DNA repair: kinetics and thresholds.

DNA damage is a critical factor in the initiation of chemically induced toxicities (including cancer), and the repair of this damage represents the cell's first line of defense against the deleterious effects of these agents. The various mechanisms of DNA repair are reviewed briefly and the actions of the DNA repair protein O6-alkylguanine DNA alkyltransferase (ATase) are used to illustrate how DNA repair can protect cells against alkylating agent-induced toxicities, mutagenesis, clastogenesis, and carcinogenesis. The effectiveness of this repair protein can be measured based on its ability to deplete levels of its promutagenic substrate O6-methylguanine (O6-meG) in the DNA of cells. These studies reveal that the repair of O6-meG from DNA occurs heterogeneously, both intra- and intercellularly. Even in cells that repair O6-meG hyperefficiently, certain regions of chromatin DNA are repaired with difficulty, and in other regions they are not repaired at all; most likely this lack of repair is a result of the location of the lesion in the DNA sequence. When individual cells are compared within a tissue, some cells are clearly repair deficient, because the O6-meG can persist in DNA for many weeks, whereas in other cells, it is removed within a matter of hours. The role of these repair-deficient cells as targets for alkylating agent induced carcinogenesis is considered. The mechanisms of the homeostatic control of DNA repair function in mammalian cells are not yet well understood. Because there are now indications of the mechanisms by which the level of DNA damage may be sensed (and so influence the activity of the ATase repair protein), this is an important area for future study.

A novel and consistent amplicon at 13q31 associated with alveolar rhabdomyosarcoma.

Rhabdomyosarcomas are the most common soft-tissue sarcoma found in children. The alveolar subtype is clinically more aggressive than the embryonal subtype. In addition to the presence of specific chromosome translocations and associated fusion gene products in a high proportion of the alveolar subtype, we previously showed that tumors with this histology frequently show evidence of genomic amplification. Here, we substantially extended the number of alveolar rhabdomyosarcoma samples examined by comparative genomic hybridization analysis. Regions of loss were noted, including the smallest overlapping regions corresponding to 16q, 17/17p, and 9q32-34, in 16%, 10%, and 10% of cases, respectively (44 primary samples/6 cell lines). Amplification or gain at 12q13-15 in the region of the MDM2/GLI1/SAS/CDK4 loci and 2p24 at the MYCN locus was found in 28% and 32% of cases, respectively. Single amplicons were found at locations that in other samples showed consistent gain, including the regions 5q15-23, 7q21-31, 11p11-14, 17q23-24, and 20q13, and amplification was found in two cases at 15q24-26. However, most striking was a novel region of amplification or gain at 13q31 in 19% of cases (51 primary samples/6 cell lines). This indicates that a gene or genes at 13q31 are significant in the development or progression of alveolar rhabdomyosarcoma.

A role for molecular radiobiology in radiotherapy?

As we learn more about the cellular response to radiation and its genetic control, new avenues are opened up that have the potential to have a significant impact on radiotherapy practice. The recognition of the importance of the control of DNA damage induction and repair, cell cycle arrest and apoptosis gives us the primary areas to investigate, and the improvements in molecular technology make the application of our new knowledge more feasible. It can only be hoped that specific means can be found to assist in the prediction of normal tissue and tumour radiosensitivity and to manipulate sensitivity when that is desirable.

Cell-cycle variation in DNA migration in pulsed-field gel electrophoresis.

Pulsed-field electrophoresis is being used extensively in the gene mapping studies and in the analysis of DNA strand breakage by ionizing radiation. We have evaluated the relationship between the fraction of S phase DNA in a cell population and its ability to modify the migration of DNA in pulsed-field gel electrophoresis. We have shown that increasing the proportion of S phase DNA reduced the effective rate of migration of MGH-U1 cellular DNA. This effect was observed after treatment with ionizing radiation or the restriction enzyme Not I. However, when radiation-induced damage was studied using intact cells, only the DNA with 70 percent S phase showed apparent differences in damage induction. These studies therefore provide data to indicate the percentage of S phase cells at which overall DNA migration might be affected significantly.

Adenoviral ocular isolates demonstrate serotype-dependent differences in in vitro infectivity titers and clinical course.

The purpose of this study was to evaluate clinical ocular adenoviral isolates for differences among and within serotypes with respect to in vitro infectivity titers and clinical course. The study design included a retrospective chart review and the determination of in vitro infectivity titers (TCID90s) of 90 clinical ocular isolates of various adenoviral serotypes. Adenovirus serotype 8 (AD8) was recovered in significantly greater numbers of patients in the second week of infection compared to all other serotypes (p < 0.002). AD3 and AD4 presented with the highest infectivity titers during the first week of acute infection. Up to 4 logs of variation was demonstrated in TCID90s among isolates of the same serotype. Among the clinical parameters studied, eyelid edema was significantly more common among AD8-infected patients as compared to all other serotypes (p < 0.04). For the first time, specific, but limited serotype differences with respect to infectivity titers and clinical course were demonstrated for adenoviral ocular isolates. Important variations in isolate virulence within a given serotype were also observed.