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neu overexpression interferes with retinoblastoma protein-mediated cell cycle regulation in human pancreatic carcinoma cells
GASTROENTEROLOGYVol. 114, No. 4
A1420 SSATABSTRACTS • S0209
S0207
LAPAROSCOPIC HELLER MYOTOMY: THE GOLD STANDARD THERAPY FOR ACHALASIA. WS Richardson. JS Bolton. GM Fuhrman. JC Bowen, Ochsner Medical Institutions, New Orleans, Louisiana.
THE p21 CYCLIN-DEPENDENT KINASE PROTECTS ESOPHAGEAL CARCINOMA CELLS FROM RADIATION. D.A. Ri~berg, F.S. Kim, T.A. Bliuman. J. So. D.W. McFadden. Dept of Surgery, UCLA, Los Angeles, CA.
Introduction: The application of video assisted techniques to the treatment of achalasia has resulted in greater patient and physician interest in surgery for patients who failed conventional treatments. Aim: To evaluate the Ochsner Clinic experience with thoracoscopic and laparoscopic Heller myotomy. Methods: We retrospectively studied 21 patients who underwent thoracoscopic or laparoscopic Heller myotomy for Achalasia between 8/93 and 8/97. The average age was 37 -+9 years. The average length of symptoms was 53 -+47 months. Preoperative evaluation included EGD (21), esophageal motility (20), barium swallow (19), and 24 hour ambulatory pH probe (1). 18 patients had failed prior treatments (11 balloon dilations, 7 simple dilations, 7 botulin toxin injections). 5 myotomies were performed thoracoscopically and one was converted to laparoscopy. 16 were performed laparoscopically, one of which was a redo. Outcomes were measured by symptom severity patient questionnaire (0=none to 4=incapacitating) for dysphagia and heartburn. Results: Operating time was 2 2 2 - 49 min for thoracoscopic, 164-+49 laparoscopic, 100 redo, and 267 thoracoscopic converted to laparoscopic surgery. Average length of hospital stay was 2.25 days (range 1-8) for laparoscopic and 3 (1-6) thoracoscopic. One thoracoscopic case was converted to laparoscopic approach secondary to 02 desaturation. There was one gastroesophageal perforation which was repaired intraoperatively laparoscopically. Postoperative complications were 4 patients with dysphagia (1 required dilation). 17 patients were available for late follow-up (mean follow-up 19 months). Mean postoperative dysphagia score was 0.4 for laparoscopic and 1 thoracoscopic approach. Only 2 (9.5%) patients complained of frequent dysphagia (symptom score 3). Mean postoperative heartburn score was 0.6 for laparoscopic and 1 for thoracoscopic approach. 1 patient (4.7%) complained of frequent heartburn Condusion: Minimally invasive Heller myotomy achieves similar results to it's open counterpart with shorter hospital stay. Laparoscopic Heller myotomy is now preferred to thoracoscopic repair in our institution and in this study had shorter procedure time and length of hospital stay. Laparoscopic Heller myotomy successfully improved patients with achalasia in 90.4% of cases.
The p21 cyclin-dependent kinase inhibitor arrests the cell cycle in response to DNA damage, but its role in apoptosis following such injury is unclear. Previous studies have demonstrated that p21 may either prolong or diminish survival depending on specific cell type. The aim of this study was to determine if p21 mRNA blockade would affect esophageal squamous cell carcinoma (ESCC) survival following irradiation. METHODS: The ESCC line KYSE 150 was transiently transfected with either antiseuse p21 mRNA oligonucleotides or 20-mer p21 control oligonucleotides using a lipofectant reagent. Ceils were then exposed to 6 Gy doses of radiation or used as controls. Total protein was extracted from some cells, and p21 levels determined via ELISA. Other cells were allowed to grow for 7 days, at which time clonogenic survival was determined via Coulter machine. Student's t-test was used to compare survival and protein levels. RESULTS: Mean -+ SEM, *p<0.05 vs. controls
• S0208
N-ACETYLCYSTEINE ABROGATES THE SYSTEMIC RESPONSE TO MURINE INTESTINAL ISCHEMIA/REPERFUSION. D.A. Ribbers,. T.A. Blinman. J.S. Lane. F.S. Kim. O.J. Hines. D.W. McFadden. C.F. Chandler, Division of General Surgery, UCLA, Los Angeles, California. Intestinal ischemia/reperfusiou (IR) produces a severe systemic inflammatory response marked by high levels of serum cytokines as well as distant organ injury, particularly neutrophil-mediated lung injury. Cytokine expression and neutrophil activation in ischemia have been linked to oxygen radical formation that accompanies reperfusion. We hypothesized that the antioxidant agent N-acetylcysteine (NAC) would decrease both the systemic inflammatory cytokine response and pulmonary neutrophil sequestration in a murine model of intestinal IR. Methods: Forty female Swiss-Webster mice were divided into four groups. Group 1 underwent laparotomy alone (S). Group 2 underwent 45 minutes of cephalic mesentery artery occlusion followed by 3-hour reperfusion (I). Group 3 underwent sham operation plus NAC treatment (50 mg/kg ip, 1 hour preop) (S/N). Group 4 received IR with NAC (I/N). Animals were sacrificed 3 hours after reperfusion. Serum cytokines were determined by ELISA (pg/ml). Lung tissue was harvested for myeloperoxidase activity (U/gin). Histological scoring of fixed jejunal sections was performed by two blinded investigators. Results:
20001 ~, 200, ~, 5immm~ ~% S
I S/N I/N
S
I
S/N I/N
S
I
S/N I/N
2.5
..................................................................................
5000"r................................................................................ :
¢
:32500
r, i m
mm
ui,
F
o
,
o Bt l___m_.J CONCLUSIONS: p21 protein levels fell following transfection of the KYSE 150 cells. In addition, transfected ceils demonstrated decreased survival when exposed to radiation. This study suggests that p21 protein offers a survival advantage to KYSE 150 cells following radiation-induced DNA damage, possibly by allowing DNA repair during cell-cycle arrest. • S0210
HER2/NEU OVEREXPRESSION INTERFERES WITH RETINOBLASTOMA PROTEIN-MEDIATED CELL CYCLE REGULATION IN HUMAN PANCREATIC CARCINOMA CELLS. H. Roh, J. Pippen and J.A. Drebin Department of Surgery, Washington University School of Medicine, St. Louis, MO. Introduction: Overexpression of the HER2/neu oncogene is observed in
60-70% of human pancreatic carcinomas. The effects of down-regulating the expression of the HER2/neu gene product, p185, using antisense (AS) oligonucleotides, on the proliferation and cell cycle progression of the human pancreatic carcinoma cell line Pancl were examined. Methods: Pancl cells, which overexpress HER2/neu, were obtained from ATCC. AS oligonucleotides, as well as sense (S) and scrambled antisense (SC) controls were added to cultures at a final concentration of I uM in the presence of 10 ug/ml lipofectin for 4 hours. Cell cultures were then placed in fresh media. Effects on HER2/neu expression were determined by northern and western blotting. Expression of p21CiP, p27kiP and the phosphorylated and unphosphorylated forms of the retinoblastoma (Rb) protein were determined by western blotting. Cell proliferation was examined by BRdU-uptake ELISA. Cell cycle distribution was determined using dual channel flow cytometry in the presence of propidium iodide following pulsed BRdU exposure. Results: HER2/neu AS treatment significantly inhibits HER2/neu mRNA and p185 protein expression in Pancl cells; S and SC control oligonucleotides have no effect on HER2/neu expression. AS mediated down-regulation of HER2/neu expression causes upregulation of the cyclin-dependent kinase inhibitors p21CiP and p27mP and a resulting decrease in Rb protein phosphorylation. Treatment of Pancl cells with HER2/neu AS oligonucleotides results in > 80% inhibition of Pancl cell proliferation as determined by BrDU incorporation. Control oligonucleotides have no significant effect on cell proliferation. AS oligonucleotide treatment results in a blockade of cell cycle progression with a corresponding accumulation of cells in GI:
Mean
-+ SEM; *=P
Treatment Lipofectin control HER2/NEU AS HER2/NEU S HER2/NEU SC
Cell Cycle Distribution G0/1 S G2/M 49 32 19 75 12 13 51 30 19 50 28 22
Interestingly, these effects are independent of the pl6 and p53 tumor suppressor genes, which have undergone genetic deletions in Pancl cells. Conclusions: HER2/neu overexpression contributes to the loss of cell cycle control in human pancreatic carcinoma ceils by suppressing the expression of p21CiP and p27kiP with resulting effects on Rb phosphorylation. Strategies which down-regulate HER2/neu expression may be of use in the therapy of some pancreatic tumors.
A1420 SSATABSTRACTS • S0209
S0207
LAPAROSCOPIC HELLER MYOTOMY: THE GOLD STANDARD THERAPY FOR ACHALASIA. WS Richardson. JS Bolton. GM Fuhrman. JC Bowen, Ochsner Medical Institutions, New Orleans, Louisiana.
THE p21 CYCLIN-DEPENDENT KINASE PROTECTS ESOPHAGEAL CARCINOMA CELLS FROM RADIATION. D.A. Ri~berg, F.S. Kim, T.A. Bliuman. J. So. D.W. McFadden. Dept of Surgery, UCLA, Los Angeles, CA.
Introduction: The application of video assisted techniques to the treatment of achalasia has resulted in greater patient and physician interest in surgery for patients who failed conventional treatments. Aim: To evaluate the Ochsner Clinic experience with thoracoscopic and laparoscopic Heller myotomy. Methods: We retrospectively studied 21 patients who underwent thoracoscopic or laparoscopic Heller myotomy for Achalasia between 8/93 and 8/97. The average age was 37 -+9 years. The average length of symptoms was 53 -+47 months. Preoperative evaluation included EGD (21), esophageal motility (20), barium swallow (19), and 24 hour ambulatory pH probe (1). 18 patients had failed prior treatments (11 balloon dilations, 7 simple dilations, 7 botulin toxin injections). 5 myotomies were performed thoracoscopically and one was converted to laparoscopy. 16 were performed laparoscopically, one of which was a redo. Outcomes were measured by symptom severity patient questionnaire (0=none to 4=incapacitating) for dysphagia and heartburn. Results: Operating time was 2 2 2 - 49 min for thoracoscopic, 164-+49 laparoscopic, 100 redo, and 267 thoracoscopic converted to laparoscopic surgery. Average length of hospital stay was 2.25 days (range 1-8) for laparoscopic and 3 (1-6) thoracoscopic. One thoracoscopic case was converted to laparoscopic approach secondary to 02 desaturation. There was one gastroesophageal perforation which was repaired intraoperatively laparoscopically. Postoperative complications were 4 patients with dysphagia (1 required dilation). 17 patients were available for late follow-up (mean follow-up 19 months). Mean postoperative dysphagia score was 0.4 for laparoscopic and 1 thoracoscopic approach. Only 2 (9.5%) patients complained of frequent dysphagia (symptom score 3). Mean postoperative heartburn score was 0.6 for laparoscopic and 1 for thoracoscopic approach. 1 patient (4.7%) complained of frequent heartburn Condusion: Minimally invasive Heller myotomy achieves similar results to it's open counterpart with shorter hospital stay. Laparoscopic Heller myotomy is now preferred to thoracoscopic repair in our institution and in this study had shorter procedure time and length of hospital stay. Laparoscopic Heller myotomy successfully improved patients with achalasia in 90.4% of cases.
The p21 cyclin-dependent kinase inhibitor arrests the cell cycle in response to DNA damage, but its role in apoptosis following such injury is unclear. Previous studies have demonstrated that p21 may either prolong or diminish survival depending on specific cell type. The aim of this study was to determine if p21 mRNA blockade would affect esophageal squamous cell carcinoma (ESCC) survival following irradiation. METHODS: The ESCC line KYSE 150 was transiently transfected with either antiseuse p21 mRNA oligonucleotides or 20-mer p21 control oligonucleotides using a lipofectant reagent. Ceils were then exposed to 6 Gy doses of radiation or used as controls. Total protein was extracted from some cells, and p21 levels determined via ELISA. Other cells were allowed to grow for 7 days, at which time clonogenic survival was determined via Coulter machine. Student's t-test was used to compare survival and protein levels. RESULTS: Mean -+ SEM, *p<0.05 vs. controls
• S0208
N-ACETYLCYSTEINE ABROGATES THE SYSTEMIC RESPONSE TO MURINE INTESTINAL ISCHEMIA/REPERFUSION. D.A. Ribbers,. T.A. Blinman. J.S. Lane. F.S. Kim. O.J. Hines. D.W. McFadden. C.F. Chandler, Division of General Surgery, UCLA, Los Angeles, California. Intestinal ischemia/reperfusiou (IR) produces a severe systemic inflammatory response marked by high levels of serum cytokines as well as distant organ injury, particularly neutrophil-mediated lung injury. Cytokine expression and neutrophil activation in ischemia have been linked to oxygen radical formation that accompanies reperfusion. We hypothesized that the antioxidant agent N-acetylcysteine (NAC) would decrease both the systemic inflammatory cytokine response and pulmonary neutrophil sequestration in a murine model of intestinal IR. Methods: Forty female Swiss-Webster mice were divided into four groups. Group 1 underwent laparotomy alone (S). Group 2 underwent 45 minutes of cephalic mesentery artery occlusion followed by 3-hour reperfusion (I). Group 3 underwent sham operation plus NAC treatment (50 mg/kg ip, 1 hour preop) (S/N). Group 4 received IR with NAC (I/N). Animals were sacrificed 3 hours after reperfusion. Serum cytokines were determined by ELISA (pg/ml). Lung tissue was harvested for myeloperoxidase activity (U/gin). Histological scoring of fixed jejunal sections was performed by two blinded investigators. Results:
20001 ~, 200, ~, 5immm~ ~% S
I S/N I/N
S
I
S/N I/N
S
I
S/N I/N
2.5
..................................................................................
5000"r................................................................................ :
¢
:32500
r, i m
mm
ui,
F
o
,
o Bt l___m_.J CONCLUSIONS: p21 protein levels fell following transfection of the KYSE 150 cells. In addition, transfected ceils demonstrated decreased survival when exposed to radiation. This study suggests that p21 protein offers a survival advantage to KYSE 150 cells following radiation-induced DNA damage, possibly by allowing DNA repair during cell-cycle arrest. • S0210
HER2/NEU OVEREXPRESSION INTERFERES WITH RETINOBLASTOMA PROTEIN-MEDIATED CELL CYCLE REGULATION IN HUMAN PANCREATIC CARCINOMA CELLS. H. Roh, J. Pippen and J.A. Drebin Department of Surgery, Washington University School of Medicine, St. Louis, MO. Introduction: Overexpression of the HER2/neu oncogene is observed in
60-70% of human pancreatic carcinomas. The effects of down-regulating the expression of the HER2/neu gene product, p185, using antisense (AS) oligonucleotides, on the proliferation and cell cycle progression of the human pancreatic carcinoma cell line Pancl were examined. Methods: Pancl cells, which overexpress HER2/neu, were obtained from ATCC. AS oligonucleotides, as well as sense (S) and scrambled antisense (SC) controls were added to cultures at a final concentration of I uM in the presence of 10 ug/ml lipofectin for 4 hours. Cell cultures were then placed in fresh media. Effects on HER2/neu expression were determined by northern and western blotting. Expression of p21CiP, p27kiP and the phosphorylated and unphosphorylated forms of the retinoblastoma (Rb) protein were determined by western blotting. Cell proliferation was examined by BRdU-uptake ELISA. Cell cycle distribution was determined using dual channel flow cytometry in the presence of propidium iodide following pulsed BRdU exposure. Results: HER2/neu AS treatment significantly inhibits HER2/neu mRNA and p185 protein expression in Pancl cells; S and SC control oligonucleotides have no effect on HER2/neu expression. AS mediated down-regulation of HER2/neu expression causes upregulation of the cyclin-dependent kinase inhibitors p21CiP and p27mP and a resulting decrease in Rb protein phosphorylation. Treatment of Pancl cells with HER2/neu AS oligonucleotides results in > 80% inhibition of Pancl cell proliferation as determined by BrDU incorporation. Control oligonucleotides have no significant effect on cell proliferation. AS oligonucleotide treatment results in a blockade of cell cycle progression with a corresponding accumulation of cells in GI:
Mean
-+ SEM; *=P
Treatment Lipofectin control HER2/NEU AS HER2/NEU S HER2/NEU SC
Cell Cycle Distribution G0/1 S G2/M 49 32 19 75 12 13 51 30 19 50 28 22
Interestingly, these effects are independent of the pl6 and p53 tumor suppressor genes, which have undergone genetic deletions in Pancl cells. Conclusions: HER2/neu overexpression contributes to the loss of cell cycle control in human pancreatic carcinoma ceils by suppressing the expression of p21CiP and p27kiP with resulting effects on Rb phosphorylation. Strategies which down-regulate HER2/neu expression may be of use in the therapy of some pancreatic tumors.