MMTV/LTR Promoter-Driven Transgenic Expression of EpCAM Leads to the Development of Large Pancreatic Islets

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

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Transgenic Expression of Hsc70 in Pancreatic Islets Enhances Autoimmune Diabetes in Response to β Cell Damage

The Journal of Immunology ◽

10.4049/jimmunol.0901288 ◽

2009 ◽

Vol 183 (9) ◽

pp. 5728-5737 ◽

Cited By ~ 17

Author(s):

Masih-ul Alam ◽

Julie A. Harken ◽

Anna-Maria Knorn ◽

Alisha R. Elford ◽

Kip Wigmore ◽

...

Keyword(s):

Pancreatic Islets ◽

Autoimmune Diabetes ◽

Cell Damage ◽

Β Cell ◽

Transgenic Expression

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The MMTV LTR Promoter is Induced by Progesterone and Dihydrotestosterone but not by Estrogen

Molecular Endocrinology ◽

10.1210/mend-2-2-143 ◽

1988 ◽

Vol 2 (2) ◽

pp. 143-147 ◽

Cited By ~ 68

Author(s):

Anne D. Otten ◽

Michel M. Sanders ◽

G. Stanley McKnight

Keyword(s):

Mmtv Ltr ◽

Ltr Promoter

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Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2303-2314.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2303-2314

Author(s):

H M Friedman ◽

A Yee ◽

H Diggelmann ◽

J C Hastings ◽

R Tal-Singer ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Mammalian Cells ◽

Murine Sarcoma Virus ◽

Sarcoma Virus ◽

Simplex Virus ◽

Mmtv Ltr ◽

Murine Sarcoma ◽

Ltr Promoter

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

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Reduced Toxicity of Trichothecenes, Isotrichodermol, and Deoxynivalenol, by Transgenic Expression of the Tri101 3-O-Acetyltransferase Gene in Cultured Mammalian FM3A Cells

Toxins ◽

10.3390/toxins11110654 ◽

2019 ◽

Vol 11 (11) ◽

pp. 654 ◽

Cited By ~ 3

Author(s):

Nozomu Tanaka ◽

Ryo Takushima ◽

Akira Tanaka ◽

Ayaki Okada ◽

Kosuke Matsui ◽

...

Keyword(s):

Mammalian Cells ◽

Wild Type ◽

Transgenic Expression ◽

Ic50 Values ◽

In The Wild ◽

Acetyl Group ◽

A Cell ◽

Reduced Toxicity ◽

Mmtv Ltr ◽

Fm3a Cells

In trichothecene-producing fusaria, isotrichodermol (ITDol) is the first intermediate with a trichothecene skeleton. In the biosynthetic pathway of trichothecene, a 3-O-acetyltransferase, encoded by Tri101, acetylates ITDol to a less-toxic intermediate, isotrichodermin (ITD). Although trichothecene resistance has been conferred to microbes and plants transformed with Tri101, there are no reports of resistance in cultured mammalian cells. In this study, we found that a 3-O-acetyl group of trichothecenes is liable to hydrolysis by esterases in fetal bovine serum and FM3A cells. We transfected the cells with Tri101 under the control of the MMTV-LTR promoter and obtained a cell line G3 with the highest level of C-3 acetylase activity. While the wild-type FM3A cells hardly grew in the medium containing 0.40 μM ITDol, many G3 cells survived at this concentration. The IC50 values of ITDol and ITD in G3 cells were 1.0 and 9.6 μM, respectively, which were higher than the values of 0.23 and 3.0 μM in the wild-type FM3A cells. A similar, but more modest, tendency was observed in deoxynivalenol and 3-acetyldeoxynivalenol. Our findings indicate that the expression of Tri101 conferred trichothecene resistance in cultured mammalian cells.

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Plasmalemma localisation of inorganic phosphate in B cells pancreas

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083515 ◽

1978 ◽

Vol 36 (2) ◽

pp. 528-529

Author(s):

F. B. P. Wooding ◽

K. Pedley ◽

N. Freinkel ◽

R. M. C. Dawson

Keyword(s):

Electron Microscope ◽

B Cells ◽

B Cell ◽

Pancreatic Islets ◽

High Glucose ◽

Lead Acetate ◽

Inorganic Phosphate ◽

Slight Modification ◽

Uranyl Acetate ◽

Lead Phosphate

Freinkel et al (1974) demonstrated that isolated perifused rat pancreatic islets reproduceably release up to 50% of their total inorganic phosphate when the concentration of glucose in the perifusion medium is raised.Using a slight modification of the Libanati and Tandler (1969) method for localising inorganic phosphate by fixation-precipitation with glutaraldehyde-lead acetate we can demonstrate there is a significant deposition of lead phosphate (identified by energy dispersive electron microscope microanalysis) at or on the plasmalemma of the B cell of the islets (Fig 1, 3). Islets after incubation in high glucose show very little precipitate at this or any other site (Fig 2). At higher magnification the precipitate seems to be intracellular (Fig 4) but since any use of osmium or uranyl acetate to increase membrane contrast removes the precipitate of lead phosphate it has not been possible to verify this as yet.

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Glucose regulation of secretory and metabolic pathway genes in human pancreatic islets

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-2005-862882 ◽

2005 ◽

Vol 113 (S 1) ◽

Author(s):

BJ Manfras ◽

P Hengster ◽

BO Böhm

Keyword(s):

Pancreatic Islets ◽

Metabolic Pathway ◽

Glucose Regulation ◽

Human Pancreatic Islets

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IONIC EFFECTS ON THE UPTAKE OF CHLOROMERCURIBENZENE-p-SULPHONIC ACID BY PANCREATIC ISLETS

Acta Endocrinologica ◽

10.1530/acta.0.0860552 ◽

1977 ◽

Vol 86 (3) ◽

pp. 552-560 ◽

Cited By ~ 3

Author(s):

Monica Söderberg ◽

Inge-Bert Täljedal

Keyword(s):

Pancreatic Islets ◽

Islet Cell ◽

Choline Chloride ◽

Inorganic Ions ◽

Sulphonic Acid ◽

Cell Uptake ◽

Β Cells ◽

Sulphydryl Groups ◽

Microdissected Pancreatic Islets ◽

Ionic Effects

ABSTRACT Effects of inorganic ions on the uptake of chloromercuribenzene-p-sulphonic acid (CMBS) were studied in microdissected pancreatic islets of non-inbred ob/ob-mice. Na2SO4 stimulated the total islet cell uptake of CMBS but decreased the amount of CMBS remaining in islets after brief washing with L-cysteine. CaCl2 stimulated both the total and the cysteine-non-displaceable uptake; the stimulatory effect of CaCl2 on the cysteine-non-displaceable CMBS uptake was counteracted by Na2SO4. NaCl, KCl or choline chloride had no significant effect on the total islet cell uptake of CMBS, whereas LiCl was stimulatory. It is concluded that β-cells resemble erythrocytes in having a permeation path for CMBS that is inhibited by SO42−. By analogy with existing models of the erythrocyte membrane, it is suggested that the SO42−-sensitive path leads to sulphydryl groups controlling monovalent cationic permeability in β-cells.

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