BRCA1 required for transcription-coupled repair of oxidative DNA damage.

The breast and ovarian cancer susceptibility gene BRCA1 encodes a zinc finger protein of unknown function. Association of the BRCA1 protein with the DNA repair protein Rad51 and changes in the phosphorylation and cellular localization of the protein after exposure to DNA-damaging agents are consistent with a role for BRCA1 in DNA repair. Here, it is shown that mouse embryonic stem cells deficient in BRCA1 are defective in the ability to carry out transcription-coupled repair of oxidative DNA damage, and are hypersensitive to ionizing radiation and hydrogen peroxide. These results suggest that BRCA1 participates, directly or indirectly, in transcription-coupled repair of oxidative DNA damage.

Growth retardation, DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice.

BRCA1 is a nuclear phosphoprotein expressed in a broad spectrum of tissues during cell division. The inheritance of a mutant BRCA1 allele dramatically increases a woman's lifetime risk for developing both breast and ovarian cancers. A number of mouse lines carrying mutations in the Brca1 gene have been generated, and mice homozygous for these mutations generally die before day 10 of embryonic development. We report here the survival of a small number of mice homozygous for mutations in both the p53 and Brca1 genes. The survival of these mice is likely due to additional unknown mutations or epigenetic effects. Analysis of the Brca1(-/-) p53(-/-) animals indicates that BRCA1 is not required for the development of most organ systems. However, these mice are growth retarded, males are infertile due to meiotic failure, and the mammary gland of the female mouse is underdeveloped. Growth deficiency due to loss of BRCA1 was more thoroughly examined in an analysis of primary fibroblast lines obtained from these animals. Like p53(-/-) fibroblasts, Brca1(-/-) p53(-/-) cells proliferate more rapidly than wild-type cells; however, a high level of cellular death in these cultures results in reduced overall growth rates in comparison to p53(-/-) fibroblasts. Brca1(-/-) p53(-/-) fibroblasts are also defective in transcription-coupled repair and display increased sensitivity to DNA-damaging agents. We show, however, that after continued culture, and perhaps accelerated by the loss of BRCA1 repair functions, populations of Brca1(-/-) p53(-/-) fibroblasts with increased growth rates can be isolated. The increased survival of BRCA1-deficient fibroblasts in the absence of p53, and with the subsequent accumulation of additional growth-promoting changes, may mimic the events that occur during malignant transformation of BRCA1-deficient epithelia.

Differential involvement of the human mismatch repair proteins, hMLH1 and hMSH2, in transcription-coupled repair.

Defects in DNA mismatch repair have been associated with both hereditary and sporadic forms of cancer. Recently, it has been shown that human cell lines deficient in mismatch repair were also defective in the transcription-coupled repair (TCR) of UV-induced DNA damage. We examined whether TCR of ionizing radiation-induced DNA damage also requires the genes involved in DNA mismatch repair. Cells defective in the hMSH2 gene were deficient in the removal of oxidative damage, including thymine glycols, from the transcribed strand of an active gene. However, an hMLH1 mutant showed normal levels of TCR. By comparison, defects in either hMSH2 or hMLH1 resulted in reduced TCR of UV damage. Introducing chromosomes carrying either hMSH2 or hMLH1 into these cell lines restored their ability to carry out TCR. Deficiencies in either hMSH2 or hMLH1 did not result in decreased overall genomic levels of repair or lead to an increased sensitivity to either UV or ionizing radiation. Our results provide the first evidence for a protein that is absolutely required for the preferential removal of UV-induced DNA damage but not oxidative DNA damage from the transcribed strand of an active human gene.

Requirement for DNA mismatch repair proteins in the transcription-coupled repair of thymine glycols in Saccharomyces cerevisiae.

Defects in DNA mismatch repair have been shown to lead to increased genomic instability and mutability. We recently found that human cells defective in the DNA mismatch repair gene, hMSH2, were deficient in the transcription-coupled repair (TCR) of both oxidative DNA damage, including thymine glycols, and UV-induced DNA damage. However, in a hMLH1 mutant, only a reduction in the TCR of UV damage was observed. In this study, we examined whether TCR of thymine glycols in Saccharomyces cerecisiae also requires the genes involved in DNA mismatch repair. We found that yeast cells containing mutations in MSH2 were deficient in the removal of thymine glycols from the transcribed strand of the RPB2 gene, while cells with mutations in either MLH1 or PMS1 alone showed near normal levels of TCR of thymine glycols. Interestingly, double mutants in the MLH1 and PMS1 genes were deficient in TCR of thymine glycols. Taken together, these results suggest that these two MutL homologues can act independently of each other, but that they have overlapping roles in TCR. Overall levels of thymine glycol removal were not reduced in the mismatch repair mutants. In contrast to the results with thymine glycols, no defects in TCR of pyrimidine dimers were found in cells with mutations in MSH2, MLH1, PMS1, and MLH1/PMS1.

Measurement of oxidative DNA damage and repair in specific DNA sequences.

We describe two methods that were developed in our laboratory to measure the production and repair of oxidative DNA damage in specific DNA sequences. Both of these methods rely on the use of monoclonal antibodies against modified nucleotides to separate DNA sequences that contain damage from those in which repair has occurred. In one case, the modified base is bromodeoxyuridine, which is inserted into the DNA during the repair synthesis step of excision repair. An antibody against this modified base is used to detect the production of the bromodeoxyuridine in the repair patch. This approach allows for the measurement of repair of any DNA lesion whose removal is accompanied by the production of a DNA repair patch. In the other case, the modified base is thymine glycol, an oxidized base that is produced by hydrogen peroxide and ionizing radiation. Using a monoclonal antibody that recognizes this altered base, detection of the production and repair of a specific base damage in a DNA sequence can be accomplished. These approaches are used in our laboratory to examine the transcription-coupled repair of oxidative DNA damage in both yeast and mammalian cells.

Influence of base composition on membrane binding and cellular uptake of 10-mer phosphorothioate oligonucleotides in Chinese hamster ovary (CHRC5) cells.

A key problem in antisense therapeutics is the relatively poor cell uptake of oligonucleotides and subsequent transport to the cytoplasm and nucleus. Although the chemical characteristics of oligonucleotides seem likely to affect their uptake by cells, little is known about this issue. In this article we explore the effect of base composition on oligonucleotide uptake. We show that phosphorothioate homo-G oligomers have a distinctly greater cellular uptake than other phosphorothioate homooligomers. This is probably due to a greater initial association with the plasma membrane, because homo-G oligomers show the greatest binding to liposome membranes, when tested at physiological ionic strength. Under different buffer conditions appreciable differences in membrane binding to liposomes were detected for the various homooligonucleotides.

Radiolabeling of methylphosphonate and phosphorothioate oligonucleotides and evaluation of their transport in everted rat jejunum sacs.

PURPOSE: The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work. METHODS: We have developed a simple procedure for radiolabeling oligonucleotides by reductive alkylation with 14C-formaldehyde. We have utilized this approach, as well as 5' addition of fluorophores, to prepare labeled methylphosphonate and phosphorothioate oligonucleotides for use in intestinal transport studies. An everted rat gut sac model was employed to compare the transport of oligonucleotides to that of model compounds whose permeation properties are better understood. RESULTS: We demonstrate that both methylphosphonate and phosphorothioate oligonucleotides are passively transported across the intestinal epithelium, probably by a paracellular route. The rates of transport for both types of oligonucleotides were similar, and were significantly greater than that of the very high MW polymer blue dextran, but were lower than the transport rate of valproic acid, a low MW compound known to have high oral availability. CONCLUSIONS: A significant degree of permeation of oligonucleotides across the gastrointestinal epithelium does occur, but it is still unclear whether this is sufficient to permit effective oral administration of oligonucleotides as drugs.

Evaluation of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides.

PURPOSE: A factor limiting the effectiveness of antisense (AS) deoxyoligonucleotides (ODNs) is inefficient transport to their sites of action in the cytoplasm and in the nucleus. The extent of ODN transfer from endosomes to cytosol seems to be an important determinant of ODN effects. Consequently, the development of compounds (adjuvants) that enhance endosome to cytosol transfer may be vital in AS ODN therapeutics. METHODS: In this report, we evaluated compounds for their potential to enhance the effects of phosphorothioate ODNs. The test system used a CHO cell line expressing the enzyme chloramphenicol acetyl-transferase (CAT) under the control of an inducible promoter. Several potential endosomal disrupting adjuvants were screened, including: (a) fusogenic peptides; (b) a pH sensitive polymer; (c) polymeric dendrimers, (d) cationic liposomes and (e) a pH sensitive surfactant N-dodecyl 2-imidazole-propionate (DIP). ODN effects were evaluated at the protein level by quantitating levels of CAT. RESULTS: The use of AS ODN in co-incubation with the GALA peptide, cationic liposomes or 5th generation dendrimers resulted in a 35-40% reduction in CAT expression. The mis-matched ODN had no effect on CAT expression. Only modest effects were observed with the other adjuvants. DIP did not increase ODN activity by itself; however, when the liposomal form was used a significant reduction (48%) in CAT activity was seen. CONCLUSIONS: We found the fusogenic peptide GALA, dendrimers, as well as the liposomal form of DIP, could significantly enhance the effects of ODNs.