Human fetal and adult chondrocytes. Effect of insulinlike growth factors I and II, insulin, and growth hormone on clonal growth.

Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life.

Biochemical analysis of callus tissue in osteogenesis imperfecta type IV. Evidence for transient overmodification in collagen types I and III.

We analyzed tissue and cells from a stationary and a rapidly growing hyperplastic callus from a patient with osteogenesis imperfecta (OI) type IV and compared the results with those of compact bone and skin fibroblasts of an age-matched control. Collagen and protein contents per cell were low in the callus tissues and collagen I and III were overmodified as evidenced by an elevated level of hydroxylysine. The degree of lysyl hydroxylation was highest in those regions that appeared most immature by histological examination. Lysyl hydroxylation approached normal levels in collagen from the stationary callus and from the center of the growing callus. Overmodification of collagen was not seen in compact bone or cell cultures (neither skin fibroblasts nor callus cells) from the patient. Elevation of hydroxylysine in collagen from OI patients is generally attributed to mutations that delay triple helix formation. Our observations suggest that the varying degree of collagen modifications may occur in consequence of regulatory mechanisms during bone development and tissue repair. These mechanisms may be defective in some patients with OI as seen in this case with hyperplastic callus formation.

Long-term risk of second malignancy in survivors of Hodgkin's disease treated during adolescence or young adulthood.

PURPOSE: To quantify the long-term risk of second primary cancers (SCs) in patients diagnosed with Hodgkin's disease (HD) during adolescence or young adulthood. PATIENTS AND METHODS: The risk of SCs was assessed in 1,253 patients diagnosed with HD before the age of 40 years and treated in two Dutch cancer centers between 1966 and 1986. The median follow-up duration was 14.1 years. RESULTS: In all, 137 patients developed SCs, compared with 19.4 cases expected on the basis of incidence rates in the general population (relative risk [RR] = 7.0; 95% confidence interval, 5.9 to 8.3). The 25-year actuarial risk of SC overall was 27.7%. The RR of solid tumors increased greatly with younger age at the first treatment of HD, not only for breast cancer but also for all other solid tumors, with RRs of 4.9, 6.9, and 12.7 for patients first treated at ages 31 to 39 years, 21 to 30 years, and

Skeletal growth in fetal rats. Effects of glucose and amino acids.

The modified hyperglycemia-hyperinsulinism hypothesis, which characterizes intrauterine growth of diabetic pregnancy, was studied in fetal rats. From day 19 to day 21 postconception, pregnant rats were constantly infused with saline, amino acids, or glucose. In the fetus, serum somatomedin activity was determined, with the porcine bioassay and the incorporation of 3H-thymidine into rib cartilage and isolated chondrocytes in vivo in response to serum from normal maternal or fetal rats. In comparison with control fetuses, body weights were decreased in glucose-exposed fetuses (4.66 +/- 0.25 versus 3.75 +/- 0.99, N = 121; P less than 0.001), and increased (4.87 +/- 0.57, N = 105; P less than 0.05) in amino acid-exposed fetuses. Serum somatomedin activity (U/ml) was higher in glucose-treated (0.79 +/- 0.40, N = 11; P less than 0.05) and amino acid-treated animals (0.90 +/- 0.16, N = 10; P less than 0.001) than in controls (0.55 +/- 0.04, N = 13). In vivo labeling with thymidine resulted in a higher radioactivity of cartilage in small fetuses compared with large fetuses when the dams had been infused with saline (r = -0.531, N = 56; P less than 0.001) or amino acids (r = -0.292, N = 52; P less than 0.01). Opposite results were obtained in hyperglycemic animals (r = 0.542, N = 54; P less than 0.001). When isolated chondrocytes were incubated with serum from normal fetal rats, the incorporation of thymidine was about 10 times higher into cells from small fetuses than from large fetuses, irrespective of the infusion regimen.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of insulin during cell cycle in a myeloid cell line.

The characteristics of insulin receptor binding and structure and its proliferative and metabolic action in a human leukemic cell line was investigated during the cell cycle. Exponentially growing cells were separated by counterflow centrifugation which fractionates cells primarily on the basis of size into subpopulations representing G0/G1, S, and G2 + M cells. This method avoids disturbance of the cellular metabolism. After separation the cells showed a viability of at least 92%, underwent further proliferation, and remained morphologically unchanged, which was shown by electron microscopy. The cells could be enriched to 70-90% purity for G0/G1 phase and 50-60% purity for S and G2 + M phase, respectively, which was shown by DNA flow-cytometry. Specific binding of insulin could be demonstrated in G0/G1, S, and G2 + M enriched cells. Insulin binding sites decreased from 20-25,000 per cell in G0/G1 to 1-2,000 in S and increased to 30-50,000 in G2 + M. The affinity of insulin binding remained nearly constant during the cell cycle. The specificity of the insulin receptor could also be demonstrated by covalent crosslinking of the receptor to radiolabeled ligand in all enriched cell fractions. Glucose transport was stimulated by insulin independently of cell cycle. An increase to 140% of control was observed at an insulin concentration of 10 ng/ml. In contrast, glycogen synthesis could only be stimulated by insulin in the G0/G1 phase. An increase to 140% of control was already reached at 0.25 ng/ml insulin. Insulin in concentrations of 1 and 10 ng/ml stimulated the transit to S-phase in cycling, but not in resting, cells. The growth promoting action of insulin could be investigated by consecutive DNA analysis of the separated cells which had been stimulated by insulin.

Heterogeneity of insulin and insulin-like growth factor I binding in a human Burkitt type ALL cell line during the cell cycle and in three Burkitt type ALL sublines.

The expression of hormonal and growth factor receptors in leukemic cell lines might be heterogeneous due to the admixture of different cell cycle phases and the presence of yet unidentified sublines. Therefore, cell cycle-specific separation of Burkitt type ALL cells was performed by counterflow elutriation, and by limited dilution procedure three sublines of this cell line were obtained. Counterflow elutriation enriched to 60-80% purity for G1-, S-, and G2-phase which was shown by DNA flow cytometry. Insulin and insulin-like growth factor I (IGF-I) binding was investigated in the G1, S, and G2 phase. Insulin binding sites decreased from 10 to 15,000 per cell in G1 to 1,000-5,000 in S and increased to 40-50,000 in G2. The affinity of insulin binding remained constant during the cell cycle. IGF-I binding sites increased from 2,000 per cell in G1 to 5,000 in S and 15,000 in G2. The affinity of IGF-I binding decreased from G1 toward S and then remained constant in G2. The three isolated clonal sublines differed in numbers of insulin and IGF-I binding sites/cell without differences in affinity. The fact that IGF-I shows higher affinity binding during G1 than during S and G2 Burkitt type ALL cells suggests that IGF-I might be important for initiation of proliferation. The reduction in insulin binding sites during S-phase may indicate refractoriness of the cell to insulin during DNA replication.

Expression of osteoblastic markers in cultured human bone and fracture callus cells.

We compared the expression of osteoblastic markers in cultured human cells isolated from fracture calluses of various histological states of development with that in cells from adult and fetal bone. Adult osteoblasts and all callus cells produced almost exclusively type I collagen, whereas fetal osteoblasts produced also considerable amounts of type III collagen in vitro. 1,25-Dihydroxyvitamin D3 induced the synthesis of osteocalcin in all bone and callus cells but to varying extents. Fetal bone cells and early-stage callus cells synthesized less than 10% the amount of osteocalcin produced by adult bone cells. Late-stage callus cells produced intermediate levels of osteocalcin. Fetal bone cells and early-stage callus cells responded to parathyroid hormone with a less pronounced increase in intracellular cAMP than did adult bone cells. Late-stage callus cells showed the best response to parathyroid hormone. The activity of alkaline phosphatase was highest in fetal bone cells. These observations show that cells isolated from fetal bone and from fracture callus tissues express a pattern of markers clearly relating them to the osteoblastic lineage. On the basis of the different patterns of osteoblastic markers expressed in vitro we conclude that functionally distinct subtypes of osteoblasts do exist in different mineralized tissues and at different developmental stages.

Determination of gradient elastic tensors: stress and strain dependencies of electric field gradients in cubic and hexagonal systems.

We present ab-initio calculations of the independent components of gradient elastic tensors, so-called gradient elastic constants, which relate electric field gradient tensors to stress or strain tensors. The constants of cubic and hexagonal metals, MAX phases, and zinc oxide were determined within the framework of density functional theory by using the augmented plane waves plus local orbitals method implemented in the WIEN2k code. Comparison with experimental gradient elastic constants and electric field gradients' stress dependencies suggest an accuracy of about 30% of the calculated constants, independent of the probe that detects the field gradient being self- or foreign-atom. Changes in the electric field gradient take place by strain-induced asymmetric occupations of the p and d states in the valence region for all investigated materials. Volume and structural dependencies of the electric field gradient can directly be determined from this fundamental approach and are, for hexagonal closed packed metals, consistent with vanishing electric field gradients around ideal close packing and volume dependencies larger than one. The concept of these calculations is applicable in any hyperfine interaction method and, thus, can be used to gain information about intrinsic strains in systems where the experimental gradient elastic constants are inaccessible.

Extracellular matrix stoichiometry in osteoblasts from patients with osteogenesis imperfecta.

In previous work, we compared the steady-state levels of specific matrix components in human bone cells derived from patients with osteogenesis imperfecta (OI) to those of age-matched controls. A remarkable finding was the observation that there was a reduction not only in the total levels of collagen, but also in osteonectin and three proteoglycans (a large chondroitin sulfate proteoglycan, biglycan, and decorin). This pattern was observed in patients with and without detectable collagen defects. More recent analysis of extracellular matrix composition have yielded that, compared with age-matched controls, bone cells from OI patients produced higher steady-state levels of fibronectin and thrombospondin. The percentage of these two proteins incorporated into the cell layer pool was also higher in OI than in age-matched controls. In addition, the steady-state levels of hyaluronan and a heparan sulfate proteoglycan were analyzed in both OI and age-matched controls. Although the total (medium + cell layer) steady-state levels of hyaluronan were reduced by 1/3, the percentage of the hyaluronan in the cell layer pool of patients with OI increased between 100-250% of age-matched control. Thus the matrix elaborated by human OI bone cells is not only quantitatively different but also qualitatively distinct from that of age-matched controls. Not only have specific bone cell matrix components (collagen, osteonectin, the large chondroitin sulfate proteoglycan, biglycan, and decorin) been found to be present in reduced levels in OI bone cells, but some matrix components (thrombospondin, fibronectin, and hyaluronan) have also been found to be present in elevated levels in the matrix of OI cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular matrix formation by osteoblasts from patients with osteogenesis imperfecta.

Extracellular matrix proteins synthesized by bone cells isolated from 16 patients with different forms of osteogenesis imperfecta (OI) were analyzed in vitro. Specific components of the extracellular matrix by OI and age-matched cultures were investigated by steady-state radiolabeling followed by quantitation of label into specific proteins and comparison of OI cultures to those of age-matched controls. The in vitro proliferation of OI bone cells was found to be lower than that of control cells. In seven patients, abnormalities of the alpha 1(I) and/or alpha 2(I) chains of type I collagen were detected by gel electrophoresis. In two of these patients, the mutations in the COLIA1 and COLIA2 genes have been previously identified. Although the amount of total protein synthesized by the cells in culture was the same for OI bone cells and age-matched control cells, OI bone cells showed a significantly reduced synthesis of not only collagen but also other bone matrix glycoproteins. The synthesis of osteonectin (SPARC/BM40) and three proteoglycans [a large chondroitin sulfate proteoglycan, biglycan (PGI), and decorin (PGII)] was found to be decreased in OI cells. The reduction was most pronounced at the developmental age at which these macromolecules reach maximal levels during normal development.

Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta.

Urinary excretion of type I collagen cross-linked N-telopeptides was studied in 52 children and adolescents with osteogenesis imperfecta (OI) and found to be above the 75th percentile of controls in 44 of the patients. OI patients suffering from fractures during the preceding 6 months had significantly higher values (p < 0.05). In contrast, patients with better motor performance tended to have lower values (p = 0.059). The concentration of urinary type I collagen cross-linked N-telopeptides was positively correlated with urinary calcium excretion (p < 0.05), which was found to be elevated in 20 of the patients. Our results show that during childhood and adolescence in OI not only the synthesis but also the turnover of mature cross-linked type I collagen is disturbed and provide evidence that bone resorption rates are elevated.

Collagen crosslinks and mineral crystallinity in bone of patients with osteogenesis imperfecta.

In cortical bone samples from patients with osteogenesis imperfecta (OI), the concentrations of hydroxypyridinium cross-linking amino acids in collagen were measured by reversed-phase HPLC and the x-axis crystallinity of the apatite mineral phase was determined by x-ray diffraction. Bone samples from three patients with type I, nine patients with type III, and eight patients with type IV OI were analyzed and compared with human bone from nine controls. The concentration of the two chemical forms of the mature collagen crosslinking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and the ratio HP/LP were found to be alike in bone collagen of OI patients and healthy controls. However, the c-axis crystallinity of the apatite was found to be reduced in the type III and IV OI patients compared with controls. Regression analysis of crosslink concentrations and c-axis crystallinity in OI bones did not show any correlation. Therefore, collagen molecules deposited in the extracellular matrix of OI bone appear to fulfill the structural requirements for the action of the enzyme lysyl oxidase, such that a normal concentration of intermolecular crosslinks is formed compared with healthy bone. Consequently, crosslink formation and apatite crystal growth seem to be regulated independently in OI bone.

Osteogenesis imperfecta: changes in noncollagenous proteins in bone.

The noncollagenous proteins osteonectin, bone sialoprotein, osteocalcin, the small proteoglycan decorin (PG II), and alpha 2-HS glycoprotein (which is synthesized in the liver but highly concentrated in bone) were measured in extracts of cortical bone from 3 type I, 2 type II, 8 type III and 13 type IV patients with osteogenesis imperfecta (OI) and from 7 control subjects. Osteonectin was found to be reduced in the bone of all OI patients. The bone from severely affected type III OI patients contained the lowest levels of osteonectin. In contrast, bone sialoprotein was found to be elevated in the bones of OI patients. The highest levels were found in individuals classified as type IV patients. Osteocalcin and alpha 2-HS glycoprotein concentrations were increased in all OI patients. Decorin levels were not significantly altered in OI bones compared to controls. These changes in the concentrations of the noncollagenous proteins may contribute to the fragility of the OI bone by interfering with complete mineralization and/or normal tissue architecture.

Structure and molecular regulation of bone matrix proteins.

The organic matrix of bone contains several protein families, including collagens, proteoglycans, and glycoproteins, all of which may be extensively modified by posttranslational events, such as phosphorylation and sulfation. Many of the glycoproteins contain Arg-Gly-Asp (RGD), the integrin-binding sequence, within their structure, whereas other constituent proteins contain gamma-carboxyglutamic acid. The deposition of bone matrix by cells in the osteoblastic lineage is regulated by extrinsic factors, such as systemic and local growth factors and physical forces, and factors that are intrinsic to the cell, such as position in the cell cycle, maturational stage, and developmental age of the donor. Recent studies of several bone matrix gene promoters have identified cis- and trans-acting elements that are responsible for gene activity, although the precise sequence of regulatory events is not known. Development of in vitro assays, coupled with studies of the appearance of these proteins during development in vivo, provides insight into the functions of these proteins during the various stages of bone metabolism. Potential roles for these proteins include proliferation and maturation of stem cells, formation of matrix scaffolding elaborated by bone-forming cells, modeling, and remodeling. Changes in the functional properties of the extracellular matrix may be involved in a variety of disease processes, including osteoporosis and oral bone loss.

Ultrastructural studies of bones from patients with osteogenesis imperfecta.

Bone samples from patients suffering from osteogenesis imperfecta (OI) types I, II, III and IV, as well as normal controls, were studied by scanning (SEM) and transmission electron microscopy (TEM). SEM views of normal bone at low magnification show coherent structure, with regular striations due to a lamellar plywood-like arrangement of the mineralized collagen fibrils. Compact lamellar bone was also found in various OI specimens, but in limited disconnected regions separated by open spaces. Furthermore, some OI, but not normal, bones have regions of loose unconnected fibers and others of apparently abnormally dense mineral deposition. High resolution TEM studies of OI bone fragments have served to elucidate the structures of these different textures. There appears to be a substantial, though reduced, proportion of normal lamellar bone even in quite severe OI. However, the regions of loose fibers are largely unmineralized and probably contain abnormal collagen. Other regions are overmineralized, with generally small unorganized apatite crystals deposited onto fibril surfaces or in separate clusters. These structural abnormalities, together with the paucity of normal bone, may explain the fragility of OI bones.

Expression of p53 in human leukemic cell lines.

The cell-encoded p53 antigen seems to be tightly associated with various human malignancies. We have analyzed biochemical properties of p53 in two different cell lines derived from patients with ALL or ANLL. p53 was found in elevated levels in both leukemic cell lines compared to unstimulated or stimulated normal lymphocytes. High levels of p53 in these cell lines are due to an extended stability of p53 protein rather than to different rates of synthesis. p53 from both cell lines formed low- and high-molecular weight oligomers which revealed that p53 exists in a heterogenous population in these tumor cells. The presence of immunologically different subsets of p53 was demonstrated by sequential immunoprecipitation experiments with different p53 specific monoclonal antibodies. Our results showed structural and immunological variabilities of p53 in cell lines derived from human tumors and may thus provide an insight into the role p53 may play in human malignancies.

Human leukemic cells: receptor binding and biological effects of insulin and insulin-like growth factors.

Receptor binding and biological effects of insulin and insulin-like growth factors I and II (IGF I/II) were assessed in three human malignant cell lines: a Burkitt-type ALL-cell line, a ANLL-cell line and a Hodgkin's disease-cell line. Insulin receptor binding could be demonstrated in Burkitt-type ALL cells and ANLL cells, whereas no insulin receptor binding was detectable in Hodgkin cells. IGF I and IGF II binding could be demonstrated in all leukemic cells. Insulin stimulated glycogen synthesis in the insulin receptor bearing cell lines. DNA synthesis was stimulated by insulin, IGF I and II. IGF I was more active in stimulating DNA synthesis than IGF II.

Osteogenesis imperfecta and hyperplastic callus formation: light- and electron-microscopic findings.

In rare cases of osteogenesis imperfecta, an "overshoot" growth of new bone may occur, which, clinically gives the impression of a tumour. This condition is known as hyperplastic callus formation. Morphology showed an excessive mixed desmal-chondral osteoneogenesis. Atypical collagen fibrils in non-callus tissue represent an indicator for this callus formation in individual patients.

Effects of transforming growth factor beta on cells derived from bone and callus of patients with osteogenesis imperfecta.

We studied the influence of transforming growth factor beta (TGF-beta) on cultured bone cells derived from two patients with osteogenesis imperfecta (OI) and from human controls. Additionally, cells from a hyperplastic callus that had developed spontaneously at the femur of the patient in Case 1 and cells from a normal fracture callus were included in the study. TGF-beta increased the synthesis of total protein and collagen of all cells without changing the pattern of interstitial collagens. Proliferation was stimulated by TGF-beta in the OI bone cells from Case 1, in cells from the central part of the hyperplastic callus, and in cells from the fracture callus. In Case 2, proliferation of bone cells was decreased by low concentrations of TGF-beta. Alkaline phosphatase (AP) activity was enhanced by TGF-beta in normal human bone cells, not affected in bone cells from the patient in Case 2 or in cells from the central part of the hyperplastic callus, and inhibited in bone cells and cells from the peripheral part of the hyperplastic callus of Case 1 and in cells from the fracture callus. We conclude that TGF-beta has common and specific effects on cultured human cells derived from different types of skeletal tissues. Simultaneous stimulation of collagen synthesis and AP activity by TGF-beta was restricted to normal human bone cells and might reflect their mature state of osteoblastic differentiation. Cells derived from bone of both patients with OI, from the hyperplastic callus, and from the fracture callus showed a different response pattern to TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Glibenclamide stimulates growth of human chondrocytes by IGF I dependent mechanisms.

In rats and men the sulfonylurea glibenclamide augmented skeletal growth. However, with the design of the in vivo studies it was not possible to distinguish whether the growth promoting effect of glibenclamide was mediated by the augmented peripheral insulin or IGF-I levels or if the sulfonylurea had a direct effect on chondrocytes. We therefore measured clonal growth of isolated human chondrocytes in response to glibenclamide in vitro. Cells were isolated from human nose septal cartilage and incubated in a semi-solid medium. Colony formation in response to glibenclamide and IGF-I was determined. Glibenclamide stimulated clonal growth of chondrocytes in a bell-shaped fashion (p < 0.001). 50 ng/ml glibenclamide as the maximal dose augmented colony formation to 144 +/- 9% compared to clonal growth without glibenclamide in the incubation medium, which was designated as 100%. Basal values were obtained with 200 ng/ml glibenclamide. Insulin-like growth factor-I (IGF-I) at 3 ng/ml (118 +/- 4%) and 25 ng/ml (149 +/- 8%, p < 0.02) stimulated growth of chondrocytes. To elucidate the possible mechanism of glibenclamide on clonal growth, chondrocytes were incubated with the sulfonylurea and the IGF-I receptor antibody alpha IR-3. The antibody completely abolished the effect of glibenclamide on colony formation. The results suggest that the growth promoting effect of glibenclamide on isolated human chondrocytes is mediated by IGF-I dependent mechanisms.