Induction of senescence in MYCN amplified neuroblastoma cell lines by hydroxyurea

Abstract

Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei-inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cell-associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associated-beta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure

“…It has to be considered, however, that dmin are per se unstable structures. They can be eliminated spontaneously or after addition of certain drugs such as hydroxyurea (Ambros et al 1997;Narath et al 2007;Shimizu et al 2007). Their retention in tumor cells is, very likely, under strong selective pressure, as it has been demonstrated for dihydrofolate reductase dmin originated in response to methotrexate treatment (Martinsson et al 1982).…”

“…However, there is no doubt that MYCN is the target gene of amplifications of the 2p4.3 domain present in all dmin and hsr amplicons. Amplicon erosion can lead to tumor cell revertance and tumor cell senescence in vitro (Ambros et al 1997;Narath et al 2007). The MYCNOS gene partially overlaps with MYCN because it lies on the opposite strand (Armstrong and Krystal 1992).…”

“…STA-NB cell lines were provided by Cold Spring Harbor Laboratory Press on May 8, 2018 -Published by genome.cshlp.org Downloaded from P.F. Ambros, and some of them have been previously described (Ambros et al 1997;Narath et al 2007;Stock et al 2008). STA-NB-10/hsr and STA-NB-10/dmin are subclones of the same cell line, showing MYCN [v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)] amplification in form of hsr or dmin, respectively.…”

The antibacterial substance taurolidine exhibits anti-neoplastic action based on a mixed type of programmed cell death

“…26,47,48 Senescence has lately attracted interest in cancer research, because limitless replicative potential is a feature that is essential for the development of malignancy. 47 Using SA-β-gal for visualizing senescent cells, a 4-fold increase of SA-β-gal positive cells at a TAU concentration of 20 μg/ml could be demonstrated. However, at a TAU concentration of 50 μg/ml there were less senescent cells as compared to a TAU concentration of 20 μg/ml (Fig.…”

FOXO Transcription Factors as Potential Therapeutic Targets in Neuroblastoma

“…To analyze the connection between deregulated PI3K/PKB survival signaling and impaired apoptotic cell death in neuroblastoma, we determined the protein levels of PKB, FOXO3, phospho-PKB (serine-473) and phospho-FOXO3 (threonine-32) by immunoblot in different neuroblastoma cell lines and found that PKB and FOXO3 were phosphorylated, suggesting the inactivation of FOXO3. To study the function of FOXO3 we infected SH-EP and STA-NB15 neuroblastoma cells (Narath et al, 2007) with retroviruses coding for a 4OH-tamoxifen (4OHT)-regulated FOXO3(A3)ER tm transgene. In the untreated condition this fusion protein is expressed in the cytoplasm.…”