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A monoclonal antibody specific for paneth and neuroendocrine cell in the human intestine
Cell Biology
International
Reports,
Vol. 74, Abstracts
AND F’ROLIFERATfON IN A CULTURED HUMAN EPfTf+ELlLfM Yves Poumay, Annie Degen, Franpoise Boucher, Robert Leloup. DBpartement d’Histologie. Facult& Universitaires Notre-Dame de la Paix, Namur, Belgium. In vivo, only human keratinocytes (HK) present in Lhe basal and first suprabasal layer of a stratified epithelium are able to synthesize DNA. However, mechanisms involved to keep cells of the basal layers in a highly proliferative and undifferentiated state are certainly not fully understood. Studying cultured HK in context of their clinical use. which means detachment of intact epithelial sheets from culture substratum by a 30-60 min. incubation with dispase, we were interested to investigate whether cultured cells were still able 10 enter the S-phase of DNA synthesis in these conditions. In the present work. we first investigated whether proliferating cells are preferentially those present in the basal layer and then whether anchorage lo the culture substratum is necessary for this capacity. Confluent cultured epithelia were detached by incubation with dispase and then reincubated in their growth medium for 0 to 24 h. The S-phase was detected by 60 min. of incubation with bromodeoxyuridine (Brdlf) and the immunofluorescence staining of paraffin sections allowed
P669
BAsALcEuf? ANCMORAGE
determination of the labelling index (LI). Incorporation of 3Hthymkfine was also determined in the same conditions. First, the immunofluorescence revealed that only HK of the basal layer, previously anchored to the culture substratum, were positive for BrdU incorporation. Second, our experiments revealed that detachment from the substratum produced a progressive fall in the LI, together with a decreased incorporation of 3H-thymidine. These results suggest that basal HK enclosed in an epithelial sheet have lost their proliferating activity when unanchored to culture substratum. This confirms that cell attachment and spreading is essential for the proliferative potential of HK Whether the vicinity of basement membrane components cooperates to keep basal HK as proliferating cells remains to be established.
Supplement
1990
P690
251
TKE EPITHELIAL PDV AND PDVCSiB CELL LINES: A SYSl3.l TO STUDY TRANSFORMATION .+WIGENS
Albert0 Gandarillas, Carlos Caulin, &nparo Cane, Miguel Quintanilla. Instituto de Investigaciones Biomidicas CSIC. Dpto Bioquimica U&f. 28029 adrid Spain The PDV cell line was obtained from a primary culture of normal mouse euidermis [strain: CSiBU61 treated in vitro by dime&vlbenz(ajnthracene (Fusehi: NE. et al. Bull.Cancer, 1978, 65:2X-230). PDV cells are tumorogenic in nude mice but rarely give tumors in syngeneic mice. From one PDV-induced tumor in a CSiB1/6 mouse the PDVCSiB cell line was derived, after two cycles of tUrnor induction. PDVCSiB cells are highly tumorogenic in both nude and syngeneic rice. Using an antisenrm raised against whole cells growth in suspension, we have detected tir, antigens: p46 and ~110, both present in PDV but absent in FDVCSiB cells. p46 is a membrane antigen which disappear in tumors induced by PDV either in syn_ zeneic or nude mice, suggesting a role as a tLrmOr transplantation or tumor rejection antigen. p110 could be detected in the high-salt insoluble fraction of the cells and, apparently,is associated to the intermediate filament component of cytoskeleton. We are now in the process of developing mono clonal antibodies in order to study biochemical and functional characteristics of these antigens. Supported
P692
by EWXI’
(PM88-0004)
DIFFERENTIATION-SPE4XFIC INRATANDPIGSMALL-
ANTIGENS
Timothy P King’, Denise Kelly’, Andrea Quaroni2. ‘Nutrition Division, Rowett Research Institute, Aberdeen, Scotland, AB2 9SB. UK. ‘Section of Physiology, Division of Biological Sciences, Cornell University, Ithaca, NY 14853 USA. Monoclonal antibody FBB2/29 was prepared against luminal membranes purified from rat intestinal cells at day 19 of gestation. it has been previously demonstrated that in the rat FBB2/29 recognizes high molecular mass glycoprotein antigens expressed during fetal intestinal development and retained by intestinal crypt cells at all times. The antigens are also expressed by rat intestinal turnours, human colonic adenocarcinomas and the CaCo-2 human tumour colonic cell line. The structure of the FBB2/29 antigen has not yet been determined. In the present study electron microscope immunogold Lytochemistry was used to determine the ,&cellular distribution of the FBB2EJ antigen in adult rat and suckling pig small intestines. In undifferentiated crypt cells FBBZiL9 antigen was associated with Golgi body cisternae and apical secretion granules and was also located on the luminai surCaces of the microvilli. The ultrastructural cytochemistry iidditionally revealed the presence of FBB2/2’) antigen in trans Golgi cisternae of villus goblet cells. Double labelling with FBB2129 antibody and other defined monoclonal antibodies and lectins specific for blood-group related antigens suggests that the FBB2/29 determinant is structurally similar to the oligosaccharide backbone sequences of such glycoconjugates.
International
Reports,
Vol. 74, Abstracts
AND F’ROLIFERATfON IN A CULTURED HUMAN EPfTf+ELlLfM Yves Poumay, Annie Degen, Franpoise Boucher, Robert Leloup. DBpartement d’Histologie. Facult& Universitaires Notre-Dame de la Paix, Namur, Belgium. In vivo, only human keratinocytes (HK) present in Lhe basal and first suprabasal layer of a stratified epithelium are able to synthesize DNA. However, mechanisms involved to keep cells of the basal layers in a highly proliferative and undifferentiated state are certainly not fully understood. Studying cultured HK in context of their clinical use. which means detachment of intact epithelial sheets from culture substratum by a 30-60 min. incubation with dispase, we were interested to investigate whether cultured cells were still able 10 enter the S-phase of DNA synthesis in these conditions. In the present work. we first investigated whether proliferating cells are preferentially those present in the basal layer and then whether anchorage lo the culture substratum is necessary for this capacity. Confluent cultured epithelia were detached by incubation with dispase and then reincubated in their growth medium for 0 to 24 h. The S-phase was detected by 60 min. of incubation with bromodeoxyuridine (Brdlf) and the immunofluorescence staining of paraffin sections allowed
P669
BAsALcEuf? ANCMORAGE
determination of the labelling index (LI). Incorporation of 3Hthymkfine was also determined in the same conditions. First, the immunofluorescence revealed that only HK of the basal layer, previously anchored to the culture substratum, were positive for BrdU incorporation. Second, our experiments revealed that detachment from the substratum produced a progressive fall in the LI, together with a decreased incorporation of 3H-thymidine. These results suggest that basal HK enclosed in an epithelial sheet have lost their proliferating activity when unanchored to culture substratum. This confirms that cell attachment and spreading is essential for the proliferative potential of HK Whether the vicinity of basement membrane components cooperates to keep basal HK as proliferating cells remains to be established.
Supplement
1990
P690
251
TKE EPITHELIAL PDV AND PDVCSiB CELL LINES: A SYSl3.l TO STUDY TRANSFORMATION .+WIGENS
Albert0 Gandarillas, Carlos Caulin, &nparo Cane, Miguel Quintanilla. Instituto de Investigaciones Biomidicas CSIC. Dpto Bioquimica U&f. 28029 adrid Spain The PDV cell line was obtained from a primary culture of normal mouse euidermis [strain: CSiBU61 treated in vitro by dime&vlbenz(ajnthracene (Fusehi: NE. et al. Bull.Cancer, 1978, 65:2X-230). PDV cells are tumorogenic in nude mice but rarely give tumors in syngeneic mice. From one PDV-induced tumor in a CSiB1/6 mouse the PDVCSiB cell line was derived, after two cycles of tUrnor induction. PDVCSiB cells are highly tumorogenic in both nude and syngeneic rice. Using an antisenrm raised against whole cells growth in suspension, we have detected tir, antigens: p46 and ~110, both present in PDV but absent in FDVCSiB cells. p46 is a membrane antigen which disappear in tumors induced by PDV either in syn_ zeneic or nude mice, suggesting a role as a tLrmOr transplantation or tumor rejection antigen. p110 could be detected in the high-salt insoluble fraction of the cells and, apparently,is associated to the intermediate filament component of cytoskeleton. We are now in the process of developing mono clonal antibodies in order to study biochemical and functional characteristics of these antigens. Supported
P692
by EWXI’
(PM88-0004)
DIFFERENTIATION-SPE4XFIC INRATANDPIGSMALL-
ANTIGENS
Timothy P King’, Denise Kelly’, Andrea Quaroni2. ‘Nutrition Division, Rowett Research Institute, Aberdeen, Scotland, AB2 9SB. UK. ‘Section of Physiology, Division of Biological Sciences, Cornell University, Ithaca, NY 14853 USA. Monoclonal antibody FBB2/29 was prepared against luminal membranes purified from rat intestinal cells at day 19 of gestation. it has been previously demonstrated that in the rat FBB2/29 recognizes high molecular mass glycoprotein antigens expressed during fetal intestinal development and retained by intestinal crypt cells at all times. The antigens are also expressed by rat intestinal turnours, human colonic adenocarcinomas and the CaCo-2 human tumour colonic cell line. The structure of the FBB2/29 antigen has not yet been determined. In the present study electron microscope immunogold Lytochemistry was used to determine the ,&cellular distribution of the FBB2EJ antigen in adult rat and suckling pig small intestines. In undifferentiated crypt cells FBBZiL9 antigen was associated with Golgi body cisternae and apical secretion granules and was also located on the luminai surCaces of the microvilli. The ultrastructural cytochemistry iidditionally revealed the presence of FBB2/2’) antigen in trans Golgi cisternae of villus goblet cells. Double labelling with FBB2129 antibody and other defined monoclonal antibodies and lectins specific for blood-group related antigens suggests that the FBB2/29 determinant is structurally similar to the oligosaccharide backbone sequences of such glycoconjugates.