Apoptosis may be either suppressed or enhanced with strategic combinations of antineoplastic drugs or anti-IgM.

A variety of drugs have been used to treat B-lymphocyte neoplasms, including both cell cycle-specific (CCS) and non-cell-cycle-specific drugs. Although the therapy for such cancers is complex and can include both types of drugs, the efficacy of these drugs in inducing cell death remains unclear. In this paper we have concentrated on specific CCS drugs and have examined their ability to induce programmed cell death (apoptosis) in Burkitt's lymphoma cell lines derived from patients. The CCS drugs chosen were hydroxyurea and aphidicolin (active in late G1, early S phase), the topoisomerase poisons camptothecin and etoposide (S, early G2 phase) and vincristine and Taxol (late G2, M phase). These choices allow comparison of two drugs with differing modes of action for each of the various phases of the cell cycle. Our results indicate that the variation in apoptosis between drugs that act at the same phase of the cell cycle is negligible. Both S/G2 and G2/M blockers are very potent at inducing apoptosis whereas G1/S blockers are ineffective in the induction of apoptosis. In addition, marked kinetic variations in the rate of apoptosis induction were observed, etoposide and camptothecin being more rapid in their action than the other agents. The order of effectiveness in inducing apoptosis on a kinetic basis was S/G2 agents >> G2/M agents >> G1/S agents. In this study we have also found that growth inhibition was induced by all the CCS agents chosen and by anti-IgM in various Burkitt's lymphoma lines. Furthermore c-myc was down-regulated under similar conditions. Since apoptosis was only selectively induced by some of the CCS agents, it implies c-myc expression is associated with growth regulation and c-myc down-regulation is an insufficient condition for the induction of apoptosis. In addition, cotreatments using the CCS and other agents revealed the following: Cotreatment using two CCS drugs which act at the same stage in the cell cycle showed either no change or only additivity to the effects seen with either agent alone. However, cotreatment with CCS drugs showed that an inhibitory effect is found between G1/S and G2/M drugs or S/G2 and G2/M drugs. No effect was found between G1/S and S/G2 drugs. Anti-IgM, which by itself was capable of inducing apoptosis, was observed to augment apoptosis induced by very low concentrations of G2/M-acting drugs but it has little effect on G1/S or the S/G2 drugs. The inhibitory effect of anti-CD40 or TNF-alpha on anti-IgM-induced apoptosis did not carry over to an effect on apoptosis induction by the CCS agents. Thus specific combinations of agents may lead to either enhancement, inhibition, or no interactive effect on apoptosis.

Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro.

The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytes maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL-11 (10 ng/ml), or IL-6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum-free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin-3 (IL-3), LIF, IL-6, and IL-11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-11 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3+IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-11, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and IL-11 promote murine and human Mk maturation in vitro, although the effect of IL-11 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on Mk ploidy, IL-11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3.

Characteristics of a novel rat anti-mouse platelet monoclonal antibody: application to studies of megakaryocytes.

Using murine platelets as an immunogen, a rat monoclonal antibody (designated 4A5) that recognizes only murine blood platelets and marrow megakaryocytes was developed. The extent of binding of 4A5 to platelets was dependent upon their state of activation. Following phorbol ester, ionophore, or thrombin stimulation of resting platelets, a decrease of > 50% in the binding of 4A5 was observed by flow cytometry. This decrease in antibody binding to the platelets was accompanied by an increase in antibody released into the platelet-free supernatant following platelet activation. When platelets were first radioiodinated, followed by activation and incubation of the platelet-free supernatant with 4A5-derivatized beads, no precipitable counts were observed compared with control resting platelets. This suggests that antibody release was related to an activation-dependent conformational change in the 4A5 epitope. Following solubilization of biotinylated platelets, 4A5 bound to an 80-kd membrane protein. Immunohistochemical studies with 4A5 showed that megakaryocytes could be identified both in vitro and ex vivo. When marrow was first stained histochemically with 4A5 followed by staining for acetylcholinesterase, the distribution of stained cells was similar. Flow cytometric analysis using 4A5 and propidium iodide showed that the antibody could be used to identify megakaryocytes for ploidy analysis in vivo or in vitro. 4A5 was capable of inducing a moderate thrombocytopenia in mice compared with polyclonal anti-platelet serum. When bound to plastic or to magnetic beads, 4A5 could be used to purify murine megakaryocytes to homogeneity. The data suggest that monoclonal antibody 4A5 will be useful in quantitative studies of murine platelets and megakaryocytes.

Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6.

Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long-term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.

Predictive validity of a national examination for medical graduates in the People's Republic of China.

National examinations for medical graduates were introduced on an experimental basis in the People's Republic of China in 1982. To estimate the predictive validity of the National Medical Examination (NME), an investigation of the postgraduate competence of a sample of the participating examinees was conducted in 1984. The sample consisted of 1,717 of the 4,995 graduates from 13 medical colleges who had taken the initial NME. Their scores on the NME and the ratings given them by directors of postgraduate programs in nine aspects of clinical competence were compared by frequency distribution and product-moment correlation coefficients. Scores on the NME were consistent with measures of postgraduate clinical competence and, as a whole, correlated significantly with the ratings of clinical competence, supporting the use of the score on the NME as a predictor of postgraduate clinical competence. However, the extent of the relationship between the NME score and postgraduate clinical competence varied according to the specialty program of postgraduate medical training.