Stathmin Overexpression Inhibits Megakaryocyte Maturation and Platelet Production David C Gajzer1, M.D. Camelia Iancu-Rubin2, Ph.D. 1Cardiovascular Research Center, Department of Medicine and 2Division of Hematology and Medical Oncology,

1
Stathmin Overexpression Inhibits Megakaryocyte
Maturation and Platelet ProductionDavid C
Gajzer1, M.D. Camelia Iancu-Rubin2, Ph.D.
1Cardiovascular Research Center, Department of
Medicine and 2Division of Hematology and Medical
Oncology, Department of Medicine and The Tisch
Cancer Institute, Mount Sinai School of Medicine,
New York

• Original research article was published as
  Stathmin downregulation is required for
  megakaryocyte maturation and platelet production
  - Blood, 117(17)4580-9 (2011)
• Iancu-Rubin C1, Gajzer D1, Tripodi J1, Najfeld
  V1,2, Gordon RE2, Hoffman R1,3 and Atweh GF4
• 1 Division of Hematology and Medical Oncology,
  Department of Medicine and The Tisch Cancer
  Institute, Mount Sinai School
• of Medicine, New York, NY
• 2 Department of Pathology, Mount Sinai School of
  Medicine, New York, NY
• 3 Department of Gene and Cell Medicine, Mount
  Sinai School of Medicine, New York, NY
• 4 Division of Hematology/Oncology, Department of
  Medicine and UC Cancer Institute, University of
  Cincinnati College of
• Medicine, Cincinnati, OH

2
Background and Rationale

• Megakaryocytopoiesis
• The maturation process of the hematopoietic
  progenitor cells that go on to form blood
  platelets
• Microtubules in megakaryocytopoiesis
• Microtubules help maintain cell structure,
  provide platforms for intracellular transport and
  form the spindle during mitosis. In
  megakaryocytes, they make up the multipolar
  mitotic spindle and thus enable endomitosis (a
  form of abortive mitosis, leading to gt4N DNA
  content) (Nagata et al., 1997, Vitrat et al.
  1998)
• Proplatelet extension and platelet formation
• In megakaryocytes, microtubules provide the
  structural scaffold required for extension of
  proplatelets and ensure the transport of
  cytoplasmic organelles and specific granules into
  nascent platelets (Italiano JE et al., 1999
  Richardson JL et al., 2005)

3
Stathmin
Promotes microtubule depolymerization
Sequesters tubulin heterodimers and prevents
polymerization

• Stathmin is a microtubule-regulatory protein that
  destabilizes microtubules and regulates
  microtubule dynamics. It is important for
  cellular proliferation and cell motility.
  Interfering with its expression results in
  abnormal mitotic spindles and aberrant exit from
  mitosis
• All cell types express stathmin in their
  proliferative stages but, with the exception of
  neurons, they do not express stathmin in the
  mature stages
• Stathmin is overexpressed in a wide variety of
  solid tumors and hematological malignancies

4
Stathmin Expression During Megakaryocyte
Maturation

• In murine MK and in human leukemic cell lines
  with MK-like features (K562 and HEL), stathmin is
  downregulated during differentiation and its
  level of expression correlates inversely with
  the level of ploidy (Chang et al. 2001,
  Iancu-Rubin et al. 2003)
• Alterations of stathmin expression in these cell
  lines are associated with abnormal mitotic
  spindles and changes in their propensity to
  become polyploid (Iancu-Rubin et al. 2003, 2005)
• Stathmin is expressed at very low levels in
  normal bone marrow megakaryocytes and is not
  found in platelets (Brattsand G. 1993, Rowlands
  DC, 1995, Iancu-Rubin et al. 2003)

5
Is Stathmin Important For Megakaryocytopoiesis
and Platelet Production?

• Hypothesis
• Stathmin may be important in primary
  megakaryocytopoiesis where its expression may be
  necessary for proliferation of megakaryocyte
  progenitors in the early stages while its
  suppression may be required for megakaryocyte
  maturation in the later stages.
• In our report, we assessed the influence of
  stathmin expression on human primary MK
  development and identified a role for stathmin in
  MK maturation and platelet formation.

6
Methods

• Primary megakaryocyte culturesTwo-step liquid
  culture systemHuman peripheral blood
  (PB)-derived CD34 hematopoietic stem cells were
  cultured in a two-step liquid culture system for
  14 days. hSCF and hTPO were added to the culture
  medium for the first six days, then cells were
  cultured in the absence of hSCF for an additional
  7-10 days.Semi-solid collagen-based
  culturesPB-derived CD34 cells were cultured in
  semi-solid collagen-based cultures in MegaCult
  medium supplemented with hTPO, hIL-3, hIL-6 for
  16 days until microscopic analysis.
• FIV-based vectors and lentiviral
  transductionsSTMN-WT or STMN-4A stathmin cDNA
  was excised from pTRE2-STMN plasmids and cloned
  into FIV-based pCDF1-MCS2-EF1-copGFP plasmid
  backbone. STMN-4A cDNA was generated by
  site-directed mutagenesis which replaced serine
  for alanine residues at the four phosphorylation
  sites (4A). An empty control vector expressing
  only copGFP was used as a control. The transfer
  vectors were co-transfected with pCPR-?Env and
  pCI-VSV plasmids into the 293T packaging cell
  line using lipofectamine 2000. Viral supernatants
  were harvested on day 3 and viral titers
  determined by flow cytometric analysis.
  Successful packaging and viral functionality was
  confirmed by transduction of HEK293 cells and
  subsequent gene and protein expression analysis.
• Gene expression analysisTotal RNA was purified
  using the RNeasy purification kit (Qiagen, MD).
  1 µg total RNA was used for cDNA synthesis with
  the Omniscript kit (Qiagen, MD). One tenth of the
  cDNA was used for quantitative real-time PCR on a
  RealPlex MasterCycler (Eppendorf, NY). Primers
  used were specific for human stathmin, GATA-1 and
  platelet factor 4 (SABiosciences, MD). cDNA
  templates were mixed with 1X IQ SYBR Green
  Supermix (Bio-Rad Laboratories, CA) and 0.2 mM of
  each primer in a total volume of 50 µl in PCR
  plates (Fisher Scientific, PA) in duplicates.

7
Methods

• Protein expression analysisCells were lysed (50
  mM Tris-Cl, 15 mM NaCl, 1 Triton-X, 40 mg/ml
  protease inhibitor cocktail, Roche Molecular
  Biochemicals, IN), then 50 µg of protein were
  separated on 12.5 SDS-PAGE gels and transferred
  to a polyvinylidene difluoride (PVDF) membrane.
  Polyclonal anti-stathmin antibodies (Calbiochem,
  CA) and goat anti-rabbit IgG-horseradish
  peroxidase conjugated (HRP) secondary antibodies
  (Pierce, IL) were used to detect stathmin.
  Monoclonal anti-actin antibodies (Oncogene
  Research Products, MA) and goat anti-mouse
  IgM-HRP secondary antibodies (Calbiochem, CA)
  were used to detect actin. The proteins were
  visualized by enhanced chemiluminescence
  detection (ECL, Amersham Pharmacia Biotech, NJ).
• Microscopic analysisCells were placed on slides
  using a Shandon centrifuge (Life Sciences
  International, England), fixed in methanol for 5
  minutes and stained with Wright-Giemsa (Sigma,
  MO) for 20 minutes. MegaCult cultures were fixed
  and stained using the manufacturers staining kit
  (StemCell Technologies, Canada).
  Immunofluorescence labeling of cells fixed in 4
  paraformaldehyde was performed using
  phycoerythrin (PE)-conjugated anti-CD41
  antibodies (Becton-Dickinson, CA) and 1.5 µM
  Hoechst 33342 (Sigma, MO) for nuclear staining.
  The slides were mounted with Vectashield mounting
  solution (Vector Laboratories, CA) and analyzed
  using a Zeiss Axiophot 2 fluorescence microscope
  using a 63X/1.25 oil objective. Image acquisition
  was performed using a Hammamatsu Orca CCD camera
  and OpenLab software.
• Flow cytometric analysisCultured cells were
  labeled with PE-conjugated anti-CD41 antibodies
  and allophycocyanin (APC)-conjugated anti-CD42b
  antibodies (Becton-Dickinson, CA ) for 45 minutes
  then incubated with 5 µl 7-aminoactinomycin
  (7-AAD) for 15 additional minutes. The data was
  acquired and analyzed using a FACSCanto II flow
  cytometer and FACS Diva software
  (Becton-Dickinson, CA).

8
Methods

• Analysis of culture-derived plateletsMK cultures
  were harvested and centrifuged at 800 rpm for 5
  minutes and supernatants were collected and
  further centrifuged at 3500 rpm for 10 minutes.
  The pellet containing culture-derived platelets
  was re-suspended in phosphate buffered saline
  (PBS) with 0.1 fetal bovine serum (FBS) and
  labeled with CD41-PE and CD42-APC antibodies for
  30 minutes at room temperature. Reticulated
  platelets were identified following incubation
  with 2 µg/ml thiazole orange (TO) (Sigma, MO) for
  additional 15 minutes at room temperature in the
  dark. The cells were then washed, re-suspended in
  PBS with 0.1 FBS and analyzed by flow cytometry.
  PB-derived human platelets were immunolabeled in
  the same manner and used as control.
• Fluorescence in situ hybridization
  (FISH)Uninfected and lentivirus-infected MK were
  fixed then labeled with two different DNA probes,
  the alpha satellite sequence of the centromeric
  region of chromosome X (Xp11.1-q11.1), and the
  satellite III sequence of chromosome Y (Yq12).
  These DNA Probes were obtained from Abbott
  Molecular (Des Plaines, IL) and labeled with
  SpectrumOrange and SpectrumGreen, respectively.
  FISH probe hybridization signals were visualized
  using an Axioplan 2 fluorescence microscope
  (Zeiss, Germany) at 100X magnification and imaged
  with CytoVision software (Genetix Corp., CA).
• Statistical analysisData are expressed as means
  SD and analyzed by using students unpaired
  t-test. P values of lt 0.05 were considered
  statistically significant.

9
Results

• Characterization of primary MK culturesBy the
  end of the maturation step (day 14 in liquid
  culture), 80-90 of cells were MK, out of which
  40-50 were mature MK.

MK
CD42b
Wright-Giemsa Staining
MK
PTL
PTL
CD42b
GPIIb/IIIa Staining
Flow Cytometric Analysis of Cultured Cells
10
Results

• Stathmin expression is downregulated during ex
  vivo megakaryocytopoiesis

11
Results

• Lentiviral-mediated forced stathmin expression
  prevents physiological stathmin downregulation in
  maturing megakaryocytes
• After confirming physiological downregulation of
  stathmin during megakaryocytopoiesis, cells
  cultured in our two-step liquid culture system
  were transduced with either FIV-GFP (empty
  control), FIV-STMN or FIV-STMN4A. FIV-STMN4A,
  expressing mutant non-phosphorylatable stathmin,
  was used because it was shown previously that
  ectopically-expressed wild-type stathmin activity
  is often lost due to phosphorylation by cellular
  kinases. Use of the mutant form of stathmin
  allowed for analysis of the effects of a
  constitutively active stathmin protein on
  microtubules.

12
Results

• Sustained stathmin expression inhibits
  megakaryocyte maturation

Uninfected cells were used as negative controls
to set a gate for detection of infected cells
based on GFP fluorescence. Subsequently, the
fraction of CD41/CD42b cells within the GFP
population was quantified.
SSC
CD42b-APC
CD41-PE
GFP
Expression of wild-type stathmin did not
significantly alter CD41/CD42b express-ion while
expression of the constitutively active form of
stathmin resulted in over 50 reduction in the
fraction of mature MK.
13
Results

• Sustained stathmin expression is associated with
  low GATA-1 and PF-4 expression

PF4 expression was barely detectable in CD34
cells whereas it was markedly up-regulated in
mature MK infected with the control lentiviral
vector. A small but not statistically significant
increase in PF4 expression was observed in MK
that were transduced with the wild-type
lentiviral vector. However, up-regulation of PF4
was not observed in MK transduced with the
lentivirus that encodes the constitutively active
form of stathmin.
Increased GATA-1 levels were detected in mature
MK transduced with control lentiviruses compared
to primary CD34 cells. MK transduced with
lentiviruses expressing wild-type stathmin were
also characterized by up-regulation of GATA-1
expression. Up-regulation of GATA- 1 was not
observed in MK transduced with lentiviruses
expressing the constitutively active form of
stathmin.
14
Results

• Sustained stathmin expression inhibits
  polyploidization

A typical human cell with 2N DNA content (i.e.
diploid) has one copy of each chromosome X and Y
(or two copies of an X-chromosome if the cell
originated from a female), a cell with 4N DNA
content (i.e. tetraploid) has two copies of each
chromosome, a cell with 8N DNA has four copies of
each chromosome and so on. Representative
examples of MK with ploidy levels ranging from 2N
to 32N are presented. An increase in ploidy
levels correlates with the nuclear complexity
detected by DAPI staining (blue fluorescence) and
with nuclear size. In order to assess the effects
of stathmin on the generation of MK with high
ploidy levels, we analyzed by FISH cells with 8N
DNA and categorized them in three ploidy classes
based on the number of X and Y chromosome copies.
Sustained stathmin expression was associated with
a decrease in the fraction of MK with 16N and 32N
DNA in both wild type- and constitutively active
stathmin-expressing cultures. This was
accompanied by an accumulation of MK with lower
ploidy (i.e. 8N DNA content). These results
indicate that sustained expression of stathmin
impaired the ability of MK to achieve high ploidy
levels.
15
Results

• Sustained stathmin expression interferes with
  platelet production by primary MK

PTL
FSC
CD42b
CD41
GFP
Following transduction with control or
stathmin-expressing lentiviruses, MK cultures
were inspected by microscopy to visualize
proplatelet formation. By day 14, control
cultures contained large MK displaying long
cytoplasmic extensions resembling proplatelets.
The frequency of proplatelet-bearing MK was much
lower in the cultures infected with stathmin
expressing lentiviruses. Because the number of
platelets derived in culture is directly
proportional to the number of proplatelets
formed, we collected and labeled culture-derived
platelets from MK transduced with control or
stathmin-expressing lentiviruses and quantified
them by flow cytometry. Platelets derived from
uninfected MK cultures were used as negative
control for GFP expression. As illustrated above,
platelet-sized particles derived from lentivirus
infected cultures are GFP, indicating that they
were derived from the GFP positive MK. From the
platelet-sized GFP events, we quantified only
those expressing CD41. The results presented in
the bar graph show a two-fold reduction in the
fraction of platelets derived in vitro from MK
expressing wild-type or phosphorylation-deficient
stathmin as compared to those produced by control
MK. This finding indicates that sustained
stathmin expression has a negative effect on in
vitro platelet production, suggesting that
alterations of expression of an MT-regulatory
protein can interfere with platelet production by
primary MK.
16
Conclusions

• Downregulation of stathmin is required for normal
  megakaryocytopoiesis
• Sustained levels of active stathmin inhibit
  megakaryocyte maturation, the ability to achieve
  high ploidy and the capacity to generate
  platelets in vitro
• The results of this study validate the importance
  of microtubules for megakaryocytopoiesis
• Dysregulation of stathmin might contribute to
  alterations of the megakaryocyte lineage in
  hematological malignancies such as leukemia and
  myelodysplastic syndrome which express very high
  levels of stathmin deficient platelet production
  and bleeding from low platelet counts is one of
  the most common causes of death in these patients
  
• This study supports the development of small
  stathmin-like molecules capable of targeting
  megakaryocyte-specific b1-tubulin to limit
  excessive thrombocytosis. As it accounts for more
  than 90 of platelet microtubules and its
  expression is restricted to the MK lineage, the
  ß1-isoform of tubulin may be an excellent
  therapeutic target for disrupting microtubule
  function exclusively in megakaryocytes