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From the Department of Pathology and Kaplan Comprehensive CancerCenter,* New York University School of Medicine, New York,New York; the Department of Pathology,University of Torino, Torino, Italy; the Department of Pathology andExperimental Medicine Research Center,University of Verona, Verona, Italy; and the Division of Morphology andMolecular Pathology, Catholic UniversityLeuven, Leuven, Belgium
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Abstract |
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40-amino acid long motif called F-box.
Skp1, Cul1, and Roc1/Rbx1 associate with different Fbps to form E3
ubiquitin ligase complexes known as SCF. The Fbp subunits of the SCF
complex ensure the specific recognition and ubiquitination of a large
number of substrates that are in turn degraded by the
proteasome.10 Skp2 was first identified to interact with the cyclin A-Cdk2 complex in immortalized fibroblasts and transformed cells and required for G1 to S transition.11 In vitro and in vivo studies have demonstrated that the SCF complexes containing Skp2, as Fbp (SCFSkp2), bind to p27, which is specifically ubiquitinated by the ubiquitin-conjugating enzyme Ubc3.4-7 Skp2-deficient mice have smaller organs than littermate controls, their cells grow more slowly, and they display centrosome overduplication together with the accumulation of p27 and free cyclin E.7 Importantly, in Skp2 transgenic mice, the forced expression of Skp2 in T cells cooperates with the N-ras oncogene leading to lymphomagenesis.8 Finally, the tumorigenic role of Skp2 has been suggested by several investigators.9,12
The deregulated expression of p27 plays a critical role in the pathogenesis of many human tumors.13 The protein levels of p27 in normal cells are mainly regulated by an ubiquitin-mediated degradation.14 Moreover, an enhanced protein degradation via the ubiquitin-proteasome pathway is also responsible for the low levels of p27 protein in aggressive human tumors15-17 and in mantle cell lymphomas (MCLs).1 We and others have demonstrated that the protein levels of p27 expression are different in specific subsets of human lymphomas, being high in low-grade and low in high-grade lymphomas.1,3,18 More importantly, the partial or total loss of p27 function defines a group of patients that exhibit low overall survival and poor outcome.1,3
In this study we have analyzed the expression of Skp2 in cell lines and human lymphomas and compared it to other proliferation indexes and to the expression of its substrate p27. Our findings demonstrate that Skp2 protein levels tightly correlate with the late G1 and S phase in neoplastic cell lines and tumors. We have found that there is a direct correlation among Skp2 and Ki-67 and cyclin A and an inverse relationship between Skp2 and p27 protein levels in the majority of human lymphomas. However, in a subset of lymphomas this correlation is lost, suggesting that alternative pathways of p27 inactivation might exist.
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Materials and Methods |
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A panel of 244 well-characterized non-Hodgkin’s lymphomas (NHLs) was retrieved from the archives of the Division of Hematopathology of the New York University School of Medicine, from the Division of Morphology and Molecular Pathology of the Catholic University of Leuven in Belgium, and the Surgical Pathology Departments of the Universities of Torino and Verona in Italy. NHLs were classified according to the International Lymphoma Study Group, based on hematoxylin and eosin and immunoperoxidase stains, and clinical and molecular data, as previously described.1 The NHLs characterized in this study included: 28 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLLs), 51 follicular lymphomas (FL), 34 mucosa-associated lymphoid tissue B-cell lymphomas, 34 MCLs, and 76 diffuse large B-cell lymphomas (DLCLs), 15 lymphoblastic lymphomas (6 B-lymphoblastic lymphoma and 9 T-lymphoblastic lymphomas), 25 Burkitt’s lymphomas, and 5 Burkitt-like lymphomas. FL were classified based on the criteria of the revised World Health Organization classification19 and of Mann and Berard.20 A single case of plasma cell leukemia (>90% plasma cells) was also included for the flow cytometry staining. The following cell lines were also used: Namalwa, Jurkat, Karpas 299, DHL, JB, KM512, and K562.
Antibodies
The monoclonal antibodies (mAbs) applied for the immunohistochemistry in this study included: anti-p27 (KIP-1, 1:1000; Transduction Laboratories, Lexington, KY, USA), anti-p53 (DO-1, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Ki-67 (MIB-1, 1:1000; Immunotech, Marseille, France), anti-cyclin E (1:50; Novocastra, CA, USA) and cyclin A (1:100, Santa Cruz Biotechnology). Mouse mAbs to Skp2 were produced in collaboration with Zymed Inc., South San Francisco, CA, USA. Immunohistochemical staining and Western blotting for Skp2 were performed using a cocktail of four different affinity-purified mAbs (clones 2C8D9, 2C2B12, 4A9B2, and 2G12E9) or a single mAb (clone 4A9B2),8 as indicated.
Western Blot Analysis and in Vitro Protein Degradation Assay
For Western Blotting, cells were lysed (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Triton X-100, 5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L Na3VO4, and 1 mmol/L phenylmethyl sulfonyl fluoride and protease inhibitors) and 20 to 30 µg of proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto nitrocellulose membranes. The membranes were first blocked and then incubated with the primary antibody (anti-Skp2, clone 4A9B2, 1:500; anti-p27, KIP-1, 1:2000; and anti-cyclin A, 1:1000) for 1 hour at room temperature. After three washes, filters were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (1:2000; Amersham, Arlington Heights, IL, USA) for 1 hour at room temperature. Detection of immunocomplexes was performed with an enhanced chemiluminescence system (ECL, Amersham).1
In vitro degradation assays were performed on cryopreserved tissue samples.1 Frozen tissues of MCLs (expressing low, intermediate, and high protein levels of p27), with >80% of tumor cells were sectioned and quickly disrupted by nitrogen decompression in 100 µl of lysing buffer (50 mmol/L Tris-HCl, pH 8.3, 5 mmol/L MgCl2, and 1 mmol/L dithiothreitol). Samples were then frozen and thawed three consecutive times. Lysates were spun down at 15,000 rpm and supernatants were collected and frozen at -80°C. Histidine-tagged p27 (100 µg) was incubated at 37°C for different intervals in 60 µl of degradation mix containing 30 µg of protein tissue homogenates, 50 mmol/L Tris-HCL (pH 8.0), 5 mmol/L MgCl2, 1 mmol/L dithiothreitol, 2 mmol/L ATP, 10 mmol/L creatine phosphokinase, and 10 mmol/L creatine phosphatase. The in vitro degradation of p27 was analyzed by immunoblotting with anti-p27 mAb.
Immunohistochemical Staining
For immunohistochemistry, slides were subjected to
microwaving for 20 minutes in 10 mmol/L of citrate buffer (pH 8.0 for
Skp2, cyclin E, and cyclin A; pH 6.0 for p27, p53, and Ki67).
Immunostaining were performed on formalin-fixed, paraffin-embedded
tissues using the avidin biotin peroxidase complex method and a
semiautomated immunostainer (DAKO, Carpinteria, CA, USA or Ventana
Systems, Tucson, AZ, USA) as described.1
Three independent
pathologists (RC, YF, and GI) evaluated the immunostaining. At least 10
high-power fields were randomly chosen and at least 100 cells/field
were counted. Tumors were scored as percentage of positive cells for
each antigen. The statistical significance of the means was calculated
using a Student’s t-test. The statistical significance of
Spk2 versus cyclin A expression was calculated with the
chi-square test. P 
0.05 was required for statistical
significance.
Flow Cytometry and BrdU Labeling
For flow cytometric staining, untreated cell lines or lovastatin-treated (24 hours, 50 µmol/L; Sigma Chemical Co., St. Louis, MO, USA) cells were fixed and permeabilized according to the manufacturer’s instruction (Fix and Perm; Caltag, Burlingame, CA). Cells were subsequently stained using a cocktail of anti-Skp2 mAbs (1:100, Zymed). Anti-Skp2 mAbs were detected using phycoerythrin-conjugated horse anti-mouse antibody (1:200; Biosource, Camarillo, CA, USA). For DNA content determination, cells were fixed for 1 hour in 70% ethanol at 4°C. After washing, cells were treated with RNase (0.25 mg/ml) and stained with propidium iodide (50 µg/ml). The S-phase fraction was calculated using the Modfit program from Becton-Dickinson, Mountain View, CA.
For BrdU labeling, 1 x 106 cells or 1-mm-thick fresh tissues (three normal tonsils and seven randomly selected NHLs) were incubated in complete cell-culture media (RPMI-1640 with 10% fetal calf serum) in the presence of 10 µmol/L of BrdU for 30 minutes (suspension cells) or for 2 hours (tissue samples) at 37°C in 5% CO2. For BrdU determination using flow cytometry, cells were subsequently washed, fixed in 70% alcohol for 30 minutes, denatured with 2 N HCl, and then incubated with an anti-BrdU fluorescein isothiocyanate-labeled mAb (1:10, Becton-Dickinson) and propidium iodide (50 µg/ml). For BrdU determination using immunohistochemistry, tissue samples were rinsed in phosphate-buffered saline and fixed overnight in 10% buffered formalin. The incorporation of BrdU in proliferating cells was then detected by enzymatic digestion (0.05% proteinase K for 20 minutes at 37°C), denaturation (2 N HCl for 30 minutes at 37°C), and incubation with an anti-BrdU fluorescein isothiocyanate-conjugated antibody (30 minutes at room temperature, Becton-Dickinson). Bound anti-BrdU fluorescein isothiocyanate-conjugated antibodies were detected using an alkaline-conjugated anti-fluorescein isothiocyanate antibody (1:200; Roche, Indianapolis, IN) and NBT/BCIP as a substrate.
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Results |
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The specificity of our anti-Skp2 antibodies was first evaluated in
cell lines derived from human lymphomas by Western blotting. Skp2 mAbs
detected only a single band migrating slightly faster than a tagged
Skp2 recombinant protein (Figure 1A)
. We
subsequently evaluated by immunohistochemistry Skp2 protein expression
in normal primary and secondary lymphoid tissues. In primary lymphoid
organs, Skp2 staining was restricted to the nuclei of a subset of
cortical thymocytes as well as of precursor myeloid and erythroid cells
in the bone marrow (Figure 1, B and C)
. In peripheral lymphoid organs,
centroblasts or large centrocytes, within the dark zone of the germinal
centers, were preferentially positive (Figure 1, D and E)
. In contrast,
small resting cells within the mantle of the B-cell follicle and
interfollicular T lymphocytes were negative. Nonetheless, in the
interfollicular areas some of the intermediate large cells were also
Skp2-positive. In the oral mucosa as well as in the epidermis,
Skp2-positive cells were confined, similarly to the Ki-67-positive
cells, within the basal layer of the epithelium (data not shown).
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Since in normal cells the levels of Skp2 vary during the cell
cycle, with a peak of expression at G1 to S
through S phase,11
we investigated the kinetics of Skp2
during cell-cycle progression in neoplastic lymphoid cells to seek for
a possible aberrant pattern and/or deregulation. First, we compared
Skp2 expression to the fraction of cells in S phase calculated by DNA
content and BrdU incorporation in multiple and T-lymphoblastoid cell
lines and in a case of plasma cell leukemia. In all of the cell lines
the percentage of Skp2-positive cells always corresponded with the
percentage of cells in the S phase (Table 1
, Figure 2A
). Moreover, flow cytometric
analysis demonstrated two distinct subpopulations of Skp2-positive
cells, with intermediate and high intensity of expression, suggesting
that different levels of expression of Skp2 are achieved in different
phases of the cell cycle (Figure 2B)
. To further confirm the relation
between Skp2 and S phase, we blocked the progression into the S phase
of multiple lymphoid cell lines by inducing an early
G1 cell-cycle arrest. In cells arrested by
lovastatin in G1 phase, both the percentage of
cells in S phase and those of Skp2-positive cells decreased in a
comparable manner (Figure 2B)
. These results were confirmed in all
lymphoma-derived cell lines, demonstrating that Skp2 expression in
neoplastic lymphoid cells as well in normal nonlymphoid
cells,11
peaks in late G1 to S phase
and is maintained throughout the S phase. Next, we correlated the
percentage and distribution of Skp2-positive cells with BrdU
incorporation in fresh lymphoma samples. This approach showed a close
correspondence between Skp2- and BrdU-positive cells (Figure 2C)
,
confirming that the majority of Skp2-positive cells are actively
synthesizing the DNA in normal as well in neoplastic lymphoma cells.
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The expression of Skp2 was subsequently evaluated in B-cell NHLs
representing all grades of malignancy. In CLLs and Skp2-positive cells
were primarily localized within the proliferation centers primarily
restricted to para-immunoblasts. (Figure 3B)
. In mucosa-associated lymphoid tissue
B-cell lymphomas only 5% of the neoplastic cells were found to be
positive for Skp2. Skp2 expression in FL was restricted to a fraction
of centroblasts and large centrocytes, but a variable number of
interfollicular cells were also found positive. More importantly, the
percentage of Skp2-positive cells significantly correlated with the
histological grade of these lymphoma (mean percentage: FL grade I,
3.5%; grade II, 10.4%, and grade III, 20.1%). Notably the sole
expression of Skp2 statistically correlated with the FL grading
obtained using conventional cytological criteria20
(grade
I versus grade II, P = 0.0007; grade II
versus grade III, P = 0.0006; grade I
versus grade III, P < 0.0001) (Table 2)
. A positive correlation was also found
when the percentages of Ki-67-positive cells were correlated to the
same groups of FL. In MCLs, Skp2-positive tumor cells were uniformly
distributed (Figure 4, B and E)
, with
clusters of Skp2-positive cells corresponding to nonneoplastic cells
within residual normal germinal centers. As expected, the blastoid and
large cell variants of MCL21
showed the highest
percentages of Skp2-positive cells (Figure 4E)
. Interestingly, among
high-grade lymphomas, a very high percentage of Skp2-positive cells was
found in Burkitt’s lymphomas (87 ± 8.8%) (Figure 5)
. By contrast, in DLCL and Burkitt-like
and lymphoblastic lymphomas the percentage of Skp2-positive cells
ranged from 10 to 65%. These values were statistically different and
we were able to separate these tumors from Burkitt’s lymphomas
(P < 0.0001). Finally, when all types of
lymphomas were compared, the percentage of Skp2-positive cells strictly
correlated with the distinct groups of NHLs being low in low-grade
lymphomas and higher in high-grade tumors (P <
0.001) (Table 2)
.
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We next compared the expression of Skp2 and Ki-67, the most
commonly used proliferation marker, with those of p27, cyclin E, and
cyclin A. Skp2 expression showed a statistical correlation with Ki-67
and cyclin E both in low-grade lymphomas, such as CLLs
(r = 0.47, P = 0.01), and in
high-grade lymphomas such as DLCL (r = 0.63 and
r = 0.48, respectively; P < 0.0001).
Nevertheless, the percentage of Skp2-positive cells always corresponded
to a subset of Ki-67-positive cells. As previously described, in
high-grade lymphomas (lymphoblastic lymphoma, DLCL, Burkitt’s and
Burkitt-like lymphomas), Ki-67 was expressed by the very large majority
of tumor cells (mean percentage 
64%), whereas the percentage of
Skp2-positive cells varied among these categories (Table 2)
.
Interestingly, in lymphoblastic lymphomas, DLCL, and Burkitt-like
lymphomas anti-Skp2 mAb identified only a fraction (mean percentage
36% of the total tumors cells) of the Ki-67-positive cells.
Conversely, the percentages of Skp2- and Ki-67-positive cells were very
similar in Burkitt’s lymphomas (87% versus 92.6%,
respectively). As shown above, the expression of Skp2 in lymphoid cell
lines highly correlates with the percentage of cells within the S phase
and BrdU-positive cells. To corroborate this finding, we further
investigated the relationship between Skp2 expression and the
expression of cyclin A. A total of 38 cases, including FL
(n = 10), MCL (n = 18),
and DLCL (n = 10) lymphomas, were studied. We
decided to study the expression of cyclin A because its expression is
largely limited to S to G2
phases22,23
and it correlates with the percentages of S to
G2 and BrdU in normal and transformed
cells.24
To identify whether the p27, and cyclin A and
Skp2 expression were correlated, we selected p27-negative
(n = 14) and p27-positive (n =
4) MCL lymphomas. Using this approach, we demonstrated that Skp2 and
cyclin A expression directly correlated in all tumors, including
p27-negative lymphomas (Figure 6A)
.
Overall, these findings further support the hypothesis that in human
lymphomas the percentage of Skp2-positive cells is closely correlated
to the fraction of proliferating cells.
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(r = 0.81,
P = 0.01). Overall, these findings parallel and expand
our previous results obtained from the analysis of Ki-67 and p27
expression in human NHLs.1
However in MCLs and in a subset
of DLCLs and lymphoblastic lymphomas this relationship was not found.
In fact, the percentage of p27-negative cells did not always correlate
with that of Skp2-positive cells, being MCL mostly negative for p27
expression and showing a relatively low percentage of Skp2-positive
cells (Figure 4)
. Moreover, a fraction of cells of DLCLs was negative
for both p27 and Skp2 (Figure 3, E and F)
. Because the protein levels
of p27 in MCL correlate with the degree of p27 protein degradation
in vitro,1
we compared the expression of p27
and Skp2 and p27 degradation in vitro in a small number of
MCLs with high, intermediate, and low levels of p27. As shown in Figure 6
, the protein levels of Skp2 were quite similar despite the different
expression of p27 and they correlated quite closely to those of cyclin
A and to the percentage of positive cells stained with anti-cyclin A
and anti-Ki-67 antibodies (data not shown). These findings further
support the hypothesis that Skp2 is not overexpressed in tumors lacking
p27 and that p27 protein levels in these lymphomas may be regulated via
alternative mechanisms. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In human lymphomas p27 has been shown to inversely correlate with the proliferation index and to have prognostic significance.1,3 Since p27 degradation in normal cells is dependent on the expression of Skp2 and because the loss of p27 expression in some lymphomas is due to an increased rate of p27 degradation,1 we tested whether the expression of Skp2 could correlate with the levels of p27 protein in human lymphomas. We analyzed a large series of B-cell lymphomas derived from different B cells frozen at different stages of differentiation and representing unique histological and clinical grades. As hypothesized, we could observe an inverse correlation between p27 and Skp2 in most human lymphomas. Overall, these findings are in agreement with the notion that the protein levels of p27 are—at least in normal cells—primarily regulated by the SCFSkp2 ligase. On the other hand MCLs, in which p27 levels are often undetectable and do not correlate with the cell-cycle progression,1 showed only a low percentage of Skp2-positive cells. Moreover 30 to 35% of the cells of high-grade lymphomas displayed no staining for both Skp2 and p27. These findings argue in favor of a down-regulation of p27 that is not solely dependent on Skp2. In favor of this hypothesis are data that indicate that other factors might influence p27 protein levels, such as decreased transcription or translation, abnormal localization or sequestration,13 and cleavage via nonapoptotic caspases.26 Finally the recent demonstration that p27 can also be degraded via a Skp2-independent pathway sheds new light on the high and complex regulation of this cell-cycle inhibitor.27
By comparing the expression of Skp2 with that of other cell-cycle proteins, such as Ki-67, cyclin E, and cyclin A, we have also shown that Skp2 directly correlates with the proliferative index in all types of lymphomas. Interestingly, the fraction of cells stained with anti-Skp2 antibody was always lower than the percentage of cells stained using anti-Ki-67 mAb. These findings are in accordance with the different patterns of expression of these proteins during the cell cycle. In cycling cells, in fact, the Ki-67 protein is expressed throughout the cell cycle, ie, from the G1 phase, through the entire S phase, up to the G2 to M phase,28 whereas Skp2 is expressed only during the late G1 and S phase.11,29 Thus, Skp2 expression defines a narrower window in the cell-cycle progression compared with that identified by anti-Ki-67 mAbs. This difference may offer a clear advantage in the analysis of neoplastic processes. It is not simple, in fact, to separate lymphoma subtypes or to predict biological evolutions within specific groups considering only the percentage of Ki-67-positive cells. This is particularly true in tumors with high proliferating indexes such as Burkitt’s lymphoma, Burkitt-like lymphomas, and many DLCLs, whose percentages of Ki-67-positive cells are uniformly too high. By contrast, Skp2 determination could provide a real advantage in clinical settings permitting discrimination between Burkitt’s lymphomas (87%), and other high-grade neoplasms (Burkitt-like lymphomas and DLCLs, 34% and 21%, respectively). Nonetheless, the mechanisms responsible for these differences are unclear. Toward this end, we could speculate that the high expression of Skp2 in Burkitt’s lymphomas might result from its deregulated expression or might simply reflect a very fast cell-cycle progression.
The analysis of Skp2 levels may also provide a powerful tool for a more precise grading of follicular cell lymphomas. So far, the grading system of follicular cell lymphomas remains still imprecise and often irreproducible, being dependent on the subjective count of centroblasts or large centrocytes.19,30 Because Skp2 staining gives the precise measurement of cells committed to proliferating its quantification, particularly by using automatic microscopic scanning, it might offer a more objective grading. A reproducible classification of FL will allow studying the efficacy of conventional and new therapeutic approaches, such as anti-idiotype vaccine strategies in the treatment of lymphomas.
Finally, flow cytometry has also shown that the percentage of Skp2-positive cells matches with the number of cells in S phase. Because Skp2 has a precise concordance with S phase, it could be the best candidate to replace Ki-67, proliferating cell nuclear antigen, and other commonly used markers for the determination of the proliferation index in human tumors. In particular, it could be extremely convenient in the determination of the fraction of proliferating cells in tumors in which BrdU incorporation still has an important prognostic value, such as multiple myeloma. BrdU incorporation analysis, in fact, is technically demanding, time consuming, and limited only to specialized laboratories. The reproducibility and technical simplicity of the flow cytometry and immunohistochemical stains using anti-Skp2 antibodies gives a real edge for the analysis of many human tumors in routine clinical settings.
In conclusion, we have shown that Skp2 is a useful and precise marker to quantify cells committed to or in S phase. Thus, the analysis of Skp2 expression may represent a useful tool with both diagnostic and possibly prognostic applications in lymphomas. Notably, Kudo and colleagues31 have recently demonstrated that Skp2 expression can be used as a prognostic marker in oral squamous cell carcinomas. We also found that the expression of Skp2 seems not to be deregulated in the large majority of human lymphomas. Nonetheless, more studies are requested to confirm our observations, particularly in lymphomas with very high Skp2 expression, ie, Burkitt’s lymphomas. In fact, because Skp2 overexpression may occur in other neoplasms and high levels of Skp2 seem to play an important role in tumorigenesis8,9,12,31 a more extensive analysis of Skp2 may be necessary. Finally, the apparent discrepancy between low levels of Skp2 and p27 seen in MCLs suggests that an enhanced degradation of p27 may occur via alternative F-box proteins or additional E3 ligase(s). In fact, recent studies have pointed out that the protein regulation of p27 and other SCF substrates may be achieved by different pathways involving multiple players.27,32,33 The discovery of the molecular mechanisms regulating the protein expression of p27 in lymphomas might bring new light in the understanding of the pathogenesis of human tumors. Moreover, the recognition of these defects will allow the design of new and more specific approaches for the treatment of human malignancies.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants ROI-CA90773 (to G.I.), ROI-CA76584, and ROI-GM57587 (to M.P.).
R. C. and Y. F. both contributed equally to this work.
R. C. is on leave from the University of Torino, Department of Pathology, Via Santena, 10126, Torino, Italy.
Present address of J. S.: Department of Pediatrics, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115.
Accepted for publication January 18, 2002.
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E.-H. Shim, L. Johnson, H.-L. Noh, Y.-J. Kim, H. Sun, C. Zeiss, and H. Zhang Expression of the F-Box Protein SKP2 Induces Hyperplasia, Dysplasia, and Low-Grade Carcinoma in the Mouse Prostate Cancer Res., April 1, 2003; 63(7): 1583 - 1588. [Abstract] [Full Text] [PDF]
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G. Z. Rassidakis, F.-X. Claret, R. Lai, Q. Zhang, A. H. Sarris, T. J. McDonnell, and L. J. Medeiros Expression of p27Kip1 and c-Jun Activation Binding Protein 1 Are Inversely Correlated in Systemic Anaplastic Large Cell Lymphoma Clin. Cancer Res., March 1, 2003; 9(3): 1121 - 1128. [Abstract] [Full Text] [PDF]
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M. Sanchez-Beato, A. Sanchez-Aguilera, and M. A. Piris Cell cycle deregulation in B-cell lymphomas Blood, February 15, 2003; 101(4): 1220 - 1235. [Abstract] [Full Text] [PDF]
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P. L. Bergsagel Prognostic Factors in Multiple Myeloma: It's in the Genes Clin. Cancer Res., February 1, 2003; 9(2): 533 - 534. [Full Text] [PDF]
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 D. Sitry, M. A. Seeliger, T. K. Ko, D. Ganoth, S. E. Breward, L. S. Itzhaki, M. Pagano, and A. Hershko Three Different Binding Sites of Cks1 Are Required for p27-Ubiquitin Ligation J. Biol. Chem., October 25, 2002; 277(44): 42233 - 42240. [Abstract] [Full Text] [PDF]
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M. S. Lim, A. Adamson, Z. Lin, B. Perez-Ordonez, R. C. K. Jordan, S. Tripp, S. L. Perkins, and K. S. J. Elenitoba-Johnson Expression of Skp2, a p27Kip1 ubiquitin ligase, in malignant lymphoma: correlation with p27Kip1 and proliferation index Blood, September 26, 2002; 100(8): 2950 - 2956. [Abstract] [Full Text] [PDF]
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