p53-Mediated downregulation of H ferritin promoter transcriptional efficiency via NF-Y

The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53–NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis. These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation

Toward optimization of postremission therapy for residual disease-positive patients with acute myeloid leukemia

Purpose:Despite the identification of several baseline prognostic indicators, the outcome of patients with acute myeloid leukemia (AML) is generally heterogeneous. The effects of autologous (AuSCT) or allogeneic stem-cell transplantation (SCT) are still under evaluation. Minimal residual disease (MRD) states may be essential for assigning patients to therapy-dependent risk categories. Patients and Methods: By multiparametric flow cytometry, we assessed the levels of MRD in 142 patients with AML who achieved complete remission after intensive chemotherapy. Results: A level of 3.5 x 10(-4) residual leukemia cells (RLCs) after consolidation therapy was established to identify MRD-negative and MRD-positive cases, with 5-year relapse-free survival (RFS) rates of 60% and 16%, respectively (P <.0001) and overall survival (OS) rates of 62% and 23%, respectively (P=.0001). Of patients (n = 77) who underwent a transplantation procedure (56 AuSCT and 21 SCT procedures); 42 patients (55%) were MRD positive (28 patients who underwent AuSCT and 14 patients who underwent SCT) and 35 patients (45%) were MRD negative (28 patients who underwent AuSCT and seven who underwent SCT). MRD-negative patients had a favorable prognosis, with only eight (22%) of 35 patients experiencing relapse, whereas 29 (69%) of 42 MRD-positive patients experienced relapse (P <.0001). In this high-risk group of 42 patients, we observed that 23 (82%) of 28 of those who underwent AuSCT experienced relapse, whereas six (43%) of 14 who underwent SCT experienced relapse (P=.014). Patients who underwent SCT also had a higher likelihood of RFS (47% v 14%). Conclusion A threshold of 3.5 x 10(-4) RLCs postconsolidation is critical for predicting disease outcome. MRD-negative patients have a good outcome regardless of the type of transplant they receive. In the MRD-positive group, AuSCT does not improve prognosis and SCT represents the primary option

Muscle Oxygen Changes following Sprint Interval Cycling Training in Elite Field Hockey Players

This study examined the effects of Sprint Interval Cycling (SIT) on muscle oxygenation kinetics and performance during the 30-15 intermittent fitness test (IFT). Twenty-five women hockey players of Olympic standard were randomly selected into an experimental group (EXP) and a control group (CON). The EXP group performed six additional SIT sessions over six weeks in addition to their normal training program. To explore the potential training-induced change, EXP subjects additionally completed 5 x 30s maximal intensity cycle testing before and after training. During these tests near-infrared spectroscopy (NIRS) measured parameters; oxyhaemoglobin + oxymyoglobin (HbO2+ MbO2), tissue deoxyhaemoglobin + deoxymyoglobin (HHb+HMb), total tissue haemoglobin (tHb) and tissue oxygenation (TSI %) were taken. In the EXP group (5.34±0.14 to 5.50±0.14m.s-1) but not the CON group (pre = 5.37± 0.27 to 5.39±0.30m.s-1) significant changes were seen in the 30-15IFTperformance. EXP group also displayed significant post-training increases during the sprint cycling: ΔTSI (-7.59±0.91 to -12.16±2.70%); ΔHHb+HMb (35.68±6.67 to 69.44 ±26.48μM.cm); and ΔHbO2+ MbO2 (-74.29±13.82 to -109.36±22.61μM.cm). No significant differences were seen in ΔtHb (-45.81±15.23 to -42.93±16.24). NIRS is able to detect positive peripheral muscle oxygenation changes when used during a SIT protocol which has been shown to be an effective training modality within elite athletes

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

Introduction: Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods: We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results: Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion: These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer

A Model of Brain Circulation and Metabolism: NIRS Signal Changes during Physiological Challenges

We construct a model of brain circulation and energy metabolism. The model is designed to explain experimental data and predict the response of the circulation and metabolism to a variety of stimuli, in particular, changes in arterial blood pressure, CO2 levels, O2 levels, and functional activation. Significant model outputs are predictions about blood flow, metabolic rate, and quantities measurable noninvasively using near-infrared spectroscopy (NIRS), including cerebral blood volume and oxygenation and the redox state of the CuA centre in cytochrome c oxidase. These quantities are now frequently measured in clinical settings; however the relationship between the measurements and the underlying physiological events is in general complex. We anticipate that the model will play an important role in helping to understand the NIRS signals, in particular, the cytochrome signal, which has been hard to interpret. A range of model simulations are presented, and model outputs are compared to published data obtained from both in vivo and in vitro settings. The comparisons are encouraging, showing that the model is able to reproduce observed behaviour in response to various stimuli

BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20–45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC50) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC50 : 30–40 μM) compared with MCF-7 (IC50 : 60–70 μM) and MDA-MB231 (IC50 : 90–100 μM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC50 : 45–50 μM) compared with MCF-7 (IC50 : 1–5 μM) and MDA-MB231 (IC50 : 5–10 μM) (P<0.02), as well as to paclitaxel (Tax) (IC50 : >2 μM for HCC1937, 0.1–0.2 μM for MCF-7 and 0.01–0.02 μM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/WTBRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting

The G67E mutation in hMLH1 is associated with an unusual presentation of Lynch syndrome

Germline mutations in the mismatch repair (MMR) genes are associated with Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Here, we characterise a variant of hMLH1 that confers a loss-of-function MMR phenotype. The mutation changes the highly conserved Gly67 residue to a glutamate (G67E) and is reminiscent of the hMLH1-p.Gly67Arg mutation, which is present in several Lynch syndrome cohorts. hMLH1-Gly67Arg has previously been shown to confer loss-of-function (Shimodaira et al, 1998), and two functional assays suggest that the hMLH1-Gly67Glu protein fails to sustain normal MMR functions. In the first assay, hMLH1-Gly67Glu abolishes the protein's ability to interfere with MMR in yeast. In the second assay, mutation of the analogous residue in yMLH1 (yMLH1-Gly64Glu) causes a loss-of-function mutator phenotype similar to yMLH1-Gly64Arg. Despite these molecular similarities, an unusual spectrum of tumours is associated with hMLH1-Gly67Glu, which is not typical of those associated with Lynch syndrome and differs from those found in families carrying the hMLH1-Gly67Arg allele. This suggests that hMLH1 may have different functions in certain tissues and/or that additional factors may modify the influence of hMLH1 mutations in causing Lynch syndrome