The Role of HIPRPL29 in Prostate Cancer

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The Role of HIP/RPL29 in Prostate Cancer

• Lynn Schwarting-Opdenaker
• Advisor Cindy Farach-Carson

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HIP/RPL29Heparin/Heparan Sulfate Interacting
Protein/ Ribosomal Protein L29

• 159 amino acids (human mouse and pig have 160)
• Apparent MW of 24-28kDa on SDS-PAGE
• Highly basic
• Contains polyanionic binding sites
• 84.4 similarity at the protein level between
  mouse and human
• No prokaryotic ortholog

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Predicted Structure and Functions of HIP/RPL29

• Polyanion binding
• Translational regulation (RNA binding?)
• Modulation of blood coagulation via heparin
  binding
• High affinity HP/HS binding
• Adhesion and invasion of trophoblasts and cancer
  cells
• Inhibition of heparanase
• Antagonist of HB growth factor response

Basic residues in blue
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HIP/RPL29 Potential FunctionsProtein Synthesis
The mammalian ribosome reconstructed by electron
microscopy

• Peripheral protein?
• Stabilizes RNA-protein interactions?
• Increases ribosome translational efficiency?

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HIP/RPL29 Protein Expression Increases with
Phenotypic Progression in Various Prostate Cancer
Cell Lines
PREC LNCaP C4-2 C4-2B PC3 DU145
49 kD
B-actin
28 kD
HIP
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HIP/RPL29 Protein Expression in Different Bone
Marrow Cells
HIP/RPL29 Nuclei
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Hypotheses

• Modifying HIP/RPL29 expression in lineage related
  prostate cancer cells will alter their growth and
  progression.
• By reducing HIP/RPL29 expression in prostate
  cancer cells, we predict that this will
  negatively regulate growth and tumor progression.

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Comparison of HIP/RPL29 Expression in Human Cell
Lines
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FACS Analysis of Cell Surface HIP/RPL29
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Ribosome Isolation of HIP/RPL29
Lift cells with PBS 10mM EDTA
Dounce Homogenize 100 strokes
Spin 15 min at 13,000 x G
Supernatant
Pellet (nuclei, large
membrane fragments, cell debris)
Spin 2 hours at 78,000 x G
Pellet
Supernatant (Cytoplasmic fraction)
Add Triton-X and Sodium Deoxycholate
Spin 15 min at 13,000 x G
Supernatant
Pellet (insoluble material)
Layer over 0.5M sucrose
Spin 2.5 hours at 105,000 x G
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Subcellular Fractionation

• There is little or no HIP/RPL29 on the cell
  surface of the LNCaP and C4-2B cell lines in
  standard culture conditions

• HIP/RPL29 in these cells is intracellular and
  follows the pattern of ribosomal protein L4

• The intracellular function of HIP/RPL29 in
  prostate cancer is the focus of further work for
  this project

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Strategy to Study HIP/RPL29

• Used a ribozyme as a tool to reduce expression
• Created C4-2B cell lines stably expressing
  HIP/RPL29 ribozyme
• Assay for knockdown and affect of HIP/RPL29
  reduced expression

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Initial Look at Knockdown Clones
Relative HIP/RPL29 Expression
49
48
58
61
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Soft Agar Assay
Parental
Clone 9
Clone 20
Clone 22
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Rebound of Knockdown Clones
Relative HIP/RPL29 Expression Determined by
Western Blot
HIP/b-actin ratio
Cell Line
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Lentiviral titering
HIP ribozyme Lenti Clone 3 concentrated with
polybrene C4-2B cells
HIP ribozyme Lenti Clone 3 concentrated C4-2B
cells
10-2
10-3
0
10-3
10-2
0
10-4
10-5
10-6
10-5
10-4
10-6
10-3
10-2
0
10-3
10-2
0
10-6
10-5
10-4
10-6
10-5
10-4
HIP ribozyme Lenti Clone 3 with polybrene C4-2B
cells
HIP ribozyme Lenti Clone 3 ATDC5 cells
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Lentivirus yields HIP/RPL29 ribozyme knockdown
clones
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Conclusions

• Creation of stable HIP/RPL29 knockdown clones has
  not been feasible due to rebound effect
• C4-2B cells do not express the receptors
  necessary to transduce at a high rate with
  lentivirus
• Alternatives include
• Adenovirus (transient)
• Inducible promoter in C4-2B

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Use of Adenovirus to deliver HIP/RPL29 Ribozyme

• Cannot create stables must use transient
  transfections
• Could not obtain a knockdown of HIP/RPL29 using
  this method
• Never saw more than a 28 knockdown
• Higher MOIs caused cell death

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Creation of an Inducible System to Deliver the
HIP/RPL29 Ribozyme

• Chose to use an ecdysone-inducible system
• Advantages include low background expression,
  high induction, can be used in animal models as
  well
• Creation of double stable clones needed

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Ecdysone-Inducible System
GR glucocorticoid receptor EcR ecdysone
receptor VgEcR fusion of ligand-binding and
dimerization domain of EcR, DNA binding domain of
GR and transcriptional activation domain of HSV
VP16 Minimal promoter 3 SP1 binding sites and
minimal heat shock promoter (mHSP)
http//www.stratagene.com/manuals/217468.pdf
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Vectors needed to create Ecdysone System
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Ongoing Work

• Create second set of stable clones using the
  C4-2B cell lines expressing the highest levels of
  the receptors (clones 7 and 34)
• Analyze the HIP/RPL29 knockdown mice that we have
  recently obtained for changes in prostate
  size/morphology