Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord.

Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted 'decoy' receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.

Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors.

Dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting step in pyrimidine degradation, was studied in two cell lines of murine neuroblastoma (MNB-T1 and MNB-T2) that were derived from C-1300 MNB tumor carried in A/J mice. The MNB-T2 (low malignancy) cell line was originally derived from the in situ tumor and carried in tissue culture for more than 100 passages; the MNB-T1 (high malignancy) line consisted of a new sub-culture that was also established from the in situ MNB tumor. DPD activity was determined in cytosolic preparations of MNB utilizing high performance liquid chromatography to separate the radiolabeled substrate ([2-14C]thymine) from [2-14C]dihydrothymine. The apparent affinity of DPD for NADPH in MNB cells (Km approximately 0.08 mM) was identical to that of A/J mouse brain and liver. The DPD activity of the high malignancy (MNB-T1) cell line was 14.3% of that observed in the low malignancy (MNB-T2) line. In situ tumors formed after implantation of high malignancy (MNB-T1) cells into A/J mice had only 25.2% of the DPD activity observed in tumors derived from low malignancy (MNB-T2) cells. When MNB-T2 cells were injected into naive A/J mice, tumors developed in only 68% of animals, the tumor growth rate was slow and a mortality of 20% was observed. In contrast, tumors derived from injected MNB-T1 cells showed a faster growth rate and 100% mortality. Most MNB-T2 derived tumors were not lethal and ultimately resolved while the MNB-T1 derived tumors were invariably lethal. These studies support the concept that the levels of DPD activity in neoplastic cells are inversely related to their malignant expression and also provide a model to study differences between neuroblastoma cell lines derived from the same in situ tumor but which manifest different neoplastic behavior.

Effects of uridine and thymidine on the degradation of 5-fluorouracil, uracil, and thymine by rat liver dihydropyrimidine dehydrogenase.

The kinetic properties and control mechanisms of 5-fluorouracil (5-FU), uracil, and thymine degradation by rat liver dihydropyrimidine dehydrogenase were studied in vitro. The calculated Michaelis constant (Km) for 5-FU was 3.49 +/- 0.41 (SE) microM, similar to those for uracil (2.26 +/- 0.28 microM) and for thymine (2.23 +/- 0.34 microM). However, the reduction of 5-FU appears to be most sensitive to the inhibitory effects of increased substrate concentration. The specific activities of dihydropyrimidine dehydrogenase (nmol/min/mg of protein) for 5-FU, uracil, and thymine were 0.82, 0.68, and 0.56, respectively. Uridine was found to be a potent noncompetitive inhibitor of pyrimidine base degradation in vitro, displaying an inhibition constant (Ki) for 5-FU of 0.71 microM. Total inhibition of 5-FU degradation occurred at a uridine concentration of 10 microM, whereas thymidine was found to be a much less potent noncompetitive inhibitor of pyrimidine base degradation (Ki 24 microM). This paper provides the first documentation of in vitro inhibition of dihydropyrimidine dehydrogenase activity by nucleosides. The concomitant utilization of uridine and 5-FU in clinical situations might prove useful by decreasing 5-FU catabolism to toxic metabolites as well as enhancing 5-FU cytotoxicity.

Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain.

This paper describes a model of tumor-induced bone destruction and hyperalgesia produced by implantation of fibrosarcoma cells into the mouse calcaneus bone. Histological examination indicates that tumor cells adhere to the bone edge as early as post-implantation day (PID) 3, but osteolysis does not begin until PID 6, correlating with the development of hyperalgesia. C3H/He mice exhibit a reproducible hyperalgesia to mechanical and cold stimuli between PID 6 and 16. These behaviors are present but significantly reduced with subcutaneous implantation that does not involve bone. Systemic administration of morphine (ED(50) 9.0 mg/kg) dose-dependently attenuated the mechanical hyperalgesia. In contrast, bone destruction and hypersensitivity were not evident in mice implanted with melanoma tumors or a paraffin mass of similar size. A novel microperfusion technique was used to identify elevated levels of the putative algogen endothelin (ET) in perfusates collected from the tumor sites of hyperalgesic mice between PID 7 and 12. Increased ET was evident in microperfusates from fibrosarcoma tumor-implanted mice but not from melanoma tumor-implanted mice, which are not hyperalgesic. Intraplantar injection of ET-1 in naive and, to a greater extent, fibrosarcoma tumor-bearing mice produced spontaneous pain behaviors, suggesting that ET-1 activates primary afferent fibers. Intraplantar but not systemic injection of the ET-A receptor antagonist BQ-123 partially blocked tumor-associated mechanical hyperalgesia, indicating that ET-1 contributes to tumor-induced nociception. This model provides a unique approach for quantifying the behavioral, biochemical, and electrophysiological consequences of tumor-nerve interactions.

Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain.

The cancer-related event that is most disruptive to the cancer patient's quality of life is pain. To begin to define the mechanisms that give rise to cancer pain, we examined the neurochemical changes that occur in the spinal cord and associated dorsal root ganglia in a murine model of bone cancer. Twenty-one days after intramedullary injection of osteolytic sarcoma cells into the femur, there was extensive bone destruction and invasion of the tumor into the periosteum, similar to that found in patients with osteolytic bone cancer. In the spinal cord, ipsilateral to the cancerous bone, there was a massive astrocyte hypertrophy without neuronal loss, an expression of dynorphin and c-Fos protein in neurons in the deep laminae of the dorsal horn. Additionally, normally non-noxious palpation of the bone with cancer induced behaviors indicative of pain, the internalization of the substance P receptor, and c-Fos expression in lamina I neurons. The alterations in the neurochemistry of the spinal cord and the sensitization of primary afferents were positively correlated with the extent of bone destruction and the growth of the tumor. This "neurochemical signature" of bone cancer pain appears unique when compared to changes that occur in persistent inflammatory or neuropathic pain states. Understanding the mechanisms by which the cancer cells induce this neurochemical reorganization may provide insight into peripheral factors that drive spinal cord plasticity and in the development of more effective treatments for cancer pain.

Three years of experience with random urinary homovanillic and vanillylmandelic acid levels in the diagnosis of neuroblastoma.

During the last 3 years, random urine samples from 408 patients were tested for elevated homovanillic acid (HVA) and vanillylmandelic acid (VMA) levels to rule out the diagnosis of neuroblastoma. Thirty-seven of these patients had elevated HVA and/or VMA levels, and neuroblastoma was subsequently diagnosed. In three additional patients with negative test results (normal HVA and VMA levels), tumors were subsequently diagnosed (false-negative rate of 7.5%). Ten percent of the patients with neuroblastoma had normal HVA and 27.5% had normal VMA levels at the time of diagnosis. Only one patient (2.5%) with neuroblastoma had elevated VMA levels in the presence of normal HVA levels. More than 60% of the patients with neuroblastoma had urinary HVA and/or VMA levels higher than twice the upper limit of normal. No false-positive results were encountered. Age and stage distributions of the patients are shown, and the significance of the results is discussed.

Value of random urinary homovanillic acid and vanillylmandelic acid levels in the diagnosis and management of patients with neuroblastoma: comparison with 24-hour urine collections.

Urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) levels were determined in random samples and in 24-hour collections from 13 patients with neuroblastoma and 22 patients without neuroblastoma. Random sample levels were compared with levels in 24-hour collections and showed a positive correlation of 95% for HVA (N = 59) and 93% for VMA (N = 52). No false positives or false negatives occurred using random samples for diagnosis. Nonneuroblastoma (normal) HVA (N = 126) and VMA (N = 119) levels are reported for different age groups. Sequential random HVA and VMA determinations in patients with neuroblastoma during and after therapy are shown. Random urinary HVA and VMA levels are shown to be adequate for utilization in the diagnosis of neuroblastoma and sequential determinations of random HVA and VMA are shown to be helpful in the follow-up of those patients.

Assessment of the diurnal variations in urinary homovanillic and vanillylmandelic acid excretion for the diagnosis and follow-up of patients with neuroblastoma.

The diurnal variation of urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) was studied in neuroblastoma patients and in a control group. Urinary HVA and VMA levels in four sequential 6-hour urine collections within a 24-hour period were compared. HVA and VMA levels were expressed in microgram/mg of urinary creatinine (UCr) and in mg/6h specimens. No statistically significant variations between the four time intervals were found when expressed in microgram/mg UCr or mg/6h. The small variations that exist in the excretion of HVA and VMA during different periods of the day are due to variations in renal excretion rather than variations in production. The results from this study indicate that a random urine sample should be as good as a 24-hour collection for diagnosis and follow-up of neural crest tumors.

Determination of urinary homovanillic and vanillylmandelic acids from dried filter paper samples: assessment of potential methods for neuroblastoma screening.

Urinary homovanillic (HVA) and vanillylmandelic (VMA) acids were analyzed on 200 random urine samples from patients with neuroblastoma and controls, after the samples had been dried onto absorbent filter paper. The acids were determined quantitatively by gas chromatography (GC) and qualitatively by thin layer chromatography (TLC). The results were analyzed for correlation between liquid urine samples and urine dried on filter paper and between TLC and GC methods. A high overall correlation for HVA and VMA (99%) was found between liquid and dried filter samples analyzed by GC. The correlations were more significant for samples with elevated levels of these acids than for those with normal levels. Normalization of the results to the urinary creatinine concentration (UCr) is indicated due to variations in urine concentration. Results from TLC analysis showed a false positive rate of 3.5% and a false negative rate of 0.5% compared to GC analysis. This work suggests that a combination of a sensitive TLC method with a rapid quantitative GC method would be suitable for mass neuroblastoma screening in infants.

Osteoclast formation during tumor osteolysis does not require proliferating osteoclast precursor cells.

The cellular mechanism or mechanisms through which tumors induce osteoclast formation at sites of tumor osteolysis is unknown. To test the hypothesis that osteoclast formation at sites of tumor osteolysis reflects influences that tumors have on proliferating osteoclast precursor cells, a novel in vivo experimental model was developed that produced mice that were deficient in osteoclasts (op/op) and were depleted (by way of total body irradiation) of proliferating osteoclast precursor cells. The femora of irradiated op/op mice were injected with tumor cells (2472 clone) that had been previously shown to form osteolytic tumors and to induce focal osteoclastogenesis, and the influence of these tumor cells on osteoclast formation was determined in op/op mice that were depleted of proliferating osteoclast precursor cells. The results indicated that 2472 tumor cells induced osteoclast formation in op/op mice despite the absence of proliferating osteoclast precursor cells. This finding disproved the hypothesis under investigation and suggests that osteoclast formation at sites of tumor osteolysis reflects influences of tumors on postmitotic, not proliferating, osteoclast precursor cells.

Osteoclasts are required for bone tumors to grow and destroy bone.

It has been hypothesized that bone resorption during tumor osteolysis is performed by osteoclasts. Data supporting this hypothesis have been provided from analysis of human biopsy specimens obtained from sites of tumor osteolysis, as well as from experimentation with in vivo animal models. Experiments in this report take this concept one step further by testing the hypothesis that osteoclasts are required for bone tumors to grow and destroy bone. To test this hypothesis, the influence of an osteolytic sarcoma tumor, NCTC clone 2472 (2472), on bone was studied in animals that are osteoclast deficient (microphthalmic, strain B6C3Fe-a/a-Mitf(mi)) but whose osteoclast deficiency can be reversed following bone marrow transplantation. Femora of these mice and unaffected wild-type siblings were injected with 10(5) 2472 cells, and after 14 days the femora were analyzed by radiographic and histomorphometric analysis. Macroscopic tumor, tumor-induced osteolysis, and increased osteoclast number were noted in femora of normal mice but not in femora of osteoclast-deficient mice (p < 0.001). Bone marrow transplantation converted osteoclast-deficient mice to mice with femora that contained osteoclasts in 4 weeks. Femora of these mice were then injected with 10(5) 2472 tumor cells; after 14 days, in contrast to the findings in the original osteoclast-deficient mice, macroscopic tumor was present, tumor-induced osteolysis was noted on roentgenograms, and osteoclast number was increased when tumor-bearing limbs were compared with sham-injected limbs (p < 0.001). These data prove the hypothesis that osteoclasts are required for 2472 tumor-induced osteolysis, and they introduce the exciting possibility that osteoclasts are also required for tumors to grow in bone.

Pamidronate decreases tumor-induced osteoclastogenesis in osteopetrotic mice.

Recent studies indicate that the bisphosphonate pamidronate reduces skeletal complications caused by tumor osteolysis. In this investigation, the cellular mechanism through which pamidronate affects tumor-induced osteoclastogenesis is studied in osteopetrotic mice. A unique animal model is employed which studies the effect of pamidronate on a tumor (2472 sarcoma) which induces osteoclastogenesis in osteoclast-deficient mice (oplop). This model provides opportunity to specifically study effects on osteoclast formation and findings suggest that pamidronate decreases the number of osteoclasts at sites of 2472 tumor by decreasing the number of osteoclast precursor cells at the level of myeloid precursors.

Tumor osteolysis in osteopetrotic mice.

Details of the cellular and biochemical mechanisms involved in focal destruction of bone at sites of tumor osteolysis are unknown. It has been shown that tumors from sarcoma (2472) cell lines induce focal osteolysis in mice by stimulating formation and activation of osteoclasts. In this report, the influence of 2472 tumors on the skeletons of osteoclast-deficient animals (op/op) was studied. After op/op femora had been inoculated with 2472 cells, tumors developed and focal osteolysis occurred. There were more osteoclasts per histologic section in sham-injected femora (19 +/- 5) than in tumor-bearing femora (412 +/- 129) (p < 0.05). The size of the osteoclasts also increased from 304 +/- 81 microns 2 in sham-injected limbs to 407 +/- 62 microns 2 in tumor-bearing limbs (p < 0.001). Conditioned media from 2472 op/op tumor explants contained macrophage colony-stimulating factor. A deficiency of osteoclasts in op/op mice is the result of the absence of this factor; therefore, these data introduce the possibility that macrophage colony-stimulating factor derived from 2472 tumor may be responsible for directing osteoclast-mediated osteolysis at sites of the tumor.

Localized, tumor-associated osteolysis involves the recruitment and activation of osteoclasts.

The cellular and biochemical mechanisms that direct destruction of bone at the site of tumor osteolysis are unknown. In order to understand this process better, a murine model designed for the study of tumor osteolysis was developed and the influence of osteolytic and nonosteolytic tumors on bone was investigated. Tumors developed following femoral intramedullary injection of sarcoma (2472) and melanoma (G3.26) cell lines; however, only tumors from the 2472 cell line caused osteolysis. It was determined that 2472 tumor-induced osteolysis commenced 6 days after the femora had been inoculated with 2472 cells. There were more osteoclasts per millimeter of bone surface in 2472 tumor-bearing limbs (16.7 +/- 5.0) than in sham-injected limbs (3.8 +/- 0.9) (p < 0.015). In addition, an increase in the osteoclast size (area) was detected in 2472 tumor-bearing limbs: 412 +/- 65 micron2 compared with 187 +/- 17 micron2 (p < 0.01). In vitro bone resorption experiments indicated that 2472 tumor cells had a limited ability to destroy bone in comparison with macrophages and osteoclasts. Taken in total, these findings define a model that is useful for the study of tumor osteolysis, and the data from analyses of the model demonstrate that the cellular mechanisms responsible for 2472 tumor-induced osteolysis include both an increase in the number of osteoclasts and activation of mature osteoclasts.

Human breast cancer induces osteoclast activation and increases the number of osteoclasts at sites of tumor osteolysis.

The cellular mechanism through which osseous breast cancer metastases induce the focal destruction of bone (tumor osteolysis) is unknown. An athymic mouse model designed for the study of tumor osteolysis was developed and the influence of two human breast cancer tumors on bone was studied. Tumor-induced osteolysis occurred between 7 and 10 weeks after inoculation of mouse femora with MDA-MB-231 or MDA-MB-435s breast cancer cells. An increase in osteoclast number and an increase in osteoclast size (area) were detected when tumor-bearing and sham-injected limbs were compared. In vitro analysis of the influence of the tumor-conditioned medium on osteoclast-mediated bone resorption revealed that this conditioned medium stimulated the resorption by increasing both the number of osteoclasts bound to bone and the number of bone resorption pits formed per osteoclast. In addition, in vitro analysis of the influence of breast cancer tumor cells on osteoclast formation or survival, or both, demonstrated that breast cancer cells induced a dramatic increase in the number of osteoclasts detected in culture. Taken in total these findings suggest that human breast cancer tumors induce osteolysis by enhancing osteoclast adherence to bone, stimulating osteoclast-mediated bone resorption and either prolonging the survival of osteoclasts or increasing osteoclast formation.

Osteoprotegerin inhibits tumor-induced osteoclastogenesis and bone tumor growth in osteopetrotic mice.

Osteoprotegerin and osteoprotegerin ligand have recently been identified as novel proteins that inhibit and stimulate, respectively, osteoclast formation. We examined the possibility that osteoprotegerin would inhibit cancer-induced osteoclastogenesis and cancer growth in bone. An experimental model was used in which osteolytic tumors are known to stimulate osteoclastogenesis and grow in femora of osteoclast-deficient mice (op/op). Osteoprotegerin treatment decreased the number of osteoclasts by 90% (p < 0.0007) at sites of tumor in a dose-dependent manner and decreased bone tumor area by greater than 90% (p < 0.003). The mechanisms through which osteoprotegerin decreased osteoclast formation in tumor-bearing animals included (a) an osteoprotegerin-mediated, systemic reduction in the number of splenic and bone marrow-residing osteoclast precursor cells, (b) a decrease in the number of osteoclast precursor cells at sites of tumor as detected by cathepsin K and receptor activator of NFkappaB mRNA expression, and (c) a decrease in osteoprotegerin ligand mRNA at sites of tumor. These findings suggest that osteoprotegerin treatment, in addition to having direct antagonistic effects on endogenous osteoprotegerin ligand, decreases the number of osteoclast precursors and reduces production of osteoprotegerin ligand at sites of osteolytic tumor.

Release of immunosuppressive substances from tissue culture cells infected with bovine viral diarrhea virus.

Bovine fetal lung tissue culture cells were infected with bovine viral diarrhea virus. The cells released substances into the supernatant fluid that suppressed the proliferative response of bovine peripheral blood mononuclear cells which had been stimulated with concanavalin A. Activity could be found in low molecular weight fractions (less than or equal to 2,000 daltons). During the assay, these fractions were required to inhibit the response of leukocytes to concanavalin A. Addition of indomethacin to the infected tissue culture cells inhibited generation of immunosuppressive supernatants; addition of indomethacin to the suppressive supernatants did not block their activity. The possible involvement of prostaglandins in this immunosuppressive activity is discussed.

Cytotoxic effect of Pasteurella haemolytica on bovine polymorphonuclear leukocytes and impaired production of chemotactic factors by Pasteurella haemolytica-infected alveolar macrophages.

Pasteurella haemolytica exerted a cytotoxic effect on bovine polymorphonuclear neutrophil leukocytes. This effect was less than that seen with cultured alveolar macrophages or peripheral blood monocytes. When alveolar macrophages were cultured with Pasteurella haemolytica, macrophages produced less chemotactic factors for polymorphonuclear leukocytes than did noninfected controls. This effect was reversible, in that removal of the bacteria permitted remaining macrophages to elaborate more chemotactic factors than was seen in controls. The possible consequences of this impairment of function of alveolar macrophages are discussed.

Aromatic and nitrogen-containing acid separation by reversed-phase high performance liquid chromatography and identification by photodiode array detector.

A new method of separation and identification of aromatic and nitrogen-containing acids is presented. Fifty-seven different acid standards were separated by reversed-phase high performance liquid chromatography (HPLC). Detection and identification of the acids by ultraviolet (UV) spectra using a photodiode array detector (DAD) is demonstrated. Peak height ratios are reported and complete UV spectra of these acids are displayed. Possible applications of this technique are demonstrated using patients' urines.

Determination of urinary succinylacetone by capillary gas chromatography.

The average analytical recovery of succinylacetone added to urine and separated by capillary gas chromatography was 69% for solvent extraction and 72% for anion exchange separation. Treating succinylacetone with hydroxylamine hydrochloride at a pH of less than 5 caused formation of a derivative separated by capillary gas chromatography into two isomers: 3-methyl-5- isoxazole propionate and 5-methyl-3- isoxazole propionate as their trimethylsilyl derivatives (molecular weight 227). In a pH greater than or equal to 5, succinylacetone dioxime was formed and separated into 3 isomers as their trimethylsilyl derivatives (molecular weight 404). Succinylacetone dioxime was converted to 3(5)-methyl-(3)5- isoxazole propionate whenever the pH of the solution was dropped to less than 5. Mass spectra of both derivatives are shown. This study demonstrates that capillary gas chromatography is suitable for use in urinary succinylacetone determination.