The detection of early micrometastasis or disseminated single tumor cells poses a problem for conventional diagnosis procedures. Using a panel of monoclonal antibodies against cytokeratin and the 17-lA epithelial antigen we identified immunocytochemically tumor cells in bone marrow of patients with breast cancer (n = 155) and colorectal cancer (n = 57) at the time of surgery of the primary tumor. Monoclonal antibody CK2, recognizing the human cytokeratin component 18 in simple epithelia, appeared to be the most suitable reagent because of its negative reaction with bone marrow samples of the noncarcinoma patients (n = 75). Its specificity was further demonstrated in a double-marker staining procedure using an anti-leukocyte common antigen monoclonal antibody (T200) as counterstain. A comparative analysis showed that immunocytology was clearly superior to conventional cytology (n = 212) and histology (n = 39). In 9.5-20.5% of patients without distant metastasis, tumor cells could be detected in bone marrow. We found a significant correlation between tumor cells in bone marrow and conventional risk factors, such as distant metastasis or lymph node involvement. In a first approach toward immunotherapy we demonstrated in 3 patients that infused monoclonal antibody 17-lA can label single tumor cells in bone marrow in vivo. We then used this approach to follow up 7 patients undergoing 17-4A therapy in an adjuvant clinical trial.

No abstract

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 ...

No abstract