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Cellular characterization and successful transfection of serially subcultured normal human esophageal keratinocytes
GASTROENTEROLOGY VoL 114, No. 4
A65a AGA ABSTRACTS • G2711 RELATIONSHIP BETWEEN THE AGE AND THE SITE DISTRIBUTION OF COLORECTAL CANCER. M Okamot0. Y Yamaji, J Kato, T Ikenoue, M Takahashi, H Yoshida, T Kawabe, Y Shiratori and M Omata. Second Department of Internal Medicine, University of Tokyo, Japan. BACKGROUND: Colorectal cancer is one of the common neoplasms in the United States, Europe, and also Japan, Since the number of older people is increasing, the clinical feature of colorectal cancer in the older patients were analysed in this study. PATIENTS AND METHODS: From September 1995 to October 1997, we have performed total colonoscopy in 2228 patients consecutively (1554 males and 674 females, mean age 62 years old (range 11 to 93)). The characters of detected colorectal cancer proven histopathlogically by endoscopic polypectomy or biopsy were analysed in relation to the site and the stage. RESULTS: 171 colorectal cancer lesions were found in 165 patients. 13 lesions were found in the patients with <49 y.o., 34 in the patients aged 50s, 60 in aged 60s, 36 in aged 70s, and 28 in the patients with > 80 y.o.. The ratio of right side colon cancer (cecum, ascending colon, and transverse colon) was increased in association with the age, reached 61% in the patients with >80y.o. (Table) (p<0.05). Early colorectal cancer (carcinoma in situ and invading within the submucosa) was found in aproximately 50-60% in each age group. However, when these early colorectal cancers were classified as flat type and protruding type, the ratio of flat type was found to be increased in relation to the age, reached 46% in the patients with > 80y.o. (Table). age Rt side Ca. fiat type early Ca.
< 49 23% (3/13) 12% (1/8)
50s 12% (4/34) 17% (3/18)
60s 28% (17/60) 26% (11/42)
70s 47% (17/36) 33% (6/18)
> 80 61% (17/28) 46% (6/13)
CONCLUSION: In the older patients, fight side colon cancer was found to be increased, and flat type of early colorectal cancer was found to be increased. Total colonoscopy may be needed in the older people. • G2712 CELLULAR CItARACTERIZATION AND SUCCESSFUL TRANSFECTION OF SERIALLY SUBCULTURED NORMAL HUMAN ESOPHAGEAL KERATINOCYTES. OG 0piIz 1, CC Compton23, G Warland 2,3, H Nakagawa l, K Togawa 1, AK RustgiL IGastrointestinal Unit and 2Department of Pathology, Massachusetts General Hospital, Harvard Medical School and -~Shriner's Burn Institute, Boston, MA INTRODUCTION: The establishment of normal human esophageal epithelium in vitro would greatly enhance the ability to study the basic biological mechanisms regulating its proliferation and differentiation. Since normal human epidermal keratinocytes are known to be easily propagated in vitro when grown on feeder layers of irradiated 3T3 mouse fibroblasts, we adopted this strategy to successfully establish primary cultures of esophageal basal ceils and to expand this population through serial subcultivations. The cytokeration and growth factor profiles of confluent tertiary cultures were then analyzed by morphological, immunohistochemical and molecular techniques to define the basic properties of these cells. METHODS: Normal human esophagus specimens were received within 5-6 hr post mortem, and ensured to be free of infection, malignancy, or inflammation by gross and microscopic inspection. The mucosa was immediately dissected away from the muscularis propria and incubated in Dispase. The epithelium was pealed off with forceps and incubated in a mixture of trypsin-EDTA with collection of the supernatant. All cells recovered were plated onto feeder layers of irradiated 3T3-J2 mouse fibroblasts. Esophageal basal ceils were grown in a 3:1 mixture of Dulbecco's Modified Eagles Medium: Ham's F12 medium supplemented with 10% serum, hydrocortisone, cholera toxin, transferrin, triiodo-L-thyronine, insulin and human recombinant epidermal growth factor. Sections of fresh-frozen keratinocyte sheets were stained via the avidin-biotin peroxidase complex method with antibodies against keratins and growth factors. Cells were transiently transfected by the calcium phosphate method with a Rous-sarcoma virus (RSV)-luciferase reporter gene. RESULTS: Normal human esophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers and expanded through four serial subcultivations. Postconfluent cultures displayed a predominantly basal cell phenotype with limited stratification, widespread expression of proliferation markers such as keratins 5 and 14, and production of attachment of ~6[~4integrin and collagen VII. The ceils also expressed transforming growth factor-I~ and interleukin-ll3. Tertiary cultures are successfully transfected with an RSV luciferase reporter gene construct. CONCLUSIONS: Primary cultures of normal human esophageal cells can be successfully established with serial passages through extended numbers of cell generations. This is the first extensive characterization of such cells which can be transiently transfected as demonstrated for the first time. These cultures of normal esophageal cells can be ultimately used as models of differentiation, immortalized with retroviral vectors harboring oncogenes, and used to identify stem ceils.
• G2713 THE HUMAN KERATIN 4 PROMOTER IS REGULATED BY ESOPHAGEAL-SPECIFIC AND UBIQUITOUS TRANSCRIPTIONAL ACTIVATORS. OG Opitz 1, TD Jenkins l, T Inomoto l, AK Rustgi1,2. 1Gastrointestinal and 2Hematology/Oncology Units, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 INTRODUCTION: Keratin 4 is highly expressed in the suprabasal compartment of the esophageal squamous epithelium. We previously demonstrated that targeted disruption of the keratin 4 gene in mice leads to impaired differentiation with basal cell hyperplasia in the esophageal epithelium. To delineate molecular mechanisms underlying transctiptional regulation of keratin 4, we identified critical cis-regulatory elements within the human keratin 4 promoter and interacting trans-activating nuclear factors in human esophageal cells. METHODS: The human keratin 4 promoter was cloned using a PCR based strategy and sequenced. Seven deletion constructs were generated employing PCR based strategies and naturally occurring restriction enzyme sites. The constructs were subcloned into the pXP2 vector which contains an enhancedess luciferase reporter gene. All constructs were transfected in esophageal squamous epithelial cell lines by the calcium phosphate method and harvested for luciferase assays. Electromobility shift assays (EMSAs) were performed with wild-type and mutant versions of identified cis-regulatory elements and nuclear extracts from esophageal cell lines. RESULTS: A 1.1 kilobase fragment of the 5' flanking region of the human keratin 4 gene was cloned and sequenced. This region was found to be active in human esophageal cell lines. Functional dissection of the human keratin 4 promoter by deletion analysis revealed that a region from -163 to -140 basepairs (bp) contributed significantly to promoter activity. Further deletion of this region resulted in a 100-fold decrease in promoter activity. -163 to -140 contains as possible cis-regulatory elements overlapping E-box and AP-1 binding sites and a binding site for the keratinocyte specific factor (KSF), previously shown by us to be a novel zinc dependent transcriptional activator uniquely expressed in esophageal squamous epithelium. EMSA competitor experiments, along with transfection studies, suggest KSF and AP-1 transactivate region -163 to -140 of the human keratin 4 promoter in esophageal squamous epithelial cells. CONCLUSION: These data indicate that the human keratin 4 promoter is active in human esophageal cells with critical cis-regulatory elements between -163 and -140 bp. This region interacts with KSF and possibly AP-1, esophageal-specific and ubiquitous trans-activating nuclear factors, respectively. Taken together, promoter analysis and targeted deletion in mice strongly imply a role for keratin 4 in differentiation in the esophageal squamous epithelium. • G2714 MECHANISM OF INHIBITION OF BENZO(a)PYRENE-INDUCED FORESTOMACH CANCER IN MICE BY DIETARY CURCUMIN. J.L. Orchard, X. Hu, H. Xia, S.K. Srivastava, S.V. Singh, Pittsburgh, PA. The present study was undertaken to elucidate the mechanism(s) by which curcumin (diferuloylmethane), the major yellow pigment in turmeric, inhibits benzo(a)pyrene (BP)-induced forestomach cancer in mice. BP is a widespread environmental pollutant, abundant in cigarette smoke, auto exhaust etc., and believed to be a human carcinogen. Previous studies have shown that while cytochrome P4501A1 (CYP1A1) and epoxide hydrolase (EH) play an important role in the conversion of BP to its ultimate carcinogenic form, anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE), the detoxification of anti-BPDE is accomplished by glutathione S-transferases (GST). Therefore, it is reasonable to postulate that curcumin may exert anticarcinogenic activity either by inhibiting conversion of BP to anti-BPDE or (and) by enhancing the detoxification of anti-BPDE. The ethoxyresorufin O-deethylase (EROD) activity, which is a measure of CYP1A1 expression, was reduced by about 30% (/9 < 0.05, control vs. curcumin fed) in the liver of mice fed 2% curcumin diet for 14 days as compared with the mice fed control diet. The EROD activity could not be detected in the forestomach of either control or curcumin fed mice. Curcumin feeding caused a statistically significant increase in Eli activity in the liver (about 2.3-fold), but not in the forestomach of mice. While curcumin feeding did not alter forestomach GST activity, this activity in the liver of curcumin fed mice was significantly higher (about 2.4-fold) as compared with the mice fed control diet. Even though the levels of various hepatic GST isoenzymes were increased upon curcumin administration, maximum induction was noticed for the pi class GST isoenzyme, which among murine GSTs in highly efficient in the detoxification of anti-BPDE. On the other hand, the levels of forestomach GST isoenzymes were not affected by curcumin feeding. In conclusion, the results of the present study suggest that curcumin may inhibit BP-induced forestomach cancer in mice by affecting both the activation as well as inactivation pathways of BP metabolism in the liver. This study was supported in part by a NIH grant CA 55589. • G2715 CELL PROLIFERATION ANALYSIS OF FLAT AND POLYPOID ADENOMAS OF COLORECTUM. S. Orii, N. Murata, *S. Nakamura, M. Sugawara, H. Takahasi, Y. Fujino, C. Amano, M. Endo, S. Suzuki, Y. Takano, S. Sato. The First Department of Internal Medicine, *the Division of Pathology, Central Clinical Laboratory, Iwate Medical University, School of Medicine, Mofioka, Iwate, Japan. [Purpose] Recently, in Japan, numerous flat (elevated, depressed and mixed) adenomas of the colorectum have been diagnosed and resected
A65a AGA ABSTRACTS • G2711 RELATIONSHIP BETWEEN THE AGE AND THE SITE DISTRIBUTION OF COLORECTAL CANCER. M Okamot0. Y Yamaji, J Kato, T Ikenoue, M Takahashi, H Yoshida, T Kawabe, Y Shiratori and M Omata. Second Department of Internal Medicine, University of Tokyo, Japan. BACKGROUND: Colorectal cancer is one of the common neoplasms in the United States, Europe, and also Japan, Since the number of older people is increasing, the clinical feature of colorectal cancer in the older patients were analysed in this study. PATIENTS AND METHODS: From September 1995 to October 1997, we have performed total colonoscopy in 2228 patients consecutively (1554 males and 674 females, mean age 62 years old (range 11 to 93)). The characters of detected colorectal cancer proven histopathlogically by endoscopic polypectomy or biopsy were analysed in relation to the site and the stage. RESULTS: 171 colorectal cancer lesions were found in 165 patients. 13 lesions were found in the patients with <49 y.o., 34 in the patients aged 50s, 60 in aged 60s, 36 in aged 70s, and 28 in the patients with > 80 y.o.. The ratio of right side colon cancer (cecum, ascending colon, and transverse colon) was increased in association with the age, reached 61% in the patients with >80y.o. (Table) (p<0.05). Early colorectal cancer (carcinoma in situ and invading within the submucosa) was found in aproximately 50-60% in each age group. However, when these early colorectal cancers were classified as flat type and protruding type, the ratio of flat type was found to be increased in relation to the age, reached 46% in the patients with > 80y.o. (Table). age Rt side Ca. fiat type early Ca.
< 49 23% (3/13) 12% (1/8)
50s 12% (4/34) 17% (3/18)
60s 28% (17/60) 26% (11/42)
70s 47% (17/36) 33% (6/18)
> 80 61% (17/28) 46% (6/13)
CONCLUSION: In the older patients, fight side colon cancer was found to be increased, and flat type of early colorectal cancer was found to be increased. Total colonoscopy may be needed in the older people. • G2712 CELLULAR CItARACTERIZATION AND SUCCESSFUL TRANSFECTION OF SERIALLY SUBCULTURED NORMAL HUMAN ESOPHAGEAL KERATINOCYTES. OG 0piIz 1, CC Compton23, G Warland 2,3, H Nakagawa l, K Togawa 1, AK RustgiL IGastrointestinal Unit and 2Department of Pathology, Massachusetts General Hospital, Harvard Medical School and -~Shriner's Burn Institute, Boston, MA INTRODUCTION: The establishment of normal human esophageal epithelium in vitro would greatly enhance the ability to study the basic biological mechanisms regulating its proliferation and differentiation. Since normal human epidermal keratinocytes are known to be easily propagated in vitro when grown on feeder layers of irradiated 3T3 mouse fibroblasts, we adopted this strategy to successfully establish primary cultures of esophageal basal ceils and to expand this population through serial subcultivations. The cytokeration and growth factor profiles of confluent tertiary cultures were then analyzed by morphological, immunohistochemical and molecular techniques to define the basic properties of these cells. METHODS: Normal human esophagus specimens were received within 5-6 hr post mortem, and ensured to be free of infection, malignancy, or inflammation by gross and microscopic inspection. The mucosa was immediately dissected away from the muscularis propria and incubated in Dispase. The epithelium was pealed off with forceps and incubated in a mixture of trypsin-EDTA with collection of the supernatant. All cells recovered were plated onto feeder layers of irradiated 3T3-J2 mouse fibroblasts. Esophageal basal ceils were grown in a 3:1 mixture of Dulbecco's Modified Eagles Medium: Ham's F12 medium supplemented with 10% serum, hydrocortisone, cholera toxin, transferrin, triiodo-L-thyronine, insulin and human recombinant epidermal growth factor. Sections of fresh-frozen keratinocyte sheets were stained via the avidin-biotin peroxidase complex method with antibodies against keratins and growth factors. Cells were transiently transfected by the calcium phosphate method with a Rous-sarcoma virus (RSV)-luciferase reporter gene. RESULTS: Normal human esophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers and expanded through four serial subcultivations. Postconfluent cultures displayed a predominantly basal cell phenotype with limited stratification, widespread expression of proliferation markers such as keratins 5 and 14, and production of attachment of ~6[~4integrin and collagen VII. The ceils also expressed transforming growth factor-I~ and interleukin-ll3. Tertiary cultures are successfully transfected with an RSV luciferase reporter gene construct. CONCLUSIONS: Primary cultures of normal human esophageal cells can be successfully established with serial passages through extended numbers of cell generations. This is the first extensive characterization of such cells which can be transiently transfected as demonstrated for the first time. These cultures of normal esophageal cells can be ultimately used as models of differentiation, immortalized with retroviral vectors harboring oncogenes, and used to identify stem ceils.
• G2713 THE HUMAN KERATIN 4 PROMOTER IS REGULATED BY ESOPHAGEAL-SPECIFIC AND UBIQUITOUS TRANSCRIPTIONAL ACTIVATORS. OG Opitz 1, TD Jenkins l, T Inomoto l, AK Rustgi1,2. 1Gastrointestinal and 2Hematology/Oncology Units, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 INTRODUCTION: Keratin 4 is highly expressed in the suprabasal compartment of the esophageal squamous epithelium. We previously demonstrated that targeted disruption of the keratin 4 gene in mice leads to impaired differentiation with basal cell hyperplasia in the esophageal epithelium. To delineate molecular mechanisms underlying transctiptional regulation of keratin 4, we identified critical cis-regulatory elements within the human keratin 4 promoter and interacting trans-activating nuclear factors in human esophageal cells. METHODS: The human keratin 4 promoter was cloned using a PCR based strategy and sequenced. Seven deletion constructs were generated employing PCR based strategies and naturally occurring restriction enzyme sites. The constructs were subcloned into the pXP2 vector which contains an enhancedess luciferase reporter gene. All constructs were transfected in esophageal squamous epithelial cell lines by the calcium phosphate method and harvested for luciferase assays. Electromobility shift assays (EMSAs) were performed with wild-type and mutant versions of identified cis-regulatory elements and nuclear extracts from esophageal cell lines. RESULTS: A 1.1 kilobase fragment of the 5' flanking region of the human keratin 4 gene was cloned and sequenced. This region was found to be active in human esophageal cell lines. Functional dissection of the human keratin 4 promoter by deletion analysis revealed that a region from -163 to -140 basepairs (bp) contributed significantly to promoter activity. Further deletion of this region resulted in a 100-fold decrease in promoter activity. -163 to -140 contains as possible cis-regulatory elements overlapping E-box and AP-1 binding sites and a binding site for the keratinocyte specific factor (KSF), previously shown by us to be a novel zinc dependent transcriptional activator uniquely expressed in esophageal squamous epithelium. EMSA competitor experiments, along with transfection studies, suggest KSF and AP-1 transactivate region -163 to -140 of the human keratin 4 promoter in esophageal squamous epithelial cells. CONCLUSION: These data indicate that the human keratin 4 promoter is active in human esophageal cells with critical cis-regulatory elements between -163 and -140 bp. This region interacts with KSF and possibly AP-1, esophageal-specific and ubiquitous trans-activating nuclear factors, respectively. Taken together, promoter analysis and targeted deletion in mice strongly imply a role for keratin 4 in differentiation in the esophageal squamous epithelium. • G2714 MECHANISM OF INHIBITION OF BENZO(a)PYRENE-INDUCED FORESTOMACH CANCER IN MICE BY DIETARY CURCUMIN. J.L. Orchard, X. Hu, H. Xia, S.K. Srivastava, S.V. Singh, Pittsburgh, PA. The present study was undertaken to elucidate the mechanism(s) by which curcumin (diferuloylmethane), the major yellow pigment in turmeric, inhibits benzo(a)pyrene (BP)-induced forestomach cancer in mice. BP is a widespread environmental pollutant, abundant in cigarette smoke, auto exhaust etc., and believed to be a human carcinogen. Previous studies have shown that while cytochrome P4501A1 (CYP1A1) and epoxide hydrolase (EH) play an important role in the conversion of BP to its ultimate carcinogenic form, anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE), the detoxification of anti-BPDE is accomplished by glutathione S-transferases (GST). Therefore, it is reasonable to postulate that curcumin may exert anticarcinogenic activity either by inhibiting conversion of BP to anti-BPDE or (and) by enhancing the detoxification of anti-BPDE. The ethoxyresorufin O-deethylase (EROD) activity, which is a measure of CYP1A1 expression, was reduced by about 30% (/9 < 0.05, control vs. curcumin fed) in the liver of mice fed 2% curcumin diet for 14 days as compared with the mice fed control diet. The EROD activity could not be detected in the forestomach of either control or curcumin fed mice. Curcumin feeding caused a statistically significant increase in Eli activity in the liver (about 2.3-fold), but not in the forestomach of mice. While curcumin feeding did not alter forestomach GST activity, this activity in the liver of curcumin fed mice was significantly higher (about 2.4-fold) as compared with the mice fed control diet. Even though the levels of various hepatic GST isoenzymes were increased upon curcumin administration, maximum induction was noticed for the pi class GST isoenzyme, which among murine GSTs in highly efficient in the detoxification of anti-BPDE. On the other hand, the levels of forestomach GST isoenzymes were not affected by curcumin feeding. In conclusion, the results of the present study suggest that curcumin may inhibit BP-induced forestomach cancer in mice by affecting both the activation as well as inactivation pathways of BP metabolism in the liver. This study was supported in part by a NIH grant CA 55589. • G2715 CELL PROLIFERATION ANALYSIS OF FLAT AND POLYPOID ADENOMAS OF COLORECTUM. S. Orii, N. Murata, *S. Nakamura, M. Sugawara, H. Takahasi, Y. Fujino, C. Amano, M. Endo, S. Suzuki, Y. Takano, S. Sato. The First Department of Internal Medicine, *the Division of Pathology, Central Clinical Laboratory, Iwate Medical University, School of Medicine, Mofioka, Iwate, Japan. [Purpose] Recently, in Japan, numerous flat (elevated, depressed and mixed) adenomas of the colorectum have been diagnosed and resected