Shourong Zhao

Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies

T‐cell cytotoxicity is primarily mediated by two cell surface proteins, Fas ligand (FasL) and tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL), and intracellular perforin and granzyme granules. FasL‐deficient and perforin‐deficient T lymphocytes maintain cytotoxicity but fail to induce graft‐versus‐host disease (GVHD) when transplanted into mice, suggesting that GVHD and graft‐versus‐tumour (GVT) effects can be dissociated, and that TRAIL is not involved in the pathogenesis of GVHD. Because TRAIL could mediate a favourable GVT effect it became important to study the spectrum of its activity and to investigate factors that can dissociate its expression from FasL. TRAIL induced apoptosis in 11/41 (27%) tumour specimens of haematological origin compared to 16/41 (39%) induced by FasL. Although eight specimens were sensitive to both FasL and TRAIL, no synergism was observed between these two ligands. TRAIL induced apoptosis in a dose and time dependent manner with an ED50 of 0.5 μg/ml and EDmax of 1 μg/ml. TRAIL activity was not reduced by the over‐expression of the multidrug resistant (MDR) protein, and was not enhanced by 9‐cis retinoic acid (RA), which can down‐regulate bcl‐2 protein. Both ligands were simultaneously up‐regulated in normal peripheral blood lymphocytes in response to IL‐2, IL‐15 and anti‐CD3 antibody, whereas IL‐10 had no effect. Together, our data show that (1) TRAIL can mediate cell death in a variety of human haematological malignancies, (2) resistance to TRAIL is not mediated by MDR protein, (3) the lack of synergy between TRAIL and FasL suggests that either one is sufficient to mediate T‐cell cytotoxicity, and (4) within the panel of cytokines tested, the expression of TRAIL and FasL could not be dissociated.

Expression of Bcl-2-related genes in normal and AML progenitors: changes induced by chemotherapy and retinoic acid

The expression of Bcl-2 family members was examined in normal and leukemic hematopoietic cells. Immature hematopoietic progenitor cells (CD34+/33−/13−) did not express Bcl-2 but Bcl-XL, the majority of CD34 cells expressed Bcl-2, Bcl-XL and BAD, and normal promyelocytes (CD34−/33+) lacked expression of both Bcl-2 and Bcl-XL, while leukemic CD34+progenitors and promyelocytes expressed these anti-apoptotic proteins. In AML, Bcl-2 expression was higher on CD34+ than on all AML cells, however, expression of Bcl-2 or Bcl-XL did not predict achievement of complete remission. Surprisingly, low Bcl-2 content was associated with poor survival in a group of patients with poor prognosis cytogenetics. The anti-apoptotic BAD protein was found to be expressed in AML, but was phosphorylated in 41/42 samples. Phosphorylation was found at both sites, Ser 112 and Ser 136. During induction chemotherapy, Bcl-2 levels of CD34 cells increased significantly. In the context of evidence for small numbers of leukemic CD34+ cells expressing very high levels of Bcl-2 prior to therapy, this finding is interpreted as a survival advantage of Bcl-2 overexpressing progenitors and rapid elimination of cells with low Bcl-2. Bcl-2 and Bcl-XL were both expressed in minimal residual disease cells. Downregulation of Bcl-2 mRNA and protein was observed by ATRA and the combination of Ara-C, followed by ATRA, resulted in markedly increased cytotoxicity in HL-60 cells, as compared to Ara-C alone or ATRA followed by Ara-C. Implications of these findings for the development of new therapeutic strategies for AML are discussed.

The anti-apoptotic genes Bcl-XL and Bcl-2 are over-expressed and contribute to chemoresistance of non-proliferating leukaemic CD34+ cells

Summary. In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti‐apoptotic genes Bcl‐2 and Bcl‐XL have been implicated in cell cycle regulation, we investigated the expression of these genes in non‐proliferating (Q) and proliferating (P) AML and normal CD34+ progenitor cells. Using reverse transcription polymerase chain reaction, Bcl‐XL and Bcl‐2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl‐XS and Bax expression was found. Furthermore, the Bcl‐XL/XS but not the Bcl‐2/Bax ratio was higher in Q AML compared with normal CD34+ Q cells (P = 0·001). An inverse correlation between Bcl‐2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all‐trans retinoic acid (ATRA) reduced Bcl‐2 and Bcl‐XL expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara‐C). These findings demonstrate overexpression of the anti‐apoptotic proteins Bcl‐XL and Bcl‐2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl‐2 and proliferation suggests a role for Bcl‐2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.

Phase I study of mitoxantrone plus etoposide with multidrug blockade by SDZ PSC-833 in relapsed or refractory acute myelogenous leukemia.

PURPOSE Expression of the multidrug resistance gene (MDR1) p170 protein is frequent in leukemic blasts from patients with relapsed acute myelogenous leukemia (AML). A phase I study using the nonimmunosuppressive MDR1 blocker SDZ PSC-833 (PSC) in combination with mitoxantrone (MITO) and etoposide (VP) was performed. PATIENTS AND METHODS Starting doses (LVL0) of MITO (3.25 mg/m2/d on days 1 and 3 to 6) and VP (210 mg/m2/d on days 1 and 3 to 5) were 40% of the maximal-tolerated dose (MTD) from a prior study. A 1.5-mg/kg loading dose of PSC was followed by a 120-hour continuous infusion of 10 mg/kg/d on days 2 to 6. Blood samples for PSC, MITO, and VP pharmacokinetics (PK) were taken on days 1 and 3, and samples for MDR1 expression were taken on day 0. RESULTS Severe mucositis developed in all patients at LVL0; therefore, MITO and VP doses were reduced to 2.5 and 170 mg/m2 (LVL-1) for the next seven patients, and this dose proved to be MTD. All LVL0 and three LVL-1 patients had transient elevations in the serum bilirubin level to > or = 4 mg/dL. Serum creatinine level increased to greater than 2 mg/dL in one case. There were no other grade 3 or 4 nonhematologic toxicities observed. The peripheral blood was cleared of leukemia in three LVL0 and four LVL-1 patients. The marrow was cleared of leukemic cells in one LVL0 and five LVL-1 patients, and a significant reduction in marrow leukemic infiltrate was observed in eight of 10. No patient achieved complete remission (CR), and all died of progressive disease (n = 8) or infection (n = 2). MDR1 expression was detected by fluorescent-activated cell sorter (FACS) analysis in five of seven cases. An elevated MDR1 mRNA level was detected by quantitative polymerase chain reaction (Q-PCR) in six of eight cases studied. Clearing of leukemia cells from the marrow occurred in four of six MDR1-positive and one of three MDR1-negative patients. Despite the fact that LVL0 doses had to be reduced due to toxicity, coadministration of PSC did not produce a consistent effect on MITO PK; however, it did repeatedly lead to increased levels of VP in the serum. CONCLUSION We conclude that PSC-MITO-VP is a tolerable regimen with antileukemic activity. Addition of PSC necessitated a 66% reduction in MITO and VP doses from a prior study without PSC.

CD30 ligand is expressed on resting normal and malignant human B lymphocytes

CD30, a member of the tumour necrosis receptor superfamily, is physiologically expressed on a subpopulation of T helper cells in normal individuals but is also expressed on several malignant and virally transformed cells. Its ligand (CD30L) is a pleiotropic cellular transmembrane protein that can induce cell death in several CD30+ cell lines. CD30L expression has been reported on activated human peripheral blood T lymphocytes and macrophages but not on B cells. Here we show that the CD30L is expressed on resting normal and on malignant B cells in addition to both CD4+ and CD8+ subsets of activated T cells, making it the second tumour necrosis family member, in addition to the CD27 ligand, that can be expressed on both T and B cells. These findings raise the possibility that the CD30L has a role in B‐cell/T‐cell communication and that B and T cells are likely to be involved in the growth regulation of CD30+ tumours.

Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells.

CD40 ligand (CD40L) is involved in the T‐cell‐dependent regulation of B‐cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro. We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B‐cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B‐cell lymphomas (P  < 0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T–B‐cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo.

Expression of bcr-abl mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification

We present the results of a novel method developed for evaluation of in situ amplification, a molecular genetic method at the cellular level. Reverse transcription polymerase chain reaction (RT‐PCR) was used to study bcr–abl transcript levels in individual cells from patients with chronic myelogenous leukaemia (CML). After hybridizing a fluorochrome‐labelled probe to the cell‐bound RT‐PCR product, bcr–abl mRNA‐positive cells were determined using image analysis. A dilution series of bcr–abl‐positive BV173 into normal cells showed a good correlation between expected and actual values. In 25 CML samples, the percentage of in situ PCR‐positive cells showed an excellent correlation with cytogenetic results (r = 0·94, P < 0·0001), interphase fluorescence in situ hybridization (FISH) (r = 0·95, P = 0·001) and hypermetaphase FISH (r = 0·81, P < 0·001). The fluorescence intensity was higher in residual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0·004), and was highest in late‐stage CML resistant to IFN therapy and lowest in CML blast crisis (P = 0·001). Mean fluorescence values correlated with bcr–abl protein levels, as determined by Western blot analysis (r = 0·62). Laser scanning cytometry allowing automated analysis of large numbers of cells confirmed the results. Thus, fluorescence in situ PCR provides a novel and quantitative approach for monitoring tumour load and bcr–abl transcript levels in CML.

Functional expression of TRAIL by lymphoid and myeloid tumour cells

TRAIL is a potent death protein that favours the killing of various types of cancer cells to normal cells, but under the right conditions TRAIL can also kill activated human T cells. TRAIL mRNA is widely expressed by normal cells but its expression by primary tumour cells is not known. In this study, primary tumour cells of haemopoietic origin constitutively expressed TRAIL mRNA and protein and were capable of inducing the apoptosis of target Jurkat cells in a dose‐dependent manner. This killing effect was reversed by anti‐TRAIL antibody. The functional expression of TRAIL by lymphoid and myeloid malignant cells raises the possibility of its involvement in tumour cell evasion of immunosurveillance, and could be related to spontaneous tumour cell death and necrosis.

Cell-surface exposure of phosphatidylserine correlates with the stage of fludarabine-induced apoptosis in chronic lymphocytic leukemia and expression of apoptosis-regulating genes.

BACKGROUND Programmed cell death (PCD) is characterized by a sequence of tightly regulated events that result in the activation of caspases and in internucleosomal DNA cleavage. Late apoptotic events such as DNA-strand breaks can be assayed by in situ end labeling (ISEL) and DNA measurement (sub G1) using flow cytometry. Phosphatidylserine (PS) redistribution from the inner plasma membrane leaflet to the outer leaflet, an early event in PCD, can be detected by annexin V (AxV) binding to PS. AxV-fluorescein isothiocyanate (FITC) fluorescence intensity is variable and characterizes different cell populations, denoted here as AxV-negative (AxV(neg)), AxV-low-positive (AxV(lo)), and AxV-high-positive (AxV(hi)). METHODS We investigate the correlation of three methods (ISEL, sub G1 DNA content, and AxV assay) for detecting apoptosis with focus on differences between populations with different levels of PS. We also examined the expression of PCD-regulating Bcl-2 family members in these cell populations by reverse transcription-polymerase chain reaction (RT-PCR). Chronic lymphocytic leukemia (CLL) cells exposed to fludarabine (FAMP) were used as an in vitro model. Cells with different PS/AxV levels were separated using fluorescence-activated cell sorting (FACS). RESULTS Only purified AxV(hi) cells had high positivity in the ISEL and sub G1 assays (94 +/- 0.6%, 88.6 +/- 6.6%, and 98.6 +/- 0.6%, respectively), indicating that late apoptotic cells are detected equally by all three methods. In the AxV(lo) population, ISEL was positive in 21% +/- 13% and DNA sub G1 in 20% +/- 6.6% of cells, suggesting that AxV identifies early apoptotic cells better than the other assays. Anti-apoptotic Bcl-2 and Bcl-X(L) were upregulated by FAMP when cells entered apoptosis (AxV(lo)), as was pro-apo- ptotic Bcl-X(S), which was undetectable in nonapoptotic AxV(neg) cells. Pro-apoptotic Bax was only expressed in AxV(neg) and AxV(lo) cells. Late apoptotic AxV(hi) cells did not express Bcl-X(S) or Bax. RESULTS (1) AxV staining is more sensitive than sub G1 or ISEL in detecting early apoptotic cells; (2) only late apoptotic cells are equally detected by all assays; (3) AxV is a valuable tool in the detection and isolation of apoptotic cells at different stages of PCD; and (4) pro-apoptotic Bcl-X(S) and Bax are expressed at early, not late, stages of apoptosis.

Low incidence of MDR1 expression in acute promyelocytic leukaemia

Summary. The MDR1 gene product, P‐glycoprotein, functions as a transmembrane efflux pump for certain cytotoxic agents including anthracyclines. Based upon the clinical observation that patients with acute promyelocytic leukaemia (APL) respond favourably to anthracyclines, we hypothesized that APL cells may have low levels of MDR1 expression. We therefore investigated MDR1 expression in 10 patients with APL and compared results with those obtained in 18 patients with other subtypes of acute myelogenous leukaemia (AML). Prior to reverse transcriptase polymerase chain reaction with MDR1 specific primers, leukaemic cells were purified by fluorescence activated cell sorting to exclude normal haemopoietic cells, in particular lymphocytes, from the MDR1 analysis. In sorted APL cells, MDR1 expression was detected in only two of 10 patients, which was significantly different from findings in other AML subtypes (MDR1 expression in 14/18 patients; P<0.01). When unsorted specimens from APL patients were studied, five of six cases were MDR1 positive, whereas sorted APL cells were shown to express MDR1 mRNA in only one of these cases. MDR1 mRNA levels expressed as MDRl/beta‐2 microglobulin ratios were significantly lower in APL (0‐24 –0‐2, mean –SD) than in AML (0‐75 –0‐48; P<0.01). We conclude that low or absent expression of MDR1 in APL cells may contribute to the efficacy of anthracyclines in the treatment of APL.