The differentiation/retrodifferentiation program of human U937 leukemia cells is accompanied by changes of VCP/p97

<p>Abstract</p> <p>Background</p> <p>Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.</p> <p>Results</p> <p>Separation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G₀/G₁cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G₀/G₁ growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.</p> <p>Conclusion</p> <p>The findings demonstrated that monocytic differentiation and G₀/G₁growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.</p

Lenalidomide in the context of complex karyotype or interrupted treatment: case reviews of del(5q)MDS patients with unexpected responses

Lenalidomide has particular activity in patients with transfusion-dependent del(5q) myelodysplastic syndromes (MDS), but mechanistic information is limited regarding the relationship between erythroid and cytogenetic responses. We reviewed medical records from three distinct subgroups of del(5q) MDS patients who had unexpected effects with lenalidomide treatment: 1. two patients with complex karyotypes who achieved both cytogenetic remissions and transfusion independence; 2. two patients with 5q- syndrome who took lenalidomide for less than 12 weeks but remained transfusion independent for 15+ months still displaying del(5q) metaphases after 6 and 12 months; and 3. one patient who was a non-responder on lenalidomide during treatment but became transfusion independent for 13+ months after discontinuation. All but the latter patient in this series had reduction of affected metaphases, suggesting that erythroid responses might be mediated by result from partial or complete suppression of the malignant clone, either directly or indirectly through modulation of the bone marrow microenvironment. These clinical observations illustrate the heterogeneity of del(5q)MDS pathogenesis and the diversity of lenalidomide responses within this patient subset

Analysis of Array-CGH Data Using the R and Bioconductor Software Suite

Background. Array-based comparative genomic hybridization (array-CGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs) are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data. Results. We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection, and comparative analysis of array-CGH data which allows the accurate and sensitive detection of CNAs. Conclusion. The implemented option for the determination of minimal altered regions (MARs) from a series of tumor samples is a step forward in the identification of new tumor suppressor genes or oncogenes

Polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and susceptibility to pediatric acute lymphoblastic leukemia in a German study population

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) has a major impact on the regulation of the folic acid pathway due to conversion of 5,10-methylenetetrahydrofolate (methylene-THF) to 5-methyl-THF. Two common polymorphisms (677C>T and 1298A>C) in the gene coding for MTHFR have been shown to reduce MTHFR enzyme activity and were associated with the susceptibility to different disorders, including vascular disease, neural tube defects and lymphoid malignancies. Studies on the role of these polymorphisms in the susceptibility to acute lymphoblastic leukemia (ALL) led to discrepant results. METHODS: We retrospectively evaluated the association of the MTHFR 677C>T and 1298A>C polymorphisms with pediatric ALL by genotyping a study sample of 443 ALL patients consecutively enrolled onto the German multicenter trial ALL-BFM 2000 and 379 healthy controls. We calculated odds ratios of MTHFR genotypes based on the MTHFR 677C>T and 1298A>C polymorphisms to examine if one or both of these polymorphisms are associated with pediatric ALL. RESULTS: No significant associations between specific MTHFR variants or combinations of variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement. CONCLUSION: Our findings suggest that the MTHFR 677C>T and 1298A>C gene variants do not have a major influence on the susceptibility to pediatric ALL in the German population

Systematic genetic analysis of pediatric patients with autoinflammatory diseases

Monogenic autoinflammatory diseases (AID) encompass a growing group of inborn errors of the innate immune system causing unprovoked or exaggerated systemic inflammation. Diagnosis of monogenic AID requires an accurate description of the patients’ phenotype, and the identification of highly penetrant genetic variants in single genes is pivotal. We performed whole exome sequencing (WES) of 125 pediatric patients with suspected monogenic AID in a routine genetic diagnostic setting. Datasets were analyzed in a step-wise approach to identify the most feasible diagnostic strategy. First, we analyzed a virtual gene panel including 13 genes associated with known AID and, if no genetic diagnosis was established, we then analyzed a virtual panel including 542 genes published by the International Union of Immunological Societies associated including all known inborn error of immunity (IEI). Subsequently, WES data was analyzed without pre-filtering for known AID/IEI genes. Analyzing 13 genes yielded a definite diagnosis in 16.0% (n = 20). The diagnostic yield was increased by analyzing 542 genes to 20.8% (n = 26). Importantly, expanding the analysis to WES data did not increase the diagnostic yield in our cohort, neither in single WES analysis, nor in trio-WES analysis. The study highlights that the cost- and time-saving analysis of virtual gene panels is sufficient to rapidly confirm the differential diagnosis in pediatric patients with AID. WES data or trio-WES data analysis as a first-tier diagnostic analysis in patients with suspected monogenic AID is of limited benefit

Prevalence of pathogenic BRCA1/2 germline mutations among 802 women with unilateral triple-negative breast cancer without family cancer history

Background: There is no international consensus up to which age women with a diagnosis of triple-negative breast cancer (TNBC) and no family history of breast or ovarian cancer should be offered genetic testing for germline BRCA1 and BRCA2 (gBRCA) mutations. Here, we explored the association of age at TNBC diagnosis with the prevalence of pathogenic gBRCA mutations in this patient group. Methods: The study comprised 802 women (median age 40 years, range 19–76) with oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2 negative breast cancers, who had no relatives with breast or ovarian cancer. All women were tested for pathogenic gBRCA mutations. Logistic regression analysis was used to explore the association between age at TNBC diagnosis and the presence of a pathogenic gBRCA mutation. Results: A total of 127 women with TNBC (15.8%) were gBRCA mutation carriers (BRCA1: n = 118, 14.7%; BRCA2: n = 9, 1.1%). The mutation prevalence was 32.9% in the age group 20–29 years compared to 6.9% in the age group 60–69 years. Logistic regression analysis revealed a significant increase of mutation frequency with decreasing age at diagnosis (odds ratio 1.87 per 10 year decrease, 95%CI 1.50–2.32, p &lt; 0.001). gBRCA mutation risk was predicted to be &gt; 10% for women diagnosed below approximately 50 years. Conclusions: Based on the general understanding that a heterozygous mutation probability of 10% or greater justifies gBRCA mutation screening, women with TNBC diagnosed before the age of 50 years and no familial history of breast and ovarian cancer should be tested for gBRCA mutations. In Germany, this would concern approximately 880 women with newly diagnosed TNBC per year, of whom approximately 150 are expected to be identified as carriers of a pathogenic gBRCA mutation