The MYCN oncogene is the major negative prognostic marker in neuroblastoma with important roles in both the pathogenesis and clinical behavior of this aggressive malignancy. MYC oncogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis. To identify novel transcriptional targets mediating the MYCN-dependent phenotype, we screened a MYCN-amplified neuroblastoma cell line by using chromatin immunoprecipitation (ChIP) cloning. We identified the essential p53 inhibitor and protooncogene MDM2 as a putative target. (1), and improvement in outcome clearly requires a better understanding of the pathogenesis and pathophysiology of this disease. The MYCN oncogene is amplified in 25% of neuroblastomas and is the most powerful clinical prognostic marker for poor survival (2). Tissue-targeted expression of MYCN is sufficient to induce neuroblastoma in transgenic mice (3), suggesting this oncogene plays an important role in the pathogenesis of neuroblastoma. In vitro studies demonstrate that MYCN overexpression induces an aggressive phenotype with decreased contact inhibition, decreased growth factor dependence, and increased metastatic potential (4, 5). These findings correlate with the malignant clinical behavior of MYCN-amplified tumors in children.MYCN is a transcription factor with well defined mechanisms of both transcriptional activation, when bound to promoter E-boxes as a MYCN͞Max heterodimer, and transcriptional repression, when bound as a heterodimer with Mnt, Mxi, Mad, or other negative cofactors (6, 7). MYCN is involved in many aspects of normal and oncogenic cellular physiology, including proliferation, cell cycle regulation, apoptosis, and genomic instability (8, 9). Clinically and therapeutically relevant insight into neuroblastoma biology thus can be achieved through identification of the downstream transcriptional targets of MYCN involved in these pathways.In vitro studies of neuroblastoma cell lines demonstrate that overexpression of MYCN induces the conflicting cellular processes of rapid proliferation and apoptotic cell death (10). MYCN shortens the G 1 -S phase transition, increases cell proliferation rates, and decreases cell dependence on paracrine growth factors (5,11,12). Concurrently, MYCN suppresses Bcl-2, activates Bax, and sensitizes cells to genotoxicity-mediated apoptosis through intrinsic apoptosis pathways (13). MYCC has been shown to activate the ARF tumor suppressor, leading to p53 activation and apoptosis through Bcl-x and Bcl-2-dependent and -independent pathways (14). Less than 2% of neuroblastomas have mutated p53, and the p53 pathways are functionally active in the majority of de novo tumors (15, 16). Therefore, for MYCN-expressing neuroblastoma precursor cells to escape p53-mediated cell death, proliferate, and progress to invasive malignancy, a balance must be struck between MYCN-driven proliferation and MYCN-driven apoptosis.In this study we use chromatin immunoprecipita...

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There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.