Substantial evidence indicates that clustered damages are difficult for cellular enzymes to repair, and thus clusters pose a threat to genomic integrity. Although several cultured human cell lines were found to contain very low levels of endogenous oxidized base and abasic clusters, two were shown to accumulate substantial levels of oxidized base clusters [10
]. It was not clear, therefore, whether DNA in human tissue would accumulate measurable levels of endogenous clusters.
Human skin offers an ideal system for examination of endogenous clusters, as skin tissue, monolayer primary cultures of cells derived from skin, and three-dimensional model skin cultures are readily available. Although based on a limited number of specimens, the data in show that the dermis and epidermis of skin from two individuals contain few if any endogenous clusters. However, cultured monolayer epidermal keratinocytes derived from one of these donors as well as monolayer fibroblast cultures from other individual accumulated detectable levels of all three classes of clustered damages (Figs. and ). Similar levels of clusters were observed in AGO1522 human fibroblasts (Sutherland and Bennett, manuscript in preparation).
Cells from skin can also be grown in three-dimensional models that have many similarities to skin tissue. We therefore asked if the low level of clusters seen in human skin tissue would also be observed in a 3-D skin model culture. To avoid possible interindividual variability, we used cells of the same origin and grew them both as monolayer cultures and in a 3-D model. Although the 3-D model culture might be expected to have levels of clusters similar to those in human skin tissue, the data in show that the 3-D model culture contained significant cluster levels, similar to those in the companion monolayer keratinocytes as well as to cultured skin keratinocytes (initiated in this laboratory) derived from other individuals.
Systematic investigations of the levels of endogenous isolated base lesions have shown that the measured levels depend on the method of analysis [35
]. The levels of 8-oxo-dGuo in human peripheral lymphocytes were found to be 4.24/Mb by high-performance liquid chromatography (HPLC) and estimated to be 0.34/Mb Fpg protein-sensitive sites by alkaline comet assays. Similarly, the level of 8-oxo-dGuo in HeLa cells was found to be 2.78/Mb by HPLC and only 0.5/Mb Fpg-sensitive sites by alkaline comet determinations.
Rodriguez et al. used gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry to determine the levels of several DNA biomarkers in a tissue-engineered skin (TestSkin II; Organogenesis), along with those in several cultured human cell lines, human peripheral lymphocytes, and commercial calf thymus DNA [36
]. These markers included both oxidized pyrimidines and oxidized purine lesions. DNAs from both the tissue-engineered skin and the cell lines and strains contained similar levels of endogenous oxidized bases, ~1 – 15 lesions per million bases. Calf thymus DNA contained higher levels of several oxidized bases. Comparing our results on cluster levels in engineered skin with those for lesion levels in similar cell constructs of Rodriguez et al., the levels of endogenous clustered damages we measure are approximately a thousandfold lower than those of the isolated oxidized lesions. However, the cluster data support a similar conclusion: that there is no intrinsic difference in oxidative damage—at the level of either lesions/Mb or clusters/Gbp—between 3-D tissue models and primary human cells or cell lines. The cluster data reveal, however, that DNA in human skin can contain lower levels of complex damage than DNA from cultured skin cells growing either as monolayers or in three-dimensional cultures.
The steady-state level of clustered damages probably reflects the net result of the cluster level induced by cellular metabolism or by exogenous factors in the cellular environment minus the level that the cells processed. As a first approximation, it can be assumed that cells use the same paths for processing radiation-induced and endogenous clusters. However, it must be noted that radiation-induced and endogenous clusters may differ in complexity (number of individual lesions per cluster), composition (identity of lesions comprising the cluster), and geometry (polarity and interlesion spacing). Thus cellular processing of clusters induced by endogenous factors might differ from that of clustered damages induced by environmental agents such as radiation.
Human cells can deal with radiation-induced clustered damages by several paths. TK6 cells apparently convert radiation-induced clusters to DSBs [21
]. However, these cells show higher radiation sensitivity than related cells and have been reported to be defective in double-strand break repair [37
]. Thus DSB generation by these cells may be a consequence of defective repair. Radiation-resistant, repair-proficient 28SC cells exposed to X-rays apparently do not generate measurable levels of repair-related DSBs [20
]. In these cells, bistranded abasic clusters persist until DNA replication and are then presumably converted to unistranded abasic lesions upon DNA synthesis. In addition, evidence from in vitro studies of complex clusters by extracts of radiation-resistant MRC5-V1 fibroblasts showed a hierarchical processing of the lesions comprising the cluster so that DSB induction was avoided [38
]. Likewise, Malyarchuk and Harrison found that the majority of plasmids containing clustered damages that were transfected into HeLa cells did not suffer DSB induction, suggesting the presence of alternate paths of cluster processing [39
The endogenous cluster data for skin-derived cultured cells—accumulation of all three cluster classes (Figs. and )—differ strikingly from those in TK6 and WI-L2-NS cells, which contained measurable levels of oxidized base clusters but no detectable abasic clusters. However, these data are consistent with a unified hypothesis of cluster processing based on several lines of evidence: first, irradiated, repair-proficient human 28SC cells produce de novo abasic clusters [20
], probably from glycosylase action without lyase action (consistent with the much higher glycosylase than lyase activity of several glycosylases [40
]). TK6 and WI-L2-NS cells have lower glycosylase activities than 28SC cells [10
] and do not effectively convert oxybase clusters to abasic clusters. Thus TK6 and WI-L2-NS cells accumulate endogenous oxybase clusters but no abasic clusters [10
]. In normal, repair-proficient cells growing under low-stress conditions the levels of induced endogenous clusters are low; those that are induced can be processed successfully and thus these cells do not accumulate measurable levels of endogenous clusters of any kind [10
]. In cells growing under conditions of higher oxidative stress, significant levels of oxidized base endogenous clusters are induced, and a portion of these clusters is processed to repair-intermediate abasic clusters, which are highly refractory to repair. Thus under conditions of stress, even normal human cells can accumulate significant levels of endogenous clusters of all classes.
Endogenous clusters apparently do accumulate and are likely to be repair refractory. In dividing cells, the levels of endogenous bistranded clusters should be “reset” to zero, because DNA synthesis would send lesions on one strand to one daughter DNA molecule and any lesions on the opposing strand to the companion daughter DNA molecule. Such DNA synthesis could be carried out by an error-prone polymerase (e.g., a Y-family polymerase [43
] or POLQ polymerase [44
]). Any resulting misincorporated bases could be removed by mismatch repair, and the remaining isolated lesion could then be repaired faithfully by BER (base excision repair). This cellular strategy would allow conversion of a repair-refractory bistranded clustered damage to unistranded lesions that can be readily repaired by normal BER pathways, circumventing both DSB formation and mutation induction.
If the hypothesis that the observed endogenous cluster levels result from the net of induced minus processed clusters is correct, then it should be possible to change the level of endogenous clusters by changing the cellular environment or the ability of cells to process them. The data in indeed show that addition of Se to the medium or an increase in cellular repair enzyme levels can affect the net levels of endogenous clusters. Selenomethionine has anticarcinogenic properties in experimental animals [45
] and reduces the levels of radiation-induced cellular oxidative stress in cultured cells [46
], probably through its role as a component of critical repair enzymes. It also increases the level of expression of the ATR gene [46
], whose gene product ATR is an important component of the DNA damage response path [47
]. In addition to these possibly indirect paths, increasing the cellular levels of the glycosylase Fpg should facilitate cluster processing. These data suggest that it should be possible to alter the level of endogenous clusters accumulated in cells, providing useful avenues for determining the mechanisms of endogenous cluster induction and cellular paths for dealing with them.