Marked increased expression of cyclooxygenase 2 (COX-2), a prostaglandin-synthesizing enzyme that is pharmacologically inhibited by nonsteroid anti-inflammatory-type drugs, is a major early oncogenic event in the genesis of human colon neoplasia. We report that, in addition to inducing expression of COX-2, colon cancers further target the prostaglandin biogenesis pathway by ubiquitously abrogating expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme that physiologically antagonizes COX-2. We find that 15-PGDH transcript and protein are both highly expressed by normal colonic epithelia but are nearly undetectable in colon cancers. Using gene transfection to restore 15-PGDH expression in colon cancer cells strongly inhibits the ability of these cells to form tumors in immune-deficient mice and demonstrates 15-PGDH to have functional colon cancer tumor suppressor activity. In interrogating the mechanism for 15-PGDH expression loss in colon cancer, we determined that colonic 15-PGDH expression is directly controlled and strongly induced by activation of the TGF-␤ tumor suppressor pathway. These findings thus delineate an enzymatic pathway that induces colon cancer suppression, a pathway that is activated by TGF-␤ and mediated by 15-PGDH.colon ͉ gastric

Bms1p and Tsr1p define a novel family of proteins required for synthesis of 40S ribosomal subunits in Saccharomyces cerevisiae. Both are essential and localize to the nucleolus. Tsr1p shares two extended regions of similarity with Bms1p, but the two proteins function at different steps in 40S ribosome maturation. Inactivation of Bms1p blocks at an early step, leading to disappearance of 20S and 18S rRNA precursors. Also, slight accumulation of an aberrant 23S product and significant 35S accumulation are observed, indicating that pre-rRNA processing at sites A0, A1, and A2 is inhibited. In contrast, depletion of Tsr1p results in accumulation of 20S rRNA. Because processing of 20S to 18S rRNA occurs in the cytoplasm, this suggests that Tsr1p is required for assembly of a transport- or maturation-competent particle or is specifically required for transport of 43S pre-ribosomal particles, but not 60S ribosome precursors, from the nucleus to the cytosol. Finally, Bms1p is a GTP-binding protein, the first found to function in ribosome assembly or rRNA processing.

A new protein with translational activity has been identified on the basis of its ability to stimulate translation in an in vitro globin synthesis assay deficient in eukaryotic initiation factor (eIF) 4B and eIF4F. This protein has been purified to greater than 80% homogeneity from rabbit reticulocyte lysate and has been given the name eIF4H. eIF4H was shown to stimulate the in vitro activities of eIF4B and eIF4F in globin synthesis, as well as the in vitro RNA-dependent ATPase activities of eIF4A, eIF4B, and eIF4F. Three tryptic fragments of eIF4H yielded amino acid sequences that were 100% identical to a human sequence found in the GeneBank TM that codes for a previously uncharacterized protein (HUMORFU_1). The calculated molecular weight of the protein encoded by this sequence, its predicted cyanogen bromide fragmentation, and calculated isoelectric point are all consistent with those determined experimentally for eIF4H. Also, the presence of an RNA recognition motif within HUMORFU_1 suggests that eIF4H may interact with mRNA. We conclude that this newly characterized protein, eIF4H, functions to stimulate the initiation of protein synthesis at the level of mRNA utilization, and is encoded by the gene for HUMORFU_1.Protein synthesis is the process by which an mRNA sequence is translated into protein on ribosomes using aminoacyl-tRNAs as substrates. In eukaryotes, this process involves many accessory proteins, which are transiently associated with the ribosome and function either in the initiation, elongation, or termination of protein synthesis. The steps involving delivery of the mRNA to the ribosome and positioning of the first initiating methionyl-tRNA (Met-tRNA i ) 1 at the AUG start codon is termed initiation. This process involves at least 11 protein initiation factors (for a recent review on translation, see Merrick and Hershey (1)).The first step in the initiation of the majority of mRNAs is the specific recognition of the 7-methylguanosine (m 7 G) cap of the mRNA by the multisubunit complex eukaryotic initiation factor (eIF) 4F. eIF4F is composed of three protein subunits, eIF4E (25 kDa), eIF4A (46 kDa), and eIF4G (150 kDa). The interaction of eIF4F with the m 7 G cap is facilitated specifically through the eIF4E subunit (2). Following binding of the eIF4F complex to the 5Ј end of mRNA, the initiation factors eIF4A (46 kDa) and the eIF4B dimer (160 kDa) interact with the mRNA and facilitate "melting" of mRNA secondary structure through the helicase activity of eIF4A (3). This mechanism facilitates binding of the 40 S ribosomal subunit to the 5Ј end of the mRNA and may also provide the motor for the ATP-dependent process of scanning.The other major step in the initiation of protein synthesis involves binding of the first aminoacyl-tRNA, Met-tRNA i , to the 40 S ribosomal subunit. This is facilitated by the translation factors eIF1A, 2 eIF2, and eIF3. The Met-tRNA i forms a ternary complex (3°) with eIF2 and GTP (3°ϭ eIF2⅐GTP⅐Met-tRNA i ) that will then bind to the 40 S ribosomal subunit to form the 43...

, Mol. Cell. Biol. 8:381-392, 1988). Bi was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. Bi is identical to the proto-oncogene Spi-1IPU.1 (Spi-l), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-I locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-) mRNA to approximately 20%o of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-I mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-l mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-) locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.We have previously identified by electrophoretic mobility shift analysis (EMSA) several factors in murine erythroleukemia (MEL) cells that bind to specific sequences within the mouse n-major (3M)-globin intervening sequence 2 (IVS2) (27). The region encompassing these sites has been observed by others to be a tissue-specific DNase I-hypersensitive site and the border of nucleosome phasing on the globin gene in erythroid cells (7,39,78). This region includes two homologous sites for factor B1 (B1-A and B1-B) and one site each for factors Oct-1 (40) and B2 (now designated We report here the cloning of the cDNA for MEL factor B1 by using a modification of the approach taken by Tsai et al. (83), involving a mammalian expression system combined with EMSA analysis of extracts from the transfected COS cells as the detection method. The cDNA sequence revealed that factor B1 is identical to the proto-oncogene/DNAbinding factor Spi-1/Pu.1ISfpi-1 (Spi-1 stands for spleen focus-forming virus [SFFV] proviral integration 1) (33,47,55,65), an ets gene family member which had been previously cloned by different methodologies. On the basis of analysis of the protein sequence and protein-DNA contacts, we propose that there are structural similarities between the DNA-protein interactions of the ETS domain, in particular that of Spi-1, and of the helix-turn-helix (HTH) motif of homeodomain proteins.MEL cells are generated by transformation with the Friend virus which is a complex of a replication-defective SFFV and a replication-competent Friend murine leukemia virus (F-MuLV) helper (for a review, see reference 43) and are thought to be blocked from further differentiation a...