Relation of skin polyamines to the hairless phenotype in transgenic mice overexpressing spermidine/spermine N-acetyltransferase.

We recently generated a transgenic mouse line with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase. Phenotypic changes in these animals included permanent loss of hair at the age of 3 wk. We have now further explored development of hair loss during early postnatal life. The first hair cycle appeared to be completed normally in the transgenic animals. At postnatal day 15, although macroscopically indistinguishable from their syngenic littermates, the transgenic animals already showed microscopically signs of hair follicle degeneration. Wild-type mice started their second anagen phase at day 27, whereas the transgenic animals did not display functional hair follicles at that time. Hair follicles were replaced by dermal cysts and epidermal utriculi. Analysis of skin polyamines revealed that the transgenic animals continuously overaccumulated putrescine. The view that an overaccumulation of putrescine was related to the disturbed hair follicle development was strengthened by the finding that doubly transgenic mice overexpressing, both spermidine/spermine N1-acetyltransferase and ornithine decarboxylase and with extremely high levels of putrescine in the skin, showed distinctly more severe skin changes compared with the singly transgenic animals. Interest ingly, in spite of their hairless phenotype, the spermidine/spermine N1-acetyltransferase transgenic mice, were significantly more resistant to the development of papillomas in response to the two-stage skin carcinogenesis. Analysis of skin polyamines indicated that the syngenic mice tripled their spermidine content when exposed to promotion, whereas the transgenic animals showed only modest changes. These results suggest that putrescine plays a pivotal part in normal hair follicle development.

HSV-tk gene therapy for human renal cell carcinoma in nude mice.

We have treated Caki-2 human renal cell carcinoma in vivo using herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Both stably transduced Caki-2 tumors, generated using retrovirus-mediated ex vivo HSV-tk gene transfer and direct intratumoral adenovirus-mediated HSV-tk gene transfer of wild type tumors, were tested. Similar treatments with LacZ containing retro- and adenoviruses were used as controls. The outcome was evaluated by imaging the tumors before and after the treatment with magnetic resonance imaging, and using histology, immunocytochemistry, and survival analysis. When implanted orthotopically into nude mouse kidneys, Caki-2 cells formed reproducible cystic papillary kidney carcinomas. In vivo magnetic resonance imaging provided an important tool for the evaluation of tumor growth. Transduction efficiency of wild-type tumors in vivo with adeno-LacZ was 22+/-14%. Significant tumor regression was achieved with direct intratumoral adeno-HSV-tk transduction followed by intraperitoneal ganciclovir (GCV) (P<.001). Also, the treatment of stably transduced Caki-2 tumors with intraperitoneal GCV resulted in a significant treatment response in the HSV-tk group as compared to the LacZ group (P<.009). Increased apoptosis and macrophage infiltrations, reduced proliferation, and degenerative changes were observed in the tumors treated with HSV-tk and GCV. Also, significant prolongation in survival was achieved with adeno-HSV-tk- and GCV-treated mice as compared to the controls. It is concluded that adeno-HSV-tk gene therapy may be useful for the treatment of renal cell carcinoma in vivo.

Irregular expression of hyaluronan and its CD44 receptor is associated with metastatic phenotype in laryngeal squamous cell carcinoma.

The distributions of hyaluronan (HA) and its CD44 receptor were studied in 24 normal, 27 dysplastic samples of laryngeal epithelium and in 172 squamous cell carcinomas (LSCC), using a specific probe prepared from cartilage proteoglycan (bHABC, biotinylated hyaluronan binding complex) and a monoclonal antibody (Hermes 3). HA and CD44 were expressed similarly in all normal and about 90% of dysplastic and neoplastic laryngeal epithelia. In the normal epithelium HA and CD44 were homogeneously distributed throughout the epithelium, whereas the most superficial layers were negative. This was in contrast to the picture in dysplastic epithelium and well-differentiated invasive carcinomas, which were entirely HA and CD44 positive. Local areas with a low signal for HA and CD44 were present in 11% and 22% of the samples with dysplasia, and in 27% and 28% of those with carcinoma, respectively. The presence of this staining irregularity was associated with poor differentiation of the carcinoma, a significantly elevated mitotic index and a high frequency of nodal spreading and metastases. Furthermore, the irregular staining showed a trend towards poor disease-free survival, suggesting that an altered metabolism of HA is a common feature in LSCC and is associated with an aggressive growth pattern.

Local stimulation of proteoglycan synthesis in articular cartilage explants by dynamic compression in vitro.

Cultured bovine articular cartilage was subjected to 50 ms, 0.5-1.0 MPa compressions repeated at intervals of 2-60 s for 1.5 h and simultaneously labeled with 35SO4. The compression was delivered with a 4-mm-diameter nonporous loading head on an 8-mm-diameter cartilage explant. This method created directly compressed (central) and uncompressed (border) areas within the tissue. Analysis of the whole explant under a 0.5 MPa load showed significantly increased 35SO4 incorporation by compression repeated at 2- and 4-s but not at 20- and 60-s intervals. When the incorporation was studied separately in the border and central areas, a statistically significant stimulation was noticed in the central area with a 4-s cycle, while the border area was stimulated with a 2-s cycle. Autoradiography of the central area showed that the stimulation with 0.5 MPa and a 4-s cycle occurred through the whole depth of the cartilage, while raising the pressure to 1 MPa or the frequency to 2 s reduced the stimulation, particularly in the superficial cartilage. In the border area the stimulation with 0.5 MPa and a 2-s cycle was noted in the superficial zone only. The stimulation of proteoglycan synthesis is thus limited to certain loading frequencies and pressures and occurs in specific areas under and around the loaded site. Its rapid appearance suggests enhanced glycosylation or sulfation of core proteins or enhanced speed of posttranslational processing.

Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes.

The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure.

A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants.

An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.

Characterization of a new animal model for human renal cell carcinoma.

BACKGROUND: Human renal cell carcinoma (RCC) is the most common kidney malignancy with significant mortality. Human tumor xenograft models are important tools for cancer research. MATERIALS AND METHODS: We have established and characterized a new animal model for human RCC using Caki-2 cells implanted into the renal subcapsule (RSC) of nude mice. Histology, immunocytochemistry, in situ hybridization and magnetic resonance imaging (MRI) were used to analyze the tumors. RESULTS: The implantations generated reproducible carcinomas which closely resemble human RCC. The tumors showed cystic-papillary structures, rich capillary network and fibro-septa formations. Proliferation varied from 0-5% and from 1-60% in cystic and solid areas, respectively. Apoptosis was less than 1%. Macrophages and other inflammatory cell infiltrations were detected in the tumors. VEGF-A and angiopoietin I were expressed in a small number of cells in large tumors. Tumors did not metastasize outside peritoneal cavity. Survival of the tumor bearing animals was 23 +/- 3 weeks. CONCLUSIONS: It is concluded that Caki-2 carcinomas implanted into renal subcapsule of nude mice resemble human RCC in several aspects and represent a good animal model for studies regarding the pathogenesis and treatment of human RCC.

Subchondral bone remodeling increases in early experimental osteoarthrosis in young beagle dogs.

We evaluated subchondral bone remodeling and structure in the condyles of the femur and the patellar surface of the femur in early experimental osteoarthrosis of young female beagle dogs. 14 littermate (twin) dogs were divided into operation (n 7) and control groups (n 7). The dogs in the operation group underwent surgically a 30 degrees valgus angulation of the right tibia to induce osteoarthrotic articular cartilage lesions in the knee (stifle) joint. 7 months postoperatively, bone samples were harvested from both condyles and the patellar surface of the femur and evaluated by histomorphometry of subchondral bone. Cartilage samples from the same areas were taken for histology. In the operated dogs, subchondral bone remodeling increased strikingly in the patellar surface of the femur; osteoid thickness and osteoblast surface/bone surface increased up to 42% and 94% (p < 0.05), as compared to controls. Total and active erosion depths increased by 14% and 30% in the same area (p < 0.05). However, in bone structural parameters no significant difference could be observed between the groups. In the medial condyle of the femur, the trabecular number decreased in operated dogs, as compared to controls (p < 0.05). The lateral condyle of the femur in operated animals did not differ from controls in the parameters tested. In the operated dogs, histology from cartilage samples showed initial osteoarthrotic changes in the patellar surface and the medial condyle of the femur. Histologic changes were greatest in the patellar surface of the femur, as assessed by the Mankin scores. At the very onset of osteoarthrosis, subchondral bone remodeling increases, but the bone structural changes are indistinct. It seems that in this osteoarthrosis model, cartilage lesions precede major subchondral changes in the structure of the bone.

A high sensitivity dot-blot assay for proteoglycans by cuprolinic blue precipitation.

A highly sensitive blot-assay was developed for glycosaminoglycans (GAGs) and proteoglycans (PGs) utilizing a precipitation reaction by a cationic dye Cuprolinic Blue. The precipitates were deposited into 1-2 mm2 spots on nitrocellulose membrane by using a 96-well filtration apparatus. The dried sheet was digitized by a flat bed scanner and the intensity of the dots was quantitated by an image analysis software. The working range for chondroitin sulfate was 10-300 ng. The response of various GAGs differed according to the number of anionic groups, both sulphate and carboxyl groups being able to bind the dye. The sensitivity of the assay was decreased by high concentrations of GuC, CsC and protein, but not by nonionic detergents, common buffers and 8 M urea. Contact exposure to autoradiography film enabled quantitation of 25-250 DPM, and 1-10 DPM, of 35SO4-radioactivity in precipitated PGs after overnight and 14 days' exposures, respectively.

Activation of polyamine catabolism in transgenic rats induces acute pancreatitis.

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine/spermine N(1)-acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of N(1)-acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [N(1),N(2)-bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue N(1),N(11)-diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of N(1), N(11)-diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.

Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants.

Primary chondrocyte cell cultures and explants of bovine articular cartilage were subjected to cyclic hydrostatic pressure in a novel computer-controlled pressure chamber designed for this purpose. The cultures were labeled with 5 microCi/ml 35SO4 and simultaneously pressurized with 5 MPa load for 1.5 or 20 h with pressure cycles of 0.0167, 0.05, 0.25, and 0.5 Hz. The chondrocyte cell cultures were also subjected to 0.0082 and 0.0034 Hz cycles. Sulfate incorporation was significantly inhibited in cell cultures subjected to the 0.5, 0.25, or 0.05 Hz cyclic loads for 1.5 h, but stimulated in explant cultures with a 0.5 Hz cyclic 1.5-h load. Chondrocyte cultures subjected to longer (20 h) loading showed a stimulation of sulfate incorporation with 0.5 and 0.25 Hz cycles, but an inhibition with 0.0167 Hz. The results indicate that cyclic hydrostatic pressures of presumably physiological magnitude have significant influences on proteoglycan synthesis in articular cartilage chondrocytes. Comparison of the cell and explant cultures under identical pressure conditions suggested that chondrocyte interactions with extracellular matrix are involved in this regulation by cyclic hydrostatic pressure. The responses of the chondrocytes to pressurization also varied according to the total length of the treatment, a finding compatible with the idea of multiple metabolic steps in chondrocytes, both pre- and post-translational, controlled by the ambient hydrostatic pressure.

Effects of long-term running exercise on canine femoral head articular cartilage.

After long-term running program (40 km/day) anterior and posterior tissue samples from canine femoral head were labeled ex vivo in the presence of 35S-SO4. Sulfate incorporation rates did not differ between runner and control groups. The statistically significant changes in runners included a decreased uronic acid concentration (p < or = 0.02) and proportion of extractable proteoglycans (p < or = 0.05) as well as increased concentration of tissue uronic acid after 4 M GuCl extraction (p < or = 0.05) in the posterior area. These results support an idea of strengthened cartilage tissue after this kind of motion and load.

Lifelong moderate running training increases the incidence and severity of osteoarthritis in the knee joint of C57BL mice.

BACKGROUND: Inbred C57BL male mice express a high incidence of spontaneous osteoarthritis of the knee joint at the age of 18 months. We used this strain of mice to find out the effects of life-long, moderate running exercise on the health of articular cartilage and the incidence of osteoarthritis. METHODS: Male mice (294) were divided into controls and runners. The runners were trained daily between 2 and 18 months of age. The speed was 13.3 m/min and the distance on a flatbelt treadmill was 1,000 m/day. The mice were sacrificed at the ages of 2, 6, 10, 14, and 18 months. The knee joints were sectioned in frontal direction and the osteoarthritic changes were graded using a conventional light microscope. The reproducibility of the grading method was tested by calculating the extended kappa-coefficient for the results of six researchers. RESULTS: The incidence of osteoarthritis at the age of 18 months increased from 72% in controls to 88% in runners in the medial tibial condyles (P < 0.05), and from 80 to 96% in the lateral tibial condyles (P < 0.001). The incidence of the most severe osteoarthritic changes rose from 16% in controls to 36% in runners in the medial tibial condyles, and from 4 to 36% in the lateral tibial condyles. CONCLUSION: According to our results, the moderate, long-lasting running exercise accelerates the development of osteoarthritis in the knee joints of C57BL mice.

Polyamine-dependent alterations in the structure of microfilaments, Golgi apparatus, endoplasmic reticulum, and proteoglycan synthesis in BHK cells.

The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a beta-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [3H]glucosamine and [35S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation.

Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes.

OBJECTIVES: Articular cartilage proteoglycan content is controlled by joint loading. This study aimed to elucidate the role of hydrostatic pressure in this regulation. METHODS: Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure, applied continuously or cyclically at 0.125 or 0.05 Hz. The Golgi apparatus was visualised either by a fluorochrome coupled wheat germ agglutinin or by transmission electron microscopy. Proteoglycan synthesis was studied by the incorporation of sulphur-35 labelled sulphate. RESULTS: After 30 MPa continuous hydrostatic pressure, the Golgi apparatus was observed in a compact form with a concomitant decrease in proteoglycan synthesis. The normal stacked appearance of the Golgi apparatus was no more visible in the electron microscopy preparation of the pressurised chondrocytes. This effect was reversible and was also noticed after 15 MPa continuous load, though to a minor extent. Cyclic pressures (5-30 MPa) caused no apparent change in the Golgi apparatus. The shape of some cells changed to a more retracted form after 30 MPa continuous pressure. Nocodazole, which causes disassembly of the microtubules, blocked the compacting influence of pressurisation on the Golgi apparatus, and reduced proteoglycan synthesis to about half of the control level. CONCLUSIONS: The packing of the Golgi apparatus is dependent on microtubules and may contribute to the inhibition of proteoglycan synthesis observed in articular cartilage subjected to high hydrostatic pressure.

Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer.

A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with 35S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of 35S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.

Adaptation of canine femoral head articular cartilage to long distance running exercise in young beagles.

OBJECTIVE: To study the effects of long term (one year), long distance (up to 40 km/day) running on the metabolism of articular cartilage the biosynthesis of proteoglycans was examined by in vitro labelling of anterior (weight bearing) and posterior (less weight bearing) areas of the femoral head from young beagles. METHODS: Total sulphate incorporation rates were determined and distribution of the incorporated sulphate was localised by quantitative autoradiography. Concentration and extractability of the proteoglycans were determined, and proteoglycan structures were investigated by gel filtration chromatography, agarose gel electrophoresis, and chemical determinations. RESULTS: In the less weight bearing area the amount of extractable proteoglycans was decreased (p < or = 0.02), simultaneously with an increased concentration of residual glycosaminoglycans in the tissue after 4 M GuCl extraction (p < or = 0.05). In control animals proteoglycan synthesis was most active in the deep zone of the cartilage, whereas exercise increased synthesis in the intermediate zone. There was a tendency to a lower keratan: chondroitin sulphate ratio in the running dogs. No macroscopical or microscopical signs of articular degeneration or injury were visible in any of the animals. CONCLUSION: The articular cartilage of the femoral head showed a great capacity to adapt to the increased mechanical loading. The reduced proteoglycan extractability in the less weight bearing area changed it similar to the weight bearing area, suggesting that the low extractability of proteoglycans reflects the long term loading history of articular cartilage. The congruency of the femoral head with acetabulum seems to protect the cartilage from the untoward alterations that occur in the femoral condyles subjected to a similar running programme.

Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.

The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.