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AQABAAMAAQACAAIAAQABAAIAAgACAAQAAAAuAQAABAAAAC0BBAAEAAAA8AEDAAMAAAAAAA== ------=_NextPart_01CAE6B4.BCB59900 Content-Location: file:///C:/A4FBBE50/Hellmich1.6.10_files/pres.xml Content-Transfer-Encoding: quoted-printable Content-Type: text/xml; charset="utf-8" ------=_NextPart_01CAE6B4.BCB59900 Content-Location: file:///C:/A4FBBE50/Hellmich1.6.10_files/editdata.mso Content-Transfer-Encoding: base64 Content-Type: application/x-mso QWN0aXZlTWltZQAAAfAEAAAA/////xAAA/AJAAAAAAAlBAEAAAABAAAL8AQAAAASNFZ4 ------=_NextPart_01CAE6B4.BCB59900 Content-Location: file:///C:/A4FBBE50/Hellmich1.6.10_files/slide0023.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Slide 1
Objectives:
1) To understand basic molecular mechanisms mediati= ng peptide hormone receptor desensitization. 
2) To gain knowledge of some modern molecular biolo= gy techniques used to conduct biomedical research.
“Mechanisms of peptide hormone receptor desensitization: implications
Prese= nter: Mark Hellmich, PhD
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Gastrin and CCK
Gastrin 1-17 (G-17) :      H2NNH2
Cholecystokinin (CCK) :        = ;            &n= bsp;           H2N-D-Y-M-G-W-M-D-F-NH2
•<= /span> Stomach
­ acid secretion from Parietal cells
­ histamine release fr= om ECL cells
­ pepsinogen from Chief cells
­normal mucosal growth
­ risk of gastric cancer in presence= of inflammation
  = ;
•<= /span> Colon
­expression in colon polyps and tumor
­ growth and tumor progression <= /span>
–
•<= /span>Pancreas
­ islet cell differentiation
­pancreatic cancer ce= ll growth
–
      
Gastrin  in the GI Tract
 
–<= /span> G protein-coupled receptor superfamily
–<= /span>Shares 50%  sequence identity with CCK-AR (CCK1R)
–<= /span> Bind= s CCK and amidated G-17 (2-6 nM)
–<= /span>Expression regulated by Wnt/APC/β-cat pathway and Kras
–<= /span>Increased expression in colon, gastric and pancrea= tic cancers
–
–
Cholecystokinin (CCK2)/Gastrin Receptor
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vector
CCK2R sv
CCK2R
3D"NCM356 3D"NCM356 3D"Copy
B
C
D
A
CCK2R sv<= /div>
CCK2R
Gastrin mRNA
vector
E
Figure 2.  Expression of human recombinant CCK2 receptor splice variants in NCM356 cells increases cellular adhesion and proliferation in vitro. A)  Reverse transcriptase/polymerase chain reaction assay demonstrated that all three <= /span>transduced cell lines expressed gastrin mRNA , B) NCM356 cells stably transduced with the pFbIRES retroviral expression vector exhibited a small round, non-<= span style=3D'position:absolute;top:39.5%;left:66.85%;width:35.39%;height:3.0%'= >adherent phenotype indistinguishable from the parental line.=   In contrast,  NCM356 <= /span>cells transduced either with pIRES containing recombinant human CCK2R (C) = or CCK2i4svR (D) exhibit a spread adherent phenotype with few floating cells.  E) CCK2R (open circles) and CCK2i4svR (closed circles) receptor variant-expressing cells proliferate significantly faster than cells transduced with the expression vector alone (closed triangles), suggesting autocrine stimulation of proliferation by endogenous gastrin.
CCK2R
CCK2R sv
vector
3Dmouse2
A
B
C
D
D<= /b>
= Figure 4.  CCK2 receptor variant expression is sufficient to transform NCM356 cells.   The athymic nude mouse model (A) was used to <= /span>assess whether acquisition of either CCK2 receptor variant was sufficient to transform non-tumorigenic NCM356 cells into tumor forming cells.&nbs= p; Subcutaneous injection of  either CCK2R-  (open circles) or CCK2R sv-expressing NCM356 cells (closed circles) produced large tumors over a 33 day time course (B).  In contrast, vector transduced cells (closed triangles) did form tumors (B), like the original NCM356 cell line (data not shown).  Examination of H&E stained tissue sections from CCK2R- (C) and CCK2R sv-= = expressing (D) NCM356 tumors at day 33 showed a similar histology.