Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol.

Flavopiridol (NSC 649890; Behringwerke L86-8275, Marburg, Germany), is a potent inhibitor of cyclin dependent kinases (CDKs) 1, 2, and 4. It has potent antiproliferative effects in vitro and is active in tumor models in vivo. While surveying the effect of flavopiridol on cell cycle progression in different cell types, we discovered that hematopoietic cell lines, including SUDHL4, SUDHL6 (B-cell lines), Jurkat, and MOLT4 (T-cell lines), and HL60 (myeloid), displayed notable sensitivity to flavopiridol-induced apoptosis. For example, after 100 nmol/L for 12 hours, SUDHL4 cells displayed a similar degree of DNA fragmentation to that shown by the apoptosis-resistant PC3 prostate carcinoma cells only after 3,000 nmol/L for 48 hours. After exposure to 1,000 nmol/L flavopiridol for 12 hours, typical apoptotic morphology was observed in SUDHL4 cells, but not in PC3 prostate carcinoma cells despite comparable potency (SUDHL4: 120 nmol/L; PC3: 203 nmol/L) in causing growth inhibition by 50% (IC50). Flavopiridol did not induce topoisomerase I or II cleavable complex activity. A relation of p53, bcl2, or bax protein levels to apoptosis in SUDHL4 was not appreciated. While flavopiridol caused cell cycle arrest with decline in CDK1 activity in PC3 cells, apoptosis of SUDHL4 cells occurred without evidence of cell cycle arrest. These results suggest that antiproliferative activity of flavopiridol (manifest by cell cycle arrest) may be separated in different cell types from a capacity to induce apoptosis. Cells from hematopoietic neoplasms appear in this limited sample to be very susceptible to flavopiridol-induced apoptosis and therefore clinical trials in hematopoietic neoplasms should be of high priority.

Detectable inhibition of heparin-binding growth factor activity in sera from patients treated with pentosan polysulfate.

BACKGROUND: Previous studies indicate that the heparinoid pentosan polysulfate (PPS) can inhibit heparin-binding growth factors (HBGFs) released from tumor cells and thus block tumor growth in animal models. However, because of its heparin-like activity, the major toxic effect expected for PPS is its inhibition of coagulation. PURPOSE: Our purpose was to determine if anti-HBGF activity could be achieved in patients without causing complications from anticoagulation. METHODS: We initiated a phase I trial in cancer patients and developed a cell proliferation assay to detect PPS in human serum based on its antigrowth factor activity. Blood samples from six healthy volunteers were collected in tubes containing different concentrations of PPS (FIBREZYM; concentration range, 0-10 micrograms/mL). Additional samples were obtained from four patients in the phase I trial before and after subcutaneous treatment with 15 mg/m2 of PPS. The activated partial thromboplastin time (aPTT), which is associated with coagulation, was measured in all blood samples. Serum prepared from the blood samples was heat inactivated and then incubated for 4-5 days with proliferating SW-13 cells, allowing determination of antigrowth factor activity. RESULTS: PPS added to blood samples increased aPTT only at concentrations above 1 microgram/mL, whereas HBGF-dependent proliferation was inhibited at less than 0.1 microgram/mL. Sera obtained from patients up to 4 hours after PPS treatment specifically inhibited HBGF-dependent cell proliferation by more than 65% even at a 1:10 dilution. At the same time, the aPTT was not altered in these patients, indicating no significant effect on coagulation by this dose of the heparinoid. CONCLUSIONS: HBGF-inhibitory concentrations of PPS can be achieved in patients' sera without significant effects on coagulation. IMPLICATION: The assay presented here could be useful to determine doses and scheduling of treatment in studies evaluating PPS as an antitumor agent.

Trichomonas vaginalis: alternative pathway activation of complement.

Serum from adult human donors lysed Trichomonas vaginalis. The lytic effect was eliminated by heat-inactivation of serum (56 degrees C, 30 min). No serum donor exhibited significantly increased antibody against the parasite as measured by indirect immunofluorescence. Treatment of serum with ethylene glycol-bis (B-aminoethyl ether)-N, N-tetraacetic acid to eliminate classical pathway complement activation did not prevent C3 leads to C3i conversion in serum incubated with T. vaginalis. Release of complement products during alternative pathway activation may contribute to pathogenesis of trichomonal vaginitis.

Immunization against Leishmania donovani: glucan as an adjuvant with killed promastigotes.

Mice were immunized by a series of intravenous injections of formalin-killed Leishmania donovani promastigotes alone and combined with glucan, a beta 1,3 polyglucose derivative of baker's yeast. In three separate experiments animals were challenged with viable parasites on day 21, 40 or 80 after immunization. Mice which received dead parasites and glucan exhibited resistance against challenge up to 80 days after immunization. Animals which had been injected with glucan alone exhibited a lesser degree of resistance but injections of killed promastigotes alone conferred no measurable resistance against infection.

Glucan-enhanced immunogenicity of killed erythrocyte stages of Plasmodium berghei.

Intravenous injections of glucan simultaneously with Formalin-killed erythrocytic stages of Plasmodium berghei elicited a greater degree of resistance in mice against subsequent infection with viable parasites than injections of killed erythrocytic stages alone. In two experiments with P. berghei strain NK 65, 100% of mice immunized with the glucan-dead parasite preparation survived challenge, whereas only 28.6% of mice receiving dead parasites alone survived. In the third experiment, using P. berghei strain NYU-2, the same proportion of mice survived after immunization with glucan and dead parasites as with dead parasites alone (i.e., 10 of 11 in each group), but mice immunized with the glucan-dead parasite preparation experienced parasitemias of significantly less intensity and shorter duration than mice which received only dead parasites before infection. Inoculation of glucan alone or with normal erythrocytes conferred no protection against challenge.

Activation of the alternative complement pathway by Naegleria fowleri.

Naegleria fowleri amoebae were lysed by adult fresh human serum, and their multiplication was inhibited in culture medium supplemented with 10% fresh human serum. Heat inactivation (56 degrees C, 30 min) of serum abrogated these lytic and inhibitory effects. Absorption of human serum with amoebae failed to reduce immunoglobulin levels, and no specific antibody was detected in untreated or treated sera by counterimmunoelectrophoresis. Conversion of C3 and C3i occurred after incubation of n. fowleri with serum which had been treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid, indicating activation of complement via the alternative pathway.

Naegleria fowleri in chick embryos. Effects of embryo age and incubation temperature, and the infectivity of embryo-derived amebae for mice.

Chick embryos were infected with Naegleria fowleri which was initially isolated from an ultimately fatal human case. Following inoculation of equivalent numbers of amebae on the chorioallantoic membrane, younger embryos died earlier than older embryos infected at the same time. Incubation of infected embryos at 32 degrees C prolonged survival only slightly in comparison with those at 37 degrees C. N. fowleri maintained for more than 25 serial passages in chick embryos retained infectivity for mice and the ability to convert to the biflagellate form in vitro.

The target tissue of limb-bud polarizing activity in the induction of supernumerary structures.

When polarizing mesoderm from the posterior border of the 4-day chick limb bud is placed adjacent to anterior limb mesoderm and ectodermal ridge, the anterior ridge thickens and mesodermal outgrowth ensues, resulting in supernumerary limb structures. This apposition of anterior and posterior limb tissues can be accomplished by cutting off the apical one third of the limb bud and reimplanting it on the stump with its anteroposterior axis reversed. The preaxial response to polarizing activity can be obtained after only 12--18 h in the reoriented position. Reversed apical mesoderm develops supernumerary digits when combined with untreated ectoderm. The reciprocal combination, reversed ectoderm and untreated mesoderm, fails to develop supernumerary structures. We have interpreted this as evidence that, in inducing supernumerary limb structures, polarizing activity acts only on the mesoderm.

Polarizing activity in the developing limb of the Syrian hamster.

The transplantation of small pieces of tissue from the limb buds of 9 1/2 -10 day hamster embryos to the wing bud of the chick results in the induction of supernumerary wing structures. The anteroposterior polarity of these induced structures is under the control of the transplanted hamster tissue. The developing hamster limb thus has limb polarizing activity similar to that found in avian species and, as in the chick, the activity is found primarily in the posterior region of the limb bud.