The Myc/Max/Mad network of transcriptional regulatory proteins plays an essential role in cell proliferation, growth, apoptosis, and di erentiation. Whereas Myc proteins a ect cell cycle progression positively, Mad proteins are negative regulators of cell proliferation. It has been shown in several in vitro systems that Mad proteins antagonize c-Myc functions. In this report we describe the inhibition of tumor cell outgrowth in vivo by Mad1 expression. Transformed cell lines were generated by co-transfection of c-myc, c-H-ras, and a chimeric mad1ER construct into primary rat embryo cells (MRMad1ER cells). Activation of Mad1 by 4-Hydroxy-Tamoxifen (OHT) resulted in abrogation of telomerase activity, reduced cloning e ciency, and decreased proportion of cells in S phase. Injection of MRMad1ER cells into syngenic rats induced aggressively growing tumors after a short latency period. This tumor growth was inhibited by OHT-treatment of animals, with the extent of inhibition correlating with the amount of OHT injected. No e ect of OHT on tumor growth was observed with similarly transformed Myc/Ras cell lines which did not express Mad1ER. These data demonstrate that Mad1 is able to suppress Myc/Ras-mediated transformation under in vivo conditions.

Anaplastic large-cell lymphomas are highly associated with a chromosomal translocation t(2;5). This condition results in a chimeric protein NPM/ALK (nucleophosmin/anaplastic lymphoma kinase), in which the shuttle protein NPM is fused to the catalytic domain of the tyrosine receptor kinase ALK. Because the oncogenic potential of NPM/ALK is not well understood, we analyzed the effects of NPM/ALK and the specific contribution of ALK in a standardized cell culture system by using primary rat embryo cells (REC) as cellular targets. We demonstrate by several biological parameters that NPM/ALK is an immortalizing oncogene that provides unlimited, yet normal, growth potential to REC and, with cooperation with a c-H-ras oncogene, induces cellular transformation. Targeting NPM/ALK to the nucleus diminishes its oncogenicity, which indicates that the fusion protein exerts its action mainly in the cytoplasm. Expression of the mere catalytic domain of ALK is insufficient with regard to immortalization and transformation. However, reestablishing the potential of ALK to homodimerize by fusing the bacterial dimerization domain of the tetracycline repressor to ALK reenforces the immortalizing function. Collaboration of dimeric ALK with c-H-ras converted primary REC into aggressively growing tumor cell lines. These studies identify ALK as a new member of immortalizing oncoproteins that exerts its function within the cytoplasm.Key words: NPM/ALK • NPM/ALK constructs • primary rat embryo cells • immortalization • transformation • tumor formation naplastic large-cell lymphoma (ALCL) represents a distinct subgroup of non-Hodgkin's lymphomas (NHL) acknowledged in the Revised European and American Lymphoma classification. This entity constitutes ~5% of all NHL and accounts for ~30% of pediatric large-cell lymphomas (1). It is estimated that every year about 1000 new ALCL cases occur in the United States and 1200 new cases occur in Europe.(2). The majority of ALCL cases arising A in childhood (3, 4) have a characteristic chromosomal translocation t(2;5)(p23;q35). Morris et al. have cloned the genes affected by the chromosomal aberration (5). The rearrangement results in the fusion of a novel tyrosine kinase gene anaplastic lymphoma kinase (ALK) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35. The physiologic NPM encodes a highly conserved nonribosomal RNA-binding nucleolar phospho-protein, which is thought to function as a shuttle protein that transports ribosomal ribonucleoproteins between the nucleolus and the cytoplasm for assembly of ribosomes (6, 7). Both transcription and translation of ubiquitous NPM are cell cycle-regulated, which reach peak levels when cells prepare to enter S-phase, with a decrease to baseline values just before the onset of G2. In addition to its function as a shuttle protein, NPM was shown to bind to double-stranded DNA and to serve in the initiation and early elongation steps of DNA replication (8). The other fusion partner, ALK, shows sequence similarities to members of the insulin ...

um of healthy rats. We collected cells lining the anterior nasal passages, as confirmed by light microscopy, into 200 µl of Trizol reagent by controlled intranasal instillation. We performed gene expression analysis using Clontech complementary DNA Rat Atlas 1.2 arrays (approximately 1,200 genes) and verified selected transcripts using the polymerase chain reaction with reverse transcription (Taqman). The percentages of genes within specific average expression ranges, on the basis of phosphorimager signal intensities, were 1.4% at 3,000-45,000, 3.3% at 1,000-3,000, 30.1% at 100-1,000, and 65.2% below 100. The most highly expressed genes included those involved in phase I (for example, cytochrome Ps) and phase II (for example, glutathione S-transferases) xenobiotic metabolism, bioenergetics (for example, cytochrome oxidase), osmotic balance (for example (Na + +K +)ATPase) and epithelial ionic homeostasis (for example, ion channels). These findings are consistent with normal nasal epithelial functions. Such baseline data will contribute to a further understanding of the functions of this region of the respiratory tract and facilitate the interpretation of treatment-or disease-related responses by the nasal epithelial transcriptome.

No abstract