Mg 2+ -free Dulbecco's posphate buffered saline (PBS(-)), and cell suspension obtained was inoculated into 25 cm 2 plastic culture flasks containing Eagle's minimum essential medium (Nissui) supplemented with MEM sodium pyruvate solution (GIBCO), nonessential amino acid solution (GIBCO), and 10% inactivated fetal bovine serum (FBS; Hyclone).…”

“…After 6 hr of incubation, E. coli cells were removed Fragile sites are non-random chromosomal breaks and gaps observed in the cells under the folate-deficient condition [23] as well as those treated with particular mutagen, carcinogen and clastogens [30]. We have recently detected novel fragile sites in the feline fibroblasts treated with fluorodeoxyuridine and caffeine [13]. Though the exact mechanism for the expression of the fragile site is still unknown, the drug-induced lack of thymidine nucleotides or resultant nucleotide pool imbalance may be included in the mechanism.…”

“…After incubated for 2 days, the cells were subjected to drug treatment by the procedure described by Yunis et al with slight modifications [13,29]. Briefly, the exponentially growing cells were treated with 25 ng/ml fluorodeoxyuridine (FUdR) for 18 hr.…”

“…Alternatively, the cells were treated only with FUdR for 24 hr. For chromosome analysis, colcemid was added at 0.05 µg/ml 30 min prior to the termination of the drug treatments [13,28]. The Q-banded chromosomes were analyzed as described previously [12][13][14].…”

“…For chromosome analysis, colcemid was added at 0.05 µg/ml 30 min prior to the termination of the drug treatments [13,28]. The Q-banded chromosomes were analyzed as described previously [12][13][14]. DNA was extracted from drug-treated fibroblasts by the neutral guanidine thiocyanate phenol-chloroform (GPC) method [1,9].…”