Involvement of DownRegulation of Cdk2 Activity in Hepatocyte Growth FactorInduced Cell Cycle Arrest

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Involvement of Down-Regulation of Cdk2 Activity
in HepatocyteGrowth FactorInduced Cell Cycle
Arrest at G1 in the HumanHepatocellular
Carcinoma Cell Line HepG2Yu-ichi Tsukada,
Toshiaki Tanaka, Keiji Miyazawa and Naomi
KitamuraJ. Biochem. 136, 701709 (2004)
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Growth Factor Receptor Signaling Gene
Transcription
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(No Transcript)
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Hepatocyte Growth Factor (HGF) signaling in
normal cells
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Tsukuda, et al., 2001J. Biol. Chem., Vol. 276,
Issue 44, 40968-40976,

• Low HGF conc. increase cell no. HGF conc higher
  than 30ng/ml decreases cell no.
• However, high HGF conc still increases ERK
  phosphorylation. Inhibited by MEK inhibitor at
  10mM.
• ERK activity is required for both the stimulation
  and inhibition of proliferation of HepG2 cells by
  HGF.
• Expression of dominant active Ras inhibits cell
  proliferation.
• The level of ERK activity determines the opposing
  proliferation responses.
• HGF-induced proliferation inhibition is caused
  by cell cycle arrest, which results from pRb
  (retinoblastoma protein) being maintained in its
  active hypophosphorylated form via a
  high-intensity ERK signal in HepG2 cells.

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• Hypothesis In hepatocyte cancer cells high ERK
  activity in response to HGF leads to cell cycle
  arrest.
• Objective Identify signaling intermediates of
  HGF that reduce pRb phosphorylation and induces
  cell cycle arrest.

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Transforming Growth Factor beta Signaling
Induction of p15 results in inhibition of
Cdk4,6/cyclin D complexes and cell cycle arrest
at G1.
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Fig. 1. Both HGF and TGFb reduce cell number of
HepG2 hepatocellular carcinoma cell line
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Measurement of DNA content indicates cell cycle
stage
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Fig. 2. Cell cycle distribution of HepG2 cells
after treatment with HGF or TGF
Four days following treatment, both HGF (solid
circles) and TGFb (solid triangles) increases
number of cells in G1 and decreases number of
cells in S compared to control (open circles) .
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Addition of MEK inhibitor PD98059 decreases G1
peak in response to HGF
White bars, control Black bars , HGF Grey bars,
PD98059HGF
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Cell cycle progression
Hyperphosphorylation of pRb leads to cell cycle
progression Hypophosphorylation of Rb leads to G1
arrest. Therefore, check for Rb phosphorylation
status - HGF
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4-42
SDS-Gel Electrophoresis
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(No Transcript)
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Immunoblotting
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Fig. 3. HGF and TGFb reduces Rb phosphorylation
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Co-immunoprecipitation
Cell lysate
Binding partners
antigen
antibody
Centrifugation
Protein A
Pellet binding partners/antigen/antibody/Protein
A-Sepharose
Sepharose
Boil in Laemmli Sample Buffer SDS, reducing agent
SDS-PAGE and immunoblotting for binding partners
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Fig. 4. What is the cyclin and cylin dependent
kinase (cdk) complex regulated by HGF in HepG2
cells?
A. HGF reduced Rb phosphorylation by CdK2
activity. B. HGF reduced Rb phosphorylation by
Cyclin A/CdK2 activity.
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Fig. 5. Determination of the specific
phospho-serine residues on Rb that are affected
by HGF
Kinase assay of Cdk2 immune complexes with Rb
fusion protein as substrate. At 48 h pRb Ser 780,
795, 807/811 are reduced by treatment with HGF.
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Is HGF-induced hypophosphorylation of Rb
regulated by ERK?
C, control H, HGF PD, HGFPD 98059 T, TGFb
Addition of PD 98059 (MEK inhibitor) restores Rb
phosphorylation even in the presence of
HGF. HGF-induced dephopshorylation of Rb is
regulated via ERK/MAPK pathway.
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Fig.6. Does HGF reduce Rb phosphorylation by
altering cyclin A or p21 levels?
HGF and TGFb reduces Cyclin A expression. HGF and
TGFb increases p21 expression. Both HGF-induced
decreased cyclin A and increased p21 levels are
regulated by ERK signaling.
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Fig.7. Does HGF signaling alter E2F-1 levels?
HGF signaling induces E2F-1 expression via
ERK/MAPK pathway.
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HGF-induced high intensity ERK signal leads
to 1. Decrease in cyclin A expression 2.
Increase in p21expression 3. Suppression of Rb
phosphorylation by Cdck2 4. Decrease in E2F1
expression Cell cycle arrest at G1 of HepG2
cells.
Conclusion
Gene transcription Cell cycle progression