Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine

Abstract

Cell cycle kinetics after X-irradiation were studied in a solid rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine (BrdUrd) in cells in which the DNA was labeled by BrdUrd. It could be shown that this tumor was composed of about 80% diploid host cells, and only 20% of the cells in the dissociated tumor were actually tetraploid tumor cells. When rats were injected intraperitoneally with BrdUrd to label S-phase cells in the tumor, only a fraction of both tqpes of cells became labeled with BrdUrd during Sphase, even 24 h after injection. The diploid BrdUrd-labeled cells progressed rapidly into cycle; 4 h after injection of BrdUrd, labeled diploid G1-phase cells could be observed. Only 25% of the tetraploid S-phase cells could be labeled by a single injection of BrdUrd (160 mgkg body weight). These labeled tetraploid cells progressed through the cell cycle with similar velocities as did labeled diploid cells. Using a "Mini Osmotic Pump" containing bromodeoxycytidine (BrdCyd) at high concentration (0.3 mol/L) that released BrdCyd continuously into the organism where it was converted to BrdUrd, it could be shown that after 2 days about 60% of cells in S-phase and 70% of cells in G2-phase were labeled. The fraction of labeled G2-phase cells in irradiated tumors (D = 10 and 20 Gy) was enhanced between 10 and 50 h after irradiation due to a radiation-induced G2 block in cycling tetraploid tumor cells. No effect of irradiation on incorporation of BrdUrd in S-phase could be observed in the continuous labeling experiments in contrast to the pulse labeling with RrdUrd, where a small retardation of DNA synthesis was observed in the irradiated tumors during the first 15 h after irradiation. When dissociated cells of the tumor were plated in medium containing 20 pmol/L BrdUrd, only the tetraploid tumor cells were able to incorporate BrdUrd and to enter cycle.Key terms: Tumor cell cycle kinetics, flow cytometry, monoclonal antibody to BrdUrd, X-ray irradiation, continuous labeling with bromodeoxycytidineThe response of a rat rhabdomyosarcoma system to irradiation with heavy charged particles and X-rays has been extensively investigated previously with experiments conducted both in vivo and in vitro (5)(6)(7)(22)(23)(24). The radiobiological end-points studied included tumor volume response (22,231, cell survival after tumor irradiation in situ (24), and survival of oxic and hypoxic cells irradiated and assayed in vitro (7). It is known that the redistribution of cells within the cell cycle after irradiation is a n important factor affecting the speed of tumor regrowth. Therefore, measurement of the movement of both the total and the clonogenic population of tumor cells in vivo through the cell cycle as a function of time after irradiation is of special interest in this context. Blocks at certain stages of the cycle, recruitment of resting cells into the proliferating component, and variations in the length of the G1-, S-and G2-phases are important effects induced in the tumor by irradiation. The knowledge of s...

The Fates of Cells in the Developing Cerebral Cortex of Normal and Methylazoxymethanol Acetate‐lesioned Mice

“…Although BrdU has been used quite widely in vitro and in invertebrates (e.g. Nusse et al, 1985;Wilson et al, 1985;Bissen and Weisblat, 1989), its use in vivo for labelling proliferating neuronal precursors in mammals is a relatively recent development (Miller and Nowakowski, 1988;Gillies et al, 1989;del Rio and Soriano, 1989;Wood et al, 1992). Like the more extensively used tracer, tritiated thymidine, BrdU has a short half-life in the blood (Uzman, 1960;Kriss and Revesz, 1962;Packard et al, 1973); its advantages are (i) its increased sensitivity, since the emulsion used in autoradiography is only sensitive to cells labelled in the most superficial few micrometres of a section (Miller and Nowakowski, 1988), and (ii) the rapidity of the immunohistochemical processing (Gillies et al, 1989).…”

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90° and forward scatter measurements

“…Scatter parameters (forward scatter or 90" scatter) are also used to resolve subpopulations in tumors (4) or to discriminate mitotic cells from interphase cells (11,13,16,21,35). Quantification of proliferating S-phase cells can be achieved by the BrdUrd/ DNA technique using monoclonal antibodies to bromodeoxyuridine (BrdUrd) incorporated into DNA during S-phase (5,15,18,24).…”

“…Usually, three compartments of the cell cycle can be resolved after pulse labeling with BrdUrd: unlabeled cells in GIand Gz + M-phase and labeled cells in S-phase. Under special conditions (in vivo tumors or plateau-phase cells in vitro), unlabeled S-phase cells can be observed (1,24,33). With a sensitive technique worked out to detect small amounts of BrdUrd uptake into DNA, Beisker and Hittelman (3) were able to measure unscheduled DNA synthesis associated with the repair of DNA damage of G1-phase cells induced by UV irradiation.…”

Epithelial cell dynamics in rabbit cecum and proximal colon P1

“…The kinetics of BrdU labeling have been investigated by Nusse et al (1985), in which rat rhabdomyosarcoma cells were found to reach a plateau of BrdU-labeled cells 4 hr after pulse-labeling, and remained at that level for 15 hr. This information, combined with an estimated cell cycle time of 30 hr for colonic epithelial cells (Potten et al, 1992b) validates the technique used in the present study.…”