The tyrosine kinase inhibitor STI571 is a promising agent for the treatment of advanced Philadelphia chromosome positive (Ph ؉ ) acute lymphoblastic leukemia (ALL), but resistance develops rapidly in most patients after an initial response. To identify mechanisms of resistance to STI571, 30 complementary DNAs (including 9 matched samples) obtained from the bone marrow of individuals with Ph ؉ ALL were analyzed by direct sequencing of a 714-base pair region of ABL encoding for the adenosine triphosphate (ATP)-binding site and the kinase activation loop. A single point mutation was found at nucleotide 1127 (GI6382056) resulting in Glu255Lys. This mutation occurred in 6 of 9 patients (67%) following their treatment with STI571 but not in the samples from patients before beginning treatment with STI571. Glu255Lys is within the motif important for forming the pocket of the ATP-binding site in ABL and it is highly conserved across species. In conclusion, Ph ؉ ALL samples resistant to STI571 have a unique mutation Glu255Lys

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5 end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3 splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3 splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.T he vast majority of eukaryotic mRNAs are translated via a mechanism in which the 40S ribosomal subunit engages the mRNA at its methylguanosine-capped 5Ј end (1). Upon associating with the transcript, these subunits are believed then to scan in the 5Ј to 3Ј direction for an appropriately situated AUG at which to begin translation (2, 3). A smaller number of mRNAs are translated by a 5Ј end-and cap-independent mechanism wherein ribosomes are recruited to the transcript at an interior location through an internal ribosome entry site (IRES).IRESs were first discovered in the picornaviruses encephalomyocarditis virus (EMCV) and poliovirus (4, 5). The RNA of these viruses possesses very long 5Ј UTRs bearing many unutilized upstream AUGs (uAUGs) and, unlike cellular mRNA, is uncapped (6). Soon after the identification of picornaviral IRESs, a number of cellular mRNAs were also reported to contain IRESs. To date, at least 85 cellular IRESs have been described (7). The experimental grounds on which proof of most cellular IRESs rest, however, has been the subject of dispute (8-10).A primary criticism of the data presented as establishing the existence of cellular IRESs concerns the plasmid-based dicistronic assay, the standard method of ascertaining IRES activity. In this assay, the candidate sequence is inserted between two reporter genes (5) so that both the upstream and downstream cistron are transcribed on the same RNA. If the test insert causes increased expression of the downstream cistron relative to the upstream cistron, the result is considered evidence for internal ribosome entry. However, the generation of even low levels of monocistronic RNAs from dicistronic constructs has the potential to falsely indicate IRES activity (8, 9, 11). One way that such RNAs could arise is through splicing of the dicistronic transcript due to the presence of a 3Ј splice site (ss) in the test sequence [see supporting information (SI) Fig....

Cysteine-rich protein 61 (Cyr61) is a growth factorinducible, immediate-early gene that has multifaceted activities in various cancers. In a previous study, we found that Cyr61 inhibited the growth of the H520 and H460 non-small-cell lung cancer (NSCLC) cell lines. In further studies, we now report that p53 plays a pivotal role in Cyr61-dependent cellular growth arrest. Blocking Cyr61 with a Cyr61 antibody resulted in the downregulation of expression of p53 and p21, as well as partially reversing the growth suppression of H520-Cyr61 cells. Proliferation of NSCLC cell lines (NCI-H157, H125, H1299), having a mutant p53, were not suppressed by Cyr61. Inhibition of wild-type p53, by either human papilloma virus type 16 E6 or a dominant-negative p53, resulted in the rescue of the growth suppression mediated by Cyr61 in the H520-Cyr61 cells. The enhanced levels of p21 WAF1 and p130/RB2, in the Cyr61-expressing H520-Cyr61 cells, were also inhibited by blocking p53 showing that p21 and p130 were induced by p53 in these cells. In addition, levels of both c-myc and b-catenin increased in Cyr61 stably transfected H520 cells. Moreover, b-catenin was translocated into the nucleus in these cells. Inhibition of c-myc expression in the H520-Cyr61 cells with antisense c-myc resulted in their decreased levels of p53. Transfecting cells with a dominant-negative T-cell factor (TCF4), the specific inhibitor of the b-catenin/TCF4 complex, downregulated the expression of c-myc. Taken together, the data suggest that Cyr61 suppressed the growth of NSCLC cells by triggering a signal transduction pathway through bcatenin. In this pathway, Cyr61 activated the b-catenin/ TCF4 complex, which promoted the expression of c-myc and the latter induced expression of p53, and p53 upregulated p21 WAF1 and p130/RB2, resulting in growth arrest.

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