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Increased sensitivity to gemcitabine following Bcl-xL antisense treatment in pancreatic cancer cells
April 2000
2771 INCREASED SENSITIVITY TO GEMCITABINE FOLLOWING BCL-XL ANTISENSE TREATMENT IN PANCREATIC CANCER CELLS. Zhongwei Xu, Helmut Friess, Stefan Aebi, Murray Korc, Jorg H. Kleeff, Markus W. Buchler, Univ of Bern, Bern, Switzerland; Univ of CA, Irvine, CA. Bel-xL prevents apoptotic cell death induced by a intrinsic and extrinsic signals. It has been shown recently that Bel-xL is expressed in pancreatic cancer in vivo and that pancreatic cancer patients with moderate expression of Bel-xL have shorter survival times than patients whose tumors express low levels of this gene. In this study, we have designed sequence-specific antisense oligonucleotides, which target the coding region of BcI-xL, but not of its splice variant BcI-xS, which promotes apoptosis. BcI-xL expression was reduced after exposure to BcI-xL antisense oligonucleotides in ASPC-I pancreatic cancer cells, both at the mRNA and protein level. Using FACScan flow cytometer and DAPI staining we show that antisense but not sense oligonucleotides cause apoptotic and not necrotic cell death in these cells. Furthermore, antisense oligonucleotides caused apoptosis in several additional pancreatic cancer cell lines. Interestingly, in ASPC-I cells, Bcl-xL antisense oligonucleotides significantly potentiated the cytotoxic effects of gemcitabine. In conclusion, our results indicate that Bel-xL antisense oligonucleotides reduce Bcl-xL protein levels thereby inhibiting pancreatic cancer cell growth and causing apoptosis. Importantly, Bcl-xL antisense oligonucleotides increase the chemosensitivity of pancreatic cancer cells, suggesting that this approach might be useful in the future therapy of this disease.
2772 ASSESSMENT OF HEALTH RELATED QUALITY OF LIFE (HRQL) IN PATIENTS WITH MALT-LYMPHOMA IN GI-TRACT. (PRELIMINARY RESULTS OF A MULTI-CENTER STUDY). Michael R. Kraus, Maria-Elisabeth Goebeler, Arne Schaefer, Wolfgang Fischbach, Med Poliklinik, Univ of Wuerzburg, Wuerzburg, Germany; II Med Clin, Clin Aschaffenburg, Aschaffenburg, Germany. Background: Clinicians are increasingly interested in impact of illness and therapy on their patients' HRQL. An assessment of HRQL in MALTlymphoma has not been carried out yet. Aims: To prospectively evaluate HRQL in MALT-lymphoma. Methods: 155 patients with MALT-lymphoma HRQL were evaluated by means of the "Gastrointestinal Quality of Life Index" (GIQLI, Eypasch 1995). Scores were obtained before, at the end and one year after therapy. Strategy of treatment (H.p.-eradication, gastrectomy, chemotherapy, radiotherapy) was defined according to stage of disease and histology (high-grade [h.g.] or low-grade [I.g.]). Results: Compared to healthy controls (120,8 SD 15) patients with MALT-lymphoma show a reduced HRQL (95,4 SD 19,5). Before therapy GIQLI scores of patients with h.g. MALT-lymphoma were significantly lower (92,7 SD 18,8) than in I.g. (101,4 SD 19,8; P = .01). The observed difference between these histological subgroups is mainly based on psychological items (emotions) and items of physical functions. No significant differences were found in the remaining subscales (core symptoms, social functions, disease-specific items). Stage of disease shows no significant influence on HRQL. The largest subgroup (st. E I, E II, h.g., n = 88) with gastrectomy and chemotherapy (6 x CHOP) shows a significant reduction of GIQLI scores at the end of treatment (p = .02). One year after therapy there is a significant increase of GIQLI scores compared to the end of treatment and a tendency of improvement regarding pretreatment scores. Deterioration of GIQLI scores after therapy is mainly due to subscales core symptoms and disease-specific items. Values of emotion related items however were continually increasing during observation period. Conclusions:Pretherapeutic GIQLI in h.g. MALT-lymphoma is significantly lower than in I.g. lymphoma. After a significant deterioration during therapy recovering can be observed I year after therapy.
2773 PHOTODYNAMIC THERAPY (PDT) WITH 5-AMINOLEVULINIC ACID (ALA) INDUCED PROTOPORPHYRIN IX (PPIX): THE INTRACELLULAR LOCALIZATION INFLUENCES CELLULAR DAMAGES. Rene C. Krieg, Joachim Rauch, Klaus Schlottmann, Esther Endlicher, Juergen Schoelmerich, Ferdinand Hofstaedter, Ruth Knuechel, Helmut Messmann, Dept of Internal Medicine I and Institute of Pathology, Regensburg, Germany; Institute of Pathology, Univ of Regensburg, Regensburg, Germany; Dept of Internal Medicine I, Univ of Regensburg, Regensburg, Germany. Aim: PDT is a non thermal treatment which induces tissue destruction after sensitization and illumination with light of appropriate wavelength in the presence of oxygen. In order to optimize photodynamic therapy (PDT)
AGAA521
effects in vivo, in vitro testing of gastrointestinal cell lines was applied. Methods: Three human colon carcinoma cell lines (SW480, HT29, CaC02) in plateau growth were incubated with 5-aminolevulinic acid (ALA, 200JA,g/ml, 3h). Protoporphyrin IX (PPIX) accumulation which is formed out of the precursor ALA, was quantified after extraction and localization was visualized by fluorescence microscopy. PDT was performed with an incoherent light source (,\=590-700 nm). For further PDT experiments, each cell line was incubated with defined ALA concentrations, resulting in identical intracellular PPIX. Cells, irradiated with LDso, were examined by fluorescence microscopy for morphological alterations using a double staining technique. To distinguish between apoptosis and necrosis, besides morphology, analysis of DNA strand breaks was performed by the laddering method, and caspase-3 activity was checked by immunolabelling. Results: The cell line CaC02 obtained the highest PPIX content (467+/174 ng/mg protein), followed by HT29 (255+/-11 ng/mg protein) and SW480 (124+/-58 ng/mg protein) after incubation with ALA. In HT29 PPIX is localized in association to the outer cell membrane, in SW480 and CaC02 PPIX colocalizes with mitochondria. For all cells, PDT leads to a complete kill at a dose of 15 J/cm 2 . For lower doses (3-9 J/cm 2) , CaC02 as well as SW480 show a stronger response to PDT than HT29. Performing PDT with similar PPIX content, SW480 as well as CaC02 still showed higher PDT response than HT29. Phototoxic effects lead to specific cellular morphological alterations. Preliminary data indicate a higher rate of apoptosis in SW480 and CaC02 in comparison to HT29. Conclusions: It is shown for the first time that besides absolute PPIX concentration also intracellular localization of the photosensitizer plays an important role for resulting PDT effects. This work was supported by the Wilhelm-SanderStiftung (96.081.2)
2774 PRE·OPERATIVE RADIOTHERAPY UP·REGULATES MATRILYSIN GENE EXPRESSION IN RECTAL CANCER. Adarsh Kumar, Hilary M. Collins. Mike Sokal, John H. Scholefied, Susan A. Watson, Univ of Nottingham, Nottingham, United Kingdom. Background: Matrilysin (MMP-7), a member of the MMP family, is believed to playa significant role in the growth and proliferation of colon cancer cells, in addition to promotion of angiogenesis and invasion. Overexpression of the matrilysin gene has been shown to correlate with Dukes stage and increased metastatic potential in colorectal cancer. Pre-operative radiotherapy has been shown to up-regulate gelatinase expression in rectal cancer. Aim: To determine the effect of pre-operative high dose short-term radiotherapy (5x5 Gy) on matrilysin gene expression in patients with resectable rectal cancer by competitive-based RT-PCR. Methods: Biopsy samples of tumour (n=30) and normal mucosa (n= 12) from 15 patients with resectable rectal cancer were obtained pre and post radiotherapy. Messenger RNA was extracted from tissues using guanidium thiocyanate, purified and reverse transcribed to double stranded cDNA. Competitive RT-PCR was performed with primer pairs for matrilysin and the house keeping gene GAPDH. This is a competitive reaction in which a multicompetitor cDNA standard with priming sites for all the MMPs, is coamplified with cellular cDNA in the same PCR reaction. Matrilysin mRNA levels were expressed relative to GAPDH. Immunocytochemistry was performed, using monoclonal anti-human MMP-7 antibody, for cellular localisation of matrilysin protein. Results: Messenger RNA was successfully extracted from all the samples. 14/15 tumour samples expressed matrilysin mRNA. Although all 6 paired mucosal samples expressed matrilysin, the levels were 10 fold lower as compared to those seen in tumour samples. Pre-operative radiotherapy led to a significant 6 to 7 fold (p=O.OOI, Wilcoxon paired non parametric test) increase in the levels of matrilysin mRNA of tumour samples. In contrast there was no significant change in the expression of matrilysin mRNA of normal mucosal samples after radiotherapy. Conclusions: Pre-operative high dose radiotherapy upregulates matrilysin gene expression in rectal cancer. This may be responsible for growth and proliferation and promotion of angiogenesis of the remaining viable cancer cells in the pelvis, enhancing recurrence rate, although future studies are needed to validate these findings at the protein level. In future, matrilysin inhibition may be a useful preventive or therapeutic adjunct to radiotherapy in rectal cancer.
2771 INCREASED SENSITIVITY TO GEMCITABINE FOLLOWING BCL-XL ANTISENSE TREATMENT IN PANCREATIC CANCER CELLS. Zhongwei Xu, Helmut Friess, Stefan Aebi, Murray Korc, Jorg H. Kleeff, Markus W. Buchler, Univ of Bern, Bern, Switzerland; Univ of CA, Irvine, CA. Bel-xL prevents apoptotic cell death induced by a intrinsic and extrinsic signals. It has been shown recently that Bel-xL is expressed in pancreatic cancer in vivo and that pancreatic cancer patients with moderate expression of Bel-xL have shorter survival times than patients whose tumors express low levels of this gene. In this study, we have designed sequence-specific antisense oligonucleotides, which target the coding region of BcI-xL, but not of its splice variant BcI-xS, which promotes apoptosis. BcI-xL expression was reduced after exposure to BcI-xL antisense oligonucleotides in ASPC-I pancreatic cancer cells, both at the mRNA and protein level. Using FACScan flow cytometer and DAPI staining we show that antisense but not sense oligonucleotides cause apoptotic and not necrotic cell death in these cells. Furthermore, antisense oligonucleotides caused apoptosis in several additional pancreatic cancer cell lines. Interestingly, in ASPC-I cells, Bcl-xL antisense oligonucleotides significantly potentiated the cytotoxic effects of gemcitabine. In conclusion, our results indicate that Bel-xL antisense oligonucleotides reduce Bcl-xL protein levels thereby inhibiting pancreatic cancer cell growth and causing apoptosis. Importantly, Bcl-xL antisense oligonucleotides increase the chemosensitivity of pancreatic cancer cells, suggesting that this approach might be useful in the future therapy of this disease.
2772 ASSESSMENT OF HEALTH RELATED QUALITY OF LIFE (HRQL) IN PATIENTS WITH MALT-LYMPHOMA IN GI-TRACT. (PRELIMINARY RESULTS OF A MULTI-CENTER STUDY). Michael R. Kraus, Maria-Elisabeth Goebeler, Arne Schaefer, Wolfgang Fischbach, Med Poliklinik, Univ of Wuerzburg, Wuerzburg, Germany; II Med Clin, Clin Aschaffenburg, Aschaffenburg, Germany. Background: Clinicians are increasingly interested in impact of illness and therapy on their patients' HRQL. An assessment of HRQL in MALTlymphoma has not been carried out yet. Aims: To prospectively evaluate HRQL in MALT-lymphoma. Methods: 155 patients with MALT-lymphoma HRQL were evaluated by means of the "Gastrointestinal Quality of Life Index" (GIQLI, Eypasch 1995). Scores were obtained before, at the end and one year after therapy. Strategy of treatment (H.p.-eradication, gastrectomy, chemotherapy, radiotherapy) was defined according to stage of disease and histology (high-grade [h.g.] or low-grade [I.g.]). Results: Compared to healthy controls (120,8 SD 15) patients with MALT-lymphoma show a reduced HRQL (95,4 SD 19,5). Before therapy GIQLI scores of patients with h.g. MALT-lymphoma were significantly lower (92,7 SD 18,8) than in I.g. (101,4 SD 19,8; P = .01). The observed difference between these histological subgroups is mainly based on psychological items (emotions) and items of physical functions. No significant differences were found in the remaining subscales (core symptoms, social functions, disease-specific items). Stage of disease shows no significant influence on HRQL. The largest subgroup (st. E I, E II, h.g., n = 88) with gastrectomy and chemotherapy (6 x CHOP) shows a significant reduction of GIQLI scores at the end of treatment (p = .02). One year after therapy there is a significant increase of GIQLI scores compared to the end of treatment and a tendency of improvement regarding pretreatment scores. Deterioration of GIQLI scores after therapy is mainly due to subscales core symptoms and disease-specific items. Values of emotion related items however were continually increasing during observation period. Conclusions:Pretherapeutic GIQLI in h.g. MALT-lymphoma is significantly lower than in I.g. lymphoma. After a significant deterioration during therapy recovering can be observed I year after therapy.
2773 PHOTODYNAMIC THERAPY (PDT) WITH 5-AMINOLEVULINIC ACID (ALA) INDUCED PROTOPORPHYRIN IX (PPIX): THE INTRACELLULAR LOCALIZATION INFLUENCES CELLULAR DAMAGES. Rene C. Krieg, Joachim Rauch, Klaus Schlottmann, Esther Endlicher, Juergen Schoelmerich, Ferdinand Hofstaedter, Ruth Knuechel, Helmut Messmann, Dept of Internal Medicine I and Institute of Pathology, Regensburg, Germany; Institute of Pathology, Univ of Regensburg, Regensburg, Germany; Dept of Internal Medicine I, Univ of Regensburg, Regensburg, Germany. Aim: PDT is a non thermal treatment which induces tissue destruction after sensitization and illumination with light of appropriate wavelength in the presence of oxygen. In order to optimize photodynamic therapy (PDT)
AGAA521
effects in vivo, in vitro testing of gastrointestinal cell lines was applied. Methods: Three human colon carcinoma cell lines (SW480, HT29, CaC02) in plateau growth were incubated with 5-aminolevulinic acid (ALA, 200JA,g/ml, 3h). Protoporphyrin IX (PPIX) accumulation which is formed out of the precursor ALA, was quantified after extraction and localization was visualized by fluorescence microscopy. PDT was performed with an incoherent light source (,\=590-700 nm). For further PDT experiments, each cell line was incubated with defined ALA concentrations, resulting in identical intracellular PPIX. Cells, irradiated with LDso, were examined by fluorescence microscopy for morphological alterations using a double staining technique. To distinguish between apoptosis and necrosis, besides morphology, analysis of DNA strand breaks was performed by the laddering method, and caspase-3 activity was checked by immunolabelling. Results: The cell line CaC02 obtained the highest PPIX content (467+/174 ng/mg protein), followed by HT29 (255+/-11 ng/mg protein) and SW480 (124+/-58 ng/mg protein) after incubation with ALA. In HT29 PPIX is localized in association to the outer cell membrane, in SW480 and CaC02 PPIX colocalizes with mitochondria. For all cells, PDT leads to a complete kill at a dose of 15 J/cm 2 . For lower doses (3-9 J/cm 2) , CaC02 as well as SW480 show a stronger response to PDT than HT29. Performing PDT with similar PPIX content, SW480 as well as CaC02 still showed higher PDT response than HT29. Phototoxic effects lead to specific cellular morphological alterations. Preliminary data indicate a higher rate of apoptosis in SW480 and CaC02 in comparison to HT29. Conclusions: It is shown for the first time that besides absolute PPIX concentration also intracellular localization of the photosensitizer plays an important role for resulting PDT effects. This work was supported by the Wilhelm-SanderStiftung (96.081.2)
2774 PRE·OPERATIVE RADIOTHERAPY UP·REGULATES MATRILYSIN GENE EXPRESSION IN RECTAL CANCER. Adarsh Kumar, Hilary M. Collins. Mike Sokal, John H. Scholefied, Susan A. Watson, Univ of Nottingham, Nottingham, United Kingdom. Background: Matrilysin (MMP-7), a member of the MMP family, is believed to playa significant role in the growth and proliferation of colon cancer cells, in addition to promotion of angiogenesis and invasion. Overexpression of the matrilysin gene has been shown to correlate with Dukes stage and increased metastatic potential in colorectal cancer. Pre-operative radiotherapy has been shown to up-regulate gelatinase expression in rectal cancer. Aim: To determine the effect of pre-operative high dose short-term radiotherapy (5x5 Gy) on matrilysin gene expression in patients with resectable rectal cancer by competitive-based RT-PCR. Methods: Biopsy samples of tumour (n=30) and normal mucosa (n= 12) from 15 patients with resectable rectal cancer were obtained pre and post radiotherapy. Messenger RNA was extracted from tissues using guanidium thiocyanate, purified and reverse transcribed to double stranded cDNA. Competitive RT-PCR was performed with primer pairs for matrilysin and the house keeping gene GAPDH. This is a competitive reaction in which a multicompetitor cDNA standard with priming sites for all the MMPs, is coamplified with cellular cDNA in the same PCR reaction. Matrilysin mRNA levels were expressed relative to GAPDH. Immunocytochemistry was performed, using monoclonal anti-human MMP-7 antibody, for cellular localisation of matrilysin protein. Results: Messenger RNA was successfully extracted from all the samples. 14/15 tumour samples expressed matrilysin mRNA. Although all 6 paired mucosal samples expressed matrilysin, the levels were 10 fold lower as compared to those seen in tumour samples. Pre-operative radiotherapy led to a significant 6 to 7 fold (p=O.OOI, Wilcoxon paired non parametric test) increase in the levels of matrilysin mRNA of tumour samples. In contrast there was no significant change in the expression of matrilysin mRNA of normal mucosal samples after radiotherapy. Conclusions: Pre-operative high dose radiotherapy upregulates matrilysin gene expression in rectal cancer. This may be responsible for growth and proliferation and promotion of angiogenesis of the remaining viable cancer cells in the pelvis, enhancing recurrence rate, although future studies are needed to validate these findings at the protein level. In future, matrilysin inhibition may be a useful preventive or therapeutic adjunct to radiotherapy in rectal cancer.