Charles Allen Smith

DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall

DNA repair was measured in the dihydrofolate reductase gene in Chinese hamster ovary cells, amplified for the gene, by quantitating pyrimidine dimers with a specific UV-endonuclease. More than two thirds of the dimers had been removed from a 14.1 kb restriction fragment of the gene by 26 hr after irradiation (20 J/m2), while little removal was detected in fragments upstream of the gene and only 15% were removed from the genome overall. This suggests that damage processing can vary according to function or activity of affected sequences, which has general implications for correlations of DNA repair with survival and mutagenesis. Perhaps preferential repair of vital sequences facilitates UV-resistance of these cells despite low overall repair levels.

Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells Introduction of T4 endonuclease V into frozen and thawed cells

Endonuclease V from E. coli infected with phage T4 was used to evaluate the frequency and the removal of pyrimidine dimers from DNA in cultured mammalian cells. Cellular membranes were made permeable to the enzyme by two cycles of rapid freezing and thawing. The number of endonuclease-sensitive sites in DNA was assayed by sedimentation in alkaline sucrose gradients upon which the cells were lysed directly. Comparison of the frequency of endonuclease-sensitive sites with the frequency of pyrimidine dimers determined by chromatographic analysis of hydrolysed DNA indicated that about 50% of the dimers in the permeabilized cells were substrates for T4 endonuclease V. This was confirmed by observation that when DNA treated with the enzyme in situ was purified, it contained the expected additional number of endonuclease-sensitive sites if again treated with the enzyme. The percentage of pyrimidine dimers recognized by T4 endonuclease V was enhanced to nearly 100% by exposing the permeabilized cells to 2 M NaCl before the enzyme was introduced. This method allowed the measurement of frequencies of endonuclease-sensitive sites after doses of UV irradiation at low as 0.5 J/m2. Loss of endonuclease sites from cellular DNA was observed during post-irradiation incubation of V79 Chinese hamster cells and several human cell strains. A comparison of the results obtained in human cells with or without the high-salt exposure before endonuclease treatment suggested that the dimers recognized under low-salt conditions may be removed slightly faster than those recognized only after high-salt exposure.

Repair replication in human cells. Simplified determination utilizing hydroxyurea.

A simplified and shortened procedure has been developed for the determination of repair replication of DNA in cultured mammalian cells. The procedure, using the bromodeoxyuridine density label and a radio-isotopic label has been applied to normal diploid human cells (WI38) and to their SV40 transformants (VA13). After incubation with the repair label the cells are lysed and digested for two hours at 50 degrees C with proteinase K. This digest can then be immediately subjected to alkaline cesium chloride density gradient centrifugation with no need for DNA extraction. Hydroxyurea is used to reduce the level of semi-conservative synthesis that a quantitative determination of repair replication can be accomplished by a single centrifugation. The method is not affected by variation in the effectiveness of the inhibitor although a small amount of semi-conservative synthesis normally occurs in the presence of the drug. The time course of repair replication in WI38 cells is unaffected by the drug. The apparent amount of repair synthesis in ultraviolet irradiated cells is increased 25 to 40% in the presence of hydroxyurea when thymidine is used as tracer. Under certain conditions in which the level of semiconservative synthesis is low (e.g., contact inhibited cells, high ultraviolet doses) the use of hydroxyurea is unnecessary.

Deficient repair of chemical adducts in α DNA of monkey cells

Abstract We have examined excision repair of DNA damage in the highly repeated α DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in α DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for α DNA and bulk DNA. In cells treated with furocoumarins and long-wavelength ultraviolet light, however, repair synthesis in α DNA was only 30% of that in bulk DNA, although it followed the same time course. We found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as we found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from α DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in α DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences.

Repair replication in cultured normal and transformed human fibroblasts.

Repair replication in response to ultraviolet irradiation has been studied in normal human diploid fibroblast cultures, W138, and an SV40 transformant, VA13. Quantitative comparisons have been made using the combined isotopic and density labeling method for assaying repair replication. We find no significant difference in the amount of repair replication performed its dose response, or the time course between growing and confluent W138 cells, early passage and senescent cells, or normal W138 cells and the transformed VA13 cells. When [3H]dThd was employed as the isotopic label in the presence of a 30-200 fold excess of unlabelled BrdUrd, apparent differences in repair replication were seen between W138 cells shortly after subcultivation and cells which had been allowed to reach confluence. These differences were the same over a wide dose range and regardless of the passage number of the cells, but could be influenced by using different serum lots. The differences were not seen, however, when [3H]BrdUrd provided the isotopic label; thus they reflect either impurities in the [3H]dThd or a slight discrimination by some cellular process.

DNA Repair in Specific Sequences in Mammalian Cells

SUMMARY To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of a number of adducts in the non-transcribed, heterochromatic alpha DNA of monkey cells (by physically isolating the DNA) and also the removal of pyrimidine dimers in a number of genes in rodent and human cells (by an indirect assay using a dimer-specific endonuclease). In confluent cells, psoralen and aflatoxin B1 (AFB1) adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha is severely deficient. Adducts of N-acetoxyacetylaminofluorene (NA-AAF) are formed in slightly higher frequencies in alpha, and removal is slightly deficient. The removal of thymine glycols from alpha DNA in gamma-irradiated cells is proficient, as is repair synthesis elicited by exposure to methyl methane sulphonate, dimethyl sulphate, or 254nm ultraviolet light (u.v.). Removal of AFB1 and NA-AAF adducts from alpha is enhanced by small doses of u.v. but not by X-rays or DMS. The quantum efficiency of conversion of psoralen monoadducts to crosslinks is much lower in alpha DNA. Taken together, these results suggest that the highly condensed chromatin structure of alpha hinders access of the repair system that acts on bulky adducts but not of systems for repair of specific base damage, u.v. damage may alter this chromatin structure directly or facilitate the action of some system that changes accessibility of chromatin to repair. The repair deficiencies are not observed in actively growing cells, in which chromatin structure may be less condensed due to DNA replication. We have also demonstrated preferential excision repair of pyrimidine dimers in active genes. Dimers are efficiently removed from the essential dihydrofolate reductase (DHFR) and hydroxy-methylglutaryl CoA reductase genes in Chinese hamster ovary (CHO) cells and from the transcribed c-ab1 proto-oncogene in the mouse cells. Both cell types remove few dimers from their overall genomes or from sequences distal to the DHFR gene; dimers are also removed poorly from the non-transcribed mouse c-mos gene. In human cells, dimers are removed more rapidly from the DHFR gene than from the genome as a whole. However, repair is as deficient in this gene in XP-C cells as it is in the entire genome. These results suggest that resistance to DNA damage correlates better with repair of vital or active sequences than with .overall repair levels and that mutagenic efficiency may vary according to the activity of the gene under study.

Response to trastuzumab, erlotinib, and bevacizumab, alone and in combination, is correlated with the level of human epidermal growth factor receptor-2 expression in human breast cancer cell lines

Human epidermal growth factor receptor-2 (HER2) and epidermal growth factor receptor (EGFR) heterodimerize to activate mitogenic signaling pathways. We have shown previously, using MCF7 subcloned cell lines with graded levels of HER2 expression, that responsiveness to trastuzumab and AG1478 (an anti-EGFR agent), varied directly with levels of HER2 expression. HER2 and EGFR up-regulate vascular endothelial growth factor (VEGF), a growth factor that promotes angiogenesis and participates in autocrine growth-stimulatory pathways that might be active in vitro. Here, we show that trastuzumab, erlotinib, and bevacizumab, individually and in combination, inhibit cell proliferation in a panel of unrelated human breast cancer cell lines, in proportion to their levels of HER2 expression. The combination of all three drugs provided a greater suppression of growth than any single drug or two-drug combination in the high HER2–expressing cell lines (P < 0.001). Combination index analysis suggested that the effects of these drugs in combination were additive. The pretreatment net level of VEGF production in each cell line was correlated with the level of HER2 expression (r = 0.883, P = 0.016). Trastuzumab and erlotinib each reduced total net VEGF production in all cell lines. Multiparameter flow cytometry studies indicated that erlotinib alone and the triple drug combination produced a prolonged but reversible blockade of cells in G1, but did not increase apoptosis substantially. These studies suggest that the effects of two and three-drug combinations of trastuzumab, erlotinib, and bevacizumab might offer potential therapeutic advantages in HER2-overexpressing breast cancers, although these effects are of low magnitude, and are likely to be transient. [Mol Cancer Ther 2007;6(10):2664–74]

RECONSTRUCTING TUMOR PHYLOGENIES FROM HETEROGENEOUS SINGLE-CELL DATA

Studies of gene expression in cancerous tumors have revealed that tumors presenting indistinguishable symptoms in the clinic can be substantially different entities at the molecular level. The ability to distinguish between these genetically distinct cancers will make possible more accurate prognoses and more finely targeted therapeutics, provided we can characterize commonly occurring cancer sub-types and the specific molecular abnormalities that produce them. We develop a new method for identifying these common tumor progression pathways by applying phylogeny inference algorithms to single-cell assays, taking advantage of information on tumor heterogeneity lost to prior microarray-based approaches. We combine this approach with expectation maximization to infer unknown parameters used in the phylogeny construction. We further develop new algorithms to merge inferred trees across different assays. We validate the expectation maximization method on simulated data and demonstrate the combined approach on a set of fluorescent in situ hybridization (FISH) data measuring cell-by-cell gene and chromosome copy numbers in a large sample of breast cancers. The results further validate the proposed computational methods by showing consistency with several previous findings on these cancers and provide novel insights into the mechanisms of tumor progression in these patients.

Excision-repair patch lengths are similar for transcription-coupled repair and global genome repair in UV-irradiated human cells

We have used the buoyant density shift method to measure excision-repair patch lengths in UV-irradiated repair-proficient human cells and in primary fibroblasts belonging to xeroderma pigmentosum complementation group C (XP-C), in which excision repair of UV-induced photoproducts is dependent upon transcription. The patch size was found to be about 30 nucleotides for both cell types. This agrees with the size of the DNA fragments excised in vitro by the dual incisions of the structure-specific nucleases XPG and ERCC1-XPF. We conclude that the XPC protein is not required to target the excision nucleases to sites of DNA cleavage in transcribed strands of expressed genes or to protect the newly incised DNA from further processing by exonucleases.

Repair replication schemes in bacteria and human cells.

Publisher Summary The chapter discusses the schemes that remove damage from the DNA and attempt restoration of the original nucleotide sequence. The excision-repair of damaged DNA must involve a coordinated sequence of steps beginning with recognition of the lesion. After phosphodiester bonds are broken, the integrity of the interrupted DNA strand is restored through the enzymic processes of degradation, repair resynthesis, and ligation. Pathways for excision-repair in E.coli are elucidated through analysis of mutants deficient in various enzyme activities. A pathway mediated by DNA polymerase I that results in short repair patches approximately 20 nucleotides in extent; a pathway that results in a somewhat larger average patch size, probably due to intrinsic properties of DNA polymerases II and III; and a pathway that is induced in response to DNA damage and is dependent upon protein synthesis, requires recA+ and lexA+, and leads to much longer patches. Short patches are initiated and completed earlier than the long patches produced by the inducible pathway. In UV-irradiated human cells, only a single class of repair patches comparable in size to the short patches in E.coli, is synthesized at both early and late times after irradiation. The same patch size distribution also results from the treatment of human cells by angelicin or 8-methoxypsoralen plus long-wavelength UV, or by activated aflatoxin B1.