1. Volume 116, Issue 5, Pages 1202-1216 (May 1999)
Bone morphogenetic protein 2 exerts diverse effects on cell growth in vitro and is expressed in human pancreatic cancer in vivo 
Jörg Kleeff*, Haruhisa Maruyama*, Toshiyuki Ishiwata*, Harneet Sawhney*, Helmut Friess‡, Markus W. Büchler‡, Murray Korc* 
Gastroenterology 
Volume 116, Issue 5, Pages (May 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Expression of BMP-2 and its receptors in human pancreatic cancer. Total RNA (20 μg/lane) from 6 normal and 8 cancerous human pancreatic tissues were subjected to Northern blot analysis using 32P-labeled cDNA probes (500,000 cpm/mL) for BMP-2, BMPR-IA, and BMPR-II. A 7S cDNA probe (50,000 cpm/mL) was used as a loading and transfer control. Exposure times were 4 days for BMP-2, BMPR-IA, and BMPR-II and 6 hours for 7S.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Expression of BMP-2 in pancreatic tissues. (A) Total RNA (20 μg/lane) from 10 normal and 14 chronic pancreatitis (CP) tissues were subjected to Northern blot analysis using a 32P-labeled BMP-2 cDNA probe (500,000 cpm/mL) and a 7S cDNA probe (50,000 cpm/mL) as a loading and transfer control. Exposure times were 4 days for BMP-2 and 6 hours for 7S. (B) Relative expression of BMP-2 mRNA. Autoradiographs of Northern blots for BMP-2 and 7S RNA from 12 normal (●), 14 chronic pancreatitis (▴), and 16 cancerous (■) pancreatic tissue samples were analyzed by densitometry, and the level of BMP-2 expression was calculated as the ratio of BMP-2 and 7S RNA. Data are expressed as median BMP-2 scores (●) ± SD. The median BMP-2 score of the cancer samples was significantly greater than the medians from normal and chronic pancreatitis tissues (P < 0.01).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Immunohistochemistry and in situ hybridization of pancreatic cancer tissues. (A and B) Moderate to strong BMP-2 immunoreactivity was present in the cytoplasm of the cancer cells. (C) Preabsorption with a BMP-2–specific blocking peptide abolished immunoreactivity. (D) Moderate to strong BMP-2 in situ hybridization signals were present in the cancer cells from serial sections of the cancer sample shown in A. (E) In situ hybridization with the BMP-2 sense probe did not produce any specific signal (bar = 25 μm).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
In situ hybridization of pancreatic cancer and chronic pancreatitis tissues. (A) Moderate to strong BMP-2 in situ hybridization signals were present in the cytoplasm of the cancer cells. (C) Moderate BMP-2 in situ hybridization signals were also present in the acinar cells in the areas adjacent to the cancer cells with chronic pancreatitis–like alterations (solid arrowheads). In contrast, BMP-2 in situ hybridization signals were absent in the epithelial cells forming large hyperplastic ducts (open arrows). (E) In some of the pancreatitis samples, moderate to strong BMP-2 in situ hybridization signals were present in foci of proliferating acinar cells (solid arrowheads), whereas the epithelial cells forming larger ducts did not exhibit BMP-2 in situ hybridization signals (open arrows). (B, D, and F) In situ hybridization with the BMP-2 sense probe did not produce any specific signal (bar = 50 μm).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Survival curves: Kaplan–Meier plots of the postoperative survival period in two groups of patients. In one group, immunohistochemistry revealed positive BMP-2 immunostaining in most cancer cells (solid line), whereas in the other group, BMP-2 immunostaining was absent (broken line). Log rank analysis of the postoperative survival period indicated that patients whose tumors did not express BMP-2 lived longer (P < 0.01) than those whose tumors expressed this ligand.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Expression of BMP-2, BMPR-IA, and BMPR-II in cultured human pancreatic cancer cell lines. Northern blots of total RNA (20 μg/lane) were hybridized with 32P-labeled cDNA probes (500,000 cpm/mL) specific for BMP-2, BMPR-IA, and BMPR-II and with a 7S cDNA probe (50,000 cpm/mL). Exposure times were 4 days for BMP-2, BMPR-IA, and BMPR-II and 6 hours for 7S. Approximate sizes of the respective bands are shown on the left.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Effects of BMP-2 on the growth of pancreatic cancer cell lines. (A) ASPC-1, (B) CAPAN-1, and (C) COLO-357 human pancreatic cancer cells were incubated for 72 hours in serum-free medium in the absence or presence of the indicated concentrations of BMP-2. Data are expressed as percent increase or decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Effects of BMP-2, TGF-β1, and EGF on the growth of pancreatic cancer cell lines. (A) CAPAN-1 and (B) COLO-357 cells were incubated for 72 hours in serum-free medium in the absence or presence of BMP-2 (100 ng/mL), TGF-β1 (1 nmol/L), EGF (1 nmol/L), or the indicated combinations. Data are expressed as percent increase or decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Effects of BMP-2 on the growth of TAKA-1 immortalized pancreatic ductal cells. Cells were incubated for 72 hours in serum-free medium in the absence or presence of the indicated concentrations of BMP-2. Data are expressed as percent decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig. 10
Effects of BMP-2 and EGF on MAPK activation in CAPAN-1 and COLO-357 cells. (A) CAPAN-1 cells were incubated in the absence or presence of 100 ng/mL BMP-2 for the indicated times. (B) COLO-357 cells were incubated in the absence or presence of (100 ng/mL) BMP-2 or (1 nmol/L) EGF for the indicated times. Immunoblotting was carried out with a specific antiactive MAPK antibody as described in Materials and Methods. To confirm equal loading of lanes, the membranes were stripped and reprobed with an anti–ERK-1 antibody that also recognizes ERK-2. The positions of MAPKs ERK-1 (p44) and ERK-2 (p42) are indicated on the right.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

12. Fig. 11
Effects of PD98059 on BMP-2 actions. (A) CAPAN-1 cells were incubated for 72 hours in serum-free medium in the absence (−) or presence (+) of 100 ng/mL BMP-2 and 5 μmol/L PD98059 as indicated. Cell growth was assayed by MTT. Data are expressed as percent increase or decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments. (B) CAPAN-1 cells were incubated for 2 hours in the absence (−) or presence (+) of PD98059 (5 μmol/L) and subsequently incubated in the absence (−) or presence (+) of BMP-2 (100 ng/mL) for 10 minutes. Immunoblotting was carried out with a specific antiactive MAPK antibody. To confirm equal loading of lanes, the membrane was stripped and reprobed with the anti–ERK-1 antibody as in Figure 10. The positions of MAPKs ERK-1 (p44) and ERK-2 (p42) are indicated on the right. (C) COLO-357 cells were incubated for 72 hours in serum-free medium in the absence (−) or presence (+) of 100 ng/mL BMP-2 and 5 μmol/L PD98059 as indicated. Cell growth was assayed by MTT. Data are expressed as percent increase or decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

13. Fig. 12
Effects of Smad4 on BMP-2–induced cell proliferation and MAPK activation in ASPC-1 and CAPAN-1 cells. Northern blot analysis of sham-transfected (Neo) and wild-type Smad4-transfected (A) ASPC-1 and (B) CAPAN-1 clones (left panel) using a 32P-labeled Smad4 cDNA probe (500,000 cpm/mL). Cell growth assays: sham-transfected and Smad4-transfected (A) ASPC-1 and (B) CAPAN-1 clones were incubated for 72 hours in serum-free medium in the absence or presence of the indicated concentrations of BMP-2. Data are expressed as percent increase or decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments (A and B, right panel). (C) Smad4-transfected CAPAN-1 clones were incubated in the absence or presence of BMP-2 (100 ng/mL) or EGF (1 nmol/L) for the indicated times. Immunoblotting was done with a specific antiactive MAPK antibody. To confirm equal loading of lanes, the membranes were stripped and reprobed with an anti–ERK-1 antibody that also recognizes ERK-2. The positions of MAPKs ERK-1 (p44) and ERK-2 (p42) are indicated on the right.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

14. Fig. 13
Effects of Smad4 on BMP-2–induced inhibition of cell proliferation in COLO-357 cells. Northern blot analysis of sham-transfected (Neo) COLO-357 cells and two COLO-357 clones transfected with a truncated Smad4 (upper panel) using a 32P-labeled Smad4 cDNA probe (500,000 cpm/mL). Cell growth assays: sham-transfected and Smad4-transfected COLO-357 clones were incubated for 72 hours in serum-free medium in the absence or presence of the indicated concentrations of BMP-2 and TGF-β1. Data are expressed as percent increase or decrease of untreated controls and are the means ± SEM of 8 determinations from 3 separate experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions