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Programmed cell death, also known as apoptosis, is a normal physiologic process which occurs during embryonic development as well as in maintenance of tissue homeostasis. Increasing evidence suggests that alterations in cell death contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases and acquired immunodeficiency syndrome (AIDS). The extraordinary research activity of the past few years has resulted in the characterization of the principal proteins involved in the apoptosis machinery. An area of particular interest has been the induction of apoptosis by two death receptor/ligand pairs, Fas/Fas Ligand and DR4-DR5/TRAIL. The identification of these molecules with the recruited signaling pathways could clarify their physiopathological implications, having a significant impact upon potential therapeutic interventions in diseases associated with cell survival alterations.

We have previously shown that Prolactin (PRL) activates the native and the in vitro acquired cytotoxicity and the DNA synthetic activity of Natural Killer (NK) cells. Here we show that the supernatant and the cell lysate of NK cells express a 35S-labelled 50 kDa peptide specifically immunostained by two different PRL-antisera. The supernatant of NK cells was biologically active in a Nb2 assay and the activity could be adsorbed by an anti-PRL antiserum. The production of the PRL-like peptide only occurred when NK cells were isolated through binding to immobilized immunocomplexes, the biological ligand for CD16, and was positively modulated by exogenous PRL. These results indicate that PRL, produced by NK cells following stimulation, may act in an autocrine fashion to maintain and/or activate the NK cell function.

PURPOSE In view of the pleomorphic role cytokines play in human lymphoproliferative disorders, we investigated the possible involvement of tumor necrosis factor-alpha (TNF) in hairy cell leukemia (HCL). PATIENTS AND METHODS The levels of TNF were measured in the serum of untreated patients, and in the culture supernatants of unstimulated and stimulated enriched hairy cells (HC). Furthermore, the presence of TNF mRNA transcripts in HC was analyzed. The possibility that HC could inhibit the in vitro growth of normal erythroid progenitors via the release of TNF was also investigated. Finally, in an attempt to correlate the circulating levels of TNF with the course of the disease, these were retested during and after treatment with interferon-alpha (IFN). RESULTS Significantly increased levels of TNF were found in the sera of untreated HCL patients compared with normal control sera were seen from patients with other diseases (P less than .001), with values greater than 10 pg/mL in 21 of 42 samples tested. A significant decrease (P less than .01) of TNF levels was recorded following IFN-2a administration in 16 cases with detectable pretreatment serum levels of TNF. In two cases, an increase in TNF values was associated with persistence or progression of disease. The likelihood that the circulating levels of TNF were caused by the pathologic cells is supported by the evidence that purified HC may release TNF spontaneously. The values can be markedly increased following in vitro activation with the phorbol ester 12-0-tetradecanoylphorbol-13 acetate (PMA), with B-cell growth factor (BCGF), and, to a further extent, with the combination of PMA and BCGF. Furthermore, the constitutive mRNA for TNF was found in seven of eight HC samples analyzed. Although supernatants of enriched HC, were capable of reducing the growth of normal bone marrow erythroid progenitors by 50%, duplicate experiments using an anti-TNF antibody produced an almost complete disappearance of the inhibitory effect. CONCLUSION The results of this study suggest that TNF plays an important role in the pathogenesis of the cytopenia(s) characteristically associated with HCL.

Pretreatment of acute myeloblastic leukemia cells with the hemopoietic growth factor interleukin 3 (IL3) increased their susceptibility to lymphokine activated killing (LAK) but did not affect their constitutive resistance to native natural killer activity. In addition, IL3 treatment did not alter the LAK cell-mediated killing of CD34+ hemopoietic progenitors present in normal bone marrow. Increased 3H-thymidine uptake was generally observed after IL3 treatment. However, failure to proliferate in response to IL3, observed in some cases, did not prevent changes in LAK susceptibility. Enhanced lysis of IL3-treated leukemic cells was accompanied by a moderate increase of the effector-target binding. Increased LAK susceptibility was already observed at 18 h, while optimal cytolysis and expression of the cell adhesion molecule (CAM) LFA-3 (CD58) by IL3-treated AML cells were concomitantly observed at later culture times. In contrast, the CAM ICAM-1 (CD54) was not modulated by IL3, nor were significant changes in the expression of either CAMs observed in normal hemopoietic cells. Blocking experiments with the anti-CD58 monoclonal antibody demonstrated a variable neutralizing effect on the IL3-induced increase of LAK activity, depending on the leukemia cell studied. The effect described here, together with the known role of IL3 in normal hemopoiesis makes it a factor of potential therapeutic value for the treatment of leukemic patients.

Freshly isolated B cells from chronic lymphocytic leukaemia patients (B-CLL) have been previously shown to induce a strong proliferative response and high levels of NK-like activity in lymphocytes from healthy donors. The present paper deals with the origin, mitotic state, target spectrum and cell surface phenotype of the NK-like effectors generated after stimulation with B-CLL. Experiments using large granular lymphocytes (LGL) and T cells as responders demonstrated that most of the precursors of the newly generated NK-like effectors express the CD3 antigen. The induction of NK-like activity paralleled cell activation, as judged by blast transformation, thymidine uptake and appearance of cell surface activation markers. The newly generated NK-like effectors displayed a T cell phenotype and a broader target repertoire than native NK cells.

Triggering of large granular lymphocyte (LGL) Fc receptor with a specific monoclonal antibody (AB8.28) linked to an insoluble matrix induces cell activation, as witnessed by expression of HLA class II (DR and DQ) molecules and interleukin 2 receptor. Moreover, this event is accompanied by a concomitant release of platelet-activating factor by LGL. We conclude that the Fc receptor molecule identified by mAb AB8.28 represents a trigger for LGL activation.

Natural Killer (NK) activity has been shown to be depressed under stressful conditions. Glucocorticoids, which are known to increase during stress, seem to negatively regulate the activity of NK cells. In the present study we have explored the effect of cortisol (hydrocortisone, HC) on NK activity. A significant inhibitory effect could be observed as early as 6 hr after the addition of HC at the concentration of 0.5 microM, corresponding to the upper physiological circulating level. Both the lysis and the binding of the K562 target cells were affected by HC, indicating that the hormone acts on the target recognition phase. The HC-mediated inhibition of the NK activity was fully reversed after 6 hr incubation in a HC-free medium. The observation of comparable levels of NK-inhibition using unseparated PBL or purified LGL, show that HC acts directly on LGL to inhibit their cytotoxic function. The effect of HC on the responsiveness of NK cells to the modifiers beta-interferon (beta-IFN) and recombinant interleukin 2 (rIL2) was also studied. Pre-incubation with HC did not alter the enhancement of the activity induced by beta-IFN, demonstrating that the HC- and beta-IFN-mediated effects occur in separate NK subsets. By contrast the increase of NK cytotoxicity induced by rIL2 was lower in the HC-treated compared to the untreated cell cultures (35.8 +/- 6.2 and 20.7 +/- 4.3% respectively, p less than 0.05) which could indicate that a portion of the cells triggered by rIL2 belong to the HC-sensitive NK subpopulation.

Multiple genetic aberrations contribute to the development of biologically aggressive, clinically malignant colorectal carcinomas (CRCs). Some of these have been linked to inappropriate signaling through the tyrosine kinase moieties of growth factor receptors. We have described previously (G. Bellone et al., J. Cell. Physiol., 172: 1-11, 1997) that human CRCs overexpress both the receptor tyrosine kinase c-kit and its ligand, stem cell factor (SCF), relative to normal mucosa cells, thus establishing an autocrine c-kit-mediated loop. In addition, we noted that exogenous SCF contributes to anchorage-independent growth of HT-29 colon carcinoma cells in semisolid medium. Here, we investigated possible roles of the c-kit/SCF autocrine/paracrine system in survival and invasive capacity of DLD-1 colon carcinoma cells. We report that SCF was required for migration and invasion of DLD-1 cells through reconstituted basement membranes (Matrigel) and up-regulated gelatinase (matrix metalloproteinase-9) activity in DLD-1 cells. Furthermore, we describe that SCF supported survival of DLD-1 cells in growth factor-deprived conditions. These results suggest multiple roles of c-kit activation in support of the malignant phenotype of DLD-1 cells related to growth, survival, migration, and invasive potential.

Here we present the characterization of a new antigen called CB02 which is present on B cells and on a subset of CD16+ large granular lymphocytes. The pattern of reactivity of this molecule on normal cells, B cell lines, leukemic cells and tissue sections suggests that the CB02 molecule is expressed by B cells before isotype switch and is lost during terminal differentiation. Furthermore, the CB02+ population encompassed natural killer (NK) cells exerting significant cytotoxic activity: in fact, peripheral blood lymphocytes depleted of the CB02+ subset display a marked reduction in NK activity.

Atractyligenin (1) and several synthetic derivatives were tested and found to be active against tumor cell replication. Compound 1 was readily converted to the 2,15-diketo (3) or 15-keto (4) derivatives, which contain an alpha,beta-unsaturated ketone. Compounds 3 and 4 showed significant cytotoxic activity against all six tested cancer cell lines and were most potent against 1A9 ovarian cancer cells with EC50 values of 0.2 and 0.3 microM, respectively. These two 1-analogues are promising lead compounds for further investigation.