K Ganeshaguru

Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro

Leukaemic cells from most cases of B‐chronic lymphocytic leukaemia die rapidly by apoptosis in vitro unless they are cultured in the presence of interleukin‐4 or interferon α or γ. We now report prolonged survival of purified CLL cells cultured on bone marrow (BM) derived stromal cells in the absence of exogenous growth factors. In 10 cases of CLL examined 0–61% (mean 14.7%) of the cells were viable after 10 d culture in medium alone, whereas in the presence of BM stromal cells 10–102% (mean 47.0%) of cells were recovered alive (P < 0.005) in 7/10 cases of CLL, cells remained viable after 30 d of culture in BM stromal cells with cell recovery of 12–65%. These long‐term cultured CLL cells were Epstein Barr virus negative, shown by the failure to detect the ENBA‐2 and BZLF1 genes of EBV by PCR analysis. Identity between day 0 and day 30 CLL cells was demonstrated by sequence analysis of their clonal IgH CDR3 region. Adherence of CLL cells to BM stromal cell layers was critical for their protection from apoptosis. Separation of CLL cells from stroma by 0.45 μm culture filters resulted in loss of the protective effect of the stromal cells. Stromal cells were also able to protect CLL cells from hydrocortisone‐induced apoptotic cell death. Our findings provide an in vitro system that can be used to analyse the growth requirements of CLL cells and their chemosensitivity in an in vitro environment that mimics the in vivo milieu.

Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic leukaemia cells in vitro.

Summary. When monoclonal B cells from B‐chronic lymphocytic leukaemia (B‐CLL) patients are cultured in vitro, they die by apoptosis. Apoptotic cell death occurred in the B cells from 20/24 B‐CLL patients after 26–30 h in in vitro culture, with 14.3–59.0% (mean 33.6%) of their DNA being fragmented in ∼180 base pair multimers. After 8–10 d culture, 90–100% of the B‐CLL cells were dead. Cell death and DNA fragmentation were inhibited in the presence of 0.5–5 ng/ml human recombinant interleukin‐4 (IL‐4) and viable monoclonal B cells could be maintained in culture up to 3 weeks. At 5 ng/ml. IL‐4 reduced DNA fragmentation after a 26–30 h culture to 2.2–33.3% (mean 14.9%). IL‐4 inhibited apoptosis without stimulating cell proliferation. In four patients the cells were resistant to apoptosis in vitro and they could be maintained for up to 4 weeks in culture medium alone. DNA fragmentation in all patients was increased in the presence of the RNA synthesis inhibitor actinomycin‐D. Western blot analysis of cell lysates showed expression of the bcl‐2 protein in all 11 B‐CLL patients studied. However, during culture, bcl‐2 protein levels were preserved only in patients resistant to apoptosis and were reduced in those susceptible to apoptosis. Reduction of bcl‐2 protein levels was inhibited in cells cultured in the presence of IL‐4. These data offer an explanation for the difference between the long life in vivo and rapid death in vitro of B‐CLL cells and indicate that IL‐4 may participate in the extended survival of these non‐dividing cells in vivo.

Terminal Transferase Enzyme Assay and Immunological Membrane Markers in the Diagnosis of Leukaemia: a Multiparameter Analysis of 300 Cases

Multiparameter analyses have been carried out with recently developed enzyme and membrane markers in 300 patients with various leukaemias including ALL, AML, but excluding Ph1 positive leukaemias.

Terminal Deoxynucleotidyl Transferase Expression in Acute Non-lymphoid Leukaemia: an Analysis by Immunofluorescence

Summary. Indirect immunofluorescence for terminal transferase enzyme (TdT) was used to study the blasts of 64 patients with acute non‐lymphoid leukaemia (ANLL). In 32 patients no TdT positive cells were seen. In 19 cases a small subpopulation of cells expressing TdT was detected; these constituted up to 5% of total nucleated cells, and it was not clear whether these TdT positive cells were part of the leukaemic process or represented residual normal bone marrow lymphoid cells.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD50 for PTL was 6.2 μM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1–3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34+ haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IκB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

Alpha‐interferon (α‐IFN) protects B‐chronic lymphocytic leukaemia cells from apoptotic cell death in vitro

B‐chronic lymphocytic leukaemia cells (CLL) are prone to apoptotic cell death when cultured in vitro. Apoptosis and loss of the bcl‐2 protein is prevented in CLL cells cultured in the presence of interleukin‐4. In this study we analysed the effects of α‐IFN on the DNA fragmentation, bcl‐2 protein levels and cell survival in purified B‐cells from 16 CLL patients. α‐IFN (103 U/ml) reduced the degree of spontaneous DNA fragmentation of CLL cells after a 30 h culture period (from a mean of 22·2% in control cultures to 10·5%, P<0·01). This inhibition was accompanied by preservation of bcl‐2 protein and an increased survival of CLL cells compared to control cultures. In parallel, α‐IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of α‐IFN on DNA synthesis of CLL cells were variable (in two patients a decrease and in seven an increase in 3H‐thymidine uptake) and did not correlate with the effect on DNA fragmentation. In conclusion, our data suggest that α‐IFN, like IL‐4 and γ‐IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to α‐IFN cannot be explained by a direct cytotoxic effect of α‐IFN on circulating CLL cells. Alternatively, α‐IFN may inhibit the proliferation of the small fraction of clonogenic CLL progenitors, or interfere with cellular interactions necessary for the survival and growth of CLL cells.

Alpha interferon in the treatment of chronic hepatitis C infection in thalassaemia major

Summary. Hepatitis C virus (HCV) is responsible for the majority of cases of post transfusion non‐A non‐B (NANB) hepatitis in thalassaemia major (TM). Twelve multi‐transfused TM patients with serological, biochemical, histological and molecular biological evidence of HCV infection have been treated for 6 months with recombinant α‐interferon (IFN). Ten (83%) responded as assessed by a fall of at least 50% of pre‐treatment serum transaminase levels. Histological improvement was observed in 6/7 responders tested. Natural killer (NK) cell activity 24 h after the first dose of IFN was significantly increased in responders as compared to non‐responders (P<0.05). HCV RNA disappeared from serum in 5/12 and from liver tissue in 2/5 of the responders. The degree of induction of peripheral blood mononuclear cell 2′5’oligoadenylate synthetase messenger RNA (2‐5 OAS mRNA), an enzyme induced by IFN, after the first dose of IFN did not correlate with response. IFN was generally well tolerated. We conclude that the response rate in multi‐transfused TM patients infected with HCV and treated with IFN is similar to that in non‐multi‐transfused patients.

Immunofluorescent and biochemical studies of terminal deoxynucleotidyl transferase in treated acute leukaemia.

Summary. Indirect Immunofluorescence (IF) for terminal deoxynucleotidyl trans‐ferase (TdT) was used in conjunction with the biochemical assay of TdT enzymatic activity to study human leukaemias before and during therapy. In addition, non‐leukaemic marrows were analysed to compare the enzyme expression on normal cells. An excellent correlation was observed between the IF and biochemical methods when specimens contained greater than 5% TdT+ cells (by IF); below this level the biochemical assay was less reliable, while the sensitive IF test could detect isolated TdT+ cells among greater than 10 000 TdT negative cells. The IF method also had the advantage of allowing further immunological characterization of TdT+ cells, by simultaneous labelling of membrane antigens with appropriate antiscra. TdT+ cells expressing la‐like antigens (but lacking other antigens associated with B‐ and T‐ lymphoid differentiation) were frequently found in low numbers in remission marrows from acute lymphoblastic leukaemia (ALL) patients. However, similar cells were also observed in remission acute myeloid leukaemia, as well as in non‐leukaemic regenerating marrows, and marrow from normal donors. The presence of these normal TdT+ precursor cells therefore precluded the use of either IF or biochemical TdT tests for estimating the degree of residual disease or predicting early relapse in patients with non‐T, non‐B ALL. In contrast, the detection of TdT+ cells with T lymphoid antigens (HuTLA+) but lacking Ia antigens, in thymic (T‐cell) ‐ALL, but not in normal marrow, allowed the use of this combination of markers to detect minimal residual disease in T‐ALL.

Circulating monoclonal B lymphocytes in multiple myeloma

Summary. Peripheral blood mononuclear cells from 28 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of unknown significance (MGUS) were studied by immunoglobulin gene analysis. Clonal immunoglobulin gene rearrangements in peripheral blood mononuclear cells (PBIGRA) were demonstrated in 10 of the 28 MM patients (36%). Bone marrow and peripheral blood mononuclear cells were studied simultaneously in five of these 10 patients, and identical gene rearrangements were demonstrated in both. The incidence of such gene arrangements was higher in patients with active disease (cases at presentation or relapsed = 10/19 [47%]) compared to remission status (0/9) and higher in untreated (47%) compared to treated patients (11%) (P<0.05). Patients with this phenomenon had higher serum calcium levels (P<0.001), and higher bone marrow plasma cell counts (P<0.05). Serum creatinine and β2‐microglobulin were also higher but did not reach statistical significance. None of the patients with monoclonal gammopathy of uncertain significance had gene arrangements. Our findings confirm that circulating B lymphocytes are part of the malignant clone in MM and their presence correlates with high tumour volume.

Studies on the Biochemical Sequelae of Therapy in Thy-Acute Lymphoblastic Leukaemia with the Adenosine Deaminase Inhibitor 2’Deoxycoformycin

Summary. In four patients with Thy‐acute lymphoblastic leukaemia changes in blast cell deoxynucleoside triphosphate concentrations and, in three, changes in blast cell S‐adenosyl homocysteine hydrolase activity were measured during treatment with 2’deoxycoformycin, a potent inhibitor of adenosine deaminase. These studies were aimed at identifying the molecular basis of cell killing by this drug. In three patients an increase in blast deoxyadenosine triphosphate (dATP) concentration occurred which was found to be temporally related to cell killing and was accompanied by decreased concentrations of the other three deoxyribonucleoside triphosphates. In the one patient with Thy‐ALL who responded poorly to treatment, the increase in dATP concentration was delayed and was not accompanied by a fall in the concentrations of the other deoxyribonucleoside triphosphates. Progressive inactivation of blast cell S‐adenosyl homocysteine hydrolase was found to occur in the three patients tested but was maximal only after a substantial reduction of peripheral blast cell count. These results show that 2’deoxycoformycin has a potent cytoreductive effect in Thy‐ALL and suggest that the molecular basis of this toxicity is related both to the intracellular accumulation of dATP with inhibition of ribonucleotide reductase. Inactivation of S‐adenosyl homocysteine hydrolase may be of importance as an additional mechanism.