Effect of hepatectomy on mitotic activity in the rat spleen

Mitotic activity in the rat spleen was studied in 60 animals by DNA uptake of [³H]thymidine and autoradiographic nuclear labeling. Sham operation did not affect these parameters. After partial splenectomy, there was a marked increase in DNA uptake of [³H]thymidine and nuclear labeling that was maximal at 48 hr and remained elevated throughout the first postoperative week. After 70% hepatectomy, there was a similar increase in DNA activity at 24 hr but this increase returned toward control values at 48 and 72 hr. The distribution of nuclear labeling was similar after splenectomy and hepatectomy. These studies suggest that common control mechanisms may regulate regenerative responses in both liver and spleen.     

In vitro and in vivo responses of a murine transitional cell carcinoma to doxorubicin,mitoxantrone and aclacinomycin-A

Summary In vitro and in vivo effects of mitoxantrone, aclacinomycin-A and doxorubicin were examined in a transplantable murine transitional bladder carcinoma, FCB. The in vitro parameters used included monolayer growth kinetics, tumor stem-cell colony formation and autoradiographic analysis of thymidine labeling. Monolayer growth kinetics revealed that both mitoxantrone and aclacinomycin-A resulted in reductions in FCB cell growth, which were significantly higher (41% and 65%, respectively) than those seen with doxorubicin treatment (22%). Similarly, by the stem-cell assay, an increased reduction in colony formation was seen in aclacinomycin-A (98%) and mitoxantrone (91%) treated cultures when compared with doxorubicin (51%) treated cultures. Autoradiographic data revealed that 24-h exposure with both aclacinomycin-A and mitoxantrone significantly inhibited thymidine incorporation (98% and 80% respectively), which was an increase over doxorubicin (19%). In vivo studies revealed that aclacinomycin-A treatment increased the mean life span of C57BL mice by 60.6% when compared with a 33.6% increase in doxorubicin-treated animals and a 19.7% increase in mitoxantrone-treated animals. Both the in vitro and in vivo data suggest that aclacinomycin-A is a superior drug when used against this specific murine bladder tumor cell and that further testing of this agent for its efficacy in other urologic tumors is justified.     

Soft agarose colony formation assay for human renal cell carcinoma: comparison of optical colony counting versus tritiated thymidine incorporation

Use of the Hamburger-Salmon soft agar assay method for in vitro chemotherapy sensitivity testing of samples of renal cell carcinoma has been somewhat limited by a relatively low proliferation/evaluability rate for this tumor type (approximately 50%). The tritiated thymidine ([ 3H]-TdR) incorporation assay method of Tanigawa et al. (Cancer Res., 42: 2159, 1982) was compared to a standard optical colony counting assay technique. Fifty-seven different primary and five metastatic fresh samples of human renal cell carcinoma were studied. Evaluability rate by the [3H]-TdR assay was 90% (greater than or equal to 300 cpm control). In comparison, evaluability rate by optical colony counting was 43% for this group of tumors. [3H]-TdR incorporation increased with increasing tumor grade and increasing stage. Spindle cell tumors showed significantly higher cpm than other cell types. Twenty-three primary tumors were evaluable by both [3H]-TdR and colony counting methods. The correlation coefficient ("r") for regression lines for drug sensitivity data points (optical counting vs. [3H]-TdR) of these individual experiments ranged from 0.50 to 0.99 with a mean r +/- S.D. of 0.76 +/- 0.15. For all 260 paired drug response observations of 23 tumors exposed to different drugs, the correlation was very good with r = 0.71. Since the [3H]-TdR assay has an evaluability rate of approximately 90% for renal cell carcinoma, gives drug sensitivity information which correlates well with the colony counting endpoint and yields chemotherapy sensitivity information four days after sample accession, the [3H]-TdR assay may be a more useful method for study of human renal cell carcinoma in vitro chemotherapy sensitivity testing than standard colony counting techniques.     

High-frequency low-level diode laser irradiation promotes proliferation and migration of primary cultured human gingival epithelial cells

In periodontal therapy, the use of low-level diode lasers has recently been considered to improve wound healing of the gingival tissue. However, its effects on human gingival epithelial cells (HGECs) remain unknown. The aim of the present study was to examine whether high-frequency low-level diode laser irradiation stimulates key cell responses in wound healing, proliferation and migration, in primary cultured HGECs in vitro. HGECs were derived from seven independent gingival tissue specimens. Cultured HGECs were exposed to a single session of high-frequency (30 kHz) low-level diode laser irradiation with various irradiation time periods (fluence 5.7–56.7 J/cm²). After 20–24 h, cell proliferation was evaluated by WST-8 assay and [³H]thymidine incorporation assay, and cell migration was monitored by in vitro wound healing assay. Further, phosphorylation of the mitogen-activated protein kinase (MAPK) pathways after irradiation was investigated by Western blotting. The high-frequency low-level irradiation significantly increased cell proliferation and [³H]thymidine incorporation at various irradiation time periods. Migration of the irradiated cells was significantly accelerated compared with the nonirradiated control. Further, the low-level diode laser irradiation induced phosphorylation of MAPK/extracellular signal-regulated protein kinase (ERK) at 5, 15, 60, and 120 min after irradiation. Stress-activated protein kinases/c-Jun N-terminal kinase and p38 MAPK remained un-phosphorylated. The results show that high-frequency low-level diode laser irradiation promotes HGEC proliferation and migration in association with the activation of MAPK/ERK, suggesting that laser irradiation may accelerate gingival wound healing.     

Antineoplastic activity of honey in an experimental bladder cancer implantation model: In vivo and in vitro studies

OBJECTIVES: The antitumor effect of bee honey against bladder cancer was examined in vitro and in vivo. Methods: Three human bladder cancer cell lines (T24, 253J and RT4) and one murine bladder cancer cell line (MBT-2) were used in these experiments. In an in vitro study, the antitumor activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, 5-Bromodeoxyuridine (BrdU) labeling index and flowcytometry (FCM). In the in vivo study, cancer cells were implanted subcutaneously in the abdomens of mice, and the effects were assessed by the tumor growth. RESULTS: In vitro studies revealed significant inhibition of the proliferation of T24 and MBT-2 cell lines by 1-25% honey and of RT4 and 253J cell lines by 6-25% honey. BrdU labeling index was significantly lower. FCM showed lower S-phase fraction, as well as absence of aneuploidy compared with control cells. In the in vivo studies, intralesional injection of 6 and 12% honey as well as oral ingestion of honey significantly inhibited tumor growth. CONCLUSION: Bee honey is an effective agent for inhibiting the growth of T24, RT4, 253J and MBT-2 bladder cancer cell lines in vitro. It is also effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation models. Our results are promising, and further research is needed to clarify the mechanisms of the antitumor activity of honey.     

Elongation of fetal chick long bonein vitro is formed by a mitogenic activity preparation from porcine bone

Summary Elongation of fetal chick long bone rudiments is formed by a mitogenic activity from porcine bonein vitro. Fractions of mitogenic activity from a heat- and acid-treated extract and from sequential chromatography on hydroxyapatite and from gelfiltration in 4 M guanidine-HCl increase diaphyseal elongation of metatarsals. The bone elongation-forming activity is associated with the mitogenic activity estimated by the incorporation of [³H]-methyl thymidine into the DNA of cells from embryonic chick periosteum. Histological examination of the mitogen-treated embryonic chick long bone shows that the partially purified fractions with a preferential effect on osteogenic cells increase diaphyseal elongation via cartilage cell proliferation. DNA of primary cells [15, 17] or cell lines of mesodermal origin in monolayer culture [14, 16]. Another approach to studying the effect of growth promotion is the use of ektopic bone formation. Bone morphogenetic protein BMP [18], osteogenin [19], and other distinct bone-inducing factors [20] induce bone formation locally in subcutaneouous sitesin vivo. The purification and characterization, however, have been hampered by the cumbersomein vivo assay. In view of this we used a mitogenic assay on primary embryonic cells to accompany purification, and further analyzed mitogenic fractions for their ability to induce elongation of long bones in culture.     

The role of DNA synthesis in the responses of fetal rat calvariae to cortisol

To determine the extent to which the effects of cortisol on collagen synthesis in 21 day fetal rat calvariae are linked to its effects on cell replication, calvariae were cultured for 24-72 h with 0.1 and 1 microM cortisol in the presence or absence of 1 mM hydroxyurea (HU) or 30 microM aphidicolin (APC), inhibitors of DNA synthesis. The incorporation of [3H]proline into collagenase-digestible protein (CDP) and [3H]thymidine into DNA were measured during the last 2 h of culture. At 24 h HU and APC decreased thymidine incorporation by greater than 90%, and this remained low for the duration of culture. In contrast, cortisol reduced thymidine incorporation by only 44% at 72 h. Although cortisol caused a 24 h stimulatory effect and a 48 and 72 h inhibitory effect on CDP labeling and the percentage of collagen being synthesized (PCS), HU, and APC had no effect on basal CDP labeling or PCS over the 72 h culture period. Cortisol caused parallel alterations in the steady-state levels of alpha-1(I) procollagen mRNA, suggesting that its effects occur at the pretranslational level. At 24 h HU and APC did not prevent the stimulatory effect of cortisol on CDP labeling and PCS. At 48 h the inhibitory effects of cortisol on CDP labeling and PCS were observed in the presence of APC but not in the presence of HU. At 72 h the inhibitory effects of cortisol on CDP labeling and PCS were still observed in the presence of HU and APC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concomitant alteration in number and affinity of P<Subscript>2</Subscript>X and muscarinic receptors are associated with bladder dysfunction in early stage of diabetic rats

Objectives

To investigate time course of bladder dysfunction and concurrent changes in number and affinity of the muscarinic and P₂X receptor in the early stage of streptozotocin (STZ)-induced diabetic rats.

Materials and methods

Diabetic rats were prepared by the intraperitoneal injection of 50 mg/kg of STZ to 7-week-old female Wistar rats. We performed recording of 24-h voiding behavior and cystometry at 1, 4, 8, and 12 weeks after the induction of diabetes. A muscle strip experiments with electrical field stimulation (EFS), carbachol, and α,β-methylene adenosine 5′-triphosphate (α,β-MeATP) were also performed at the same time-points. Additionally, concurrent changes in number and affinity of bladder muscarinic and P₂X receptor were measured by a radioreceptor assay using [N-methyl-³H] scopolamine methyl chloride ([³H]NMS) and α,β-methylene-ATP (2,8-³H) tetrasodium salt ([³H]α,β-MeATP).

Results

In STZ-induced diabetic rats, polydipsic polyuric pollakiuria were noted on recording of 24-h voiding behavior from early stage. Also, the residual urine volume markedly increased in diabetic rats on cystometry. In the muscle strip experiment, the detrusor contractions induced by EFS, carbachol, and α,β-MeATP were enhanced in STZ-induced diabetic rats. Based on the radioreceptor assay, the maximum number of sites (Bmax) for the specific binding of [³H]NMS and [³H]α,β-MeATP was concurrently increased in the bladder from diabetic rats.

Conclusion

Increased bladder contractility is found in early stage of diabetic rats. Then, bladder dysfunction is associated with increased number of muscarinic and P₂X receptors in STZ-induced diabetic rats.

     

Correlation of lysozyme activity with proteoglycan biosynthesis in epiphyseal cartilage

Summary Pig epiphyseal cartilage (proximal ulna epiphysis) previously incubated in vitro in the presence of sodium [³⁵S]sulfate or [³H]thymidine was either analyzed by autoradiography or separated into 9 morphologically defined consecutive layers and investigated for³⁵S-incorporation into the guanidinium chloride-extractable proteoglycans and for lysozyme activity.The lowest³⁵S incorporation and lysozyme activity were determined in the zone of resting cells, but there is a consecutive increase in the rate of proteoglycan synthesis and lysozyme activity toward the diaphyseal cartilage-bone junction, with the maximum at the lower columnar cell zone and a sharp reduction of both parameters at the hypertrophic zone. The maxima of³⁵S incorporation and [³H]thymidine incorporation do not coincide.The guanidinium chloride-soluble proteoglycans exhibit macromolecular polydispersity. Fractions excluded from as well as retarded by Sepharose 2B gel could be separated and were detected in all zones.The results indicate a correlation of proteoglycan biosynthesis and lysozyme activity in epiphyseal cartilage.     

Effect of ciprofloxacin on the proliferation of osteoblast-like MG-63 human osteosarcoma cells in vitro

Locally applied antibiotic therapy is gaining popularity for the treatment of infections associated with open fractures and posttraumatic osteomyelitis. With use of local techniques, ciprofloxacin levels as high as 1,300 μg/ml. or over 200 times the bone levels achieved with intravenous administration, have been reported. To study the possible effects of ciprofloxacin on bone, osteoblast-like cells from the MG-63 human osteosarcoma cell line were studied. The cells were grown in antibiotic-free media and exposed to concentrations of ciprofloxacin at 0, 10, 100, 200, and 1,000 μg/ml to establish an initial dose-response curve. Media containing the appropriate dose of ciprofloxacin were changed every 24 hours. Cell number and [³H]thymidine incorporation per cell were determined at 0, 24 and 72 hours. A second dose-response curve was performed at concentrations of 0, 10, 20, 40, and 80 μg/ ml. Three experiments, each with four observations, were performed. The results of this study demonstrated that ciprofloxacin caused significant decreases (p < 0.05) in cell number at 40 μg/ml at 24 hours and 20 μg/ml at 72 hours. [³H]thymidine incorporation per cell decreased significantly at levels of 80 μg/ml at 24 hours and 20 μg/ml at 72 hours. The authors conclude that reported local levels of ciprofloxacin seen in vivo inhibit the proliferation of human osteoblast-like cells in vitro.     

Proliferative and synthetic response of bovine growth plate chondrocytes to various capacitively coupled electrical fields

In vitro monolayer cultures of growth plate chondrocytes isolated from newborn calf costochondral junctions were subjected to capacitively coupled electrical fields for 48 h. In part A, the electrical signal was a 60-kHz sine wave applied at different voltages so as to produce electrical fields at the pericellular level of 7, 20, 50, and 126 mV/cm. Incorporations of [3H]thymidine and [35S]sulfate were assayed to determine the effect of the above fields on cells proliferation and matrix synthesis, respectively. Proliferation was increased by 47% in the 20 mV/cm field whereas the same field decreased [35S]sulfate incorporation by 21%. These changes were significant at p less than 0.05 in both instances. In part B, the 20 mV/cm field was applied in a pulsed fashion to produce daily duty cycles of 100, 25, 2, and 0.25%. Incorporation of [3H]thymidine, [35S]sulfate, and [14C]proline per DNA were assayed. Results indicated that the 100, 25, and 0.25% percent duty cycles showed significantly (p less than 0.01-0.05) increased proliferation, whereas the 0.25% signal (5 ms on/495 ms off for 6 h/day) significantly decreased [14C]proline incorporation. We conclude that the biologic response of cells in vitro is signal specific, and that the total amount of electrical energy required to stimulate the growth plate chondrocyte to increased proliferation is very small since the total time the 0.25% duty cycle signal was only 3.6 min of a 24-h period.     

Stimulation of DNA synthesis by epidermal growth factor in osteoblast-like cells

Summary Normal and malignant osteoblast-like cells in culture have been shown to possess specific, high affinity receptors for epidermal growth factor (EGF). In this study, the mitogenic response to EGF was examined in a clonal line of a rat osteogenic sarcoma (UMR 106) and in osteoblast-rich newborn rat calvarial cells. Twenty-four hour treatment of UMR 106 cells with EGF in doses ranging from 10⁻¹² m to 2 × 10⁻⁸ m stimulated the incorporation of [³H]thymidine and DNA synthesis in a dose-dependent manner. This short-term stimulatory effect was sustained in long-term culture with a dose-dependent increase in cell proliferation by calvarial cells. A lag period of 8 h occurred before significant stimulation of [³H]thymidine incorporation was observed. Commitment to increased incorporation of [³H]thymidine required a minimum of 6 h continuous incubation with EGF. These results establish the osteoblast as a target cell for EGF action on bone.     

Verapamil enhancement of chemotherapeutic efficacy in human bladder cancer cells

The calcium influx blocker verapamil has been used to overcome drug resistance in several tumor systems. The possible in vitro enhancement of drug efficacy was assessed in bladder cancer cell line T24. Combination of thiotepa and doxorubicin hydrochloride with verapamil significantly reduced the survival and growth of T24 cells after as little as 1 hour of drug exposure. An increase in doxorubicin hydrochloride-induced inhibition of [3H]thymidine uptake resulted when verapamil was administered. However, this trend was not demonstrated when combined with thiotepa. It appears that verapamil enhances thiotepa-induced cytotoxicity while it potentiates the antimitotic nature of doxorubicin hydrochloride. The data presented is consistent with the postulate that verapamil alters active efflux of drug from malignant cells and suggests that verapamil has a role in the clinical management of bladder cancer.     

Application of targeted radiotherapy/gene therapy to bladder cancer cell lines

OBJECTIVES: A targeted radiotherapy/gene therapy strategy for transitional cell carcinoma of bladder is described, using [131I]meta-iodobenzylguanidine ([131I]MIBG), a radionuclide combined with a tumour-seeking drug. The aim is to decrease side effects from radiation toxicity, while increasing radiation dose to tumour. This tumour cell kill approach is augmented by radiological bystander effects. METHODS: The bladder cancer cell line EJ138 was transfected with a gene encoding the noradrenaline transporter (NAT) under the control of tumour-specific telomerase promoters. Resulting uptake of [131I]MIBG was assessed by gamma-counting of cell lysates, and NAT transgene expression by real-time RT-PCR. Cell kill of monolayers and disaggregated spheroids, dosed with [131I]MIBG, was assessed by clonogenic assay. RESULTS: NAT gene transfected cells exhibited a significantly increased active uptake of [131I]MIBG, leading to dose-dependent cell kill. Clonogenic assay of disaggregated spheroids, a three-dimensional model, suggested cell kill via bystander effects. CONCLUSIONS: Expression of a functional NAT after in vitro transfection of bladder cancer cells with the NAT gene under the control of telomerase promoters leads to active uptake of [131I]MIBG and dose-dependent cell kill. This strategy could produce a promising new treatment option for bladder cancer.     

Sustained proliferation accompanies distraction osteogenesis in the rat

These studies were conducted to compare the local cellular proliferation patterns in the rat tibia during distraction osteogenesis with those during nondistracted fracture healing. Bone specimens from distraction osteogenesis and nondistracted fracture groups were analyzed 2, 10, and 20 days after surgery. Proliferation was determined by metabolic labeling with [³H]thymidine and by immunocytochemistry with an antibody to proliferating cell nuclear antigen. Videomicroscopy was used to count the cells staining positively within specified regions. The number of cells incorporating [³H]thymidine was positively correlated (r² = 0.78) with the number of proliferating cell nuclear antigen positive cells on alternating serial slides. At day 2, the latter cells were largely confined to the bone marrow and periosteum in both groups, and the cell numbers per mm² were also equivalent. At days 10 and 20, the proliferating cell nuclear antigen positive cells predominated in both the proximal and distal primary matrix front zones in the distraction osteogenesis group. In contrast, the proliferating cell nuclear antigen positive cells in the nondistracted fracture group were scattered throughout the healing area. Significantly more of these cells were in the primary matrix front zones than in any location within the nondistracted fracture-healing area. The number of these cells in the bone marrow adjacent to the surgical area declined from day 2 to day 10 in both groups. The results suggest that (a) proliferating cell nuclear antigen immunostaining is a reliable indicator of cycling cells; (b) by day 10, distraction osteogenesis is characterized by a zone-specific pattern of proliferating cells; and (c) distraction osteogenesis prolongs the stimulation of proliferation within the gap after fracture.     

Inhibitory effects of somatostatin on rat hepatocyte proliferation are mediated by cyclic AMP.

Somatostatin (SS-14) is known as an antigrowth factor for a variety of cell types, including gastrointestinal mucosa, exocrine pancreas, lymphocytes, and some tumors. We have recently identified and biochemically characterized SS-14-binding protein on rat liver plasma membranes (S. E. Raper, P. C. Kothary, and J. DelValle, Gastroenterology 96: A408, 1989; P. C. Kothary et al., Digestion 46 (Suppl 1): 58, 1990). We hypothesized that SS-14 may affect liver growth as well and investigated cellular mechanisms of this phenomenon focusing on the second messenger cAMP. Freshly isolated rat hepatocytes were plated on tissue culture dishes coated with Matrigel (laminin, heparan sulfate, and type IV collagen). The medium was not supplemented with serum or hormones. Either dibutyryl-cAMP (1 mM) or isobutylmethylxanthine (IBMX, 0.1 mM) was added in the presence or absence of SS-14 (10 nM). DNA synthesis was estimated by the rate of [3H]thymidine incorporation into DNA and by the labeling index (an autoradiographic measurement of the number of labeled nuclei). SS-14 significantly inhibited both [3H]thymidine incorporation and labeling index of rat hepatocytes stimulated by dibutyryl-cAMP or IBMX. SS-14 also inhibited intracellular cAMP accumulation stimulated by IBMX. We conclude that SS-14 exerts at least part of its antiproliferative effects via the adenylate cyclase system. Further study using other signal transduction systems may yield more information about mechanisms of hepatocyte growth.     

Effects of deflazacort on aspects of bone formation in cultures of intact calvariae and osteoblast-enriched cells.

Deflazacort, a synthetic glucocorticoid reported to have bone-sparing properties in vivo, and cortisol were compared for their effects on bone formation in vitro. Deflazacort and cortisol were studied for their effects on DNA and collagen synthesis in cultures of intact fetal rat calvariae and of osteoblast-enriched (Ob) cells from 21- to 22-day-old fetal rat parietal bone. Both steroids were also examined for their effects on skeletal insulin-like growth factor (IGF) I production, which is decreased by cortisol and appears relevant to its mode of action. After 24 h of culture, deflazacort and cortisol had limited effects on the parameters studied, although cortisol at 100 nM decreased [3H]proline incorporation into collagen in intact calvariae. In contrast, after 72 h deflazacort and cortisol at 1-100 nM inhibited the incorporation of [3H]thymidine into DNA and at 100 nM decreased the incorporation of [3H]proline into collagen and noncollagen protein in intact calvariae. Deflazacort and cortisol at 10-1000 nM decreased calvarial collagen degradation to a similar extent. Both steroids had a similar activity, and at 100 nM for 72 h they decreased IGF-I production by calvariae; however, cortisol at 10 nM was somewhat more effective than deflazacort in decreasing IGF-I levels. Deflazacort and cortisol had analogous effects in Ob cell cultures. After 24 h of treatment, deflazacort at 100-1000 nM and cortisol at 10-1000 nM decreased the labeling of DNA, and both steroids at 100-1000 nM caused a similar decrease in [3H]proline incorporation into collagen in Ob cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukocyte (3H) thymidine uptake in short-term whole-blood cultures of human and canine renal allograft recipients

Serial studies of human and canine renal allograft recipients, using a simple and reproducible whole-blood culture technique, demonstrated that measurements of in vitro leukocyte [³H]thymidine uptake were not useful as a means of predicting and monitoring the onset of early graft rejection.During the first 15 days after transplantation, significant increases in leukocyte incorporation of [³H]thymidine were observed among all nine human recipients studied. However, only three patients showed clinically detectable episodes of allograft rejection.In contrast, canine renal allograft recipients, allowed to undergo rejection of the transplanted organs, showed no significant changes in leukocyte [³H]thymidine uptake during the 5–13 days prior to death.     

Characterization of insulin-like growth factor I binding sites in human bladder cancer cell lines

Summary The role of insulin-like growth factor I (IGF-I) in the growth and development of bladder cancer cells was investigated using cultured human cell lines representing differentiated (RT-4, 5637) or undifferentiated (T-24, J-82, TCC-SUP) transitional cell carcinoma (TCC). In the presence of 2% serum, IGF-I significantly stimulated the growth of all cell lines. The proliferation of T-24, 5637, and RT-4 cells was more sensitive to IGF-I than that of J-82 and TCC-SUP cells. [¹²⁵I]IGF-I binding to 5637 and J-82 cells was significantly higher than that to T-24 and TCC-SUP cells (P<0.001). RT-4 cells possessed the lowest binding capacity among the cell lines tested. Scatchard analysis of [¹²⁵I]IGF-I binding to four of the five cell lines indicated a single binding site for IGF-I, with apparent dissociation constants (K _d) of 1.27, 1.18, 1.34, and 1.39 nmol/l for TCC-SUP, J-82, 5637, and T-24, respectively. Therefore, the difference observed in [¹²⁵I]IGF-I binding among the bladder cancer cell lines was attributed to the difference of IGF-I binding sites and not to a change in receptor binding affinity. Cross-linking studies supported the suggestion that [¹²⁵I]IGF-I was bound to a receptor on these cells. The results indicate that cultured human bladder cancer cells contain functional IGF-I receptors. A differentiated cell line, RT-4, possesses significantly fewer IGF-I receptors than other cell lines. This suggests that the overexpression of IGF-I receptor may reflect the malignant potential of bladder cancer cells.     

Periosteum responds to dynamic fluid pressure by proliferating in vitro.

Periosteum provides a source of undifferentiated chondrocyte precursor cells for fracture healing that can also be used for cartilage repair. The quantity of cartilage that can be produced, which is a determining factor in fracture healing and cartilage repair, is related to the number of available stem cells in the cambium layer. Cartilage formation during both of these processes is enhanced by motion of the fracture or joint in which periosteum has been transplanted. The effect of dynamic fluid pressure on cell proliferation in periosteal tissue cultures was determined in 452 explants from 60 immature (2-month-old) New Zealand White rabbits. The explants were cultured in agarose suspension for 1-14 days. One group was subjected to cyclic hydrostatic pressure, which is referred to as dynamic fluid pressure, at 13 kPa and a frequency of 0.3 Hz. Control explants were cultured in similar chambers without application of pressure. DNA synthesis ([3H]thymidine uptake) and total DNA were measured. The temporal pattern and distribution of cell proliferation in periosteum were evaluated with autoradiography and immunostaining with proliferating cell nuclear antigen. Dynamic fluid pressure increased proliferation of periosteal cells significantly, as indicated by a significant increase in [3H]thymidine uptake at all time points and a higher amount of total DNA compared with control values. On day 3, when DNA synthesis reached a peak in periosteal explants, [3H]thymidine uptake was 97,000+/-5,700 dpm/microg DNA in the group exposed to dynamic fluid pressure and 46,000+/-6,000 dpm/microg in the controls (p < 0.001). Aphidicolin, which blocks DNA polymerase alpha, inhibited [3H]thymidine uptake in a dose-dependent manner in the group subjected to dynamic fluid pressure as well as in the positive control (treated with 10 ng/ml of transforming growth factor-beta1) and negative control (no added growth factors) groups, confirming that [3H]thymidine measurements represent proliferation and dynamic fluid pressure stimulates DNA synthesis. Total DNA was also significantly higher in the group exposed to dynamic fluid pressure (5,700+/-720 ng/mg wet weight) than in the controls (3,700+/-630) on day 3 (p < 0.01). Autoradiographs with [3H]thymidine revealed that one or two cell cycles of proliferation took place in the fibrous layer prior to proliferation in the cambium layer (where chondrocyte precursors reside). Proliferating cell nuclear antigen immunophotomicrographs confirmed the increased proliferative activity due to dynamic fluid pressure. These findings suggest either a paracrine signaling mechanism between the cells in these two layers of the periosteum or recruitment/migration of proliferating cells from the fibrous to the cambium layer. On the basis of the data presented in this study, we postulate that cells in the fibrous layer respond initially to mechanical stimulation by releasing growth factors that induce undifferentiated cells in the cambium layer to divide and differentiate into chondrocytes. These data indicate that cell proliferation in the early stages of chondrogenesis is stimulated by mechanical factors. These findings are important because they provide a possible explanation for the increase in cartilage repair tissue seen in joints subjected to continuous passive motion. The model of in vitro periosteal chondrogenesis under dynamic fluid pressure is valuable for studying the mechanisms by which mechanical factors might be involved in the formation of cartilage in the early fracture callus and during cartilage repair.