tretinoin and Rhabdomyosarcoma

Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours.. We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS.. The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Child; DNA Methylation; Enhancer of Zeste Homolog 2 Protein; Enzyme Inhibitors; Humans; Rhabdomyosarcoma; Tretinoin

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current treatment strategies do not cure most children with recurrent or high-risk disease, underlying the need for novel therapeutic approaches. Retinoic acid has been shown to induce differentiation in a variety of cells including skeletal myoblasts and neuroblasts. In the setting of minimal residual disease, retinoic acid improves survival in neuroblastoma, another poorly differentiated childhood tumor. Whether such an approach is useful for rhabdomyosarcoma has not yet been investigated. Several in vitro studies have demonstrated an appreciable effect of retinoic acid on human RMS cellular proliferation and differentiation.. We assessed the efficacy of ATRA on rhabdomyosarcoma, in vitro and in vivo, using cell lines and xenografts.. ATRA slowed RMS cell proliferation, and promoted a more differentiated myogenic phenotype in both alveolar and embryonal RMS cell lines. Treatment of cultured murine myoblasts with retinoids increased Myogenin expression, but did not induce cell cycle arrest. Despite the favorable in vitro effects, ATRA failed to delay relapse of minimal residual disease using human RMS xenografts in immuno-suppressed NOD-SCID (NSG) mice. Interestingly, tumors that recurred after ATRA treatment showed evidence of enhanced muscle differentiation.. Our results indicate that ATRA could increase the expression of some genes associated with muscle differentiation in rhabdomyosarcoma cells, but there was no benefit of single-agent therapy in an MRD model, likely because cell cycle arrest was uncoupled from the pro-differentiation effects of retinoids.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Cycle Checkpoints; Cell Differentiation; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Myoblasts, Skeletal; Rhabdomyosarcoma; Tretinoin; Xenograft Model Antitumor Assays

Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Histone Deacetylases; HL-60 Cells; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Rhabdomyosarcoma; RNA Splicing Factors; RNA, Small Interfering; Transcription Factors; Transcriptional Activation; Tretinoin

Rhabdomyosarcoma (RMS) is the most frequent sporadic soft tissue sarcoma of childhood and adolescence. The overall 5-year survival rate for patients with RMS is 70% with the use of surgery, radiation, and chemotherapy. Novel therapeutic approaches are necessary to improve on these outcomes particularly among the more aggressive alveolar RMS (ARMS) and late stages of disease, where 5-year survival is less than 20%. Retinoids have been successfully used in the treatment of acute promyelocytic leukemia (APML) and neuroblastoma.. However, analysis of retinoids as a differentiating agent for RMS has been incomplete. This work examined the ability of retinoic acid (RA) to promote differentiation of RMS cell lines by examining the expression of myogenic proteins in five RMS cell lines in response to All-trans Retinoic Acid (ATRA) or 9-cis retinoic acid (CRA).. Analysis of growth curves indicates that both retinoids suppress cell growth of Rh4 and Rh28. RD cells only responded to-CRA whereas Rh30 and Rh18 did not respond. Following treatment with ATRA FACS analysis showed an altered cell cycle with the same pattern as the growth curves. ATRA altered cellular morphology of two cell lines, Rh4 and Rh28, and induced Troponin T expression in these cells suggesting a differentiating effect.. These studies suggest that retinoids are effective inducers of growth arrest and differentiation in some RMS cell lines, and offer a basis for further in vivo testing in mice of ATRA as a potential approach to ARMS treatment.

Topics: Alitretinoin; Antineoplastic Agents; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Ethanol; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mutation; Rhabdomyosarcoma; Sensitivity and Specificity; Tretinoin; Troponin T; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Differentiation therapy with retinoic acid has been considered a potential approach for treating rhabdomyosarcoma. Analysis of retinoids as differentiating agents for rhabdomyosarcoma is, however, rendered incomplete by the fact that some rhabdomyosarcoma cell lines are retinoic acid resistant. Therefore, the aim of the present work was to study the effect of all-trans-retinoic acid on two rat tumour cell lines, derived from the same rhabdomyosarcoma tumour model (i.e. the moderately differentiated low metastatic F21 cell line and the poorly differentiated high metastatic S4MH cell line), to discover how degree of differentiation and glutathione metabolism influence response to this retinoic acid derivative. We observed that whereas in the S4MH cell line all-trans-retinoic acid induced a significant inhibition of tumorigenic potential, in F21 cells all-trans-retinoic acid enhanced tumour growth and only at a higher dose was there a slight antiproliferative effect. These effects were in consonance with the activity level of gamma-glutamyltranspeptidase, which was significantly increased in F21 cells, but not in S4MH cells, in response to the all-trans-retinoic acid-induced increase in reactive oxygen species. The pro-tumour effect observed in F21 cells was reversed by adding buthionine sulphoximide, a specific cellular glutathione-depleting agent, to the all-trans-retinoic acid treatment. This combination produced a decrease in gamma-glutamyltranspeptidase activity, and an increase in oxidative stress and apoptosis. Our findings suggest that the response to all-trans-retinoic-acid of the tumour cell lines studied is influenced by the strong relationship between intracellular glutathione content, gamma-glutamyltranspeptidase activity and degree of differentiation of the rhabdomyosarcoma cell line, and that this relationship should be taken into account when identifying 'retinoid-sensitive' tumours.

Topics: Animals; Antineoplastic Agents; Apoptosis; Buthionine Sulfoximine; Cell Differentiation; Cell Proliferation; Drug Combinations; Female; gamma-Glutamyltransferase; Glutathione; Rats; Rats, Wistar; Reactive Oxygen Species; Rhabdomyosarcoma; Transplantation, Homologous; Tretinoin; Tumor Burden; Tumor Cells, Cultured

All-trans-retinoic acid (ATRA) promotes cell differentiation. We have studied its effect on the local recurrence and metastatic spreading of an experimental rhabdomyosarcoma in rats.. syngenic rhabdomyosarcoma cells (S4MH) were inoculated s.c. in male WAG/RijCrl rats. After 25 days tumors were excised and a 40% hepatectomy was performed for all animals. Ten days later the rats were sacrificed and a thorough necropsy was performed. The animals were randomly allocated to receive daily doses of ATRA (5 mg/kg, i.p.) or its solvent (Clinoleic/ethanol 90/10), starting three days before surgery until the end of the experiment.. ATRA reduced the incidence of local recurrence from 70 to 33% (p < 0.05), but the tumor size was not altered (1.8 vs. 2.0 cc). Regarding inguinal metastasis, there was a six-fold decrease (0.2 vs. 1.2 cc; p < 0.05) in mean tumor volume, although the rate of this proliferation increased sharply (86 vs. 29%; p < 0.05) for treated animals. The volume of the retroperitoneal tumor masses also decreased with ATRA (0.7 vs. 5.1 cc; p < 0.05), but the difference in rate was not significant (71 vs. 67%). Lung metastases, which were present in 100% of control animals, were found in only 33% of treated rats, while the mean number of metastatic foci dropped from 26.7 to 5.7 (p < 0.05).. Protocols including retinoid administration prior to and following primary tumor excision could help in controlling both recurrence and metastatic progression in surgically treated rhabdomyosarcoma.

Topics: Animals; Antineoplastic Agents; Disease Progression; Liver Neoplasms, Experimental; Male; Muscle Neoplasms; Neoplasm Recurrence, Local; Neoplasm Transplantation; Rats; Rats, Inbred Strains; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Mice; Muscle, Skeletal; Retinoid X Receptor beta; Retinoid X Receptor gamma; Rhabdomyosarcoma; Signal Transduction; Transcription, Genetic; Transfection; Tretinoin

Human rhabdomyosarcoma cells produce autocrine and paracrine growth factors that can sustain their growth and malignancy. Here we report constitutive production of stem cell factor (SCF) by 5 of 5 human rhabdomyosarcoma cell lines both of alveolar and embryonal histotype. SCF production, ranging from 30 to 162 pg/ml, was independent from the degree of myogenic differentiation and was not modulated by exogenous addition of retinoic acid (RA) or tumor necrosis factor-alpha (TNF-alpha). Four of 5 rhabdomyosarcoma cell lines expressed the mRNA for SCF receptor c-kit, while the 5th cell line became weakly positive for c-kit mRNA only after stimulation with retinoic acid. On the cell surface, c-kit protein was detectable at very low levels in only 1 of 5 rhabdomyosarcoma cell lines and was not up-regulated by RA or TNF-alpha. Addition of anti-c-kit and anti-SCF blocking antibodies, or of exogenous SCF did not alter the in vitro growth ability of rhabdomyosarcoma cells. In conclusion, our data show that rhabdomyosarcoma cells produce consistent amounts of SCF but did not demonstrate autocrine growth modulation. SCF secretion may thus have a paracrine, rather than an autocrine activity in this tumor.

Topics: Autocrine Communication; Cell Division; Humans; Paracrine Communication; Proto-Oncogene Proteins c-kit; Rhabdomyosarcoma; RNA, Messenger; Stem Cell Factor; Tretinoin; Tumor Cells, Cultured

Human stromelysin-3 and interstitial collagenase are matrix metalloproteinases whose expression by stromal cells in several types of carcinomas has been associated with cancer progression. We compared here the regulation of the expression of both proteinases by retinoids in human fibroblasts. Physiological concentrations of retinoic acid were found to simultaneously induce stromelysin-3 and repress interstitial collagenase. In both cases, the involvement of a transcriptional mechanism was supported by run-on assays. Furthermore, in transient transfection experiments, the activity of the stromelysin-3 promoter was induced by retinoic acid through endogenous receptors acting on a DR1 retinoic acid-responsive element. The ligand-dependent activation of the receptors was also investigated by using selective synthetic retinoids, and we demonstrated that retinoic acid-retinoid X receptor heterodimers were the most potent functional units controlling both stromelysin-3 induction and interstitial collagenase repression. However, specific retinoids dissociating the transactivation and the AP-1-mediated transrepression functions of the receptors were found to repress interstitial collagenase without inducing stromelysin-3. These findings indicate that such retinoids may represent efficient inhibitors of matrix metalloproteinase expression in the treatment of human carcinomas.

Topics: Carcinoma; Collagenases; Drug Synergism; Enzyme Repression; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 11; Metalloendopeptidases; Promoter Regions, Genetic; Receptors, Retinoic Acid; Rhabdomyosarcoma; Transcription Factor AP-1; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

A newly established cell line, designated NRS-1, was derived from an alveolar rhabdomyosarcoma that developed in the left forearm of a 7-year-old girl. The cell line had a t(2; 13) chromosomal translocation. Reverse transcription-polymerase chain reaction demonstrated that 5' PAX3-3' FKHR chimeric transcript was expressed in NRS-1 cells. NRS-1 cells showed myogenic differentiation without any particular stimulus in vitro and exhibited various kinds of muscle markers. All-trans retinoic acid promoted cell differentiation in the myogenic direction. Transforming growth factor-beta (TGF-beta) inhibited myogenic differentiation of those cells and promoted cell proliferation.

Topics: Base Sequence; Cell Differentiation; Cell Division; Cell Line, Transformed; Child; Female; Forearm; Humans; Karyotyping; Molecular Sequence Data; Rhabdomyosarcoma; Skin Neoplasms; Transforming Growth Factors; Tretinoin

The clonal rat rhabdomyosarcoma cell line BA-HAN-1C is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. This cell line has been shown to be susceptible to differentiation induction with all-trans retinoic acid (RA). Since it is still unknown whether exclusively all-trans RA itself or also its metabolites can act as inductive compounds in our cell line, we exposed BA-HAN-1C cells to the metabolites 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA. Exposure to these RA metabolites resulted in a significant inhibition of proliferation (P < 0.001) and induction of cellular differentiation, as evidenced by a significant increase in the number of myotube-like giant cells (P < 0.05) and a significant increase in creatine kinase activity (P < 0.05). However, differences in the inductive potency of these RA metabolites became apparent. Furthermore, RA metabolites exhibited a significantly weaker (P < 0.05) inductive activity when compared to all-trans RA. Summarizing our results we could demonstrate that the endogenous metabolites 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA are not merely deactivated cellular excretion products of all-trans RA, but potent inducers of differentiation and inhibitors of proliferation, possibly contributing to the complex physiological actions of retinoic acid.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line, Transformed; Creatine Kinase; Microscopy, Electron; Microscopy, Electron, Scanning; Rats; Rhabdomyosarcoma; Tretinoin

Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 10 mM promoted myogenic differentiation of the human alveolar rhabdomyosarcoma cell line KFR. These concentrations inhibited DNA synthesis of the cells in a dose-dependent manner without significant effect on cell viability. The morphological differentiation of small mononuclear elements to terminal, elongated multinuclear structures resembling myotubes was accompanied by the expression of skeletal muscle myosin. The proportion of differentiated myosin-positive cells which was around 0.8-1.7% in control cultures 12 days after seeding was increased by NaPA treatment up to 47%. In the cytoplasm of differentiated cells, features of sarcomerogenesis were observed. These results suggest that NaPA is an effective inducer of rhabdomyosarcoma cell differentiation at concentrations that have been achieved in humans with no significant adverse effects.

Topics: Cell Differentiation; Humans; Microscopy, Electron; Muscles; Myosins; Phenylacetates; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

Three clonal subpopulations (A, B, C) isolated from the same rhabdomyosarcoma of the rat and differing in their degree of spontaneous differentiation were tested for their invasive potential before and after differentiation induction with retinoic acid (RA), N-monomethylformamide (NMF) and sodium butyrate (NaBut). Invasive potential was analysed in an in vitro assay using embryonic chick heart fragments in organotypic culture. In standard culture medium, all three subpopulations were shown to be invasive, progressively replacing the chick heart fragments within 7-11 days after confrontation. After exposure to RA, NMF or NaBut, marked differences in the invasive potential of these subpopulations were, however, observed. Subpopulation C exhibited a pronounced decline in invasive potential, as evidenced by a significant decrease (P = 0.005) in the proportion of chick heart fragments with advanced stages of invasion. This response, however, was confined to the differentiation-inducing agents RA and NaBut, which had also produced a marked increase in morphological and/or biochemical differentiation (P = 0.0001). In contrast, NMF, which had only minor effects on differentiation, failed to affect the invasive potential of subpopulation C. In subpopulation B, a transient inhibition of single cell invasion became evident after exposure to RA, whereas NMF and NaBut failed to affect the invasive potential in spite of minor effects on differentiation. In the least differentiated subpopulation A, which was shown to be refractory to the differentiation-inducing effects of RA, NMF and NaBut, there was also no observation of any reduction of invasive potential. The results of our study demonstrate that differentiation-inducing agents can significantly reduce the invasive potential of malignant tumors, although marked differences of response are to be expected between the different subpopulations of a tumor.

Topics: Animals; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Clone Cells; Creatine Kinase; Formamides; Isoenzymes; Neoplasm Invasiveness; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Division; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Isoxazoles; Phenylacetates; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

We have been evaluating the role of all-trans-retinoic acid (RA) in the differentiation and growth of human rhabdomyosarcoma (RMS) cell lines. Treatment of both embryonal (RD) and alveolar (RH30) human RMS cell lines with all-trans-RA resulted in a dose-dependent inhibition of cell growth with a maximal inhibition of 92 and 66%, respectively, at 5 x 10(-6) M. When 13-cis-RA was used under identical experimental conditions, maximal growth inhibition was 41 and 37%, respectively. This stereo-specific growth inhibition was not associated with morphological or biochemical evidence of myogenic differentiation. Furthermore, all-trans-RA demonstrated no evidence of competition with binding of insulin-like growth factor II (IGF-II), an autocrine growth factor in RMS, to its membrane receptor as evaluated by an [125I]IGF-I-receptor-binding assay. Attempts to rescue all-trans-RA growth-inhibited RMS cells with exogenous IGF-II resulted in no increase in growth compared to cells treated with all-trans-RA alone. We conclude that RA inhibits the growth of human RMS cell lines in a dose-dependent, stereo-specific manner, is not associated with differentiation, and does not appear to be directly related to IGF-II.

Topics: Base Sequence; Cell Differentiation; Cell Division; Humans; Insulin-Like Growth Factor II; Molecular Sequence Data; Rhabdomyosarcoma; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

The clonal rat rhabdomyosarcoma cell line BA-HAN-1C is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. Both the induction of differentiation by retinoic acid (RA) and by sodium butyrate (NaBut), as well as the inhibition of proliferation by fetal calf serum (FCS)-depleted medium uniformly resulted in the same effects. There was a significant (p less than 0.001) inhibition of proliferation and induction of cellular differentiation, as evidenced by a significant (p less than 0.05) increase in creatine kinase activity. Furthermore, after exposure to RA-supplemented or FCS-depleted medium, a significant (p less than 0.001) increase in the number of myotube-like giant cells was observed. These effects were preceded by a uniform enhancement of c-raf mRNA expression, which became evident 6 h after exposure to RA, NaBut and FCS-depleted media. C-raf mRNA expression persisted at an elevated level throughout the observation period of 5 days after exposure to RA or NaBut, whereas the increased expression of c-raf mRNA observed after FCS-depletion declined near to the basal level after only 24 h. Furthermore, a transient c-fos mRNA expression was observed 15 and 30 min after exposure to RA-supplemented and FCS-depleted medium but not after exposure to NaBut. The present results suggest a possible role of c-raf in the regulation of differentiation and proliferation of this cell line. Since all our experiments with RA, NaBut and FCS-depletion resulted in an early peak of c-raf mRNA expression, it is suggested that this early peak may be sufficient to trigger events crucial for differentiation and proliferation of BA-HAN-1C tumor cells.

Topics: Animals; Blotting, Northern; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Cell Fusion; Creatine Kinase; Gene Expression Regulation, Neoplastic; Microscopy, Electron; Proto-Oncogenes; Rats; Rhabdomyosarcoma; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

BA-HAN-IC is a clonal rat rhabdomyosarcoma cell line consisting of proliferating mononuclear tumor cells, some of which spontaneously fuse to form terminally differentiated post-mitotic myotubes. Exposure of BA-HAN-IC cells to retinoic acid (RA) or N-methylformamide (NMF) resulted in a significant inhibition of proliferation (p less than 0.001) and in cellular differentiation, as evidenced by a significant increase in the creatine kinase (CK) activity (p less than 0.05) and the number of terminally differentiated post-mitotic myotubes (p less than 0.001). Furthermore, between 5% (NMF) and 30% (RA) of the mononuclear tumor cells exhibited ultrastructural features of rhabdomyogenic differentiation, not observed in their mononuclear counterparts under standard growth conditions. Although BA-HAN-IC cells responded to both inducers of differentiation, differences in time course and magnitude of both increase of differentiation and growth inhibition were observed. These effects of RA and NMF were preceded by a marked enhancement of c-raf expression which became evident 6 and 12 hr after exposure to RA and NMF, respectively, and which persisted throughout the observation period of 5 days. Furthermore, a transient expression of c-fos could be observed 15 and 30 min after exposure to RA. Our results suggest that c-raf expression might be implicated in the differentiation process of BA-HAN-IC tumor cells.

Topics: Animals; Antineoplastic Agents; Formamides; Gene Expression Regulation, Neoplastic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-raf; Rats; Rhabdomyosarcoma; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

BA-HAN-1C is a clonal rat rhabdomyosarcoma cell line consisting of proliferating mononuclear tumor cells, some of which spontaneously fuse to form terminally differentiated postmitotic myotubelike giant cells. Exposure to retinoic acid resulted in an inhibition of proliferation and a marked increase in cellular differentiation. The number of myotubelike giant cells significantly increased, and about 30% of the mononuclear tumor cells exhibited morphological features of rhabdomyogenic differentiation which were not observed in the mononuclear cells of untreated cultures. Morphological differentiation was paralleled by an increase in total creatine kinase activity as a biochemical marker of differentiation. These effects of retinoic acid were preceded by an increased expression of proto-oncogene raf and transient expression of proto-oncogene fos. The maximum level of fos expression was observed at 15 min and of raf at 12 hr after exposure to retinoic acid. No expression of the proto-oncogenes src, myb, myc, ros, mos, erbA, and erbB was detected.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Gene Expression Regulation, Neoplastic; Proto-Oncogenes; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

Retinoic acid causes a significant inhibition of cell growth of the tumor cell line BA-HAN-1C. This growth inhibition is the same whether the cells are treated with a pulse dose of retinoic acid (RA) or continuously expand to RA. The determination of RA and its degradation products within the culture medium and in the cells showed that after 24 hours 13-cis-RA was the major retinoid in all cells (96 ng/10(6) cells); all-trans-RA represented 56 ng/10(6) cells. After 48 hours 4-hydroxy-RA and a small amount of 5,6-epoxy-RA was found in the cells and also in the culture medium. 4-hydroxy-RA increased up to 96 hours, whereas 13-cis- and all-trans-RA were not detectable in the cells after 96 hours. We conclude that the BA-HAN-1C cells take up and metabolize RA. Nonlinear fit analysis of the time behavior of the RA concentration in medium demonstrates that the RA uptake unexpectedly follows a mono-exponential time function. Discussion of the experimental results in connection with a proper compartment model shows that uptake and metabolism of RA cannot be described really by a first order kinetics. The mathematical analysis leads to a more complicated kinetic model with certain restrictions for the corresponding rate constants.

Topics: Animals; Cell Compartmentation; Cell Transformation, Neoplastic; Kinetics; Mathematics; Models, Theoretical; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

Three clonal subpopulations (A, B, C) isolated from the same rhabdomyosarcoma of the rat were tested and compared for their susceptibility to differentiation induction using retinoic acid (RA), dimethylformamide (DMF), and N-monomethylformamide (NMF). These subpopulations differ in that a block to spontaneous differentiation is imposed at different stages which are characteristic for each subpopulation. Whereas tumor cell proliferation was significantly inhibited (P less than 0.001) in all three subpopulations, the effects of RA, DMF, and NMF on tumor cell differentiation were strikingly heterogeneous. The response was most marked in subpopulation C, as evidenced by a significant increase in the number of terminally differentiated myotube-like giant cells (P less than 0.001) and in biochemical differentiation, as indicated by the creatine kinase activity (P less than 0.05). Between 5% (DMF and NMF) and 30% (RA) of the mononuclear cells in subpopulation C exhibited thick and thin myofilaments, which were never observed in the mononuclear cells of the control. In contrast, subpopulation A and B responded to RA, DMF, and NMF quite heterogeneously with an increase in biochemical differentiation, whereas terminally differentiated myotube-like giant cells were never observed. These results demonstrate that the therapeutic potential of differentiation induction in malignant tumors may be impaired by tumor heterogeneity.

Topics: Animals; Cell Differentiation; Cell Division; Cell Fusion; Clone Cells; Creatine Kinase; Dimethylformamide; Formamides; Isoenzymes; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

We report on the establishment of a model for differentiation induction in sarcomas, using the clonal rhabdomyosarcoma cell line BA-HAN-1C. This rhabdomyosarcoma cell line is composed of morphologically undifferentiated mononuclear stem cells, some of which spontaneously fuse to form terminally differentiated multinuclear myotube-like giant cells. The deprivation of fetal calf serum (FCS) or the exposure to retinoic acid, respectively, resulted in a significant inhibition of proliferation (P less than 0.001) and a marked increase in cellular differentiation as shown by a significant increase in the number of myotube-like giant cells (P less than 0.001) and in the creatine kinase activity (P less than 0.05) used as a biochemical marker of differentiation. Furthermore, after exposure to retinoic acid about 30% of the mononuclear tumour cells exhibited morphological features of rhabdomyogenic differentiation, such as bundles of thick and thin myofilaments, which had never been observed in the mononuclear cells of untreated cultures. These results confirm that the inverse linkage between proliferation and differentiation known from embryonic myogenesis is preserved in our rhabdomyosarcoma cell line. The failure to induce terminal differentiation by exposure to retinoic acid in all the cells of our clonal cell line indicates that some tumour cells might epigenetically be blocked from responding to retinoic acid. The temporary growth retardation observed after FCS-deprivation suggests that autocrine stimulation of proliferation may be operating in our cell line, too.

Topics: Animals; Cattle; Cell Differentiation; Cell Division; Cell Line; Fetal Blood; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

Topics: Animals; Blood; Cell Differentiation; Culture Media; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

BA-HAN-1C is a clonal rat rhabdomyosarcoma cell line composed of proliferating mononuclear cells, which partly fuse to terminally differentiated postmitotic myotube-like giant cells. The exposure to retinoic acid in vitro resulted in time- and dose-dependent changes of both cell differentiation and cell growth. The mononuclear cells revealed bundles of newly formed thick and thin myofilaments, never observed in untreated cultures, and exhibited signs of contact inhibition. In addition, there was a statistically significant increase (P less than 0.001) in the number of terminally differentiated postmitotic myotube-like giant cells and in the creatine kinase activity (P less than 0.05) which was used as a biochemical differentiation marker. At the same time cell growth was significantly inhibited (P less than 0.001) in vitro and a decrease in plating efficiency, as well as in saturation density, was observed. These data demonstrate that retinoic acid can suppress cell growth and simultaneously initiate differentiation in a malignant mesenchymal tumor cell line. However, despite the clonal nature of BA-HAN-1C, the complete status of terminal differentiation was not achieved by all tumor cells. The reason why not all tumor cells responded to retinoic acid is unknown at the present time and will have to be the subject of further studies.

Topics: Animals; Cell Differentiation; Cell Division; Cell Fusion; Cell Line; Creatine Kinase; Microscopy, Electron; Rats; Rhabdomyosarcoma; Tretinoin

N-homocysteine thiolactonyl retinamide was synthesized from trans retinoic acid and the free base of homocysteine thiolactone. In doses of 90-1800 mg/kg given i.p. in mixed lipid vehicle over nine weeks, the compound decreased to 60% of controls the number of lung tumors which was induced in A/J mice by 20 mg of ethyl carbamate. The highest dose also decreased the mean volume of lung tumors to 50% of controls, resulting in a total tumor volume of 30% of controls. Retinoic acid itself at 450 mg/kg was toxic, and no chemopreventive activity was observed. The free base and the lipophilic perchlorate salt of homocysteine thiolactone both increased the number of lung tumors to 114-117% of controls, indicating a co-carcinogenic effect. In C57BL/6N mice with transplanted MUO4 rhabdomyosarcoma, N-homocysteine thiolactonyl retinamide in a dose of 1000 mg/kg given over 11-21 days decreased the weight of the tumors to 30-70% of controls. These results show that N-homocysteine thiolactonyl retinamide has chemopreventive activity against chemical carcinogenesis and antineoplastic activity against a transplanted neoplasm.

Topics: Animals; Antineoplastic Agents; Homocysteine; Lung Neoplasms; Mice; Mice, Inbred A; Mice, Inbred C57BL; Neoplasm Transplantation; Rhabdomyosarcoma; Solubility; Tretinoin; Urethane