Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other ''immortalizing'' genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNAbinding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P ¼ 0.001, Fisher's exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.

A hydroxypridinone derivative of desferrioxamine (Na-DFO-HOPO), a dihydroxamic derivative of diethylenetriaminepenta-acetic acid (ZnNa-DTPA-DX), and DTPA (CaNa3- and ZnNa3-DTPA) were tested at dosages of 30 mumol kg-1 for their ability to remove 238Pu or 241Am from rats after their intravenous injection as citrate or inhalation as nitrate. The most effective treatment regimen for injected Pu was the repeated administration of DFO-HOPO; by 7 days the body content was reduced to 8% of that in untreated animals. Repeated dosages of 3 mumol kg-1 DFO-HOPO were as effective as those of 30 mumol kg-1 DTPA. After inhalation of Pu nitrate, repeated treatment with DTPA, DTPA-DX or DFO-HOPO reduced the body content by 7 days to, respectively, 10, 15 and 31% of those in untreated animals. After inhalation of Am, DTPA-DX and DTPA were equally effective, the body contents being reduced to 7% of control values with repeated treatment. Injection of DFO-HOPO was ineffective for enhancing the elimination of inhaled or injected Am. The results confirm the strategy of examining the use of siderophore analogues for the decorporation of Pu or Am. However, at present DTPA should remain the agent of choice, particularly after inhalation.

No abstract

Normal tissue reactions to radiation therapy vary in severity among patients and cannot be accurately predicted, limiting treatment doses. The existence of heritable radiosensitivity syndromes suggests that normal tissue reaction severity is determined, at least in part, by genetic factors and these may be revealed by differences in gene expression. To test this hypothesis, peripheral blood lymphocyte cultures from 22 breast cancer patients with either minimal (11) or very severe acute skin reactions (11) have been used to analyse gene expression. Basal and post-irradiation expression of four radiation-responsive genes (CDKN1A, GADD45A, CCNB1, and BBC3) was determined by quantitative real-time PCR in T-cell cultures established from the two patient groups before radiotherapy. Relative expression levels of BBC3, CCNB1, and GADD45A 2 h following 2 Gy X-rays did not discriminate between groups. However, post-irradiation expression response was significantly reduced for CDKN1A (Po0.002) in severe reactors compared to normal. Prediction of reaction severity of B91% of individuals sampled was achieved using this end point. Analysis of TP53 Arg72Pro and CDKN1A Ser31Arg single nucleotide polymorphisms did not show any significant association with reaction sensitivity. Although these results require confirmation and extension, this study demonstrates the possibility of predicting the severity of acute skin radiation toxicity in simple tests.

With DTPA as a comparison, the siderophore analogue code named 3,4,3-LIHOPO has been tested for its ability to remove 238Pu and 241Am from rats after their inhalation or intravenous injection as nitrate. The most effective treatment regimen for inhaled Pu was the repeated administration of 30 mumol kg-1 3,4,3-LIHOPO. By 7 days after exposure, the Pu contents of the lungs and total body were reduced respectively to 2 and 4% of those in untreated animals. These values were six and three times less than when DTPA was administered using the same protocol. For inhaled Am, 3,4,3-LIHOPO and DTPA were considered equally effective, the lung and total body contents being reduced respectively to 13 and 10% of those in controls. Some animals showed slight degenerative changes in the liver and proximal tubules of the kidneys after the repeated administration of 30 mumol kg-1 of 3,4,3-LIHOPO; however these changes were less marked than after DTPA treatment. After the intravenous injection of Pu, the most effective regimen was the single administration of 3 mumol kg-1 3,4,3-LIHOPO. The body content at 7 days was reduced to 7% controls compared with 19% after the repeated administration of 30 mumol kg-1 DTPA. At a dosage of 30 mumol kg-1, 3,4,3-LIHOPO was less effective owing to the higher retention of Pu in the liver. With repeated dosages of 30 mumol kg-1 3,4,3-LIHOPO was more effective than DTPA for the decorporation of Am; the body contents were 16 and 31% of those in controls respectively. Importantly, the body content was still reduced to 28% of control after a single administration of 3 mumol kg-1. The ligand 3,4,3-LIHOPO, which is also superior to other siderophore analogues, could represent a most significant development in the decorporation of Pu and Am.

With DTPA as a comparison, the siderophore analogue 3,4,3-LIHOPO has been examined for its ability to remove 238Pu and 241Am from the rat after subcutaneous (s.c.) and intramuscular (i.m.) injection of about 200 Bq of each actinide (0.3 ng Pu, 1.6 ng Am). After the s.c. deposition of 238Pu and 241Am, both ligands were more effective after local administration than (in decreasing order) their repeated interperitoneal (i.p.) injection, single i.p. injection and continuous infusion. Dosages of 3 mumol kg-1 of 3,4,3-LIHOPO were at least as effective as 30 mumol kg-1 DTPA after each mode of administration. The most effective regimen of those investigated for s.c. 238Pu and 241Am involved local administration of 30 mumol kg-1 of 3,4,3-LIHOPO at 30 min followed by i.p. injections at 6 h, 1, 2 and 3 day. By day 7 after exposure, the amounts of 238Pu and 241Am retained in the body were 2 and 7% of those in controls, respectively and 10 and four times less than when DTPA was administered using the same regimen. The ligand 3,4,3-LIHOPO was more effective for 238Pu and 241Am after their i.m. injection. This was attributed to the greater retention of these actinides at the wound site (97 versus 67%) when treatment commenced. After a single local injection of 30 mumol kg-1 at 30 min, the amounts of 238Pu and 241Am retained in the body at 7 day were 0.9 and 0.8% of controls. These values were 34 and 27 times less than after local and repeated i.p. injections of DTPA at dosages of 30 mumol kg-1. It is concluded that the administration of 3,4,3-LIHOPO represents potentially a most significant advance in the treatment of wound contamination by 238Pu and 241Am by chelating agents.

The pure carboxylated catechoyl amide LICAM(C) and the calcium and zinc salts of diethylenetriaminepenta-acetic acid (DTPA), were tested for efficacy for removing 238Pu and 241Am from rats after inhalation of the nitrate or intravenous injection of the citrate. The results were compared with the efficacy of methylated LICAM(C) used in previous experiments. It was shown that: (1) after inhalation of 238Pu nitrate, DTPA was far superior to pure LICAM(C); (2) after intravenous injection of 238Pu citrate, the infusion of DTPA plus LICAM(C) was only marginally more effective than DTPA alone; and (3) after inhalation or intravenous injection of 238Pu plus 241Am, the efficacy of pure LICAM(C) was only marginally more effective than the methylated form and neither form was effective for the decorporation of 241Am. It was concluded that DTPA, at present, remains the chelating agent of choice for treating persons accidentally contaminated with transportable forms of Pu and Am.