This paper describes the determination of the effect of IFN-beta on U-251 MG cells using the bromodeoxyuridine (BrdU/DNA) analysis technique. The cell cycle perturbation of exponentially growing cells was estimated by a newly developed two-dimensional analysis of sequential BrdU/DNA distributions measured at 4-hr intervals after IFN-beta administration. The U-251 MG cell line was sensitive to IFN-beta, and cell proliferation was inhibited by 50.0% at 48 hr. Analysis of DNA histograms indicated that IFN-beta accumulated the cells in the S-phase, from 16 to 48 hr after treatment. In the two-dimensional analysis, labeled cells treated with IFN-beta moved from the S-phase through the G2M-phase and then entered the G1-phase within 12 hr after the initial treatment, in a pattern similar to labeled cells untreated with IFN-beta. After 16 hr, labeled cells treated with IFN-beta began to accumulate in the S-phase and remained there even after 48 hr. These results imply that IFN-beta may have an effect on the G1-phase, thereby inducing S-phase accumulation of human glioma cell line U-251 MG.

The purpose of this study was to investigate the cell cycle perturbation of cultured Ct; rat glioma cells induced by 1-(4-aminod-methyl-5-pyrimidil) methyl -3-(2-chloroethyl) 3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdUlabeled cells with m i d 4 phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (T,) and S phase time (Ts) were estimated from a manually plotted F I S curve at 18 and 6 hr, respectively.The major effect of ACNU on the cell cycle Malignant gliomas are highly infiltrative and generally defy complete excision due to the close proximity of vital brain areas. Since the median survival time of patients who only undergo surgical intervention is only around 14 weeks, postoperative chemoradiotherapy is an important treatment to prolong survival rates (13J4).Nitrosoureas, such as BCNU, 1-(4-amino-2-methyl-5-pyrimidil)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), and methyl-CCNU are the major line of chemotherapeutic drugs for the treatment of malignant gliomas. ACNU has been widely used in Japan, as a single agent or in combination with radiation andlor other chemotherapeutic drugs (12).The knowledge of the effect of various chemotherapeutic agents on cell cycle perturbation is essential for a rational regimen of combined chemotherapy and chemoradiotherapy (6,10,11). Flow cytometric analysis of DNA profiles has demonstrated that ACNU increases the number of cells in the G2M phase. However, it was not clear what cell cohort contributes to G2M phase accumulation (10). The advent of monoclonal antibody against BrdU enabled more detailed cell cycle analyses; the antibody facilitated detection of the DNA-synthesizwas an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to GZM traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the GI phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.Key terms: Flow cytometry, two-dimensional display, BrdU ing cells that had incorporated BrdU (3). Dolbeare et a1 reported a method to analyze cell kinetics using PI (propidium iodide) and FITC (fluorescein isothiocyanate) staining (2). We have recently developed a graphic presentation of multidimensional flow histograms using hexagonal segmentation by the principle of vector quantization (5). The method provides a n accurate presentation of the distribution with a small number of bins and samples, and also permits the quantitative analysis of the cell cycle phase. In this paper, we...