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NEOPLASIA
From the Department of Pathology and ARUP Institute for
Clinical and Experimental Pathology, University of Utah, Salt Lake
City; the Department of Anatomic Pathology, Sunnybrook Women's College
Health Sciences Centre, University of Toronto, ON, Canada; and
the Department of Oral Pathology, University of California, San
Francisco.
Reduced levels of p27Kip1 are frequent in human cancers
and have been associated with poor prognosis. Skp2, a component of the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, has been implicated in p27Kip1 degradation. Increased Skp2 levels
are seen in some solid tumors and are associated with reduced
p27Kip1. We examined the expression of these proteins using
single and double immunolabeling in a large series of lymphomas to
determine if alterations in their relative levels are associated with
changes in cell proliferation and lymphoma subgroups. We studied the
expression of Skp2 in low-grade and aggressive B-cell lymphomas
(n = 86) and compared them with p27Kip1 and the
proliferation index (PI). Fifteen hematopoietic cell lines and
peripheral blood lymphocytes were studied by Western blot analysis. In
reactive tonsils, Skp2 expression was limited to proliferating germinal
center and interfollicular cells. Skp2 expression in small lymphocytic
lymphomas (SLLs) and follicular lymphomas (FCLs) was
low (mean percentage of positive tumor cells, less than 20%) and was
inversely correlated (r = Reduced levels of p27Kip1, a
cyclin-dependent kinase inhibitor, are frequently observed in human
cancers1-4 and are associated with aggressive tumor
biology5 and poor patient prognosis.6-8 Low
levels of the p27Kip1 protein are thought to contribute to
malignant cell proliferation owing to its role as a negative regulator
of the protein kinases cyclin-dependent kinase 2 (cdk2)/cyclin
E and cdk2/cyclin A.9-11 Regulation of
p27Kip1 occurs predominantly at the posttranscriptional and
posttranslational level,12 through sequestration by either
cyclin D1 or cyclin D3 and by phosphorylation and degradation
via the ubiquitin-mediated proteolysis
pathway.11-15 Skp2, a member of the
substrate-recognition subunit of Skp1-Cul1-F-box (SCF) ubiquitin ligase
complex, has been implicated in targeting p27Kip1
for degradation.16 In cultured cells, Skp2 protein levels
are cell-cycle regulated,17 showing an inverse pattern to
that of p27Kip1protein.18 Skp2 overexpression
can promote S-phase entry in serum-starved cells by inducing the
accumulation of cyclin A, cyclin E,19 and cyclin A-cdk2
activation20 and phosphorylation-dependent degradation of
p27Kip1.21 Skp2 has also been implicated in
the ubiquitination of other cell-cycle regulatory proteins, including
cyclin E and the transcription factor E2F-1.21,22
Thus, deregulation of Skp2 may contribute to neoplastic transformation
through accelerated p27Kip1 proteolysis. Furthermore,
recent studies from our group and others,23,24 using
colony-forming assays in soft agar and tumor formation in nude
mice,23 have shown that Skp2 has oncogenic
potential. Skp2 in cooperation with
H-Ras can lead to malignant transformation of primary rodent
fibroblasts. Transgenic mice expressing Skp2 targeted to
T-lymphoid-lineage-developed T-cell lymphomas in cooperation with
activated N-Ras.23 The observation
that Skp2 can promote the ubiquitin-mediated proteolysis of
p27Kip1 led us to examine the relationship between the
expression of Skp2 and p27Kip1 proteins in different
categories of non-Hodgkin lymphomas (NHLs) with different histologic
grades and proliferative indices. Our studies demonstrate that Skp2
expression is strongly correlated with the proliferation index in
reactive lymphoid tissues. These data provide evidence for the
p27Kip1-degradative function of Skp2 in low-grade B-cell
lymphomas but suggest the existence of other molecular abnormalities,
in addition to Skp2, in the deregulation of p27Kip1
expression in aggressive lymphomas.
Tissue samples
Isolation and activation of peripheral blood lymphocytes
Cell cultures Human malignant lymphoma cell lines were obtained from American Type Culture Collection (Rockville, MD) or from the laboratory of Dr N. Berinstein, University of Toronto (ON, Canada). All cell lines (Table 1) were maintained in RPMI 1640 (GibCo BRL) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin mixture (GibCo BRL) at 37°C and 5% CO2.
Preparation of cell lysates and immunoblot analysis Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl [tris(hydroxymethyl)aminomethane-HCl], 0.1% sodium dodecyl sulfate [SDS], 1% triton-× 100, 1% sodium deoxycholate, 0.15 M NaCl, 1 mM EDTA [ethylenediaminetetraacetic acid], and 0.1% protease inhibitor cocktail (Sigma Chemical, St Louis, MO). Fifteen micrograms of protein was resolved in a 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel by means of a BioRad (Hercules, CA ) mini-gel system. Separated proteins were then transferred onto a nitrocellulose film by means of a semidry transfer apparatus (BioRad). The following antibodies were used for immunoblot analysis: mouse monoclonal antibody against p27Kip1 (Transduction Laboratories, Lexington, KY), polyclonal Skp2 (Santa Cruz Biotechnology, CA), and monoclonal antibody 1 (MIB1; Novocastra, Newcastle, United Kingdom), which recognizes an epitope of the Ki-67 antigen. Protein bands were visualized by means of the enhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL). Because of significant differences in the molecular weights of p27Kip1 (27 kDa) and Skp2 (45 kDa), the same blot was reused after stripping, and signals were developed for an equal period of time. Densitometry was performed by means of the Molecular Dynamics Imaging system and ImageQuant 3.3 software (Amersham Biosciences, Sunnyvale, CA) to quantify relative amounts of protein detected on Western blots.Preparation of nuclear and cytoplasmic fractions Cells were subfractionated as previously described,3 with minor modifications. In brief, cells at subconfluency conditions were collected and resuspended in hypertonic buffer supplemented with 0.1% protease inhibitor cocktail (Sigma Chemical). After swelling in ice for 20 minutes, plasma membranes were disrupted by repeated pipetting. Cell breakage was assessed by microscopic observation. Samples were microcentrifuged at 3000 rpm for 10 minutes at 4°C to collect cytoplasmic fractions (supernatant). The pellets were washed once with hypertonic buffer and resuspended in RIPA buffer. The nuclear fraction (supernatant) was recovered by microcentrifugation at top speed at 4°C for 10 minutes.Immunoprecipitation For immunoprecipitation followed by immunoblotting, 200 µg proteins in 500 µL RIPA buffer was mixed with 2 µg antibodies and incubated overnight at 4°C with gentle shaking. Immune complexes were collected on protein G-Agarose beads (GibCo BRL) and washed 5 times in lysis buffer.Immunohistochemistry Serial sections that were 5-µm thick were mounted on glass slides coated with 2% aminopropyltrioxysilane (APES; Sigma Chemical) in acetone. Sections were dewaxed in xylene and rehydrated in graded ethanols. Endogenous peroxidase activity was blocked by immersion in 0.3% methanolic peroxide for 15 minutes. Immunoreactivity of p27Kip1 was enhanced by microwaving by incubating the tissue sections for 10 minutes in 0.1 M citrate buffer. Expression of p27Kip1 protein was detected by incubating the tissue sections with a monoclonal antibody to p27Kip1 (Transduction Laboratories) diluted 1:500. The proliferation index was assessed by using an MIB1 monoclonal antibody (Novocastra) that recognizes a formalin-resistant epitope of the Ki-67 antigen. Skp2 protein expression was detected by incubating a monoclonal antibody to Skp2 (Santa Cruz Biotechnology) at a dilution of 1:50 for 32 minutes. The sections were then incubated with a biotinylated peroxidase agent (BioGenex, San Ramon, CA). Antigen-antibody reactions were visualized with the chromogen diaminobenzadine. Normal mouse serum containing mixed immunoglobulins at a concentration approximating that of the primary antibody was used as a negative control. Sections were counterstained with hematoxylin. A normal tonsil was used as a positive control for all antibodies. Double immunohistochemistry was performed with the use of citrate buffer, pH 6.0, for 3 minutes. The primary antibody p27Kip1 was used at a dilution of 1:30 for 32 minutes and was detected with the use of diaminobenzadine. Antibody to Skp2 was used at a dilution of 1:10 for 32 minutes and blocked with levamisole for 16 minutes, after which the antigen-antibody reaction was visualized with the use of alkaline phosphatase red and was counterstained with hematoxylin.Interpretation of immunohistochemical stains The percentages of neoplastic cells expressing p27Kip1, Skp2, and Ki-67 by immunohistochemical stains were assessed in each case by counting 400 cells from at least 3 different representative fields with the use of the 50 × or 100 × oil objectives. Distinct nuclear staining that was appreciably above background was considered positive. Expression of p27Kip1 and Skp2 by less than 20% of the total cell population was considered low, and expression exceeding 20% was considered high.23Statistical analyses Descriptive statistics were used to present the distribution of the measured parameters. The Pearson rank correlation coefficient was used to describe correlation between Skp2 and p27Kip1; between Skp2 and Ki-67; and between p27Kip1 and Ki-67 expression. The 2-tailed Student t test was used to evaluate the significance of these correlations. P < .05 was used as the criterion for statistical significance.
Expression of Skp2 and p27Kip1 in cell lines derived from hematopoietic neoplasms Western blot analysis of total cell lysates obtained from cell lines of myeloid, B-lymphoid, and T-lymphoid origin (Table 1) demonstrated Skp2 protein expression in all cell lines. Negligible levels were detected in the Hodgkin-derived cell line L428 and the diffuse large B-cell lymphoma (DLBCL) cell line Karpas 422. In comparison with p27Kip1 levels, which were abundantly expressed in most cell lines (10 of 15, 67%), the levels of Skp2 protein were low (Figure 1). Although quiescent peripheral blood lymphocytes (PBLs) expressed high levels of p27Kip1 protein that decreased with activation, the levels of Skp2 protein were not detectable in either cell population.
Expression of Skp2 in reactive lymphoid tissue and non-Hodgkin lymphomas We studied the expression of Skp2, p27Kip1, and Ki-67 in reactive tonsils and lymph nodes and in 86 B-lineage non-Hodgkin lymphomas composed of 12 cases of small lymphocytic lymphoma (SLL), 19 cases of follicular lymphoma (FCL), 6 cases of nodal marginal zone lymphoma (MZL), 29 cases of mantle cell lymphoma (MCL) and 20 cases of diffuse large B-cell lymphoma (DLBCL). The results are presented in Figures 2 and 3 and summarized in Tables 2 and 3. In reactive lymphoid tissues (Figure 2A), Skp2 was characteristically expressed in a subset of the proliferating germinal center (GC) cells and in rare interfollicular cells. This staining pattern was similar to that of Ki-67 but with significantly lower intensity. By contrast, strong p27Kip1 expression was seen in the quiescent cells of the follicular mantle zone and in interfollicular T lymphocytes, whereas only rare cells in the GC were positive.
In B-CLL/SLL, Skp2 expression was limited to prolymphocytes and
paraimmunoblasts within the proliferation centers (Figure 2B). The mean
Skp2 expression in 12 CLL/SLLs was 4% (Table 2). The
p27Kip1 protein showed a staining pattern inverse to that
of Skp2, with numerous neoplastic lymphocytes demonstrating
intense expression (mean, 69%). A strong inverse relationship was seen
between Skp2 expression and p27Kip1 expression
(r = In FCL, Skp2 expression was seen in large centroblasts of the
neoplastic follicles, whereas the majority of the small-cleaved cells
were negative (Figure 2C). The mean Skp2 expression in FCL was 6%
(range, 2% to 12%). The p27Kip1 expression was
predominantly seen within nonneoplastic mantle zones and paracortical T
cells. An inverse correlation was observed between Skp2 and
p27Kip1 expression (r = Of 6 patients with MZL, the mean Skp2 expression was 10%, with
staining observed in large transformed centroblasts (data not shown).
The mean p27Kip1 expression was high (61%), with most
immunoreactivity observed within small neoplastic and reactive
lymphocytes. Skp2 expression correlated inversely with
p27Kip1 (r = As a group, MCLs had low levels of Skp2 expression (mean, 9%) (Figure
2D); a poor negative correlation that was not statistically significant (r = Skp2 levels exhibited the most variability among DLBCLs, with 11 cases showing low (less than 20%) expression while 9 cases demonstrated high (greater than 20%) expression (Figure 2E). The mean percentage of Skp2 expression (25%) did not correlate with p27Kip1 (r = 0.17; P < .001) expression but did correlate with expression of Ki-67 (r = 0.55; P < .0001). Notably, cytoplasmic Skp2 was seen in a subset of the DLBCLs but not in the other lymphoma groups (data not shown). Double immunolabeling was performed in a selected number of cases (Figure 2F). Skp2 expression, indicated by alkaline phosphatase (pink), and p27Kip1 expression, indicated by diaminobenzadine (brown), confirm the findings seen by single-immunohistochemical studies. In all cases of NHLs analyzed, the percentage of cells expressing Skp2 never exceeded that of Ki-67. In the low-grade B-cell lymphomas, CLL/SLL, FCL, and MZL, the vast majority (92%) of cases showed lower than 10% Skp2 expression with high p27Kip1 expression, and a clear-cut inverse relationship was observed between the 2 proteins. Overall, the highest percentage of Skp2+ cells were seen in the DLBCL group (mean, 25%) as well as the blastic variants of MCL (mean, 64%). Skp2 is expressed in the cytoplasm and complexes with p27Kip1 We observed cytoplasmic expression of Skp2 in 2 cases of DLBCLs by immunohistochemistry (data not shown). We sought to determine whether complexes of Skp2 and p27Kip1 were present in subcellular fractions of lymphoma cells. Figure 4 demonstrates that immunoprecipitation of Jurkat, CEM-6, Raji, and Nalm-6 cells with anti-Skp2 followed by immunoblotting with p27Kip1 showed a physical relationship between the 2 proteins within both the nuclear and the cytoplasmic fractions. Western blot analysis of nuclear and cytoplasmic fractions with an antibody against Ki-67, however, showed that its expression was restricted to the nuclear fractions.
Reduced levels of p27Kip1 have been associated with malignant transformation and prognosis of a variety of cancers. The p27Kip1 can be down-regulated by several mechanisms. In addition to phosphorylation-dependent ubiquitin-mediated degradation of p27Kip1,26 an N-terminal proteolytic processing resulting in a reduced molecular mass of 22 kDa has also been described.27 The F-box protein Skp2 is a component of the ubiquitin protein ligases28,29 that play a critical role in the regulation of G0 to S phase progression.30 The targets of Skp2 include cell cycle proteins such as free cyclin E,18 cyclin D1,31 and p27Kip1.32 Recent studies have shown that Skp2 functions as an oncogene and induces lymphomagenesis in transgenic mice24 as well as malignant transformation of primary rodent fibroblasts.23 A comprehensive study of Skp2 expression in a large series of human malignant lymphoma cell lines and tissues has not been previously reported, although, during the preparation of this manuscript, Latres and coworkers24 reported that Skp2 expression in CLL and DLBCL correlated directly with grade. We have analyzed the expression pattern and levels of Skp2 in 15 hematopoietic cell lines, PBLs as well as a large series of B-lineage NHLs of varying histologic types and proliferation indices. All cell lines expressed Skp2 protein except for the Hodgkin-derived cell lines L428, which expressed negligible levels of both Skp2 and p27Kip.1 Most of the cell lines (10 of 15; 67%) demonstrated abundant p27Kip1 protein levels while Skp2 levels were low. In contrast, quiescent PBLs expressed negligible levels of Skp2 even after mitogenic stimulation. In keeping with the proposed role of Skp2 as a positive regulator of the G1/S transition, our results show that expression of Skp2 was limited largely to the nuclei of proliferating cells. In reactive lymphoid tissues, Skp2 expression was seen within proliferating GC cells and large transformed cells of the interfollicular areas. We found a strong inverse correlation between Skp2 and p27Kip1 expression, with an inverse topographical distribution of these 2 proteins within the reactive lymphoid tissues. In NHLs, we observed increasing levels of Skp2 with increasing grades of NHL. Low-grade lymphomas (CLL, FCL, and MZL) and most MCLs showed low Skp2 expression (less than 20%). Nine (45%) of aggressive, intermediate-grade DLBCLs and all blastic variants of MCLs demonstrated high Skp2 expression (greater than 20%). Chiarle et al33 attributed the low level of p27Kip1 expression in MCL to increased proteasome degradation. Other investigators have attributed the low p27Kip1 expression in MCL and other aggressive B-cell lymphomas to sequestration by cyclin D1 and cyclin D3.11,13 In this study, our aim was to determine whether Skp2 overexpression was associated with decreased p27Kip1 expression in aggressive B-cell lymphomas. Our data indicate that in most MCLs, Skp2 expression was low and is thus not the sole determinant of p27Kip1 levels. In blastic MCL and other aggressive B-cell lymphomas, high Skp2 levels are associated with low p27Kip1 levels and may be indicative of ubiquitin-mediated degradation of this protein. In addition to ubiquitin-mediated degradation, other parallel modes of p27Kip1 down-regulation have been described. Proteolytic processing of p27Kip127 resulting in a 22-kDa intermediate band may contribute to the reduced p27Kip1 expression in these neoplasms. Indeed, our immunoblot analysis of cell lysates of PBLs revealed a significant protein band of approximately 22 kDa (Figure 1). The absence of this lower-molecular-weight species in the lysates of lymphoma cell lines is of interest, and may be a reflection of the homogeneity of cells in different phases of the cell cycle in lymphoma cell lines relative to those of PBLs. Alternatively, the work of Yaroslavskiy et al34 suggests the presence of a 24-kDa variant p27Kip1 protein expressed in PBLs that is rapidly degraded via a proteasome-mediated mechanism. Our data suggest that in a large subset of lymphomas, Skp2 may be responsible for the regulation of p27Kip1 levels. This was most evident in low-grade lymphomas, but in DLBCLs the correlation was not as strong. Thus, overexpression of Skp2 may represent an important mechanism in the deregulation of p27Kip1 that contributes to malignant transformation in NHLs. In a subset of lymphomas, high Skp2 protein was not correlated with increased proliferation, suggesting that elevated Skp2 levels are not due merely to increased proliferation and that modulation of Skp2 expression levels may contribute to malignant phenotype without affecting cell proliferation. This finding has also been observed in a subset of oral cancers.23 In those cases in which there was high p27Kip1 and high Skp2 expression, we can postulate that degradation of other proteins, such E2F,22 cyclin D1, B-Myb,35 and cyclin E,19 by Skp2 may be involved in neoplastic transformation. Previous studies have shown that cytoplasmic displacement of p27Kip1 could potentially abrogate its function as a cdk inhibitor.3,13 Immunoprecipitation data provide support for the physical complexing of Skp2 and p27Kip that was observed in both the nuclear and the cytoplasmic subfractions of 4 cell lines. The significance of this is uncertain, however, since splice variants of Skp2 that result in cytoplasmic localization were found to render it inactive for ubiquitination and degradation of cyclin D1.31 These results suggest that abnormal cellular localization of Skp2 may provide an additional level of regulation of the F-box protein, resulting in deregulated ubiquitin-mediated control of cell cycle progression. In conclusion, we show that Skp2 expression is strongly correlated with the proliferation index in reactive lymphoid tissues and is expressed primarily in proliferating germinal centers. Among malignant NHLs, low Skp2 protein expression was observed in SLL/CLL and FCL, and was positively correlated with the proliferation index but inversely correlated with p27Kip1 expression. Within aggressive B-cell lymphomas, including MCL and DLBCL, Skp2 levels positively correlated with the proliferation index but did not correlate with p27Kip1 expression. Our findings are in agreement with those reported by Chiarle et al36 and provide evidence for the p27Kip1-degradative function of Skp2 in low-grade B-cell lymphomas, but suggest the existence of other molecular abnormalities in addition to Skp2 in the deregulation of p27Kip1 expression in aggressive lymphomas.
Submitted November 7, 2001; accepted May 28, 2002.
Supported by a grant from the Sunnybrook Trust for Research to M.S.L. and grant CA 83984-01 from the National Institutes of Health to K.S.J.E.-J.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Megan S. Lim, Department of Pathology, University of Utah, 50 North Medical Dr, Rm A 565, Salt Lake City, UT 84132; email: megan.lim{at}path.utah.edu.
1. Catzavelos C, Bhattacharya N, Ung YC, et al. Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nat Med. 1997;3:227-230[CrossRef][Medline] [Order article via Infotrieve]. 2. Sgambato A, Ratto C, Faraglia B, et al. Reduced expression and altered subcellular localization of the cylin-dependent kinase inhibitor p27(Kip1) in human colon cancer. Mol Carcinog. 1999;26:172-179[CrossRef][Medline] [Order article via Infotrieve].
3.
Singh SP, Lipman J, Goldman H, et al.
Loss or altered subcellular localization of p27 in Barrett's associated adenocarcinoma.
Cancer Res.
1998;58:1730-1735 4. Slingerland J, Pagano M. Regulation of the Cdk inhibitor p27 and its deregulation in cancer. J Cell Physiol. 2000;183:10-17[CrossRef][Medline] [Order article via Infotrieve].
5.
Thomas GVSK, Murphy M, Draetta G, Pagano M, Loda M.
Down-regulation of p27 is associated with development of colorectal adenocarcinoma metastases.
Am J Pathol.
1998;153:681-687
6.
Esposito V, Baldi A, De Luca A, et al.
Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer.
Cancer Res.
1997;57:3381-3385
7.
Erlanson M, Portin C, Linderholm B, Lindh J, Roos G, Landberg G.
Expression of cyclin E and the cyclin-dependent kinase inhibitor p27 in malignant lymphomas: prognostic implications.
Blood.
1998;92:770-777
8.
Tsihlias J, Kapusta LR, DeBoer G, et al.
Loss of cyclin-dependent kinase inhibitor p27Kip1 is a novel prognostic factor in localized human prostate adenocarcinoma.
Cancer Res.
1998;58:542-548 9. Koff A, Polyak K. p27KIP1, an inhibitor of cyclin-dependent kinases. Prog Cell Cycle Res. 1995;1:141-147[Medline] [Order article via Infotrieve]. 10. Morisaki HFA, Ando A, Nagata Y, Ikeda K, Nakanishi M. Cell cycle-dependent phosphorylation of p27 cyclin-dependent kinase (Cdk) inhibitor by cyclin E/Cdk2. Biochem Biophys Res Commun. 1997;240:386-390[CrossRef][Medline] [Order article via Infotrieve].
11.
Quintanilla-Martinez L, Thieblemont C, Fend F, et al.
Mantle cell lymphomas lack expression of p27Kip1, a cyclin-dependent kinase inhibitor.
Am J Pathol.
1998;153:175-182 12. Hengst L, Reed SI. Translational control of p27Kip1 accumulation during the cell cycle. Science. 1996;271:1861-1864[Abstract].
13.
Sanchez-Beato M, Camacho FJ, Martinez-Montero JC, et al.
Anomalous high p27/KIP1 expression in a subset of aggressive B-cell lymphomas is associated with cyclin D3 overexpression: p27/KIP1-cyclin D3 colocalization in tumor cells.
Blood.
1999;94:765-772 14. Barnouin K, Fredersdorf S, Eddaoudi A, et al. Antiproliferative function of p27Kip1 is frequently inhibited in highly malignant Burkitt's lymphoma cells. Oncogene. 1999;18:6388-6397[CrossRef][Medline] [Order article via Infotrieve]. 15. Allessandrini A, Chiau DS, Pagano M. Regulation of the cyclin-dependent kinase inhibitor p27 by degradation and phosphorylation. Leukemia. 1997;11:342-345[CrossRef][Medline] [Order article via Infotrieve]. 16. Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H. p27 (Kip1) ubitquitin and degradation is regulated by the SCF (Skp2) complex through phosphorylated Thr187 in p27. Curr Biol. 1999;9:661-664[CrossRef][Medline] [Order article via Infotrieve]. 17. Bilodeau M, Talarmin H, Ilyin G, et al. Skp2 induction and phosphorylation is associated with the late G1 phase of proliferating rat hepatocytes. FEBS Lett. 1999;452:247-253[CrossRef][Medline] [Order article via Infotrieve]. 18. Nakayama K, Nagahama H, Minamishima YA, et al. Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication. EMBO J. 2000;19:2069-2081[CrossRef][Medline] [Order article via Infotrieve]. 19. Yeh KH, Kondo T, Zheng J, Tsvetkov LM, Blair J, Zhang H. The F-box protein SKP2 binds to the phosphorylated threonine 380 in cyclin E and regulates ubiquitin-dependent degradation of cyclin E. Biochem Biophys Res Commun. 2001;281:884-890[CrossRef][Medline] [Order article via Infotrieve]. 20. Zhang H, Kobayashi R, Galaktionov K, Beach D. p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell. 1995;82:915-925[CrossRef][Medline] [Order article via Infotrieve]. 21. Nakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G(1)-S transition by proteolysis of cyclin E and p27(Kip1). Biochem Biophys Res Commun. 2001;282:853-860[CrossRef][Medline] [Order article via Infotrieve]. 22. Marti A, Wirbelauer C, Scheffner M, Krek W. Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nat Cell Biol. 1999;1:14-19[CrossRef][Medline] [Order article via Infotrieve].
23.
Gstaiger M, Jordan R, Lim M, et al.
Skp2 is oncogenic and overexpressed in human cancers.
Proc Natl Acad Sci U S A.
2001;98:5043-5048
24.
Latres E, Chiarle R, Schulman BA, et al.
Role of the F-box protein Skp2 in lymphomagenesis.
Proc Natl Acad Sci U S A.
2001;98:2515-2520 25. Diebold J. Classification of malignant lymphoma. WHO international proposal [in French]. Ann Pathol. 1998;18:361-367[Medline] [Order article via Infotrieve]. 26. Vlach J, Hennecke S, Amati B. Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. EMBO J. 1997;16:5334-5344[CrossRef][Medline] [Order article via Infotrieve].
27.
Shirane M, Harumiya Y, Ishida N, et al.
Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing.
J Biol Chem.
1999;274:13886-13893 28. Deshaies RJ. Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast. Curr Opin Genet Dev. 1997;7:7-16[CrossRef][Medline] [Order article via Infotrieve]. 29. Kipreos ET, Pagano M. The F-box protein family. Genome Biol. 2000;1:REVIEWS3002[Medline] [Order article via Infotrieve]. 30. Sutterluty H, Chatelain E, Marti A, et al. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol. 1999;1:207-214[CrossRef][Medline] [Order article via Infotrieve]. 31. Ganiatsas S, Dow R, Thompson A, Schulman B, Germain D. A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT. Oncogene. 2001;20:3641-3650[CrossRef][Medline] [Order article via Infotrieve]. 32. Carrano AC, Eytan E, Hershko A, Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol. 1999;1:193-199[CrossRef][Medline] [Order article via Infotrieve].
33.
Chiarle R, Budel LM, Skolnik J, et al.
Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is associated with a decreased overall survival in mantle cell lymphoma.
Blood.
2000;95:619-626 34. Yaroslavskiy B, Watkins SC, Alber S, Steinman RA. Dynamic changes in p27kip1 variant expression in activated lymphocytes. J Cell Biochem. 2001;83:380-389[CrossRef][Medline] [Order article via Infotrieve]. 35. Charrasse S, Carena I, Brondani V, Klempnauer KH, Ferrari S. Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway. Oncogene. 2000;19:2986-2995[CrossRef][Medline] [Order article via Infotrieve].
36.
Chiarle R, Fan Y, Piva R, et al.
S-phase kinase-associated protein 2 expression in non-Hodgkin's lymphoma inversely correlates with p27 expression and defines cells in S phase.
Am J Pathol.
2002;160:1457-1466
© 2002 by The American Society of Hematology.
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 A. Agarwal, T. G. P. Bumm, A. S. Corbin, T. O'Hare, M. Loriaux, J. VanDyke, S. G. Willis, J. Deininger, K. I. Nakayama, B. J. Druker, et al. Absence of SKP2 expression attenuates BCR-ABL-induced myeloproliferative disease Blood, September 1, 2008; 112(5): 1960 - 1970. [Abstract] [Full Text] [PDF]
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 O. A. O'Connor Mantle Cell Lymphoma: Identifying Novel Molecular Targets in Growth and Survival Pathways Hematology, January 1, 2007; 2007(1): 270 - 276. [Abstract] [Full Text] [PDF]
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 H.-Y. Huang, H.-Y. Kang, C.-F. Li, H.-L. Eng, S.-C. Chou, C.-N. Lin, and C.-Y. Hsiung Skp2 Overexpression Is Highly Representative of Intrinsic Biological Aggressiveness and Independently Associated with Poor Prognosis in Primary Localized Myxofibrosarcomas Clin. Cancer Res., January 15, 2006; 12(2): 487 - 498. [Abstract] [Full Text] [PDF]
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 O. A. O'Connor Targeting Histones and Proteasomes: New Strategies for the Treatment of Lymphoma J. Clin. Oncol., September 10, 2005; 23(26): 6429 - 6436. [Abstract] [Full Text] [PDF]
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J. S. Knight, N. Sharma, and E. S. Robertson SCFSkp2 Complex Targeted by Epstein-Barr Virus Essential Nuclear Antigen Mol. Cell. Biol., March 1, 2005; 25(5): 1749 - 1763. [Abstract] [Full Text] [PDF]
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Y. H. Min, J.-W. Cheong, M. H. Lee, J. Y. Kim, S. T. Lee, J. S. Hahn, and Y. W. Ko Elevated S-Phase Kinase-Associated Protein 2 Protein Expression in Acute Myelogenous Leukemia: Its Association with Constitutive Phosphorylation of Phosphatase and Tensin Homologue Protein and Poor Prognosis Clin. Cancer Res., August 1, 2004; 10(15): 5123 - 5130. [Abstract] [Full Text] [PDF]
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 J. Yun, A. Tomida, T. Andoh, and T. Tsuruo Interaction between Glucose-regulated Destruction Domain of DNA Topoisomerase II{alpha} and MPN Domain of Jab1/CSN5 J. Biol. Chem., July 23, 2004; 279(30): 31296 - 31303. [Abstract] [Full Text] [PDF]
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C. Q. Zhu, F. H. Blackhall, M. Pintilie, P. Iyengar, N. Liu, J. Ho, T. Chomiak, D. Lau, T. Winton, F. A. Shepherd, et al. Skp2 Gene Copy Number Aberrations Are Common in Non-Small Cell Lung Carcinoma, and Its Overexpression in Tumors with ras Mutation Is a Poor Prognostic Marker Clin. Cancer Res., March 15, 2004; 10(6): 1984 - 1991. [Abstract] [Full Text] [PDF]
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L. Quintanilla-Martinez, T. Davies-Hill, F. Fend, J. Calzada-Wack, L. Sorbara, E. Campo, E. S. Jaffe, and M. Raffeld Sequestration of p27Kip1 protein by cyclin D1 in typical and blastic variants of mantle cell lymphoma (MCL): implications for pathogenesis Blood, April 15, 2003; 101(8): 3181 - 3187. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
0.67;
P < .0001) with p27Kip1 and positively
correlated with the PI (r = 0.82;
P < .005). By contrast, whereas most mantle cell
lymphomas (MCLs) demonstrated low expression of
p27Kip1 and Skp2, a subset (n = 6) expressed high
Skp2 (exceeding 20%) with a high PI (exceeding 50%). Skp2 expression
was highest in diffuse large B-cell lymphomas (DLBCLs) (mean,
22%) and correlated with Ki-67 (r = 0.55;
P < .005) but not with p27Kip1. Cytoplasmic
Skp2 was seen in a subset of aggressive lymphomas. Our data provide
evidence for p27Kip1 degradative function of Skp2 in
low-grade lymphomas. The absence of this relationship in aggressive
lymphomas suggests that other factors contribute to deregulation of
p27Kip1 expression in these tumors.
(Blood. 2002;100:2950-2956)
