Low-dose radiation exposure induces a HIF-1-mediated adaptive and protective metabolic response.

Because of insufficient understanding of the molecular effects of low levels of radiation exposure, there is a great uncertainty regarding its health risks. We report here that treatment of normal human cells with low-dose radiation induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis resulting in increased radiation resistance. This metabolic change is highlighted by upregulation of genes encoding glucose transporters and enzymes of glycolysis and the oxidative pentose phosphate pathway, concomitant with downregulation of mitochondrial genes, with corresponding changes in metabolic flux through these pathways. Mechanistically, the metabolic reprogramming depends on HIF1α, which is induced specifically by low-dose irradiation linking the metabolic pathway with cellular radiation dose response. Increased glucose flux and radiation resistance from low-dose irradiation are also observed systemically in mice. This highly sensitive metabolic response to low-dose radiation has important implications in understanding and assessing the health risks of radiation exposure.

Regulation of normal cell cycle progression by flavin-containing oxidases.

Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21(Waf1) and phospho-p38(MAPK). When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G(1) delay was observed with concomitant activation of p53/p21(Waf1) signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, gamma-radiation or t-butyl-hydroperoxide, attenuated the G(1) delay. Whereas the DPI-induced G(1) checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G(1) to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G(2). We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G(1).

[Nontargeted effects of ionizing radiation: implications for low-dose exposures].

The traditional thinking has been that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. This implies that: 1) biological effects occur only in irratiated cells, 2) radiation traversal through the nucleus of the cell is a prerequisite to produce a biological response, and 3) DNA is the target molecule in the cell. Evidence has been emerging, however, for non-DNA targeted effects of radiation; that is, effects including mutations, chromosomal aberrations, and changes in gene expression which occur in cells that in themselves receive no radiation exposure. Two of these phenomena will be described in this paper. The first is radiation-induced genomic instability whereby biological effects, including elevated frequencies of mutations and chromosomal aberrations, arise in the distant descendants of irradiated cells. The second phenomenon has been termed the "bystander effect", whereby in a mixed population of irradiated and nonirradiated cells, biological effects arise in those cells that receive no radiation exposure. The damage signals are transmitted from cell to cell through gap junction channels, and the genetic effects observed in bystander cells appear to result from an upregulation of oxidative stress. The possible influence of these non-targeted effects of radiation of the respounse to low-dose exposures is discussed.

Levels of gamma-H2AX Foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual.

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks, as a possible means for identifying individuals who are mildly hypersensitive to ionizing radiation, such as some ATM heterozygotes. We compared levels of gamma-H2AX foci after irradiation in cells from six apparently normal individuals as well as from individuals from two separate AT families including the proband, mother, father and three unaffected siblings in each family. After a 1-Gy single acute (high-dose-rate) gamma-ray dose delivered to noncycling contact-inhibited monolayers of cells, clear differences were seen between samples from normal individuals (ATM(+/+)) and probands (ATM(-/-)) at nearly all sampling times after irradiation, but no clear distinctions were seen for cells from normal compared to obligate heterozygotes (ATM(+/-)). In contrast, after 24 h of continuous irradiation at a dose rate of 10 cGy/h, appreciable differences in numbers of foci per cell were observed for cells from individuals for all the known ATM genotypes compared with controls. Four unaffected siblings had mean numbers of foci per cell similar to that for the obligate heterozygotes, whereas the other two had mean values similar to that for normal controls. We determined independently that those siblings with mean numbers of foci per cell in the range of ATM heterozygotes carried the mutant allele, while both siblings with a normal number of foci per cell after irradiation had normal alleles. A more limited set of experiments using lymphoblastoid cell strains in the low-dose-rate assay also revealed distinct differences for normal compared to ATM heterozygotes from the same families and opens the possibility of using peripheral blood lymphocytes as a more suitable material for an assay to detect mild hypersensitivities to radiation among individuals.

gamma-H2AX foci after low-dose-rate irradiation reveal atm haploinsufficiency in mice.

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks (DSBs), as a possible means for identifying individuals who may be intermediate with respect to the extremes of hyper-radiosensitivity phenotypes. In this case, cells were studied from mice that were normal (Atm+/+), heterozygous (Atm+/-), or homozygous recessive (Atm-/-) for a truncating mutation in the Atm gene. After single acute (high-dose-rate) exposures, differences in mean numbers of gamma-H2AX foci per cell between samples from Atm+/+ and Atm-/- mice were clear at nearly all sampling times, but at no sampling time was there a clear distinction for cells from Atm+/+ and Atm+/- mice. In contrast, under conditions of low-dose-rate irradiation at 10 cGy/h, appreciable differences in the levels of gamma-H2AX foci per cell were observed in synchronized G1 cells derived from Atm+/- mice relative to cells from Atm+/+ mice. The levels were intermediate between those for cells from Atm+/+ and Atm-/- mice. After 24 h exposure at this dose rate, measurements in cells from four different mice for each genotype yielded mean frequencies of foci per cell of 1.77 +/- 0.13 (SEM) for Atm+/+ cells, 4.75 +/- 0.20 for the Atm+/- cells, and 11.10 +/- 0.33 for the Atm-/-cells. The distributions of foci per G1 cell were not significantly different from Poisson. To the extent that variations in sensitivity with respect to gamma-H2AX focus formation reflect variations in radiosensitivity for biological effects of concern, such as carcinogenesis, and that similar differences are seen for other genetic DNA DSB processing defects in general, this assay may provide a relatively straightforward means for distinguishing individuals who may be mildly hypersensitive to radiation such as we observed for Atm heterozygous mice.

Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations.

The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of alpha particles such that < 1% of the nuclei were traversed by an alpha particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.

Stress signaling from irradiated to non-irradiated cells.

Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and non-irradiated cells in the population. This phenomenon, termed the "bystander response", has been shown to occur both in vitro and in vivo. Experiments have indicated that genetic alterations, changes in gene expression and lethality occur in bystander cells that neighbor directly irradiated cells. Furthermore, cells recipient of growth medium harvested from irradiated cultures exhibit responses similar to those of the irradiated cells. Several mechanisms involving secreted soluble factors, gap-junction intercellular communication and oxidative metabolism have been proposed to regulate the radiation-induced bystander effect. In this review, our current knowledge of this phenomenon and its potential impact both on the estimation of risks of exposure to low doses/low fluences of ionizing radiation and on radiotherapy is discussed.

Differing effects of breast cancer 1, early onset (BRCA1) and ataxia-telangiectasia mutated (ATM) mutations on cellular responses to ionizing radiation.

PURPOSE: The ataxia-telangiectasia mutated (ATM) gene encodes a protein kinase, the activation of which is an early event in the cellular response to ionizing radiation. One of the many substrates of ATM is BRCA1 (breast cancer 1, early onset gene), which has been associated with susceptibility to breast and ovarian cancer, and has been implicated in DNA repair processes. Various cellular responses to radiation were analysed in cells with mutations in ATM or BRCA1 in an attempt to clarify which effects of ATM can be mediated through BRCA1. MATERIALS AND METHODS: The response to radiation of cells with mutations in ATM or BRCA1 was examined, as were BRCA1-mutant tumour cells transfected with an exogenous wild-type BRCA1 allele. Assays included cell-survival curves, studies of potentially lethal damage repair, measurement of chromosomal aberrations and of G1 arrest, and Western blot analysis of lysates of irradiated cells to determine the phosphorylation of the product of the human Mdm2 gene (HDM2). RESULTS: Both ATM and BRCA1 mutations were associated with sensitivity to ionizing radiation, deficient repair of potentially lethal damage and markedly increased chromosomal aberrations. A BRCA1-mutated tumour cell line HCC1937, like ATM mutant cells, did not exhibit a normal G1 arrest but, unlike ATM mutant cells, did exhibit phosphorylation of HDM2. Expression of wild-type BRCA1 in HCC1937 cells partially restored radioresistance, restored repair of potentially lethal damage and markedly reduced radiation-induced chromosomal aberrations. G1 arrest, however, was not restored by expression of BRCA1. CONCLUSIONS: The results are consistent with a model in which ATM phosphorylation of BRCA1 regulates DNA repair functions, particularly those involved in potentially lethal damage repair and chromosomal integrity, but not other aspects of the cellular response to radiation such as G1 cell cycle arrest. To the authors' knowledge, this is the first demonstration of the ability of exogenously expressed BRCA1 to restore the ability to perform potentially lethal damage repair and maintain chromosomal integrity in irradiated cells.

Increased bystander mutagenic effect in DNA double-strand break repair-deficient mammalian cells.

PURPOSE: We have shown previously that when monolayer cultures of Chinese hamster ovary (CHO) cells are exposed to very low fluences of alpha-particles, HPRT mutations are induced in non-irradiated 'bystander' cells in the population. The present investigation was designed to examine the role of DNA repair in this process. MATERIALS AND METHODS: The DNA double-strand repair-deficient mutant cell line xrs-5 was exposed to mean doses of alpha-particles as low as 0.04 cGy whereby less than 1% of the nuclei were traversed by an alpha track and thus received any radiation exposure. RESULTS: With this very low alpha-particle fluence, most of the cells in the xrs-5 population appeared to be at risk for the induction of mutations, indicating a much larger bystander effect than observed with wild-type CHO cells. Molecular structural analyses showed that xrs-5 mutants primarily involved partial and total gene deletions as opposed to wild-type cells where point mutations predominated in bystander cells. CONCLUSIONS: These results indicate a very large bystander effect in xrs-5 cells. They support the hypothesis that unrepaired or misrepaired double-strand breaks (DSB), arising from opposed DNA lesions, enhance the sensitivity of bystander cells in xrs-5 cultures to the induction of mutations.

Low dose ionizing radiation-induced activation of connexin 43 expression.

PURPOSE: Connexin 43 has been implicated in the cellular response to ionizing radiation by enabling cell-to-cell communication. It is established here that the expression of connexin 43 is affected by ionizing radiation and the mechanism involved is investigated. MATERIALS AND METHODS: The human connexin 43 promoter was cloned into a Luciferase reporter plasmid and activation by ionizing radiation was measured in normal human fibroblasts as well as HeLa cells. The regions responsible for the radiation inducibility were defined using deletion and point mutations of the construct. The results were confirmed by Northern and Western blotting. RESULTS: Ionizing radiation activates the human connexin 43 promoter in a time- and dose-dependent manner with a maximal induction (4.2-fold +/-0.58) after 6 h and a dose of 0.5 Gy. Higher doses up to 5 Gy led to a less marked increase (2-fold) over the same period. This promoter activation was associated with comparable increases in both connexin 43 mRNA and protein levels. The low dose radiation response of the promoter is mainly dependent on consensus binding sites for nuclear factor of activated T-cells (NFAT) and activator protein (AP1) in a region -2537 and -2110 bp from the transcriptional start site as determined by mutation analysis. CONCLUSIONS: Low doses of ionizing radiation induce the transcriptional upregulation of connexin 43 expression employing NFAT and AP1 sites.

Bystander effects: intercellular transmission of radiation damage signals.

Biological effects were examined in confluent cultures of fibroblasts and epithelial cells exposed to very low mean doses of alpha radiation, doses by which only 1-2% of the cells were actually traversed by an alpha particle. Enhanced frequencies of sister chromatid exchanges and HPRT mutations occurred in the non-irradiated, 'bystander' cells associated with a similar increase in the frequency of micronuclei, indicating the induction of DNA damage in these cells. In order to gain information concerning molecular pathways, changes in gene expression were examined in bystander cells by western analysis and in situ immunofluorescence staining. The expression levels of p53, p21 and MDM2 were significantly modulated in bystander cells; the damage signals leading to these changes were transmitted from irradiated to bystander cells by gap junction mediated intercellular communication. The bystander response was suppressed by incubation with superoxide dismutase as well as an inhibitor of NADPH oxidase, suggesting the effect may be mediated by oxidative stress. To examine other signalling pathways responsive to oxidative stress, the activation of stress-related kinases and their downstream transcription factors were analysed in bystander cells by western blotting and electrophoretic mobility shift assays; a 2-4-fold increase in the phosphorylation levels of JNK, ERK1/2, p90RSK, Elk-1 and ATF2 was observed. These changes were detected by 15 min after irradiation and persisted for at least 1 h. These findings indicate the activation of multiple signal transduction pathways in bystander cells, involving signals arising from the plasma membrane as well as from DNA damage.

Multiple manifestations of X-ray-induced genomic instability in Chinese hamster ovary (CHO) cells.

Carcinogenesis is postulated to follow a multistep cascade in which the first genetic event may destabilize cells and thereby facilitate the induction of subsequent mutations within the same cell. It has recently been shown that exposure to ionizing radiation can in itself induce a persistent, heritable genetic instability in cells. To further investigate this phenomenon, we utilized a mutationally unstable population derived from a single Chinese hamster ovary (CHO) cell that survived X irradiation. We exposed these cells to a second dose of radiation, selected hypoxanthine phosphoribosyl transferase (HPRT) mutant subclones, and identified the type of mutations involved. We found complete deletions, continuous tract partial deletions, single-exon deletions, discontinuous-exon deletions ("skip mutations"), and point mutations (changes of less than 100 bp) among the isolated HPRT mutants. We hypothesized that the skip mutation clones might be more likely to demonstrate genomic instability. To test this hypothesis, mutant subclones were screened for three markers of genetic instability: alteration of minisatellite sequences, change in telomere length, and induction of chromosomal aberrations. Clones with skip mutations and single-exon deletions possessed elevated frequencies of minisatellite alterations and chromosomal aberrations, particularly rings and dicentrics. All mutant clones showed longer telomere terminal restriction fragment lengths than did wild-type cells. These results are consistent with the hypothesis that irradiation may induce a global instability phenotype, since the multiple alterations observed are mechanistically distinct, heritable cellular modifications that arose in the clonogenic progeny of the irradiated cells. Skip mutations may be one manifestation of this instability, but their presence was not specifically associated with the other genetic alterations.

HPRT mutants induced in bystander cells by very low fluences of alpha particles result primarily from point mutations.

We have shown previously that damage signals may be transmitted from irradiated cells to nonirradiated cells in monolayer cultures, leading to changes in gene expression and an enhanced frequency of mutations in these "bystander" cells. The present study was designed to test the hypothesis that mutations occurring in bystander cells result from a different mechanism than those occurring in irradiated cells, and thus show differences in molecular structure. Structural changes in the HPRT gene of Chinese hamster ovary (CHO) cells were determined by multiplex PCR analysis. A total of 790 mutant clones derived from monolayer cultures exposed to mean doses of 0, 0.5 or 10 cGy of alpha-particle radiation (0, 3% or 44%, respectively, of nuclei traversed by one or more alpha particles) were examined. Whereas mutations induced by 10 cGy included a high frequency of deletions, nearly all mutations occurring in bystander cells in cultures irradiated with 0.5 cGy involved point mutations, confirming our hypothesis that they are induced by a different mechanism.

X-ray induction of microsatellite instability at autosomal loci in human lymphoblastoid WTK1 cells.

Many models of carcinogenesis posit that multiple genetic events are required for a normal cell to become cancerous. As the mutation rate of a single gene is in the range of 10(-8) to 10(-5) per cell division, a central question remains, how does a single cell acquire multiple mutations? One hypothesis, originally articulated by Loeb [10], proposed that some mutations may not be isolated events, but are associated with a mutator phenotype that leads to the occurrence of additional mutations elsewhere in the cellular genome. To test this hypothesis, we utilized a human lymphoblastoid cell line (WTK1) that is known to be hypermutable at the autosomal thymidine kinase (TK) locus. We isolated 139 independent clones which were selected for new TK mutations that arose either spontaneously or as the result of a single X-ray exposure of 1.5Gy. These clones were examined for second-site alterations in several microsatellite loci scattered throughout the genome using polymerase chain reaction (PCR) amplification followed by both denaturing gel electrophoresis and single-stranded conformational polymorphism (SSCP) analysis. Of these clones, 21 exhibited second-site mutations primarily involving loss of heterozygosity, 17 arose from irradiated cells whereas the remaining four arose from non-irradiated cells. We further examined the 17 clones which exhibited alterations specifically at the D16S265 locus; alterations at this site were associated with an enhanced frequency of mutations at other loci in the same region of chromosome 16q, but were not associated with additional mutations at other sites in the genome. Furthermore, new mutations arose in loci on 16q when these clones were propagated for 6 months in culture. Overall, these results support the hypothesis that radiation can induce a type of genetic instability which may facilitate the occurrence of multiple mutations throughout the genome in a small population of exposed cells. Furthermore, some cells may possess localized regions in the genome which are highly sensitive to the induction of instability.

The bystander effect in radiation oncogenesis: II. A quantitative model.

There is strong evidence that biological response to ionizing radiation has a contribution from unirradiated "bystander" cells that respond to signals emitted by irradiated cells. We discuss here an approach incorporating a radiobiological bystander response, superimposed on a direct response due to direct energy deposition in cell nuclei. A quantitative model based on this approach is described for alpha-particle-induced in vitro oncogenic transformation. The model postulates that the oncogenic bystander response is a binary "all or nothing" phenomenon in a small sensitive subpopulation of cells, and that cells from this sensitive subpopulation are also very sensitive to direct hits from alpha particles, generally resulting in a directly hit sensitive cell being inactivated. The model is applied to recent data on in vitro oncogenic transformation produced by broad-beam or microbeam alpha-particle irradiation. Two parameters are used in analyzing the data for transformation frequency. The analysis suggests that, at least for alpha-particle-induced oncogenic transformation, bystander effects are important only at small doses-here below about 0.2 Gy. At still lower doses, bystander effects may dominate the overall response, possibly leading to an underestimation of low-dose risks extrapolated from intermediate doses, where direct effects dominate.

Persistent genetic instability in cancer cells induced by non-DNA-damaging stress exposures.

A hallmark of cancer cells is their pronounced genetic instability, which has been implicated in both tumor development and negative treatment outcomes. Recently, it has been reported that ionizing radiation may induce a persistent state of hypermutability in mammalian cells that lasts for many (>30) cell divisions. In this study, we examined whether other stress signals (both DNA-damaging non-DNA-damaging) can initiate a similar process. We show that persistent genetic instability was induced by nongenotoxic stress exposures such as heat treatment, serum starvation, or the tumor microenvironment, as well as genotoxic stresses such as ionizing radiation and exposure to hydrogen peroxide. Progeny of 10-20% of surviving cells exhibited persistent and pronounced genetic instability at both an artificially transfected gene and a genomic minisatellite locus 23 cell divisions after the initial exposure. Stress-induced persistent genetic instability may be a general response of tumor cells to a wide range of genotoxic or nongenotoxic stress conditions.

Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells.

It has generally been considered that important biological effects of ionizing radiation arise as a direct consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by exposing cells to very low fluences of alpha-particles, similar to those emitted by radon gas, such that as few as 1% of the cells in a population are traversed by a particle and thus receive any radiation exposure. By using the endpoints of changes in gene expression and induction of DNA damage, we show that nonirradiated "bystander" cells participate in the overall response of confluent density-inhibited populations of cultured fibroblast and epithelial cells. By in situ immunofluorescence techniques and the use of cells genetically compromised in their ability to perform gap junction intercellular communication, we present direct evidence for the involvement of connexin43-mediated intercellular communication in the transmission of damage signals to nonirradiated cells. Induction of the stress-inducible p21(Waf1) protein in aggregates of neighboring cells far exceeding the fraction of cells whose nucleus has been traversed occurred in gap junction-competent cells only. These changes in p21(Waf1) expression correlated with both the induction of DNA damage (as measured by micronucleus formation) as well as increased Ser-15 phosphorylation of p53.

Molecular events in radiation transformation.

Studies of human tumor cell lines have revealed alterations in the regulation of a number of cell cycle-related genes, associated in some cases with a TP53-independent loss of the radiation-induced G(1)-phase arrest. It is not clear, however, whether these are early or late events in tumor development, or they arise in tumor cell lines during growth in culture. Since the oncogenic transformation of an individual cell is thought to be an early event in tumor development, we have used a model system of normal and radiation-transformed C3H 10T(1/2) mouse fibroblast cell clones to address this issue. Transformed clones derived from type III foci were compared with clones derived from parental, wild-type cells. Approximately 25% of transformed clones showed Trp53 mutations in exon 5; however, preliminary results based on in situ immunofluorescence studies with an antibody recognizing mutant Trp53 indicate that the appearance of such mutations in transformed clones occurs late in the process of transformation and is unlikely to represent an initiating event. The remaining transformed clones and all clones derived from parental cells expressed wild-type Trp53. Radiation-induced G(1)-phase arrest was either absent or significantly reduced in all of the transformed clones, independent of Trp53 status. Constitutive expression of Cdkn1a protein was significantly increased in most of the transformed clones. Also, the majority of transformed clones showed elevated levels of cyclin D1, and two clones overexpressed cyclin E. These results indicate that loss of G(1)-phase checkpoint control, independent of Trp53 status, and altered expression of cell cycle regulatory proteins may represent early events in the process of radiation-induced carcinogenesis that are associated with the malignant transformation of individual cells.

Requirement of wild-type p53 protein for maintenance of chromosomal integrity.

Chromosomal double-strand breaks (DSBs) occurring in mammalian cells can initiate genomic instability, and their misrepairs result in chromosomal deletion, amplification, and translocation, common findings in human tumors. The tumor-suppressor protein p53 is involved in maintaining genomic stability. In this study, we demonstrate that the deficiency of wild-type p53 protein may allow unrepaired DSBs to initiate chromosomal instability. The human lymphoblastoid cell line TK6-E6 was established by transfection with human papilloma virus 16 (HPV16) E6 cDNA into parental TK6 cells via a retroviral vector. Abrogation of p53 function by E6 resulted in an increase in the spontaneous mutation frequencies at the heterozygous thymidine kinase (TK) locus but not at the hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus. Almost all TK-deficient mutants from TK6-E6 cells exhibited loss of heterozygosity (LOH) with the hemizygous TK allele. LOH analysis with microsatellite loci spanning the long arm of chromosome 17, which harbors the TK locus, showed that LOH extended over half of 17q toward the terminal end. Cytogenetic analysis of LOH mutants by chromosome painting indicated a mosaic of chromosomal aberrations involving chromosome 17, in which partial chromosome deletions, amplifications, and multiple translocations appeared heterogeneously in a single mutant. We speculate that spontaneous DSBs trigger the breakage-fusion bridge cycle leading to such multiple chromosome aberrations. In contrast, no chromosomal alterations were observed in TK-deficient mutants from TK6-20C cells expressing wild-type p53. In wild-type p53 cells, spontaneous DSBs appear to be promptly repaired through recombination between homologous chromosomes. These results support a model in which p53 protein contributes to the maintenance of genomic integrity through recombinational repair.

Heat-induced gene expression as a novel targeted cancer gene therapy strategy.

One of the main advantages of gene therapy over traditional therapy is the potential to target the expression of therapeutic genes in desired cells or tissues. To achieve targeted gene expression, we experimented with a new approach based on the long-established phenomenon of the heat shock response. By using the green fluorescence protein as a reporter gene, it was demonstrated that expression of a heterologous gene with a heat shock protein 70 promoter could be elevated to 500-1000-fold over background by moderate hyperthermia (39 degrees C to 43 degrees C) in tissue cultured cells. The heat-induced green fluorescence protein expression was first detectable at 3 h after heating and reached a maximum at 18-24 h. The expression dropped back to baseline within 72 h. In addition, when cells were infected with adenovirus vectors containing the heat-inducible interleukin 12 or tumor necrosis factor alpha genes and then heated (42 degrees C, 30 min), expression was at least 13,600- or 6.8 x 10(5)-fold over background, respectively. Intralesion injection of the interleukin-12-carrying adenovirus vector in a mouse melanoma tumor model caused significant tumor growth delay only with hyperthermia treatment. Our results therefore support heat-induced gene expression as a feasible approach for targeted cancer gene therapy.