Role of Smac in Human Leukaemic Cell Apoptosis and Proliferation

Oncogene. Mar, 2003  |  Pubmed ID: 12642862

Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.

BH3-domain Mimetic Compound BH3I-2' Induces Rapid Damage to the Inner Mitochondrial Membrane Prior to the Cytochrome C Release from Mitochondria

British Journal of Haematology. Apr, 2003  |  Pubmed ID: 12694257

The Bcl-2 family proteins are major regulators of cell survival and death in human leukaemia. BH3-containing peptides induce apoptosis by binding to the hydrophobic pocket of the anti-apoptotic proteins, such as Bcl-2 or Bcl-XL. A small cell-permeable compound, BH3I-2' (3-iodo-5-chloro-N-[2-chloro-5-((4-chlorophenyl)sulphonyl)phenyl]-2-hydroxybenzamide), has been recently reported to have a function similar to Bak BH3 peptide. BH3I-2' induces apoptosis by disrupting interactions mediated by the BH3 domain, between pro-apoptotic and anti-apoptotic members of the Bcl-2 family. This study found that BH3I-2' induced cytochrome c release from the mitochondrial outer membrane in a Bax-dependent manner and that this correlated with the sensitivity of leukaemic cells to apoptosis. Moreover, it also induced rapid damage to the inner mitochondrial membrane, represented by a rapid collapse of mitochondrial membrane potential (DeltaPsim), prior to the cytochrome c release. This occurred both in whole cells and isolated mitochondria, and was not associated with the sensitivity of cells to BH3I-2'-induced apoptosis. Exogenous Bcl-2 or Bcl-XL neutralized BH3I-2'in vitro and diminished its effect on the inner mitochondrial membrane. Our results indicate that BH3I-2' not only induces cytochrome c release from the outer mitochondrial membrane but also damages the inner mitochondrial membrane, probably by interacting with Bcl-2 family proteins.

PAD Combination Therapy (PS-341/bortezomib, Doxorubicin and Dexamethasone) for Previously Untreated Patients with Multiple Myeloma

British Journal of Haematology. Jun, 2005  |  Pubmed ID: 15953001

Bortezomib (formerly PS-341) has significant activity in patients with relapsed multiple myeloma (MM), its efficacy is increased with the addition of dexamethasone and it demonstrates synergy with doxorubicin, thus providing the rationale for combination therapy with bortezomib, doxorubicin and dexamethasone (PAD). Patients with untreated MM received four 21-d cycles of PAD, comprising bortezomib 1.3 mg/m(2) on days 1, 4, 8 and 11, along with dexamethasone 40 mg on days 1-4, 8-11 and 15-18 during cycle 1 and days 1-4 during cycles 2-4. During days 1-4, patients also received 0, 4.5 or 9 mg/m(2) of doxorubicin at dose levels 1, 2, and 3 respectively. Following peripheral blood stem cell (PBSC) collection, patients received high-dose melphalan (MEL200) with PBSC transplantation (PBSCT). After PAD induction alone, 20 of 21 patients (95%) achieved at least a partial response (PR), including complete response (CR) in five patients (24%). Twenty of 21 had PBSC mobilized, and 18 of 20 received MEL200/PBSCT. In an intention-to-treat analysis, response rates were: CR 43%, near CR 14%, very good PR 24%, PR 14% and stable disease 5%. PAD was effective, did not prejudice subsequent PBSC collection, and should be further evaluated in prospective randomized trials.

Long-term Follow-up After Reduced-intensity Conditioning Allogeneic Transplantation for Acute Myeloid Leukemia/myelodysplastic Syndrome: Late CNS Relapses Despite Graft-versus-host Disease

Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. May, 2006  |  Pubmed ID: 16682729

Comparison of DNA Extraction Methods for Aspergillus Fumigatus Using Real-time PCR

Journal of Medical Microbiology. Sep, 2006  |  Pubmed ID: 16914647

Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze-thaw method, a freeze-boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.

Assessing the Total Costs of Blood Delivery to Hospital Oncology and Haematology Patients

Current Medical Research and Opinion. Oct, 2006  |  Pubmed ID: 17022848

To determine direct costs associated with a blood transfusion session in two hospital settings.

New Flow Cytometric Technique for the Evaluation of Circulating Endothelial Progenitor Cell Levels in Various Disease Groups

Journal of Immunological Methods. Oct, 2006  |  Pubmed ID: 17027849

Circulating endothelial progenitor cells (EPC) localise to sites of ischaemia and play a role in vascular repair and re-endothelialisation of injured blood vessels. Low levels of EPCs are associated with cardiovascular disease (CVD) in the general population. It is not clear at present whether and how the numbers of circulating EPCs vary in diseases other than CVD. We have enumerated EPCs by the flow cytometric analysis of whole blood by using a novel cocktail of monoclonal antibodies. This consisted of CD2FITC, CD13FITC and CD22FITC to eliminate non-progenitor cells and VEGFR2PE and CD133-streptavidin-PeCy7 to include only EPCs. We analysed 250 patients with varying stages of uraemia, 36 patients with inflammatory bowel disease (IBD) and 9 patients with acute respiratory distress syndrome and compared this to 74 healthy controls. Using flow cytometry we were able to measure the circulating levels of EPCs, with a result available within hours of the sample being obtained. Circulating EPC numbers vary in different patient groups and healthy controls. In uraemic patients, irrespective of disease severity, there are lower numbers of circulating EPC numbers compared to normal controls (46.6+/-3.7 vs. 66.1+/-4.7; p=0.03). This new technique provides a means of monitoring patients and shows a reduction in circulating EPCs in uraemic patients; this abnormality may be a target of novel therapies.

Let Cost Effectiveness Models Be Open to Scrutiny

BMJ (Clinical Research Ed.). Oct, 2007  |  Pubmed ID: 17932167

Safe and Efficacious Use of Recombinant Human Erythropoietin in Malignancy

Clinical Medicine (London, England). Dec, 2007  |  Pubmed ID: 18193714

Anti-CD38 Antibody-mediated Clearance of Human Repopulating Cells Masks the Heterogeneity of Leukemia-initiating Cells

Blood. Aug, 2008  |  Pubmed ID: 18523148

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34(+)CD38(+) fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

Dietary Flavonoids Inhibit the Anticancer Effects of the Proteasome Inhibitor Bortezomib

Blood. Nov, 2008  |  Pubmed ID: 18633129

Dietary flavonoids have many health-promoting actions, including anticancer activity via proteasome inhibition. Bor-tezomib is a dipeptide boronate proteasome inhibitor that has activity in the treatment of multiple myeloma but is not effective in chronic lymphocytic leukemia (CLL). Although CLL cells are sensitive in vitro to bortezomib-induced apoptosis when cultured in medium, the killing activity was blocked when cultured in 50% fresh autologous plasma. Dietary flavonoids, quercetin and myricetin, which are abundant in plasma, inhibited bortezomib-induced apoptosis of primary CLL and malignant B-cell lines in a dose-dependent manner. This inhibitory effect was associated with chemical reactions between quercetin and the boronic acid group, -RB(OH)2, in bortezomib. The addition of boric acid diminished the inhibitory effect of both quercetin and plasma on bortezomib-induced apoptosis. The protective effect was also reduced when myeloma cell lines, but not B-cell lines, were preincubated with quercetin, indicating a direct effect of quercetin on myeloma cells. At high doses, quercetin itself induced tumor cell death. These data indicate that dietary flavonoids limit the efficacy of bortezomib, whereas supplemental inorganic boric acid is able to reverse this. The complex interactions between quercetin, tumor cells, and bortezomib mean caution is required when giving dietary advice to patients.

Increased Proteasomal Degradation of Bax is a Common Feature of Poor Prognosis Chronic Lymphocytic Leukemia

Blood. Mar, 2008  |  Pubmed ID: 18160666

Many biologic markers are associated with poor prognosis in chronic lymphocytic leukemia (CLL), but their mechanistic role remains unclear. Bax is an essential proapoptotic protein and decreased levels in malignant cells lead to resistance to apoptosis. Using a Bax degradation activity (BDA) assay, CLL cells were found to show variable Bax instability. However, BDA did not correlate with Bax protein levels: BDA positive and negative cases had high and low baseline Bax levels. BDA positive cases showed a marked accumulation of poor prognostic markers-unmutated immunoglobulin heavy chain variable genes, ZAP-70/CD38 positivity, 11q22/17p13 deletion, and short lymphocyte doubling time. Patients with BDA positive cells had a shorter median overall survival (OS; 126 months vs not reached, P = .011) and time to first treatment (16 vs 156 months, P = .029) than BDA negative cases. Dual BDA and ZAP-70 positivity had a median OS of 84 months (P = .012). The BDA assay measures the intrinsic ubiquitin/proteasome activity of CLL cells and dynamic changes in Bax protein levels over time. Mechanistically, Bax instability may represent a final common pathway for disparate prognostic markers, as well as being itself an indicator of poor prognosis.

Bortezomib Blocks Bax Degradation in Malignant B Cells During Treatment with TRAIL

Blood. Mar, 2008  |  Pubmed ID: 18160669

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

Treatment of Respiratory Syncytial Virus Infection in Haemopoietic Stem Cell Transplant Recipients with Aerosolized Ribavirin and the Humanized Monoclonal Antibody Palivizumab: a Single Centre Experience

British Journal of Haematology. Sep, 2009  |  Pubmed ID: 19538531

Ficoll-Paque Versus Lymphoprep: a Comparative Study of Two Density Gradient Media for Therapeutic Bone Marrow Mononuclear Cell Preparations

Regenerative Medicine. Sep, 2009  |  Pubmed ID: 19761394

Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium, following acute myocardial infarction, may be, in part, due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials.

Association of Mannose-binding Lectin Deficiency with Acute Invasive Aspergillosis in Immunocompromised Patients

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Nov, 2009  |  Pubmed ID: 19827955

Invasive aspergillosis is a devastating infection with attributable mortality of 40% despite antifungal therapy. In animal models of aspergillosis, deficiency of mannose-binding lectin (MBL), a pattern recognition receptor that activates complement, is a susceptibility factor. MBL deficiency occurs in 20%-30% of the population. We hypothesized that MBL deficiency may be a susceptibility factor for invasive aspergillosis in humans.

Leukemia-initiating Cells from Some Acute Myeloid Leukemia Patients with Mutated Nucleophosmin Reside in the CD34(-) Fraction

Blood. Mar, 2010  |  Pubmed ID: 20053758

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

CD160 Signaling Mediates PI3K-dependent Survival and Growth Signals in Chronic Lymphocytic Leukemia

Blood. Apr, 2010  |  Pubmed ID: 20164468

B-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against-mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)-induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8(+) T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.

A Patient with CLL and a Dry Cough

BMJ (Clinical Research Ed.). 2010  |  Pubmed ID: 20591962

Optimizing Management of Invasive Mould Diseases

The Journal of Antimicrobial Chemotherapy. Jan, 2011  |  Pubmed ID: 21177403

We describe an integrated care pathway (ICP) for the optimal management of invasive mould disease (IMD). The ICP is for use by health professionals involved in the care of patients with haematological malignancies and haematopoietic stem cell transplant recipients who are at increased risk of IMD. The ICP is not intended for use in other patient groups where the evidence base is more limited. The ICP involves the patient and their carers, as well as describing the roles and the complex interaction of healthcare professionals in different departments. Therefore, the management of IMD as described in the ICP must be appropriate for the overall organization, and will be dependent on the facilities [e.g. high-efficiency particulate air (HEPA) filtration] and services available. The ICP deals with risk stratification, diagnostic tests, prophylactic and treatment strategies and how to incorporate these into the ICP. Outpatient drug management after hospital discharge and cessation of therapy are outlined. Local implementation of this ICP will vary from centre to centre: the ICP is a generic template for guidance indicating the requirements for optimal IMD management and as such provides a standard against which local practice can be audited. For clinical governance, to minimize variation in practice and, ultimately, to improve patient outcomes, each centre should regularly monitor and document compliance with the local ICP, from provision of patient information, appropriate prescribing and diagnostic investigation to clinical outcomes.

Differential and Tumor-specific Expression of CD160 in B-cell Malignancies

Blood. Aug, 2011  |  Pubmed ID: 21715317

CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.

Impact of the Revised (2008) EORTC/MSG Definitions for Invasive Fungal Disease on the Rates of Diagnosis of Invasive Aspergillosis

Medical Mycology : Official Publication of the International Society for Human and Animal Mycology. Nov, 2011  |  Pubmed ID: 22074309

Diagnosis of invasive aspergillosis (IA) remains a challenge as the clinical manifestations are not specific, and a histological diagnosis is often unfeasible. The 2002 European Organization for Research and Treatment of Cancer (EORTC) and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (MSG) criteria for classification of cases into possible, probable or proven were revised in 2008. Our objective was to analyze the impact of these revisions on the diagnosis of IA. A retrospective analysis of 589 high risk patient-episodes revealed that 125 of 155 ?possible? (81%) and 12 of 16 ?probable? (75%) cases of IA should be changed to ?non-classifiable? when the new criteria were applied. We concluded, as expected, that the 2008 EORTC/MSG revised definitions reduced the number of cases classified as ?possible? IA, but additionally, there has been a dramatic reduction in ?probable? cases. These changes have significant implications on the interpretation of clinical trial data based on EORTC/MSG classifications.

Bile Acid Malabsorption in Patients with Graft-versus-host Disease of the Gastrointestinal Tract

British Journal of Haematology. Jan, 2012  |  Pubmed ID: 22225419

A Practical Critique of Antifungal Treatment Guidelines for Haemato-oncologists

Critical Reviews in Microbiology. Feb, 2012  |  Pubmed ID: 22324737

The management of invasive fungal disease (IFD) in the haemato-oncology setting remains a challenge. This article reviews recent guidelines relating to IFD for their similarities and differences, as well as applying the Appraisal of Guidelines Research and Evaluation (AGREE) criteria. The guidelines' recommendations on antifungal prophylaxis, empirical and definitive treatment of candidiasis and aspergillosis are summarized; also, minimum standards for diagnosis and follow-up are discussed. This critique of the reviewed guidelines is a practical guide to physicians and commissioners in making local policies for IFD management.

Whole Blood Stem Cell Re-infusion and Escalated Dose Melphalan in Castration-resistant Prostate Cancer: a Phase 1 Study

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2012  |  Pubmed ID: 22392912

PURPOSE: Non-taxane based chemotherapeutic options in castrate resistant prostate cancer (CRPC) are limited despite the long natural history of the disease. We performed a phase 1 dose escalation study of the alkylating agent melphalan with autologous stem cell transplantation, comparing rapid changes in circulating tumor cells (CTCs) and PSA as a measure of response.EXPERIMENTAL DESIGN: Cohorts of individuals with advanced CRPC received high dose intravenous melphalan and autologous blood was returned to patients during treatment. The efficacy endpoints were the PSA reduction rate, CTC response, survival parameters, toxicity and whether re-induction of endocrine sensitivity occurred.RESULTS: 24 patients were recruited. Dose-escalation was feasible with the highest dose cohort being reached. Of 23 individuals evaluable for response, 16 had a PSA response greater than 30%; of 11 patients with soft tissue disease, 4 achieved a partial response and 7 had stable disease. Patients with CTC counts that fell to less than 5 within 2 weeks from the start of therapy had a longer overall survival (30.6 months vs 15.3 months, p=0.03) Treatment was associated with myelosuppression and frequent hospitalizations. In 20 patients after the study, hormone therapy was re-introduced when PSA rose again; response rates were high.CONCLUSIONS: Autologous transplantation following high dose alkylating agent chemotherapy induces responses but proved toxic, although dose escalation proved possible. The possibility of using CTCs to identify responders at two weeks may be used to justify such an intensive approach. Many individuals went on to further respond to both docetaxel and hormonal therapy.