Kay Oliver Kliche

Overexpression of Bax gene sensitizes K562 erythroleukemia cells to apoptosis induced by selective chemotherapeutic agents

Bax and Bcl-2 are a pair of important genes that control programmed cell death, or apoptosis, with Bax being the apoptosis promoter and Bcl-2 the apoptosis protector. Although the detailed mechanism is unknown, the protein products of these two genes form protein dimers with each other and the relative ratio of the two proteins is believed to be a determinant of the balance between life and death. In our preliminary study, we found that K562 erythroleukemia cells have an extremely low level of endogenous Bcl-2 expression and a fairly high level of endogenous Bax expression. We constructed Bax and Bcl-2 expression vectors and transfected them into K562 cells. We found that transfection of Bax vector increased the expression of Bax protein; a shortened form of Bax also appeared. Cell death analysis using the Annexin V assay showed that the Bax vector caused significantly more apoptotic cells that the Bcl-2 or pCI-neo vector did. After selection with G418, Bax, Bcl-2 and pCI-neo stably transfected cells were established. These three cell lines were examined for their response to the chemotherapeutic agents ara-C, doxorubicin, etoposide and SN-38. Bax-K562 cells showed significantly higher fractions of apoptotic cells than pCI-neo-K562 cells when treated with ara-C, doxorubicin or SN-38. No sensitization effect was seen when etoposide was used. In contrast, Bcl-2-K562 cells had fewer apoptotic cells than pCI-neo-K562 cells after treatment with all these agents. Therefore, Bax may sensitize K562 cells to apoptosis induced by a wide range of, but not all, chemotherapeutic agents.

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.

CD30 ligand in lymphoma patients with CD30+ tumors.

PURPOSE CD30 ligand (CD30L), which is expressed on resting B and activated T lymphocytes, can induce cell death in several CD30+ cell lines. Patients with CD30+ tumors (Hodgkins disease and Ki-1+ non-Hodgkins lymphoma) frequently have elevated soluble CD30 (sCD30) levels in their serum, which correlates with a poor prognosis. The role of sCD30 in protecting tumor cells from CD30L-mediated cell death and the pattern of CD30L expression on human peripheral-blood lymphocytes (PBLs) of normal donors and patients with CD30+ tumors are investigated. MATERIALS AND METHODS CD30L surface protein expression was determined by two-color flow cytometry on PBLs of patients with CD30+ tumors and normal individuals. CD30L levels were determined on subsets of PBLs before and after stimulation with phytohemagglutinin (PHA), anti-CD3 antibody, or CD40L. sCD30 was measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic activity of membrane-bound CD30L was tested in a CD30+ cell line by the annexin V-binding method. RESULTS Unstimulated T lymphocytes of normal donors and patients with lymphoma rarely expressed CD30L surface protein, but were able to express it after stimulation with PHA or anti-CD3 antibody. Resting B cells of patients with CD30+ tumors had lower levels of detectable surface CD30L compared with normal donors (mean, 55% and 80.6%, respectively; P = .0008). Patients with high levels of serum sCD30 had lower detectable levels of CD30L on their PBLs (R2 = .72, P = .0008) and exogenous sCD30 blocked membrane-bound CD30L-mediated apoptosis in a CD30+ cell line. CONCLUSION In patients with CD30+ tumors, sCD30 can decrease the availability of CD30L on PBLs. Blocking the apoptosis-inducing activity of CD30L by its soluble receptor may explain how CD30+ tumors escape immunosurveillance and may be related to the reported poor prognosis of patients who have elevated sCD30 levels.

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.

Elevated serum levels of IL-10 are associated with inferior progression-free survival in patients with Hodgkin's disease treated with radiotherapy.

Elevated pretreatment serum interleukin-10 (IL-10) is associated with inferior progression-free survival (PFS) in patients with Hodgkins disease (HD) treated with ABVD or equivalent regimens. Therefore, we explored the association of serum IL-10 with presenting features and PFS in HD patients treated only by radiotherapy (RT) with curative intent. Eligible patients were previously untreated, had biopsy-proven HD, were older than 16 years, HIV-negative, and had unthawed pretreatment serum. Serum IL-10 levels were measured with ELISA and were considered high if > or = 10 pg/ml. We identified 69 patients with median age of 34 years (range 16 - 74), of who 52% were males, and 3% had B-symptoms. Ann Arbor Stage was I in 35%, II in 58%, and III in 7% of the patients. Histology was lymphocyte predominance in 26%, and classical HD in 74% of the patients. Serum IL-10 was elevated in 35% of the patients. After a median follow-up of 67 months for survivors, the 5-year PFS of patients with high vs. normal serum IL-10 was 50% vs. 81% (all patients, P = 0.006), and 43% vs. 77% for the subset with classical HD (P = 0.008). Multivariate analysis revealed that high serum IL-10 and beta2-microglobulin were independently associated with inferior PFS. Patients with none, 1, or 2 adverse features comprised 57%, 36%, and 7% of the population, and their 5-year PFS was 80%, 63%, and 0%, respectively (P < 0.0001). In conclusion, high serum IL-10 is independently associated with inferior PFS in patients with HD treated with RT.