Regulation of protein turnover versus growth state. III. Growth cessation is associated with activation of autophagy in Yoshida ascites hepatoma AH-340

Yoshida AH-130 ascites hepatoma cells were grown in rats and were examined by quantitative electron microscopy 4 days (exponential growth phase) and 10 days (stationary phase) after intraperitoneal inoculation. No significant differences between growing and growth-inhibited tumors were found in the composition of the cytoplasm, except for a slight increase in the cytoplasm, except for a slight increase in the volume fraction of mitochondria (from 9.6 to 12.1%) and, in particular, a prominent (4.2-fold) increase in the volume fraction of early stage of autophagic vacuoles (from 0.31 x 10(-4) at day 4 to 1.37 x 10(-4) at day 10; P less than 0.001). At the same time, the rate of cell protein degradation was increased twofold, namely from 0.67%/h at day 4 to 1.37%/h at day 10, as measured in vitro after labeling cells with 3H-leucine in vivo. Such elevated proteolytic activity was entirely suppressed by ammonia, which inhibits the lysosomal pathway for protein degradation. The data show that: (i) the regulation of autophagic degradation of cytoplasmic constituents depending on the growth state was maintained in these tumor cells, and (ii) the increase in autophagy measured by morphometric analysis contributed to, yet did not quantitatively explain, the acceleration of protein degradation characterizing the transition from the logarithmic growth phase to the growth-inhibited state.     

Regulation of protein turnover versus growth state. Studies on the mechanism(s) of initiation of acidic vacuolar proteolysis in cells of stationary ascites hepatoma.

1. After transplantation, the rat AH-130 Yoshida ascites hepatoma enters a phase of exponential (log) growth, followed by a quasi-stationary (sta) state. Combining measurements made in vivo and in vitro, cessation of protein accumulation (growth) in sta phase has previously been shown to result from convergent reduction of protein synthesis and enhancement of protein breakdown [Tessitore, Bonelli, Cecchini, Amenta & Baccino (1987) Arch. Biochem. Biophys. 255, 372-384]. 2. One day after labelling in the animal with [3H]leucine, AH-130 cells were processed for short-term assays in vitro to measure rates of endogenous protein breakdown. 3. Exposure of AH-130 cells to inhibitors interfering with different steps of the acidic vacuolar pathway (AVP) showed that: (i) in log tumour cells the AVP was extensively suppressed; (ii) in sta tumour cells virtually all of the proteolytic acceleration was accounted for by activation of the AVP. 4. Treating log tumour cells with glucagon, cyclic AMP, or nutritional deprivation failed to elevate substantially the proteolytic rates. Nor could the elevation in proteolysis be explained by changes in free amino acids, which were more concentrated in the ascitic fluid of sta tumours. 5. The enhanced proteolysis in sta tumour cells was not associated with any increase in the intracellular activity levels of lysosomal cathepsins B, D, H, and L. 6. The above growth-related modulation of protein breakdown in AH-130 cells was probably a reflection of the tumour growth state rather than the direct effect of environmental stimuli.     

Antineoplastic effect of hydrogel prospidin on seidel ascites hepatoma used as a model

Antineoplastic effect of hydrogel dextran phosphate, hydrogel prospidin, and prospidin in an injectable preparation has been assessed using Seidel ascites hepatoma as a model. Injectable and hydrogel prospidin in doses from 250, 500 to 1000 mg/kg and hydrogel phosphate dextran in doses of 500 and 1000 mg/kg were administered to rats intraperitoneally in a single dose in a volume of 1 or 2 ml per each 100 g of animal body weight. The study has shown that irrespective of rats with Seidel ascites hepatoma and significantly increase in the dosage of prospidin preparations and hydrogel dextran phosphate results in a longer average life expectancy of rats Compared with its injectable variant, hydrogel prospidin appears to produce more than twice as high antineoplastic effect, and is found to provide prolonged therapeutic effects, as well as cure of animals in more than 60 % of cases.     

Regulation of the phospholipid turnover rate and protein kinase C activity as a necessary stage in the realization of the growth-inhibiting effect of dexamethasone on hepatoma 22 cells]

The role of protein kinase C and phospholipid turnover in the realization of the cytostatic effect of dexamethasone on hormone-sensitive cells of mouse hepatoma 22 has been studied. It was found that dexamethasone added to hepatoma cells induces a rapid (within 30 min) inhibition of the protein kinase C activity with a simultaneous decrease of the 32P incorporation into the major phospholipids (phosphatidylglycerol, phosphatidylcholine, and phosphoinositides). Analysis of correlation between the protein kinase C activity and phospholipid turnover rate revealed that phosphatidylglycerol and phosphatidylcholine synthesis is under the positive control of protein kinase C, whereas that of phosphoinositides is not controlled by the enzyme. A proportional decrease in the rates of metabolism of all the three major phospholipids after addition of the hormone to hepatoma cells suggests that inhibition of phospholipid turnover is one of the primary manifestations of the dexamethasone effect. The hormone-induced decrease in the protein kinase C activity may be regarded as being due to these changes.     

Stringent requirement for the C protein of wild-type measles virus for growth both in vitro and in macaques

The P gene of measles virus (MV) encodes the P protein and three accessory proteins (C, V, and R). However, the role of these accessory proteins in the natural course of MV infection remains unclear. For this study, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C-), by using a reverse genetics system (M. Takeda, K. Takeuchi, N. Miyajima, F. Kobune, Y. Ami, N. Nagata, Y. Suzaki, Y. Nagai, and M. Tashiro, J. Virol. 74:6643-6647). When 293 cells expressing the MV receptor SLAM (293/hSLAM) were infected with wtMV(C-) or parental wild-type MV (wtMV), the growth of wtMV(C-) was restricted, particularly during late stages. Enhanced green fluorescent protein-expressing wtMV(C-) consistently induced late-stage cell rounding and cell death in the presence of a fusion-inhibiting peptide, suggesting that the C protein can prevent cell death and is required for long-term MV infection. Neutralizing antibodies against alpha/beta interferon did not restore the growth restriction of wtMV(C-) in 293/hSLAM cells. When cynomolgus monkeys were infected with wtMV(C-) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C-) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV but dramatically reduced expression in the same tissues in a cynomolgus monkey infected with wtMV(C-). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.     

Effect of the growth state on protein turnover in two lines of cultured BHK cells

The adherence, phagocytic activity and buoyant density of mouse peritoneal exudate colony forming units (CFU-PE) were investigated. There was a significant enrichment in the proportion of CFU-PE in the adherent cells population, defined as cells adhering to a plastic surface within 30 minutes of incubation. The phagocytic activity of CFU-PE was studied by incubating exudate cells with iron particles for 45 minutes. The cells were then separated into phagocytic and non-phagocytic cell fractions by passing the incubation mixture through a magnetic field. A significant enrichment of CFU-PE was seen in the phagocytic cell fraction. When exudate cells were fractionated in a Ficoll discontinuous density gradient, more than 88% of CFU-PE were recovered at the 16/18% and 18/20% interfaces. It is concluded that CFU-PE are adherent cells, have strong phagocytic activity and have a buoyant density between 1.0562 and 1.0703. When bone marrow cells were studied by these techniques, the committed stem cells for both granulocytes and macrophages (CFU-C) were enriched in both non-adherent cell and non-phagocytic cells populations. In the Ficoll density gradient, CFU-C banded at a heavier density region than CFU-PE.     

Characterization of a novel Xenopus tropicalis cell line as a model for in vitro studies

Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.     

Regulation of protein turnover by recombinant human insulin-like growth factor-I in L6 myotube cultures

Muscle cell culture experiments were conducted to determine the relative regulatory effects of insulin-like growth factors (IGF) on protein turnover. The effects of recombinant (rc) human IGF-I, ovine somatomedin (oSm/oIGF-I), and insulin on rates of protein labeling and degradation in L6 myotube cultures were evaluated. Myotube cultures were treated with growth factors following a 4-h serum-free incubation period. Protein labeling was measured by determining the rate of [3H] leucine incorporation into cell protein. Protein degradation was measured by a pulse-chase procedure using [3H] leucine. The apparent half maximal stimulation of protein labeling (12%, 8%, 7%) occurred at approximately .1 nM rcIGF-I, 1 nM oSm/oIGF-I and 15 nM insulin, respectively. The apparent half maximal inhibition of proteolysis (18%, 15% and 11%) occurred at .4 nM rcIGF-I, .6 nM oSm/oIGF-I and 4 nM insulin, respectively. The magnitude of the response for protein labeling and degradation was greatest for rcIGF-I. The results provide additional evidence that IGFs play a primary role in regulating protein turnover in muscle.     

Properties of protein polymers as substratum for cell growth in vitro

The behavior of two established cell lines was found to vary when subcultivated on protein polymers covered with either negatively or positively charged substances. Results indicate that the influence on cell behavior is conditioned by the charge rather than by structural differences or degree of attachment of the substances to the polymer. Furthermore, the substratum seems to have just a physical effect at the cell membrane without any direct influence on cell metabolism. Cells were also seeded on a calf serum polymer and it was found that serum loses its properties on cultivated cells when used as substratum.     

A sheep model for fracture treatment in osteoporosis: benefits of the model versus animal welfare

Animal models are necessary to evaluate new options for the treatment of fractures in osteoporotic bone. They permit both the biological response of a living system and the influence of the pathological processes to be taken into account. A sheep model for osteoporosis was established by combining oestrogen deficiency, calcium and vitamin D-deficient diet with steroid medication. Bone mineral density (BMD) was reduced by >30% after 12 weeks of combined treatment. Osteoporosis similar to the human situation with corresponding changes in the micro-architecture and mechanical properties of bone was observed. This publication focuses on the impressive results obtained with the model and contrasts them with considerations of animal welfare. Considerable side-effects associated with steroid medication became manifest. Animals in the treatment groups showed signs of infection of various degrees due to the immunosuppressive effect of the medication. The infections were mostly caused by Corynebacterium pseudotuberculosis. Antibody testing revealed a 100% prevalence of infection in this breed of sheep. A modification of the steroid treatment, i.e. less-frequent injections, reduced the incidence of side-effects. This sheep model shows a significant and reproducible reduction in cancellous BMD of >30%, including relevant changes in biomechanical properties and increased fracture risk. However, the severity of the side-effects cannot be overlooked. The model must be improved if it is to be used in the future. Options to reduce the side-effects are discussed.     

The guinea pig as an animal model for developmental and reproductive toxicology studies

BACKGROUND : Regulatory guidelines for developmental and reproductive toxicology (DART) studies require selection of “relevant” animal models as determined by kinetic, pharmacological, and toxicological data. Traditionally, rats, mice, and rabbits are the preferred animal models for these studies. However, for test articles that are pharmacologically inactive in the traditional animal models, the guinea pig may be a viable option. This choice should not be made lightly, as guinea pigs have many disadvantages compared to the traditional species, including limited historical control data, variability in pregnancy rates, small and variable litter size, long gestation, relative maturity at birth, and difficulty in dosing and breeding. METHODS : This report describes methods for using guinea pigs in DART studies and provides results of positive and negative controls. Standard study designs and animal husbandry methods were modified to allow mating on the postpartum estrus in fertility studies and were used for producing cohorts of pregnant females for developmental studies. RESULTS : A positive control study with the pregnancy-disrupting agent mifepristone resulted in the anticipated failure of embryo implantation and supported the use of the guinea pig model. Control data for reproductive endpoints collected from 5 studies are presented. CONCLUSION : In cases where the traditional animal models are not relevant, the guinea pig can be used successfully for DART studies. Birth Defects Res (Part B)86: 92-97, 2009. © 2009 Wiley-Liss, Inc.     

Electrophysiology and pathology evaluation of the Yucatan pig as a non-rodent animal model for regulatory and mechanistic toxicology studies

Six male and six female Yucatan pigs were utilized to investigate the feasibility of this species as a non-rodent model for routine regulatory and mechanistic toxicology studies. This study evaluated disease surveillance and computerized electrophysiology, along with possible gross and micropathology changes. Two pigs were used as sentinel animals to evaluate the microbiological status of the vendor upon arrival; the other pigs were maintained as biomonitors and to provide baseline clinical chemistry, urinalysis, pathology and electrophysiology data. The electrophysiology tests conducted included electrocardiography (ECG), electroretinography (ERG) and quantitative electroencephalography (qEEG), which achieved consistent baseline values with acceptable intrasubject variation. Tissue cholinesterase and histochemical staining were done to determine their suitability for testing cholinesterase compounds. Evaluation of the serum chemistry profile demonstrated increased CPK and LDH, which was likely associated with slight haemolysis or minor subclinical muscle stress during handling. There were no additional clinical chemistry changes or findings in haematology, urinalysis parameters or gross pathology. Micropathology found an absence of background lesions which would interfere with routine toxicology studies, except for a mild rhinitis. The aetiological agent was identified by electron microscopy as being consistent with inclusion body rhinitis of swine, previously unreported in miniature swine. This would most notably interfere with inhalation studies. The anatomical and physiological similarities of the Yucatan pig, along with its ability to accept the performance of electrophysiology tests allow this species to be considered as a suitable model for organ system testing in toxicology studies.