Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures

Abstract

DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called "attached ovarian epispheres". The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three "normal" levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development.

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer

“…In our culture system, normal breast epithelium forms 3D clusters called attached "epispheres," consisting of 30 to 100 rounded epithelial cells (36) (Fig. 1A).…”

“…Based on tissue engineering techniques, we have developed a reliable and unique method to culture breast epithelial cells (33,34). This system allows for direct analysis of the somatic changes occurring during breast oncogenesis, rather than relying on a surrogate tissue, that may be affected by tissuespecific differences in DNA repair (35,36). Through functional analysis of such primary explants, we now report a ubiquitous deficiency of NER activity in a series of sporadic stage I breast tumors, suggesting that loss of NER capacity is an intrinsic element in the development of breast neoplasia.…”

Regulation and Disregulation of Mammalian Nucleotide Excision Repair: A Pathway to Nongermline Breast Carcinogenesis

“…We have previously demonstrated differential NER capacity in the human breast, ovary and lymphocytes (Fig. ) . The low level of repair manifested in peripheral blood lymphocytes relative to foreskin fibroblasts may not have a biological impact, because these cells are terminally differentiated and turn over every 3 months .…”

“…We have previously shown that NER capacity is tissue‐specific during mouse embryogenesis . We have extended this work to the analysis of NER capacity in human tissues. We have therefore established the “range of normal” for human fibroblasts and epithelial cells from breast and ovarian tissue, which is necessary in a species as outbred as man.…”

Analysis of Actively Transcribed DNA Repair Using a Transfection-Based System

“…It is important to consider the fact that NER has been shown to be tissue specific [30–32] and that the choice of a positive control should reflect this tissue specificity, if possible. In addition, when a putative disease specimen is being tested, this sample should be placed into the range of normal by running several normal controls for comparison or by comparing with a previously established range of normal [3].…”