Characterization of human multicentric osteosarcoma using newly established cells derived from multicentric osteosarcoma.

PURPOSE: Human multicentric osteosarcoma (HMOS) is a rare, aggressive variant of osteosarcoma, and its etiology is not clear. We used newly established HMOS cells, which were derived from primary (HMOS-A) and secondary (HMOS-P) lesions, respectively, to perform a basic study analyzing the cellular biology and gene expression of HMOS. METHODS: We performed a cell growth assay, an invasion assay, DNA microarray analysis, quantitative real-time RT-PCR (Qrt-PCR), and a telomerase assay and compared the results between HMOS-A, HMOS-P, and human osteosarcoma (HOS) cell lines (MNNG-HOS and Saos-2). RESULTS: The cell biological analysis revealed that HMOS-A and HMOS-P had similar characteristics to Saos-2, and the invasion assay showed that they had similar characteristics to MNNG-HOS. The DNA microarray study showed that the gene expression profiles of HMOS-A and HMOS-P were similar to that of MNNG-HOS, but the overexpression of MMP-2, MMP-9, and MT1-MMP was observed in HMOS-A and HMOS-P, which was correlated with the invasiveness of the extracellular matrix, and collagen type-4 (COL-4) and VEGF were also detected. HMOS-A and HMOS-P showed low telomerase activity similar to Saos-2, which are known to be telomerase negative, but a similar telomere length and telomerase protein to MNNG-HOS. CONCLUSIONS: HMOS-A and HMOS-P demonstrated strong invasive ability, and their gene expression profiles correlated with the invasiveness of the extracellular matrix. Their telomerase activity was low, but they did not shown the typical features of alternative lengthening of telomeres (ALT). HMOS-A and HMOS-P are useful models for further study of various biological aspects and therapeutic manipulation of HMOS.

Presence of prostacyclin receptor in arteriosclerotic human femoral artery.

AIM: Prostacyclin, which is mainly synthesized by vascular endothelial cells, exerts antiplatelet and smooth-muscle-relaxant effects, thereby maintaining cardiovascular homeostasis. Prostacyclin analogues have been clinically proven to improve ischemic symptoms and prevent the occurrence of vascular events in the lower extremities of patients with arteriosclerosis obliterans. We examined the presence of prostacyclin receptor (IP receptor) in an arteriosclerotic human femoral artery. METHODS: Specimens of the femoral artery were obtained at the time of limb amputation from an 83-year-old woman. Atherosclerotic lesions and associated changes such as calcification were evident. The specimens were stained with hematoxylin and eosin, and processed for immunohistochemistry. RESULTS: A monolayer of cells was observed on the luminal side of the femoral artery. Single immunohistochemistry showed the presence of the IP receptors on cells of the luminal side of the femoral artery. Triple-immunofluorescence staining revealed colocalization of IP-receptor-positive cells and cells positive for von Willebrand factor, a marker of vascular endothelial cells. CONCLUSIONS: We investigated the presence of the IP receptor in the human femoral artery immunohistochemically, and demonstrated their strong expression in endothelial cells. This finding suggests that prostacyclin or prostacyclin analogues may act on their receptors on endothelial cells in patients with arteriosclerosis obliterans.

Hip-shelf procedure in the treatment of osteonecrosis of the transpositioned acetabulum after rotational acetabular osteotomy.

Necrosis of the transpositioned acetabulum after rotational acetabular osteotomy (RAO) is a major complication characteristic of this procedure. This complication, although rare, has been thought difficult to treat. We report a patient with acetabular osteonecrosis and subsequent collapse after RAO that was effectively treated with a shelf operation, providing satisfactory remodeling of the hip joint. A 16-year-old female had undergone RAO for the treatment of developmental acetabular dysplasia. Postoperative radiography showed that the osteotomized acetabular fragment was unusually thin, and that the osteotome entered the hip joint during the surgery. Five months after the RAO, X-rays revealed significant collapse of the transpositioned acetabulum, and femoral head subluxation caused by postoperative osteonecrosis. Seven months after the RAO, the patient underwent a hip-shelf procedure. The remaining acetabular fragment was used in this procedure, according to the Spitzy method. Seven years after the second operation, favorable remodeling of the hip joint was observed; however, early osteoarthritic changes, including slight joint space narrowing, bone sclerosis of the new acetabulum, and bone cysts within the femoral head, were seen.

Expression of keratan sulfate at the arthroplasty surface after cup arthroplasty.

Fibrous tissue which regenerated on the acetabular arthroplasty surface was obtained from a 52-year-old woman who underwent total hip replacement after cup arthroplasty. The histological features of this newly formed fibrous tissue and expression of keratan sulfate, which is a characteristic matrix component of articular cartilage, were studied. Microscopic observation revealed that the arthroplasty surface consisted mainly of fibrous tissue which did not show metachromasia with toluidine blue staining, but there were many nodular structures communicating with the bone marrow. Immunostaining for keratan sulfate revealed clear positive staining around the cells of the nodular structures communicating with the bone marrow, while only weakly positive staining was observed in the superficial layer of the loose fibrous tissue. The present study revealed marked formation of articular cartilaginous tissue in areas having good communication with the bone marrow, which indicates that maintenance of this communication may be necessary to improve the outcome of cup arthroplasty.

Hyaluronan in synovial fluid of patients with loose total hip prosthesis. Comparison with hyaluronan in patients with hip osteoarthritis and idiopathic osteonecrosis of femoral head.

The concentration and molecular weight of hyaluronan (HA) in the synovial fluid of the hip joint were determined in 13 patients (aged 62.8 +/- 9.4 years) who had undergone prior total hip arthroplasty(THA), 23 patients (aged 65.0 +/- 8.2 years) with osteoarthritis of the hip joint (OA), and 13 patients (aged 40.2 +/- 2.7 years) with idiopathic osteonecrosis of the femoral head (ION). A sample of synovial fluid was obtained during revision THA because of loosening of the total hip prosthesis for the THA group, and during the first replacement surgery or osteotomy for the OA and ION groups. The concentration of HA in the synovial fluid was 0.64 +/- 0.42 mg/ml in the THA group, 1.07 +/- 0.28 mg/ml in the OA group, and 1.30 +/- 0.56 mg/ml in the ION group. The concentration of HA in the synovial fluid of the THA patients was significantly lower than that of the OA and ION patients (P = 0.0156 vs OA, P = 0.003 vs ION). The molecular weight of HA was 309 +/- 88.3 x 10(4) Da in the THA group, 377 +/- 201 x 10(4) Da in the OA group, and 240 +/- 148 x 10(4) Da in the ION group; these values do not differ significantly (P = 0.259 vs OA, P = 0.174 vs ION). Among the THA patients, there was no relation between the concentration of HA and the age of the patient, length of time since the first operation, or type of prosthesis fixation; there was also no relation between the molecular weight of HA and each of these factors. These results suggest that a pseudosynovial membrane is regenerated after THA, and that it produces HA of the same molecular weight as that in patients with OA and ION, although in smaller quantities.

Content and sulfation pattern of keratan sulfate in hip osteoarthritis using high performance liquid chromatography.

OBJECTIVE: To determine the content and sulfation pattern of keratan sulfate (KS) in synovial fluid (SF) from patients with hip osteoarthritis (OA) and investigate its significance as a marker of cartilage matrix metabolism. METHODS: Hip SF samples were aspirated from 50 patients with OA. KS in the samples was digested to 2 disaccharide isomers, beta-galactosyl-(1-4)-6-0-sulfo-N-acetylglucosamine (L2) and beta-6-0-sulfo-galactosyl-(I-4)-6-0-sulfo-N-acetylglucosamine (LA) by keratanase II. Concentrations of these disaccharide isomers were determined by high performance liquid chromatography (HPLC), and their levels were investigated in relation to radiological stage of disease. RESULTS: Analysis of covariance (age as covariate) showed that the L2 levels in advanced stage OA were significantly lower than in early stage OA (p < 0.0001). L2 levels in terminal stage OA were also significantly lower than in early stage OA (p < 0.0001); however, no significant difference was observed between the L2 levels in advanced and terminal stage OA (p = 0.516). There were no significant differences in the levels of L4, L2 + L4, or the ratio of L4 to L2 at each disease stage. CONCLUSION: The levels of KS related disaccharide isomer vary with severity of disease in hip OA. Analysis of these KS related disaccharide isomers by HPLC provides information on both the content and sulfation pattern of KS in SF, reflecting the metabolism of cartilage aggrecan.

Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease.

It has been well documented that the liver is an exceptional organ in which the monoallelic expression of insulin-like growth factor 2 (IGF2) due to genomic imprinting is relaxed during the postnatal period, resulting in biallelic expression thereafter. In the present study, changes in the status of genomic imprinting were examined in 15 hepatocellular carcinomas (HCCs) as well as in 29 liver biopsies of chronic hepatitis or liver cirrhosis without clinical evidence of HCC, following screening for heterozygotes with an ApaI polymorphism in IGF2 in 34 HCCs and 80 such non-HCC cases. Extreme allelic-expression imbalance, leading to restoration of monoallelic IGF2 expression, was observed in 15 (100%) of 15 informative HCCs for the polymorphism with this monoallelic IGF2 expression appearing to be non-random from the paternal allele. Interestingly, the same allelic-expression imbalance was also present in a significant fraction of noncancerous liver specimens of patients with underlying disease known to be associated with HCC development. In contrast, the status of genomic imprinting of H19, another gene closely mapped at 11p15 under opposite imprinting, was strictly maintained in seven (100%) of seven cases informative for an RsaI polymorphism of H19. Together with the previous reports on altered genomic imprinting of IGF2 and H19 in embryonal lesions such as Wilms tumors as well as in lung cancers, the results suggest that perturbations of imprinting status occur as locus and tumor-type specific events in the development of human cancers.

Prognostic significance of abnormal p53 accumulation in primary, resected non-small-cell lung cancers.

PURPOSE: This study was conducted to evaluate the prognostic significance of p53 abnormalities in primary, resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Methodologic validation of immunohistologic detection of p53 abnormalities in routine pathology sections was assessed using 31 lung cancer specimens for which p53 gene status was known from our previous molecular biologic studies. Applying the optimized cutoff value, we evaluated the prognostic significance of p53 abnormalities in an independent cohort of 208 NSCLC patients with complete follow-up data, whose resections were consecutively performed between January 1984 and December 1988. RESULTS: Immunohistologic detection of p53 abnormalities appeared to be reliable and showed approximately 90% concordance with the p53 gene status. Using the selected cutoff value of 10%, 46% of 208 NSCLCs showed p53 abnormalities. There was no relationship between p53 abnormalities and clinical outcome in the entire cohort, which represented all histologic subtypes of NSCLC (P = .58). Based on the reasoning that the influence of p53 abnormalities may have been obscured by distinct biologic roles depending on histologic subtypes, we also separately analyzed subsets of patients with adenocarcinomas (n = 100) and with squamous cell carcinomas (n = 88) and found that it may be a useful prognosticator only in adenocarcinoma patients (P = .04). CONCLUSION: p53 abnormalities are not a significant prognostic factor in primary, resected NSCLC when all histologic subtypes are combined, but may be a useful prognosticator for adenocarcinomas. Additional studies are warranted for further evaluation, specifically of adenocarcinomas.

In vivo occurrence of p16 (MTS1) and p15 (MTS2) alterations preferentially in non-small cell lung cancers.

Frequent homozygous deletions of the p16 (MTS1) gene encoding a cyclin-dependent kinase inhibitor were recently reported in various tumor cell lines including examples derived from lung cancers, but direct evidence for their occurrence in lung cancer patients has not been reported thus far. In the present study, alterations of p16 and/or p15, a p16-related cyclin-dependent kinase, were observed not only in lung cancer cell lines but also in the corresponding tumor specimens in vivo, excluding the possibility of in vitro artifacts. Interestingly, a clear specificity was also noted in terms of the affected histological subtype; i.e., only non-small cell lung cancers carried alterations (6 of 20 as compared to 0 of 20 small cell lung cancer cell lines).

Expression of CD44 variant isoforms in normal and neoplastic cells of the lung.

CD44 is a cell surface receptor that has been implicated in lymphocyte homing, hematopoiesis, cell migration and possibly also tumor metastasis. In the present study, expression of CD44 variant (CD44v) isoforms was analyzed in 23 lung cancer specimens together with corresponding normal lung tissues by Southern blot analysis coupled with reverse transcription-polymerase chain reaction amplification. We found that CD44v isoforms were expressed in all lung cancer specimens, suggesting a possible role in the establishment of metastases by these highly malignant tumors, but normal tissues were also positive. This is in marked contrast to the previous reports of essentially negligible expression of CD44v isoforms in normal colon and breast, and suggests a physiological function in the lung.