Radiobiological rationale and clinical implications of hypofractionated radiation therapy.

Recent clinical trials of hypofractionated radiation treatment have provided critical insights into the safety and efficacy of hypofractionation. However, there remains much controversy in the field, both at the level of clinical practice and in our understanding of the underlying radiobiological mechanisms. In this article, we review the clinical literature on hypofractionated radiation treatment for breast, prostate, and other malignancies. We highlight several ongoing clinical trials that compare outcomes of a hypofractionated approach versus those obtained with a conventional approach. Lastly, we outline some of the preclinical and clinical evidence that argue in favor of differential radiobiological mechanisms underlying hypofractionated radiation treatment. Emerging data from the ongoing studies will help to better define and guide the rational use of hypofractionation in future years.

A proposed methodology to select radioisotopes for use in radionuclide therapy.

The American Journal of Neuroradiology has played a seminal role in the history of vertebral augmentation (VA). Because VA is increasingly being included in the multidisciplinary management of malignant vertebral compression fractures (VCFs), combined therapeutic approaches that include strategies to treat metastatic disease along with the fracture have become appealing options for patients. To that end, we recently investigated the dosimetric feasibility of treating malignant VCFs with radionuclide therapy. The goal would be to provide local control of the systemic disease beyond the pain relief and structural support provided by polymethylmethacrylate cement. The purpose of this article is to propose a methodology for evaluating radionuclides for use in radiation therapy that takes into account a number of factors including radiation characteristics, biochemical effects, production capacity, and safety. The goal of such a methodology is to introduce a systematic approach to selecting radionuclides in designing treatment regimens and future investigations and also to stimulate discussion and experimentation involving new radionuclides that may provide more effective treatments than the current isotopes in widespread use.

ATM variants 7271T>G and IVS10-6T>G among women with unilateral and bilateral breast cancer.

Recent reports suggest that two ATM gene mutations, 7271T>G and IVS10-6T>G, are associated with a high risk of breast cancer among multiple-case families. To assess the importance of these two mutations in another 'high-risk' group, young women (under age 51) with multiple primaries, we screened a large population-based series of young women with bilateral breast cancer and compared the frequency of these mutations among similar women diagnosed with unilateral breast cancer. The 1149 women included were enrolled in an ongoing population-based case-control study of the genetic factors that contribute to bilateral breast cancer; they were not selected on the basis of family history of cancer. Screening for 7271T>G and IVS10-6T>G ATM gene mutations was conducted using DHPLC followed by direct sequencing. The 7271T>G mutation was detected in one out of 638 (0.2%) women with unilateral breast cancer and in none of the bilateral cases, and the IVS10-6T>G mutation in one out of 511 (0.2%) bilateral and in eight out of 638 (1.3%) unilateral breast cancer cases. Carriers of either mutation were not limited to women with a family history. Given the likelihood that young women with bilateral breast cancer have a genetic predisposition, the observed mutation distribution is contrary to that expected if these two mutations were to play an important role in breast carcinogenesis among individuals at high risk.

Screening breast cancer patients for ATM mutations and polymorphisms by using denaturing high-performance liquid chromatography.

All 62 coding exons of the ATM gene, along with 10-20 bases of the intronic region flanking each exon, were screened for DNA base sequence alterations by using denaturing high-performance liquid chromatography (DHPLC) in a series of 52 breast cancer patients. Six (12%) of these patients exhibited a total of eight different novel germ-line mutations that do not represent common polymorphisms. Of these, three patients possessed four nonconservative missense mutations while two conservative missense and two synonymous mutations were detected in the other three patients. In addition, 43 patients were found to have a total of 141 DNA sequence variations representing 21 different common polymorphisms and rare variants. An analysis of the relationship between the presence of a novel ATM mutation and either patient demographics or tumor properties demonstrated a significant difference between African Americans (3/7 = 43%) and other ethnic groups (3/45 = 7%, P = 0.026). None of the other characteristics examined was found to be related to mutation status.

ATM heterozygosity and breast cancer: screening of 37 breast cancer patients for ATM mutations using a non-isotopic RNase cleavage-based assay.

Based upon the results of several epidemiologic studies, it has been suggested that women who are carriers for a mutation in the ataxia telangiectasia-mutated (ATM) gene are susceptible for the development of breast cancer. Therefore, 37 consecutive breast cancer patients were screened for the presence of a germline ATM mutation using a non-isotopic RNase cleavage-based assay (NIRCA). This paper reports the first use of NIRCA for detection of ATM mutations in breast cancer patients. Using this assay, no ATM mutations were found in our patient population. This result is similar to the findings of other studies that have employed approaches complementary to NIRCA.

p53 mutations in basal cell carcinomas arising in routine users of sunscreens.

Sun exposure histories were obtained from a series of patients age 35 or younger following diagnosis and removal of a basal cell carcinoma (BCC). The DNA was extracted from tumor biopsy samples derived from BCC of 10 patients who reported that they did not use sunscreens during youth (age 18 or younger) and 10 patients who routinely employed sunscreens during this age period. Exons 5-9 of the p53 gene were then amplified in three fragments from these samples using a nested polymerase chain reaction (PCR) approach and screened for mutations using an RNA heteroduplex assay. All PCR products displaying evidence of a mutation were sequenced. It was found that 6 of the 10 patients who were not routine sunscreen users displayed mutations in these p53 exons. All of the mutations were located at dipyrimidine sites, five of the six were C-->T transitions and one mutation was a tandem double mutation, consistent with a role for solar UVB in BCC formation. In contrast, only one p53 mutation was detected in the group of 10 patients who routinely employed sunscreens during childhood and adolescence. Hence, a significantly (P = 0.029) lower level of p53 mutations was detected in the BCC obtained from sunscreen users compared with tumors derived from nonusers. These findings suggest that the mechanisms involved in the etiology of skin carcinogenesis differ in sunscreen users compared with people who did not routinely employ sunscreens. These data are also indicative of a protective effect associated with sunscreen use against the formation of p53 mutations. It is possible that the patients who were diagnosed with BCC despite their use of sunscreens possessed a genetic susceptibility for skin cancer formation and developed BCC through a p53-independent pathway. Alternatively, solar UVA wavelengths, that were generally not blocked by the suncare products employed by the sunscreen users, may have played a significant role in BCC development through induction of a mutation(s) in an oncogene and/or a tumor suppressor gene, other than p53, for these patients.

Transmittance spectra and theoretical sun protection factors for a series of sunscreen-containing sun care products.

Sun care products containing sunscreens are widely used, but consumers are generally unaware of the important differences in the ability of these lotions to block exposure to the ultraviolet A (UVA) portion of the solar spectrum. The purpose of this study was to determine the transmittance spectra, with particular emphasis on the UVA portion of the spectrum, for a variety of commercially available sun care products, to determine Commission Internationale d'Eclairage (CIE) erythema effectiveness spectra and to compare these with information in the product label. The transmittance spectra for a sample of sun care products were measured spectrophotometrically. These values were convoluted with the CIE erythema action spectrum and the sunlight spectra determined for solar noon on June 21 at 0 degrees and 50 degrees N latitude to produce CIE effectiveness spectra. The UVA transmitted through the sun care products that claimed UVA protection on the bottle label varied from as little as 6% to as much as 52%. In addition, it was determined from the CIE effectiveness spectra that any erythema induced following application of the tested lotions would be caused by the UVA portion of the solar spectrum for all, but one, of the products examined. The results of this study emphasize the necessity for better guidance to the consumer as to the ability of sun care products to provide protection against UVA exposure.

Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair.

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.

The involvement of topoisomerase I in the induction of DNA-protein crosslinks and DNA single-strand breaks in cells of ultraviolet-irradiated human and frog cell lines.

Exposure of GM 4390 human skin fibroblasts and ICR 2A frog cells to 10 kJ m(-2) of ultraviolet B (UVB) radiation resulted in the formation of DNA-protein crosslinks (DPCs) and DNA single-strand breaks (SSBs). However, upon incubation, there were rapid increases in the yields of both DPCs and SSBs. An enhancement in these DNA alterations was detected within 12 min after irradiation and their levels continued to rise by 5-8-fold within 15 h after exposure to UV radiation. Using an antibody-based assay that measures covalent complex formation between topoisomerase (topo) I and genomic DNA, it was found that topo I is one of the proteins involved in these DPCs induced by UV radiation. The levels and rate of increase of topo I-DNA covalent complexes were similar to the UV-radiation-dependent formation of DPCs and SSBs. A UV-radiation-sensitive mutant frog cell line, DRP 153, was also examined and was found to be deficient in this induction of DPCs and SSBs by UV radiation. When these cells were transfected with the human SUVCC3 gene, the resulting transformant displayed kinetics for the induction of DPCs and SSBs similar to the human and parental frog cells. However, human topo I was not defected in the transformed frog cells, indicating that SUVCC3 does not encode topo I. It is likely that SUVCC3 encodes an associated enzymatic activity which permits normal stimulation of topo I-DNA covalent complexes in UV-irradiated cells.

Induction of 8-oxo-7,8-dihydro-2'-deoxyguanosine by ultraviolet radiation in calf thymus DNA and HeLa cells.

The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10(5) 2'-deoxyguanosine (dGuo) per kJ/m2 for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10(5) dGuo per kJ/m2, respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irradiated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.

Identification of possible reactive oxygen species involved in ultraviolet radiation-induced oxidative DNA damage.

We have previously demonstrated that each region of the ultraviolet (UV) spectrum (UVA, UVB, and UVC) induces the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells in a fluence-dependent manner. In the present study, we further characterize the possible reactive oxygen species (ROS) that are involved in the induction of 8-oxodGuo by UV radiation. Sodium azide, a singlet oxygen (1O2) scavenger though its quenching effect on HO. was also reported, inhibited 8-oxodGuo production in calf thymus DNA exposed to UVA, UVB, or UVC in a concentration-dependent fashion with maximal quenching effect of over 90% at a concentration of 10 mM. Catalase, at a concentration of 50 U/ml, reduced the yields of UVA- and UVB-induced 8-oxodGuo formation by approximately 50%, but had little effect on UVC-induced 8-oxodGuo production. In contrast, 50 U/ml of superoxide dismutase (SOD) did not affect induction of 8-oxodGuo by any portion of the UV spectrum. Hydroxyl radical (HO.) scavengers mannitol and dimethylsulfoxide (DMSO) moderately reduced the levels of 8-oxodGuo induced by UVA and UVB, but not those by UVC. Instead, mannitol and DMSO enhanced the formation of 8-oxodGuo induced by UVC. These results suggest that certain types of ROS are involved in UV-induced 8-oxodGuo formation with 1O2 playing the predominant role throughout the UV spectrum. Except for UVC, other ROS such as hydrogen peroxide (H2O2) and HO. may also be involved in UVA- and UVB-induced oxidative DNA damage. Superoxide anion appears not to participate in UV-induced oxidation of guanosine in calf thymus DNA, as SOD did not display any quenching effects.

Molecular cloning of the human gene SUVCC3 associated with the formation of DNA-protein crosslinks following exposure to solar UV radiation.

DRP 153 cells, which are hypersensitive to solar UV and deficient in the formation of DNA-protein crosslinks (DPC) following irradiation, were transfected with human DNA and a secondary transformant obtained in which a normal DPC response and solar UV sensitivity reestablished. DNA from this secondary transformant was used to construct a genomic DNA library from which a recombinant phage was isolated containing the human gene capable of restoring a normal DPC response and solar UV sensitivity to DRP 153. This gene has been designated SUVCC3 to denote solar UV cross-complementing gene number 3.

Molecular cloning of the human gene SUVCC2 associated with mutagenesis following the induction of non-dimer DNA damages by solar UV radiation.

A mutant cell line, DRP 512, sensitive to the induction of non-dimer DNA damages produced by solar UV radiation was derived from ICR 2A frog cells. In addition, the DRP 512 cells exhibited an abnormally high level of ouabain-resistant mutants after exposure to solar UV. A level of 1.1. mutants per 10(6) survivors per kJ m-2 was measured for ICR 2A whereas the yield was 4.2 mutants per 10(6) survivors per kJ m-2 for the solar-UV-sensitive cell line. The DRP 512 cells were transfected with human DNA and a secondary transformant obtained in which normal solar UV sensitivity and mutation induction were restored. DNA from this secondary transformant was used to construct a genomic DNA library from which a recombinant phage was isolated containing the human gene capable of restoring normal solar UV sensitivity and mutation induction to DRP 512. This gene has been designated SUVCC2.

Molecular cloning of the human gene SUVCC1 associated with the repair of nondimer DNA damage induced by solar UV radiation.

A mutant cell line, DRP 287, sensitive to solar UV radiation and deficient in the repair of solar UV-induced nondimer DNA damage, was derived from ICR 2A frog cells. These cells were transfected with human DNA and a secondary transformant obtained in which normal solar UV sensitivity was restored and the repair defect corrected. The DNA from this secondary transformant was used to construct a genomic DNA library from which a recombinant phage was isolated containing the human gene capable of restoring normal solar UV sensitivity and correcting the repair defect in the DRP 287 cells. This represents the first human gene which has been isolated that is specifically involved in the repair of nondimer DNA damage induced by solar UV radiation. It has been designated SUVCC1 to denote solar UV cross-complementing gene number 1.

Survival, excision repair and inhibition of DNA synthesis in normal human skin fibroblasts exposed to simulated sunlight.

The responses of normal human skin fibroblasts exposed to simulated sunlight produced by a solar simulator were examined. The parameters investigated were cellular survival, excision repair and the inhibition and recovery of DNA synthesis. The latter two effects were examined using the bromodeoxyuridine photolysis assay and the alkaline step elution assay respectively. The results of these experiments are consistent with the conclusion that the lesions induced by simulated sunlight represent a mixture of damage which elicits cellular responses and repair mechanisms similar to those manifested by cells irradiated with UVC and UVA radiation.

Repair of DNA damage induced in systemic lupus erythematosus skin fibroblasts by simulated sunlight.

Skin fibroblasts derived from three normal individuals and three patients exhibiting the disease systemic lupus erythematosus (SLE) were exposed to the simulated sunlight produced by a solar simulator. The induction and repair of DNA damage induced by this treatment were examined. The total number of lesions repaired by excision, as well as the removal of pyrimidine dimers and E. coli endonuclease III--sensitive sites did not differ significantly in the three SLE cell strains compared with normal cells. However, abnormalities in the formation and maintenance of DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) were found in SLE-4 and SLE-5 following simulated sunlight exposure. In contrast, SLE-3 cells exhibited responses similar to normal cells in reference to SSB and DPC formation. These findings correlate well with the previously determined UV sensitivity of these SLE cell strains.

The repair of DNA damages induced in normal human skin fibroblasts exposed to simulated sunlight.

The induction and repair of DNA damages produced by exposure of normal human skin fibroblasts to the simulated sunlight produced by a solar simulator were examined. The photoproducts measured were pyrimidine dimers, E. coli endonuclease III-sensitive sites, 6-4 photoproducts, Dewar isomers, DNA-protein crosslinks, and DNA single-strand breaks. The results of these experiments serve to form a basis for the quantitation of damages induced by exposure to sunlight.

Induction of DNA strand breaks and DNA-protein cross-links in normal human skin fibroblasts following exposure to 254 nm UV radiation.

Levels of DNA strand breaks and DNA-protein cross-links (DPCs) were measured using the alkaline elution assay in normal human skin fibroblasts irradiated with 0-200 J m-2 of 254 nm UV radiation and incubated for 0-24 h. On incubation, the yields of both single-strand breaks (SSBs) and DPCs increased with similar kinetics and remained elevated. In addition, when SSBs were measured under conditions in which DPCs were not eliminated by treatment with proteinase K, a measurable yield of SSBs could not be detected. Hence, the SSBs that form in the UV-irradiated cells following incubation appear to be associated with the DPCs.

Inhibition and recovery of semiconservative DNA synthesis in normal and solar UV sensitive ICR 2A frog cell lines following the induction of non-dimer DNA damage by sunlamp UV greater than 315 nm.

Cultures of the solar UV-sensitive cell lines, DRP 36 and DRP 153, and of the parental ICR 2A cell line, were exposed to 150 kJ/m2 of sunlamp UV greater than 315 nm plus photoreactivating light. This treatment resulted in the induction primarily of non-dimer DNA damage. Following either a 0, 3, 6, 12 or 24 h incubation, the cultures were pulse-labelled with [3H]thymidine, and the synthesis of different size classes of replicon intermediates measured using the alkaline step elution assay. For all three cell lines tested, an immediate depression of low molecular weight DNA synthesis was observed. This was followed by an inhibition of all size classes of replicon intermediates. Within 12 h following irradiation, recovery of DNA synthesis was observed, which was generally most apparent for low molecular weight DNA. The ICR 2A cells exhibited a nearly full recovery in all size classes of DNA synthesized by 24 h. However, a much smaller recovery of DNA synthesis was detected for the DRP 36 and DRP 153 cultures. This continued inhibition was primarily in the synthesis of full replicon size DNA, and was most pronounced for the DRP 36 cells. Hence, it appears that replicon chain elongation continues to be inhibited in these solar UV-sensitive cell lines long after irradiation.

DNA-protein crosslinking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation.

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5-7 DPC per 10(10) dalton were induced in these cells by either 150 kJ/m2 of sunlamp UV greater than 315 nm plus photoreactivating light (PRL) or 10 kJ/m2 of sunlamp UV greater than 295 nm. The irradiated cells were then incubated for 0-24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV greater than 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV greater than 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.