Effects of PKCα activation on Ca2+ pump and K(Ca) channel in deoxygenated sickle cells

We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deoxygenation-induced K+ loss and Ca2+ uptake and prevented call dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999- 2007, 1995). The present study explores the detailed mechanism of this PMA- induced inhibition. The main findings are, first, the detection of PKCα PKCζ in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The α-isoform only is translocated to the membrane and activated by PMA and by elevation of cytosolic Ca2+. Second, PMA is demonstrated to activate Ca2+ efflux in deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly indirect and explained by the reduc

Stathmin 1 deficiency induces erythro-megakaryocytic defects leading to macrocytic anemia and thrombocythemia in Stathmin 1 knock out mice

Stathmin 1 (STMN1) is a cytosolic phosphoprotein that was discovered as a result of its high level of expression in leukemic cells. It plays an important role in the regulation of mitosis by promoting depolymerization of the microtubules that make up the mitotic spindle and, aging has been shown to impair STMN1 levels and change microtubule stability. We have previously demonstrated that a high level of STMN1 expression during early megakaryopoiesis is necessary for proliferation of megakaryocyte progenitors and that down-regulation of STMN1 expression during late megakaryopoiesis is important for megakaryocyte maturation and platelet production. In this report, we examined the effects of STMN1 deficiency on erythroid and megakaryocytic lineages in the mouse. Our studies show that STMN1 deficiency results in mild thrombocytopenia in young animals which converts into profound thrombocythemia as the mice age. STMN1 deficiency also lead to macrocytic changes in both erythrocytes and megakaryocytes that persisted throughout the life of STMN1 knock-out mice. Furthermore, STMN1 knock-out mice displayed a lower number of erythroid and megakaryocytic progenitor cells and had delayed recovery of their blood counts after chemotherapy. These studies show an important role for STMN1 in normal erythro-megakaryopoietic development and suggests potential implications for disorders affecting these hematopoietic lineages

The role of stathmin, a regulator of mitosis, in hematopoiesis

Steel, JC ORCiD: 0000-0003-3608-7542Introduction Stathmin is a 17KDa cytosolic protein that plays an important role in the regulation of microtubule dynamics, mitotic spindle formation, cell cycle progression and cell differentiation. Stathmin knockout (KO) mice were initially reported to have a normal phenotype but were subsequently shown to develop an age-related neurological phenotype with axonopathy evident in both central and peripheral nervous systems. These mice were also shown to have a defect in recovery from acute ischemic renal injury. We had previously shown that stathmin plays an important role in the differentiation and proliferation of megakaryocytes (MK) and that down-regulation of stathmin is necessary for the maturation of MK and platelet production in vitro. In this study, we investigated the role of stathmin in megakaryopoiesis and hematopoiesis in vivo using the stathmin KO mouse as an experimental model. Results Stathmin KO mice had lower platelet (PLT) counts at 3 weeks of age when compared to WT mice. The WT mice had a mean PLT count of 662 ± 27 K/μL while KO mice had a mean PLT count of 543 ± 37 K/μL. This correlated with larger and fewer MK in the bone marrow of KO mice (WT: 4.2 ± 0.7 MK/40X field; KO: 3.6 ± 0.2 MK/40X field). Furthermore, in the spleen, there was a 10 fold decrease in the number of MK in KO mice compared to WT mice (6.6 ± 0.6 vs 0.7 ± 0.1 MK/40X field). By 8 weeks, PLT counts and MK size and numbers in the bone marrow and spleen were similar in WT and KO mice. Interestingly, by 16 weeks, the mean PLT of KO mice became significantly higher than that of WT and by 40 weeks, the mean PLT count of KO mice was 1379 ± 100K/μL compared to 1045 ± 120K/μL in WT mice (P<0.05). Microscopic analysis of the bone marrow at 46 weeks of age showed approximately 50% more MK in KO mice compared to WT mice. Differences in red blood cell counts (RBC) were also observed. While at 3 weeks, there were no significant differences between the 2 groups, at 8 weeks, KO mice had significantly lower RBC counts, hemoglobin levels (Hb) and hematocrit (HCT). This trend continued until the last measurement recorded at 40 weeks. Mean RBC in WT mice was 10.5 ± 0.1M/μL compared to 8.9 ± 0.2M/μL in KO mice. The mean corpuscular volume (MCV) and the red blood cell distribution width (RDW) were consistently higher in KO mice than in WT mice. No significant differences were noted in white blood cell counts. Bone marrow cell counts were significantly lower in KO mice when compared to WT mice at different ages from 3–40 weeks. Progenitor cell assays from 10–12 week old animals have shown that bone marrow from KO mice produce significantly fewer BFU-E and Pre-B colonies while no differences were observed in CFU-GMs. Conclusions The phenotypic characteristics of stathmin KO mice confirmed our prior in vitro findings that suggested a role for stathmin in megakaryopoiesis. We expected to see a decrease in the number of platelets and MK coupled with an increase in MK size. This was confirmed in stathmin KO mice at 3 weeks of age. However, we did not expect to see the marked increase in the number of platelets and MK that was observed as the mice aged. The exact mechanism for this has not been identified. Interestingly, the stathmin KO mice exhibited characteristic features of megaloblastic anemia including mild anemia and a significant increase in MCV and RDW. The megaloblastic anemia that is seen in the presence of B12 and folate deficiency results from interference with DNA synthesis resulting in asynchronous maturation of the nucleus and the cytoplasm. We believe a similar phenomenon is occurring in the stathmin KO mice. The deficiency of stathmin results in aberrant exit from mitosis, thereby delaying nuclear maturation and resulting in the megaloblastic features. Thus, the deficiency of stathmin in the KO mice results in two hematopoietic phenotypes that are seen in humans, megaloblastic anemia and thrombocytosis. It is unclear whether mutations of stathmin in humans might result in similar phenotypes. This is a question that will require further investigation. Future studies will investigate the compensatory mechanisms that result in the switch from decreased to increased platelet production as the mice age. Furthermore, examining the effects of hematopoietic stress (e.g. response to chemotherapy or bleeding) in stathmin KO mice might also elucidate a role for stathmin in the recovery from hematopoietic injury as was seen in acute ischemic renal injury

Does Molecular Profiling of KRAS-Mutant Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Help in Treatment Strategy Planning?

Background: Several studies suggest that patients with KRAS-mutant NSCLC fail to benefit from standard systemic therapies and do not respond to EGFR inhibitors. Most recently, KRAS 12c data suggest specific treatment for improving ORR and OS. There is a clear need for therapies specifically developed for these patients. Moreover, data that might be suggestive of a response to specific therapies, such as BRCA1, are needed, and two mutations that were studied in other malignancies show more response to PARP inhibitors. Molecular profiling has the potential to identify other potential targets that may provide better treatment and novel targeted therapy for KRAS-mutated NSCLC. Methods: We purified RNA from archived tissues of patients with stage I and II NSCLC with wild-type (wt) and mutant (mt) KRAS tumors; paired normal tissue adjacent to the tumor from 20 and 17 patients, respectively, and assessed, using real-time reverse transcriptase&ndash;polymerase chain reaction (RT-PCR), the expression of four genes involved in DNA synthesis and repair, including thymidylate synthase (TS), BRCA1, ECCR1, RAP80, and the proto-oncogene SRC. Additionally, we assessed the expression of PD-L1 in mt&nbsp;KRAS tumors with immunohistochemistry using an antibody against PD-L1. Results: Our results show that in mtKRAS tumors, the level of expression of ERCC1, TS, and SRC was significantly increased in comparison to paired normal lung tissue (p &le; 0.04). The expression of BRCA1 and RAP80 was similar in both mt&nbsp;KRAS tumors and paired normal tissue. Furthermore, the expression of BRCA1, TS, and SRC was significantly increased in wt&nbsp;KRAS tumors relative to their expression in the normal lung tissue (p &lt; 0.044). The expression of ERCC1 and RAP80 was similar in wt&nbsp;KRAS tumors and paired normal tissue. Interestingly, SRC expression in mtKRAS tumors was decreased in comparison to wt&nbsp;KRAS tumors. Notably, there was an expression of PD-L1 in the tumor and stromal cells in a few (5 out of 20) mtKRAS tumors. Our results suggest that a greater ERCC1 expression in mt KRAS tumors might increase platinum resistance in this group of patients, whereas the greater expression of BRCA1 in wt&nbsp;KRAS tumor might be suggestive of the sensitivity of taxanes. Our data also suggest that the combination of an SRC inhibitor with a TS inhibitor, such as pemetrexed, might improve the outcome of patients with NSCLC and in particular, patients with wt&nbsp;KRAS tumors. PD-L1 expression in tumors, and especially stromal cells, suggests a better outcome. Conclusion: mt&nbsp;KRAS NSCLC patients might benefit from a treatment strategy that targets KRAS in combination with therapeutic agents based on pharmacogenomic markers, such as SRC and BRCA1. mtKRAS tumors are likely to be platinum-, taxane-, and pemetrexed-resistant, as well as having a low level of PD-L1 expression; thus, they are less likely to receive single-agent immunotherapy, such as pembrolizumab, as the first-line therapy. wt&nbsp;KRAS tumors with BRCA1 positivity tend to be sensitive to taxane therapy and, potentially, platinum. Our results suggest the need to develop targeted therapies for KRAS-mutant NSCLC or combine the targeting of oncogenic KRAS in addition to other therapeutic agents specific to the molecular profile of the tumor

Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin

Butyrate is a prototype of histone deacetylase inhibitors that is believed to reactivate silent genes by inducing epigenetic modifications. Although butyrate was shown to induce fetal hemoglobin (HbF) production in patients with hemoglobin disorders, the mechanism of this induction has not been fully elucidated. Our studies of the epigenetic configuration of the β-globin cluster suggest that DNA methylation and histone H3 acetylation are important for the regulation of developmental stage-specific expression of the β-like globin genes, whereas acetylation of both histones H3 and H4 seem to be important for the regulation of tissue-specific expression. These studies suggest that DNA methylation may be important for the silencing of the β-like globin genes in nonerythroid hematopoietic cells but may not be necessary for their silencing in nonhematopoietic cells. Furthermore, our studies demonstrate that butyrate exposure results in a true reversal of the normal developmental switch from γ- to β-globin expression. This is associated with increased histone acetylation and decreased DNA methylation of the γ-globin genes, with opposite changes in the β-globin gene. These studies provide strong support for the role of epigenetic modifications in the normal developmental and tissue-specific regulation of globin gene expression and in the butyrate-mediated pharmacologic induction of HbF production