Adenovirus-Mediated Wt-P16 Reintroduction Induces Cell Cycle

D2001 Nature Publishing Group 0929-1903/01/$17.00/+0 www.nature.com/cgt

Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer Joaquim Calbo´,1,3 Mario Marotta,1,3 Manel Cascallo´,1 Josep Maria Roig,1 Josep Lluis Gelpı´,1 Juan Fueyo,2 and Adela Mazo1 1Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain; and 2Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030.

Pancreatic cancer has long carried poor prognosis. The development of new therapeutic approaches is particularly urgent. Inactivation of the tumor-suppressor gene p16 INK4a / CDKN2, a specific inhibitor of the cyclin-dependent kinases CDK4 and CDK6, is the most common genetic alteration in human pancreatic cancer, making it an ideal target for gene replacement. Here we transfected tumor cells using a recombinant adenovirus containing the wt-p16 cDNA (Ad5RSV-p16). The overexpression of p16 decreased cell proliferation in all four human pancreatic tumor cell lines (NP-9, NP-18, NP-29, and NP-31). However, G1 arrest and senescence were observed in only three. In contrast, the fourth (NP-18) showed a significant increase in apoptosis. This differential behavior may be related to the differences found in the expression level of E2F-1. Experiments on subcutaneous pancreatic xenografts demonstrated the effectiveness of p16 in the inhibition of pancreatic tumor growth in vivo. Taken together, our results indicate that approaches involving p16 replacement are promising in pancreatic cancer treatment. Cancer Gene Therapy (2001) 8, 740–750

Key words: p16; apoptosis; senescence; adenovirus; pancreatic cancer.

ancreatic cancer is one of the most aggressive human The pRb protein inhibits genes required for S-phase entry Ptumors and the fourth leading cause of cancer death in and stimulates genes that are active in resting cells.13 The Europe and North America.1 Nowadays, surgery is the only function of pRb is regulated by phosphorylation, which is curative therapy. Despite the improvements in diagnosis and carried out by cyclin-dependent kinases CDK4/6 and 10,13,14 surgical treatment, most patients are not candidates for CDK2, sequentially acting in the G1 phase. The surgery and less than 5% of patients survives for 5 years.2,3 activity of these kinases is positively regulated by D-type The development of new therapeutic approaches is therefore or E-type cyclins, respectively, and can be down- particularly urgent for pancreatic tumors. modulated by kinase inhibitors (CKIs).15,16 Changes in One of the most promising approaches to emerge from the the activity of the pRb protein are accompanied by improved understanding of cancer at the molecular level is variations in the expression level and the state of the possibility of using gene therapy to selectively target and phosphorylation of the other components of the retino- destroy tumor cells.4–6It has become clear that tumorigenesis blastoma family, p107 and p130.17 Complex interrelations is driven by alterations in genes that control cell growth or cell between these pocket proteins and the regulation of death.7,8 Despite the multiplicity of these lesions within a transcription factors belonging to the E2F family may cancer cell, the correction of a single critical genetic lesion determine the final destination of a cycling cell.16 –18 may be sufficient to abrogate tumorigenicity if the function Among the different CKIs, the most altered in tumori- restored succeeds in the reestablishment of growth control.9 genesis is p16.19,20 The p16 gene (INK4a, CDKN2, MTS) Research in cell cycle regulation has revealed a striking localized to the 9p21 chromosomal region encodes for a new regulatory network involving various tumor-suppres- CDK4/6–cyclin D complex inhibitor.21 By preventing sor genes and oncogenes. This network tightly regulates the formation of cyc/CDK complexes, the p16 protein entry into the cell cycle in normal cells and it is probably inhibits the phosphorylation of pRb, which remains bound to 10 deregulated in every tumor. Functional inactivation of E2F transcription factor, causing an arrest in the G1 phase of the p53/MDM2/p14ARF and pRb/p16/CDK4/cycD path- the cell cycle.22 ways appears to be a fundamental requirement for the It has been demonstrated that p16 level increases genesis of most human cancers because it results in gradually as cells proceed toward senescence, and when disruption of cell cycle regulation and deactivation of the expressed ectopically in mouse fibroblasts, it can elicit sen- apoptotic response.11,12 escence.23,24 Moreover, p16 is probably the most important mediator of the induction of senescence in response Accepted July 13, 2001. to Ras activation, and this action probably constitutes the basis of its role as a tumor-suppressor gene in many Address correspondence and reprint requests to Dr. A. Mazo, Depart- 25,26 ment of Biochemistry and Molecular Biology, C/ Martı´ i Franque´s 1, cancers. 08028 Barcelona, Spain. E-mail address: [email protected] Intriguingly, recent experimental evidence from p16 3These two authors contributed by equal to the study. overexpression experiments suggests additional biological

740 Cancer Gene Therapy, Vol 8, No 10, 2001: pp 740–750 CALBO´ , MAROTTA, CASCALLO´ , ET AL: P16 IN HUMAN PANCREATIC TUMORS 741 functions for p16. Apart from cell cycle control and (30 minutes at 568C). Mock-infected cells were incubated senescence,27 –31 p16 has been implicated in other funda- with serum-free medium. mental cellular processes, such as angiogenesis,32 tumor invasion,33 cell spreading,34,35 and apoptosis.36 – 39 Immunofluorescence assay Taking into account the importance of p16 as a tumor Cells were seeded on 10-mm coverslides in 60-mm suppressor, and its high frequency of alterations in pancreatic dishes (5–10 coverslides per dish). Infected cell mono- tumors,20 we planned a strategy based on adenovirus- layers were fixed 48 hours after infection with parafor- mediated transduction of the p16 gene into a panel of human maldehyde 4% in phosphate buffered saline (PBS); after pancreatic tumor cells. Our results indicate that restoration of washing in PBS, cells were permeabilized by incubation the p16 function caused significant inhibition of cell for 10 minutes in 0.2% Triton X-100 in PBS. Immuno- proliferation in all cell lines assayed and, depending on the detection was performed after blocking with PBS–BSA genetic background of tumor cells, it even induces cell death. 3% using a mouse antihuman monoclonal p16 antibody Moreover, experiments on human pancreatic tumors devel- (G175-405; Pharmingen, San Diego, CA) as primary oped in the subcutaneous tissue of Balb/c mice have antibody. Mouse IgG was used as control. The secondary demonstrated that the reintroduction of wt-p16 gene also antibody was an Alexa 488–conjugated goat antimouse reduces tumor growth in vivo. (Molecular Probes Europe, Leiden, the Netherlands). All the antibodies were diluted in washing solution containing MATERIALS AND METHODS 0.5% BSA and 0.05% Tween-20 in PBS. Immunofluore (ICN, Costa Mesa, CA) was used as mounting medium. Cell lines Photomicrographs were obtained in a Leica DMRB FLUO NP-9, NP-18, NP-29, and NP-31 cell lines were derived Microscope. from human pancreatic adenocarcinomas, which had been perpetuated as xenografts in nude mice.40 NP-9 and NP-29 Western blotting analysis were grown in DMEM:F12 medium (1:1) and NP-18 and Total cell lysates were obtained by incubating cells in RIPA NP-31 in RPMI 1640 medium, both supplemented with 10% buffer (150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, fetal bovine serum (FBS) and antibiotics, in a humidified 20 mM EDTA, 1% Igepal CA-630, and 50 mM Tris, pH 7.4) atmosphere containing 5% CO2 at 378C. The status of p16, for 1 hour at 48C and sonicating at 50 W for three cycles of p53, and p21 genes and their expression levels in these cell 30 seconds. After centrifugation at 14,000 rpm, supernatant lines are summarized in Table 1. proteins (40 g/lane determined by BCA protein assay; Pierce Chemicals, Rockfield, IL) were subjected to electro- Adenovirus and infection conditions phoresis on 8–12% SDS-PAGE and transferred to a Replicative-deficient recombinant type 5 adenoviruses were nitrocellulose membrane (Schleicher and Schuell, Dassel, used as vectors containing the complete p16 cDNA under a Germany). Membranes were blocked with Blotto-Tween Rous Sarcoma Virus promotor (Ad5RSV-p16,Ad-p16), or (3% BSA, 0.05% Tween 20, 0.9% NaCl, and 50 mM Tris, the cDNA of -galactosidase under the Cytomegalovirus pH 7.4) and incubated with the primary antibodies: mouse promotor (Ad5CMV-lacZ,Ad-lacZ), or without insert antihuman p16 (G175-405; Pharmingen), mouse antihuman (Ad-control). All viruses were cloned and amplified using E2F-1 (KH95; Santa Cruz Biotechnology, Santa Cruz, CA) the 293 cell line. Titration was done by plaque assay. and mouse antihuman PARP (7D3-6; Pharmingen) mono- Optimal infection conditions had been previously deter- clonal antibodies, goat antihuman pRb (C-15; Santa Cruz), mined by transfection with Ad-lacZ and X-gal staining.41 and rabbit antihuman p107 (C-18; Santa Cruz) polyclonal Cells were plated 24 hours before infection with Ad-p16 or antibodies, and with the secondary antibodies: HRP- Ad-control. The viral stock was diluted to reach a conjugated antimouse IgG (DAKO, Carpinteria, CA), multiplicity of infection (MOI) of 50, added to cell HRP-conjugated antigoat IgG (Santa Cruz), and HRP- monolayer, and incubated for 2 hours in serum-free medium conjugated antirabbit IgG (Sigma Chemicals, St. Louis, at 378C. Infection was stopped by adding medium MO). Membranes were developed according to Amersham’s supplemented with 10% heat-shock inactivated FBS ECL protocol (Amersham, Arlington Heights, IL).

Table 1. Status of Several Genes Involved in G1 Checkpoint in Human Pancreatic Tumor Cell Lines

Cell line p16 p16 protein p53* p53 protein p21 p21 protein

NP-9 11 bp deletion (codons 27–31) À 175 CGC–CAC ++ wt + NP-18 wild type + 246 ATG–GTG ++ wt ± NP-29 1 bp deletion (codon 36) À wt ± wt + NP-31 Homo (exon 1,2,3)yÀexon 7z ++ wt + *CGC, arginine; CAC, histidine; ATG, methionine; GTG, valine. yHomozygous deletion. zAn abnormal SSCT pattern was observed in multiple experiments; no mutation was detected by direct sequencing and/or sequencing of the PCR product.

Cancer Gene Therapy, Vol 8, No 10, 2001 742 CALBO´ , MAROTTA, CASCALLO´ , ET AL: P16 IN HUMAN PANCREATIC TUMORS

Cell growth curves performed by incubation in 0.1 M HCl, 0.5% Triton X-100 Cells were seeded at 10,000 cell/well in 24-well culture for 30 minutes at room temperature followed by 10 minutes 8 plates. Cells were infected with Ad-p16 or Ad-control at at 90 C. After blocking with FBS, 5-BrdU incorporated into MOI of 50. Serum-free culture medium was used for mock the DNA was immunodetected using an FITC-conjugated infection. Quadruplicate wells of each treatment were anti–5-BrdU (Caltag Laboratories, Burlingame, CA) and counted periodically until the eighth day after infection in DNA was stained with 20 g/mL PI and 10 g/mL an auto-analyzer Multisizer (Coulter, Hialeah, FL). ribonuclease A in PBS. Analysis was performed on an EPICS-XL flow cytometer (Coulter). Cell cycle analysis Apoptosis assays All measurements of cell cycle distribution were performed on an EPICS-XL flow cytometer (Coulter) equipped with Apoptosis was assessed by Annexin V-FITC binding an air-cooled argon ion laser to give 488 nm light. (Genzyme, Cambridge, MA). Supernatant and plated cells were collected 48 and 72 hours after infection, washed in Triplicates of cell cultures were harvested at different times 6 after viral infections and stained in TBS containing PBS, and resuspended in binding buffer. A total of 1Â10 propidium iodide (PI; 50 g/mL), ribonuclease A (10 cells were collected, washed in PBS, and resuspended in g/mL), and Igepal CA-630 (0.1%) for 1 hour at 48C. 1 mL binding buffer containing a final concentration of Data from at least 10,000 cells were collected and analysed 0.5 g/mL Annexin V-FITC and 5 g/mL PI. Cells were by Multicycle software (Phoenix Flow Systems, San incubated at room temperature for 1 hour in the dark. Diego, CA). Triplicate dishes of each sample were analysed with an EPICS-XL flow cytometer (Coulter). PI-positive cells were considered as the necrotic population, and only PI- 5-BrdU assay negative, Annexin V-FITC–positive cells were considered To measure DNA synthesis, cells were incubated for 4 hours as the apoptotic population. with 5-BrdU–containing medium (30 M). After the Apoptosis was also assessed by Hoechst 33342 pulse incubation, medium was aspirated and cells, at a concen- labeling protocol, as described by Darzynkiewicz et al.42 tration of 106 cells/mL, were fixed on ice-cold 70% ethanol. Chromatin condensed cells incorporate this dye more Fixed cells were pelleted, and DNA denaturation was rapidly than viable or necrotic cells. Infected cells were

A. NP-18 NP-9NP-31 NP-29 Ad-p16 - + --+++- 16 KDa -

B. a b

Figure 1. Detection of exogenous expressed p16 in transduced pancreatic tumor cells. (A) Western blot analysis NP-9 of the expression of p16 protein after infection with Ad-p16. Total extracts of mock-infected ( À ) and infected ( + ) cells with 50 MOI of Ad-p16 were collected 3 days after infection, electro- phoretically separated, and analyzed using a monoclonal antibody against c d p16. (B) p16 immunocytofluorescent detection on NP-9 and NP-18 cells 3 days after adenovirus-mediated p16 gene transfer. Cells were seeded on NP-18 glass cover slides and, 3 days after transfection, were fixed and immunos- tained against p16 (Â400). Ad-control (a) and Ad-p16 (b) transfected NP-9 cells; Ad-control (c) and Ad-p16 (d) transfected NP-18 cells. Infections were done at MOI 50 of each virus. Ad-control Ad-p16

Cancer Gene Therapy, Vol 8, No 10, 2001 CALBO´ , MAROTTA, CASCALLO´ , ET AL: P16 IN HUMAN PANCREATIC TUMORS 743 collected at days 1, 2, 3, and 4 after transfection, washed et al.43 Photomicrographs were obtained in an Olympus in PBS, counted, and resuspended at 106 cells/mL in IMT-2 Inverted Microscope. Hoechst 33342 containing PBS. After a 30-second incubation, cells were analysed in an Epics Elite FACS Experiments in vivo (Coulter) equipped with a UV laser lamp. Stained cells Animal care and use were in accordance with were also observed and photomicrographed with an recommendations for the Proper Care and Use of Olympus BH-2 Microscope. Laboratory Animals. A total of 2Â107 NP-9 or NP- 18 tumor pancreatic cells were injected subcutaneously Senescence-associated -gal staining (s.c.) into each posterior flank region of BALB/c nude Six days after infection, cell monolayer was washed in PBS mice. Tumors were allowed to grow for 16 days and and fixed for 30 minutes in 4% paraformaldehyde in PBS. experiments were started with tumors of 46±9 mm3 for The method for senescence-associated -galactosidase NP-9 and 103±49 mm3 for NP-18. The tumors were (SA- -gal) staining was performed as described by Dimri divided into four groups: groups 1 (NP-9 control) and

A. B. a b

NP-9

NP-18

Figure 2. Effects of p16 gene transfer on e f tumor pancreatic cell lines. (A) Cell growth curves. 104 cells of each cell line were seeded per well in 24-well dishes 1 day before being infected at MOI 50 of NP-29 Ad-control (&), Ad-p16 (~), or mock- infected (5) (day 0). Cell growth was monitored by counting cells from the monolayer in a Coulter Multisizer over the following 8 days. Results are the mean±SD of four separate samples. (B) Morphological analysis. Phase-con- g h trast photomicrographs showing changes in morphology 3 days after adenovirus-mediated p16 gene transfer NP-31 (Â100); Ad-control (a) and Ad-p16 (b) transfected NP-9 cells; Ad-control (c) and Ad-p16 (d) transfected NP-18 cells; Ad-control (e) and Ad-p16 (f) transfected NP-29 cells; Ad-control (g) and Ad-p16 (h) transfected NP-31 cells. Infections were done at MOI 50 of Ad-control Ad-p16 each virus.

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A. NP-9 NP-18

%G:53,8 %G: 48,9 ab%G1: 57,9 %G1: 86,1 cd1 1 % S: 15,1 %S: 5,1 %S: 32,3 %S: 35,3 %G: 14,7 %G: 15,9 %G2: 23,6 %G2:8,8 2 2

B. e % S: 13.8 f %S:1.7g %S:20.2h %S:19.8

Ad-control Ad-p16 Ad-control Ad-p16 Figure 3. Analysis of cell cycle in NP-9 and NP-18 transduced with Ad-p16. (A)Frequency histograms of DNA content (cell cycle profile). Two days after infection, cells were collected, permeabilized, and stained with PI, and DNA content was measured by flow cytometry. Ad-control (a) and Ad-p16–infected (b)NP-9 cell line; Ad-control (c)and Ad-p16–infected (d)NP-18 cell line. The analysis of the percentage of cells in each cell cycle phase was determined using Multicycle software. (B) Percentage of cells in synthesis phase of the cell cycle determined by 5-BrdU incorporation and flow cytometric analysis. Ad-control (e) and Ad-p16–infected (f) NP-9 cell line; Ad-control (g) and Ad-p16–infected (h) NP-18 cell line. 5-BrdU incorporated into the DNA was detected with an FITC-conjugated antibody (y axis, green fluorescence) and total DNA content was labeled with PI (x axis, red fluorescence).

2 (NP-18 control) received only saline; groups 3 (NP- respectively, and volumes were calculated according to 9Ad-p16) and 4 (NP-18 Ad-p16) received Ad-p16. the formula V (mm3 )=larger diameter (mm)Âsmaller Treatment was administered three times weekly (on diameter2 (mm)2Â/6. days 1, 8, and 15). Each administration consisted of 4Â5 L injections of 1010 pfu/mL Ad-p16 or saline serum in different tumor areas using a Hamilton Statistical analysis syringe. Tumors were measured every other day over The Mann-Whitney nonparametric test was used on paired the next 38 and 80 days in NP-18 and NP-9 tumors, data, and significance was set at P<.05.

Table 2. p16 Overexpression in Four Tumoral Pancreatic Cell Lines

G1 population S-phase Apoptosis Apoptosis PI staining population 5-BrdU Annexin V Hoechst pulse Cell Inhibition (%)y (%)z (%)x (%){ Senescence destination of growth acidic -gal after p16 (%)* Ad-c Ad-p16 Ad-c Ad-p16 Ad-c Ad-p16 Ad-c Ad-p16 staining ( reintroduction NP-9 66 57.9 86.1 13.8 1.7 3.7 4.1 10 13 + + + Senescence NP-18 85 53.1 48.9 20.2 19.8 8.0 28.6 8 26 À Apoptosis NP-29 30 57.4 65.4 15.5 8.2 n.d. n.d. 9 11 + + Senescence NP-31 50 66.8 80.9 21.3 7.6 n.d. n.d. 12 13 + + Senescence This table summarizes the effects of Ad-p16 transfection on NP-9, NP-18, NP-29, and NP-31. *Percentage of growth inhibition at day 8 after Ad-p16 respect to Ad-control infection, as measured by cell counting in a Coulter Multisizer.

yG1 population determined by flow cytometry analysis of PI-stained cells. zS-phase population determined by flow cytometry analysis of 5-BrdU incorporation. xApoptosis measurement by flow cytometry analysis of Annexin V incorporation. {Apoptosis measurement by flow cytometry analysis of Hoechst 33342 pulse-labeled cells. (Presence of SA- -Gal activity in transduced cells. y, z, x, { obtained at day 2 after infection; ( obtained at day 6 after infection.

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Immunohistochemistry of tumor sections Subcellular localization was assessed by immunocyto- Some of the tumors were treated a fourth time on day 21 in chemistry on the third day after infection. Fluorescence order to obtain tumoral tissue samples. Three days after the observed in Ad-p16–infected cells indicates that the last injection, mice were sacrificed and tumors were excised, exogenous protein localized both in the nucleus and in the fixed in formalin, embedded in paraffin, and stored for cytoplasm (Fig 1B). further experiments. Five micrometer paraffin sections was Cell growth and morphology after infection with Ad-p16 mounted on coated glass slides, baked for 20 minutes at 608C, and dewaxed. Immunodetection of p16 was performed Cell growth curves of the four cell lines showed a significant after heat antigen retrieval using a mouse antihuman growth inhibition after the second day postinfection (Fig monoclonal p16 antibody (G175-405; Pharmingen) as 2A). The inhibitions ranged from 30% in NP-29 cells to primary antibody. Mouse IgG was used as control. The 85% in NP-18 cultures. The progressive slowing of cell secondary antibody was an ENVISION antimouse (DAKO). proliferation was accompanied by morphological changes in Slides were developed with DAB and counterstained with the transduced tumor cells (Fig 2B). In three of the four cell hematoxylin. lines assayed, cells acquired a more refractile cytoplasm and showed a flattened and enlarged morphology, characteristic of arrested or senescent cells. In contrast, a significant proportion of NP-18 cells showed typical features of RESULTS apoptotic status; cells shrank, separated from their neighbors, Infection conditions and p16 expression appeared refringent, and finally detached from the monolayer by day 3 after infection. The four human adenocarcinoma pancreatic cell lines (NP- 9, NP-18, NP-29, and NP-31) were efficiently transduced with the Ad-p16 vector. Cells were infected with Ad-p16 at Cell cycle and senescence analysis variable MOI to identify a dose that would produce In order to determine the mechanism of growth inhibition detectable p16 expression and a clear functionality of the elicited by p16 overexpression in our pancreatic cell lines, expressed protein without cytotoxic effects in cells trans- the cell cycle was analyzed at different times after viral duced with Ad-control (data not shown). For most experi- infection. In NP-9 cells, the expression of exogenous p16 ments, the virus concentration was fixed at 50 MOI. led to a marked G1 arrest. The ratio G1 /S in transduced The expression of exogenous p16 was analyzed by NP-9 cells varied from an initial value of 2.5 to 16.9 after Western blot (Fig 1A). Exogenous p16 was detected in all infection. In contrast, NP-18 cells did not show cell cycle four cell lines 24 hours after the infection and reached its arrest in G1 phase after Ad-p16 infection, whereas a maximum at 48–96 hours, decreasing progressively there- significant population was observed to be in sub-G1 phase, after. In all cell lines, p16 expression was at least 50% of the suggesting that the exogenous p16 expression causes maximum on the eighth day after infection (data not shown). apoptosis in these cells (Fig 3A). To corroborate the

ab NP-9

c d NP-18

Ad-control Ad-p16 Figure 4. Induction of senescence by p16 overexpression. Six days after infection, cells were fixed and stained with X-gal to detect a senescence-associated acidic -galactosidase activity (Â100). Noninfected (a) and Ad-p16–infected (b) NP-9 cell line; noninfected (c) and Ad-p16–infected (d) NP-18 cell line.

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A. NP-9 NP-18 256 ab256 310 c128 d 8.0 % 28.6 % 3.7 % 4.1 % Events Events Events Events

0 0 0 0 1 2 3 1 2 3 1 2 3 1 2 3 11010 FITC log 10 11010 FITC log 10 11010 FITC log 10 11010 FITC log 10 Ad-control Ad-p16 Ad-control Ad-p16

Ad-p16 Ad-control ab B. 1 2 3 4 1 2 3 4 Day C.

-- 116 KDa NP-9 -- 85 KDa NP-18 -- 116 KDa -- 85 KDa Ad-control Ad-p16 Figure 5. Apoptosis induction in Ad-p16 transduced cells. (A) Annexin V binding to the apoptotic cells. Two days after infection, cells were collected, washed in PBS, and directly labeled with FITC-conjugated Annexin V and PI. Analysis was carried out by flow cytometry and necrotic cells (PI-positive) were discarded. Ad-control (a) and Ad-p16 (b) transfected NP-9 cells; Ad-control (c) and Ad-p16 (d) transfected NP-18 cells. (B) PARP cleavage. Total extracts of Ad-control and Ad-p16–infected cells were collected at days 1, 2, 3, and 4 after infection, run in 8% SDS-PAGE gels to optimally separate the cleaved form (85 kDa), and analyzed using a monoclonal antibody against PARP. (C) DNA staining with Hoechst 33342. Ad-control (a) and Ad-p16 (b) infected NP-18 cells were stained with Hoechst 33342 and observed in a fluorescence microscope. Arrows mark chromatin condensation in apoptotic nuclei (Â100).

absence of G1 arrest in NP-18 cells, 5-BrdU assays were To check the apoptosis observed in p16-transduced NP- performed. Results from these experiments clearly indicate 18 cells by other methods, cell extracts were prepared at that in Ad-p16–transduced NP-9 cells, DNA synthesis different times postinfection (days 1, 2, 3, and 4) and was stopped, whereas transduced NP-18 cells did not examined for evidence of poly(ADP-ribose) polymerase show variations in the percentage of cells in S phase (Fig (PARP) cleavage. In the Western blot shown in Figure 5B, 3B). NP-29 and NP-31 cells did not show such a clear the 85-kDa cleavage product was only observed in extracts arrest in their cell cycle profile as NP-9 cells when from NP-18 cells infected with Ad-p16, confirming that transduced with Ad-p16. However, significant decreases in these cells underwent apoptosis when p16 was overex- S phase percentage were observed in 5-BrdU incorpo- pressed. ration experiments (Table 2). Apoptosis induced in NP-18 cells was also visualized by We also determined the presence of senescence features in incubation with the vital DNA dye Hoechst 33342. p16-transduced cells. Acidic X-gal staining indicated that Transduced NP-18 cells showed characteristic morpholog- NP-9, NP-29, and NP-31 cells were committed to a ical patterns with condensed chromatin in the nucleus and senescent phenotype, as assessed by the increase in their the appearance of apoptotic bodies, whereas in control cells, SA- -gal activity. NP-18 cells did not show any increase in these features were not seen (Fig 5C). SA- -gal activity, corroborating the different destination of these cells when p16 is overexpressed (Fig 4). Effect of the reintroduction of wt-p16 on pRb, p107, and E2F-1 expression Apoptosis determination Western blot analysis performed with NP-9 and NP-18 We determined the percentage of apoptotic cells in cell lines cell extracts at different times postinfection with Ad-p16 infected with Ad-p16 by two methods: Annexin V and showed that, concomitant to high levels of p16 expression, Hoechst 33342 pulse labeling. Both analyses showed that the pRb protein exhibited hypophosphorylation (Fig 6). In p16 overexpression increased the apoptosis level only in addition, a marked decrease in the level of total pRb was NP-18 cells (Fig 5A). In NP-9, NP-29, and NP-31 cell observed by day 3 postinfection. Similar results were lines, no significant increases in apoptosis were found (Fig obtained with NP-29 and NP-31 cell lines (data not 5A, Table 2). shown).

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Ad-p16 Ad-cont Ad-p16 Ad-cont Western blots of the same cell extracts using antibodies 0 2 3 2 3 0 2 3 2 3 against p107 and E2F-1. For both proteins, we observed a dramatic decrease in expression in NP-9 cell extracts, α-pRb ]pRbpp -pRb whereas no significant variations were observed in NP-18 cell extracts (Fig 6). α-p107 in vivo α-E2F-1 Experiments We have previously determined the efficiency of adenoviral α-p16 transduction in vivo by using a vector containing a marker NP-9 NP-18 gene, Ad-lacZ. -gal–expressing areas were evaluated after intratumoral injection of 2.4Â108 pfu of this vector. At Figure 6. Western blot analysis of the expression of pRB, p107, and 2 days after virus administration, X-gal staining showed that E2F-1 in pancreatic tumor cells transduced with Ad-p16. Immuno- the expression of -galactosidase was restricted to patches blotting of pRb, p107, and E2F-1 in NP-9 and NP-18 cells at days 0, of -gal–expressing cells around the injection points in the 2, and 3 after infection at MOI 50 of Ad-p16 or Ad-control. In the lower tumors.44 No more than 20% of reduction in tumor growth panel is an immunoblot showing p16 overexpression in both cell lines. was observed in animals treated with Ad-lacZ at the same dose as for Ad-p16 (data not shown). To check differences in the expression levels of other Here, we examined the capacity of Ad-p16 to inhibit the proteins involved in the cell cycle arrest, we performed growth of tumors in vivo. Human tumors were developed in

A NP-9 NP-18 1400 7000 1200 a 6000 b 1000 5000 800 4000 600 3000 400 2000 % tumor growth

% tumor growth 200 0 1000 -200 0 0 20406080 0 10203040 Day 1 Day 1 B NP-9 tumors c d

α-p16

Figure 7. Response to p16 gene transfer in vivo. (A) Tumor growth curves. Xenografts established in the s.c. tissue of BALB/c nude mice were treated with Ad-p16 (~) or saline buffer (5) at days 1, 8, and 15 (indicated by arrows) and tumor growth was measured over the next 80 days in NP-9 tumors (a)and 38 days in NP-18 tumors (b). Difference between groups was statistically significant from day 21 for NP-9 tumors and from day 10 for NP-18 tumors (P<.05). Data are expressed as the median of each group. Bars indicate first and third quartiles, respectively. (B) p16 immunohistochemistry on NP-9 tumor sections. Paraffin sections were obtained 3 days after the last saline (c) or Ad-p16 (d) injection. p16 protein was immunodetected on sections of these tumors using a monoclonal -p16 primary antibody and an ENVISION antimouse secondary antibody. Immunohistochemistry was developed with DAB and sections were counterstained with hematoxylin.

Cancer Gene Therapy, Vol 8, No 10, 2001 748 CALBO´ , MAROTTA, CASCALLO´ , ET AL: P16 IN HUMAN PANCREATIC TUMORS the s.c. tissue of nude mice and treated as described in cell cytoplasm, such as cell membrane–associated activities Materials and Methods. Marked reductions in tumor during cell proliferation and migration.33 – 35 volumes were observed in the Ad-p16–treated groups In all cell lines, the newly expressed p16 was functional. corresponding to both NP-18 and NP-9 when compared Inhibition of cell growth was observed in all treated cells, with the control groups. Statistically significant differences and was significant from day 2 after infection. In three of were found from day 10 after the start of treatment in NP-18 four cell lines assayed, this inhibition was associated with G1 tumors, and from day 21 in NP-9 tumors. In spite of the arrest and subsequent acquisition of the senescence pheno- market inhibition of the growth observed in NP-18 tumors, type. This behavior, observed in all p16-negative cells, is in no absolute decreases in volume were observed. NP-9 agreement with previous results of Ad5RSV-p16–treated tumors treated with Ad-p16 showed greater inhibition of cells 28,29,31 and plasmid-p16 transfection experiments.49 growth than NP-18 tumors. Moreover, NP-9 tumors showed The only cell line that contains endogenous wt-p16 did a pronounced reduction in the size after the third admin- not exhibit G1 arrest and senescence phenotype. This istration of Ad-p16. This decrease was observed for 15 days difference in behavior strongly suggests that these cells have (Fig 7A). the ability to overcome the cell cycle arrest imposed by p16, Immunohistochemistry of p16 on tumor sections revealed probably because of an additional alteration in the same exogenous p16 expression in localized groups of cells, pathway. This is consistent with the current hypothesis that confirming the difficulty of delivering the therapeutic gene to whole pathways should be considered, rather than alterations the whole tumor (Fig 7B). To determine the impact of p16 to specific genes. In this respect, there is substantial evidence on the proliferation index of tumors, we performed Ki67 in favor of alterations in different components of the main immunodetection. NP-18 tumors had a much bigger pathways involved in tumorigenesis (p53 and p16/pRb proliferation rate than NP-9 tumors because they presented pathways), which allow transformation of the cell by an 60–80% vs 2–5% Ki67-positive cells, respectively, but no oncogenic stimulus.11,50 relevant differences between groups of treatment were found Interestingly, NP-18 cells suffered the greatest growth (data not shown). inhibition when p16 was overexpressed, and the results demonstrated that this inhibition was due to the induction of apoptosis. In the last years, emerging evidence has been DISCUSSION reported showing that apoptosis may be induced by p16 overexpression, particularly if p53 is also overexpressed, but The availability of new human pancreatic cell lines with no clear explanation for the mechanisms of such apoptosis marked differences in the status of several genes involved in has been provided until now.36 – 39,51,52 the control of the cell cycle and apoptosis, such as p16 and One hypothesis that relates the inhibition of CDK4/CycD p53 (Table 1), makes it possible to study the impact of p16 complexes with apoptosis is that it arises from a decrease in overexpression in different models of human pancreatic the pRb levels observed when p16 blocks this pathway.53 cancer both in vitro and in vivo. Low pRb levels may be enough to cancel the protective Adenoviruses are excellent tools to transfer genes to effect of pRb, releasing the E2F-1 factor, which in turn human tumors owing to their efficiency in infecting human would induce apoptosis by p53-dependent or p53-inde- cells and their low toxicity in vitro and in vivo.45 However, pendent pathways.54,55 Indeed, the results obtained with the several problems still stand in the way of optimal results, different cell lines may support this hypothesis. While all such as their immunogenicity, which limits virus dissem- four cell lines showed a reduction of pRb levels over time, ination.46 Another limitation for their use in therapy is the important differences in the levels of p107 and E2F-1 were lack of specificity, which may be overcome by using targeted observed between NP-9 and NP-18 cell lines. In NP-9 cells, vectors and/or regional perfusion.47,48 p107 and E2F-1 disappeared concomitantly to the reduc- Nevertheless, until more potent and specific vectors (ideal tions in pRb levels, whereas in NP-18 cells, this disappear- vectors) are developed, preclinical assays are needed to ance was not observed. These differences might determine identify the most suitable genes for therapeutic approaches. whether cells enter the normal program of cell cycle arrest In the present study, we have addressed this last point. As a and subsequent senescence or are committed to other final very high percentage of pancreatic tumors have lost p16 destinations. However, we cannot rule out that p16 may expression,20 it is predictable that reintroduction of func- induce apoptosis by other very recently described mecha- tional p16 gene could be an effective molecular approach to nisms, such as a regulation of cell–cell and/or cell– the treatment of these tumors. extracellular matrix interactions.56 When wt-p16 gene was delivered to our pancreatic cell In vivo experiments are also informative because the lines by using Ad5RSV-p16, Western blot analysis demon- results indicate that p16 overexpression is also able to elicit strated that expression of the p16 protein was significantly an important inhibition of tumor growth. In spite of the low increased in all infected cells and maintained long enough to extent of tumor areas transduced, the differences in tumor achieve notable effects on cell proliferation. Immunocyto- growth are statistically significant from day 10 in NP-18 chemical analysis performed on the third day after infection model and from day 21 in NP-9 tumors (P<.05). revealed that the newly expressed p16 was located both in the Surprisingly, the impact on tumor growth achieved in Ad- nucleus and in the cytoplasm of all transduced cell lines. This p16–treated NP-18 tumors was lower than that reached in result could be consistent with the additional roles attributed NP-9 tumors. These differences may be related to the slower to p16 other than control of cell cycle, which are fulfilled in growth of NP-9 tumors, correlating with the doubling times

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