RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells

Background

Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.

Result

Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.

Conclusion

Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.     

Evaluation of CD4+CD25+FoxP3+ T cell populations,IL-10 production,and their correlation with clinical and biochemical parameters in sickle cell anemia patients with leg ulcers

Leg ulcers (LUs) are a debilitating complication of sickle cell anemia (SCA), with inflammation known to play a crucial role in their pathogenesis. Many studies have described the roles of T helper type 1 (Th1) and Th2 pathways in SCA; however, defects in anti-inflammatory responses are poorly understood. We evaluated interleukin (IL)-10 levels in serum and peripheral blood mononuclear cells (PBMCs) in SCA patients with leg ulcers (SCALU) and without leg ulcers (SCAWH) in addition to CD4⁺ CD25⁺FoxP3⁺ T cell populations and their its IL-10 expression. In stimulated and unstimulated PBMC cultures, SCALU patients produced higher levels of IL-10 than those in the SCAWH group. Higher levels of IL-10 in SCALU patients correlated with a history of osteonecrosis in stimulated and unstimulated cultures when compared with those in SCAWH. Immunophenotyping revealed that SCALU patients had a higher proportion of CD4⁺CD25⁺FoxP3⁺, Tr1 and CD4⁺CD25⁺FoxP3⁺IL-10⁺ T cells than other groups. Our findings revealed that IL-10 levels were increased in unstimulated cells from the SCALU group, and that this group also presented with a predominant CD4⁺ CD25⁺FoxP3⁺ cell population despite many of those cells being IL-10 negative.     

Differential role of Kruppel like factor 1 (KLF1) gene in red blood cell disorders

The master erythroid regulator KLF1,plays a pivotal role during erythroid lineage development by regulating the expression of many erythroid genes. Variations in the KLF1 gene are found to be associated with varied erythroid phenotypes. With the aim of determining the role of KLF1 gene variations in HbF induction and their genotype phenotype relationship, in this study, we screened 370 individuals with different hemoglobinopathy condition. Hematological analysis was carried out using automated blood cell counter and Variant II HPLC (Biorad). KLF1 gene mutations were screened using automated DNA sequencing. Expression analysis was carried out using q-RT PCR of KLF1, BCL11A and γ-globin after selective enrichment and culturing of CD 34 +ve cells into an erythroid lineage. Over all 14 KLF1 gene variations were identified, of which six variants were novel. The incidence of KLF1 gene mutations was found to be 8.1%. It was seen that KLF1 mutations contributed in borderline HbA₂ levels as 7.6% of our borderline HbA₂ cases showed presence of KLF1 variations. It also contributed in induction of HbF levels under stress erythropoietic conditions. Gene expression studies revealed inverse correlation of KLF1, BCL11A (reduced) with γ-globin gene expression (increased) in patients showing KLF1 gene mutations, thus indicating the role of KLF1 gene in regulating the γ-globin gene expression. The identification of genomic variants of the KLF1 may help in determining the functionally active domain of this protein and will facilitate in understanding the wide spectrum of phenotypes generated by these variants.     

Ubiquitin‐like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway

Ubiquitin and ubiquitin‐like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre‐mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5‐deficient cells display spliceosome‐independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5–FANCI binding, we generated separation‐of‐function mutants to demonstrate that key aspects of FA pathway function, including FANCI–FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5–FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.     

Progression of central nervous system disease from pediatric to young adulthood in sickle cell anemia

Silent cerebral infarcts and arteriopathy are common and progressive in individuals with sickle cell anemia. However, most data describing brain lesions in sickle cell anemia are cross-sectional or derive from pediatric cohorts with short follow-up. We investigated the progression of silent cerebral infarct and cerebral vessel stenosis on brain MRI and MRA, respectively, by describing the incidence of new or worsening lesions over a period of up to 25 years among young adults with sickle cell anemia and explored risk factors for progression. Forty-four adults with sickle cell anemia (HbSS or HbSβ⁰thalassemia), exposed to chronic transfusions (n = 12) or hydroxyurea (n = 32), median age 19.2 years (range 18.0–31.5), received a screening brain MRI/MRA and their results were compared with a clinical exam performed during childhood and adolescence. We used exact log-rank test to compare MRI and MRA progression among any two groups. The hazard ratio (HR) and 95% confidence interval (CI) were calculated from Cox regression analyses. Progression of MRI and MRA occurred in 12 (27%) and 4 (9%) young adults, respectively, relative to their pediatric exams. MRI progression risk was high among participants with abnormal pediatric exams (HR: 11.6, 95% CI: 2.5–54.7) and conditional or abnormal transcranial Doppler ultrasound velocities (HR: 3.9, 95% CI: 1.0–15.1). Among individuals treated with hydroxyurea, high fetal hemoglobin measured in childhood was associated with lower hazard of MRI progression (HR: 0.86, 95% CI: 0.76–0.98). MRA progression occurred more frequently among those with prior stroke (HR: 8.6, 95% CI: 1.2–64), abnormal pediatric exam (P = 0.00084), and elevated transcranial Doppler ultrasound velocities (P = 0.004). Brain MRI/MRA imaging in pediatrics can identify high-risk patients for CNS disease progression in young adulthood, prompting consideration for early aggressive treatments.     

Anemia and the transition of nomadic hunter-gatherers to a sedentary life-style: Follow-up study of a Kalahari community

Iron profiles of communities of hunter-gatherers and former hunter-gatherers conducted between 1969 and 1987 at Dobe in the Kalahari Desert of Botswana exhibited pronounced differences during periods of rapid culture change. The loss of good health and particularly the increase in anemia through time was attributed to notable changes in diet, although changes in mobility patterns were considered a secondary cause. In 1988 and 1989, studies were conducted at Kutse, also in the Kalahari Desert of Botswana, to ascertain the frequency of anemia at a recently sedentary community in which residents still relied primarily on wild animals for meat. Although not identical, the hematological presentation in 1989 was similar to that in 1988. The studies together suggest that our findings characterize the pattern of health and disease at Kutse, which is unrelated to any specific year or to diet. Additional measures of disease, specifically ESR (erythrocyte sedimentation rate) and oral temperatures, support an interpretation of anemia of chronic disease as the cause of hypoferremia at Kutse. Morbidity is high, in spite of adequate diets, because the residents are transitional from a nomadic to a sedentary lifestyle and from a relatively dispersed to an aggregated settlement pattern. These changes have introduced new health problems. © 1996 Wiley-Liss, Inc.     

Echinococcus multilocularis: responses to infection in cotton rats (Sigmodon hispidus)

Various aspects of the responses of cotton rats to intraperitoneal infections with Echinococcus multilocularis were examined to determine if they could be related to the progress of the infection. At 14 weeks post-infection, infected animals had enlarged spleens; there was a slight decrease in packed cell volume, but no reticulocytosis. The number of all four types of leukocytes in the peripheral blood (lymphocytes, monocytes, neutrophils and eosinophils) increased during the course of the infection. In the peritoneal fluid, the numbers of neutrophils increased, monocytes and basophils decreased, and lymphocytes and eosinophils remained unchanged. Antibodies to E. multilocularis were detected in the serum of infected cotton rats as early as 2 weeks post-infection. The mean levels of transaminases (SGOT and SGPT) in the serum of infected animals were higher than in controls, and 5'-nucleotidase levels were elevated in heavily infected animals. There were no differences in responses between male and female animals. Comparison with results previously obtained suggest that both the outcome of the infection, and responses to it, may be under host control.     

Volume-stimulated,Cl−-dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS

Summary We report here that a Cl^–-dependent K^– (KCl) efflux, which is stimulated by N-ethylmaleimide, (NEM) and by increased red cell volume, exists in young red cells of individuals with normal hemoglobin A (AA) and in those homozygous for hemoglobin S (SS). We have investigated this KCl efflux in several density-defined red cell fractions obtained from Percoll-Stractan continuous density gradients. We found high activity of the NEM-stimulated KCl transport in reticulocytes and young red cells from nine sickle cell (SS) patients (43±27 mean±sd mmol K⁺/liter of cells/hr=flux units (FU)) and in the young cell fraction of three AA individuals with high reticulocytosis recuperating from nutritional anemias (41.7±10 FU). In addition, we observed significant interindividual variation of this KCl efflux in the discocyte fraction of SS blood. Cell swelling markedly stimulated the KCl efflux, in SS whole blood (9.8±7.4 FU, in SS young cells (13±13 FU), and in AA young cells (21.4±11 FU). The activity of the Na–K–Cl cotransport, as estimated by the bumetanide sensitive K⁺ efflux was not found to be cell-age dependent in either AA or SS cells.Measurements of red cell density by isopycnic gradients indicated that 27% of the young cells reduce their volume by a Cl^–-dependent process in hypotonic or low pH-induced swelling.The large volume-stimulated KCl efflux in AA young cells raises the possibility that these fluxes may be involved in the maturation of erythropoietic precursors. The high activity in the red cells of sickle cell anemia patients and its interindividual variation may have pathophysiological consequences since it reverses the decrease in the intracellular concentration of hemoglobin which occurs in response to low pH or osmolarity, an unwelcome pro-sickling event.