The dependence of cell growth on methionine aminopeptidase (MetAP) function in bacteria and yeast is firmly established. Here we report experimental evidence that the control of cell proliferation in mammalian cells is directly linked and strictly dependent on the activity of both MetAP-1 and MetAP-2. The targeted downregulation of either methionine aminopeptidase MetAP-1 or MetAP-2 protein expression by small interfering RNA (siRNA) significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) (70%-80%), while A549 human lung carcinoma cell proliferation was less inhibited (20%-30%). The cellular levels of MetAP-2 enzyme were measured after MetAP-2 siRNA treatment and found to decrease over time from 4 to 96 h, while rapid and complete depletion of MetAP-2 enzyme activity was observed after 4 h treatment with two pharmacological inhibitors of MetAP-2, PPI-2458 and fumagillin. When HUVEC and A549 cells were treated simultaneously with MetAP-2 siRNA and PPI-2458, or fumagillin, which irreversibly inhibit MetAP-2 enzyme activity, no additive effect on maximum growth inhibition was observed. This strongly suggests that MetAP-2 is the single critical cellular enzyme affected by either MetAP-2 targeting approach. Most strikingly, despite their significantly different sensitivity to growth inhibition after targeting of either MetAP-1 or MetAP-2, HUVEC, and A549 cells, which were made functionally deficient in both MetAP-1 and MetAP-2 were completely or almost completely inhibited in their growth, respectively. This closely resembled the observed growth inhibition in genetically double-deficient map1map2 yeast strains. These results suggest that MetAP-1 and MetAP-2 have essential functions in the control of mammalian cell proliferation and that MetAP-dependent growth control is evolutionarily highly conserved.

Nitric oxide (NO˙) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO˙-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO˙-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO˙resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO˙, which indicates that normal processing of abasic sites is critical for defense against NO˙. In addition, recA mutant cells were exquisitely sensitive to NO˙-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO˙, indicating that both SOS and recombinational repair play important roles in defense against NO˙. Furthermore, strains specifically lacking doublestrand end repair (recBCD strains) were very sensitive to NO˙, which suggests that NO˙exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO˙induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO˙, it is also important to consider recombination events among the spectrum of genetic changes that NO˙can induce. Furthermore, the importance of recombinational repair for cellular survival of NO˙exposure reveals a potential susceptibility factor for invading microbes.

Purpose: Fumagillin and related compounds have potent antiproliferative activity through inhibition of methionine aminopeptidase-2 (MetAP-2). It has recently been reported that MetAP-2 is highly expressed in germinal center B cells and germinal center^derived non^Hodgkin's lymphomas (NHL), suggesting an important role for MetAP-2 in proliferating B cells. Therefore, we determined the importance of MetAP-2 in normal and transformed germinal center B cells by evaluating the effects of MetAP-2 inhibition on the form and function of germinal centers and germinal center^derived NHL cells. Experimental Design: To examine the activity of PPI-2458 on germinal center morphology, spleen sections from cynomolgus monkeys treated with oral PPI-2458 were analyzed. Antiproliferative activity of PPI-2458 was assessed on germinal center^derived NHL lines in culture. A MetAP-2 pharmacodynamic assay was used to determine cellular MetAP-2 inhibition following PPI-2458 treatment. Finally, inhibition of MetAP-2 and proliferation by PPI-2458 was examined in the human SR NHL line in culture and in implanted xenografts. Results: Oral PPI-2458 caused a reduction in germinal center size and number in lymphoid tissues from treated animals. PPI-2458 potently inhibited growth (GI 50 = 0.2-1.9 nmol/L) of several NHL lines in a manner that correlated with MetAP-2 inhibition. Moreover, orally administered PPI-2458 significantly inhibited SR tumor growth, which correlated with inhibition of tumor MetAP-2 (>85% at 100 mg/kg) in mice. Conclusions: These results show the potent antiproliferative activity of PPI-2458 on NHL lines in vitro and oral antitumor activity in vivo and suggest the therapeutic potential of PPI-2458 as a novel agent for treatment of NHL should be evaluated in the clinical setting.Non-Hodgkin's lymphoma (NHL) is the sixth leading cause of cancer death in the United States and has increased in incidence by >80% since 1973 (1, 2). NHL can be divided into at least 29 different subtypes of malignancy of either T-or Blymphocyte origin. The vast majority of NHL malignancies are B cell derived. Moreover, a large number of these B-cell malignancies exhibit phenotypic characteristics consistent with a germinal center origin, the site where B cells undergo proliferation and somatic hypermutation.Germinal center -derived B-cell lymphomas can be divided into at least three groups. Follicular lymphoma is responsible for 22% of all NHLs, and almost always exhibits aberrant expression of the antiapoptotic gene 3,4). Follicular lymphoma typically follows an indolent course but often transforms into the more aggressive diffuse large B-cell lymphoma (DLBCL; ref. 5). Whereas at least half of all DLBCLs express germinal center markers (Bcl-6 + , CD10 + ), the remainder may more closely resemble activated B lymphocytes and potentially deserve a distinct classification (6, 7). Under the current classification, DLBCLs represent 40% of all NHLs (8). A third type of germinal center -derived B-cell neoplasm, Burkitt's lymphoma, is highly aggre...

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.