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 LD(50) for PTL was 6.2 muM (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 IkappaB. 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.

CCK2 gastrin receptor as a potential target for therapy in leukaemia cell lines.

The gastrin CCK2 pathway has been implicated in the development of various cancers including leukaemia. An autocrine or intracrine pathway may exist in the leukaemia cell that is involved in stimulating proliferation. We tested four leukaemia cell lines, KU812, ML-1, MOLT-4 and U937 for the existence of the CCK2 receptor and gastrin precursor protein using immunoblotting. We also assessed the effect of CCK2 antagonist PD 135 and both gastrin 17 and glycine-extended gastrin on the proliferation of the cell lines. We found immunoreactive CCK2 and gastrin precursors present in all 4 cell lines. We also observed a stimulatory effect on proliferation by gastrin and glycine-extended gastrin on 2 and 3 of the cell lines respectively and an inhibitory effect of PD 135 on all 4 cell lines. These results demonstrate that the gastrin-gastrin receptor axis is a potential target for new therapeutic strategies.

Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.

Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells.

We have studied the actions of autologous plasma on both basal and DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis-specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and gamma-radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons alpha or gamma or stromal cell-derived factor 1, each of which have been shown to protect B-CLL cells from apoptosis in vitro. Plasma addition to B-CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti-apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3'-kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B-CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective-signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B-CLL treatment.

Expression of the human major vault protein LRP in acute myeloid leukemia.

Overexpression of a 110-kD protein (lung resistance-related protein [LRP]) may predict a poor response to chemotherapy in patients with acute myeloid leukemia (AML) and ovarian carcinoma. The LRP gene has recently been mapped to chromosome 16, close to the multidrug resistance-associated protein (MRP) gene. Seventy-seven samples from 67 patients with AML were examined for expression of LRP, MRP, and multidrug resistance (MDR1) mRNA using a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Results were compared with 29 normal samples (11 normal peripheral blood and 18 normal bone marrow). Thirty-three patients with untreated AML were evaluable for response to chemotherapy. Levels of LRP, but not of MRP or MDR1 mRNA, were significantly higher in eight patients who failed to achieve complete remission (CR) compared with 25 patients who achieved CR (p = 0.033). A positive correlation was demonstrated between LRP and MRP (R = 0.368, p = 0.001) and between MRP and MDR1 mRNA levels (R = 0.301, p = 0.01) in the 77 clinical samples analyzed. In AML samples, a significant difference in MDR1 mRNA levels was found between presentation (47 samples) and relapse (30 samples) (p = 0.031). No significant difference was seen in LRP mRNA levels between these two groups or in eight patients studied sequentially at both presentation and relapse. Thirteen samples (10 at presentation, 3 at relapse) were analyzed for LRP protein expression by flow cytometry. Eight (5 at presentation, 3 at relapse) displayed greater than 10% positive cells (range 15-86%). These data suggest that LRP gene overexpression may constitute a novel mechanism of multidrug resistance.

Lack of clonal BCRA2 gene deletion on chromosome 13 in chronic lymphocytic leukaemia.

Chromosome 13q deletion is among the most common cytogenetic abnormalities in chronic lymphocytic leukaemia (CLL). We investigated the 13q14.3 deletion in 44 CLL patients by Southern blotting following purification of clonal B CLL cells to >90%. Two sets of probes were used to investigate the site of clonal deletion, the D13S25 and D13S319 markers (at 13q14.3) and probes for exons 11 and 26-27 of the BRCA2 gene (at 13q12). Homozygous and heterozygous deletion at the 13q14.3 region was found in five and 17 patients, respectively. Despite the recent report of the BRCA2 gene involvement in >80% of CLL patients, we failed to detect a single case of homozygous or heterozygous deletion involving the 13q12 region. Our data support previous findings that the 13q14.3, and not the 13q12 region, is the major site of candidate tumour suppressor gene(s) in CLL.

Role of double-stranded RNA-activated protein kinase in human hematological malignancies.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is one of many genes induced by IFN. The PKR sequentially undergoes autophosphorylation and activation on binding to dsRNA. Previous studies have shown that PKR may be an important factor in the regulation of viral and cellular protein synthesis. Recent studies suggest that PKR may function as a tumor suppressor gene. The role of PKR in various human leukemic cells was therefore investigated. PKR mRNA levels by reverse transcription-PCR, protein expression by Western blot and FACScan analysis, and activity by phosphorylation status were studied. The expression of a known inhibitor of PKR, p58, was also investigated at mRNA and protein levels. A total of 24 samples from normal mononuclear cells (MNCs), 26 samples of acute lymphoblastic leukemia, 26 samples of acute myelogenous leukemia, 32 samples of chronic lymphocytic leukemia, and 5 samples of hairy cell leukemia was investigated. Mean mRNA levels were increased in acute lymphoblastic leukemia and acute myelogenous leukemia and decreased in chronic lymphocytic leukemia compared to normal MNCs. The mRNA levels in hairy cell leukemia were similar to those of normal MNCs. PKR protein was detectable in normal MNCs and leukemic cell extracts, and on FACScan analysis, more than 70% of cells stained positive for PKR. PKR activity was detectable in all samples investigated and was enhanced 4-23-fold in the presence of the synthetic dsRNA, poly(I) x poly(C). Protein expression of a known PKR inhibitor, p58, was barely detectable in normal MNCs and leukemic cells, with high expression in the HeLa cell line. These findings provide no evidence to support the hypothesis that PKR acts as a tumor suppressor in human leukemic cells.

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 alpha or gamma. 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 micron 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.

Expression and function of the FAS antigen in B chronic lymphocytic leukemia and hairy cell leukemia.

The expression and function of the FAS antigen was analyzed in 21 patients with B chronic lymphocytic leukemia (CLL) and four with hairy cell leukemia (HCL) using a specific IgM monoclonal antibody and FACS analysis. The FAS antigen was expressed in a minority (5-41%, mean 15.6%) of the CLL cells in 10 of 21 CLL patients and this expression was not modified during spontaneous or hydrocortisone-induced apoptosis of CLL cells. In contrast, culture with gamma-interferon (gamma-IFN) upregulated the expression of FAS in all CLL patients, with 65-100% (mean 84.8%) of the cells being positive after 2 days in vitro culture. Culture with alpha-IFN induced FAS expression in 15 of 19 CLL patients tested, with 15-74% (mean 34%) of the cells being FAS+ after 2 days culture. IL-4 and IL-10, lymphokines that inhibit and promote CLL apoptosis respectively, did not modify the expression of FAS. These results from FACS analysis were consistent with FAS mRNA analysis of fresh and cultured CLL cells, using a semi-quantitative reverse transcriptase (RT)-PCR technique. Although IL-4 and IFNs prevent apoptotic cell death of CLL cells in vitro, the present results show that IFNs induce the expression of the apoptosis-inducing protein FAS. However, FAS+ CLL cells were not killed in the presence of anti-FAS monoclonal antibody (while the FAS+ Jurkat and four lymphoblastoid cell lines were killed). This resistance is not due to a mutated FAS protein, since only wild-type FAS cDNA was demonstrated in the leukemic cells of three CLL patients. In four HCL patients 34-53% (mean 44.5%) of the leukemic cells were FAS+ and they were also resistant to the anti-FAS mediated cytotoxicity. The combination of high bcl-2 protein levels and resistance to anti-FAS mediated cytotoxicity may contribute to the extended in vivo survival of CLL and HCL cells.

Deletion of chromosome 13 (band q14) but not trisomy 12 is a clonal event in B-chronic lymphocytic leukaemia (CLL).

Chromosomal abnormalities are detected by conventional cytogenetic or FISH analysis in 50% of chronic lymphocytic leukaemias (CLL). Trisomy 12 and del 13q14 account for 70% of these abnormalities. The incidence of these two abnormalities was studied in CLL patients by Southern blot analysis using a highly purified B-cell malignant population (CD5 > 95%, CD3 < 5%). Probes for the D13S25 marker on chromosome 13 band q14 and for the RBTN3 gene on chromosome 12 band p12-13, were used. Deletion of the D13S25 was detected in 17/42 patients (43%) in a homozygous (9.5%) or heterozygous (30%) configuration. Deletion of the D13S25 marker appears to be a clonal and early event in CLL development since it is detected in > 95% of the malignant clonal population. Conversely, trisomy 12 is rarely a clonal event (5/33 patients, 15%) and a varying proportion of cells carrying this abnormality can be demonstrated in 30% of CLL patients (10/33 patients).

Expression of the multidrug resistance-associated protein (MRP) in acute leukaemia.

A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to investigate and compare transcription levels of the human multidrug resistance gene (MDR1) and the recently described multidrug resistance-associated protein (MRP) in 105 samples from patients with acute leukaemia at presentation and relapse. MRP gene expression was significantly greater in samples from patients with acute lymphoblastic leukaemia (ALL) compared with samples from normal peripheral mononuclear cells (PBMC) and patients with de novo acute myeloid leukaemia (AML). MRP gene expression was found to be higher in patients with relapsed de novo AML compared to those at presentation but prior therapy did not affect MRP gene expression in ALL. MDR1 gene expression was significantly lower in ALL patients compared to normal PBMC and AML samples. Samples from patients with secondary AML had higher levels of MDR1 expression than those of de novo AML. No changes of MDR1 expression were observed in AML or ALL at relapse. No correlation was observed between MDR1 and MRP gene expression in this group of patients. Our results suggest that MRP expression may be of prognostic importance in AML but the significance of the increased levels we have detected remain unclear.

Quantitation of multidrug resistant MDR1 transcript in acute myeloid leukaemia by non-isotopic quantitative cDNA-polymerase chain reaction.

Drug resistance in acute myeloid leukaemia (AML) may be caused by overexpression of the P glycoprotein (PGP), an efflux pump encoded by the multidrug resistance mdr 1 gene. Previous studies have suggested that increased PGP expression in the leukaemic blasts is of prognostic significance, and that use of PGP antagonists may be beneficial in treatment. We describe preliminary results with a non-isotopic quantitative MDR 1 cDNA-PCR assay, using an artificial RNA construct sharing primer recognition sites with the target MDR 1 mRNA (MDR 1 nucleic acids 483-504 and 624-644) as an internal control. KB 3.1 parent and KB 8.5 MDR positive cell lines expressed 0.004 and 1.96 molecules MDR 1 mRNA/pg total RNA. Semiquantitative screening of 60 RNA samples from 53 AML cases detected MDR 1 transcript ranging from 0 to 1.81 molecules per pg RNA. The median value at presentation (33 patients) was 0.055 and was higher in 14 patients at relapse (0.13) and in seven patients with refractory disease (0.14). Quantitation of MDR 1 transcript in serial samples in seven treated patients between presentation and relapse showed the decrease in three patients (0.18-0.02 x) to be as marked as the increase in three other patients (3-16 x). The method described is well suited for the study of clinical samples because it is sensitive, specific, rapid and requires small amounts of clinical material.

Alpha-interferon (alpha-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 alpha-IFN on the DNA fragmentation, bcl-2 protein levels and cell survival in purified B-cells from 16 CLL patients. Alpha-IFN (10(3) 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, alpha-IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of alpha-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 alpha-IFN, like IL-4 and gamma-IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to alpha-IFN cannot be explained by a direct cytotoxic effect of alpha-IFN on circulating CLL cells. Alternatively, alpha-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.

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

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 approximately 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.

Flow cytometric assessment of multidrug resistance (MDR) phenotype in acute myeloid leukaemia.

Forty one patients with acute myeloid leukemia (AML), including 27 at presentation and 14 relapsed or resistant cases, were assessed for laboratory evidence of the MDR phenotype. Leukaemic cells from all 41 cases were studied by immunocytochemistry using the JSB-1 monoclonal antibody and simultaneously by reverse transcription polymerase chain reaction (RT-PCR) to evaluate expression of the mdr 1 gene. Cells from 32/41 cases were also assessed for daunorubicin (DNR) accumulation and retention by flow cytometry (FC). Nineteen of the 41 (46%) patients were positive for MDR by JSB-1 immunocytochemistry (11/27 [41%] at presentation and 8/14 [57%] relapsed or resistant cases). Nine of the 19 (47%) P-gp positive, de novo patients achieved complete remission. 22 patients were negative by JSB-1 immunocytochemistry (16/27 [59%] at presentation and 6/14 [43%] of the relapsed or resistant cases) and 11/22 (50%) P-gp negative patients achieved a complete remission. Of the 32 patients assessed by FC, 7 (22%) were positive for the MDR phenotype with increased DNR accumulation and retention in the presence of the MDR reversing agent verapamil (VPM). 6/7 of the FC positive cases were also JSB-1 positive, and 6 had additional poor risk features. Of the twenty five FC negative patients, 6 had received previous chemotherapy and 15 (60%) achieved complete remission. Mdr 1 mRNA levels were increased in all seven FC positive cases whereas only 7/19 JSB-1 positive cases had raised mdr 1 mRNA levels. These results suggest that the assessment of MDR status by immunocytochemistry using JSB-1 is not predictive of response to chemotherapy. Flow cytometric analysis of blast cells appears to correlate well with mdr 1 mRNA levels and may be a better predictor of treatment outcome.

Interferon-alpha inducible 2'-5' oligoadenylate synthetase transcripts in lymphoid and myeloid leukemias.

Alpha-interferon (IFN) is effective in the treatment of a proportion of patients with hairy cell leukemia (HCL). B-cell chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and multiple myeloma (MM). One of the proteins induced by IFN is the enzyme 2'-5' oligoadenylate synthetase (2-5 AS). Peripheral blood or bone marrow samples treated with IFN in vitro, or from patients treated with IFN were studied for expression of the different 2-5 AS mRNA transcripts. A total of four normal individuals and 31 patients (nine HCL, five CLL, six MM, nine acute myeloid leukemia (AML) and two T-cell acute lymphoblastic leukemia (T-ALL) have been investigated. In normal peripheral blood lymphocytes, only the 1.8 kb transcript was induced with IFN in vitro. In HCL, CLL, and MM all four transcript sizes were induced by IFN in vivo and in vitro. The 1.6 and 1.8 kb forms were equally and predominantly expressed in HCL and B-CLL. On the other hand, the 1.8 kb transcript was predominantly expressed in MM and this increased expression was statistically significant. In acute leukemia, the majority of samples expressed all four transcripts equally but four of eleven samples expressed only the 1.8 kb transcript. These results suggest that the pattern of induction of specific 2-5 AS mRNA transcripts may be related to the underlying disease. Whether these different patterns of 2-5 AS induction have implications for response to IFN treatment, remains to be determined.

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

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 alpha-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.