Subclassification of patients with acute myelogenous leukemia based on chemokine responsiveness and constitutive chemokine release by their leukemic cells

Background and Objectives Chemokines are soluble mediators involved in angiogenesis, cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells.Design and Methods Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation, colony formation), chemokine receptor expression, constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells.Results Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors, but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression, but, compared to CD34− AML cells, CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore, a broad constitutive chemokine release profile was detected for most patients, and the following chemokine clusters could be identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFκB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased.Interpretation and Conclusions Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile

A Pilot Study of Circulating Monocyte Subsets in Patients Treated with Stem Cell Transplantation for High-Risk Hematological Malignancies.

Background and Objectives: Autologous and allogeneic stem cell transplantation is used in the treatment of high-risk hematological malignancies, and monocytes are probably involved in hematological reconstitution as well as posttransplant immunoregulation. The aim of our study was to investigate the levels of circulating monocyte subsets in allotransplant recipients. Materials and Methods: The levels of the classical, intermediate, and nonclassical monocyte subsets were determined by flow cytometry. Sixteen patients and 18 healthy controls were included, and the levels were analyzed during pretransplant remission (n = 13), early posttransplant during cytopenia (n = 9), and early reconstitution (n = 9). Results: Most patients in remission showed a majority of classical monocytes. The patients showed severe early posttransplant monocytopenia, but the total peripheral blood monocyte counts normalized very early on, and before neutrophil and platelet counts. During the first 7–10 days posttransplant (i.e., during cytopenia) a majority of the circulating monocytes showed a nonclassical phenotype, but later (i.e., 12–28 days posttransplant) the majority showed a classical phenotype. However, the variation range of classical monocytes was wider for patients in remission and during regeneration than for healthy controls. Conclusions: The total peripheral blood monocyte levels normalize at the very early stages and before neutrophil reconstitution after stem cell transplantation, and a dominance of classical monocytes is reached within 2–4 weeks posttransplant

Circulating monocyte subsets in multiple myeloma patients receiving autologous stem cell transplantation – a study of the preconditioning status and the course until posttransplant reconstitution for a consecutive group of patients

Background Induction therapy of multiple myeloma patients prior to autologous stem cell transplantation has changed from conventional chemotherapy to treatment based on proteasome inhibitors or immunomodulatory drugs. We used flow cytometry to analyze total monocyte and monocyte subset (classical, intermediate and non-classical monocytes) peripheral blood levels before and following auto-transplantation for a consecutive group of myeloma patients who had received the presently used induction therapy. Results The patients showed normal total monocyte concentrations after induction/stem cell mobilization, but the concentrations of classical monocytes were increased compared with healthy controls. Melphalan conditioning reduced the levels of total CD14+ as well as classical and non-classical monocytes, whereas intermediate monocytes were not affected. Thus, melphalan has a non-random effect on monocyte subsets. Melphalan had a stronger effect on total and classical monocyte concentrations for those patients who had received induction therapy including immunomodulatory drugs. Total monocytes and monocyte subset concentrations decreased during the period of pancytopenia, but monocyte reconstitution occurred before hematopoietic reconstitution. However, the fractions of various monocyte subsets varied considerably between patients. Conclusions The total level of circulating monocytes is normalized early after auto-transplantation for multiple myeloma, but pre- and post-transplant levels of various monocyte subsets show considerable variation between patients

A Pilot Study of Circulating Monocyte Subsets in Patients Treated with Stem Cell Transplantation for High-Risk Hematological Malignancies.

Background and Objectives: Autologous and allogeneic stem cell transplantation is used in the treatment of high-risk hematological malignancies, and monocytes are probably involved in hematological reconstitution as well as posttransplant immunoregulation. The aim of our study was to investigate the levels of circulating monocyte subsets in allotransplant recipients. Materials and Methods: The levels of the classical, intermediate, and nonclassical monocyte subsets were determined by flow cytometry. Sixteen patients and 18 healthy controls were included, and the levels were analyzed during pretransplant remission (n = 13), early posttransplant during cytopenia (n = 9), and early reconstitution (n = 9). Results: Most patients in remission showed a majority of classical monocytes. The patients showed severe early posttransplant monocytopenia, but the total peripheral blood monocyte counts normalized very early on, and before neutrophil and platelet counts. During the first 7–10 days posttransplant (i.e., during cytopenia) a majority of the circulating monocytes showed a nonclassical phenotype, but later (i.e., 12–28 days posttransplant) the majority showed a classical phenotype. However, the variation range of classical monocytes was wider for patients in remission and during regeneration than for healthy controls. Conclusions: The total peripheral blood monocyte levels normalize at the very early stages and before neutrophil reconstitution after stem cell transplantation, and a dominance of classical monocytes is reached within 2–4 weeks posttransplant

Immunomodulatory Drugs Alter the Metabolism and the Extracellular Release of Soluble Mediators by Normal Monocytes.

Immunomodulatory drugs (IMiDs) are used in the treatment of hematological malignancies, especially multiple myeloma. IMiDs have direct anticancer effects but also indirect effects via cancer-supporting stromal cells. Monocytes are a stromal cell subset whose metabolism is modulated by the microenvironment, and they communicate with neighboring cells through extracellular release of soluble mediators. Toll-like receptor 4 (TLR4) is then a common regulator of monocyte metabolism and mediator release. Our aim was to investigate IMiD effects on these two monocyte functions. We compared effects of thalidomide, lenalidomide, and pomalidomide on in vitro cultured normal monocytes. Cells were cultured in medium alone or activated by lipopolysaccharide (LPS), a TLR4 agonist. Metabolism was analyzed by the Seahorse XF 96 cell analyzer. Mediator release was measured as culture supernatant levels. TLR4 was a regulator of both monocyte metabolism and mediator release. All three IMiDs altered monocyte metabolism especially when cells were cultured with LPS; this effect was strongest for lenalidomide that increased glycolysis. Monocytes showed a broad soluble mediator release profile. IMiDs decreased TLR4-induced mediator release; this effect was stronger for pomalidomide than for lenalidomide and especially thalidomide. To conclude, IMiDs can alter the metabolism and cell–cell communication of normal monocytes, and despite their common molecular target these effects differ among various IMiDs

p53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML

p53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML

Knockdown of Human Nα-Terminal Acetyltransferase Complex C Leads to p53-Dependent Apoptosis and Aberrant Human Arl8b Localization▿

Protein Nα-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes, NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex, containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes, and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo. Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability in human cells

Acute response in circulating microRNAs following a single bout of short-sprint and heavy strength training in well-trained cyclists

Background: Heavy strength (HS) and short-sprint (SS) are commonly used training methods for competitive road cyclists, with the aim to improve the anaerobic power and short time cycling performance. Knowledge of how such training methods affects biochemical as well as molecular factors, are particularly important for determining individual recovery and long-term adaptations. The primary aim of the current study was to investigate the expression levels of small non-coding RNAs in response to HS and SS training in elite cyclists as potential biomarkers for individual optimal restitution time.Methods: Eleven well trained cyclists performed one session of HS training and one session of SS training on separate days. Blood samples were taken at baseline and 5 min, 1 h and 21 h post training. Along with physiological measurements and biochemical factors (serum creatine kinase, myoglobin, human growth hormone and plasma lactate), real-time quantitative PCR was used to explore whether HS and/or SS training influenced the abundance of 24 circulating miRNAs, in serum, associated with muscle development, angiogenesis, and/or inflammation.Results: Based on complete miRNA profiles from nine cyclists, the miRNAs showing most altered expression after both training sessions included the three striated muscle-specific miRNAs (myomiRs) miR-1-3p, 133a-3p and 133b-3p. While all three miRNAs showed significantly highest expression at 1 h post HS session, the acute effect of the SS session included a significantly higher level of miR-1-3p alone, at 5 min (highest), as well as at 1 h and 21 h post session. Correlation (negative) with biochemical markers was only shown for miR-133a-3p and CK (r = −0.786, p = 0.041) and between miR-133b-3p and [La−] (r = −0.711, p = .032), at 21 h post SS session.Conclusion: Our findings support that unique myomiRs are regulated by HS and SS training. Such knowledge may be important for individually adjusted restitution times