Paradoxical Increase in Retinoblastoma Protein in Colorectal Carcinomas May Protect Cells from Apoptosis1

Vol. 5, 1805–1815, July 1999 Clinical Cancer Research 1805

Hirofumi Yamamoto, Jae-Won Soh, growth inhibition and apoptosis, perhaps by counterbalanc- Takushi Monden, Micheal G. Klein, ing potentially toxic effects of excessive E2F activity. Li Ming Zhang, Haim Shirin, Nadir Arber, INTRODUCTION Naohiro Tomita, Ira Schieren, C. A. Stein, and 2 Studies done about 10 years ago on cases of hereditary I. Bernard Weinstein Rbs3 led to the discovery of the Rb gene, the first tumor Herbert Irving Comprehensive Cancer Center [H. Y., J-W. S., suppressor gene to be identified (1–3). Subsequent studies in- M. G. K., H. S., C. A. S., I. B. W.], Center for Neurobiology and Behavior [I. S.], Department of Medicine [L. M. Z., C. A. S., dicated that the function of this gene is also lost, at the somatic I. B. W.], and Department of Pharmacology [C. A. S.], Columbia level, in a variety of nonhereditary human cancers, including University, College of Physicians and Surgeons, New York, New cancers of the breast, lung, soft tissue, bladder, and prostate. On York 10032; Department of Surgery II, Osaka University Medical the other hand, deletions and loss of expression of the Rb gene School, Osaka, 565-0871, Japan [H. Y., T. M., N. T.]; and are rare in human colorectal carcinomas (for review see Ref. 4). Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv, 64239, Israel [N. A.] Indeed, in these cancers, and in cell lines derived from these cancers, there is often increased expression of the Rb gene (5–8), and in some tumors, there are additional copies of the Rb ABSTRACT gene due to amplification of the corresponding DNA region on The retinoblastoma (Rb) gene is inactivated in a variety chromosome 13 or nonrandom increases in chromosome 13 (4). of human cancers, but in colorectal carcinomas there is This increase in the expression of a tumor suppressor gene in frequently increased expression of this gene. This is para- colorectal cancers seems to be paradoxical, and its biological doxical in view of the known role of Rb as a tumor suppres- significance is not known. sor gene. In the present study, we compared the levels of Studies done within the past few years indicate that the expression of the Rb protein (pRb) in normal human colo- protein encoded by the Rb gene, pRb, normally plays a key role rectal mucosa, adenomatous polyps, and carcinomas by im- as a negative regulator of the G1 to S transition in the cell cycle munohistochemistry. In vitro studies were also done to ex- by binding the transcription factor E2F and preventing it from amine the phenotypic effects of an antisense oligo- activating the transcription of genes required for the S phase. deoxynucleotide (AS-Rb) targeted to Rb mRNA in the Phosphorylation of pRb by cyclin D1/CDK4 or cyclin D1/ HCT116 colon carcinoma cell line that expresses a relatively CDK6 complexes, and possibly by the cyclin E/CDK2 complex, high level of pRb. The incidence of pRb-positive cells was during the latter part of G1 relieves this inhibitory effect, thus increased during multistage colorectal carcinogenesis. In activating E2F (for review see Ref. 9). Human colorectal car- vitro treatment of HCT116 cells with AS-Rb decreased the cinomas frequently display abnormalities in the expression of level of pRb by about 70% and also decreased the levels of several genes that play either positive or negative roles in this the cyclin D1 protein and cyclin D1-associated kinase activ- pathway. Thus, there is frequently increased expression of the ity. AS-Rb inhibited growth of HCT116 cells and induced cyclin D1, cyclin E, CDK1, and CDK2 proteins (7, 10, 11), and apoptosis. Reporter assays indicated about a 17-fold in- in a subset of tumors and cell lines, there is amplification of the crease in E2F activity. These findings suggest that the in- cyclin E and CDK2 genes (12, 13). Alterations in cyclin decreased expression of pRb in colorectal carcinoma cells may pendent kinase inhibitors are also seen in colorectal carcinomas. provide a homeostatic mechanism that protects them from The p16Ink4 gene is inactivated in about 40% of the cases due to de novo methylation of a 5Ј CpG island (14), and expression of the p21Waf1 gene is frequently decreased (15, 16). Decreased levels of p27Kip1 due to proteolytic degradation have been seen Received 12/23/98; revised 3/31/99; accepted 3/31/99. in a subset of cases, and this is associated with a poor prognosis The costs of publication of this article were defrayed in part by the or high-grade tumors (17, 18). payment of page charges. This article must therefore be hereby marked To further explore the role of pRb in colon cancer, in the advertisement in accordance with 18 U.S.C. Section 1734 solely to present study, we have examined the expression of pRb by indicate this fact. 1 Supported by an award for cancer research from the FUSO Pharma- immunohistochemistry, and we found that there was an increase ceutical Company (Osaka, Japan; to H. Y.), a Deutshes Krebsforschun- in the expression of pRb during the multistage process of gszentrum (DKFZ) award (to N. A.), and by National Cancer Institute Grant CA 63467 and an award from the National Foundation for Cancer Research (to I. B. W.). 2 To whom requests for reprints should be addressed, at Herbert Irving Comprehensive Cancer Center, Columbia University, College of Phy- 3 The abbreviations used are: Rb, retinoblastoma; pRb, Rb protein; sicians and Surgeons, 701 West 168th Street, New York, NY 10032. oligo, oligodeoxynucleotide; TUNEL, terminal deoxynucleotide trans- Phone (212) 305-6921; Fax: (212) 305-6889; E-mail: weinstein@ ferase; DAPI, 4Ј,6-diamidino-2-phenylindole; AS-Rb, antisense Rb cuccfa.ccc.columbia.edu. oligo; S-Rb, sense Rb oligo.

colorectal carcinogenesis. To examine the phenotypic and bio- Rb mRNA: 5Ј-GUCAUGCCGCCCAAAACC (AUG is the chemical effects of decreasing the level of expression of pRb in translation start site). colon cancer cells, we carried out in vitro studies using the These 18 mer oligos are identical or similar to those used in HCT116 human colon carcinoma cell line because it expresses previous studies (20–22). a relatively high level of pRb. Our strategy was to transfect into Transfection of Oligos. Cells were plated at the appro- these cells an antisense phosphorothioate oligonucleotide tar- priate numbers, according to the plate size, and grown for 48 h geted to the 5Ј region of Rb mRNA. Our results provide evi- in the standard medium, at which time they were in the expo- dence that the increased expression of pRb has a positive selec- nential growth phase and the monolayer was 40–50% confluent. tion value in HCT116 colon carcinoma cells because it protects They were then rinsed once with serum-free medium and trans- them from growth inhibition and apoptosis. Furthermore, our fected with a 1 ␮M solution of the indicated oligo together with studies provide evidence that cellular levels of pRb can mark- the lipofectin reagent (Life Technologies, Inc., Gaithersburg, edly influence the expression of other proteins involved in cell MD), which were premixed for 15 min after dilution, as recom- cycle control. mended by the manufacturer, in the medium (DMEM for HCT116 cells and RPMI 1640 for WI38 cells). The amount of lipofectin (19 ␮g/ml) and volume of medium (3–4 ml for a 6-cm MATERIALS AND METHODS dish) used were proportional to the plate area. Four hours later, Cell Lines and Tissues. The HCT116 human colon car- fetal bovine serum was added at a final concentration of 10%. cinoma and the WI38 human lung fibroblast cell lines were The toxicity of the lipofectin was monitored by cell viability obtained from the American Type Culture Collection. The using trypan blue dye exclusion and found to be minimal be- HCT116 cell line was grown in DMEM, and the fibroblasts cause the viability for HCT116 and WI38 cells at 48 h after were grown in RPMI 1640 plus 10% fetal bovine serum, 100 transfection was over 96%. ␮ Western Blot Analysis. Western blot analysis was per- units/ml penicillin, and 100 g/ml streptomycin in 5% CO2 at 37°C. Fifty colorectal carcinomas and paired adjacent normal formed, as described previously (23). Equal loading of the mucosa samples, 50 samples of adenomatous colorectal polyps protein samples was confirmed either by Coomassie blue stain- (37 tubular and 13 tubulo-villous adenomas), and 15 polyps ing or by immunoreactivity with an antiactin antibody (Sigma). containing focal carcinomas were collected during surgery or The intensities of the bands were quantitated with an image during endoscopic polypectomy in the Department of Surgery II scanner (Molecular Dynamics). in Osaka University Medical School from 1994–1996. The Antibodies. The following antibodies were used for detection of various human proteins, using the concentrations samples of normal mucosa were cut in the longitudinal direction recommended by the manufacturer: (a) mouse monoclonal an- to examine the top, middle, and bottom parts of the glands, and tibodies: Rb (G3-245) and p16Ink4 (Pharmingen), and p21Waf1 the polyps and carcinomas were cut across the maximum diam- (Ab-1; Oncogene Science); (b) rabbit polyclonal antibodies: eter. These samples were fixed in buffered formalin at 4°C cyclin A, cyclin D1, CDK2, CDK4, and cyclin E (UBI); actin overnight and embedded in paraffin. For Western blot analyses, (Sigma); p27Kip1 (C-19) and CDK6 (Santa Cruz). a piece of tissue was immediately frozen in liquid nitrogen and Flow Cytometric Analysis. Flow cytometric analysis stored at Ϫ80°C. was performed, as described previously (23). Immunostaining. Immunostaining was performed by an In Vitro Assay for Cyclin D1, Cyclin E, and CDK2- avidin-biotin complex method, as described previously (7). An associated Kinase Activities. In vitro assays for cyclin D1-, anti-pRb antibody (G3-245; Pharmingen) was applied to the cyclin E-, and CDK2-associated kinase activities were per- sections at a dilution of 1:50. A negative control section, to formed as described previously (23). For the cyclin D1-associ- which normal mouse serum had been applied rather than the ated kinase assay, 200 ␮g of the cell extracts were immunopre- specific antibody, was included in each staining procedure. The cipitated with 2 ␮g of the cyclin D1 antibody. The GST-Rb entire series of samples was stained at least twice, with sepa- fusion protein (1 ␮g) was incubated with the immunoprecipitate rately prepared sections, and similar results were obtained. With plus 10 ␮Ci [␥-32P]ATP for 30 min at 30°C. For the cyclin E- each section, 10 fields were randomly picked under high power or CDK2-associated kinase assay, 100 ␮gor50␮g of total cell magnification of the microscope, more than 700 cells were extracts, respectively, were immunoprecipitated with 2 ␮gof scored for positive or negative nuclear staining, and the percent- the cyclin E antibody or 1 ␮g of the CDK2 antibody. These age of pRb-positive cells was determined. If a nucleus displayed immunoprecipitates were incubated with 5 ␮g of Histone H1 brown staining, it was considered positive. (Sigma) as the substrate and 5 ␮Ci of [␥-32P]ATP for 30 min at Construction of Antisense and Sense Oligos to Rb 30°C. The reaction mixtures were then subjected to SDS-PAGE, mRNA. An antisense phosphorothioate oligo to Rb mRNA and the intensities of phosphorylation of the substrates were (AS-Rb) was designed to target the translation start site. It was determined by autoradiography. synthesized by standard methods using a 380B DNA synthesizer In Situ Apoptosis Detection Using the TUNEL Assay (Applied Biosystems Inc., Foster City, CA; Ref. 19). As a and DAPI Staining. For the detection of apoptotic cells, an in control, the corresponding S-Rb was synthesized. The se- situ apoptosis detection kit ApopTag (Oncor, Gaithersburg, quences of these oligos and the corresponding region of the MD) was used, as recommended by the manufacturer (20). For human Rb mRNA are shown below. the DAPI staining, the cells were incubated with DAPI solution Antisense oligo: 3Ј-CAGTACGGCGGGTTTTGG-5Ј; (Boehringer Mannheim, Indianapolis, IN) at a concentration of sense oligo: 5Ј-GTCATGCCGCCCAAAACC-3Ј; 1.5 ␮g/ml PBS for 30 min and then washed in PBS for 2 h. For

Fig. 1 Immunohistochemical staining for pRb expression with an antihuman pRb antibody in normal colonic mucosa (A), an adenomatous colonic polyp (B), a focal cancer in an adenomatous polyp (C), and a colon carcinoma (D). In the normal mucosa, the positive staining for nuclear pRb was confined to the lower portion of the crypts and the germinal center of lymphoid follicles (A). The upper regions of the normal crypt (not shown here) did not show pRb staining. In the polyps and carcinomas, pRb- positive cells were scattered throughout the lesions. Magni- fication: A, ϫ66; B, ϫ66; C, ϫ50; D, ϫ50.

quantitation of apoptotic cells, 10 microscopic fields were ran- Statistical Analyses. Statistical analyses were performed domly chosen at ϫ25 magnification, and a series of pictures using In Stat version 2.01. To determine correlations between were taken under light (for whole cells) or fluorescence (for the expression of pRb and histological types, Fisher’s exact test TUNEL-positive cells or for DAPI-positive cells) microscopy. was used. The differences obtained between the S-Rb and The total number of the cells and the number of apoptotic cells AS-Rb treatment of the HCT116 cells was determined by the (those with a fluorescent signal by TUNEL staining and those unpaired t test. with a nuclear fragmentation or a condensation by DAPI staining) were counted in each field. Over 700 total cells were counted in each sample, and the percentage of the apoptotic cells RESULTS was calculated. pRb Expression during Multistage Colorectal Carcino- Luciferase Reporter Assay for E2F Activity. E2F ac- genesis. The expression of pRb was examined by immunohis- tivity was determined by a luciferase reporter assay. The lucif- tochemistry in the following set of colorectal samples: normal erase reporter plasmid E2F-Luc contains four repeats of the mucosa (n ϭ 50), adenomatous polyps (n ϭ 50), focal carcino- wild-type E2F site (TTTCGCGC) upstream of a minimal TATA mas within adenomatous polyps (n ϭ 15), and various stages of box, followed by the luciferase coding sequence (24). To nor- carcinomas (n ϭ 50; Fig. 1 and Table 1). The extent of expres- malize the transfection efficiency, the E2F-Luc plasmid was sion of pRb was divided into three categories, based on the cotransfected with a CMV-␤ gal (galactosidase) plasmid at a percentage of cells that were positive for pRb, as follows: ratio of 2:1. At the indicated time, the cell extracts were pre- Ͻ10%, 10–50%, and Ͼ50%. In the normal mucosa samples, pared and assayed for luciferase and ␤-galactosidase, as de- pRb-positive nuclei were seen only in the transitional zone of scribed previously (25). The luciferase level in each sample was the crypt in the lower portion of the glands and not the upper normalized by the corresponding value for ␤-gal activity. portions of the crypts, and also in the germinal centers of Colony Efficiency Assay. Cells were seeded at a density lymphoid follicles (Fig. 1A). The percentage of pRb-positive of 1 ϫ 105/well in 6-cm dishes. Twenty four hours later, the epithelial cells in all of the normal mucosa samples was Ͻ10% vectors encoding wild-type E2F (CMV-Ap12-Stu) or mutant (Table 1). When compared with the normal mucosa samples, 14 type E2F (CMV-Ap12⌬DS) under CMV promoter and the of 50 (28%) adenomatous polyps, 13 of 15 (87%) focal carci- corresponding control vector (26) were cotransfected with the nomas in polyps, and 39 of 50 (78%) carcinomas displayed pBabe plasmid encoding puromycin resistance at a ratio of 5:1, increased expression of pRb. When a cut off of Ͼ50% was used using the lipofectin reagent. Forty-eight hours after transfection, to define very high expressors of pRb, none of the normal the cells were subplated into 15-cm dishes and then selected in mucosa samples, 8% of the adenomatous polyps, 47% of the the presence of 0.5 ␮g/ml puromycin in complete medium for focal carcinomas, and 34% of the carcinomas fit this criterion 2 weeks. Individual drug-resistant clones were stained with (Table 1). The differences between the normal mucosa and Giemsa solution (Sigma). tumor samples and between the adenomatous polyps and focal

Table 1 pRb expression during multistage colorectal carcinogenesis In the polyps group of samples, the percentage of pRb-positive cells showed no significant correlation with the following clinicopathologic parameters: location in the colorectum (right side, transverse, left side, or rectum), size (Ͻ10 mm, 10–20 mm, or Ͼ20 mm), shape (pedunculate, pseudosessile, or sessile), histology (tubular or tubulo- villous). In the carcinomas group of samples, the percentage of pRb- positive cells showed no significant correlation with tumor size (Ͻ3.9 cm or Ͼ4.0 cm), depth of invasion, Dukes’ stage, site, or metastasis to lymph nodes or the liver. pRb expression (% of cells positive) Ͻ10% 10–50% Ͼ50% Total Normal mucosa 50 (100%) 0 0 50 Adenomatous polyps 36 (72%) 10 (20%) 4 (8%) 50 Focal cancers in 2 (13%) 6 (40%) 7 (47%) 15 adenomatous polyps Carcinomas 11 (22%) 22 (44%) 17 (34%) 50

cancers were significant (P ϭϽ0.0001 and 0.0018, respectively), but the differences between the focal carcinomas and carcinomas was not significant (P ϭ 0.381). These findings indicate that during the transition from normal mucosa to adenomatous polyps to carcinomas there is a progressive increase in pRb expression. No significant correlations were found between the extent of pRb expression and other clinicopathologic parameters (Table 1), but we should emphasize that only a relatively small number of cases were available in each data set. Phosphorylation status of pRb was also examined by West- ern blot analyses with the same antibody that was used for immunostaining. Ten normal mucosa expressed solely underphosphorylated pRb, whereas 9 of 10 cancer tissues exhibited both hyper- and underphosphorylated pRb. In the adenomatous polyps, seven of eight samples displayed mainly the underphosphorylated pRb (data not shown). Fig. 2 Effects of AS-Rb and S-Rb on the expression of pRb (A) and Effects of AS-Rb on pRb Expression and Cell Growth. on growth (B) in HCT116 cells. A, cells were plated at 1–2 ϫ 105 in a An antisense phosphorothioate oligo to Rb mRNA (AS-Rb) was 6-cm diameter dish or at 3–5 ϫ 105 in a 10-cm dish (for the confluent designed to target the translation start site, and the correspond- cultures 5 ϫ 105 cells/6-cm diameter dish were seeded) and then ing S-Rb served as a control, as described previously (20–22). transfected with 1 ␮M AS-Rb, S-Rb, or lipofectin alone. At 46 h after The lipofectin reagent (Life Technologies, Inc.) was used as the transfection, extracts were prepared and examined by Western blot analysis for the level of expression of pRb. B, for the growth studies, vehicle. To assess the self-association properties of these oligos, 2 ϫ 104 cells were plated in 24-well plates (1.5-cm diameter) in 0.5 OD of the AS-Rb or S-Rb was subjected to PAGE in a triplicate for each treatment 2 days before transfection, and the cell nondenaturing gel (12%), and the gels were stained with number was about 8 ϫ 104 at the time of transfection. At 24, 36, and ethidium bromide solution. A single band was seen, which 48 h after transfection, the cells were trypsinized and counted in a Coulter counter to provide growth curves. The data indicate the mean migrated at the appropriate rate for a monomer. No higher-order values and SDs for triplicate assays. structures were observed (data not shown). To determine the effects of reducing the level of pRb expression in the HCT116 colon cancer cell line, the cells were transfected with 1 ␮M AS-Rb, 1 ␮M S-Rb, or only lipofectin, as described in “Mate- (Ͻ10%) reduction in the level of pRb. In the growing condition, rials and Methods.” Cell extracts were harvested 46 h later when the control culture treated only with lipofectin displayed a the cells were confluent or still growing and examined by prominent doublet, consisting of both the hyperphosphorylated Western blot analysis with a pRb antibody. With extracts of the (higher molecular weight) and underphosphorylated (lower mo- confluent cultures only single Rb bands were detected that were lecular weight) forms of pRb (Fig. 2A, bottom). Similar results about 110 kDa (underphosphorylated forms of pRb; Fig. 2A, were obtained with untreated HCT116 cells (data not shown). top). The treatment with AS-Rb led to a marked reduction in the The treatment with AS-Rb reduced the total level of pRb by level of pRb. Densitometric analysis indicated that the residual about 70%, and the residual pRb protein was mainly the hyper- level was only about 30% that of the control culture, which was phosphorylated form. Equal loading of these lanes was con- treated only with lipofectin. In contrast, treatment of the cells firmed either by Coomassie blue staining or by parallel Western with S-Rb, a sense Rb oligonucleotide, led to only a slight blotting with an antiactin antibody (data not shown).

Fig. 3 A, morphologic changes in HCT116 cells. a, at 48 h after transfection with AS-Rb, the HCT116 cells displayed cytoplasmic enlargement and irregular shaped cell morphologies. b, comparable cultures transfected with S-Rb exhibited no morphological change. Magnification, ϫ100. B, in situ TUNEL assays for apoptosis. For these assays, 1–2 ϫ 104 cells were plated in duplicate in 24-well plates (1.5-cm diameter) and were treated with the oligos or only with lipofectin. Forty-eight hours later, floating cells and adherent cells were collected, pooled, and centrifuged. TUNEL assays were performed using an in situ apoptosis detection kit, ApopTag (Oncor, Gaithersburg, MD), as recommended by the manufacturer. A representative fluorescence microscopy photograph of the cells at 48 h after treatment with AS-Rb displays chromatin condensation and nuclear fragmentation characteristic of apoptosis. Magnification, ϫ80. C, cell kinetics in HCT116 cultures treated with the AS-Rb. Exponential cultures of HCT116 cells were transfected with either AS-Rb or S-Rb and, 48 h later, analyzed by DNA flow cytometry, as described in “Materials and Methods.” The figure also indicates the mean percentage of the cell population in each phase of the cell cycle from three independent experiments. Control cultures gave results similar to those seen with the S-Rb-treated culture (data not shown). The AS-Rb-treated cells displayed a subdiploid DNA fraction characteristic of apoptosis.

Growth curves indicated that the culture treated with Effects of AS-Rb on Cell Morphology, Apoptosis, and AS-Rb displayed marked growth inhibition when compared Cell Cycle Kinetics in HCT116 Cells. Treatment of mono- with the lipofectin-treated control culture; but, the growth of the layer cultures of HCT116 cells with AS-Rb under the conditions culture treated with S-Rb was similar to that of the control described above not only inhibited growth but also increased the culture (Fig. 2B). These effects of AS-Rb, with respect to number of nonadherent cells. At 48 h, the number of floating causing a decrease in the level of pRb expression and markedly cells, expressed as percentage of the total cell culture, for the inhibiting the growth of HCT116 cells, were reproduced in three cultures treated with lipofectin alone, S-Rb or AS-Rb, were: additional experiments and with three independent preparations 4.8 Ϯ 0.01%, 7.3 Ϯ 0.36%, and 13.3 Ϯ 0.92%, respectively. of AS-Rb and S-Rb. In contrast to the effects obtained with the The difference between AS-Rb- and S-Rb-treated cultures was HCT116 cells, when cultures of WI38 human lung fibroblasts significant (P ϭ 0.014). In addition, by 48 h some adherent cells were treated under identical conditions with AS-Rb, a reproduc- treated with AS-Rb displayed triangular or irregular shaped cell ible 1.5–2.0 fold increase in growth was seen when compared morphologies, and other adherent cells had an enlarged cyto- with the lipofectin-treated control culture at 48 h after transfec- plasm (Fig. 3Aa). The S-Rb-treated adherent cells were round tion, but the S-Rb had no significant effect on growth (data not and had a thin cytoplasm (Fig. 3Ab); their morphology was shown). The latter result is consistent with previous evidence similar to that of cultures treated only with lipofectin or the that AS-Rb stimulates the growth of HEL human lung fibro- untreated HCT116 cultures (data not shown). blasts and keratinocytes (21, 22). To investigate whether the above-described effects of

AS-Rb on growth inhibition and morphological changes in HCT116 cells were associated with apoptosis, we carried out in situ TUNEL assays to assess the extent of DNA cleavage. The culture treated with AS-Rb for 48 h displayed numerous positive cells. A representative photograph of the AS-Rb-treated cells is shown in Fig. 3B. The changes of the fluorescein-stained cells were characteristic of apoptosis (i.e., nuclear condensation and fragmentation). The cultures treated with lipofectin or S-Rb displayed only an occasional positive staining cell, and control samples in which PBS was added instead of the TdT enzyme gave negative results (data not shown). The percentage of TUNEL-positive cells was calculated (see “Materials and Meth- ods”), and the values for the cultures treated with lipofectin alone, S-Rb or AS-Rb, were: 1.64 Ϯ 0.81%, 2.87 Ϯ 0.64%, and 17.39 Ϯ 0.47%, respectively. Thus, when compared with the lipofectin-treated culture, S-Rb caused less than a 2-fold increase, but AS-Rb caused about a 10-fold increase in apoptosis. The difference between AS-Rb- and S-Rb-treated cultures was significant (P ϭ 0.0015). These TUNEL assays were repeated three times, and similar results were obtained. DAPI staining also confirmed this difference in apoptosis (P ϭ 0.003), with the Fig. 4 A, effects of AS-Rb on the expression of cyclins, CDKs, and CKIs. HCT116 cells were transfected with S-Rb or AS-Rb, and cell following values of percentage of apoptotic cells: lipofectin, extracts were prepared 24, 32, and 46 h later. Protein (50 ␮g) was 2.24 Ϯ 0.25%; S-Rb, 4.12 Ϯ 0.17%; and AS-Rb, 12.94 Ϯ applied to each lane and subjected to PAGE and Western blot analysis 0.61%. with the appropriate antibodies. The relative intensities of the protein To further assess the mechanism of growth inhibition of bands were determined by densitometry. For additional details see “Materials and Methods.” B, in vitro kinase assays. Cells (3–5 ϫ 105) HCT116 cells by AS-Rb, we analyzed cell cycle kinetics by were plated in a 10-cm dish and then treated with oligos. Forty-six h DNA flow cytometry of cultures treated for 48 h with either later, the cells were harvested and lysed with kinase lysis buffer [50 mM S-Rb or AS-Rb. The results obtained with the S-Rb were similar HEPES, 150 mM NaCl, 2.5 mM EGTA, 1.0 mM EDTA, 1.0 mM DTT, to those obtained with cultures treated only with lipofectin (data 0.1% Tween 20, 10% Glycerol, 10 mM glycerophosphate, 1.0 mM NaF, 0.1 mM Na VO ,10␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 1.0 mM not shown). The mean values and SD for each cell cycle phase 3 4 PMSF (pH 7.5)]. After sonification, the extracts were clarified at were determined from three independent experiments and are 15,000 ϫ g for 10 min at 4°C, and the supernatant fraction was shown in Fig. 3C. There was no significant difference in the collected. In vitro assays for cyclin D1-, cyclin E-, and CDK2-associ- fraction of cells in each phase of the cell cycle between AS-Rb- ated kinase activities were performed as described previously (23). and S-Rb-treated cells. However, the AS-Rb-treated cells displayed an increase in the early sub-G1 DNA fraction, providing further evidence for an increase in apoptotic cells (Fig. 3C). Effects of AS-Rb on the Expression of Other Cell Cycle- other hand, there was no major change in cyclin-E-associated or Related Genes. It was of interest to examine the effects of CDK2-associated histone H1 kinase activity. The studies shown reducing the level of pRb in HCT116 cells on the expression of in Fig. 4 were repeated three times and gave similar results. various positive and negative regulators of the cell cycle. E2F Reporter Assays. Because it seems that the major

HCT116 cells were transfected with either S-Rb or AS-Rb, and function of pRb in regulating the G1 to S transition of the cell 24, 32, or 46 h later, extracts were prepared and examined by cycle is to inhibit activity of the transcription factor E2F, it was Western blot analyses for levels of expression of several pro- of interest to use an E2F-luciferase reporter to examine E2F teins (Fig. 4A). The intensities of the various bands were quan- activity in HCT116 cells cotransfected with S-Rb or AS-Rb. The titated by densitometry. luciferase activity was normalized by performing simultaneous Treatment of the cells with AS-Rb led to about a 50% cotransfection assays for ␤-galactosidase (see “Materials and reduction in the levels of cyclin D1 and p21Waf1 and about a Methods”). Treatment with S-Rb caused a slight (Ͻ3.5-fold) 30% reduction in the levels of p27Kip1, cyclin E, and CDK4. increase in E2F activity, but treatment with AS-Rb caused about Slight or no major changes were seen in the levels of cyclin A, a 17-fold increase in E2F activity (P ϭ 0.003) (Fig. 5A). Similar CDK6, or CDK2. We did not detect the p16Ink4 protein in any results were obtained in two additional studies (data not shown). of the HCT116 cultures, presumably reflecting inactivation of The finding of a marked increase in E2F activity in the this gene, although when used as a positive control, WI38 cells AS-Rb-treated cells suggested that this might be the reason for gave a strong p16Ink4 band on a Western blot. the induction of apoptosis because enhancement of E2F activity In vitro kinase assays were also performed with the 46-h can induce apoptosis in other types of carcinoma (27). There- protein extracts (Fig. 4B). Assays for cyclin D1-associated ki- fore, we examined the direct effect of E2F overexpression on the nase activity using a GST-Rb fusion protein as substrate indi- growth of HCT116 cells, using the following plasmids; CMV cated that there was an appreciable reduction of this activity in wild-type E2F, CMV mutant E2F, and a CMV vector control the AS-Rb-treated cells. This activity was only 48 Ϯ 5% that plasmid. To determine the level of E2F activity that could be obtained with the extract from the S-Rb-treated cells. On the induced with the wild-type E2F plasmid, we first carried out

Fig. 6 Colony efficiency assay with overexpression of wild type, or mutant type E2F. Cells were seeded at a density of 1 ϫ 105/well in 6-cm dishes. Twenty-four h later, the vectors encoding wild-type E2F, or mutant type E2F under CMV promoter, and the corresponding control vector were cotransfected with the pBabe plasmid encoding puromycin Fig. 5 A, luciferase reporter assays for E2F activity. The HCT116 resistance, at a ratio of 5:1 (2.5 ␮g of each plasmid and 0.5 ␮g of pBabe cells were plated at 0.5 ϫ 105/well (3.5-cm diameter) in triplicate 48 h ␮ ␤ ␮ plasmid), using the lipofectin reagent. Forty-eight hours after transfec- before transfection. The E2F-Luc (2 g) and CMV- gal (1 g) plas- tion, the cells were subplated into 15-cm dishes and then selected in the mids were cotransfected together with either AS-Rb or S-Rb oligos or presence of 0.5 ␮g/ml of puromycin in complete medium, for 2 weeks. with only lipofectin. Twenty-four hours after transfection, the cells were ␮ Individual drug-resistant clones were stained with Giemsa solution washed twice with cold PBS and lysed with 300 l of reporter lysis (Sigma). buffer (Promega) for 15 min at room temperature. After centrifugation, 30 ␮l of supernatant fraction was used for the luciferase assays, as described previously (25). ␤-gal activity was determined using the ␤-Galactosidase Enzyme Assay System (Promega), as recommended by the manufacturer. The luciferase level in each sample was normalized irregular-shaped cell morphologies, were frequently seen in the ␤ by the corresponding value for -gal activity. The increase in E2F cultures transfected with the wild-type E2F plasmid, but not in activity in the AS-Rb-treated cells when compared with the S-Rb-treated cells was highly significant (P ϭ 0.0031). B, induction of E2F activity the cultures transfected with the mutant E2F or vector control by forced expression of wild-type E2F. One ␮g of wild type E2F, plasmids (data not shown). These assays on colony formation mutant E2F, or control vector was cotransfected with 1 ␮gofE2F were repeated three times, and similar results were obtained. reporter plasmid and 0.5 ␮g of the CMV-␤ gal plasmid. Forty-eight h later, cells were harvested and then examined for the luciferase activity. Luciferase assays indicated that the E2F wild type construct yielded DISCUSSION increased E2F activity by about 20-fold. In the present study, we performed immunostaining of the pRb protein in normal human colonic mucosa, adenomatous polyps, early carcinoma in polyps, and advanced colon cancers. To our knowledge, this is the first immunohistochemical anal- reporter assays. We found that cotransfection of the wild-type ysis for pRb expression using boundary lesions such as adenoma E2F plasmid and the E2F reporter plasmid into HCT116 cells and carcinoma in adenoma. In the normal mucosa, pRb was yielded about a 25-fold increase in E2F activity when compared located in the transitional zone of the crypt at the lower part of with the vector control plasmid, but this marked induction was the glands where there is a homeostatic state of equilibrium not seen by cotransfection with the mutant E2F plasmid (Fig. between cell proliferation and cell differentiation (28). It was of 5B). In an additional assay, the level of E2F activity using the interest that in the normal crypt, lack of expression of pRb was wild-type E2F plasmid was again about 17-fold higher than that observed in the upper portion of crypt where growth arrest and with vector control plasmid. This high level was similar to that apoptosis occurs (29, 30). This is consistent with the findings of induced with AS-Rb on growth (Fig. 5A). We then performed our in vitro study (Figs. 2–4), indicating that decreased expres- colony efficiency assays to examine the effects of this marked sion of pRb, induced by AS-Rb, leads to growth inhibition and increase in E2F activity. The cells were transfected with each of apoptosis in HCT116 colon cancer cells. Thus, if loss of Rb the above plasmids, transfectants were selected with puromycin expression is mechanistically associated with growth inhibition for 2 weeks, and the puromycin-resistant colonies were stained. and apoptosis in the normal crypt, the maintenance of increased A clear inverse correlation was seen between the ability to form Rb in the colonic epithelial cell may prevent this negative colonies and the levels of E2F activity (Fig. 6). Furthermore, growth process from occurring. Previous studies (5–8) have during selection, morphological changes similar to those seen in found that there is often increased expression of pRb in human the AS-Rb-treated cells, including cytoplasmic enlargement and colorectal carcinomas. The present study confirms this finding

and, furthermore, demonstrates that this increase is progressive Furthermore, it is clear that our AS-Rb exerted the expected during the multistage process of colorectal carcinogenesis be- biochemical effects because the treated cells displayed a marked cause we found a progressive increase in pRb expression during decrease in the level of pRb and a marked increase in E2F the transition from normal mucosa to adenomatous polyps to activity, and these effects were not seen with the S-Rb construct carcinomas (Table 1 and Fig. 1). This is surprising because in (Figs. 2 and 5). In addition, it is unlikely that the phenotypic previous studies (10, 17) we found that during colorectal carci- effects of the AS-Rb on HCT116 cells were due to nonspecific nogenesis there is an increase in staining for the proliferation toxicity because, as mentioned below, treatment of WI38 cells marker Ki67, and also an increase in the expression of cyclin with the same AS-Rb construct actually stimulated the growth D1, which would be expected to enhance cell proliferation. of these cells. Western blot analyses for pRb with the same antibody used for Previous studies indicated that introduction of a full-length immunostaining indicated that this antibody reacts with both the Rb cDNA or micro injection of pRb into Rb-deficient osteosar- hyper- and underphosphorylated forms of pRb, as described coma cells or melanoma cells inhibited growth (32, 38) and that previously (7, 31). It is known that the percentage of phospho- antisense oligos to Rb stimulated the growth of human fibroblast rylation of pRb is higher in colon carcinoma tissues than in and keratinocytes (21, 22). We also confirmed that transfection normal mucosa, thus suggesting a mechanism for inactivation of of our AS-Rb into WI38 human lung fibroblasts under the pRb in the colon carcinomas. However, in a subset of colon identical conditions used with HCT116 cells stimulated growth. carcinomas, there is a significant increase in the absolute These findings are what would be expected with a tumor sup- amount of underphosphorylated pRb (6, 7), which could bind to pressor gene. The growth inhibition seen when the level of pRb and inactivate E2F. These findings seem paradoxical for a tumor was reduced in HCT116 cells is, therefore, somewhat surprising.

suppressor gene, which in its active form, inhibits the G1 to S This situation is not, however, unique since a previous study transition in the cell cycle (32). indicated that an antisense Rb oligonucleotide also inhibited the In an attempt to understand this paradox, we carried out growth of rat hepatocytes (20). In addition, although introduc- mechanistic studies in vitro to examine the effects of decreasing tion of a wild-type Wilms’ tumor suppressor gene (WT1) cDNA the level of pRb expression in the human colon cancer cell line into NIH3T3 cells inhibited proliferation (39), treatment of HCT116, which expresses a high level of this protein. For this chronic leukemia cells with an WT1 antisense oligonucleotide purpose, we used an antisense oligonucleotide sequence (AS- inhibited growth (19, 40). Thus, the phenotype effects of de- Rb) targeted to Rb mRNA because previous investigators had creasing the expression of certain tumor suppressor genes is shown the specificity of this AS-Rb in other cell systems (20– highly dependent on cell context. 22). We found that introduction into the HCT116 cell line of the The reduction in the level of pRb in the HCT116 cells was AS-Rb caused about a 70% inhibition in the level of expression associated with changes in several other cell cycle control genes. of pRb. This was associated with a marked inhibition of cell The most striking change was a decrease in the level of cyclin growth in vitro, changes in cell morphology, induction of apo- D1 (Figure 4A). This, plus a decrease in the level of CDK4, ptosis, altered levels of expression of several cell cycle related probably explains the marked decrease in cyclin D1-associated proteins, inhibition of cyclin D1 associated kinase activity, and kinase activity (Fig. 4B). The latter effect may be responsible, at a marked increase in E2F activity. These pleiotropic effects least in part, for the overall growth inhibition seen in the were highly reproducible and seem to be secondary to the AS-Rb-treated HCT116 cells. Our finding that decreased ex- reduced levels of pRb because of their time course and the fact pression of pRb was associated with decreased expression of that they occurred with relatively low concentrations of AS-Rb cyclin D1 and p27Kip1 is consistent with previous evidence for (1 ␮M). A similar sense Rb oligonucleotide, S-Rb, had a slight the existence of feedback control mechanisms that coordinate effect on some of these parameters, perhaps due to partial the levels of these three proteins. Thus, pRb-deficient tumors inhibition of translation of Rb mRNA, but the effects of the often have decreased levels of cyclin D1 (41), and increased AS-Rb were always much more dramatic (Figs. 2–5). In addi- expression of cyclin D1 is often associated with increased levels tion, a preliminary study on tumorigenicity in nude mice showed of pRb and p27Kip1 in human esophageal and colon carcinoma an apparent growth inhibition of HCT116 cells when treated cells (42, 43). It is of interest that the AS-Rb-treated cells also with AS-Rb (data not shown). displayed some reduction in the levels of p21Waf1 (Fig. 4A). The As previously emphasized (33), studies using antisense decreased levels of p21Waf1, and possibly CDK4, in the AS-Rb- oligos must be interpreted with caution because of the possibil- treated cells might be secondary to the decrease in cyclin D1 ity of obtaining nonspecific effects with these materials. In the because there is evidence that cyclin D1 can induce p21Waf1 present study, we followed several guidelines (34, 35) to min- expression (44). Furthermore, in unpublished recent studies, we imize such effects. These included using a relatively short (18 have found that retrovirus-mediated transduction of a cyclin D1 mer) oligomer that does not have apparent higher order struc- cDNA sequence into HCT116 cells results in a marked increase Kip1 Waf1 ture, using a low concentration of the oligomer (1 ␮M), and in the expression of pRb, p27 , p21 , and CDK4. It is also using the cationic lipid carrier lipofectin (which allows us to use of interest that increased expression of pRb, cyclin D1, and a lower concentration of the oligomer and also reduces the p27Kip1 is often seen in primary colorectal carcinomas (5–8, 10, possibility of degradation of the oligomer to toxic derivatives). 17–18), and concurrent overexpression of cyclin D1 and CDK4 The use of lipofectin as a carrier (36, 37) also permits egress of is seen in the intestinal adenomas of patients with familial charged oligos from the intracellular vesicular compartment. adenomatous polyposis coli (45). Thus, the abrupt drop in Thus, the extracellular concentration of phosphorothioate oligos cellular levels of pRb in the HCT116 cells, which occurred in can be kept low, and nonsequence specificity can be avoided. the present study after transfection of AS-Rb, could disrupt a

complex network of feedback control mechanisms in colon upstream pRb-regulatory genes such as p16 (67). Taken to- cancer cells. gether, these findings suggest that the multistage process of It is also of interest that treatment of the HCT116 cells with carcinogenesis does not simply involve the step-wise activation AS-Rb induced apoptosis, as assessed by the TUNEL assay, of growth-promoting proto-oncogenes and inactivation of DAPI staining, and DNA flow cytometry (Fig. 3). These results growth inhibitory tumor suppressor genes. The evolving popu- suggests that the high level of expression of pRb in HCT116 lation of tumor cells may also have to upregulate the expression colorectal carcinoma cells might have a selective advantage by of certain negative-acting genes to maintain a homeostatic bal- protecting them from apoptosis. This finding is consistent with ance that favors optimal growth and viability. This concept evidence that pRb can protect osteosarcoma cells, bladder car- would help to explain the complex and heterogeneous pheno- cinoma, and hepatic carcinoma cells from apoptosis induced by types of cancer cells and might also have implications with ceramide, INF-␥, ionizing radiation and transforming growth respect to novel approaches to cancer chemoprevention and factor-␤ (20, 46–48). Using an E2F-luciferase reporter assay, therapy. we found that treatment of HCT116 cells with AS-Rb led to a 17-fold increase in E2F activity as compared with control cul- ACKNOWLEDGMENTS tures treated with lipofectin (Fig. 5A). Although E2F can func- We thank Dr. S. Chellappan (Columbia University, New York, tion as either an oncogene or tumor suppressor gene, depending NY) for providing the plasmid used for the E2F luciferase reporter on the cellular environment (49, 50), our findings of the toxicity assay; Dr. W-H. Lee (The University of Texas Health Science Center at and morphological changes with forced expression of wild-type- San Antonio, San Antonio, TX) for providing the wild type and mutant E2F (Figs. 5B and 6) suggest that dysregulation of E2F activity E2F expression plasmids; Y. J. Zhang, E. Okin, and W-Q. Xing for may be responsible for the induction of apoptosis and also excellent assistance; and other members of the Weinstein Laboratory for contribute to the growth inhibition seen in the AS-Rb-treated helpful advice and discussions. We are also grateful to B. Castro and P. HCT116 cells. Similar effects of E2F on apoptosis were seen in Jean-Louis for valuable assistance in the preparation of this manuscript. several cell lines, including breast and ovarian cancer cells (27, 50, 51). However, we do not know the mechanism that mediates REFERENCES the apoptosis because the AS-Rb-treated HCT116 cells showed 1. Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. 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Hirofumi Yamamoto, Jae-Won Soh, Takushi Monden, et al.

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