Xu-Rong Jiang

Expression of functional antigens on neutrophils: Effects of preparation

We have studied how different conditions of cell labelling and isolation affect the expression of five functional antigens on neutrophils from healthy subjects. Fluorescein isothiocyanate conjugated (FITC) antisera specific for the C3bi receptor CR3 (CD11b), aminopeptidase N (CD13), the LPS:LPS binding protein receptor (CD14) and the receptors for human IgG (Fc gamma RII CDw32 and Fc gamma RIII CD16) were incubated with (i) unfixed whole blood at 4 degrees C and at room temperature (RT, approximately 20 degrees C), and the leukocytes prepared for analysis using the Coulter Q-Prep system, (ii) leukocytes which had obtained following the removal of erythrocytes from whole blood by dextran sedimentation and which had been washed or left unwashed at RT, and (iii) leukocytes which had been prepared from whole blood that had been formaldehyde fixed immediately following venesection. The amount of fluorescence associated with the cells was determined by flow cytometry. The expression of CD14 was low under all conditions. However the expression of CD11b, CD16 and CDw32 was significantly higher (p less than 0.05) on neutrophils obtained by dextran sedimentation (n = 4) than on cells which had been fixed with formaldehyde ex vivo; the increase in expression was even greater if the cells had been washed. In contrast, the expression of CD13 on formaldehyde fixed cells was higher than on cells which had been labelled at 4 degrees C or at room temperature and was similar to or slightly lower than that on cells obtained by dextran sedimentation. Increasing the time between 10 and 60 min for which the whole blood was incubated with antisera at RT or at 4 degrees C, resulted in progressive increases in the expression of CD11b and CD13. When neutrophils which had been obtained by dextran sedimentation were incubated with unlabelled antibodies to CD16 or CDw32 and FITC labelled antibodies to CD11b there was a marked increase in the expression of CD11b. Altogether these findings indicate that the analysis of functional molecules on neutrophils (which may be rapidly up-regulated during activation) should be performed under clearly defined and controlled conditions. Dual fluorescence studies may, in some circumstances, produce misleading results.

Circumvention of P-glycoprotein-mediated drug resistance in human leukaemic cells by non-immunosuppressive cyclosporin D analogue, SDZ PSC 833

Summary. Cyclosporin A (CSA) exhibits greater multidrug resistance (MDR) modulating activity in vitro than other MDR modulators such as verapamil and quinidine. However, the immunosuppressive and nephrotoxic effects of CSA may limit its clinical use. PSC 833, a new cyclosporin D derivative, exerts a higher MDR reversal activity but lacks toxic or immunosuppressive effects. The drug‐resistant sublines K/DAV10.,, K/DAU200. K/DAU30o. K/DAU40o, K/ DAU500 and K/DAU60o have been derived from the drug‐sensitive parental cell line, K562 cl.6 and CEM/VLB100 is a drug‐resistant derivative of CCRF‐CEM. We report a comparison of the effects of PSC 833 and CSA on daunorubicin (DAU) transport kinetics and chemosensitivity in these cell lines. Both CEM/VBL100 and KS62 cl.6 DAU‐resistant cells displayed high levels of P‐glycoprotein (PGP), decreased DAU accumulation and increased DAU efflux when compared to their parental cells. PSC 833 was 1‐6‐, 3‐4‐, 4‐9‐ and 4‐6‐fold more effective than CSA in reversing DAU resistance in higher resistance CEM/VLB10o, K/DAU40o, K/DAU500 and K/DAU60o cells respectively. DAU transport kinetics showed that PSC 833 was more effective than CSA in increasing cellular DAU accumulation and decreasing DAU efflux in higher resistant leukaemia subclones. PSC 833 could restore DAU retention at lower doses and was more active than CSA in all the resistant cells. A 89‐100% restoration of intracellular DAU retention were gained by PSC 833 at 1‐0ftM in K562 cl.6 DAU‐resistant sublines, whereas a 73‐100% restoration of DAU retention was obtained by CSA only at 30‐0 β, M in the same resistant sublines. PSC 833 at 3‐0/XM is sufficient to restore full DAU retention in all resistant cells. CSA, however, even at 30‐0 pM, cannot confer full restoration of DAU retention in higher resistance K562 cl.6/DAU sublines. By measuring MDR modulator‐mediated short‐term inhibition of PGP function, PSC 833 was found to be at least 10‐30 times more active than CSA. As no effect on DAU retention and sensitivity has been found in sensitive parental cells with PSC 833, it is suggested that PSC 833 may act by blocking the effluxing function of PGP in the resistant leukaemia cells.

Adriamycin stimulates proliferation of human lymphoblastic leukaemic cells via a mechanism of hydrogen peroxide (H2O2) production.

It is becoming clear that adriamycin cytotoxicity may be mediated by semiquinone‐free radicals derived from the drug itself and reactive oxygen species (ROS). Recent evidence supports the concept that low concentrations of ROS are able to stimulate cell proliferation, and, based on the observation that subtoxic concentrations of adriamycin can also induce cell proliferation, we hypothesize that low concentrations of adriamycin stimulate cell proliferation by a ROS generation mechanism. We have employed spin‐trapping and electron spin resonance (ESR) spectroscopy to investigate the nature of the adriamycin‐generated ROS. The spin trap 3,5‐dibromo‐4‐nitrosobenzenesulphonate (DBNBS), which is oxidized in the presence of H2O2 and peroxidase enzymes, was used to produce a characteristic three‐line spectrum, and it was found that an identical spectrum was produced by human lymphoblastic leukaemic cells (CCRF‐CEM cells) after exposure to adriamycin. We tested our hypothesis further by exposing CCRF‐CEM cells to subtoxic concentrations of adriamycin (10−8, 10−9 and 10−10 M) and low concentrations of H2O2 (10−8, 10−9 and 10−10 M) and subsequently monitored cell proliferation. We found that low concentrations of both adriamycin and H2O2 significantly stimulate CCRF‐CEM cell proliferation. We therefore conclude that subtoxic concentrations of adriamycin are likely to induce cell proliferation via an H2O2 mediated mechanism.

Upregulation of lipocortin 1 inhibits tumour necrosis factor‐induced apoptosis in human leukaemic cells: a possible mechanism of resistance to immune surveillance

The signal transduction pathway through which tumour necrosis factor (TNF) induces apoptosis in leukaemic cells may involve activation of cytosolic phospholipase A2 (cPLA2). The steroids dexamethasone (Dex) and 1,25(OH)2 D3 both render U937 leukaemic cells resistant to TNF‐induced apoptosis. In this study, we found that Dex inhibited both spontaneous and TNF‐induced activation of cPLA2. Dex had no direct effect on cellular cPLA2 levels, but facilitated cPLA2 degradation upon subsequent stimulation of cells with TNF. In addition, Dex increased synthesis of the endogenous cPLA2 inhibitor lipocortin 1 (LC1). An antisense oligonucleotide to LC1 could completely abrogate Dex‐induced resistance to the cytotoxic action of TNF. Constitutive LC1 levels were relatively higher in myeloid leukaemic blasts showing resistance to TNF than TNF‐sensitive myeloid leukaemic cell lines. Our data suggest that Dex confers the resistance of U937 cells to TNF‐induced apoptosis by upregulating intracellular levels of LC1 and by facilitating a negative‐feedback loop, which is activated upon stimulation with TNF. High constitutive levels of LC1 in leukaemic blasts may protect them against immune‐mediated killing.

The anti-leukaemic effects and the mechanism of sodium selenite

The anti-proliferative effects of selenium were studied both in vivo and in vitro. At a selenium concentration of 0.6 micrograms/ml, cells from patients with ALL-L1, L2 and AML-M1, M3 and M5 were more sensitive than cells from patients with CML. Cells from patients with AML-M2, CLL and leukaemic lymphoma were least sensitive. Normal bone marrow or peripheral blood cells were not sensitive to selenium at this concentration. In the mouse leukaemia models (L797, L615, L7712), the sensitivity of leukaemic cells were: L797 (93% cytotoxicity) greater than L615 (49.7% cytotoxicity) greater than L7712 (4.4% cytotoxicity). Sodium selenite injected i.p. increased the longevity of L797-inoculated mice. Administration of 40 micrograms selenium daily for 7 days resulted in a significant increase in the longevity of mice inoculated with 10(5) L797 cells. However, no remarkable increase of the longevity was observed in either L615- or L7712-inoculated mice after treatment with sodium selenite for 7 days. Treatment of the HL-60 leukaemic cell line with selenium caused a dose- and time-related decrease in DNA, RNA and protein syntheses as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. The inhibitory effect of selenium on DNA synthesis was reversed when selenium was removed from the medium, demonstrating that selenium-induced inhibition of DNA synthesis was due to interference with DNA biosynthesis rather than DNA template damage. These results suggest that the anti-leukaemic effect of sodium selenite is associated with inhibition of DNA replication, transcription and translation.

Modulation of surface TNF expression by human leukaemic cells alters their sensitivity to exogenous TNF.

In this study, U937 leukaemic cells underwent apoptotic cell death following exposure to TNF. Pre-incubation of cells for 48 h with VitD(3) (10(-8)M) induced resistance to TNF, whereas incubation with tau-IFN or GM-CSF increased susceptibility to TNF. Resistance to exogenous TNF (exTNF) following culture with VitD(3) was associated with increased expression of endogenous TNF (enTNF). The TNF inhibitors pentoxifylline(PTF) and dichloroisocoumarin (DCI) inhibited TNF synthesis by U937 cells and abrogated the increase in resistance to TNF seen with VitD(3). The tau-IFN increased TNF expression, whereas GM-CSF had little effect. The data show that the sensitivity of leukaemic cells to exTNF can be modulated by cytokines. The protective effect of VitD(3) is mediated in part by directly upregulating enTNF synthesis.

TNF-mediated killing of human leukaemic cells: Effects of endogenous antioxidant levels and TNFα expression in leukaemic cell lines

Using the human erythroleukaemic cell line K562 cl.6 and its daunorubicin-resistant subline K/DAU600, and the human T-lymphoblastic leukaemic cell line CCRF-CEM and its vinblastine-resistant subline CEM/VLB100, we have shown that the drug-resistant cell lines were more sensitive to cytotoxicity induced by tumour necrosis factor-alpha (TNF alpha). Drug-resistant cell lines showed increased activities of copper/zinc superoxide dismutase (Cu/ZnSOD) and catalase compared with their parental drug-sensitive cell lines. However, the greater susceptibility of drug-resistant cells to TNF alpha cytotoxicity was, in part, related to their decreased activities of manganese superoxide dismutase (MnSOD). Persistence of this differential sensitivity when MnSOD is inhibited by sodium nitroprusside (SNP) suggests that the greater susceptibility of drug-resistant cells to TNF alpha was not entirely due to their decreased level of MnSOD activity. K562 cl.6 and K/DAU600, which were more resistant to TNF alpha, both expressed greater levels of endogenous plasma membrane-bound TNF alpha than the CCRF-CEM cell line. All cell lines examined were (more or less) equal in susceptibility to the cytolytic effect of exogenous O2-. generated by xanthine/xanthine oxidase. These results demonstrate that both MnSOD and endogenous TNF alpha play a role in protecting leukaemic cells against TNF alpha cytotoxicity, but there is an unknown mechanism that causes drug-resistant cells to be more susceptible to TNF alpha cytotoxicity.

Characterization and modulation of drug transport kinetics in K562 cl.6 daunorubicin‐resistant cell line

Summary. The effects of cyclosporin A (CSA) and cellular energy depletion on daunorubicin (DAU) transport kinetics were investigated in a human erythroid leukaemia cell line K562 c1.6 selected for resistance to daunorubicin. K562 c1.6/DAU resistant cells displayed high levels of P‐glycoprotein and a high level of multidrug resistance against several antitumour drugs. The resistance factors of K562 c1.6/DAU cells to DAU, doxorubicin, vinblastine and etoposide were 106, 114, 85 and 13 respectively. A 1·6‐fold decrease (P<0·01, n= 8) in DAU accumulation and a 4‐fold increase (P<0·001, n= 8) in DAU efflux were shown in the resistant cells when compared to K562 c1.6 drug‐sensitive parental cells. K562 c1.6/DAU cells were also shown to reach a DAU saturation level (SL) 8‐fold faster (P<0·001, n= 8) than the parental cells. Addition of CSA to the resistant cells led to a dose‐dependent increase in cellular DAU retention, while no such effect was observed in the sensitive cells by the introduction of CSA. Resistance to the antitumour drugs could be reduced to various extents by CSA. The patterns of changes and modulations of DAU transport kinetics, as well as chemosensitivity in K562 c1.6/DAU cells were found to be similar to a vinblastine‐resistant leukaemia cell line CEM/VLB100. However, K562 cl.6/DAU cells were mere resistant to DAU, doxorubicin and etoposide than the CEM/VLB100 cells. An increase in DAU accumulation, intracellular SL and the time to reach 90% saturation level (SL90), and a decrease in DAU efflux in the resistant but not the sensitive cells were found in response to ATP depletion by sodium azide. These effects could be completely reversed by addition of glucose. Our results suggest that the presence of an energy‐dependent effluxing mechanism responsible for the decreased drug accumulation and enhanced drug efflux may make a major contribution to the mechanism of resistance in K562 c16/DAU resistant cells.

Telomeres, Telomerase and Leukaemia.

There is increasing evidence supporting the hypothesis that telomere shortening both in vitro and in vivo is the clock that counts cell divisions and determines the onset of cellular senescence. Cells overcome the senescence mechanisms by stabilising telomere length, probably due to the activity of telomerase that specifically elongates telomeres. The striking observation that almost all malignant cancers have telomerase activity indicate that there is intensive selective pressure of telomerase activation with the progression of malignancy. Indirect support for this view is that benign or pre-cancerous lesions are telomerase silent. The fact that telomerase activity is observed in over 85% of human primary malignancies raises the possibilities that it may be a new marker of cancer with diagnostic and therapeutic potentials. Can such ideas be applied to leukaemias and preleukaemias? Since normal haematopoietic stem cells and their progeny express telomerase activity, it is important to consider whether or not telomere shortening and telomerase activity play any role in leukaemic progression. Telomere reduction has been observed in various leukaemias including in ALL, AML, transformed leukaemias from MDS and late stage of CML and CLL and might be indicative of the length of the disease. Elevated telomerase activity has also been found in ALL, AML, CML and CLL. In AML, patients with chromosomal abnormalities 11q, -5, -7 had higher telomerase activity and unfavourable prognosis; while those with favourable cytogenetics such as t(8:21), inversion 16 showed low levels of telomerase. This suggests that telomerase activity may be a marker for poor prognosis of AML. Unanswered at present is the potential role of telomeres and telomerase in the progression of benign to malignant tumours. Further studies on the expression and regulation of the individual components of telomerase may enable us to clarify the diagnostic and therapeutic potential of telomerase in leukaemias.