scholarly journals A novel method for isolation and culture of primary swine gastric epithelial cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Henry Bautista-Amorocho ◽  
Jorge Alexander Silva-Sayago ◽  
Diego A. Goyeneche-Patino ◽  
Tania Liseth Pérez-Cala ◽  
Fabio Macías-Gómez ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Growth Factors ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Protease Inhibitors ◽  
Cell Lines ◽  
Cell Phenotype ◽  
Swine Model ◽  
Fundic Gland ◽  
Gastric Epithelial Cells

Abstract Background Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach. Results Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively. Conclusions A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

scholarly journals A novel method for isolation and culture of primary swine gastric epithelial cells

2020 ◽  
Author(s):  
Henry Bautista-Amorocho ◽  
Jorge Alexander Silva-Sayago ◽  
Diego A. Goyeneche-Patino ◽  
Tania Liseth Pérez-Cala ◽  
Fabio Macías-Gómez ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Growth Factors ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Protease Inhibitors ◽  
Cell Lines ◽  
Cell Phenotype ◽  
Swine Model ◽  
Fundic Gland ◽  
Gastric Epithelial Cells

Abstract Background: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.Results: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.Conclusions: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

scholarly journals A novel method for isolation and culture of primary swine gastric epithelial cells

2021 ◽  
Author(s):  
Henry Bautista-Amorocho ◽  
Jorge Alexander Silva-Sayago ◽  
Diego A. Goyeneche-Patino ◽  
Tania Liseth Pérez-Cala ◽  
Fabio Macías-Gómez ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Growth Factors ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Protease Inhibitors ◽  
Cell Lines ◽  
Cell Phenotype ◽  
Swine Model ◽  
Fundic Gland ◽  
Gastric Epithelial Cells

Abstract Background: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.Results: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.Conclusions: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

scholarly journals A Novel Methodology For Isolation and Primary Culture of Swine Gastric Epithelial Cells.

2020 ◽  
Author(s):  
Henry Bautista-Amorocho ◽  
Jorge Alexander Silva-Sayago ◽  
Diego A. Goyeneche-Patino ◽  
Tania Liseth Pérez-Cala ◽  
Fabio Macías-Gómez ◽  
...  
Keyword(s):  
Growth Factors ◽  
Epithelial Cells ◽  
Protease Inhibitors ◽  
Swine Model ◽  
Type I ◽  
Fundic Gland ◽  
Gastric Epithelial Cells ◽  
Gland Area ◽  
Fundic Gland Area ◽  
Functional Phenotype

Abstract Background: Culture of primary epithelial cells has a great advantage over tumor-derived or immortal cells lines since functional phenotype and genetic makeup are preserved. Swine model has proved to be helpful and reliable as a surrogate model in human diseases. Several porcine cell lines have been established from a variety of tissues and shown to extensively contribute to the current understanding of several pathologies, including cancer. However, few protocols for the isolation and culture swine gastric epithelial cells with phenotype preservation have been described. Therefore, the objective of this research was to develop a new methodology for establishing a primary cell culture from the fundic gland area of the porcine stomach.Results: Enzymatic disaggregation of gastric tissue by using a combination of collagenase type I and dispase II, protease inhibitors (soybean trypsin inhibitor and bovine serum albumin), and antioxidants (Dithiothreitol) allowed the isolation of gastric epithelial cells from the fundic gland area with viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM HG, and DMEM/F12 media did not lead to cell adhesion, cluster formation and cell proliferation. By contrast, Williams’ medium supplemented with growth factors supports the confluence and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC in a 5% CO2 incubator. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining as well as the expression of MUC1 and MUC20 genes by RT-PCR and DNAc sequencing. Swine Gastric epithelial cells also showed origin-specific markers such as epithelial membrane antigen (EMA), cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) detected by immunohistochemistry and immunofluorescence, respectively. Conclusions: A new methodology was successfully established for the isolation of primary gastric epithelial cells from the fundic gland area in a swine model, based on a combination of tissue specific proteases, protease inhibitors, and antioxidants. The formulation of Williams’ medium with growth factors for epithelial cells supports the adherence and maintains the functional phenotype of primary cells, which was confirmed by mucin production and expression of typical epithelial markers in the long term.

Human gastric epithelial cell lines derived from primary cultures of normal gastric epithelial cells

2000 ◽  
Vol 118 (4) ◽  
pp. A540-A541 ◽  
Author(s):  
Duane T. Smoot ◽  
Cornell R. Allen ◽  
Pamela Barnes ◽  
Milton Brown ◽  
Suhas Phadnis ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Primary Cultures ◽  
Gastric Epithelial Cell ◽  
Gastric Epithelial Cells ◽  
Epithelial Cell Lines

scholarly journals Modulation of cell adhesion and migration by poly-dispersed-acid-functionalized-single-walled carbon nanotubes in lung epithelial cells

2020 ◽  
Author(s):  
Sushreesangita P. Behera ◽  
Rajiv K. Saxena
Keyword(s):  
Carbon Nanotubes ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Single Walled Carbon Nanotubes ◽  
Lung Epithelial ◽  
And Migration ◽  
Walled Carbon Nanotubes

AbstractEpithelial cell lining of the lung alveoli is under constant onslaught of airborne pathogens and pollutants that may cause injury and disruption of the epithelial lining. Repair mechanisms involve proliferation and migration of nearby healthy epithelial cells to the site of injury. Using murine LA4 and human A549 lung epithelial cell lines, and in vitro models of cell migration we have examined the modulation of cellular adhesion and migration by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs). Flow cytometric and confocal microscopy studies indicated that AF-SWCNTs were efficiently internalized by both cell lines and were localized essentially in the cytoplasmic area. In the scratch wound repair model, exposure to AF-SWCNTs blocked the filling of the scratched area of the cellular monolayers in both cells. Behaviour of the cells around the scratch area was examined in by live-cell imaging time-lapse micrography. The results indicated active cell proliferation around the scratch area that was totally blocked by AF-SWCNTs in LA4 cells and significantly inhibited in A549 cells. Cell migration across a porous membrane in transwell assay system also indicated a marked inhibition of migration of both cells across the membrane. Effect of AF-SWCNTs on the expression levels of important cell proteins involved in cell migration and adhesion were examined by western blotting and immunofluorescence staining. Expressions of proteins like β-Catenin, NM-Myosin and Vimentin that play crucial role in cell migration were suppressed in AF-SWCNTs-exposed cells whereas the expression levels of E-cadherin and Claudin-1, involved in cell-cell adhesion remained unaltered. Our results provide an insight into the mechanism of repair of lung epithelial cell layers.

Assessment of Proliferation Induced in Fibroblasts and Rabbit Corneal Epithelial Cells by a Platelet Lysate Formulation: A Stability Study

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4072-4072
Author(s):  
Carla Caramella ◽  
Maria Cristina Bonferoni ◽  
Silvia Rossi ◽  
Giuseppina Sandri ◽  
Sara Gibin ◽  
...  
Keyword(s):  
Growth Factors ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Growth Medium ◽  
Storage Time ◽  
Platelet Lysate ◽  
Corneal Epithelial Cell ◽  
Corneal Epithelial ◽  
Proliferative Effect

Abstract Introduction. Platelets contain a mixture of growth factors (GF). These can be released from platelet lysate (PL) whose application to wounds is supposed to favour the healing process. The application of PL in a suitable formulation can improve therapeutic efficacy and patient compliance. A formulation of PL with a selected biocompatible vehicle has been developed for application in buccal mucosal damages (mucositis), that are the most common complications of the nonsurgical therapy of cancer. The same vehicle can be proposed also for ophthalmic formulations as several studies have shown that topical application of the growth factors to the injured cornea facilitates wound repair by rapid regrowth of the epithelial cells. Aim of the present work was to develop in vitro tests for a fast evaluation of PL activity in the formulation to evidence excipient compatibility, and the effect of formulative parameters on PL stability. The proliferative effect on two different models, fibroblasts and a corneal epithelial cell line, was evaluated. The stability of PL in the formulation has been assessed after two weeks at 4–8 °C storage. Methods. PL was mixed in a 1:1 ratio to the selected vehicle and stored at 4–8 °C. Immediately after preparation (time zero, T0) and after 2, 7, 10 and 15 days (T2–T15), aliquots were used to assess proliferative effect on cell cultures. Both fibroblast (primary human cell lines) and RCE (Rabbit corneal epithelial cell lines, ECACC) were seeded in 96-well plates with area of 0.34 cm2 at the concentrations of either 10000 or 5000 cells/well. After 24 hours the cells were added with either complete growth medium (Reference), minimal medium not supplemented with foetal calf serum and for REC cells without also EGF (Control), PL formulation diluted 1/20 (containing PL at 5%) and PL formulation diluted 1/40 (containing PL at 2.5%). After 24 hours, neutral red (NR) test was performed (Tox Kit 4, Sigma-Aldrich, Milano I). The NR solution absorbance was determined by means of a plate reader (Perkin Elmer, Milan, I) at wavelength of 490 nm. The absorbance read for each sample was compared with that of Reference, whose proliferation was assumed as 100%. Results. At time zero, the percentage of proliferation showed no significant differences with respect to the cells in complete growth medium (Reference) in the case of fibroblasts, both at 5000 and 10000 seeding level. The two cell models gave comparable results although slightly higher values of proliferation were obtained in fibroblasts. This can be attributed to the epithelial nature of the RCE and to the possible different percentage in the platelet derived mixture of FGF and EGF. In both cases it can be argued that the vehicle is compatible with cell growth. Both for fibroblasts and for RCE no decrease of activity with time was observed, and no statistical differences with respect to the control have been obtained until 15 days storage. (See figure 1). The results obtained suggest that the two cell culture models evaluated can be suitable to assess the activity of PL in easy and fast way. This will be useful to develop formulations intended for the treatment of mucositis and of damages of corneal epithelia. In particular, the formulation tested seems to be stable and to allow the release of GF from PL until 15 days of storage. Figure 1. Proliferation (5000 cells/well) induced by the PL formulation as a function of storage time. The dotted line indicates the proliferation of the Reference (assumed as 100%). Figure 1. Proliferation (5000 cells/well) induced by the PL formulation as a function of storage time. The dotted line indicates the proliferation of the Reference (assumed as 100%).

Redistribution and dysfunction of integrins in cultured renal epithelial cells exposed to oxidative stress

1993 ◽  
Vol 264 (1) ◽  
pp. F149-F157 ◽  
Author(s):  
J. Gailit ◽  
D. Colflesh ◽  
I. Rabiner ◽  
J. Simone ◽  
M. S. Goligorsky
Keyword(s):  
Oxidative Stress ◽  
Cell Adhesion ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Apical Cell ◽  
Flow Cytometric Analysis ◽  
Adhesion Properties ◽  
Renal Tubular Cells ◽  
Renal Tubular

Tubular obstruction by detached renal tubular epithelial cells is a major cause of oliguria in acute renal failure. Viable renal tubular cells can be recovered from urine of patients with acute tubular necrosis, suggesting a possible defect in cell adhesion to the basement membrane. To study this process of epithelial cell desquamation in vitro, we investigated the effect of nonlethal oxidative stress on the integrin adhesion receptors of the primate kidney epithelial cell line BS-C-1. Morphological and functional studies of cell adhesion properties included the following: interference reflection microscopy, intravital confocal microscopy and immunocytochemistry, flow cytometric analysis of integrin receptor abundance, and cell-matrix attachment assay. High levels of the integrin subunits alpha 3, alpha v, and beta 1 were detected on the cell surface by fluorescence-activated cell sorting (FACS) analysis, as well as lower levels of alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, and beta 3. Exposure of BS-C-1 cells to nonlethal oxidative stress resulted in the disruption of focal contacts, disappearance of talin from the basal cell surface, and in the redistribution of integrin alpha 3-subunits from predominantly basal location to the apical cell surface. As measured in a quantitative cell attachment assay, oxidative stress decreased BS-C-1 cell adhesion to type IV collagen, laminin, fibronectin, and vitronectin. Defective adhesion was not associated with a loss of alpha 3-, alpha 4-, or alpha v-integrin subunits from the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

2011 ◽  
Vol 301 (2) ◽  
pp. C522-C529 ◽  
Author(s):  
Justine Elliott ◽  
Nadezhda N. Zheleznova ◽  
Patricia D. Wilson
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Collecting Duct ◽  
Specific Inhibitor ◽  
Cell Phenotype ◽  
Epithelial Cell Proliferation ◽  
Matrix Adhesion ◽  
Cyst Lining

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY418)-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY418-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α2β1-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase–CD26 interaction

2002 ◽  
Vol 361 (2) ◽  
pp. 203-209 ◽  
Author(s):  
Silvia GINÉS ◽  
Marta MARIÑO ◽  
Josefa MALLOL ◽  
Enric I. CANELA ◽  
Chikao MORIMOTO ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Adhesion ◽  
T Cell ◽  
B Cells ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Adenosine Deaminase ◽  
Cell To Cell Adhesion ◽  
Lymphocyte Cell

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA—CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50–70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA—CD26 interaction in the lymphocyte—epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA—CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA—CD26 interaction on the cell surface has a role in lymphocyte—epithelial cell adhesion.

scholarly journals Alterations of p53 and c-myc in the clonal evolution of malignant lymphoma

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 452-459 ◽  
Author(s):  
H Chang ◽  
S Benchimol ◽  
MD Minden ◽  
HA Messner
Keyword(s):  
Growth Factors ◽  
T Cell ◽  
Cell Lines ◽  
P53 Protein ◽  
Clonal Evolution ◽  
Cdna Probe ◽  
Cell Phenotype ◽  
Coding Region ◽  
P53 Genes

Abstract We derived the lymphoma cell lines OCI-Ly 13.1 and OCI-Ly 13.2 from a patient with non-Hodgkin's lymphoma at the time of presentation and during chemotherapy-resistant relapse. These lines were of T-cell phenotype and contained the identical T-cell receptor beta-chain rearrangement, indicating that both lines were members of the same malignant clone. The lines differed in their growth characteristics; OCI-Ly 13.1 grew slowly and required growth factors for colony formation, whereas OCI-Ly 13.2 grew rapidly and formed colonies without addition of growth factors. To test whether or not these biologic differences were associated with specific genetic changes, we evaluated the status of the c-myc and p53 genes of both cell lines. The p53 and c- myc genes of OCI-Ly 13.1 were in germline configuration and produced normal-sized transcripts. The p53 protein expressed in OCI-Ly 13.1 was recognized by the anti-p53 monoclonal antibody, PAb240, indicating a conformation typical of p53 proteins expressed by p53 alleles containing a missense mutation. However, sequencing studies of the entire p53 coding region did not reveal any point mutations. In contrast, the cell line OCI-Ly 13.2 contained structural abnormalities of both the c-myc and p53 genes. In addition, one of the p53 alleles was lost as determined by a cDNA probe for the p53 gene (17p 13.1) and the YNZ22.1 probe (17p 13.3). These changes resulted in the absence of p53 protein and mRNA in OCI-Ly 13.2 as detected by immunoprecipitation and Northern blot analysis, respectively. They may be a reflection of disease progression and may be associated with the altered behavior of the malignant cell population within the patient and in vitro.

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