Human Embryonal Rhabdomyosarcoma Cell Lines Research Articles

Berberine and palmatine inhibit the growth of human rhabdomyosarcoma cells.

A natural isoquinoline alkaloid, berberine, has been known to exhibit anti-tumor activity in various cancer cells via inducing cell cycle arrest. However, it has not been investigated whether berberine and its analogs inhibit the growth of rhabdomyosarcoma (RMS), which is the most frequent soft tissue tumor in children. The present study examined the anti-tumor effects of berberine and palmatine on expansions of three human embryonal RMS cell lines; ERMS1, KYM1, and RD. Intracellular incorporation of berberine was relatively higher than that of palmatine in every RMS cell line. Berberine significantly inhibited the cell cycle of all RMS cells at G1 phase. On the other hand, palmatine only suppressed the growth of RD cells. Both of berberine and palmatine strongly inhibited the growth of tumorsphere of RD cells in three-dimensional culture. These results indicate that berberine derivatives have the potential of anti-tumor drugs for RMS therapy.Abbreviations: ARMS: alveolar rhabdomyosarcoma; ERMS: embryonal rhabdomyosarcoma; RMS: rhabdomyosarcoma.

Inhibition of differentiation by transforming growth factor ß1 in rhabdomyosarcoma cells

OBJECTIVE To study the effect of transforming growth factor s1 (TGF-β1) on differentiation of rhabdomyosarcoma (RMS) cells METHODS RD (human embryonal RMS cell line) cells, cultured in differentiation medium containing 9-cis retinoic acid (9CRA), were treated with TGF-β1. Proliferation of the cells was examined by the MTT assay. The differentiation specific proteins (sarcomeric actin and MyHC) and myogenic transcription factors (MyoD1 and myogenin) in the RD cells were assessed by immunofluorescence staining. RESULTS Compared to control cells, treatment with lower concentra-tions of TGF-s1 (0.1 and 0.2 ng/mL) induced an increase in OD values after 4d ( P < 0.01), whereas higher concentrations of TGF-s1 (2 and 5 ng/mL) led to a reduction of cell viability ( P < 0.01). After exposure to 9CRA, the viability of the cells decreased significantly ( P < 0.01), while sarcomeric actin, MyHC and myogenin were induced. These changes were antagonized by TGF-β1 (0.1 ng/mL). No changes were observed in expression of MyoD1. CONCLUSION The RMS cells, derived from myogenic progenitors are committed to a myogenic fate, but are arrested in the differentiation course by the addition of TGF-s1 which represses some of the myogenic transcription factors.

Multifactor complex containing B element binding factor, BBF, and repressors regulate the human alpha 1(III) collagen gene (COL3A1).

Type III collagen is found in fetal skin and blood vessels. Previously, we characterized the proximal promoter of the human alpha1(III) collagen gene (COL3A1) using the human rhabdomyosarcoma cell line, A204, and NIH3T3 cells (Yoshino et al., Biochim Biophys Acta, 2005). In the present study, we further analyzed this promoter using additional cell lines, namely a human embryonal rhabdomyosarcoma cell line (RD) and bovine vascular smooth muscle cells (vSMCs), both of which show high expression of type III collagen. Using a luciferase assay, electrophoretic mobility shift assays (EMSA), and DNase footprinting assay, 2 types of multifactor complexes were shown to bind to the DNA region in the vicinity of the B element (- 80 to - 58), depending on the cell type. Next, we used cells stably transfected with a GFP-linked type III collagen promoter fragment for analysis of promoter expression. Usually, transfected cells retained the characteristics of the original cells. However, in several clones derived from RD cells, promoter expression as well as cell shape changed to patterns characteristic of the A204 cell line. Nuclear factors expressed by these clones were also characteristic of the A204 line.

Identification of new genes related to the myogenic differentiation arrest of human rhabdomyosarcoma cells

Rhabdomyosarcoma is a soft tissue tumor committed to the myogenic lineage but arrested prior to terminal differentiation. To identify new genes implicated in the block in myogenic differentiation of rhabdomyosarcoma cells and those responsible for their proceeding along the myogenic pathway we used cDNA microarrays to compare gene expression profiles of two clones of the human embryonal rhabdomyosarcoma cell line RD with different myogenic potentials: RD/12, which is unable to differentiate, and RD/18, which shows elements able to terminally differentiate, as defined by the expression of myosin heavy chain (up to 50% of the population) and the formation of multinucleated myotube-like structures. We identified 80 genes differentially expressed by the two clones. Differentiating RD/18 cells overexpressed the myogenic transcription factor myogenin along with known myogenic markers; myogenin transfection into undifferentiated RD/12 cells was able to revert the phenotype giving rise to 94% of clones displaying a differentiated morphology. RD/18 cells also expressed several genes not known to be expressed in rhabdomyosarcoma or muscle cells, such as pigment-epithelium derived factor and endothelin-3. Poorly differentiated RD/12 cells, along with genes related to mesenchymal lineage or early myogenic commitment, also expressed genes not previously known to be related to the differentiation block of human rhabdomyosarcoma, such as monocyte chemotactic protein-1, connective tissue growth factor and insulin-like growth factor binding protein-5. Differential expression of these genes in a time course of differentiation suggested their potential roles as either new myogenic markers or repressors of differentiation. These results identify a cluster of new genes related to the aberrant myogenic differentiation program of human rhabdomyosarcoma cells.

Characterization of a new human embryonal rhabdomyosarcoma cell line, RMS-GR.

A human tumor cell line designated RMS‐GR was established from an embryonal rhabdomyosarcoma. The monolayer cells were polygonal, round or spindle‐shaped. The RMS‐GR cell line became stable with a doubling time of 42 h. Tumorigenicity of the cells was confirmed by hetero‐transplantion into nude mice. Electron microscopic images showed typical cytoplasmic inclusion of aggregated intermediate filaments and myofibril‐like thin filaments. The expression of desmin, vimentin, actin and human myoglobin was recognized by cytofluorometric analyses, and a large fraction of CK‐MM and small fractions of CK‐BB and MCK‐1 isoenzymes were found. Chromosomal analysis showed that the modal chromosome number was consistently near triploid with structural abnormalities mostly involving chromosomes 1, 3 and 8, and additional unidentified markers. No alteration of chromosome 2 was observed. The RMS‐GR cell line may provide a system to identify genes which are involved in the pathogenic mechanism of rhabdomyosarcomas, and to investigate the modulation of myogenic differentiation.

Actinomycin D treatment leads to differentiation and inhibits proliferation in rhabdomyosarcoma cells

Human embryonal cell line RD is derived from rhabdomyosarcoma, a tumor of childhood that arises from rhabdomyoblasts probably arrested somewhere along their pathway to maturation. Because actinomycin D is a drug of choice in the treatment of rhabdomyosarcomas, and because it has been used to induce differentiation as an alternative therapy for myeloproliferative syndromes, we treated RD cells with different concentrations of actinomycin D and evaluated the effects on growth and differentiation. Actinomycin D treatment in vitro caused time- and dose-dependent growth inhibition. Interestingly, RD cells treated with low doses (2.85 and 5.7 nmol/L) of actinomycin D for 6 days showed morphologic and phenotypic differentiation, with increased expression of desmin, α-actinin, and tropomyosin. However, treatment with 11.4 nmol/L actinomycin D strongly inhibited growth and had cytotoxic effects that prevented the cells from attaining myogenic differentiation. We conclude that exposure of this human embryonal rhabdomyosarcoma cell line to low concentrations of actinomycin D released the neoplastic cells from their blockade, allowing them to recover normal myogenic development. We suggest a potential role for differentiation therapy in the treatment of rhabdomyosarcomas.

Chemical modifications on the acyclic moiety of 3-(2-hydroxyethoxy)-1-alkoxypropyl nucleobases. 2. Differentiation and growth inhibition in rhabdomyosarcoma cells after exposure to a novel 5-fluorouracil acyclonucleoside

Abstract A series of new 5FU acyclonucleoside analogues has been synthesized and tested for their in vitro cytotoxicity versus HT-29 colon carcinoma. The only active compound is 14 , which is 8-fold less active than 5-fluorouracil. The rest of the newly prepared compounds showed no significant activity. We selected 14 as the drug in the treatment of an human embryonal cell line RD derived from rhabdomyosarcoma. Such treatment caused time-dependent growth inhibition. Interestingly, RD cells treated with 14 at a concentration of 90 μM for 6 days showed phenotypic differentiation, with increased expression of desmin, α-actinin and tropomyosin. We conclude that exposure of this human embryonal rhabdomyosarcoma cell line to a 90 μM concentration released the neoplastic cells from their blockade, allowing them to recover normal myogenic development.

Ara-C Treatment Leads to Differentiation and Reverses the Transformed Phenotype in a Human Rhabdomyosarcoma Cell Line

Rhabdomyosarcoma (RMS) is an embryonal tumor of childhood that arises from primitive skeletal muscle-forming cells (rhabdomyoblasts) probably arrested and transformed along the normal myogenic pathway to maturation. Since Ara-C is an antitumor agent known to induce differentiation in human acute myelogenous leukemia, also presumably a disorder of cellular maturation, we treated RD, a human embryonal RMS cell line, with Ara-C and evaluated its effect on growth and differentiation. Ara-C treatment of RD cells in vitro caused a dose-dependent growth inhibition in the absence of cytotoxicity. Interestingly, RD cells treated with 5 × 10 -7 M Ara-C for 4 days were able to recover logarithmic growth after the removal of the drug from the media. A reexposure of these cells to Ara-C led to morphological and biochemical changes related to differentiation, including the appearance of an increased number of multinucleated cells that expressed muscle-specific actin and skeletal muscle myosin heavy chain (MHC) (fast). In vivo studies demonstrated that RD cells pretreated with 5 × 10 -7 M Ara-C lost their ability to form tumors in nude mice. We conclude that treatment of this human embryonal RMS cell line with Ara-C results in marked growth inhibition in vitro, loss of tumorigenicity in vivo, and the expression of biochemical markers present in a more differentiated phenotype. These data suggest a potential role for differentiation therapy as a therapeutic approach in RMS.

Cytogenetic and cellular characteristics of a human embryonal rhabdomyosarcoma cell line, RMS-YM

A human tumour cell line, designated RMS-YM, was established from a childhood rhabdomyosarcoma. The monolayer cells were polygonal, round or spindle-shaped. The cells became multilayered and formed many focal piles when confluent. RMS-YM became stable with a doubling time of about 30 h and has been maintained for 104 passages to date. Tumourigenicity of the cells was confirmed by heterotransplantation into nude mice. Morphological features were similar to those of the primary tumour, and myofibrils were found by electron microscopy. The expression of desmin and human myoglobin, and high levels of striated muscle system specific enzymes were recognised. Chromosomal analysis revealed possible gene amplification in the form of homogeneously staining regions. Oncogene analysis was performed on the primary tumour and the cell line, but neither N-myc nor N-ras genes were amplified, nor were Ki-ras, Ha-ras or N-ras genes mutated at the 12th, 13th and 61st codons. The RMS-YM cell line may provide a system to identify novel genes which are amplified in rhabdomyosarcoma.

Karyotypic characterization of a new human embryonal rhabdomyosarcoma cell line

Chromosomal analysis of an advanced recurrent rhabdomyosarcoma of the embryonal type was performed on cell cultures in the 9th passage of in vitro cultivation. This tumor showed a modal karyotype of 54 and was characterized by multiple numerical and structural chromosome abnormalities, all present in high frequencies. Abnormalities observed in 100% of the cells included a der(1) chromosome with a short unidentified insertion between q31 and q32; a der(1) chromosome, arising from insertion at the same breakpoint of a longer segment with a duplicated 1q31 band and translocation of 13q23→qter to 1p36, a deleted tetrasomic 13q23→qter, and a der(4) chromosome showing 1p36→pter translocated to 4p13. Other common abnormalities included trisomy of chromosomes 8, 13, and 9p, deletions of chromosomes 6, 10, 11, and 12, and presence of marker chromosomes. Characterization of the established line at the 38th passage evidenced the persistence of both the modal karyotype and all the numerical and structural abnormalities previously found. The results of this study provide further evidence of the major involvement of alterations in chromosome 1 in the progression of rhabdomyosarcoma.

High intrinsic radiosensitivity of a newly established and characterised human embryonal rhabdomyosarcoma cell line.

A new human rhabdomyosarcoma cell line (HX170c) has been established from a paratesticular embryonal tumour in a 5-year-old male. The cells grew as an adherent monolayer with a doubling time of 32 h and showed pleomorphic features. Intermediate filament analysis revealed the line to be mesenchymal in origin (reactivity to vimentin and desmin antibodies). The line was tumorigenic in nude mice, possessed elevated levels of creatine phosphokinase (mainly of the MM isoenzyme form) and had a near diploid mean chromosome number of 50. In vitro cell cloning determinations gave colony forming efficiencies of 0.01% in soft agar and 24% in a monolayer anchorage-dependent assay. Radiosensitivity determinations using a monolayer clonogenic assay with feeder layer support showed the cells to be among the more radiosensitive human tumour cell types (surviving fraction at 2 Gy of 0.26) that have been investigated. Furthermore, experiments utilising continuous low dose rate radiation at 3.2 cGy min-1, showed that, under these experimental conditions, the cells possessed only a very low capacity to recover from radiation-induced damage (dose reduction factor at 1% cell survival of 1.07 for 150 versus 3.2 cGy min-1). As other human tumour cells of an embryonal cell origin (e.g. neuroblastoma and germ cell tumours of the testis) have also been shown to be radiosensitive it appears that sensitivity to radiation may be a common property of this group of tumours.