p53 cellular localization and function in neuroblastoma: evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells

Abstract

This study investigated the hypothesis that p53 accumulation in neuroblastoma, in the absence of mutation, is associated with functional inactivation, which interferes with downstream mediators of p53 function. To test this hypothesis, p53 expression, location, and functional integrity was examined in neuroblastoma by irradiating 6 neuroblastoma cell lines and studying the effects on p53 transcriptional function, cell cycle arrest, and induction of apoptosis, together with the transcriptional function of p53 after irradiation in three ex vivo primary, untreated neuroblastoma tumors. p53 sequencing showed five neuroblastoma cell lines, two of which were MYCN-amplified, and that all of the tumors were wild-type for p53. p53 was found to be predominantly nuclear before and after irradiation and to up-regulate the p53 responsive genes WAF1 and MDM2 in wild-type p53 cell lines and a poorly-differentiated neuroblastoma, but not a differentiating neuroblastoma or the ganglioneuroblastoma part of a nodular ganglioneuroblastoma in short term culture. This suggests intact p53 transcriptional activity in proliferating neuroblastoma. Irradiation of wild-type p53 neuroblastoma cell lines led to G(1) cell cycle arrest in cell lines without MYCN amplification, but not in those with MYCN amplification, despite induction of WAF1. This suggests MYCN amplification may alter downstream mediators of p53 function in neuroblastoma.

Figure 1.

Western blots of p53 and related proteins in all cell lines studied (50 μg protein/well)

Figure 2.

a: Immunocytochemistry (ICC) before and 6 hours after 4 Gy irradiation of IMR32 cells prepared as cytoblocks. A and B: controls without primary antibodies. Note the mitotic cell in A (arrow). C and D: p53 DO-7 ICC showing an increase in nuclear immunostaining (brown) after treatment. E and F: WAF1 ICC showing an increase in nuclear immunostaining after treatment. Note the negative mitotic cell in E (arrow) (original magnification, ×500). b: Immunocytochemistry of SKNSH cells grown on glass slides before and 6 hours after 4 Gy irradiation. A and B: controls without primary antibodies. Note nonspecific brown staining in the cytoplasm of some cells especially N types. C and D: p53DO-7 immunostaining (brown) in both nucleus and cytoplasm of controls and increased positivity predominantly in nucleus after treatment. E and F: WAF1 immunostaining showing nuclear positivity in S type cells before and nuclear positivity in N type cells after treatment. G and H: BCL2 immunostaining of SKNSH cells grown on collagen slides before and 6 hours after 4 Gy irradiation showing cytoplasmic positivity in N type cells but not S type. I and J: Ki67 immunostaining before and 6 hours after irradiation showing nucleolar enhancement (×300). K and L: apoptosis using Hoechst (33258) nuclear staining before and 24 hours after irradiation. Apoptotic cells arrowed (×200).

Figure 3.

a: Western blots of SHSY5Y (wild-type p53) cells and SKNBE(2c) (mutant p53) cells for p53 and p53 regulated genes after 4 Gy irradiation. Protein expression quantitated by densitometry and measured relative to loading control actin and untreated controls (0 hours) or 1 hour if not detectable at 0 hours. Less than a twofold change in values is not stated (50 μg protein/well). p53+ con = positive control for p53 (recombinant human p53). MDM2 +con = positive for MDM2 (recombinant human MDM2); M, marker, n/d, not detectable. bi: G₁/S phase ratio before and 24 hours after 4 Gy irradiation. Mean ± SD of three experiments. bii: Proportion of cells in G₂ before and 24 hours after 4 Gy irradiation. Mean ± SD of three experiments. biii: DNA histograms of wild-type p53 SKNSH and IMR32, and mutant p53 SKNBE(2c) neuroblastoma cells, before and 24 hours after 4 Gy irradiation. G₁ arrest is shown in SKNSH cells, G₂ arrest in IMR32 (MYCN-amplified) cells and no cell cycle arrest in SKNBE(2c) cells.

Figure 4.

A and B: Hematoxylin and eosin stained sections of tumors 2 and 3 showing mitotic and apoptotic cells in A (arrows). C and D: Control immunostained sections without primary antibodies of tumors 2 and 3 showing apoptotic cells in C (arrows). E and F: Immunostaining of tumors 2 and 3 with p53 DO-7 antibody showing many cells with heterogeneous nuclear immunostaining in E and a few weakly positive cells in F (brown staining) (original magnification, ×200).

Figure 5.

p53 and related protein expression in three short-term primary neuroblastoma tumor cell cultures before and 6 hours after 4 Gy irradiation. Protein expression quantitated by densitometry and measured relative to loading control actin and untreated controls. If no there was no detectable protein in control, then the absolute value for treated is shown. We used IMR32 cells 6 hours after irradiation as a positive control (50 μg protein/well). Rx, treatment; n/d, not detectable.