289 research outputs found
Efficacy of second line agents for the treatment of metastatic castration-resistant prostate cancer progressing after docetaxel: Systematic review and meta-analysis.
No full textCryopreserved whole blood and isolated peripheral blood mononuclear cells as alternative sample types for the cytokinesis-block micronucleus assay
No full textBackground
The cytokinesis-block micronucleus (MN) assay is widely used in basic radiobiological research, environmental biomonitoring studies, biological dosimetry and in vitro radiosensitivity testing. Fresh whole blood cultures are generally used for the MN assay, limiting the application of this assay in studies where immediate processing of fresh samples is logistically challenging. Therefore, we standardized the MN assay on cryopreserved whole blood samples and isolated peripheral blood mononuclear cells (PBMCs). The reliability of both MN assays was thoroughly evaluated for use in radiosensitivity assessment.
Materials and methods
Blood of healthy donors and radiosensitive patients was collected and the MN assay was performed on fresh whole blood, cryopreserved whole blood and isolated PBMCs. MN yields were scored after irradiation with 220 kV X-rays, with doses ranging from 0.5 - 2 Gy.
Results and conclusion
Fresh whole blood samples showed the highest MN values, while both cryopreserved PBMCs and cryopreserved whole blood cultures showed similar but slightly lower MN values. Inter-individual and intra-individual variabilities were acceptable for all three sample types. The cryopreservation period of PBMCs or whole blood has no significant impact on MN yields, analyzed up to six months and one year, respectively. In addition, radiosensitive patients could successfully be identified using these alternative sample types. Here, we demonstrate the possibility of using cryopreserved isolated PBMCs or whole blood for the MN assay. Depending on the application, the optimal sample type can be selected. PBMCs are highly advantageous when multiple other assays need to be performed on a single blood sample, while cryopreserved whole blood is favorable when a quick and simple sample storing procedure is required in large-scale multicenter studies.
References
Sioen S, Cloet K, Vral A, Baeyens A. The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells. J Pers Med. 2020 Sep 14;10(3):125. doi: 10.3390/jpm10030125.
Beyls E, Baeyens A, Vral A. The cytokinesis-block micronucleus assay for cryopreserved whole blood. Int J Radiat Biol. 2021 Jul 1:1-9. doi: 10.1080/09553002.2021.1941378
The cellular response to different radiation qualities in relation to breast cancer screening and treatment
No full textPolymorphisms in DNA double-strand break repair genes: link with breast cancer susceptibility and in vitro chromosomal radiosensitivity.
No full textBreast cancer is one of the most common types of neoplasia in females in Western industrialised countries. In Belgium, breast cancer is the leading cause of death by cancer in females and the risk of being diagnosed with breast cancer before the age of 75 years in Belgium is 11.5 %. One of the strongest risk factors is a family history of the disease, indicating a genetic predisposition to breast cancer. However, only 6% of all breast cancer cases can be linked to monogenic, germline mutations in the breast cancer predisposing genes BRCA1, BRCA2, ATM, CHK2, PTEN and TP53. Epidemiological analyses suggest that the remaining breast cancer cases can be explained by a polygenic model that states that the combined effect of many individual weak genetic variants is responsible for an enhanced breast cancer risk. As enhanced in vitro chromosomal radiosensitivity is a hallmark for breast cancer and results from non‐ or misrepaired double strand breaks (DSBs), single nucleotide polymorphisms (SNPs) in DSB repair genes, could be involved in in vitro chromosomal radiosensitivity and genetic predisposition to breast cancer. Several population based case‐control studies have already shown a link between SNPs in DSB repair genes and breast cancer risk. Moreover, the breast is a selected micro‐environment, vulnerable to endogenous oxidative stress through hormone exposure. Especially oestrogen has attracted considerable attention, as it induces DSBs during its metabolism and may act as a complete carcinogen. In the studies presented in this thesis, we investigated whether SNPs in the core repair genes of DSBs are associated with an enhanced breast cancer susceptibility and/or in vitro chromosomal radiosensitivity. Genes from the two main DSB repair pathways were studied: Ku70, Ku80 and DNAPKCS of the non homologous end‐joining (NHEJ) pathway and RAD51, XRCC3, BRCA1 and BRCA2 of the homologous recombination (HR) pathway.
The results demonstrate that the c.‐1310C>G SNP in the promoter region of Ku70 is significantly associated with breast cancer risk in an unselected patient population, comprising mainly of sporadic patients. Additionally, the combination of the variant “G” allele of this polymorphism with a hormonal breast cancer risk factor, reflecting susceptibility to oestrogen exposure, is associated with a more pronounced increase in breast cancer risk. The c.2099‐2408G>A SNP in Ku80 shows a positive association with breast cancer risk in a group of patients with a known or putative genetic predisposition to the disease. Both the c.‐1310C>G (Ku70) and c.2099‐2408G>A (Ku80) point‐variations can be considered risk alleles for breast cancer and they also show a positive association with chromosomal radiosensitivity. The combination of 2 or 4 putative high‐risk genotypes in RAD51 and XRCC3 resulted in a significant association with breast cancer risk in a patient population selected for a genetic predisposition, which is in agreement with the polygenic model for breast cancer initiation. Our results are also indicative of a modifying effect of SNPs in RAD51, XRCC3, BRCA1 and BRCA2 on breast cancer penetrance and phenotype in patients carrying a pathological mutation in BRCA1 or BRCA2. We also showed that the c.190T>C variation in the BRCA1 RING finger domain may induce modifications of the protein structure which could disrupt the BRCA1‐BARD1 interaction and hence predispose to breast cancer. The studies performed in the frame of this thesis contribute to the ongoing research concerning the genetic profiles associated with an enhanced breast cancer risk. A better understanding of the underlying genetic factors, responsible for breast cancer predisposition will improve our understanding of the mechanisms underlying breast cancer aetiology and this will influence the approach to breast cancer prevention and treatment
Analysis of radiosensitivity in South African cervical and breast cancer patients
No full textIntroduction: Ionising radiation can cause DNA double strand breaks (DSB), that result in chromosomal aberrations if un- or mis-repaired. Individuals with compromised DNA damage repair mechanisms display increased chromosomal radiosensitivity. The G0-micronucleus assay (MN assay) and the γ-H2AX assay are two assays used in radiobiology to study DNA DSB and repair.
Breast cancer is the leading cancer amongst South African women, with a lifetime risk of 1 in 34. Since most cancer patients in South Africa present with late-stage disease, chemotherapy and radiotherapy are commonly-used treatments. Several international studies have shown breast cancer patients to be more chromosomally radiosensitive than healthy controls. These studies have not been confirmed on a cancer population living in South Africa.
Cervical cancer is the second most common cancer in South Africa; however, it is the leading cancer amongst black women with a lifetime risk of 1/35 compared to 1/82 in white women. Studies show a genetic link to cervical cancer susceptibility and DNA damage repair genes. International studies on radiation-induced DNA damage in lymphocytes of cervical cancer patients remain inconclusive and have never been performed on a South African population. Cervical cancer is caused by infection with the Human Papilloma Virus (HPV). Human Immunodeficiency Virus (HIV), HPV and cervical cancer are epidemiologically linked. Due to the high rate of HIV in South Africa, a significant proportion of cervical cancer patients receiving radiotherapy treatment will be HIV-positive. Studies show an effect of HIV on chromosomal radiosensitivity, however this has not been confirmed on a cancer population. The MN assay on the biopsies and exfoliated cervical cells of cervical cancer patients could be used as a predictive test for response to radiotherapy. The overall aim was to study chromosomal radiosensitivity in South African cervical and breast cancer patients.
Materials and methods: Chromosomal radiosensitivity of lymphocytes of cervical and breast cancer patients was examined using the MN assay with the Metafer 4 of Metasystems. Different scoring methods for the Metafer system were compared to each other. The effect of HIV, HPV, ethnicity, clinical parameters and age on micronuclei (MN) values in lymphocytes was investigated. The MN assay was attempted on cells from cervical biopsies and exfoliated cervical cells. The γ-H2AX was performed on the lymphocytes of a group of cervical cancer patients.
Results: A new scoring method for the Metafer 4 system that is more reliable in patients with late-stage disease was introduced. Cervical cancer patients had significantly higher MN values with HIV patients having the highest values. HPV, clinical parameters and age had a limited effect on MN values. The MN assay was unsuccessful on biopsies and exfoliated cervical cells of cervical cancer patients. There was no difference in double strand break induction and repair between cervical cancer patients and controls. In breast cancer patients, ethnicity had an effect on MN values, with only white breast cancer patients having significantly higher MN counts.
Conclusion: The study showed increased chromosomal radiosensitivity in cervical cancer and white breast cancer patients. Results highlight how such studies are important within the South African context, where factors like HIV, disease stage and ethnicity can have an effect on chromosomal radiosensitivity and where unique genes/polymorphisms may play a role in cancer risk
From human genetics to radiobiology : in vitro radiosensitivity in individuals with a germline defect in DNA damage response genes
No full textAll currently known high to intermediate risk “breast cancer genes”, including BRCA1 and BRCA2, are involved in the DNA damage response pathway. Heterozygous germline mutations in these genes predispose to breast and ovarian cancer. In addition, such mutations may also result in enhanced radiosensitivity mediated by chromosomal instability after exposure to ionizing radiation, leading to a higher risk to develop radiation-induced breast cancer. However, results of currently available clinical studies evaluating carcinogenesis and in vitro studies comparing chromosomal radiosensitivity in mutation carriers and non-carriers are inconclusive. Nevertheless, insights into the radiosensitive phenotype of healthy tissues of mutation carriers is of the utmost importance for the safe use of ionizing radiation for
diagnostic purposes or radiotherapy treatment. In this thesis, we evaluated in vitro radiosensitivity in carriers of a mutation in DNA damage response genes by means of two different assays.
The first assay, the G2 micronucleus assay, is a cytogenetic assay in which MN are analyzed in cells irradiated in the G2 phase of the cell cycle. This assay was developed to evaluate radiosensitivity in cells with a heterozygous BRCA1 or BRCA2 mutation. BRCA1 and BRCA2 have a function in homologous recombination (HR), the main DNA double strand break repair pathway activated in late S and G2 phase of the cell cycle. Furthermore, BRCA1 is also involved in the G2/M cell cycle checkpoint. The G2 micronucleus assay allows evaluation of both functions by means of two distinct endpoints: (1) the radiation-induced micronucleus yield, which reflects DNA double strand break repair capacity and (2) the G2/M checkpoint efficiency ratio, which allows evaluation of the G2 arrest capacity.
Before applying the G2 micronucleus assay on BRCA mutation carriers, the assay was validated in a patient with Ataxia Telangiectasia (AT). AT patients are characterized by a manifest increased radiosensitivity. AT patients show biallelic inactivation of ATM, involved in both DNA double strand break repair by means of HR and G2/M checkpoint activation. We demonstrated a severely increased radiosensitivity with both endpoints when applying the G2 micronucleus assay in lymphocytes of this AT patient. In lymphocytes of healthy relatives with a heterozygous ATM mutation the radiosensitivity observed with this assay was intermediate between the AT patient and the control cohort.
When applying the G2 micronucleus assay on lymphocytes of healthy BRCA1/2 mutation carriers, we demonstrated significantly enhanced radiation-induced MN yields in both BRCA1 and BRCA2 germline mutation carriers, pointing to an impaired DNA double strand break repair capacity in both groups. Furthermore, an impaired G2 arrest capacity was observed in BRCA1 mutation carriers. In healthy relatives who did not inherit the familial mutation, no enhanced radiosensitivity was observed. Although a significantly enhanced radiosensitivity was demonstrated for the cohort of BRCA1 and BRCA2 mutation carriers compared to the control cohort, individual radiosensitivity evaluation was less straightforward due to overlap in micronucleus yields between both cohorts. Therefore, a scoring system to evaluate individual radiosensitivity was implemented.
As both BRCA1 and BRCA2 are involved in HR, we evaluated if the accumulation of RAD51, a key protein involved in this pathway, at the double strand break site can be used to assess HR functionality and radiosensitivity. To this end, a radiation-induced RAD51 foci assay was optimized in a breast epithelial cell line (MCF10A) expressing ±50% reduced BRCA1 and BRCA2 protein levels, obtained by RNA interference. RAD51 foci were analyzed in cells synchronized in S phase by aphidicolin as HR is upregulated during this phase of the cell cycle. We demonstrated significantly reduced RAD51 foci formation, and thus impaired HR capacity, in response to the induction of radiation-induced double strand breaks in the BRCA knockdown cells compared to control cells. As no overlap in RAD51 foci distribution is observed between knockdown and control cells, we think that this assay could better differentiate between normal cells and cells with a heterozygous BRCA1 or BRCA2 mutation than the G2 micronucleus assay. This will be further explored in synchronized lymphocytes of heterozygous germline mutation carriers.
In addition to the detection of unequivocal deleterious mutations in BRCA1 and BRCA2, variants of unknown clinical significance (VUS) are detected during diagnostic screening. The associated breast cancer risk is unknown, which creates a challenge for genetic counselling. mRNA analysis to assess variants that might impair proper RNA splicing, a highly regulated process, are widely used. We evaluated the outcome at cDNA level of 21 putative splicing variants in BRCA1 and BRCA2 and demonstrated aberrant splicing for 12 variants, suggesting that these are likely pathogenic. Furthermore, we demonstrated that in silico prediction tools might assist in the evaluation of these putative splicing variants. However, further optimization is warranted to allow reliable application outside the highly conserved consensus splice sites.
The results obtained in this thesis may indicate that care should be taken when applying ionizing radiation for diagnostic or therapeutic purposes in individuals with a germline mutation in BRCA1 or BRCA2 as they may be at higher risk of developing radiation-induced breast cancer
The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons
No full textAbstract Background A better understanding of the underlying mechanisms of DNA repair after low- and high-LET radiations represents a research priority aimed at improving the outcome of clinical radiotherapy. To date however, our knowledge regarding the importance of DNA DSB repair proteins and mechanisms in the response of human cells to high-LET radiation, is far from being complete. Methods We investigated the radiosensitizing effect after interfering with the DNA repair capacity in a human mammary epithelial cell line (MCF10A) by lentiviral-mediated RNA interference (RNAi) of the Ku70 protein, a key-element of the nonhomologous end-joining (NHEJ) pathway. Following irradiation of control and Ku-deficient cell lines with either 6 MV X-rays or p(66)+Be(40) neutrons, cellular radiosensitivity testing was performed using a crystal violet cell proliferation assay. Chromosomal radiosensitivity was evaluated using the micronucleus (MN) assay. Results RNAi of Ku70 caused downregulation of both the Ku70 and the Ku80 proteins. This downregulation sensitized cells to both X-rays and neutrons. Comparable dose modifying factors (DMFs) for X-rays and neutrons of 1.62 and 1.52 respectively were obtained with the cell proliferation assay, which points to the similar involvement of the Ku heterodimer in the cellular response to both types of radiation beams. After using the MN assay to evaluate chromosomal radiosensitivity, the obtained DMFs for X-ray doses of 2 and 4 Gy were 2.95 and 2.66 respectively. After neutron irradiation, the DMFs for doses of 1 and 2 Gy were 3.36 and 2.82 respectively. The fact that DMFs are in the same range for X-rays and neutrons confirms a similar importance of the NHEJ pathway and the Ku heterodimer for repairing DNA damage induced by both X-rays and p(66)+Be(40) neutrons. Conclusions Interfering with the NHEJ pathway enhanced the radiosensitivity of human MCF10A cells to low-LET X-rays and high-LET neutrons, pointing to the importance of the Ku heterodimer for repairing damage induced by both types of radiation. Further research using other high-LET radiation sources is however needed to unravel the involvement of DNA double strand break repair pathways and proteins in the cellular response of human cells to high-LET radiation.</p
Zebrafish as a model to study DNA repair and developmental defects following loss of Brca2 or Atm
No full textIn vitro chromosomal radiosensitivity in breast cancer patients
No full textIn vitro chromosomal radiosensitivity has been investigated in patients with inherited cancer prone syndromes (e.g. Ataxia Telangiectasia, Nijmegen breakage syndrome) and also in different types of cancer patients (e.g. head and neck-, colorectal cancer, breast cancer). In breast cancer patients enhanced chromosomal radiosensitivity is observed in several independent studies. In most of these studies chromosomal radiosensitivity was analysed by using the G2 and G0-MN assay for peripheral blood lymphocytes and it has been suggested that enhanced chromosomal radiosensitivity of PBL may be a marker for breast cancer predisposing genes of low penetrance. In the first part of this thesis we investigated the suitability of the G2 and G0-MN assay as biomarkers of individual radiosensitivity. Therefore we analysed the reproducibility and sensitivity of these assays. Our studies showed that the intra-individual variability of both assays is high and not different from the inter-individual variability. Although it was suggested in literature that the inter-individual variability might be attributable to the hormonal status in females, we could not demonstrate any influence of the blood serum levels of estradiol or progesterone on the intra-individual and inter-individual variability obtained in the G0-MN assay. In most of the studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients the G0-MN assay has been performed on fresh blood cultures. Because the use of fresh blood cultures allows no repeated testing, we have investigated the suitability of EBVtransformed cell lines and IL-2 cultures for the analysis of chromosomal radiosensitivity in breast cancer patients. As we were not able to demonstrate the radiosensitive response in the cell cultures derived from the original PBL, care has to be taken when EBV-transformed cell lines or IL-2 cultures are used to assess chromosomal radiosensitivity in breast cancer patients.In the second part of this thesis, we analysed chromosomal radiosensitivity of different breast cancer populations with the G2 and G0-MN assay. We have demonstrated that a group of breast cancer patients with a known or putative genetic predisposition is more radiosensitive than a group of healthy women and this as well with the G2 and G0-MN assay. We showed that this enhanced in vitro chromosomal radiosensitivity is not correlated with the age of onset of the disease in breast cancer patients and that the enhanced chromosomal radiosensitivity in breast cancer patients with a mutation in BRCA1 or BRCA2 is not due to the presence of a
mutation in these two genes. Analysis of the in vitro chromosomal radiosensitivity of a small group of breast cancer patients with a CHEK2 1100delC mutation revealed no correlation between the CHEK2 1100delC variant and chromosomal radiosensitivity. No correlation is found between the G2 and G0 chromosomal radiosensitivity, pointing to the fact that these two assays reflect different underlying mechanisms of chromosomal radiosensitivity. In conclusion, we performed a thorough analysis of the suitability of the G2 and G0-MN assay as biomarkers of individual radiosensitivity and we applied these assays to determine the chromosomal radiosensitivity of different population groups of breast cancer patients. We can conclude that both assays are very valuable to analyse chromosomal radiosensitivity in breast cancer populations. Further studies have to be performed to analyse the underlying mechanisms of chromosomal radiosensitivity
The role of the Ku heterodimer in the response of human MCF10A cells to low-and high-LET radiation qualities
No full textSeveral observations suggest that the mechanisms involved in DNA double-strand break (DSB) repair are of particular importance during breast tumorigenesis and chromosomal radiosensitivity. An enhanced chromosomal radiosensitivity has been observed in a large number of breast cancer patients by means of the G0 micronucleus (MN) and G2 chromatid break assay. As micronuclei and chromatid breaks are the result of non-repaired or misrepaired DNA DSBs, mutations and/or polymorphisms in DNA DSB repair genes could account for the observed elevation in chromosomal radiosensitivity in breast cancer patients and breast cancer risk. Recently, our research group showed that single nucleotide polymorphisms (SNPs) in the DSB repair genes, Ku70 and Ku80, are associated with an enhanced breast cancer susceptibility and in vitro chromosomal radiosensitivity. To further unravel the underlying mechanisms responsible for an enhanced chromosomal radiosensitivity in breast cancer patients and breast cancer risk, we investigated in a first section of this dissertation the role of the Ku70/80 (Ku) heterodimer, in the response of human breast epithelial cells to low-LET (linear energy transfer) 60Co γ-rays. For this purpose, we generated a stable Ku-knockdown by lentiviral-mediated RNAi of Ku70 in human breast epithelial MCF10A cells. Analysis of chromosomal radiosensitivity by means of the MN assay in both knockdown and control MCF10A cells showed that knockdown of Ku70/80 caused a significant increase in the number of micronuclei, which supports the hypothesis that defects in DNA DSB repair underlie chromosomal radiosensitivity. We further demonstrated that this increase in chromosomal radiosensitivity was accompanied by a significant decrease in cell survival, measured by both the crystal violet cell proliferation assay and the classic colony formation assay. To further understand the mechanisms that lead to this radiosensitive phenotype, DSB induction and repair was investigated using γH2AX foci as a marker of DNA DSBs. These results showed that downregulation of Ku resulted in a higher number of foci present at early post-irradiation times, which could be further indicative of less efficient repair mechanisms. A profound knowledge of the pathways involved in the cellular response to radiation-induced damage is not only important for an improved understanding of the mechanisms underlying breast cancer susceptibility, it also has important implications for the use of ionizing radiation in radiotherapy as the ability to repair radiation-induced damage will determine therapeutic outcome of both normal and malignant cells. Besides the use of conventional low-LET radiation in radiotherapy, high-LET radiation qualities are increasingly implemented. Based on the argument that depending on the radiation quality, the degree of complexity of radiationinduced DSBs differs, it could be anticipated that the relative importance of pathways involved in the repair of DNA damage also varies. Therefore, in a second section of this dissertation, we investigated the influence of Ku-knockdown in the cellular response to low-LET X-rays and high-LET neutrons, both routinely used in clinical practice. The results of this study showed that knockdown of the Ku heterodimer enhanced the chromosomal and cellular radiosensitivity of MCF10A cells to both low-LET X-rays and high-LET neutrons. Moreover, we observed that the Ku heterodimer is of similar importance for repairing radiation damage induced by both radiation qualities. These results may open perspectives towards the use of strategies to enhance radioresponsiveness of neoplastic tissues and improve the outcome of radiotherapy using both low-LET and high-LET radiation types
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