1Clinical Research Unit for Gastrointestinal Endocrinology, 2Institute of Pathology, and 3Institute of Medical Biometry and Epidemiology, University of Marburg, 35033 Marburg, Germany
Submitted 16 January 2004 ; accepted in final form 12 August 2004
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ABSTRACT |
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tumor growth; cell cycle; tumor suppressor gene
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MATERIALS AND METHODS |
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Western blot. Total proteins were extracted from cells, and protein concentration was determined with a protein assay from Bio-Rad according to the manufacturer's protocol. Proteins (30 µg) were separated by SDS-PAGE (10% gel) and electrotransferred onto nitrocellulose membrane (pore size 0.45 µm). Immunodetection was carried out with different antibodies in 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, followed by HRP-conjugated secondary antibodies. Detection was performed with an ECL kit (Amersham Bioscience). Except for anti-V5-antibodies (HRP coupled; Invitrogen) all antibodies were from Santa Cruz (Actin-HRP, cdc2p34, RbIF8, p21, Wee 1) and Cell Signaling (secondary antibodies, cdc2 Tyr15, cdc2 Thr161, pRb Ser807/811, pRb Ser795, p53, E2F1, cdk4, p-cdc25C Ser216, p27/Kip1, cyclin D1).
RT-PCR.
Total RNA from mock- and pdcd4-transfected Bon-1 cells was isolated with the RNeasy Mini Kit (Qiagen), and semiquantitative RT-PCR was performed with the One Step RT-PCR kit (Qiagen) according to the supplier's protocol. As an internal control -actin/3 and /6 primers (Promega) were used, yielding a 285-bp product. Primers for amplification of CDK1/cdc2 were synthesized by Metabion, yielding a 203-bp product (5-cdc2: CAG TCT TCA GGA TGT GCT TAT GC; 3-cdc2: GAG GTT TTA AGT CTC TGT GAA GAA CTC). After 15, 20, 25, and 30 cycles, aliquots from each sample were removed and analyzed on a 2% agarose gel.
Gene expression analysis.
The 4.6K cDNA chips contained the GF200 set of Research Genetics cDNAs. Each cDNA was spotted in duplicate [www.imt.uni-marburg.de (Research, Microarray Unit)]. Chips were generated by the group of M. Krause (Institute of Molecular Biology and Tumor Research, Marburg, Germany). One microgram of total RNA was amplified with the MessageAmp aRNA kit (Ambion), yielding 20 µg of aRNAs. Cy3- and Cy5-labeled cDNAs were synthesized with the CyScribe cDNA Post-Labelling kit (Amersham Biosciences). A flip-color experiment was included. The appropriate Cy3- and Cy5-labeled samples were pooled and hybridized to microarrays under a glass coverslip for 16 h at 55°C and washed with 0.1x SSC. Chips were scanned with a GMS 418 fluorescent scanner (MWG-Biotech), and the images were analyzed with IMAGENE 3.0 software. The raw data were evaluated as described previously (18).
Methylthiazoletetrazolium assay. To test the effect of the CDK inhibitor roscovitine on tumor cells, cells were incubated for 24 h in 500 µl of culture medium in the absence or presence of different concentrations of roscovitine. Subsequently, 100 µl of methylthiazoletetrazolium (MTT; 5 mg/ml phosphate-buffered saline) was added and incubated for an additional 2 h. After removal of the medium, cells were treated with 200 µl of DMSO for 1 h. Samples were centrifuged, and the absorbance at 550 nm was determined in the supernatants with a multiwell plate reader. Percent inhibition of cell proliferation was calculated with the following formula: 100 (extinction of treated sample x 100/extinction of control sample). To test whether cell viability is affected by overexpression of pdcd4, mock-transfected or pdcd4-overexpressing cells were detached from culture plates, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting (FACS). Analysis revealed that there was no difference between cell lines in cell viability (data not shown).
Anti-pdcd4 antibodies. Polyclonal anti-pdcd4 antibodies were generated by Peptide Specialty Laboratories (Heidelberg, Germany). For this, rabbits were immunized with synthetic peptide corresponding to the amino-terminal 20 amino acids of the human pdcd4 protein coupled to keyhole limpet hemocyanin. Specific antibodies were affinity purified from the resulting serum with nitrocellulose-bound antigen as described by Hammerl et al. (10).
Histology.
The present study included carcinomas of colon (n = 11), lung (n = 3), breast (n = 10), pancreas (n = 3), and prostate (n = 7) and adjacent tumor-free tissue, which were surgically resected when indicated. Specimens were fixed for diagnostic purposes in formalin (10%) and embedded in paraffin, and subsequently sections of 5 µm were prepared. Sections were mounted on glass slides and stained with hematoxylin and eosin for routine purposes by standard protocols. Consecutive sections of the specimens were used for immunohistochemical examination with antibodies.
Immunohistochemistry. Expression of pcdc4 was detected with a polyclonal rabbit antibody (dilution 1:10) by means of the standard avidin-biotin complex (ABC)-peroxidase method (ABC Elite Kit; Vector, Burlingame, CA) with 3,3-diaminobenzidine (DAB) as chromogen after microwave pretreatment performed by heating the deparaffinized and rehydrated sections, immersed in 10 mM sodium citrate buffer (pH 6.0), in a microwave oven at 600 W three times for 5 min. Sections were counterstained with Mayer's hemalum. After dehydration in graded alcohols, sections were cleared in xylene and coverslipped with Entellan.
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RESULTS |
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Cells of normal tissues showed intense nuclear staining for pdcd4 in the majority of epithelia, whereas cytoplasmic staining was not observed. Normal colonic mucosa showed a characteristic local distribution of epithelia with nuclear pdcd4 staining, in that epithelia of the middle and apical portions of the crypts disclosed intensely stained nuclei. In contrast, epithelia in the lower portion of the crypt showed no pdcd4 staining, neither nuclear nor cytoplasmic. In normal prostate, breast, and lung, most epithelia disclosed intense nuclear staining and no pdcd4 in cytoplasm. In addition to epithelial cells, intense pdcd4 staining was also found in the nuclei of endothelia, stromal fibrocytes, and lymphocytes. No cytoplasmic staining was observed in these cells.
We investigated a total of 30 carcinomas obtained from different anatomic sites and immunohistochemically compared pdcd4 staining with that observed in adjacent tumor-free tissue. Depending on the primary tumor site and grade of differentiation, a decrease of nuclear pdcd4 staining accompanied by a gain of cytoplasmic pdcd4 staining was found (Table 1). Six of seven colon carcinomas showed a complete loss of nuclear pdcd4 staining (Fig. 4), and in four cases cytoplasmic pdcd4 staining was found. All colonic adenomas investigated showed a clear shift from nuclear pdcd4 localization to cytoplasmic staining.
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DISCUSSION |
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The transcription of the CDK1/cdc2 gene is regulated by pRb/E2F/DP. Hypophosphorylated pRb recruits E2F-DP complexes, converting them from sequence-specific transcription activators to sequence-specific repressors (29). Despite the lack of change in total protein amounts, in pdcd4-overexpressing cells pRb was found to be hypophosphorylated, which might explain the repressed transcrip-tion of the CDK1/cdc2 gene. This assumption is sup-ported by the previous finding that Rb negatively regulates the cdc2 promoter (5). pRb is substrate for CDK4/6 and CDK2, and, accordingly, we found a reduction of CDK4/6 protein in pdcd4-overexpressing cells. Furthermore, the expression of p21Waf1/Cip1 is drastically increased. p21Waf1/Cip1 is a CDK inhibitor that inhibits CDK4/6 and CDK2 (11). Obviously, pdcd4 reduces the activity of CDKs via induced p21Waf1/Cip1. It is unlikely that other CDK1/cdc2 regulators are involved, because, analyzing gene arrays and protein expression pattern using Western blotting, we did not find any effect of pdcd4 on expression of p16, p27, Wee1, Cdc25C, CHK1, CHK2, or cyclin D1, D2, and D3. The induction of p21Waf1/Cip1 seems to be independent of p53, because we found no change or even a minor decrease in p53 protein. Hence, pdcd4 might suppress tumor progression even in p53-defective cells. Interestingly, we found previously (9) that pioglitazone, an activator of the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR)-, upregulates the expression of p21Waf1/Cip1, inhibits cell growth, and sensitizes NCI-H727 carcinoid cells to tumor necrosis factor-related apoptosis-inducing ligand TRAIL-induced apoptosis. Influencing the p21Waf1/Cip1 signal pathway may be a promising therapeutic strategy. Moreover, in p53-arrested cells p21Waf1/Cip1 associates with CDK1/cdc2/cyclin B, representing an additional inhibitory mechanism (27) that might also play a role in pdcd4-overexpressing cells. We summarize the proposed pdcd4 signal pathway in Fig. 5.
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Because overexpression of pdcd4 in Bon-1 carcinoid cells repressed CDK1/cdc2, resulting in reduced cell proliferation, we speculated that CDK1 inhibitors should exert a similar effect. To test this hypothesis, different endocrine tumor cell lines were treated with the CDK1 inhibitor roscovitine. As expected, roscovitine concentration-dependently inhibited the growth of all endocrine tumor cell lines tested. This is of special interest because most endocrine tumors are resistant to radiation and common chemotherapeutic agents. In our experiments the IC50 of roscovitine was quite high (80 µM). However, CDK inhibitors more potent than roscovitine might represent a new approach for the treatment of endocrine tumors.
Previously, immunohistochemical studies revealed that CDK1/cdc2 is overexpressed in a subset of colonic adenoma (14 of 59; 28%), which was more obvious in focal carcinoma (13 of 15; 86.7%) (13, 30). These data suggested an upregulation of CDK1/cdc2 accompanied by a malignant change. In accordance with these findings, we show here that expression of the CDK1/cdc2 suppressor pdcd4 is lost in progressed carcinomas of lung, breast, colon, and prostate. Furthermore, it seems that both pdcd4 localization and expression level directly correlate with tumor progression. Our data indicate that pdcd4 might represent a novel target for antineoplastic therapies.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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