Cytotoxicity in patients with different clinical forms of Chagas' disease

There are few studies on cell-mediated cytotoxicity in human Chagas' disease. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) cytotoxicity activity from chagasic patients with different clinical forms of disease. To verify the cytotoxic response, we performed cell lysis assays using 51Cr-labelled K562 cells as targets. Results are reported as lytic units (LU = number of cells required for 30% lysis) per million PBMC. Exposure of patients' PBMC to Trypanosoma cruzi antigen led to an increase in cytotoxic activity compared with unstimulated patient cells against K562. Asymptomatic cardiomyopathy patients had higher responses (37.8 +/- 5.0 LU/10(6) PBMC; mean +/- s.d.) than indeterminate (11.5 +/- 3.6 LU/10(6) and symptomatic cardiomyopathy (7.8 +/- 2.5 LU/10(6)). Normal control PBMC stimulated with T. cruzi antigen had 4.36 +/- 1.31 LU/10(6)) PBMC against K562. Addition of recombinant interferon-gamma (IFN-gamma) did not lead to significant increase in cytotoxicity in any group of patients. On the other hand, recombinant human IL-12 significantly increased cytotoxic responses from symptomatic cardiomyopathy patients and normal controls who presented low levels of cytotoxicity induced by T. cruzi antigen. The combined use of IL-12 and a neutralizing anti-IFN-gamma antibody did not change IL-12-induced cytotoxic responses, showing the direct role of this cytokine on natural killer (NK) cells. NK cells were the main cells responsible for the lysis of K562 target cells as evidenced by testing cell subsets following magnetic cell sorting. These data demonstrate that chagasic patients with different clinical forms of disease have PBMC which respond to T. cruzi antigen with a cytotoxic response, and this response is up-regulated by IL-12.     

Teratogenic effects and distribution of cadmium (Cd2+) administered via osmotic minipumps to gravid CF-1 mice

Cytotoxic T lymphocytes (CTL) specific against autologous human cervical cancer cells were generated in vitro from peripheral blood leukocytes (PBL) from four patients with non-keratinized epidermoid carcinoma. For this purpose, these patients' PBL were co-cultured for 28 days either with IL-2 or a mixture of IL-2, IFN-gamma and TNF-alpha in the presence of autologous tumour cells (ATC). Our results showed that these CTL were highly cytotoxic for ATC, weakly cytotoxic for heterologous cervical cancer tumour cells, and not cytotoxic for carcinoma cell lines, normal cervix cells nor autologous PBL. Proliferation and cytotoxicity against ATC were greater when the PBL were activated with the three cytokines. These CTL had a CD4:CD8 ratio of 1:1, were CD16- and CD45RO+ and their killing activity was inhibited by antibodies against CD3, CD8 and MHC-class I but not by antibodies against CD4, CD16 or HLA-class II. The possibility of generating specific CTL in long term cultures for cervical cancer therapy is also discussed.     

Single- and multiple-dose pharmacokinetic comparison of a sustained-release tablet and conventional tablets of naproxen in healthy volunteers

We investigated the cytotoxicity and cellular pharmacology of idarubicin (IDA), idarubicinol (IDAol) and daunorubicin (DNR) in K562/VP-H2 cells, which show topoisomerase II-related multidrug resistance but do not overexpress P-glycoprotein. K562/VP-H2 cells were less resistant to IDA and IDAol than to DNR. There was no significant difference in the accumulation of each drug between K562 and K562/VP-H2 cells. The cleavage of DNA induced by each drug was decreased in K562/VP-H2 cells, however, the decrease in cleavage in K562/VP-H2 cells was less with IDA and IDAol than with DNR. These results suggest that IDA and IDAol have more cytotoxic potency than DNR in topoisomerase II-related multidrug-resistant leukemia cells.     

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.     

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.     

Dietary iron overload as a risk factor for hepatocellular carcinoma in Black Africans

We analyzed the effect of high temperature (a 1-h incubation at 43 degrees C) on the accumulation and cytotoxicity of vinblastine and docetaxel in two model cell lines, K562 and MESSA, and their multidrug resistance (MDR) counterparts, K562/R7 and MESSA/Dx5. High temperature increased the amount of intracellular vinblastine and docetaxel significantly in MESSA cell and, to a much lesser extent, in K562 cells. MDR-positive cells retained a profound drug accumulation defect at 43 degrees C. Hyperthermia enhanced the cytotoxic effect of vinblastine (but not docetaxel) in both K562 and MESSA cells, but not in the MDR-positive variants. PSC833, a potent modulator of P-glycoprotein, induced high levels of drug accumulation in the two MDR-positive cell lines at both 37 degrees C and at 43 degrees C. PSC833 also significantly reduced the resistance levels of the two MDR-positive lines at both 37 degrees C and at 43 degrees C. The effect of hyperthermia on drug accumulation thus seems to depend on the cell line, whereas the effect on cytotoxicity depends on the type of compound. The MDR phenotype remains a therapeutic obstacle at 43 degrees C but is accessible to modulation.     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.     

Effect of intravenous immunoglobulin G on natural killer cell cytotoxicity in vitro in women with recurrent spontaneous abortion

Intravenous immunoglobulin (IVIg) has been used to treat women with recurrent spontaneous abortion (RSA), particularly for women with elevated natural killer (NK) cells. We investigated the effect of IVIg on peripheral blood NK cell activity in vitro in women with RSA. 51Cr-release assays using K562 in the presence of varying concentrations of IVIg were performed using PBL from 16 women with RSA. Antibody dependent cellular cytotoxicity (ADCC) was evaluated using Daudi cells. Effectors and targets were preincubated with IVIg. Binding of IVIg to K562 and Daudi was evaluated by flow cytometry. The effect of K562 absorbed IVIg on NK activity was compared to that of non-absorbed IVIg. NK cytotoxicity and ADCC in the presence of F(ab')2 fragments were compared with those in the presence of intact IVIg. IVIg produced a significant, dose dependent inhibition of NK activity in vitro. Inhibition of NK activity occurred when effectors but not targets were preincubated with IVIg. IVIg binds to K562 and Daudi. IVIg increased ADCC when targets but not effectors were incubated with IVIg. K562 absorbed IVIg produced more inhibition of NK cytotoxicity than non-absorbed IVIg. Suppression of NK cytotoxicity by F(ab')2 was as effective as that of IVIg. However, F(ab')2 did not increase ADCC. IVIg effectively reduces peripheral blood NK cytotoxicity in vitro. Inhibition of NK cytotoxicity is mediated at the effector cell level through the antigen binding portion of the immunoglobulins. Women with RSA and elevated NK cells may benefit from IVIg treatment.     

Halogen-substituted trimetoquinol analogs as thromboxane A2 receptor antagonists in platelets and aorta

Trimetoquinol (TMQ) is a non-prostanoid compound that blocks prostaglandin H2/thromboxane A2 (TXA2) receptor-mediated responses initiated by a prostaglandin (PG) H2 analog, U46619, in human platelets and rat aorta. Ring fluorine-substituted TMQ analogs selectively antagonized PG-dependent human platelet activation induced by U46619, arachidonic acid, collagen, ADP or epinephrine; and were about 300-fold less potent as inhibitors of PG-independent responses mediated by thrombin or bacterial phospholipase C. For each inducer of the PG-dependent pathway, the rank order of inhibitory potency was identical (TMQ > 8-fluoro-TMQ > 5-fluoro- TMQ). Iodine substitution yielded a similar rank order of antagonism against U46619-induced platelet activation (TMQ > 8-iodo-TMQ > 5-iodo-TMQ), and all TMQ analogs inhibited platelet aggregation in whole blood as well as in platelet-rich plasma. Inhibition of specific [3H]SQ 29,548 binding by TMQ analogs was highly correlated with inhibition of functional responses to U46619. Radioligand binding experiments using TMQ analogs with rat platelets showed no interspecies difference in comparison with human platelets. The rank order of inhibitory potencies for the fluorinated (but not iodinated) TMQ analogs changed in rat thoracic aorta with 8-fluoro-TMQ > TMQ > or = 5-fluoro-TMQ as antagonists of U46619-induced vascular contraction. These findings demonstrate that the primary mechanism of antiplatelet action of TMQ analogs is related to a blockade of TXA2 receptor sites, and ring-halogenated TMQ analogs distinguish between TXA2-mediated functional responses in vascular smooth muscle and platelets.     

A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood

Myeloablative chemo-/radiotherapy supported by transplantation of autologous bone marrow or blood progenitor cells for acute leukemia, lymphoma, or myeloma continues to be associated with a high relapse rate because of the infusion of malignant stem cells and the lack of an in vivo graft-vs.-leukemia (GVL) effect. Although various methods of purging are established for marrow, purging procedures for blood progenitor cell preparations have not been widely used primarily because of the technical challenges to process a higher number of cells. As a broadly applicable method for immunological purging, we tested whether highly cytotoxic cells from a natural killer (NK) cell line characterized previously (NK-92) could be used for immunological purging of blood preparations. The NK-92 cell line, which was established from a patient with non-Hodgkin's lymphoma, can lyse in vitro a broad range of leukemia, lymphoma, and myeloma cell lines even at very low effector:target (E:T) ratios; this lysis is superior to cytotoxicity obtained from normal peripheral blood mononuclear cells (PBMCs) stimulated for 4 days with interleukin (IL)-2. In an attempt to quantitate the purging achievable with NK-92 cells, normal PBMCs were spiked with 10% K562 cells that had been transfected with the neo(r) marker gene (K562-neo(r). Various numbers of NK-92 cells were then added to the cell mixtures, which were incubated for 4 or 48 hours at 37 degrees C with or without IL-2 (500 U/mL). In order to prevent their proliferation, NK-92 cells were irradiated with 1000 cGy (cesium source). This radiation dose was determined to suppress proliferation of NK-92 cells, but at the same time maintain full cytotoxic activity. After co-culture, the cells were plated in methylcellulose containing 0.8 mg/mL G418. The number of surviving K562-neo(r) colonies was counted under the microscope 7 days later and the results were considered a quantitative readout for the purging efficacy of NK-92 cells. No neomycin-resistant K562 colonies could be detected up to a ratio of NK-92:K562-neo(r) cells of 5:1 (effective NK-92:PBMC ratio of 0.5:1). The presence or absence of IL-2 during the culture period did not affect the results. At this ratio of NK-92:PBMC, the growth of normal clonogenic hematopoietic progenitor cells was not compromised as determined by a standard methylcellulose assay. Considering that K562 is a rapidly proliferating cell line and that the input number of K562 cells (10%) tested here was high, the data suggest that the cytotoxic NK-92 clone (after irradiation to prevent proliferation) could be used effectively for immunological ex vivo purging without compromising hematopoietic cell function.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

Attachment of Trypanosoma cruzi epimastigotes to hydrophobic substrates and use of this property to separate stages and promote metacyclogenesis

Sphingosine and its analogs, which inhibit protein kinase C (PKC), are known to be potent inducers of apoptosis in tumor cells. However, we were concerned that sphingosine might also interfere with anti-tumor cells of the immune system. Therefore, we evaluated the effect of sphingosine on activation of human monocytes by interleukin-2 (IL-2) for killing of leukemic cells. Monocytes, purified by elutriation and adherence, were activated with IL-2 or interferon-gamma (IFN-gamma) in the presence or absence of sphingosine or another inhibitor for 18 h. Then the monocytes were washed and the culture medium was replaced with fresh medium to remove the sphingosine. HL- 60 and K562 leukemic cells were added to the monocyte cultures. Over the next 48 h, the cytotoxic activity of the monocytes towards the leukemic cells was assessed by means of an 111-indium-releasing assay. IL-2-activated monocytes lysed 48 +/- 3% of HL-60 cells and 44 +/- 3% of K562 cells. Sphingosine, dihydrosphingosine, N,N-dimethylsphingosine, and the PKC inhibitor H7 inhibited the activation of monocytes by IL-2, blocking cytotoxic activity against the leukemic cells by approximately 75%. These inhibitors were not toxic to monocytes at the concentrations used. In a PKC assay, sphingosine and H7 inhibited PKC activity in IL-2-treated monocytes. Thus, sphingosines, by inhibiting PKC activity, inhibited activation of monocytes by IL-2, which inhibited the killing of leukemic cells.     

SR 4233 (tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide, WIN 59075, tirapazamine) is the lead compound in a new class of bioreductive anticancer drugs, the benzotriazine di-N-oxides. It is currently undergoing Phase I clinical testing. The preferential tumour cell killing of SR 4233 is a result of its high specific toxicity to cells at low oxygen tensions. Such hypoxic cells are a common feature of solid tumours, but not normal tissues, and are resistant to cancer therapies including radiation and some anticancer drugs. The killing of these tumour cells by SR 4233, particularly when given on multiple occasions, can increase total tumour cell killing by fractionated irradiation by several orders of magnitude without increasing toxicity to surrounding normal tissues. Topics covered in this review include the rationale for developing a hypoxic cytotoxic agent, the cytotoxicity of SR 4233 as a function of oxygen concentration, the mechanism of action of the drug and its intracellular target and the in vivo evidence that the drug may be useful as an adjunct both to radiotherapy and chemotherapy. Finally, the major unanswered questions on the drug are outlined.     

Pseudolesions in liver sonography

Two different molecular forms of cholecystokinin (CCK) were studied with respect to their modulatory effect on non-MHC restricted or natural killer (NK) cell cytotoxicity. NK activity of peripheral blood mononuclear cells was found to be hardly affected by co-incubation with either CCK-8 or CCK-33 within a physiological concentration range against K-562 or Caco-2 tumour target cells. NK activity by lamina propria mononuclear cells isolated from histologically normal intestinal mucosa was found to be enhanced dose-dependently by incubation with CCK-8 in the 4-h assay against K-562, but not in the prolonged 18-h assay or against Caco-2 targets. In contrast, CCK-8 showed a tendency to inhibit NK activity in the prolonged 18-h assay against K-562; however, these alterations were not found to be statistically significant. CCK-33 was not found to modulate the NK activity of lamina propria mononuclear cells. It is suggested that NK activity by lamina propria mononuclear cells may be stimulated preferentially by CCK-8 because this molecular form of CCK in particular predominates in the nervous tissue of the intestinal mucosa.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

Novel cytotoxic diterpenes from the stem of Dysoxylum kuskusense

Three novel diterpenes, dysokusones A (1), B (2), and C (3), were isolated from the stem of Dysoxylum kuskusense as cytotoxic substances. The structures were established by spectroscopic examinations. Compounds 1, 2, and 3 were cytotoxic toward HL-60(TB) cells with EC50 values of 2.25, 6.35, and 2.37 microM, respectively. Compound 1 also displayed cytotoxicity against K-562 and NCI-H522 cells with EC50 values of 5.04 and 4.80 microM, respectively.     

Stimulation of NK-like YT cells via leukocyte function-associated antigen (LFA)-1. Possible involvement of LFA-1-associated tyrosine kinase in signal transduction after recognition of NK target cells

The YTA-1 anti-LFA-1 alpha mAb activates protein tyrosine kinase (PTK), augments NK cytotoxicity, and induces proliferation of fresh CD3- large granular lymphocytes. We demonstrate here that LFA-1 is physically associated in the YT human NK-like cell line cells with a PTK(s) that is distinct from Src family PTKs such as Lck, Fyn, or Lyn. In vitro kinase assays revealed similar association of protein kinase activity with LFA-1 in normal CD3- large granular lymphocytes. Tyrosine phosphorylation of the proteins associated with LFA-1 drastically increased in YT cells after stimulation with NK-sensitive K562 cells but not with NK-resistant P815 cells. Furthermore, pretreatment of YT cells with TS1/22 anti-LFA-1 alpha and TS1/18 anti-LFA-1 beta mAbs abrogated not only the cytotoxicity against K562 cells but also an increase in tyrosine phosphorylation of LFA-1-associated molecules induced by K562 stimulation. These results provide biochemical evidence that the PTK(s) associated with LFA-1 is involved in the signal transduction that follows the recognition of NK target cells.     

The influences of height and age on waist circumference as an index of adiposity in adults

Using a doxorubicin-resistant subline (K562/ADM) of human leukaemia K562 cells (Tsuruo et al, 1986), the effect of immunoliposomes that targeted a cellular transferrin receptor (TFR) was examined by neutralization of doxorubicin (DOX) resistance. OKT9-CIL, prepared by conjugation of DOX-encapsulated liposome with an anti-TFR monoclonal antibody, OKT9 (Aisenberg and Wilkes, 1980), showed similar binding to both K562 and K562/ADM. Although an 80-fold higher sensitivity to free DOX on cell growth inhibition in K562 than in K562/ADM was found, the difference was clearly diminished after OKT9-CIL treatment through the increased sensitivity of K562/ADM. The cellular DOX level 30 min after the exposure of free DOX was 45-fold lower in K562/ADM than in K562, whereas nearly equivalent DOX levels were detected in K562 and K562/ADM after OKT9-CIL treatment. In addition, DOX in K562/ADM in the free DOX treatment was efficiently excreted by 54% within 120 min of incubation, whereas almost all DOX supplied by OKT9-CIL remained uncleared. Fluorescence microscopic observation showed that OKT9-CIL was internalized into juxtanuclear vesicles in K562/ADM cells. These results suggest that OKT9-CIL has a potency to accumulate DOX, resulting in augmentation of DOX cytotoxicity in DOX-resistant tumour cells.     

Modulation of NK-target cell interaction by a monoclonal antibody to K562 cells

In order to identify the target cell recognition molecules involved in the interaction between natural killer (NK) cells and target cells, we have generated monoclonal antibodies to K562, NK-sensitive target cells. After screening by FACScan for the reactivity to K562, one monoclonal antibody (mAb), 4A60, was selected. MAb 4A60 was found to inhibit the proliferation of NK cells induced by IL-2 and K562 cells. However, this monoclonal antibody could not significantly block the conjugate formation between NK and target cells. Moreover, mAb 4A60 only slightly inhibited the cytotoxicity of NK cells induced by IL-2. Protein analysis showed that mAb 4A60 recognized a 53-kDa protein of K562 cells. Taken together, these data suggest that mAb 4A60 inhibits the proliferation of NK cells induced by IL-2 and target cells, and the 53-kDa protein, a tentative ligand of this mAb of K562, may be involved in this process.     

Homocamptothecins: synthesis and antitumor activity of novel E-ring-modified camptothecin analogues

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered beta-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-mediated activity, is an attractive template for the elaboration of new anticancer agents. Like CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of Topo I. The preparation and biological screening of racemic hCPT analogues are described. The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c, d,f,g were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts. In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of 0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor cells and promising in vivo efficacy.