HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 16/7/754 01-0829fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GOUKASSIAN, D. A. Articles by GILCHREST, B. A. Search for Related Content PubMed PubMed Citation Articles by GOUKASSIAN, D. A. Articles by GILCHREST, B. A.

DNA oligonucleotide treatment corrects the age-associated decline in DNA repair capacity¹

Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA

²Correspondence: Department of Dermatology, Boston University School of Medicine, 609 Albany St., Boston, MA 02118, USA. E-mail: bgilchre{at}bu.edu

SPECIFIC AIMS

With increasing age, there is decreased DNA repair capacity, increased mutation rate, and increased skin cancer incidence. We have shown that small single-stranded DNA fragments activate p53, induce DNA repair proteins, and enhance DNA repair as measured by host cell reactivation assays. We sought to determine the magnitude of the oligonucleotide effects in fibroblasts cultured from donors of different ages and whether oligonucleotide treatment could compensate for age-associated losses in DNA repair capacity.

PRINCIPAL FINDINGS

1. Treatment with oligonucleotides compensates for age-associated decreases in the levels of DNA repair and cell cycle proteins
To determine whether treatment with oligonucleotides could compensate for the previously determined age-associated decrease in levels of DNA repair and cell cycle proteins, fibroblasts from newborns and donors aged 17 to 72 years were treated with thymidine dinucleotide (pTT), a 5' phosphorylated 9 base oligonucleotide (p9mer), or diluent alone for 48 h. After 48 h, total cellular proteins from simultaneously processed triplets of newborn, young adult, and old adult donor samples were subjected to Western blotting (Fig. 1 A). As reported earlier, constitutive protein levels of p53, PCNA, ERCC1, RPA, XPA, and p21 were comparable in newborn and young adult donors but decreased in fibroblasts from old adults. Compared with paired newborn samples, old adult levels of each protein were significantly decreased: p53 by 85 ± 11% (P<0.002), PCNA by 78 ± 6% (P<0.0001), ERCC1 by 70 ± 13% (P<0.005), RPA by 65 ± 9% (P<0.003), XPA by 80 ± 10% (P<0.004), and p21 by 20 ± 12% (P<0.03) (Fig. 1B-G ). In all age groups, there was an up-regulation of NER and cell cycle regulatory proteins 48 h after treatment with oligonucleotides (Fig. 1) . In old adults, the increased levels approached or exceeded the constitutive protein levels of newborn and young adults except for PCNA, which increased to 20–50% of newborn levels.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of age and oligonucleotide treatment on NER protein levels. Fibroblasts from newborn, young, and adult donors were supplemented with 100 µM pTT, 40 µM p9mer, or diluent (DME) alone and total cellular protein was collected 48 h after oligonucleotide supplementation. Protein (50 µg per lane) was loaded on the gel and processed for Western blot analysis. Coomassie blue-stained gels demonstrate protein loading. A) A representative Western blot (1 of 3 complete experiments, each with different donors) shows an age-associated decrease in the constitutive levels of NER proteins in control diluent-treated cells. B–G) After densitometric analysis of band intensity, in each triplet of donors processed together, the newborn diluent-treated cells were set at 100%; all other treatments for each donor were calculated as a % of this value. Each graphed, normalized value (mean±stdev) is the average of three independent experiments. For all proteins examined in each age group, treatment with oligonucleotides lead to a statistically significant 200–650% up-regulation of NER and cell cycle regulatory proteins. Asterisks indicate values that are statistically greater than diluent control values in the same age group (P<0.03, ANOVA Fisher PLSD).

2. Treatment with small DNA oligonucleotides compensates for age-associated decreases in DNA repair capacity
To determine whether pretreatment with oligonucleotides can correct the age-associated decrease in DNA repair capacity, we supplemented fibroblast cultures from older adult donors with pTT, p9mer, or diluent alone as control for 48 h, then sham- or UV-irradiated the cultures using a solar simulator. Total cellular DNA was collected for slot blot analysis immediately postirradiation and at intervals over 24 h to examine the removal rate of thymine dimers (Fig. 2 A). By 3 h, diluent pretreated control cells (black bars) removed 37 ± 9% of thymine dimers, whereas pTT pretreated cells (gray bars) removed 50 ± 15% and p9mer pretreated cells (clear bars) removed 63 ± 3% of thymine dimers, significantly faster rates (P<0.05) (Fig. 2B ). By 24 h, 40 ± 5% of photoproducts remained in diluent pretreated cells whereas only 20 ± 9% and 15 ± 11% remained in the genome of pTT and p9mer pretreated cells, respectively, indicating that both oligonucleotides significantly accelerated repair of UV-induced DNA damage during this critical period (P<0.04 and P<0.02, respectively).

View larger version (21K): [in this window] [in a new window]   Figure 2. Effect of oligonucleotide pretreatment on DNA repair rate in old adult cells. A) DNA from paired irradiated cultures pretreated for 48 h with pTT, p9mer, or diluent alone was harvested and slot blots were prepared with 100 ng DNA per slot. Each blot was hybridized with thymine dimer antibodies, then with 32P-labeled genomic DNA for loading control. There is relatively even loading, maximum binding immediately after irradiation (time 0) and progressively less binding over time, as expected. A representative slot blot from adult donor (69 years old) is shown. B) After densitometric analysis of band intensity of 4 adult donors (51–72 years old), % of remaining thymine dimers for each donor was determined as the ratio of the amount of bound antibody at that time compared with that immediately after irradiation (time 0), normalized for loading of DNA in each lane. For each donor, time 0 was set as 100%; all time points for each donor were calculated as a % of these values. Each graphed, normalized time point value (mean±stdev) is the average of 4 independent experiments (4 donors).

CONCLUSIONS AND SIGNIFICANCE

Several studies using direct and indirect methods to measure DNA damage repair have documented a decline in DNA repair capacity with donor age in human dermal fibroblasts, lymphocytes, and transformed lymphoblastoid cells. A major DNA repair mechanism in mammalian cells is nucleotide excision repair (NER), known to be principally responsible for correction of damage resulting from sun exposure. The age-associated slowing of removal of UV-induced DNA photoproducts appears due at least in part to decreases in the basal and UV-induced mRNA and protein levels of NER and cell cycle proteins that include XPA, ERCC3, p21, PCNA, RPA, and p53.

Many studies have shown that p53 and genes regulated by p53 contribute to NER in human cells through modulation of global genome repair and transcription-coupled repair. Considerable evidence also implicates the adequacy of NER protein levels and rate of DNA repair in skin cancer risk. For example, cell lines derived from patients with xeroderma pigmentosum (XP), with inactivating mutations in genes that encode any one of at least eight NER proteins, have severely compromised DNA damage repair after UV irradiation, and the patients are markedly prone to skin cancer. Compared with age-matched controls, individuals with a history of basal cell carcinoma at an early age have decreased DNA repair capacity comparable to that of more cancer-prone older adults. Finally, an exogenously provided prokaryotic endonuclease that increases the cell’s complement of DNA repair enzymes can accelerate removal of DNA photoproducts in cultured human cells and animal skin; topical application of this endonuclease to skin of XP patients was recently shown to decrease the incidence of skin cancer and precancerous lesions during a 1 year vehicle-controlled double-blinded clinical trial. These findings strongly suggest that the age-associated decline in DNA repair capacity, a consequence at least in part of decreased expression and/or functional activity of NER proteins, is largely responsible for the greatly increased skin cancer risk among the elderly.

Inducible DNA repair was first recognized in bacteria. This so-called SOS response, described by Radman in 1975, is known to be initiated by partially processed damaged DNA and mediated by transcriptional up-regulation of genes encoding DNA repair enzymes. Several recent reports have suggested that NER is also inducible in mammalian cells. For example, carcinogen treatment and low-dose UV irradiation enhance the repair rate for subsequent DNA damage in repair-proficient as well as repair-deficient mammalian cells. We have shown that certain DNA fragments, particularly thymidine dinucleotides, also induce NER proteins and increase the rate of DNA repair in cultured human cells, as determined by host cell reactivation assays. These effects are mediated at least in part through induction and activation of p53, suggesting that the oligonucleotides mimic a physiological signal generated during the course of DNA damage or its repair.

Because cells from older, statistically cancer prone, individuals have reduced levels of NER proteins and reduced rates of DNA repair compared with newborns and young adults and because oligonucleotides can induce NER protein levels and repair rates, at least in young donors, we wanted to determine whether oligonucleotide treatment of older donor cells could restore their NER protein levels and DNA repair rate after UV irradiation to that characteristic of newborns and young adults. We found that pTT and p9mer increased NER protein levels in old adult cells by 200–600%, sufficient to bring the stimulated levels of five of the six repair proteins to the basal levels observed in newborn and young adult cells. DNA repair rates in oligonucleotide-treated old adult cells were enhanced to that previously observed in young donor cells under baseline conditions.

Cyclobutane pyrimidine dimers, particularly thymine dimers, constitute nearly 75% of all UV-induced DNA photoproducts. In earlier studies, we demonstrated virtually no removal of thymine dimers in fibroblasts derived from older adults within 24 h. We examined the effect of oligonucleotide pretreatment on the removal rate of thymine dimers in irradiated cells from older adult donors. As observed in newborn donor cells using the same slot blot assay to measure persistent dimers by antibody binding, as well as a host cell reactivation assay, oligonucleotide pretreated cells removed DNA damage more rapidly than diluent-treated control cells. The proportionate acceleration observed for oligonucleotide-treated adult donor cells was similar to that observed in newborn donors, an 50% reduction in remaining photoproducts at 24 h vs. control cells. These results demonstrate that the inducible DNA repair response is preserved with age, despite the substantial reduction in baseline repair capacity, and that oligonucleotide treatment can double the rate of DNA repair in all age groups. That this inducible enhancement of DNA repair capacity is biologically significant is suggested by the fact that differences of less than half this magnitude in constitutive repair rates distinguish young adult from old adult donor cells and cancer-prone from normal subjects.

DNA damage such as that after UV irradiation activates p53 and thus triggers multiple pathways involved in DNA repair, cell cycle arrest, and apoptosis through trans-activation of genes and by protein–protein interactions. Oligonucleotide-induced p53 activation occurs within 6–8 h and (as shown in this and earlier studies) is accompanied by p53 protein induction, events presumably responsible in large part for the improvement seen in DNA repair in oligonucleotide-treated cells. However, the response also includes induction of gene products such as XPA and ERCC1, not known to be p53 regulated; this suggests that the inducible SOS-like response is mediated as well through p53-independent pathway(s).

In summary, our results show that the age-related decline in DNA repair capacity is substantially reversible, at least in fibroblasts, by treatment with oligonucleotides, which appear to act by increasing NER protein levels (although additional mechanisms of action cannot be excluded). Moreover, because the oligonucleotides appear to mimic a physiological DNA damage signal, the data suggest that inducible DNA damage responses are an important mechanism throughout life by which cells manage recurrent DNA insults. Our results also suggest that topical application of such oligonucleotides may enhance DNA reparative capacity in human skin in the absence of actual DNA damage (which normally induces this protective response)and thus reduce the carcinogenic risk from solar UV irradiation, particularly in the elderly (Fig. 3 ).

View larger version (20K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0829fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0829fje

This article has been cited by other articles:

				S. Arad, E. Zattra, J. Hebert, E. H. Epstein Jr., D. A. Goukassian, and B. A. Gilchrest Topical Thymidine Dinucleotide Treatment Reduces Development of Ultraviolet-Induced Basal Cell Carcinoma in Ptch-1+/- Mice Am. J. Pathol., May 1, 2008; 172(5): 1248 - 1255. [Abstract] [Full Text] [PDF]

				V. Gorbunova, A. Seluanov, Z. Mao, and C. Hine Changes in DNA repair during aging Nucleic Acids Res., December 3, 2007; 35(22): 7466 - 7474. [Abstract] [Full Text] [PDF]

				S. Arad, N. Konnikov, D. A. Goukassian, and B. A. Gilchrest T-oligos augment UV-induced protective responses in human skin FASEB J, September 1, 2006; 20(11): 1895 - 1897. [Abstract] [Full Text] [PDF]

				V. Marwaha, Y.-H. Chen, E. Helms, S. Arad, H. Inoue, E. Bord, R. Kishore, R. D. Sarkissian, B. A. Gilchrest, and D. A. Goukassian T-oligo Treatment Decreases Constitutive and UVB-induced COX-2 Levels through p53- and NF{kappa}B-dependent Repression of the COX-2 Promoter J. Biol. Chem., September 16, 2005; 280(37): 32379 - 32388. [Abstract] [Full Text] [PDF]

				N. PURI, M. S. ELLER, H. R. BYERS, S. DYKSTRA, J. KUBERA, and B. A. GILCHREST Telomere-based DNA damage responses: a new approach to melanoma FASEB J, September 1, 2004; 18(12): 1373 - 1381. [Abstract] [Full Text] [PDF]

				D. A. Goukassian, E. Helms, H. van Steeg, C. van Oostrom, J. Bhawan, and B. A. Gilchrest Topical DNA oligonucleotide therapy reduces UV-induced mutations and photocarcinogenesis in hairless mice PNAS, March 16, 2004; 101(11): 3933 - 3938. [Abstract] [Full Text] [PDF]

				M. S. ELLER, G.-Z. LI, R. FIROOZABADI, N. PURI, and B. A. GILCHREST Induction of a p95/Nbs1-mediated S phase checkpoint by telomere 3' overhang specific DNA FASEB J, February 1, 2003; 17(2): 152 - 162. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 16/7/754 01-0829fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GOUKASSIAN, D. A. Articles by GILCHREST, B. A. Search for Related Content PubMed PubMed Citation Articles by GOUKASSIAN, D. A. Articles by GILCHREST, B. A.