BUdR Dose Research Articles

Cell kinetics of human brain tumors: In vivo study with bromodeoxyuridine and flow cytometry

Bromodeoxyuridine (BUDR) is a thymidine analog which is incorporated into the DNA of proliferating cells. Since the dose of BUDR needed to label cells is not toxic, cell labelling can be accomplished in vivo, by infusing the substance in patients. A monoclonal antibody against BUDR is then used to identify BUDR-labelled cells. The same cell population can also be stained for DNA content with propidium iodide (PI). Using bivariate flow cytometry (FCM) for measurements, both the percentage of BUDR-labelled cells and their total DNA content can be evaluated. This technique allows one to obtain the labelling index (LI) and the DNA synthesis time (TS). The potential doubling time (T pot ) and the fractional turnover rate (FTR) can be mathematically derived, so that a complete picture of tumor growth can be obtained. Our aim was to ascertain whether this method is clinically applicable and whether the kinetic values obtained are reliable. We studied 22 patients with benign and malignant brain tumors, and observed no immediate toxicity from BUDR administration. The BUDRLI obtained ranged from 0.9% to 3.9% (median: 2.0%) in meningiomas and from 3.8% to 7.6% (median: 6.3%) in malignant gliomas (Pš 0.01). The fraction of S-phase cells determined with the BUDR FCM technique was statistically similar to that found by single DNA flow cytometric analysis performed on duplicate samples of both benign and malignant brain tumors. The TS obtained in malignant gliomas ranged from 10.5 to 227 h (median: 12.8). The calculated T pot ranged from 7.6 to 26.8 days (median: 11.6), and the calculated FTR ranged from 3.7 to 13.1 cells/ 100 cells/day (median: 8.8). These data suggest that in vivo BUDR infusion coupled with FCM can be performed in clinical settings, and it is reliable and can easily be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and in planning tumor treatment.

The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo.

The effects of 500 and 300 mg/kg bromodeoxyuridine (BUdR) on the process of fusion of the neural folds were tested after injection into pregnant mice on day 8 of gestation (192 hours postcoitum). Various doses of the natural nucleoside, thymidine (TdR), were also tested. Both doses of BUdR retarded growth to the same extent, but only the larger dose caused neural tube defects in 28.8% of embryos. Treatment with the larger dose also caused extensive cell necrosis to appear in the neuroepithelium of the neural folds between 12 and 15 hours after treatment. No changes were detectable with the light microscope up to this time. Measurement of the cell generation time in treated and control embryos indicated that the BUdR prolonged the cycle by about 2 hours and that the dying cells were in the second DNA synthetic phase following incorporation of the analog. Treatment with the smaller dose of BUdR caused minimal cell necrosis. This was taken as evidence for the importance of cell necrosis in the pathogenesis of BUdR-induced neural tube defects. Treatment with excess TdR did not cause either neural tube defects or cell necrosis, and a dose of TdR equimolar with the large dose of BUdR (400 mg/kg TdR) did not retard growth. Doses of 800 and 1,200 mg/kg TdR retarded growth to the same extent as BUdR. The administration of an equimolar amount of TdR, along with the teratogenic dose of BUdR, prevented the occurrence of cell necrosis and neural tube defects. When treatments were given on day 9 of gestation, 500 mg/kg BUdR caused cell necrosis in the neuroepithelium about 15 hours after treatment but no neural tube defects were produced by day 9 after treatment. It is suggested that in this case cell necrosis occurred too late to interfere with neural fold fusion. It was concluded that the ability of BUdR to cause exencephaly in mouse embryos was due to cell necrosis in the neuroepithelium.

A phase i study of intermittent intravenous bromodeoxyuridine (BUdR) with conventional fractionated irradiation

A Phase I trial of intravenous bromodeoxymidine (BUdR) and conventional fractionated radiation therapy was performed in 14 patients with gliobiastoma multiforme and 7 patients with other poorly radioresponsive tumors. The BUdR was given as a constant intravenous infusion for 12 hr/day for up to 14 days. Thirteen patients received a second 14 day infusion following a 10 to 14 day interruption for bone marrow recovery. Local toxicity (within the radiation field) was minor, with 7 of the 21 patients requiring a brief treatment break for moist skin desquamation. There was no significant CNS toxicity noted clinically nor by autopsy examination. Additionally, no significant enhancement of radiation injury was noted to bowel or liver. However, one patient treated for multiple pulmonary metastases experienced a clinical and radiographic pattern consistent with radiation pneumonitis. Dose-dependent systemic toxicity occurred in bone marrow and skin. Moderate myelosuppression, especially thrombocytopenia, was found following a 14 day cycle of BUdR at and above 650 mg/m 2/12 hr infusion. Approximately one-third of patients developed a maculo-papular erythematous rash to the scalp, neck and upper chest. In two patients, the rash became generalized with evidence of epidermolysis on skin biopsy. Pharmacology studies revealed steady-state arterial plasma levels of 2 x 10 −6 M/1 during the 12 hr infusion of 650 to 700 mg/m 2. Radiosensitization was measured by a change in the D 0 of radiation survival curves of human bone marrow CFUc prior to and following the 14 day infusion in 4 patients. A trend of increasing radiosensitization was noted in most patients as the infusion rate of BUdR was increased from 500 to 870 mg/m 2/12 hr. We conclude that the maximum tolerable dose of BUdR is 650 to 700 mg/m 2/12 hrs when given as a 2 week intermittent intravenous infusion. Local toxicity is acceptable. The major systemic toxicities are myelosuppression and a maculopapular skin rash.

Radiosensitization of hematopoietic precursor cells (CFUc) in glioblastoma patients receiving intermittent intravenous infusions of bromodeoxyuridine (BUdR)

The potential use of bromodeoxyuridine (BUdR) as a radiosensitizer given by an intermittent intravenous route is being studied in a Phase I/II trial at the National Cancer Institute. In order to assess the extent of radiosensitization, we have studied the radiation response of human bone marrow cells CFUc taken from 6 patients prior to and after a 14-day infusion of BUdR. Varying concentrations (1000–1500 mg) of BUdR were infused for 12 hours every 24 hours for up to 14 consecutive days. Cell survival was determined by colony formation of CFUc in soft agar suspensions. X ray survival curves were generated over a dose range of 0–300 rad and the slopes of the survival curves (D 0) before and after BUdR infusion were compared. Radiation enhancement ratios (ER) (D 0 pre-BUdR/D 0 post-BUdR) ranged from 1.0–2.2 and appeared to be BUdR dose dependent. Above 650 mg/m 2 the radiation ER was ≥ 1.5. Dose dependent systemic toxicity to bone marrow and skin was also observed with intermittent intravenous infusions of BUdR. From our study, it appears that an intravenous dose of <700 mg/m 2/12 hours is well tolerated and may result in radiosensitization of CFUc in man.

Incorporation of 5-bromo-2’-deoxyuridine into mesenchymal limb-bud cells destined to die: relationship to polydactyly induction in rats

The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), given at the proper dose and time to pregnant rats produces preaxial hindlimb polydactyly in a high proportion of near term foetuses. The lack of physiological cell death in an area of preaxial mesenchyme known as the foyer primaire preaxial (fpp) is thought to be important in the pathogenesis of this defect. This study addresses the question of whether BUdR's well-known antidifferentiative effects, which are due in some way to drug incorporation into DNA, are operative in this in vivo system. The dose and temporal response of BUdR for the induction of preaxial polydactyly inversely parallels the frequency of embryonic hindlimbs with an fpp. Incorporation of BUdR into degenerative fragments within the fpp of these treated limbs is demonstrated with indirect immunofluorescence using an antibody to bromouridine. Hindlimbs exposed to a threshold dose of BUdR at the optimal time for producing polydactyly have incorporated the drug into degenerative fragments within the fpp. This suggests that a higher, teratogenic dose of BUdR might likewise be incorporated. The resulting higher level of incorporation presumably alters the normal course of terminal differentiation for these cells originally destined to die. Teratogenic doses of BUdR injected at later than the optimal time are also incorporated into dead cell fragments within the fpp, suggesting that presumptive dead cells have additional rounds of DNA synthesis which are BUdR-insensitive. Approximately 12 h prior to overt death presumptive fpp cells no longer incorporate the drug. Results reported support the hypothesis that incorporation of teratogenic levels of BUdR prevent cell death in the fpp. The extra cells are thought to contribute directly or indirectly to the added digit. Contrary to other views, it is suggested that BUdR-induced teratogenesis can be a result of the drug's antidifferentiative effects on specific, 'sensitive', populations of cells.

A simple technique for in vivo observation of SCE in mouse ascites tumor and spermatogonial cells

A simplified technique to observe sister chromatid exchange (SCE) in ascites tumor cells and spermatogonial cells of the mouse was described. The technique consisted of a single intraperitoneal injection of 5-bromo-2′-deoxyuridine (BUdR) absorbed on activated charcoal that assured continuous release of BUdR throughout two rounds of the replication period. The minimum dose of BUdR to detect SCE was found to be 5 mg/body, the SCE frequency being 4.50 – 4.90/ cell in tumor cells and 1.30 – 1.88/cell in spermatogonia, respectively.

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 × 10 −2 M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 μg per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.

Bromodeoxyuridine (BUdR) treatment of melanoma cells decreases cellular proteolytic activity

Summary Neutral and acidic protease levels of B16 mouse melanoma cells were studied before and after cells were treated with bromodeoxyuridine (BUdR) levels sufficient to suppress tumorigenicity and differentiation. Treatment of cells with BUdR for up to 2 weeks produced a 33% decrease in pH 3.4 protease activity and an 80% decrease in pH 7.4 protease activity. Changes in protease levels were also found to be proportional to the BUdR dose after two generations of treatment. Previous results show that high neutral and acidic protease levels seem to be associated with increased tumorigenicity in variant cell lines selected in vivo for increased implantation. The present study documents that proteolytic activity decreases proportionally in the same cells treated with BUdR to lower tumorigenicity and differentiation.

Toxicity of 5-Bromo 2'-Deoxyuridine to Malignant Lymphoid Cell Lines

SummaryMalignant lymphoid cell lines are unusually sensitive to the toxic effects of BUDR. Incubation of mouse myeloma MOPC 315 with 1 and 0.1 μg of BUDR/ml results in an acute toxic effect by Days 3 and 5, respectively. Subcultivation of MOPC 315 in 0.01 and 0.001 μg of BUDR/ml typically results in on Days 12 and 18, respectively. Similar patterns of acute toxicity occur with mouse lymphoma S49.1, human leukemia CCRF/CEM, and mouse myeloma MPC 11. The dose of BUDR required to cause a 50% inhibition of proliferation (ID50) ranges from 0.09 to 0.3 μg of BUDR/ml. In contrast, the ID50 for a variety of cell lines of alternate origins range from 3.14 μg of BUDR/ml for human cervical adenocarcinoma HeLa to 6.23 μg of BUDR/ml for mouse fibroblast L929. Fibroblasts L929 and HeLa cells were grown for 20 and 25 days, respectively, in 2 μg of BUDR/ml with no toxicity.The toxicity of 1 μg of BUDR/ml to MOPC 315 and MPC 11 is partially prevented by 1 μg of thymidine/ml and completely prevented by 10 μg of thymidine/m...