In vitro and in vivo Evaluation of 111In-labeled E. coli Heat-Stable Enterotoxin Analogs for Specific Targeting of Human Breast Cancers

2006 ◽  
Vol 98 (1) ◽  
pp. 7-15 ◽  
Author(s):  
Michael F. Giblin ◽  
Hariprasad Gali ◽  
Gary L. Sieckman ◽  
Nellie K. Owen ◽  
Timothy J. Hoffman ◽  
...  
Keyword(s):  
Breast Cancers ◽  
Human Breast ◽  
In Vivo Evaluation ◽  
E Coli ◽  
Specific Targeting ◽  
Heat Stable

In vitro and in vivo evaluation of 177Lu- and 90Y-labeled E. coli heat-stable enterotoxin for specific targeting of uroguanylin receptors on human colon cancers

2006 ◽  
Vol 33 (4) ◽  
pp. 481-488 ◽  
Author(s):  
Michael F. Giblin ◽  
Gary L. Sieckman ◽  
Tiffani D. Shelton ◽  
Timothy J. Hoffman ◽  
Leonard R. Forte ◽  
...  
Keyword(s):  
Human Colon ◽  
In Vivo Evaluation ◽  
E Coli ◽  
Specific Targeting ◽  
Colon Cancers ◽  
Heat Stable

scholarly journals Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

2000 ◽  
Vol 68 (7) ◽  
pp. 4064-4074 ◽  
Author(s):  
Isabelle Batisson ◽  
Maurice Der Vartanian ◽  
Brigitte Gaillard-Martinie ◽  
Michel Contrepois
Keyword(s):  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Biological Properties ◽  
Leader Peptide ◽  
Dependent Manner ◽  
Reporter Protein ◽  
E Coli ◽  
Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

Human breast cancers respond to growth factors in vivo but not in vitro

1994 ◽  
Vol 85 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Jason Yang ◽  
Nikolay K. Popnikolov ◽  
Ramasamy Sakthivel ◽  
Satyabrata Nandi
Keyword(s):  
Growth Factors ◽  
Breast Cancers ◽  
Human Breast

scholarly journals Immunohistochemical detection of GHRH and its receptor splice variant 1 in primary human breast cancers

2004 ◽  
pp. 391-396 ◽  
Author(s):  
I Chatzistamou ◽  
AV Schally ◽  
H Kiaris ◽  
E Politi ◽  
J Varga ◽  
...  
Keyword(s):  
Splice Variant ◽  
Polyclonal Antibodies ◽  
Breast Tumors ◽  
In Vivo Studies ◽  
Breast Cancers ◽  
Human Breast ◽  
Ghrh Receptor ◽  
Primary Human Breast

OBJECTIVE: GHRH is secreted by the hypothalamus and, upon binding to specific GHRH receptors in the pituitary, stimulates growth hormone (GH) production and release from the pituitary. In addition to this neuroendocrine action, accumulated evidence implies additional roles for GHRH in carcinogenesis in non-pituitary tissues. In vitro and in vivo studies have shown that splice variant 1 (SV1) of the GHRH receptor, which is widely expressed in non-pituitary tissues and cancers, can mediate the proliferative effects of GHRH. The aim of the present study was to investigate the operation of an autocrine stimulatory loop between GHRH and SV1 in primary breast tumors. DESIGN: Fifty-three primary breast tumors were evaluated for GHRH and SV1 expression. METHODS: Expression of GHRH and SV1 was assessed by immunohistochemistry using anti-GHRH SV95 and anti-SV1 2317/5 polyclonal antibodies. RESULTS: About 40% of the specimens tested express GHRH and/or SV1 (approx. 25% each), while in 35% of these positive specimens co-expression of these antigens was detected (P<0.01). Furthermore, a correlation of GHRH, but not SV1, expression was detected in lobular compared with ductal carcinomas. CONCLUSIONS: These results constitute the first demonstration for the expression of GHRH and SV1 in primary breast cancers, and provide evidence for the operation of an autocrine stimulatory loop between GHRH and SV1 in primary cancers. Our findings indicate that GHRH analogs could have diagnostic and therapeutic applications for the management of breast cancer.

How to target estrogen receptor-negative breast cancer?

2003 ◽  
pp. 261-266 ◽  
Author(s):  
H Rochefort ◽  
M Glondu ◽  
M E Sahla ◽  
N Platet ◽  
M Garcia
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Cancer Cells ◽  
Targeted Therapies ◽  
Cathepsin D ◽  
Human Breast Cancer ◽  
Breast Cancers ◽  
Human Breast

Estrogen receptor (ER)-positive breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy, which is the first example of a successful therapy targeted on a specific protein, the ER. Unfortunately ER-negative breast cancers are more aggressive and unresponsive to anti-estrogens. Other targeted therapies are thus urgently needed, based on breast cancer oncogene inhibition or suppressor gene activation as far as molecular studies have demonstrated the alteration of expression, or structure of these genes in human breast cancer. Using the MDA-MB.231 human breast cancer cell line as a model of ER-negative breast cancers, we are investigating two of these approaches in our laboratory. Our first approach was to transfect the ER or various ER-deleted variants into an ER-negative cell line in an attempt to recover anti-estrogen responsiveness. The unliganded receptor, and surprisingly estradiol, were both found to inhibit tumor growth and invasiveness in vitro and in vivo. The mechanisms of these inhibitions in ER-negative cancer cells are being studied, in an attempt to target the ER sequence responsible for such inhibition in these cancer cells. Another strategy is trying to inhibit the activity or expression of an oncogene specifically overexpressed in most breast cancers. This approach was recently shown by others to be efficient in breast cancer therapy with HER2-Neu oncogene amplification using Herceptin. Without excluding other molecular putative targets, we have focused our research on cathepsin D as a potential target, since it is often overexpressed in aggressive human breast cancers, including ER-negative tumors, and rarely associated with HER2-Neu amplification. Our first results obtained in vitro on cell lines and in vivo in tumor xenografts in nude mice, illustrate that the mode of action of cathepsin D in breast cancer is useful to guide the development of these therapies. In the past 20 years we have learned that the action of cathepsin D is complex and involves both intracellular and extracellular activities due to its proteolytic activity and to interactions with membrane components without catalytic activity. Each of these mechanisms could be potentially inhibited in an attempt to prevent tumor growth. Breast cancer is a very heterogeneous and multigenic disease and different targeted therapies adapted to each category of breast cancer are therefore required. Validated assays in the primary tumor of molecular markers such as ER, HER2-Neu and cathepsin D should help to predict which targeted therapy should be applied to cure breast cancer patients.

scholarly journals Estradiol Regulates Different Genes in Human Breast Tumor Xenografts Compared with the Identical Cells in Culture

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 700-713 ◽  
Author(s):  
Djuana M. E. Harvell ◽  
Jennifer K. Richer ◽  
D. Craig Allred ◽  
Carol A. Sartorius ◽  
Kathryn B. Horwitz
Keyword(s):  
Gene Expression ◽  
Breast Tumor ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Tumor Model ◽  
Class Ii ◽  
Breast Cancers ◽  
Human Breast

In breast cancers, estrogen receptor (ER) levels are highly correlated with response to endocrine therapies. We sought to define mechanisms of estrogen (E) signaling in a solid breast tumor model using gene expression profiling. ER+ T47D-Y human breast cancer cells were grown as xenografts in ovariectomized nude mice under four conditions: 1) 17β-estradiol for 8 wk (E); 2) without E for 8 wk (control); 3) E for 7 wk followed by 1 wk of E withdrawal (Ewd); or 4) E for 8 wk plus tamoxifen for the last week. E-regulated genes were defined as those that differed significantly between control and E and/or between E and Ewd or control and Ewd. These protocols generated 188 in vivo E-regulated genes that showed two major patterns of regulation. Approximately 46% returned to basal states after Ewd (class I genes); 53% did not (class II genes). In addition, more than 70% of class II-regulated genes also failed to reverse in response to tamoxifen. These genes may be interesting for the study of hormone-resistance issues. A subset of in vivo E-regulated genes appears on lists of clinical ER discriminator genes. These may be useful therapeutic targets or markers of E activity. Comparison of in vivo E-regulated genes with those regulated in identical cells in vitro after 6 and 24 h of E treatment demonstrate only 11% overlap. This indicates the extent to which gene expression profiles are uniquely dependent on hormone-treatment times and the cellular microenvironment.

scholarly journals THE FATE OF BACTERIA WITHIN PHAGOCYTIC CELLS

1964 ◽  
Vol 120 (5) ◽  
pp. 869-883 ◽  
Author(s):  
Zanvil A. Cohn
Keyword(s):  
Alveolar Macrophages ◽  
Peritoneal Macrophages ◽  
Immune Serum ◽  
Soluble Form ◽  
Phagocytic Cells ◽  
E Coli ◽  
Heat Stable ◽  
Immune Globulins

The fate of a heat-stable Escherichia coli agglutinogen within three types of rabbit phagocytic cells was examined. A system is described whereby quantitative ingestion of viable E. coli by suspensions of PMN leucocytes, BCG-induced alveolar macrophages, and oil-induced peritoneal macrophages took place in vitro. After various periods of intracellular residence aliquots were injected intraperitoneally into NCS mice and the resulting agglutinins assayed. The loss of immunogenicity within phagocytes was estimated by comparison with a dose-response titration prepared with bacteria alone. Under these conditions no increase in immunogenic mass occurred in vivo or in vitro when viable organisms were employed. PMN leucocytes and alveolar macrophages destroyed the majority of the immunogen within 2 hours of intracellular residence. In contrast, the immunogenicity of E. coli was maintained within peritoneal macrophages for periods up to 5 hours. The use of heat-killed bacilli or specific immune serum did not significantly influence the intracellular fate of the immunogen. Residual immunogenicity was associated with a particle having the same centrifugal properties as the intact organism and essentially none was released in a soluble form. Intracellular residence within phagocytic cells did not influence the resulting temporal sequence of antibody formation nor the proportions of mercaptoethanol-sensitive and resistant immune globulins.

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