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Cell Growth & Differentiation Vol. 12, 19-27, January 2001
© 2001 American Association for Cancer Research

Articles

Morphologic Conversion of a Neuroblastoma-derived Cell Line by E6-mediated p53 Degradation1

Supriya V. Gaitonde, Wenqing Qi, Ryan R. Falsey, Neil Sidell and Jesse D. Martinez2

Graduate Program, Cancer Biology Interdisciplinary Program [S. V. G.], and Arizona Cancer Center, Department of Radiation Oncology [W. Q., R. R. F., J. D. M.], University of Arizona, Tucson, Arizona 85724; and Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322 [N. S.]

Abstract

Neuroblastoma-derived tumor cells, unlike cells from other tumor types, characteristically express a wild-type but cytoplasmically sequestered p53 protein. To ascertain whether the p53 in these cells retained any physiological activity, we inactivated it in SK-N-SH cells, a neuroblastoma-derived cell line, by introducing the human papilloma virus type 16 E6 expression plasmid. Parent SK-N-SH cell cultures are composed of two cell types exhibiting characteristic morphologies designated neuroblastic (N-type) or substrate-adherent fibroblastic (S-type) cells, both of which have been shown to spontaneously transdifferentiate or interconvert. We report here that down-regulation of p53 resulted in conversion of SK-N-SH cells to the substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, and a lack of responsiveness to retinoic acid-induced neuronal differentiation. Importantly, we did not observe N-type cells in the E6-transfected cell population, suggesting that they were incapable of transdifferentiating to the N-type morphology. We also tested the ability of these E6-transfected S-type cells to form colonies in soft agar and observed a markedly reduced capacity of these cells to do so when compared with the parent and mutant E6-transfected cells. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype.

Introduction

Neuroblastomas are one of the most common solid tumors found in children (1) . In culture, human neuroblastoma cell lines are typically comprised of heterogeneous cellular subpopulations (2 , 3) . These include neuroblastic (N-type) and substrate-adherent/Schwannian (S-type) cells that can be distinguished by their characteristic morphologies and the expression of differentiation-associated antigens. Each of the two different cell types is capable of transdifferentiation or interconversion into the other type (3) . The N-type cells have properties of noradrenergic or cholinergic neurons, whereas the S-type cells have properties of embryonic Schwann/glial/melanocytic cells of the neural crest (4 , 5) . Neuronal differentiation can be induced in vitro in neuroblastoma cells by exposure to a variety of agents, with RA3 being the most commonly used compound (6, 7, 8) . Differentiation is associated with an increase in the number and length of neurite extensions as well as an increase in the expression of differentiation markers such as neurofilament (8) .

Inactivation of p53 protein is observed in over 50% of human tumors, and the most common mechanism is gene mutation (9) . Other mechanisms of p53 inactivation include its enhanced degradation by cellular proteins such as mdm2 and by viral proteins such as HPV-16 E6 (10 , 11) . Surprisingly, in neuroblastoma-derived tumors, the incidence of p53 gene mutation as the mechanism of p53 inactivation is very low (12, 13, 14) . Instead, in these tumors, p53 is physically sequestered in the cytoplasmic compartment of the cell under normal growth conditions (15 , 16) . The functional role of p53 as a transcription factor requires that it be capable of nuclear translocation, therefore suggesting that cytoplasmic sequestration may be very effective in abrogating the function of p53 in spite of a genotypically wild-type protein. Reports have also shown that this wild-type p53 in neuroblastoma cells is overexpressed due to enhanced stability (13 , 17 , 18) . Moll et al. (15) have correlated such increased levels of wild-type p53 protein with poorly differentiated neuroblastomas. These latter findings are in direct contrast to the known regulatory role p53 plays in the differentiation process in a broad range of cell lineages (19, 20, 21, 22, 23, 24, 25) . Increased expression of p53 protein can induce differentiation in such varied cell types such as glioma, thyroid, hematopoietic, and muscle cells. However, in transgenic mice overexpressing wild-type p53 protein, defects in the differentiation of the ureteric bud were observed, suggesting that p53 can also play a negative role in the differentiation process (26) . The observation that embryonic development is normal in p53 knockout mice suggests that p53 is not required for normal mammalian development. However, p53 function in these mice may be compensated for by the recently discovered p53-related proteins p63 and p73, members of the p53 family of proteins, which have been found to display overlapping functions (27, 28, 29) . Recent reports have identified a role for p73 in neuronal differentiation and a role for p63 in ectodermal differentiation (30 , 31) .

In the present study, we demonstrate that p53 can directly influence the differentiation of SK-N-SH cells, a neuroblastoma-derived cell line that expresses wild-type p53 protein that is cytoplasmically sequestered under nonstressed conditions. Normally, these cells are present as a heterogeneous mix of N- and S-type cells. In response to RA, the N-type cells undergo neuronal differentiation associated with neurite extension and the increased expression of neurofilament protein, a marker specific for neuronal differentiation. Reduction of p53 protein levels by stable introduction of a plasmid encoding the HPV-16 E6 protein resulted in the conversion of SK-N-SH cells from a predominantly N-type cell line to an exclusively S-type cell line that was resistant to RA treatment. Our results suggest that expression of wild-type p53 protein is necessary for the transdifferentiation between N- and S-type cells.

Results

Elimination of p53 Protein by Introduction of HPV-16 E6.
A role for p53 in cell differentiation has been indicated in many different cell types. In SK-N-SH cells, the p53 has been sequenced previously and found to be wild type (13) . To determine whether p53 is involved in the differentiation response of neuroblastoma cells, we inactivated it by enhancing its degradation by introducing the HPV-16 E6 protein. SK-N-SH cells were stably transfected with either the HPV-16 E6-expressing plasmid shown previously to degrade wild-type p53 protein or a mutant E6 plasmid that is identical to the wild-type E6 except for a 3-amino acid substitution (residues 8, 9, and 10) in the conserved region of the NH2 terminus of the E6 protein that makes it nononcogenic by abrogating its ability to interact with p53 (32) . Colonies resulting from the transfection were pooled together, and the entire pooled population was maintained in 500 µg/ml G418 to ensure neomycin resistance. RT-PCR analysis (Fig. 1A)Citation , using primers specific for E6, was used to confirm the expression of E6 and mutant E6 in the transfected cell populations. The relative level of p53 protein was determined by Western blotting. As expected, E6-transfected SK-N-SH cells (SK-E6 cells) had reduced levels of p53 protein compared with the parent SK-N-SH cells and the mutant E6-transfected cells (SK-mutE6 cells; Fig. 1BCitation ). Expression of {alpha}-tubulin was determined as a loading control (Fig. 1C)Citation .



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  		Fig. 1. Characterization of SK-N-SH cells transfected with wild-type or mutant HPV E6. SK-N-SH cells were transfected with either wild-type HPV E6- or mutant E6-expressing plasmids, and stably transfected colonies were pooled together and maintained in G418-containing medium. A, reverse transcription-PCR was performed on the pooled populations using primers specific for HPV E6, and the PCR products were resolved on a 1% agarose gel containing ethidium bromide to stain DNA. B, expression of p53 protein was determined by Western blotting of 50 µg of total cell protein extracts obtained from parent SK-N-SH and E6-transfected (SK-E6) and mutant E6 (SK-mutE6)-transfected SK-N-SH cells with a p53-specific antibody (DO-1). C, expression of {alpha}-tubulin as loading control. D, SK-N-SH, SK-E6, and SK-mutE6 cells were exposed to 5 Gy of ionizing radiation (IR), and the level of p21 protein induction 12 h after exposure was determined by Western blot analysis. E, the apoptotic response of SK-N-SH, SK-E6, and SK-mutE6 cells to 5 Gy ionizing radiation and 25 µM menadione was determined 12 h after treatment. Apoptosis was quantitated using a fluorescence dye method that stains viable cell nuclei green and apoptotic cell nuclei red. A total of 200 cells were counted for each time point, and the experiment was repeated two times in duplicate.

 
Previous reports have shown that neuroblastoma cells are capable of activating p53 in response to stress signals and inducing either growth arrest by p21 induction, an inhibitor of cyclin-dependent kinase activity, or apoptosis in a p53dependent manner (33 , 34) . To determine whether the p53 protein in the E6-transfected cells could be functionally activated in response to genotoxic stress, we exposed the cells to 5 Gy of ionizing radiation and compared the level of p21 induction and apoptosis in these cells to those in the parent SK-N-SH cells and the mutant E6-transfected cells. We observed a lack of p21 induction in the E6-transfected cells after exposure to ionizing radiation as well as a complete abrogation of the apoptotic response in these cells (Fig. 1, D and E)Citation . When exposed to menadione, a vitamin K3 analogue that induces apoptosis in cells in a fas-dependent and p53-independent manner (35) , all three cell lines responded by undergoing approximately 80% apoptosis within 12 h, indicating that the effects of E6 were specific to p53. Our data indicated that p53 protein levels as well as functionality had been successfully down-regulated by E6 overexpression.

Reduction of p53 Protein Levels in SK-N-SH Cells Affects Cell Morphology.
During the characterization of SK-N-SH cells transfected with the E6 plasmid, we noticed a complete change in the morphology of these cells. The E6-transfected cells (SK-E6), which expressed low levels of p53 protein, were exclusively (100%) S-type cells as judged by morphology compared with the parent SK-N-SH cells and SK-mutE6 cells, which expressed approximately 10% and 15% S-type cells, respectively, in their population (Table 1Citation , Fig. 2Citation ).


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  		Table 1 Percentage of S-type cells in SK-N-SH, SK-E6, and SK-mutE6 cells

 


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  		Fig. 2. Morphology of parent SK-N-SH cells compared with wild-type E6- and mutant E6-transfected SK-N-SH cells. A–C represent phase-contrast images of SK-N-SH, SK-E6, and SK-mutE6 cells, respectively. All images were acquired at the same magnification using a x10 objective lens.

 
We further characterized this change in morphology by Western blot analysis as well as by indirect immunofluorescence using vimentin and neurofilament as specific markers for the two cell types. As shown in Fig. 3ACitation , we were unable to detect the expression of neurofilament protein in SK-E6 cells by both Western analysis and immunofluorescence staining. Interestingly, in the parent SK-N-SH cell population and the mutant E6-transfected cells, one can observe S-type cells in the light micrographs, which also do not express any neurofilament protein (Fig. 3B)Citation . Hence, consistent with previous reports, S-type cells in both SK-N-SH and SK-derived populations lack neurofilament expression. Vimentin protein expression was slightly increased in SK-E6 cells compared with the parent SK-N-SH and SK-mutE6 cells as judged by Western analysis (Fig. 3A)Citation . Indirect immunofluorescence demonstrated that all the cells in the SK-E6 cell population expressed vimentin. However, in the parent SK-N-SH cells and the SK-mutE6 cells, only a few of the N-type cells in the mixed population expressed vimentin, and they did so at low levels compared with the S-type cells of this population (Fig. 3C)Citation . Taken together, the Western analyses and immunofluorescence staining data demonstrate that both parent and SK-mutE6 cells are comprised of a mixed population of N- and S-type cells, whereas SK-E6 cells are exclusively S-type in morphology. This phenotype of the E6-transfected cells has persisted up to passage 20.



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  		Fig. 3. Characterization of E6- and mutant E6-transfected SK-N-SH cells. Cells were plated on 60-mm2 dishes and harvested 4 days later, and 50 µg of the protein extract were used for Western blot analysis. For immunofluorescence, cells were plated onto coverslips and harvested 48 h later, fixed, and used for staining. A, Western blot analysis was performed to determine the expression of neurofilament protein, vimentin, and {alpha}-tubulin in the total cell extracts obtained from SK-N-SH, SK-E6, and SK-mutE6 cells. B, indirect immunofluorescence was performed on SK-N-SH (2), SK-E6 (4), and SK-mutE6 cells (6) to determine the expression of neurofilament protein and compared with reflective light pictures (1, 3, and 5, respectively) obtained of the same field by confocal microscopy. C, expression of vimentin in the two different cell types within each cell line was also determined by indirect immunofluorescence using an antibody specific for vimentin. Note that N-type cells in SK-N-SH and SK-mutE6 populations also express vimentin. 1 and 2 are reflective immunofluorescence images of SK-N-SH cells, 3 and 4 are images of SK-E6, and 5 and 6 are images of SK-mutE6 cells, respectively. All images were taken using a x40 objective and at the same brightness and contrast settings for each panel. The white arrowheads in B and C are pointed toward S-type cells.

 
Differential Expression of p53 in N-type versus S-type SK-N-SH Cells.
To determine whether there was differential expression of p53 in N- and S-type cells in the parent SK-N-SH cultures, these two phenotypes were separated based on their differences in substrate adhesion properties and p53 expression as determined by Western blotting. Enrichment of the two populations was accomplished by separately reculturing the cells that lost adherence after mild to moderate shaking (enriched for N-type) versus those cells that remained adherent after vigorous shaking (enriched for S-type). This technique was used a total of three times to yield cultures that were approximately 99% pure for N-type cells and 95% pure for S-type cells, as judged by their microscopic morphology. As shown in Fig. 4Citation , Western blotting for p53 indicated that there was a 1.5-fold increase in the level of p53 in the S-type cells compared to the N-type cells, as determined by densitometry analysis using the Stratagene Eagle Eye II. p53 expression in the mixed population of parent SK-N-SH cells was similar to that found in S-type cells. To confirm our enrichment procedure for the cell types, we assessed the expression of two phenotype-specific markers. To confirm the N-type morphology, we examined the expression of the neurofilament protein, and to confirm the S-type morphology, we used vimentin expression as a marker. Fig. 4Citation shows increased expression of neurofilament in the N-type cultures, whereas increased expression of vimentin was observed in cells from the S- versus N-type cultures. Expression of {alpha}-tubulin was determined as a loading control. These results suggest that the p53 in the two cell types may be differentially regulated.



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  		Fig. 4. Expression of p53 protein levels in N- and S-type cells derived from SK-N-SH cells. Enrichment of the two cell types was achieved by vigorous shaking off of the N-type cells from a mixed population of cells and subsequent culturing. When populations were approximately 99% pure for N-type cells and 95% pure for S-type cells, cells were lysed, and 50 µg of total cell protein were used for Western blot analysis. The mixed population of cells used for these experiments contained approximately 80% N-type cells and 20% S-type cells. Membranes were probed with DO-1, an antibody specific for p53. Western analysis of the same extracts was also performed to determine the expression of neurofilament, vimentin, and {alpha}-tubulin, respectively.

 
E6-transfected SK-N-SH Cells Are Refractory to Retinoic Acid Treatment.
Previous reports have demonstrated that SK-N-SH neuroblastoma cells can undergo neuronal differentiation in response to treatment with RA as reflected by the formation of neurite projections and increased expression of differentiation markers such as neurofilament expression. Fig. 5Citation demonstrates the induced increase in neurite projections when SK-N-SH cells were treated with 5 µM RA for 1 week. We observed a marked increase in the number of neurite projections from cells and an overall increase in the length of these projections indicative of neuronal differentiation of these cells in response to RA treatment. However, the morphologic transformation was not complete because examination by microscopy revealed that S-type cells were still present in the RA-treated population. The number of S-type cells in the basal layer increased with time, indicating that these cells were still growing, albeit slowly. Hence, RA appeared to be causing the N-type cells to differentiate and cease proliferation but had little or no effect on the morphology of the S-type cells.



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  		Fig. 5. Response of SK-N-SH cells to RA treatment. SK-N-SH cells were treated with 5 µM RA for 1 week, and the morphological response was determined by microscopy. A and B represent phase-contrast micrographs of SK-N-SH before and after RA treatment, respectively. The arrow in B points toward neurite extensions, whereas the white arrowheads in A and B-point to the S-type cells present in both the untreated and treated populations.

 
To determine whether SK-E6 cells were capable of responding to RA and transdifferentiating into N-type cells, we exposed them to 5 µM RA for 1 week and characterized their morphological response as well as the expression of neurofilament protein. Light micrographs showed an increase in neurite extensions in the parent SK-N-SH cells and SK-mutE6 cells along with an increase (by Western blot analysis) in the expression of neurofilament protein in these cells in response to RA treatment (Fig. 6, A and B)Citation . In contrast, no neurite extensions or expression of neurofilament protein was observed in the E6-transfected cells. No differences in the level of p53 protein expression were observed by Western analysis in SK-N-SH or SK-mutE6 cells after RA treatment (data not shown). Over the course of the RA treatment, we observed an increase in the overall number of SK-E6 cells as well as an increase in the number of S-type cells growing below the N-type population in SK-N-SH and SK-mutE6 cultures, again suggesting that the S-type cells are resistant to the antiproliferative effects of RA (Fig. 6, A and C)Citation . We conclude from these experiments that SK-E6 cells are resistant to the antiproliferative effects of RA treatment, as are the S-type cells of the parent SK-N-SH population.



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  		Fig. 6. Characterization of response of SK-N-SH, SK-E6, and SK-mutE6 cells to RA treatment. After 1 week of 5 µM RA treatment, cells were harvested, and protein extracts were used to determine neurofilament and vimentin expression by Western blot analysis. Untreated cells were harvested after 4 days due to confluence of the plates. For immunofluorescence staining, cells were plated onto coverslips, and 5 µM RA was added 48 h later. Coverslips were harvested 1 week later, and indirect immunofluorescence was performed to determine the level of neurofilament and vimentin expression. A, expression of neurofilament, vimentin, and {alpha}-tubulin was determined by Western blot analysis in the three cell lines before and after treatment with RA. B shows the expression of neurofilament in the three different cell lines by immunofluorescence staining. 1 and 2 are reflective light and immunofluorescence confocal images of RA-treated SK-N-SH cells, respectively. 3 and 4 are confocal images of SK-E6 cells, whereas 5 and 6 are confocal images of SK-mutE6 cells after RA treatment. Note the expression of neurofilament protein in the neurite extensions after RA treatment, which was not observed in Fig. 4BCitation , 1 and 2. C, expression of vimentin was also determined by indirect immunofluorescence in the three cell types. 1 and 2 are reflective light and immunofluorescence images of SK-N-SH cells after treatment with RA, respectively. 3 and 4 are images of SK-E6 cells, whereas 5 and 6 are images of SK-mutE6 cells after treatment with RA. White arrowheads in B and D point to S-type cells within the cell population. All images were taken using a x40 objective lens and are at the same brightness and contrast settings within each panel.

 
E6-transfected SK-N-SH Cells Lack Anchorage-independent Growth.
Previous studies have shown that S-type cells isolated from the mixed neuroblastoma cell population are less tumorigenic, based on growth properties in soft agar assays and tumor formation in nude mice (36) . To further test whether the E6-transfected SK-N-SH cells displayed characteristics similar to those of S-type cells clonally expanded from mixed neuroblastoma populations, we compared their colony-forming ability in 0.6% soft agar to that of the parent and the mutant E6-transfected SK-N-SH cells. Consistent with previous reports of the S-type cells, the E6-transfected cells were also unable to form colonies in soft agar assays when compared with the parent and mutant E6-transfected SK-N-SH cells, both of which are predominantly N-type (Table 2)Citation . These results indicate that the outcome of the reduction of p53 in the E6-transfected cells affects characteristics other than morphology, including the ability to exhibit anchorage-independent growth.


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  		Table 2 Anchorage-independent growth properties of SK-N-SH, SK-E6, and SK-mutE6 cells assessed by colony formation in soft agar

 
Discussion

Our results indicate that p53 plays an important role in the transdifferentiation process of SK-N-SH cells, a neuroblastoma-derived cell line with elevated levels of wild-type p53 protein (13) . Inactivation of p53 in these cells by HPV-16 E6-mediated degradation led to a striking change in their phenotype. SK-E6 cells adopted a more flattened, fibroblast-like morphology and showed increased vimentin and decreased neurofilament expression characteristic of transdifferentiation from an N- to an S-type lineage.

S-type cells have been shown to spontaneously occur in N-type neuroblastoma populations, and it has been suggested that both cell types share a common heritage (3 , 37) . Although these spontaneously occurring S-type cells and those that are induced by HPV-E6 transfection appear to be phenotypically identical, we have determined that the S-type cells resulting from the E6 transfection show marked down-regulation of p53, whereas the S-type cells derived from spontaneous transformation of SK-N-SH cells actually show an increase in the levels of p53 relative to the N-type cells. Thus, it appears that although N- to S-type transdifferentiation does not require loss of p53, reduced p53 protein levels are sufficient to induce this alteration. Such a phenomenon would be predicted if other proteins, in addition to p53, played a more direct role in maintaining the neuroblastic phenotype and are more readily inactivated during routine culturing. Nevertheless, the SK-E6 cells have one critical difference that distinguishes them from the spontaneously derived S-type cells: they lack the capacity to transdifferentiate from S- to N-type cells. This is supported by the observation that no N-type cells have appeared spontaneously in the SK-E6 cell population over several passages, and no neuronal differentiation was induced by RA treatment. Thus, it appears that the N-type morphology is fostered by the presence of p53.

HPV-16 E6 is known to have pleiotropic effects and can interact with a number of other cellular proteins. Hence, it is possible that the effects we observed in the E6-transfected SK-N-SH cells could be due to nonspecific activity brought about by the interaction between E6 and other cellular proteins rather than a reduction in the levels of p53 (38, 39, 40) . However, this is unlikely for several reasons. First, the control mutant E6 transfected into SK-N-SH cells is identical to the wild-type E6 protein except for substitutions in 3 amino acids at residues 7, 8, and 9 in the NH2 terminus of E6. These changes made to the conserved region of the NH2 terminus of HPV-16 E6 protein compromise its ability to associate with p53 but presumably leave other protein/protein interactions unaffected (32) . Second, E6 associates with other proteins such as E6-AP independent of its interactions with p53, and it has been shown that the control mutant E6 used in our experiments can still associate with E6-AP, suggesting that interactions with proteins other than p53 remain unaffected in the mutant E6 (41) . Finally, the E6 protein is toxic when introduced into some neuroblastoma cell lines with elevated levels of N-myc, and we found that the wild-type E6 and mutant E6 are equally toxic in these cells.4 Hence, we conclude that the change in morphology seen in wild-type E6-transfected SK-N-SH cells is due to the degradation of p53. However, it remains possible that degradation of some proteins by wild-type E6 may be dependent on their association with p53. Consequently, the morphologic conversion that we observed may be attributed to increased degradation of such proteins as mediated through the interaction with E6 by p53.

Previously, it was reported that a reduction in the level of p53 protein occurs after RA treatment of neuroblastoma cells (42 , 43) . However, in our hands, neither the parent SK-N-SH cells nor the SK-mutE6 cells exhibited a change in p53 levels after RA treatment. This may be due to the presence of S-type cells in the population that are resistant to the antiproliferative effects of RA and have elevated levels of p53, which compensates for the low levels of p53 in the N-type cells that are initially predominant in the cultures. Induction of differentiation by RA in the parent SK-N-SH cells resulted in neuronal differentiation of the N-type cells, whereas the S-type cells formed a basal layer of flattened fibroblast-like cells that did not possess the morphological characteristics of neuronal differentiation. Also, we did not observe an increase in the expression of neurite-like projections or neurofilament protein in E6-transfected S-type cells similar to S-type cells present in the parent SK-N-SH population. Spontaneous S-type cells showed elevated p53 levels in comparison to N-type cells, whereas E6-derived S-type cultures showed reduced p53 levels, suggesting that the level of p53 expression does not play a role in the RA resistance exhibited by cells of the S phenotype. Thus, our results with the E6-transfected SK-N-SH cells did not support the results observed by Schlett and Madarasz (44) , who demonstrated neuronal differentiation by RA in neuroepithelial cell lines generated from mice lacking functional p53. The difference in outcome between our experiments and those reported by Schlett and Madarasz (44) may be due in part to the fact that their cells were derived from cerebral vesicles of mouse embryos lacking functional p53, whereas the SK-N-SH cells are neuroblastoma cells derived from a bone marrow metastasis and presumably have additional genetic alterations.

When tested for their ability to form colonies in soft agar assays, we observed that the E6-transfected cells were unable to grow in an anchorage-independent manner. Anchorage-independent growth of cells has been used classically as a marker for cell tumorigenicity, along with the ability to form tumors in nude mice. As such, our results suggest that reduction of wild-type p53 protein expression leads to a less tumorigenic cell type. In light of the role of p53 as a tumor suppressor protein, these findings are contradictory to what might be expected in cells in which wild-type p53 levels are reduced. One possible explanation for these results is suggested by what is known about the oncogenic mechanism of mutant p53 (45 , 46) . The role of mutant p53 as an oncogene is due in part to its dominant negative behavior and subsequent inactivation of wild-type p53. However, mutant p53 has also been shown to behave as an oncogene in cells lacking expression of wild-type p53, indicating oncogenic effects of mutant p53 independent of its dominant negative interactions. In this regard, overexpressed mutant p53 protein that is cytoplasmically sequestered can cause cells lacking wild-type p53 to become tumorigenic in nude mice (47) . Given these observations, it is tempting to speculate that the wild-type cytoplasmically sequestered p53 protein observed at a high incidence in neuroblastoma cells could be playing a role similar to that of mutant p53 by enhancing tumorigenic potential. Our laboratory will investigate this possibility in more detail in future experiments.

Materials and Methods

Cell Cultures, Antibodies, and Treatments.
The human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection. Cells were grown in DMEM containing 15% fetal bovine serum, 4 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The SK-E6 and SK-mutE6 cell lines were generated in the laboratory from SK-N-SH cells at passage 36 by transfecting the pCMV-Bam-Neo-E6 and pCMV-Bam-Neo-mutE6 plasmids, respectively. Transfections were performed using LipofecTAMINE (Life Technologies, Inc.), and stable clones generated by G418 (500 µg/ml) selection were pooled together and maintained in medium containing 500 µg/ml G418.

Antibodies using for Western blotting and immunofluorescence analysis include anti-p53 (PAb 421; provided by Dr. Arnold Levine, (Rockefeller University, New York, NY); anti-p21 (Ab-6; Oncogene Science); anti-neurofilament (M+H; Ventana Medical Systems); and anti-vimentin (Dako Corp.).

SK-N-SH, SK-E6, and SK-mutE6 cells were plated in 60-mm2 tissue culture plates at a cell density of 2 x 105 cells/plate. After 48 h, the cells were treated for 7 days (refed every third day) with 5 µM all-trans RA (Sigma) and allowed to differentiate. Cells were exposed to 5 Gy of ionizing radiation using a 60Co source (Atomic Energy of Canada) with an average dose of 60 cGy/min and 25 µM menadione (Sigma).

Generation of Plasmids.
The wild-type HPV-16 E6 (p1221) and mutant E6 (p2022) plasmids were obtained from P. M. Howley (Harvard Medical School, Boston, MA) (32) . The mutant E6 plasmid has amino acid substitutions at residues 7 (Arg->Ser), 8 (Pro->Ala), and 9 (Arg->Thr). The full-length E6 and mutant E6 inserts were amplified by PCR using 5'-ACGGATCCATGTTTCAGGACCCACAGGAG-3' as the forward primer and 5'-CGGATCCTTACAGCTGGGTTTCTCTACG-3' as the reverse primer, and the products were cloned into the pCMV-BAM-NEO vector. Appropriate cloning of both plasmids (pCMV-Neo-E6 and pCMV-Neo-mutE6) was confirmed by automated sequence analysis.

Reverse Transcription-PCR Analysis.
Reverse transcription of 2 µg of total RNA from SK-N-SH, SK-E6, and SK-mutE6 cells was carried out using 0.5 gram of oligo(dT)12–18, 2 mM deoxynucleotide triphosphate mixture, 1 mM DTT, 0.5 mM MgCl2, 10x reverse transcription buffer, and 50 units of Superscript II reverse transcriptase in a 20-µl reaction (Life Technologies, Inc.). Full-length E6 or mutant E6 was amplified by using 5'-ACGGATCCATGTTTCAGGACCCACAGGAG-3' as the upstream primer and 5'-TCGGATCCTTACAGCTGGGTTTCTCTACG-3' as the reverse primer to generate a PCR product of 450 bp. PCR conditions were as follows: denaturation at 95°C for 5 min followed by 30 cycles of 94°C for 40 s, 55°C for 30 s, and 72°C for 1 min. The PCR product was separated on a 1% agarose gel, and an image was acquired using Stratagene EagleEye II.

Quantitation of S-type Cells in Cell Populations.
Cells were plated onto coverslips at a density of 3 x 105 cells/35-mm2 plate. After 48 h, the cells were harvested by rinsing twice in PBS containing 3 mM KCl, 1 mM KH2PO4, 0.2 mM MgCl2, 137 mM NaCl, and 8 mM Na2HPO4 (pH 7.5) and fixed in ice-cold methanol:acetone (1:1) for 5 min before air drying. Coverslips were mounted on the slides with Mowiol, and the percentage of S-type cells was counted based on cell morphology using a Nikon phase-contrast microscope.

Indirect Immunofluorescence.
Cells were plated onto coverslips at a density of 1 x 105 cells/35-mm2 plate. Forty-eight h after plating, 5 µM RA was added to the plates, and cells were incubated for 7 days. Untreated cells were harvested 48 h after plating onto coverslips to ensure cell attachment. Coverslips were harvested by rinsing twice in PBS containing 3 mM KCl, 1 mM KH2PO4, 0.2 mM MgCl2, 137 mM NaCl, and 8 mM Na2HPO4 (pH 7.5) and incubated in ice-cold methanol:acetone (1:1) for 5 min before air drying. When the coverslips were ready to be stained, cells were rehydrated in PBS for 10 min and blocked in PBS containing 0.1% BSA for 10 min. Primary and secondary antibody reactions were done at room temperature for 1 h and 30 min, respectively, and the antibodies were dissolved in PBS containing 0.1% BSA. After the secondary antibody was applied, nuclei were counterstained with 4', 6-diamidino-2-phenylindole for 5 min, and the coverslips were rinsed twice. The secondary antibodies used were goat antimouse Cy3 and goat antimouse FITC conjugates (Jackson Immunoresearch).

Western Blot Analysis.
Protein expression was determined by Western blot analysis using the Laemmli method. Briefly, cells were lysed in lysis buffer [50 mM Tris-Cl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% NP40, 1 mM phenylmethylsulfonyl fluoride, and 1 mM aprotinin, leupeptin, and pepstatin A, Sigma, and protein concentration was determined using the DC Protein Assay kit (Bio-Rad). Fifty µg of protein were loaded per lane and resolved on a 10% acrylamide gel, followed by transfer onto nitrocellulose membrane (Pall Gelman Corp.). The membrane was blocked overnight in Blotto (TBS containing 0.05% Tween 20 and 5% milk (Bio-Rad). Primary antibody reaction was carried out at room temperature for 1.5 h, and secondary antibody reaction was carried out at room temperature for 45 min. Chemiluminescence was observed using the enhanced chemiluminescence reagent kit (Amersham).

Apoptosis Assay Using Fluorescent Dye Method.
After treatment with apoptosis-inducing agents, the percentage of apoptosis was determined using a fluorescent dye staining protocol. Briefly, cells were collected from the plates by collecting the floating cells and removing the substrate adherent cells with trypsin. Cells were centrifuged at 2000 rpm for 5 min at 4°C and resuspended in a small volume of medium (approximately 1 ml). From this cell suspension, 10 µl were removed and mixed with 1 µl of fluorescent dye mix containing acridine orange and propidium iodine (Sigma). Acridine orange stains the nuclei green in viable cells, and the propidium iodide is excluded. However, in apoptotic or necrotic cells, the nuclei are stained red by the propidium iodide, and the morphology of the nucleus easily determines whether the cell is apoptotic or necrotic. A total of 200 cells were counted per time point, and the percentage apoptosis was determined.

Soft Agar Colony-forming Assay.
A stock 1.2% low melting point (LMP) agarose (Life Technologies, Inc.) was autoclaved and maintained at 37°C overnight to allow the temperature to equilibrate. Two ml of 1 1:1 solution of LMP agarose and DMEM (supplemented as described above) were poured into each well of a 6-well plate, and the basal layer was allowed to solidify for 5 min at 4°C and equilibrated at room temperature for 30 min. The top layer was similar to the basal layer but contained 4000 cells/well. The top layer was allowed to solidify at room temperature for approximately 15 min, and the plates were transferred to a 37°C incubator with 5% CO2. The following day, 1 ml of medium was added to each well, and the wells were refed every 3–4 days for 2 weeks. Two sets of experiments were performed in triplicate. The total number of cells and colonies was counted, and the percentage of colony formation was determined.

Acknowledgments

We are grateful to Dr. P. M. Howley for plasmids p1221 and p2022, from which the E6 and mutant E6 plasmids expressing neomycin resistance were generated, respectively. We are also grateful to the University of Arizona College of Medicine, Department of Pathology and in particular to Dr. Anne Cress for use and help with the LSM-10 confocal microscope in these studies. We also thank M. Pennington for help with acquiring digital phase-contrast images.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant CA64842. This work is submitted by S. V. G. in partial fulfillment of the requirements of the Cancer Biology Interdisciplinary program for the Ph.D. degree in Cancer Biology offered by the University of Arizona. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, University of Arizona, P.O. Box 245024, 1515 North Campbell Avenue, Tucson, AZ 85724. Phone: (520) 626-4250; Fax: (520) 626-4480; E-mail: jmartinez{at}azcc.arizona.edu Back

3 The abbreviations used are: RA, retinoic acid; HPV, human papilloma virus. Back

4 Unpublished data. Back

Received for publication 9/ 1/00. Revision received 11/28/00. Accepted for publication 11/30/00.

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