70 research outputs found
Evolving DNA methylation and gene expression markers of B-cell chronic lymphocytic leukemia are present in pre-diagnostic blood samples more than 10 years prior to diagnosis
Background
B-cell chronic lymphocytic leukemia (CLL) is a common type of adult leukemia. It often follows an indolent course and is preceded by monoclonal B-cell lymphocytosis, an asymptomatic condition, however it is not known what causes subjects with this condition to progress to CLL. Hence the discovery of prediagnostic markers has the potential to improve the identification of subjects likely to develop CLL and may also provide insights into the pathogenesis of the disease of potential clinical relevance.
Results
We employed peripheral blood buffy coats of 347 apparently healthy subjects, of whom 28 were diagnosed with CLL 2.0–15.7 years after enrollment, to derive for the first time genome-wide DNA methylation, as well as gene and miRNA expression, profiles associated with the risk of future disease. After adjustment for white blood cell composition, we identified 722 differentially methylated CpG sites and 15 differentially expressed genes (Bonferroni-corrected p < 0.05) as well as 2 miRNAs (FDR < 0.05) which were associated with the risk of future CLL. The majority of these signals have also been observed in clinical CLL, suggesting the presence in prediagnostic blood of CLL-like cells. Future CLL cases who, at enrollment, had a relatively low B-cell fraction (<10%), and were therefore less likely to have been suffering from undiagnosed CLL or a precursor condition, showed profiles involving smaller numbers of the same differential signals with intensities, after adjusting for B-cell content, generally smaller than those observed in the full set of cases. A similar picture was obtained when the differential profiles of cases with time-to-diagnosis above the overall median period of 7.4 years were compared with those with shorted time-to-disease. Differentially methylated genes of major functional significance include numerous genes that encode for transcription factors, especially members of the homeobox family, while differentially expressed genes include, among others, multiple genes related to WNT signaling as well as the miRNAs miR-150-5p and miR-155-5p.
Conclusions
Our findings demonstrate the presence in prediagnostic blood of future CLL patients, more than 10 years before diagnosis, of CLL-like cells which evolve as preclinical disease progresses, and point to early molecular alterations with a pathogenetic potential
An Active Site Aromatic Triad in Escherichia coli DNA Pol IV Coordinates Cell Survival and Mutagenesis in Different DNA Damaging Agents
DinB (DNA Pol IV) is a translesion (TLS) DNA polymerase, which inserts a
nucleotide opposite an otherwise replication-stalling
N2-dG lesion in vitro, and
confers resistance to nitrofurazone (NFZ), a compound that forms these lesions
in vivo. DinB is also known to be part of the cellular
response to alkylation DNA damage. Yet it is not known if DinB active site
residues, in addition to aminoacids involved in DNA synthesis, are critical in
alkylation lesion bypass. It is also unclear which active site aminoacids, if
any, might modulate DinB's bypass fidelity of distinct lesions. Here we
report that along with the classical catalytic residues, an active site
“aromatic triad”, namely residues F12, F13, and Y79, is critical for
cell survival in the presence of the alkylating agent methyl methanesulfonate
(MMS). Strains expressing dinB alleles with single point
mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains
show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in
addition to its role in TLS, modulates DinB's accuracy in bypassing
distinct lesions. The high bypass fidelity of prevalent alkylation lesions is
evident even when the DinB active site performs error-prone NFZ-induced lesion
bypass. The analyses carried out with the active site aromatic triad suggest
that the DinB active site residues are poised to proficiently bypass distinctive
DNA lesions, yet they are also malleable so that the accuracy of the bypass is
lesion-dependent
Dietary acrylamide intake and risk of breast cancer in the UK women's cohort
No studies to date have demonstrated a clear association with breast cancer risk and dietary exposure to acrylamide.Methods:A 217-item food frequency questionnaire was used to estimate dietary acrylamide intake in 33,731 women aged 35-69 years from the UK Women's Cohort Study followed up for a median of 11 years
INVIVO FORMATION AND REPAIR OF O-6-METHYLGUANINE IN HUMAN-LEUKOCYTE DNA AFTER INTRAVENOUS EXPOSURE TO DACARBAZINE
Blood leukocyte DNA obtained from 11 Hodgkin’s disease patients
undergoing ABVD chemotherapy was analysed for the presence of the
precarcinogenic adduct O6-methyl-guanine (O6-meG) at various times (1-2
h up to 49 h) after i.v. treatment with the methylating drug
dacarbazine. Adduct formation was detected in all but one of the
patients examined at levels ranging up to 0.45 fmol/mu-g DNA (7.2 x
10(-7) mol/mol guanine). The levels of the adduct decreased by
approximately 30% over the 24 h following exposure and were usually not
detectable 49 h after exposure. In five out of seven individuals
examined after more than one treatment, consistent methylation responses
were noted, while in the remaining two cases the responses were mixed.
No correlation between the extent of adduct formation and lymphocyte
levels of the repair enzyme O6-alkylguanine - DNA alkyltransferase was
observed. The average extent of O6-meG formation 1 h after dacarbazine
treatment was (4.3 +/- 3.1) x 10(-2) fmol/mu-g DNA per mg/kg dose
[(1.2 +/- 0.8) x 10(-3 fmol/mu-g DNA per mg/m2 dose)]. Following
exposure of rats to similar doses of dacarbazine, the corresponding
levels of adduct in blood leukocyte DNA were 1.1 x 10(-2) fmol/mu-g DNA
per mg/kg dose (2.6 x 10(-3) fmol/mu-g DNA per mg/m2 dose)
Intra- and intercellular variations in the repair efficiency of O-6-methylguanine, and their contribution to kinetic complexity
Following administration to rats of various doses of
N-nitrosodimethylamine (NDMA), O-6-methylguanine (O-6-meG) was lost from
the DNA of four tissues (liver, white blood cells, lymph nodes, bone
marrow) over two, sharply demarcated phases with substantially differing
repair rates. Repair during each phase followed approximately
first-order kinetics in O-6-meG, even after a high dose of NDMA which
caused substantial depletion of O-6-alkylguanine-DNA alkyltransferase
(AGT), a suicide repair protein. This is compatible with
rate-determining adduct repair being brought about by a distinct, minor
pool of AGT molecules which is rapidly replenished by de novo AGT
synthesis. Similar biphasic repair kinetics were also observed in HepG2
cells treated in vitro with NDMA. In this case, the first phase of
repair was inhibited by alpha-amanitin, an inhibitor of RNA polymerase
II-mediated transcription. However, no dependence on transcriptional
activity was found when O-6-meG repair in specific gene sequences with
different transcriptional status in rat liver was examined, suggesting
that the effects of a-amanitin in HepG2 cells did not reflect inhibition
of preferential repair of transcribed sequences.
Repair was also examined in rat liver hepatocytes and non-parenchymal
cells separately after administration of NDMA at non-AGT depleting
doses. Within each cell-population, the repair followed single phase,
first-order kinetics, with adduct loss from AGT-rich hepatocytes being
significantly faster than from the relatively AGT-deficient
non-parenchymal cells.
In conclusion, differences in the AGT content of different cell
subpopulations in the liver (and probably in other tissues), as well as
additional cellular factors affecting repair efficiency, appear to
determine the observed variation in the kinetics of repair of O-6-meG.
The additional cellular factors involved appear not to be related to the
transcriptional state of the sequences being repaired, but may reflect
different states of chromatin condensation. (C) 2004 Elsevier B.V. All
rights reserved
Ubiquitous presence of O-6-methylguanine in human peripheral and cord blood DNA
O6-Methylguanine (O6-meG) is a powerful premutagenic lesion that can arise from exposure to methylating agents. Although it has been reported to occur in human DNA, no systematic epidemiological analysis of its occurrence in populations suffering general environmental exposure is available. We report here results from a study of the presence of O6-meG in maternal and cord blood leukocyte DNA of women not knowingly exposed to methylating agents. Using a modification of an already existing method capable of detecting the lesion at levels as low as 16 nmol/molG, the adduct was detected in 31 of 36 maternal and 30 of 36 cord samples, at levels ranging up to 192 nmol/molG. Adduct levels in maternal blood DNA were significantly higher than those in cord blood DNA (P < 0.05), and there was a strong correlation between adduct levels in the two tissues (P < 0.001). In bivariate analysis, no significant association of adduct levels in either tissue and residence air pollution, active and passive smoking status, or eating habits was found. However, intake of fruits/vegetables and of vitamin supplements showed nonstatistically significant trends toward being associated with lower adduct levels in both maternal and cord blood DNA. The same trend was observed after multivariate analysis where all the above variables were controlled for. These findings indicate that premutagenic methylation DNA damage is commonplace in individuals not known to have suffered excessive exposure to environmental methylating agents or their precursors and are compatible with an endogenous origin of this damage, possibly associated with endogenous nitrosation processes
COMPARATIVE DOSIMETRY OF O-6-METHYLGUANINE IN HUMANS AND RODENTS TREATED WITH PROCARBAZINE
The accumulation of O-6-methylguanine (O-6-meG) in the DNA of blood
leukocytes of 21 Hodgkin’s lymphoma patients (followed for up to 12
cycles of treatment) treated in the context of MOPP combination
chemotherapy with 150 mg procarbazine daily for 10 days was examined and
compared to that observed in rats treated with different doses of
procarbazine as a single agent once per day for 10 days. In humans, the
adduct accumulated in a dose-related fashion and appeared to approach a
steady-state after 7-8 days of treatment. Adduct levels on day 10 of the
treatment cycle averaged 0.25 +/- 0.09 (mean +/- SD) mu mol/ molG and,
for different individuals, covered a 3-fold range. Intra-individual
variability between different treatment cycles was much more limited
than inter-individual variability, the two parameters accounting for
8.9% and 84.5% respectively of adduct variance at a constant
cumulative dose. Comparison of the dose-response relationships for
humans and rats indicates that, under conditions of no depletion of
O-6-alkylguanine-DNA alkyltransferase (AGT), O-6-meG accumulates in
blood leukocyte DNA of humans at a rate which is only approximately
2-fold lower than in rats, implying that, to the extent to which O-6-meG
contributes to the genotoxic activity of procarbazine, human
susceptibility to it is likely to be comparable to that of the rat. This
is likely to be true also of the bone marrow (the tissue of interest as
a target tissue for leukaemogenesis), since the tissue distribution of
O-6-meG induced by low doses of procarbazine in rats, mice and rabbits
indicated that blood leukocyte levels of this adduct closely reflect
those in the bone marrow. Based on these results, it is estimated that
by the end of a MOPP chemotherapy cycle O-6-meG reaches levels of the
order of 0.2-0.3 fmol/mu g DNA (0.3-0.5 mu mol/molG) in human bone
marrow (the target tissue of leukaemogenesis observed after such
treatment)
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