Alterations of Sexual and Erectile Functions after Brachytherapy for Prostate Cancer Based on Patient-Reported Questionnaires.

The aim of the study was to compare the side effects of high-dose-rate brachytherapy (HDRBT) and low-dose-rate brachytherapy (LDRBT), with a particular focus on the effects on sexual functions and sexual well-being (PROMOBRA study, NCT02258087). Localized low-risk and low-intermediate-risk prostate cancer patients were treated with mono LDR (N = 123, 145 Gy dose) or mono HDR brachytherapy (N = 117, 19/21 Gy). Prior to the treatment and during follow-up (at 3, 6, 9, 12, 18, and 24 months after treatment, and then annually after two years), patients completed patient-reported outcome measurement (PROM) questionnaires EORTC QLQ-PR-25, International Index of Erectile Function (IIEF), and IIEF-5 (SHIM). We compared the patients in different group breakdowns (HDR vs. LDR, hormone naïve and hormone-receiving HDR vs. LDR, hormone naïve and hormone-receiving patients in general, and 19 Gy HDR vs. 21 Gy HDR). In the hormone-naive LDR group, erectile function, orgasm function, sexual desire, satisfaction with intercourse, and overall satisfaction functions significantly decreased compared to baseline throughout the whole follow-up period. However, there were significant decreases in function at a maximum of three time points after HDR therapy without hormone therapy. In hormone-receiving patients, the orgasm function was significantly better in the HDR group at multiple time points compared to the baseline, and sexual desire improved at four time points. According to our results, both LDRBT and HDRBT can be safely administered to patients with localized prostate cancer. In hormone-naive patients, the HDR group showed only recovering decreases in sexual functions, while the LDR group showed a lasting decline in multiple areas. Thus, HDR appears to be more advantageous to hormone-naive patients.

Malignant Transformation and Long-Term Outcome of Oral and Laryngeal Leukoplakia.

BACKGROUND: Oral or laryngeal leukoplakia has an increased risk for malignant transformation but the risk of the two anatomical sites has not been compared to each other yet. MATERIALS AND METHODS: Clinical data of 253 patients with leukoplakia (oral = 221 or laryngeal = 32) enrolled from January 1996 to January 2022 were analyzed. One hundred and seventy underwent biopsy and 83 did not. The mean follow-up time was 148.8 months. Risk factors for the malignant transformation of leukoplakia were identified using Cox proportional hazard models. RESULTS: In the oral or laryngeal group, the rate of cancer was 21.7% and 50% (p = 0.002), respectively. The 10-year estimated malignant transformation was 15.1% and 42% (p < 0.0001), respectively. The laryngeal group had an increased risk of malignant transformation (p < 0.0001). The 5-year estimated survival with leukoplakia-associated cancer for the oral or laryngeal group was 40.9% and 61.1% (p = 0.337), respectively. Independent predictors of malignant transformation in the oral group were dysplasia and the grade of dysplasia of the leukoplakia, and in the laryngeal group, dysplasia had a significant impact. The malignant transformation rate was low for oral patients without biopsy or with no dysplasia, 3.9% and 5.1%, respectively. The malignant transformation occurred over 10 years. CONCLUSIONS: Patients with dysplastic leukoplakia have an increased risk of malignant transformation, but the risk is higher with laryngeal than with oral leukoplakia. There is no significant difference between the groups regarding survival with leukoplakia-associated cancer. Oral patients with no dysplastic lesions have a low risk of malignant transformation. A complete excision and long-term follow up are suggested for high-risk patients to diagnose cancer in an early stage and to control late (over 10 years) malignant events.

Chromosomal breakage tests in the differential diagnosis of Fanconi anemia and aplastic anemia.

BACKGROUND: FA patients are hypersensitive to preconditioning of bone marrow transplantation. OBJECTIVE: Assessment of the power of mitomycin C (MMC) test to assign FA patients. METHODS: We analysed 195 patients with hematological disorders using spontaneous and two types of chromosomal breakage tests (MMC and bleomycin). In case of presumed Ataxia telangiectasia (AT), patients' blood was irradiated in vitro to determine the radiosensitivity of the patients. RESULTS: Seven patients were diagnosed as having FA. The number of spontaneous chromosomal aberrations was significantly higher in FA patients than in aplastic anemia (AA) patients including chromatid breaks, exchanges, total aberrations, aberrant cells. MMC-induced ≥10 break/cell was 83.9 ± 11.4% in FA patients and 1.94 ± 0.41% in AA patients (p < .0001). The difference in bleomycin-induced breaks/cell was also significant: 2.01 ± 0.25 (FA) versus 1.30 ± 0.10 (AA) (p = .019). Seven patients showed increased radiation sensitivity. Both dicentric + ring, and total aberrations were significantly higher at 3 and 6 Gy compared to controls. CONCLUSIONS: MMC and Bleomycin tests together proved to be more informative than MMC test alone for the diagnostic classification of AA patients, while in vitro irradiation tests could help detect radiosensitive-as such, individuals with AT.

Prostate Cancer Survivors Present Long-Term, Residual Systemic Immune Alterations.

BACKGROUND: The development of cancer and anti-tumor therapies can lead to systemic immune alterations but little is known about how long immune dysfunction persists in cancer survivors. METHODS: We followed changes in the cellular immune parameters of prostate cancer patients with good prognostic criteria treated with low dose rate brachytherapy before and up to 3 years after the initiation of therapy. RESULTS: Patients before therapy had a reduced CD4+ T cell pool and increased regulatory T cell fraction and these alterations persisted or got amplified during the 36-month follow-up. A significant decrease in the total NK cell number and a redistribution of the circulating NK cells in favor of a less functional anergic subpopulation was seen in patients before therapy but tumor regression led to the regeneration of the NK cell pool and functional integrity. The fraction of lymphoid DCs was increased in patients both before therapy and throughout the whole follow-up. Increased PDGF-AA, BB, CCL5 and CXCL5 levels were measured in patients before treatment but protein levels rapidly normalized. CONCLUSIONS: while NK cell dysfunction recovered, long-term, residual alterations persisted in the adaptive and partly in the innate immune system.

Spontaneous Chromosomal Aberrations in Lymphocytes and Development of Tumor in Hospital Workers.

BACKGROUND: Cytogenetic analysis of chromosomes in blood lymphocytes can be used to reveal biomarkers of tumor risk. The frequency of chromosomal aberrations (CAs) appears to correlate with the later incidence of cancer. PATIENTS AND METHODS: In our work, a total of 515 healthy Hungarian medical workers and 725 controls were enrolled in our investigation. The CAs in peripheral blood lymphocytes were analyzed. RESULTS: The frequency of CAs was significantly higher in the groups working with ionizing radiation and with cytostatic agents compared to unexposed controls and in male smokers rather than non-smokers. The frequency of dicentric chromosomes, however, was not significantly different between control and exposed groups. Among 82 cancer cases (6.6%), the most frequent types were cancer of the breast (20.5%), colon (12.8%), lung and thyroid gland (9-9%). Our analysis showed 8.1% cancer cases in smokers compared to 5.7% in non-smokers. CONCLUSION: The potential exposure to carcinogens did not modify the effect of CAs on cancer risk but tobacco smoking did increase risk.

Relationship between biodosimetric parameters and treatment volumes in three types of prostate radiotherapy.

Brachytherapy (BT) and external beam radiotherapy (EBRT) apply different dose rates, overall treatment times, energies and fractionation. However, the overall impact of these variables on the biological dose of blood is neglected. As the size of the irradiated volume influences the biological effect as well, we studied chromosome aberrations (CAs) as biodosimetric parameters, and explored the relationship of isodose surface volumes (ISVs: V1%, V1Gy, V10%, V10Gy, V100%, V150%) and CAs of both irradiation modalities. We performed extended dicentrics assay of lymphocytes from 102 prostate radiotherapy patients three-monthly for a year. Aberration frequency was the highest after EBRT treatment. It increased after the therapy and did not decrease significantly during the first follow-up year. We showed that various types of CAs 9 months after LDR BT, 3 months after HDR BT and in a long time-range (even up to 1 year) after EBRT positively correlated with ISVs. Regression analysis confirmed these relationships in the case of HDR BT and EBRT. The observed differences in the time points and aberration types are discussed. The ISVs irradiated by EBRT showed stronger correlation and regression relationships with CAs than the ISVs of brachytherapy.

[Trimodal, organ-preserving treatment of organ-confined, muscle-invasive bladder cancer]. / Szervre lokalizált, izominvazív húgyhólyagrák trimodális, szervmegtartó kezelése.

Radical cystectomy is the gold standard treatment in localized muscle-invasive bladder cancer according to today's guidelines. However, in many cases, surgery is not possible due to the patient's general condition, or the patient refuses bladder removal. In such cases, as well as in some selected patients suitable for surgery, trimodal organ preservation therapy is an alternative, which provides the patient with similar survival, local tumor control, so that 80% of patients retain their bladder. In some cases, due to complications or a muscle-invasive local recurrence in the bladder, the bladder may not be retained. At this point, a salvage cystectomy can still save the patient's quality of life and life. Adequate patient selection is a prerequisite for effective trimodal therapy. We summarize the components of organ-preserving treatment, including radiation therapy, its state-of-the-art technology, results and side effects. The results and toxicity of trimodal treatment are compared with those of radical cystectomy.

Influence of sample preparation optimization on the accuracy of dose assessment of an automatic non-fluorescent MN scoring system.

PURPOSE: Automatizing the scoring of the cytokinesis-blocked micronucleus assay spares a lot of valuable time. The dose-effect relationship can be applied reliably for dose estimation if the quality of the slides is the same from the perspective of the used image processing algorithm. This aspect brings in additional requirements against the quality of the slides compared to the conventional visual scoring. MATERIALS AND METHODS: An add-in software was created to the non-fluorescent RS-MN automatic MN scoring system which is capable of measuring quantitatively the degree of typical anomalies. The image processing is less reliable when the presence of these anomalies is more frequent. The behavior of the designed sample quality parameters (SQPs) was tested on in vitro irradiated peripheral blood samples (0, 1, and 2 Gy) obtained from a healthy donor and also on samples from patients undergoing low dose-rate brachytherapy. RESULTS: We examined 20 different SQPs and identified two that are independent and correlate significantly with the error of the fully automatic MN frequency. One is related to the size of the cells and the other reflects the homogeneity of the environment. An equation was established which presents a connection between the error of the auto MN frequency and the SQPs. By adding a fourth cleaning step to the conventional sample preparation and changing the pre-dripping temperature of the slide, the SQP can be modified, and consequently, the sample quality can be improved. The gain in accuracy is 54 ± 10 MN per 1000 binucleated cells, which corresponds to the effects of 0.5 Gy. Around the lowest limit of detection (<0.5 Gy), it means a 50-100% drop in the error of dose, which is significant. With sample quality harmonization, the positive predictive value was raised to 80-93% depending on the dose. CONCLUSIONS: With the technique described in this paper, the suitability for automated scoring of a micronucleus slide can be tested quantitatively and objectively. A method is presented with which in some cases the uncertainty of the assessed doses due to variance in sample quality can be decreased or if it is not possible its bias can be predicted. The proposed protocol leads to more reliable estimation of dose. The SQPs are designed in a way that they have the potential to be adapted to similar systems.

Smoking, chromosomal aberrations, and cancer incidence in healthy subjects.

Chromosomal aberrations (CAs) in peripheral blood lymphocytes can be used as biomarkers of cancer risk. Cytogenetic tests were conducted on 2396 healthy Hungarian individuals and cancer incidence was followed up from 1989 to 2018. Venous blood samples were obtained from the subjects and metaphases from lymphocyte cultures were prepared. We compared the CA frequencies of the various smoking (1-5; 6-10; 11-19; or 20-40 cigarettes/day) and exposure (irradiation; chemical industry; chemical research laboratory) groups. Chromatid break (p = 0.0002), total aberration (p = 0.002), and aberrant cell (p = 0.001) frequencies were higher in smokers than in non-smokers. For very heavy smokers, total CAs were significantly higher than for non-smokers (<0.001) or less intensive smokers (p = 0.003-0.0006). Intensity of smoking was a predictor of chromosomal aberrations, while duration was not. During follow-up, 177 (7.3 %) cancer cases were found. A Cox-regression model showed that subjects with cell values ≥2 CAs developed cancer more frequently (hazard ratio = 1.39; 95 % CI, 1.02-1.90). The relative risks of cancer were 1.06 (95 % CI 0.53-2.06) for light smokers and 1.74 (95 % CI 1.08-2.77) for very heavy smokers. The distributions of cancer sites showed differences between smoker and non-smoker groups: in male smokers, lung cancer, in non-smokers, prostate, and in females (both groups) breast cancer were most common. Cancer incidence correlated with chromosome aberrations; smoking was not a confounder in this relationship.

[And what about doctors? Associations between spontaneous chromosomal aberrations and cancer morbidity in healthcare workers]. / És mi van az orvosokkal? Egészségügyi dolgozók spontán kromoszómaaberrációi és rákmorbiditása közti kapcsolat.

Healthcare workers may be occupationally exposed to low dose rate radiation or different chemicals during their work. There are strong associations between the increased frequency of spontaneous chromosomal aberrations in blood lymphocytes and the risk of cancer. Cytogenetic tests were conducted on 1240 healthy medical workers and cancer incidence was followed up between 1997-2018. Both structural and numerical chromosome aberrations were evaluated and the results were compared taking into account gender, age, and smoking. The frequency of aberrant cells was significantly higher in smoker males than in non-smokers (p=0.009). Within the same study period, there was no significant difference in chromosome aberrations between the potentially exposed group of workers and the control group. Among 82 cancer cases (6.6%) the most common tumors were breast (16), colon (12), lung (7) and thyroid gland cancers (7). Our analysis showed 7.3% cancer occurrence among smokers compared to 6.2% among non-smokers. These results suggest that in our cases cytogenetic effects of smoking are more deleterious than occupational exposures.

[Biological dose estimation for photons of different qualities in radiation therapy]. / Biológiai dózisbecslés a sugárterápiában alkalmazott különbözo sugárminoségu fotonnyaláboknál.

Flattening filter free mode (FFF) has been introduced in radiotherapy during the past decades, however, not much has been reported on its radiobiological effect. The purpose of our study was to compare the radiobiological effects of flattening filter and flattening filter free photon beams on chromosomal aberrations in peripheral blood lymphocytes. In our study the blood of the same healthy donor was irradiated with linear accelerator using both conventional flattening filter (FF) and FFF photon beams at dose rate of 3.57-23.08 Gy/min, using 6 or 10 MV. The dose-response calibration curves for dicentric + ring chromosomes induced by irradiation were fitted with linear-quadratic model. CABAS (Chromosomal Aberration Calculation Software) was used to prepare the curves. The coefficients and equations of the curves were calculated and compared with the results of other authors. We found significant differences in the number of aberrations at different irradiation parameters. Based on our results, FFF mode has a 10-20% higher biological effect than FF mode. These results can be used during radiotherapy or to estimate the biological doses in case of an accidental exposure to radiation.

[Mutagen sensitivity and risk of second cancer in young patients with head and neck squamous cell cancer]. / Mutagénérzékenység és második primer daganat kialakulása fiatal fej-nyaki laphámrákos betegeknél.

Head and neck cancer patients are at high risk for secondary primary cancer (SPC) development. Mutagen hypersensitivity may be associated with elevated risk of SPC. A survey was made of SPC among 124 young (≤50 years) patients with squamous cell carcinoma of the head and neck who were enrolled in a pretreatment mutagen sensitivity investigation during 1996-2006. Mutagen sensitivity was assessed by exposing lymphocytes to bleomycin in vitro and quantitating the bleomycin-induced chromatid breaks per cell (b/c). Patients were classified as hypersensitive (>1 b/c) or not hypersensitive (≤1 b/c). The mean follow-up time was 64 months (range: 5-244 months). Eighteen patients (15%) developed a SPC. The 10-year estimated rate of SPC for hypersensitive (n=65) or not hypersensitive (n=59) patients were 17% and 30%, respectively (p=0.4272). Thirty-nine percent of SPC was developed after 10-year follow-up. The 5-year cancer-specific survival was 17% following the development of SPC. According to our findings, mutagen hypersensitivity does not increase the risk of developing SPC.

Chromosomal Aberrations in Blood Lymphocytes as Predictors of Respiratory Function After Stereotactic Lung Irradiation.

Due to the profound difference in radiosensitivity of patients and various side effects caused by this phenomenon, a radiosensitivity marker is needed. Prediction by a marker may help personalise the treatment. In this study, we tested chromosomal aberrations (CA) of in vitro irradiated blood as predictor of pulmonary function decrease of nonsmall cell lung cancer (NSCLC) patients and also compared it with the CAs in the blood of irradiated patients. Peripheral blood samples were taken from 45 lung cancer patients before stereotactic radiotherapy (SBRT) and immediately after the last fraction and 3, 6, 9, 12, 15, 18, 21, and 24 months later. Respiratory function measurements were performed at the same time. Diffusing capacity of lung for carbon monoxide (DLCO), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1s), and FEV1s/FVC (FEV1%) were monitored. Metaphase preparations of lymphocytes were made with standard procedures, and chromosome aberrations were analysed. In our cohort, the 36-month local relapse-free survival was 97.4%, and the distant metastasis-free survival was 71.5% at 36 months. There was no change in the mean of the pulmonary function tests (PFTs) after the therapy. However, there was a considerable variability between the patients. Therefore, we subtracted the baseline and normalised the PFT values. There were significant decreases at 12-24 months in relative FEV1s and relative FEV1%. The tendentious decrease of the PFTs could be predicted by the in vitro chromosome aberration data. We also found connections between the in vitro and in vivo CA values (i.e., dicentrics plus rings after 3 Gy irradiation predicts dicentric-plus-ring value directly after the radiotherapy/V54 Gy (p = 0.001 24.2%)). We found that-after further validation-chromosome aberrations resulted from in vitro irradiation before radiotherapy can be a predictive marker of pulmonary function decrease after lung irradiation.

A scanning and image processing system with integrated design for automated micronucleus scoring.

Purpose: Our aim was to design a compact and cost-effective optical microscopic system for automated non-fluorescent micronucleus (MN) scoring whose performance can reach the accuracy of visual scoring with the help of minimal user interaction and also gives an option for fully automatic scoring with an accuracy suitable for triage purposes.Materials and methods: The concept of Radometer MN-Series (RS-MN) microscopic system designed by Radosys was to develop hardware and software layers in parallel in order to optimize the performance in automated MN scoring. A MN assay slide is automatically scanned by the RS-MN then the binucleated cells and micronuclei are automatically identified. Processing 1000 cells takes 10-60 minutes by automatic scoring (scanning plus image processing) depending on sample quality and required accuracy. The manual revision of the cell gallery takes an extra 10 minutes per sample. Dose response curves are determined for manual, automatic and semi-automatic scoring methods.Results: The combination of object-sensitive autofocusing method and the multi-layer image acquisition is able to reduce the minimum resolvable dose by 14%. The MN yields obtained from the manual, semi-automatic and automatic scoring methods are well correlating (Pearson's correlation coefficients are between 0.977 and 0.998). In order to compare the reliability of the results of visual and automatic scoring, an extended analysis on uncertainty contributors was performed. For a dose of 1 Gy, the estimated relative uncertainty from the Poisson characteristics of MN yield is 17-19% for the manual and 20-38% for the automated scoring. Other uncertainty factors (differences in donor radiosensitivity, scorer performance, and sample preparations) can contribute to this error fall within a similar range: 3-16%. Taking into account all of the possible uncertainties, the minimum resolvable dose for the manual (0.48 Gy) is the two-thirds of that of the automatic scoring (0.61 Gy).Conclusions: The results verify that the fully automatic mode of RS-MN is suitable for triage purposes. The performance of the user interacted semi-automatic mode is comparable with the reference manual scoring. Its performance reaches up to other non-fluorescent automatic systems and offers a compact and cost-effective alternative.

Quantifying biological effects of radiation from high-energy linear accelerators on lymphocytes.

The aim of this study was to investigate the radiobiological effects of flattening filter (FF) and flattening filter-free (FFF) modes of linear electron accelerators and to understand whether there is any difference between the effects of these modes. We evaluated the number of chromosome aberrations following irradiation of lymphocytes from healthy volunteers with X-ray photons at two energy levels, 6 and 10 MV; the dose rate ranged between 5.50 and 23.08 Gy/min and absorbed doses ranged between 0.5 and 8 Gy. A 60Co curve was employed for comparison. Metaphases from the lymphocyte cultures were prepared using standard cytogenetic techniques and chromosome analysis was performed. Our results allow the performance of biodosimetry at higher energies and doses than the currently used reference dosimetry. We observed significant differences in aberration frequencies when different irradiation techniques were used. FFF mode has a higher radiobiological effect than the FF mode. Linear-quadratic dose response calibration curves were constructed and relative biological effectiveness (RBE) values were calculated. Average RBE values using 6 MV (5.50 Gy/min) as a reference radiation were 1.28 for 60Co γ irradiation, 1.11 for 6 FFF and 0.79-0.92 for 10 FFF. Since there are compelling differences between radiation modalities in cases of hypofractionation, these results may be even more important in a therapeutic situation. In case of an accidental overdose of a patient, use of the appropriate calibration curves for biodosimetry are also essential for quantifying the overdose.

Radiotherapy-Induced Changes in the Systemic Immune and Inflammation Parameters of Head and Neck Cancer Patients.

Though radiotherapy is a local therapy, it has systemic effects mainly influencing immune and inflammation processes. This has important consequences in the long-term prognosis and therapy individualization. Our objective was to investigate immune and inflammation-related changes in the peripheral blood of head and neck cancer patients treated with radiotherapy. Peripheral blood cells, plasma and blood cell-derived RNA were isolated from 23 patients before and at two time points after radiotherapy and cellular immune parameters, plasma protein changes and gene expression alterations were studied. Increased regulatory T cells and increased CTLA4 and PD-1 expression on CD4 cells indicated an immune suppression induced by the malignant condition, which was accentuated by radiotherapy. Circulating dendritic cells were strongly elevated before treatment and were not affected by radiotherapy. Decreased endoglin levels in the plasma of patients before treatment were further decreased by radiotherapy. Expression of the FXDR, SESN1, GADD45, DDB2 and MDM2 radiation-response genes were altered in the peripheral blood cells of patients after radiotherapy. All changes were long-lasting, detectable one month after radiotherapy. In conclusion we demonstrated radiotherapy-induced changes in systemic immune parameters of head and neck cancer patients and proposed markers suitable for patient stratification worth investigating in larger patient cohorts.

Progressive breast fibrosis caused by extreme radiosensitivity: Oncocytogenetic diagnosis and treatment by reconstructive flap surgery.

BACKGROUND: Fibrosis, a proliferative response of fibrocytes after tissue injury, is a common sequela of external radiotherapy and can vary greatly among patients even in the absence of DNA repair syndromes, due to their different intrinsic radiosensitivity. Fibrosis is also a serious cosmetic problem for patients, and in some cases it can also imply pain. CASE: Here, we report a case of a severe fibrosis 2 years after breast conserving surgery and postoperative 3D conformal breast irradiation. Furthermore, our patient had the suspicion of tumour recurrence. Our examinations were aimed at diagnosing recurrence or the lack of recurrence and investigating whether the symptoms occurred due to overdosing or extreme intrinsic radiosensitivity. Therefore, examining the patients' radiosensitivity, a cytogenetic test was performed, which revealed the patient's increased susceptibility to ionizing radiation, and therefore we rejected the prospect of overdosage. As a solution for the fibrosis, mastectomy was effectuated, and a latissimus dorsi musculocutaneous flap was used for reconstruction. CONCLUSIONS: We suggest a multi-disciplinary approach to manage fibrosis and propose cytogenetic markers to be used as predictors to identify patients who most likely benefit from a certain therapeutic regimen in terms of reduction of therapy-related side effects.

Relationship between spontaneous frequency of aneuploidy and cancer risk in 2145 healthy Hungarian subjects.

Numerical and structural chromosomal abnormalities are the hallmarks of cancer. Whereas the structural chromosome aberrations got more substantial attention for cancer risk assessment in a healthy population, the role of aneuploidy is much less understood in this respect. We analysed the frequency of numerical (and structural) aberrations in peripheral blood lymphocytes of 2145 healthy individuals between 1989 and 2010, taking into account different biological- and exposure-conditions. We also studied to what extent chromosome gains or losses may predict the probability of cancer. The average frequency of all aneuploid cells was 1.78±0.06% in the entire study population, which increased linearly with age. Gender and smoking did not influence the values, however, occupational exposures did. The highest frequency of aneuploidy was found in chemical industry-workers (1.89±0.05%) compared with the lowest value of medical radiation workers (1.44±0.10%), respectively. No correlation was found between numerical and structural chromosomal aberrations. Cancer incidence followed for 1-23 years after the chromosome analysis showed a 1.26-fold relative risk (confidence interval: 1.02-1.58; P = 0.04) for those with higher frequency of aneuploid cells (1.82% vs. 1.44% in controls). Hypodiploidy had higher impact on the cancer risk than hyperdiploidy (1.72% vs. 0.10%). Our findings on the frequency of numerical aberrations in a healthy cohort represent the largest cytogenetic database from one laboratory with an unchanged mechanistic scoring method during a 30-year period, and provide basic information not only for genotoxicological studies but also confirm the association between numerical aberrations and cancer risk.

Recent adaptations of fluorescence techniques for the determination of mechanistic parameters of helicases and translocases.

Helicases and translocases are nucleic acid (NA)-based molecular motors that use the free energy liberated during the nucleoside triphosphate (NTP, usually ATP) hydrolysis cycle for unidirectional translocation along their NA (DNA, RNA or heteroduplex) substrates. Determination of the kinetic and thermodynamic parameters of their mechanoenzymatic cycle serves as a basis for the exploration of their physiological behavior and various cellular functions. Here we describe how recent adaptations of fluorescence-based solution kinetic methods can be used to determine practically all important mechanistic parameters of NA-based motor proteins. We outline practically useful analysis procedures for equilibrium, steady-state and transient kinetic data. This analysis can be used to quantitatively characterize the enzymatic steps of the NTP hydrolytic cycle, the binding site size, stoichiometry and energetics of protein-NA interactions, the rate and processivity of translocation along and unwinding of NA strands, and the mechanochemical coupling between these processes. The described methods yield insights into the functional role of the enzymes, and also greatly aid the design and interpretation of single-molecule experiments as well as the engineering of enzymatic properties for biotechnological applications.

A nucleotide-dependent and HRDC domain-dependent structural transition in DNA-bound RecQ helicase.

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.