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Results for “Serial Passage”

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PubMed1980

Pulmonary blastoma. An ultrastructural study of a case and its transplanted tumor in athymic nude mice.

A pulmonary blastoma in a 52-year-old Japanese man was serially transplanted into athymic nude mice. This report emphasizes the characteristics of the neoplasm seen on light and electron microscopic examination. The original tumor consisted of branching tubular structures and primitive mesenchymal components with a few smooth-muscle cell bundles, and resembled fetal lung tissue. Electron microscopic examination revealed that the immature stromal cells resembled epithelial cells. After serial passages in nude mice, the stromal element of the tumor disappeared, and the epithelial element showed squamous metaplasia and occasional mucus production. Endocrine-type granules were observed in a few tumor cells. Histogenetic considerations, including a suggestion that the tumor was derived from entodermal cells, are presented.

Animals↗

PubMed1980

Human yolk sac tumor serially transplanted in nude mice: its morphologic and functional properties.

Three human yolk sac tumors (TE, OE, and TT-1) were serially transplanted into nude mice. The histology of the transplanted tumors is moderately different from that of the original tumor; the transplants had mainly a tubular structure with an inconspicuous endodermal sinus structure due to a decreased mesenchymal element surrounding the vascular core. Ultrastructural features of the transplanted tumor cells, however, were identical to those of the original tumor. Immunochemical survey of the blood plasma of nude mice bearing these tumors demonstrated the production of adult type human plasma proteins including prealbumin, albumin (Alb), alpha 1-antitrypsin (alpha AT), transferrin (Tf), hemopexin, and alpha 1-fetoprotein (AFP). The types of adult plasma proteins that were produced varied, depending on the tumor line but were the same between serial passages within the same tumor line. The intracellular localization of AFP, alpha AT, Tf, and Alb was also investigated by immunoelectron microscopy. Plasma proteins of the adult type and AFP were localized mainly within the membrane systems of the endoplasmic reticulum and Golgi complex and occasionally on the microvilli. These results suggest that transplanted human yolk sac tumors have almost all of the protein-producing function possessed by their normal counterparts.

Animals↗

PubMed1983

Tyrosine hydroxylase and choline acetyltransferase activity in human neuroblastoma. Correlations with clinical features.

Two neurotransmitter-synthesizing enzymes, tyrosine hydroxylase (TH) and choline acetyltransferase (CAT), were assayed in neuroblastoma tissues from 24 children, in human neuroblastoma tissues serially transplanted in nude mice, and in human neuroblastoma cells in culture. Among tissues from 24 children, five showed an adrenergic pattern with significant TH activity alone, seven showed a cholinergic pattern with significant CAT activity alone, and the remaining 12 specimens showed a "both-active" pattern with both TH and CAT activity. Enzymatic activities were maintained through many serial passages in vitro and in nude mice. All four specimens from children under one year of age exhibited the adrenergic pattern. In general, enzymatic activity was not correlated with degree of differentiation histologically. Among four cases of paravertebral dumb-bell type in this series, two were cholinergic, one was adrenergic, and the last was both-active. These results suggest that dumb-bell type tumors may arise from either sympathetic ganglia or dorsal root ganglia. This study supports the concept that neuroblastomas are a composite of adrenergic and cholinergic cells. Significant changes in the relative proportions of these two cell types were observed with time, and after extensive therapy. Different metastatic sites often exhibited important differences in enzymatic activity. These results help to account for clinical discrepancies between urinary VMA levels and tumor growth. Assays for TH and CAT can be useful for confirming a diagnosis of neuroblastoma, and have important potential for helping to clarify the natural history of neuroblastoma.

Animals↗

PubMed1986

The morphology and biologic behavior of human glioblastoma growing in nude mice.

Seven human glioblastomas (five small cell glioblastomas [SCG] and two anaplastic astrocytomas with giant cells [AA]) grown in serial passage in BALB/c nude mice and nude rats, were studied histologically and compared to human donor tumors. Four SCG maintained many basic features seen in donor tissue, i.e., cell type, tendency to produce micronecrotic palisading (MNP), high cellularity, numerous mitoses. Significant vascular proliferation was seen only in nude rat hosts of one of the SCG lines transplanted from mice at passage 11. Giant capillaries, cyst formation and hemorrhages were features of large (1.0 cm) heterotransplanted SCG. One SCG altered morphology from first passage, showing an adenoid pattern and mucinous change. Both AA preserved original tissue characteristics in initial passages. Later dedifferentiation occurred with small cells predominating. These small cells were larger and rounder than those of the SCG. Large AA showed central necrosis but rarely MNP, hemorrhages and focal inflammatory infiltrates.

Adult↗

PubMed1988

Differences between subcutaneous and intraperitoneal forms of three human testicular teratocarcinomas in nude mice.

Three human testicular teratocarcinomas were serially passaged following subcutaneous transplantation into nude mice. Tumor cell suspensions from selected passages were injected intraperitoneally. The subcutaneous transplants of each tumor conserved the morphological characteristics of one component of the primary tumor, namely an embryonal carcinoma in one case and a yolk sac tumor in two. The latter maintained the capacity to synthesize alpha-fetoprotein (AFP). After intraperitoneal injection of cell suspensions, tumors, either attached to or even invading abdominal organs or in the form of free-floating tumor spheroids, were observed. AFP could be localized within the attached growths but not in the spheroids. A critical tumor volume and/or vascularization seemed to be necessary for AFP production in tumor cells. In spheroids from one tumor, cytogenetic analysis revealed both human and murine cells. Thus, these spheroids, apparently composed of tumor cells in the center and murine cells at the periphery, can not be considered to be embryoid bodies.

Animals↗

PubMed1989

Therapy-induced drug resistance in a human leukemia line (LALW-2). A clinically relevant model.

A human leukemic T-cell line, LALW-2, established by xenografting in nude mice, has been maintained through 14 serial passages. The cells display consistent morphologic features, immunophenotype, and karyotypic aberrations (including an 11;14 translocation) and exhibit rearrangement of the T-cell receptor beta-chain gene. The growth rate of LALW-2 xenografts was differentially affected by drugs administered to host mice, the cells being resistant to cytotoxic agents (particularly methotrexate and doxorubicin) used in treatment of the donor patient. In short-term in vitro culture, LALW-2 cells exhibited extreme resistance to methotrexate and were also resistant to vincristine, vinblastine, dactinomycin, and doxorubicin. The findings differ from those obtained with laboratory-derived methotrexate or multidrug-resistant cell lines. The response of LALW-2 cells, in both the nude mouse model and in vitro, is consistent with acquisition of drug-resistance as a result of clinical treatment.

Animals↗

PubMed1992

Two cell lines established from mixed müllerian tumors of the uterus. Morphologic, immunocytochemical, and cytogenetic analyses.

To clarify the cellular origin and characteristics of malignant mixed müllerian tumor (MMMT), the authors investigated two cell lines (designated as FU-MMT-1 and FU-MMT-2) established from two patients with heterologous MMMT of the uterus. Both cell lines propagated continuously for 83 and 55 serial passages over 1.5 years, respectively. Morphologically, FU-MMT-2 was a mixture of carcinoma cells and sarcoma cells with predominance of carcinoma cells; FU-MMT-1 only had a sarcomatous element with distinct rhabdomyoblastic differentiation. Immunocytochemically, the sarcoma cells of each cell line expressed, not only myogenic and mesenchymal antigens (desmin, myoglobin, and vimentin), but also epithelial antigens, including epithelial membrane antigen and keratin. The carcinoma cells in FU-MMT-2 were positive for the epithelial antigens and vimentin and negative for desmin and myoglobin. Both lines had abnormal karyotypes; the modal chromosome numbers of FU-MMT-1 and FU-MMT-2 were 47 and 80, respectively. In addition, FU-MMT-1 had trisomy 8, and FU-MMT-2 had complex structural abnormalities. When transplanted into nude mice, FU-MMT-1 reproduced and maintained the characteristics of the original tumor. These cell lines and xenografts appear to provide a useful system for studying the biologic behavior, cytogenetic features, and histogenesis of MMMT. In conclusion, the presence of epithelial antigens in the sarcomatous and carcinomatous elements seemed to support the hypothesis that both elements are derived from a common stem cell.

Aged↗

PubMed1993

Growth patterns of human neuroblastoma xenografts and their relationship to treatment outcome.

BACKGROUND: Several investigators have reported the ability to establish xenografts in nude mice from children with neuroblastomas, but a correlation of prognosis with this establishment and the growth patterns of the neuroblastomas has not been reported. METHODS: Tumor specimens from 58 children with neuroblastomas were heterotransplanted into BALB/c nude mice. In 34 patients, heterotransplantation was done before therapy; in 24 patients, tumors were obtained after at least one course of chemotherapy or radiation therapy. The histology, cytogenetics, and growth characteristics of serial passages of the xenografts were studied. RESULTS: The engraftment rate was 34%. Neuroblastomas with diploid chromosome numbers did not engraft. Chromosomal abnormalities involving 1p were seen in more than 50% of the xenografts. Cytogenetic features were retained between original tumors and resultant xenografts. Xenografts could be established only from tumors with unfavorable histology, as defined by Shimada classification criteria. The histology of each xenograft line was strikingly similar, and each was highly undifferentiated. Engraftment rates, doubling times, and lag times did not vary appreciably between xenografts established from treated tumors compared with xenografts established from untreated tumors. There was no correlation between doubling or lag times and prognosis. Patients whose tumors engrafted had only a 5% 3-year survival rate. CONCLUSIONS: From these results, it appears that successful engraftment is the most important prognostic indicator for patients with neuroblastomas. Because of the commonality of the histologic features and the stability of the tumor clones from patients before and after heterotransplantation, these xenografts may be useful as an in vivo model for studying drug resistance and for designing treatment regimens.

Animals↗

PubMed2000

Tumor necrosis factor alpha promotes the expression of stem cell factor in synovial fibroblasts and their capacity to induce mast cell chemotaxis.

OBJECTIVE: To investigate the expression of the stroma cell product stem cell factor (SCF) in synovial fibroblasts (SFB) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and to analyze the capacity of SFB to induce mast cell (MC) chemotaxis. METHODS: Synovial tissue was obtained from 29 patients with RA and 25 patients with OA. Tissue was dispersed by enzymatic digestion using collagenase. SFB were grown in serial passage and exposed to tumor necrosis factor alpha (TNFalpha) or control medium. Expression of SCF in cultured SFB was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunostaining. The ability of SFB (supernatants) to induce MC migration was analyzed using a double-chamber chemotaxis assay and the human mast cell line HMC-1. In situ expression of SCF in synovial tissue from patients with RA (n = 6) and OA (n = 6) was examined by double immunohistochemistry using antibodies against SCF and the fibroblast-specific antibody AS02. RESULTS: In both RA and OA, cultured SFB were found to express SCF messenger RNA, as assessed by RT-PCR. In addition, the SCF protein was detectable in cell lysates and supernatants of SFB by ELISA. Incubation of SFB with TNFalpha resulted in an increased expression and release of SCF. Recombinant human SCF (rHuSCF) and SFB supernatants induced significant migration of HMC-1 cells above control levels. In addition, exposure of SFB to TNFalpha led to an increased migration of HMC-1, and a blocking anti-SCF antibody inhibited the rHuSCF- and SFB-induced migration of HMC-1. In situ double immunostaining revealed expression of SCF in AS02-positive SFB in the synovium of patients with RA. CONCLUSION: Our results show that SFB (in RA and OA) express SCF and induce MC chemotaxis. Furthermore, TNFalpha was found to augment SCF expression in SFB. It is hypothesized that these cellular interactions play an important role in MC accumulation and related events in RA.

Arthritis, Rheumatoid↗

PubMed2001

Multipotent mesenchymal stem cells from adult human synovial membrane.

OBJECTIVE: To characterize mesenchymal stem cells (MSCs) from human synovial membrane (SM). METHODS: Cell populations were enzymatically released from the SM obtained from knee joints of adult human donors and were expanded in monolayer with serial passages at confluence. Cell clones were obtained by limiting dilution. At different passages, SM-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatments, phenotypes of cell cultures were analyzed by histo- and immunohistochemistry and by semiquantitative reverse transcription-polymerase chain reaction for the expression of lineage-retated marker genes. RESULTS: SM-derived cells could be expanded extensively in monolayer, with limited senescence. Under appropriate culture conditions, SM-derived cells were induced to differentiate to the chondrocyte, osteocyte, and adipocyte lineages. Sporadic myogenesis was also observed. Five independent cell clones displayed multilineage potential. Interestingly, only 1 clone was myogenic. Donor age, cell passaging, and cryopreservation did not affect the multilineage potential of SM-derived cells. In contrast, normal dermal fibroblasts under the same culture conditions did not display this potential. CONCLUSION: Our study demonstrates that human multipotent MSCs can be isolated from the SM of knee joints. These cells have the ability to proliferate extensively in culture, and they maintain their multilineage differentiation potential in vitro, establishing their progenitor cell nature. SM-derived MSCs may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.

Adipocytes↗

PubMed1979

Transmission of chronic spongiform encephalopathy with kuru plaques from humans to small rodents.

An unusual case of spongiform encephalopathy was transmitted directly from a human to rats and mice. After serial passages, incubation periods were shortened to about six months in rats and four months in mice. Clinical symptoms were similar in rats and mice, including ruffled fur, arched back, bradykinesia, and hind limb paralysis. Pathologically, a spongy state, proliferation of astrocytes, and neuronal changes were observed. Electron microscopic observation of the parietal cortex of rats and mice disclosed many membrane-bound vacuoles in the neuropil, predominantly in dendrites. In the pons of mice, intramyelinic vacuoles and accumulation of extracellular fluid were prominent. The clinicopathological symptoms of the affected animals resembled those in other experimental spongiform encephalopathies, especially scrapie.

Animals↗

PubMed1989

Cytochrome c oxidase--deficient myogenic cell lines in mitochondrial myopathy.

In a patient with mitochondrial myopathy, the defect of cytochrome c oxidase activity was restricted to some muscle fibers. To isolate cell lines with or without oxidase activity from a single muscle sample, primary cultured cells were transformed by replication origin-defective simian virus 40, and then cloned. The clones were examined by cytochemical staining for cytochrome c oxidase activity. Eight myogenic clones were completely devoid of activity, while the other myogenic and nonmyogenic clones were not. Deficiency of cytochrome c oxidase was stable in culture for at least a year after serial passaging. The amount of mitochondrial DNA in cytochrome c oxidase-deficient cells was the same as in control cells, and no deletion in the mitochondrial DNA was detected. Protein synthesis in mitochondria of the subunits of cytochrome c oxidase and subunit 6 of the ATP synthase complex was markedly decreased, whereas synthesis of the other subunits encoded by mitochondrial DNA was normal. These cloned cell lines provide an excellent system for clarifying the cause of mitochondrial myopathy and for investigating nuclear-mitochondrial genetic interaction.

Adolescent↗

PubMed1986

The fate of the primary diploid population during spontaneous transformation of growth factor-supplemented murine cell cultures.

Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine. Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a "crisis"; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings. They were replaced, either gradually or precipitously, by various heteroploid cell fractions. In three of four independent cultures, these "established" cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages. In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern. Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements. These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.

Animals↗

PubMed1975

Radioactive antigen suicide of an anti-DNP (2,4-dinitrophenyl) clone. I. Recovery and escape from clonal dominance by suicide resistant precursors.

An anti-2,4-dinitrophenyl (DNP) clone line, maintained in vivo by serial passage through irradiated syngeneic recipients, was exposed to 125I-labeled dinitrophenylated L-Tyr-L-Glu-L-Lys copolymer antigen of high specific activity. Hapten-specific suicide was obtained, though with more difficulty than for a heterogeneous secondary response. The recovery was followed and was shown to occur either with the same clones or with new clones, depending on conditions. After suicide at a late passage of the clone, recovery by new clones occured very rapidly. This indicated that there was a background heterogeneity in the clone system, normally suppressed by clonal dominance and that some of these new clones were suicide resistant.

Animals↗

PubMed2004

Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR.

Despite the high incidence of EGFR amplification in patient glioblastoma multiforme (GBM) tissues, only a single GBM cell line, of the many described in the literature, is known to contain and maintain amplified EGFR. Because EGFR mutations in GBM manifest primarily, if not exclusively, in amplified form, it follows that the availability of cell lines with mutation of endogenous EGFR would also be in short supply. In fact, there are no GBM cell lines harboring the common EGFR mutants described in patient GBMs. These observations suggest that in vivo environments select for EGFR amplification, whereas in vitro environments, specifically cell cultures, select against this gene alteration. To contrast directly the fates of EGFR amplification in vivo and in vitro, as well as to examine potential relationships between EGFR amplification and mutation, we have established and maintained GBM explants as xenografts by serial passaging in nude mice. Analysis of EGFR copy number and EGFR mutation status in 11 patient tumors and their corresponding xenografts, as well as the monitoring of EGFR copy number during the establishment of a GBM cell line from a xenograft with amplified EGFR, indicated that selection for EGFR amplification is an in vivo phenomenon. Furthermore, our data indicated that EGFR mutation occurs only in tumors with EGFR amplification and showed that the selection of amplified mutant EGFR over amplified wild-type EGFR as a xenograft occurred rapidly and completely during tumor propagation.

Animals↗

PubMed1992

Losses of 3p, 11p, and 13q in EJ/ras-transformable simian virus 40-immortalized human uroepithelial cells.

Five independent clones of Simian virus 40 (SV40)-immortalized human uroepithelial cells (CK/SV-HUC) were established after transfection of HUC cultures from the same tissue donor with plasmids encoding SV40 large T and small t antigen genes. Each CK/SV-HUC clone contained a unique SV40 integration site, and all expressed similar levels of SV40 mRNA. All five clones were nontumorigenic, but clones 2, 4, and 5 tumorigenically transformed after transfection at P19 with mutant EJ/ras and also spontaneously after 40 serial passages in vitro. In contrast, CK/SV-HUC clones 1 and 3 did not transform when either approach was used. These differences in transformability among CK/SV-HUC clones could not be predicted based on differences in SV40 gene expression nor on any in vitro growth property tested. In cytogenetic analyses, a transformable clone showed losses of three chromosome arms containing putative cancer suppressor gene regions, including 3p14----pter, 13q, and 11p, whereas the nontransformable clones showed none of these losses. Thus these data indicate that genetic losses on 3p, 11p, and 13q may contribute to tumorigenic transformation of SV40-immortalized human uroepithelial cells.

Cell Division↗

PubMed1994

Nonrandom loss of human chromosome 3 fragments from mouse-human microcell hybrids following progressive growth in SCID mice.

Microcell hybrid lines of A9 mouse fibrosarcoma containing complete or partially deleted human chromosomes 3 (chr. 3) were inoculated into SCID mice. Cell lines derived from the tumors were examined by fluorescent in situ hybridization for the status of the transferred human chromosome and by PCR for marker loss. The SCID tumors arising after the inoculation of 10(5) cells were passaged serially in vivo and regularly showed loss of four markers; D3S1029 (3p21.3-21.2), AP20R (3p22-21.3, D3S32 (3p21.3-p21.2), and THRB (3p24). This regularly deleted region is bordered by markers GNA12 (3p21.1-p21.3) and VHL (3p25) that were maintained in a fraction of tumors. Fragments derived from the long arm of chromosome 3 and corresponding markers in the 3q26-q28 region were retained in all tumors. Our findings may be related to the postulated presence of tumor suppressor genes in the 3p24-p21 region as indicated by the frequent deletion of this region in renal and small cell lung carcinomas and other solid tumors. The technically cumbersome identification of suppressor genes may be supplemented by an "elimination test" based on analogous principles.

Animals↗

PubMed1979

The differentiation of clonal rat yolk sac tumor cell lines cultivated with dibutyryl-cyclic 3', 5'-adenosine monophosphate.

Five rat yolk sac tumor cell lines were cloned from a yolk sac tumor line which originally arose following fetectomy. The doubling time of each of the cloned tumor lines was about 50 h. All of the cloned tumor cell lines synthesized and secreted AFP and albumin but there was a gradual decrease in the synthesis of these proteins during serial passage. The cells formed clusters which looked like vitelline ducts or the parietal yolk sac and they also had basement membranes which closely resembled Reichert's membrane. When the cloned cell lines were cultivated in the presence of 1 mM dibutyryl cyclic-AMP, myotube-like and neuron-like cells appeared. Acetylcholine esterase and creatine phosphokinase activity were present when myotube-like cells were present whereas acetylcholine esterase activity predominated when neuron-like cells were present.

Acetylcholinesterase↗