Pluripotential mesenchymal cells repopulate bone marrow and retain osteogenic properties.

Precursor cells, isolated from bone marrow, can develop into various cell types and may contribute to skeletal growth, remodeling, and repair. The D1 cell line was cloned from a multipotent mouse bone marrow stromal precursor and has osteogenic, chondrogenic, and adipogenic properties. The osteogenic phenotype of these precursor cells is relevant to the process of fracture healing and osteointegration of prosthetic implants. The D1 cells were labeled genetically using a replication incompetent retroviral vector encoding beta-galactosidase, an enzyme which is used as a marker. Labeled cells are readily identifiable by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside and by flow cytometry, and retain the desired osteogenic characteristics in vivo as shown by von Kossa staining, alkaline phosphatase assay, an increase in cyclic adenosine monophosphate in response to parathyroid hormone, osteocalcin messenger ribonucleic acid production, and bone formation in diffusion chambers. In addition, the cells cloned from marrow stroma repopulate the marrow of host mice, persist for several weeks, and retain their osteogenic potential ex vivo. The data suggest that such cells may be used to replenish the number of osteoprogenitors in marrow, which appear to decrease with age, thereby leading to recovery from bone loss and improved bone growth and repair. Labeling these cells creates a model in which to study the potential of such cells to participate in fracture repair, ingrowth around prosthetic implants, treatment of osteoporosis, and to explore the possibility of gene delivery to correct mutations or defects in metabolism that are responsible for certain skeletal abnormalities.

Metacarpophalangeal pattern profile analysis in Noonan syndrome.

Metacarpophalangeal pattern (MCPP) analysis is an application of an anthropometric technique that provides a quantitative assessment of the amount and direction of abnormality in the hand skeleton. MCPP analysis was undertaken on 15 individuals (9 males, 6 females) with Noonan syndrome ranging in age from 0.1 to 36 years with a mean age at 11.6 years. The overall average Z score for the MCPP variables was -2.1 and the range was -2.5 (for metacarpal two) and -1.5 (for middle phalanx 5). The average hand pattern variability index, a measure of hand bone length relationships, was abnormal. A Pearsonian correlation analysis was used to assess similarity between the mean pattern and each of the 15 individual patterns. Nine (60%) of the fifteen individuals with Noonan syndrome had significant positive correlations (P < 0.05), indicating homogeneity or similarity in the hand patterns. A stepwise discriminant analysis was performed on Z score data from the individual hand bone measurements on the 15 subjects with Noonan syndrome and 41 healthy controls (24 females, 17 males; mean age = 13.1 years with age range of 9.6 to 18 years). This analysis produced a discriminant function with two MCPP variables (metacarpal 1 and middle phalanx 3) entering into the function and producing a correct classification rate of 93%. The two MCPP variables contributed to the overall difference between individuals with Noonan syndrome and the normative sample. The hand pattern variability index was outside of the normal range, indicating an abnormal MCPP with multivariate analysis. The MCPP analysis may be useful as a tool for diagnosis in screening subjects for Noonan syndrome.

Cytogenetic, telomere, and telomerase studies in five surgically managed lumbosacral chordomas.

Lumbosacral chordomas are rare skeletal sarcomas of the spine that originate from the remnant notochord. The understanding of this human cancer is limited to observations of its clinical behavior and its embryonic link. Thus, we performed chromosome and molecular analyses from five surgically harvested chordomas in an effort to document genetic and biochemical abnormalities which might aid in understanding the tumor biology of this understudied neoplasm. Cytogenetic analysis of the five chordomas revealed normal results in four patients and random abnormalities in only one tumor cell in the 100 cells studied from the fifth patient. A repeat telomeric probe (TTAGGG)50 was hybridized to genomic DNA isolated from chordoma cells (and HeLa cells) and digested with HinfI. The tumor DNA was paired with leukocyte DNA from age-matched controls and revealed telomere elongation in four of the four chordoma patients studied with molecular genetic techniques. Conversely, telomere length reduction has been reported during in vitro senescence of human fibroblasts, giant cell tumor of bone, colon cancer, intracranial tumors, childhood leukemia, Wilms tumor, and in HeLa cells. Telomerase activity (telomerase is required to maintain telomere integrity) was also determined by visualizing the extension of radioactive telomeric repeats on DNA sequencing gels. The telomeric fragments were assembled during incubation of the cytoplasmic extract containing telomerase. Telomerase activity was observed in HeLa (positive control and commercially available cell line), giant cell tumor of bone (positive control tumor cells from living patients), and in chordoma cells from one of the two chordoma patients (but to a lesser degree compared with HeLa). As expected, the chordoma patients' fibroblasts exhibited no telomerase activity.

Dosage and allelic restriction fragment studies and PCR analysis of the H-ras locus in giant cell tumor of bone.

Several studies have shown that giant cell tumor of bone frequently exhibits telomeric associations, commonly at chromosome 11p, which is also the location of the H-ras oncogene. In addition, rare H-ras alleles are more common among cancer patients than among healthy controls and point mutations of this oncogene have also been reported in several malignancies. These data led us to investigate gene dosage, restriction fragment-length size, and point mutations for H-ras in giant cell tumor of bone. Quantitative Southern blot analysis revealed no amplification of the H-ras oncogene in tumor DNA compared with DNA from peripheral blood in the same patient or from control subjects. In addition, no point mutations were detected in codons 12, 13, or 61 (mutations of these codons have been reported in other neoplasms) of the H-ras gene. No differences were noted in restriction fragment-length polymorphisms between tumor and peripheral blood in the same patient and no loss of heterozygosity was detected. In addition, there was no increased frequency of rare H-ras alleles (8% of alleles) in giant cell tumor patients compared to controls (21% of alleles) in our study. However, large allele sizes (> 8.5 kb) were significantly overrepresented in GCT patients compared with healthy controls. In our study, three of 12 alleles were found to be rare in the healthy controls but were common among GCT patients. Our data suggest that the H-ras oncogene is unlikely to be the site of a biologically significant primary lesion in GCT tumorigenesis.

Telomere reduction in giant cell tumor of bone and with aging.

Giant cell tumor of bone is a benign, primary skeletal neoplasm that has an unpredictable pattern of biologic aggressiveness, and cytogenetically demonstrates genetic instability by exhibiting telomeric associations. Molecular analysis of telomeres from giant cell tumor of bone demonstrated reduction of telomere length (average loss of 500 base pairs) in eight individuals when compared with their leukocyte DNA. Those tumors which exhibited telomeric associations were found to have a greater reduction in telomere length than tumors not exhibiting them. For comparison, eleven cytogenetically healthy control individuals (7 females and 4 males, age range 2 weeks to 70 years) were included in this study. They demonstrated loss of telomere size (average 40 base pairs per year) with advancing age and the greatest rate of telomere reduction was identified in the young. Thus, the functional consequences of telomere shortening in a neoplastic cell may prove fundamental to sustaining the transformed phenotype in giant cell tumor of bone.

Metacarpophalangeal pattern profile analysis in Brachmann-de Lange syndrome.

We analyzed the metacarpophalangeal pattern profile (MCPP) of 19 individuals with Brachmann-de Lange syndrome (BDLS) and calculated a mean syndrome profile. Fourteen of 19 individuals with BDLS had significant positive correlations which indicated clinical homogeneity. Discriminant analysis of individuals with BDLS compared with a sample of normal individuals produced a correct classification rate of 100% based on a function of 2 MCPP variables that may provide a useful tool for assisting in the diagnosis of BDLS. An average pattern variability index calculated for the BDLS patients was 1.9 indicating an abnormal hand pattern in this syndrome.

Molecular analysis of transforming growth factor beta in giant cell tumor of bone.

Giant cell tumor of bone (GCT) is a primary bone neoplasm with unique cytogenetic findings including telomeric associations. Elevated expression of message RNA for transforming growth factor beta (TGF beta), but not transforming growth factor alpha (TGF alpha), has been reported in this tumor. Further investigation of GCT was undertaken to determine whether genetic loci for TGF beta in GCT patients with and without chromosome abnormalities are altered. Due to the reported TGF beta overexpression in GCT, qualitative and quantitative Southern blot analyses with TGF beta 1 and TGF beta 2 and an internal control probe (p3-21) were performed with tumor DNA and DNA from normal tissue on ten patients with GCT and control individuals. No obvious TGF beta 1 or TGF beta 2 gene alterations were detected. Normal copy numbers were calculated when comparing tumor and normal DNA from GCT patients as well as DNA from control individuals. Abnormal chromosome findings, including telomeric associations, marker chromosome, double minutes, chromosome fragments, ring chromosomes (possibly representing intra-chromosome telomeric associations), and polyploid cells were observed in seven of the ten patients with GCT. Chromosomes 11, 16, 19, 20, and 21 were most commonly observed in telomeric associations, with the terminus of the long arm of chromosome 19 being the most frequent. We conclude that there are no TGF beta 1 or TGF beta 2 gene alterations detected in GCT with the methodologies described, and that telomeric associations are a reproducible cytogenetic characteristic of this neoplasm.