Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotypeselective HDAC inhibitor. We have developed an isotypeselective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted. [Mol Cancer Ther 2008;7(4):759 -68]

There is an important need for clinically relevant animal models for human cancers. Toward this goal, histologically intact human colon-cancer specimens derived surgically from patients were implanted orthotopically to the colon or cecum of nude mice. We have observed extensive orthotopic growth in 13 of 20 cases of implanted patient colon tumors. These showed various growth patterns with subsequent regional, lymph-node, and liver metastasis, as well as general abdominal carcinomatosis. Thus, models for human colon cancer have been developed that show (i) local growth, (it) abdominal metastasis, (iiM) general abdominal carcinomatosis with extensive peritoneal seeding, (iv) lymph-node metastasis, (v) liver metastasis, and (vW) colonic obstruction. These models permit the passage of the tumors to form large cohorts. They will facilitate research into the biology of colon cancer metastatic capability and the development of new drugs active against metastatic cancer. These models may also predict the clinical course and the in vivo response to drugs of the cancer of individual patients.There is a need for the development of better animal models for human cancer. Models based on athymic nude mice have been used for this purpose. However, metastatic rates from subcutaneous or intramuscular xenografts have been low or nonexistent even from tumors that were highly metastatic in the patient from whom the tissue was derived (1-5).Recent work from a number of laboratories has indicated that implanting human tumor cells orthotopically in the corresponding organ of nude mice resulted in much higher metastatic rates. For example, a human renal-cell carcinoma obtained from a surgical specimen was dissociated by enzymatic treatment and subcutaneously injected into the renal capsule of nude mice as well as other sites. The injection of human renal-cell carcinoma cells into the kidney ofnude mice produced the highest incidence oftumor establishment and of metastasis to the lungs and other peritoneal organs. The nude-mouse renal capsule appears to be a most advantageous site for implantation of human renal-cell carcinoma (6-8). However, the subrenal capsule may be an advantageous implant site for other tumor types also (9). Human coloncancer cells were dissociated, grown in culture, and subsequently injected into the cecum of nude mice to produce tumors that eventually metastasized to the liver, demonstrating that orthotopic implantation can enhance the metastatic capability of human tumor cells in nude mice (5, 10-13). Similar results also have been achieved for orthotopic implantation of cell lines of human lung cancer (14), human pancreatic cancer (15), bladder cancer (16, 17), melanoma (18, 19), breast cancer (20-22), and head and neck cancer (23). It should be noted, however, that the effects of orthotopicity have not been fully evaluated in that, at least in some cases, metastasis may arise from nonorthotopic sites.Our approach is to avoid disruption of tumor integrity and to orthotopically implant histologically int...

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The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14 C]sucrose . Initial experiments revealed that cellular accumulation of chromatographically repurified [14 C]sucrose was not linear with incubation time . Deviation from linearity was shown to be due to constant exocytosis of accumulating marker . Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium . A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [ 1 'C] sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium . Results indicated that modeling the process of fluidphase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t 1/2 = 5 min in macrophages, 6-8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t 1/2 = 180 min in macrophages, 430-620 min in fibroblasts) . Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics . Moreover, the sizes of the compartments and magnitude of the intercom partment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis . This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations . Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation . We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min .Pinocytosis may represent the afferent arm of a homeostatic mechanism for recycling the plasmalemma . The foundation for this hypothesis was laid by the work of Steinman et al . (37) . Their stereological analysis of pinocytosis in macrophages and L-cells revealed that the volume and surface area of incoming pinocytic vesicles was ten times greater than that which the secondary lysosomal compartment was shown to accommodate and, therefore, they postulated : (a) that interiorized pinocytic vesicle fluid must rapidly egress from the vacuolar system and (b) that pinocytic vesicle membrane is recycled back to the cell surface . Recently, the latter prediction has received experimental support both in mammalian (25, 34) and nonmammalian (6, 42) cells . These studies provided evide...