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doi:10.1182/blood-2008-02-139139
Prepublished online September 2, 2008;
Tibaldi, Cristina Gattazzo, Renato Zambello, Gianpietro Semenzato and Anna M. Brunati
Livio Trentin, Martina Frasson, Arianna Donella-Deana, Federica Frezzato, Mario A. Pagano, Elena
lymphocytic leukemia
cytosolic complex is an early event in apoptotic mechanisms in B-chronic
Geldanamycin-induced Lyn dissociation from aberrant HSP90-stabilized
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GELDANAMYCIN-INDUCED LYN DISSOCIATION FROM ABERRANT HSP90-
STABILIZED CYTOSOLIC COMPLEX IS AN EARLY EVENT IN APOPTOTIC
MECHANISMS IN B-CHRONIC LYMPHOCYTIC LEUKEMIA
Livio Trentin,1,2 * Martina Frasson, 3 * Arianna Donella-Deana,3 Federica Frezzato,1,2 Mario A.
Pagano, 3 Elena Tibaldi, 3 Cristina Gattazzo,1,2 Renato Zambello,1,2 Gianpietro Semenzato,1,2 and
Anna M. Brunati3
1Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology
Branch, Padua University School of Medicine, Padua, Italy.
2Venetian Institute for Molecular Medicine (VIMM), Centro di Eccellenza per la Ricerca
Biomedica.
3Department of Biological Chemistry, University of Padua, Padua, Italy.
*These two authors contributed equally to the work
Running Title: Lyn Hsp90-stabilized cytosolic complex in B-CLL
Correspondence author:
Gianpietro Semenzato, Department of Hematology and Clinical and Experimental Medicine,
University of Padova, Via Giustiniani 2, 35128 Padova, Italy.
Phone: 011-39-049-821-2298
Fax: 011-39-049-821-1970
E-mail: g.semenzato@unipd.it
Blood First Edition Paper, prepublished online September 2, 2008; DOI 10.1182/blood-2008-02-139139
Copyright © 2008 American Society of Hematology
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Abstract
Lyn, a tyrosine kinase belonging to the Src family, plays a key role as a switch molecule that
couples the B cell receptor to downstream signalling. In B-CLL cells, Lyn is over-expressed,
anomalously present in the cytosol and displays a high constitutive activity, as compared to normal
B lymphocytes. The aim of this work was to gain insights into the molecular mechanisms
underlying these aberrant properties of Lyn, which have already been demonstrated to be related to
defective apoptosis in B-CLL cells. Herein, Lyn is described to be in an active conformation as
integral component of an aberrant cytosolic 600 kDa multiprotein complex in B-CLL cells,
associated with several proteins, such as Hsp90 through its catalytic domain, and HS1 and SHP-1L
through its SH3 domain. In particular, Hsp90 appears tightly bound to cytosolic Lyn (CL), thus
stabilizing the aberrant complex and converting individual transient interactions into stable ones.
We also demonstrate that treatment of B-CLL cells with geldanamycin, an Hsp90 inhibitor already
reported to induce cell death, is capable of dissociating the CL complex in the early phases of
apoptosis and thus inactivating CL itself. These data identify the CL complex as a potential target
for therapy in B-CLL.
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Introduction
B cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world
and is characterized by the accumulation of relatively mature B cells with aberrant co-expression of
CD5+ in blood, bone marrow and peripheral lymphoid organs (1). The clinical course of B-CLL is
heterogeneous, varying from stable, long-lasting indolent form to rapidly progressive disease and
death. Several biological parameters have been proposed to account for this clinical heterogeneity,
including the mutational status of immunoglobulin variable region genes (IgVH), expression of 70-
kDa zeta-associated protein (ZAP-70) and CD38+ antigen, as well as specific cytogenetic alterations
(2,3). On the basis of the mutational status of IgVH, B-CLL patients can be classified into two major
groups with different outcomes: one with mutated IgVH genes (M-CLL) and a relatively stable
disease course, and one with unmutated IgVH configuration (U-CLL) and a more aggressive clinical
behaviour. The latter correlates with the up-regulation of ZAP–70 (4,5). Despite the extensive
molecular characterization of B-CLL cells, little is known on the molecular mechanisms involved in
its neoplastic transformation and proliferation. In the past, it was assumed that CLL is caused by the
accumulation of slowly proliferating cells with defective apoptosis, but more recently B-CLL has
been described as a dynamic disorder, in which the accumulation of cells is consequent to increased
cell replication associated with decreased cell death, the high replication rate resulting in a rapidly
progressive disease (6,7).
One approach toward the discovery of new therapeutic targets is to explore the nature of the
intracellular pathways responsible for modulating the proliferation and/or apoptotic rate of B-CLL
cells. In this regard, B Cell Receptor (BCR) engagement is known to play an important role by
triggering a signaling cascade mediated by Lyn, a tyrosine kinase belonging to the Src family
(SFK), which plays a key role in many downstream pathways (8-10). The activity of SFKs is
mainly modulated by the phosphorylation of two critical tyrosine residues in the activation loop
(Tyr396 of Lyn) and the C-terminus (Tyr508 of Lyn) (11-13). Phosphorylation of C-terminal
tyrosine induces an inactive closed conformation of the protein kinase through two major
intramolecular inhibitory interactions: binding of phosphorylated C-terminal tyrosine to the SH2
domain, and interaction of a polyproline type II helical motif (PPII) in the SH2-kinase linker with
the SH3 domain (14-16). The activation of SFKs involves disruption of these inhibitory interactions
through multiple mechanisms, such as dephosphorylation of the tail, displacement of the tail from
the SH2 domain, displacement of the PPII motif from the SH3 domain, mutations in the SH3-SH2
connector, and/or mutations in the SH2-kinase linker (17-23). It is known that, following activation,
SFK level is regulated by the balance of two opposing mechanisms, degradation by ubiquitinylation
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or rescue by association with Hsp90, a chaperone interacting with the N-terminal lobe of the SFK
catalytic domain (24-27).
We have recently demonstrated in B-CLL cells that Lyn is remarkably overexpressed and
anomalously localized in the cytosol, displaying remarkable constitutive activity that leads to
increased basal tyrosine protein phosphorylation and poor responsiveness to BCR ligation.
Restoration of apoptosis by treatment of leukemia cells with specific Lyn inhibitors also points out
the importance of high basal Lyn activity in altering the balance between cell survival and apoptosis
signals in B-CLL cells (28). The aim of this work was to gain deeper insight into the molecular
mechanisms which give rise to aberrant properties of Lyn in this disease. We herein demonstrate
that Lyn is an integral component of an aberrant cytosolic 600 kDa complex, where Lyn is
associated both with Hsp90 through its catalytic domain and with HS1 and SHP-1L through its SH3
domain. Moreover, Hsp90 stabilizes the complex by contributing to converting a network of
transient interactions into permanent ones, thus maintaining Lyn in an active conformation and
preventingits degradation. Geldanamycin (GA), an apoptotic compound which directly binds and
inhibits Hsp90, causes disruption of the aberrant cytosolic complex and in turn inactivation of Lyn
in the early phases of apoptosis in B-CLL cells.
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Materials and Methods
Materials
Geldanamycin (GA), 17-AAG [17-(allylamino)-17-demethoxy-geldanamycin), lactacystin, polymer
polyGlu4Tyr, λPPase, and phosphatase inhibitor cocktail 1 and 2 were from Sigma-Aldrich (St.
Louis, MO). [
γ
32P]ATP was from Amersham Bioscience (NJ, USA).
Anti-IgM–FITC and anti-IgD–FITC antibodies were from DakoCytomation (Glostrup, Denmark).
Anti-phospho-Lyn (Tyr-507) (pYT), anti-phospho-SFK (Tyr-416) (pYA) and anti-PARP polyclonal
antibodies from Cell Signaling Technology (Danvers, MA). Anti-Lyn, anti-Akt, anti-Cbl, anti-SHP-
2, anti-SHP-1, a polyclonal antibody raised against the C-terminal tail of SHP-1 and Hsp90
α
/
β
were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HS1 polyclonal antibody was
obtained as elsewhere reported (42). Monoclonal antibodies CD3–FITC, CD19–FITC, CD5–PE,
CD16–PE, CD23–PE, CD38–PE and CD79b–PE, the monoclonal anti-SHP-1/1L, were from BD
Biosciences (San Diego, CA). The monoclonal anti-phosphotyrosine (clone PY-20) was from MP
Biomedicals (Aurora, OH). Anti-STAT3 antibody and monoclonal anti-ZAP-70 antibody were from
Upstate Biotechnology (Lake Placid, NY). Anti-
β
-actin antibody (clone AC-15) was from Sigma-
Aldrich.
Patients, cell separation and culture conditions
After obtaining their informed consent, 40 untreated patients, according to the criteria for diagnosis
of B-CLL (29), were enrolled in the study. The study was approved by the scientific board from the
Department of Clinical and Experimental Medicine, Padua University. Informed consent was
provided in accordance with the Declaration of Helsinki.
Patient characteristics are listed in Table 1. Peripheral blood mononuclear cells (PBMC) were
isolated from the blood of B-CLL patients by density gradient centrifugation through a Ficoll-
Hypaque cushion, as previously reported (30). B cells were isolated as already described (31). As
assessed by flow cytometry, the content of CD19+ B cells was higher than 95% in all samples.
Untouched peripheral blood B cells were isolated from the PBMC of 5 healthy donors by negative
selection with a B-cell isolation kit and MACS separation columns (Milteny Biotec, Bergisch
Gladbach, Germany). The purity of isolated peripheral blood B cells was at least 95% (CD19+), as
assessed by flow cytometry.
Purified B-CLL cells were either used immediately or cultured (2 x 106 cells/ml) in RPMI-1640
medium (Sigma- Aldrich) supplemented with 10% heated inactivated fetal calf serum, 2 mM L-
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glutamine, 100 U/ml penicillin and 100
μ
g/ml streptomycin, at 37° C in a humidified atmosphere
containing 5% CO2.
Flow cytometry analysis
The following monoclonal antibodies were used for direct immunofluorescence staining: CD3–
FITC, CD19–FITC, anti-IgM–FITC, anti-IgD–FITC, CD5–PE, CD16–PE, CD23–PE, CD38–PE,
and CD79b–PE. Cells were scored on a FACScalibur analyzer (BD Biosciences, Immunocytometry
Systems) and data were processed by CellQuest software (BD).
Recombinant proteins
The recombinant GST-Lyn/SH3 domain was expressed and purified according to the protocol
described in (32). The GST-Lyn/SH2 domain was expressed and purified as previously reported
(33).
Recombinant HS1
Δ
N-Term (
Δ
1-207), HS1
Δ
SH3 (
Δ
403-486) and HS1-
Δ
Pro-rich (
Δ
324-393) were
expressed and purified as previously described (34).
Cell lysis and subcellular fractionation
For total lysates, normal and B-CLL cells (5 x 105 for each assay) were rapidly lysed in 62mM
Tris/HCL buffer, pH6.8, 5% glycerol and 0.5%
β
-mercaptoethanol containing 0.5% SDS.
For subcellular fractionation, B-CLL cells (15 x 106 for each assay) were disrupted on ice by
sonication (3 cycles of 5 seconds at 22 Hz intervalled by 15 seconds) or alternatively in a Dounce
homogenizer (20 strokes) in 350
μ
l of isotonic buffer (50 mM Tris/HCl, pH 7.5, 0.25 M saccharose,
1 mM sodium orthovanadate, and protease inhibitor cocktail). Homogenates were centrifuged 10
min at 10,000g, and the supernatant was further centrifuged 1 hour at 105,000g to separate cytosol
from microsomes. Protein concentration was determined by the Bradford method.
Treatment of cytosol with
λ
PPase
300 μg of the cytosolic protein were obtained from B-CLL cells as described above, with isotonic
buffer without sodium orthovanadate. The sample was supplemented with 20 mM MnCl2 and
λPPase buffer. The mixture was divided into two equal aliquots and 300 units of λPPase were added
to one of them, The samples were incubated 45 min at 30°C and blocked by phosphatase inhibitor
cocktails 1 and 2 and 1mM sodium orthovanadate.
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Fractionation by centrifugation on glycerol gradient
150
μ
g of cytosolic protein from B-CLL cells were loaded on a 3.9-ml glycerol (10-40%) linear
gradient in 25 mM Hepes, pH 7.4, 1 mM EDTA. The tubes were centrifuged 18 h at 100,000g in a
SW60Ti rotor (Beckman) at 4°C, and fractionated from the top into 18 fractions.
Western blotting
Samples, from different cell fractions or immunoprecipitates, were run in 10% SDS-PAGE and
transferred to nitrocellulose membranes. After treatment with 3% BSA at 4° C overnight,
membranes were incubated with the appropriate antibodies for 2 h and treated as reported in (28).
Membranes, when required, were reprobed with other primary antibodies after stripping with 0.1 M
glycine (pH 2.5), 0.5 M NaCl, 0.1% Tween 20, 1%
β
-mercaptoethanol and 0.1% NaN3 for 2 x 10
min.
In vitro tyrosine kinase assays
Lyn activity from various samples was assayed on 200 µM cdc2(6-20) peptide or 1mg/ml
polyGlu4Tyr as described in (28).
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Results
The remarkable basal activity of Lyn is related to its aberrant phosphorylation state in B-CLL
cells.
To understand the molecular mechanisms by which Lyn becomes constitutively active in resting B-
CLL cells, cell lysates as well as microsomes and cytosol from untreated B-CLL patients in
different clinical stages (Table 1), compared with B lymphocytes from healthy donors, were tested
for the autophosphorylation site common to all SFKs (Tyr-396 of Lyn, and referred to as pYA) and
for phospho-Lyn site recognizing the C-terminal regulatory tyrosine of Lyn (Tyr-508 of Lyn, and
referred to as pYT), diagrammed as shown in Figure 1A, by Western blot analysis. The relative
purity of the subcellular compartments was assessed by using antibodies against specific markers
(Figure 1B). Figure 1C shows the representative results obtained on the sample from one normal
donor (left panel) in comparison with that from patient #15 (right panel), whereas Figure 1D
displays the densitometric analysis of the western blot bands corresponding to Lyn on B-cells from
5 normal donors (left panel) compared to the data obtained in B-CLL cells from all the 40 B-CLL
patients (right panel). As expected in normal B cells, Lyn appeared exclusively in the microsomal
fraction and immunoreacted only with anti-pYT antibody, demonstrating that the enzyme was in the
inactive conformation (Figure 1C and D). As previously observed (28), B-CLL samples showed
overexpression of Lyn compared with control cells (Figure 1C and D, compare lane 1 with lane 4)
and a remarkable fraction of the kinase (more than 30% of total protein) abnormally localized in the
cytosol (Figure 1C and D, compare lanes 2 and 3 with lanes 5 and 6, respectively). Immunostaining
of the sub-cellular fractions revealed that the anti-pYT antibody reacted only with microsomal Lyn,
whereas anti-pYA reacted with both microsomal and cytosolic Lyn, indicating that cytosolic Lyn
was in its fully active conformation in unstimulated leukemia cells.
Cytosolic Lyn (CL) participates in a complex stabilized by interactions mediated by its SH3 and
catalytic domains
Because Lyn cumulates in an active conformation in the cytosolic fraction of B-CLL cells, we
examined the role of its interactions with potential partners, which might lead to an aberrant state of
Lyn.
Irrespective of the method of cell disruption, either by sonication (Figure 2A, S) or by douncing
(Figure 2A, D), Lyn was detected by western blot analysis after fractionation of the cytosol of
leukemia cells from B-CLL patients on a glycerol gradient in the correspondence of a molecular
weight of around 600 kDa, suggesting its taking part in a multiprotein complex.
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To assess whether phosphorylation of the components, including Lyn, of the cytosolic Lyn (CL)
complex played a role in its stabilization, the cytosol was fractionated, under the conditions already
described above, on a glycerol gradient as such or after treatment with the broad-specificity lambda
protein phosphatase (λ-PPase). Figure 2B shows that the molecular weight of the CL complex was
not modified by λ-PPase treatment, demonstrating that neither Lyn pYA nor phosphorylation of
other protein partners had a role in the maintenance of the CL complex.
Furthermore, the distribution profile of CL activity of the gradient fractions tested in vitro on the
Src-specific peptide cdc2(6-20) showed that, after
λ
-PPase treatment, the dephosphorylated form of
Lyn displayed lower kinase activity, this finding being directly related to the tyrosine
dephosphorylation in the activation loop of the kinase (Figure 2B, lower panel compared with upper
panel). In parallel experiments, PP2, a selective SFK inhibitor (35), abolished the kinase activity
assayed on the non-specific substrate poly(Glu4Tyr) random polymer, indicating that the tyrosine
kinase activity of the cytosolic complex was exclusively catalyzed by Lyn (data not shown).
To highlight the nature of the interactions in the CL complex, the fractions 13 and 14 of the glycerol
gradient containing Lyn (Figure 2A) were collected, differently treated, and fractionated on a
further glycerol gradient. The results are shown in Figure 2C. This procedure did not affect protein
interactions, as demonstrated by sedimentation of the CL complex at the expected molecular mass
and the kinase activity fully coincided with the CL complex. The addition of SDS to a final
concentration of 0.05% to the CL complex under non-reducing conditions showed that disulfide
bridges did not mediate Lyn binding to the interacting proteins, as proven by the detergent-induced
dissociation of Lyn and its appearance as uncomplexed and inactive protein in fractions 1-2.
To analyze the interactions of Lyn with hypothetical binding proteins, we performed competition
assays by using compounds capable of disrupting their binding to SH3, SH2 and catalytic domains
of Lyn itself, thus altering the structure of the CL complex. Western blotting with anti-Lyn antibody
showed that addition of the GST-Lyn/SH3 domain induced complex dissociation and the
appearance of CL in the fractions 6-10, suggesting that the enzyme interacted with other proteins by
means of its SH3 domain. Conversely, the addition of the GST-Lyn/SH2 domain, which is capable
of binding phosphotyrosine-containing motifs, did not induce an apparent complex dissociation.
The CL complex was also treated with geldanamycin (GA), a compound that directly binds to and
inhibits Hsp90, thereby causing proteolytic degradation of its client proteins (36, 37). This treatment
promoted the dissociation of Lyn from the 600-kDa complex, as revealed by the immunodetection
of Lyn in the gradient fractions at lower molecular weight, suggesting that CL may associate with
Hsp90. As for the activity of Lyn, it was unaltered when the GST-Lyn/SH2 domain was used in the
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competition assay, whereas strongly reduced and totally abolished after treatment with the GST-
Lyn/SH3 domain and GA, respectively.
Lyn SH3 domain promotes the interaction of CL with HS1 and SHP-1
To identify the protein partners binding to Lyn through the Lyn SH3 domain, we analyzed the CL
complex for the presence of already known proteins interacting with the SH3 domain of SFKs,
namely Cbl, Akt, SHP-1L, SHP-2, HS1 and STAT3 (38-43). The CL complex purified from the
cytosol of leukemia cells obtained from each of 40 patients was thus subjected to a further glycerol
gradient, and aliquots of the resulting fractions were probed with antibodies against Lyn and the
above mentioned potential protein ligands, demonstrating that only SHP-1L, HS1 and STAT3 co-
sedimented with Lyn (Figure 3A).
SHP-1L, the C-terminal alternatively spliced form of SHP-1 with a unique C-terminal tail
containing the proline-rich motif PVPGPPVLSP, was identified with an antibody recognizing the
protein-tyrosine phosphatase domain shared by SHP-1L and SHP-1 (40-41). The presence of the
latter was ruled out, because the specific antibody raised against its C-terminal tail showed no
immunoreactivity (Figure 3A). SHP-1L physically interacted with Lyn in the CL complex, as
demonstrated by immunoprecipitating SHP-1L or Lyn from the glycerol fractions containing the CL
complex and analyzing the immunocomplex with anti-Lyn or anti-SHP1/1L antibody, respectively.
The addition of either the synthetic Pro-rich peptide (KGGRSRLPLPPLPPPG) known to interact
with Lyn SH3 domain (44) or the GST-Lyn/SH3 domain, during anti-Lyn or anti-SHP-1/1L
immunoprecipitation, completely abolished the binding between Lyn and SHP-1L (Figure 3B).
The interaction between HS1 and Lyn in the CL complex was demonstrated by similar
immunoprecipitation assays (Figure 3C). Since HS1 contains a variety of structurally significant
motifs, including a proline-rich region (aminoacids 324-393) and an SH3 domain located at the C-
terminus (32), we performed competition assays by adding recombinant truncated forms of HS1
during anti-Lyn or anti-HS1 immunoprecipitation, to identify the HS1 region interacting with Lyn.
As shown in Figure 3C, while HS1ΔSH3 completely abolished the interaction between Lyn and
HS1, the addition of the HS1 mutant lacking the Pro-rich sequence proved to be ineffective,
indicating that the Lyn SH3 domain interacted with the HS1 Pro-rich sequence. This finding was
further confirmed by adding the Pro-rich peptide, which abolished Lyn-HS1 co-
immunoprecipitation (Figure 3C) performed on the fractions containing the CL complex, under the
same experimental conditions.
On the other hand, no interaction between STAT3 and Lyn was evidenced, as the former was not
revealed after immunoprecipitation with anti-Lyn antibody and the latter was not detected after
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immunoprecipitation with anti-STAT3 antibody, showing that the two proteins are not part of the
same complex (data not shown).
Lyn catalytic domain interacts with the chaperone Hsp90 in the CL complex
To explore the role played by the catalytic domain of Lyn in the CL complex, and on the basis of
data obtained with GA, we verified whether Hsp90 takes part in the cytosolic complex (Figure 2C).
The CL complex purified from the cytosol of leukemia cells obtained from each of 40 patients was
fractionated on a second glycerol gradient, and aliquots of the resulting fractions were revealed with
antibodies to Lyn and Hsp90. Hsp90 and Lyn sedimented in the same glycerol gradient fractions
(Figure 4A), and the interaction between the two proteins was demonstrated by probing Hsp90- or
Lyn-immunoprecipitates with anti-Lyn and anti-Hsp90 antibodies, respectively (Figure 4B).
Treatment of CL complex with GA or its derivative 17-allylamino-17-demethoxygeldanamycin (17-
AAG) induced the expected dissociation of Lyn from Hsp90 (Figure 4B), whereas addition of Pro-
rich peptide or GST- Lyn/SH3 domain did not disrupt Lyn-Hsp90 interaction. Experiments aimed at
investiganting the potential binding of Hsp90 with the membrane-anchored Lyn in B-CLL cells
showed that the chaperone was not bound to the microsomal tyrosine kinase (data not shown).
Synergistic effect of Lyn domains in maintaining the CL complex
To better define the mechanisms regulating the stabilization of the CL complex, the cytosol of
leukemia cells obtained from 16 B-CLL patients, 8 U-CLL and 8 M-CLL, was incubated with
compounds capable of destabilizing interactions mediated by either the Lyn SH3 domain (GST-
Lyn/SH3 domain and the Pro-rich peptide) or the catalytic domain (GA), in the presence or absence
of lactacystin, a specific proteasome inhibitor (45). After treatment with each compound, both Lyn
activity and protein level of CL were tested.
In all the samples analyzed, the addition of GST-Lyn/SH3 domain and GA clearly led to
degradation of CL (Figure 5A, lanes 3 and 5). In the presence of lactacystin, although Lyn activity
was markedly reduced, its protein level was not altered; conversely, treatment with the Pro-rich
peptide enhanced the kinase activity of Lyn and protected it from degradation. It was the inhibitory
effect of lactacystin indeed to enable us to examine the interactions between CL and its protein
ligands on the CL immunoprecipitated from B-CLL cytosol after treatment with GST-Lyn/SH3
domain or GA. GA abolished the interaction between Hsp90 and the CL complex, leaving the
association between Lyn with HS1 and SHP-1L nearly intact (Figure 5B). In parallel, treatment
with the GST-Lyn/SH3 domain disrupted the interaction of HS1 and SHP-1L with Lyn and led to
partial dissociation of Hsp90 from the CL complex.
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These results demonstrate that not only Hsp90 contributes to preventing Lyn degradation, but also
that interactions between Lyn SH3 domain and its protein ligands play a role in the stabilization and
preservation of the CL complex, maintaining the Lyn active conformation, regardless of the protein
partners containing the SH3-cognate Pro-rich sequence, such as HS1 or SHP-1L.
CL complex becomes destabilized in parallel with GA-induced apoptosis
Since inhibition of Lyn activity, obtained by treating B-CLL cells with specific inhibitors of
tyrosine kinases is sufficient to restore cell apoptosis (28), we investigated the fate of the CL
complex in GA-induced apoptosis (46).
Freshly isolated leukemic B-cells obtained from 16 B-CLL patients, 8 U-CLL and 8 M-CLL, were
incubated in the presence of GA at different times. The cleavage of poly-ADP-ribose polymerase
(PARP-1), which is indicative of apoptosis, was observed after 12 h of GA treatment, in both U-
CLL (Figure 6A left panels) and M-CLL (Figure 6A, right panels) samples. On the other hand, CL
specific activity started to be decreased 2 h after GA treatment, reaching 73% and 84% of
inhibition in U-CLL and M-CLL, respectively (Figure 6C), whereas microsomal Lyn specific
activity decreased only after 24 h treatment (Figure 6D). It is noteworthy that the aberrant high
basal protein Tyr-phosphorylation in B-CLL lysates (Figure 6B) was found to be decreased in
parallel with the inhibition of CL activity induced by GA treatment. These data led us to infer that
the increased basal tyrosine phosphorylation could be accounted for by the stabilization resulting
from synergistic interactions between Lyn and other partners.
To confirm this hypothesis, freshly isolated B-CLL cells from 8 patients, 4 U-CLL and 4 M-CLL,
were treated with GA at different times, and the cytosol was fractionated on a glycerol gradient. The
resulting fractions were tested for Lyn activity and protein level. This analysis showed that Lyn
activity, detected only in the correspondence of the 600-kDa complex, decreased over time in
parallel with GA treatment (Figure 7A). This treatment changed the distribution profile of CL over
a wider range of molecular weights in the glycerol gradient, as revealed by Western blotting,
suggesting that CL activity is directly correlated with the stability of the complex (Figure 7A). CL
immunoprecipitates from the cytosol of the same samples also displayed decreasing amounts of
HS1, SHP-1L and Hsp90 after GA treament, supporting the hypothesized involvement of the
interaction domains of CL in the stabilization of the CL complex (Figure 7B).
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Discussion
In this report, we demonstrate that the Src kinase Lyn, which is abnormally present in the cytosol of
B-CLL cells, is an integral component of an aberrant cytosolic complex of 600 kDa (Figure 2). In
this complex, cytosolic Lyn (CL), which represents 30% of total Lyn, is present in an active
conformation and is associated with Hsp90, HS1 and SHP-1L (Figures 3 and 4). These proteins are
likely to account for some of the anomalous properties of Lyn in B-CLL cells in comparison to
normal B cells, such as overexpression with altered turnover, atypical localization in the cytosol and
remarkable constitutive activity (28). Geldanamycin (GA), a compound known to bind and inhibit
Hsp90, is shown to cause the disruption of the aberrant cytosolic complex and consequently the
inactivation of Lyn in the early phases of apoptosis.
Hsp90 targets client protein-kinases, including SFKs (47-48), stabilizing the kinase active
conformation and counterbalancing the opposing mechanism of degradation by ubiquitination.
Although the association of SFKs with Hsp90 is difficult to detect, likely because of low-affinity
binding and repeated cycles of association and release (49-51), in B-CLL cells CL appears tightly
bound to Hsp90, suggesting an abnormal behaviour of both proteins or other types of interaction
that synergistically contribute to stabilize of the complex. Hsp90 has been recently reported to
exhibit an activated conformation in tumor cells which, in contrast with normal tissues, results in
higher affinity to its inhibitors (52-53). This increased affinity appears to be due to co-chaperone-
induced changes in the ATP binding site of Hsp90, which lead to high ATPase activity, whereas
Hsp90 from normal tissues is in a latent, apparently unactivated state. Notably, activated Hsp90 is
described to bind to and stabilize ZAP-70, which acts as a Hsp90 client protein only in B-CLL cells
(54). Because we detected neither Hsp70 nor cdc37, essential co-chaperones of two known
multichaperone Hsp90 complexes, in association with Hsp90 in the CL complex purified on a
glycerol gradient (data not shown), Hsp90 does not appear to be in a traditionally known activation
state, suggesting that Hsp90 might be in an altered condition accounting for its tight binding to Lyn.
The data presented in this work actually support the view that the high affinity between Hsp90 and
Lyn may be due to the interaction of Lyn with specific ligands through its non-catalytic domains.
It is well established that ligands binding to the SH3 domain of SFKs can efficiently activate them
by directly disrupting the intramolecular inhibitory interaction between polyproline linker and the
kinase SH3 domain (11). In this regard, we demonstrate that the interaction between the SH3
domain of Lyn and the Pro-rich regions of discrete proteins, including HS1 and SHP-1L,
contributes to stabilize the complex and protect Lyn from degradation (Figures 3 and 5). In
particular, HS1 is a 79-kDa intracellular protein expressed in cells of lymphohematopoietic origin
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with a pivotal role in the signaling cascade triggered by BCR stimulation, upon which Lyn
phosphorylates HS1 by "sequential" mechanism (55); SHP-1L is a 70 kDa cytoplasmic spliced
variant of the protein-tyrosine phosphatase SHP-1, predominantly expressed in human
hematopoietic tissues and involved in the regulation of hematopoietic signal transduction (40,41).
HS1 and SHP-1L may be replaced by the Pro-rich peptide KGGRSRLPLPPLPPPG which contains
the optimal motif for binding to the SH3 domain of Lyn without altering the stability of the CL
complex (Figure 5B). Furthermore, interaction between the SH3 domain of Lyn and the Pro-rich
peptide stimulates the kinase activity of Lyn leaving the protein level of Lyn unchanged in the
cytosol (Figure 5A). We also show that treatment with GA destabilizes the CL complex by
disrupting the interaction between Lyn and Hsp90 and by weakening the interactions mediated by
the SH3 domain, as observed in the cytosolic fraction of B-CLL cells (Figure 5). However, our
results indicate that, in B-CLL cells, Lyn is maintained in an active conformation and preserved
from degradation due to the interaction not only with Hsp90 but also with several ligands of the Lyn
SH3 domain in a synergistic manner with Hsp90, regardless of the partners containing the Pro-rich
sequence, such as HS1 and SHP-1L. In this view, we can hypothesize that the CL complex, which
is detected in all B-CLL patients, may display a wide variability of the proteins binding to Lyn SH3,
without ruling out the possibility the composition of the CL complex result from the expression
level of the interacting proteins.
As emphasized by studies on regulatory mechanisms of SFKs, interaction of the SH3 domain with
proteins containing Pro-rich sequences may be modulated by ligands targeting SFKs themselves,
e.g. C-terminal Src kinase-homologous (CHK), which has been shown to destabilize this binding by
a non-catalytic inhibitory mechanism (12,13,56). Conversely, we demonstrate that Hsp90 stabilizes
the complex in which the SH3 domain is engaged and maintains the kinase in an active
conformation. Hence, interaction of Hsp90 and SH3 ligands with their respective binding domains
synergistically converts individual transient interactions into permanent ones, making the complex
difficult to degrade (Figure 7C). Further studies are needed to establish a model for sequential
binding of ligands in the assembly of the CL complex and why specific proteins are recruited to it.
Notably, HS1 has already been shown to play a role related to its phosphorylation state in B-CLL
(57): finding a link between the two events may shed light on the pathogenesis of B-CLL.
We observed that a relationship exists between the stability of the CL complex and hence the
activated state of Lyn and the defective apoptosis of B-CLL cells. We had previously demonstrated
that the high basal activity of Lyn, related to the reduced ability of B-CLL cells to enter apoptosis,
results from the tyrosine kinase activity of microsomal and cytosolic Lyn (28). Treatment with GA,
which triggers programmed cell death, enabled us to differentiate these two subpopulations of Lyn,
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by disrupting the CL complex and thus inactivating CL. It is noteworthy that inactivation of CL by
GA occurs earlier than its degradation, indicating that the activity of CL is directly dependent on the
stability of the complex. These data, observed in freshly isolated B-CLL cells, are in agreement
with those obtained in cytosol extracts treated with compounds capable of destabilizing the
interactions of the CL complex mediated by the Lyn SH3 domain (GST-Lyn/SH3) or by the
catalytic domain (GA), in the presence of lactacystin, which, as other proteasome inhibitors (e. g.
MG-132 and PS-341), can induce apoptosis in B-CLL cells, although there is a variation in the
sensitivity to treatment of the cells (58-59). Despite the loss of activity caused by the dissociation of
the complex in the presence of GA or GST-Lyn/SH3, Lyn protein level is preserved by the use of
lactacystin, confirming that CL activity is related to the stabilization of the CL complex and not to
its expression level (Figure 5).
GA-induced inactivation of CL parallels the decrease in the aberrant basal protein Tyr-
phosphorylation detectable in B-CLL lysates, indicating that CL, by contrast to microsomal Lyn, is
bound to Hsp90, and that its activity relies on the stability of the complex (Figures 6 and 7).
Instead, degradation of Lyn is a late event, involving both the cytosolic and microsomal fractions of
Lyn (Figure 6C and D).
These results highlight the prominent role played by CL in aberrant high basal protein Tyr-
phosphorylation detected in both Zap+ and Zap- B-CLL samples (Figure 6B), and seem to exclude a
correlation between apoptosis induced by Hsp90 inhibitors and ZAP-70, as is already elsewhere
reported (60). Moreover, the aberrant activation of Lyn through the interaction with specific protein
ligands appears to contribute to the pathogenesis of B-CLL and has led us to regard the proteins
stabilizing the CL complex as potential targets for a possible therapeutic approach for B-CLL.
Hence, inactivation of CL complex can be proposed as a further mediator of apoptosis, in addition
to depletion of Akt ( 46) and alteration in the expression of p53 (61) due to Hsp90 inhibition in B-
CLL, and this is corroborated by the intense work carried out in the last few years focused on
Hsp90 inhibitors (46, 54, 60-62). In addition, the interactions of the SH3 domain of SFKs could be
considered as potential targets in the development of novel drugs capable of disrupting the
interaction with protein ligands, also suggesting a new approach in the treatment of pathologic
processes in which SFKs are directly involved.
Acknowledgements
This work was supported by grants from: Ministero dell’Istruzione dell’Università e della Ricerca
to A. Donella-Deana. (Prin 2005) and University of Padova to A.M. Brunati (Progetto di Ateneo
2005), A.I.R.C. (Milan) to G. Semenzato, Fondazione Berlucchi per la Ricerca sul Cancro on
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16
“Approccio clinico/biologico ai pazienti con leucemia linfatica cronica B” and by Regione Veneto
on Chronic Lymphocytic Leucemia.
Author contribution:
Livio Trentin-contributed clinical patient samples, performed some of the in vitro research,
analyzed the data and wrote parts of the manuscript.
Martina Frasson performed the majority of the in vitro research, analyzed the data and wrote parts
of the manuscript.
Arianna Donella-Deana provided intellectual input into the phosphorylation studies and reviewed
the manuscript.
Federica Fredazzo contributed clinical patient samples and performed some of the in vitro research.
Mario A. Pagano, performed some of the in vitro research and reviewed the manuscript.
Elena Tibaldi, performed some of the in vitro research.
Cristina Gattazzo, contributed clinical patient samples and performed some of the in vitro research.
Renato Zambello, contributed clinical patient samples.
Gianpietro Semenzato, provided intellectual input into the lymphocyte studies and reviewed the
manuscript.
Anna M. Brunati designed the research, reviewed all of the data, participated in analysis of data and
wrote the manuscript.
Conflict of interest discosure: All authors declare no competing financial interests.
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Legends
Figure 1. Aberrant phosphorylation state of Lyn in B-CLL. A) Schematic representation of
domain structure and functional properties of Lyn along with the two phosphorylation sites
recognized by the specific antibodies. B) Whole B cell lysates (lanes 1 and 4), microsomes (lanes 2
and 5) and cytosol (lanes 3 and 6) were assayed by Western blot analysis with anti-LDH (cytosolic
marker), anti-calnexin (microsomal marker), anti-lamin (nuclear marker) and anti-aconitase
(mitochondrial marker) antibodies. Western blots are representative of samples from 5 normal
donors (left, lanes 1-3) and of those from 10 CLL patients (right, lanes 4-6) are shown. C) Whole B
cell lysates (lanes 1 and 4), microsomes (lanes 2 and 5) and cytosol (lanes 3 and 6) from one normal
donor (lanes 1-3) and from CLL patient #15 (lanes 4-6) were analyzed by immunoblotting with
anti-pYA, anti-pYT and, after stripping, reprobed with anti-Lyn antibody. Molecular weight (kDa)
corresponding to p53 and p56 isoforms of Lyn are indicated in the middle. D) Densitometric
analysis (arbitrary units) of anti-pYA, anti-pYT and anti-Lyn bands of whole cell lysates (lanes 1 and
4), microsomes (lanes 2 and 5) and cytosol (lanes 3 and 6) from 5 normal (lanes 1-3) and 40 B-CLL
samples (lanes 4-6) is shown. Data are expressed as means ± SD from 3 separate
experiments.Whole cell lysates, microsomes and cytosol were prepared as detailed in Materials and
Methods.
Figure 2. Purification and characterization of Lyn-complex from cytosol of B-CLL. A) Cytosol
from 15 x 106 freshly isolated B-CLL cells lysed by sonication (upper panel, S) or, alternatively, by
douncing (lower panel, D) was loaded on top of a linear glycerol gradient (10-40%) and centrifuged
18 h at 100000g in a SW60Ti rotor (Beckman) at 4° C. 18 fractions (200 μL each) were collected
from top and analyzed by immunoblotting with anti-Lyn antibody. The figure is representative of
experiments performed in triplicate on samples from each of 5 B-CLL patients. B) Cytosol, from 15
x 106 freshly isolated B-CLL cells lysed by sonication, was treated without (upper panel, –
λ
PPase)
or with
λ
PPase (lower panel, +
λ
PPase), and subjected to the separation procedure described in A).
18 fractions (200 μL each) were collected from top, assayed for Lyn activity tested on Src-specific
peptide substrate cdc2(6-20), and analyzed by immunoblotting with anti-pYA antibody and, after
stripping, with anti-Lyn antibody. The figure is representative of experiments performed in
triplicate on samples from each of 40 B-CLL patients. C) Fractions 13 and 14 (CL complex) of the
cytosol purified from 75 x 106 B-CCL cells and subjected to a linear glycerol gradient under the
conditions described in A) were collected and split into 5 aliquots, which were incubated for 30 min
at 4° C in the absence (control) or presence of 0.05% SDS, 0.1
μ
M GST-Lyn/SH3, 0.1
μ
M GST-
Lyn/SH2 and 0.1
μ
M geldanamycin, respectively. The treated samples were then subjected
separately to glycerol gradient centrifugation as described in A) and aliquots of the resulting
fractions assayed for Lyn activity tested on Src-specific peptide substrate cdc2(6-20), and analyzed
by immunoblotting, with anti-Lyn antibody. The figure is representative of experiments performed
in triplicate on samples from 10 B-CLL patients. Downward arrows: position of molecular weight
standards on glycerol gradients, glutamate dehydrogenase (62 kDa), alcohol dehydrogenase (150
kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa) (Sigma-Aldrich) are indicated to estimate
the molecular weight of the protein complexes on parallel gradient runs.
Figure 3. SH3 domain of cytosolic Lyn binds to HS1 and SHP-1L proteins. A) Cytosol from 15
x 106 freshly isolated B-CLL cells lysed by sonication was subjected to the separation procedure
described in figure 2A. Fractions 13 and 14 (CL complex) were collected and re-submitted to an
additional centrifugation step on a glycerol gradient. Aliquots of the gradient fractions were
analyzed by immunoblotting with anti-Lyn, anti-SHP-1/1L, anti-SHP-1, anti-HS1, anti-STAT3,
anti-Akt, anti-Cbl and anti-SHP-2 antibodies. Whole cell lysates from 2 x 105 B-CLL cells were
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23
probed with the same antibodies as positive controls. Downward arrows: position of molecular
weight standards on glycerol gradients, glutamate dehydrogenase (62 kDa), alcohol dehydrogenase
(150 kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa) (Sigma-Aldrich) are indicated to
estimate the molecular weight of the protein complexes on parallel gradient runs. B) CL complex
purified after two centrifugation steps on a glycerol gradient, as described in A), was collected and
aliquots were treated for 30 min at 4o C in the absence or presence of 0.1
μ
M GST/SH3-Lyn and 0.1
μ
M Pro-rich peptide, further subjected to immunoprecipitation by anti-SHP-1/1L and anti-Lyn
antibodies, and assayed for Lyn and SHP-1/1L, respectively. C) Same fractions as in B) were
collected and aliquots were treated for 30 min at 4° C in the absence or presence of 0.1
μ
M
GST/SH3-Lyn, 0.1
μ
M HS1ΔN-term., 0.1
μ
M HS1ΔSH3, 0.1
μ
M HS1-ΔPro-rich, 0.1
μ
M Pro-rich
peptide, further subjected to immunoprecipitation by anti-HS1 and anti-Lyn antibodies, and assayed
for Lyn and HS1, respectively.
Figure 4. Hsp90 is detectable in Lyn-complex and interacts with Lyn. A) Cytosol from 15 x 106
freshly isolated B-CLL cells lysed by sonication, was subjected to the separation procedure
described in figure 2A. Fractions 13 and 14 (CL complex) were collected and re-submitted to an
additional centrifugation step on a glycerol gradient. Aliquots of the gradient fractions were
analyzed by immunoblotting with anti- Lyn and anti-Hsp90 antibodies. Downward arrows: position
of molecular weight markers on glycerol gradient. B) CL complex purified after two centrifugation
steps on a glycerol gradient, as described in A), were collected and aliquots were treated for 30 min
at 4oC in the absence or presence of 0.1
μ
M geldanamycin (GA), 0.1
μ
M 17-allylamino-17-
demethoxygeldanamycin (17-AAG), 0.1
μ
M GST/SH3-Lyn, and 0.1
μ
M Pro-rich peptide,
respectively, and further subjected to immunoprecipitation by anti-Hsp90 or anti-Lyn antibodies.
Immunoprecipitates were subsequently assayed both for Lyn and Hsp90, respectively. The figure is
representative of experiments performed in triplicate on samples from 40 B-CLL patients.
Figure 5. Stabilization of cytosolic Lyn complex by synergic cooperation of SH3 and catalytic
domains. Cytosol from freshly isolated B-CLL cells lysed by sonication was subjected to the
separation procedure described in figure 2A and treated without or with 0.1
μ
M geldanamycin
(GA), 0.1
μ
M GST/SH3-Lyn and 0.1
μ
M Pro-rich peptide, respectively, in the absence or presence
of 10
μ
M lactacystin for 1 h at 37°C. A) Aliquots of each treated sample were analyzed for in vitro
Lyn activity on Src-specific peptide substrate cdc2(6-20) and by Western blotting for Lyn. B)
Aliquots of each sample were immunoprecipitated with anti-Lyn antibody and the
immunocomplexes probed with anti-HS1, anti-SHP-1/1L, anti-Hsp90 antibodies, respectively.
Blots were then stripped and re-probed with anti-Lyn antibody. The bar graph above the blot panels
represent the values of a densitometric analysis (arbitrary units) of anti-HS1, anti-SHP-1/1L and
anti-Hsp90 bands, expressed as mean ± SD. The statistical analyses were performed by employing a
one-way ANOVA with post test, and the significance is indicated as a P value. *, P< 0.05 and **,
P< 0.001, compared with control (bar 1) Data are representative of experiments performed in
triplicate on samples from 16 B-CLL patients.
Figure 6. Analysis of Lyn activity and protein level during GA-mediated apoptosis of B-CLL
cells. Unmutated CLL/ZAP+ (U-CLL/ZAP+) and and mutated CLL/ZAP- (M-CLL/ZAP-) cells were
cultured for the indicated times, in the presence of 0.1
μ
M GA. A) After GA treatement, U-
CLL/ZAP+ and M-CLL/ZAP- cells were lysed and analyzed by immunostaining with antibodies
raised against PARP. Blots were stripped and reprobed with anti-
β
-actin antibody as loading
control. B) After GA treatment U-CLL/ZAP+ and M-CLL/ZAP- cells were lysed and analyzed by
immunostaining with antibody raised against phospho-Tyr (pY). Molecular mass of protein
standards are indicated in the middle. Blots were stripped and reprobed with anti-
β
-actin antibody
as loading control. C) and D) After GA treatment U-CLL/ZAP+ and M-CLL/ZAP- cells were lysed
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24
by sonication in an isotonic buffer and subjected to differential centrifugation to separate cytosolic
(C) and microsomal (D) fractions. Comparable aliquots were assayed for in vitro Lyn activity on
Src-specific peptide substrate cdc2(6-20) and by Western blotting for Lyn. Lyn specific activity is
calculated as ratio of Lyn activity (bar graphs) over densitometric values of western blot analysis
for Lyn (panel below bar graphs) by standardizing the ratios of each control to the value of 100. All
calculated SD values are <10%. Data are representative of experiments performed in triplicate on
samples from each of 16 B-CLL patients.
Figure 7. Monitoring of Lyn-complex degradation during GA treatment. B-CLL cells were
cultured in the presence of GA for different times, as described in Figure 6. A) Cells were lysed by
sonication in an isotonic buffer and subjected to differential centrifugation to separate microsomal
and cytosolic fractions. Cytosol underwent glycerol gradient centrifugation, as described above.
Fractions were collected from top and assayed for in vitro Lyn activity on Src-specific peptide
substrate cdc2(6-20) and by Western blotting for Lyn. B) Cytosol, isolated from B-CLL cells as in
(A), were immunoprecipitated with anti-Lyn antibody. Immunocomplexes were then probed with
anti-HS1, anti-SHP-1/1L and anti-Hsp90 respectively. Blots were then stripped and re-probed with
anti-Lyn antibody. The bar graph above the blot panels represent the values of a densitometric
analysis (arbitrary units) of anti-HS1, anti-SHP-1/1L and anti-Hsp90 bands, expressed as mean ±
SD Data are representative of 3 experiments performed with 8 B-CLL samples. C) Proposed model
for sequential binding of ligands in the assembly of the CL complex. Step 1: SH3 binding proteins
(X) can promote displacement of the PPII motif in the SH2-kinase linker from the SH3 domain,
thus inducing an “open” conformation. Step 2: Association of Hsp90 with the N-terminal lobe of
Lyn catalytic domain stabilizes the complex and maintains the kinase in an active conformation.
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25
Table 1 Biological and clinical characteristic of the patients
AStaging system developed by K.R. Rai (63). B“Mutated was defined as having a frequency of mutations greater than 2% from
germline VH sequence. CAs determined by immunoblot analysis on purified B cells (purity at least 98%). ND, not determined;
NEG, negative; POS, positive.
.
Patient
no.
Age Sex Rai stage
A
Wbc count
(/mm
3
) Lymphocytes
(%)
V
H
mutational
status
B
ZAP70
Expression
c
1 51 M 0 33,300 87.1 Mutated NEG
2 64 F 0 49,600 83.6 Mutated POS
3 46 F 0 25,000 74.4 Mutated NEG
4 58 F 0 37,500 84.0 Unmutated POS
5 67 M 0 18,600 82.8 Mutated ND
6 64 F I 68,900 85.5 Mutated NEG
7 72 F I 16,600 92.4 Mutated NEG
8 68 M I 29,600 81.2 Mutated NEG
9 44 F I 50,200 89.5 Mutated NEG
10 70 F I 15,200 76,3 Mutated POS
11 74 M I 17,700 68.6 Unmutated POS
12 75 F I 14,200 60.0 Unmutated POS
13 63 M I 28,800 84.0 ND NEG
14 56 M I 77,000 82.5 Mutated POS
15 49 F I 37,200 83.4 Unmutated POS
16 60 M I 11,500 77.6 Mutated POS
17 69 M I 18,500 81.5 Unmutated POS
18 74 M II 28,000 85.0 Mutated NEG
19 59 M II 156.200 75.9 ND NEG
20 65 M II 27,680 79.7 Mutated NEG
21 65 M II 142,000 80.2 Unmutated ND
22 63 M II 33,000 85.1 Unmutated POS
23 66 M III 29,700 88.6 Mutated NEG
24 60 M III 39,400 74.0 Unmutated POS
25 60 M III 73,640 88.7 Mutated NEG
26 60 M III 33,500 79.5 Mutated POS
27 68 M III 128,800 96.4 Unmutated POS
28 84 M I
V
160,500 76.1 Unmutated POS
29 80 F I
V
27,300 84.6 Mutated POS
30 77 F I
V
46,700 88.8 Mutated NEG
31 77 M I
V
120,600 79.1 Unmutated POS
32 74 F I
V
31,700 85.6 Unmutated POS
33 85 M I
V
36,800 91.6 Mutated NEG
34 63 M I
V
106,100 85.6 Mutated NEG
35 80 M I
V
41,300 87.8 Unmutated POS
36 82 M I
V
150,800 76.0 Mutated NEG
37 58 M I
V
32,800 89.0 ND NEG
38 60 M I
V
87,600 94.4 Unmutated POS
39 33 F I
V
21,700 81.6 Unmutated POS
40 64 M I
V
51,700 82.1 Mutated ND
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