Immune-Deficient Animals

DOI: 10.1159/000408646

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Models for Human Tumor Therapy in Nude Mice

David P. Houchens¹,

Artemio A. Ovejera²

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Evaluation Of Antineoplastic Effects1

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“…The growing tumors were measured twice a week with a caliper, and tumor volumes were calculated using width (a) and length (b) measurements (a 2 b/2, where a Ͻ b) (37). For intracerebral stereotactic implantation, 1 ϫ 10 5 -5 ϫ 10 5 cells in 5 l of PBS were inoculated into the corpus striatum in the right hemisphere of the nude mouse brain (38).…”

Section: Methodsmentioning

confidence: 99%

The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling

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et al. 1997

Journal of Biological Chemistry

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Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named ⌬EGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active ⌬EGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of ⌬EGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation. Ligand binding to wild-type epidermal growth factor receptor (wt EGFR)1 results in receptor dimerization, kinase activation, and autophosphorylation that provides both docking sites for proteins involved in signal transduction and exposure of endocytic and lysosomal targeting sequence codes required for receptor internalization and down-regulation (1). The biochemical and biological roles of each autophosphorylation site in wt EGFR have been explored by mutational analysis, and mutation of any single autophosphorylation site does not significantly abrogate binding of the activated receptor to specific SH2-containing proteins associated with distinct signaling pathways (2). Likewise, such single mutations are generally incapable of reducing the biological functions of the receptor in in vitro models (3, 4). Correspondingly, the mitogenic and transforming activities of wild-type receptor were diminished only when combinations of favorable autophosphorylation sites (i.e. Tyr-1068, Tyr-1148, and Tyr-1173) were mutated (3), suggesting that the autophosphorylation sites of wt EGFR may have less specificity for signaling proteins and can compensate for each other. Sites of tyrosine phosphorylation may be provided via heterodimerization with other members of the erb B family of receptors (5-7). Point mutations that inactivated the tyrosine kinase activity of wt EGFR eliminated occupancyinduced receptor internalization (8,9,40,41), whereas mutant receptors lacking multiple autophosphorylation sites also lacked the ability to undergo ligand-induced endocytosis, suggesting that kinase-regulated re...

“…The growing tumors were measured twice a week with a caliper, and tumor volumes were calculated using width (a) and length (b) measurements (a 2 b/2, where a Ͻ b) (37). For intracerebral stereotactic implantation, 1 ϫ 10 5 -5 ϫ 10 5 cells in 5 l of PBS were inoculated into the corpus striatum in the right hemisphere of the nude mouse brain (38).…”

Section: Methodsmentioning

confidence: 99%

The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling

¹

,

²

,

³

et al. 1997

Journal of Biological Chemistry

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Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named ⌬EGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active ⌬EGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of ⌬EGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation. Ligand binding to wild-type epidermal growth factor receptor (wt EGFR)1 results in receptor dimerization, kinase activation, and autophosphorylation that provides both docking sites for proteins involved in signal transduction and exposure of endocytic and lysosomal targeting sequence codes required for receptor internalization and down-regulation (1). The biochemical and biological roles of each autophosphorylation site in wt EGFR have been explored by mutational analysis, and mutation of any single autophosphorylation site does not significantly abrogate binding of the activated receptor to specific SH2-containing proteins associated with distinct signaling pathways (2). Likewise, such single mutations are generally incapable of reducing the biological functions of the receptor in in vitro models (3, 4). Correspondingly, the mitogenic and transforming activities of wild-type receptor were diminished only when combinations of favorable autophosphorylation sites (i.e. Tyr-1068, Tyr-1148, and Tyr-1173) were mutated (3), suggesting that the autophosphorylation sites of wt EGFR may have less specificity for signaling proteins and can compensate for each other. Sites of tyrosine phosphorylation may be provided via heterodimerization with other members of the erb B family of receptors (5-7). Point mutations that inactivated the tyrosine kinase activity of wt EGFR eliminated occupancyinduced receptor internalization (8,9,40,41), whereas mutant receptors lacking multiple autophosphorylation sites also lacked the ability to undergo ligand-induced endocytosis, suggesting that kinase-regulated re...

“…In vivo effects of GC on MCT cell growth in nude mice: In vivo inhibitory effects of PRD on the growth of MCT cells inoculated into nude mice were evaluated according to the protocol of Battle Columbus Laboratories [11], which was proposed for screening of antitumor drugs using nude mice.…”

Section: Mct Cells and Culture Methodmentioning

confidence: 99%

Inhibitory Effects of Glucocorticoids on Proliferation of Canine Mast Cell Tumor.

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et al. 1997

The Journal of Veterinary Medical Science

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ABSTRACT. The inhibitory effect of glucocorticoids (GCs) on proliferation of canine mast cell tumor (MCT) was studied using two types of MCT cells; JuMC cells and LuMC cells derived from spontaneous canine cutaneous and intestinal MCT, respectively. In in vitro study, growth of JuMC cells was significantly inhibited with more than 1 nM GCs and apoptotic-like cell death was seen, while that of LuMC cells was never inhibited even with 10 µM GCs. Growth rate of masses in nude mice developed by inoculation of JuMC cells was reduced in a dose-dependent manner by administration of GC, while growth inhibition of masses developed by inoculation of LuMC cells was minimal with increasing GC doses. We have established both cutaneous and intestinal types of canine MCT cells from the spontaneous canine cases [18,19]. In this experiment, in vitro and in vivo inhibitory effects of GCs on these tumor cells were investigated and cytoplasmic GC receptors of these cells were measured. MATERIALS AND METHODS MCT cells and culture method:JuMC cell was derived from spontaneous canine cutaneous MCT clinically categorized as intermediately-differentiated MCT, which had been treated with prednisolone (PRD) as one of the chemotherapeutic agents. This cell was maintained in our laboratory by in vivo passage (subcutaneous inoculation) in female nude mice (5 week old, BALB/c-nu/nu Slc, Nippon SLC Inc., Shizuoka, Japan) in every 6 to 10 weeks. This cell was characterized as rat connective tissue type mast cell by morphology and the manner of degranuration by various stimulations [19]. Doubling time of this cell in vitro was over 300 hr. In this study JuMC cells with 20 to 24 passages were used.JuMC cells were obtained by excision of the mass from the nude mouse. The mass was minced in phosphate buffered saline (PBS( )), filtrated through a 53-µm mesh, washed and centrifuged at 1,000 rpm for 5 min. Then it was resuspended and cultured in RPMI-1640 medium (2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, 50 µM 2-mercaptoethanol) with 10% heat-inactivated fetal bovine serum (FBS) (JRH Biosciences, U.S.A.) at 37°C in humidified air with 5% CO 2 .LuMC cells were derived from spontaneous canine intestinal MCT. The case also had a history of receiving PRD treatment for indistinct eosinophilia. This cell was Mast cell tumor (MCT) accounts for 7 to 21% of all skin tumors and 11 to 27% of all malignant skin tumors in dogs [9,23]. Canine MCT occurs primarily in the dermis and subcutaneous tissues, which is called as cutaneous MCT. Mast cell tumor cells have cytoplasmic granules containing several vasoactive substances like histamine and heparin, which often cause various clinical symptoms such as gastric ulcer and bleeding tendency in the tumor patients. Surgical excision is the choice of treatment for cutaneous MCT, however it often metastasizes to the adjacent and/or distant skin and visceral lymph nodes. In addition, the vasoactive substances of this tumor affect the systemic conditions. Another MCT, called as intestinal MCT, is derived f...

“…We estimated the weight of the tumors by the formula W = (a2 X b)/2, where W is the weight in mg, a is the width in mm, and b is the length in mm (Geran et al, 1972;Houchens and Ovejera, 1984). To evaluate the antineoplastic effects, we used the T/C ratio, where T is the mean tumor weight in treated mice, and C is the corresponding mean tumor weight in control mice.…”

Section: Evaluation Of Antineoplastic Effectsmentioning

confidence: 65%

Heavy water enhances the antineoplastic effect of 5‐fluoro‐uracil and bleomycin in nude mice bearing human carcinoma

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1990

Intl Journal of Cancer

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The antineoplastic effect of heavy water on the growth of xenotransplanted human carcinoma was compared to that of 2 cytostatic drugs. Seven-week-old BALB/c-nu/nu mice were inoculated subcutaneously with a poorly differentiated oropharyngeal squamous-cell carcinoma, or with variants of carcinoma of the large intestine. After tumor inoculation, 3 subgroups of mice were treated by: (i) moderately deuterated drinking water; (ii) i.p. injections of 5-Fluoro-uracil (5-FU) or Bleomycin; (iii) a combination of deuterated drinking water and of the cytostatic drugs. Control mice were not treated. Heavy water delayed growth of all carcinoma variants. The cytostatic drugs slowed down the growth of the 2 poorly differentiated tumor variants. Conversely, 5-FU did not retard the growth of the moderately well differentiated colon carcinoma. Heavy water combined with either cytostatic drug showed synergistic effects in the 2 poorly differentiated tumor variants. The tumors of treated animals weighed 36% to 90% less than those of control animals. The antineoplastic effects were more conspicuous in poorly than in moderately well differentiated tumor variants. Cytokinetic parameters such as labelling indices following application of 5-125I-Iodo-2'deoxyuridine, and of Ki-67, a monoclonal antibody (MAb) directed against an antigen of proliferating cells, or mitotic indices and tumor volume doubling time, combined with the results of histologic evaluation of the tumors, suggested an underlying deuterium-induced prolongation of tumor-cell cycle times and a reduction of the growth fraction.

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