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Pancreatic reg and a conserved bioactive fragment are mitogenic through the MAPK P38 pathway
Vol. 191, No. 4S, October 2000
Surgical Forum Abstracts
S29
temic illness during AP through direct activation of systemic inflammatory cells.
inhibited (MP-2: 1.5 vs 0.4 cm3; HP-2: 1.6 vs 0.9 cm3, p ⬍ 0.05). Growth suppression in vivo was accompanied by suppression of tumor neoangiogenesis as measured by microvessel density.
Pancreatic reg and a conserved bioactive fragment are mitogenic through the MAPK P38 pathway
Conclusions: Selective inhibition of FGF and VEGF receptors impair growth of human pancreatic cancer by a strong anti-mitogenic effect in vitro and, in addition, an antiangiogenic effect in vivo. Addressing angiogenic factors central to the progression of pancreatic cancer may offer a useful therapeutic strategy.
Michael E Zenilman, MD, Qing-hu Zheng, MS, Haiyan Wu, MD, Padmanabha Rengabashyam MD. Department of Surgery, Albert Einstein College of Medicine, 1825 Eastchester Road, Bronx, NY, 10461, USA. Phone 718-904-2260, Fax 718-904-4183; Supported By: NIH RO1 DK 54511-01 Introduction: Pancreatic reg I and reg III proteins are mitogenic to pancreatic ductal and -cells, but the mechanism of their action is unknown. Reg proteins are calcium-dependant lectins, which may confer the proliferative activity. We postulated that the action of reg would be through activation of mitogen-activated protein kinases (MAPK). Methods: We isolated reg from human pancreatic juice, and synthesized a 6 amino acid peptide highly conserved in the lectin domain of rat, mouse bovine and human reg proteins (RegP). Rat ARIP ductal cells (7.5 ⫻ 103 per well) were cultured in DMEM ⫹ 1% serum replacement media and inoculated with protein, and mitogenesis assayed at 48 hours using a tetrazolium assay. A time course of phophorylation of the mitogen-activated kinases (MAPK) P44-42 (ERK1/2), P38 and SAPK/JNK was assessed on 10ug of cellular protein by Western analysis. Results: In Figure 1, data are expressed as Mean ⫾ SEM O.D. units (n ⫽ 3 for each point). Both reg and RegP were mitogenic to ARIP cells in a dose related fashion. It was also active on -cells (not shown). Two random 15 amino acid peptides were not active. Western analysis of cellular protein showed activation of MAPK P38 in the first 24 hours (Figure 2); P44-42 and SAPK/JNK were not activated. Conclusions: We identified a highly conserved 6 amino acid portion of reg which confers proliferative bioactivity. The exocrine protein reg and its bioactive peptide stimulates proliferation of pancreatic-derived cells via phosphorylation of the mitogen-activated protein kinase P38.
Selective inhibition of angiogenic receptors for bFGF and VEGF in pancreatic cancer in vitro and in vivo Peter Bu¨chler, MD, Howard A Reber, MD, M. Bu¨chler, H Friess, MD, Mendel M Roth, Mark Shiroishi, O. Joe Hines, MD. UCLA School of Medicine, Department of Surgery, PO Box 956904, 10833 Le Conte, Los Angeles, CA, 90095-6904, USA, Tel (310) 206-0441. Introduction: VEGF and bFGF regulate neoangiogenesis, are mitogenic for pancreatic cancer (PaCa) cells and, along with their receptors, are overexpressed in human PaCa. In the present study, we selectively inhibited both FGF and VEGF receptors in vitro and in vivo, thus targeting both neoangiogenesis and mitogenesis. Methods: Five human PaCa cell lines were studied: AsPc-1 (A1), Capan-1 (C1), HPAF-2 (HP2), PANC-1 (P1) and MIA PaCa-2 (MP2). PD173074 was used to selectively inhibit FGR-I and VEGF-II receptor signaling. RT-PCR and Western blot analysis were performed to analyze receptor expression and MAP-kinase activation. MTT and Anchorage Independent Growth Assay were used to study cell growth. Flow cytometry was used for cell cycle analysis. In vivo 2 cell lines (HP2 & MP2) were used to induce tumors in the pancreas of nude mice. Animals received 10mg/kg PD173074 i.p. for 8 wks. Immunohistochemistry (anti CD31) was used to quantify microvessel density. Results: Receptors were expressed in all cell lines tested. Highest levels of FGF-RI were found in MP2 cells, and lowest in HP2 cells. Cell growth was significantly inhibited in all cell lines (greatest inhibition in those with highest levels of FGF-RI). Growth inhibition appeared to be due to suppression of S-phase. MAP-kinase activity was lower in all cell lines in vitro. In vivo orthotopic tumor growth in both cell lines was
Retinoid signaling directs secondary lineage selection in pancreatic organogenesis Jinu Kim, M.D., Alan Kadison, M.D., Thomas Maldonado, M.D., Christopher Crisera, M.D., Krishna Prasadan, PhD., Pradip Manna, PhD., Barry Preuett, B.A., Mark Hembree, B.A., Michael P Longaker, M.D., George K Gittes, M.D. Children’s Mercy Hospital, 2401 Gilham Rd., Kansas City, MO, 64112, USA. (816) 802-1472. Introduction: The mechanism for secondary lineage selection (acinar vs. ductal and mature islet architecture vs. primitive endocrine differentiation) in the developing pancreas is unknown. Retinoic acid is a known regulator of carbonic anhydrase II (CA II) expression, a pancreatic duct marker, and has been used clinically as a differentiating agent in many types of cancer, including pancreatic ductal carcinoma. The purpose of this study was to examine the role of retinoid signaling in normal pancreatic development. Methods: Pancreata were dissected from mice at embryonic days 12 (E12), 15, 18 and adult mice, and then were either processed immediately for immunohistochemistry (IHC) or placed into collagen gel culture for 7 days with all-trans retinoic acid (ATRA), and/or 9-cis retinoic acid (9cRA) added to the media. IHC was performed for retinoic acid receptors RAR␣,,␥, and RXR␣,,␥ in freshly harvested tissue. IHC was also performed for insulin, glucagon, amylase and CA II in both freshly harvested tissue and cultured tissue. Results: RAR␣ is expressed in mesenchymal cells at E12 and subsequently localizes to acini at E15 and E18, whereas RXR␥ is expressed in epithelial cells at E12 and then subsequently localizes to acini at E15 and E18. Thus, RAR␣ and RXR␥ may play a role in pancreatic exocrine differentiation. Addition of ATRA to cultured embryonic pancreas led to a dramatic preponderance of mature acini, while 9cRA and the combination ATRA ⫹ 9cRA led to the formation of predominantly pancreatic ducts. IHC in these retinoid-treated cultured pancreases showed amylase expressed in the acini and CA II in the ducts, implying that both of these structures were mature. In 9cRA- and ATRA ⫹ 9cRAtreated pancreases there were multiple clusters of endocrine cells with central insulin and peripheral glucagon, thus representing mature islet architecture. Conclusions: Retinoids seem to induce normal embryonic pancreatic exocrine differentiation. In particular ATRA and 9cRA appear to selectively induce acinar vs. ductal differentiation. Also, ATRA seems to be the first known molecule to induce acinar-specific exocrine differentiation. Furthermore, results in the 9cRA-treated cultured pancreas represent the first demonstration of an exogenous agent inducing mature islet formation in vitro from embryonic tissue.
Disruption of TGF- signaling allows for expansion of endocrine cells derived from embryonic pancreatic ducts in vitro Thomas S Maldonado, M.D., Christopher A Crisera, M.D., Michael T Longaker, M.D., F.A.C.S., George K Gittes, M.D. The Laboratory of Developmental Biology and Repair, New York University Medical Center, 550 First Avenue, Room H-169, New York, NY, 10016, USA. (212) 263-8745 Introduction: TGF- signaling is important in pancreatic differentiation. TGF-s are known to function as inhibitors of growth and development in multiple organ systems. In the developing pancreas, TGF- expression is upregulated late in gestation. Concomitantly, the type II TGF- receptor (TRII), localizes to the pancreatic ductal epithelium. Previous studies in transgenic mice have suggested that cells within the pancreatic ductal epithelium are able to proliferate and differentiate into other mature pancreatic cellular lineages (endocrine and acinar). We hypothesized that TGF- signaling in the late embryonic pancreas
Surgical Forum Abstracts
S29
temic illness during AP through direct activation of systemic inflammatory cells.
inhibited (MP-2: 1.5 vs 0.4 cm3; HP-2: 1.6 vs 0.9 cm3, p ⬍ 0.05). Growth suppression in vivo was accompanied by suppression of tumor neoangiogenesis as measured by microvessel density.
Pancreatic reg and a conserved bioactive fragment are mitogenic through the MAPK P38 pathway
Conclusions: Selective inhibition of FGF and VEGF receptors impair growth of human pancreatic cancer by a strong anti-mitogenic effect in vitro and, in addition, an antiangiogenic effect in vivo. Addressing angiogenic factors central to the progression of pancreatic cancer may offer a useful therapeutic strategy.
Michael E Zenilman, MD, Qing-hu Zheng, MS, Haiyan Wu, MD, Padmanabha Rengabashyam MD. Department of Surgery, Albert Einstein College of Medicine, 1825 Eastchester Road, Bronx, NY, 10461, USA. Phone 718-904-2260, Fax 718-904-4183; Supported By: NIH RO1 DK 54511-01 Introduction: Pancreatic reg I and reg III proteins are mitogenic to pancreatic ductal and -cells, but the mechanism of their action is unknown. Reg proteins are calcium-dependant lectins, which may confer the proliferative activity. We postulated that the action of reg would be through activation of mitogen-activated protein kinases (MAPK). Methods: We isolated reg from human pancreatic juice, and synthesized a 6 amino acid peptide highly conserved in the lectin domain of rat, mouse bovine and human reg proteins (RegP). Rat ARIP ductal cells (7.5 ⫻ 103 per well) were cultured in DMEM ⫹ 1% serum replacement media and inoculated with protein, and mitogenesis assayed at 48 hours using a tetrazolium assay. A time course of phophorylation of the mitogen-activated kinases (MAPK) P44-42 (ERK1/2), P38 and SAPK/JNK was assessed on 10ug of cellular protein by Western analysis. Results: In Figure 1, data are expressed as Mean ⫾ SEM O.D. units (n ⫽ 3 for each point). Both reg and RegP were mitogenic to ARIP cells in a dose related fashion. It was also active on -cells (not shown). Two random 15 amino acid peptides were not active. Western analysis of cellular protein showed activation of MAPK P38 in the first 24 hours (Figure 2); P44-42 and SAPK/JNK were not activated. Conclusions: We identified a highly conserved 6 amino acid portion of reg which confers proliferative bioactivity. The exocrine protein reg and its bioactive peptide stimulates proliferation of pancreatic-derived cells via phosphorylation of the mitogen-activated protein kinase P38.
Selective inhibition of angiogenic receptors for bFGF and VEGF in pancreatic cancer in vitro and in vivo Peter Bu¨chler, MD, Howard A Reber, MD, M. Bu¨chler, H Friess, MD, Mendel M Roth, Mark Shiroishi, O. Joe Hines, MD. UCLA School of Medicine, Department of Surgery, PO Box 956904, 10833 Le Conte, Los Angeles, CA, 90095-6904, USA, Tel (310) 206-0441. Introduction: VEGF and bFGF regulate neoangiogenesis, are mitogenic for pancreatic cancer (PaCa) cells and, along with their receptors, are overexpressed in human PaCa. In the present study, we selectively inhibited both FGF and VEGF receptors in vitro and in vivo, thus targeting both neoangiogenesis and mitogenesis. Methods: Five human PaCa cell lines were studied: AsPc-1 (A1), Capan-1 (C1), HPAF-2 (HP2), PANC-1 (P1) and MIA PaCa-2 (MP2). PD173074 was used to selectively inhibit FGR-I and VEGF-II receptor signaling. RT-PCR and Western blot analysis were performed to analyze receptor expression and MAP-kinase activation. MTT and Anchorage Independent Growth Assay were used to study cell growth. Flow cytometry was used for cell cycle analysis. In vivo 2 cell lines (HP2 & MP2) were used to induce tumors in the pancreas of nude mice. Animals received 10mg/kg PD173074 i.p. for 8 wks. Immunohistochemistry (anti CD31) was used to quantify microvessel density. Results: Receptors were expressed in all cell lines tested. Highest levels of FGF-RI were found in MP2 cells, and lowest in HP2 cells. Cell growth was significantly inhibited in all cell lines (greatest inhibition in those with highest levels of FGF-RI). Growth inhibition appeared to be due to suppression of S-phase. MAP-kinase activity was lower in all cell lines in vitro. In vivo orthotopic tumor growth in both cell lines was
Retinoid signaling directs secondary lineage selection in pancreatic organogenesis Jinu Kim, M.D., Alan Kadison, M.D., Thomas Maldonado, M.D., Christopher Crisera, M.D., Krishna Prasadan, PhD., Pradip Manna, PhD., Barry Preuett, B.A., Mark Hembree, B.A., Michael P Longaker, M.D., George K Gittes, M.D. Children’s Mercy Hospital, 2401 Gilham Rd., Kansas City, MO, 64112, USA. (816) 802-1472. Introduction: The mechanism for secondary lineage selection (acinar vs. ductal and mature islet architecture vs. primitive endocrine differentiation) in the developing pancreas is unknown. Retinoic acid is a known regulator of carbonic anhydrase II (CA II) expression, a pancreatic duct marker, and has been used clinically as a differentiating agent in many types of cancer, including pancreatic ductal carcinoma. The purpose of this study was to examine the role of retinoid signaling in normal pancreatic development. Methods: Pancreata were dissected from mice at embryonic days 12 (E12), 15, 18 and adult mice, and then were either processed immediately for immunohistochemistry (IHC) or placed into collagen gel culture for 7 days with all-trans retinoic acid (ATRA), and/or 9-cis retinoic acid (9cRA) added to the media. IHC was performed for retinoic acid receptors RAR␣,,␥, and RXR␣,,␥ in freshly harvested tissue. IHC was also performed for insulin, glucagon, amylase and CA II in both freshly harvested tissue and cultured tissue. Results: RAR␣ is expressed in mesenchymal cells at E12 and subsequently localizes to acini at E15 and E18, whereas RXR␥ is expressed in epithelial cells at E12 and then subsequently localizes to acini at E15 and E18. Thus, RAR␣ and RXR␥ may play a role in pancreatic exocrine differentiation. Addition of ATRA to cultured embryonic pancreas led to a dramatic preponderance of mature acini, while 9cRA and the combination ATRA ⫹ 9cRA led to the formation of predominantly pancreatic ducts. IHC in these retinoid-treated cultured pancreases showed amylase expressed in the acini and CA II in the ducts, implying that both of these structures were mature. In 9cRA- and ATRA ⫹ 9cRAtreated pancreases there were multiple clusters of endocrine cells with central insulin and peripheral glucagon, thus representing mature islet architecture. Conclusions: Retinoids seem to induce normal embryonic pancreatic exocrine differentiation. In particular ATRA and 9cRA appear to selectively induce acinar vs. ductal differentiation. Also, ATRA seems to be the first known molecule to induce acinar-specific exocrine differentiation. Furthermore, results in the 9cRA-treated cultured pancreas represent the first demonstration of an exogenous agent inducing mature islet formation in vitro from embryonic tissue.
Disruption of TGF- signaling allows for expansion of endocrine cells derived from embryonic pancreatic ducts in vitro Thomas S Maldonado, M.D., Christopher A Crisera, M.D., Michael T Longaker, M.D., F.A.C.S., George K Gittes, M.D. The Laboratory of Developmental Biology and Repair, New York University Medical Center, 550 First Avenue, Room H-169, New York, NY, 10016, USA. (212) 263-8745 Introduction: TGF- signaling is important in pancreatic differentiation. TGF-s are known to function as inhibitors of growth and development in multiple organ systems. In the developing pancreas, TGF- expression is upregulated late in gestation. Concomitantly, the type II TGF- receptor (TRII), localizes to the pancreatic ductal epithelium. Previous studies in transgenic mice have suggested that cells within the pancreatic ductal epithelium are able to proliferate and differentiate into other mature pancreatic cellular lineages (endocrine and acinar). We hypothesized that TGF- signaling in the late embryonic pancreas