Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation.

We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgV(H) mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.

In vitro differentiation characteristics of human skin fibroblasts: correlations with radiotherapy-induced breast fibrosis in patients.

PURPOSE: To determine whether there is an association between dermal fibroblast differentiation characteristics in vitro and breast fibrosis developing in patients following radiotherapy for breast cancer. MATERIALS AND METHODS: Three hundred and eighty-five patients had been characterized for the degree of breast fibrosis and the level of clinical risk factors for fibrosis as established by logistic regression. Early-passage fibroblasts from 79 patients with a high (HR) or low (LR) level of risk factors were studied in vitro. The percentage differentiated cells (%DC) 7 days after 0 and 8 Gy was scored, and unirradiated colonies were scored for the ratio of early:late fibroblast differentiation stages (E:L ratio). RESULTS: %DC: For the 0 Gy data there was a significant interpatient variation (CoV = 55%, p = 0.0001). HR patients with breast fibrosis had a higher %DC compared with patients without (p = 0.017). E:L ratio: for HR patients there was a significant interpatient variation (82%, p = 0.0030) and a lower E:L ratio for patients with fibrosis compared with those without (p = 0.086), but for LR patients this relationship was reversed (p = 0.079) CONCLUSIONS: There was a true interpatient variation in the in vitro parameters of fibroblast differentiation but insufficient correlation with observed fibrosis after radiotherapy for use as a predictive test.

Low predictive value of intrinsic fibroblast radiosensitivity for fibrosis development following radiotherapy for breast cancer.

PURPOSE: To test whether the intrinsic radiosensitivity of skin fibroblasts from breast cancer patients correlates with the degree of breast fibrosis after breast conserving therapy. METHODS: In a systematic study design, 79 patients were selected from an earlier study group of 385 patients based on observed fibrosis and seven identified clinical risk factors for fibrosis development. In vitro radiosensitivity of patients' dermal fibroblasts was determined by clonogenic assay of early passage cultures. Survival was determined after irradiation at 0, 2 and 4 Gy, given in two fractions of 2 Gy with a 6 h interval. RESULTS: There was a significant inter-patient variation for SF2 values (coefficient of variation = 40%). The ratio of SF2 values for fibroblasts from patients with breast fibrosis versus those without was 0.80 (95% CI: 0.60-1.07). This was a statistically non-significant trend (p = 0.13). The same ratio for a derived value for SF2 ((SF2 + square root of SF4)/2) was 0.88 (p = 0.19). CONCLUSIONS: A significant variation in intrinsic radiosensitivity of breast cancer patients' dermal fibroblasts was observed. However, the degree of radiosensitivity did not show a significant correlation with fibrosis development. This indicates that the use of fibroblast radiosensitivity will have a limited usefulness for predicting fibrosis following breast irradiation.

Chemical characterization and comparative cellular effects of meta-iodobenzyl guanidine and benzyl guanidine.

meta-Iodobenzyl guanidine (MIBG) combines the structural properties of the neuron-blocking agents bretylium and guanethidine and is being used increasingly for various clinical applications. Different samples of MIBG were assayed for possible contamination with benzyl guanidine (BG). Fast-atom-bombardment mass spectrometry (FAB-MS) analysis showed a prominent but variable m/z 150 signal, corresponding to a protonated BG. The MS/MS fragmentation pattern of these [M + H]+ ions was similar to that obtained from FAB-MS-generated, protonated BG, confirming the proposed molecule and associated structures. RP-HPLC analysis of both guanidines, however, excluded the possibility of contamination of MIBG with BG. It was therefore concluded that the BG signal was an artifact of the FAB-MS procedure. In addition, the importance of the meta-substituted iodine for the biological activity of MIBG was investigated. Three different biochemical and cell-biological properties of MIBG were compared with those of its precursor MIBA and BG. The assays used were: inhibition of the catecholamine "Uptake I" system in SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells, inhibition of mitochondrial respiration, and general cytotoxicity in L1210 leukemia cells. Of the drugs tested, MIBG was the most efficient in Uptake I inhibition and was more toxic in survival assays, but as compared with BG it was almost equipotent in inhibiting mitochondrial respiration. These findings contribute to a further elucidation of the mechanism by which MIBG exerts its various actions.