Can radiation-induced apoptosis be modulated by inhibitors of energy metabolism?

PURPOSE: To determine the effect of the inhibitors of energy metabolism, 2-deoxyglucose (2DG) and sodium azide, on radiation-induced apoptosis. MATERIALS AND METHODS: Radiation-induced apoptosis was determined in U937 monocytic leukaemia cells exposed to energy inhibitors post-irradiation. Apoptosis was scored microscopically using morphological criteria. Glycolysis was determined by assessing glucose consumption and lactate production. Adenine nucleotide levels were measured using a luciferase assay after enzymatic conversion to ATP. Respiration was measured using a Clark-type oxygen electrode. RESULTS: In addition to their apoptosis-inducing properties, both 2DG and azide modified post-irradiation apoptosis. 2DG induced apoptotic radiosensitization after exposure to lower concentrations (5 mM, 10 mM) up to 20 h post-irradiation while a level of radioprotection was found after 5 h exposure to higher doses up to 100 mM. By contrast, all doses of azide examined (5-50 mM) induced apoptotic radioprotection at all times examined. Glycolytic flux and ATP levels fell rapidly with increasing 2DG dose but energy charge remained unchanged. Glycolysis was less influenced by azide, with ATP levels being initially maintained after exposure but decreasing in a dose-dependent manner at 3 h post-irradiation. However, energy charge was unaffected by azide at the concentrations examined. CONCLUSIONS: Both 2DG and azide can influence radiation-induced apoptosis possibly through their effects on glycolysis and ATP levels. We suggest that modulation of energy metabolism provides mechanistic insight into radiation-induced apoptotic pathways.

Conserved 12-bp element downstream from mRNA polyadenylation sites.

Sequences downstream from the AATAAA motif in a number of cellular and viral transcription units have been compared. A 12-bp conserved element was identified in approximately half of the cases studied, and a consensus sequence TTGANNNTTTTTT was derived from a comparison of 74 such sequences. This element is located immediately (5-20 bp) downstream from the poly(A)-addition site in every case where this is known, and it is suggested that this element may be involved in the cleavage/polyadenylation reaction. This proposal is consistent with published studies on deletion mutants of downstream regions.

Does NF-kappa B relieve the transcription block in c-myc?

In this report, it is shown that the mRNA of the c-myc oncogene is capable of forming an extensive stem-and-loop structure, with a free energy of delta G (25 degrees C) = -34 kcal. This secondary structure is situated at the 3' end of the first exon, immediately upstream of an elongation block. It is shown that this region contains potential binding sites for 3 different activator proteins, namely AP-1, AP-2, and nuclear factor-kappa B (NF-kappa B). From an analysis of the properties of these proteins, NF-kappa B could be identified as a candidate for the trans-acting factor involved in relieving the block to transcription.

Sequence homologies in the control regions of c-myc, c-fos, HTLV and the interleukin-2 receptor.

Homologies in the control regions of 2 cellular oncogenes have been identified in this study. Both oncogenes (c-myc and c-fos) are known to be transiently induced by mitogens. We suggest that transcriptional activators bind to these putative control sequences, thus de-regulating gene expression in a coordinated and cell-cycle specific manner. In addition, we report on homologous sequences in the control regions of the human T-cell leukemia viruses types I and II, and in the flanking region of the gene coding for the interleukin-2 receptor. These, and other experimental data, lead to the formation of a model in terms of which the unlimited proliferation of cells infected with HTLV-I and -II may be explained. The differing biological effects of HTLV-I, -II and -III are also examined and discussed at a molecular level.

Cancer genes: current status, future prospects, and applications in radiotherapy/oncology.

There is a sense of excitement in contemporary cancer research, generated largely by the discovery, and subsequent characterization, of oncogenes. These genes are part of the normal complement of cells, and become altered in their structure or expression, during the development of the neoplastic phenotype. In this review, I highlight some of the important advances in the field, starting with the relationships between viral oncogenes and their cellular homologs. I illustrate some of the molecular mechanisms whereby a harmless, or quiescent, cellular gene can be converted ("activated") by radiation or by other carcinogens to a full-blown oncogene involved in carcinogenesis. Next, I discuss two areas where oncogene research has specific relevance for professionals working with radiation, namely the question of radiation-induced cancer, and the issue of the radiocurability of tumors. I also assess the important role of tumor-suppressor genes in oncogenesis. I then describe a genetic model, to illustrate the current status of our understanding of carcinogenesis. Finally, I discuss potential applications of specific interest to oncologists: topics such as prognostic indicators, novel therapeutic strategies, and gene-replacement techniques, are critically reviewed.

Increased radioresistance of tumor cells exposed to metallothionein-inducing agents.

In this study, we have determined the radiosensitivity parameters of cells exposed in vitro to metallothionein-inducing agents. Three well-characterized tumor cell lines were chosen for investigation: HeLa, B16, and WHFIB. We have shown that exposure of cells in vitro to a heavy metal (cadmium), followed by irradiation, enhances cell survival for two out of three cell lines studied. As measured by the mean inactivation dose, the radioresistance increases by a factor of 1.6 for HeLa cells, 1.4 for WHFIB, and a negligible factor for B16 cells. An additional effect was noted when different classes of metallothionein inducers (such as serum factors, cadmium, and dexamethasone) were allowed to act together. Also, we found that the increase in radioresistance exhibits a peak at exposure times of approximately 10 h; longer exposure to inducing agents results in a reduction in radioresistance.

The influence of a high-bran diet on trace element retention in primates.

Proton-induced x-ray emission (PIXE) has become an established analytical method for the determination of trace elements in biomedical samples. The standard PIXE technique has been modified, resulting in a considerable improvement in detection limits. Refinements include the use of an external proton beam, the incorporation of a triggered beam-pulsing system, and the backward angle location (135 degrees to the incident ions) of the Si(Li) x-ray detector. The technique is now clearly capable of rapid, multielemental investigations, and requires a minimum of target preparation. Samples of liver and oesophagus tissue were obtained from 26 baboons at autopsy. Of these, 13 had been maintained on a semi-synthetic (high-bran) diet, with the remaining 13 forming the control group. Some 15 elements were analysed quantitatively; optimum detection limits were in the range 10-20 PPB (1 billion = 10(9). Clear differences were obtained: the high-bran diet is shown to produce imbalances in biologically active elements such as S, K, Ca, Ni Cu and Zn. These results are discussed on the basis of the phytate hypothesis, in terms of which trace elements in the diet are rendered physiologically unavailable to the subject.

Conserved elements in the 3' untranslated regions of c-fos and actin mRNAs.

In this study, the nucleotide sequences of the 3' untranslated regions (UTR) of the mouse and human c-fos genes, and the rat and human beta-actin genes were examined. It is shown (i) that the 3' UTR of c-fos is highly conserved between mouse and man, (ii) that multiple copies of a 12 bp element occur, in clusters, in the 3' UTR both of c-fos and of beta-actin. This conserved 12 bp element is analogous to the putative repressor binding site previously identified (Renan, Bioscience Reports, 5 (1985), 739-753). These findings provide additional support for the proposal that regulatory signals are located in the 3' UTR's of certain genes.

Putative repressor binding sites in the regions mediating transcriptional control of viral and cellular genes.

In this study, the sequences of several cellular genes (c-myc, c-fos, c-sis, c-mos, and the genes for urokinase, heat shock proteins, interleukin-2 and its receptor), thought to be controlled by negative regulatory factors, were examined. As a result of this comparison, multiple (and often clustered) copies of a 12 basepair (bp) element were identified in the flanking regions of these genes. Moreover, sequences with close homology to this 12 bp element were identified in specific control regions of some DNA and RNA tumor viruses. A consensus sequence (TTG nnn TTTTTT) was derived from an analysis of 111 of these elements. These sequence homologies have yielded a coherent first hypothesis, namely that this 12 bp element is the binding site of a transcriptional repressor protein.

How infections with non-retroviral RNA viruses may be involved in the development of neoplasia.

There have been reports of associations of infections with non-retroviral RNA viruses and tumour development. A hypothesis is proposed as to how non-retroviral RNA viruses may play a role in the development of neoplasia. It is based on a recent report of the detection of complementary DNA (cDNA) of the RNA virus lymphocytic choriomeningitis virus in mouse and hamster cells. This lends credence to the claim made in 1975 of the detection of cDNA copies of genomic DNA of three non-retroviral RNA viruses integrated into the DNA of host cells. Briefly, the hypothesis proposes that at least one cDNA fragment of a non-retroviral RNA virus is synthesized and integrated into the genome of the host cell in a way that could lead or contribute to tumour development. General approaches for testing the hypothesis are outlined.

Chromosomal localization of human endogenous retroviral element ERV1 to 18q22----q23 by in situ hybridization.

From a clone containing the entire locus of human endogenous retroviral element ERV1, we have obtained a DNA probe that is specific for the 3' long terminal repeat (LTR) sequence. This probe was used to map the LTR of ERV1 by in situ hybridization to chromosomes from normal human blood lymphocytes. The LTR was found to be localized to the distal portion of the long arm of human chromosome 18, within bands q22----q23. This chromosome locus is near the constitutive fragile site at band q21.3 on chromosome 18 associated with the 14;18 translocations seen in follicular lymphomas.

Dietary wheaten bran in baboons: long-term effect on the morphology of the digestive tract and aorta, and on tissue mineral concentrations.

Two groups of 13 young baboons, each consisting of 8 males and 8 females, were fed on either high- or low-bran diets (based on wheat of either high or low extraction rate) for a period of 26 months. All animals grew well and remained in good condition throughout. Male (but not female) baboons on the high-bran diet had lower (p less than 0.05) concentrations of zinc in serum and bone, despite a low phytate: zinc molar ratio and a high intake of zinc. Particle-induced X-ray emission analysis showed there to be lower concentrations of calcium, copper, zinc, sulphur, potassium and nickel in the livers of baboons on the high-bran diet (P less than 0.005). Baboons on the low-bran diet passed smaller quantities of softer faeces, they had fewer nodules of lymphoid tissue in the distal portion of the colon (P less than 0.05), and within mucosal microherniations of their ileo-caecal valves the epithelial cells showed a greater tendency to squamous transformation (P less than 0.05). Mucosal microherniations of the ileo-caecal valves tended to be more frequent and larger in size (P less than 0.05) in animals of the low-bran group. Morphometrical studies did not reveal any differences in the general structural development of the digestive tract. The high-bran diet had no effect on serum cholesterol concentrations, nor on the incidence or severity of atherosclerotic lesions of the aorta.