Adenovirus-Mediated Gene Transfer of P16INK4/CDKN2 Into Bax-Negative

Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo Ingo Tamm,1,2 Axel Schumacher,3 Leonid Karawajew,2 Velia Ruppert,2 Wolfgang Arnold,4 Andreas K Nu¨ssler,5 Peter Neuhaus,5 Bernd Do¨rken,1,2 and Gerhard Wolff 2,4 1Department of Hematology and Oncology, Charite´, Campus Virchow, Humboldt University of Berlin, Berlin, Germany; 2Department of Hematology, Oncology and Tumor Immunology, Robert-Ro¨ssle-Klinik, University Medical Center Charite´, Humboldt University of Berlin, Berlin, Germany; 3Department of Cell Biology, Institute of Biology, Humboldt University of Berlin, Berlin, Germany; 4Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany; and 5Department of General, Visceral, and Transplantation Surgery, Charite´, Campus Virchow, Humboldt University of Berlin, Berlin, Germany.

The tumor-suppressor gene p16INK4/CDKN2 (p16) is a cyclin-dependent kinase (cdk) inhibitor and important cell cycle regulator. Here, we show that adenovirus-mediated gene transfer of p16 (AdCMV.p16) into colon cancer cells induces uncoupling of S phase and mitosis and subsequently apoptosis. Flow cytometric analysis revealed that cells infected with AdCMV.p16 showed an initial G2-like arrest followed by S phase without intervening mitosis (DNA >4N). Using microscopic analysis, deformed polyploid cells were detectable only in cells infected with AdCMV.p16 but not in control-infected cells. Subsequently, AdCMV.p16-infected polyploid cells underwent apoptosis, as assessed by AnnexinV staining and DNA fragmentation, suggesting that cell cycle dysregulation is upstream of the onset of apoptosis. Treatment of mice with subcutaneously transplanted tumors of colorectal cancer cells with AdCMV.p16 but not AdCMV.p53 resulted in significantly reduced tumor volume and prolonged survival. Using an orthotopic model of liver metastasis, we observed both reduced local tumor growth and secondary intrahepatic metastasis after AdCMV.p16 treatment. Importantly, induction of apoptosis in vitro and reduction of tumor growth in vivo by p16 was p53- as well as bax-independent because identical results were obtained using cancer cells, either wild type or mutant for p53 or bax. The studies suggest that an AdCMV.p16-based treatment may be especially effective in patients with bax-negative colon cancer where overexpression of p53 appears not to be of therapeutic value. Cancer Gene Therapy (2002) 9, 641–650 doi:10.1038/sj.cgt.7700480 Keywords: gene therapy; adenovirus; p16INK4/CDKN2; apoptosis; cell cycle

olorectal cancer is the second leading cause of cancer 10–15 months.1,5,6 Therefore, colon cancer represents an Cmortality in most Western countries.1–3 Early stages are important target for new therapeutic strategies. treated surgically, with high curability. However, locally Over the last years it became evident from studies of advanced cases and patients with distant metastases at patients with hereditary forms of colon cancer (hereditary diagnosis share a very poor prognosis. Recurrence following nonpolyposis colorectal cancer) that besides the known surgery, which occurs in 50–65% of the cases, is a major causes for neoplastic growth due to mutations in either problem especially in patients with lymph-node–positive proto-oncogenes or tumor-suppressor genes there exists a disease and often the ultimate cause of death.1,4 An estimated third class of genes that can contribute to tumor growth when 50,000 patients continue to die annually of this disease in the mutated. These genes encode enzymes involved in DNA United States, two-thirds of whom will have liver metastases mismatch repair and their mutational inactivation leads to as their first site of disease dissemination and 40% of whom genomic instability, which results in frameshift mutations of will have liver as the only site of metastatic disease.1,2 short nucleotide repeat sequences called microsatellites.7,8 Response rates to systemic therapy have remained in the Whereas this microsatellite mutator phenotype affects repeat range of 25–40%, with median survival times in the range of sequences throughout the genome, some appear to be preferentially targeted. One of these targets is the bax gene, a promoter of apoptosis, which is frequently mutated in 9 Received April 29, 2002. patients of the mutator phenotype. The absence of bax has been correlated with poor prognosis in nonhereditary colon Address correspondence and reprint requests to: Dr Ingo Tamm, 10,11 Department of Hematology and Oncology, Charite´, Campus Virchow, cancer. Humboldt University of Berlin, Forschungshaus, R. 2.0315, Augustenbur- Currently, most cancer gene therapy studies employ the ger Platz 1, 13353 Berlin, Germany. E-mail: [email protected] transfer of tumor-suppressor genes like p53 12,13 or the p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 642 transfer of genes that activate prodrugs into active chemo- with 10% fetal calf serum (FCS) and 2 mM glutamine. Cells therapeutic agents (e.g., herpes simplex virus thymidine were cultured at 378C in a humidified atmosphere of 5% CO2. kinase/ganciclovir system).14 – 17 In addition, immunologi- The cell lines LoVo and LS174T express wild-type p53 and cal approaches are under investigation using the transfer p21 but are negative for the expression of bax or p16 (Fig 3 of cytokine genes like IL-2 or IL-12,18 – 20 tumor specific and data not shown).9,47,48 The SW620 cells line express antigens like CEA,21 –24 or costimulatory molecules like mutant p53 but wild-type p21, bax, and p16 (data not B7-1 25,26 to induce tumor regression. Adenoviral vectors are shown).11,49 In accordance with institutional guidelines, mostly used for these experiments because they have a human hepatocytes were isolated from normal liver wedge tropism for a broad range of cell types, can be produced at resections by collagenase-P (Roche Diagnostics, Grenzau, high titers leading to a higher transduction efficiency, and Germany) digestion as recently described.50,51 Briefly, after infect both dividing and nondividing cells.27 enzymatic digestion hepatocytes were separated from non- The tumor suppressor p16 is a member of the INK4 family parenchymal cells by differential centrifugation at 50Âg and of cyclin-dependent kinase (cdk) inhibitor proteins.28 P16 then passed over a 30% Percoll (Pharmacia, Freiburg, inhibits cdk4 and cdk6, which promote cell cycle progression Germany) gradient at a concentration of 106 cells/mL through G1 phase by phosphorylation of the retinoblastoma Percoll to obtain a highly purified cell population. Hepatocyte gene product (Rb). Inhibition of cdk4 and cdk6 by plasmid- purity and viability assessed by microscopy was greater than borne p16 leads to hypophosphorylated Rb, which, in turn, 95% and viability consistently exceeded 90% by trypan blue blocks cell cycle transition from G1 to S phase.29 –31 In exclusion.51 The primary hepatocytes were cultured in addition, p16 plays a role in the regulation of apoptosis. Williams Medium E, supplemented with 10% heat-inacti- Adenovirus-mediated overexpression of p16 was reported to vated (90 minutes at 708C) FBS, 1% penicillin/streptomy- be cytotoxic to p53+ cancer cells,29 and p16 sensitized p53+ cin, 15 mM Hepes buffer (all Life Technologies, Karlsruhe, carcinoma cells to proapoptotic stimuli.32,33 Adenoviral gene Germany), 2.0 g/mL insulin, and 0.35 nM hydrocortisone transfer of p16 was shown to induce inhibition of tumor (both Sigma, Deisenhofen, Germnay) at 378C, 5% CO2 in a growth and apoptosis in vitro.32 – 34,36,37 Moreover, adeno- humidified atmosphere. viral delivery of p16 may reduce the metastatic potential of tumor cells in vitro.38 Interestingly, p16 is often mutated or Construction and propagation of recombinant adenovirus deleted in tumor cell lines and primary tumors.39 –43 Other vector mechanisms of p16 gene silencing have been described, in- The replication-deficient recombinant adenovirus (Ad 44 – 46 cluding hypermethylation and chromatin condensation. vector) constructs carry the cDNAs of p16 (AdCMV.p16), Mutations in the p16 gene are strongly associated with p21 (AdCMV.p21), or p53 (AdCMV.p53) and E. coli tumorigenesis because mice that are nullizygous for p16 -galactosidase (AdCMV. -gal), respectively, under the 35 develop spontaneous tumors. control of the cytomegalovirus (CMV) promoter, as The aim of this study was to elucidate the potential clinical described.32,52,53 The Ad vector AdRSV-hAAT.2 carries significance of adenovirus-mediated overexpression of p16 the cDNA of the human alpha-1 antitrypsin, driven by the protein in bax-negative colon carcinoma cells as a means of Rous sarcoma virus promoter (RSV).54 All vectors were blocking metastatic disease in vivo. We demonstrate that propagated on 293 cells, purified, and stored at À708C, as adenovirus-mediated overexpression of exogenous p16 described previously.55,56 Each vector preparation was tested induces apoptosis in colon cancer cells as a result of for the presence of replication competent virus by a modified uncoupling of S phase and mitosis (S/M uncoupling). This supernatant rescue assay. Briefly, 5Â105 nonpermissive cells effect was caused by an initial G2-like arrest followed by S (LoVo) were infected with 50 multiplicity of infection phase without intervening mitosis and subsequent p53- (MOI) of the respective vector and incubated for 6 days with independent apoptosis. Significant inhibition of tumor medium change on day 4. DNA was isolated from aliquots of growth could be observed in vivo. Using an orthotopic in the culture supernatant and assayed by PCR for the presence vivo model of liver metastasis, we show that the intratumoral of sequences from the E1a and E4 regions of the adenoviral application of p16 by adenovirus-mediated gene transfer genome. Only vector batches that were tested twice negative results in inhibition of local tumor growth and intrahepatic for E1a were subsequently used for experiments. metastasis as well as prolonged survival in a p53/bax- pathway–independent manner. Hence, the data support Detection of intracellular transgene expression clinical studies employing adenovirus-mediated gene trans- Cells were infected with various Ad vectors at the indicated fer of the cdk inhibitor p16 in patients with bax-negative MOI. Harvested cells were spun onto slides (5Â105 cells) colon cancer where overexpression of p53 appears not to be and air dried. Cells were fixed with 4% paraformaldehyde in effective therapeutically. PBS for 10 minutes and washed three times with TBS and incubated with TBS/BSA (0,2%) containing either anti-p16 Material and methods mAb (1:100; G175–405) or IgG1 mAb (1:100; MOPC-21; both Pharmingen, La Jolla, CA), and incubated for Cell lines and cell culture 60 minutes at room temperature. Following washing with All cell lines were purchased from ATCC. The human colon TBS cells were incubated with a bridging antibody (1:20; carcinoma cell lines LS174T and SW620 were maintained in rabbit anti-mouse Ig, Z259, Dako, Glostrup, Denmark) RPMI 1640; LoVo and 293 cells (human embryonal kidney) for 30 minutes in TBS/BSA. After additional three were cultured in DMEM. Both media were supplemented wash steps with TBS, cells were incubated with mouse

Cancer Gene Therapy p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 643 APAAP complexes (D651, Dako) in TBS/BSA (1:40) for model) after tumor cell challenge, respectively. Immediately 30 minutes. Following further washing slides were stained before vector injection, the tumor size was measured in two for 30 minutes with a New Fuchsine substrate solution, dimensions and the volume (v) was calculated according to counterstained with haemalaun, mounted, and analyzed v=(ab 2)/2 (with ab). Only tumors measuring 18–32 mm3 microscopically. (subcutaneous model) or 12–20 mm3 (orthotopic model) before the start of the treatment were included. The Ad Flow cytometric analysis of apoptosis and cell cycle vector dose administered was adjusted to the tumor size 6 3 5 assuming 10 cells/mm . Animals were inspected daily and A total of 8Â10 cells were seeded in triplicates in 10-cm the subcutaneous tumors were measured twice per week. dishes. Twenty-four hours later, cells were infected with 53 In the orthotopic model, the mice were killed and autopsied various Ad vectors at the indicated MOI as described. Cells before they reached the final cachectic phase. The size of were harvested by trypsinization at the indicated time points. the tumor at the injection site was measured as well as the For detection of apoptosis, cells were stained with FITC- number and size of metastases determined separately for conjugated AnnexinV and propidium iodide (PI) using a the remainder of the liver and extrahepatic sites. commercial kit (Immunotech, Marseille, France) as described by the manufacturer. Samples were subsequently analyzed by flow cytometry (FACScan, Becton-Dickinson, Results San Jose, CA) for the presence of viable (FITC-negative and PI-negative) and apoptotic (FITC-positive and PI- AdCMV.p16 leads to efficient gene transfer of p16 into negative) cells. For analysis of cell cycle distribution, FITC- colon cancer cells in vitro AnnexinV–stained samples were fixed and permeabilized as To confirm Ad vector–mediated overexpression of the 57,58 described. Briefly, after washing two times with PBS, a transgene, we performed immunocytological staining for 5 pellet of 5Â10 cells was fixed after addition of 2 mL ice- p16 24 hours after infection of LoVo colon cancer cells. Due cold 70% ethanol for 1 hour at 48C. After washing, cells to hypermethylation of its promoter region, LoVo cells are were resuspended in 0.5 mL PBS containing 50 g/mL PI negative for p16 expression at the mRNA and protein level (pH 7.5). After treatment with 10 l of 10 mg/mL RNase A (Fig 1C and data not shown).47,48 No p16 expression was (Boehringer Mannheim, Mannheim, Germany) at room detectable in control-vector–infected (Fig 1B) or mock- temperature in the dark for 30 minutes, cells were stored at treated cells (Fig 1C), whereas AdCMV.p16-infected cells 48C until flow cytometric analysis. Cell cycle analysis was stained strongly positive for p16 (Fig 1A). performed using CellQuest software (Becton Dickinson). Adenovirus-mediated overexpression of p16 leads to a G2/ Microscopic analysis of apoptosis M-like arrest in vitro Apoptosis was assessed microscopically using an immuno- Endogenous p16 is well known to induce G1 arrest by peroxidase-based detection kit for digoxigenin-labeled binding and inhibiting cdk4 and cdk6.28 To study the effects DNA strand breaks as described by the manufacturer of Ad vector–mediated overexpression of p16 (AdCMV. 1 (ApopTag Plus In Situ Apoptosis Detection Kit, ONCOR, p16) on cell cycle in colon cancer cells, LoVo cells were Gaithersburg, MD) with the following modifications: On slides, cells (5Â105 cells) were air-dried and incubated with 4% paraformaldehyde for 10 minutes at room temperature. After washing three times with PBS, cells were incubated in 0.1% Triton X-100 for 10 minutes at room temperature. Cells were washed again three times with PBS, then the manufacturers protocol was followed.

Animal studies C.B17/BlnA-Scid/Scid mice (SCID mice) were obtained from the animal core facility at the Max Delbru¨ck Center (Berlin, Germany). Mice of the same sex, age (8–10 weeks) and weight (20–22 g) were used for the experiments. Tumor cells (106 cells in 30 l of PBS) were inoculated subcutaneously into the left flank (subcutaneous model of xenotransplantation) or intrahepatically into the tip of the left lateral lobe of the liver (orthotopic model of xenotransplantation). For intrahepatic tumor cell injection, animals were anesthetized before laparatomy. After tumor cell inoculation, Figure 1 Intracellular localization of p16 protein after adenovirus- mediated gene transfer (AdCMV.p16) into p16-negative LoVo cells. the puncture was closed by hemoclip (Weck, Research LoVo cells were infected with 50 MOI of AdCMV.p16 (A,D), control Triangle Park, NC), the peritoneal wall was sutured with vector (AdRSV-hAAT.2) (B), or mock treated (C), respectively. catgut and the skin clamped (Michel 12 mm). Ad vectors Twenty-four hours after treatment, cells were harvested and were administered intratumorally in anesthetized animals analyzed for p16 expression (A–C); (D) isomatch IgG2 control 5 days (orthotopic model) and 14 days (subcutaneous antibody. Representative slides are shown (magnification Â200).

Cancer Gene Therapy p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 644 infected with either AdCMV.p16, AdCMV.p21, various reporter-gene constructs (AdRSV-hAAT.2, AdCMV. -gal), or treated with medium alone (mock). Because Ad vector infection itself can induce a modest reduction of cells in G1 phase compared to mock-treated cells,37,59 the effects of AdCMV.p16 and AdCMV.p21 were analyzed as change of cell cycle distribution compared to an irrelevant reporter gene construct (AdRSV-hAAT.2). Flow cytometric analysis revealed that infection with AdCMV.p16 resulted in a dramatic reduction of cells in the G1 phase of cell cycle after 96 hours, which was paralleled by a corresponding increase of cells in G2/M phase compared to control-infected and mock-treated cells (Fig 2) and data not shown). In contrast to AdRSV-hAAT.2-infected cells, infection with AdCMV. p21 led to a sustained G1 arrest (Fig 2).

Adenovirus-mediated overexpression of p16 causes apoptosis downstream of AdCMV.p16-induced cell cycle dysregulation in colon cancer cells but not in normal hepatocytes AdCMV.p16-infection of colon cancer cells resulted in a significantly higher number of apoptotic cells (P<.001) (Fig 3C) compared to control-infected (AdRSV-hAAT2, AdCMV.p21, Fig 3, B and D) or mock-treated (Fig 3A) samples, as assessed by a TUNEL-based assay. Similar results were obtained when apoptosis was assessed using AnnexinV staining (data not shown). To exclude cytotoxic effects on nonmalignant cells after Ad vector–mediated gene transfer, we performed additional studies using human primary hepatocytes. No significant differences in viability between AdCMV.p16, AdCMV.p21, and control (AdCMV. -gal, AdRSV-hAAT2) infected or mock-treated nonmalignant human hepatocytes were observed suggesting

Figure 3 AdCMV.p16 induces apoptosis in colon cancer cells but not in cultured human primary hepatocytes. LoVo cells were mock treated (A) or infected with 50 MOI of control vector (AdRSV-hAAT.2) (B), AdCMV.p16 (C), AdCMV.p21 (D). After 24 hours, cells were harvested and apoptosis was assessed using a Tunel-based assay as described in Material and methods. Representative slides are shown (magnification Â200). E: Primary human hepatocytes were cultured in vitro and either mock treated or infected with 20 MOI of AdCMV.p16 or control vector (AdCMV. -gal). After 48 hours, cells were harvested and viability was assessed by flow cytometry of AnnexinV-stained cells.

that AdCMV.p16 is nontoxic to normal liver tissue, at least at the MOI (20–50) tested in the experiments (Fig 3E and data not shown). To elucidate the AdCMV.p16-mediated proapoptotic effects on colon cancer cells in more detail, we analyzed Figure 2 Cell cycle distribution after adenovirus-mediated gene AdCMV.p16-infected nonapoptotic cells separately using transfer of the cell cycle regulators p16 or p21. LoVo cells were flow cytometry (Fig 4, A–C). Cells infected with AdCMV. infected with 50 MOI of either AdCMV.p16, AdCMV.p21, or control p16 accumulated significantly more hyperdiploid cells with vector (AdRSV-hAAT.2). At the indicated time points, Annexin- V-negative cells were analyzed for cell cycle distribution by flow a DNA content >4N (Fig 4C) compared to control-infected cytometry. Bars denote the relative changes of the fraction of (Fig 4B) or mock-treated (Fig 4A) cells. When the entire cells in G1 after infection with AdCMV.p16 (black columns) or cell population was analyzed, the occurrence of hyperdiploid AdCMV.p21 (white columns) compared to control Ad vector AdRS- cells was accompanied by a substantial increase in the V-hAAT.2 (set to 0). number of apoptotic cells (DNA content

Cancer Gene Therapy p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 645 cycle analysis. Again, the analysis of nonapoptotic sub- populations revealed that hyperdiploid cells (DNA content >4N) were exclusively observed in cells infected with AdCMV.p16 but not in mock-treated or control-infected cells. The occurrence of hyperdiploid cells was accompanied by a substantial increase in the number of apoptotic cells (DNA content

Adenovirus-mediated gene transfer of p16 inhibits tumor growth of colon cancer cells in vitro and prolongs survival Figure 4 Cell cycle distribution after adenovirus-mediated gene in tumor-bearing mice transfer of p16 in p53 mut SW 620 colon cancer cells. SW620 cells To analyze whether induction of apoptosis by S/M un- were infected with 200 MOI of AdCMV.p16, control vector (AdRSV- coupling after Ad vector–mediated gene transfer of p16 hAAT.2), or mock treated, respectively. Ninety-six hours after may also be effective in inhibiting tumor growth in vivo,we infection, cells were harvested and analyzed for apoptosis by injected 1Â106 p53 wt/bax neg LoVo cells or p53 mut/bax AnnexinV staining and cell cycle distribution by flow cytometry. One wt SW620 subcutaneously into SCID mice. After 14 days, representative analysis (of triplicate cultures) is shown (A to C: AnnexinV-negative subpopulation; D to F: entire cell population, the resulting tumors were subjected to in vivo gene transfer AnnexinV-negative and –positive cells. A,D=mock; B,E=AdRSV- by a single intratumoral injection of Ad viruses at an MOI of hAAT.2; C,F=AdCMV.p16). 30. In contrast to various control viruses (AdRSV-hAAT.2, AdCMV. -gal) or mock treatment, intratumoral administration of AdCMV.p16 resulted in a substantially reduced growth of both p53 wt/bax neg (Fig 5A and data not shown) significant apoptosis was detectable in control-vector– and p53 mut/bax wt tumors (Fig 5B and data not shown). In infected (Fig 4E) or mock-treated (Fig 4D) cells. addition, as suggested by our in vitro experiments (Figs 2 In parallel, using microscopic analysis of nonapoptotic and 3), adenovirus-mediated gene transfer of another cdk colon carcinoma cells, we detected deformed polyploid inhibitor, p21, did not inhibit tumor growth compared to nuclei exclusively in cells infected with AdCMV.p16 but not control vectors (data not shown). Further support for the in control-infected cells. Furthermore, only polyploid cells observation that inhibition of tumor growth by AdCMV.p16 underwent apoptosis suggesting that cell cycle deregulation overexpression is independent of p53-mediated proapop- preceded cell death (data not shown). Thus, colon carcinoma totic signaling in vivo comes from experiments where p53 cells can be killed effectively by Ad vector–mediated overexpression of p16. Following transient arrest in G2/M phase, infection with AdCMV.p16 induces uncoupling of S phase from subsequent mitosis (S/M uncoupling) leading to polyploid cells (N >4) and, finally, cell death by apoptosis.

Induction of apoptosis by AdCMV.p16 is independent of the genetic status of p53 and bax The tumor-suppressor gene p53 is an important upstream regulator of cell cycle and apoptosis.28 Because p53 is mutated in approximately 50% of malignant tumors10,11 and its downstream effector bax is also frequently mutationally inactivated, a gene therapy construct to be effective clinically Figure 5 Reduced tumor growth of subcutanously transplanted p53 should function in a p53/bax-independent manner. We wt / bax neg and p53 mut / bax wt colon carcinoma cells after investigated whether the observed effects of AdCMV.p16 are adenovirus-mediated gene transfer of p16 in vivo. LoVo cells (p53 p53- and bax-independent. For these experiments we used wt/bax neg) (A) or SW620 cells (p53 mut/bax wt) (B) were two colon carcinoma cell lines: LoVo cells are wild-type p53 inoculated subcutaneously into the left inguinal region of SCID mice (1Â106 cells in 30 l of PBS per animal). Fourteen days after (p53 wt) but bax negative (bax neg) and SW620 cells are inoculation of tumor cells, Ad vectors were injected intratumorally at p53 mutant (p53 mut) and wild-type bax (bax wt) (data not an MOI of 30. Tumor growth was analyzed as described in Materials 9,47,49 shown). Using the same experimental procedures as and methods. Data are given as mean±SEM. (o) AdCMV.p16, (Å) described above for LoVo cells, SW620 cells were infected AdRSV-hAAT.2, (4) AdCMV. -gal or ( — ) mock treated. Six with AdCMV.p16 or control vectors and subjected to cell animals were used per experimental group.

Cancer Gene Therapy p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 646 Table 1 Reduced metastatic tumor growth of colon tumors after adenovirus-mediated gene transfer of p16 in vivo

Mock AdRSV-hAAT.2 AdCMV.p16 Site of metastasis (%) (%) (%)

Peritoneal 33 27 0 carcinomatosis Diaphragms 83 82 18 Stomach 50 36 9 Spleen 58 64 9 Pancreas 67 27 18 Mesenterial 83 73 64 lymph nodes Pelvic 92 100 64 lymph nodes Peritoneal wall 0 27 9 Kidneys 42 55 18 Adrenal glands 25 64 0

LoVo cells (1Â106 cells per animal) were injected in the tip of the Figure 6 Reduced tumor growth of subcutaneously transplanted p53 left lateral lobe of the liver of SCID mice. Five days after inoculation, wt, bax neg LS174T colon cancer cells after adenovirus-mediated Ad vectors (30 MOI) were administered intratumorally. When the gene transfer of p16 in vivo. Fourteen days after inoculation of mice were sacrificed, the number of extrahepatic metastases was LS174T cells in the left inguinal region of SCID mice (1Â106 cells in assessed by whole-body autopsy of tumor-challenged animals as 30 l PBS per animal), Ad vectors were injected intratumorally as described in Materials and methods. The percentages of animals with described in Materials and methods. Ad vectors were administered metastases according to the affected anatomic site are given. intratumorally at an MOI of 30 and tumor growth was analyzed as Eleven/twelve animals were used per experimental /mock group. described in Materials and methods. Data are given as mean±SEM. (o) AdCMV.p16, (6) AdCMV.p53, (4) AdCMV.p21, (Å) AdCMV. -gal, or ( — ) mock treated. Eight animals were used per tumors in AdCMV.p16-infected mice revealed only very experimental group. few tumor cells within fibrotic tissue. Adenovirally mediated expression of the transgene was detectable in xenograft wild type, but bax-negative LS174T colon cancer cells were tumors for 80 days (data not shown). These results investigated in an analogous xenotransplant model. A single demonstrate that adenovirus-mediated gene transfer of injection of 30 MOI AdCMV.p16 into subcutaneous p16, but not of p21 or p53, by single intratumoral injection tumors led to a substantially reduced tumor growth in mice induces a significant reduction of tumor growth in both (Fig 6). In contrast, neither treatment with AdCMV. -gal, p53-mutant but bax-positive and p53-wild type but bax- AdCMV.p53, nor AdCMV.p21 significantly inhibited tumor negative tumors in vivo. Most importantly, AdCMV.p16 growth. All AdCMV.p16-treated animals were alive at day increased overall survival in tumor-bearing mice compared 83 (end of the experiment). In contrast, all mice in the to control vectors. control groups had to be euthanized until day 35 due to tumor growth. Histological examination of the remaining Adenovirus-mediated gene transfer of p16 inhibits tumor growth in an orthotopic model of liver metastasis in vivo Two-thirds of patients with colorectal cancer will have liver metastases as their first site of disease dissemination and 40% of whom will have liver as the only site of metastatic disease.1,2 Because liver metastases can be targeted by direct intratumoral injection, they represent a reasonable target for gene therapy strategies in vivo. To further investigate whether AdCMV.p16 infection of liver metastases from colon cancer can block secondary metastasis in vivo,we designed an orthotopic murine model of liver metastasis by xenotransplantation of 1Â106 LoVo colon cancer cells into Figure 7 Reduced tumor growth of colon tumors transplanted into the the tip of the left lateral lobe of the liver. At day 5, hepatic liver of SCID mice after adenovirus-mediated gene transfer of p16 in tumors were either injected with 30 MOI of adenoviral 6 vivo. LoVo cells (1Â10 cells per animal) were injected in the tip of the vectors AdCMV.p16, control vector (AdRSV-hAAT.2), or left lateral lobe of the liver of SCID mice. Five days after inoculation, mock treated. Tumor size at the site of injection and tumor Ad vectors (30 MOI/25–35 l) were administered intratumorally. volume of all liver metastases were evaluated when the When the animals had to be killed, local tumor volume at the site of injection (A)and total volume of liver metastases (B)were assessed animals were sacrificed (before they reached the final as described in Materials and methods.(5) mock treated, (y) control cachectic phase). vector (AdRSV-hAAT.2), (&) AdCMV.p16. Eleven/twelve animals Intratumoral administration of AdCMV.p16 significantly were used per experimental/mock group. inhibited local hepatic tumor growth compared to control

Cancer Gene Therapy p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 647 vectors (Fig 7A and data not shown) as well as secondary independent of proapoptotic signaling pathways downstream intrahepatic metastases (Fig 7B and data not shown). All of p53, i.e., pathways including bax. Our data underline the mock-treated and control-vector–infected mice developed significance of adenoviral vectors overexpressing members secondary liver metastases as assessed by macroscopic of the p16/Rb/E2F regulatory pathway such as E2F-1 69 or evaluation. In contrast, only 18% of animals injected with p16 as a means of bypassing p53-/bax-dependent apoptosis AdCMV.p16 developed secondary liver metastases (data not in vitro and bax-negative colon cancer in vivo, respectively. shown). Moreover, we observed a dramatic reduction in the Similar to p53, p16 is frequently altered in human cancers number of extrahepatic metastases in p16-infected mice in and represents a multifunctional key element in cell cycle comparison to control animals, as assessed by whole body regulation and apoptosis.28 Regarding the latter, coinfection autopsy of tumor-challenged animals (Table 1). For of AdCMV.p16 and AdCMV.p53 has been shown to be example, peritoneal carcinogenesis developed in 0%, 27%, cytotoxic to Rb+ cancer cells and to induce apoptosis. and 33% of p16, alpha-1 antitrypsin, and mock-treated However, the underlying mechanism remains elusive.29,32 animals, respectively. These results suggest that adenovirus- After infection of LoVo colon cancer cells with AdCMV.p16, mediated gene transfer of p16 blocks hepatic tumor growth we observed accumulation of infected cells in G2/M and metastatic disease in vivo. phase, subsequent S phase without intervening mitosis and, finally, apoptosis, as assessed by AnnexinV staining and DNA fragmentation. Our data suggest that S/M uncoupling Discussion is located upstream of apoptotic cell death and may represent a mechanism of how AdCMV.p16 induces cell death in the Among the different approaches to treat colon cancer by infected tumor cell. gene therapy is the ‘‘replacement strategy’’ where a mutated Because of their ability to inhibit phosphorylation and or deleted tumor-suppressor gene is substituted by its inactivation of Rb by cyclin/cdk complexes and their ability corresponding wild-type counterpart.60 The classical candi- thereby to induce a G1 growth arrest, the various cdk date gene is p53, which plays a central role in the regulation inhibitors are potential therapeutic tumor suppressors.37 of cell cycle and apoptosis.28 The latter is mediated, at least After infection of colon cancer cells with AdCMV.p16, we in part, by transcriptional activation of the proapoptotic bax observed only a transient G1 arrest, which was followed by a gene.61 However, as demonstrated by frame shift mutations G2-like block. The well-known ability of p16 to induce G1 of bax in colon cancer of the mutator phenotype,9 p53- arrest by binding and inhibiting cdk4 and cdk6 is usually controlled proapoptotic pathways may be blocked in tumor seen in experiments employing either endogenous or cells and this blockade cannot be overcome by over- plasmid-born p16. In contrast, we used adenovirus- expression of p53 as shown here in LS174T cells (bax mediated overexpression of p16 resulting in G2 arrest and neg) in vivo (Fig 6). Thus, in addition to results from other apoptosis, which may be caused by the intracellular gene therapy studies,28 our data provide further evidence that interaction of adenovirus and transgene. However, this effect alternative strategies are needed to improve the efficacy of in may also be dependent on the employed cell lines since vivo gene therapy for tumors expressing wild-type p53 and Schreiber et al observed cells accumulating in G2/M after mutated bax. adenovirus-mediated expression of various INK4 family Selective induction of apoptosis using transfer of pro- members in MT1A2 cells, but not in A549 cells, where the apoptotic genes like bax,62 apoptin,63 or caspases 64,65 di- same experimental procedure resulted in a G1 arrest.37 The rectly into tumor cells in vivo appears to be a promising authors were able to correlate the ability of the cdk inhibitors treatment strategy. However, a therapeutic effect using this to inhibit pRb phosphorylation in the different cell lines with approach requires intact downstream signal transduction their ability to arrest cells in G1.37 In another study, mRNA pathways. Unfortunately, death effectors like proapoptotic and protein levels of INK4 family members were found to bax 9 or caspases 66 are frequently mutated in tumor cells oscillate during the cell cycle, accumulating during S phase potentially blocking the therapeutic effect. Gene transfer of and remaining high throughout G2/M.70 In response to cdk inhibitors like p16 into tumor cells is a promising nonlethal ultraviolet irradiation, HeLa cells accumulated alternative antitumor approach that has been tested by elevated levels of p16. The accumulation of p16 correlated several groups.32,37,60,67,68 There are several advantages to with S phase and G2 delays. The UV-induced cell cycle choose p16 as therapeutic gene. We show that intratumoral delay was absent in cell lines derived from HeLa that did not application of AdCMV.p16 was superior to p53 or p21 in express p16 and in a melanoma cell line deleted for p16.71 controlling local tumor growth. The strong antitumor effect Thus, p16 may play an important role during later stages of of AdCMV.p16 was detectable as long as 12 weeks after a the cell cycle and its proapoptotic effects seem to depend on single intratumoral application, resulting in a prolonged an adenoviral delivery.37 survival of AdCMV.p16-treated animals. Moreover, we It has been shown that Ad virus infection of cells can detected AdCMV.p16-induced apoptosis in both p53 wt cause cell cycle dysregulation, which is characterized by a LoVo and p53 mut SW620 colon cancer cells, suggesting G2-like cell cycle arrest and deregulated expression of that AdCMV.p16-induced cell death is independent of the cyclins A, B1, D, and cdk p34cdc2 protein levels.59,72 genomic p53 status. Similar antitumor effects after over- However, the cell cycle disturbances observed in our expression of AdCMV.p16 were seen in LS174T cells, which experiments were clearly due to the overexpression of p16 are like LoVo wild type for p53, but negative for bax, and not the adenoviral infection itself because irrespective of suggesting that AdCMV.p16-induced apoptosis is also the genomic status of p16, p53, p21,orbax of tumor cells,

Cancer Gene Therapy p16 induces cell cycle alteration and apoptosis in colon cancer I Tamm et al 648 infection with various control vectors did not induce In summary, we demonstrate for the first time that significant cell cycle disturbances and apoptosis. overexpression of exogenous p16 induces apoptosis in bax- To investigate adenovirus-mediated gene transfer of p16 negative colon cancer cells as a result of uncoupling of S as a potential new treatment option for bax-negative phase and mitosis (S/M uncoupling). AdCMV.p16 infection metastatic colon cancer, we used an orthotopic model of is safe, applicable, and highly efficient in inhibiting liver liver metastasis in SCID mice. Similar to the results of the metastases of colon cancer in a p53/bax-pathway–inde- subcutaneous tumor model, in vivo gene transfer of p16, but pendent manner. Moreover, even a single injection of neither of p21 nor of p53, was superior to control vectors in AdCMV.p16 is capable of controlling tumor growth and inhibiting secondary liver metastasis. Most strikingly, we metastasis, which is accompanied by a prolonged survival of observed a significantly reduced number of metastases in tumor-challenged animals. These results indicate that other organs as well. Thus, the proapoptotic effect of AdCMV.p16 but not overexpression of p53 holds significant AdCMV.p16 in vitro is paralleled by an antitumor effect in promise as a therapeutic agent especially for patients with vivo. In concordance with other in vivo models,37,68 the bax-negative colon cancer with metastatic disease or at high animal studies presented in this paper demonstrate a low risk of relapse after first-line therapy. toxicity of p16 in vivo, because infection of normal hepatocytes with p16 did not cause more apoptosis than control vector or mock treatment, suggesting that the clinical Acknowledgments application of this treatment approach should be safe. The ability to promote cell death is not a universal property of all The authors thank Dr Mark Kay (Stanford University, cdk inhibitors because expression of p21 did not induce Stanford, CA) Dr Ronald Crystal (Cornell University, New apoptosis in cancer cells either in vitro or in vivo. In fact, York, NY), and Dr Michael Strauss (Max Delbru¨ck Center various researchers reported that p21 has a protective effect for Molecular Medicine, Berlin, Germany, deceased) for against apoptosis induced by p53 as well as other stimu- 37,73,74 generously providing the adenoviral vectors. Furthermore, li. In addition, despite extensive searches, only very we thank B Durieux, M Gries, K Ja¨ger, and P Pierschalek for few tumors have been shown to have mutations in their p21 75 expert technical assistance and M Dittmar for help in genes, and mice null for p21 develop normally with no preparing the manuscript. increased incidence of neoplasia.76 In our hands, only p16 inhibited tumor growth in vivo, demonstrating that the cdk inhibitors vary in their ability to promote cell death. Kim et al reported that adenoviral-mediated overexpression of wild- References type p16 in vitro induces cell death more efficiently in cancer cells expressing mutant forms of p16 than in cancer cell lines 1. 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