WIN55,212-2 induces cytoplasmic vacuolation in apoptosis-resistant MCL cells.

Cannabinoid receptors 1 (CB1) and/or 2 (CB2) are overexpressed in many types of human malignancies including mantle cell lymphoma (MCL). Agonists to CB1 and CB2 promote ceramide de novo synthesis, p38-mitogen-activated protein kinase-dependent activation of caspase-3 and apoptotic cell death in most MCLs. However, in this report we describe that in some MCLs the response to treatment with cannabinoids decreased cell viability as assessed by metabolic activity but did not involve the caspase-3 cascade or loss of plasma membrane integrity. Both primary cells from one MCL patient and the MCL cell line Granta519 responded to treatment with cannabinoids by formation of cycloheximide-sensitive cytoplasmic vacuoles, but did not enter apoptosis. The persistent expression of mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3 II (LC3 II) and p62, as well as the lack of protection from chloroquine, indicates that lysosomal degradation is not involved in this cytoplasmic vacuolation process, distinguishing from classical autophagy. Transmission electron microscopy images and immunofluorescence staining of endoplasmic reticulum (ER) chaperone calreticulin showed that the vacuoles were of ER origin and that chromatin remained normal. These features resemble paraptosis-like cell death-a third type of a programmed cell death not previously described in response to cannabinoids.

Freezing technique and quality of fresh-frozen plasma.

Cell-poor plasma was prepared by apheresis from 10 donors. From each donor, an amount of 200 ml was frozen rapidly to -40 degrees C in standard blood bags, and a further 200 ml was frozen slowly to -20 degrees C. Before freezing and after thawing, plasma samples were collected and frozen to -70 degrees C pending analysis. Coagulation factor VIII activity was reduced to 90% by rapid freezing and to 80% by slow freezing. Factor V was not influenced by rapid freezing, but slow freezing reduced the levels to 92% of the pre-freezing levels. In some of the plasma bags a slight increase in fibrinopeptide A occurred. However, soluble fibrin, thrombin-antithrombin complexes and spontaneous proteolytic activity were not altered by freezing. The beta-thromboglobulin increased slightly with slow freezing. Moreover, in a separate experiment, evaluating the possible effects of refreezing plasma samples, an increase in beta-thromboglobulin was also recorded, while the levels of factors VIII and V and von Willebrand factor were not affected. The changes in some variables, which were recorded in the cell-poor plasma, frozen soon after the blood donation at a slow freezing rate, must be regarded as insignificant in most clinical situations.