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Cell Growth & Differentiation Vol. 11, 373-384, July 2000
© 2000 American Association for Cancer Research

Articles

Adenovirus-mediated Overexpression of p15INK4B Inhibits Human Glioma Cell Growth, Induces Replicative Senescence, and Inhibits Telomerase Activity Similarly to p16INK4A

Jonas Fuxe, Göran Akusjärvi, Helena M. Goike, Göran Roos, V. Peter Collins1 and Ralf F. Pettersson2

Ludwig Institute for Cancer Research, Stockholm Branch, S-17177 Stockholm [J. F., H. M. G., V. P. C., R. F. P.]; Department of Medical Immunology and Microbiology, Uppsala University, 75123 Uppsala [G. A.]; and Department of Pathology, Umeå University, 90187 Umeå [G. R.], Sweden

Abstract

The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40–50% of primary tumors display homozygous deletions of these two loci. Although the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth-suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. After infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251 MG and U373 MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251 MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth-arrested, showed no increased apoptosis, and displayed a markedly altered cellular morphology and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated ß-galactosidase, thus fulfilling criteria for replicative senescence. In contrast, the growth and morphology of U373 MG, which expresses p16 and p15 endogenously, but undetectable levels of RB protein, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence, and to inhibit telomerase activity as p16 in glioma cells with an intact RB protein pathway.

Introduction

Cell cycle progression is regulated by sequentially acting CDKs.3 Different CDKs are activated by binding to a set of cyclins, the regulatory subunits in the active kinases (1, 2) . In G1 phase, the level of D-type cyclins (D1, D2, and D3) increases, which results in their binding to, and activation of, CDK4/6. This leads to the phosphorylation of the pRB and its dissociation from E2F transcription factors. The release of E2F induces the transcription of a series of genes involved in S-phase entry (2, 3) .

During recent years, a number of CDKIs, which are able to block the action of CDKs have been identified (4) . The CDKIs fall into two families. The p21 family includes the prototype p21 (also known as WAF1/Cip1), as well as p27Kip1 and p57Kip2 (4) . These inhibitors have the capacity to bind to and inhibit a broad range of CDK-cyclin complexes, which indicates that they are general inhibitors of cell cycle progression (2) . In contrast, members of the INK4 family, which includes the prototype p16INK4A (p16, encoded by CDKN2A; Refs. 5, 6 ) as well as p15INK4B (p15, encoded by CDKN2B; Ref. 7 ), p18INK4C (8, 9) , and p19INK4D (9, 10) , are specific for CDK4 and CDK6. The binding of p16 and p15 to CDK4/6 prevents the formation of the active cyclin D-CDK complex (11) . This keeps pRB in an inactive hypophosphorylated state and results in G1-S arrest.

The CDKIs have attracted much attention because of their potential as tumor suppressors (1, 12, 13) . This is particularly the case with the p16 and p15 genes, which are closely linked on chromosome 9p21 and are frequently homozygously deleted in a broad range of tumor cell lines and primary tumors (14–17) . In a majority of human grade IV astrocytomas, the RB pathway is inactivated by genetic lesions in one of the regulatory components. The most frequent alteration occurs in the CDKN2A and CDKN2B loci, which display homozygous deletions in primary tumors in 40–50% of cases (18–22) and an even higher percentage in glioma cell lines (14, 15, 23) . In addition, heterozygous deletions have been observed in an additional 30% of primary tumors (19) . Alternatively, G1 control may be abrogated by amplification of CDK4. In cases with no deletion in the CDKN2A/B locus, 50% showed CDK4 amplification (19, 24) . Finally, the RB gene may be mutated in cases with an intact CDKN2A/B locus and no CDK4 amplification (23, 25) .

CDKN2A has also been found to be inactivated, albeit less frequently, by methylation of CpG islands in the promoter region and by point mutations (20, 26–29) . Mice carrying a targeted deletion of CDKN2A spontaneously develop tumors at an early age and are highly sensitive to carcinogenic treatments (30) . Embryonic fibroblasts derived from CDKN2A-/- mice grow faster, reach a higher cell density, form colonies in soft agar, and do not exhibit a senescent phase after a high number of population doublings.

Overexpression of p16 inhibits cell growth attributable to the arrest of the cell cycle in G1 (8, 31–35) . The ability of p16 to inhibit cell proliferation is dependent on a functional pRB (31, 33, 34, 36) . Overexpression of p16 causes marked morphological changes reminiscent of replicative senescence (37, 38) . Human keratinocytes (39) or fibroblasts (40, 41) , undergoing replicative senescence, display a markedly increased p16 expression.

Although the role of the p16 as a tumor suppressor has thus been amply documented, the role of p15 in tumorigenesis remains less clear. CDKN2B is also frequently deleted, usually together with CDKN2A, in tumor cell lines and primary tumors (17) , but only rare cases of inactivation through mutations or hypermethylation have been reported (42, 43) . The level of p15 has been found to dramatically increase on treatment with TGF-ß (7) , and p15 seems to cooperate with p27 to cause G1 arrest and quiescence after TGF-ß treatment (44) .

To compare the effects of p15 and p16 on cell proliferation, morphology, and telomerase activity, we have overexpressed these inhibitors by using recombinant adenoviruses. As model targets, we chose two glioma cell lines, one carrying homozygously deleted CDKN2A and CDKN2B genes but an intact RB gene (U251 MG), or one with intact CDKN2A and CDKN2B genes but with no detectable pRB expression (U373 MG). Our results show that p15 and p16 both completely arrested cell proliferation in vitro, caused phenotypic changes characteristic of replicative senescence, and inhibited telomerase activity. These effects were seen only in U251 MG (pRB+) but not in U373 MG (pRB-). Thus, by the criteria used here, p15 is indistinguishable from p16 in the in vitro biological effects and, therefore, has the potential to function as a bona fide tumor suppressor.

Results

Characterization of Target Glioma Cell Lines—Clarification of the Identity of Cell Lines U251 MG and U373 MG.
To compare the effect of p15 with that of p16 on cell proliferation and other phenotypic parameters, we chose the three established glioblastoma cell lines U251 MG, U373 MG, and T98G. These cell lines have been widely used for more than 2 decades and have been found to differ in the status of some of the relevant cell-cycle-regulating genes (23, 24) .

During the course of our analyses, we found some discrepancies between the genetic status of two of the above cell lines used in our laboratory and those described in the literature. This prompted us to reanalyze these cell lines (Table 1Citation ). The mutations found in the TP53 gene [G818A transition (codon 273; Ref. 24 )] and the PTEN gene [an insertion of two thymidine residues at position 725 (codon 241) in exon 7 (45, 46) ] have been reported to be identical in the U251 MG and U373 MG cell lines. Different groups working with these cell lines have also cited the above reports (e.g., Refs.47, 48 ). As summarized in Table 1Citation , we found by direct sequencing that the two cell lines harbor different mutations in both genes. The TP53 and PTEN mutations that were found in the U373 MG cell line used in our laboratory were identical to the ones found in an early passage obtained from the laboratory at Uppsala University in which the cell line was originally isolated (49) . p53 was mutated in codon 248 (G743A transition, leading to an Arg248Gln substitution), whereas the PTEN gene had a deletion of six nucleotides (404–409del in exon 5), leading to replacement of Ile-Cys-Ala (residues 135–137) by threonine. In contrast, U373 MG obtained from ATCC, U251 MG used in our laboratory, and U251 MG from an early passage obtained from Uppsala University had the same TP53 and PTEN mutations, identical with the ones reported in the literature (Refs. 24, 45, and 46 ; Table 1Citation ). In summary, the most conceivable explanation for these findings is that U373 MG from ATCC in fact is the original cell line U251 MG from Uppsala University.


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  		Table 1 Status of TP53 and PTEN mutations in cell lines U373MG and U251MG

 
We next analyzed the status of the genes encoding p15, p16, pRB, CDK4, cyclin D1, and p53 in the three cell lines. Table 2Citation summarizes the results obtained by Southern blotting, RT-PCR, immunohistochemistry, and genomic sequencing. We found that U251 MG had a homozygous deletion of the CDKN2A and CDKN2B loci, whereas the RB, CDK4, and cyclin D1 (CCND1) genes were intact. U373 MG had intact CDKN2A and CDKN2B genes but expressed a very low level of RB mRNA as determined by RT-PCR, and pRB was undetectable by immunohistochemistry. However, the RB gene was retained as determined by Southern blotting. The only alteration found thus far was a point mutation in the 5' noncoding region of the mRNA encoded by exon 1 at nucleotide -133 upstream of the ATG translation initiation codon. Whether this mutation is the actual cause of the absent pRB expression, or represents an innocent polymorphism, remains unclear. No amplification of the CDK4 or cyclin D1 genes were found (Table 2)Citation . Finally, T98G is a cell line of particular interest, because it turned out to have intact CDKN2B alleles but a homozygously deleted CDKN2A gene. The RB1, CDK4, and cyclin D1 genes and their expression were unaltered, whereas TP53 was mutated.


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  		Table 2 Status of different cell-cycle-regulating genes in three human glioma cell lines

 
Construction of Recombinant Adenoviruses Expressing p15 and p16.
To achieve a high transduction rate and level of expression, we chose the adenovirus expression system. cDNAs encoding p15 and p16 under the CMV promoter were inserted into the E1 region of E1- and E3-deleted adenovirus type 5 DNA. Replication-defective recombinant viruses, designated Ad5CMVp15 and Ad5CMVp16 were isolated by plaque purification, and high-titer virus stocks were produced. Preparations with a particle/p.f.u. ratio below 150 were used in all of the experiments. We have noted that preparations with a higher ratio exhibit increased cytotoxicity to glioma cells using a m.o.i. above 50 p.f.u./cell.

The time course of expression of p15 and p16 was determined in U251 MG cells by immunoblotting using anti-p15 and -p16 antisera after infection with Ad5CMVp15 and Ad5CMVp16 at a m.o.i of 50 p.f.u./cell (Fig. 1ACitation and B, respectively). An antiserum to the endoplasmic reticulum protein calnexin was used as an internal standard to monitor that the same amount of protein was loaded onto each lane. Both proteins were readily detected from 24 h p.i. onwards, the expression reaching a maximum at 2–3 days p.i., at which level it remained until about day 8. Both of the proteins exhibited a similar level and time course of expression. The level of protein was almost undetectable 2 weeks p.i. However, despite the high m.o.i. used, a minor fraction of the cells did not become infected initially. These cells were able to proliferate and expand over a 2-week period. The low level of p15 and p16 expression observed at day 14 is, therefore, to a large extent, a consequence of a dilution of p15- and p16-positive cells with uninfected ones.



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  		Fig. 1. Time course of recombinant adenovirus-directed expression of p15 and p16. U251 MG glioma cells were infected with Ad5CMVp15 (A) or Ad5CMVp16 (B) at a m.o.i. of 50 p.f.u./cell. Cytoplasmic extracts were prepared at the indicated time points after infection, and 20 µg of protein from each lysate was resolved by SDS-PAGE and probed with polyclonal antisera against p15 (A) or p16 (B). As an internal control for the amount of protein loaded, the same filters were also immunoblotted with an anticalnexin antiserum. Immunoreactive proteins were visualized by ECL. The positions of p15, p16, calnexin, and molecular weight markers are indicated.

 
As noted previously by others (7, 44) , p15 migrated as a doublet (Fig. 1ACitation ). We have recently determined that the more slowly migrating form (p15.5) is derived from an alternative translation initiation codon located upstream from the main AUG codon (50) .

The level of expression and time course of p15 and p16 in U373 MG cells were similar to those in U251 MG cells, whereas no expression was observed in T98G cells. Infection with a recombinant adenovirus expressing ß-galactosidase (Ad5CMVlacZ) showed that only a few T98G cells (<1%) became infected.

Association of Overexpressed p15 and p16 with CDK4 and CDK6.
We next analyzed whether overexpressed p15 and p16 could form complexes with CDK4 and CDK6, which would indicate that they are functional. U251 MG cells were infected with Ad5CMVp15 or Ad5CMVp16, and cell lysates prepared 48 h later were subjected to immunoprecipitation with antisera against CDK4 or CDK6. The precipitates were separated by SDS-PAGE, and proteins were blotted onto a nitrocellulose filter and probed with antisera against p15 (Fig. 2ACitation ) or p16 (Fig. 2BCitation ). As shown in Lanes 2 and 4, both forms of p15, as well as p16 were found in complex with CDK4/6. No p15 or p16 was recovered from mock-infected cells (Lanes 1 and 3).



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  		Fig. 2. Formation of complexes between overexpressed p15 and p16 with CDK4 and CDK6. Lysates from Ad5CMVp15-infected (A, Lanes 2 and 4), Ad5CMVp16-infected (B, Lanes 2 and 4), or mock-infected U251 MG cells (A and B, Lanes 1 and 3) were prepared 48 h after infection. Lysates were incubated with anti-CDK4 (Lanes 1 and 2) or anti-CDK6 (Lanes 3 and 4) antisera, and the immunoprecipitates were analyzed by SDS-PAGE, followed by transfer onto nitrocellulose filters. The filters were probed with antisera against p15 (A), or p16 (B). The positions of p15, p16, and molecular weight markers are indicated.

 
Overexpression of p15 or p16 in Human p15/p16-negative and pRb-positive Glioma Cells Results in Growth Arrest.
To compare the growth-inhibitory effect of overexpressed p15 and p16, U251 MG cells were infected with Ad5CMVp15 or with Ad5CMVp16 at a m.o.i. of 50 p.f.u./cell. As shown in Fig. 3ACitation , both inhibitors were equally effective in causing complete growth arrest during a 7-day period. The control Ad5CMVlacZ virus showed some growth-inhibitory effect at the end of the period as compared with mock-infected cells, probably attributable to toxicity of the virus. These results were reproduced by using another similar glioma cell line, Tp483 MG, which is also RB+, p15-, and p16- (Ref. 23 and our unpublished results4 ; Fig. 3CCitation ). We, thus, conclude that in two glioma cell lines lacking endogenous expression of p15/p16 but having an intact pRB gene, cell proliferation can be arrested equally efficiently by overexpressing either p15 or p16. We also determined whether p15 or p16 induced apoptosis of U251 MG cells. Using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, no increase in the apoptotic rate was observed (data not shown).



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  		Fig. 3. Overexpression of p15 or p16 inhibits U251 MG and Tp483 MG, but not U373 MG or SW1783 glioma cell proliferation. U251 MG (A), U373 MG (B), Tp483 MG (C), or SW1783 (D) glioma cells were seeded at 5 x 104 cells/well in six-well plates 1 day before infection with Ad5CMVlacZ, Ad5CMVp15, or Ad5CMVp16 at a m.o.i. of 50 p.f.u./cell. Control cells were mock-infected. The medium was changed every 2nd day throughout the experiment. At the indicated times, cells in triplicate wells were collected and counted. Bars, ±SD.

 
In contrast to U251 MG cells, the proliferation of U373 MG cells (Fig. 3BCitation ), as well as the cell line SW1783 (Fig. 3DCitation ), which also has intact CDKN2A and CDKN2B loci but is pRBnegative (Ref. 23 and our unpublished results4), could not be inhibited by overexpressing p15 or p16. The slight inhibition of U373 MG cells observed with Ad5CMVp15 and Ad5CMVp16 was similar to that of the control virus (Ad5CMVlacZ) and was likely attributable to the toxicity of the adenovirus particles (Fig. 3BCitation ).

Overexpression of p15 and p16 Induces Morphological Changes of U251 MG Cells.
To determine the fraction of cells overexpressing p15 and p16, U251 MG cells were infected with Ad5CMVp15 or Ad5CMVp16 with an m.o.i. of 50 p.f.u./cell. As shown in Fig. 4Citation , >90% of the cells were strongly p15 (Fig. 4ECitation ) and p16 (Fig. 4GCitation ) immunoreactive at 48 h p.i. Staining was predominantly nuclear, but a diffuse cytoplasmic staining was also evident. Already at this time point, both p15- and p16-expressing cells showed clear morphological changes as compared with mock-infected or Ad5CMVlacZ control virus-infected cells. Transduced cells were enlarged and flattened. This effect was much more pronounced at 7 days p.i. (Fig. 4, F and H)Citation . The cytoplasmic immunoreactivity showed a reticular and trabecular pattern, reminiscent of cytoskeletal structures. Mock-infected and control virus-infected cells were negative at both time points with either anti-p15 or anti-p16 antisera (Fig. 4A–DCitation ).



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  		Fig. 4. Morphological changes of U251 MG cells induced by overexpressed p15 and p16. U251 MG cells were infected with Ad5CMVp15 (E and F), Ad5CMVp16 (G and H), or Ad5CMVlacZ (C and D) at a m.o.i. of 50 p.f.u./cell. A and B, mock-infected cells. At 48 h (A, C, E, and G), or 7 days (B, D, F, and H) after infection, cells were fixed and permeabilized, and incubated with antisera against p15 (A, B, E, and F), or p16 (C, Di, G, and H) and FITC-conjugated secondary antibody. Scale bar, 50 µm.

 
In contrast to U251 MG cells, U373 MG cells infected with Ad5CMVp15 or Ad5CMVp16 showed no morphological changes even after 7 days p.i. (Fig. 5Citation , E and F, and G and H, respectively). It should be noted that the morphology of uninfected or Ad5CMVlacZ-infected U373 MG and U251 MG cells differ from each other. As evident from Fig. 5, A–DCitation , U373 MG cells are larger and more flat than U251 MG (Fig. 4, A–D)Citation .



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  		Fig. 5. p15 and p16 do not cause morphological changes of U373 MG cells. U373 MG cells were infected with Ad5CMVp15 (E and F), Ad5CMVp16 (G and H), or Ad5CMVlacZ (C and D) at a m.o.i. of 50 p.f.u./cell. A and B, mock-infected cells. At 48 h (A, C, E, and G) or 7 days (B, D, F, and H) after infection, cells were fixed and permeabilized, and incubated with antisera against p15 (A, B, E, and F) or p16 (C, D, G, and H), and FITC-conjugated secondary antibody. Scale bar, 20 µm.

 
Induction of SA-ß-gal in p15- and p16-transduced Cells.
The morphological changes induced in U251 MG cells infected with Ad5CMVp15 or Ad5CMVp16 were reminiscent of those seen in replicative senescence. A ß-galactosidase activity, detectable at pH 6.0 and referred to as SA-ß-gal, has been found to be expressed in senescent human fibroblasts but not in presenescent or quiescent cells (38, 51) . When U251 MG cells infected with Ad5CMVp15 or Ad5CMVp16 were stained for the SA-ß-gal marker 8 days p.i., the morphologically changed, enlarged, and flattened cells were strongly positive (Fig. 6Citation , D and F, respectively) in contrast to mock-infected (Fig. 6CCitation ) or to Ad5CMVlacZinfected cells (Fig. 6ECitation ). The latter cells were strongly positive if stained at pH 7.5 (data not shown). Normal human foreskin fibroblasts (AG1523) grown at low passage (Fig. 6ACitation ) or high passage (Fig. 6BCitation ) were used as negative and positive controls, respectively, for the specificity of SA-ß-gal activity (38) .



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  		Fig. 6. Induction of SA-ß-gal expression in U251 MG cells but not in U373 MG cells, by p15 and p16. Early (A) and late (B) passage primary human foreskin fibroblasts (AG1523) were used as negative and positive controls for SA-ß-gal activity. U251 MG (C–F) or U373 MG (G and H) cells were infected with Ad5CMVlacZ (E), Ad5CMVp15 (D and G), or Ad5CMVp16 (F and H) at a m.o.i. of 50 p.f.u./cell. Control cells (C) were mock-infected. Cells were grown for 8 days before staining for ß-galactosidase activity at pH 6.0 using X-gal as substrate. Scale bars: A–F, 50 µm; G and H, 100 µm.

 
The U373 MG cell line did not become SA-ß-gal-positive after infection by adenoviruses expressing p15 or p16 (Fig. 6, G and H)Citation . This is in conformity with the observation that neither p15 nor p16 induced a morphological change or could arrest cell growth in this cell line.

Inhibition of Telomerase Activity in U251 MG Cells Overexpressing p15 and p16.
Several reports have shown a correlation between cell proliferation and telomerase activity (52) . To find out whether U251 MG cells express telomerase and whether this activity could be inhibited by overexpressing p15 or p16, we carried out a semiquantitative analysis of telomerase activity levels in mock-infected and recombinant virus-infected cells at 2, 4, and 6 days p.i. Telomerase activity was readily detected in proliferating mock-infected and Ad5CMVlacZ-infected cells, and the activity increased somewhat during the 6-day follow-up period (Fig. 7Citation , A and B). In contrast, telomerase activity was efficiently repressed in both Ad5CMVp15- and Ad5CMVp16-infected cells, reaching an undetectable level at day 6. Telomerase activity was not repressed by p15 or p16 in U373 MG cells (Fig. 7CCitation ). In a set of independent experiments, these results were fully reproducible (data not shown).



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  		Fig. 7. Telomerase activity in U251 MG and U373 MG cells overexpressing p15 and p16. Cells were seeded in six-well plates 1 day before infection with Ad5CMVlacZ, Ad5CMVp15, or Ad5CMVp16 at a m.o.i. of 50. Control cells were mock-infected. The medium was changed every 2nd day. Telomerase activity was measured by the TRAP assay. A, a representative gel, showing a gradual decrease in telomerase activity in U251 MG cells expressing p15 and p16. B and C, graphic representation of relative telomerase activity as determined from triplicate samples prepared from 105 U251 MG (B) and U373 MG (C) cells 0, 2, 4, and 6 days after infection. All activities were related to the activity measured on day 0, which was set at 1.

 
Discussion

Whereas the role of p16 as a bona fide tumor suppressor has been amply documented, the contribution of p15 to tumorigenesis is still unclear. Because CDKN2A and CDKN2B are frequently lost concomitantly (14, 15, 17–19) and p15 mutations have very infrequently been detected in tumors (2, 18, 42) , it has been concluded that p15 is unlikely to play a significant role in tumorigenesis.

Here, we have compared the effects of p16 and p15, overexpressed from adenovirus recombinants, on proliferation and some phenotypic characteristics. As models, we used glioma cell lines the status of which in regard to relevant cell-cycle-regulating genes was determined. We found that both p16 and p15 efficiently inhibited proliferation of U251 MG having a p16-/p15-/pRb+/CDK4+/cyclin D1+/p53mt genotype, whereas neither protein inhibited U373 MG with a p16+/p15+/pRb-/CDK4+/cyclin D1+/p53mt genotype. Thus, although both of the CDKN2A and CDKN2B genes are deleted in U251 MG, it was sufficient to inhibit cell proliferation by restoring the activity of either of the two inhibitors. These results were fully reproduced in another pair of glioma cell lines, Tp483 MG and SW1723, which have the same RB, CDKN2A/2B status as that of U251 MG and U373 MG, respectively. Both proteins also similarly induced the profound morphological and phenotypic changes of U251 MG, including enlargement and flattening of the cells, induction of SA-ß-gal expression, and down-regulation of telomerase activity. These changes were not observed in U373 MG cells. Thus, the most important conclusion from our present results is that both p16 and p15 have a similar ability to arrest cell proliferation, to induce replicative senescence, and to inhibit telomerase activity in a glioma cell line provided that the pRB pathway is intact.

Whether the two inhibitors are equally efficient, on a molar basis, in exerting these biological effects could not be fully assessed using the adenovirus expression system. The mRNAs encoding p15 and p16 were both expressed from the CMV promoter, and the same titer of recombinant adenoviruses was used in each experiment comparing the biological effects. In addition, we carried out extensive control experiments to ascertain that the level of expression of p15 and p16 was the same in each experiment. These included careful titration of the virus, the use of different m.o.i., and titration of the antisera used. All of the results from these experiments suggested that p15 and p16 both qualitatively and quantitatively have the same biological effects.

The U373 MG and U251 MG glioma cell lines have been used as models in several recent publications (24, 45–48, 53, 54) . During the course of our studies, we found that the U373 MG obtained from ATCC had mutations in the TP53 and PTEN genes identical to those in U251 MG obtained from Uppsala, Sweden, where the U-series of cell lines were originally established (49) . In addition, the original U373 MG cell line displayed mutations that differed from those in U373 MG from ATCC. The fact that U373 MG (ATCC) and U251 MG (Uppsala) harbored identical mutations in two critical genes involved in oncogenesis suggests that they are derived from the same original clone, as was also pointed out recently by Ishii et al. (55) . We, thus, conclude that U373 MG distributed by ATCC is in fact U251 MG, and that a mix-up has occurred at some point.

Overexpression of p16, including the use of recombinant adenovirus as a vector, in glioma cells (32, 38, 51, 52, 54) and other types of tumor (36, 56–58) that lack endogenous p16 expression has been shown to inhibit proliferation. This growth-inhibitory effect is dependent on an intact RB gene (31, 33, 34, 36, 54, 59) . Our results in regard to p16 are in agreement with these conclusions. Together with the frequent loss of the p16 gene in primary tumors and tumor cell lines, as well as the increased incidence of tumors in mice with a targeted inactivation of the p16 gene (30) , these results all underscore the critical role of p16 as a suppressor of tumorigenesis.

The impressive body of data obtained in support of the role of p16 as a tumor suppressor is in sharp contrast to the relative lack of comparable data for p15. A p15encoding cDNA, stably transfected into a melanoma cell line, was shown to reduce the formation of colonies (60) . Results similar to those presented here were recently reported by Arap et al. (54) , who showed that p15 inhibited the growth of glioma cells possessing an intact RB pathway. Expression of p15, but not of p16, is regulated by exogenous growth-inhibitory factors such as TGF-ß (7) , or IFN-{alpha} (61) . TGF-ß-induced expression of p15 seems to cooperate with p27 to cause G1 arrest and quiescence (44) . Although p15 may not generally function as a tumor suppressor, it may do so under special circumstances, e.g., after exposure of cells to inhibitory factors such as TGF-ß and IFN-{alpha}. Loss of p15 expression could render cells resistant to the action of such suppressing cytokines.

Hayflick and Moorhead (62) originally reported that human fibroblasts grow for a limited number of cell divisions, followed by a state of senescence. Since then, the finite life span in vitro (Hayflick limit) has been documented for a variety of somatic cells. The intrinsic loss of cell-proliferative capacity (replicative or cellular senescence) is accompanied by characteristic morphological, phenotypic, and biochemical changes (63) . The drastic morphological and phenotypic changes of U251 MG cells observed as a consequence of overexpression of p16 or p15 are reminiscent of replicative senescence. Apart from the enlarged and flattened morphology, the cells expressed SA-ß-gal, shown to be specific for senescent cells (38, 51) . Some of the changes observed by us have also been reported by others, who have overexpressed p16 in glioma cell lines (38, 53, 59) . Overexpression of p16 or p15 in primary fibroblasts has also been shown to induce replicative senescence (64) , but the same effect on glioma cells has not been described before.

Several reports have pointed to a strong correlation between increased p16 expression and replicative senescence (37, 39–41, 65) . Human fibroblasts showed a 40-fold increase in p16 expression during senescence as compared with early-passage cells. (40) . Similar results were obtained with mouse fibroblasts. Whereas the level of p15 was unchanged, that of p16 increased with increasing population doublings (37) . Mouse embryo fibroblasts from p16-/- mice did not exhibit a senescent phase at a stage when normal cells entered senescence (41) . Human head and neck keratinocytes showed an up-regulation of p16 undergoing replicative senescence, whereas immortalized keratinocytes showed loss of p16 expression (39) . Recently, the transcriptional repressor bmi-1 was shown to negatively regulate p16 transcription. Mouse fibroblasts deficient in bmi-1 underwent premature senescence and showed a markedly increased p16 and p19ARF expression (66) .

Similar reports concerning a role of p15 in replicative senescence are largely lacking. However, the expression of both p15 and p16 have been shown to increase as Tlymphocytes approach senescence in vitro (67) . The fact that we could show that p15 caused a replicative senescence phenotype indistinguishable from that caused by p16 suggests that p15 under some situations could also play a role in senescence, and that the two cell cycle inhibitors may work through a similar mechanism.

Finally, as for most other tumors, we found that U251 MG and U373 MG cells expressed telomerase activity. This activity was down-regulated by overexpressing p15 and p16, the loss of activity correlating with the appearance of the morphologically altered senescent cells. To our knowledge, this effect of p15 and p16 has not been reported previously. The importance of telomere length in determining the life span of cells, as well as the role played by sustained telomerase activity for tumor cell proliferation, has been debated for more than a decade (68, 69) . Telomere length is shortened as cells approach replicative senescence (70, 71) , and senescence can be overcome by expressing the catalytic subunit of telomerase in telomerase-negative cells. Such cells show a dramatically extended life span (71) . However, human keratinocytes or mammary epithelial cells were not immortalized by expressing telomerase, unless the pRB/p16 pathway was simultaneously inactivated or p16 expression was down-regulated (72) . The mechanism by which p15 and p16 caused telomerase down-regulation in U251 MG cells possessing an intact RB pathway and a mutated p53 is unclear. Our results are supported by the finding of Xu et al. (73) , which show that re-expression of pRB in pRB/p53-defective tumor cells results in growth arrest and induces both replicative senescence and down-regulation of telomerase activity. These effects were not seen when p53 was reintroduced and suggest that there is an inverse relationship between p16/p15/pRB-mediated G1 arrest and telomerase activity.

Materials and Methods

Cell Cultures.
The human glioma cell lines U251 MG, U373 MG, and T98G, which have been grown in our laboratory for many years, were originally obtained from the Department of Pathology, Uppsala University (49) . To verify their identity, early passages of U251 MG (passage 43) and U373 MG (passage 51), were obtained from Dr. Monica Nistér (Department of Pathology, Uppsala University, Uppsala, Sweden). U373 MG was also obtained from the ATCC. Tp483 MG was established in our laboratory by V. P. C., and SW1783 was obtained from ATCC. The status of the RB, CDKN2A, and CDKN2B loci has been determined previously by He et al. (23) and in our laboratory. The cells were grown at 37°C in Ham’s F10 medium (Life Technologies Inc., Renfrewshire, Scotland) supplemented with 10% FCS (Life Technologies) and 1% penicillin and streptomycin. The normal human foreskin fibroblast cell line AG1523 was kindly provided by Dr. Lene Uhrbom (Uppsala University, Uppsala, Sweden) and was grown in Eagle’s MEM, supplemented with 10% FCS and 1% each of penicillin, streptomycin, and L-glutamine. The human embryonic kidney cell line 293 (from ATCC) was grown in DMEM supplemented with 10% FCS and 1% each of penicillin, streptomycin, and L-glutamine.

Plasmids and Construction of Recombinant Adenoviruses.
The transfer plasmids pAd5CMVp15 and pAd5CMVp16 containing an expression cassette consisting of the CMV promoter/enhancer, the cDNAs encoding p15 or p16, and the polyadenylation signal from the human ßglobin gene were constructed using standard recombinant DNA techniques. These cassettes were flanked on the 5' and 3' sides, respectively, by the left inverted terminal repeat (ITR) and a sequence corresponding to map units 9–5 of Ad5. The recombinant plasmids were cotransfected into 293 cells together with plasmid pJM17 obtained from Microbix Biosystems Inc. (Toronto, Ontario, Canada; Ref. 74 ). This plasmid contains the complete genome of the dl309 Ad5 mutant carrying an altered E3 region (75) . The cells were overlayed by agarose containing 1.5% low melting agarose (Seaplaque GTG; FMC Bioproducts, Rockland) in complete medium and were incubated at 37°C for 10–14 days. Recombinant adenovirus plaques were isolated, and the virus was amplified in 293 cells and finally purified by two consecutive CsCl gradient centrifugations. Ad5CMVlacZ{Delta}E1/{Delta}E3 (Ad5CMVlacZ) encoding ß-galactosidase was purchased from Quantum Biotechnologies, Inc. (Montreal, Quebec, Canada), and was similarly amplified and purified. Virus titers, determined by plaque assay on 293 cells under an agar overlay, varied between 1 x 1010 and 5 x 1010 p.f.u./ml. The concentration of virus particles was determined by measuring absorbance at 260 nm. Virus stocks with a particle:infectious particle ratio <150 were used in all of the experiments.

Infection with Recombinant Adenoviruses.
Prior to infection of glioma cells, the medium was changed to Ham’s F10 medium without serum. Glioma cells were infected at the desired m.o.i. with the different recombinant adenoviruses. The virus was allowed to adsorb for 60 min at 37°C, after which the unadsorbed virus was aspirated, and the cells were incubated with fresh medium for the desired time period.

DNA and RNA Analyses.
Extraction of genomic DNA and total RNA, Southern blotting, hybridization, exposure to storage phosphor screens, and densitometric assessment of allele dosage using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) were performed as described previously (76) . DNA probes for CDKN2A (exons 1 and 2), CDKN2B (exon 2), RB, CDK4, and CCND1 (cyclin D1) were generated by PCR amplification as reported previously (19, 25, 77, 78) . Plasmid probes for D12S7 (pDL32B) and D2S6 (pXG-18) were obtained from ATCC. SSCP analysis of the RB gene, including promoter region and all of the coding exons and flanking regions, was performed as described by Ichimura et al. (25) . PCR products showing abnormal SSCP band patterns were sequenced by the dideoxy method as described by Ichimura et al. (25) . Exons 5–8 of the TP53 gene and exons 5 and 7 of the PTEN gene were directly sequenced from PCR-amplified products (79) . Analysis of the expression of p16, p15, and pRB mRNAs using RT-PCR was performed as described previously (20, 25) . Amplification of ß-actin cDNA by RT-PCR was used as a control to assess mRNA amounts (20) . PCR products were analyzed on 1% ethidium bromide-containing agarose gels and were visualized and recorded using the Eagle Eye II system (Stratagene, La Jolla, CA).

Cell Lysates, Immunoblotting, and Immunoprecipitation.
Cell lysates were prepared by incubating U251 MG cells on ice for 30 min in a solubilization buffer [1% Triton X-100, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA (pH 8.0), 100 IU of aprotinin (Trasylol; Bayer AG, Leverkusen, Germany)). Nuclei were removed by centrifugation at 15,000 x g for 5 min. Protein concentrations were measured by the Bradford method (Bio-Rad Protein Assay; Bio-Rad Laboratories GmbH, Munich, Germany) and equal amounts of protein loaded on a 15% SDS-polyacrylamide gel followed by blotting on a nitrocellulose membrane (Hybond ECL; Amersham Life Science). Membranes were blocked for 1 h with 5% nonfat milk in TPBS (0.1% Tween 20 in PBS), probed with polyclonal antisera against p15 (N-20), p16 (N-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or calnexin prepared in our laboratory (80) . After several washings, membranes were incubated with antirabbit IgG conjugated to horseradish peroxidase, washed, and analyzed by ECL detection reagents (Amersham).

For immunoprecipitations, cell lysates prepared as described above, were precleared by incubating with normal rabbit serum and protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) at 4°C for 60 min, followed by centrifugation at 15,000 x g for 30 min. Supernatants were incubated with antisera against CDK4 (H22) or CDK6 (C21; Santa Cruz) for 3 h at 4°C. Immunocomplexes were collected by incubating with protein A-Sepharose at 4°C overnight. Immunoprecipitates were washed three times with solubilization buffer, dissociated in SDS-sample buffer, and analyzed on a 15% SDS-gel. Protein bands were identified by immunoblotting as described above.

Immunofluorescence.
Cells were seeded on coverslips in six-well plates the day before infection with recombinant adenoviruses. At 48 h or 7 days after infection, cells were fixed in 3% paraformaldehyde for 20 min and quenched with 10 mM glycine in PBS for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 30 min, incubated with anti-p15 or anti-p16 antiserum followed by detection with FITC-conjugated antirabbit IgG (Sigma Immunochemicals, St. Louis, MO). Cells were analyzed with an Axiophot microscope (Zeiss) equipped for immunofluorescence.

Cell Proliferation Analysis.
Cells to be counted were washed with PBS, trypsinized in trypsin-EDTA (Life Technologies, Inc.), and diluted in cell culture medium. The concentration of cells were then measured in a Coulter Counter (ZM; Scientific Instruments, Hialeah, FL).

SA-ß-gal Staining.
Mock- or adenovirus-infected cells were cultured for 8 days at 37°C and assayed for SA-ß-gal activity. Cells were washed twice with PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde for 5 min at room temperature, washed again twice with PBS, and stained with an X-gal staining solution at pH 6.0 as described previously (50) . Early (passage 18) and late passage (passage 35) human foreskin fibroblasts (AG1523 cell line) were used as negative and positive controls, respectively. Staining for recombinant adenovirus-directed ß-galactosidase expression was done after fixation with 3% paraformaldehyde at pH 7.5 using X-gal as substrate. Cells were viewed in a Nikon TMS light microscope and photographed with a Nikon F3 camera.

Telomerase Assay.
Cells were seeded at 1.5 x 105 (U251 MG) or 2 x 105 (U373 MG) cells per 10-cm dish 1 day before infection with different recombinant adenoviruses. At the day of infection (day 0), cells from triplicate dishes were collected and counted, and 1 x 105 cells were frozen at -80°C and were stored until assayed. Cells from infected, or mock-infected, triplicate dishes were collected and counted 2, 4, or 6 days after infection, and 1 x 105 cells were frozen. Cytoplasmic extracts were prepared by solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, and telomerase activity was determined using the telomeric repeat amplification protocol (TRAPeze kit; Oncor, Gaithersburg, MD). Telomerase activity levels were expressed as total-product-generated values.

Acknowledgments

We thank Anita Bergström and Elisabeth Raschperger for excellent technical assistance. We are grateful to Monica Nistér and Lene Uhrbom for cell lines, and to Koichi Ichimura for critical comments on the manuscript.

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 Present address: Department of Histopathology, Addenbrooke’s Hospital, CB2 2QQ Cambridge, England. Back

2 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-17177 Stockholm, Sweden. Phone: 468-310701; Fax: 468-332812; E-mail: rpet{at}licr.ki.se Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; CDKI, CDK inhibitor; RB, retinoblastoma; pRB, RB protein; ATCC, American Type Culture Collection; RT, reverse transcription; m.o.i., multiplicity/multiplicities of infection; p.f.u., plaque-forming unit(s); p.i., postinfection; SA-ß-gal, senescence-associated ß-galactosidase; mt, mutant; CMV, cytomegalovirus; TGF, transforming growth factor; Ad5, adenovirus type 5; SSCP, single-stranded conformational polymorphism; ECL, enhanced chemiluminescence; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside. Back

4 H. Goike, K. Ichimura, and V.P. Collins, unpublished observations. Back

Received for publication 9/13/99. Revision received 2/28/00. Accepted for publication 5/10/00.

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