Early outcomes after allogeneic hematopoietic SCT in pediatric patients with hematologic malignancies following single fraction TBI.

Fractionated TBI (FTBI) followed by allogeneic hematopoietic SCT results in donor engraftment and improves survival in children with high-risk hematologic malignancies. However, acute toxicities (skin, lung and mucosa) are common after FTBI. Late complications include cataracts, endocrine dysfunction, sterility and impaired neurodevelopment. Instead of FTBI, we used low-dose single fraction TBI (550 cGy) with CY as transplant conditioning for pediatric hematologic malignancies. GVHD prophylaxis included CYA and short-course MTX; methylprednisolone was added for unrelated donor transplants. A total of 55 children in first (40%) or second remission and beyond (60%) underwent transplantation from BM (65%) or peripheral blood; 62% from unrelated donors; 22% were mismatched. Median follow-up was 18.5 months (1-68). Overall survival and disease-free survival at 1 year were 60 and 47%, respectively. Acute toxicities included grade 3-4 mucositis (18%), invasive infections (11%), multiorgan failure/shock (11%), hemolytic anemia (7%), veno-occlusive disease (4%) and renal failure (4%). TRM was 11% at 100 days. Non-relapse mortality was 6% thereafter. Graft rejection occurred in 2%. Three patients (5%) died of GVHD. The regimen was well tolerated even in heavily pretreated children and supported donor cell engraftment; long-term follow up is in progress.

Glutathione peroxidase and viral replication: implications for viral evolution and chemoprevention.

It is likely that several of the biological effects of selenium are due to its effects on selenoprotein activity. While the effects of the anti-oxidant selenoprotein glutathione peroxidase (GPx) on inhibiting HIV activation have been well documented, it is clear that increased expression of this enzyme can stimulate the replication and subsequent appearance of cytopathic effects associated with an acutely spreading HIV infection. The effects of GPx on both phases of the viral life cycle are likely mediated via its influence on signaling molecules that use reactive oxygen species, and similar influences on signaling pathways may account for some of the anti-cancer effects of selenium. Similarly, selenium can alter mutagenesis rates in both viral genomes and the DNA of mammalian cells exposed to carcinogens. Comparisons between the effects of selenium and selenoproteins on viral infections and carcinogenesis may yield new insights into the mechanisms of action of this element.

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase.

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.

Low grade gliomas treated with adjuvant radiation therapy in the modern imaging era.

The purpose of this study is to evaluate tumor control and failure patterns in patients with low grade gliomas treated with surgery and conventional adjuvant radiation therapy. Twenty-eight patients with low grade gliomas (7 grade I, 21 grade II) were retrospectively evaluated. Extent of resection was gross total (3), subtotal (17), and biopsy alone (8). All grade I tumors underwent subtotal resection. Median radiation therapy dose was 54 Gy delivered to localized fields. Tumor control and patterns of failure were determined from follow-up computed tomography and/or magnetic resonance scans. Median follow-up was 86 months (range, 2.4-177 months). Thirteen patients (46%) (four grade I, nine grade II) developed tumor progression. The 5-year actuarial progression-free survival rates for grade I and grade II patients were 86% and 51%, respectively. Corresponding 5-year actuarial survival rates were 100% and 70%. All recurrences were within the treated volume. Our results reveal that conventional adjuvant radiation therapy is associated with high rates of local tumor progression in both grade II and incompletely resected grade I low grade gliomas. Alternative strategies need to be explored in these patients in an effort to improve tumor control and outcome.

Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines.

To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines.

What is the optimal treatment volume in Hodgkin's disease patients undergoing high-dose chemotherapy and adjuvant radiation therapy?

To determine the optimal treatment volume in Hodgkin's disease patients undergoing high-dose chemotherapy (HDCT) and radiation therapy (RT), failure sites were reviewed in 56 patients. Twenty-one (38%) received involved-field RT (IFRT) before or after HDCT encompassing sites of prior disease. Failure sites were designated as previously involved (old) or uninvolved (new) sites. Seven patients (12%) died in the immediate post-HDCT period, leaving 49 evaluable (median follow-up, 41 months). Twenty-five patients (51%) relapsed (14 HDCT, 11 HDCT + IFRT): seven (28%) in old, eight (32%) in new, and ten (40%) in old and new sites. Six of the seven who relapsed in old sites received HDCT alone, whereas seven of the eight who relapsed in new sites received IFRT. Relapse in old sites was particularly common in patients failing to achieve a complete response. The most common new failure site was nodal, occurring in 11 patients and was primarily (10/11) adjacent to an old site. Although it controls prior disease, IFRT is insufficient in Hodgkin's disease patients undergoing HDCT. Relapse is common in new nodal sites and is primarily adjacent to prior sites. These results suggest that extended-field RT encompassing old and adjacent uninvolved nodal sites may be the optimal treatment volume in these patients.

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.

Enzyme cytochemical localization of alkaline phosphatase in cultures of chondrocytes derived from normal and rachitic rats.

Epiphyseal growth plate cartilages were removed from rats which had been maintained on normal laboratory chow or a rachitogenic diet. Chondrocytes were released from the growth plates by collagenase digestion and cultured in tissue chamber slides. After 7, 10 and 12 days of culture, the chondrocytes were removed as intact multilayers and processed for electron microscopical enzyme cytochemical studies. Alkaline phosphatase activity in the cultures was visualized by means of a cerium based capture method. Electron-dense cerium phosphate deposits were localized on the membrane of matrix vesicles and plasma membranes of chondrocytes derived from normal and rachitic animals. The appearance of first crystals within matrix vesicles was characterized by a concomitant decrease in alkaline phosphatase activity in the membrane of these structures. Calcification was initiated at approximately the same time in cultures of chondrocytes derived from normal or rachitic animals. The results suggest that rickets has no serious effects on the capacity of chondrocytes to support matrix calcification in vitro. Additionally, the evidence indicates that alkaline phosphatase-positive matrix vesicles play a significant role in the initiation of this process.