Regulation of haem biosynthesis in normoblastic erythropoiesis: role of 5-aminolaevulinic acid synthase and ferrochelatase

The development of haem biosynthetic enzyme activity during normoblastic human erythropoiesis was examined in seven patients. The first and last enzymes of the haem biosynthetic pathway, ALA synthase and ferrochelatase, were assayed by radiochemical/high performance liquid chromatographic (HPLC) methods. An assay for ferrochelatase activity in human bone marrow was developed. Enzyme substrates were protoporphyrin IX and ⁵⁹Fe²⁺ ions. ⁵⁹Fe-labelled haem was isolated by organic solvent extraction/sorbent extraction followed by reversed-phase HPLC. Optimal activity occurred at pH 7.3 in the presence of ascorbic acid, in darkness and under anaerobic conditions. Haem production was proportional to cell number and was linear with time to 30 min. The assay was sensitive to the picomolar range of haem production. ALA synthase and ferrochelatase activity was assayed in four highly purified age-matched erythroid cell populations. ALA synthase activity was maximal in the most immature erythoid cells and diminished as the cells matured with an overall five fold loss of activity from proerythroblast to late erythroblast development. Ferrochelatase activity was, however, more stable with less than a two fold change in activity observed during the same period of erythroid differentiation. Maximal activity occurred in erythroid fractions enriched with intermediate erythroblasts. These results support sequential rather than simultaneous appearance of these enzymes during normoblastic erythropoiesis. Quantitative analysis of relative enzyme activity however indicates that at all times during erythroid differentiation ferrochelatase activity is present in excess to that theoretically required relative to ALA synthase activity since ALA and haem are not produced in stoichiometric amounts. The lability of ALA synthase versus the stability and gross relative excess of ferrochelatase activity indicates a far greater role for ALA synthase in the regulation of erythroid haem biosynthesis than for ferrochelatase.     

Chemokines and dendritic cells: a crucial alliance

Dendritic cells (DC) are bone marrow-derived professional antigen-presenting cells that function as sentinels of the immune system. Their importance in immunity resides in their unique ability to prime or tolerize T lymphocytes, thereby initiating or inhibiting immune responses. They reside in all tissues and organs and upon appropriate activation, migrate to secondary lymphoid organs to present antigen to T lymphocytes in the T cell zones. Because of this central role in T cell activation, there is a great deal of interest in using DC therapeutically to deliver positive or negative signals to the immune system. The DC system is critically dependent on the ability of DC at different stages of maturation to respond to a range of soluble and cell-bound signals, including members of the chemokine gene superfamily. This review will describe the interactions between DC and the chemokine system.     

Studies on gastric mucosal IgA: separation of immunoglobulin rich fraction from gastric mucoproteins.

Human gastric mucosal scrapings were subjected to fractionation on an isopycnic CsCl gradient. Immunoglobulin A was found between the 5th and 10th ml from the top of the tube. (Total volume 12ml). After two-fold fractionation the combined IgA containing fraction accounted for 4%–7% of the total carbohydrate content of the original gastric mucosal scrapings. Gas liquid chromatography of sugars showed the fraction to be enriched in Mannose and N-Acetyl glucosamine. The total carbohydrate content of the material was 5.5%–7% by weight. Immunodiffusion against specific anti Secretory component serum failed to demonstrate the presence of the secretory component in this fraction. It is concluded that gastric mucosal IgA, which appears to differ from a typical sIgA in lacking the characteristic secretory component activity, can be separated from the carbohydrate-rich gastric mucoproteins by CsCl fractionation. This indicates the absence of covalent bonding between IgA and the mucoproteins of gastric mucus.     

Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP₃ receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP₃Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP₃R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.Lung cancer accounts for 12% of all new cancers worldwide and is a leading cause of cancer-related mortality in the United States.^{1, 2, 3} Although small cell lung cancer (SCLC) comprises only 15% of lung cancer cases,^{2, 3} it has an annual mortality rate approaching that of breast and prostate cancer.⁴ Compared with the more common non-small cell lung cancer (NSCLC), SCLC is more aggressive and associated with rapid development of metastasis.² Moreover, although SCLC is more responsive to chemotherapy and radiation therapy initially, it typically relapses quickly with treatment-resistant disease.² In contrast to dramatic advances in chemotherapy and personalized medicine in other malignancies, the life expectancy of SCLC patients has remained <2 years for decades and is <1 year for patients with extensive disease.^{5, 6} The lethality of SCLC is attributed in part to the development of resistance to standard combination chemotherapies, underscoring the need to develop novel therapeutic approaches based on understanding the molecular and cellular biology of SCLC.^{5, 6}Evasion from apoptosis is a major hallmark of cancer and a prominent factor underlying drug resistance in SCLC.³ Multiple mechanisms contribute to apoptosis resistance in SCLC, including elevated expression of the antiapoptotic Bcl-2 protein³ (Supplementary Figure S1). Tsujimoto and colleagues discovered elevated levels of Bcl-2 mRNA and protein in SCLC cells not long after their identification of Bcl-2 as the protein product of the bcl-2 gene in follicular lymphoma.^{7, 8} Subsequently, immunohistochemistry of 164 primary SCLC samples revealed 76% were positive for Bcl-2, a finding substantiated by microarray detection of increased BCL-2 mRNA levels in 84% of SCLC samples^{9, 10} and by genomic sequencing of circulating SCLC tumor cells.¹¹ Moreover, proteomic profiling documented that Bcl-2 is more highly expressed in SCLC than in NSCLC, reflecting the vastly different biology of these lung cancer subtypes.¹²The major known function of Bcl-2 is to bind and sequester BH3-only proteins such as Bim, preventing these proteins from inducing apoptosis.^{13, 14, 15} Therefore, a major investment has been made in targeting this interaction for cancer treatment. The interaction takes place in a hydrophobic groove on Bcl-2 and the therapeutic strategy for targeting this interaction has been to develop small molecules, BH3-mimetic agents, which bind in the hydrophobic groove and induce apoptosis by displacing the BH3-only proteins. This approach has been reviewed in detail.^{14, 15, 16}Among BH3-mimetic agents advancing through clinical trials for both hematological malignancies^{15, 17} and solid tumors¹⁸ are ABT-737 and its orally bioavailable derivative ABT-263 (Navitoclax). Reported studies of ABT-199, a selective inhibitor of Bcl-2, are at present limited to hematological malignancies.¹⁸ In screening a large number of cancer cell lines, the pioneering work of Oltersdorf et al.¹⁹ demonstrated potent single-agent activity of ABT-737 against cell lines representative of lymphoid malignancies and SCLC. Clinical trials of ABT-263, an orally bioavailable version of ABT-737, achieved overall response rates ranging from as high as 35% in relapsed/refractory chronic lymphocytic leukemia (CLL) and 22% in follicular lymphoma.¹⁷ Reported responses are generally less in solid tumors with the notable exception of SCLC.¹⁸ But even in SCLC, activity of ABT-263 is limited in comparison to hematological malignancies, with 1 of the 39 (3%) of patients achieving a partial response to ABT-263 and 9 of the 37 (23%) achieving stable disease in a phase I clinical trial.²⁰ This experience suggests a need to develop additional ways of targeting Bcl-2 for cancer treatment.A potential alternative therapeutic target for Bcl-2-positive malignancies involves interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP₃R), an IP₃-gated Ca²⁺ channel located on the endoplasmic reticulum (ER). Bcl-2 is located not only on the outer mitochondrial membrane but also on the ER, and at both of these locations, it functions as a potent inhibitor of apoptosis.^{21, 22, 23} ER-localized Bcl-2 interacts with IP₃Rs and inhibits apoptosis by preventing excessive IP₃R-mediated Ca²⁺ transfer from the ER lumen into the cytoplasm and nearby mitochondria.^{24, 25, 26} Notably, regions of Bcl-2 involved in binding BH3-only proteins and IP₃Rs are entirely different. Whereas BH3-only proteins and their BH3-mimetic counterparts bind in a hydrophobic groove composed of BH3 domains 1–3 of Bcl-2,^{13, 14} the BH4 domain of Bcl-2 is necessary for interaction with IP₃Rs.²⁷ To develop a peptide inhibitor of Bcl-2–IP₃R interaction, we identified the Bcl-2-binding region on the IP₃R and developed a small synthetic 20 amino-acid peptide corresponding to this region.²⁸ This peptide, when fused to the cell-penetrating peptide of HIV TAT, binds to the BH4 domain of Bcl-2 and functions as a decoy peptide, inhibiting Bcl-2–IP₃R interaction.^{29, 30} We currently refer to this peptide as BIRD-2 (Bcl-2-IP₃ Receptor Disruptor-2), having formerly named it TAT-IDP_DD/AA.³¹ By disrupting the Bcl-2–IP₃R interaction, BIRD-2 abrogates Bcl-2 control over IP₃R-mediated Ca²⁺ elevation and induces Ca²⁺-mediated apoptosis in primary human CLL cells²⁹ and diffuse large B-cell lymphoma cells.³² Notably, BIRD-2 does not kill normal cells, including human lymphocytes isolated from peripheral blood²⁹ and normal murine embryonic fibroblasts (F Zhong and C Distelhorst, unpublished data).The present investigation was undertaken to determine whether Bcl-2–IP₃R interaction is a potentially useful therapeutic target in SCLC. In support of this concept, we find the majority of SCLC lines tested are sensitive to BIRD-2-induced apoptosis and that BIRD-2 induces apoptosis in several ABT-263-resistant SCLC lines. BIRD-2, we find, lacks generalized cytotoxicity as it does not induce cell death in NSCLC lines or a normal lung epithelial line. On the other hand, we find that BIRD-2 and ABT-263 synergize in killing SCLC cells. These findings for the first time provide preclinical evidence of the potential value of targeting both antiapoptotic mechanisms of Bcl-2 for the treatment of SCLC.     

Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans

Lithium (Li(+)) has been used to treat mood affect disorders, including bipolar, for decades. This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the one or more underlying mechanisms of its therapeutic action are not understood. Here we describe a pharmacogenetic study of Li(+) in the nematode Caenorhabditis elegans. Exposure to Li(+) at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li(+) treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference of T08D10.2 is sufficient to extend longevity ( approximately 25% median increase), suggesting Li(+) regulates survival by modulating histone methylation and chromatin structure.     

Biosynthesis of proteins by human gastric mucosa in vitro.

Incorporation of L-[U-14C]leucine and of D[U-14C]glucose into proteins of fresh human gastric mucosa in vitro was studied after incubation of homogenized tissue and of intact mucosal pieces. CsCl centrifugation was used to separate high-density mucus glycoproteins from other mucosal proteins, and the macromolecular nature of radioactive mucosal glycoprotein fractions was confirmed by SDS/polyacrylamide-gel electrophoresis and autoradiography of the polyacrylamide gels. In all experiments a substantial proportion of total incorporated radioactivity was associated with gastric-mucosal glycoprotein fractions (CsCl fraction L3), indicating their biosynthesis. Radioactivity of these fractions was shown to co-chromatograph with carbohydrates when fractionated either directly or after reduction and alkylation (1) Sephadex G-200 chromatography in the excluded fractions and (2) by DEAE-cellulose ion-exchange chromatography. On incubation of intact mucosa, the major portion of radioactivity associated with the glycoprotein fractions of both leucine- and glucose-labelled specimens was secreted into the mucosal media during the course of the experiment. It is suggested that biosynthesis of mucus in vivo by gastric mucosa may be associated with rapid secretion of the synthesized macromolecules into the lumen of the stomach and that investigations of the metabolic processes within the mucosa should consider the products of secretion of the tissue. Incorporation of L-[U-14C]leucine implies biosynthesis of the polypeptide components of the macromolecules.     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.Key words: autophagy, calcium, Fyn, IP₃ receptor, dexamethasone     

Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2

A cytochrome c-enhanced green fluorescent protein chimera (cyt-c.EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release of endogenous cytochrome c from mitochondria into the cytoplasm was detected in Bcl-2-negative cells treated with the kinase inhibitor staurosporine or the calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochrome c was evident in Bcl-2-positive cells, consistent with earlier evidence that Bcl-2 overexpression inhibits cytochrome c release from mitochondria. Cyt-c.EGFP appeared to be localized to the mitochondria in Bcl-2-negative cells and to be released into the cytoplasm following treatment with either staurosporine or thapsigargin. However, in Bcl-2-positive cells the pattern of distribution of cytochrome c-EGFP was inconsistent with that of endogenous cytochrome c, due to accumulation of both cyt-c.EGFP and free EGFP in the cytoplasm of both treated and untreated cells. In summary, cyt-c.EGFP may be useful for monitoring cytochrome c release in living cells that do not express high levels of Bcl-2 but is an unreliable marker of cytochrome c release in cells that overexpress Bcl-2.