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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 619-626
NEOPLASIA
Increased proteasome degradation of cyclin-dependent kinase
inhibitor p27 is associated with a decreased overall survival in
mantle cell lymphoma
Roberto Chiarle,
Leo M. Budel,
Jeffrey Skolnik,
Glauco Frizzera,
Marco Chilosi,
Alessandra Corato,
Gianni Pizzolo,
Jory Magidson,
Alessia Montagnoli,
Michele Pagano,
Brigitte Maes,
Christine De Wolf-Peeters, and
Giorgio Inghirami
From the Department of Pathology and the Kaplan Comprehensive Cancer
Center, New York University School of Medicine; the Institute of
Pathology, Erasmus University of Rotterdam, the Netherlands; the
Department of Pathological Anatomy and Histology and the Department of
Medicine, University of Verona, Italy, the Department of Pathology,
Morristown Memorial Hospital, NJ; and the Department of
Surgical Pathology, University of Leuven, Belgium.
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Abstract |
Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized
by the deregulated expression of cyclin D1 by t(11;14). The molecular
mechanisms responsible for MCL's clinical behavior remain unclear. The
authors have investigated the expression of p53, E2F-1, and the CDK
inhibitors p27 and p21 in 110 MCLs, relating their expression to
proliferative activity (Ki-67). For comparison, they have similarly
analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL)
lymphomas. p53 was detected more frequently in large-cell MCL
(l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of
19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high,
correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs
(12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27
positive. Reverse transcription-polymerase chain reaction and in vitro
protein degradation assays demonstrated that MCLs have normal p27 mRNA
expression but increased p27 protein degradation activity via the
proteasome pathway. Correlation of MCL p53 and p27 expression with
clinical data showed an association between reduced overall survival
rates and the overexpression of p53 (P = .001), the loss of
p27 (P = .002), or both. Loss of p27 identified patients
with a worse clinical outcome among p53 negative cases
(P = .002). These findings demonstrated that MCL has a
distinct cell cycle protein expression similar to that of high-grade
lymphoma. The loss of p27 and the overexpression of p53 in MCL are
prognostic markers that identify patients at high risk. The
demonstration that low levels of p27 in MCL result from enhanced
proteasome-mediated degradation should encourage additional clinical
trials. (Blood. 2000;95:619-626)
(Blood. 2000;95:619-626)
© 2000 by The American Society of Hematology.
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Introduction |
Mantle cell lymphoma (MCL) is a lymphoproliferative
disorder that, by morphologic, immunophenotypic, and genotypic
findings, is thought to be derived from the mature B cells of the
follicular mantle of the follicles (ie, the "mantle zone"). At
the molecular level, MCL is characterized by the juxtaposition of
bcl-1 and the heavy chain immunoglobulin gene (PRAD-1/CCND1),
resulting in the deregulation of bcl-1
expression.1-10 Based on morphologic and genetic features
and its unique clinical behavior, MCL is now recognized as a distinct
type of non-Hodgkin's lymphoma (NHL) with a very aggressive course. In
fact, patients with MCL (along with T-lymphoblastic and peripheral
T-cell lymphoma) have one of the lowest 5-year survival rates among all
types of lymphomas.11
Most cases of MCL have a strikingly similar appearance, characterized
by a uniform population of relatively small neoplastic cells with
irregular nuclear contours, dispersed chromatin, and relatively little
cytoplasm. However, the morphologic spectrum of MCL is wide. The
architectural features comprise a growth pattern that can be
perifollicular, nodular, or diffuse; the latter is associated with a
worse prognosis.12-17 Furthermore, though uncommon, several
cytologic variants have been described, all of which share the classic
MCL immunophenotype/genotype (IgM+, IgD±, CD5+, CD10 , CD23,
cyclin D1 overexpression, and t(11;14) association) but demonstrate a
distinct clinical course.12,18-21 These variants include
the blastoid variant, characterized by slightly larger cells than
classical s-MCL, with fine dusty chromatin, inconspicous nucleoli, and
a high proliferative rate 12; the pleomorphic variant,
characterized by large cells with a pleomorphic nucleus, rarefied
chromatin, a single central nucleolus and scant
cytoplasm18; and the large-cell variant, characterized by a
high proliferation rate, a more aggressive clinical course, and
decreased median survival time.12,18,22
Only a few studies have investigated the molecular differences among
these MCL subtypes. We and others have reported a higher incidence of
p53 anomalies18,23-25 and of bcl-1 translocations of the major translocation cluster locus among the MCL
variants.19,22 Recently, loss-of-function mutations in cell
cycle-negative regulatory elements, including point mutations and
deletions or rearrangements of the cyclin-dependent kinase (CDK)
inhibitors p15, p16, and p18 genes, have been described in a subset of
MCLs and have been associated with an aggressive clinical course,
blastic morphology, and extranodal dissemination.26,27
Deregulated expression of cell cycle genes plays a significant role in
oncogenesis. In the past few years, many genetic aberrations of cell
cycle regulators, such as cyclin D1, the retinoblastoma gene (pRB),
p53, and CDK inhibitors p15 and p16 have been described in a variety of
malignancies, including lymphoma.28 The role of cyclin D1
in the pathogenesis of MCL is strongly indicated by its
overexpression/deregulation in this neoplasm. Cyclin D1-Cdk4 functions
as a sensor for mitogenic signals. The only known major substrate of
cyclin D1-Cdk4 is pRb, which, when phosphorylated, allows the release
and activation of E2F.29,30 Cyclin/Cdks complexes are negatively regulated by two families of inhibitors the INK4 family, which includes p15, p16, p18, and p19, and the CIP family,
which includes p21, p27, and p57. Of the latter group, p21 is induced
by the tumor suppressor p53 in response to DNA damage.31,32
p27 can produce cell-cycle arrest in response to inhibitory stimuli
such as transforming growth factor- or cyclic adenosine
monophosphate, lack of adhesion, and cell-contact inhibition.33 Recently, the deregulation of p27 protein
expression has been demonstrated in several human neoplasms, and its
low levels correlate with poor survival rates,34-37
high-grade neoplasms, or both.
The aim of this study was to compare the expression of key cell cycle
regulators in different subsets of NHL to identify unique protein
expression profiles that may correlate with biologic and clinical
features. We specifically studied the function of p21, p27, p53, and
E2F-1 gene expressions because they play a key role in G1-S transition
and they ultimately regulate cell proliferation and cell growth.
Moreover, we investigated whether the expression patterns of p53 and
p27 could allow the identification of a subset of patients with MCL
whose clinical outcomes are worse.
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Materials and methods |
Pathologic samples
A panel of 157 well-characterized cases of NHL were selected from
among those processed in the surgical pathology laboratories of the New
York University School of Medicine, the University of Leuven, Belgium,
and the University of Verona, Italy. The lymphoproliferative disorders
characterized in this study included MALT-type lymphoma (12 cases),
chronic lymphocytic leukemia/prolymphocytic leukemia/small lymphocytic
lymphoma (16 cases), high-grade large B-cell lymphoma (19 cases of
DLCL), and 110 cases of MCL. The l-MCL cases included 1 transformed
large, 2 blastoid, and 4 pleomorphic variant cases that were classified
using morphologic, immunophenotypic, and genotypic criteria, according
to criteria previously described.12,18,19,38 The lymphomas
were classified according to the International Lymphoma Study
Group,20 based on hematoxylin-eosin staining and
immunoperoxidase stains for B- and T-cell markers (CD3, CD5, CD10,
CD20, and CD23 antigens; DAKO, Santa Barbara, CA). In select cases,
frozen tissue sections also were stained (kappa, lambda, IgG, IgD, and
IgM), and extensive flow cytometric analysis was performed. Gene
rearrangement18 and/or cytogenetic studies38,39
were performed in all cases of MCL to demonstrate the presence of
bcl-1-hIg chimeric products or t(11;14) translocation.
Overexpression of Bcl-1 was documented by immunohistochemistry
(anti-cyclin D1; Biotechnology, Santa Cruz, CA) or by Western blot
analysis in several cases.
Monoclonal antibodies and immunohistochemical staining
The monoclonal antibodies (mAb) used in this study were anti-p21
(WAF-1, 1:25; Calbiochem, La Jolla, CA); anti-p27 (KIP-1, 1:1000;
Transduction Laboratories, Lexington, KY), anti-p53 (DO-1, 1:200; Santa
Cruz Biotechnology, Santa Cruz, CA), anti-Ki-67 (MIB-1, 1:1000;
Immunotech, Marseilles, France), and anti-E2F-1 (E2F-1, 1:25; tissue
culture supernatant KH95, kindly provided by Dr Kristian Helin40,41).
All mAbs required antigen retrieval by microwave
pretreatment for 20 minutes in citrate buffer (10 mmol/L, pH 6). The
immunostaining was performed on formalin-fixed or B5-fixed,
paraffin-embedded tissue using the avidin-biotin-peroxidase complex
(ABC) method and semiautomated immunostainers (Optimax; BioGenex, San
Ramon, CA; Ventana-ES; Ventana, Tucson,
AZ).42,43 For E2F-1 staining, a modification of the ABC
technique was used to enhance the signal detection (Tyramide Signal
Amplification Products; New England Nuclear, Boston, MA). This
modification comprised two additional steps. Specifically, after
incubation with ABC complex, the slides were incubated with tyramide
(1:100, 8 minutes at room temperature [RT]) followed by 15 minutes of
a second ABC incubation step at RT. The DAB was subsequently applied (5 minutes at RT), and the slides were washed and counterstained with hematoxylin.
Purification of neoplastic B cells
Cryopreserved mononuclear cells (1 × 107/mL) from
selected patients with CLL/SLL or MCL, containing a high percentage of
neoplastic B cells (ratio of neoplastic to normal B cells, >50:1)
were incubated with anti-CD19 magnetic beads (80 µL; Immunotech) for
45 minutes on ice, harvested using a magnet, washed twice, and
harvested again. The percentage of positive B cells was determined by
flow cytometry using anti-CD3 and anti-CD20 mAbs (Becton-Dickinson, Mountain View, CA). Harvested populations always contained more than
95% B cells.
Western blot and immunoprecipitation analysis
Enriched B cells (1 × 106/sample) were lysed (20 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, 1% Triton X100, 5 mmol/L EDTA,
1 mmol/L Na3VO4 and 1 mmol/L PMSF)
and spun. Ten microgram of total protein cell lysates were
electrophoresed in SDS-PAGE gel and transferred to nitrocellulose
membranes. Filters first were blocked (5% low-fat milk in PBS with
0.1% Tween 20) and subsequently incubated with anti-p27 (1:250;
Transduction, Lexington, KY; 1-hour RT) or CDK2 (1:500)
antibody.44 After three washes, the filters were incubated with HRPO-conjugated goat antimouse antibody (1:2000; Amersham, Arlington Heights, IL; 1 hour at RT) or with HRPO-conjugated goat antirabbit antibody (1:2000; Armesham; 1 hour at RT). The detection of
immunocomplexes was performed with chemiluminescence (ECL; Amersham).
Purified recombinant histidine-tagged p27 was incubated (30°C, 1 hour) with HeLa extract obtained as described above. The reaction mix
contained 0.1 µmol/L histidine-tagged p27, 0.2 µg/µL HA-tagged
Ubiquitin, 20 mmol/L Tris-HCl (pH, 7.2), 2 mmol/L dithiothreitol 0.25 mmol/L EDTA, 0.2 mmol/L adenosine triphosphate, 10 mmol/L creatine
phosphate, 70 U/mL creatine phosphokinase, 100 µmol/L hemin, and 20 µg cellular extract. The reaction products were immunoprecipitated
with a rabbit anti-HA antibody (3 µg/mL; Santa Cruz) or with a rabbit
anti-ubiquitin (2 µL/mL)45 followed by immunoblotting
with the p27 mAb.
RNA extraction and cDNA preparation
Total mRNA was obtained from CD19 positive B cells
(1 × 106) using a total RNA isolation kit (Qiagen,
Valencia, CA) according to the manufacturer's instructions. cDNA was
obtained from total RNA (1-5 µg) after reverse transcription using
hexanucleotide random primers and Moloney murine leukemia virus reverse
transcriptase (Gibco-BRL, Gaithersburg, MD). Briefly, genomic DNA was
digested with DNAse (Boehringer-Mannheim, Indianapolis, IN) in the
presence of MgCl2 (1 mmol/L) for 10 minutes at room
temperature. Total RNA first was heated in the presence of oligoprimers
(50 ng for 10 minutes, at 70°C) and then quenched on ice (2 minutes).
The volume of the RNA/primer mixture was adjusted to 20 µL, giving the following final concentration: 0.5 mmol/L each dATP, dCTP, dGTP,
and dTTP; 10 mmol/L dithiothreitol; 50 mmol/L Tris-HCl, pH 8.3; 3 mmol/L MgCl2; 75 mmol/L KCl; and 200 U Moloney murine leukemia virus RNAse H- reverse transcriptase (Gibco-BRL). The reaction
mixture was incubated at 42°C for 2 hours and then the reverse
transcriptase was inactivated at 70°C for 10 minutes.
Polymerase chain reaction analysis
The efficiency and quality of each cDNA preparation was tested by
polymerase chain reaction (PCR) amplification using specific oligonucleotides recognizing human 2-microglobulin.46
The presence of p27 mRNA transcripts were investigated using specific
oligonucleotides recognizing p27 (p27-forward:
5'-ATGTCAAACGTGCGAGTGTCT, bp 1-21; p27-backward:
5'-TTACGTTTGACGTCTTCTGA, bp 577-597, NM_004064, giving a PCR product of
597 bp) and spanning the intron area within the first and second exons.
Two microliters cDNA was amplified under appropriate conditions (10 pmol/L each primer, 250 µmol/L dNTP, 10 mmol/L Tris-HCl, pH 8.8, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.01% gelatin, 0.5 U Taq
polymerase, in a final volume of 25 µL) for 10, 25, and 30 cycles
(denaturing 94°C, 30 seconds; annealing 57°C, 1 minute; extension
72°C, 1.5 minutes) using a Cetus Perkin-Elmer (Norwalk, CT)
thermocycler apparatus.
In vitro p27 protein degradation assay
Each frozen lymphoid tissue sample (10 different cases of MCL) was
sectioned and quickly disrupted by nitrogen decompression in 100 mol/L
lysing buffer (50 mmol/L Tris-HCl, pH 8.3, 5 mmol/L MgCl2,
and 1 mmol/L dithiothreitol). The lysates were spun at 15 000 rpm, and
supernatants were collected and frozen at 80°C. Histidine-tagged
p27 (100 ng) was incubated at 37°C for different intervals in 60 µL
degradation mix (containing 30 µg protein tissue homogenates, 50 mmol/L Tris-HCl, pH 8, 5 mmol/L MgCl2, 1 mmol/L dithrothreitol, 2 mmol/L adenosine triphosphate, 70 U/mL creatine phosphokinase, and 10 mmol/L creatine phospatase).47
Degradation of p27 was analyzed by immunoblotting with anti-p27 mAb.
The optical intensity of the protein bands was calculated using ID
Image Analysis Software (Kodak Digital Science; Eastman Kodak,
Rochester, NY) and scored (ratio of band intensity of p27 at time 0 hours/band intensity of p27 at time 6 hours: <1.25 = ;
1.25 to 2 = +; >2 = ++. The inhibition of proteasome
activity was achieved by incubating the degradation mixture with hemin
(100 µmol/L).48
Score and statistical analysis
Neoplasms were considered positive or strongly positive when nuclear
staining was detected in at least 15% or more than 50% of the tumors
cells, respectively. With the E2F-1 mAb, a discrete subpopulation of
neoplastic cells showed moderate to high-density cytoplasmic staining.
In these instances, the neoplasms were considered positive only when
nuclear staining also was observed in at least 15% of the tumor cells.
p27 nuclear positivity was scored by counting the percentage of
positive nuclei, after correction of the percentage of nonneoplastic CD3+ T cells on serial sections and by evaluating the intensity of
anti-p27 immunostaining (compared to internal control positive cells).
We also decided to score the relative nuclear intensity of p27 because
a partial loss of p27 protein expression may have a pathogenetic role
in tumorigenesis.49 In our semiquantitative scoring system,
neoplastic cells were scored as negative () when nuclear
immunostaining was negative or barely evident within the neoplastic
cells. Neoplastic cells clearly expressing nuclear p27, but at levels
intermediate between negative and normal cells, were scored ±, and
cells at levels comparable to those seen in normal lymphoid elements
were scored as +. Statistical significance was calculated using the
Fisher exact test and the Kaplan-Meier (Mantel-Cox) method.
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Results |
Cell cycle regulatory protein expression
Our goals were to study the expression in MCL of 4 key cell cycle
regulators (p21, p27, p53, and E2F-1), which primarily regulate the
G1 phase of the cell cycle, or S-phase entry, or both, to identify any aberrant expression pattern(s) and to compare the cell
cycle profile of MCL with that of other NHL. Because these proteins are
expressed in a well-defined stage of the cell cycle, we first
investigated the overall fraction of tumor cells committed to
proliferation (G1b) or proliferating (S and
G2-M). This was accomplished using the MIB-1 mAb, which
specifically recognizes a protein (Ki-67) expressed by all cells within
G1 and G2-M phases. As previously
described,17,18,50 most MCLs and high-grade lymphomas had a
large number of proliferating cells. However, the analysis of Ki-67
positive s-MCL versus l-MCL tumors demonstrated a significant
difference (P = .004) when strongly positive tumors (>50%
positive cells) were compared. In contrast, only a small subset (25%)
of low-grade lymphomas was Ki-67 positive, with positivity primarily
restricted to the proliferation centers (>80%) in CLL/SLL and
prolymphocytic lymphoma. When the percentage of positive cases among
low-grade lymphomas was compared with that of high-grade lymphomas, a
statistically significant difference was identified (P < .001).
Immunohistochemical analysis for p27 protein expression demonstrated
that most MCLs showed a total loss (54%) or a partial loss (29%) of
detectable p27. Only in a minority of cases (17%) did the neoplastic
cells display p27 levels similar to those seen in the nuclei of normal
surrounding CD3+ T cells (Table 1).
Expression of p53 was highest in l-MCL. Among 7 cases of 1-MCL with
aggressive cytology, 5 cases demonstrated p53 overexpression (1 blastoid, 2 transformed, and 2 pleomorphic cases; 71%) compared with
only 13 of 103 cases of s-MCL (12.6%; P = .02). No MCL
expressed detectable p21 nuclear staining. When comparisons were made
between p27 and Ki-67 expression or between p27 and p53 expression, no
significant correlation(s) could be demonstrated in MCL. However, when
the same analyses were performed including all lymphoma cases, p27 and
Ki-67 expression showed a significant reverse correlation (P = .003). Furthermore, E2F-1 was highly expressed in most
MCLs (38 of 50 cases; 76%).
When a similar analysis was performed in DLCL, these tumors overall
showed a profile similar to that of MCL, particularly when
l-MCL and DLCL were compared. More than 60% of cases of DLCL displayed
low or negative levels of p27 (12 of 19). Interestingly, neoplastic
cells of DLCL demonstrated a slight degree of intratumoral heterogeneity for p27 nuclear expression in contrast to MCL tumors cells, which showed a more homogeneous pattern of expression. Similarly
to l-MCL, a substantial subset (8 of 19) of DLCL overexpressed p53. In
only two cases were p21 and p53 concomitantly expressed. Finally, more
that 70% (14 of 19) of these neoplasms showed strong nuclear staining
for E2F-1 (Figures 1 and
2); only 2 cases of all DLCL failed to express any detectable E2F-1 protein
despite their high rates of proliferation.
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| Fig 1.
Expression of p21, p53, and E2F in 102 cases of B-cell
non-Hodgkin's lymphoma.
Neoplasms expressing the indicated antigens in >15% of the nuclei
were considered positive. Numbers of positive cases are indicated on
the top of each bar. *p27 and p53 analysis were performed in 103 s-MCL.
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| Fig 2.
Immunohistochemical characterization of cell cycle
regulators in non-Hodgkin's lymphoma.
(A) p27 expression analysis in l-MCL demonstrates that the neoplastic
cells are negative and that rare intratumor reactive cells show strong
nuclear reactivity. (B) The same case also shows overexpression of p53.
The expression of Ki-67 (C) in l-MCL and E2F-1 (s-MCL, Panel D) are
shown. (E) p27 is highly expressed in resting CLL/SLL cells. However,
E2F-1 is detected primarily in proliferating cells of CLL/SLL within
proliferation centers (inset; magnification ×400).
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CLL/SLL and MALT cases displayed similar profiles that differed from
the corresponding profiles of MCL and DLCL. All cases expressed very
high levels of p27 (Table 1), and strong nuclear p27 staining was
detected in >90% of the tumor cells on average. Paraimmunoblasts and
prolymphocytes within proliferation centers proved to be an exception;
they were only weakly positive or negative (Figure 1). Detectable
staining was never observed in more than 15% of the neoplastic cells
for p21 and p53 antigens. Among CLL/SLL cases, only one (classified as
a paraimmunoblastic variant) expressed E2F-1 in >50% of the cells.
When all neoplasms were compared, overall E2F-1 expression showed a
positive correlation with Ki-67 (P < .0001) and a negative
correlation with p27 (P = .0003). This was confirmed by the
reverse pattern of expression in normal and neoplastic cells within
normal germinal centers and proliferation centers of CLL/SLL, in which
Ki-67 expression was high and p27 expression was low.
p27 expression and protein degradation in mantle cell lymphoma
To study the mechanism leading to the decrease or loss of p27
expression in MCL, we studied the presence of p27 protein and mRNA
expression in highly enriched neoplastic B cells (>95% CD19+ B
cells) by Western blotting and RT-PCR, respectively. The relative amount of p27 protein was normalized with that of CDK2 protein expression. We used CDK2 because its levels are stable along the cell
cycle.44 By Western blot analysis, p27 protein levels in MCL cells were undetectable or considerably lower than p27 protein levels in cells comprising CLL/SLL (Figure
3A) and in normal T cells (data not shown).
These results confirmed our immunohistochemical findings and
demonstrated that the low levels of p27 detected by
immunohistochemistry were not caused by possible steric inhibition by
other cellular proteins (such as cyclin D1). In view of the fact that
p27 protein expression can be regulated at a transcriptional level, we
performed semiquantitative RT-PCR using purified CD19+ MCL cells. No
difference could be demonstrated among representative MCL and CLL
control cases (Figure 3B). Similar data were found when cDNA was
subjected to a subliminar amplification (15 and 20 cycles
only, data not shown).
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| Fig 3.
p27 protein and mRNA expression in CLL and MCL B cells.
Highly enriched neoplastic B cells from patients with CLL and MCL were
used to prepare total protein lysates and total RNA (see Material and
Methods). Total protein lysates were electrophoresed, blotted, and
incubated with anti-p27 (A top row) or with anti-CDK2 (A, bottom row).
cDNA transcribed from total RNA was amplified using oligonucleotides
recognizing p27 (B, top row) or 2-microglobulin (B, bottom row).
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To investigate whether the low levels of p27 in MCL were caused by
enhanced proteasome-mediated degradation, as in colon and lung
carcinomas,35,36 the degradation kinetics of recombinant p27 in the presence of protein lysates derived from 10 MCL fresh-frozen tissue samples were examined. The percentages of neoplastic B cells and
normal intratumor T cells first were calculated by immunohistochemistry on fresh-frozen tissue samples. Using this approach, we were able to
demonstrate that extracts from p27-negative MCL were able to degrade
recombinant p27 rapidly and efficiently (Table
2 and Figure
4A). Conversely, extracts from those
neoplasms with high p27 expression displayed considerably slower
degradation kinetics. Intermediate kinetics were observed primarily in
those cases containing a large number of p27-positive T cells. To study
whether the p27 degradation of MCL was caused by a proteasome-dependent
pathway, we analyzed the effect of hemin, a specific proteasome
inhibitor.48 Recombinant p27 rapidly disappeared over time
in the presence of hemin. Conversely, new polyubiquitinated p27
products were readily identified (Figure 4B). To confirm the presence
of polyubiquinated high-molecular-weight p27 products, recombinant
histidine-tagged p27 was incubated in the presence of HA-ubiquiting
over time and then immunoprecipated with anti-HA or
anti-ubiquitin mAbs. Ubiquitaned p27 products were successfully
identified using anti-p27 mAb (Figure 4C).
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| Fig 4.
Kinetics of p27 degradation in MCL.
Purified recombinant p27 was incubated for the indicated intervals with
the extracts from three representative MCL cases (a, high p27
expression; b, low expression of p27 by the tumor cells, but the
lymphoid tissue sample contained a relative large number of p27+
reactive T cells; c, low expression of p27 by the tumor cells, and the
lymphoid tissue sample contained a small number of p27+ reactive
T-cells) (A). To demonstrate that p27 degradation is mediated by
proteasome the MCL lysates were incubated with and without hemin (B).
p27 was polyubiquitinated before proteosome degradation (C). Purified
recombinant p27 was incubated for 1 hour with the cell extract (HeLa)
in the presence of HA-ubiquitin and subsequently was immunoprecipated
with an anti-HA or anti-ubiquitin mAbs. After transfer,
polyubiquitinated p27 forms were identified using anti-p27 mAb.
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Loss of p27 protein expression is a predictor for overall
survival
To analyze the prognostic significance of p27 expression on overall
survival, MCLs were stratified based on the intensity of p27 expression
(group 1, 44 patients; group 2, 35 patients). Clinical data were
obtained in 79 patients with MCL with a minimum follow-up of 4 months.
The mean follow-up was 39 months (SD, ±24 months; range, 106 months;
minimum, 4 months; maximum, 110 months). No significant differences
were demonstrated when the age, sex, LDH level, and stage (stage 3 vs.
stage 4; P = .5) of these two groups were compared. However,
the overall survival times from the date of diagnosis were
significantly shorter in patients with tumor cells expressing no
detectable p27 (median survival time, 44 months) than in patients with
tumors in which the neoplastic cells expressed on
intermediate or high levels of p27 expression (median survival time, 67 months; P = .002). We also compared the overall survival
times of patients with MCL overexpressing p53 (10 of 78). Despite the
relatively small number (10), p53+ patients showed a survival time
considerably shorter than p53 patients (P = .001),
regardless of p27 expression (P = .7). Finally, when
p27 expression and survival of p53 patients were correlated, loss of
p27 was found to be associated with a decreased overall survival time
(P = .002) in patients with p53-negative tumors (Figure
5B).
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| Fig 5.
Loss of p27 is associated with poor survival in mantle
cell lymphoma.
Kaplan-Meier curve for overall survival of p27+ (open squares) and
p27 (open circles) in MCL. Survival curves in p27+/p53 (open
squares), p27/p53 (open circles), p27/p53+ (full triangles),
and p27+/p53+ (open diamonds) in patients with MCL (panel B).
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Discussion |
In this study, we investigated the expression of multiple cell cycle
regulators in MCL and in a representative panel of B-cell NHL,
including low- and high-grade neoplasms. Overall, our findings demonstrated that MCL has an expression pattern similar to that observed in DLCL (high Ki-67, p53, E2F-1; low p27); decreased levels or
total loss of p27 protein expression are common features of MCL; low
protein levels of p27 in MCL do not correlate with proliferation rates
and are caused by ubiquitin- and proteasome-dependent degradation; and
loss of p27 levels correlates with a decreased rate of overall
survival. Taken together, these findings indicate that, in combination
with p53 overexpression, p27 expression may allow a more precise
characterization of patients with MCL.
To ascertain whether the expression of key cell cycle regulators in MCL
could allow a better stratification of these neoplasms, we investigated
the expression of p53, p21, p27, and E2F-1 in MCL and compared the
expression of these regulators with that seen in low- and high-grade
NHLs. In keeping with data published by other investigators, MCLs are
characterized by high proliferation rates as determined by the high
number of Ki-67- and E2F-1-positive tumors. We investigated the
expression of E2F-1 because it is a key regulator of cell
growth.29 Members of the E2F transcription factor family
(E2F-1-E2F-5) act as critical positive regulators of cell cycle
progression.29 Two groups independently have reported that
E2F-1 knockout mice show a high incidence of tumors, including large-cell lymphoma.51,52 Relatively little is known about E2F-1 expression and its correlation with other key cell cycle regulators in lymphoma.53,54 Here, a positive correlation
between the proliferation rate and E2F-1 expression was demonstrated in cases of MCL and DLCL. We observed a similar correlation when we
compared the expression of Ki-67 and E2F-1 of proliferation centers in
CLL/SLL (Figures 2E, 2F) and in normal residual germinal centers. In
contrast, none of the MALT-type lymphomas and only 2 of the
CLL/SLL cases showed a significant number of E2F-1-positive tumor
cells. Moreover, we identified rare cases of DLCL in which the neoplastic cells did not express detectable E2F-1 despite high
proliferation rates. Characterization of the E2F-1 locus in these
neoplasms is to demonstrate whether deletions or somatic mutations
ultimately are responsible for these findings. Alternatively, other
mechanisms acting upstream from E2F-1, or posttranscriptional mechanisms, may be operational. One may speculate that an aberration involving cellular protein degradation may alter E2F-1 levels because,
like p27, levels of E2F-1 also are regulated by ubiquitination and
proteasome degradation.
Overexpression of p53 is the most common defect identified in human
tumors. It often is seen within high-grade NHL, in which it is
associated with cellular transformation. We and
others18,23,25 have demonstrated that p53 is a prognostic
indicator in MCL and that its overexpression is more frequently seen in
MCL variants than in classical s-MCL18,23,24 (71% in l-MCL
vs. 11% in s-MCL; P = .002). We also have confirmed, despite
the relatively small number of cases, that p53 expression in l-MCL does
not appear significantly different from p53 protein expression in DLCL.
The considerably lower incidence of p53 overexpression in s-MCL
compared with l-MCL and DLCL tend to indicate that p53 somatic
mutations may occur during the transformation of MCL and therefore may
be associated with a more aggressive clinical course. The fact that only a relatively small subset of s-MCL carries p53 genetic aberrations suggests that factors other than p53 anomalies may be responsible for
their aggressive clinical course. Our data demonstrate that the level
of p27 protein expression may have a crucial role in the pathogenesis
and biologic behavior of these neoplasms. In fact, total or partial
loss of p27 appears to be a consistent feature of MCL and one that
differentiates MCL from low- and even high-grade lymphomas. Because p27
expression appears to correlate inversely with the cell proliferation
index, an observation supported by our findings in normal germinal
centers and in the proliferation centers of CLL/SLL, the fact that high
p27 expression is seen in low-grade NHL is not surprising if one
considers that in these neoplasms only a small number of cells is
committed to cell division. That a larger percentage of MCLs than DLCLs
loses p27 is intriguing because the fraction of proliferating cells in
MCL is approximately the same or even less than that seen in most
DLCLs. In addition, though classical and variant MCLs do not differ
significantly regarding the percentage of p27 positive cases
(P = .7), these two groups are considerably different
regarding their percentages of proliferating tumor cells (classical
MCL, MIB-1 >50 = 14.3%; variant MCL, MIB-1 >50 = 71.4%).
Thus, the deregulation of p27 in MCL appears not to be correlated with
the cell cycle proliferation.50 On the other hand, the
neoplastic cells of some DLCL appear to express different levels of
p27, indicating that in these cases p27 protein levels are still under
the physiological regulation of cell cycle progression.
The precise mechanisms regulating the amount of p27 in tumor cells have
been only partially elucidated. The overall consensus is that levels of
p27 in normal and neoplastic cells are regulated by multiple
mechanisms. These include increased degradation through the
ubiquitin-proteasome machinery,47 reduced mRNA
expression,55 decreased protein
translation,56,57 and the acquisition of rare somatic
mutations resulting in premature protein termination.58,59 In addition, the loss of the p27 locus has been demonstrated in some
cases of acute lymphoblastic leukemia with and without the concomitant
presence of p27 somatic aberration(s) on the remaining second
allele.58,60-62 Among all these possible mechanisms, an enhanced ubiquitin proteosome-mediated degradation is the most common
mechanism regulating the availability of p27 in normal cells.47 In the past few years, several investigators have
demonstrated that the deregulation of p27 expression is a relatively
common feature of many solid tumors.34-37,55 Total or
partial loss of p27 expression in tumor cells may have important
implications. This is supported by several studies on breast, colon,
and esophageal carcinomas demonstrating that loss of p27 correlates
with poor prognosis and high-grade neoplasms.34-37
Furthermore, p27 knockout mice63-65 and heterozygous mice
(p27+/ ), in which the loss of a single p27 allele is
associated with decreased levels of p27 protein expression, are more
prone to spontaneous and induced tumors.49 Thus, decreased
levels of p27 resulting from the loss of heterozygosity may be
sufficient to give a growth advantage to tumor cells. Toward this end,
recent studies in Rb//p27+/ mice have
shown that the loss of p27 allows the growth and escape of cells that
have acquired additional genetics defects (biallelic loss of Rb),
resulting in the generation of more aggressive neoplasms, higher
incidence of neoplastic transformation, or both.66 Thus, it
is possible that MCL lymphomas that have acquired p27 deregulation may
not only lose the inhibitory action of p27 on cyclin E/cdk2 complex,
they may be prone to accumulate additional mutations. The high
rate of p53 abnormalities18,23,25 and the loss of p16
function26,27 seen in aggressive forms of MCL tend to
support this hypothesis.
In this article, we have shown that levels of p27 mRNA are similar in
normal and neoplastic cells (MCL and CLL/SLL), but that in MCL cells,
there is a positive correlation between p27 protein levels and the
degree of p27 degradation in vitro. This enhanced protein degradation
is blocked by a proteasome-specific inhibitor and requires the
generation of ubiquitinated forms. Our data not only support the
concept that p27 regulation is achieved primarily through proteasome
degradation, they expand previous findings by demonstrating for the
first time that the loss of p27 in NHL can occur as a result of
enhanced protein degradation. Furthermore, we have demonstrated that,
as in solid human neoplams, the loss of p27 in MCL is associated with a
more aggressive biologic phenotype and decreased overall survival.
These findings are particularly important given the relatively few
clinically prognostic indicators in MCL. Among the histopathologic
criteria, a nodular pattern is associated with a better outcome than is
a diffuse pattern; a worse prognosis also is associated with a high
proliferation index and with some cytologic MCL
variants.12,18,22 More recently, the overexpression of p53
has been shown to be a marker of poor prognosis.23,25
However, p53 anomalies occur only in a small subset of MCL and
therefore are of limited value. In contrast, the loss of p27 involves
most cases of MCL,50 and therefore it appears to be a more
useful prognostic marker. Our findings clearly demonstrated that the
analysis of p27 expression allows a more reliable and comprehensive
stratification of patients with MCL and possibly of patients with other
forms of NHL into prognostically significant subgroups suitable for ad
hoc chemotherapeutic approaches. We can envision alternative
therapeutic protocols designed to modulate pharmacologically the
degradation of p27, thereby providing new avenues in the management
of MCL.
| |
Acknowledgments |
The authors thank Elena Mazzone for her excellent technical assistance.
They also thank Kristian Helin for supplying the anti-E2F-1 antibody
and J. Tiesinga for helpful discussion and critical review of the manuscript.
| |
Footnotes |
Received June 15, 1999; accepted September 3, 1999.
Supported in part by the Dutch Cancer and the Johan Vermeij Foundations
and by National Institutes of Health grants CA66229, CA14462, CA76584,
CA76584, and GM57587; by HFSPO grant RG0229/98-M; and by
the Associazione Italiana Ricerca sul Cancro, Milan, Italy.
R.C. and L.M.B. contributed equally to this work.
Reprint: Giorgio Inghirami, New York University, Department of
Pathology and Kaplan Comprehensive Cancer Center, 550 First Avenue, New
York, NY 10016; e-mail:
inghig01{at}mcrcr6.med.nyu.edu.
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.
| |
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Top
Abstract
Introduction
