Different formins restrict localization of distinct tropomyosins on dorsal stress fibers in osteosarcoma cells.

Formins and tropomyosins (Tpms) are two central components of the microfilaments. Formins are involved in the nucleation and polymerization of actin filaments, and Tpms form along the actin stress fibers to regulate their dynamics. However, the correlation between formins and Tpms remains unclear. Here, we elucidated the function of distinct formins and their specific regulation to the subcellular-localization of Tpm isoforms on dorsal stress fibers in human osteosarcoma cells. Knockdown of individual formin isoform led to varied defects in actin stress fiber network, but did not affect the expression level of other formin isoforms and Tpms. Further investigation showed that different formins regulated distinct Tpm isoforms in decorating dorsal stress fibers. Specifically, DAAM1 and FHOD1 restricted the distal end expression of Tpm3.1; INF2 controlled the approximate localization of Tpm4.2; and Dia1 partially modulated the dorsal localization of Tpm1.6. Taken together, these data provide microscopy experimental evidence that different formins restrict the localization of distinct Tpm isoforms on dorsal actin stress fibers.

Proinflammatory MG-63 cells response infection with Enterococcus faecalis cps2 evaluated by the expression of TLR-2, IL-1ß, and iNOS mRNA.

OBJECTIVE: We have previously demonstrated that unencapsulated Enterococcus faecalis cps2 inhibits biofilm formation of Candida albicans, a fungus commonly found with E. faecalis in periapical lesion. In this study, we compared encapsulated and unencapsulated E. faecalis cps2 strains relationship with osteoblastic (MG-63) cells, whereas E. faecalis ATCC 29212 were used as a reference strain. RESULTS: The binding capacity of E. faecalis to MG-63 cells as shown by each tested strain was comparable, but the unencapsulated strain was less invasive compared to the encapsulated and the reference strains. Moreover, quantitative real time-PCR (qPCR) results showed that infecting unencapsulated E. faecalis cps2 is a stronger stimulator for toll like receptor 2 (TLR2) and interleukin-1ß (IL-1ß) mRNAs, but not for inducible nitric oxide synthase (iNOS) mRNA in osteoblastic cells. In conclusion, the performance of unencapsulated E. faecalis cps2 when the bacterium interacts with osteoblastic cells is quite different from that of encapsulated E. faecalis cps2 and reference strains. It appears that the unencapsulated strain might contribute to the persistence of the periapical inflammatory response, depending on down-regulation of iNOS mRNA expression.

Tumor-targeting Salmonella typhimurium A1-R regresses an osteosarcoma in a patient-derived xenograft model resistant to a molecular-targeting drug.

Osteosarcoma occurs mostly in children and young adults, who are treated with multiple agents in combination with limb-salvage surgery. However, the overall 5-year survival rate for patients with recurrent or metastatic osteosarcoma is 20-30% which has not improved significantly over 30 years. Refractory patients would benefit from precise individualized therapy. We report here that a patient-derived osteosarcoma growing in a subcutaneous nude-mouse model was regressed by tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R, p<0.001 compared to untreated control). The osteosarcoma was only partially sensitive to the molecular-targeting drug sorafenib, which did not arrest its growth. S. typhimurium A1-R was significantly more effective than sorafenib (P <0.001). S. typhimurium grew in the treated tumors and caused extensive necrosis of the tumor tissue. These data show that S. typhimurium A1-R is powerful therapy for an osteosarcoma patient-derived xenograft model.

A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: 'the race to the surface' studied in vitro.

Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing.

Zoledronic acid affects the cytotoxic effects of Chlamydia pneumoniae and the modulation of cytokine production in human osteosarcoma cells.

Chlamydia pneumoniae is an obligate intracellular Gram-negative bacterium that causes persistent infections with a tendency to chronicize, which might motivate the resistance of chlamydiae to some commonly used antibiotics. The bisphosphonates are an emerging class of drugs mostly used in the palliative care of cancer patients to inhibit proliferation and metastasis of cancer cells but their role in modulating immune responses remains unknown. We investigated the in vitro activity of a highly potent bisphosphonate, zoledronic acid, on the cytotoxic effects of C. pneumoniae in human SaOS-2 osteoblast-like cells and the consequent immune response carried out by this cell line. We have reported that zoledronic acid showed a significant anti-proliferative effect on SaOS-2 cell line infected by C. pneumoniae in a time- and dose-dependent manner. We have also found that zoledronic acid induced growth inhibition of C. pneumoniae. Our data showed that C. pneumoniae-infection of SaOS-2 cells induced a significant gene expression of proinflammatory cytokines TNF-α, IL-6, IL-8 and IL-12, detected by RT-PCR, and confirmed by protein release assay. Our results demonstrated that zoledronic acid could facilitate C. pneumoniae-mediated immune response, thus reprofiling this traditional anti-tumor drug as a novel immune regulator in promoting host defense against C. pneumoniae infection.

A case report of osteogenic sarcoma with leprosy.

This is a rare case report of osteosarcoma with lepromatous leprosy. A 15 year old male patient presented with swelling around the right knee joint. Imaging and biopsy were consistent with osteosarcoma. After his first cycle of adjuvant chemotherapy (ACT), the patient developed fever, erythematous nodules, perichondritis of ear lobe, and thickened nerves. His slit-skin smear examination showed acid-fast bacilli in clumps, and a diagnosis of multibacillary leprosy was made. He was treated with anti-leprosy medications with steroids, and once his condition stabilized, his ACT was continued. On follow-up, his skin lesions completely recovered.

Fine-needle aspiration biopsy of chondroblastic osteosarcoma of the vertebral column complicated by Corynebacterium infection: a case report.

We describe a case of chondroblastic osteosarcoma of the vertebral column in a 67-yr-old male in whom the preoperative diagnosis was made by fine-needle aspiration biopsy (FNAB). This diagnosis was subsequently confirmed in the T8 corpectomy specimen. Although the smears of the aspirate revealed only occasional markedly atypical spindle-shaped nuclei, the cell block was diagnostic of malignancy. It showed a well-preserved fragment of neoplastic cartilage populated by markedly atypical hyperchromatic cells and a crushed fragment of anaplastic spindle-shaped cells surrounded by opaque collagenous matrix reminiscent of osteoid. The surgically resected specimen exhibited comparable histological features as well as colonies of gram-positive bacilli within the necrotic tumor. Culture confirmed the presence of Corynebacterium species. It is likely that these skin organisms were introduced at the time of FNAB. This case demonstrates the value of FNAB in the diagnosis of primary bone tumors and reports a rare complication of this procedure.

The effect of GM-CSF and G-CSF on the growth of human osteosarcoma cells in vitro and in vivo.

The human osteosarcoma cell line, MG63, responds both to GM-CSF and to G-CSF in vitro. To assess the significance of these observations to tumor growth in vivo, MG63 cells were engineered by retroviral infection to produce human GM-CSF or G-CSF. These retrovirally infected cells become autostimulatory as measured by increased [3H]-thymidine incorporation (3- to 7-fold) and anchorage-independent colony formation (7- to 10-fold) as compared with uninfected MG63 cells or cells infected with control (neor) retrovirus. The increased proliferation induced by exogenous GM-CSF or G-CSF on uninfected MG63 cells in both assays could be completely inhibited by anti-GM-CSF or anti-G-CSF antibodies, while the same antibodies only partially abrogated proliferation by the growth-factor-producing cells. None of 34 nude or SCID mice developed tumors when injected s.c. with uninfected or neor-virus-infected cells. In contrast, all 30 mice injected with GM-CSF- or G-CSF-producing MG63 cells developed tumors which were G418-resistant and factor-producing. Tumor cell DNA showed a polyclonal retroviral integration pattern indistinguishable from that in the DNA of cells injected into mice. Tumors that formed following injection of a mixture of G418-resistant, GM-CSF-producing cells and cells infected with virus containing only the hygror gene contained hygromycin-resistant cells in the same proportion as was present in the original cell mixture. These data indicate that GM-CSF and G-CSF can support the growth of an osteosarcoma cell line both in vitro and in vivo whether the factor is supplied by autocrine production or from exogenous sources.

Bovine natural killer activity against virally infected cells inhibited by monoclonal antibodies.

Forty-four monoclonal antibodies (mAbs) were evaluated for their ability to alter natural killer (NK) cell lysis of virally infected target cells. Six of the mAbs inhibited the lysis of the target cells, while one of the mAbs enhanced lysis. Four of the inhibitory mAbs, CACT26A, CACT16A, CACTB45A and MUC76A, had very marked activity. These mAbs with inhibitory or enhancing activity recognized (1) WC2 molecules (CACTB44A, CACT16A, CACT26A) present on putative NK cells, (2) molecules on granulocytes, monocytes, a subpopulation of lymphocytes (CACTB45A and TH2A), (3) CD11a (MUC76A), and a protein of CD3 (MM1A).

Interactions between HIV-1 and cytomegalovirus in human osteosarcoma cells carrying both viruses.

Cytomegalovirus (CMV) and the human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. We compared CMV replication in human osteosarcoma (HOS) cells to that in HOS cells genetically engineered to contain an envelope-deficient HIV-1 proviral construct (designated HOS-HXG). Following acute CMV infection of each cell line, HOS-HXG cells contained higher numbers of intranuclear CMV nucleocapsids than did HOS cells. Infectious CMV could be persistently detected in culture supernatant fluids of the CMV-infected HOS-HXG cells, whereas CMV was lost over several weeks from HOS cells infected with CMV in parallel. HIV-1 CMV pseudotypes were not detected in supernatant fluids from CMV-infected HOS-HXG cells. On day 119 after CMV infection, these cultures were superinfected with HIV-1. These dually infected HOS-HXG cells produced infectious HIV-1 and exhibited markedly enhanced CMV replication compared to parental CMV-infected HOS-HXG cells. Two different HIV-1 tat gene function antagonists, Ro24-7429 and chemically modified antibodies to the Tat protein, did not inhibit the replication of CMV in either acute or persistent infections of HOS-HXG cells at concentrations that inhibited HIV-1 replication.

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells.

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenicity of BALB/c-derived N-tropic murine leukemia viruses.

N-tropic murine leukemia viruses have been observed in connection with radiation-induced osteosarcomagenesis in BALB/c mice. We have investigated the bone disease-inducing potential of molecularly cloned, BALB/c-derived N-tropic viruses in the random-bred NMRI mouse strain. The germ-line virus and an exogenous virus isolate were found to induce high incidences of osteopetrosis and lymphomas and a lower incidence of osteomas. Two viruses derived from somatically acquired proviruses of independent radiation-induced osteosarcomas induced lower incidence of osteopetrosis and lymphomas. Nucleotide sequence analysis of the long terminal repeat regions and RNase T1 fingerprint analysis revealed only few differences between the isolates. The possible involvement of N-tropic murine leukemia viruses in radiation-induced osteosarcomagenesis in the BALB/c mouse strain is discussed.

Down-regulation of MHC class I antigens is not a general mechanism for the increased tumorigenicity caused by c-myc amplification.

Previous studies have shown that increased expression of oncogenes from the myc-family can down-regulate the level of MHC class I antigens in tumor cells. This has suggested a mechanism by which amplification/overexpression of myc-genes may contribute to the malignancy development of certain tumors. Earlier published data from the murine SEWA tumor, a polyomavirus-induced osteosarcoma, have correlated the degree of tumorigenicity of different sublines to their level of c-myc amplification. Here I present a quantitative and qualitative analysis of MHC class I antigens from five sublines of this tumor system. No differences could be found, between sublines with different degrees of tumorigenicity, regarding MHC class I antigen expression. Thus, in the SEWA tumor, the enhancement of the tumorigenicity caused by c-myc amplification is not mediated through down-regulation of MHC class I antigens.

Retroviral particles in radionuclide-induced murine osteosarcomas: mouse strain-related differences.

Twenty-six osteosarcomas from strain NMRI mice and twenty-six osteosarcomas from (C3H x 101)F1 hybrid mice were investigated by electron microscopy for the presence of retroviral particles. The bone tumors had been induced by the incorporation of the short-lived bone-seeking radionuclides 224Ra, 223Ra, or 227Th. All of the 26 osteosarcomas from NMRI mice contained retrovirus-like intracisternal type A particles in varying quantities. No type C retrovirus particles could be detected. In contrast, most of the 26 osteosarcomas from (C3H x 101)F1 mice contained budding, immature, and mature type C virus particles predominantly in large quantities. Intracisternal type A particles were also present in all tumors from these mice, but were found only occasionally after extensive search. The mouse strain-related differences in the presence of intracisternal type A particles and type C virus particles in the bone tumors do not provide evidence of an etiological relation of these particles to radionuclide-induced osteosarcomagenesis. Some of the mature type C virus particles observed in osteosarcoma tissue from the hybrid mice were atypical in structure and size. Such pleomorphic particles are probably associated with the spontaneous osteomagenesis which occurs in untreated old (C3H x 101)F1 mice.

Establishment and characterization of osteogenic cell lines from a spontaneous murine osteosarcoma.

Five clonal cell lines were established from a spontaneous BALB/c mouse osteosarcoma, and characterized. Four of these lines showed some similarities in morphology, in vitro growth properties, production of collagenous and noncollagenous extracellular matrix proteins and osteogenic differentiation. The cells formed colonies with characteristic differences in size and morphology in soft agar, and osteogenic sarcomas and metastases in syngeneic mice after transplantation. Ultrastructurally, cells in the transplant tumours showed marked osteogenic features. There were no osteoclast-like cells. The fifth cell line had somewhat different characteristics. All five lines expressed infectious endogenous murine leukemia viruses. Increased c-myc protoon-cogene expression was found in one cell line and c-fos expression at different levels in all lines. There was only very low expression of c-Ha-ras and no expression of c-Ki-ras and c-sis. DNA analysis showed the presence of newly acquired proviral genomes integrated at different sites in the cellular DNA. The results show that distinct osteogenic neoplastic subclones can be obtained from a primary mouse osteosarcoma. Although the clones exhibited an appreciable morphological, functional, and molecular diversity they retained the basic pathogenic properties of the tumour from which they were derived.

Dexamethasone effects on induction of neoplastic transformation by Fujinami sarcoma virus in an in vitro chick embryo periosteal model for osteosarcoma.

Recently we have developed a model in vitro system for the study of factors regulating the histogenesis of osteosarcoma. In this system, Fujinami sarcoma virus (FSV) induces osteosarcomatous changes such as increased cell proliferation and altered patterns of bone and nonmineralized matrix (osteoid) formation. Such changes can be quantitated at the individual cell level, by computer-assisted morphometry. Here we report on the effects of dexamethasone (DEX) on FSV-induced neoplastic transformation and osteogenesis in chick embryonic periosteum cultures, as reflected by a series of histopathological parameters. Most significantly, it was found that compared to 10(-9) M DEX treated cultures, in 10(-7) M DEX pretreated cultures, the bone/osteoid ratio was increased due to a relative increase in the area of bone and a decrease in the area of osteoid. The number of [3H]thymidine-labeled cells decreased significantly, while the proportion of alkaline phosphatase positive cells increased. Double-label immunohistochemistry (with anti-P140gag-fps) and histochemistry for alkaline phosphatase activity was performed, to demonstrate production of the oncogene-encoded protein, and osteoblastic differentiation, respectively. In an in vitro transformation assay single cells derived from 10(-9) M DEX treated cultures formed a significantly higher number of colonies than those obtained from 10(-7) M DEX pretreated cultures. Taken together, the data indicate that in the chick embryonic periosteum culture system, pretreatment with 10(-7) M DEX inhibits the ability of FSV to induce neoplastic transformation. This effect is probably the result of DEX-induced cell differentiation, prior to infection with FSV.

Skin papillomas and other neoplasms induced by murine sarcoma viruses in mid-gestation-infected mice.

During extensive investigations on the effects of oncogenic retroviruses in developing rodents, the ability of MSV to mount a neoplastic response in CD-I Swiss mouse embryos was determined. By infecting the animals directly in utero at selected stages of post-implantation development, we detected a peculiar reaction of the embryonal tissues to certain MSVs: when mice were exposed to KiMSV at mid-gestation, the newborn developed characteristic tumors, in addition to mesenchymal cell sarcomas, not induced in fetuses and neonates. These included pulmonary alveologenic tumors and skin papillomas and were seen in mice infected on days 8 and 10 of pregnancy, roughly corresponding to 15 and 35 somites, respectively. To determine the specificity of these events, other 8- and 10-day-old embryos were infected with retroviruses of the same or different families. HaMSV and MoMSV also induced mesenchymomas and a low incidence of skin papillomas (10% and 15% compared to 40% in the KiMSV group) but not pulmonary tumors. In contrast, FBRMSV was inactive in this respect and only osteogenic sarcomas were detected in the offspring. Infecting the embryos on day 7 of pregnancy produced no tumors. Later infections (in 15-day-old fetuses and neonates) mainly induced mesenchymal sarcomas. No congenital malformations were detected in the embryos exposed to MSV during organogenesis, although some abortions and resorptions were seen.

Amplification of endogenous proviral MuLV sequences in radiation-induced osteosarcomas.

The endogenous ecotropic provirus of BALB/c mice was found to be amplified in 17 out of 29 radiation-induced osteosarcomas. In contrast, 19 clonal cell lines established from bone-marrow cells of a tumor-bearing mouse, which were used as controls, did not reveal newly acquired ecotropic proviruses. Ecotropic viral RNA was expressed in tumors that showed reintegrated proviruses. DNA probes from 2 tumors, derived from cellular sequences flanking the newly integrated proviruses, did not detect DNA rearrangements in any of the other tumors. The possible role of activated endogenous retroviruses in the development of radiation-induced osteosarcomas is discussed.