Christopher N. Parris

Prolonged expression of the γ-H2AX DNA repair biomarker correlates with excess acute and chronic toxicity from radiotherapy treatment.

The normal tissue tolerance levels to fractionated radiotherapy have been appreciated by a century of careful clinical observations and radiobiological studies in animals. During clinical fractionated radiotherapy, these normal tissue tolerance levels are respected, and severe sequelae of radiotherapy are avoided in the majority of patients. Notwithstanding, a minority of patients experience unexpectedly severe normal tissue reactions. The ability to predict which patients might form this minority would be important. We have conducted a study to develop a rapid and reliable diagnostic test to predict excessive normal tissue toxicity (NTT) in radiotherapy patients. A flow cytometric immunocytochemical assay was used to measure DNA damage in peripheral blood lymphocytes (PBL) from cancer patients exposed to 2‐Gy gamma radiation. DNA damage and repair was measured by induction of cellular γ‐H2AX in unirradiated and exposed cells at specific time points following exposure. In 12 cancer patients that experienced severe atypical NTT following radiotherapy, there was a failure to repair DNA double‐strand breaks (DSB) as measured by γ‐H2AX induction and persistence. In ten cancer patients that experienced little or no NTT and in seven normal (noncancer controls), efficient repair of DNA DSB was observed in the γ‐H2AX assay. We conclude that a flow cytometric assay based on γ‐H2AX induction in PBL of radiotherapy patients may represent a robust, rapid and reliable biomarker to predict NTT during radiotherapy. Further research is required with a larger patient cohort to validate this important study.

Telomerase activity in melanoma and non-melanoma skin cancer

SummaryTelomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen’s disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found.

The manipulation of chromosomes by mankind: the uses of microcell-mediated chromosome transfer

Microcell-mediated chromosome transfer (MMCT) was a technique originally developed in the 1970s to transfer exogenous chromosome material into host cells. Although, the methodology has not changed considerably since this time it is being used to great success in progressing several different fields in modern day biology. MMCT is being employed by groups all over the world to hunt for tumour suppressor genes associated with specific cancers, DNA repair genes, senescence-inducing genes and telomerase suppression genes. Some of these genomic discoveries are being investigated as potential treatments for cancer. Other fields have taken advantage of MMCT, and these include assessing genomic stability, genomic imprinting, chromatin modification and structure and spatial genome organisation. MMCT has also been a very useful method in construction and manipulation of artificial chromosomes for potential gene therapies. Indeed, MMCT is used to transfer mainly fragmented mini-chromosome between cell types and into embryonic stem cells for the construction of transgenic animals. This review briefly discusses these various uses and some of the consequences and advancements made by different fields utilising MMCT technology.

A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a patient with xeroderma pigmentosum

Background Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair. Aim To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells. Methods Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and γ-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed. Results Transfer of chromosome 8 (location of DNA-PKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene. Conclusion We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.

Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines

The measurement of γ‐H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ‐H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5‐SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA‐PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a 60Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5‐SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24‐hr period was due to the failure in the repair of DNA DSB. However, in the DNA‐PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through‐put analysis of foci induction in a variety of cell types.

Exercise intensity modulates the appearance of circulating microvesicles with pro-angiogenic potential upon endothelial cells.

The effect of endurance exercise on circulating microvesicle dynamics and their impact on surrounding endothelial cells is unclear. Here we tested the hypothesis that exercise intensity modulates the time course of platelet (PMV) and endothelial-derived (EMV) microvesicle appearance in the circulation through hemodynamic and biochemical-related mechanisms, and that microvesicles formed during exercise would stimulate endothelial angiogenesis in vitro. Nine healthy young men had venous blood samples taken before, during, and throughout the recovery period after 1 h of moderate [46 ± 2% maximal oxygen uptake (V̇o2max)] or heavy (67 ± 2% V̇o2max) intensity semirecumbent cycling and a time-matched resting control trial. In vitro experiments were performed by incubating endothelial cells with rest and exercise-derived microvesicles to examine their effects on cell angiogenic capacities. PMVs (CD41+) increased from baseline only during heavy exercise (from 21 ± 1 × 103 to 55 ± 8 × 103 and 48 ± 6 × 103 PMV/μl at 30 and 60 min, respectively; P < 0.05), returning to baseline early in postexercise recovery (P > 0.05), whereas EMVs (CD62E+) were unchanged (P > 0.05). PMVs were related to brachial artery shear rate (r2 = 0.43) and plasma norepinephrine concentrations (r2 = 0.21) during exercise (P < 0.05). Exercise-derived microvesicles enhanced endothelial proliferation, migration, and tubule formation compared with rest microvesicles (P < 0.05). These results demonstrate substantial increases in circulating PMVs during heavy exercise and that exercise-derived microvesicles stimulate human endothelial cells by enhancing angiogenesis and proliferation. This involvement of microvesicles may be considered a novel mechanism through which exercise mediates vascular healing and adaptation.

Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis

Background:The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients.Methods:Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells.Results:Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis.Conclusion:We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.

Heterogeneity of dimer excision in young and senescent human dermal fibroblasts

We have examined the relationship between nucleotide excision of the main UV‐induced photoproduct, the cyclobutane pyrimidine dimer and in vitro cellular senescence. An in situ semiquantitative immunocytochemical assay has demonstrated that, following a UV‐C dose of 15 J m−2, young human dermal fibroblasts maintained in a high level of serum are more efficient than senescent fibroblasts in the removal of dimers. However, in G0‐arrested cultures (serum‐starved), young fibroblasts are compromised in their ability to remove dimers and are significantly less efficient than senescent cells in this process. Supplementation of the culture medium with 0.1 mm deoxyribonucleosides enhances the removal of dimers in both young and senescent fibroblasts in proliferating or serum‐starved cells. These data indicate that overall there is a modest but significant reduction in nucleotide excision of dimer photoproducts in cells as they age in vitro. In addition, G0‐arrested young cells exhibit reduced removal of dimers, although this can be complemented by deoxyribonucleoside addition. In addition, this in situ assay has revealed heterogeneity in both susceptibility to UV‐C‐induced damage and excision. Overall, we provide evidence of reduced UV‐induced damage excision in senescent compared with young fibroblasts, and demonstrate modulation of these processes in young and senescent cells under specific growth conditions.

Radiosensitivity of human breast cancer cell lines expressing the breast tumor kinase (Brk)

This work was supported by a grant from The Bart’s Charity (Grant Number: 419/2071), 12 Cock Lane, London EC1A 9BU.

Increased γ-H2AX and Rad51 DNA repair biomarker expression in human cell lines resistant to the chemotherapeutic agents nitrogen mustard and cisplatin.

Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms. However, alkylating agents which induce DNA interstrand crosslinks (ICL) are amongst the most effective anticancer agents and often form the mainstay of many anticancer therapies. The effectiveness of these drugs can be limited by the development of drug resistance in cancer cells and many studies have demonstrated that alterations in DNA repair kinetics are responsible for drug resistance. In this study we developed two cell lines resistant to the alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow cytometry to quantitate the number of γ-H2AX and Rad51 foci in the nuclei of cells after drug exposure. γ-H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases and Rad51 was used to measure the activity of homologous recombination in the repair of ICL. In the drug-resistant derivative cell lines there was overall a significant increase in the number and persistence of both γ-H2AX and Rad51 foci in the nuclei of cells over a 72-hour period, when compared to the non-resistant parental cell lines (ANOVA p < 0.0001). In a Pt-resistant ovarian cancer cell line (A2780cisR) a similar enhancement of DNA repair was observed when compared to the non-drug-resistant wild-type ovarian cancer cells (A2780) following exposure to HN2. Our data suggest that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and human tumours may be of experimental and clinical benefit. We concede, however, that examination of a larger population of cell lines and tumours is required to fully evaluate the validity of this approach.