The main purpose of this work was to determine which of the multiple isoforms of acetylcholinesterase (AChE) are associated with clathrin-coated vesicles (CVs) from developing and adult rat skeletal muscles. CV-enriched preparations were obtained by subcellular fractionation/equilibrium sedimentation and further purified by immunoadsorption to anti-clathrin IgG-coated Staphylococcus aureus cells. Analysis of individual AChE isoforms by velocity sedimentation ultracentrifugation showed that a) while both globular (G-forms) and asymmetric (A-forms) AChE were detected in all subcellular fractions evaluated, the CV-enriched fraction contained a higher proportion of A-forms (mainly the A12 species); b) most of the AChE activity contained in such a CV fraction was recovered following immunoadsorption; c) alkaline extraction conditions (pH 8.5) which depolymerize clathrin were necessary to detect a large proportion of A-forms in both the CV-enriched and immunoprecipitated preparations, while most of the G-forms (especially G1 + G2 AChE) were detected following extraction at pH 6.8; and d) comparison of AChE isoform profiles from neonate and adult muscle CV-enriched fractions showed a greater concentration of A-forms in the former. These data suggest that considerable amounts of A12 and, to a lesser extent, G4 AChE are sequestered within muscle CVs which may be destined for the plasmalemma. Our findings also indicate that the relative proportions of AChE isoenzymes in rat muscle CVs vary according to the extent of muscle development and lend support to the contention that CVs participate in the externalization of functionally important AChE isoenzymes.
N-methyl-N-nitrosourea (MNU) induces thymic lymphomas in AKR mice after a 2-3 month latency. This study shows that hormonal factors profoundly influence MNU-induced lymphomagenesis. Tumor development is accelerated in females compared to males, regardless of whether a single high dose or multiple low doses of MNU are administered. Testosterone is implicated in this phenomenon, since castrated mice develop MNU-induced lymphomas with the same latency as intact females, while ovariectomized females have the same pattern of tumor development as intact females. Furthermore, reconstitution experiments demonstrated that testosterone replacement suppresses MNU-induced lymphoma development in castrated males. Although tumor development is delayed in male compared to female mice, sex does not influence tumor immunophenotype, clonality or the frequency of ras mutations in animals given identical MNU treatment protocols. In contrast, the frequency of ras mutations is dramatically altered depending on whether the animals are treated with a single high dose or multiple low doses of MNU. Nevertheless, there is no correlation between the presence of an activated K-ras allele and tumor latency. These data demonstrate that sex has a more profound influence on the progression of MNU-induced lymphomas than does the presence of an activated K-ras allele.
The mechanism of action of the novel anti-cancer compound CI-994 was studied in C26 murine colon tumor and HCT-8 human colon adenocarcinoma cells. Treatment of either cell line resulted in the specific loss of a 16-kDa phosphoprotein in a time- and concentration-dependent manner. Treatment with salicylanilide, CI-940, mimosine, aphidicolin, quercetin or ciclopirolxalamine, which, like CI-994, block cells in the G1-S phase of the cell cycle, did not affect the production of this protein. Loss of the 16-kDa protein preceded the block in cell proliferation induced by CI-994 treatment, and recovery of this protein was evident prior to the resumption of cell growth. Cellular fractionation studies demonstrated that the 16 kDa phosphoprotein is confined to the nuclear compartment. Our data indicate that loss the 16-kDa nuclear phosphoprotein appears to be a direct effect of CI-994 treatment and that the inhibition of this phosphoprotein may play a critical role in the mechanism of action of CI-994.
A major concern in autologous bone marrow transplantation (ABMT) is the possible contamination of the graft with tumor cells. Transplantation of malignant cells, along with normal hematopoietic stem and progenitor cells, may contribute to relapse of disease. Therefore, a growing strategy is to subject autologous marrow to some type of purging procedure to eliminate tumor cells selectively. Transplantation of purged marrow, however, often results in a delayed engraftment associated with (specific or nonspecific) loss of normal stem and progenitor cells during manipulations related to the purging process. A new and burgeoning field in the area of clinical bone marrow transplantation is the ex vivo production of stem and progenitor cells. Several advantages accrue to this strategy. First, this technology makes it possible to expand the stem and progenitor cell population of a small volume of bone marrow or mobilized peripheral blood (MPB), thus lessening the initial tumor burden to be purged. Secondly, ex vivo marrow or MPB expansion may overcome the significant problem of delayed engraftment by rebuilding the numbers of normal stem and progenitor cells necessary for both early and durable engraftment. To accomplish these and other objectives, an automated and closed, clinical-scale bioreactor system, based on continuous perfusion technology, is being developed and will soon enter clinical trials.
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