Stephen M. Kelsey

Inhibition of autophagy abrogates tumour necrosis factor α induced apoptosis in human T-lymphoblastic leukaemic cells

The pattern and the sequence of tumour necrosis factor‐α (TNFα) induced cell death in the acute T‐lymphoblastic leukaemic cell line CCRF‐CEM and its vinblastine‐resistant subline CEM/VLB100 have been studied. Previously, we found that the CEM/VLB100 cell line was more sensitive to TNFα‐induced killing than its parental CCRF‐CEM cell line. TNFα‐induced cell death showed an apoptotic pattern, as detected by agarose electrophoresis, flow cytometry and transmission electron microscopy (TEM). TEM images revealed that autophagy and condensed mitochondria occurred earlier than nuclear fragmentation. The specific inhibitor of autophagy, 3‐methyladenine (3MA), inhibited the formation of autophagosomes. TNFα‐induced DNA fragmentation and cytolysis were completely inhibited by 10 mM 3MA. Inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNFα‐induced apoptosis. In addition, amino acid and protein deprivation enhanced TNFα‐induced autophagy but not apoptosis. We propose that the early stages of autophagy are required for, but do not necessarily result in, TNFα‐induced apoptosis.

In vivo 'purging' of residual disease in CLL with Campath-1H

We assessed the role of human CD52 antibody (Campath‐1H) in six patients with chronic lymphocytic leukaemia (CLL) treated to maximal response with purine analogues (fludarabine/deoxycoformycin) in whom persistent leukaemic infiltration of blood and bone marrow had precluded autologous stem cell transplantation. Five patients achieved haematological and histological complete remission following Campath‐1H and one had minimal focal residual CLL in a trephine biopsy. Autologous transplantation was performed in two patients without complications and with rapid haemopoietic engraftment. Treatment with Campath‐1H may be of value in eradicating residual disease in CLL and may facilitate high‐dose therapy in young patients.

Role of DNA methylation in the suppression of Apaf-1 protein in human leukaemia.

Apaf-1 protein deficiency occurs in human leukaemic blasts and confers resistance to cytochrome-c-dependent apoptosis. Demethylation treatment with 5-aza-2′-deoxycytidine (5aza2dc) increased the sensitivity of the K562 leukaemic cell line to UV light-induced apoptosis in association with increased Apaf-1 protein levels. There was no correlation between Apaf-1 protein expression and Apaf-1 mRNA levels after the demethylation treatment. Methylation-specific polymerase chain reaction was used to show that the methylation can occur within the Apaf-1 promoter region in leukaemic blasts. Apaf-1 DNA methylation was demonstrated in acute myeloid leukaemia, chronic myeloid leukaemia and acute lymphoid leukaemia, suggesting that it is not specific to a particular leukaemia subtype. Apaf-1 protein expression did not correlate with Apaf-1 mRNA levels in human leukaemic blasts. Some leukaemic cells expressed high levels of Apaf-1 mRNA but low levels of Apaf-1 protein. This study suggests that Apaf-1 DNA promoter methylation might contribute to the inactivation of Apaf-1 expression. However, Apaf-1 protein levels might also be controlled at post-transcription level.

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis.

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (ΔΨm). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.

G-CSF after peripheral blood stem cell transplantation in lymphoma patients significantly accelerated neutrophil recovery and shortened time in hospital: results of a randomized BNLI trial.

We have undertaken a prospective randomized study in 90 patients with relapsed or resistant lymphomas to assess the value of G‐CSF (lenograstim) in the acceleration of myeloid recovery after peripheral blood stem cell transplantation (PBSCT). A common regimen of cyclophosphamide 1.5 g/m2 on day 1 and lenograstim 263 μg s.c. on days 2–10 with two aphereses on days 10 and 11 was used for stem cell mobilization. 77% of patients achieved an adequate PBSC collection in two harvests (>2 × 108 MNC/kg or >2 × 106 CD34+ cells/kg). 65 patients went on to receive high‐dose BEAM chemotherapy and engraftment data was available for 62. 34 patients had been randomized to receive lenograstim 263 μg/d s.c. and 28 to no growth factor. The median time to ANC > 0.5 × 109/l was 9 d in the lenograstim arm versus 12.5 d in the no‐lenograstim arm (P = 0.0001). This was associated with a median duration of time in hospital post PBSCT of 13 d in the lenograstim arm versus 15.5 d in the no‐lenograstim arm (P = 0.0002). Median days to platelet independence, platelet transfusions, incidence of infection and red cell transfusion were the same in both arms. These data indicate that lenograstim significantly accelerated myeloid recovery after PBSCT and shortened the duration of hospital stay.

A British National Lymphoma Investigation randomised trial of single agent chlorambucil plus radiotherapy versus radiotherapy alone in low grade, localised non-Hodgkins lymphoma.

Local radiotherapy (RT) alone was compared with radiotherapy plus continuous oral chlorambucil (RT + CHL) for the treatment of localised, low grade non-Hodgkins lymphoma (NHL) in a prospective randomised study of 148 patients. After a maximum of 18 years follow up there was no significant difference in overall survival or disease free survival between the two treatment groups. Age greater than 50 years and low serum albumin at diagnosis correlated with a poor prognosis in the series overall. Over one third of patients with Iocalised, low grade NHL may be cured by RT alone and adiuvant chlorambucil as initial therapy confers no survival advantage.

Quantitative determination of apoptosis on leukemia cells by infrared spectroscopy

Quantitation of apoptotic cell death in vivo has become an important issue for patients with acute leukemia. We describe herein a new analytical method, based on infrared (IR) spectroscopy, to estimate the percentage of apoptotic leukemic cells in two different cell lines (CEM and K562), induced with etoposide (VP-16). As the percentage of apoptosis increases, the protein structure shifts from dominantly β-sheet to unordered (random coil), the overall lipid content increases and the amount of detectable DNA decreases. These changes can be directly related to the percentage of apoptosis as determined by two standard reference methods: flow cytometry and DNA ladder formation. The correlation between the significant IR spectral changes and the percentage of apoptotic leukemia cells in the two cell lines was optimal up to 24 h following etoposide treatment (r = 0.99 for CEM cells and r = 0.96 for K562 cells). Furthermore, IR spectroscopy is able to detect apoptotic changes in these cells already after 4 h treatment with VP-16, compared to flow cytometry which needs 6 h to observe significant changes. Our study suggests that IR spectroscopy may have potential clinical utility for the early, fast and reagent free assessment of chemotherapeutic efficacy in patients with leukemia.

Role of Smac in human leukaemic cell apoptosis and proliferation.

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.

The role of vitamin D derivatives and retinoids in the differentiation of human leukaemia cells

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.

Molecular detection of clonally rearranged cells in peripheral blood progenitor cell harvests from multiple myeloma patients.

Summary. Peripheral blood progenitor cells (PBPC) are increasingly used for autologous reconstitution following high‐dose chemotherapy in multiple myeloma but it is unclear whether these cells are less likely to be contaminated with malignant cells than bone marrow (BM). We have investigated this using immunoglobulin heavy‐chain (IgH) gene fingerprinting, a polymerase chain reaction based technique with a sensitivity of 0.1–0.01% (10‐3 ‐ 10‐4). We have looked for patient‐specific IgH rearrangements in leukapheresis samples from eight myeloma patients undergoing PBPC harvest. Seven were in first remission (six partial, one complete) and one in second complete remission. Mobilization of PBPC was accomplished using cyclophosphamide (4 or 7 mg/m2) and rhG‐ or GM‐CSF. Between two and five leukaphereses were performed in each patient. Patient‐specific IgH rearrangements were identified in diagnostic BM in all patients and bands of identical size were found in one or more leukaphereses from 6/8 patients. Overall, 14/32 leukaphereses were shown to be contaminated. Two patients who showed contamination of at least one PBPC harvest had BM harvests in which contaminating cells were not detectable, suggesting that PBPC are not necessarily less likely to be contaminated than marrow stem cells.