Beata Biesaga

Low-dose radiation response of primary keratinocytes and fibroblasts from patients with cervix cancer

Abstract Słonina, D., Biesaga, B., Urbański, K. and Kojs, Z. Low-Dose Radiation Response of Primary Keratinocytes and Fibroblasts from Patients with Cervix Cancer. Radiat. Res. 167, 251–259 (2007). The aim of the present study was to examine, using the micronucleus (MN) assay, the low-dose radiation response of normal skin cells from cancer patients and to determine whether the hyper-radiosensitivity (HRS)-like phenomenon occurs in cells of these patients. Primary skin fibroblasts and keratinocytes derived from 40 patients with cervix cancer were studied. After in vitro γ irradiation with single doses ranging from 0.05 to 4 Gy, MN induction was assessed. For each patient, the linear-quadratic (LQ) model and the induced repair (IR) model were fitted over the whole data set. In fits of the IR model, an HRS-like response after low doses (seen as the deviation over the LQ curve) was demonstrated for the fibroblasts of two patients and for the keratinocytes of four other patients. The αs/αr ratio for the six patients ranged from 2.7 to 15.4, whereas the values of the parameter dc ranged from 0.13 to 0.36 Gy. No relationship was observed between chromosomal radiosensitivity of fibroblasts and keratinocytes derived from the same donor in the low-dose (0.1–0.25 Gy) region. In conclusion, the fact that low-dose chromosomal hypersensitivity was observed for cells of only six of the patients studied suggests that it is not a common finding in human normal cells and can represent an individual characteristic.

The response of primary keratinocytes and fibroblasts from cancer patients to multiple low-dose irradiations.

Abstract Słonina, D., Biesaga, B., Urbański, K. and Kojs, Z. The Response of Primary Keratinocytes and Fibroblasts from Cancer Patients to Multiple Low-Dose Irradiations. Radiat. Res. 168, 631–636 (2007). In our previous study, using the micronucleus (MN) assay, a hyper-radiosensitivity (HRS)-like phenomenon was observed after single low doses for fibroblasts from two and keratinocytes from four of the 40 patients studied. In this paper, we report the response of primary keratinocytes from 23 and fibroblasts from 21 of these cancer patients to multiple low-dose irradiations and answer the question regarding whether the patients with an HRS-like response after single low doses also demonstrate chromosomal hypersensitivity after multiple low doses. The cells were irradiated with three doses of 0.25 Gy separated by 4-h intervals, and MN induction was compared with that after the same total dose given as a single fraction of 0.75 Gy. Similarly, the effect of three doses of 0.5 Gy was compared with that of a single dose of 1.5 Gy. For fibroblasts from two and keratinocytes from four patients who demonstrated a single-dose HRS-like response, a significant inverse effect of fractionation (greater MN induction after three doses of 0.25 Gy than after a single dose of 0.75 Gy) was observed, which suggests a repeated hypersensitive response after each dose of 0.25 Gy. Such an effect was not seen for the cells from 19 patients who were single-dose HRS-like negative. In conclusion, an inverse fractionation effect for MN induction that was observed in fibroblasts from two and keratinocytes from four patients after three doses of 0.25 Gy (but not 3 × 0.5 Gy) reflects the chromosomal hyper-radiosensitivity seen in the same patients in response to single low doses.

Low-Dose Hyper-Radiosensitivity Is Not a Common Effect in Normal Asynchronous and G2-Phase Fibroblasts of Cancer Patients

PURPOSE In our previous study, using the micronucleus assay, a low-dose hyper-radiosensitivity (HRS)-like phenomenon was observed for normal fibroblasts of 2 of the 40 cancer patients investigated. In this article we report, for the first time, the survival response of primary fibroblasts from 25 of these patients to low-dose irradiation and answer the question regarding the effect of G2-phase enrichment on HRS elicitation. METHODS AND MATERIALS The clonogenic survival of asynchronous as well as G2-phase enriched fibroblast populations was measured. Separation of G2-phase cells and precise cell counting was performed using a fluorescence-activated cell sorter. Sorted and plated cells were irradiated with single doses (0.1-4 Gy) of 6-MV x-rays. For each patient, at least 4 independent experiments were performed, and the induced-repair model was fitted over the whole data set to confirm the presence of HRS effect. RESULTS The HRS response was demonstrated for the asynchronous and G2-phase enriched cell populations of 4 patients. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, G2-phase enrichment had no effect on HRS elicitation. CONCLUSIONS The fact that low-dose hyper-radiosensitivity is not a common effect in normal human fibroblasts implies that HRS may be of little consequence in late-responding connective tissues with regard to radiation fibrosis.

An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients.

In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1-2Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells.

HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.

Physical state of human papillomavirus type 16 in cervical intraepithelial lesions and cancers determined by two different quantitative real-time PCR methods.

The aim of this study was to analyse the correlation between a new multiplex qPCR assay and a reference qPCR assay for assessment of the human papillomavirus (HPV16) load and the viral genome status. The study was performed on 100 HPV16 positive samples containing premalignant lesions and carcinomas. HPV16 E2 and E6 gene loads were assessed by two PCR methods. The load of E2 and E6 was normalized to the cell number by qPCR targeting the RNase P open reading frame. The physical state of the viral genome was determined as a ratio of E2/E6 copies number per cell. Among 100 samples analysed, there were no statistically significant differences in the E2 and E6 viral load evaluated by multiplex qPCR and qPCR, the correlation coefficients were 0.98 and 0.97, respectively. There were 19% of samples with the integrated, 73% with mixed and 8% with episomal state of viral genome detected by multiplex qPCR and 17%, 79%, 4%, respectively, found by qPCR. Prevalence of integrated and episomal forms estimated by multiplex qPCR was higher than the one obtained by qPCR (Chi2, p < 0.0001), but in samples with premalignant and malignant diagnoses no significant differences were demonstrated regardless of the methods used. Sensitivity and specificity of multiplex qPCR were 93.7% and 100% as compared with qPCR, the positive predictive value was 100%. In summary, the multiplex qPCR assay in respect of HPV16 load and the frequency of viral genome status was shown to be a sensitive and specific reference method. Simultaneous estimation of E2 and E6 genes in one reaction tube reduces the cost of testing.

Positive effect of single nucleotide RAD51 135G>C polymorphism and low Ku70 protein expression on female rectal cancer patients survival after preoperative radiotherapy.

BACKGROUND/AIMS This is a retrospective analysis of 103 patients having locally advanced rectal cancer who received short-course radiotherapy (SCRT). The objective of the study was to check whether a polymorphism in the RAD51 gene (135 G>C), Ku70 protein expression, and tumor microenvironment: proliferation rate measured by BrdUrdLI and Ki-67LI, hypoxia (glucose transporter-1 expression), P53 protein expression, and DNA ploidy can influence DNA repair capacity, the factors contributing to patient overall survival (OS) and the incidence of recurrences and metastases. MATERIALS AND METHODS RAD51 (135 G>C) polymorphism was evaluated using restriction fragment length polymorphism polymerase chain reaction, and proteins were identified using immunohistochemistry. RESULTS There were 3 (2.9%) tumors with RAD51 CC, 75 (72.8%) with GG, and 25 (24.3%) with GC genotypes. The median follow-up time was 63.1 months (range 2-120). Patients with CC genotype survived significantly longer than those with GG and GC genotypes and did not develop any recurrences or distant metastases. Female patients with Ku70 expression (<75.1) or RAD51CC genotype (impaired DNA damage repair and radiosensitive) had significantly longer OS (p=0.013) than those with Ku70>75.1 % or RAD51GG,GC (radioresistant phenotype) and male patients in the log-rank test. In multivariate analysis, positive prognostic factors for OS in the male patients were grade=1 and <17 days break in the treatment, whereas in the female subgroup, only radiosensitive phenotype (Ku70 <75.1% or RAD51CC genotype). CONCLUSION To the best of our knowledge, this is the first study to provide evidence for the positive effect of CC genotype of RAD51 or low Ku70 expression on OS in females with rectal cancer after SCRT.